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1.
bioRxiv ; 2024 Jul 13.
Artigo em Inglês | MEDLINE | ID: mdl-39026748

RESUMO

Targeted protein degradation (TPD) modulates protein function beyond inhibition of enzyme activity or protein-protein interactions. Most degraders function by proximity induction, and directly bridge an E3 ligase with the target to be degraded. However, many proteins might not be addressable via proximity-based degraders, and other challenges, such as resistance acquisition, exist. Here, we identified pseudo-natural products derived from (-)-myrtanol, termed iDegs, that inhibit and induce degradation of the immunomodulatory enzyme indoleamine-2,3-dioxygenase 1 (IDO1) by a distinct mechanism. iDegs induce a unique conformational change and, thereby, boost IDO1 ubiquitination and degradation by the cullin-RING E3 ligase CRL2 KLHDC3 , which we identified to also mediate native IDO1 degradation. Therefore, iDegs supercharge the native proteolytic pathway of IDO1, rendering this mechanism of action distinct from traditional degrader approaches involving proteolysis-targeting chimeras (PROTACs) or molecular-glue degraders (MGDs). In contrast to clinically explored IDO1 inhibitors, iDegs reduce formation of kynurenine by both inhibition and induced degradation of the enzyme and should also modulate non-enzymatic functions of IDO1. This unique mechanism of action may open up new therapeutic opportunities for the treatment of cancer beyond classical inhibition of IDO1.

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.
Angew Chem Int Ed Engl ; 63(12): e202318849, 2024 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-38239128

RESUMO

Cyanamides have emerged as privileged scaffolds in covalent inhibitors of deubiquitinating enzymes (DUBs). However, many compounds with a cyanopyrrolidine warhead show cross-reactivity toward small subsets of DUBs or toward the protein deglycase PARK7/DJ-1, hampering their use for the selective perturbation of a single DUB in living cells. Here, we disclose N'-alkyl,N-cyanopiperazines as structures for covalent enzyme inhibition with exceptional specificity for the DUB UCHL1 among 55 human deubiquitinases and with effective target engagement in cells. Notably, transitioning from 5-membered pyrrolidines to 6-membered heterocycles eliminated PARK7 binding and introduced context-dependent reversibility of the isothiourea linkage to the catalytic cysteine of UCHL1. Compound potency and specificity were analysed by a range of biochemical assays and with a crystal structure of a cyanopiperazine in covalent complex with UCHL1. The structure revealed a compound-induced conformational restriction of the cross-over loop, which underlies the observed inhibitory potencies. Through the rationalization of specificities of different cyanamides, we introduce a framework for the investigation of protein reactivity of bioactive nitriles of this compound class. Our results represent an encouraging case study for the refining of electrophilic compounds into chemical probes, emphasizing the potential to engineer specificity through subtle chemical modifications around the warhead.


Assuntos
Inibidores Enzimáticos , Ubiquitina Tiolesterase , Humanos , Inibidores Enzimáticos/farmacologia
4.
J Am Chem Soc ; 145(38): 20801-20812, 2023 09 27.
Artigo em Inglês | MEDLINE | ID: mdl-37712884

RESUMO

Post-translational modifications with ubiquitin (Ub) and ubiquitin-like proteins (Ubls) are regulated by isopeptidases termed deubiquitinases (DUBs) and Ubl proteases. Here, we describe a mild chemical method for the preparation of fluorescence polarization substrates for these enzymes that is based on the activation of C-terminal Ub/Ubl hydrazides to acyl azides and their subsequent functionalization to isopeptides. The procedure is complemented by native purification routes and thus circumvents the previous need for desulfurization and refolding. Its broad applicability was demonstrated by the generation of fully cleavable substrates for Ub, SUMO1, SUMO2, NEDD8, ISG15, and Fubi. We employed these reagents for the investigation of substrate specificities of human UCHL3, USPL1, USP2, USP7, USP16, USP18, and USP36. Pronounced selectivity of USPL1 for SUMO2/3 over SUMO1 was observed, which we rationalize with crystal structures and biochemical assays, revealing a SUMO paralogue specificity mechanism distinct from SENP family deSUMOylases. Moreover, we investigated the recently identified Fubi proteases USP16 and USP36 and found both to act as bona fide deFubiylases, harboring catalytic activity against isopeptide-linked Fubi. Surprisingly, we also noticed the activity of both enzymes toward ISG15, previously not identified in chemoproteomics, which makes USP16 and USP36 the first human DUBs with specific isopeptidase activity toward three distinct modifiers. The methods described here for the preparation of isopeptide-linked, fully folded substrates will aid in the characterization of further DUBs/Ubl proteases. More broadly, our findings highlight possible limitations associated with fluorogenic substrates and Ubl activity-based probes and stress the importance of isopeptide-containing reagents for validating isopeptidase activities and quantifying substrate specificities.


