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1.
Nat Commun ; 12(1): 5212, 2021 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-34471133

RESUMO

The autophagic degradation of misfolded and ubiquitinated proteins is important for cellular homeostasis. In this process, which is governed by cargo receptors, ubiquitinated proteins are condensed into larger structures and subsequently become targets for the autophagy machinery. Here we employ in vitro reconstitution and cell biology to define the roles of the human cargo receptors p62/SQSTM1, NBR1 and TAX1BP1 in the selective autophagy of ubiquitinated substrates. We show that p62 is the major driver of ubiquitin condensate formation. NBR1 promotes condensate formation by equipping the p62-NBR1 heterooligomeric complex with a high-affinity UBA domain. Additionally, NBR1 recruits TAX1BP1 to the ubiquitin condensates formed by p62. While all three receptors interact with FIP200, TAX1BP1 is the main driver of FIP200 recruitment and thus the autophagic degradation of p62-ubiquitin condensates. In summary, our study defines the roles of all three receptors in the selective autophagy of ubiquitin condensates.


Assuntos
Autofagia/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas de Neoplasias/metabolismo , Ubiquitina/metabolismo , Proteínas Relacionadas à Autofagia/metabolismo , Proteínas de Transporte , Linhagem Celular , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Neoplasias/genética , Domínios Proteicos , Proteínas de Ligação a RNA/metabolismo , Proteína Sequestossoma-1/metabolismo , Proteínas Ubiquitinadas/genética , Proteínas Ubiquitinadas/metabolismo
2.
Adv Exp Med Biol ; 1288: 215-240, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34453739

RESUMO

Ubiquitination is one of the most diverse forms of protein post-translational modification that changes the function of the landscape of substrate proteins in response to stimuli, without the need for "de novo" protein synthesis. Ubiquitination is involved in almost all aspects of eukaryotic cell biology, from the best-studied role in promoting the removal of faulty or unnecessary proteins by the way of the ubiquitin proteasome system and autophagy-lysosome pathway to the recruitment of proteins in specific non-proteolytic signaling pathways, as emerged by the more recent discoveries about the protein signature with peculiar types of ubiquitin chains. Spermatogenesis, on its own, is a complex cellular developmental process in which mitosis, meiosis, and cell differentiation coexist so to result in the continuous formation of haploid spermatozoa. Successful spermatogenesis is thus at the same time a mixed result of the precise expression and correct intracellular destination of structural proteins and enzymes, from one hand, and the fine removal by targeted degradation of unfolded or damaged proteins as well as of obsolete, outlived proteins, from the other hand. In this minireview, I will focus on the importance of the ubiquitin system all over the spermatogenic process, discussing both proteolytic and non-proteolytic functions of protein ubiquitination. Alterations in the ubiquitin system have been in fact implicated in pathologies leading to male infertility. Notwithstanding several aspects of the multifaceted world of the ubiquitin system have been clarified, the physiological meaning of the so-called ubiquitin code remains still partially elusive. The studies reviewed in this chapter provide information that could aid the investigators to pursue new promising discoveries in the understanding of human and animal reproductive potential.


Assuntos
Espermatogênese , Ubiquitina , Animais , Humanos , Masculino , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Ubiquitina/metabolismo , Ubiquitinação
3.
FASEB J ; 35(9): e21825, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34383978

