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1.
Mol Biol (Mosk) ; 53(4): 638-647, 2019.
Artigo em Russo | MEDLINE | ID: mdl-31397437

RESUMO

The ubiquitin-proteasome system (UPS) performs proteolysis of most intracellular proteins. The key components of the UPS are the proteasomes, multi-subunit protein complexes, playing an important role in cellular adaptation to various types of stress. We analyzed the dynamics of the proteasome activity, the content of proteasome subunits, and the expression levels of genes encoding catalytic subunits of proteasomes in the human histiocytic lymphoma U937 cell line immediately, 2, 4, 6, 9, 24, and 48 h after a heat shock (HS). The initial decrease (up to 62%) in the proteasome activity in cellular lysates was revealed, then 10 h after HS the activity began to recover. The amount of proteasomal α-subunits in the cells decreased 2 h after HS, and was restored to 24-48 h after HS. Fluctuations in the levels of mRNAs encoding proteasome catalytic subunits with the maximum expression 2 h after HS and a gradual decrease to 48 h after HS were observed. The average estimated number of mRNA copies per cell ranged from 10 for weakly to 150 for highly expressed proteasome genes. Thus, the recovery efficiency of UPS functionality after HS, which reflects the important role of proteasomes in maintaining cell homeostasis, was evaluated.


Assuntos
Resposta ao Choque Térmico , Complexo de Endopeptidases do Proteassoma/química , Complexo de Endopeptidases do Proteassoma/metabolismo , Subunidades Proteicas/metabolismo , Humanos , Complexo de Endopeptidases do Proteassoma/genética , Subunidades Proteicas/genética , Proteólise , Células U937 , Ubiquitina/metabolismo
2.
Biomed Khim ; 65(4): 306-310, 2019 Jun.
Artigo em Russo | MEDLINE | ID: mdl-31436171

RESUMO

It becomes increasingly clear that ubiquitination of cellular proteins is not an indispensable prerequisite of their degradation in proteasomes. There are a number of proteins to be eliminated which are not pre-ubiquitinated for their recognition by regulatory subcomplex of 26S proteasome, but which directly interact with the 20S proteasome core particle (20S proteasome). The obligatory precondition for such interaction consists in existence of disordered (hydrophobic) fragments in the target protein. In this study we have investigated the interaction of a number of multifunctional (moonlighting) proteins (glyceraldehyde-3-phosphate dehydrogenase (GAPDH), aldolase, pyruvate kinase) and neurodegeneration-related proteins (a-synuclein, myelin basic protein) with 20S proteasome immobilized on the SPR-biosensor chip and stabilized by means of a bifunctional agent dimethyl pimelimidate (in order to prevent possible dissociation of this subcomplex). Only two of all investigated proteins (aldolase and pyruvate kinase) interacted with the immobilized 20S proteasome (Kd of 8.17´10-7 M and 5.56´10-7 M, respectively). In addition to earlier detected GAPDH ubiquitination, mass spectrometric analysis of the studied proteins revealed the presence of the ubiquitin signature (Lys-e-Gly-Gly) only in aldolase. Oxidation of aldolase and pyruvate kinase, which promotes elimination of proteins via their direct interaction with 20S proteasome, caused a 2-3-fold decrease in their Kd values as comparison with this parameter obtained for the intact proteins. The results of this study provide further evidence for direct interaction of both ubiquitinated proteins (aldolase), and non-ubiquitinated proteins (pyruvate kinase) with the 20S proteasome core particle (20S proteasome). The effectiveness of this interaction is basically equal for the ubiquitinated proteins and non-ubiquitinated proteins.


