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1.
Biochemistry (Mosc) ; 84(Suppl 1): S159-S192, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31213201

RESUMO

In the middle of the 20th century, it was postulated that degradation of intracellular proteins is a stochastic process. More than fifty years of intense studies have finally proven that protein degradation is a very complex and tightly regulated in time and space process that plays an incredibly important role in the vast majority of metabolic pathways. Degradation of more than a half of intracellular proteins is controlled by a hierarchically aligned and evolutionarily perfect system consisting of many components, the main ones being ubiquitin ligases and proteasomes, together referred to as the ubiquitin-proteasome system (UPS). The UPS includes more than 1000 individual components, and most of them are critical for the cell functioning and survival. In addition to the well-known signaling functions of ubiquitination, such as modification of substrates for proteasomal degradation and DNA repair, polyubiquitin (polyUb) chains are involved in other important cellular processes, e.g., cell cycle regulation, immunity, protein degradation in mitochondria, and even mRNA stability. This incredible variety of ubiquitination functions is related to the ubiquitin ability to form branching chains through the ε-amino group of any of seven lysine residues in its sequence. Deubiquitination is accomplished by proteins of the deubiquitinating enzyme family. The second main component of the UPS is proteasome, a multisubunit proteinase complex that, in addition to the degradation of functionally exhausted and damaged proteins, regulates many important cellular processes through controlled degradation of substrates, for example, transcription factors and cyclins. In addition to the ubiquitin-dependent-mediated degradation, there is also ubiquitin-independent degradation, when the proteolytic signal is either an intrinsic protein sequence or shuttle molecule. Protein hydrolysis is a critically important cellular function; therefore, any abnormalities in this process lead to systemic impairments further transforming into serious diseases, such as diabetes, malignant transformation, and neurodegenerative disorders (multiple sclerosis, Alzheimer's disease, Parkinson's disease, Creutzfeldt-Jakob disease and Huntington's disease). In this review, we discuss the mechanisms that orchestrate all components of the UPS, as well as the plurality of the fine-tuning pathways of proteasomal degradation.


Assuntos
Doenças Neurodegenerativas/metabolismo , Complexo de Endopeptidases do Proteassoma , Proteólise , Ubiquitinas , Humanos , Complexo de Endopeptidases do Proteassoma/química , Complexo de Endopeptidases do Proteassoma/fisiologia , Transdução de Sinais , Ubiquitinação , Ubiquitinas/química , Ubiquitinas/fisiologia
2.
Cell ; 177(2): 286-298.e15, 2019 04 04.
Artigo em Inglês | MEDLINE | ID: mdl-30929903

RESUMO

The 26S proteasome is the principal macromolecular machine responsible for protein degradation in eukaryotes. However, little is known about the detailed kinetics and coordination of the underlying substrate-processing steps of the proteasome, and their correlation with observed conformational states. Here, we used reconstituted 26S proteasomes with unnatural amino-acid-attached fluorophores in a series of FRET- and anisotropy-based assays to probe substrate-proteasome interactions, the individual steps of the processing pathway, and the conformational state of the proteasome itself. We develop a complete kinetic picture of proteasomal degradation, which reveals that the engagement steps prior to substrate commitment are fast relative to subsequent deubiquitination, translocation, and unfolding. Furthermore, we find that non-ideal substrates are rapidly rejected by the proteasome, which thus employs a kinetic proofreading mechanism to ensure degradation fidelity and substrate prioritization.


Assuntos
Complexo de Endopeptidases do Proteassoma/metabolismo , Complexo de Endopeptidases do Proteassoma/fisiologia , Anisotropia , Sítios de Ligação/fisiologia , Ativação Enzimática , Cinética , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Processamento de Proteína Pós-Traducional/fisiologia , Proteólise , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Especificidade por Substrato/fisiologia , Ubiquitina/metabolismo
3.
Gene ; 702: 66-74, 2019 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-30930224

