Category Archives: Protein Folding

As you age, your dementia risk increases rapidly. In fact, according to the Alzheimer's Association, your risk of Alzheimer's doubles every five years after the age of 65. However, there are some things you can do to lower your risk of developing cognitive impairmentincluding one you may be able to do in your very own bathroom. Experts say that by doing this one thing roughly three times per week, you can slash your odds of dementia in half over a 20 year period. Read on to find out which habit may stave off dementia, and how to do it safely.

RELATED:This Heartburn Medication Raises Your Dementia Risk 44 Percent, Study Says.

According to a 2020 study published in the journal Preventive Medicine Reports, "repeated heat exposure like sauna bathing" seems to be beneficial in preventing dementia development. The cohort study, which was conducted in Finland, utilized surveys and existing medical records from 13,994 middle-aged men and women who had not previously been diagnosed with dementia.

When the team compared the health data from those who took a sauna between nine and 12 times per month with that of those who did so four or fewer times per month, they found that those who regularly took a sauna had lower dementia risk. "During the first 20 years of follow-up, the dementia risk of those reporting 912 sauna baths per month (i.e., approximately three per week) was less than a half of the risk of those who had sauna baths only 04 times per month," the team wrote. "The reduction in the dementia risk was attenuated during the follow-up, but the decrease of the risk was still evident after nearly 40 years. Accordingly, a sauna bathing frequency of three times per week may be associated with a reduced risk of dementia," they added, noting that further research is required to verify the benefits.

RELATED:This Could Be Your First Sign of Dementia Years Before Diagnosis, Study Says.

Because sauna bathing is considered commonplace in Finland, nearly all of the study participants practiced the habit, and did so on average 6.03 times per month. These sessions typically lasted under 15 minutes at a temperature below 100 degrees Celsius (212 degrees Fahrenheit), the researchers wrote.

They found that "a straight stay in heat for five to 14 minutes per heat session vs. less than 5 minutes was suggestively related to a reduced risk [of dementia]. The most favorable sauna temperature for dementia protection was 8099 degrees Celsius [176-210 degrees Fahrenheit]," they wrote.

Wondering why a sauna would lower your dementia risk? The Finnish team said that while more research is needed on the matter, there are several possible answers related to "physiological, metabolic, and cellular changes which may affect brain function."

In particular, they say that a sudden elevation in temperature causes heat shock, which leads to the creation of something called "heat shock proteins." The researchers explained that these "are important regulators in normal cell functions and have an essential role in guarding and controlling protein formation. Because disturbances of protein construction and folding are central to the development of neurological diseases, heat shock proteins may be important in maintaining protein homeostasis in the brain."

Additionally, saunas may improve vascular and cardiovascular function, which increases cerebral blood flow and lowers inflammation. "It is possible that some of the effects of sauna in the brain are conveyed via reduced inflammation," the team wrote.

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Though the researchers found a lower risk of dementia in those who took a sauna on average three times per week, they also observed that those who did so in extremely hot temperatures were at significantly elevated risk of the brain disease. "Sauna heat which is too high may not be good for the brain. The dementia risk of those bathing in sauna temperatures higher than 100 degrees Celsius [212 degrees Fahrenheit] doubled compared to those bathing at temperatures lower than 80 degrees Celsius [176 degrees Fahrenheit] during the first twenty years of follow-up," the team warned.

However, most saunas in the U.S.like the one you may have at your gym, or, if you're lucky, in your homeare heated to temperatures between 150 and 195 degrees Fahrenheit. They typically include a thermometer and temperature controls. Be sure to check the sauna's settings before entering, and limit your time inside to no more than 15 minutes.

RELATED:If You Do This in Your Sleep, Get Checked for Dementia, Says Mayo Clinic.

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Doing This in the Bathroom Can Reduce Your Dementia Risk Best Life - Best Life

Significance

This study reveals a mechanism for effector perception by a plant NLR immune receptor that contains an integrated domain (ID) that mimics an authentic effector target. The Arabidopsis immune receptors RRS1 and RPS4 detect the Pseudomonas syringae pv. pisi secreted effector AvrRps4 via a WRKY ID in RRS1. We used structural biology to reveal the mechanisms of AvrRps4CWRKY interaction and demonstrated that this binding is essential for effector recognition in planta. Our analysis revealed features of the WRKY ID that mediate perception of structurally distinct effectors from different bacterial pathogens. These insights could enable engineering NLRs with novel recognition specificities, and enhance our understanding of how effectors interact with host proteins to promote virulence.

Plants use intracellular nucleotide-binding domain (NBD) and leucine-rich repeat (LRR)containing immune receptors (NLRs) to detect pathogen-derived effector proteins. The Arabidopsis NLR pair RRS1-R/RPS4 confers disease resistance to different bacterial pathogens by perceiving the structurally distinct effectors AvrRps4 from Pseudomonas syringae pv. pisi and PopP2 from Ralstonia solanacearum via an integrated WRKY domain in RRS1-R. How the WRKY domain of RRS1 (RRS1WRKY) perceives distinct classes of effector to initiate an immune response is unknown. Here, we report the crystal structure of the in planta processed C-terminal domain of AvrRps4 (AvrRps4C) in complex with RRS1WRKY. Perception of AvrRps4C by RRS1WRKY is mediated by the 2-3 segment of RRS1WRKY that binds an electronegative patch on the surface of AvrRps4C. Structure-based mutations that disrupt AvrRps4CRRS1WRKY interactions invitro compromise RRS1/RPS4-dependent immune responses. We also show that AvrRps4C can associate with the WRKY domain of the related but distinct RRS1B/RPS4B NLR pair, and the DNA-binding domain of AtWRKY41, with similar binding affinities and how effector binding interferes with WRKYW-box DNA interactions. This work demonstrates how integrated domains in plant NLRs can directly bind structurally distinct effectors to initiate immunity.

Plants coevolve with their pathogens, resulting in extensive genetic variation in host immune receptor and pathogen virulence factor (effector) repertoires (1). To enable host colonization, pathogenic microbes deliver effector proteins into host cells that suppress host immune responses and elevate host susceptibility by manipulating host physiology (2, 3). Plants have evolved surveillance mechanisms to detect and then activate defenses that combat pathogens, and detect host-translocated effectors via nucleotide-binding domain (NBD) and leucine-rich repeat (LRR)containing receptors (NLRs) (4). NLR genes are highly diverse, showing both copy-number and presence/absence of polymorphisms, and different alleles can exhibit distinct effector recognition specificities (5, 6). As described by the gene-for-gene model, plant NLRs usually recognize a single effector (7). However, NLRs capable of responding to multiple effectors are known (5, 8, 9).

NLRs typically contain an N-terminal Toll/interleukin-1 receptor (TIR) or coiled-coil (CC) domain, a central NBD (NB-ARC [NBD shared with APAF-1, various R proteins, and CED-4]), and a C-terminal LRR domain (6). In addition to these canonical domains, some NLRs have evolved to carry integrated domains that mimic effector virulence targets and facilitate immune activation by directly binding effectors (1015). Interestingly, integrated domain-containing NLRs (NLR-IDs) usually function with a paired helper NLR, which is required for immune signaling (10, 16).

The Arabidopsis NLR pair RRS1-R/RPS4 is a particularly interesting NLR-ID/NLR pair that confers resistance to bacterial pathogens Pseudomonas syringae and Ralstonia solanacearum, and also to a fungal pathogen (Colletotrichum higginsianum) where the effector is unknown (1720). RRS1-R contains an integrated WRKY domain near its C terminus (RRS1WRKY), which interacts with two structurally distinct type III secreted bacterial effectors, AvrRps4 from P. syringae pv. pisi and PopP2 from R. solanacearum (13, 14, 21, 22). The RRS1WRKY domain may mimic the DNA-binding domain of WRKY transcription factors (TFs), the putative virulence targets of AvrRps4 and PopP2, to enable immune perception of these effectors (13). Two alleles of RRS1 have been identified that differ in the length of the C-terminal extension after the WRKY domain (SI Appendix, Fig. S1). RRS1-R, from the accession Ws-2, has a 101amino acid C-terminal extension beyond the end of the WRKY domain, and can perceive AvrRps4 and PopP2, while RRS1-S from Col-0, which perceives AvrRps4 but not PopP2, is likely a derived allele with a premature stop codon, and has only an 18amino acid C-terminal extension (23). Most Arabidopsis ecotypes also carry a paralogous and genetically linked RRS1B/RPS4B NLR pair, which only perceives AvrRps4 (24). RRS1B/RPS4B share a similar domain architecture with RRS1/RPS4, including 60% sequence identity in the integrated WRKY domain.

AvrRps4 is proteolytically processed in planta to produce a 133amino acid N-terminal fragment (AvrRps4N) and an 88amino acid C-terminal fragment (AvrRps4C) (25, 26). Previous studies have highlighted the role of AvrRps4C in triggering RRS1/RPS4-dependent immune responses (25, 26). AvrRps4N has been reported to potentiate immune signaling from AvrRps4C (27, 28). PopP2 is sequence and structurally distinct from AvrRps4 and has an acetyltransferase activity that is likely related to its role in virulence. The structural basis of PopP2 perception by RRS1WRKY has been determined (29), but how RRS1WRKY binds AvrRps4C and whether this is via a shared or different interface to PopP2 is unknown.

Here, we determined the structural basis of AvrRps4C recognition by the integrated WRKY ID of RRS1. The recognition of AvrRps4C is mediated by the 2-3 segment of RRS1WRKY, the same region used to bind PopP2. This segment interacts with surface-exposed acidic residues of AvrRps4C. Structure-informed mutagenesis at the AvrRps4CRRS1WRKY interface identifies AvrRps4 residues required for proteinprotein interactions invitro and in planta and AvrRps4 perception and immune responses. Residues mediating the interaction of AvrRps4C and RRS1WRKY are conserved in both the RRS1BWRKY and the DNA-binding domain of WRKY TFs, and AvrRps4C mutants that prevent interaction with RRS1WRKY also disrupt binding to AtWRKY41. This supports the hypothesis that the RRS1WRKY mimics host WRKY TFs through a shared effector-binding mechanism. We also show that AvrRps4C prevents the interaction of RRS1WRKY and AtWRKY41 with W-box DNA, most likely via steric blocking, at the same WRKY domain site acetylated by PopP2.

To investigate how AvrRps4C interacts with the RRS1WRKY domain, constructs comprising residues 134 to 221 of AvrRps4C (the in planta processed C-terminal fragment) and residues 1194 to 1273 of RRS1-R (corresponding to the RRS1WRKY domain) were separately expressed in Escherichia coli and proteins were purified via a combination of immobilized metal-affinity chromatography (IMAC) via 6His tags and gel filtration (Superdex 75 26/60 and Superdex S75 16/60) (see SI Appendix, Materials and Methods for full details). We qualitatively assessed the interaction of purified AvrRps4C with RRS1WRKY using analytical gel filtration chromatography. Individually, the proteins displayed well-separated elution profiles. RRS1WRKY eluted at a volume (Ve) of 14.9 mL and AvrRps4C eluted at a Ve of 12.1 mL (Fig. 1A). Following incubation of a 1:1 molar ratio of the proteins, we observed a new elution peak with an earlier Ve of 11.8 mL, and a lack of absorption peaks for the separate proteins (Fig. 1A). This demonstrates complex formation invitro and suggests a 1:1 stoichiometry of the AvrRps4CRRS1WRKY complex.

AvrRps4C interacts with the WRKY domain of RRS1 invitro. (A) Analytical gel filtration traces (using a Superdex 75 10/300 column) for AvrRps4C alone (gold), RRS1WRKY alone (green), and AvrRps4C with RRS1WRKY (blue) with sodium dodecyl sulfatepolyacrylamide gels of relevant fractions. An equimolar ratio of AvrRps4C and RRS1WRKY was used for the analysis. AvrRps4C runs as a dimer invitro. Poor absorbance for AvrRps4C at 280 nm is due to its low molar extinction coefficient. (B) ITC titrations of AvrRps4C with RRS1WRKY. (B, Upper) Raw processed thermogram after baseline correction and noise removal. (B, Lower) The experimental binding isotherm obtained for the interaction of AvrRps4C and RRS1WRKY together with the global fitted curves (displayed in red) were obtained from three independent experiments using AFFINImeter software (61). Kd and binding stoichiometry (N) were derived from fitting to a 1:1 binding model.

We then determined the binding affinities of the interaction using isothermal titration calorimetry (ITC). Titration of AvrRps4C into a solution of RRS1WRKY resulted in an exothermic binding isotherm with a fitted dissociation equilibrium constant (Kd) of 0.103 M (Fig. 1B) and stoichiometry of 1:1. The thermodynamic parameters of the interaction are given in SI Appendix, Table S1. As RRS1WRKY may be a mimic of WRKY TFs, we explored the binding kinetics of AvrRps4C with AtWRKY41 and AtWRKY70 by ITC [previous reports have shown that AvrRps4 interacts with these proteins in yeast two-hybrid assay and by in planta coimmunoprecipitation (13, 30)]. We chose AtWRKY41 for further study as this protein expressed and purified stably from E. coli. AvrRps4C interacted with AtWRKY41 with a Kd of 0.02 M, and with similar thermodynamic parameters as RRS1WRKY (SI Appendix, Fig. S2 and Table S1).

To reveal the molecular basis of the AvrRps4C and RRS1WRKY interaction, we coexpressed the proteins in E. coli, purified the complex, and obtained crystals that diffracted with 2.65- resolution at the Diamond Light Source (SI Appendix, Materials and Methods). The crystal structure of the AvrRps4CRRS1WRKY complex was solved by molecular replacement using the structure of RRS1WRKY (from the PopP2RRS1WRKY complex, Protein Data Bank [PDB] ID code 5W3X) and AvrRps4C (PDB ID code 4B6X) as models (SI Appendix, Materials and Methods). X-ray data collection, refinement, and validation statistics are shown in SI Appendix, Table S2.