Assuntos
Endopeptidases , Peptídeo Hidrolases , Humanos , Ubiquitina , Azidas , Enzimas Desubiquitinantes , Peptidase 7 Específica de Ubiquitina , Ubiquitina Tiolesterase
5.
Nat Chem Biol ; 19(11): 1394-1405, 2023 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-37443395

RESUMO

Ubiquitin and ubiquitin-like proteins typically use distinct machineries to facilitate diverse functions. The immunosuppressive ubiquitin-like protein Fubi is synthesized as an N-terminal fusion to a ribosomal protein (Fubi-S30). Its proteolytic maturation by the nucleolar deubiquitinase USP36 is strictly required for translationally competent ribosomes. What endows USP36 with this activity, how Fubi is recognized and whether other Fubi proteases exist are unclear. Here, we report a chemical tool kit that facilitated the discovery of dual ubiquitin/Fubi cleavage activity in USP16 in addition to USP36 by chemoproteomics. Crystal structures of USP36 complexed with Fubi and ubiquitin uncover its substrate recognition mechanism and explain how other deubiquitinases are restricted from Fubi. Furthermore, we introduce Fubi C-terminal hydrolase measurements and reveal a synergistic role of USP16 in Fubi-S30 maturation. Our data highlight how ubiquitin/Fubi specificity is achieved in a subset of human deubiquitinases and open the door to a systematic investigation of the Fubi system.


Assuntos
Ubiquitina Tiolesterase , Ubiquitina , Humanos , Ubiquitina/metabolismo , Ubiquitina Tiolesterase/metabolismo , Ubiquitinas/metabolismo , Nucléolo Celular/metabolismo , Endopeptidases/metabolismo
6.
Nat Commun ; 13(1): 5950, 2022 10 10.
Artigo em Inglês | MEDLINE | ID: mdl-36216817

RESUMO

Ubiquitination regulates protein homeostasis and is tightly controlled by deubiquitinases (DUBs). Loss of the DUB UCHL1 leads to neurodegeneration, and its dysregulation promotes cancer metastasis and invasiveness. Small molecule probes for UCHL1 and DUBs in general could help investigate their function, yet specific inhibitors and structural information are rare. Here we report the potent and non-toxic chemogenomic pair of activity-based probes GK13S and GK16S for UCHL1. Biochemical characterization of GK13S demonstrates its stereoselective inhibition of cellular UCHL1. The crystal structure of UCHL1 in complex with GK13S shows the enzyme locked in a hybrid conformation of apo and Ubiquitin-bound states, which underlies its UCHL1-specificity within the UCH DUB family. Phenocopying a reported inactivating mutation of UCHL1 in mice, GK13S, but not GK16S, leads to reduced levels of monoubiquitin in a human glioblastoma cell line. Collectively, we introduce a set of structurally characterized, chemogenomic probes suitable for the cellular investigation of UCHL1.


