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1.
Nat Chem ; 11(7): 644-652, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31182821

RESUMO

A promising approach in cancer therapy is to find ligands that directly bind ubiquitin (Ub) chains. However, finding molecules capable of tightly and specifically binding Ub chains is challenging given the range of Ub polymer lengths and linkages and their subtle structural differences. Here, we use total chemical synthesis of proteins to generate highly homogeneous Ub chains for screening against trillion-member macrocyclic peptide libraries (RaPID system). De novo cyclic peptides were found that can bind tightly and specifically to K48-linked Ub chains, confirmed by NMR studies. These cyclic peptides protected K48-linked Ub chains from deubiquitinating enzymes and prevented proteasomal degradation of Ub-tagged proteins. The cyclic peptides could enter cells, inhibit growth and induce programmed cell death, opening new opportunities for therapeutic intervention. This highly synthetic approach, with both protein target generation and cyclic peptide discovery performed in vitro, will make other elaborate post-translationally modified targets accessible for drug discovery.


Assuntos
Lisina/química , Peptídeos Cíclicos/metabolismo , Bibliotecas de Moléculas Pequenas/metabolismo , Ubiquitinas/metabolismo , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Cisteína Endopeptidases/metabolismo , Inibidores de Cisteína Proteinase/metabolismo , Inibidores de Cisteína Proteinase/farmacologia , Células HeLa , Humanos , Estrutura Molecular , Peptídeos Cíclicos/farmacologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma/metabolismo , Inibidores de Proteassoma/farmacologia , Ligação Proteica , Bibliotecas de Moléculas Pequenas/farmacologia , Ubiquitinas/síntese química , Ubiquitinas/química
2.
Nat Commun ; 10(1): 2576, 2019 06 12.
Artigo em Inglês | MEDLINE | ID: mdl-31189900

RESUMO

Mitochondrial quality control is essential in highly structured cells such as neurons and muscles. In skeletal muscle the mitochondrial fission proteins are reduced in different physiopathological conditions including ageing sarcopenia, cancer cachexia and chemotherapy-induced muscle wasting. However, whether mitochondrial fission is essential for muscle homeostasis is still unclear. Here we show that muscle-specific loss of the pro-fission dynamin related protein (DRP) 1 induces muscle wasting and weakness. Constitutive Drp1 ablation in muscles reduces growth and causes animal death while inducible deletion results in atrophy and degeneration. Drp1 deficient mitochondria are morphologically bigger and functionally abnormal. The dysfunctional mitochondria signals to the nucleus to induce the ubiquitin-proteasome system and an Unfolded Protein Response while the change of mitochondrial volume results in an increase of mitochondrial Ca2+ uptake and myofiber death. Our findings reveal that morphology of mitochondrial network is critical for several biological processes that control nuclear programs and Ca2+ handling.


Assuntos
Dinaminas/metabolismo , Mitocôndrias Musculares/patologia , Dinâmica Mitocondrial/fisiologia , Miopatias Mitocondriais/patologia , Músculo Esquelético/patologia , Animais , Cálcio/metabolismo , Núcleo Celular/metabolismo , Modelos Animais de Doenças , Dinaminas/genética , Homeostase/fisiologia , Humanos , Camundongos , Camundongos Knockout , Miopatias Mitocondriais/genética , Miopatias Mitocondriais/mortalidade , Músculo Esquelético/citologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Ubiquitinas/metabolismo , Resposta a Proteínas não Dobradas/fisiologia
3.
Int J Mol Sci ; 20(9)2019 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-31067743

RESUMO

Questions have been raised since the discovery of UBA6 and its significant coexistence with UBE1 in the ubiquitin-proteasome system (UPS). The facts that UBA6 has the dedicated E2 enzyme USE1 and the E1-E2 cascade can activate and transfer both ubiquitin and ubiquitin-like protein FAT10 have attracted a great deal of attention to the regulational mechanisms of the UBA6-USE1 cascade and to how FAT10 and ubiquitin differentiate with each other. This review recapitulates the latest advances in UBA6 and its bispecific UBA6-USE1 pathways for both ubiquitin and FAT10. The intricate networks of UBA6 and its interplays with ubiquitin and FAT10 are briefly reviewed, as are their individual and collective functions in diverse physiological conditions.


