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1.
J Mol Biol ; 436(11): 168589, 2024 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-38677494

RESUMO

UvrD is a helicase vital for DNA replication and quality control processes. In its monomeric state, UvrD exhibits limited helicase activity, necessitating either dimerization or assistance from an accessory protein to efficiently unwind DNA. Within the DNA mismatch repair pathway, MutL plays a pivotal role in relaying the repair signal, enabling UvrD to unwind DNA from the strand incision site up to and beyond the mismatch. Although this interdependence is well-established, the precise mechanism of activation and the specific MutL-UvrD interactions that trigger helicase activity remain elusive. To address these questions, we employed site-specific crosslinking techniques using single-cysteine variants of MutL and UvrD followed by functional assays. Our investigation unveils that the C-terminal domain of MutL not only engages with UvrD but also acts as a self-sufficient activator of UvrD helicase activity on DNA substrates with 3'-single-stranded tails. Especially when MutL is covalently attached to the 2B or 1B domain the tail length can be reduced to a minimal substrate of 5 nucleotides without affecting unwinding efficiency.


Assuntos
DNA Helicases , Proteínas MutL , DNA/química , DNA Helicases/química , DNA Helicases/genética , Proteínas MutL/química , Proteínas MutL/genética , Ligação Proteica , Domínios Proteicos , Mesilatos/química , Reagentes de Ligações Cruzadas/química
2.
Life Sci Alliance ; 7(4)2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38355287

RESUMO

The ubiquitin-specific protease (USP) family of deubiquitinases (DUBs) controls cellular ubiquitin-dependent signaling events. This generates therapeutic potential, with active-site inhibitors in preclinical and clinical studies. Understanding of the USP active site is primarily guided by USP7 data, where the catalytic triad consists of cysteine, histidine, and a third residue (third critical residue), which polarizes the histidine through a hydrogen bond. A conserved aspartate (fourth critical residue) is directly adjacent to this third critical residue. Although both critical residues accommodate catalysis in USP2, these residues have not been comprehensively investigated in other USPs. Here, we quantitatively investigate their roles in five USPs. Although USP7 relies on the third critical residue for catalysis, this residue is dispensable in USP1, USP15, USP40, and USP48, where the fourth critical residue is vital instead. Furthermore, these residues vary in importance for nucleophilic attack. The diverging catalytic mechanisms of USP1 and USP7 are independent of substrate and retained in cells for USP1. This unexpected variety of catalytic mechanisms in this well-conserved protein family may generate opportunities for selective targeting of individual USPs.


Assuntos
Histidina , Proteases Específicas de Ubiquitina , Proteases Específicas de Ubiquitina/genética , Proteases Específicas de Ubiquitina/metabolismo , Peptidase 7 Específica de Ubiquitina/genética , Peptidase 7 Específica de Ubiquitina/metabolismo , Catálise
3.
Nucleic Acids Res ; 51(3): 1173-1188, 2023 02 22.
Artigo em Inglês | MEDLINE | ID: mdl-36715327

RESUMO

The DNA mismatch repair protein MutSα recognizes wrongly incorporated DNA bases and initiates their correction during DNA replication. Dysfunctions in mismatch repair lead to a predisposition to cancer. Here, we study the homozygous mutation V63E in MSH2 that was found in the germline of a patient with suspected constitutional mismatch repair deficiency syndrome who developed colorectal cancer before the age of 30. Characterization of the mutant in mouse models, as well as slippage and repair assays, shows a mildly pathogenic phenotype. Using cryogenic electron microscopy and surface plasmon resonance, we explored the mechanistic effect of this mutation on MutSα function. We discovered that V63E disrupts a previously unappreciated interface between the mismatch binding domains (MBDs) of MSH2 and MSH6 and leads to reduced DNA binding. Our research identifies this interface as a 'safety lock' that ensures high-affinity DNA binding to increase replication fidelity. Our mechanistic model explains the hypomorphic phenotype of the V63E patient mutation and other variants in the MBD interface.


