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1.
Cell ; 169(3): 470-482.e13, 2017 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-28431247

RESUMO

Aging is attended by a progressive decline in protein homeostasis (proteostasis), aggravating the risk for protein aggregation diseases. To understand the coordination between proteome imbalance and longevity, we addressed the mechanistic role of the quality-control ubiquitin ligase CHIP, which is a key regulator of proteostasis. We observed that CHIP deficiency leads to increased levels of the insulin receptor (INSR) and reduced lifespan of worms and flies. The membrane-bound INSR regulates the insulin and IGF1 signaling (IIS) pathway and thereby defines metabolism and aging. INSR is a direct target of CHIP, which triggers receptor monoubiquitylation and endocytic-lysosomal turnover to promote longevity. However, upon proteotoxic stress conditions and during aging, CHIP is recruited toward disposal of misfolded proteins, reducing its capacity to degrade the INSR. Our study indicates a competitive relationship between proteostasis and longevity regulation through CHIP-assisted proteolysis, providing a mechanistic concept for understanding the impact of proteome imbalance on aging.


Assuntos
Envelhecimento , Antígenos CD/metabolismo , Receptor de Insulina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Caenorhabditis elegans , Drosophila melanogaster , Endocitose , Humanos , Longevidade , Lisossomos/metabolismo , Proteólise , Proteoma , Transdução de Sinais , Somatomedinas , Ubiquitinação
2.
Mol Cell ; 82(17): 3239-3254.e11, 2022 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-36027913

RESUMO

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/genética
3.
Bioessays ; 40(5): e1700223, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29611634

RESUMO

The insulin/insulin-like growth factor-1 (IGF-1) signaling (IIS) pathway is a pivotal genetic program regulating cell growth, tissue development, metabolic physiology, and longevity of multicellular organisms. IIS integrates a fine-tuned cascade of signaling events induced by insulin/IGF-1, which is precisely controlled by post-translational modifications. The ubiquitin/proteasome-system (UPS) influences the functionality of IIS through inducible ubiquitylation pathways that regulate internalization of the insulin/IGF-1 receptor, the stability of downstream insulin/IGF-1 signaling targets, and activity of nuclear receptors for control of gene expression. An age-related decline in UPS activity is often associated with an impairment of IIS, contributing to pathologies such as cancer, diabetes, cardiovascular, and neurodegenerative disorders. Recent findings identified a key role of diverse ubiquitin modifications in insulin signaling decisions, which governs dynamic adaption upon environmental and physiological changes. In this review, we discuss the mutual crosstalk between ubiquitin and insulin signaling pathways in the context of cellular and organismal homeostasis.


Assuntos
Homeostase/fisiologia , Insulina/metabolismo , Ubiquitinação/fisiologia , Animais , Homeostase/genética , Humanos , Fator de Crescimento Insulin-Like I/genética , Fator de Crescimento Insulin-Like I/metabolismo , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Ubiquitina/genética , Ubiquitina/metabolismo , Ubiquitinação/genética
4.
J Vis Exp ; (171)2021 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-34057440

RESUMO

The covalent attachment of ubiquitin (Ub) to internal lysine residue(s) of a substrate protein, a process termed ubiquitylation, represents one of the most important post-translational modifications in eukaryotic organisms. Ubiquitylation is mediated by a sequential cascade of three enzyme classes including ubiquitin-activating enzymes (E1 enzymes), ubiquitin-conjugating enzymes (E2 enzymes), and ubiquitin ligases (E3 enzymes), and sometimes, ubiquitin-chain elongation factors (E4 enzymes). Here, in vitro protocols for ubiquitylation assays are provided, which allow the assessment of E3 ubiquitin ligase activity, the cooperation between E2-E3 pairs, and substrate selection. Cooperating E2-E3 pairs can be screened by monitoring the generation of free poly-ubiquitin chains and/or auto-ubiquitylation of the E3 ligase. Substrate ubiquitylation is defined by selective binding of the E3 ligase and can be detected by western blotting of the in vitro reaction. Furthermore, an E2~Ub discharge assay is described, which is a useful tool for the direct assessment of functional E2-E3 cooperation. Here, the E3-dependent transfer of ubiquitin is followed from the corresponding E2 enzyme onto free lysine amino acids (mimicking substrate ubiquitylation) or internal lysines of the E3 ligase itself (auto-ubiquitylation). In conclusion, three different in vitro protocols are provided that are fast and easy to perform to address E3 ligase catalytic functionality.


