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1.
Nat Commun ; 15(1): 2005, 2024 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-38443338

RESUMO

Antimicrobial resistance is a global health threat that requires the development of new treatment concepts. These should not only overcome existing resistance but be designed to slow down the emergence of new resistance mechanisms. Targeted protein degradation, whereby a drug redirects cellular proteolytic machinery towards degrading a specific target, is an emerging concept in drug discovery. We are extending this concept by developing proteolysis targeting chimeras active in bacteria (BacPROTACs) that bind to ClpC1, a component of the mycobacterial protein degradation machinery. The anti-Mycobacterium tuberculosis (Mtb) BacPROTACs are derived from cyclomarins which, when dimerized, generate compounds that recruit and degrade ClpC1. The resulting Homo-BacPROTACs reduce levels of endogenous ClpC1 in Mycobacterium smegmatis and display minimum inhibitory concentrations in the low micro- to nanomolar range in mycobacterial strains, including multiple drug-resistant Mtb isolates. The compounds also kill Mtb residing in macrophages. Thus, Homo-BacPROTACs that degrade ClpC1 represent a different strategy for targeting Mtb and overcoming drug resistance.


Assuntos
Mycobacterium smegmatis , Mycobacterium tuberculosis , Proteólise , Dimerização , Descoberta de Drogas
2.
Cell ; 186(10): 2176-2192.e22, 2023 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-37137307

RESUMO

The ClpC1:ClpP1P2 protease is a core component of the proteostasis system in mycobacteria. To improve the efficacy of antitubercular agents targeting the Clp protease, we characterized the mechanism of the antibiotics cyclomarin A and ecumicin. Quantitative proteomics revealed that the antibiotics cause massive proteome imbalances, including upregulation of two unannotated yet conserved stress response factors, ClpC2 and ClpC3. These proteins likely protect the Clp protease from excessive amounts of misfolded proteins or from cyclomarin A, which we show to mimic damaged proteins. To overcome the Clp security system, we developed a BacPROTAC that induces degradation of ClpC1 together with its ClpC2 caretaker. The dual Clp degrader, built from linked cyclomarin A heads, was highly efficient in killing pathogenic Mycobacterium tuberculosis, with >100-fold increased potency over the parent antibiotic. Together, our data reveal Clp scavenger proteins as important proteostasis safeguards and highlight the potential of BacPROTACs as future antibiotics.


Assuntos
Antituberculosos , Mycobacterium tuberculosis , Antituberculosos/farmacologia , Proteínas de Bactérias/metabolismo , Endopeptidase Clp/metabolismo , Proteínas de Choque Térmico/metabolismo , Mycobacterium tuberculosis/efeitos dos fármacos , Proteostase
3.
Commun Biol ; 6(1): 301, 2023 03 21.
Artigo em Inglês | MEDLINE | ID: mdl-36944713

RESUMO

Mycobacterium tuberculosis Clp proteases are targeted by several antitubercular compounds, including cyclomarin A (CymA). CymA exerts its toxicity by binding to AAA + chaperone ClpC1. Here, we show that CymA can also bind a partial homologue of ClpC1, known as ClpC2, and we reveal the molecular basis of these interactions by determining the structure of the M. tuberculosis ClpC2:CymA complex. Furthermore, we show deletion of clpC2 in Mycobacterium smegmatis increases sensitivity to CymA. We find CymA exposure leads to a considerable upregulation of ClpC2 via a mechanism in which binding of CymA to ClpC2 prevents binding of ClpC2 to its own promoter, resulting in upregulation of its own transcription in response to CymA. Our study reveals that ClpC2 not only senses CymA, but that through this interaction it can act as a molecular sponge to counteract the toxic effects of CymA and possibly other toxins targeting essential protease component ClpC1 in mycobacteria.


Assuntos
Proteínas de Choque Térmico , Mycobacterium tuberculosis , Proteólise , Proteínas de Choque Térmico/metabolismo , Chaperonas Moleculares/metabolismo , Mycobacterium tuberculosis/genética , Antituberculosos/farmacologia
4.
Cell ; 185(13): 2338-2353.e18, 2022 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-35662409

RESUMO

Hijacking the cellular protein degradation system offers unique opportunities for drug discovery, as exemplified by proteolysis-targeting chimeras. Despite their great promise for medical chemistry, so far, it has not been possible to reprogram the bacterial degradation machinery to interfere with microbial infections. Here, we develop small-molecule degraders, so-called BacPROTACs, that bind to the substrate receptor of the ClpC:ClpP protease, priming neo-substrates for degradation. In addition to their targeting function, BacPROTACs activate ClpC, transforming the resting unfoldase into its functional state. The induced higher-order oligomer was visualized by cryo-EM analysis, providing a structural snapshot of activated ClpC unfolding a protein substrate. Finally, drug susceptibility and degradation assays performed in mycobacteria demonstrate in vivo activity of BacPROTACs, allowing selective targeting of endogenous proteins via fusion to an established degron. In addition to guiding antibiotic discovery, the BacPROTAC technology presents a versatile research tool enabling the inducible degradation of bacterial proteins.