Assuntos
Reparo de Erro de Pareamento de DNA , Reparo do DNA , Proteína 2 Homóloga a MutS , Animais , Camundongos , DNA/química , Mutação , Proteína 2 Homóloga a MutS/metabolismo
4.
J Struct Biol ; 214(3): 107862, 2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-35605756

RESUMO

Ubiquitin specific protease USP15 is a deubiquitinating enzyme reported to regulate several biological and cellular processes, including TGF-ß signaling, regulation of immune response, neuro-inflammation and mRNA splicing. Here we study the USP15 D1D2 catalytic domain and present the crystal structure in its catalytically-competent conformation. We compare this apo-structure to a previous misaligned state in the same crystal lattice. In both structures, mitoxantrone, an FDA approved antineoplastic drug and a weak inhibitor of USP15 is bound, indicating that it is not responsible for inducing a switch in the conformation of active site cysteine in the USP15 D1D2 structure. Instead, mitoxantrone contributes to crystal packing, by forming a stack of 12 mitoxantrone molecules. We believe this reflects how mitoxantrone can be responsible for e.g. nuclear condensate partitioning. We conclude that USP15 can switch between active and inactive states in the absence of ubiquitin, and that this is independent of mitoxantrone binding. These insights can be important for future drug discovery targeting USP15.


Assuntos
Mitoxantrona , Proteases Específicas de Ubiquitina , Domínio Catalítico , Ligação Proteica , Ubiquitina/metabolismo , Proteases Específicas de Ubiquitina/química , Proteases Específicas de Ubiquitina/genética , Proteases Específicas de Ubiquitina/metabolismo
5.
PLoS One ; 16(9): e0257688, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34591877

RESUMO

BRCA1-associated protein 1 (BAP1) is a tumor suppressor and its loss can result in mesothelioma, uveal and cutaneous melanoma, clear cell renal cell carcinoma and bladder cancer. BAP1 is a deubiquitinating enzyme of the UCH class that has been implicated in various cellular processes like cell growth, cell cycle progression, ferroptosis, DNA damage response and ER metabolic stress response. ASXL proteins activate BAP1 by forming the polycomb repressive deubiquitinase (PR-DUB) complex which acts on H2AK119ub1. Besides the ASXL proteins, BAP1 is known to interact with an established set of additional proteins. Here, we identify novel BAP1 interacting proteins in the cytoplasm by expressing GFP-tagged BAP1 in an endogenous BAP1 deficient cell line using affinity purification followed by mass spectrometry (AP-MS) analysis. Among these novel interacting proteins are Histone acetyltransferase 1 (HAT1) and all subunits of the heptameric coat protein complex I (COPI) that is involved in vesicle formation and protein cargo binding and sorting. We validate that the HAT1 and COPI interactions occur at endogenous levels but find that this interaction with COPI is not mediated through the C-terminal KxKxx cargo sorting signals of the COPI complex.


Assuntos
Complexo I de Proteína do Envoltório/metabolismo , Citoplasma/metabolismo , Histona Acetiltransferases/metabolismo , Neoplasias/metabolismo , Proteômica/métodos , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina Tiolesterase/genética , Ubiquitina Tiolesterase/metabolismo , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Técnicas de Inativação de Genes , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Espectrometria de Massas , Mutação , Proteínas Recombinantes/metabolismo
6.
Cell Chem Biol ; 28(2): 191-201.e8, 2021 02 18.
Artigo em Inglês | MEDLINE | ID: mdl-33238157

RESUMO

Functional analysis of lysine 27-linked ubiquitin chains (K27Ub) is difficult due to the inability to make them through enzymatic methods and due to a lack of model tools and substrates. Here we generate a series of ubiquitin (Ub) tools to study how the deubiquitinase UCHL3 responds to K27Ub chains in comparison to lysine 63-linked chains and mono-Ub. From a crystal structure of a complex between UCHL3 and synthetic K27Ub2, we unexpectedly discover that free K27Ub2 and K27Ub2-conjugated substrates are natural inhibitors of UCHL3. Using our Ub tools to profile UCHL3's activity, we generate a quantitative kinetic model of the inhibitory mechanism and we find that K27Ub2 can inhibit UCHL3 covalently, by binding to its catalytic cysteine, and allosterically, by locking its catalytic loop tightly in place. Based on this inhibition mechanism, we propose that UCHL3 and K27Ub chains likely sense and regulate each other in cells.


