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1.
Proc Natl Acad Sci U S A ; 115(12): 2982-2987, 2018 03 20.
Artigo em Inglês | MEDLINE | ID: mdl-29514959

RESUMO

Succinate:quinone oxidoreductase (SQR) functions in energy metabolism, coupling the tricarboxylic acid cycle and electron transport chain in bacteria and mitochondria. The biogenesis of flavinylated SdhA, the catalytic subunit of SQR, is assisted by a highly conserved assembly factor termed SdhE in bacteria via an unknown mechanism. By using X-ray crystallography, we have solved the structure of Escherichia coli SdhE in complex with SdhA to 2.15-Å resolution. Our structure shows that SdhE makes a direct interaction with the flavin adenine dinucleotide-linked residue His45 in SdhA and maintains the capping domain of SdhA in an "open" conformation. This displaces the catalytic residues of the succinate dehydrogenase active site by as much as 9.0 Å compared with SdhA in the assembled SQR complex. These data suggest that bacterial SdhE proteins, and their mitochondrial homologs, are assembly chaperones that constrain the conformation of SdhA to facilitate efficient flavinylation while regulating succinate dehydrogenase activity for productive biogenesis of SQR.


Assuntos
Complexo II de Transporte de Elétrons/metabolismo , Proteínas de Escherichia coli/química , Flavoproteínas/química , Proteínas de Bactérias , Cristalização , Cristalografia por Raios X , Complexo II de Transporte de Elétrons/genética , Escherichia coli , Proteínas de Escherichia coli/ultraestrutura , Flavoproteínas/ultraestrutura , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Estrobilurinas
2.
Mol Cell ; 44(5): 811-8, 2011 Dec 09.
Artigo em Inglês | MEDLINE | ID: mdl-22152483

RESUMO

The mitochondrial inner membrane harbors the complexes of the respiratory chain and translocase complexes for precursor proteins. We have identified a further subunit of the carrier translocase (TIM22 complex) that surprisingly is identical to subunit 3 of respiratory complex II, succinate dehydrogenase (Sdh3). The membrane-integral protein Sdh3 plays specific functions in electron transfer in complex II. We show by genetic and biochemical approaches that Sdh3 also plays specific functions in the TIM22 complex. Sdh3 forms a subcomplex with Tim18 and is involved in biogenesis and assembly of the membrane-integral subunits of the TIM22 complex. We conclude that the assembly of Sdh3 with different partner proteins, Sdh4 and Tim18, recruits it to two different mitochondrial membrane complexes with functions in bioenergetics and protein biogenesis, respectively.


Assuntos
Transporte de Elétrons , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Membranas Mitocondriais/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Succinato Desidrogenase/metabolismo , Complexo II de Transporte de Elétrons/metabolismo , Membranas Mitocondriais/enzimologia , Proteínas do Complexo de Importação de Proteína Precursora Mitocondrial , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/enzimologia
3.
FASEB J ; 28(4): 1794-804, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24414418

RESUMO

Mutations in succinate dehydrogenase (SDH) subunits and assembly factors cause a range of clinical conditions. One such condition, hereditary paraganglioma 2 (PGL2), is caused by a G78R mutation in the assembly factor SDH5. Although SDH5(G78R) is deficient in its ability to promote SDHA flavinylation, it has remained unclear whether impairment to its import, structure, or stability contributes to its loss of function. Using import-chase analysis in human mitochondria isolated from HeLa cells, we found that the import and maturation of human SDH5(G78R) was normal, while its stability was reduced significantly, with ~25% of the protein remaining after 180 min compared to ~85% for the wild-type protein. Notably, the metabolic stability of SDH5(G78R) was restored to wild-type levels by depleting mitochondrial LON (LONM). Degradation of SDH5(G78R) by LONM was confirmed in vitro; however, in contrast to the in organello analysis, wild-type SDH5 was also rapidly degraded by LONM. SDH5 instability was confirmed in SDHA-depleted mitochondria. Blue native PAGE showed that imported SDH5(G78R) formed a transient complex with SDHA; however, this complex was stabilized in LONM depleted mitochondria. These data demonstrate that SDH5 is protected from LONM-mediated degradation in mitochondria by its stable interaction with SDHA, a state that is dysregulated in PGL2.