Assuntos
Proteínas de Bactérias , Chaperonas Moleculares , Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Chaperonas Moleculares/metabolismo , Proteólise
5.
Elife ; 102021 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-34328418

RESUMO

In Gram-positive bacteria, the McsB protein arginine kinase is central to protein quality control, labeling aberrant molecules for degradation by the ClpCP protease. Despite its importance for stress response and pathogenicity, it is still elusive how the bacterial degradation labeling is regulated. Here, we delineate the mechanism how McsB targets aberrant proteins during stress conditions. Structural data reveal a self-compartmentalized kinase, in which the active sites are sequestered in a molecular cage. The 'closed' octamer interconverts with other oligomers in a phosphorylation-dependent manner and, unlike these 'open' forms, preferentially labels unfolded proteins. In vivo data show that heat-shock triggers accumulation of higher order oligomers, of which the octameric McsB is essential for surviving stress situations. The interconversion of open and closed oligomers represents a distinct regulatory mechanism of a degradation labeler, allowing the McsB kinase to adapt its potentially dangerous enzyme function to the needs of the bacterial cell.


Assuntos
Bacillus subtilis/genética , Proteínas de Bactérias/metabolismo , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Bacillus subtilis/enzimologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Regulação Bacteriana da Expressão Gênica , Fosforilação , Proteínas Quinases/química
6.
FEBS J ; 288(1): 111-126, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-32301575

RESUMO

In Mycobacterium tuberculosis (Mtb), the Clp protease degradation pathway, mediated by the modular ClpCP and ClpXP protease complexes, is essential for growth and presents an attractive drug target. Employing a bacterial adenylate cyclase two-hybrid (BACTH) screening approach that we adapted to screen the proteome of an Mtb ORF library, we identify protein interaction partners of the ClpC1 chaperone on a genome-wide level. Our results demonstrate that bipartite type II toxin-antitoxin (TA) systems represent a major substrate class. Out of the 67 type II TA systems known in Mtb, 25 appear as ClpC1 interaction partners in the BACTH screen, including members of the VapBC, MazEF, and ParDE families, as well as a RelBE member that was identified biochemically. We show that antitoxins of the Vap and Rel families are degraded by ClpCP in vitro. We also demonstrate that ClpCP is responsible for mediating the N-end rule pathway, since the adaptor protein ClpS supports ClpC-dependent degradation of an N-end rule model substrate in vitro.


Assuntos
Antitoxinas/genética , Toxinas Bacterianas/genética , Endopeptidase Clp/genética , Regulação Bacteriana da Expressão Gênica , Mycobacterium tuberculosis/genética , Antitoxinas/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/metabolismo , Clonagem Molecular , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Endopeptidase Clp/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Biblioteca Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Genoma Bacteriano , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/metabolismo , Mycobacterium tuberculosis/metabolismo , Mycobacterium tuberculosis/patogenicidade , Fases de Leitura Aberta , Mapeamento de Interação de Proteínas , Estabilidade Proteica , Proteólise , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Sistemas Toxina-Antitoxina/genética
7.
PLoS One ; 10(5): e0125345, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25933022

RESUMO

Clp chaperone-proteases are cylindrical complexes built from ATP-dependent chaperonerings that stack onto a proteolytic ClpP double-ring core to carry out substrate protein degradation.Interaction of the ClpP particle with the chaperone is mediated by an N-terminal loop and a hydrophobic surface patch on the ClpP ring surface. In contrast to E. coli, Myco bacterium tuberculosis harbors not only one but two ClpP protease subunits, ClpP1 and ClpP2,and a homo-heptameric ring of each assembles to form the ClpP1P2 double-ring core. Consequently,this hetero double-ring presents two different potential binding surfaces for the interaction with the chaperones ClpX and ClpC1. To investigate whether ClpX or ClpC1 might preferentially interact with one or the other double-ring face, we mutated the hydrophobicchaperone-interaction patch on either ClpP1 or ClpP2, generating ClpP1P2 particles that are defective in one of the two binding patches and thereby in their ability to interact with their chaperone partners. Using chaperone-mediated degradation of ssrA-tagged model substrates, we show that both Mycobacterium tuberculosis Clp chaperones require the intact interaction face of ClpP2 to support degradation, resulting in an asymmetric complex where chaperones only bind to the ClpP2 side of the proteolytic core. This sets the Clpproteases of Mycobacterium tuberculosis, and probably other Actinobacteria, apart from the well-studied E. coli system, where chaperones bind to both sides of the protease core,and it frees the ClpP1 interaction interface for putative new binding partners [corrected].


Assuntos
Adenosina Trifosfatases/química , Proteínas de Bactérias/química , Endopeptidase Clp/química , Proteínas de Escherichia coli/química , Escherichia coli/enzimologia , Proteínas de Choque Térmico/química , Chaperonas Moleculares/química , Mycobacterium tuberculosis/enzimologia , Subunidades Proteicas/química , ATPases Associadas a Diversas Atividades Celulares , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Clonagem Molecular , Endopeptidase Clp/genética , Endopeptidase Clp/metabolismo , Escherichia coli/química , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Expressão Gênica , Regulação Bacteriana da Expressão Gênica , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Modelos Moleculares , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Dados de Sequência Molecular , Mycobacterium tuberculosis/química , Mycobacterium tuberculosis/genética , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Estrutura Secundária de Proteína , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Transdução de Sinais
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