Assuntos
Ubiquitina Tiolesterase/metabolismo , Ubiquitinas/metabolismo , Regulação Alostérica , Cristalografia por Raios X , Humanos , Cinética , Modelos Moleculares , Conformação Proteica , Especificidade por Substrato , Ubiquitina Tiolesterase/antagonistas & inibidores , Ubiquitina Tiolesterase/química , Ubiquitinação , Ubiquitinas/química
7.
Nat Commun ; 10(1): 231, 2019 01 16.
Artigo em Inglês | MEDLINE | ID: mdl-30651545

RESUMO

USP7 is a highly abundant deubiquitinating enzyme (DUB), involved in cellular processes including DNA damage response and apoptosis. USP7 has an unusual catalytic mechanism, where the low intrinsic activity of the catalytic domain (CD) increases when the C-terminal Ubl domains (Ubl45) fold onto the CD, allowing binding of the activating C-terminal tail near the catalytic site. Here we delineate how the target protein promotes the activation of USP7. Using NMR analysis and biochemistry we describe the order of activation steps, showing that ubiquitin binding is an instrumental step in USP7 activation. Using chemically synthesised p53-peptides we also demonstrate how the correct ubiquitinated substrate increases catalytic activity. We then used transient reaction kinetic modelling to define how the USP7 multistep mechanism is driven by target recognition. Our data show how this pleiotropic DUB can gain specificity for its cellular targets.


Assuntos
Processamento de Proteína Pós-Traducional , Peptidase 7 Específica de Ubiquitina/metabolismo , Ubiquitina/metabolismo , Isótopos de Carbono/química , Domínio Catalítico/genética , Ensaios Enzimáticos/métodos , Cinética , Modelos Químicos , Mutagênese Sítio-Dirigida , Isótopos de Nitrogênio/química , Ressonância Magnética Nuclear Biomolecular/métodos , Peptídeos/química , Peptídeos/metabolismo , Ligação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Ressonância de Plasmônio de Superfície , Proteína Supressora de Tumor p53/química , Ubiquitina/química , Peptidase 7 Específica de Ubiquitina/química , Peptidase 7 Específica de Ubiquitina/genética , Peptidase 7 Específica de Ubiquitina/isolamento & purificação
8.
EMBO Rep ; 19(10)2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30150323

RESUMO

A common strategy for exploring the biological roles of deubiquitinating enzymes (DUBs) in different pathways is to study the effects of replacing the wild-type DUB with a catalytically inactive mutant in cells. We report here that a commonly studied DUB mutation, in which the catalytic cysteine is replaced with alanine, can dramatically increase the affinity of some DUBs for ubiquitin. Overexpression of these tight-binding mutants thus has the potential to sequester cellular pools of monoubiquitin and ubiquitin chains. As a result, cells expressing these mutants may display unpredictable dominant negative physiological effects that are not related to loss of DUB activity. The structure of the SAGA DUB module bound to free ubiquitin reveals the structural basis for the 30-fold higher affinity of Ubp8C146A for ubiquitin. We show that an alternative option, substituting the active site cysteine with arginine, can inactivate DUBs while also decreasing the affinity for ubiquitin.


Assuntos
Enzimas Desubiquitinantes/genética , Endopeptidases/genética , Proteínas de Saccharomyces cerevisiae/genética , Transativadores/genética , Proteases Específicas de Ubiquitina/genética , Alanina/genética , Substituição de Aminoácidos/genética , Proteínas de Transporte/química , Proteínas de Transporte/genética , Catálise , Cisteína/genética , Enzimas Desubiquitinantes/química , Endopeptidases/química , Humanos , Mutação/genética , Conformação Proteica , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Transativadores/química , Ubiquitina/química , Ubiquitina/genética , Proteases Específicas de Ubiquitina/química , Ubiquitinação/genética
9.
Mol Cell ; 70(1): 165-174.e6, 2018 04 05.
Artigo em Inglês | MEDLINE | ID: mdl-29576528

