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• W3124388718 abstract "Article20 January 2021free access Transparent process Biogenic amine neurotransmitters promote eicosanoid production and protein homeostasis Kishore K Joshi Kishore K Joshi Department of Genetics, The Waksman Institute, Rutgers The State University of New Jersey, Piscataway, NJ, USA Search for more papers by this author Tarmie L Matlack Tarmie L Matlack Department of Genetics, The Waksman Institute, Rutgers The State University of New Jersey, Piscataway, NJ, USA Search for more papers by this author Stephanie Pyonteck Stephanie Pyonteck Department of Genetics, The Waksman Institute, Rutgers The State University of New Jersey, Piscataway, NJ, USA Search for more papers by this author Mehul Vora Mehul Vora orcid.org/0000-0002-1239-6458 Department of Genetics, The Waksman Institute, Rutgers The State University of New Jersey, Piscataway, NJ, USA Search for more papers by this author Ralph Menzel Ralph Menzel Institute of Biology and Ecology, Humboldt University Berlin, Berlin, Germany Search for more papers by this author Christopher Rongo Corresponding Author Christopher Rongo [email protected] orcid.org/0000-0002-1361-5288 Department of Genetics, The Waksman Institute, Rutgers The State University of New Jersey, Piscataway, NJ, USA Search for more papers by this author Kishore K Joshi Kishore K Joshi Department of Genetics, The Waksman Institute, Rutgers The State University of New Jersey, Piscataway, NJ, USA Search for more papers by this author Tarmie L Matlack Tarmie L Matlack Department of Genetics, The Waksman Institute, Rutgers The State University of New Jersey, Piscataway, NJ, USA Search for more papers by this author Stephanie Pyonteck Stephanie Pyonteck Department of Genetics, The Waksman Institute, Rutgers The State University of New Jersey, Piscataway, NJ, USA Search for more papers by this author Mehul Vora Mehul Vora orcid.org/0000-0002-1239-6458 Department of Genetics, The Waksman Institute, Rutgers The State University of New Jersey, Piscataway, NJ, USA Search for more papers by this author Ralph Menzel Ralph Menzel Institute of Biology and Ecology, Humboldt University Berlin, Berlin, Germany Search for more papers by this author Christopher Rongo Corresponding Author Christopher Rongo [email protected] orcid.org/0000-0002-1361-5288 Department of Genetics, The Waksman Institute, Rutgers The State University of New Jersey, Piscataway, NJ, USA Search for more papers by this author Author Information Kishore K Joshi1, Tarmie L Matlack1, Stephanie Pyonteck1, Mehul Vora1, Ralph Menzel2 and Christopher Rongo *,1 1Department of Genetics, The Waksman Institute, Rutgers The State University of New Jersey, Piscataway, NJ, USA 2Institute of Biology and Ecology, Humboldt University Berlin, Berlin, Germany *Corresponding author. Tel: +1 848 445 0955; Fax: +1 732 445 5735; E-mail: [email protected] EMBO Reports (2021)22:e51063https://doi.org/10.15252/embr.202051063 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Metazoans use protein homeostasis (proteostasis) pathways to respond to adverse physiological conditions, changing environment, and aging. The nervous system regulates proteostasis in different tissues, but the mechanism is not understood. Here, we show that Caenorhabditis elegans employs biogenic amine neurotransmitters to regulate ubiquitin proteasome system (UPS) proteostasis in epithelia. Mutants for biogenic amine synthesis show decreased poly-ubiquitination and turnover of a GFP-based UPS substrate. Using RNA-seq and mass spectrometry, we found that biogenic amines promote eicosanoid production from poly-unsaturated fats (PUFAs) by regulating expression of cytochrome P450 monooxygenases. Mutants for one of these P450s share the same UPS phenotype observed in biogenic amine mutants. The production of n-6 eicosanoids is required for UPS substrate turnover, whereas accumulation of n-6 eicosanoids accelerates turnover. Our results suggest that sensory neurons secrete biogenic amines to modulate lipid signaling, which in turn activates stress response pathways to maintain UPS proteostasis. SYNOPSIS Protein homeostasis or proteostasis must respond to adverse physiological or environmental conditions. The Caenorhabditis elegans