Assuntos
Técnicas Biossensoriais , Complexo de Endopeptidases do Proteassoma/química , Proteínas Ubiquitinadas/química , Humanos , Ubiquitina , Ubiquitinação
3.
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
4.
Molecules ; 24(12)2019 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-31242677

RESUMO

Protein accumulation has been identified as a characteristic of many degenerative conditions, such as neurodegenerative diseases and aging. In most cases, these conditions also present with diminished protein degradation. The ubiquitin-proteasome system (UPS) is responsible for the degradation of the majority of proteins in cells; however, the activity of the proteasome is reduced in these disease states, contributing to the accumulation of toxic protein. It has been hypothesized that proteasome activity, both ubiquitin-dependent and -independent, can be chemically stimulated to reduce the load of protein in diseased cells. Several methods exist to identify and characterize stimulators of proteasome activity. In this review, we detail the ways in which protease activity can be enhanced and analyze the biochemical and cellular methods of identifying stimulators of both the ubiquitin-dependent and -independent proteasome activities.


Assuntos
Descoberta de Drogas , Complexo de Endopeptidases do Proteassoma/metabolismo , Animais , Ativação Enzimática/efeitos dos fármacos , Ensaios de Triagem em Larga Escala , Humanos , Complexo de Endopeptidases do Proteassoma/química , Bibliotecas de Moléculas Pequenas , Relação Estrutura-Atividade , Especificidade por Substrato
5.
Methods Mol Biol ; 1988: 15-29, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31147929

RESUMO

Mass spectrometry (MS) is today one of the most important analytical techniques in biosciences. The development of electro spray ionization (ESI) as a gentle method, in which molecules are not destroyed, has revolutionized the analytic of peptides. MS is an ideal technique for detection and analysis of peptides generated by purified 20S proteasomes in in vitro experiments. This approach also provides a convenient and sensitive way to monitor the different processing characteristics of proteasome isoforms. The combination of high performance liquid chromatography (HPLC) with ESI-MS allows for the analysis of complex samples with separation in their specific constituents by LC and their subsequent detection by MS.


Assuntos
Antígenos/análise , Espectrometria de Massas/métodos , Peptídeos/análise , Complexo de Endopeptidases do Proteassoma/química , Sequência de Aminoácidos , Animais , Cromatografia Líquida de Alta Pressão , Análise de Dados , Humanos , Peptídeos/química , Espectrometria de Massas por Ionização por Electrospray
6.
Biochim Biophys Acta Proteins Proteom ; 1867(9): 840-853, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31228587

RESUMO

Multiple complexes of 20S proteasomes with accessory factors play an essential role in proteolysis in eukaryotic cells. In this report, several forms of 20S proteasomes from extracts of Spodoptera frugiperda (Sf9) cells were separated using electrophoresis in a native polyacrylamide gel and examined for proteolytic activity in the gel and by Western blotting. Distinct proteasome bands isolated from the gel were subjected to liquid chromatography-tandem mass spectrometry and identified as free core particles (CP) and complexes of CP with one or two dimers of assembly chaperones PAC1-PAC2 and activators PA28γ or PA200. In contrast to the activators PA28γ and PA200 that regulate the access of protein substrates to the internal proteolytic chamber of CP in an ATP-independent manner, the 19S regulatory particle (RP) in 26S proteasomes performs stepwise substrate unfolding and opens the chamber gate in an ATP-dependent manner. Electron microscopic analysis suggested that spontaneous dissociation of RP in isolated 26S proteasomes leaves CPs with different gate sizes related presumably to different stages in the gate opening. The primary structure of 20S proteasome subunits in Sf9 cells was determined by a search of databases and by sequencing. The protein sequences were confirmed by mass spectrometry and verified by 2D gel electrophoresis. The relative rates of sequence divergence in the evolution of 20S proteasome subunits, the assembly chaperones and activators were determined by using bioinformatics. The data confirmed the conservation of regular CP subunits and PA28γ, a more accelerated evolution of PAC2 and PA200, and especially high divergence rates of PAC1.