Assuntos
Enzimas Ativadoras de Ubiquitina/metabolismo , Ubiquitinação , Ubiquitinas/metabolismo , Animais , Humanos
4.
Int J Mol Sci ; 20(7)2019 Apr 04.
Artigo em Inglês | MEDLINE | ID: mdl-30987352

RESUMO

Mast cells (MCs) are one of the first immune cells recruited to a tumor. It is well recognized that MCs accumulate in colon cancer lesion and their density is associated with the clinical outcomes. However, the molecular mechanism of how colon cancer cells may modify MC function is still unclear. In this study, primary human MCs were generated from CD34⁺ progenitor cells and a 3D coculture model was developed to study the interplay between colon cancer cells and MCs. By comparing the transcriptomic profile of colon cancer-cocultured MCs versus control MCs, we identified a number of deregulated genes, such as MMP-2, VEGF-A, PDGF-A, COX2, NOTCH1 and ISG15, which contribute to the enrichment of cancer-related pathways. Intriguingly, pre-stimulation with a TLR2 agonist prior to colon cancer coculture induced upregulation of multiple interferon-inducible genes as well as MHC molecules in MCs. Our study provides an alternative approach to study the influence of colon cancer on MCs. The transcriptome signature of colon cancer-cocultured MCs may potentially reflect the mechanism of how colon cancer cells educate MCs to become pro-tumorigenic in the initial phase and how a subsequent inflammatory signal-e.g., TLR2 ligands-may modify their responses in the cancer milieu.


Assuntos
Neoplasias do Colo/metabolismo , Mastócitos/metabolismo , Transcriptoma/genética , Células Cultivadas , Neoplasias do Colo/genética , Citocinas/metabolismo , Células HT29 , Humanos , Metaloproteinase 2 da Matriz/metabolismo , Fator de Crescimento Derivado de Plaquetas/metabolismo , Receptor Notch1/metabolismo , Ubiquitinas/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo
5.
Int J Mol Sci ; 20(8)2019 Apr 17.
Artigo em Inglês | MEDLINE | ID: mdl-30999567

RESUMO

Ubiquitin-like/ubiquitin-associated proteins (UbL-UbA) are a well-studied family of non-proteasomal ubiquitin receptors that are evolutionarily conserved across species. Members of this non-homogenous family facilitate and support proteasomal activity by promoting different effects on proteostasis but exhibit diverse extra-proteasomal activities. Dysfunctional UbL-UbA proteins render cells, particularly neurons, more susceptible to stressors or aging and may cause earlier neurodegeneration. In this review, we summarized the properties and functions of UbL-UbA family members identified to date, with an emphasis on new findings obtained using Drosophila models showing a direct or indirect role in some neurodegenerative diseases.


Assuntos
Doenças Neurodegenerativas/metabolismo , Neurônios/patologia , Ubiquitina/metabolismo , Ubiquitinas/metabolismo , Animais , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/metabolismo , Enzimas Reparadoras do DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Modelos Animais de Doenças , Drosophila , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Doenças Neurodegenerativas/patologia , Neurônios/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteostase , Fatores de Transcrição/metabolismo
6.
Int J Mol Sci ; 20(7)2019 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-30939763

RESUMO

The demyelinating canine distemper virus (CDV)-leukoencephalitis represents a translational animal model for multiple sclerosis. The present study investigated the expression of type I interferon (IFN-I) pathway members in CDV-induced cerebellar lesions to gain an insight into their role in lesion development. Gene expression of 110 manually selected genes in acute, subacute and chronic lesions was analyzed using pre-existing microarray data. Interferon regulatory factor (IRF) 3, IRF7, signal transducer and activator of transcription (STAT) 1, STAT2, MX protein, protein kinase R (PKR), 2'-5'-oligoadenylate synthetase (OAS) 1 and interferon-stimulated gene (ISG) 15 expression were also evaluated using immunohistochemistry. Cellular origin of STAT1, STAT2, MX and PKR were determined using immunofluorescence. CDV infection caused an increased expression of the antiviral effector proteins MX, PKR, OAS1 and ISG15, which probably contributed to a restricted viral replication, particularly in neurons and oligodendrocytes. This increase might be partly mediated by IRF-dependent pathways due to the lack of changes in IFN-I levels and absence of STAT2 in astrocytes. Nevertheless, activated microglia/macrophages showed a strong expression of STAT1, STAT2 and MX proteins in later stages of the disease, indicating a strong activation of the IFN-I signaling cascade, which might be involved in the aggravation of bystander demyelination.