nervous system uses a signaling axis of biogenic amine neurotransmitters and eicosanoids to promote the ubiquitin proteasome proteostasis pathway in epithelia. The C. elegans nervous system releases biogenic amine neurohormones into the body. Biogenic amines promote the expression of cytochrome P450 monooxygenases. These P450 enzymes synthesize omega-6 eicosanoids from poly-unsaturated fatty acids. Omega-6 eicosanoids promote the turnover of metastable proteins in epithelia by the ubiquitin proteasome system. Introduction Cells employ multiple signaling pathways and mechanisms for maintaining the folded state and function of their proteins, a process termed protein homeostasis (proteostasis) (Balch et al, 2008; Hipp et al, 2019). Challenges to proteostasis can come from changes in the environment (e.g., extreme temperature), altered internal physiology (e.g., reactive oxygen species or ROS from mitochondrial dysfunction), and physiologically associated decline due to aging (Shore & Ruvkun, 2013; Warnatsch et al, 2013). Proteostasis response pathways offset these challenges by either restoring proper protein folding or removing unfolded or damaged proteins from cells. The failure to maintain proteostasis in the face of such challenges results in physiological decline from the accumulation of oxidized and/or unfolded proteins. One of the key pathways that removes damaged and unfolded proteins is the ubiquitin proteasome system (UPS), which covalently attaches chains of the small protein ubiquitin to lysine side chains of protein substrates (Ciechanover & Stanhill, 2014). E3-type ubiquitin ligases either specifically recognize protein substrates or recognize unfolded proteins with the help of chaperone adapters, attaching single ubiquitin protein moieties (a process called mono-ubiquitination) to these substrates (Zheng & Shabek, 2017). E4-type ubiquitin ligases recognize mono-ubiquitinated proteins and attach additional ubiquitin molecules to the initial ubiquitin, resulting in poly-ubiquitin chains (Hoppe, 2005). Poly-ubiquitinated substrates are then degraded by proteolysis via the 26S proteasome (Bard et al, 2018). Aggregation-prone poly-ubiquitinated proteins are sometimes resistant to proteasomal degradation and must be recognized and removed by additional proteostasis response pathways, including the autophagy pathway (Kirkin et al, 2009; Ryno et al, 2013; Ciechanover & Kwon, 2015; Cortes & La Spada, 2015; Lim & Yue, 2015). It is unclear how these different pathways are coordinated or how some substrates are preferred by one pathway over another (Kocaturk & Gozuacik, 2018). Multicellularity affords organisms the ability to employ different proteostasis pathways depending on the type of tissue or the particular proteostasis challenge. In metazoans, the nervous system coordinates physiology, including proteostasis, in multiple tissues by releasing humoral signals throughout the body (Balch et al, 2008; van Oosten-Hawle & Morimoto, 2014; O'Brien & van Oosten-Hawle, 2016). Biogenic amines—a family of neurotransmitters formed from the decarboxylation of amino acids—can act as such humoral signals when released into the body from neurosecretory cells or even non-neuronal cells (Zeng et al, 2007; Ng et al, 2015; Morgese & Trabace, 2019). In the nematode Caenorhabditis elegans, thermosensory neurons release the biogenic amine neurotransmitter serotonin, which is then able to activate the expression of heat-shock proteins (HSPs) in distal tissues (Prahlad et al, 2008; Tatum et al, 2015). Similarly, octopaminergic sensory neurons modulate the unfolded protein response (UPR) in distal tissues (Sun et al, 2011; Sun et al, 2012). It is unclear whether biogenic amines directly mediate their effects on target tissues or act through additional signaling intermediates to modulate proteostasis. We previously identified novel regulators of ubiquitin-mediated proteostasis in C. elegans by using UbG76V-GFP, a ubiquitin fusion degradation (UFD) substrate expressed from a transgene, to monitor UPS activity (Liu et al, 2011; Joshi et al, 2016). UbG76V-GFP contains a non-cleavable ubiquitin placed amino-terminal to GFP (Liu et al, 2011; Segref et al, 2011), thereby mimicking a mono-ubiquitinated protein. E3- and E4-type ubiquitin