RESUMO

Hepatocellular carcinoma (HCC) is the most common primary cancer of the liver with high mortality and frequent recurrence. Although various therapies provide potential cure for HCC patients, unfortunately the five-year survival rate of advanced HCC remains dismal. It is critical to explore the pathogenesis of HCC and identify novel biomarkers for early HCC diagnosis. PSMD4 is a major receptor of the 26S proteasome involved in ubiquitindependent and proteasome-mediated protein degradation. In our study, PSMD4 was overexpressed in HCC tissues and cell lines determined by Northern blot, western blot and immunohistochemistry. The silencing of PSMD4 blocked cell proliferation and tumor growth, induced cell apoptosis and inhibited the proteasome activity. Western blot results showed that the knockdown of PSMD4 blocked the expression of cyclooxygenase 2 (COX2), phosphorylated Sarcoma tyrosine kinase (P-SRC) and Bcl-2, but improved the levels of p53 and Bax in HCC, lung cancer, colorectal cancer, breast cancer and endometrial cancer cell lines. Taken together, these findings indicated that the subunit of 26S proteasome PSMD4 exerts as an oncogene in HCC and other cancers via regulating the expression p53, Bcl-2 and Bax. These findings enriched the pathogenesis of HCC, and provided a new biomarker for cancers diagnosis and a new target for cancers therapy.


Assuntos
Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/metabolismo , Complexo de Endopeptidases do Proteassoma/fisiologia , Animais , Apoptose , Carcinogênese , Carcinoma Hepatocelular/enzimologia , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Linhagem Celular , Linhagem Celular Tumoral , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Hepáticas/enzimologia , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Camundongos Nus , Complexo de Endopeptidases do Proteassoma/biossíntese , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Interferência de RNA
4.
Neurosci Lett ; 703: 68-78, 2019 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-30890471

RESUMO

Several lines of evidence have shown that defects in the endo-lysosomal autophagy degradation pathway and the ubiquitin-proteasome system play a role in Alzheimer's Disease (AD) pathogenesis and pathophysiology. Early pathological changes, such as marked enlargement of endosomal compartments, gradual accumulation of autophagic vacuoles (AVs) and lysosome dyshomeostasis, are well-recognized in AD. In addition to these pathological indicators, many genetic variants of key regulators in the endo-lysosomal autophagy networks and the ubiquitin-proteasome system have been found to be associated with AD. Furthermore, altered expression levels of key proteins in these pathways have been found in AD human brain tissues, primary cells and AD mouse models. In this review, we discuss potential disease mechanisms underlying the dysregulation of protein homeostasis governing systems. While the importance of two major protein degradation pathways in AD pathogenesis has been highlighted, targeted therapy at key components of these pathways has great potential in developing novel therapeutic interventions for AD. Future investigations are needed to define molecular mechanisms by which these complex regulatory systems become malfunctional at specific stages of AD development and progression, which will facilitate future development of novel therapeutic interventions. It is also critical to investigate all key components of the protein degradation pathways, both upstream and downstream, to improve our abilities to manipulate transport pathways with higher efficacy and less side effects.


Assuntos
Doença de Alzheimer/metabolismo , Endossomos/fisiologia , Lisossomos/fisiologia , Complexo de Endopeptidases do Proteassoma/fisiologia , Ubiquitina/fisiologia , Doença de Alzheimer/etiologia , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/metabolismo , Animais , Autofagia , Humanos , Transdução de Sinais , Proteínas tau/metabolismo
5.
PLoS Pathog ; 15(1): e1007498, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30645648

RESUMO

The establishment of HIV-1 latency gives rise to persistent chronic infection that requires life-long treatment. To reverse latency for viral eradiation, the HIV-1 Tat protein and its associated ELL2-containing Super Elongation Complexes (ELL2-SECs) are essential to activate HIV-1 transcription. Despite efforts to identify effective latency-reversing agents (LRA), avenues for exposing latent HIV-1 remain inadequate, prompting the need to identify novel LRA targets. Here, by conducting a CRISPR interference-based screen to reiteratively enrich loss-of-function genotypes that increase HIV-1 transcription in latently infected CD4+ T cells, we have discovered a key role of the proteasome in maintaining viral latency. Downregulating or inhibiting the proteasome promotes Tat-transactivation in cell line models. Furthermore, the FDA-approved proteasome inhibitors bortezomib and carfilzomib strongly synergize with existing LRAs to reactivate HIV-1 in CD4+ T cells from antiretroviral therapy-suppressed individuals without inducing cell activation or proliferation. Mechanistically, downregulating/inhibiting the proteasome elevates the levels of ELL2 and ELL2-SECs to enable Tat-transactivation, indicating the proteasome-ELL2 axis as a key regulator of HIV-1 latency and promising target for therapeutic intervention.