Assuntos
Enzimas de Conjugação de Ubiquitina , Ubiquitina-Proteína Ligases , Lisina/metabolismo , Ubiquitina/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação
5.
F1000Res ; 92020.
Artigo em Inglês | MEDLINE | ID: mdl-32076548

RESUMO

Protein ubiquitylation is essential for the maintenance of cellular homeostasis. E3 ubiquitin ligases are key components of the enzymatic machinery catalyzing the attachment of ubiquitin to substrate proteins. Consequently, enzymatic dysfunction has been associated with medical conditions including cancer, diabetes, and cardiovascular and neurodegenerative disorders. To safeguard substrate selection and ubiquitylation, the activity of E3 ligases is tightly regulated by post-translational modifications including phosphorylation, sumoylation, and ubiquitylation, as well as binding of alternative adaptor molecules and cofactors. Recent structural studies identified homotypic and heterotypic interactions between E3 ligases, adding another layer of control for rapid adaptation to changing environmental and physiological conditions. Here, we discuss the regulation of E3 ligase activity by combinatorial oligomerization and summarize examples of associated ubiquitylation pathways and mechanisms.


Assuntos
Ubiquitina-Proteína Ligases , Ubiquitinação , Humanos , Processamento de Proteína Pós-Traducional , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo
6.
Elife ; 92020 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-33021200

RESUMO

Sex-specific synaptic connectivity is beginning to emerge as a remarkable, but little explored feature of animal brains. We describe here a novel mechanism that promotes sexually dimorphic neuronal function and synaptic connectivity in the nervous system of the nematode Caenorhabditis elegans. We demonstrate that a phylogenetically conserved, but previously uncharacterized Doublesex/Mab-3 related transcription factor (DMRT), dmd-4, is expressed in two classes of sex-shared phasmid neurons specifically in hermaphrodites but not in males. We find dmd-4 to promote hermaphrodite-specific synaptic connectivity and neuronal function of phasmid sensory neurons. Sex-specificity of DMD-4 function is conferred by a novel mode of posttranslational regulation that involves sex-specific protein stabilization through ubiquitin binding to a phylogenetically conserved but previously unstudied protein domain, the DMA domain. A human DMRT homolog of DMD-4 is controlled in a similar manner, indicating that our findings may have implications for the control of sexual differentiation in other animals as well.


Assuntos
Proteínas de Caenorhabditis elegans , Neurônios/metabolismo , Caracteres Sexuais , Fatores de Transcrição , Ubiquitina/metabolismo , Animais , Comportamento Animal , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Transtornos do Desenvolvimento Sexual , Feminino , Técnicas de Inativação de Genes , Humanos , Masculino , Transmissão Sináptica/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
7.
Regen Med ; 3(1): 23-31, 2008 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-18154460

RESUMO

OBJECTIVES: Mechanisms underpinning Gram-negative bacterial vaginosis-induced birth anomalies are obscure. Ethical issues limit such studies on peri-implantation-stage human embryos. Here we have used embryoid bodies (EBs) as an in vitro model to examine the effect of Gram-negative bacterial endotoxins/lipopolysaccharides (LPS) on the faithful induction of germ lineages during embryogenesis. The role of LPS-inducible cytokine and pluripotency-related DNA-binding protein HMGB1 was also studied in these EBs. METHODS: EBs derived from the human embryonic stem cell line HUES9 were exposed to 12.5 pg/ml of LPS for 48 h. The expression profile of the ectoderm, endoderm, mesoderm and trophectoderm lineage markers, such as beta III-tubulin, GATA4, BMP2, Brachury and beta-hCG, were studied, by RT-PCR and immunofluorescence. Inhibition of mesoderm induction was confirmed by RT-PCR analysis for hANP, cTnT, ABCG2, GATA2, BMP4 and HAND1. Osteoblast differentiation was induced in the EBs, and confirmed by von Kosa and Alizarin red staining. A comet assay was also carried out to assess the degree of apoptosis in these EBs. RESULTS AND CONCLUSIONS: We found that the LPS-treated EBs were selectively silenced for mesoderm markers and failed to differentiate into functional osteoblasts. HMGB1 expression was absent in the normal EBs and was found to be localized in the cytoplasm of the LPS-treated EBs. Overall, our data indicate that endotoxin-induced HMGB1 expression in the peri-implantation-stage embryos can bring about severe birth defects of, for example, the bone and heart. This study also indicates that HMGB1 could be involved in maintenance of pluripotency in the human embryonic stem cells by impeding their differentiation.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Endotoxinas/toxicidade , Proteína HMGB1/fisiologia , Mesoderma/efeitos dos fármacos , Células-Tronco Pluripotentes/citologia , Vaginose Bacteriana/patologia , Sequência de Bases , Primers do DNA , Feminino , Proteína HMGB1/genética , Humanos , Mesoderma/citologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa
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