Assuntos
Cinomose/genética , Imunidade Inata/genética , Interferons/farmacologia , 2',5'-Oligoadenilato Sintetase/genética , 2',5'-Oligoadenilato Sintetase/metabolismo , Animais , Cerebelo/efeitos dos fármacos , Cerebelo/metabolismo , Cinomose/imunologia , Cães , Fator Regulador 7 de Interferon/genética , Fator Regulador 7 de Interferon/metabolismo , Proteínas de Resistência a Myxovirus/genética , Proteínas de Resistência a Myxovirus/metabolismo , Fatores de Transcrição STAT/genética , Fatores de Transcrição STAT/metabolismo , Ubiquitinas/genética , Ubiquitinas/metabolismo , eIF-2 Quinase/genética , eIF-2 Quinase/metabolismo
8.
Nat Struct Mol Biol ; 26(4): 289-296, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30911187

RESUMO

Ubiquitin or ubiquitin-like proteins can be covalently conjugated to multiple proteins that do not necessarily have binding interfaces. Here, we show that an evolutionary transition from covalent conjugation to non-covalent interaction has occurred in the ubiquitin-like autophagy-related 12 (ATG12) conjugation system. ATG12 is covalently conjugated to its sole substrate, ATG5, by a ubiquitylation-like mechanism. However, the apicomplexan parasites Plasmodium and Toxoplasma and some yeast species such as Komagataella phaffii (previously Pichia pastoris) lack the E2-like enzyme ATG10 and the most carboxy (C)-terminal glycine of ATG12, both of which are required for covalent linkage. Instead, ATG12 in these organisms forms a non-covalent complex with ATG5. This non-covalent ATG12-ATG5 complex retains the ability to facilitate ATG8-phosphatidylethanolamine conjugation. These results suggest that ubiquitin-like covalent conjugation can evolve to a simpler non-covalent interaction, most probably when the system has a limited number of targets.


Assuntos
Autofagossomos/metabolismo , Proteínas Relacionadas à Autofagia/metabolismo , Retículo Endoplasmático/metabolismo , Membranas/metabolismo , Membranas/ultraestrutura , Ubiquitina/metabolismo , Animais , Autofagossomos/ultraestrutura , Proteínas Relacionadas à Autofagia/química , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Cristalografia por Raios X , Retículo Endoplasmático/ultraestrutura , Humanos , Lipossomos/química , Lipossomos/metabolismo , Lipossomos/ultraestrutura , Camundongos , Mutação , Fosfolipídeos/química , Fosfolipídeos/metabolismo , Schizosaccharomyces/metabolismo , Schizosaccharomyces/ultraestrutura , Ubiquitinas/química , Ubiquitinas/metabolismo
9.
Methods Enzymol ; 618: 229-256, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30850054

RESUMO

The ubiquitin-like modifier FAT10 (also called ubiquitin D (UBD)) interacts noncovalently with a substantial number of proteins and also gets covalently conjugated to many substrate proteins, leading to their degradation by the 26S proteasome. FAT10 comprises two loosely folded ubiquitin-like domains that are connected by a flexible linker, and this unusual structure makes it highly prone to aggregation. Here, we report methods to purify high amounts of soluble recombinant FAT10 for various uses, such as in vitro FAT10ylation assays. In addition, we describe how to generate and handle overexpressed as well as endogenous FAT10 in cellulo for use in immunoprecipitations, Western blot analyses, and FAT10 degradation studies.


Assuntos
Ubiquitinas/metabolismo , Western Blotting , Linhagem Celular , Expressão Gênica , Humanos , Imunoprecipitação , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Transfecção/métodos , Ubiquitinas/genética , Ubiquitinas/isolamento & purificação , Regulação para Cima
10.
Methods Enzymol ; 618: 357-387, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30850060

RESUMO

Protein (poly-)ubiquitination is a posttranslational modification that plays a key role in almost all cellular processes. It involves the installment of either single ubiquitin (Ub) moieties or one of eight different polyUb linkage types, each giving a distinct cellular outcome. Deubiquitinating enzymes (DUBs) reverse Ub signaling by disassembly of one or multiple poly-Ub chain types and their malfunction is often associated with human disease. The Ub system displays significant crosstalk with structurally homologous ubiquitin-like proteins (Ubls), including SUMO, Nedd8, and ISG15. This can be seen with the existence of heterogeneous chains made from Ub-Ubl mixtures as well as the proteolytic cross reactivity displayed by several DUBs toward other Ubl systems. In addition, numerous pathogens have been found to encode Ub(l)-ligases and deconjugating enzymes in order to facilitate infection and fight the host immune response. Studying the activity of DUBs and Ubl-specific proteases, both human as well as pathogen-derived, gives fundamental insights into their physiological roles. Activity-based probes (ABPs) have proven to be valuable tools to achieve this, as they report on enzyme activities by making a (often irreversible) covalent complex, rather than on their relative abundance. In this chapter, we explain the potential of ABPs to assess substrate preferences, structural features, and activity of Ub and Ubl deconjugating enzymes. We further demonstrate the practical use of ABPs to (1) characterize the activity of viral proteases toward Ub and Ubls and (2) to gain more insight in the structural determinants of substrate preference of DUBs.