Assuntos
Proteínas de Insetos/química , Chaperonas Moleculares/química , Complexo de Endopeptidases do Proteassoma/química , Spodoptera/enzimologia , Animais , Cromatografia Líquida , Proteínas de Insetos/isolamento & purificação , Espectrometria de Massas , Chaperonas Moleculares/isolamento & purificação , Complexo de Endopeptidases do Proteassoma/isolamento & purificação
7.
Int J Mol Sci ; 20(9)2019 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-31067643

RESUMO

The 26S proteasome is critical for the selective degradation of proteins in eukaryotic cells. This enzyme complex is composed of approximately 70 subunits, including the structurally homologous proteins α1-α7, which combine to form heptameric rings. The correct arrangement of these α subunits is essential for the function of the proteasome, but their assembly does not occur autonomously. Assembly of the α subunit is assisted by several chaperones, including the PAC3-PAC4 heterodimer. In this study we showed that the PAC3-PAC4 heterodimer functions as a molecular matchmaker, stabilizing the α4-α5-α6 subcomplex during the assembly of the α-ring. We solved a 0.96-Å atomic resolution crystal structure for a PAC3 homodimer which, in conjunction with nuclear magnetic resonance (NMR) data, highlighted the mobility of the loop comprised of residues 51 to 61. Based on these structural and dynamic data, we created a three-dimensional model of the PAC3-4/α4/α5/α6 quintet complex, and used this model to investigate the molecular and structural basis of the mechanism of proteasome α subunit assembly, as mediated by the PAC3-PAC4 heterodimeric chaperone. Our results provide a potential basis for the development of selective inhibitors against proteasome biogenesis.


Assuntos
Chaperonas Moleculares/química , Simulação de Acoplamento Molecular , Complexo de Endopeptidases do Proteassoma/química , Multimerização Proteica , Humanos , Chaperonas Moleculares/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo
8.
Nature ; 567(7747): 187-193, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30814737

RESUMO

Dysregulation of lipid homeostasis is a precipitating event in the pathogenesis and progression of hepatosteatosis and metabolic syndrome. These conditions are highly prevalent in developed societies and currently have limited options for diagnostic and therapeutic intervention. Here, using a proteomic and lipidomic-wide systems genetic approach, we interrogated lipid regulatory networks in 107 genetically distinct mouse strains to reveal key insights into the control and network structure of mammalian lipid metabolism. These include the identification of plasma lipid signatures that predict pathological lipid abundance in the liver of mice and humans, defining subcellular localization and functionality of lipid-related proteins, and revealing functional protein and genetic variants that are predicted to modulate lipid abundance. Trans-omic analyses using these datasets facilitated the identification and validation of PSMD9 as a previously unknown lipid regulatory protein. Collectively, our study serves as a rich resource for probing mammalian lipid metabolism and provides opportunities for the discovery of therapeutic agents and biomarkers in the setting of hepatic lipotoxicity.


Assuntos
Metabolismo dos Lipídeos/genética , Lipídeos/análise , Lipídeos/genética , Proteômica , Animais , Células HEK293 , Humanos , Metabolismo dos Lipídeos/fisiologia , Lipídeos/sangue , Lipídeos/classificação , Fígado/química , Fígado/metabolismo , Fígado/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Obesidade/genética , Obesidade/metabolismo , Complexo de Endopeptidases do Proteassoma/química , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo
9.
J Biol Chem ; 294(19): 7740-7754, 2019 05 10.
Artigo em Inglês | MEDLINE | ID: mdl-30914481

RESUMO

An efficient immunosurveillance of CD8+ T cells in the periphery depends on positive/negative selection of thymocytes and thus on the dynamics of antigen degradation and epitope production by thymoproteasome and immunoproteasome in the thymus. Although studies in mouse systems have shown how thymoproteasome activity differs from that of immunoproteasome and strongly impacts the T cell repertoire, the proteolytic dynamics and the regulation of human thymoproteasome are unknown. By combining biochemical and computational modeling approaches, we show here that human 20S thymoproteasome and immunoproteasome differ not only in the proteolytic activity of the catalytic sites but also in the peptide transport. These differences impinge upon the quantity of peptide products rather than where the substrates are cleaved. The comparison of the two human 20S proteasome isoforms depicts different processing of antigens that are associated to tumors and autoimmune diseases.