ligases in the UFD complex attach additional ubiquitins to the K48 residue of UbG76V-GFP, resulting in poly-ubiquitination and proteasomal degradation of this reporter (Butt et al, 1988; Johnson et al, 1995; Dantuma et al, 2000). UbG76V-GFP degradation was previously monitored in C. elegans intestinal or hypodermal epithelia when this UFD substrate was expressed from either the sur-5 or col-19 promoter, respectively (Liu et al, 2011; Segref et al, 2011). Using this reporter, we found that the biogenic amine dopamine non-autonomously promotes UPS activity in intestinal and hypodermal epithelia in C. elegans (Joshi et al, 2016). Whether C. elegans employs other biogenic amines to regulate the UPS is unknown. Caenorhabditis elegans has four biogenic amine neurotransmitters: serotonin (5-HT), dopamine (DA), octopamine (OA), and tyramine (TA) (Chase & Koelle, 2007). The tph-1 gene encodes the C. elegans tryptophan hydroxylase, the key enzyme for 5-HT biosynthesis from tryptophan (Sze et al, 2000). The cat-2 gene encodes the C. elegans tyrosine hydroxylase, which catalyzes the rate-limiting step in DA synthesis from tyrosine (Lints & Emmons, 1999). The biogenic amines TA and OA are also synthesized from tyrosine but along a different biochemical pathway (Alkema et al, 2005). OA is made from TA, and mutants for the tbh-1 tyramine beta hydroxylase fail to produce OA, whereas mutants for tdc-1 tyrosine decarboxylase produce neither OA nor TA. Although TA and OA are synthesized along the same pathway, they have independent functions, and all four neurotransmitters modulate multiple neural circuits involved in locomotion, egg laying, response to food, or feeding behaviors in C. elegans (Chase & Koelle, 2007). The full scope of their signaling is not known. Here, we used the UbG76V-GFP reporter to examine UPS activity in mutants that fail to synthesize the different biogenic amines in C. elegans. We found that the biogenic amine neurotransmitters DA, 5-HT, and TA modulate UbG76V-GFP turnover in epithelia, as hypodermal epithelia in mutants lacking DA, 5-HT, or TA show increased stability of the otherwise unstable UbG76V-GFP reporter protein. Using RNA-seq, we determined the expression profile of biogenic amine mutants and found that biogenic amine signaling promotes the expression of a specific subclass of cytochrome P450 monooxygenases (P450s) involved in the production of eicosanoids from poly-unsaturated fatty acids (PUFAs). Mutants for one of these regulated P450s, cyp-34A4, showed stabilized UbG76V-GFP, similar to the phenotype observed in biogenic amine mutants. Consistent with our expression profile analysis, we found that biogenic amine mutants have depressed levels of eicosanoids. As eicosanoids are derived from n-3 or n-6 long-chain PUFAs, we examined mutants impaired for PUFA synthesis and found that n-6 lipids are necessary and sufficient to promote UbG76V-GFP turnover. Our results suggest that neurons use biogenic amine neurotransmitters as neurohormonal signals to modulate eicosanoid production from PUFAs. These eicosanoids in turn regulate ubiquitin-mediated proteostasis response pathways in non-neuronal tissues. Results The UbG76V-GFP transgene reports UPS activity We sought to examine ubiquitin-mediated proteostasis in the hypodermis—the epithelial layer surrounding the nematode body cavity and responsible for body growth and synthesis of the outer barrier of the collagenous cuticle. The odIs77 integrated transgene expresses both the chimeric UbG76V-GFP protein and mRFP in the hypodermis from the col-19 promoter (Liu et al, 2011). The levels of UbG76V-GFP, which is a UPS substrate, can be compared to the levels of mRFP, which remains stable (Fig 1A). The mRFP allows researchers to quickly rule out changes in transgene expression versus changes in UbG76V-GFP protein degradation. While mRFP protein remains stable, the UbG76V-GFP protein is rapidly degraded as nematodes move from the first day of adulthood (Fig 1B and D; L4 + 24 h) into the second day of adulthood (Fig 1C and E; L4 + 48 h), and this turnover is largely prevented when either the UFD complex or the proteasome is inhibited (Joshi et al, 2016). The ratio of UbG76V-GFP to mRFP levels inversely reflects UPS activity; thus, hypodermal UPS activity