The structure comprises a 1:1 complex of AvrRps4C and RRS1WRKY (Fig. 2A), which supports the 1:1 binding model in ITC. Overall, AvrRps4C adopts the same antiparallel -helical CC structure in both free [PDB ID code 4B6X (26)] and complexed forms, with an rmsd of 0.66 over 59 C atoms (SI Appendix, Fig. S3A). Also, RRS1WRKY adopts a conventional WRKY domain fold [rmsd of 2.03 over 61 C atoms compared with AtWRKY1, PDB ID code 2AYD (31)] comprising a four-stranded antiparallel -sheet (2 to 5) stabilized by a zinc ion (C2H2 type). Comparison of RRS1WRKY in the AvrRps4CRRS1WRKY and PopP2RRS1WRKY complex (PDB ID code 5W3X) structures reveals high conformational similarity, with an rmsd of 1.81 over 64 C atoms. The characteristic WRKY sequence signature motif WRKYGQK maps to the 2-strand of RRS1WRKY and is directly involved in contacting AvrRps4C (Fig. 2B and SI Appendix, Figs. S3A and S4). The same surface, including the 2-3 strands of RRS1WRKY, forms contacts with PopP2 in the PopP2RRS1WRKY complex (29) (SI Appendix, Fig. S4), and mutants at this surface showed it to be essential for PopP2 recognition.

Structure of the AvrRps4CRRS1WRKY complex. (A) Electrostatic surface representation of AvrRps4C in the AvrRps4CRRS1WRKY crystal structure displaying a prominent negative patch in AvrRps4 at the interacting interface. (B) Schematic representation of AvrRps4CRRS1WRKY, highlighting interfacing residues. AvrRps4C is shown in gold cartoon and RRS1WRKY is shown in green with surface-exposed side chains as sticks. (C) Close-up view of the interactions of AvrRps4C with the 2-3 segment of RRS1WRKY. Hydrogen bonds are shown as dashed lines, and water molecules are depicted as red spheres. The Zn2+ ion is also displayed.

The total interface area buried in the AvrRps4CRRS1WRKY complex is 591.8 2, encompassing 12.3% (589.7 2) and 11.9% (593.9 2) of the total accessible surface areas of the effector and integrated domain, respectively [as calculated by PDBePISA (32); full details are given in SI Appendix, Table S3]. The binding interface between AvrRps4C and RRS1WRKY is largely formed by residues from the 2-3 strand of RRS1WRKY, which present a positive surface patch that interacts with acidic residues on the surface of AvrRps4C (Fig. 2A, SI Appendix, Fig. S3B, and Movie S1). The interaction between the 2-segment of RRS1WRKY, which harbors the WRKYGQK motif, and AvrRps4C includes hydrogen bonds and/or salt-bridge interactions involving Tyr1218 and Lys1221 of RRS1WRKY and AvrRps4 Glu175, Glu187, and Asn171. Notably, the side chain of RRS1WRKY Lys1221 protrudes into an acidic cleft on the surface of AvrRps4C to contact the side chains of both AvrRps4 Glu175 and Glu187 (Fig. 2 B and C and Movie S1). The OH atom of RRS1WRKY Tyr1218 forms a hydrogen bond with the ND2 atom of AvrRps4 Asn171 (Fig. 2 B and C). Additional intermolecular contacts are formed by the 2-3 loop of RRS1WRKY involving the backbone carbonyl oxygen and nitrogen of Asp1222, which form hydrogen bonds with the side chains of AvrRps4 Asn190 and Gln194. The complex between AvrRps4C and RRS1WRKY is further stabilized by the 3-strand of RRS1WRKY that forms hydrogen bonds and salt-bridge interactions via side chains of RRS1WRKY Arg1230, Tyr1232, and Arg1234 to AvrRps4 Glu175 and Asp164 (Fig. 2 B and C). A detailed interaction summary is provided in SI Appendix, Table S4.

To evaluate the contribution of residues at the AvrRps4CRRS1WRKY interface to complex formation invitro, we generated six structure-guided mutants in AvrRps4C (native amino acid to Ala) and tested the effect on protein interactions by ITC. Each AvrRps4C mutant was purified from E. coli under the same conditions as for the wild-type protein, and proper folding was evaluated by circular dichroism (CD) spectroscopy (SI Appendix, Fig. S5). ITC titrations were carried out as for the wild-type interactions. Individual ITC isotherms are shown in Fig. 3, and the thermodynamic parameters of the interactions are shown in SI Appendix, Table S1. We found that mutating AvrRps4 residues Asp164 (D164A), Glu175 (E175A), Glu187 (E187A), and double mutant Glu175/Glu187 (EE/AA) essentially abolished complex formation invitro (Fig. 3). Mutations in residues Asn171 (N171A) and Gln194 (Q194A) retained binding to RRS1WRKY, with N171A displaying wild-type levels and Q194A showing an approximately sevenfold reduction in affinity. Besides structure-guided mutants, we also tested binding of an AvrRps4 quadruple mutant, carrying mutations in the N-terminal KRVY motif (KRVY/AAAA) [previously identified to be essential for the virulence activity and perception of AvrRps4 (25)], with RRS1WRKY. Unlike most interface mutants, the AvrRps4C KRVY/AAAA mutant retained wild typelike binding affinity with RRS1WRKY (Fig. 3).

Structure-guided mutants of AvrRps4C at the AvrRps4CRRS1WRKY interface disrupt interaction with RRS1WRKY invitro. ITC titrations of wild-type AvrRps4C and mutants with RRS1WRKY. (Upper) Raw processed thermograms after baseline correction and noise removal. (Lower) Experimental binding isotherms obtained for the interaction of AvrRps4C wild type and mutants with RRS1WRKY together with the global fitted curves (displayed in red) obtained from three independent experiments using AFFINImeter software (61). Kd was derived from fitting to a 1:1 binding model. N.B., nonbinding.

Since AvrRps4C binds RRS1WRKY and AtWRKY41 with similar affinity (SI Appendix, Fig. S2), we tested the impact of the AvrRps4C EE/AA double mutant on the binding to AtWRKY41. We found that this mutant also abolishes interaction with AtWRKY41, suggesting the same AvrRps4-binding interface is shared with different WRKY proteins (SI Appendix, Fig. S2).

To validate the biological relevance of the AvrRps4CRRS1WRKY interface observed in the crystal structure, we tested the effect of the AvrRps4C mutants above on RRS1-R/RPS4mediated immunity by monitoring the cell-death response in N. tabacum. Agrobacterium-mediated transient expression of wild-type AvrRps4 triggers a hypersensitive cell-death response (HR) 5 d post infiltration when coexpressed with RRS1-R/RPS4 (Fig. 4A). The previously characterized inactive AvrRps4 KRVY/AAAA mutant (25, 26) was used as a negative control. We found that AvrRps4 mutations at positions D164, E175, and E187 and the double mutant E175/E187 prevented RRS1-R/RPS4dependent cell-death responses, consistent with their loss of binding to RRS1WRKY invitro (Fig. 4A). Interestingly, the N171A mutation, which retained its binding to RRS1WRKY invitro, displayed wild typelike cell deathinducing activity, and Q194A with an approximately sevenfold reduction in RRS1WRKY affinity consistently exhibited a weaker cell-death response. Expression of all mutants was confirmed by immunoblotting (Fig. 4B). In addition to RRS1-R/RPS4, we also explored the effect of AvrRps4 structure-based mutations on RRS1-S/RPS4dependent cell death in N. tabacum (SI Appendix, Figs. S1 and S6A). We found that AvrRps4 variants elicited similar immune responses when transiently coexpressed with RRS1-S/RPS4 or RRS1-R/RPS4.

Structure-guided mutants of AvrRps4 at the AvrRps4CRRS1WRKY interface compromise RRS1-R/RPS4mediated cell-death responses and invivo binding in Nicotiana. (A) Representative leaf images showing RRS1-R/RPS4mediated cell-death response to wild-type structure-guided mutants of AvrRps4. Agroinfiltration assays were performed in 4- to 5-wk-old N. tabacum leaves, and cell death was assessed at 4 d post infiltration. The experiment was repeated three times with similar results. (B) Coimmunoprecipitation (co-IP) of RRS1-RWRKY+83 (6His/3FLAG-tagged) with AvrRps4 and variants (4myc-tagged) in N. benthamiana. Blots show protein accumulations in total protein extracts (input) and immunoprecipitates obtained with anti-FLAG magnetic beads when probed with appropriate antisera. Empty vector was used as a control. The experiment was repeated at least three times, with similar results.

To determine whether loss of RRS1-R/RPS4mediated HR in transient assays correlates with the loss of AvrRps4 binding to RRS1WRKY invivo as well as invitro, we performed coimmunoprecipitation assays using full-length C-terminal 4myc-tagged AvrRps4 constructs and C-terminal 6His/3FLAG-tagged constructs of RRS1-RWRKY+83 (equivalent to RRS1-D5/6R as defined in ref. 23). Wild-type AvrRps4 associates with RRS1-RWRKY+83 in its in planta processed form (Fig. 4B). Consistent with the cell-death phenotype and invitro binding data, no association between AvrRps4 mutants D164A, E175A, E187A, or EE/AA and RRS1WRKY+83 was detected (Fig. 4B). Further, we observed wild-type levels of association of AvrRps4 N171A with RRS1WRKY+83, while AvrRps4 Q194A appeared to coimmunoprecipitate weakly. The AvrRps4 KRVY/AAAA mutant displayed wild typelike binding affinity toward RRS1WRKY+83, as observed previously (26).

Next, we investigated the impact of AvrRps4 structure-guided mutations on the activation of RRS1-R/RPS4dependent immune responses using HR assays in Arabidopsis. Constructs carrying full-length AvrRps4 wild type and mutants, flanked by a 126-bp native AvrRps4 promoter, were delivered into plant cells by infiltration using the Pf0-EtHAn (Pseudomonas fluorescens effector-to-host analyzer, hence Pf0) system (33). HR assays used Arabidopsis ecotype Ws-2 (encoding RRS1-R/RPS4 and RPS4B/RRS1B) and Ws-2 rrs1-1/rps4-21/rps4b-1 (RRS1-R/RPS4/RPS4B triple-knockout) lines and were scored at 20 h post infiltration. Pf0 carrying wild-type AvrRps4 triggered HR in Ws-2, but not in Ws-2 rrs1-1/rps4-21/rps4b-1, as previously reported (13, 26). AvrRps4 KRVY/AAAA, an HR inactive mutant, was used as a negative control (26). The structure-guided mutants AvrRps4 D164A, E175A, E187A, and EE/AA all showed a complete loss of HR in Ws-2, with AvrRps4 Q194A showing a weaker HR and N171A showing a wild typelike phenotype (Fig. 5A). None of the AvrRps4 variants triggered HR in Ws-2 rrs1-1/rps4-21/rps4b-1 (Fig. 5A).

Structure-guided mutants of AvrRps4 compromise RRS1-R/RPS4dependent recognition specificities and restriction of bacterial growth in Arabidopsis. (A) HR assay in different Arabidopsis accessions using P. fluorescens Pf0-1 secreting AvrRps4 wild type and structure-guided mutants. Constructs were delivered to the Arabidopsis Ws-2 and rrs1-1/rps4-21/rps4b-1 knockout background and HR was recorded 20 h post infiltration. Fraction refers to the number of leaves showing HR of 12 randomly inoculated leaves. This experiment was repeated at least three times with similar results. (B) In planta bacterial growth assays of Pto DC3000 secreting AvrRps4 wild type and mutant constructs. Bacterial suspensions with OD600 = 0.001 were pressure-infiltrated into the leaves of 4- to 5-wk-old Arabidopsis plants. Values are plotted from three independent experiments (denoted in different colors). Statistical significance of the values was calculated by one-way ANOVA followed by post hoc Tukey honestly significant difference analysis. Letters above the data points denote significant differences (P < 0.05). A detailed statistical summary can be found in SI Appendix, Table S5. CFU, colony forming unit.

In addition to Ws-2, we also performed a parallel set of experiments in Arabidopsis ecotype Col-0 (which encodes the RRS1-S allele) and the Col-0 rrs1-3/rrs1b-1 (RRS1-S/RRS1B double-knockout) line. Overall, we observed a weaker HR toward AvrRps4 wild type and mutants in Col-0 in comparison with Ws-2. Nevertheless, a similar pattern of HR phenotypes was observed in Col-0 compared with Ws-2, and none of the AvrRps4 variants triggered HR in the Col-0 rrs1-3/rrs1b-1 line (SI Appendix, Fig. S6B). The pattern of HR phenotypes conferred by the AvrRps4 interface mutants further validates the AvrRps4CRRS1WRKY structure and the role of these residues in recognition of AvrRps4 by the RRS1/RPS4 receptor pair.