RESUMO

Ubiquitination is an essential post-translational modification that regulates protein stability or function. Its substrate specificity is dictated by various E3 ligases. The human C-terminal to LisH (CTLH) complex is a newly discovered multi-subunit really interesting new gene (RING) E3 ligase with only a few known ubiquitination targets. Here, we used mass spectrometry-based proteomic techniques to gain insight into CTLH complex function and ubiquitination substrates in HeLa cells. First, global proteomics determined proteins that were significantly increased, and thus may be substrates targeted for degradation, in cells depleted of CTLH complex member RanBPM. RanBPM-dependent ubiquitination determined using diGLY-enriched proteomics and the endogenous RanBPM interactome further revealed candidate ubiquitination targets. Three glycolysis enzymes alpha-enolase, L-lactate dehydrogenase A chain (LDHA), and pyruvate kinase M1/2 (PKM) had decreased ubiquitin sites in shRanBPM cells and were found associated with RanBPM in the interactome. Reduced polyubiquitination was validated for PKM2 and LDHA in cells depleted of RanBPM and CTLH complex RING domain subunit RMND5A. PKM2 and LDHA protein levels were unchanged, yet their activity was increased in extracts of cells with downregulated RanBPM. Finally, RanBPM deficient cells displayed enhanced glycolysis and deregulated central carbon metabolism. Overall, this study identifies potential CTLH complex ubiquitination substrates and uncovers that the CTLH complex inhibits glycolysis via non-degradative ubiquitination of PKM2 and LDHA.


Assuntos
Glicólise/fisiologia , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação/fisiologia , Animais , Linhagem Celular Tumoral , Células HeLa , Humanos , L-Lactato Desidrogenase/metabolismo , Camundongos , Proteômica/métodos , Especificidade por Substrato , Ubiquitina/metabolismo
4.
Biomolecules ; 11(7)2021 07 10.
Artigo em Inglês | MEDLINE | ID: mdl-34356632

RESUMO

Ubiquitin (Ub) specifically interacts with the Ub-associating domain (UBA) in a proteasomal shuttle factor, while the latter is involved in either proteasomal targeting or self-assembly coacervation. PINK1 phosphorylates Ub at S65 and makes Ub alternate between C-terminally relaxed (pUbRL) and retracted conformations (pUbRT). Using NMR spectroscopy, we show that pUbRL but not pUbRT preferentially interacts with the UBA from two proteasomal shuttle factors Ubqln2 and Rad23A. Yet discriminatorily, Ubqln2-UBA binds to pUb more tightly than Rad23A does and selectively enriches pUbRL upon complex formation. Further, we determine the solution structure of the complex between Ubqln2-UBA and pUbRL and uncover the thermodynamic basis for the stronger interaction. NMR kinetics analysis at different timescales further suggests an indued-fit binding mechanism for pUb-UBA interaction. Notably, at a relatively low saturation level, the dissociation rate of the UBA-pUbRL complex is comparable with the exchange rate between pUbRL and pUbRT. Thus, a kinetic constraint would dictate the interaction between Ub and UBA, thus fine-tuning the functional state of the proteasomal shuttle factors.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Relacionadas à Autofagia/química , Enzimas Reparadoras do DNA/química , Proteínas de Ligação a DNA/química , Proteínas Quinases/química , Ubiquitina/química , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Relacionadas à Autofagia/metabolismo , Enzimas Reparadoras do DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Humanos , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Domínios Proteicos , Proteínas Quinases/metabolismo , Termodinâmica , Ubiquitina/metabolismo
5.
Int J Mol Sci ; 22(16)2021 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-34445496

RESUMO

Post-translational modification of the DNA replication machinery by ubiquitin and SUMO plays key roles in the faithful duplication of the genetic information. Among other functions, ubiquitination and SUMOylation serve as signals for the extraction of factors from chromatin by the AAA ATPase VCP. In addition to the regulation of DNA replication initiation and elongation, we now know that ubiquitination mediates the disassembly of the replisome after DNA replication termination, a process that is essential to preserve genomic stability. Here, we review the recent evidence showing how active DNA replication restricts replisome ubiquitination to prevent the premature disassembly of the DNA replication machinery. Ubiquitination also mediates the removal of the replisome to allow DNA repair. Further, we discuss the interplay between ubiquitin-mediated replisome disassembly and the activation of CDK1 that is required to set up the transition from the S phase to mitosis. We propose the existence of a ubiquitin-CDK1 relay, where the disassembly of terminated replisomes increases CDK1 activity that, in turn, favors the ubiquitination and disassembly of more replisomes. This model has important implications for the mechanism of action of cancer therapies that induce the untimely activation of CDK1, thereby triggering premature replisome disassembly and DNA damage.