Assuntos
HIV-1/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Latência Viral/efeitos dos fármacos , Fármacos Anti-HIV/uso terapêutico , Linfócitos T CD4-Positivos/metabolismo , Sistemas CRISPR-Cas , Linhagem Celular , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Edição de Genes/métodos , Infecções por HIV/tratamento farmacológico , Infecções por HIV/metabolismo , Soropositividade para HIV , HIV-1/patogenicidade , Humanos , Células Jurkat , Complexo de Endopeptidases do Proteassoma/fisiologia , Inibidores de Proteassoma/metabolismo , Inibidores de Proteassoma/farmacologia , Fatores de Elongação da Transcrição , Ativação Viral/efeitos dos fármacos , Latência Viral/fisiologia
6.
J Zhejiang Univ Sci B ; 20(2): 146-155, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-29770645

RESUMO

More than 80% of all cases of deafness are related to the death or degeneration of cochlear hair cells and the associated spiral ganglion neurons, and a lack of regeneration of these cells leads to permanent hearing loss. Therefore, the regeneration of lost hair cells is an important goal for the treatment of deafness. Atoh1 is a basic helix-loop-helix (bHLH) transcription factor that is critical in both the development and regeneration of cochlear hair cells. Atoh1 is transcriptionally regulated by several signaling pathways, including Notch and Wnt signalings. At the post-translational level, it is regulated through the ubiquitin-proteasome pathway. In vitro and in vivo studies have revealed that manipulation of these signaling pathways not only controls development, but also leads to the regeneration of cochlear hair cells after damage. Recent progress toward understanding the signaling networks involved in hair cell development and regeneration has led to the development of new strategies to replace lost hair cells. This review focuses on our current understanding of the signaling pathways that regulate Atoh1 in the cochlea.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/fisiologia , Cóclea/fisiologia , Diferenciação Celular , Células Ciliadas Auditivas/citologia , Células Ciliadas Auditivas/fisiologia , Perda Auditiva/etiologia , Humanos , Complexo de Endopeptidases do Proteassoma/fisiologia , Transdução de Sinais/fisiologia , Fatores de Transcrição/fisiologia , Ubiquitina/metabolismo , Via de Sinalização Wnt , beta Catenina/fisiologia
7.
Pharmacol Res ; 140: 14-20, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30223085

RESUMO

Serotonin is a neurotransmitter widely conserved from ancient organisms lacking nervous systems through man, and its presence precedes the appearance of nervous systems on both developmental and evolutionary time scales. Serotonin receptor subtypes diversified approximately at the time period during which vertebrates diverged from invertebrates. The biological and clinical importance of serotonin receptors, may benefit from studies on their evolution. Although potentially informative about their pathophysiological functions, reviews on this topic are sparse. Several observations support basic functions mediated by serotonin, both in periphery and central nervous system. In particular, 5-HT2B receptors have been implicated in embryonic development, including cell proliferation, survival, and/or differentiation, in either neural crest cell derivatives, myeloid cell lineage, or heart embryogenesis. In this review, we collected existing data about the genomic association between the RPN2 proteasome subunit gene Psmd1 and the 5-HT2B receptor gene Htr2b. We discuss about the possibility that, during genome duplications, a single copy of this pair of genes has been conserved, suggesting a strong selective pressure. Many basic physiological functions in which serotonin system is involved could be linked to the early association of these two genes in pre-vertebrates. Their evolutionary association highlights the possibility that the 5-HT2B receptor gene, Htr2b, is the common ancestor of 5-HT2A/2B/2C-receptor subfamily. Disentangling these possibilities could bring new understanding of the respective importance of these receptors in pathophysiology of serotonin.


Assuntos
Receptores de Serotonina/fisiologia , Animais , Evolução Molecular , Genômica , Humanos , Complexo de Endopeptidases do Proteassoma/fisiologia , Serotonina/metabolismo
8.
Oncogene ; 38(3): 406-420, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30115976

RESUMO

Hepatocellular carcinoma (HCC) is one of the most lethal cancers worldwide. The poor survival may be due to a high proportions of tumor recurrence and metastasis. Kinesin family member C1 (KIFC1) is highly expressed in a variety of neoplasms and is a potential marker for non-small cell lung cancer or ovarian adenocarcinoma metastasis. Nevertheless, the role of KIFC1 in HCC metastasis remains obscure. We investigated this in the present study using HCC cell lines and clinical specimens. Our results indicated that increased levels of KIFC1 were associated with poor prognosis and metastasis in HCC. In addition, KIFC1 induced epithelial-to-mesenchymal transition (EMT) and HCC metastasis both in vitro and in vivo. This tumorigenic effect depended on gankyrin; inhibiting gankyrin activity reversed EMT via activation of protein kinase B (AKT)/Twist family BHLH transcription factor 1 (AKT/TWIST1). We also found that KIFC1 was directly regulated by the microRNA miR-532-3p, whose downregulation was associated with metastatic progression in HCC. These results denote that a decrease in miR-532-3p levels results in increased KIFC1 expression in HCC, leading to metastasis via activation of the gankyrin/AKT/TWIST1 signaling pathway.