RESUMO

Deubiquitylating enzymes (DUBs) enhance the dynamics of the versatile ubiquitin (Ub) code by reversing and regulating cellular ubiquitylation processes at multiple levels. Here we discovered that the uncharacterized human protein ZUFSP (zinc finger with UFM1-specific peptidase domain protein/C6orf113/ZUP1), which has been annotated as a potentially inactive UFM1 protease, and its fission yeast homolog Mug105 define a previously unrecognized class of evolutionarily conserved cysteine protease DUBs. Human ZUFSP selectively interacts with and cleaves long K63-linked poly-Ub chains by means of tandem Ub-binding domains, whereas it displays poor activity toward mono- or di-Ub substrates. In cells, ZUFSP is recruited to and regulates K63-Ub conjugates at genotoxic stress sites, promoting chromosome stability upon replication stress in a manner dependent on its catalytic activity. Our findings establish ZUFSP as a new type of linkage-selective cysteine peptidase DUB with a role in genome maintenance pathways.


Assuntos
Neoplasias Ósseas/enzimologia , Dano ao DNA , Enzimas Desubiquitinantes/metabolismo , Instabilidade Genômica , Osteossarcoma/enzimologia , Poliubiquitina/metabolismo , Epitélio Pigmentado da Retina/enzimologia , Sítios de Ligação , Neoplasias Ósseas/genética , Linhagem Celular Tumoral , Enzimas Desubiquitinantes/genética , Células HEK293 , Humanos , Lisina , Osteossarcoma/genética , Poliubiquitina/genética , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Especificidade por Substrato , Ubiquitinação
10.
Nat Commun ; 9(1): 229, 2018 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-29335415

RESUMO

BRCA1-BARD1-catalyzed ubiquitination of histone H2A is an important regulator of the DNA damage response, priming chromatin for repair by homologous recombination. However, no specific deubiquitinating enzymes (DUBs) are known to antagonize this function. Here we identify ubiquitin specific protease-48 (USP48) as a H2A DUB, specific for the C-terminal BRCA1 ubiquitination site. Detailed biochemical analysis shows that an auxiliary ubiquitin, an additional ubiquitin that itself does not get cleaved, modulates USP48 activity, which has possible implications for its regulation in vivo. In cells we reveal that USP48 antagonizes BRCA1 E3 ligase function and in BRCA1-proficient cells loss of USP48 results in positioning 53BP1 further from the break site and in extended resection lengths. USP48 repression confers a survival benefit to cells treated with camptothecin and its activity acts to restrain gene conversion and mutagenic single-strand annealing. We propose that USP48 promotes genome stability by antagonizing BRCA1 E3 ligase function.


Assuntos
Proteína BRCA1/metabolismo , Histonas/metabolismo , Proteases Específicas de Ubiquitina/metabolismo , Ubiquitina/metabolismo , Animais , Proteína BRCA1/genética , Sequência de Bases , Linhagem Celular Tumoral , Células Cultivadas , Reparo do DNA , Células HeLa , Humanos , Cinética , Camundongos Knockout , Interferência de RNA , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Proteases Específicas de Ubiquitina/genética , Ubiquitinação
11.
Methods Enzymol ; 592: 77-101, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28668131

RESUMO

DNA mismatch repair (MMR) is necessary to prevent incorporation of polymerase errors into the newly synthesized DNA strand, as they would be mutagenic. In humans, errors in MMR cause a predisposition to cancer, called Lynch syndrome. The MMR process is performed by a set of ATPases that transmit, validate, and couple information to identify which DNA strand requires repair. To understand the individual steps in the repair process, it is useful to be able to study these large molecular machines structurally and functionally. However, the steps and states are highly transient; therefore, the methods to capture and enrich them are essential. Here, we describe how single-cysteine variants can be used for specific cross-linking and labeling approaches that allow trapping of relevant transient states. Analysis of these defined states in functional and structural studies is instrumental to elucidate the molecular mechanism of this important DNA MMR process.