Assuntos
Proteína Quinase CDC2/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Ubiquitina/metabolismo , Animais , Replicação do DNA , Humanos , Mitose , Processamento de Proteína Pós-Traducional
6.
Viruses ; 13(7)2021 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-34372566

RESUMO

Infection by RNA viruses causes extensive cellular reorganization, including hijacking of membranes to create membranous structures termed replication organelles, which support viral RNA synthesis and virion assembly. In this study, we show that infection with coxsackievirus B3 entails a profound impairment of the protein homeostasis at virus-utilized membranes, reflected by an accumulation of ubiquitinylated proteins, including K48-linked polyubiquitin conjugates, known to direct proteins to proteasomal degradation. The enrichment of membrane-bound ubiquitin conjugates is attributed to the presence of the non-structural viral proteins 2B and 3A, which are known to perturb membrane integrity and can cause an extensive rearrangement of cellular membranes. The locally increased abundance of ubiquitinylated proteins occurs without an increase of oxidatively damaged proteins. During the exponential phase of replication, the oxidative damage of membrane proteins is even diminished, an effect we attribute to the recruitment of glutathione, which is known to be required for the formation of infectious virus particles. Furthermore, we show that the proteasome contributes to the processing of viral precursor proteins. Taken together, we demonstrate how an infection with coxsackievirus B3 affects the cellular protein and redox homeostasis locally at the site of viral replication and virus assembly.


Assuntos
Enterovirus Humano B/metabolismo , Ubiquitinação/fisiologia , Replicação Viral/fisiologia , Citoplasma/metabolismo , Enterovirus Humano B/patogenicidade , Células HeLa , Humanos , Proteínas de Membrana/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica/fisiologia , RNA Viral/genética , Ubiquitina/metabolismo , Proteínas Virais/metabolismo , Vírion/metabolismo , Montagem de Vírus/genética , Montagem de Vírus/fisiologia , Replicação Viral/genética
7.
Int J Mol Sci ; 22(15)2021 Jul 21.
Artigo em Inglês | MEDLINE | ID: mdl-34360560

RESUMO

Among autophagy-related molecules, p62/SQSTM1 is an adaptor for identifying and delivering intracellular cargo for degradation. Since ubiquitination is reversible, it has a switch role in autophagy. Ubiquitination is also involved in regulating autophagy in a timely manner. This study aimed to elucidate how p62-mediated autophagy is regulated in human endothelial cells and macrophages under atherosclerotic conditions, focusing on the lysosomal and proteasomal pathways. Co-cultured HUVECs and THP-1 cells were exposed to oxLDL (50 µg/mL) and autophagy was assessed. To downregulate p62, siRNA was administered, and the E3 ligases were inhibited by Heclin or MLN4924 treatment under the condition that cellular inflammatory processes were stimulated by oxLDL simultaneously initiated autophagy. Downregulating p62 induced an alternative degradation system, and the E3 ligases were found to be involved in the progression of atherosclerosis. Collectively, the present study demonstrated that the endothelial lipid accumulation under atherosclerotic conditions was caused by lysosomal dysfunction associated with autophagy.


Assuntos
Aterosclerose/patologia , Autofagia , Células Endoteliais/patologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Proteína Sequestossoma-1/metabolismo , Ubiquitinação , Aterosclerose/metabolismo , Células Endoteliais/metabolismo , Humanos , Complexo de Endopeptidases do Proteassoma/genética , Proteína Sequestossoma-1/genética , Transdução de Sinais , Ubiquitina/metabolismo
8.
Int J Mol Sci ; 22(14)2021 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-34299332