increases significantly as nematodes enter day 2 of adulthood, a period of peak fecundity. Figure 1. The UbG76V-GFP transgene reports UPS activity A. Schematic representation of the UbG76V-GFP chimeric reporter (yellow for ubiquitin and green for GFP) and its associated mRFP internal control (red). The UbG76V-GFP chimera, which contains a mutation in the terminal residue of ubiquitin, cannot be cleaved. The resulting protein is a substrate for poly-ubiquitination (indicated by the circles labeled with “u”) and degradation by the proteasome. High levels of reporter poly-ubiquitination and/or proteasome activity result in little or no GFP fluorescence. B, C. Expression via the col-19 promoter of UbG76V-GFP in C. elegans hypodermis from a single integrated transgene at (B) L4 + 24 h and (C) L4 + 48 h. Wild-type animals are shown. Scale bar, 100 µm. D, E. Expression of mRFP in the same animals as (B, C) at (D) L4 + 24 h and (E) L4 + 48 h. Scale bar, 100 µm. F, G. Expression via the col-19 promoter of UbG76V-GFP in C. elegans hypodermis from a single integrated transgene at (F) L4 + 24 h and (G) L4 + 48 h. Mutants for skn-1(zj15) are shown. Scale bar, 100 µm. H, I. Expression of mRFP in the same animals as (F, G) at (H) L4 + 24 h and (I) L4 + 48 h. Scale bar, 100 µm. J. Quantified fluorescence of UbG76V-GFP normalized to mRFP in the hypodermis of animals from the indicated time point (in hours) after the L4 stage. Animals were exposed to the indicated knockdown RNAi bacterial strains or a strain that only contained the empty RNAi vector since hatching. *P < 0.001 using ANOVA with Dunnett’s post hoc comparison to the wild-type control equivalent time point, with N = 20 animals per genotype and time point. Error bars indicate SEM. K. Quantified epoxomicin-sensitive proteasome activity (difference in slope of turnover of fluorescent chymotrypsin substrate between epoxomicin-treated and untreated samples; see Appendix Fig S2) from lysates of animals treated as in (J). *P < 0.001, using ANOVA with Dunnett’s post hoc comparison to the empty RNAi vector control, with N = 3 trials. Error bars indicate SEM. L. Quantified fluorescence of UbG76V-GFP normalized to mRFP in the hypodermis of animals from the indicated time point (in hours) and the indicated genotypes after the L4 stage, as per (J). *P < 0.001 using ANOVA with Dunnett’s post hoc comparison to the wild-type control equivalent time point, with N = 20 animals per genotype and time point. Error bars indicate SEM. M. Quantified epoxomicin-sensitive proteasome activity, as per (K). *P < 0.001 using ANOVA with Dunnett’s post hoc comparison to the wild-type control equivalent time point, with N = 3 trials. Error bars indicate SEM. Download figure Download PowerPoint We revalidated our transgenic reporter by examining transgenic animals impaired for UPS activity. UbG76V-GFP is poly-ubiquitinated by the UFD complex and degraded by the 26S proteasome (Liu et al, 2011; Segref et al, 2011; Joshi et al, 2016). Eggs harboring the odIs77 transgene were hatched on bacterial lawns expressing RNAi constructs for either the UFD complex subunit ufd-1, the proteasome complex core particle subunit pbs-5, or an empty vector as control. UbG76V-GFP levels were elevated relative to control in the ufd-1 and pbs-5 RNAi knockdown experiments by both day 1 and day 2 adulthood (Fig 1J and Appendix Fig S1), consistent with UPS-mediated turnover of this chimeric reporter protein. We did not detect any significant impact of the odIs77 transgene, which expresses UbG76V-GFP, on development or behavior (Appendix Fig S1). We also directly examined proteasome activity by generating lysates from the animals and incubating them with the fluorescent chymotryptic substrate Suc-Leu-Leu-Val-Tyr-AMC in the presence or absence of the proteasome inhibitor epoxomicin, as previously described (Vilchez et al, 2012; Joshi et al, 2016). Knockdown of the proteasome subunit pbs-5 resulted in diminished chymotrypsin-like proteasome activity relative to empty vector control (Fig 1K). Knockdown of ufd-1, a gene involved in poly-ubiquitination but not proteasome catalytic activity, did not diminish chymotrypsin-like proteasome activity, as expected (Fig 1K). The odIs77 transgene itself did not significantly impact proteasome