RESUMO
Mechanical stress during muscle contraction is a constant threat to proteome integrity. However, there is a lack of experimental systems to identify critical proteostasis regulators under mechanical stress conditions. Here, we present the transgenic Caenorhabditis elegans model OptIMMuS (Optogenetic Induction of Mechanical Muscle Stress) to study changes in the proteostasis network associated with mechanical forces. Repeated blue light exposure of a muscle-expressed Chlamydomonas rheinhardii channelrhodopsin-2 variant results in sustained muscle contraction and mechanical stress. Using OptIMMuS, combined with proximity labeling and mass spectrometry, we identify regulators that cooperate with the myosin-directed chaperone UNC-45 in muscle proteostasis. One of these is the TRIM E3 ligase NHL-1, which interacts with UNC-45 and muscle myosin in genetic epistasis and co-immunoprecipitation experiments. We provide evidence that the ubiquitylation activity of NHL-1 regulates myosin levels and functionality under mechanical stress. In the future, OptIMMuS will help to identify muscle-specific proteostasis regulators of therapeutic relevance.
Assuntos
Animais Geneticamente Modificados , Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Optogenética , Proteostase , Estresse Mecânico , Ubiquitina-Proteína Ligases , Ubiquitinação , Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/genética , Animais , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genética , Miosinas/metabolismo , Miosinas/genética , Contração Muscular/fisiologia , Músculos/metabolismo , Chaperonas MolecularesRESUMO
Proteome integrity is fundamental for cellular and organismal homeostasis. The mitochondrial unfolded protein response (UPRmt), a key component of the proteostasis network, is activated in a non-cell-autonomous manner in response to mitochondrial stress in distal tissues. However, the importance of inter-tissue communication for UPRmt inducibility under physiological conditions remains elusive. Here, we show that an intact germline is essential for robust UPRmt induction in the Caenorhabditis elegans somatic tissues. A series of nematode mutants with germline defects are unable to respond to genetic or chemical UPRmt inducers. Our genetic analysis suggests that reproductive signals, rather than germline stem cells, are responsible for somatic UPRmt induction. Consistent with this observation, we show that UPRmt is sexually dimorphic, as male nematodes are inherently unresponsive to mitochondrial stress. Our findings highlight a paradigm of germline-somatic communication and suggest that reproductive cessation is a primary cause of age-related UPRmt decline.
Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Células Germinativas , Mitocôndrias , Reprodução , Resposta a Proteínas não Dobradas , Animais , Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/genética , Mitocôndrias/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Células Germinativas/metabolismo , Masculino , Estresse Fisiológico , FemininoRESUMO
The degradation of damaged proteins is critical for tissue integrity and organismal health because damaged proteins have a high propensity to form aggregates. E3 ubiquitin ligases are key regulators of protein quality control (PQC) and mediate the selective degradation of damaged proteins, a process termed 'PQC degradation' (PQCD). The degradation signals (degrons) that trigger PQCD are based on hydrophobic sites that are normally buried within the native protein structure. However, an open question is how PQCD-specialized E3 ligases distinguish between transiently misfolded proteins, which can be efficiently refolded, and permanently damaged proteins, which must be degraded. While significant progress has been made in characterizing degradation determinants, understanding the key regulatory signals of cellular and organismal PQCD pathways remains a challenge.
RESUMO
Many proteins self-assemble into dimers and higher-order oligomers. Therefore, the goal of this protocol is to characterize the conformational states of an endogenous protein of interest. Here, we present a protocol for assessing protein self-assembly in cell lysates using chemical cross-linking. We describe steps for chemical cross-linking with recombinant proteins as well as steps for cell culture and cell lysate preparation, chemical cross-linking, SDS-PAGE, and western blotting for the detection of endogenous proteins. For complete details on the use and execution of this protocol, please refer to Balaji et al.1.