Assuntos
Carcinoma Hepatocelular/secundário , Transição Epitelial-Mesenquimal/fisiologia , Cinesina/fisiologia , Neoplasias Hepáticas/patologia , Neoplasias Pulmonares/secundário , MicroRNAs/fisiologia , Proteínas de Neoplasias/fisiologia , RNA Neoplásico/fisiologia , Animais , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/mortalidade , Linhagem Celular Tumoral , Regulação para Baixo , Xenoenxertos , Humanos , Estimativa de Kaplan-Meier , Cinesina/antagonistas & inibidores , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/mortalidade , Neoplasias Pulmonares/fisiopatologia , Masculino , Camundongos Endogâmicos BALB C , Camundongos Nus , MicroRNAs/biossíntese , MicroRNAs/genética , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas Nucleares/fisiologia , Prognóstico , Complexo de Endopeptidases do Proteassoma/fisiologia , Proteínas Proto-Oncogênicas/fisiologia , Proteínas Proto-Oncogênicas c-akt/fisiologia , Interferência de RNA , RNA Neoplásico/biossíntese , RNA Neoplásico/genética , Transdução de Sinais , Proteína 1 Relacionada a Twist/fisiologia
9.
PLoS Biol ; 16(11): e2006145, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30500822

RESUMO

The circadian oscillator is a molecular feedback circuit whose orchestration involves posttranslational control of the activity and protein levels of its components. Although controlled proteolysis of circadian proteins is critical for oscillator function, our understanding of the underlying mechanisms remains incomplete. Here, we report that JmjC domain-containing protein 5 (JMJD5) interacts with CRYPTOCHROME 1 (CRY1) in an F-box/leucine-rich repeat protein 3 (FBXL3)-dependent manner and facilitates targeting of CRY1 to the proteasome. Genetic deletion of JMJD5 results in greater CRY1 stability, reduced CRY1 association with the proteasome, and disruption of circadian gene expression. We also report that in the absence of JMJD5, AMP-regulated protein kinase (AMPK)-induced CRY1 degradation is impaired, establishing JMJD5 as a key player in this mechanism. JMJD5 cooperates with CRY1 to repress circadian locomotor output cycles protein kaput (CLOCK)-brain and muscle ARNT-like protein 1 (BMAL1), thus linking CRY1 destabilization to repressive function. Finally, we find that ablation of JMJD5 impacts FBXL3- and CRY1-related functions beyond the oscillator.


Assuntos
Criptocromos/fisiologia , Histona Desmetilases com o Domínio Jumonji/fisiologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Fatores de Transcrição ARNTL/metabolismo , Animais , Relógios Circadianos/genética , Ritmo Circadiano/genética , Criptocromos/genética , Proteínas F-Box/fisiologia , Células HEK293 , Humanos , Histona Desmetilases com o Domínio Jumonji/genética , Camundongos , Camundongos Endogâmicos C57BL , Células-Tronco Embrionárias Murinas , Complexo de Endopeptidases do Proteassoma/fisiologia , Domínios Proteicos , Proteólise
10.
Adv Exp Med Biol ; 1088: 347-368, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30390260

RESUMO

Muscle atrophy in aging is characterized by progressive loss of muscle mass and function. Muscle mass is determined by the balance of synthesis and degradation of protein, which are regulated by several signaling pathways such as ubiquitin-proteasome system, autophagy-lysosome systems, oxidative stress, proinflammatory cytokines, hormones, and so on. Sufficient nutrition can enhance protein synthesis, while exercise can improve the quality of life in the elderly. This chapter will discuss the epidemiology, pathogenesis, as well as the current treatment for aging-induced muscular atrophy.