Assuntos
Enzimas Desubiquitinantes/metabolismo , Ubiquitinas/metabolismo , Animais , Ensaios Enzimáticos/métodos , Humanos , Inteínas , Sondas Moleculares/química , Sondas Moleculares/metabolismo , Peptídeo Hidrolases/metabolismo , Especificidade por Substrato , Ubiquitina/metabolismo , Vírus/enzimologia
11.
Mol Immunol ; 108: 111-120, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30818228

RESUMO

FAT10 is the only ubiquitin-like modifier which directly targets its substrate proteins for rapid degradation by the proteasome. While the conjugation and proteasomal targeting of FAT10 are fairly well understood, the biological functions of FAT10 have remained largely elusive. Here we have investigated the role of FAT10 in cytokine responses in mice upon viral infection. We used lymphocytic choriomeningitis virus (LCMV) infection of mice to induce the IFN-γ and TNF-α-dependent expression of FAT10. We found that TCR-stimulated splenocytes derived from LCMV-infected FAT10-/- mice secreted less IFN-γ and expressed less mRNA for IL-12 p40 but secreted more IFN-α and IFN-ß compared to FAT10+/- mice. The reduction in IFN-γ secretion could be assigned to CD8+ T cells. Nevertheless, LCMV viral clearance was similar in FAT10-/- as compared to FAT10+/- mice. Since FAT10 has previously been reported to promote influenza A virus (IAV) replication in vitro we have studied the effect of FAT10 deficiency during IAV infection in mice. Unexpectedly, IAV titers and disease symptoms were not changed in FAT10-/- mice even though the Fat10 mRNA was rapidly induced in the lung upon IAV infection. In conclusion, we find that FAT10 fine-tunes the balance of interferons during viral infection by lowering the production of type I and enhancing type II interferons.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Interferon gama/biossíntese , Ativação Linfocitária , Ubiquitina/metabolismo , Ubiquitinas/metabolismo , Animais , Vírus da Influenza A/fisiologia , Ativação Linfocitária/imunologia , Coriomeningite Linfocítica/imunologia , Coriomeningite Linfocítica/virologia , Vírus da Coriomeningite Linfocítica/fisiologia , Camundongos Endogâmicos C57BL , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Baço/patologia , Fator de Necrose Tumoral alfa/metabolismo , Ubiquitinas/deficiência , Ubiquitinas/genética , Regulação para Cima/genética
12.
Proc Natl Acad Sci U S A ; 116(11): 4983-4988, 2019 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-30804189

RESUMO

Platelets mediate primary hemostasis, and recent work has emphasized platelet participation in immunity and inflammation. The function of the platelet-specific integrin αIIbß3 as a fibrinogen receptor in hemostasis is well defined, but the roles of αIIbß3 or integrin-associated proteins in nonhemostatic platelet functions are poorly understood. Here we show that human platelets express the integrin-associated protein SHARPIN with functional consequences. In leukocytes, SHARPIN interacts with integrin α cytoplasmic tails, and it is also an obligate member of the linear ubiquitin chain assembly complex (LUBAC), which mediates Met1 linear ubiquitination of proteins leading to canonical NF-κB activation. SHARPIN interacted with αIIb in pull-down and coimmunoprecipitation assays. SHARPIN was partially localized, as was αIIbß3, at platelet edges, and thrombin stimulation induced more central SHARPIN localization. SHARPIN also coimmunoprecipitated from platelets with the two other proteins comprising LUBAC, the E3 ligase HOIP and HOIL-1. Platelet stimulation with thrombin or inflammatory agonists, including lipopolysaccharide or soluble CD40 ligand (sCD40L), induced Met1 linear ubiquitination of the NF-κB pathway protein NEMO and serine-536 phosphorylation of the p65 RelA subunit of NF-κB. In human megakaryocytes and/or platelets derived from induced pluripotent stem (iPS) cells, SHARPIN knockdown caused increased basal and agonist-induced fibrinogen binding to αIIbß3 as well as reduced Met1 ubiquitination and RelA phosphorylation. Moreover, these SHARPIN knockdown cells exhibited increased surface expression of MHC class I molecules and increased release of sCD40L. These results establish that SHARPIN functions in the human megakaryocyte/platelet lineage through protein interactions at the nexus of integrin and immune/inflammatory signaling.