Having demonstrated the role of AvrRps4 interface residues in effector-triggered HR in Arabidopsis, we next investigated their effects on bacterial growth. We performed bacterial growth assays on Arabidopsis ecotypes Ws-2, Col-0, Ws-2 rrs1-1/rps4-21/rps4b-1, and Col-0 rrs1-3/rrs1b-1 using the P. syringae pv. tomato (Pto) DC3000 strain carrying AvrRps4 wild type or structure-based mutants. Since both the single mutants AvrRps4 E175A and E187A displayed the same impaired HR as the double AvrRps4 EE/AA mutant in previous assays, we focused on AvrRps4 EE/AA only for this assay. Bacterial growth was scored at 3 d post infection. Pto DC3000 carrying wild-type AvrRps4 displayed reduced growth on Ws-2 when compared with the mutant background (Ws-2 rrs1-1/rps4-21/rps4b-1), presumably due to the activation of RRS1-R/RPS4dependent immunity (Fig. 5B). The effector mutants AvrRps4 D164A, EE/AA, and KRVY/AAAA, which displayed a complete loss of HR in Ws-2, showed a severe or complete lack of restriction of bacterial growth in Ws-2 (Fig. 5B). Pto DC3000:AvrRps4 Q194A and Pto DC3000:AvrRps4 N171A showed reduced bacterial growth (but not full restriction) when compared with wild-type AvrRps4, even though they displayed a similar cell-death phenotype in N. tabacum (albeit weaker for AvrRps4 Q194A) and HR in Arabidopsis (Figs. 4A and 5A). All the Pto DC3000:AvrRps4 variants tested displayed indistinguishable bacterial growth in the RRS1-R/RPS4 loss-of-function line (Fig. 5B). Finally, all the Pto DC3000:AvrRps4 variants displayed similar bacterial growth profiles in the Col-0 and Col-0 rrs1-3/rrs1b-1 line when compared with Ws-2 and Ws-2 rrs1-1/rps4-21/rps4b-1 (SI Appendix, Fig. S6C).

In addition to RRS1/RPS4, the RRS1B/RPS4B pair can confer recognition of AvrRps4 in Arabidopsis (24). Sequence alignment revealed an overall 60% amino acid identity of the integrated WRKY domains from RRS1 and RRS1B, with the WRKYGQK motif and all residues interfacing with AvrRps4C conserved (SI Appendix, Fig. S7). To explore AvrRps4 recognition by RRS1B/RPS4B, we performed ITC titrations of RRS1BWRKY with wild-type AvrRps4C invitro. We found that RRS1BWRKY binds to AvrRps4C three times more weakly than RRS1WRKY (SI Appendix, Fig. S7), possibly due to subtle changes imposed by residues outside the direct binding interface. When comparing the binding kinetics with the strength of immune responses in planta, we observed a weaker RRS1B/RPS4B-dependent HR to AvrRps4 compared with RRS1/RPS4. Nonetheless, both NLR pairs displayed a similar profile of immune responses toward the AvrRps4 structure-guided mutants in transient cell-death assays and in Arabidopsis HR assays (SI Appendix, Fig. S7).

To regulate gene expression, WRKY TFs bind to specific W-box DNA motifs in the promoters of their target genes (3436). Intriguingly, the majority of the AvrRps4-interacting residues are conserved within the DNA-binding domain of WRKY TFs (SI Appendix, Fig. S4 and Movie S2) and are indispensable for DNA binding (34). To test if AvrRps4 interferes with the W-box DNA-binding activity of RRS1WRKY and AtWRKY41, we preincubated increasing concentrations of AvrRps4C and the AvrRps4C EE/AA mutant (as a negative control) and studied their effect on the DNA-binding capacity of RRS1WRKY and AtWRKY41 using both electrophoretic mobility-shift assay (EMSA) and surface plasmon resonance (SPR)based assays. We found that the interaction of RRS1WRKY and AtWRKY41 with W-box DNA was reduced after preincubation with increasing concentrations of AvrRps4C but not the AvrRps4C EE/AA mutant (Fig. 6 and SI Appendix, Figs. S8S10), revealing that AvrRps4C interferes with WRKY binding to W-box DNA.

AvrRps4 interferes with W-box DNA binding by the RRS1WRKY domain. (A) EMSA of DNA binding by RRS1WRKY following preincubation of increasing concentrations of AvrRps4C or AvrRps4C EE/AA mutant. Bovine serum albumin (BSA) was used as a negative control for W-box DNA binding. Scrambled DNA was used as a negative control to test the specificity of RRS1WRKY to W-box DNA. The experiment was repeated three times with similar results. (B) An SPR ReDCaT assay was performed using W-box and scrambled DNA (as a negative control). Percentage of normalized response (% Rmax) of RRS1WRKY binding to W-box DNA and scrambled DNA (denoted by an asterisk) immobilized on a ReDCaT SPR chip. Titrations were performed following preincubation of 2 M RRS1WRKY with increasing concentrations of AvrRps4C wild type and AvrRps4C EE/AA mutant. The experiment was performed in eight replicates (each dot represents one replicate).

Despite recent advances, structural knowledge of how diverse integrated domains in plant NLRs perceive pathogen effectors is limited. Here, we investigated how the integrated WRKY domain of the Arabidopsis NLR RRS1 binds to the Pseudomonas effector AvrRps4, and how this underpins RRS1/RPS4-dependent immunity in planta. Further, through this work, we gained insights into interfaces in the RRS1WRKY domain that are crucial for perception of two structurally unrelated effectors from distinct bacterial pathogens, which may have implications for NLR integrated domain engineering.

Transcriptional reprogramming upon NLR activation is well-established as an early immune response in plants (3739), and direct interactions between NLRs and TFs have been reported (4044). WRKY TFs are important molecular players in the regulation of plant growth and development and abiotic and biotic stresses (35, 36, 45). Typically, WRKY TFs target genes by binding W-box DNA in promoters, via a signature amino acid motif, WRKYGQK, to either promote or repress transcription (34, 4648). As WRKY TFs play an important role in plant immunity, it is unsurprising that they are often found as integrated domains in NLR immune receptors (49), supporting the hypothesis that pathogen effectors enhance virulence by targeting WRKY TFs. Therefore, understanding how effectors bind to WRKY integrated domains may inform how effector/WRKY binding promotes disease. The structure of the AvrRps4CRRS1WRKY complex reveals that the effector directly interacts with the DNA-binding WRKYGQK motif, likely rendering it unavailable for binding to DNA (SI Appendix, Fig. S4 and Movie S2). AvrRps4C binds to AtWRKY41 with similar thermodynamic parameters to RRS1WRKY, and interface mutants that prevent AvrRps4C interaction with RRS1WRKY prevent interaction with AtWRKY41, supporting the hypothesis that AvrRps4C binds different WRKY proteins via a similar interface. WRKY TFs bind W-box DNA sequences in the promoters of their target genes. We used EMSAs and SPR assays to observe how the interaction of AvrRps4C with RRS1WRKY or AtWRKY41 affects the binding of these proteins to a generic W-box DNA sequence. Preincubation of RRS1WRKY and AtWRKY41 with increasing amounts of AvrRps4C reduced DNA-binding activity, whereas preincubation with AvrRps4 EE/AA showed no significant difference (Fig. 6 and SI Appendix, Figs. S8S10). WRKY domain residues interacting with AvrRps4C are well-conserved in these TFs (SI Appendix, Fig. S11), suggesting that AvrRps4 could interfere with and sterically block DNA binding of multiple WRKY TFs, thus promoting virulence. In addition to WRKY TFs, a recent publication suggests AvrRps4 can interact with BTS (nucleus-located Fe sensor BRUTUS) domains to affect pathogen colonization (50). Understanding whether these functions are related requires further investigation.

Comparing the AvrRps4CRRS1WRKY structure with that of PopP2RRS1WRKY (29) reveals an overlapping binding site for the effectors, primarily mediated by the 2-3 segment of the WRKY domain (SI Appendix, Fig. S4 and Movie S2). The second lysine of the WRKYGQK motif, Lys1221, is acetylated by PopP2, abolishing the affinity of the WRKY domain for W-box DNA (13, 14, 29). Intriguingly, this acetylation event also abolished the association of AvrRps4C with RRS1WRKY (13), highlighting the important role of this interface in mediating the association of RRS1WRKY with both effectors. It also highlights the likely shared role of these effectors in preventing interaction of WRKY domains with DNA as their virulence activity, either via enzymatic modification or steric blocking.

Studies with the NLR pair Pik from rice have shown that the strength of effector binding to integrated domains invitro can correlate with immune responses in planta (5153). Of the AvrRps4 mutants we tested to validate the RRS1WRKY interface, all except N171A and Q194A prevented binding invitro (by ITC) and in planta (by coimmunoprecipitation), and these did not give cell death in Nicotiana species when coexpressed with either RRS1-R/RPS4 or RRS1-S/RPS4. Further, they did not give HR or restrict bacterial growth in Arabidopsis Ws-2 or Col-0 ecotypes (except for a partial restriction of bacterial growth for the D164A mutation in the Col-0 background). The N171A mutant retained the same level of binding as wild type invitro, and displayed the same in planta phenotypes, although restriction of bacterial growth in Arabidopsis was reduced compared with wild type in both Ws-2 and Col-0 ecotypes. Finally, the Q194A mutant showed a reduced binding invitro (approximately sevenfold compared with wild type) but maintained an HR in Arabidopsis as well as displaying a restriction of bacterial growth in Arabidopsis, albeit reduced compared with wild type. Interestingly, this mutant consistently showed a qualitative reduction in the intensity of cell death in Nicotiana. Taken together, these AvrRps4 mutations validate the complex with RRS1WRKY in that they prevent interaction invitro and in planta, but they are not sufficient to determine whether strength of binding invitro can directly correlate with in planta phenotypes. Further studies, including additional mutants, will be required to study this in the RRS1/RPS4 system.

Structural studies of singleton NLRs have shown that interactions between effectors and multiple domains within an NLR can be essential for activation (5457). It is yet to be established whether this is also the case for effector perception involving paired NLRs with integrated domains, although the rice blast pathogen effector AVR-Pia immunoprecipitates with its sensor NLR Pia-2 (RGA5) when the integrated HMA domain has been deleted. However, this interaction does not promote immune responses in planta (58). Although unresolved in the structure of AvrRps4C alone, or in complex with RRS1WRKY, the N-terminal KRVY motif is known to be required for both the virulence activity of the effector and its perception by RRS1/RPS4 (25, 26). Here, we verified that the quadruple mutant AvrRps4 KRVY/AAAA retains interaction with RRS1WRKY at wild-type levels invitro and invivo, but did not trigger RRS1/RPS4-dependent responses in our in planta assays. This suggests that while binding of AvrRps4 to the RRS1WRKY domain is essential for immune activation, an additional interaction mediated by the N-terminal region of the effector to a region of RRS1 and/or RPS4 outside this domain is also required for initiation of defense. Further studies are required to determine how additional receptor domains outside of integrated domains in NLR-IDs contribute to receptor function.

The Arabidopsis NLR pair RRS1B/RPS4B perceives AvrRps4, but not PopP2 (24). Phylogenetically, the RRS1 WRKY belongs to group III of the WRKY superfamily, whereas RRS1BWRKY is grouped into group IIe (14, 24, 48). Here we found that AvrRps4C binds RRS1BWRKY with threefold lower affinity and RRS1B/RPS4B shows a similar pattern of recognition specificity in planta but with reduced phenotypes compared with RRS1/RPS4. A full investigation addressing why AvrRps4 shows differential interaction strength and phenotypes between RRS1 and RRS1B is beyond the scope of this work, but will be a direction for future research.

The unique ability of RRS1/RPS4 to perceive two effectors that differ both in sequence and structure, via the same integrated domain, highlights the potential for engineering of sensor NLRs to recognize diverse effectors. Recently, the range of rice blast pathogen effectors recognized by the integrated HMA domain of Pia-2 (RGA5) has been expanded by molecular engineering (58). However, this expanded recognition was toward structurally related effectors and may not be via a shared interface. Further, although cell-death responses were observed in Nicotiana benthamiana, the engineered NLR was not able to deliver an expanded disease resistance profile in transgenic rice. This suggests we still require a better understanding of how NLR-IDs interact with effectors, and their partner helper NLRs, to enable bespoke engineering of disease resistance.

For invitro studies, the gene fragments of AvrRps4C (Gly134 to Gln221), RRS1WRKY (Ser1194 to Thr1273), RRS1BWRKY (Asn1164 to Thr1241), and AtWRKY41 (Thr125 to Ile204) were cloned in various pOPIN expression vectors using an in-fusion cloning strategy as described in SI Appendix, Materials and Methods.

For transient assays in N. tabacum and N. benthamiana, domesticated genomic fragments encoding RRS1-R, RRS1-S, RRS1B, RPS4, and RPS4B were cloned into binary vector pICSL86977 under a 35S (CaMV) promoter with a C-terminal 6His/3FLAG tag using the Golden Gate assembly method as described (23). Similar cloning techniques were used to generate constructs expressing RRS1WRKY+83. Full-length AvrRps4 (P. syringae pv. pisi) was PCR-amplified from published constructs (13, 23, 26) and assembled with a C-terminal 4myc tag in binary vector pICSL86977 under the control of the 35S (CaMV) promoter using the Golden Gate assembly method. DNA encoding each mutation was synthesized and cloned into pICSL86977 as described above.

For HR and bacterial growth assays in Arabidopsis, full-length AvrRps4 and variants were cloned into a Golden Gatecompatible pEDV3 vector with a C-terminal 4myc tag.

Plasmids expressing the in planta processed C-terminal fragment of AvrRps4 (AvrRps4C) and integrated WRKY domain of RRS1 (RRS1WRKY) were expressed in E. coli SHuffle cells. The proteins were purified via IMAC followed by size-exclusion chromatography. Purified fractions were pooled and concentrated to 15 mg/mL and used for further studies. Detailed procedures are provided in SI Appendix, Materials and Methods.