Assuntos
Ubiquitina Tiolesterase , Ubiquitina , Animais , Humanos , Camundongos , Ubiquitina/metabolismo , Ubiquitina Tiolesterase/metabolismo , Ubiquitinação
7.
Elife ; 102021 10 12.
Artigo em Inglês | MEDLINE | ID: mdl-34636321

RESUMO

Lung squamous cell carcinoma (LSCC) is a considerable global health burden, with an incidence of over 600,000 cases per year. Treatment options are limited, and patient's 5-year survival rate is less than 5%. The ubiquitin-specific protease 28 (USP28) has been implicated in tumourigenesis through its stabilization of the oncoproteins c-MYC, c-JUN, and Δp63. Here, we show that genetic inactivation of Usp28-induced regression of established murine LSCC lung tumours. We developed a small molecule that inhibits USP28 activity in the low nanomole range. While displaying cross-reactivity against the closest homologue USP25, this inhibitor showed a high degree of selectivity over other deubiquitinases. USP28 inhibitor treatment resulted in a dramatic decrease in c-MYC, c-JUN, and Δp63 proteins levels and consequently induced substantial regression of autochthonous murine LSCC tumours and human LSCC xenografts, thereby phenocopying the effect observed by genetic deletion. Thus, USP28 may represent a promising therapeutic target for the treatment of squamous cell lung carcinoma.


Assuntos
Proteínas de Ligação a DNA/genética , Deleção de Genes , Neoplasias Pulmonares/genética , Neoplasias de Células Escamosas/genética , Fatores de Transcrição/genética , Ubiquitina Tiolesterase/genética , Animais , Proteínas de Ligação a DNA/metabolismo , Modelos Animais de Doenças , Humanos , Camundongos , Fatores de Transcrição/metabolismo , Ubiquitina Tiolesterase/metabolismo
8.
Life Sci Alliance ; 3(8)2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32636217

RESUMO

The mitochondrial deubiquitylase USP30 negatively regulates the selective autophagy of damaged mitochondria. We present the characterisation of an N-cyano pyrrolidine compound, FT3967385, with high selectivity for USP30. We demonstrate that ubiquitylation of TOM20, a component of the outer mitochondrial membrane import machinery, represents a robust biomarker for both USP30 loss and inhibition. A proteomics analysis, on a SHSY5Y neuroblastoma cell line model, directly compares the effects of genetic loss of USP30 with chemical inhibition. We have thereby identified a subset of ubiquitylation events consequent to mitochondrial depolarisation that are USP30 sensitive. Within responsive elements of the ubiquitylome, several components of the outer mitochondrial membrane transport (TOM) complex are prominent. Thus, our data support a model whereby USP30 can regulate the availability of ubiquitin at the specific site of mitochondrial PINK1 accumulation following membrane depolarisation. USP30 deubiquitylation of TOM complex components dampens the trigger for the Parkin-dependent amplification of mitochondrial ubiquitylation leading to mitophagy. Accordingly, PINK1 generation of phospho-Ser65 ubiquitin proceeds more rapidly in cells either lacking USP30 or subject to USP30 inhibition.


Assuntos
Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Tioléster Hidrolases/metabolismo , Células HeLa , Humanos , Proteínas de Membrana Transportadoras/metabolismo , Mitocôndrias/fisiologia , Membranas Mitocondriais/fisiologia , Proteínas do Complexo de Importação de Proteína Precursora Mitocondrial , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/fisiologia , Mitofagia/efeitos dos fármacos , Mitofagia/genética , Células-Tronco Neurais/metabolismo , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Receptores de Superfície Celular/metabolismo , Tioléster Hidrolases/fisiologia , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação
9.
Mol Cell ; 74(3): 436-451.e7, 2019 05 02.
Artigo em Inglês | MEDLINE | ID: mdl-30926242

RESUMO

The evolutionarily related deubiquitinating enzymes (DUBs) USP25 and USP28 comprise an identical overall domain architecture but are functionally non-redundant: USP28 stabilizes c-MYC and other nuclear proteins, and USP25 regulates inflammatory TRAF signaling. We here compare molecular features of USP25 and USP28. Active enzymes form distinctively shaped dimers, with a dimerizing insertion spatially separating independently active catalytic domains. In USP25, but not USP28, two dimers can form an autoinhibited tetramer, where a USP25-specific, conserved insertion sequence blocks ubiquitin binding. In full-length enzymes, a C-terminal domain with a previously unknown fold has no impact on oligomerization, but N-terminal regions affect the dimer-tetramer equilibrium in vitro. We confirm oligomeric states of USP25 and USP28 in cells and show that modulating oligomerization affects substrate stabilization in accordance with in vitro activity data. Our work highlights how regions outside of the catalytic domain enable a conceptually intriguing interplay of DUB oligomerization and activity.