RESUMO

Exposure of rodents to <20 cGy Space Radiation (SR) impairs performance in several hippocampus-dependent cognitive tasks, including spatial memory. However, there is considerable inter-individual susceptibility to develop SR-induced spatial memory impairment. In this study, a robust label-free mass spectrometry (MS)-based unbiased proteomic profiling approach was used to characterize the composition of the hippocampal proteome in adult male Wistar rats exposed to 15 cGy of 1 GeV/n 48Ti and their sham counterparts. Unique protein signatures were identified in the hippocampal proteome of: (1) sham rats, (2) Ti-exposed rats, (3) Ti-exposed rats that had sham-like spatial memory performance, and (4) Ti-exposed rats that impaired spatial memory performance. Approximately 14% (159) of the proteins detected in hippocampal proteome of sham rats were not detected in the Ti-exposed rats. We explored the possibility that the loss of the Sham-only proteins may arise as a result of SR-induced changes in protein homeostasis. SR-exposure was associated with a switch towards increased pro-ubiquitination proteins from that seen in Sham. These data suggest that the role of the ubiquitin-proteome system as a determinant of SR-induced neurocognitive deficits needs to be more thoroughly investigated.


Assuntos
Radiação Cósmica , Hipocampo/efeitos da radiação , Proteoma/metabolismo , Ubiquitina/metabolismo , Animais , Cognição/efeitos da radiação , Relação Dose-Resposta à Radiação , Meio Ambiente Extraterreno , Hipocampo/metabolismo , Masculino , Proteômica/métodos , Ratos , Ratos Wistar , Memória Espacial/efeitos da radiação
9.
Nature ; 596(7871): 285-290, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34321666

RESUMO

Ageing is driven by a loss of cellular integrity1. Given the major role of ubiquitin modifications in cell function2, here we assess the link between ubiquitination and ageing by quantifying whole-proteome ubiquitin signatures in Caenorhabditis elegans. We find a remodelling of the ubiquitinated proteome during ageing, which is ameliorated by longevity paradigms such as dietary restriction and reduced insulin signalling. Notably, ageing causes a global loss of ubiquitination that is triggered by increased deubiquitinase activity. Because ubiquitination can tag proteins for recognition by the proteasome3, a fundamental question is whether deficits in targeted degradation influence longevity. By integrating data from worms with a defective proteasome, we identify proteasomal targets that accumulate with age owing to decreased ubiquitination and subsequent degradation. Lowering the levels of age-dysregulated proteasome targets prolongs longevity, whereas preventing their degradation shortens lifespan. Among the proteasomal targets, we find the IFB-2 intermediate filament4 and the EPS-8 modulator of RAC signalling5. While increased levels of IFB-2 promote the loss of intestinal integrity and bacterial colonization, upregulation of EPS-8 hyperactivates RAC in muscle and neurons, and leads to alterations in the actin cytoskeleton and protein kinase JNK. In summary, age-related changes in targeted degradation of structural and regulatory proteins across tissues determine longevity.


Assuntos
Envelhecimento/metabolismo , Caenorhabditis elegans/metabolismo , Proteoma/metabolismo , Ubiquitina/metabolismo , Ubiquitinação , Citoesqueleto de Actina/metabolismo , Animais , Caenorhabditis elegans/citologia , Caenorhabditis elegans/microbiologia , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas do Citoesqueleto/metabolismo , Intestinos/microbiologia , Longevidade , Músculos/metabolismo , Neurônios/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Proteoma/química , Proteínas rac de Ligação ao GTP/metabolismo
10.
Nat Chem Biol ; 17(8): 843-844, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34239126
11.
Development ; 148(14)2021 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-34269385