Assuntos
Apresentação do Antígeno , Linfócitos T CD8-Positivos/enzimologia , Simulação por Computador , Complexo de Endopeptidases do Proteassoma/química , Células A549 , Animais , Linfócitos T CD8-Positivos/imunologia , Catálise , Células HeLa , Células Endoteliais da Veia Umbilical Humana , Humanos , Camundongos , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/imunologia , Células THP-1
10.
ChemMedChem ; 14(8): 842-852, 2019 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-30829448

RESUMO

The development of immunoproteasome-selective inhibitors is a promising strategy for treating hematologic malignancies, autoimmune and inflammatory diseases. In this context, we report the design, synthesis, and biological evaluation of a new series of amide derivatives as immunoproteasome inhibitors. Notably, the designed compounds act as noncovalent inhibitors, which might be a promising therapeutic option because of the lack of drawbacks and side effects associated with irreversible inhibition. Among the synthesized compounds, we identified a panel of active inhibitors with Ki values in the low micromolar or sub-micromolar ranges toward the ß5i and/or ß1i subunits of immunoproteasomes. One of the active compounds was shown to be the most potent and selective inhibitor with a Ki value of 21 nm against the single ß1i subunit. Docking studies allowed us to determine the mode of binding of the molecules in the catalytic site of immunoproteasome subunits.


Assuntos
Amidas/química , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma/química , Amidas/síntese química , Amidas/metabolismo , Doenças Autoimunes/imunologia , Doenças Autoimunes/metabolismo , Doenças Autoimunes/patologia , Sítios de Ligação , Domínio Catalítico , Humanos , Simulação de Acoplamento Molecular , Complexo de Endopeptidases do Proteassoma/química , Inibidores de Proteassoma/síntese química , Inibidores de Proteassoma/metabolismo , Subunidades Proteicas/antagonistas & inibidores , Subunidades Proteicas/metabolismo , Relação Estrutura-Atividade
11.
Nat Methods ; 16(4): 333-340, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30858598

RESUMO

Atomic-level information about the structure and dynamics of biomolecules is critical for an understanding of their function. Nuclear magnetic resonance (NMR) spectroscopy provides unique insights into the dynamic nature of biomolecules and their interactions, capturing transient conformers and their features. However, relaxation-induced line broadening and signal overlap make it challenging to apply NMR spectroscopy to large biological systems. Here we took advantage of the high sensitivity and broad chemical shift range of 19F nuclei and leveraged the remarkable relaxation properties of the aromatic 19F-13C spin pair to disperse 19F resonances in a two-dimensional transverse relaxation-optimized spectroscopy spectrum. We demonstrate the application of 19F-13C transverse relaxation-optimized spectroscopy to investigate proteins and nucleic acids. This experiment expands the scope of 19F NMR in the study of the structure, dynamics, and function of large and complex biological systems and provides a powerful background-free NMR probe.


Assuntos
Isótopos de Carbono/química , Ressonância Magnética Nuclear Biomolecular/instrumentação , Ressonância Magnética Nuclear Biomolecular/métodos , Ácidos Nucleicos/química , Proteínas/química , DNA/química , Escherichia coli/metabolismo , Flúor/química , Fluoruracila/química , Campos Magnéticos , Peso Molecular , Mutagênese Sítio-Dirigida , Complexo de Endopeptidases do Proteassoma/química , Thermoplasma/metabolismo
12.
Org Biomol Chem ; 17(10): 2734-2746, 2019 03 06.
Artigo em Inglês | MEDLINE | ID: mdl-30778435