Assuntos
Plaquetas/metabolismo , Transdução de Sinais , Ubiquitinas/metabolismo , Linhagem da Célula , Técnicas de Silenciamento de Genes , Homeostase , Humanos , Quinase I-kappa B/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Inflamação/patologia , Megacariócitos/metabolismo , Modelos Biológicos , NF-kappa B/metabolismo , Glicoproteína IIb da Membrana de Plaquetas/metabolismo , Ligação Proteica , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação
13.
FEBS J ; 286(4): 653-677, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30659753

RESUMO

Among the members of the ubiquitin-like (Ubl) protein family, neural precursor cell expressed developmentally down-regulated protein 8 (NEDD8) is the closest in sequence to ubiquitin (57% identity). The two modification mechanisms and their functions, however, are highly distinct and the two Ubls are not interchangeable. A complex network of interactions between modifying enzymes and adaptors, most of which are specific while others are promiscuous, ensures selectivity. Many domains that bind the ubiquitin hydrophobic patch also bind NEDD8 while no domain that specifically binds NEDD8 has yet been described. Here, we report an unbiased selection of domains that bind ubiquitin and/or NEDD8 and we characterize their specificity/promiscuity. Many ubiquitin-binding domains bind ubiquitin preferentially and, to a lesser extent, NEDD8. In a few cases, the affinity of these domains for NEDD8 can be increased by substituting the alanine at position 72 with arginine, as in ubiquitin. We have also identified a unique domain, mapping to the carboxyl end of the protein KHNYN, which has a stark preference for NEDD8. Given its ability to bind neddylated cullins, we have named this domain CUBAN (Cullin-Binding domain Associating with NEDD8). We present here the solution structure of the CUBAN domain both in the isolated form and in complex with NEDD8. The results contribute to the understanding of the discrimination mechanism between ubiquitin and the Ubl. They also provide new insights on the biological role of a ill-defined protein, whose function is hitherto only predicted.


Assuntos
Proteínas Culina/metabolismo , Proteína NEDD8/metabolismo , Ubiquitinas/metabolismo , Sequência de Aminoácidos , Células Cultivadas , Humanos , Proteína NEDD8/química , Proteína NEDD8/genética , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Homologia de Sequência , Ubiquitinação
14.
PLoS Pathog ; 15(1): e1007515, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30629698

RESUMO

Post-translational modification of host and viral proteins by ubiquitin (Ub) and Ub-like proteins, such as interferon stimulated gene product 15 (ISG15), plays a key role in response to infection. Viruses have been increasingly identified that contain proteases possessing deubiquitinase (DUB) and/or deISGylase functions. This includes viruses in the Nairoviridae family that encode a viral homologue of the ovarian tumor protease (vOTU). vOTU activity was recently demonstrated to be critical for replication of the often-fatal Crimean-Congo hemorrhagic fever virus, with DUB activity suppressing the type I interferon responses and deISGylase activity broadly removing ISG15 conjugated proteins. There are currently about 40 known nairoviruses classified into fourteen species. Recent genomic characterization has revealed a high degree of diversity, with vOTUs showing less than 25% amino acids identities within the family. Previous investigations have been limited to only a few closely related nairoviruses, leaving it unclear what impact this diversity has on vOTU function. To probe the effects of vOTU diversity on enzyme activity and specificity, we assessed representative vOTUs spanning the Nairoviridae family towards Ub and ISG15 fluorogenic substrates. This revealed great variation in enzymatic activity and specific substrate preferences. A subset of the vOTUs were further assayed against eight biologically relevant di-Ub substrates, uncovering both common trends and distinct preferences of poly-Ub linkages by vOTUs. Four novel X-ray crystal structures were obtained that provide a biochemical rationale for vOTU substrate preferences and elucidate structural features that distinguish the vOTUs, including a motif in the Hughes orthonairovirus species that has not been previously observed in OTU domains. Additionally, structure-informed mutagenesis provided the first direct evidence of a second site involved in di-Ub binding for vOTUs. These results provide new insight into nairovirus evolution and pathogenesis, and further enhances the development of tools for therapeutic purposes.