Assuntos
Envelhecimento/patologia , Músculo Esquelético/patologia , Atrofia Muscular/fisiopatologia , Autofagia , Citocinas/fisiologia , Humanos , Proteínas Musculares , Atrofia Muscular/epidemiologia , Atrofia Muscular/terapia , Estresse Oxidativo , Complexo de Endopeptidases do Proteassoma/fisiologia , Transdução de Sinais , Ubiquitina/fisiologia
11.
Br J Cancer ; 119(12): 1538-1551, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30385822

RESUMO

BACKGROUND: Although NGLY1 is known as a pivotal enzyme that catalyses the deglycosylation of denatured glycoproteins, information regarding the responses of human cancer and normal cells to NGLY1 suppression is limited. METHODS: We examined how NGLY1 expression affects viability, tumour growth, and responses to therapeutic agents in melanoma cells and an animal model. Molecular mechanisms contributing to NGLY1 suppression-induced anticancer responses were revealed by systems biology and chemical biology studies. Using computational and medicinal chemistry-assisted approaches, we established novel NGLY1-inhibitory small molecules. RESULTS: Compared with normal cells, NGLY1 was upregulated in melanoma cell lines and patient tumours. NGLY1 knockdown caused melanoma cell death and tumour growth retardation. Targeting NGLY1 induced pleiotropic responses, predominantly stress signalling-associated apoptosis and cytokine surges, which synergise with the anti-melanoma activity of chemotherapy and targeted therapy agents. Pharmacological and molecular biology tools that inactivate NGLY1 elicited highly similar responses in melanoma cells. Unlike normal cells, melanoma cells presented distinct responses and high vulnerability to NGLY1 suppression. CONCLUSION: Our work demonstrated the significance of NGLY1 in melanoma cells, provided mechanistic insights into how NGLY1 inactivation leads to eradication of melanoma with limited impact on normal cells, and suggested that targeting NGLY1 represents a novel anti-melanoma strategy.


Assuntos
Antineoplásicos/farmacologia , Apoptose , Interferon gama/fisiologia , Melanoma/tratamento farmacológico , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase/antagonistas & inibidores , Fator 4 Ativador da Transcrição/fisiologia , Animais , Células Cultivadas , Citocinas/análise , Perfilação da Expressão Gênica , Humanos , Interferon gama/genética , Melanoma/patologia , Camundongos , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase/fisiologia , Células-Tronco Pluripotentes/fisiologia , Complexo de Endopeptidases do Proteassoma/fisiologia , Transdução de Sinais/fisiologia , Fator de Transcrição CHOP/fisiologia
12.
Biochim Biophys Acta Gen Subj ; 1862(12): 2948-2954, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30297324

RESUMO

BACKGROUND: It has been almost three decades since the removal of oxidized proteins by the free 20S catalytic unit of the proteasome (20SPT) was proposed. Since then, experimental evidence suggesting a physiological role of proteolysis mediated by the free 20SPT has being gathered. SCOPE OF REVIEW: Experimental data that favors the hypothesis of free 20SPT as playing a role in proteolysis are critically reviewed. MAJOR CONCLUSIONS: Protein degradation by the proteasome may proceed through multiple proteasome complexes with different requirements though the unequivocal role of the free 20SPT in cellular proteolysis towards native or oxidized proteins remains to be demonstrated. GENERAL SIGNIFICANCE: The biological significance of proteolysis mediated by the free 20SPT has been elusive since its discovery. The present review critically analyzes the available experimental data supporting the proteolytic role of the free or single capped 20SPT.


Assuntos
Complexo de Endopeptidases do Proteassoma/fisiologia , Proteólise , Trifosfato de Adenosina/metabolismo , Catálise , Interações Hidrofóbicas e Hidrofílicas , Oxirredução , Ubiquitina/metabolismo
13.
Diabetes Obes Metab ; 20 Suppl 2: 88-94, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-30230178

RESUMO

ß-cell destruction in type 1 diabetes (T1D) results from the effect of inflammation and autoimmunity. In response to inflammatory signals, islet cells engage adaptive mechanisms to restore and maintain cellular homeostasis. Among these mechanisms, the unfolded protein response (UPR) leads to a reduction of the general protein translation rate, increased production of endoplasmic reticulum chaperones and the initiation of degradation by activation of the ER associated degradation pathway (ERAD) in which newly synthetized proteins are ubiquitinylated and processed through the proteasome. This adaptive phase is also believed to play a critical role in the development of autoimmunity by the generation of neoantigens. While we have previously investigated the effect of stress on transcription, translation and post-translational events as possible source for neoantigens, the participation of the degradation machinery, yet crucial in the generation of antigenic peptides, remains to be investigated in the context of T1D pathology. In this review, we will describe the relation between the unfolded protein response and the Ubiquitin Proteasome System (UPS) and address the role of the cellular degradation machinery in the generation of antigens. Learning from tumour immunology, we propose how these processes may unmask ß-cells by triggering the generation of aberrant peptides recognized by the immune cells.