Assuntos
Complexo II de Transporte de Elétrons/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Paraganglioma/metabolismo , Protease La/metabolismo , Deficiências na Proteostase/metabolismo , Complexo II de Transporte de Elétrons/genética , Estabilidade Enzimática/genética , Flavina-Adenina Dinucleotídeo/metabolismo , Células HeLa , Humanos , Immunoblotting , Proteínas Mitocondriais/genética , Paraganglioma/genética , Protease La/genética , Ligação Proteica , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Deficiências na Proteostase/genética , Especificidade por Substrato
4.
Subcell Biochem ; 66: 223-63, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23479443

RESUMO

Mitochondria are specialised organelles that are structurally and functionally integrated into cells in the vast majority of eukaryotes. They are the site of numerous enzymatic reactions, some of which are essential for life. The double lipid membrane of the mitochondrion, that spatially defines the organelle and is necessary for some functions, also creates a physical but semi-permeable barrier to the rest of the cell. Thus to ensure the biogenesis, regulation and maintenance of a functional population of proteins, an autonomous protein handling network within mitochondria is required. This includes resident mitochondrial protein translocation machinery, processing peptidases, molecular chaperones and proteases. This review highlights the contribution of proteases of the AAA+ superfamily to protein quality and activity control within the mitochondrion. Here they are responsible for the degradation of unfolded, unassembled and oxidatively damaged proteins as well as the activity control of some enzymes. Since most knowledge about these proteases has been gained from studies in the eukaryotic microorganism Saccharomyces cerevisiae, much of the discussion here centres on their role in this organism. However, reference is made to mitochondrial AAA+ proteases in other organisms, particularly in cases where they play a unique role such as the mitochondrial unfolded protein response. As these proteases influence mitochondrial function in both health and disease in humans, an understanding of their regulation and diverse activities is necessary.


Assuntos
Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Homeostase/fisiologia , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Peptídeo Hidrolases/metabolismo , Biossíntese de Proteínas , Proteólise
5.
EMBO J ; 28(12): 1732-44, 2009 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-19440203

RESUMO

The N-end rule pathway is conserved from bacteria to man and determines the half-life of a protein based on its N-terminal amino acid. In Escherichia coli, model substrates bearing an N-degron are recognised by ClpS and degraded by ClpAP in an ATP-dependent manner. Here, we report the isolation of 23 ClpS-interacting proteins from E. coli. Our data show that at least one of these interacting proteins--putrescine aminotransferase (PATase)--is post-translationally modified to generate a primary N-degron. Remarkably, the N-terminal modification of PATase is generated by a new specificity of leucyl/phenylalanyl-tRNA-protein transferase (LFTR), in which various combinations of primary destabilising residues (Leu and Phe) are attached to the N-terminal Met. This modification (of PATase), by LFTR, is essential not only for its recognition by ClpS, but also determines the stability of the protein in vivo. Thus, the N-end rule pathway, through the ClpAPS-mediated turnover of PATase may have an important function in putrescine homeostasis. In addition, we have identified a new element within the N-degron, which is required for substrate delivery to ClpA.


Assuntos
Aminoaciltransferases/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , Redes e Vias Metabólicas , Processamento de Proteína Pós-Traducional , Transaminases/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Transporte/química , Cromatografia de Afinidade , Dipeptídeos/metabolismo , Endopeptidase Clp/química , Endopeptidase Clp/metabolismo , Proteínas de Escherichia coli/química , Interações Hidrofóbicas e Hidrofílicas , Ligantes , Modelos Biológicos , Dados de Sequência Molecular , Proteínas Mutantes/metabolismo , Ligação Proteica , Especificidade por Substrato
6.
Methods Enzymol ; 686: 143-163, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37532398