Assuntos
Nairovirus/genética , Neoplasias Ovarianas/virologia , Peptídeo Hidrolases/genética , Cristalografia por Raios X/métodos , Enzimas Desubiquitinantes/metabolismo , Feminino , Variação Genética/genética , Genômica , Humanos , Nairovirus/patogenicidade , Neoplasias Ovarianas/metabolismo , Ovário/metabolismo , Peptídeo Hidrolases/metabolismo , Filogenia , Ligação Proteica , Domínios Proteicos , Processamento de Proteína Pós-Traducional/genética , Proteólise , Homologia de Sequência de Aminoácidos , Ubiquitina/metabolismo , Ubiquitinação/genética , Ubiquitinas/metabolismo , Proteínas Virais/metabolismo
15.
J Biol Chem ; 294(11): 4177-4187, 2019 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-30647135

RESUMO

Ubiquitin-specific protease 7 (USP7) regulates various cellular pathways through its deubiquitination activity. Despite the identification of a growing number of substrates of USP7, the molecular mechanism by which USP7 removes ubiquitin chains from polyubiquitinated substrates remains unexplored. The present study investigated the mechanism underlying the deubiquitination of Lys63-linked polyubiquitinated proliferating cell nuclear antigen (PCNA). Biochemical analyses demonstrated that USP7 efficiently removes polyubiquitin chains from polyubiquitinated PCNA by preferential cleavage of the PCNA-ubiquitin linkage. This property was largely attributed to the poor activity toward Lys63-linked ubiquitin chains. The preferential cleavage of substrate-ubiquitin linkages was also observed for Lys48-linked polyubiquitinated p53 because of the inefficient cleavage of the Lys48-linked ubiquitin chains. The present findings suggest a mechanism underlying the removal of polyubiquitin signals by USP7.


Assuntos
Antígeno Nuclear de Célula em Proliferação/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Peptidase 7 Específica de Ubiquitina/metabolismo , Ubiquitinas/metabolismo , Humanos , Especificidade por Substrato
16.
Int J Biochem Cell Biol ; 107: 14-26, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30529400

RESUMO

ISG15 (interferon-stimulated gene 15) exists as free ISG15 or conjugated ISG15 modifying its target proteins via ISGylation. Few proteins have been identified and studied as ISGylation targets, and their relevance is not completely clear. Here, we isolated ISG15 from MDA-MB-231 breast cancer cells using immunoprecipitation and identified non-muscle myosin IIA (NMIIA) using mass spectrometry as endogenously associated with ISG15. The identification of NMIIA as an ISG15-interacting protein was important, because levels of NMIIA mRNA were not deregulated in all breast cancers, and because our in silico analysis indicated that NMIIA was the target of different posttranslational modifications and had an interactome associated with cytoskeletal remodeling. Furthermore, our experimental assays of co-immunoprecipitation and immunofluorescence confirmed that ISG15 was covalently associated with NMIIA in the cytoplasm of breast cancer cells and that interferon γ (IFN-γ) increased this association without alterations in the NMIIA levels. Thus, NMIIA ISGylation is regulated by IFN-γ, and this modification may modulate its interactions with proteins that remodel the cytoskeleton, participating in the growth and progression of mammary tumors.


Assuntos
Neoplasias da Mama/patologia , Citocinas/metabolismo , Miosina não Muscular Tipo IIA/metabolismo , Processamento de Proteína Pós-Traducional , Ubiquitinas/metabolismo , Sequência de Aminoácidos , Linhagem Celular Tumoral , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Humanos
17.
Biomed Pharmacother ; 109: 1126-1139, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30551363