activity (Appendix Fig S2). These results confirm that the increased levels of the UbG76V-GFP chimeric reporter can reflect either decreases in UFD activity/capacity (and thus poly-ubiquitination) or proteasome activity/capacity. In addition, we examined UbG76V-GFP levels in established regulators of proteostasis (Appendix Fig S3), including daf-16, hsf-1, skn-1, and pqm-1, and found turnover depressed in these mutants, supporting the use of the odIs77 transgene as a sensitive reporter for changes in proteostasis (Ben-Zvi et al, 2009; Steinbaugh et al, 2015; O'Brien et al, 2018). The transcription factor SKN-1, which is part of the oxidative stress response and homologous to human Nrf2, directly promotes the expression of proteasome subunit genes (An & Blackwell, 2003; Li et al, 2011; Niu et al, 2011; Keith et al, 2016). We examined UbG76V-GFP levels in skn-1(zj15), a fertile hypomorphic allele, and skn-1(tm3411), a null deletion allele that results in sterile animals (Ruf et al, 2013; Tang et al, 2015). UbG76V-GFP was present at high levels in skn-1(zj15) in day 1 (L4 + 24 h) adults (Fig 1F, H and L and Appendix Fig S1) and remained elevated and stable in day 2 (L4 + 48 h) and day 3 (L4 + 72 h) adults relative to wild type (Fig 1G, I and L). UbG76V-GFP was similarly stable in skn-1(tm3411) null mutants (Fig 1L). As skn-1(zj15) animals are fertile and do not require a balancer chromosome, we were able to collect large numbers of synchronized animals to measure proteasome activity. Mutants for skn-1(zj15) showed about a 30% decrease in chymotrypsin-like proteasome activity relative to wild type in both day 1 and day 2 adults (Fig 1M and Appendix Fig S2), consistent with the decreased levels of proteasome subunit expression (Li et al, 2011; Niu et al, 2011; Keith et al, 2016) and stabilized UPS reporter (Fig 1L) observed in mutants. Thus, in addition to providing a feedback loop to activate proteasome expression during proteotoxic stress, SKN-1 maintains baseline UPS activity. Biogenic amine signaling modulates protein turnover In a prior RNAi genetic screen, we found that knockdown of dopamine receptors resulted in the stabilization of the UbG76V-GFP hypodermal UPS reporter (Joshi et al, 2016). We reasoned that other biogenic amine neurotransmitters besides dopamine might regulate UPS activity. In order to test each biogenic amine for such a role, we examined mutants for specific biosynthetic enzymes (Fig 2A). We introduced the odIs77 transgene into cat-2(e1112) mutants, which have a nonsense mutation in cat-2 and fail to synthesize DA, tph-1(mg280) mutants, which harbor a deletion of tph-1 sequences and fail to synthesize 5-HT, tdc-1(ok914) mutants, which contain a deletion of tdc-1 sequences and fail to synthesize TA and OA, and tbh-1(ok1196) mutants, which harbor a deletion of tbh-1 sequences and fail to synthesize OA (Sulston et al, 1975; Lints & Emmons, 1999; Sze et al, 2000; Alkema et al, 2005). In addition to qualitatively assessing fluorescence photomicrographs (Fig 2C–F; green and red channels merged), we quantified UbG76V-GFP and mRFP levels for each genotype (Fig 2B and K, and Appendix Fig S4). UbG76V-GFP levels in cat-2, tph-1, and tdc-1 mutants were 30–50% higher than those in wild type in day 1 (L4 + 24 h) adults and remained higher throughout our time course analysis (Fig 2B–F). We observed no difference between tbh-1 mutants and wild type, suggesting that OA is not required to regulate UbG76V-GFP turnover (Fig 2K). Figure 2. Biogenic amine signaling modulates protein turnover A. Schematic representation of the biogenic amine synthesis pathways in Caenorhabditis elegans. Final biogenic amine neurotransmitter products are shown in red. The gene encoding each biosynthesis enzyme in the pathway is indicated over the arrow representing its catalyzed reaction. B. Quantified fluorescence of UbG76V-GFP normalized to mRFP in the hypodermis of animals of the specified genotype from the indicated time point (in hours) after the L4 stage. *P < 0.001 using ANOVA with Dunnett’s post hoc comparison to the wild-type control equivalent time point, with N = 20 animals per genotype and time point. Error bars indicate SEM. C, D. Co-expression via the col-19 promoter of UbG76V-GFP (green) and mRFP (red) in C. elegans