Assuntos
Inflamação/genética , Conformação Proteica , Ubiquitina Tiolesterase/genética , Sequência de Aminoácidos/genética , Domínio Catalítico/genética , Enzimas Desubiquitinantes/química , Enzimas Desubiquitinantes/genética , Humanos , Inflamação/patologia , Mutação/genética , Ligação Proteica/genética , Domínios Proteicos/genética , Multimerização Proteica/genética , Proteínas Proto-Oncogênicas c-myb/química , Proteínas Proto-Oncogênicas c-myb/genética , Transdução de Sinais/genética , Especificidade por Substrato , Peptídeos e Proteínas Associados a Receptores de Fatores de Necrose Tumoral/genética , Ubiquitina/genética , Ubiquitina Tiolesterase/química
10.
Angew Chem Int Ed Engl ; 57(44): 14602-14607, 2018 10 26.
Artigo em Inglês | MEDLINE | ID: mdl-30129683

RESUMO

Caseinolytic protease P (ClpP) is the proteolytic component of the ClpXP protein degradation complex. Eukaryotic ClpP was recently found to act within the mitochondria-specific unfolded protein response (UPRmt ). However, its detailed function and dedicated regulation remain largely unexplored. A small molecule (D9) acts as a potent and species-selective activator of human ClpP (hClpP) by mimicking the natural chaperone ClpX. Structure-activity relationship studies highlight the importance of a halogenated benzyl motif within D9 that interacts with a unique aromatic amino acid network in hClpP. Mutational and structural studies suggest that this YYW motif tightly controls hClpP activity and regulates substrate turnover by interaction with cognate ligands. This signature motif is unique to ClpP from higher organisms and does not exist in tested bacterial homologues, allowing a species-selective analysis. Thus, D9 is a versatile tool to analyze mechanistic features of hClpP.


Assuntos
Endopeptidase Clp/metabolismo , Endopeptidase Clp/química , Ativação Enzimática , Humanos , Relação Estrutura-Atividade
11.
Nature ; 550(7677): 481-486, 2017 10 26.
Artigo em Inglês | MEDLINE | ID: mdl-29045389

RESUMO

Ubiquitination controls the stability of most cellular proteins, and its deregulation contributes to human diseases including cancer. Deubiquitinases remove ubiquitin from proteins, and their inhibition can induce the degradation of selected proteins, potentially including otherwise 'undruggable' targets. For example, the inhibition of ubiquitin-specific protease 7 (USP7) results in the degradation of the oncogenic E3 ligase MDM2, and leads to re-activation of the tumour suppressor p53 in various cancers. Here we report that two compounds, FT671 and FT827, inhibit USP7 with high affinity and specificity in vitro and within human cells. Co-crystal structures reveal that both compounds target a dynamic pocket near the catalytic centre of the auto-inhibited apo form of USP7, which differs from other USP deubiquitinases. Consistent with USP7 target engagement in cells, FT671 destabilizes USP7 substrates including MDM2, increases levels of p53, and results in the transcription of p53 target genes, induction of the tumour suppressor p21, and inhibition of tumour growth in mice.


Assuntos
Piperidinas/farmacologia , Pirazóis/farmacologia , Pirimidinas/farmacologia , Peptidase 7 Específica de Ubiquitina/antagonistas & inibidores , Animais , Apoenzimas/antagonistas & inibidores , Apoenzimas/química , Apoenzimas/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cristalografia por Raios X , Feminino , Humanos , Camundongos , Modelos Moleculares , Neoplasias/tratamento farmacológico , Neoplasias/enzimologia , Neoplasias/patologia , Piperidinas/síntese química , Proteínas Proto-Oncogênicas c-mdm2/química , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Pirazóis/síntese química , Pirimidinas/síntese química , Especificidade por Substrato , Transcrição Gênica/efeitos dos fármacos , Proteína Supressora de Tumor p53/metabolismo , Peptidase 7 Específica de Ubiquitina/química , Peptidase 7 Específica de Ubiquitina/metabolismo , Ubiquitinação/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
12.
Nat Struct Mol Biol ; 24(11): 920-930, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-28945249