RESUMO

Allosteric regulators of clinically important enzymes are gaining popularity as alternatives to competitive inhibitors. This is also the case for the proteasome, a major intracellular protease and a target of anti-cancer drugs. All clinically used proteasome inhibitors bind to the active sites in catalytic chamber and display a competitive mechanism. Unfortunately, inevitable resistance associated with this type of inhibition drives the search for non-competitive agents. The multisubunit and multicatalytic "proteolytic machine" such as the proteasome is occasionally found to be affected by agents with other primary targets. For example the immunosuppressive agent rapamycin has been shown to allosterically inhibit the proteasome albeit at levels far higher than its mTOR related efficacy. As part of an ongoing program to search for novel proteasome-targeting pharmacophores, we identified the binding domain of rapamycin as required for proteasome inhibition even without the macrocyclic context of the parent compound. By subsequent structure-activity relationship studies, we generated a pipecolic ester derivative compound 3 representing a new class of proteasome inhibitors. Compound 3 affects the core proteasome activities and proliferation of cancer cells with low micromolar/high nanomolar efficacy. Molecular modeling, atomic force microscopy imaging and biochemical data suggest that compound 3 binds into one of intersubunit pockets in the proteasomal α ring and destabilizes the α face and the gate. The α face is used as a docking area for proteasome-regulating protein modules and the gate is critical for controlling access to the catalytic chamber. Thus, the pipecolic ester template elicits a new and attractive mechanism for proteasome inhibition distinct from classical competitive drugs.


Assuntos
Ésteres/química , Ácidos Pipecólicos/química , Ácidos Pipecólicos/farmacologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma/química , Inibidores de Proteassoma/farmacologia , Domínio Catalítico , Desenho de Drogas , Concentração Inibidora 50 , Simulação de Acoplamento Molecular , Ácidos Pipecólicos/metabolismo , Complexo de Endopeptidases do Proteassoma/química , Inibidores de Proteassoma/metabolismo
13.
Cell Chem Biol ; 26(3): 340-351.e3, 2019 03 21.
Artigo em Inglês | MEDLINE | ID: mdl-30612952

RESUMO

Proteasome inhibitors (PIs) are a backbone of multiple myeloma (MM) therapy. The proteasome harbors six proteolytically active subunits (ß1, ß2, ß5), while ß5 was identified as rate-limiting and is a primary target of clinically available PIs. The most effective pattern of subunit inhibition provided by these PIs for cytotoxic activity in MM is unknown. A head-to-head comparison of clinically available PIs shows that in the clinically relevant setting only the co-inhibition of ß1 or ß2 with ß5 activity achieves meaningful functional proteasome inhibition and cytotoxicity, while the selective ß2/ß5 inhibition of both constitutive and immunoproteasome is the most cytotoxic. In the long-term setting, selective inhibition of ß5 subunit is sufficient to induce cytotoxicity in PI-sensitive, but not in PI-resistant MM, and the ß5/ß2 co-inhibition is the most cytotoxic in PI-resistant MM. These results give a rational basis for selecting individual PIs for the treatment of MM.


Assuntos
Antineoplásicos/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma/metabolismo , Idoso , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Bortezomib/metabolismo , Bortezomib/farmacologia , Bortezomib/uso terapêutico , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Pessoa de Meia-Idade , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/metabolismo , Mieloma Múltiplo/patologia , Oligopeptídeos/metabolismo , Oligopeptídeos/farmacologia , Oligopeptídeos/uso terapêutico , Complexo de Endopeptidases do Proteassoma/química , Inibidores de Proteassoma/farmacologia , Inibidores de Proteassoma/uso terapêutico , Subunidades Proteicas/antagonistas & inibidores , Subunidades Proteicas/metabolismo , Distribuição Tecidual , Células Tumorais Cultivadas
14.
Methods Mol Biol ; 1915: 149-160, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30617802

RESUMO

As opposed to proteasome-mediated proteolysis that leads to protein degradation, calpain proteases carry out limited proteolytic cleavages of their substrates. The cleavage of some substrates can produce active fragments that perform functions that are different from those performed by the full-length proteins. Therefore, cleavage by calpains can operate as a posttranslational modification and increase the functional diversity of target proteins. Nevertheless, activation of protein function by calpain cleavage is still an understudied area in molecular biology. Identifying and functionally characterizing by products generated by calpain cleavage could lead to the discovery of biomarkers and the identification of novel drug targets for the treatment of human diseases. This chapter contains a workflow designed to experimentally characterize novel calpain substrates, including identification of potential calpain targets via Western blotting, characterization of calpain cleavage sites, and the study of cellular functions played by such cleaved products. We will employ MYC as an example for these experiments.