Crystals of the AvrRps4CRRS1WRKY complex were obtained from a 1:1 solution of 15 mg/mL protein with 0.8 M potassium sodium tartrate tetrahydrate, 0.1 M sodium Hepes (pH 7.5). Diffraction data were collected at the Diamond Light Source on the i03 beamline and processed in the P61/522 space group. The structure was determined by molecular replacement using the model of a monomer of AvrRps4C (PDB ID code 4B6X) and the RRS1WRKY from the PopP2RRS1WRKY complex (PDB ID code 5W3X) as the search model. Further details are provided in SI Appendix, Materials and Methods. X-ray data collection and refinement statistics are summarized in SI Appendix, Table S2.

AvrRps4CRRS1WRKY complex formation invitro was studied using analytical gel filtration chromatography and ITC. The effect of structure-guided mutations on the AvrRps4CRRS1WRKY interaction invitro was investigated using ITC as described in SI Appendix, Materials and Methods.

Agrobacterium-mediated transient cell-death assays were performed in N. tabacum and coimmunoprecipitation assays were performed in N. benthamiana. Detailed information concerning plant materials, growth conditions, plasmid construction, and immunoblotting are provided in SI Appendix, Materials and Methods.

Bacterial strains P. fluorescens Pf0-EtHAn and Pto DC3000 were used for HR or in planta bacterial growth assays, respectively. The Arabidopsis accessions Ws-2 and Col-0 were used as wild type for all the assays in this study. Further details about plant materials, growth conditions, plasmid construction and mobilization, pathogen infection assays, and bacterial growth assays are provided in SI Appendix, Materials and Methods.

Complementary single-stranded DNA fragments encoding the W-box DNA sequence (forward strand: 5-CGCCTTTGACCAGCGC-3) were synthesized by IDT. EMSAs were performed using a Cy3-labeled W-box DNA probe in a reaction buffer containing 10 mM TrisCl (pH 7.5), 50 mM KCl, 1 mM dithiothreitol, and 5% glycerol as described in SI Appendix, Materials and Methods.

Complementary single-stranded DNA fragments encoding the W-box DNA sequence were synthesized by IDT. For SPR assays, the forward strand encoded the W-box DNA sequence (5-CGCCTTTGACCAGCGC-3) while the complementary reverse strand added an extra 20-bp ReDCaT sequence (5-CCTACCCTACGTCCTCCTGC-3) to complement the linker DNA added to the SA chip. The double-stranded DNA was then diluted to a working concentration of 1 M. SPR measurements were performed at 25C using the reusable DNA capture technique (ReDCaT) as described (59, 60) and using a Biacore 8K System (Cytiva). Further details are provided in SI Appendix, Materials and Methods.

All study data are included in the article and/or supporting information.

This work was supported by the European Research Council (Proposal 669926, ImmunityByPairDesign); the UK Research and Innovation (UKRI) Biotechnology and Biological Sciences Research Council (BBSRC) Norwich Research Park Biosciences Doctoral Training Partnership, UK (Grant BB/M011216/1); the UKRI BBSRC, UK (Grants BB/P012574 and BBS/E/J/000PR9795); and the BBSRC Future Leader Fellowship (Grant BB/R012172/1). We thank Julia Mundy and Professor David Lawson from the John Innes Centre (JIC) Biophysical Analysis and X-Ray Crystallography platform for their support with CD spectroscopy, protein crystallization, and X-ray data collection; Andrew Davies and Phil Robinson from JIC Scientific Photography for their help with leaf imaging; and Dr. Tung Lee for advice on EMSAs. We also thank Dr. Kee Hoon Sohn for helpful suggestions for triparental mating and other members of the M.J.B. and J.D.G.J. laboratories for discussions.

Author contributions: N.M., H.B., P.D., J.D.G.J., and M.J.B. designed research; N.M. and D.G. performed research; H.B., P.D., and C.E.M.S. contributed new reagents/analytic tools; N.M., A.R.B., C.E.M.S., and M.J.B. analyzed data; and N.M., J.D.G.J., and M.J.B. wrote the paper.

The authors declare no competing interest.

This article is a PNAS Direct Submission.

This article contains supporting information online at https://www.pnas.org/lookup/suppl/doi:10.1073/pnas.2113996118/-/DCSupplemental.

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Perception of structurally distinct effectors by the integrated WRKY domain of a plant immune receptor - pnas.org

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DeepMind's AI is probably best known for cracking the popular strategy game Go, but in the last few years, machine learning has proved extremely valuable in an array of applications like protein-folding and deep intuition.

Now, for the first time, the technology has been used to identify mathematical connections that have eluded researchers for decades. Teaming up with mathematicians, DeepMind's AI sought to tackle two distinct problems one in the study of symmetries and the other in knot theory.

I was very struck at just how useful the machine-learning tools could be as a guide for intuition, said Marc Lackenby, one of the mathematicians from the University of Oxford who participated in the study. I was not expecting to have some of my preconceptions turned on their head.

The study of math may turn a lot of people off but, at its core, it facilitates a greater human understanding of the fundamental properties that govern our universe. It's only through painstaking work in the area of pure mathematics that we now have revolutionary technologies like airplanes and computers.

Mathematicians try to spot patterns in large datasets which they then seek to formulate conjectures out of. These conjectures are then reviewed and tested by their peers in various hypothetical cases and, if they hold up, turn into theorems.

But the amount of data now available is impossible for any human to process. And this is where machine learning comes in. Machine learning can discover patterns much quicker than humans insights that can then guide new mathematical ideas.

Take the theory of knots for example. At a superficial level, knots describe how a piece of string or rope is entangled. But at a much deeper level, they revolve around key mathematical principles that can be applied in the realm of quantum computing.

Algebra, geometry, and quantum theory all share unique perspectives on these objects and a long-standing mystery is how these different branches relate: for example, what does the geometry of the knot tell us about the algebra? wrote the researchers.

The team of researchers created a machine learning model to probe these connections and one particular trick called saliency maps proved immensely valuable. The ML model was able to spot specific geometrical properties known as a 'signature' that researchers could then use to formulate a conjecture.

In another instance, DeepMind teamed up with mathematicians to probe a problem in symmetries one that scientists have traditionally studied using charts or graphs. But as more data is incorporated, these charts inevitably grow dauntingly large, making it nearly impossible for a human to comprehend. But DeepMind's AI discovered numerous interesting patterns that, the researchers, think could guide mathematicians toward a proof.

I was just blown away by how powerful this stuff is, said Dr Geordie Williamson from the University of Sydney. I think I spent basically a year in the darkness just feeling the computer knew something that I didn't.

DeepMind has been consistently proving that the applications of machine learning extend well beyond just games, and the latest breakthrough is another testament to the technology's growing value in solving some of humanity's most complex problems. But ultimately, due to its inherent probabilistic nature, it needs to be accompanied by human intuition and rigour. Nevertheless, the man-machine combination, the researchers believe, could inspire other scientists to incorporate AI into their own research.

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In breakthrough, DeepMind's AI has cracked two mathematical problems that have stumped experts for decades - Times Now

The next take on the battle royale genre is Rumbleverse, a 40-player brawler royale from Extinction maker Iron Galaxy. Revealed Thursday night during the Game Awards, Rumbleverse is a city full of zany-costumed pro rasslers, elbow-dropping and pile-driving one another until the last is standing.

And yes, the characters are rasslers, not wrestlers. The day that we were first jamming on the concept, Chelsea [Blasko] our co-CEO, she just goes, We should do rasslin!, said Adam Boyes, Iron Galaxys other co-CEO since 2016. Just like that, right? And then we just started, like, What would happen in a world where a wrestling match could break out anywhere on planet Earth?

Rumbleverse, to be published by Epic Games for PlayStation 4, PlayStation 5, Windows PC, Xbox One, and Xbox Series X, borrows the battle royale conventions of parachuting into a map, scarfing up loot and power-ups, and moving to stay within a steadily closing area. But because there are no guns except the ones attached to your arms, says lead designer Adam Hart Iron Galaxys developers hope the fighting will more engaging and more entertaining than the fast-twitch, shoot-or-be-shot immediacy of Fortnite or PUBG Battlegrounds.

When you see somebody in this game, theyre not a threat to you just because youve seen them, Hart said. You can kind of watch them fight or have a, you know, emote conversation with them across rooftops. Of course, at some point theyll start throwing down. While sneak attacks are possible, normally this is mutually initiated combat.

Iron Galaxy figures most events will take between 12 and 15 minutes to crown a winner. The ring shrinks tighter and more quickly to compensate for inactivity, Hart said, to force combat on anyone who is avoiding a fight. The games map, Grapital City, is quite large but more importantly, it has a lot of verticality. It packs a lot of visual appeal into the fights, and of course, supplies a lot more force to moves landed from way higher than the top rope.

I saw Hart, whose fighter was kitted out in a tuxedo under a karate gi, with a full-head cat mask (customization, of course, is very important here) pull out an old-school belly-to-back suplex on a clown (another devs costume), landing it from what looked like the observation deck of the Empire State Building.

How often do you guys imitate Jim Ross, by the way? I asked.

Every day! Boyes laughed. For the past couple of years! Hart added.

Eliminations are a simple case of draining another players health bar, which can be replenished or buffed by the power-ups strewn about Grapital City. Hart guzzled down protein powder, for example, and picked up weightlifting magazines to get himself in shape. Theres also plenty of roasted poultry, the international sign of video game health since Castlevania and Gauntlet. Its available from a drive-thru window for Squatch Chicken: The Home of Slow-Squatted Chicken.

[The cook] has all the chicken on a squat rack, and he just dips it into the fire, Hart explained.

Other pick-ups supply perks or modify one of three core attributes arms (power), core (health), and legs (stamina). Players can work up a match-to-match character build that emphasizes certain areas of their wrestling prowess. Harts character, in the playthrough, went 3-5-2, for example, with his core being the top attribute. But a max-stamina fighter could do comparable damage per second with a flurry of lesser attacks and I saw plenty of chained, juggling strikes that fighting game fans will recognize.

One thing we found out is that a lot of people that are crazy-good platform players, like Mario, Crash, Spyro fans, became the best players, very quickly, Boyes said. So its as much, I think, about how you move around the world as it is your offensive integrity.

Melee weapons are also available, like baseball bats and the de-rigueur folding chairs. But as Hart pointed out, anything a player can hold can also be knocked from their hands (and used) weapon or power-up. That means you can also improvise throwing attacks with the can of whey.

Grapital Citys environment also presents tactical possibilities for players, too. No one can swim, so if the circle is closing around an area with water, players can get rung out quickly even if theyre at maximum health. Being outside the circle doesnt inflict damage, as it does in Fortnite, but it does start a 10-second countdown, akin to disqualification matches in real pro rasslin. (Not that pro rasslin is real. Just, you know, real-life.)

Other wrestling tropes include a perk that revives a player after theyve been counted out (that is, when their health has been fully drained), much like the scripted reversals and comebacks in epic-length wrestling matches.

What I love about this is, I see someone, and I see a series of choices for me, Boyes summarized. Do I have enough stuff that Ive picked up? Do I feel like my stats are good enough? Do I have that one move I need? Can I sneak up behind him? Can I sort of stalk them? Or can I just run away, get health, and come back.

I dont want to say its casual, but because theres so much depth to the combat, it makes it less pressure, Boyes said, but more, you know, just more fun to experiment and try new things.

Rumbleverse kicks off a First Look gameplay event on Friday, Dec. 10, available to a limited number of players (the games official website has registration information and more details). Iron Galaxy expects to launch Rumbleverse in early access on the Epic Games Store on Feb. 8, 2022, as well as on PlayStation 4, PlayStation 5, Xbox One, and Xbox Series X through those consoles marketplaces. Rumbleverse will support cross-platform play and progression, Iron Galaxy said.

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Rumbleverse, a pro wrestling battle royale, announced at TGAs 2021 - Polygon

Proteostasis is critical for the function, maintenance, and long-term survival of all cells (1). Successful maintenance of the proteome entails efficacious balancing of protein synthesis and degradation, with coordinated actions of regulated gene expression and translation, the chaperone-folding network, the ubiquitin proteasome system, and autophagy all serving critical functions in overall protein quality control. Neurons are particularly susceptible to proteostasis disruption, and aggregates are a striking and common feature of neuropathology in most human neurodegenerative diseases (2). Recent studies have shown that in diseases characterized by distinctive aggregates, including Alzheimers disease, Parkinsons disease, and Huntingtons disease, aggregates can spread from neurons to neighboring cells, inducing deleterious consequences (3). The mechanisms operative for aggregate spread are unclear and remain a matter of considerable research attention.

We discovered that adult Caenorhabditis elegans neurons can select and then extrude aggregated proteins, such as expanded polyglutamine HTTQ128-CFP (related to neurotoxic Huntingtons disease protein) and an aggregation-prone, high-expression mCherry reporter (4) (see Fig. 1A). Aggregates are sent out of the neuron in strikingly large, membrane-surrounded vesicles [4-m average diameter; for comparison, the neuron soma is 6 m; this vesicle is two orders of magnitude larger than exosomes that form by a distinct mechanism (5)]. We refer to these massive cell extrusions as exophers (exo = outside and pher = carry away). In the case of the C. elegans touch receptor neurons that mediate sensitivity to gentle touch stimuli (6), exophers are passed into the surrounding hypodermis, which can partially degrade their contents. Baseline production of exophers under nonstress growth conditions is low, detected in 5 to 20% of ALMR touch neurons, and occurs at lower rates for other touch neurons. However, we found that exacerbated proteostress (for example, expressing the human A142 fragment implicated in Alzheimers disease pathology, disrupting specific autophagy genes via RNA interference [RNAi] or introducing the MG132 proteasome inhibitor) can significantly increase exopher production (4). Animals expressing toxic HTTQ128-CFP in touch neurons that extruded aggregates in exophers retained better touch sensitivity than transgenic HTTQ128-CFP animals in which the neurons did not produce exophers (4), suggesting that exopher production that helps clear the neuron of toxic aggregates is neuroprotective, at least in the short term.