RESUMO

Damaged mitochondria undergo mitophagy, a specialized form of autophagy that is initiated by the protein kinase PINK1 and the ubiquitin E3 ligase Parkin. Ubiquitin-specific protease USP30 antagonizes Parkin-mediated ubiquitination events on mitochondria and is a key negative regulator of mitophagy. Parkin and USP30 both show a preference for assembly or disassembly, respectively, of Lys6-linked polyubiquitin, a chain type that has not been well studied. Here we report crystal structures of human USP30 bound to monoubiquitin and Lys6-linked diubiquitin, which explain how USP30 achieves Lys6-linkage preference through unique ubiquitin binding interfaces. We assess the interplay between USP30, PINK1 and Parkin and show that distally phosphorylated ubiquitin chains impair USP30 activity. Lys6-linkage-specific affimers identify numerous mitochondrial substrates for this modification, and we show that USP30 regulates Lys6-polyubiquitinated TOM20. Our work provides insights into the architecture, activity and regulation of USP30, which will aid drug design against this and related enzymes.


Assuntos
Enzimas Desubiquitinantes/química , Enzimas Desubiquitinantes/metabolismo , Proteínas Mitocondriais/química , Proteínas Mitocondriais/metabolismo , Tioléster Hidrolases/química , Tioléster Hidrolases/metabolismo , Ubiquitina/química , Ubiquitina/metabolismo , Humanos , Ligação Proteica , Proteínas Quinases/metabolismo , Especificidade por Substrato , Ubiquitina-Proteína Ligases/metabolismo
13.
Chemphyschem ; 18(18): 2422-2425, 2017 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-28719100

RESUMO

Aberrant pH is characteristic of many pathologies such as ischemia, inflammation or cancer. Therefore, a non-invasive and spatially resolved pH determination is valuable for disease diagnosis, characterization of response to treatment and the design of pH-sensitive drug-delivery systems. We recently introduced hyperpolarized [1,5-13 C2 ]zymonic acid (ZA) as a novel MRI probe of extracellular pH utilizing dissolution dynamic polarization (DNP) for a more than 10000-fold signal enhancement of the MRI signal. Here we present a strategy to enhance the sensitivity of this approach by deuteration of ZA yielding [1,5-13 C2 , 3,6,6,6-D4 ]zymonic acid (ZAd ), which prolongs the liquid state spin lattice relaxation time (T1 ) by up to 39 % in vitro. Measurements with ZA and ZAd on subcutaneous MAT B III adenocarcinoma in rats show that deuteration increases the signal-to-noise ratio (SNR) by up to 46 % in vivo. Furthermore, we demonstrate a proof of concept for real-time imaging of dynamic pH changes in vitro using ZAd , potentially allowing for the characterization of rapid acidification/basification processes in vivo.


Assuntos
Adenocarcinoma/diagnóstico por imagem , Imageamento por Ressonância Magnética , Sondas Moleculares/química , Animais , Isótopos de Carbono , Concentração de Íons de Hidrogênio , Teoria Quântica , Ratos
14.
Nat Commun ; 8: 15126, 2017 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-28492229

RESUMO

Natural pH regulatory mechanisms can be overruled during several pathologies such as cancer, inflammation and ischaemia, leading to local pH changes in the human body. Here we demonstrate that 13C-labelled zymonic acid (ZA) can be used as hyperpolarized magnetic resonance pH imaging sensor. ZA is synthesized from [1-13C]pyruvic acid and its 13C resonance frequencies shift up to 3.0 p.p.m. per pH unit in the physiological pH range. The long lifetime of the hyperpolarized signal enhancement enables monitoring of pH, independent of concentration, temperature, ionic strength and protein concentration. We show in vivo pH maps within rat kidneys and subcutaneously inoculated tumours derived from a mammary adenocarcinoma cell line and characterize ZA as non-toxic compound predominantly present in the extracellular space. We suggest that ZA represents a reliable and non-invasive extracellular imaging sensor to localize and quantify pH, with the potential to improve understanding, diagnosis and therapy of diseases characterized by aberrant acid-base balance.