Assuntos
Autoimunidade/fisiologia , Diabetes Mellitus Tipo 1/imunologia , Estresse do Retículo Endoplasmático/fisiologia , Ilhotas Pancreáticas/fisiologia , Diabetes Mellitus Tipo 1/metabolismo , Estresse do Retículo Endoplasmático/imunologia , Humanos , Insulina/biossíntese , Ilhotas Pancreáticas/imunologia , Complexo de Endopeptidases do Proteassoma/fisiologia
14.
Nat Rev Mol Cell Biol ; 19(11): 697-712, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30065390

RESUMO

The proteasome degrades most cellular proteins in a controlled and tightly regulated manner and thereby controls many processes, including cell cycle, transcription, signalling, trafficking and protein quality control. Proteasomal degradation is vital in all cells and organisms, and dysfunction or failure of proteasomal degradation is associated with diverse human diseases, including cancer and neurodegeneration. Target selection is an important and well-established way to control protein degradation. In addition, mounting evidence indicates that cells adjust proteasome-mediated degradation to their needs by regulating proteasome abundance through the coordinated expression of proteasome subunits and assembly chaperones. Central to the regulation of proteasome assembly is TOR complex 1 (TORC1), which is the master regulator of cell growth and stress. This Review discusses how proteasome assembly and the regulation of proteasomal degradation are integrated with cellular physiology, including the interplay between the proteasome and autophagy pathways. Understanding these mechanisms has potential implications for disease therapy, as the misregulation of proteasome function contributes to human diseases such as cancer and neurodegeneration.


Assuntos
Neoplasias/metabolismo , Neoplasias/patologia , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/patologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Complexo de Endopeptidases do Proteassoma/fisiologia , Animais , Autofagia/fisiologia , Proliferação de Células/fisiologia , Humanos , Chaperonas Moleculares/metabolismo , Proteólise , Transdução de Sinais/fisiologia
15.
Genes Cells ; 23(10): 839-848, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30133132

RESUMO

The proteasome core particle (CP) is a cytoplasmic and nuclear protease complex and is comprised of two α-rings and two ß-rings stacked in order of αßßα. The assembly of CP proceeds by ordered recruitment of ß-subunits on an α-ring with help of assembly chaperones PAC1-PAC2, PAC3-PAC4, and UMP1. However, the mechanism of α-ring formation remains unsolved. Here, we show that α4, α5, α6, and α7 form a core intermediate as the initial process of α-ring assembly, which requires PAC3-PAC4. α1 and α3 can be incorporated independently into the core α4-α7 intermediate, whereas α2 incorporation is dependent on preceding incorporation of α1. Through these processes, PAC1-PAC2 prevents nonproductive dimerization of α-ring assembly intermediates. We also found that PAC1-PAC2 overrides the effect of nuclear localization signals of α-subunits and retains α-ring assembly intermediates in the cytoplasm. Our results first show a detailed assembly pathway of proteasomal α-ring and explain the mechanism by which CP assembly occurs in the cytoplasm.


Assuntos
Complexo de Endopeptidases do Proteassoma/metabolismo , Complexo de Endopeptidases do Proteassoma/fisiologia , Citoplasma , Células HEK293 , Humanos , Modelos Biológicos , Modelos Moleculares , Chaperonas Moleculares/metabolismo , Ligação Proteica , Subunidades Proteicas/metabolismo , RNA Interferente Pequeno
16.
Exp Cell Res ; 371(2): 353-363, 2018 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-30149001

RESUMO

Micronuclei are extra-nuclear bodies containing whole chromosomes that were not incorporated into the nucleus after cell division or damaged chromosome fragments. Even though the link between micronuclei and DNA damage is described for a long time, little is known about the functional organization of micronuclei and their contribution to tumorigenesis. We showed fusions between micronuclear membranes and lysosomes by electron microscopy and linked lysosome function to DNA damage levels in micronuclei. In addition, micronuclei drastically differ from primary nuclei in nuclear envelope composition, with a significant increase in the relative amount of nuclear envelope proteins LBR and emerin and a decrease in nuclear pore proteins. Strikingly, micronuclei lack active proteasomes, as the processing subunits and other factors of the ubiquitin proteasome system. Moreover, micronuclear chromatin shows a higher degree of compaction as compared to primary nuclei. The specific aberrations identified in micronuclei and the potential functional consequences of these defects may contribute to the role of micronuclei in catastrophic genomic rearrangements.