RESUMO

The selective serotonin reuptake inhibitor fluoxetine has been used for the treatment of depression. Although sexual disorders have been reported in male patients, few studies have demonstrated the fluoxetine effect on the reproductive histophysiology, and the target of this antidepressant in testes is unknown. We evaluated the impact of short-term treatment with fluoxetine on the adult rat testes, focusing on steroidogenesis by Leydig cells (LC) and androgen-dependent testicular parameters, including Sertoli cells (SC) and peritubular myoid cells (PMC). Since UCHL1 (ubiquitincarboxyl-terminal hydrolase L1) seems to control spermatogenesis, the immunoexpression of this hydrolase was also analyzed. Adult male rats received 20 mg/kg BW of fluoxetine (FG) or saline (CG) for eleven days. In historesin-embedded testis sections, the seminiferous tubule (ST) and epithelial (Ep) areas, and the LC nuclear diameter (LCnu) were measured. The number of abnormal ST, androgen-dependent ST, SC and PMC was quantified. Testicular ß-tubulin levels and peritubular actin immunofluorescence were evaluated. Serum testosterone levels (STL) and steroidogenesis by 17ß-HSD6 immunofluorescence were analyzed, and either UCHL1-immunolabeled or TUNEL-positive germ cells were quantified. In FG, abnormal ST frequency increased whereas ST and Ep areas, androgen-dependent ST number, LCnu, 17ß-HSD6 activity and STL reduced significantly. TUNEL-positive PMC and SC was related to decreased number of these cells and reduction in peritubular actin and ß-tubulin levels. In FG, uncommon UCHL1-immunoexpression was found in spermatocytes and spermatids, and the number of UCHL1-immunolabeled and TUNEL-positive germ cells increased in this group. These findings indicate that LC may be a fluoxetine target in testes, impairing PMC-SC integrity and disturbing spermatogenesis. The increase of UCHL1 in the damaged tubules associated with high incidence of cell death confirms that this hydrolase regulates germ cell death and may be controlled by androgens. The fertility in association with the androgenic status of patients treated with fluoxetine should be carefully evaluated.


Assuntos
Androgênios/metabolismo , Morte Celular/efeitos dos fármacos , Fluoxetina/farmacologia , Células Germinativas/efeitos dos fármacos , Túbulos Seminíferos/efeitos dos fármacos , Ubiquitina Tiolesterase/metabolismo , Animais , Células Germinativas/metabolismo , Hidrolases/efeitos dos fármacos , Hidrolases/metabolismo , Marcação In Situ das Extremidades Cortadas/métodos , Células Intersticiais do Testículo/efeitos dos fármacos , Células Intersticiais do Testículo/metabolismo , Masculino , Ratos , Túbulos Seminíferos/metabolismo , Células de Sertoli/efeitos dos fármacos , Células de Sertoli/metabolismo , Espermatogênese/efeitos dos fármacos , Testículo/efeitos dos fármacos , Testículo/metabolismo , Ubiquitinas/metabolismo
18.
Enzyme Microb Technol ; 120: 98-109, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30396406

RESUMO

The exploitation of SUMO (small ubiquitin-related modifier) fusion technology at a large scale for the production of therapeutic proteins with an authentic N-terminus is majorly limited due to the higher cost of ScUlp1 protease. Therefore, the cost-effective production of Saccharomyces cerevisiae Ulp1 protease catalytic domain (402-621 aa) was targeted via its cloning under strong T7 promoter with and without histidine tag. The optimization of cultivation conditions at shake flask resulted in ScUlp1 expression of 195 mg/L in TB medium with a specific product yield of 98 mg/g DCW. The leaky expression of the ScUlp1 protease was controlled using the chemically defined minimal medium. The Ni-NTA affinity purification of ScUlp1 was done near homogeneity using different additives (0.1% Triton X-100, 0.01 mM DTT, 0.02 mM EDTA and 1% glycerol) where a product purity of ∼95% with a recovery yield of 80% was obtained. The specific activity of purified ScUlp1 was found to be 3.986 × 105 U/mg. The ScUlp1 protease successfully cleaved the SUMO tag even at 1:10,000 enzyme to substrate ratio with high efficacy and also showed a comparable catalytic efficiency as of commercial control. Moreover, the in vivo cleavage of SUMO tag via co-expression strategy also resulted in more than 80% cleavage of SUMO fusion protein. The optimization of high cell density cultivation strategies and maintenance of higher plasmid stability at bioreactor level resulted in the ScUlp1 production of 3.25 g/L with a specific product yield of 45.41 mg/g DCW when cells were induced at an OD600 of 132 (63.66 g/L DCW).