Assuntos
Reagentes de Ligações Cruzadas , Eletroforese em Gel de Poliacrilamida , Reagentes de Ligações Cruzadas/química , Eletroforese em Gel de Poliacrilamida/métodos , Humanos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas/química , Proteínas/metabolismo , Western Blotting/métodos , Multimerização ProteicaRESUMO
The endoplasmic reticulum (ER) is central to the processing of luminal, transmembrane, and secretory proteins, and maintaining a functional ER is essential for organismal physiology and health. Increased protein-folding load on the ER causes ER stress, which activates quality control mechanisms to restore ER function and protein homeostasis. Beyond protein quality control, mRNA decay pathways have emerged as potent ER fidelity regulators, but their mechanistic roles in ER quality control and their interrelationships remain incompletely understood. Herein, we review ER-associated RNA decay pathways - including regulated inositol-requiring enzyme 1α (IRE1α)-dependent mRNA decay (RIDD), nonsense-mediated mRNA decay (NMD), and Argonaute-dependent RNA silencing - in ER homeostasis, and highlight the intricate coordination of ER-targeted RNA and protein decay mechanisms and their association with antiviral defense.
RESUMO
Most of the functions of LC3/GABARAP in macroautophagy/autophagy are considered to depend on their association with the phagophore membrane through a conjugation to a lipid. Using site-directed mutagenesis, we inhibited the conjugation of LGG-1, the single homolog of GABARAP in C. elegans. Mutants that express only cytosolic forms revealed an essential role for the cleaved form of LGG-1 in autophagy but also in an autophagy-independent embryonic function.
Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Animais , Autofagia , Proteínas Associadas aos Microtúbulos , AutofagossomosRESUMO
Ubiquitin-dependent control of mitochondrial dynamics is important for protein quality and neuronal integrity. Mitofusins, mitochondrial fusion factors, can integrate cellular stress through their ubiquitylation, which is carried out by multiple E3 enzymes in response to many different stimuli. However, the molecular mechanisms that enable coordinated responses are largely unknown. Here we show that yeast Ufd2, a conserved ubiquitin chain-elongating E4 enzyme, is required for mitochondrial shape adjustments. Under various stresses, Ufd2 translocates to mitochondria and triggers mitofusin ubiquitylation. This elongates ubiquitin chains on mitofusin and promotes its proteasomal degradation, leading to mitochondrial fragmentation. Ufd2 and its human homologue UBE4B also target mitofusin mutants associated with Charcot-Marie-Tooth disease, a hereditary sensory and motor neuropathy characterized by progressive loss of the peripheral nerves. This underscores the pathophysiological importance of E4-mediated ubiquitylation in neurodegeneration. In summary, we identify E4-dependent mitochondrial stress adaptation by linking various metabolic processes to mitochondrial fusion and fission dynamics.
Assuntos
Mitocôndrias , Proteínas Mitocondriais , Ubiquitina-Proteína Ligases , Humanos , Aclimatação , Mitocôndrias/metabolismo , Saccharomyces cerevisiae/metabolismo , Ubiquitina , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Proteínas Mitocondriais/metabolismoRESUMO
The ubiquitin-like proteins Atg8/LC3/GABARAP are required for multiple steps of autophagy, such as initiation, cargo recognition and engulfment, vesicle closure and degradation. Most of LC3/GABARAP functions are considered dependent on their post-translational modifications and their association with the autophagosome membrane through a conjugation to a lipid, the phosphatidyl-ethanolamine. Contrarily to mammals, C. elegans possesses single homologs of LC3 and GABARAP families, named LGG-2 and LGG-1. Using site-directed mutagenesis, we inhibited the conjugation of LGG-1 to the autophagosome membrane and generated mutants that express only cytosolic forms, either the precursor or the cleaved protein. LGG-1 is an essential gene for autophagy and development in C. elegans, but we discovered that its functions could be fully achieved independently of its localization to the membrane. This study reveals an essential role for the cleaved form of LGG-1 in autophagy but also in an autophagy-independent embryonic function. Our data question the use of lipidated GABARAP/LC3 as the main marker of autophagic flux and highlight the high plasticity of autophagy.
Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Humanos , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Autofagia , Autofagossomos/metabolismo , Fagocitose , Mamíferos/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismoRESUMO
Muscle is a highly dynamic tissue in which a variety of folding and degradation processes are active to maintain protein homeostasis (proteostasis) and functionality. The muscle-specific chaperone UNC-45 folds the motor protein myosin and assembles it into myofilaments. Malfunction of this chaperone leads to misfolding of myosin, disorganization of myofilaments, and degradation of misfolded myosin molecules by the proteasome. Here, we present a new muscle-specific ubiquitin fusion degradation (UFD) model substrate in C. elegans that helps clarify how UNC-45 dysfunction affects muscle proteostasis.
RESUMO
Mitochondria are central hubs for energy production, metabolism and cellular signal transduction in eukaryotic cells. Maintenance of mitochondrial homeostasis is important for cellular function and survival. In particular, cellular metabolic state is in constant communication with mitochondrial homeostasis. One of the most important metabolic processes that provide energy in the cell is amino acid metabolism. Almost all of the 20 amino acids that serve as the building blocks of proteins are produced or degraded in the mitochondria. The synthesis of the amino acids aspartate and arginine depends on the activity of the respiratory chain, which is essential for cell proliferation. The degradation of branched-chain amino acids mainly occurs in the mitochondrial matrix, contributing to energy metabolism, mitochondrial biogenesis, as well as protein quality control in both mitochondria and cytosol. Dietary supplementation or restriction of amino acids in worms, flies and mice modulates lifespan and health, which has been associated with changes in mitochondrial biogenesis, antioxidant response, as well as the activity of tricarboxylic acid cycle and respiratory chain. Consequently, impaired amino acid metabolism has been associated with both primary mitochondrial diseases and diseases with mitochondrial dysfunction such as cancer. Here, we present recent observations on the crosstalk between amino acid metabolism and mitochondrial homeostasis, summarise the underlying molecular mechanisms to date, and discuss their role in cellular functions and organismal physiology.
RESUMO
The endoplasmic reticulum (ER) coordinates mRNA translation and processing of secreted and endomembrane proteins. ER-associated degradation (ERAD) prevents the accumulation of misfolded proteins in the ER, but the physiological regulation of this process remains poorly characterized. Here, in a genetic screen using an ERAD model substrate in Caenorhabditis elegans, we identified an anti-viral RNA interference pathway, referred to as ER-associated RNA silencing (ERAS), which acts together with ERAD to preserve ER homeostasis and function. Induced by ER stress, ERAS is mediated by the Argonaute protein RDE-1/AGO2, is conserved in mammals and promotes ER-associated RNA turnover. ERAS and ERAD are complementary, as simultaneous inactivation of both quality-control pathways leads to increased ER stress, reduced protein quality control and impaired intestinal integrity. Collectively, our findings indicate that ER homeostasis and organismal health are protected by synergistic functions of ERAS and ERAD.
Assuntos
Retículo Endoplasmático , Interferência de RNA , Retículo Endoplasmático/genéticaRESUMO
Alternations of redox metabolism have been associated with the extension of lifespan in roundworm Caenorhabditis elegans, caused by moderate mitochondrial dysfunction, although the underlying signalling cascades are largely unknown. Previously, we identified transcriptional factor Krüppel-like factor-1 (KLF-1) as the main regulator of cytoprotective longevity-assurance pathways in the C. elegans long-lived mitochondrial mutants. Here, we show that KLF-1 translocation to the nucleus and the activation of the signalling cascade is dependent on the mitochondria-derived hydrogen peroxide (H2O2) produced during late developmental phases where aerobic respiration and somatic mitochondrial biogenesis peak. We further show that mitochondrial-inducible superoxide dismutase-3 (SOD-3), together with voltage-dependent anion channel-1 (VDAC-1), is required for the life-promoting H2O2 signalling that is further regulated by peroxiredoxin-3 (PRDX-3). Increased H2O2 release in the cytoplasm activates the p38 MAPK signalling cascade that induces KLF-1 translocation to the nucleus and the activation of transcription of C. elegans longevity-promoting genes, including cytoprotective cytochrome P450 oxidases. Taken together, our results underline the importance of redox-regulated signalling as the key regulator of longevity-inducing pathways in C. elegans, and position precisely timed mitochondria-derived H2O2 in the middle of it.
Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Animais , Caenorhabditis elegans/metabolismo , Longevidade/genética , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Peróxido de Hidrogênio/metabolismo , Mitocôndrias/genética , Mitocôndrias/metabolismo , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , OxirreduçãoRESUMO
Organismal physiology and survival are influenced by environmental conditions and linked to protein quality control. Proteome integrity is achieved by maintaining an intricate balance between protein folding and degradation. In Caenorhabditis elegans, acute heat stress determines cell non-autonomous regulation of chaperone levels. However, how the perception of environmental changes, including physiological temperature, affects protein degradation remains largely unexplored. Here, we show that loss-of-function of dyf-1 in Caenorhabditis elegans associated with dysfunctional sensory neurons leads to defects in both temperature perception and thermal adaptation of the ubiquitin/proteasome system centered on thermosensory AFD neurons. Impaired perception of moderate temperature changes worsens ubiquitin-dependent proteolysis in intestinal cells. Brain-gut communication regulating protein turnover is mediated by upregulation of the insulin-like peptide INS-5 and inhibition of the calcineurin-regulated forkhead-box transcription factor DAF-16/FOXO. Our data indicate that perception of ambient temperature and its neuronal integration is important for the control of proteome integrity in complex organisms.
Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Animais , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Calcineurina/genética , Calcineurina/metabolismo , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Insulina/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Proteoma/metabolismo , Células Receptoras Sensoriais/metabolismo , Fatores de Transcrição/metabolismo , Ubiquitina/metabolismoRESUMO
Branched-chain amino acid (BCAA) metabolism is a central hub for energy production and regulation of numerous physiological processes. Controversially, both increased and decreased levels of BCAAs are associated with longevity. Using genetics and multi-omics analyses in Caenorhabditis elegans, we identified adaptive regulation of the ubiquitin-proteasome system (UPS) in response to defective BCAA catabolic reactions after the initial transamination step. Worms with impaired BCAA metabolism show a slower turnover of a GFP-based proteasome substrate, which is suppressed by loss-of-function of the first BCAA catabolic enzyme, the branched-chain aminotransferase BCAT-1. The exogenous supply of BCAA-derived carboxylic acids, which are known to accumulate in the body fluid of patients with BCAA metabolic disorders, is sufficient to regulate the UPS. The link between BCAA intermediates and UPS function presented here sheds light on the unexplained role of BCAAs in the aging process and opens future possibilities for therapeutic interventions.
Assuntos
Aminoácidos de Cadeia Ramificada , Complexo de Endopeptidases do Proteassoma , Animais , Aminoácidos de Cadeia Ramificada/metabolismo , Caenorhabditis elegans/metabolismoRESUMO
The high substrate selectivity of the ubiquitin/proteasome system is mediated by a large group of E3 ubiquitin ligases. The ubiquitin ligase CHIP regulates the degradation of chaperone-controlled and chaperone-independent proteins. To understand how CHIP mediates substrate selection and processing, we performed a structure-function analysis of CHIP and addressed its physiological role in Caenorhabditis elegans and human cells. The conserved function of CHIP in chaperone-assisted degradation requires dimer formation to mediate proteotoxic stress resistance and to prevent protein aggregation. The CHIP monomer, however, promotes the turnover of the membrane-bound insulin receptor and longevity. The dimer-monomer transition is regulated by CHIP autoubiquitylation and chaperone binding, which provides a feedback loop that controls CHIP activity in response to cellular stress. Because CHIP also binds other E3 ligases, such as Parkin, the molecular switch mechanism described here could be a general concept for the regulation of substrate selectivity and ubiquitylation by combining different E3s.