Assuntos
Núcleo Celular/ultraestrutura , Cromatina/ultraestrutura , Cromotripsia , Instabilidade Genômica , Membrana Nuclear/ultraestrutura , Complexo de Endopeptidases do Proteassoma/fisiologia , Linhagem Celular , Núcleo Celular/metabolismo , Núcleo Celular/patologia , Cromatina/química , Dano ao DNA , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/ultraestrutura , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/ultraestrutura , Expressão Gênica , Humanos , Lisossomos/metabolismo , Lisossomos/ultraestrutura , Fusão de Membrana , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Testes para Micronúcleos , Nocodazol/farmacologia , Membrana Nuclear/química , Membrana Nuclear/metabolismo , Membrana Nuclear/patologia , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Complexo de Endopeptidases do Proteassoma/ultraestrutura , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Epitélio Pigmentado da Retina/efeitos dos fármacos , Epitélio Pigmentado da Retina/metabolismo , Epitélio Pigmentado da Retina/ultraestrutura
17.
Hist Philos Life Sci ; 40(2): 25, 2018 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-29560537

RESUMO

Scientific hypotheses may either predict particular unknown facts or accommodate previously-known data. Although affirmed predictions are intuitively more rewarding than accommodations of established facts, opinions divide whether predictive hypotheses are also epistemically superior to accommodation hypotheses. This paper examines the contribution of predictive hypotheses to discoveries of several bio-molecular systems. Having all the necessary elements of the system known beforehand, an abstract predictive hypothesis of semiconservative mode of DNA replication was successfully affirmed. However, in defining the genetic code whose biochemical basis was unclear, hypotheses were only partially effective and supplementary experimentation was required for its conclusive definition. Markedly, hypotheses were entirely inept in predicting workings of complex systems that included unknown elements. Thus, hypotheses did not predict the existence and function of mRNA, the multiple unidentified components of the protein biosynthesis machinery, or the manifold unknown constituents of the ubiquitin-proteasome system of protein breakdown. Consequently, because of their inability to envision unknown entities, predictive hypotheses did not contribute to the elucidation of cation theories remained the sole instrument to explain complex bio-molecular systems, the philosophical question of alleged advantage of predictive over accommodative hypotheses became inconsequential.


Assuntos
Complexo de Endopeptidases do Proteassoma/fisiologia , Biossíntese de Proteínas/fisiologia , RNA Mensageiro/fisiologia , Ubiquitinas/fisiologia , Modelos Biológicos , Proteólise
18.
Neurobiol Aging ; 66: 149-157, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29579685

RESUMO

The insulin family of growth factors plays an important role in development and function of the nervous system. Reduced insulin and insulin-growth-factor signaling (IIS), however, can improve symptoms of neurodegenerative diseases in laboratory model organisms and protect against age-associated decline in neuronal function. Recently, we showed that chronic, moderately lowered IIS rescues age-related decline in neurotransmission through the Drosophila giant fiber escape response circuit. Here, we expand our initial findings by demonstrating that reduced functional output in the giant fiber system of aging flies can be prevented by increasing proteasomal activity within the circuit. Manipulations of IIS in neurons can also affect longevity, underscoring the relevance of the nervous system for aging.


Assuntos
Envelhecimento/metabolismo , Envelhecimento/fisiologia , Insulina/metabolismo , Insulina/fisiologia , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/fisiologia , Neurônios/fisiologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Complexo de Endopeptidases do Proteassoma/fisiologia , Transdução de Sinais/fisiologia , Somatomedinas/metabolismo , Somatomedinas/fisiologia , Transmissão Sináptica/fisiologia , Animais , Células Cultivadas , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , GTP Fosfo-Hidrolases/metabolismo , Longevidade , Proteínas rab de Ligação ao GTP/metabolismo
19.
Curr Biol ; 28(4): R170-R185, 2018 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-29462587