RESUMO

The N-degron pathways are a set of proteolytic systems that relate the half-life of a protein to its N-terminal (Nt) residue. In Escherichia coli the principal N-degron pathway is known as the Leu/N-degron pathway. Proteins degraded by this pathway contain an Nt degradation signal (N-degron) composed of an Nt primary destabilizing (Nd1) residue (Leu, Phe, Trp or Tyr). All Leu/N-degron substrates are recognized by the adaptor protein, ClpS and delivered to the ClpAP protease for degradation. Although many components of the pathway are well defined, the physiological role of this pathway remains poorly understood. To address this gap in knowledge we developed a biospecific affinity chromatography technique to isolate physiological substrates of the Leu/N-degron pathway. In this chapter we describe the use of peptide arrays to determine the binding specificity of ClpS. We demonstrate how the information obtained from the peptide array, when coupled with ClpS affinity chromatography, can be used to specifically elute physiological Leu/N-degron ligands from a bacterial lysate. These techniques are illustrated using E. coli ClpS (EcClpS), but both are broadly suitable for application to related N-recognins and systems, not only for the determination of N-recognin specificity, but also for the identification of natural Leu/N-degron ligands from various bacterial and plant species that contain ClpS homologs.


Assuntos
Escherichia coli , Peptídeos , Escherichia coli/genética , Escherichia coli/metabolismo , Ligantes , Ligação Proteica , Peptídeos/química , Proteólise , Peptídeo Hidrolases/metabolismo , Especificidade por Substrato
7.
J Struct Biol ; 179(2): 193-201, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22710082

RESUMO

The mitochondrial matrix of mammalian cells contains several different ATP-dependent proteases, including CLPXP, some of which contribute to protein maturation and quality control. Currently however, the substrates and the physiological roles of mitochondrial CLPXP in humans, has remained elusive. Similarly, the mechanism by which these ATP-dependent proteases recognize their substrates currently remains unclear. Here we report the characterization of a Walker B mutation in human CLPX, in which the highly conserved glutamate was replaced with alanine. This mutant protein exhibits improved interaction with the model unfolded substrate casein and several putative physiological substrates in vitro. Although this mutant lacks ATPase activity, it retains the ability to mediate casein degradation by hCLPP, in a fashion similar to the small molecule ClpP-activator, ADEP. Our functional dissection of hCLPX structure, also identified that most model substrates are recognized by the N-terminal domain, although some substrates bypass this step and dock, directly to the pore-1 motif. Collectively these data reveal, that despite the difference between bacterial and human CLPXP complexes, human CLPXP exhibits a similar mode of substrate recognition and is deregulated by ADEPs.


Assuntos
Endopeptidase Clp/metabolismo , Animais , Endopeptidase Clp/genética , Humanos , Mutação , Ligação Proteica , Especificidade por Substrato
8.
IUBMB Life ; 63(11): 955-63, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-22031494

RESUMO

In the crowded environment of a cell, the protein quality control machinery, such as molecular chaperones and proteases, maintains a population of folded and hence functional proteins. The accumulation of unfolded proteins in a cell is particularly harmful as it not only reduces the concentration of active proteins but also overburdens the protein quality control machinery, which in turn, can lead to a significant increase in nonproductive folding and protein aggregation. To circumvent this problem, cells use heat shock and unfolded protein stress response pathways, which essentially sense the change to protein homeostasis upregulating protein quality control factors that act to restore the balance. Interestingly, several stress response pathways are proteolytically controlled. In this review, we provide a brief summary of targeted protein degradation by AAA+ proteases and focus on the role of ClpXP proteases, particularly in the signaling pathway of the Escherichia coli extracellular stress response and the mitochondrial unfolded protein response.