Assuntos
Proteínas de Caenorhabditis elegans , Ubiquitina-Proteína Ligases , Ubiquitina , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Humanos , Chaperonas Moleculares/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação/genéticaRESUMO
Temperature is an important environmental condition that determines the physiology and behavior of all organisms. Animals use different response strategies to adapt and survive fluctuations in ambient temperature. The hermaphrodite Caenorhabditis elegans has a well-studied neuronal network consisting of 302 neurons. The bilateral AFD neurons are the primary thermosensory neurons in the nematode. In addition to regulating thermosensitivity, AFD neurons also coordinate cellular stress responses through systemic mechanisms involving neuroendocrine signaling. Recent studies have examined the effects of temperature on altering various signaling pathways through specific gene expression programs that promote stress resistance and longevity. These studies challenge the proposed theories of temperature-dependent regulation of aging as a passive thermodynamic process. Instead, they provide evidence that aging is a well-defined genetic program. Loss of protein homeostasis (proteostasis) is one of the key hallmarks of aging. Indeed, proteostasis pathways, such as the heat shock response and aggregation of metastable proteins, are also controlled by thermosensory neurons in C. elegans. Prolonged heat stress is thought to play a critical role in the development of neurodegenerative protein misfolding diseases in humans. This review presents the latest evidence on how temperature coordinates proteostasis and aging. It also discusses how studies of poikilothermic organisms can be applied to vertebrates and provides new therapeutic strategies for human disease.
RESUMO
CHIP (C-terminus of Hsc70-interacting protein) and its worm ortholog CHN-1 are E3 ubiquitin ligases that link the chaperone system with the ubiquitin-proteasome system (UPS). CHN-1 can cooperate with UFD-2, another E3 ligase, to accelerate ubiquitin chain formation; however, the basis for the high processivity of this E3s set has remained obscure. Here, we studied the molecular mechanism and function of the CHN-1-UFD-2 complex in Caenorhabditis elegans. Our data show that UFD-2 binding promotes the cooperation between CHN-1 and ubiquitin-conjugating E2 enzymes by stabilizing the CHN-1 U-box dimer. However, HSP70/HSP-1 chaperone outcompetes UFD-2 for CHN-1 binding, thereby promoting a shift to the autoinhibited CHN-1 state by acting on a conserved residue in its U-box domain. The interaction with UFD-2 enables CHN-1 to efficiently ubiquitylate and regulate S-adenosylhomocysteinase (AHCY-1), a key enzyme in the S-adenosylmethionine (SAM) regeneration cycle, which is essential for SAM-dependent methylation. Our results define the molecular mechanism underlying the synergistic cooperation of CHN-1 and UFD-2 in substrate ubiquitylation.
Assuntos
Proteínas de Caenorhabditis elegans , Ubiquitina , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Chaperonas Moleculares/metabolismo , Ubiquitina/metabolismo , Enzimas de Conjugação de Ubiquitina/genética , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , UbiquitinaçãoRESUMO
The conserved Argonaute-family members ALG-1 and ALG-2 are known to regulate processing and maturation of microRNAs to target mRNAs for degradation or translational inhibition (Bouasker and Simard 2012; Meister 2013). Consequently, depletion of alg-1 and alg-2 results in multiple phenotypes. Our data describe a role of microRNA-regulation in stress resistance and proteostasis with special emphasis on ubiquitin-dependent degradation pathways, such as ubiquitin fusion degradation (UFD) and endoplasmic reticulum (ER)-associated protein degradation (ERAD).