hypodermis from a single integrated transgene at (C) L4 + 24 h and (D) L4 + 48 h. Green and red channels are merged. Wild-type animals are shown. Scale bar, 100 µm. E, F. Similar analysis as in (C, D) for cat-2(e1112) mutants. Scale bar, 100 µm. G, H. Expression of mRFP via the col-19 promoter in C. elegans hypodermis at (G) L4 + 24 h or (H) L4 + 48 h. Wild-type animals are shown. Puncta containing mRFP aggregates are indicated by arrows. Scale bar, 100 µm. I, J. Similar analysis as in (G, H) for cat-2(e1112) mutants. Scale bar, 100 µm. K, L. Quantified fluorescence of UbG76V-GFP normalized to mRFP in the hypodermis of animals of the indicated genotype at L4 + 48 h. *P < 0.001 using ANOVA with Dunnett’s post hoc comparison to the wild-type control equivalent time point, with N = 20 animals per genotype and time point. Data have been normalized to wild type. Error bars indicate SEM. Download figure Download PowerPoint Several additional experiments supported our conclusion that biogenic amines regulate UPS activity in the hypodermis. Investigation of additional alleles of each gene at day 2 (L4 + 48 h) was consistent and confirmed the above findings (Fig 2K). To confirm whether any observed regulation of UbG76V-GFP occurs at the level of protein stability rather than transcription, we quantified UbG76V-GFP and mRFP mRNA levels and detected no change between the different genotypes (Appendix Fig S5), consistent with a post-transcriptional explanation for the observed differences in protein levels. We did not detect a change in developmental timing that could alternatively explain the slowed capacity for UbG76V-GFP turnover (Appendix Fig S5). Finally, the UPS phenotype for the cat-2 and tph-1 mutants could be rescued by supplementing their growth media with exogenous DA and 5-HT, respectively, and wild-type animals supplemented with 5-HT showed accelerated UbG76V-GFP turnover (Fig EV1). Our results indicate that UPS activity is decreased in mutants that fail to synthesize the biogenic amines DA, 5-HT, and TA, but not in mutants that fail to synthesize only OA, and that supplemental levels of 5-HT can increase activity. Click here to expand this figure. Figure EV1. Exogenous biogenic amines rescue cat-2 and tph-1 mutants A, B. Quantified fluorescence of UbG76V-GFP normalized to mRFP in the hypodermis of animals of the specified genotype and supplemental drug treatment from the indicated time point (in hours) after the L4 stage. *P < 0.001, ANOVA with Dunnett’s post hoc comparison to the wild-type control equivalent time point. #P < 0.001, ANOVA with Bonferoni’s post hoc comparison between samples indicated by the bracket. N = 15–20 animals per genotype, treatment, and time point. Error bars indicate SEM. C. Quantified mRFP puncta per animal in the hypodermis of animals of the indicated genotype at the indicated time point. *P < 0.01, ANOVA with Dunnett’s post hoc comparison to the wild-type control equivalent time point. N = 20 animals per genotype and time point. Error bars indicate SEM. Download figure Download PowerPoint Although mRFP protein levels remain unchanged in all of the biogenic amine mutants, we noticed that the protein accumulated in aggregated puncta throughout the hypodermis in day 2 (L4 + 48 h) animals (Fig 2G and H). Like most derivates of DsRed, the monomeric RFP can aggregate in cells (Yanushevich et al, 2002; Snapp, 2005; Snaith et al, 2010). We reasoned that the change in mRFP aggregation from day 1 adults to day 2 adults might reflect the same change in proteostasis that augments UbG76V-GFP turnover. Indeed, tph-1(mg280) and skn-1(zj15) mutants showed a decrease in the number of visible mRFP aggregates (puncta) in day 1 and day 2 adults (Fig EV1). Mutants for cat-2(e1112) had a similar number of mRFP aggregates as wild type at day 1, but did not accumulate as many aggregates by day 2 (Figs 2I and J, and EV1). Our results suggest that in addition to promoting UPS activity, biogenic amine signaling is required for the hypodermis to sequester aggregation-prone proteins into large punctate structures. We also examined mutants that impair signaling of more than one biogenic amine. BAS-1 encodes an aromatic amino acid decarboxylase required for the synthesis of both DA and 5-HT, CAT-1 encodes a monoamine