RESUMO

Fertilization triggers significant cellular remodeling through the oocyte-to-embryo transition. In this transition, the ubiquitin-proteasome system and autophagy are essential for the degradation of maternal components; however, the significance of degradation of cell surface components remains unknown. In this study, we show that multiple maternal plasma membrane proteins, such as the glycine transporter GlyT1a, are selectively internalized from the plasma membrane to endosomes in mouse embryos by the late two-cell stage and then transported to lysosomes for degradation at the later stages. During this process, large amounts of ubiquitylated proteins accumulated on endosomes. Furthermore, the degradation of GlyT1a with mutations in potential ubiquitylation sites was delayed, suggesting that ubiquitylation may be involved in GlyT1a degradation. The clathrin inhibitor blocked GlyT1a internalization. Strikingly, the protein kinase C (PKC) activator triggered the heterochronic internalization of GlyT1a; the PKC inhibitor markedly blocked GlyT1a endocytosis. Lastly, clathrin inhibition completely blocked embryogenesis at the two-cell stage and inhibited cell division after the four-cell stage. These findings demonstrate that PKC-dependent clathrin-mediated endocytosis is essential for the selective degradation of maternal membrane proteins during oocyte-to-embryo transition and early embryogenesis.


Assuntos
Clatrina/metabolismo , Desenvolvimento Embrionário/fisiologia , Endocitose/fisiologia , Proteínas de Membrana/metabolismo , Animais , Membrana Celular/metabolismo , Embrião de Mamíferos , Endossomos/metabolismo , Feminino , Fertilização , Proteínas da Membrana Plasmática de Transporte de Glicina , Masculino , Camundongos , Oócitos , Proteína Quinase C , Ubiquitina/metabolismo , Ubiquitinação
12.
Biophys J ; 120(16): 3355-3362, 2021 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-34242591

RESUMO

TAK1-binding protein 2 (TAB2) has generally been considered to bind specifically to K63-linked polyubiquitin chains via its C-terminal Npl4 zinc-finger (NZF) domain. However, a recent study showed that the NZF domain of TAB2 (TAB2-NZF) could also interact with K6-linked polyubiquitin chains. Here, we report the crystal structure of TAB2-NZF in complex with K6-linked diubiquitin (K6-Ub2) at 1.99-Å resolution. TAB2-NZF simultaneously interacts with the distal and proximal ubiquitin moieties of K6-Ub2. By comparing the structures of TAB2-NZF in complex with K6-Ub2 and with K63-linked diubiquitin (K63-Ub2), we reveal that the binding mechanism of TAB2-NZF with K6-Ub2 is similar to that with K63-Ub2, except for the flexible C-terminal region of the distal ubiquitin. Therefore, we conclude that the C-terminal flexibility of the distal ubiquitin contributes to the dual specificity of TAB2-NZF toward K6- and K63-linked ubiquitin chains. This study provides important insights into the functions of K6-linked ubiquitin chains, which are currently unclear.


Assuntos
Poliubiquitina , Dedos de Zinco , Modelos Moleculares , Poliubiquitina/metabolismo , Ligação Proteica , Ubiquitina/metabolismo
13.
Int J Mol Sci ; 22(14)2021 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-34299191

RESUMO

Primary cilia are nonmotile cellular signal-sensing antenna-like structures composed of microtubule-based structures that distinguish them from motile cilia in structure and function. Primary ciliogenesis is regulated by various cellular signals, such as Wnt, hedgehog (Hh), and platelet-derived growth factor (PDGF). The abnormal regulation of ciliogenesis is closely related to developing various human diseases, including ciliopathies and cancer. This study identified a novel primary ciliogenesis factor Cullin 1 (CUL1), a core component of Skp1-Cullin-F-box (SCF) E3 ubiquitin ligase complex, which regulates the proteolysis of dishevelled 2 (Dvl2) through the ubiquitin-proteasome system. Through immunoprecipitation-tandem mass spectrometry analysis, 176 Dvl2 interacting candidates were identified, of which CUL1 is a novel Dvl2 modulator that induces Dvl2 ubiquitination-dependent degradation. Neddylation-dependent CUL1 activity at the centrosomes was essential for centrosomal Dvl2 degradation and primary ciliogenesis. Therefore, this study provides a new mechanism of Dvl2 degradation by CUL1, which ultimately leads to primary ciliogenesis, and suggest a novel target for primary cilia-related human diseases.