Assuntos
Endopeptidase Clp/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Mitocôndrias/metabolismo , Resposta a Proteínas não Dobradas , Animais , Parede Celular/metabolismo , Escherichia coli/enzimologia , Humanos , Mitocôndrias/enzimologia , Proteólise , Transdução de Sinais , Estresse Fisiológico
9.
EMBO Rep ; 10(5): 508-14, 2009 May.
Artigo em Inglês | MEDLINE | ID: mdl-19373253

RESUMO

In Escherichia coli, the ClpAP protease, together with the adaptor protein ClpS, is responsible for the degradation of proteins bearing an amino-terminal destabilizing amino acid (N-degron). Here, we determined the three-dimensional structures of ClpS in complex with three peptides, each having a different destabilizing residue--Leu, Phe or Trp--at its N terminus. All peptides, regardless of the identity of their N-terminal residue, are bound in a surface pocket on ClpS in a stereo-specific manner. Several highly conserved residues in this binding pocket interact directly with the backbone of the N-degron peptide and hence are crucial for the binding of all N-degrons. By contrast, two hydrophobic residues define the volume of the binding pocket and influence the specificity of ClpS. Taken together, our data suggest that ClpS has been optimized for the binding and delivery of N-degrons containing an N-terminal Phe or Leu.


Assuntos
Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Endopeptidase Clp/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Peptídeos/química , Peptídeos/metabolismo , Sequência de Aminoácidos , Leucina/química , Modelos Biológicos , Dados de Sequência Molecular , Fenilalanina/química , Ligação Proteica , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Triptofano/química
10.
Biochem Cell Biol ; 88(1): 97-108, 2010 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-20130683

RESUMO

In eukaryotes, mitochondria are required for the proper function of the cell and as such the maintenance of proteins within this organelle is crucial. One class of proteins, collectively known as the AAA+ (ATPases associated with various cellular activities) superfamily, make a number of important contributions to mitochondrial protein homeostasis. In this organelle, they contribute to the maturation and activation of proteins, general protein quality control, respiratory chain complex assembly, and mitochondrial DNA maintenance and integrity. To achieve such diverse functions this group of ATP-dependent unfoldases utilize the energy from ATP hydrolysis to modulate the structure of proteins via unique domains and (or) associated functional components. In this review, we describe the current status of knowledge regarding the known mitochondrial AAA+ proteins and their role in this organelle.


Assuntos
Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Adenosina Trifosfatases/genética , Humanos , Hidrólise , Mitocôndrias/genética , Proteínas Mitocondriais/genética , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína
11.
Biomolecules ; 10(4)2020 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-32316259

RESUMO

In Escherichia coli, SigmaS (σS) is the master regulator of the general stress response. The cellular levels of σS are controlled by transcription, translation and protein stability. The turnover of σS, by the AAA+ protease (ClpXP), is tightly regulated by a dedicated adaptor protein, termed RssB (Regulator of Sigma S protein B)-which is an atypical member of the response regulator (RR) family. Currently however, the molecular mechanism of σS recognition and delivery by RssB is only poorly understood. Here we describe the crystal structures of both RssB domains (RssBN and RssBC) and the SAXS analysis of full-length RssB (both free and in complex with σS). Together with our biochemical analysis we propose a model for the recognition and delivery of σS by this essential adaptor protein. Similar to most bacterial RRs, the N-terminal domain of RssB (RssBN) comprises a typical mixed (ßα)5-fold. Although phosphorylation of RssBN (at Asp58) is essential for high affinity binding of σS, much of the direct binding to σS occurs via the C-terminal effector domain of RssB (RssBC). In contrast to most RRs the effector domain of RssB forms a ß-sandwich fold composed of two sheets surrounded by α-helical protrusions and as such, shares structural homology with serine/threonine phosphatases that exhibit a PPM/PP2C fold. Our biochemical data demonstrate that this domain plays a key role in both substrate interaction and docking to the zinc binding domain (ZBD) of ClpX. We propose that RssB docking to the ZBD of ClpX overlaps with the docking site of another regulator of RssB, the anti-adaptor IraD. Hence, we speculate that docking to ClpX may trigger release of its substrate through activation of a "closed" state (as seen in the RssB-IraD complex), thereby coupling adaptor docking (to ClpX) with substrate release. This competitive docking to RssB would prevent futile interaction of ClpX with the IraD-RssB complex (which lacks a substrate). Finally, substrate recognition by RssB appears to be regulated by a key residue (Arg117) within the α5 helix of the N-terminal domain. Importantly, this residue is not directly involved in σS interaction, as σS binding to the R117A mutant can be restored by phosphorylation. Likewise, R117A retains the ability to interact with and activate ClpX for degradation of σS, both in the presence and absence of acetyl phosphate. Therefore, we propose that this region of RssB (the α5 helix) plays a critical role in driving interaction with σS at a distal site.