Assuntos
Reagentes de Ligações Cruzadas/química , Cisteína/química , Reparo de Erro de Pareamento de DNA , Proteínas de Escherichia coli/química , Escherichia coli/química , Transferência Ressonante de Energia de Fluorescência/métodos , Proteína MutS de Ligação de DNA com Erro de Pareamento/química , Cisteína/genética , Cisteína/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Humanos , Modelos Moleculares , Proteína MutS de Ligação de DNA com Erro de Pareamento/genética , Proteína MutS de Ligação de DNA com Erro de Pareamento/metabolismo , Mutação Puntual , Conformação Proteica
12.
DNA Repair (Amst) ; 56: 92-101, 2017 08.
Artigo em Inglês | MEDLINE | ID: mdl-28624371

RESUMO

DNA double strand breaks need to be repaired in an organized fashion to preserve genomic integrity. In the organization of faithful repair, histone ubiquitination plays a crucial role. Recent findings suggest an integrated model for DNA repair regulation through site-specific histone ubiquitination and crosstalk to other posttranslational modifications. Here we discuss how site-specific histone ubiquitination is achieved on a molecular level and how different multi-protein complexes work together to integrate different histone ubiquitination states. We propose a model where site-specific H2A ubiquitination organizes the spatio-temporal recruitment of DNA repair factors which will ultimately contribute to DNA repair pathway choice between homologous recombination and non-homologous end joining.


Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , Histonas/metabolismo , Reparo de DNA por Recombinação , Animais , DNA/metabolismo , Histonas/química , Humanos , Transdução de Sinais , Ubiquitinação
13.
EMBO J ; 36(11): 1623-1639, 2017 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-28468752

RESUMO

SMAD4 is a common intracellular effector for TGF-ß family cytokines, but the mechanism by which its activity is dynamically regulated is unclear. We demonstrated that ubiquitin-specific protease (USP) 4 strongly induces activin/BMP signaling by removing the inhibitory monoubiquitination from SMAD4. This modification was triggered by the recruitment of the E3 ligase, SMURF2, to SMAD4 following ligand-induced regulatory (R)-SMAD-SMAD4 complex formation. Whereas the interaction of the negative regulator c-SKI inhibits SMAD4 monoubiquitination, the ligand stimulates the recruitment of SMURF2 to the c-SKI-SMAD2 complex and triggers c-SKI ubiquitination and degradation. Thus, SMURF2 has a role in termination and initiation of TGF-ß family signaling. An increase in monoubiquitinated SMAD4 in USP4-depleted mouse embryonic stem cells (mESCs) decreased both the BMP- and activin-induced changes in the embryonic stem cell fate. USP4 sustained SMAD4 activity during activin- and BMP-mediated morphogenic events in early zebrafish embryos. Moreover, zebrafish depleted of USP4 exhibited defective cell migration and slower coordinated cell movement known as epiboly, both of which could be rescued by SMAD4. Therefore, USP4 is a critical determinant of SMAD4 activity.


Assuntos
Receptores de Proteínas Morfogenéticas Ósseas/metabolismo , Subunidades beta de Inibinas/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Proto-Oncogênicas/metabolismo , Transdução de Sinais , Proteína Smad4/metabolismo , Ubiquitina Tiolesterase/metabolismo , Ubiquitinação , Animais , Diferenciação Celular , Movimento Celular , Células Cultivadas , Humanos , Camundongos , Células-Tronco Embrionárias Murinas/fisiologia , Ubiquitina-Proteína Ligases/metabolismo , Proteases Específicas de Ubiquitina , Peixe-Zebra/embriologia
14.
Nat Commun ; 7: 10292, 2016 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-26739236

RESUMO

The deubiquitinating enzyme BAP1 is an important tumor suppressor that has drawn attention in the clinic since its loss leads to a variety of cancers. BAP1 is activated by ASXL1 to deubiquitinate mono-ubiquitinated H2A at K119 in Polycomb gene repression, but the mechanism of this reaction remains poorly defined. Here we show that the BAP1 C-terminal extension is important for H2A deubiquitination by auto-recruiting BAP1 to nucleosomes in a process that does not require the nucleosome acidic patch. This initial encounter-like complex is unproductive and needs to be activated by the DEUBAD domains of ASXL1, ASXL2 or ASXL3 to increase BAP1's affinity for ubiquitin on H2A, to drive the deubiquitination reaction. The reaction is specific for Polycomb modifications of H2A as the complex cannot deubiquitinate the DNA damage-dependent ubiquitination at H2A K13/15. Our results contribute to the molecular understanding of this important tumor suppressor.