A key mystery in the biology of exophers is the precise nature of the cellular conditions and stresses that induce or elevate exophergenesis. To address this question, we systematically tested external, physiological stresses for the capacity to influence the expulsion of cellular trash. Our data 1) show a clear link between specific, environmental stresses and neuronal exopher production (namely food withdrawal, osmotic stress, and oxidative stress); 2) emphasize that there exists a permissive window for exopher production in early adult life during which external stresses can elevate exopher levels; and 3) identify a previously unrecognized limit to the level of stress that can induce exophers, such that, beyond this upper stress limit, exophergenesis is not observed. We linked the fasting-induced increase in exophers (as much as 10-fold elevation in exophers) to activities of the intestinal peptide transporter PEPT-1, the transcription factors MDT-15 and SBP-1/SREBF2, and their target FASN-1/fatty acid synthase, as well as to epidermal growth factor (EGF) and fibroblast growth factor (FGF)/RAS/MAPK signaling pathways. Our data reveal how stress conditions might promote the spread of aggregates from neurons to their neighbors and suggest pathways that might be targeted to regulate analogous processes, with implications for addressing human neurodegenerative disease.

A common stress that C. elegans encounter in their natural environment is variable food type and/or food abundance, which can have a significant impact on C. elegans gene expression, development, metabolism, and longevity (79). In the laboratory, C. elegans eat a diet of Escherichia coli spread on agar plates, and food sources can be easily manipulated. We first asked whether neuronal exophergenesis levels are sensitive to food source, quantitating ALMR exophers produced by neurons expressing an mCherry protein (strain ZB4065 bzIs166[Pmec-4mCherry1]), which is avidly expelled as exopher cargo (10) (see Fig. 1 for an example). For claritys sake we refer to assay strain ZB4065 as mCherryAg2 in the text hereafter. Exophers are typically produced by young adult animals, peaking at days 2 to 3 of adulthood and returning close to baseline by adult day 4 (Ad4) (4, 11). We therefore measured exophers produced each day, Ad1 to Ad4, to compare both daily relative levels and temporal profiles.

We quantitated exopher production when mCherryAg2 animals were fed four different E. coli strains: E. coli OP50, which is the standard C. elegans food source (12); HT115, a strain that lacks RNAaseIII and is used in RNAi studies (13); HB101, a food source that promotes larger body size and faster development (14); and NA22, a strain that fosters enhanced growth in liquid culture (15). We noted no major differences among the different E. coli strains in common laboratory use, all of which support the basic pattern of peak exopher production around Ad2 that returns to the baseline around Ad4 (SI Appendix, Fig. S1A).

Complete food withdrawal markedly induces exophers. We reared mCherryAg2 animals at 20 C and at Ad1 (onset of egg laying) moved animals to unseeded plates at 20 C for either 3 or 6 h, counting ALMR exophers shortly thereafter. Continuously fed control mCherryAg2 animals generate exophers at levels that range from 5 to 20% of animals, and in this study, baseline was close to 5% on Ad1. Subsequent to a 3-h fast, we found exophers in more than 20% ALMRs; a 6-h fast increased average exopher numbers 5- to 10-fold to nearly 50% of ALMRs examined (Fig. 1B). Fasting also causes an increase in multiple-exopher events, in which more than one exopher is generated (SI Appendix, Fig. S1B). We conclude that food withdrawal can have a rapid and dramatic impact on the extrusion of exophers in animals expressing a noxious mCherry reporter.

At 20 C, in the presence of food, the young adult wave of exopher production falls narrowly within the first 4 d of adulthood (SI Appendix, Fig. S1A). We were therefore curious as to whether the fasting-induced elevation of exophers was restricted to the Ad1 to Ad4 timeframe or, alternatively, whether food withdrawal experienced at any time point could be effective for inducing exopher production. We subjected animals from a single, synchronized population to 6-h fasting regimens as L4 larvae as well as on Ad1 to Ad10; we measured exopher levels immediately after the 6-h fast on the experimental fasting day (Fig. 1C). Our analysis of mCherryAg2 ALMR exophers in this study revealed the following: 1) a 6-h fasting protocol does not induce exopher production during the L4 stage; 2) the 6-h fasting protocol elevates exopher production to peak levels at Ad1 when administered on that day; 3) fasting efficacy is slightly lower when delivered at Ad2 and lower still when delivered at Ad3; and 4) by Ad4, a fasting-induced exopher increase is no longer observed. We conclude that the impact of food withdrawal on enhancing exopher production is limited to a window of time that covers Ad1 to Ad3. The flanking of the permissive phase by exopher-recalcitrant periods suggests the existence of a licensed period, in which the expulsion of significant amounts of neuronal material is biologically feasible and during which exophergenesis can be modulated by environmental stress signals. We observed the 6-h fastinginduced elevation of exophers in strains that expressed GFP (Fig. 1D) or expanded polyglutamine (Fig. 1F) mNG:HttQ74 in touch neurons or expressed GFP in dopaminergic neurons (Fig. 1E), indicating that the fasting induction of exophers was not reporter- or cell typespecific.

Temperature is an environmental factor that dramatically influences the C. elegans reproductive life cycle time, lifespan (16), and proteostress (17). We therefore asked whether culture temperature might influence the timing or levels of adult exopher production. We reared animals continuously at 15, 20, and 25 C from the egg stage and measured exopher levels over the first 4 d of adult life (SI Appendix, Fig. S2A). Somewhat unexpectedly, exopher levels did not vary substantially with culture temperature. In an alternative experimental design, we reared synchronized populations of animals at 20 C and then split cultures at the L4 adolescent stage into three parallel cultures that were thereafter maintained at 15, 20, and 25 C (SI Appendix, Fig. S2B). We found that, in animals shifted to 25 C, the peak of exopher production occurred earlier (Ad1) than 20 C animals, but exopher levels were not significantly elevated, relative to 20 C. Our data suggest that the experience of a temperature shift, rather than continuous noxious temperature exposure, might exert the strongest impact on levels of exopher production (SI Appendix, Fig. S2C). That said, the approximate doubling of exopher scores after an L4 shift from 20 to 25 C is modest in comparison to the 5 to 10 exopher elevation measured for food withdrawal. We conclude that, within the normal confines of C. elegans laboratory culture, the temperature at which animals are grown exerts a relatively minor influence on levels of exopher production.

Hypoxia can induce protein aggregation in C. elegans models (18), raising the possibility that hypoxia might enhance exopher production. We tested the exposure to 0.1% oxygen using an adjustable hypoxia C-174 chamber (Biospherix) to generate exposures to a controlled, hypoxic environment. We subjected Ad1 to Ad5 mCherryAg2 animals to 0.1% oxygen for 6 h and monitored ALMR exophers after the removal of animals from the chamber.

We failed to find differences in exophers produced under these hypoxic conditions on any day (SI Appendix, Fig. S2D). We also tested anoxic conditions induced by replacing oxygen with nitrogen in a sealed anoxia chamber (19), measuring ALMR exophers after 6-h exposure to anoxia (SI Appendix, Fig. S2D, dark purple line). Exposure to anoxia failed to markedly increase exopher production on any exposure day. While it is possible that longer exposures or different recovery times might alter outcomes, we conclude that hypoxia/anoxia treatments, as delivered, do not elevate exopher levels.

Osmotic stress introduces proteostasis challenges (20), and thus, we were curious as to how exopher production might respond to osmotic stress conditions. We first made agar plates designed to introduce osmotic stresses based on standard C. elegans conditions, testing 250 mM concentrations of sucrose, glucose, sorbitol, and NaCl. At Ad1, we introduced mCherryAg2 animals to these osmotic stresses for 6 h and scored ALMR exophers thereafter (Fig. 2A). We found that a 6-h exposure to osmotic stress increased exopher production 4 above baseline. Since each solute-induced stress resulted in a similar elevation of exophers, the exopher response is likely grounded in osmotic stress itself, rather than the chemical nature of the specific osmolytes.

Osmotic stress and oxidative stress can increase exopher production, but extreme stress levels can decrease exophergenesis. For all panels, bars are SEM, ***P < 0.001, **P < 0.01, *P < 0.05, and CMH statistics. (A) Transient osmotic stress significantly elevates ALMR exophers on Ad1. We exposed mCherryAg2 animals to 250 mM sucrose, glucose, sorbitol, or NaCl on Ad1 and scored for exophers 6 h later: 5 trials and 50 animals per trial. (B) Animals chronically exposed to osmotic stress from Ad1 into adulthood produce elevated exophers with early onset peak production. We grew mCherryAg2 animals to the Ad1 stage under standard growth conditions and shifted to 250 mM sucrose, glucose, sorbitol, or NaCl, measuring ALMR exophers in these populations on Ad1 to Ad4: 4 trials and 50 animals per time point. CMH compares osmotic stress media to normal growth media. (C) Osmotic stress at 250 mM enhances exopher production more than at 500 mM. We exposed strain mCherryAg2 to 250 and 500 mM sucrose, glucose, sorbitol, and NaCl for 6 h on Ad1 and measured exophers shortly thereafter: 5 trials and 50 animals per trial. (DF) Increasing oxidative stress enhances exopher production to a limit, after which exopher production is suppressed. We grew mCherryAg2 animals to Ad1 at 20 C, and then, transferred animals to plates housing increasing concentrations of juglone, rotenone (rot), or paraquat (PQ) for 6 h, as indicated, measuring ALMR exophers shortly thereafter (black asterisks compare to control): 7 trials and 50 animals per trial. (G) Combined food withdrawal and osmotic stress can suppress exopher induction under conditions that individually induce high-exopher levels. We fasted Ad1 mCherryAg2 animals for 6 h on plates containing the solutes indicated (20 C) and scored for ALMR exophers shortly thereafter: 3 trials and 50 animals per trial (asterisks compare two indicated conditions at bar ends). (H) Combined food withdrawal with oxidative stress can suppress exopher induction under conditions that individually induce high-exopher levels. We fasted Ad1 mCherryAg2 animals for 6 h on juglone, rotenone, and paraquat at the concentrations indicated, scoring for ALMR exophers shortly thereafter: 5 trials and 50 animals per trial (asterisks compare two indicated conditions). (I) While anoxia does not increase exophers, combining food withdrawal and anoxic stress can suppress fasting-associated exopher induction. We fasted Ad1 mCherryAg2 animals for 6 h under anoxic conditions, scoring for ALMR exophers shortly thereafter: 4 trials and 50 animals per trial. Note that our anoxia protocol is not effective in limiting fasting-induced exopher elevation (SI Appendix, Fig. S2 D and H).

We also quantitated exopher levels under conditions of long-term, hyperosmotic stress during adult life (Fig. 2B). We raised animals under standard growth conditions until the L4 stage and then moved animals to 250 mM sucrose, glucose, sorbitol, or NaCl plates for adult life. We first measured exopher levels 24 h after initiating osmotic stress and tested the same population on Ad1 to Ad4 thereafter. For all solutes tested, osmotic stress resulted in an 5 increase in exophers over the baseline at peak and shifted the measured peak of exopher onset forward 1 d. Notably, exopher levels returned to baseline at Ad4, even in the presence of osmotic stressors. We conclude that both transient and extended exposure to hyperosmotic stress can elevate exopher production substantially in early adult life and infer that hyperosmotic conditions rapidly generate a trigger that elevates exophers.

Oxidative stress is a central player in aging biology and proteostasis (21). We tested whether well-characterized chemical inducers of mitochondrial oxidative stress can influence exopher production in ALMR neurons. We exposed Ad1 mCherryAg2 animals to 6 h of increasing concentrations of juglone (juglone is a plant-derived compound that can induce superoxide production when incorporated into nematode growth medium [NGM]); rotenone, which interacts with mitochondrial electron transport complex I to elevate reactive oxygen species (ROS); and paraquat, which increases mitochondrial superoxide production (Fig. 2 DF). For all mitochondrial ROS generators, we observed a dose-dependent increase in exophers at Ad1 (to a point, discussed in Excessive Stress Decreases Exopher Production) that ranged from 4 to 6 over untreated controls. We conclude that exposure to mitochondrial ROS induction can enhance exopher production.

We also tested conditions of continuous ROS exposure during adult life. We raised animals under standard growth conditions until the L4 stage and shifted cultures to paraquat plates, measuring ALMR exophers on Ad1 to Ad8 (SI Appendix, Fig. S2E). We find that continuous paraquat exposure increases exopher levels. Interestingly, exopher levels in the 2-mM paraquat treatment do not begin to fall at Ad3, as is typical for other stresses, but instead remain higher than control levels through Ad6, returning to baseline at Ad7. Thus, paraquat-mediated oxidative stress can extend the time period of permissive exophergenesis by 2 d, raising the possibility that paraquat might induce signals that normally promote exopher production. Overall, data from three mitochondrial ROS-generating compounds indicate that chemically-induced oxidative stress can increase exopher formation.

While initially testing the ability of stressors to modulate exopher production, we often utilized a doseresponse approach. Our studies revealed a striking commonality regardless of stressor type: excessive stress suppresses exophergenesis. For example, 6-h exposure of 240 M juglone (Fig. 2D), 25 M rotenone (Fig. 2E), or 25 mM paraquat (Fig. 2F) reduces exopher levels, even though lower levels of these ROS stressors enhance exopher production. The same pattern emerges under osmotic stress conditions; 6-h exposure to 500 mM concentrations of sucrose, glucose, sorbitol, and NaCl suppresses exopher levels, compared to 250 mM concentrations of each of these solutes (Fig. 2C). Although at Ad1 ALMR exophers modestly increase with 6-h exposures to increasing temperatures up to 30 C, 6 h at 37 C causes a collapse in exophergenesis (SI Appendix, Fig. S2F).