Assuntos
Meios de Contraste/química , Furanos/química , Rim/diagnóstico por imagem , Imageamento por Ressonância Magnética/métodos , Neoplasias Mamárias Animais/diagnóstico por imagem , Bexiga Urinária/diagnóstico por imagem , Animais , Isótopos de Carbono/química , Meios de Contraste/metabolismo , Feminino , Furanos/metabolismo , Células HeLa , Humanos , Concentração de Íons de Hidrogênio , Injeções Subcutâneas , Rim/metabolismo , Rim/patologia , Células MCF-7 , Neoplasias Mamárias Animais/metabolismo , Neoplasias Mamárias Animais/patologia , Ratos , Coloração e Rotulagem/métodos , Bexiga Urinária/metabolismo , Bexiga Urinária/patologia
15.
Chem Sci ; 8(2): 1592-1600, 2017 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-28451288

RESUMO

Caseinolytic proteases (ClpP) are important for recognition and controlled degradation of damaged proteins. While the majority of bacterial organisms utilize only a single ClpP, Listeria monocytogenes expresses two isoforms (LmClpP1 and LmClpP2). LmClpPs assemble into either a LmClpP2 homocomplex or a LmClpP1/2 heterooligomeric complex. The heterocomplex in association with the chaperone ClpX, exhibits a boost in proteolytic activity for unknown reasons. Here, we use a combined chemical and biochemical strategy to unravel two activation principles of LmClpPs. First, determination of apparent affinity constants revealed a 7-fold elevated binding affinity between the LmClpP1/2 heterocomplex and ClpX, compared to homooligomeric LmClpP2. This tighter interaction favors the formation of the proteolytically active complex between LmClpX and LmClpP1/2 and thereby accelerating the overall turnover. Second, screening a diverse library of fluorescent labeled peptides and proteins with various ClpP mutants allowed the individual analysis of substrate preferences for both isoforms within the heterocomplex. In addition to Leu and Met, LmClpP2 preferred a long aliphatic chain (2-Aoc) in the P1 position for cleavage. Strikingly, design and synthesis of a corresponding 2-Aoc chloromethyl ketone inhibitor resulted in stimulation of proteolysis by 160% when LmClpP2 was partially alkylated on 20% of the active sites. Determination of apparent affinity constants also revealed an elevated complex stability between partially modified LmClpP2 and the cognate chaperone LmClpX. Thus, the stimulation of proteolysis through enhanced binding to the chaperone seems to be a characteristic feature of LmClpPs.

16.
Nature ; 538(7625): 402-405, 2016 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-27732584

RESUMO

The post-translational modification of proteins with polyubiquitin regulates virtually all aspects of cell biology. Eight distinct chain linkage types co-exist in polyubiquitin and are independently regulated in cells. This 'ubiquitin code' determines the fate of the modified protein. Deubiquitinating enzymes of the ovarian tumour (OTU) family regulate cellular signalling by targeting distinct linkage types within polyubiquitin, and understanding their mechanisms of linkage specificity gives fundamental insights into the ubiquitin system. Here we reveal how the deubiquitinase Cezanne (also known as OTUD7B) specifically targets Lys11-linked polyubiquitin. Crystal structures of Cezanne alone and in complex with monoubiquitin and Lys11-linked diubiquitin, in combination with hydrogen-deuterium exchange mass spectrometry, enable us to reconstruct the enzymatic cycle in great detail. An intricate mechanism of ubiquitin-assisted conformational changes activates the enzyme, and while all chain types interact with the enzymatic S1 site, only Lys11-linked chains can bind productively across the active site and stimulate catalytic turnover. Our work highlights the plasticity of deubiquitinases and indicates that new conformational states can occur when a true substrate, such as diubiquitin, is bound at the active site.