Assuntos
Calpaína/genética , Proteínas de Membrana/química , Biologia Molecular/métodos , Calpaína/química , Humanos , Proteínas de Membrana/genética , Complexo de Endopeptidases do Proteassoma/química , Complexo de Endopeptidases do Proteassoma/genética , Processamento de Proteína Pós-Traducional/genética , Proteólise , Especificidade por Substrato
15.
Eur J Med Chem ; 164: 602-614, 2019 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-30639896

RESUMO

The potential of specific proteasome inhibitors to act as anti-cancer agents has attracted intensive investigations. The proteasome can be covalently inhibited by epoxyketone derivatives via a two-step reaction. Several computational approaches have been developed to mimic the covalent binding event. Compound 1 composed of a six-membered heterocyclic ring was designed by using covalent docking. With a possible different binding mode from the clinical compound Carfilzomib, it occupied the S5 pocket of 20S proteasome and showed favorable inhibitory activity. Subsequently optimization and evaluation were taken place. Among these compounds, 11h demonstrated extraordinary in vitro inhibitory activity and selectivity, and good in vivo proteasome inhibitory activity, a favorable pharmacokinetic profile and xenograft tumor inhibition. The possible binding pattern of compound 11h against proteasome was further fully explored via calculations, providing a theoretical basis for finding potent proteasome inhibitors.


Assuntos
Compostos Heterocíclicos/farmacologia , Cetonas/farmacologia , Simulação de Acoplamento Molecular , Inibidores de Proteassoma/química , Animais , Antineoplásicos/química , Sítios de Ligação , Compostos Heterocíclicos/química , Xenoenxertos , Humanos , Cetonas/química , Modelos Moleculares , Complexo de Endopeptidases do Proteassoma/química , Complexo de Endopeptidases do Proteassoma/metabolismo
17.
Biochem Biophys Res Commun ; 509(2): 590-595, 2019 02 05.
Artigo em Inglês | MEDLINE | ID: mdl-30602418

RESUMO

The Hepatitis C virus (HCV) core protein plays a crucial role in the development of chronic liver diseases such as chronic hepatitis, cirrhosis, and hepatocellular carcinoma (HCC). Its involvement in these diseases is reportedly abolished by a knockout of the proteasome activator PA28γ gene in transgenic mice, suggesting an interaction between the core protein and the PA28γ-proteasome system. This study found a direct interaction between the N-terminal 1-71 fragment of HCV core protein (Core71) and PA28γ in vitro, and that this interaction was found to enhance PA28γ-20S proteasome complex formation. While 20S proteasome activity was increased by PA28γ, it was significantly reduced by Core71 attachment in a dose-dependent manner. These results suggest that the Core-PA28γ interaction has an important role in regulating 20S proteasome activity and furthers our understanding of the pathogenesis of HCV.


Assuntos
Autoantígenos/metabolismo , Hepacivirus/metabolismo , Hepatite C/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteínas do Core Viral/metabolismo , Autoantígenos/química , Hepacivirus/química , Hepatite C/virologia , Interações Hospedeiro-Patógeno , Humanos , Modelos Moleculares , Complexo de Endopeptidases do Proteassoma/química , Mapas de Interação de Proteínas , Proteínas do Core Viral/química
18.
J Med Chem ; 62(1): 359-370, 2019 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-30452262

RESUMO

Proline- and arginine-rich peptide PR11 is an allosteric inhibitor of 20S proteasome. We modified its sequence inter alia by introducing HbYX, RYX, or RHbX C-terminal extensions (Hb, hydrophobic moiety; R, arginine; Y, tyrosine; X, any residue). Consequently, we were able to improve inhibitory potency or to convert inhibitors into strong activators: the former with an aromatic penultimate Hb residue and the latter with the HbYX motif. The PR peptide activator stimulated 20S proteasome in vitro to efficiently degrade protein substrates, such as α-synuclein and enolase, but also activated proteasome in cultured fibroblasts. The positive and negative PR modulators differently influenced the proteasome conformational dynamics and affected opening of the substrate entry pore. The resolved crystal structure showed PR inhibitor bound far from the active sites, at the proteasome outer face, in the pocket used by natural activators. Our studies indicate the opportunity to tune proteasome activity by allosteric regulators based on PR peptide scaffold.