Assuntos
Proteínas Repressoras/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina Tiolesterase/metabolismo , Sequência de Aminoácidos , Linhagem Celular , Clonagem Molecular , Escherichia coli , Regulação da Expressão Gênica/fisiologia , Histonas/metabolismo , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Proteínas Repressoras/genética , Proteínas Supressoras de Tumor/genética , Ubiquitina Tiolesterase/genética
15.
J Biol Chem ; 291(2): 630-9, 2016 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-26555268

RESUMO

FAT10 conjugation, a post-translational modification analogous to ubiquitination, specifically requires UBA6 and UBE2Z as its activating (E1) and conjugating (E2) enzymes. Interestingly, these enzymes can also function in ubiquitination. We have determined the crystal structure of UBE2Z and report how the different domains of this E2 enzyme are organized. We further combine our structural data with mutational analyses to understand how specificity is achieved in the FAT10 conjugation pathway. We show that specificity toward UBA6 and UBE2Z lies within the C-terminal CYCI tetrapeptide in FAT10. We also demonstrate that this motif slows down transfer rates for FAT10 from UBA6 onto UBE2Z.


Assuntos
Enzimas de Conjugação de Ubiquitina/química , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitinas/metabolismo , Sequência de Aminoácidos , Cristalografia por Raios X , Cinética , Dados de Sequência Molecular , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Peptídeos/metabolismo , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Especificidade por Substrato , Enzimas Ativadoras de Ubiquitina/metabolismo
16.
Elife ; 4: e06744, 2015 Jul 11.
Artigo em Inglês | MEDLINE | ID: mdl-26163658

RESUMO

To avoid mutations in the genome, DNA replication is generally followed by DNA mismatch repair (MMR). MMR starts when a MutS homolog recognizes a mismatch and undergoes an ATP-dependent transformation to an elusive sliding clamp state. How this transient state promotes MutL homolog recruitment and activation of repair is unclear. Here we present a crystal structure of the MutS/MutL complex using a site-specifically crosslinked complex and examine how large conformational changes lead to activation of MutL. The structure captures MutS in the sliding clamp conformation, where tilting of the MutS subunits across each other pushes DNA into a new channel, and reorientation of the connector domain creates an interface for MutL with both MutS subunits. Our work explains how the sliding clamp promotes loading of MutL onto DNA, to activate downstream effectors. We thus elucidate a crucial mechanism that ensures that MMR is initiated only after detection of a DNA mismatch.


Assuntos
Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , DNA/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Proteína MutS de Ligação de DNA com Erro de Pareamento/química , Proteína MutS de Ligação de DNA com Erro de Pareamento/metabolismo , Cristalografia por Raios X , Modelos Moleculares , Proteínas MutL , Ligação Proteica , Conformação Proteica
17.
Nat Commun ; 5: 5399, 2014 Nov 18.
Artigo em Inglês | MEDLINE | ID: mdl-25404403

RESUMO

Ubiquitin-specific protease USP4 is emerging as an important regulator of cellular pathways, including the TGF-ß response, NF-κB signalling and splicing, with possible roles in cancer. Here we show that USP4 has its catalytic triad arranged in a productive conformation. Nevertheless, it requires its N-terminal DUSP-Ubl domain to achieve full catalytic turnover. Pre-steady-state kinetics measurements reveal that USP4 catalytic domain activity is strongly inhibited by slow dissociation of ubiquitin after substrate hydrolysis. The DUSP-Ubl domain is able to enhance ubiquitin dissociation, hence promoting efficient turnover. In a mechanism that requires all USP4 domains, binding of the DUSP-Ubl domain promotes a change of a switching loop near the active site. This 'allosteric regulation of product discharge' provides a novel way of regulating deubiquitinating enzymes that may have relevance for other enzyme classes.


Assuntos
Ubiquitina Tiolesterase/química , Ubiquitina Tiolesterase/metabolismo , Ubiquitina/metabolismo , Regulação Alostérica , Catálise , Cristalografia por Raios X , Humanos , Cinética , Modelos Moleculares , Ligação Proteica , Ubiquitina/química , Proteases Específicas de Ubiquitina
18.
EMBO Rep ; 15(2): 142-54, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24469331

RESUMO

The RING-in-between-RING (RBR) E3s are a curious family of ubiquitin E3-ligases, whose mechanism of action is unusual in several ways. Their activities are auto-inhibited, causing a requirement for activation by protein-protein interactions or posttranslational modifications. They catalyse ubiquitin conjugation by a concerted RING/HECT-like mechanism in which the RING1 domain facilitates E2-discharge to directly form a thioester intermediate with a cysteine in RING2. This short-lived, HECT-like intermediate then modifies the target. Uniquely, the RBR ligase HOIP makes use of this mechanism to target the ubiquitin amino-terminus, by presenting the target ubiquitin for modification using its distinctive LDD region.