Assuntos
Técnicas de Cultura Celular por Lotes/métodos , Cisteína Endopeptidases/metabolismo , Escherichia coli/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Reatores Biológicos , Domínio Catalítico , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/isolamento & purificação , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/isolamento & purificação , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genética , Ubiquitinas/metabolismo
19.
Fish Shellfish Immunol ; 86: 1088-1095, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30593901

RESUMO

Protein SUMOylation (SUMO is small ubiquitin-related modifier) is a dynamic process that is strictly regulated under physiological and pathological conditions. We previously cloned and characterized two SUMO homologue genes (EcSUMO1 and EcSUMO2) from orange-spotted grouper (Epinephelus coioides). In the present study, the SUMO3 homologue from E. coioides (EcSUMO3) was cloned and its possible roles in fish immunity were analyzed. The open reading frame of EcSUMO3 contains 285 base pairs encoding a 94 amino acid protein with a predicted molecular mass of 10.73 kDa. The protein sequence of EcSUMO3 revealed similar domains with mammals, including the UBQ (ubiquitin-like proteins) domain, the hydrophobic surface, the Ulp1-Smt3 interaction sites, a VKTE motif and the C-terminal Gly residues. EcSUMO3 shares 46.83% and 89.58% identity with EcSUMO1 and EcSUMO2, respectively, and it shares 94%, 98%, and 98% identity with SUMO3 from Oreochromis niloticus, Danio rerio, and Homo sapiens, respectively. Quantitative real-time polymerase chain reaction analysis indicated that EcSUMO3 was constitutively expressed in all of the analyzed tissues in healthy grouper. EcSUMO3 expression levels were remarkably (p < 0.01) up-regulated in grouper spleen (GS) cells in response to stimulation with red-spotted grouper nervous necrosis virus (RGNNV) and Singapore grouper iridovirus (SGIV). EcSUMO3 was distributed in both the cytoplasm and nucleus in GS cells. EcSUMO3 enhanced SGIV and RGNNV replication during viral infection in vitro. These results are important for better understanding of the SUMO pathway in fish and provide insights into the regulatory mechanism of viral infection in E. coioides under farmed conditions.


Assuntos
Bass/genética , Infecções por Vírus de DNA/veterinária , Doenças dos Peixes/virologia , Infecções por Vírus de RNA/veterinária , Proteína SUMO-1/genética , Sequência de Aminoácidos , Animais , Bass/imunologia , Infecções por Vírus de DNA/imunologia , Proteínas de Peixes/química , Proteínas de Peixes/genética , Proteínas de Peixes/imunologia , Imunidade Inata/genética , Iridovirus/fisiologia , Nodaviridae/fisiologia , Proteína SUMO-1/imunologia , Ubiquitinas/metabolismo
20.
Exp Mol Pathol ; 105(1): 144-149, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-30009772

RESUMO

Hepatocellular carcinoma (HCC) is the fifth most common cancer and the second leading cause of cancer related deaths worldwide. Among others, non-alcoholic steatohepatitis (NASH) and alcoholic steatohepatitis (ASH) are the two major risk factors as both of them may develop cirrhosis and hepatocellular carcinoma (HCC) if left untreated. However, patients with NASH progress to HCC at a rate around 0.5% annually, while 3-10% ASH patients may progress to HCC annually. The present study is to demonstrate the molecular differences in oncogenesis pathway between NASH and ASH. By using immunofluorescence study and quantitating the fluorescence intensity morphometrically in liver biopsied specimens from NASH and ASH patients, the protein expression of candidate molecules within hepatocytes cytoplasm are studied, including two HCC-related molecules FAT10 and FOXO1, and one GPCR pathway related molecule ADRA2A. Compared with the control group patients, the expression levels of all the molecules were upregulated in the ASH group of patients (p < 0.001 in all molecules), while FAT10 and ADRA2A were upregulated, FOXO1 did not change in the NASH group of patients. The most important finding is that compared with the ASH group of patients, the expression levels of all three molecules were significantly lower than in the NASH group of patients (p < 0.001 in all molecules). These results confirmed our previous finding that there are significant differences of molecules change in ASH compared to NASH. Thus, we conclude that there are significantly different molecules and pathways involved during the pathogenesis of HCC development in ASH compared to NASH which could help explain why the tumorigenic rate is different in ASH and NASH.


Assuntos
Carcinogênese/genética , Carcinoma Hepatocelular/etiologia , Fígado Gorduroso Alcoólico/complicações , Neoplasias Hepáticas/etiologia , Hepatopatia Gordurosa não Alcoólica/complicações , Carcinogênese/metabolismo , Proteína Forkhead Box O1/genética , Proteína Forkhead Box O1/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Fígado/metabolismo , Fígado/patologia , Receptores Adrenérgicos alfa 2/genética , Receptores Adrenérgicos alfa 2/metabolismo , Ubiquitinas/genética , Ubiquitinas/metabolismo
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