Assuntos
Peptídeos/química , Complexo de Endopeptidases do Proteassoma/química , Regulação Alostérica , Sequência de Aminoácidos , Arginina/química , Sítios de Ligação , Desenho de Drogas , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Humanos , Peptídeos/síntese química , Peptídeos/metabolismo , Prolina/química , Complexo de Endopeptidases do Proteassoma/metabolismo
19.
Nature ; 565(7737): 49-55, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30479383

RESUMO

The proteasome is an ATP-dependent, 2.5-megadalton molecular machine that is responsible for selective protein degradation in eukaryotic cells. Here we present cryo-electron microscopy structures of the substrate-engaged human proteasome in seven conformational states at 2.8-3.6 Å resolution, captured during breakdown of a polyubiquitylated protein. These structures illuminate a spatiotemporal continuum of dynamic substrate-proteasome interactions from ubiquitin recognition to substrate translocation, during which ATP hydrolysis sequentially navigates through all six ATPases. There are three principal modes of coordinated hydrolysis, featuring hydrolytic events in two oppositely positioned ATPases, in two adjacent ATPases and in one ATPase at a time. These hydrolytic modes regulate deubiquitylation, initiation of translocation and processive unfolding of substrates, respectively. Hydrolysis of ATP powers a hinge-like motion in each ATPase that regulates its substrate interaction. Synchronization of ATP binding, ADP release and ATP hydrolysis in three adjacent ATPases drives rigid-body rotations of substrate-bound ATPases that are propagated unidirectionally in the ATPase ring and unfold the substrate.


Assuntos
Microscopia Crioeletrônica , Complexo de Endopeptidases do Proteassoma/metabolismo , Complexo de Endopeptidases do Proteassoma/ultraestrutura , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Regulação Alostérica , Holoenzimas/química , Holoenzimas/metabolismo , Holoenzimas/ultraestrutura , Humanos , Hidrólise , Modelos Moleculares , Complexo de Endopeptidases do Proteassoma/química , Conformação Proteica , Estrutura Quaternária de Proteína , Desdobramento de Proteína , Especificidade por Substrato , Ubiquitinação
20.
Biomol NMR Assign ; 13(1): 1-4, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30229448

RESUMO

The 26S proteasome is responsible for the selective, ATP-dependent degradation of polyubiquitinated proteins in eukaryotic cells. It consists of a 20S barrel-shaped core particle capped by two 19S regulatory particle at both ends. The Rpn5 subunit is a non-ATPase subunit located in the lid subcomplex of the 19S regulatory particle and is identified to inhibit the Rpn11 deubiquitinase activity in the isolated lid. The protein contains a C-terminal proteasome-CSN-eIF3 (PCI) domain and an N-terminal α-solenoid domain, the latter has been shown to be highly flexible in the isolated lid and may participate in interactions with different subunits of the proteasome. We herein report the 1H, 13C and 15N atoms chemical shift assignments of the N-terminal domain (residues 1-136) of Saccharomyces cerevisiae Rpn5, which provide the basis for further studies of the structure, dynamics and interactions of the Rpn5 subunit by NMR technique.


Assuntos
Ressonância Magnética Nuclear Biomolecular , Complexo de Endopeptidases do Proteassoma/química , Subunidades Proteicas/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/enzimologia , Isótopos de Carbono , Isótopos de Nitrogênio , Domínios Proteicos , Prótons
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