Assuntos
ATPases Associadas a Diversas Atividades Celulares/metabolismo , Proteínas de Ligação a DNA/metabolismo , Endopeptidase Clp/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Chaperonas Moleculares/metabolismo , Fatores de Transcrição/metabolismo , ATPases Associadas a Diversas Atividades Celulares/química , Proteínas de Ligação a DNA/química , Endopeptidase Clp/química , Proteínas de Escherichia coli/química , Modelos Moleculares , Chaperonas Moleculares/química , Mutação/genética , Fosforilação , Ligação Proteica , Domínios Proteicos , Espalhamento a Baixo Ângulo , Fator sigma/química , Fator sigma/metabolismo , Fatores de Transcrição/química , Difração de Raios X
12.
Commun Biol ; 3(1): 646, 2020 11 06.
Artigo em Inglês | MEDLINE | ID: mdl-33159171

RESUMO

Over a decade ago Polymerase δ interacting protein of 38 kDa (PDIP38) was proposed to play a role in DNA repair. Since this time, both the physiological function and subcellular location of PDIP38 has remained ambiguous and our present understanding of PDIP38 function has been hampered by a lack of detailed biochemical and structural studies. Here we show, that human PDIP38 is directed to the mitochondrion in a membrane potential dependent manner, where it resides in the matrix compartment, together with its partner protein CLPX. Our structural analysis revealed that PDIP38 is composed of two conserved domains separated by an α/ß linker region. The N-terminal (YccV-like) domain of PDIP38 forms an SH3-like ß-barrel, which interacts specifically with CLPX, via the adaptor docking loop within the N-terminal Zinc binding domain of CLPX. In contrast, the C-terminal (DUF525) domain forms an immunoglobin-like ß-sandwich fold, which contains a highly conserved putative substrate binding pocket. Importantly, PDIP38 modulates the substrate specificity of CLPX and protects CLPX from LONM-mediated degradation, which stabilises the cellular levels of CLPX. Collectively, our findings shed new light on the mechanism and function of mitochondrial PDIP38, demonstrating that PDIP38 is a bona fide adaptor protein for the mitochondrial protease, CLPXP.


Assuntos
Endopeptidase Clp/metabolismo , Mitocôndrias/metabolismo , Proteínas Nucleares/metabolismo , Endopeptidase Clp/genética , Regulação da Expressão Gênica , Células HeLa , Humanos , Proteínas Nucleares/genética , Proteínas Recombinantes
13.
J Cell Biol ; 163(4): 707-13, 2003 Nov 24.
Artigo em Inglês | MEDLINE | ID: mdl-14638855

RESUMO

Transport of preproteins into the mitochondrial matrix is mediated by the presequence translocase-associated motor (PAM). Three essential subunits of the motor are known: mitochondrial Hsp70 (mtHsp70); the peripheral membrane protein Tim44; and the nucleotide exchange factor Mge1. We have identified the fourth essential subunit of the PAM, an essential inner membrane protein of 18 kD with a J-domain that stimulates the ATPase activity of mtHsp70. The novel J-protein (encoded by PAM18/YLR008c/TIM14) is required for the interaction of mtHsp70 with Tim44 and protein translocation into the matrix. We conclude that the reaction cycle of the PAM of mitochondria involves an essential J-protein.