RESUMO

The maintenance of a healthy and functional mitochondrial network is critical during development as well as throughout life in the response to physiological adaptations and stress conditions. Owing to their role in energy production, mitochondria are exposed to high levels of reactive oxygen species, making them particularly vulnerable to mitochondrial DNA mutations and protein misfolding. Given that mitochondria are formed from proteins encoded by both nuclear and mitochondrial genomes, an additional layer of complexity is inherent in the coordination of protein synthesis and the mitochondrial import of nuclear-encoded proteins. For these reasons, mitochondria have evolved multiple systems of quality control to ensure that the requisite number of functional mitochondria are present to meet the demands of the cell. These pathways work to eliminate damaged mitochondrial proteins or parts of the mitochondrial network by mitophagy and renew components by adding protein and lipids through biogenesis, collectively resulting in mitochondrial turnover. Mitochondrial quality control mechanisms are multi-tiered, operating at the protein, organelle and cell levels. Herein, we discuss mitophagy in different physiological contexts and then relate it to other quality control pathways, including the unfolded protein response, shedding of vesicles, proteolysis, and degradation by the ubiquitin-proteasome system. Understanding how these pathways contribute to the maintenance of mitochondrial homeostasis could provide insights into the development of targeted treatments when these systems fail in disease.


Assuntos
Homeostase/fisiologia , Mitocôndrias/fisiologia , /fisiologia , Animais , Micropartículas Derivadas de Células/fisiologia , Humanos , Complexo de Endopeptidases do Proteassoma/fisiologia , Proteólise , Ubiquitina/fisiologia , Resposta a Proteínas não Dobradas/fisiologia
20.
Arthritis Rheumatol ; 70(7): 1030-1041, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29457374

RESUMO

OBJECTIVE: Osteoarthritis (OA) chondrocytes exhibit impairment of autophagy, one arm of the proteostasis network that coordinates proteome and organelle quality control and degradation. Deficient proteostasis impacts differentiation and viability, and inflammatory processes in aging and disease. The present study was undertaken to assess ubiquitin proteasome system proteasomal function in OA chondrocytes. METHODS: We evaluated human knee cartilage by immunohistochemistry, and assessed proteasomal function, levels of proteasomal core subunits and chaperones, and autophagy in cultured chondrocytes. Assays included Western blotting, quantitative reverse transcription-polymerase chain reaction, proteasomal protease activity assessment, and cell immunofluorescence analysis. RESULTS: Human knee OA cartilage exhibited polyubiquitin accumulation, with increased ubiquitin K48-linked polyubiquitinated proteins in situ, suggesting proteasomal impairment. Cultured OA chondrocytes demonstrated accumulation of K48 polyubiquitinated proteins, significantly reduced 20S proteasome core protease activity, and decreased levels of phosphorylated FOXO4 and proteasome 26S subunit, non-ATPase 11 (PSMD11), a FOXO4-inducible promoter of proteasomal activation. Levels of proteasome subunit ß type 3 (PSMB3), PSMB5, PSMB6, and proteasome assembly chaperone 1 were not decreased in OA chondrocytes. In normal chondrocytes, PSMD11 small interfering RNA knockdown stimulated certain autophagy machinery elements, increased extracellular nitric oxide (NO) levels, and reduced chondrocytic master transcription factor SOX9 protein and messenger RNA (mRNA) and aggrecan (AGC1) mRNA. PSMD11 gain-of- function by transfection increased proteasomal function, increased levels of SOX9-induced AGC1 mRNA, stimulated elements of the autophagic machinery, and inhibited extracellular levels of interleukin-1-induced NO and matrix metalloproteinase 13 in OA chondrocytes. CONCLUSION: Deficient PSMD11, associated with reduced phosphorylated FOXO4, promotes impaired proteasomal function in OA chondrocytes, dysregulation of chondrocytic homeostasis, and decreased levels of SOX9 mRNA, SOX9 protein, and AGC1 mRNA. Chondrocyte proteasomal impairment may be a therapeutic target for OA.


Assuntos
Agrecanas/metabolismo , Condrócitos/metabolismo , Osteoartrite do Joelho/enzimologia , Complexo de Endopeptidases do Proteassoma/fisiologia , Fatores de Transcrição SOX9/metabolismo , Cartilagem Articular/citologia , Técnicas de Cultura de Células , Humanos , Articulação do Joelho/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , RNA Mensageiro/metabolismo
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