Assuntos
Cílios/fisiologia , Proteínas Culina/metabolismo , Proteínas Desgrenhadas/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas Ligases SKP Culina F-Box/metabolismo , Ubiquitina/metabolismo , Células Cultivadas , Humanos , Ligação Proteica , Proteólise , Transdução de Sinais , Ubiquitinação
14.
J Pharm Pharm Sci ; 24: 390-399, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34319871

RESUMO

PURPOSE: SARS-CoV-2 infection is associated with substantial mortality and high morbidity. This study tested the effect of angiotensin II type I receptor blocker, losartan, on SARS-CoV-2 replication and inhibition of the papain-like protease of the virus. METHODS: The dose-dependent inhibitory effect of losartan, in concentrations from 1µM to 100µM as determined by quantitative cell analysis combining fluorescence microscopy, image processing, and cellular measurements (Cellomics analysis) on SARS-CoV-2 replication was investigated in Vero E6 cells. The impact of losartan on deubiquitination and deISGylation of SARS-CoV-2 papain-like protease (PLpro) were also evaluated.  Results: Losartan reduced PLpro cleavage of tetraUbiquitin to diUbiquitin.  It was less effective in inhibiting PLpro's cleavage of ISG15-AMC than Ubiquitin-AMC.  To determine if losartan inhibited SARS-CoV-2 replication, losartan treatment of SARS-CoV-2 infected Vero E6 was examined. Losartan treatment one hour prior to SARS-CoV-2 infection reduced levels of SARS-CoV-2 nuclear protein, an indicator of virus replication, by 80% and treatment one-hour post-infection decreased viral replication by 70%. CONCLUSION: Losartan was not an effective inhibitor of deubiquitinase or deISGylase activity of the PLpro but affected the SARS-CoV-2 replication of Vero E6 cells in vitro.  As losartan has a favorable safety profile and is currently available it has features necessary for efficacious drug repurposing and treatment of COVID-19.


Assuntos
Bloqueadores do Receptor Tipo 1 de Angiotensina II/farmacologia , Antivirais/farmacologia , Losartan/farmacologia , SARS-CoV-2/efeitos dos fármacos , Animais , COVID-19/tratamento farmacológico , Chlorocebus aethiops , Biologia Computacional , Proteases Semelhantes à Papaína de Coronavírus/antagonistas & inibidores , Proteases Semelhantes à Papaína de Coronavírus/metabolismo , Enzimas Desubiquitinantes/antagonistas & inibidores , Enzimas Desubiquitinantes/metabolismo , SARS-CoV-2/metabolismo , SARS-CoV-2/fisiologia , Ubiquitina/metabolismo , Células Vero , Replicação Viral/efeitos dos fármacos
15.
Acta Crystallogr D Struct Biol ; 77(Pt 7): 943-953, 2021 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-34196620

RESUMO

Porcine epidemic diarrhea is a devastating porcine disease that is caused by the alphacoronavirus porcine epidemic diarrhea virus (PEDV). Like other members of the Coronaviridae family, PEDV encodes a multifunctional papain-like protease 2 (PLP2) that has the ability to process the coronavirus viral polyprotein to aid in RNA replication and antagonize the host innate immune response through cleavage of the regulatory proteins ubiquitin (Ub) and/or interferon-stimulated gene product 15 (ISG15) (deubiquitination and deISGylation, respectively). Because Betacoronavirus PLPs have been well characterized, it was sought to determine how PLP2 from the alphacoronavirus PEDV differentiates itself from its related counterparts. PEDV PLP2 was first biochemically characterized, and a 3.1 Šresolution crystal structure of PEDV PLP2 bound to Ub was then solved, providing insight into how Alphacoronavirus PLPs bind to their preferred substrate, Ub. It was found that PEDV PLP2 is a deubiquitinase and readily processes a variety of di-Ub linkages, in comparison with its Betacoronavirus counterparts, which have a narrower range of di-Ub activity but process both Ub and ISG15.