We generated additional evidence in support of the idea that excessive stress can inhibit exopher formation by exposing Ad1 mCherryAg2 animals to combined two-stress conditions that, by themselves, individually enhance exopher production. For example, whereas a 6-h fast elevates exophers (Figs. 1 BF and 2G), cointroducing osmotic stress with fasting (which also normally also elevates exophers; Fig. 2 A and B) suppresses exopher levels (Fig. 2G). We also found a combined inhibitory effect for fasting + oxidative stress (Fig. 2H).

Furthermore, temperature (SI Appendix, Fig. S2G) and anoxia (Fig. 2I and SI Appendix, Fig. S2H), stresses that did not affect exophergenesis on their own (SI Appendix, Fig. S2 A, B, and D), could suppress the effects of fasting on exopher production. Data suggest that under extreme stress neurons either cannot meet molecular requirements for exopher production or might enact mechanisms that actively suppress exophergenesis (see Discussion).

In summary, food withdrawal, osmotic stress, and oxidative stress can enhance exopher production, although above specific threshold levels of these stresses (including when two individual, exopher-promoting stresses are combined), exopher production can be suppressed. Thus, in addition to a temporal constraint on when environmental stresses can elevate exopher levels, we demonstrate that there is a limit to the severity of environmental stress capable of inducing the exopher production response.

With a goal of defining molecular mechanisms by which stresses elevate neuronal exopher production, we sought to define genetic requirements for the fasting-dependent induction response. We elected to focus on the fasting response because of the robust and highly reproducible level of induction associated with 6 h food withdrawal (Fig. 1 B and C), combining genetic mutant and RNAi strategies. To probe the mechanism by which fasting elevates neuronal exopher production, we first tested mutants for well-characterized, stress-activated transcription factors: heat shock factor 1 hsf-1/HSF1, required for transcription of heat shock chaperones and proteostasis (22); hypoxia inducible factor hif-1/HIF1, required for hypoxia stress responses (23); hlh-30/TFEB, required for starvation resistance and lysosomal integration with metabolism (24); skn-1/NRF2, which promotes response to oxidative stress and xenobiotic challenge (25, 26); and daf-16/FOXO, which is activated by low insulin pathway signaling and functions in a range of stress-protective responses, including proteostasis (27).

We constructed mCherryAg2 strains with viable mutant alleles of each transcription factor and subjected mutants to 6 h food deprivation on Ad1, measuring ALMR exophers thereafter (Fig. 3A). We observed the significant elevation of exophergenesis in hsf-1, hif-1, hlh-30, and skn-1 backgrounds in response to food withdrawal, indicating that these transcription factors are not critical for the induction of exophers in response to fasting. In contrast, the daf-16 null mutant exhibited a partial defect in the food withdrawal response, with exopher levels clearly increasing over baseline in the absence of food (P < 0.001) but never reaching the levels observed in wild-type (WT) animals (P < 0.001) (Fig. 3A). The partial effect in the daf-16 null mutant background is consistent with a model in which a daf-16dependent process mediates one component of the fasting response but that a daf-16independent process works in parallel to elevate exophers when food is withdrawn.

Lipid synthesis and FGF-activated RAS/ERK signaling are required for the induction of neuronal exophers in response to fasting. For all panels, bars are SEM, ***P < 0.001, **P < 0.01, *P < 0.05, and CMH statistics. Feeding RNAi was initiated at the L4 larval stage and continued until Ad2. (A) The daf-16 null mutant has diminished exopher production in response to fasting. We tested mutants defective in major stress-responsive transcription factors (daf-16/FOXO, hsf-1/HSF-1, hif-1/HIF1, hlh-30/TFEB and skn-1/NRF2) in the mCherryA2 background, Ad2: exopher counts 6 trials, 50 animals per trial, and CMH difference between WT control and daf-16 deletion mutant ***P < 0.001. (B) Lipid synthesis is implicated in the fasting-induced boost in exophergenesis. RNAi knockdown of pept-1, mdt-15, sbp-1, and fasn-1 beginning at L4 in the mCherryAg2 strain, 6 h fast followed by exopher assay on Ad2, control is empty vector RNAi. We compared WT fed versus fasted P < 0.001 CMH, the others are not significant: 3 trials and 50 animals per trial. (C) Schematic of C. elegans MAPK signaling pathways targeted in genetic tests for fasting-induced exopher elevation: p38 MAPK signaling (purple), JUN/FOS MAP kinase signaling (orange), EGF-mediated MAPK signaling (green), and FGF-mediated MAPK signaling (blue). Black boxes highlight a common function in both EGF and FGF MAPK signaling. (D) RNAi knockdown of most components of the p38 and JUN/FOS signaling cascades does not impair fasting-induced exopher increases. The strain was mCherryAg2; RNAi was initiated at the L4 stage; and 6 h fast was followed by exopher counts at Ad2: 3 trials and 50 animals per trial. mek-1(RNAi) stood out as exceptional in failing to induce exophers. (E) Loss-of-function mutants for JUN/FOS signaling do not suppress exopher production. kgb-1; mCherryAg2 and jun-1; mCherryAg2 mutants were fasted for 6 h at Ad2 before exopher counts: 5 trials and 50 animals per trial. (F) An egl-17/FGFmediated MAPK signaling cascade is necessary for a fasting-induced increase in exopher production. RNAi for the indicated genes was initiated at the L4 stage on strain mCherryAg2, and at Ad2, animals were fasted 6 h and then scored for exophers. RNAi knockdown of let-23, ksr-2, and FGF ligand let-756 did not disrupt the fasting exopher response 3 trials and 50 animals per trial.

We also tested whether autophagy, a pathway activated by food limitation, might be critical for the fasting-induced increase in exophers. We used RNAi approaches to knockdown autophagy genes lgg-1, atg-7, and bec-1 in mCherryAg2 animals and scored for exophers on Ad2 (SI Appendix, Fig. S3A). Since all three disruptions in the autophagy pathway failed to suppress fasting-induced exopher elevation, we infer that engagement of autophagy functions is not required for fasting-induced exopher increase. Pharmacological inhibition of autophagy with 1 mM spautin or of the proteasome with 10 mM MG132 (SI Appendix, Fig. S3B) did not block the fasting-induced elevation of exophers, in further support that autophagy and proteasome contributions are not critical for fasting-induced exopher increase.

To better characterize the pathways involved in fasting-induced exopher production, we compiled a list of known genes implicated in starvation and feeding in the C. elegans literature and screened for fasting-induced exopher elevation when the candidate genes were knocked down using RNAi (28) (see SI Appendix, Table S1 for a list of candidates tested). Note that the strain we targeted with feeding RNAi, mCherryAg2, should permit efficient RNAi knockdown in all tissues except neurons, because neurons do not express a double-stranded RNAi transporter, sid-1, required for the efficacious knockdown in feeding RNAi (29). Utilizing this experimental design, we expected to identify genes operative in the nonautonomous initial events in the sensation and signaling of fasting stress to the touch neurons, rather than genes involved in neuron-intrinsic exophergenesis.

We tested positive clones from the first round of the RNAi screen in triplicate to identify intestinal peptide transporter pept-1, lipid synthesis-implicated Mediator complex factor mdt-15, MDT-15 binding partner sbp-1/SREBF2, and fatty acid synthase fasn-1, as required for robust exopher elevation in response to fasting (Fig. 3B).

pept-1 encodes a conserved intestinal di-/triamino acid transporter implicated in C. elegans nutrient sensing (30). Loss of the pept-1 function increases the intestinal absorption of free fatty acids from ingested bacteria, such that short- and medium-chain fatty acids are highly increased in the mutant, and de novo synthesis of long-chain and polyunsaturated fatty acids is greatly decreased (31). Our data suggest that sudden withdrawal of food causes a metabolic reconfiguration that signals for enhanced exopher production and that the sensing of food limitation requires PEPT-1 transporter activity.

MDT-15 has been shown to promote health and longevity by orchestrating many of the metabolic changes that occur in response to short-term fasting (32). MDT-15 encodes a subunit of the transcriptional coregulator Mediator complex that is required to express fatty acid metabolism genes and fasting-induced transcripts (3235), heavy metal and xenobiotic detoxification genes (32, 36), and oxidative stress genes (37). SBP-1, the homolog of the mammalian sterol regulatory element-binding protein (SREBF2) transcription activator that regulates fatty acid homeostasis, is a known partner of MDT-15, and together, MDT-15 and SBP-1 promote the expression of lipid synthesis genes (32). FASN-1 encodes the sole C. elegans fatty acid synthase, and its expression is regulated by MDT-15/SBP-1 (38, 39). Together, MDT-15, SBP-1, and FASN-1 may act to promote the synthesis of a lipid-based factor that signals for, or is otherwise required for, neuronal exopher production under fasting stress.

Expression of lipid synthesis genes in multiple tissues may contribute to fasting-induced exopher elevation. To address where the lipid synthesis gene group required for fasting-induced exopher elevation acts, we took a tissue-specific RNAi knockdown approach. We worked with strains that expressed mCherryAg2 in touch neurons but were defective in either the double-stranded RNA (dsRNA) transporter sid-1 (neurons, muscle, pharynx, intestine, and hypodermis) or RISC complex factor rde-1 (germline and vulva), and we reintroduced sid-1 or rde-1 using tissue-specific promoters to drive expression and restore RNAi knockdown capability only in the rescued tissue. The promoters we used to restore expression (and therefore RNAi targeting) in specific tissues were the following: pan-neuronal rgef-1, muscle myo-3, pharynx myo-2, intestine vha-6, hypodermis hyp7 semo-1, vulva lin-31, and germline sun-1. This test set of seven tissue-specific RNAi lines enabled us to target most cells of the animal. We fasted animals to ask whether RNAi disruption in individual tissues is sufficient to disrupt the exopher induction response, which would indicate that expression in that targeted tissue is necessary in the response. We found that although whole body knockdown of pept-1, mdt-15, sbp-1, and fasn-1 could disrupt the fasting-induced exopher elevation (Fig. 3B), no tissue-specific knockdown was effective in blocking this response (SI Appendix, Fig. S4A). Data are consistent with a model in which multiple tissues can contribute the required lipid biosynthesis, although we cannot rule out that RNAi targeting was ineffective or missed necessary cells.

Food limitation stresses engage multiple signaling pathways that can activate animal defenses, including RAS/MAPK pathways that transduce developmental and stress responses to activate specific transcription programs (40, 41). We tested members of three canonical, well-characterized C. elegans MAPK signaling pathwaysthe PMK-1/p38 pathway that functions in some innate immunity and oxidative stress responses; the JNK pathway, which, among other activities, functions in intermittent fasting programs (42); and the RAS/ERK pathway, which, among other things, affects vulval precursor fate and vulval development response to starvation stress (43) (pathways summarized in Fig. 3C).

The core MAP kinases in the p38 pathway are nsy-1/MAPKKK and pmk-1/MAPK (40). RNAi directed against nsy-1 and pmk-1 genes was ineffective in blocking the fasting induction of neuronal exophers (Fig. 3D), and thus, we do not find evidence supporting the engagement of the pmk-1/p38 stress pathway in fasting-induced exopher elevation.

In C. elegans, an adult intermittent fasting protocol of 2 d without food followed by 2 d of food extends lifespan via an MLK-1, MEK-1, and KGB-1 JNK pathway that converges on AP-1 (JUN-1, FOS-1)mediated transcription (42). We find that kgb-1(RNAi), kgb-1, and jun-1 genetic mutations do not block the fasting-induced elevation of exopher production (Fig. 3 D and E). This result, coupled with published data examining a transcriptional time course following food withdrawal that revealed a distinct transcription pattern for 3 to 6 h after fasting, as compared to the more chronic starvation of 9 h and longer (44), suggests that the response to short-term food withdrawal that we characterize here does not operate via the characterized, intermittent fasting pathway. Still, the positive mek-1/MAPKK and partial mlk-1/MAPK outcome (mlk-1(RNAi) fasted versus WT fasted, not significant; mlk-1(RNAi) fasted versus mlk-1(RNAi) fed, P < 0.01) (Fig. 3D), suggest possible pathway involvement or cross-talk involving these kinases. As evidence for more definitive engagement of the RAS/ERK pathway was evident in our studies (Fig. 3F), we focused on examining pathway members in more detail.

RAS/ERK signaling in C. elegans (reviewed in refs. 40 and 41; see Fig. 3C) can involve the EGF or FGF activation of receptor tyrosine kinases that interact with adaptor proteins such as SEM-5/GRB2 or SOC-1/GAB1, which recruit guanine nucleotide exchange factor SOS-1 to activate small GTPase LET-60/RAS. LET-60/RAS-GTP activates MAPKKK LIN-45/RAF. Scaffold proteins KSR-1 and/or KSR-2 collect downstream members of the MAPK cascade, so LIN-45/MAPKKK activates MEK-2/MAPKK, which in turn phosphorylates and activates MPK-1/ERK. MPK-1 can then enter the nucleus to phosphorylate transcription factors that execute a transcriptional response. We found RNAi knockdown of multiple members of the conserved core RAS/ERK signaling, namely let-60/RAS, mek-2/MAPKK, mpk-1/ERK, sem-5, and ksr-1, disrupts the fasting-induced increase of touch neuron exophers (Fig. 3F). Given the implication of five core MAPK components in fasting-induced exopher elevation, we conclude that the RAS/ERK pathway plays a critical role in the mechanism by which neurons increase mCherry expulsion (and other cell contents) under food withdrawal stress.