Assuntos
Enzimas Desubiquitinantes/metabolismo , Endopeptidases/metabolismo , Lisina/metabolismo , Poliubiquitina/metabolismo , Biocatálise , Domínio Catalítico , Cristalografia por Raios X , Enzimas Desubiquitinantes/química , Enzimas Desubiquitinantes/genética , Medição da Troca de Deutério , Endopeptidases/química , Endopeptidases/genética , Ativação Enzimática , Humanos , Espectrometria de Massas , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Especificidade por Substrato , Ubiquitinação , Ubiquitinas/metabolismo
17.
ACS Chem Biol ; 11(2): 389-99, 2016 Feb 19.
Artigo em Inglês | MEDLINE | ID: mdl-26606371

RESUMO

ClpP is a self-compartmentalizing protease with crucial roles in bacterial and mitochondrial protein quality control. Although the ClpP homocomplex is composed of 14 equivalent active sites, it degrades a multitude of substrates to small peptides, demonstrating its capability to carry out diverse cleavage reactions. Here, we show that ClpP proteases from E. coli, S. aureus, and human mitochondria exhibit preferences for certain amino acids in the P1, P2, and P3 positions using a tailored fluorogenic substrate library. However, this high specificity is not retained during proteolysis of endogenous substrates as shown by mass spectrometric analysis of peptides produced in ClpXP-mediated degradation reactions. Our data suggest a mechanism that implicates the barrel-shaped architecture of ClpP not only in shielding the active sites to prevent uncontrolled proteolysis but also in providing high local substrate concentrations to enable efficient proteolytic processing. Furthermore, we introduce customized fluorogenic substrates with unnatural amino acids that greatly surpass the sensitivity of previously used tools. We used these to profile the activity of cancer-patient- and Perrault-syndrome-derived ClpP mutant proteins.


Assuntos
Endopeptidase Clp/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Staphylococcus aureus/enzimologia , Sequência de Aminoácidos , Aminoácidos/química , Aminoácidos/metabolismo , Domínio Catalítico , Endopeptidase Clp/química , Escherichia coli/química , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Humanos , Mitocôndrias/química , Mitocôndrias/enzimologia , Mitocôndrias/metabolismo , Modelos Moleculares , Peptídeos/química , Peptídeos/metabolismo , Conformação Proteica , Staphylococcus aureus/química , Staphylococcus aureus/metabolismo , Especificidade por Substrato
18.
Chem Biol ; 22(3): 404-11, 2015 Mar 19.
Artigo em Inglês | MEDLINE | ID: mdl-25728267

RESUMO

The proteasome is responsible for the majority of protein degradation within eukaryotic cells and proteasome inhibitors have gained blockbuster status as anticancer drugs. Here, we introduce an analytical platform comprising reverse phase chromatography, intact protein mass spectrometry, and customized data analysis that allows a streamlined investigation of proteasome integrity and posttranslational modifications. We report the complete mass spectrometric assignment of all subunits of the yeast core particle, as well as of the human constitutive 20S proteasome and the human immunoproteasome, including phosphorylated isoforms of α7. Importantly, we found several batches of commercially available immunoproteasome to also contain constitutive catalytic subunits. Moreover, we applied the method to study the binding mechanisms of proteasome inhibitors, both validating the approach and providing a direct readout of subunit preferences complementary to biochemical methods. Collectively, our platform facilitates an easy, reliable and comprehensive detection of different types of covalent modifications on multisubunit protein complexes with high accuracy.


Assuntos
Complexo de Endopeptidases do Proteassoma/análise , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma/metabolismo , Processamento de Proteína Pós-Traducional , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Eletroforese em Gel Bidimensional/métodos , Humanos , Modelos Moleculares , Oligopeptídeos/química , Oligopeptídeos/metabolismo , Complexo de Endopeptidases do Proteassoma/química , Inibidores de Proteassoma/química , Ligação Proteica , Subunidades Proteicas , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo
19.
Nat Commun ; 6: 6320, 2015 Feb 19.
Artigo em Inglês | MEDLINE | ID: mdl-25695750