Assuntos
Infecções por Coronavirus/virologia , Proteases Semelhantes à Papaína de Coronavírus/química , Proteases Semelhantes à Papaína de Coronavírus/metabolismo , Vírus da Diarreia Epidêmica Suína/fisiologia , Ubiquitina/metabolismo , Animais , Cristalografia por Raios X , Ligação Proteica , Conformação Proteica , Suínos
16.
Adv Exp Med Biol ; 1322: 339-357, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34258747

RESUMO

Posttranslational modifications of targeted substrates alter their cellular fate. Ubiquitin is a highly conserved and ubiquitous covalent modifier protein that tags substrates with a single molecule or with a polyubiquitin chain. Monoubiquitination affects trafficking and signaling patterns of modified proteins. In contrast, polyubiquitination, particularly K48-linked polyubiquitination, targets the protein for degradation by the Ubiquitin-Proteasome System (UPS) resulting in a committed fate through irreversible inactivation of substrate. Given the diversity of cellular functions impacted by ubiquitination, it is no surprise that the wily pathogenic viruses have co-opted the UPS in myriad ways to ensure their survival. In this review, I describe viral exploitation of nondegradative ubiquitin signaling pathways to effect entry, replication, and egress. Additionally, viruses also harness the UPS to degrade antiviral cellular host factors. Finally, I describe how we can exploit the same proteolytic machinery to enable PROTACs (Proteolysis-Targeting Chimeras) to degrade essential viral proteins. Successful implementation of this modality will add to the arsenal of emerging antiviral therapies.


Assuntos
Antivirais , Ubiquitina , Antivirais/farmacologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação
17.
Int J Mol Sci ; 22(13)2021 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-34281157

RESUMO

Post-translational modifications play a fundamental role in regulating protein function and stability. In particular, protein ubiquitylation is a multifaceted modification involved in numerous aspects of plant biology. Landmark studies connected the ATP-dependent ubiquitylation of substrates to their degradation by the 26S proteasome; however, nonproteolytic functions of the ubiquitin (Ub) code are also crucial to regulate protein interactions, activity, and localization. Regarding proteolytic functions of Ub, Lys-48-linked branched chains are the most common chain type for proteasomal degradation, whereas promotion of endocytosis and vacuolar degradation is triggered through monoubiquitylation or Lys63-linked chains introduced in integral or peripheral plasma membrane proteins. Hormone signaling relies on regulated protein turnover, and specifically the half-life of ABA signaling components is regulated both through the ubiquitin-26S proteasome system and the endocytic/vacuolar degradation pathway. E3 Ub ligases have been reported that target different ABA signaling core components, i.e., ABA receptors, PP2Cs, SnRK2s, and ABFs/ABI5 transcription factors. In this review, we focused specifically on the ubiquitylation of ABA receptors and PP2C coreceptors, as well as other post-translational modifications of ABA receptors (nitration and phosphorylation) that result in their ubiquitination and degradation.


Assuntos
Ácido Abscísico/metabolismo , Proteína Fosfatase 2C/metabolismo , Estresse Fisiológico/fisiologia , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Fosforilação , Reguladores de Crescimento de Plantas/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Processamento de Proteína Pós-Traducional , Transdução de Sinais , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação
18.
Mol Cell ; 81(13): 2690-2692, 2021 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-34214444

RESUMO

As a component of cell-autonomous immunity, cytosolic bacterial invaders are earmarked with the protein modifier ubiquitin for targeted xenophagic destruction. Otten et al. (2021) reveal that unique bacterial lipids are unconventional sites for ubiquitination and initiate downstream pathogen clearance.