The characterized C. elegans EGF and FGF signaling pathways share the aforementioned signaling components in the conserved RAS/ERK pathways but differ in ligands, receptors, KSR-type (EGFR uses both KSR-1 and KSR-2; FGFR exclusively uses KSR-1), and FGF pathway requirement for the adaptor SOC-1/Gab1 (41). We tested MAPK components in an effort to distinguish whether FGF, EGF, or both, contribute to exopher elevation in response to fasting.

We used RNAi approaches to test for a requirement of FGF ligands egl-17 and let-756 as well as FGF receptor egl-15 in fasting-induced exopher elevation. Interventions with ligand egl-17/FGF and receptor egl-15/FGFR, but not FGF ligand let-756, disrupted the capacity to increase exophers in response to 6-h fasting, implicating a specific FGF in exopher regulation biology (Fig. 3F). Moreover, we find that FGF pathway-specific soc-1 (RNAi) diminishes fasting-induced exopher production. Our data, which implicate eight FGF pathway genes (FGF ligand egl-17, FGF receptor egl-15, FGF pathway-specific soc-1 and sem-5, and core pathway components let-60, mek-2, ksr-1, and mpk-1) in the fasting induction of exophers, identify an FGF-activated ERK/MAPK pathway that acts in response to 6-h food withdrawal, as an essential mechanistic step in neuronal exopher increase.

To address the potential source of egl-17/FGF and identify the tissue via which FGFR acts to promote fasting-elevated exopher production, we used tissue-specific RNAi approaches to disrupt all identified components in all neurons, muscle, pharynx, intestine, hypodermis, vulva, or germline (SI Appendix, Fig. S4A).

We were unable to identify single-tissue sources that were required for FGF ligand EGL-17 or FGF receptor EGL-15 activity in fasting-induced exopher elevation, suggesting that either multiple tissues can execute effective FGF signaling, that cells that we were unable to target are responsible, or that unknown technical issues might apply. We did, however, find that knockdown of the most downstream pathway targets, mek-2 and mpk-1, only in the hypodermis or only in the germline (SI Appendix, Fig. S4B) could disrupt fasting induction. Our data thus suggest that mek-2 and mpk-1 are required in both the hypodermis and in the germline for fasting-induced exopher induction.

Importantly, mek-2 and mpk-1 are downstream kinases for both the FGF and the EGF MAPK pathways. Indeed, our RNAi perturbation screens of MAPK pathway members indicated that lin-3/EGF also acts in fasting-induced exopher elevation (Fig. 3F). Tissue-specific RNAi studies reveal that germline-specific knockdown of lin-3/EGF disrupted fasting-induced exopher elevation (while hypodermal knockdown did not) (SI Appendix, Fig. S4C), implicating the germline production of LIN-3/EGF in a transgenerational influence on neuronal exophergenesis. Knockdown of mek-2 and mpk-1 are also needed in the germline for fasting-induced exophergenesis (SI Appendix, Fig. S4B).

RNAi disruption of EGF receptor let-23 and EGF pathway-specific ksr-2 did not eliminate the exopher elevation in response to fasting in our original screen (Fig. 3F). This observation raised the possibility that the EGF-responsive pathway might function in neurons (which are generally not as susceptible to RNAi as other tissues). Because let-23(gf) alleles caused extensive reproductive system development consequences that complicated the interpretation of exopher production (not shown), we tested the available strains that expressed the EGF pathway-activating EGFR/let-23(gf) in specific tissues for elevated exopher levels in the presence of food. We found that the transgenic introduction of EGFR/let23(gf) in neurons (but not muscle or intestine) elevates exophers in the presence of abundant food (Fig. 4A), suggesting a neuronal-based EGF pathway in promoting fasting-induced exopher elevation.

Molecular pathways that influence fasting-induced exopher elevation. For all panels, bars are SEM, ***P < 0.001, **P < 0.01, *P < 0.05, and CMH statistics. (A) let-23 (gain of function) increases exopher formation when expressed specifically in neurons. let23(sa62) is a gain-of-function allele that activates MAPK signaling. We measured the exopher levels in strains that expressed let-23(sa62gf) from neuronal (unc-119), intestinal (vha-6), or muscle (myo-3) promoters in the mCherryAg2 background at Ad2 in the presence of food: 3 replicates and 50 animals per trial. (B) let-60 (gain of function) increases exopher formation under abundant food conditions. let-60(n1046) is a gain-of-function RAS allele that activates MAPK signaling. We measured exopher levels in control mCherryAg2 and in let-60(n1046);mCherryAg2 strains at Ad2 in the presence of food: 3 replicates and 50 animals per trial. (C) Schematic of the logic of epistasis within the MAPK signaling context. The phenotype of a gain-of-function allele that continuously signals will not be affected by the knockdown of any effector upstream of it within the cascade. On the other hand, any actors downstream of a constitutively active component will affect the phenotype. FGF signaling is used as an example here. (D) Lipid synthesis genes act upstream of let-60(gf)/RAS to elevate neuronal exopher production. RNAi interventions were initiated at the L4 stage on gain-of-function let-60;mCherryAg2 animals. Exophers were scored at Ad2: 3 replicates and 50 animals per trial. Although mek-2 and mpk-1, known to act downstream of let-60(gf), are critical for exopher elevation, the known upstream genes egl-15, egl-17, and soc-1 are not, consistent with data that previously ordered the FGF pathway. pept-1, mdt-15, sbp-1, and fasn-1 do not suppress let-60(gf)-induced exophergenesis, indicating that the lipid synthesis genes likely act upstream of let-60/RAS. (E) Lipid synthesis genes and lin-3 act upstream of let-23(gf) activity when it is expressed in neurons. RNAi knockdown was initiated at the L4 developmental stage on gain-of-function let-23(sa62);mCherryAg2 animals. Exophers were scored at Ad2: 3 replicates and 50 animals per trial. Cascade components let-60, mek-2, and mpk-1 are known to act downstream of let-23 and effectively abrogate the MAPK signaling leading to exophers, while lin-3 works upstream of let-23 and therefore cannot suppress a downstream gain-of-function mutation. RNAi knockdown of pept-1, mdt-15, sbp-1, and fasn-1 do not suppress neuronal let-23(gf)-induced exophergenesis, indicating that the lipid synthesis genes likely act upstream of neuronal let-23(gf). (F) Model for signaling that elevates neuronal exopher formation in response to fasting. In the sudden absence of food or when dipeptide transporter PEPT-1 activity is low, a lipid-based signal is produced/released that depends upon MDT-15/SBP-1 transcriptional activity and fatty acid synthase FASN-1. The lipid signal may act on the neurons directly or may contribute to the activation of a required FGF pathway in a relay tissue (unlikely to be neurons) to promote transcription of an essential signal that acts on touch neurons to elevate exopher production. EGF pathway activation in neurons can elevate exopher production; it remains to be determined whether EGF signaling is required in touch neurons per se. In sum, transtissue stress signaling influences remote neuronal trash management through lipid, FGF, and EGF RAS/MAPK signaling. Note that we cannot distinguish between an inducible lipid signal and a static, essential lipid product required for exopher elevation.

let-60(n1046) is a well-characterized, gain-of-function RAS allele that constitutively activates MAPK signaling (45). Our RNAi data, identifying the FGF and EGF/RAS/MAPK pathways as required for fasting-induced exopher induction, predict that the let-60(gf) allele should induce higher exopher levels, even in the absence of starvation. To test this model, we constructed a let-60(n1046gf);mCherryAg2 strain and measured exopher levels at Ad2. We find that exophers are indeed elevated in the let-60(n1046gf) background in the absence of fasting, confirming that RAS activation enhances exopher production (Fig. 4B).

let-60(n1046gf) has been used extensively in epistasis pathway ordering (45), and thus we pursued an epistasis approach to clarify the relationship between the lipid synthesis gene group and the FGF/RAS/MAPK pathways (Fig. 4C). We reasoned that if the genes involved in lipid synthesis act downstream or in parallel to the RAS/MAPK pathway that elevates exophers, RNAi knockdown of the lipid synthesis genes in the let-60(gf) background should suppress constitutive exopher production. Alternatively, if the lipid synthesis branch acts upstream of LET-60/RAS activation, disruption of the upstream genes should not change the exopher elevation associated with let-60(gf) (Fig. 4C). We therefore repeated RNAi knockdowns of genes in the lipid synthesis and FGF/RAS/MAPK pathways in the let-60(gf);mCherryAg2 background, quantitating the impact on exopher formation. As expected for known downstream kinases in the RAS pathway, the RNAi disruption of mek-2/MAPKK and mpk-1/MAPK suppressed the let-60(gf) phenotype (Fig. 4D). In contrast, genes encoding egl-17/FGF, egl-15/FGFR, and soc-1 that act upstream of let-60/RAS did not suppress the let-60(gf)/RAS phenotype, consistent with the known action of these genes upstream of RAS in the signaling pathway.

Importantly, RNAi directed against pept-1, and the lipid biosynthesis genes mdt-15, sbp-1, and fasn-1 did not suppress elevated exopher levels in the let-60(gf) background (Fig. 4D). We conclude that the lipid biosynthesis branch is likely to act upstream of the FGF/RAS/MAPK to promote exopher elevation.

Lipid and FGF signaling may act upstream of EGF signaling. We also used an epistasis strategy to begin to address how lipid biosynthesis and FGF pathways might relate to the EGF pathway in fasting-induced exopher elevation. We performed the RNAi knockdown of required lipid synthesis genes and of upstream FGF pathway-specific genes egl-17/FGF, egl-15/FGFR, and soc-1 on the neuronal let-23(gf) strain in which exopher levels are elevated, reasoning that if a critical lipid or FGF pathway is normally activated downstream of neuronal EGFR activation, perturbation of lipid or FGF pathway-specific genes would block the let-23(gf) exopher elevation under well-fed conditions. Our RNAi-dependent disruptions of either pathway, however, did not suppress neuronal let-23(gf) high-exopher levels (Fig. 4E), suggesting that the essential lipid and FGF signaling required for fasting-induced exopher induction normally occurs upstream of neuronal EGF signaling. Although these studies constitute only the first rudimentary tests required to establish pathway details (caveats discussed in more detail in SI Appendix, Fig. S4), data suggest a basic framework for mechanistic evaluation (Fig. 4F).

Overall, we identify three pathways that are required for fasting-induced exopher elevation in stressed touch receptor neurons: lipid synthesis, an FGF/MAPK pathway, and an EGF/MAPK pathway that can act in neurons. Our data suggest a model for signaling that influences a dramatic expulsion of neuronal contents upon the introduction of fasting stress. Upon food withdrawal, di-/tripeptide transporter PEPT-1 plays a role in nutrient sensing and a lipid-based stress signal (the generation of which depends on MDT-15/SBP-1 transcriptional activity and fatty acid synthase FASN-1) is produced. The lipid-dependent process could act directly to nonautonomously influence exopher production in the touch neurons or could trigger/activate the required FGF/RAS/MAPK and/or EGF MAPK signaling and the likely consequent downstream transcription. Downstream, or in parallel, RAS/MAPK activity in hypodermis and germline contributes to the stress-sensing tissue network, establishing that that nonautonomous signaling directs exopher production in the touch neurons. FGFR signaling pathway genes are known to be highly expressed in the hypodermis (46). Notably, RNAi evidence supports the hypothesis that the same group of genes that mediate fasting-induced exopher increase is required for exopher elevation in response to osmotic stress (SI Appendix, Fig. S3C), indicating that general, rather than fasting-specific, mechanisms are engaged.

Although many details of this complex lipid-FGF-EGF signaling network remain to be further elucidated, our data provide documentation that aging and proteostasis-relevant stresses engage multiple pathways that can act over multiple tissues to influence a dramatic expulsion of neuronal contents. Conserved signaling molecules can modulate a process of fundamental interest in neuronal proteostasis, relevant to the understanding of neuronal degeneration.

Maintaining neuronal proteostasis is a critical goal for healthy brain aging and a fundamental challenge for diseased neurons in a range of neurodegenerative diseases (47). A recently identified facet of Alzheimers disease, Parkinsons disease, and other proteopathies is the transfer of aggregates to neighboring cells, which can seed aggregate spread and promote pathology (48, 49). In vivo dissection of the biology of protein aggregate spread is challenging to investigate in mammalian brain, but it is clear that the understanding of mechanisms that regulate autonomous and nonautonomous aggregate expulsion in relation to other neuroprotective strategies is of considerable importance in addressing potential treatment.

C. elegans touch neuron exopher production, which increases with high proteostress (4), models several aspects of aggregate/organelle transfer biology. We find that the production of neuronal exophers can be dramatically responsive to specific stress conditions, being enhanced by food withdrawal, oxidative stress, and osmotic stress but influenced relatively little by temperature or hypoxia. We also demonstrate the temporal restriction of stress-induced exopher production to the first 3 d of adult life, and we document a stress ceiling phenomenon, in which the highest levels of individual stress, or a combination of two distinct noninhibitory stresses, suspend exopher production. Finally, we show that fasting-induced exophergenesis is dependent on nonautonomous lipid biosynthesis, FGF-activated RAS/MAPK, and EGF-activated RAS/MAPK signaling pathways. Although details remain to be filled in regarding the complex interactions of the signaling steps, a major point is that environmental and genetic factors can be manipulated nonautonomously to regulate the expulsion of offensive aggregates from neurons. Given the importance of aggregate management in aging and neurodegenerative disease and the poorly understood biology of in vivo aggregate transfer, exopher-related mechanisms may suggest new strategies toward the manipulation of the analogous process in higher organisms.