Assuntos
Ubiquitina-Proteína Ligases/metabolismo , Sequência de Aminoácidos , Animais , Domínio Catalítico , Humanos , Dados de Sequência Molecular , Domínios RING Finger , Ubiquitina-Proteína Ligases/química
19.
Nucleic Acids Res ; 41(17): 8166-81, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23821665

RESUMO

The process of DNA mismatch repair is initiated when MutS recognizes mismatched DNA bases and starts the repair cascade. The Escherichia coli MutS protein exists in an equilibrium between dimers and tetramers, which has compromised biophysical analysis. To uncouple these states, we have generated stable dimers and tetramers, respectively. These proteins allowed kinetic analysis of DNA recognition and structural analysis of the full-length protein by X-ray crystallography and small angle X-ray scattering. Our structural data reveal that the tetramerization domains are flexible with respect to the body of the protein, resulting in mostly extended structures. Tetrameric MutS has a slow dissociation from DNA, which can be due to occasional bending over and binding DNA in its two binding sites. In contrast, the dimer dissociation is faster, primarily dependent on a combination of the type of mismatch and the flanking sequence. In the presence of ATP, we could distinguish two kinetic groups: DNA sequences where MutS forms sliding clamps and those where sliding clamps are not formed efficiently. Interestingly, this inability to undergo a conformational change rather than mismatch affinity is correlated with mismatch repair.


Assuntos
Pareamento Incorreto de Bases , DNA/química , Proteínas de Escherichia coli/química , Proteína MutS de Ligação de DNA com Erro de Pareamento/química , Trifosfato de Adenosina/metabolismo , DNA/metabolismo , Proteínas de Escherichia coli/metabolismo , Modelos Moleculares , Proteína MutS de Ligação de DNA com Erro de Pareamento/metabolismo , Ligação Proteica , Multimerização Proteica , Estrutura Terciária de Proteína
20.
J Biol Chem ; 287(28): 23283-93, 2012 Jul 06.
Artigo em Inglês | MEDLINE | ID: mdl-22553201

RESUMO

Smoking cessation is an important aim in public health worldwide as tobacco smoking causes many preventable deaths. Addiction to tobacco smoking results from the binding of nicotine to nicotinic acetylcholine receptors (nAChRs) in the brain, in particular the α4ß2 receptor. One way to aid smoking cessation is by the use of nicotine replacement therapies or partial nAChR agonists like cytisine or varenicline. Here we present the co-crystal structures of cytisine and varenicline in complex with Aplysia californica acetylcholine-binding protein and use these as models to investigate binding of these ligands binding to nAChRs. This analysis of the binding properties of these two partial agonists provides insight into differences with nicotine binding to nAChRs. A mutational analysis reveals that the residues conveying subtype selectivity in nAChRs reside on the binding site complementary face and include features extending beyond the first shell of contacting residues.


Assuntos
Alcaloides/química , Benzazepinas/química , Proteínas de Transporte/química , Quinoxalinas/química , Receptores Nicotínicos/química , Acetilcolina/metabolismo , Alcaloides/metabolismo , Animais , Aplysia/metabolismo , Azocinas/química , Azocinas/metabolismo , Benzazepinas/metabolismo , Sítios de Ligação/genética , Ligação Competitiva , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Linhagem Celular , Células HEK293 , Humanos , Cinética , Ligantes , Modelos Moleculares , Mutação , Nicotina/química , Nicotina/metabolismo , Agonistas Nicotínicos/química , Agonistas Nicotínicos/metabolismo , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Quinolizinas/química , Quinolizinas/metabolismo , Quinoxalinas/metabolismo , Receptores Nicotínicos/genética , Receptores Nicotínicos/metabolismo , Abandono do Hábito de Fumar , Vareniclina , Receptor Nicotínico de Acetilcolina alfa7
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