Assuntos
Proteínas de Membrana/isolamento & purificação , Proteínas de Membrana Transportadoras/isolamento & purificação , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial , Proteínas Motores Moleculares/metabolismo , Transporte Proteico/fisiologia , Proteínas de Saccharomyces cerevisiae/isolamento & purificação , Sequência de Aminoácidos/genética , Sequência de Bases/genética , Proteínas de Transporte/metabolismo , Células Cultivadas , DNA Complementar/análise , DNA Complementar/genética , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico/metabolismo , Substâncias Macromoleculares , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Proteínas do Complexo de Importação de Proteína Precursora Mitocondrial , Chaperonas Moleculares , Dados de Sequência Molecular , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
14.
Nat Struct Mol Biol ; 11(3): 226-33, 2004 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-14981507

RESUMO

Mitochondrial preproteins destined for the matrix are translocated by two channel-forming transport machineries, the translocase of the outer membrane and the presequence translocase of the inner membrane. The presequence translocase-associated protein import motor (PAM) contains four essential subunits: the matrix heat shock protein 70 (mtHsp70) and its three cochaperones Mge1, Tim44 and Pam18. Here we report that the PAM contains a fifth essential subunit, Pam16 (encoded by Saccharomyces cerevisiae YJL104W), which is selectively required for preprotein translocation into the matrix, but not for protein insertion into the inner membrane. Pam16 interacts with Pam18 and is needed for the association of Pam18 with the presequence translocase and for formation of a mtHsp70-Tim44 complex. Thus, Pam16 is a newly identified type of motor subunit and is required to promote a functional PAM reaction cycle, thereby driving preprotein import into the matrix.


Assuntos
Proteínas de Membrana Transportadoras/fisiologia , Proteínas Mitocondriais/fisiologia , Proteínas de Transporte/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Membranas Intracelulares/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Proteínas de Transporte da Membrana Mitocondrial , Proteínas do Complexo de Importação de Proteína Precursora Mitocondrial , Proteínas Mitocondriais/metabolismo , Proteínas Motores Moleculares/metabolismo , Ligação Proteica , Precursores de Proteínas/metabolismo , Transporte Proteico , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiologia , Leveduras/química , Leveduras/metabolismo , Leveduras/ultraestrutura
15.
Sci Rep ; 9(1): 18019, 2019 12 02.
Artigo em Inglês | MEDLINE | ID: mdl-31792243

RESUMO

The ClpP protease is found in all kingdoms of life, from bacteria to humans. In general, this protease forms a homo-oligomeric complex composed of 14 identical subunits, which associates with its cognate ATPase in a symmetrical manner. Here we show that, in contrast to this general architecture, the Clp protease from Mycobacterium smegmatis (Msm) forms an asymmetric hetero-oligomeric complex ClpP1P2, which only associates with its cognate ATPase through the ClpP2 ring. Our structural and functional characterisation of this complex demonstrates that asymmetric docking of the ATPase component is controlled by both the composition of the ClpP1 hydrophobic pocket (Hp) and the presence of a unique C-terminal extension in ClpP1 that guards this Hp. Our structural analysis of MsmClpP1 also revealed openings in the side-walls of the inactive tetradecamer, which may represent sites for product egress.


Assuntos
Proteínas de Bactérias/ultraestrutura , Endopeptidase Clp/ultraestrutura , Mycobacterium smegmatis/metabolismo , Multimerização Proteica , Subunidades Proteicas/metabolismo , Adenosina Trifosfatases/metabolismo , Adenosina Trifosfatases/ultraestrutura , Proteínas de Bactérias/metabolismo , Cristalografia por Raios X , Endopeptidase Clp/metabolismo , Simulação de Acoplamento Molecular , Estrutura Quaternária de Proteína , Proteólise
16.
FEBS J ; 275(7): 1400-1410, 2008 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-18279386