Acute food withdrawal, oxidative stress, and hyperosmotic stress elevate exopher production, but temperature elevation and hypoxia/anoxia are relatively ineffective at provoking similar responses. Starvation (8, 50), oxidative stress (51), and osmotic stress (52) perturb proteostasis and share ROS elevation (53, 54). The future dissection of the intersection of the genetic and physiological conditions common to these three stresses should provide insight into molecular mechanisms that promote exophergenesis. Likewise, physiological differences in stress responses to temperature and hypoxia, which are not potent inducers of exophers, may help distinguish particular conditions that are specifically correlated with exopher induction.

Exopher production, comprising the release of a large, membrane-surrounded vesicle filled with cellular contents, has the appearance of an energetically costly incident that involves the dynamic loss of organelles and aggregates. Our working model posits that exophergenesis is invoked when the levels of damaged organelles and proteins surpass the neuronal capacity for internal degradation. Consistent with this idea, increasing oxidative challenge and increasing hyperosmotic exposure both increase exophergenesis. Interestingly, conditions of extreme osmotic and oxidative stress markedly suppress the formation of exophers. Moreover, combining two stresses, either of which is sufficient to promote exopher production when introduced alone, can result in exopher suppression. This combinatorial effect can also occur with stress stimuli that themselves do not significantly induce exophers, such as anoxia and modestly elevated temperature. Together, these observations reveal a molecular summing of stress signals that appear to flip the off switch for exophergenesis. The suppression of exopher production under conditions of extreme stress may be caused by energy exhaustion, a molecular repression mechanism, or grievous loss of homeostasis, leading to physiological dysregulation.

Exopher production follows a distinctive and reproducible temporal profile in early adult life (4, 11). In the Ag2mCherry strain, exophers are not produced in larval development but begin to be detected after animals reach reproductive maturity, typically peaking in numbers around Ad2 and returning to low-baseline detection by Ad4. Data included here underscore that the temporal pattern is generally maintained, despite the continued or introduced presence of stresses. In other words, stresses definitively elevate exopher production, but for the most part, these stresses do not extend the period of exopher production later into adult life. Our findings thus define a limited temporal window in which exopher production can be modulated by stresses and suggest the existence of physiological states permissive (or restrictive) for exopher production. A link to reproduction likely defines this permissive period. For some stimuli (paraquat, rotenone, osmotic stress, and a shift to 25 C), we do report a capacity to move the peak day of exopher production ahead to Ad1 or at least to markedly enhance Ad1 levels above nonstressed controls. We infer that these stimuli reach the molecular threshold for exopher triggering faster than other conditions.

The fasting-induced elevation of exopher levels does not require stress transcription factors HSF-1, HIF-1, HLH-30/TFEB, or SKN-1/NRF2 but does depend in part on DAF-16/FOXO, a conserved stress-responsive transcription factor that drives the expression of food-sensitive, oxidative stress resistance and proteostasis genes (27, 44, 55) and is known to exert autonomous and nonautonomous impacts on stress resistance and longevity (56). Our data implicate a FOXO family member in regulation of neuronal aggregate expulsion. Our data do not rule out whether HSF-1, HIF-1, HLH-30/TFEB, or SKN-1/NRF2 might function redundantly in the exopher response to fasting.

How DAF-16 interacts with fat biosynthesis pathways and RAS/ERK signaling in exopher induction remains to be clarified. DAF-16 can control the expression of mdt-15 and has been previously implicated in transtissue benefits by interactions with MDT-15 (57). DAF-16 also intersects with FGF, ERK, and lipid biogenesis pathways and vice versa (for example, refs. 5861). The future definition of how DAF-16 integrates with these signaling pathways and the identification of the transcription factor that mediates the DAF-16independent component of the fasting-induced exopher response will add molecular understanding to what appears to be a complex regulatory network.

Mediator complex subunit MDT-15 is a transcriptional coregulator involved in lipid metabolism (34), response to fasting (32, 62), and oxidative, stress-induced expression of detoxification genes associated with the exposure to reagents like paraquat (63). The requirement for mdt-15 in fasting-induced, neuronal exophergenesis adds a new facet to the MDT-15 integration of multiple, transcriptional regulatory pathways (32), expanding the known roles of MDT-15/SBP-1 to include the activation of extrusion of remote neuronal aggregates in exophers. SBP-1/SREBF2 acts with MDT-15 to promote the expression of lipid metabolism genes (33, 34), and fatty acid synthase fasn-1 can be regulated by these (38, 39, 62, 64, 65). The requirement for multiple genes involved in lipid synthesis in fasting-induced exopher increase suggests that a lipid-based signal may be issued to ultimately direct or modulate neuronal trash expulsion. An equally plausible model is that lipid-dependent machinery is required for upstream signaling that promotes exophergenesis.

Our findings identify FGF ligand egl-17 (but not FGF ligand let-756), FGF receptor egl-15, FGF pathway specific soc-1, and pathway components let-60, mek-2, ksr-1, and mpk-1 as required for the fasting induction of exophers. These data reveal a specific FGF/RAS/ERK signaling pathway that enhances neuronal exopher production when food is withdrawn. Since the candidate RNAi screen for the factors required for fasting-induced exopher elevation was conducted in a strain background that is not readily permissive for neuronal RNAi effects and since RNAi knockdown of pathway components (specifically in neurons) does not block fasting-induced exopher increase, FGF/ERK signaling likely takes place outside of the touch receptor neurons to exert a regulatory role on neuronal exopher production. An interesting potential site of FGFR action is the hypodermis, which is necessary for some MEK-2/MPK-1 MAPK signaling. Such signaling is permissive for fasting-induced exopher production and the hypodermis is a known site of expression of FGFR pathway components (SI Appendix, Fig. S4B). Detailed, cell-specific expression studies will be required to test this model.

The conserved FGF pathway executes numerous roles in mammalian development and homeostasis (66). Interestingly, mammalian FGF21 acts as a global starvation signal that, among other things, impacts lipid metabolism. Although most FGF21 studies feature extended starvation and mousehuman differences have been noted (67), FGF21 is one of the most up-regulated rat liver genes under the conditions of an 8-h fast (68). FGF21 can cross the bloodbrain barrier to change hypothalamic neuron gene expression (69). Overall, the implication of FGF/ERK pathways in the response of neurons to food limitation across diverse metazoans suggests a mechanism that may be ancient and raises the possibility that the FGF branch of these pathways might activate extracellular trash expulsion mechanisms within mammalian neurons. If so, FGF signaling might be considered as a target for the therapeutic elimination of stored neuronal aggregates.

EGF RAS/MAPK signaling is also engaged in fasting-induced exopher elevation. The knockdown of the sole C. elegans EGF ligand lin-3 in whole animal, or only in germline, impaired fasting-induced exopher elevation.

Although RNAi knockdown of the EGFR let-23 and EGF pathway-specific ksr-2 in whole animal or only neurons was not effective, expressing activated, gain-of-function EGFR receptor allele let-23(sa62) in neurons resulted in elevated exopher levels in the absence of fasting. Epistasis studies suggest that EGFR activation in neurons could occur as a downstream target of lipid biosynthesis and FGF signaling. Although definitive establishment of fasting-induced EGFR activation in neurons remains (experimental caveats in interpretation of data are discussed in detail in SI Appendix, Fig. S4C), data are consistent with a role for EGF signaling originating in the germline as an inducer of EGFR-activated responses in neurons that promote exopher production. Future studies will need to confirm neuron requirements and to address whether EGFR directly activates exophergenesis in touch neurons or engages additional neurons as intermediatory signaling centers.

It is important that the germline serves as an EGF source needed for fasting-induced exopher elevation. Indeed, in studies of exopher production under standard growth conditions, we have defined a role for germline in the production of young adult exophers. A key point here is that food-sensing, nonautonomous growth factor signaling across generations can influence seemingly extreme neuronal proteostasis activity.

EGF- and FGF-dependent processes cooperate in development. For example, EGF signaling activates FGF production and a downstream FGF pathway required in vulval epithelial fate specification (70). Starvation conditions can influence the signaling level for the EGF/RAS/ERK pathway that specifies C. elegans vulval cell fateseither starvation or pept-1(RNAi)can enhance RAS/MAPK signaling during vulval fate specification (43). Our documentation of FGF signaling in food limitation responses that elevate neuronal proteostasis outcomes identifies a second C. elegans EGF- and FGF-regulated signaling pathway that responds to food limitation. Why food limitation might induce neuronal trash elimination is unclear. One possibility is that exophergenesis [which we track in single neurons in our study but is likely to also occur in other neurons and cells (4)] might serve as a mechanism to discard superfluous, neuronal proteins and organelles for degradative recycling in neighboring cells as resources become limited.

Our study defines the basic framework by which metabolic stresses engage a distributed network that influences a significant neuronal expulsion phenomenon. Evidence is accumulating that exopher-like extrusion capabilities are not limited to stressed C. elegans neurons [mammalian examples in refs. 71 and 72)]. For example, a recent comprehensive study of mitochondrial expulsion by mouse cardiomyocytes revealed numerous analogies between C. elegans exophers and mouse mitochondrial expulsion models (72). Although elaborating details of molecular homologies remain for the future, that related biology is likely to be conserved holds significant implications of interest with regard to mammalian aging and neurodegeneration. 1) It is interesting that stresses, particularly associated with aging (i.e., oxidative stress) or proteostasis impairment (i.e., osmotic stress), are especially potent in inducing C. elegans exopher elevation; the disruption of exopher-related biology may contribute generally to the decline/dysfunction in aging neurons across phyla. 2) Likewise, the direct demonstration that extreme stress levels, or the summation of distinct, nonconsequential stresses, can effectively shut down the exopher response suggests a type of potential excessive stress impairment relevant to pathological mechanisms. 3) Our data establish that exopher production can be responsive to specific, conserved biochemical signaling, such that chemical strategies for inducing, or limiting, the expulsion of neurotoxic material by exploiting exopher-related mechanisms in mammals might be considered targets for therapeutic manipulation.

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Discovering drugs in deadly venom. Improving transplant outcomes with better storage. Linking mannose glycosylation to eye development. Read about papers on these topics recently published in the journal Molecular & Cellular Proteomics.

The purple cone snail, Conus purpurascens, hunts fish and uses venom to immobilize its prey. Cone snail venom contains diverse toxic peptides, or conopeptides, that are biologically active, target-specific and valuable for drug discovery. One of the most powerful known painkillers, ziconotide, marketed as Prialt, was derived from a conopeptide.

Alex Holt/NIST

A purple cone snail uses its harpoon to pierce through a latex-covered tube,allowing a researcher to collect its venom.

Conopeptides vary among the more than 800 cone snail species and among members of the same species. Even a single cone snail specimen can produce unique conopeptide cocktails, called cabals, specialized for predation or defense. Cone snails also can hypermodify conopeptides at the post-translational step, increasing the diversity of the toxins and extending their range of biological targets. The extreme diversity of conopeptides provides a rich source of biologically active molecules for drug discovery.

In a recent study in the journal Molecular & Cellular Proteomics, Meghan Grandal and colleagues at the National Institute of Standards and Technology collected and analyzed the injected venom from 27 specimens of the purple cone snail. Using high-resolution mass spectrometry techniques, they discovered 543 unique conopeptides derived from 33 base peptide sequences 21 of which were newly identified base peptides. An abundant and newly discovered conopeptide named PVIIIA illustrates the complexity and diversity of modifications to the base conopeptide that occur among various purple cone snail specimens. Building on previous studies, the researchers showed that the different snail specimens produce one of two unique venom cocktails. These two cocktails correspond to what are known as the lightning strike cabal that rapidly induces paralysis of the snails prey and the motor cabal that acts more slowly to induce irreversible paralysis.

Knowing which conopeptides are co-expressed within a specific cocktail will give the researchers important clues as to the possible neural targets of newly identified conopeptides. This will be a critical step in developing new conopeptides into neural probes or therapeutics.

The demand for kidney transplants exceeds the supply of available kidneys. Some donated kidneys go unused, however, due to the prolonged time between circulatory arrest and the start of cold storage. These kidney graftsoften fail or are slow to function. Repairing such kidneys before transplant could greatly increase the available supply.

An unusual type of protein glycosylation, C-mannosylation, involves attaching a single mannose sugar to the amino acid tryptophan by a carboncarbon bond. C-mannosylation, which regulates protein secretion, folding and function, occurs at a specific sequence of four amino acids that begins with the modified tryptophan. Even though about 18% of secreted or transmembrane proteins have this sequence, few studies have looked for the modification. Consequently, researchers know of few proteins that are C-mannosylated.

In a new study in the journal Molecular & Cellular Proteomics, Karsten Cirksena of the Institute of Clinical Biochemistry and a team of researchers in Germany found numerous proteins with altered secretion levels in cells lacking the C-mannosylation machinery. One of these potentially C-mannosylated proteins, a disintegrin and metalloprotease with thrombospondin motifs, or ADAMTS16, is essential during eye development and optic fissure closure. In Chinese hamster ovary cells and Japanese rice fish, the researchers demonstrated that ADAMTS16 can be C-mannosylated, that its secretion depends on C-mannosylation and that loss of a C-mannosylation enzyme causes a developmental eye defect known as a partial coloboma a gap in the eye tissue. Their findings suggest that C-mannosylation, an understudied protein modification, plays a critical role in eye development by regulating secretion of ADAMTS16.

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From the journals: MCP - American Society for Biochemistry and Molecular Biology