RESUMO

The Clp protease complex degrades a multitude of substrates, which are engaged by a AAA+ chaperone such as ClpX and subsequently digested by the dynamic, barrel-shaped ClpP protease. Acyldepsipeptides (ADEPs) are natural product-derived antibiotics that activate ClpP for chaperone-independent protein digestion. Here we show that both protein and small-molecule activators of ClpP allosterically control the ClpP barrel conformation. We dissect the catalytic mechanism with chemical probes and show that ADEP in addition to opening the axial pore directly stimulates ClpP activity through cooperative binding. ClpP activation thus reaches beyond active site accessibility and also involves conformational control of the catalytic residues. Moreover, we demonstrate that substoichiometric amounts of ADEP potently prevent binding of ClpX to ClpP and, at the same time, partially inhibit ClpP through conformational perturbance. Collectively, our results establish the hydrophobic binding pocket as a major conformational regulatory site with implications for both ClpXP proteolysis and ADEP-based anti-bacterial activity.


Assuntos
Proteínas de Bactérias/química , Depsipeptídeos/química , Endopeptidase Clp/química , Chaperonas Moleculares/química , Sítio Alostérico , Sítios de Ligação , Calorimetria , Catálise , Domínio Catalítico , Cromatografia , Escherichia coli/química , Fluoresceína-5-Isotiocianato , Interações Hidrofóbicas e Hidrofílicas , Cinética , Luz , Listeria monocytogenes/química , Espectrometria de Massas , Mutação , Ligação Proteica , Conformação Proteica , Estrutura Secundária de Proteína , Staphylococcus aureus/química , Especificidade por Substrato
20.
EMBO J ; 34(3): 307-25, 2015 Feb 03.
Artigo em Inglês | MEDLINE | ID: mdl-25527291

RESUMO

The protein kinase PINK1 was recently shown to phosphorylate ubiquitin (Ub) on Ser65, and phosphoUb activates the E3 ligase Parkin allosterically. Here, we show that PINK1 can phosphorylate every Ub in Ub chains. Moreover, Ser65 phosphorylation alters Ub structure, generating two conformations in solution. A crystal structure of the major conformation resembles Ub but has altered surface properties. NMR reveals a second phosphoUb conformation in which ß5-strand slippage retracts the C-terminal tail by two residues into the Ub core. We further show that phosphoUb has no effect on E1-mediated E2 charging but can affect discharging of E2 enzymes to form polyUb chains. Notably, UBE2R1- (CDC34), UBE2N/UBE2V1- (UBC13/UEV1A), TRAF6- and HOIP-mediated chain assembly is inhibited by phosphoUb. While Lys63-linked poly-phosphoUb is recognized by the TAB2 NZF Ub binding domain (UBD), 10 out of 12 deubiquitinases (DUBs), including USP8, USP15 and USP30, are impaired in hydrolyzing phosphoUb chains. Hence, Ub phosphorylation has repercussions for ubiquitination and deubiquitination cascades beyond Parkin activation and may provide an independent layer of regulation in the Ub system.


Assuntos
Fosfoproteínas/metabolismo , Poliubiquitina/metabolismo , Multimerização Proteica/fisiologia , Ubiquitinação/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Regulação Alostérica/fisiologia , Endopeptidases/genética , Endopeptidases/metabolismo , Complexos Endossomais de Distribuição Requeridos para Transporte/genética , Complexos Endossomais de Distribuição Requeridos para Transporte/metabolismo , Humanos , Hidrólise , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Fosfoproteínas/genética , Fosforilação/fisiologia , Poliubiquitina/genética , Estrutura Terciária de Proteína , Serina/genética , Serina/metabolismo , Fator 6 Associado a Receptor de TNF/genética , Fator 6 Associado a Receptor de TNF/metabolismo , Tioléster Hidrolases/genética , Tioléster Hidrolases/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Ubiquitina Tiolesterase/genética , Ubiquitina Tiolesterase/metabolismo , Enzimas de Conjugação de Ubiquitina/genética , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Proteases Específicas de Ubiquitina/genética , Proteases Específicas de Ubiquitina/metabolismo
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