Assuntos
Bactérias , Ubiquitina , Bactérias/metabolismo , Lipídeos , Ubiquitina/genética , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação
19.
Int J Mol Sci ; 22(13)2021 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-34281279

RESUMO

(1) Background: Autophagy, the major cytoplasmic process of substrate turnover, declines with age, contributing to proteostasis decline, accumulation of harmful protein aggregates, damaged mitochondria and to ROS production. Accordingly, abnormalities in the autophagic flux may contribute to many different pathophysiological conditions associated with ageing, including neurodegeneration. Recent data have shown that extra-virgin olive oil (EVOO) polyphenols stimulate cell defenses against plaque-induced neurodegeneration, mainly, through autophagy induction. (2) Methods: We carried out a set of in vitro experiments on SH-SY5Y human neuroblastoma cells exposed to toxic Aß1-42 oligomers to investigate the molecular mechanisms involved in autophagy activation by two olive oil polyphenols, oleuropein aglycone (OleA), arising from the hydrolysis of oleuropein (Ole), the main polyphenol found in olive leaves and drupes and its main metabolite, hydroxytyrosol (HT). (3) Results: Our data show that the mixture of the two polyphenols activates synergistically the autophagic flux preventing cell damage by Aß1-42 oligomers., in terms of ROS production, and impairment of mitochondria. (4) Conclusion: Our results support the idea that EVOO polyphenols act synergistically in autophagy modulation against neurodegeneration. These data confirm and provide the rationale to consider these molecules, alone or in combination, as promising candidates to contrast ageing-associated neurodegeneration.


Assuntos
Doença de Alzheimer/dietoterapia , Azeite de Oliva/farmacologia , Polifenóis/farmacologia , Acetatos/administração & dosagem , Acetatos/química , Acetatos/farmacologia , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/metabolismo , Peptídeos beta-Amiloides/toxicidade , Autofagia/efeitos dos fármacos , Linhagem Celular , Monoterpenos Ciclopentânicos/administração & dosagem , Monoterpenos Ciclopentânicos/química , Monoterpenos Ciclopentânicos/farmacologia , Dieta Mediterrânea , Humanos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Modelos Neurológicos , Degeneração Neural/induzido quimicamente , Degeneração Neural/patologia , Degeneração Neural/prevenção & controle , Neurônios/efeitos dos fármacos , Neurônios/patologia , Azeite de Oliva/administração & dosagem , Azeite de Oliva/química , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/toxicidade , Álcool Feniletílico/administração & dosagem , Álcool Feniletílico/análogos & derivados , Álcool Feniletílico/química , Álcool Feniletílico/farmacologia , Polifenóis/administração & dosagem , Polifenóis/química , Complexo de Endopeptidases do Proteassoma/metabolismo , Piranos/administração & dosagem , Piranos/química , Piranos/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Ubiquitina/metabolismo
20.
Int J Mol Sci ; 22(12)2021 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-34200910

RESUMO

To increase the half-life of growth hormones, we proposed its long-lasting regulation through the ubiquitin-proteasome system (UPS). We identified lysine residues (K67, K141, and K166) that are involved in the ubiquitination of human growth hormone (hGH) using ubiquitination site prediction programs to validate the ubiquitination sites, and then substituted these lysine residues with arginine residues. We identified the most effective substituent (K141R) to prevent ubiquitination and named it AUT-hGH. hGH was expressed and purified in the form of hGH-His, and ubiquitination was first verified at sites containing K141 in the blood stream. Through the study, we propose that AUT-hGH with an increased half-life could be used as a long-lasting hGH in the blood stream.


Assuntos
Transtornos do Crescimento/tratamento farmacológico , Hormônio do Crescimento Humano/administração & dosagem , Hormônio do Crescimento Humano/química , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitina/metabolismo , Ubiquitinação , Animais , Citoplasma/metabolismo , Transtornos do Crescimento/metabolismo , Transtornos do Crescimento/patologia , Células HEK293 , Meia-Vida , Humanos , Masculino , Camundongos , Células NIH 3T3 , Ratos , Ratos Sprague-Dawley
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