transporter required for loading synaptic vesicles with all biogenic amines, and CAT-4 encodes a GTP cyclohydrolase required for the synthesis of multiple small compounds including DA and 5-HT (Sulston et al, 1975; Loer & Kenyon, 1993; Duerr et al, 1999; Hare & Loer, 2004). UbG76V-GFP levels in null mutants for all three of these genes were similar to those in wild type in day 1 (L4 + 24 h) adults but remained stable in day 2 adults (Fig 2K; Appendix Fig S4), suggesting reduced turnover. BAS-1 synthesizes 5-HT and DA from 5-hydroxytryptophan (5-HTP) and L-DOPA (Fig 2A), respectively, so this result suggests that these neurotransmitter intermediates are not sufficient to substitute in UPS regulation for their canonical biogenic amine products. Although these mutants block signaling of multiple biogenic amines, their effect on UbG76V-GFP turnover was not greater than that of mutants defective in signaling from single biogenic amines, suggesting that DA, 5-HT, and TA might regulate UPS activity through a single, shared genetic and physiological process. Finally, we characterized receptor mutants for the two best studied biogenic amines, 5-HT and DA, in C. elegans. Four DA receptors (dop-1, dop-2, dop-3, and dop-4) mediate DA signaling, whereas four 5-HT receptors (ser-1, ser-5, ser-7, and mod-1) mediate 5-HT signaling (Chase & Koelle, 2007). Mutants for dop-1 and dop-4 showed decreased turnover of UbG76V-GFP (Fig 2L), similar to previous observations (Joshi et al, 2016). Mutants for ser-1, ser-5, and mod-1, but not ser-7, also had stabilized UbG76V-GFP levels (Fig 2L and Appendix Fig S4). Serotonin regulates multiple physiological systems in C. elegans, including pharyngeal pumping rate, fat accumulation, and egg laying, through the use of distinct receptor combinations (Chase & Koelle, 2007). We did not observe defects in pumping or egg laying in cat-2 and tdc-1 mutants (Appendix Fig S5), suggesting that defects in these systems are not the cause of the UPS phenotype in these mutants. By contrast, mutants for the 5-HT receptor ser-7 are defective in pumping, egg laying, and fat accumulation, yet do not have a UPS phenotype (Carre-Pierrat et al, 2006; Hobson et al, 2006; Yemini et al, 2013). Thus, a novel combination of 5-HT receptors, distinct from those that regulate these other physiological systems, mediates UPS regulation. Biogenic amine signaling promotes protein poly-ubiquitination without perturbing proteasome chymotrypsin-like activity The stabilization of UbG76V-GFP protein in biogenic amine signaling mutants could be due to either a reduction in its poly-ubiquitination or a reduction in its proteolysis by the proteasome. We made lysates from different biogenic amine synthesis mutants at day 2 of adulthood (L4 + 48 h), separated proteins on two different SDS–PAGE gels, and then detected UbG76V-GFP protein by Western blot either with anti-GFP antibodies (Fig 3A) or anti-ubiquitin antibodies (Fig 3B). Little UbG76V-GFP protein is detected in wild-type animals or tbh-1 mutants at L4 + 48 h (quantified in Fig 3C and D). Our control skn-1(zj15) mutants accumulated non-ubiquitinated, mono-ubiquitinated, di-ubiquitinated, and tri-ubiquitinated UbG76V-GFP species, reflecting a reduction in poly-ubiquitination and/or the ability to clear poly-ubiquitinated substrates, consistent with reduced proteasome activity in these mutants (Fig 3A–D). We observed a similar pattern of ubiquitinated UbG76V-GFP accumulation in cat-2, tdc-1, and tph-1 mutants. While we did not detect proteins larger than 70 kDa in biogenic signaling mutants or skn-1(zj15) using the anti-GFP antibodies (Fig 3A), we did detect additional proteins ranging from 70 to 250 kDa in these mutants using anti-ubiquitin antibodies (Figs 3B and E, and EV2), which detects endogenous ubiquitinated proteins in addition to the UbG76V-GFP reporter. Althoug" @default.
• W3124388718 created "2021-02-01" @default.
• W3124388718 creator A5009926386 @default.
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• W3124388718 date "2021-01-20" @default.
• W3124388718 modified "2023-10-10" @default.
• W3124388718 title "Biogenic amine neurotransmitters promote eicosanoid production and protein homeostasis" @default.
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