RESUMO

Protein degradation in the cytosol of Escherichia coli is carried out by a variety of different proteolytic machines, including ClpAP. The ClpA component is a hexameric AAA+ (ATPase associated with various cellular activities) chaperone that utilizes the energy of ATP to control substrate recognition and unfolding. The precise role of the N-domains of ClpA in this process, however, remains elusive. Here, we have analysed the role of five highly conserved basic residues in the N-domain of ClpA by monitoring the binding, unfolding and degradation of several different substrates, including short unstructured peptides, tagged and untagged proteins. Interestingly, mutation of three of these basic residues within the N-domain of ClpA (H94, R86 and R100) did not alter substrate degradation. In contrast mutation of two conserved arginine residues (R90 and R131), flanking a putative peptide-binding groove within the N-domain of ClpA, specifically compromised the ability of ClpA to unfold and degrade selected substrates but did not prevent substrate recognition, ClpS-mediated substrate delivery or ClpP binding. In contrast, a highly conserved tyrosine residue lining the central pore of the ClpA hexamer was essential for the degradation of all substrate types analysed, including both folded and unstructured proteins. Taken together, these data suggest that ClpA utilizes two structural elements, one in the N-domain and the other in the pore of the hexamer, both of which are required for efficient unfolding of some protein substrates.


Assuntos
Endopeptidase Clp/química , Endopeptidase Clp/fisiologia , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/fisiologia , Chaperonas Moleculares/fisiologia , Dobramento de Proteína , Motivos de Aminoácidos/genética , Sequência de Aminoácidos , Substituição de Aminoácidos/genética , Arginina/genética , Sequência Conservada , Endopeptidase Clp/genética , Escherichia coli/enzimologia , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Choque Térmico/genética , Chaperonas Moleculares/genética , Dados de Sequência Molecular , Mutação , Desnaturação Proteica/genética , Estrutura Terciária de Proteína/genética , Especificidade por Substrato/genética
17.
FEBS Lett ; 592(1): 15-23, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29197082

RESUMO

The pupylation of cellular proteins plays a crucial role in the degradation cascade via the Pup-Proteasome system (PPS). It is essential for the survival of Mycobacterium smegmatis under nutrient starvation and, as such, the activity of many components of the pathway is tightly regulated. Here, we show that Pup, like ubiquitin, can form polyPup chains primarily through K61 and that this form of Pup inhibits the ATPase-mediated turnover of pupylated substrates by the 20S proteasome. Similarly, the autopupylation of PafA (the sole Pup ligase found in mycobacteria) inhibits its own enzyme activity; hence, pupylation of PafA may act as a negative feedback mechanism to prevent substrate pupylation under specific cellular conditions.


Assuntos
Proteínas de Bactérias/metabolismo , Mycobacterium smegmatis/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Adenosina Trifosfatases/metabolismo , Substituição de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Lisina/química , Mutagênese Sítio-Dirigida , Mycobacterium smegmatis/genética , Processamento de Proteína Pós-Traducional , Proteólise , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Complexos Ubiquitina-Proteína Ligase/química , Complexos Ubiquitina-Proteína Ligase/genética , Complexos Ubiquitina-Proteína Ligase/metabolismo
19.
Sci Rep ; 8(1): 12862, 2018 08 27.
Artigo em Inglês | MEDLINE | ID: mdl-30150665

RESUMO

The maintenance of mitochondrial protein homeostasis (proteostasis) is crucial for correct cellular function. Recently, several mutations in the mitochondrial protease CLPP have been identified in patients with Perrault syndrome 3 (PRLTS3). These mutations can be arranged into two groups, those that cluster near the docking site (hydrophobic pocket, Hp) for the cognate unfoldase CLPX (i.e. T145P and C147S) and those that are adjacent to the active site of the peptidase (i.e. Y229D). Here we report the biochemical consequence of mutations in both regions. The Y229D mutant not only inhibited CLPP-peptidase activity, but unexpectedly also prevented CLPX-docking, thereby blocking the turnover of both peptide and protein substrates. In contrast, Hp mutations cause a range of biochemical defects in CLPP, from no observable change to CLPP activity for the C147S mutant, to dramatic disruption of most activities for the "gain-of-function" mutant T145P - including loss of oligomeric assembly and enhanced peptidase activity.


Assuntos
Endopeptidase Clp/genética , Estudos de Associação Genética , Predisposição Genética para Doença , Variação Genética , Disgenesia Gonadal 46 XX/diagnóstico , Disgenesia Gonadal 46 XX/genética , Perda Auditiva Neurossensorial/diagnóstico , Perda Auditiva Neurossensorial/genética , Endopeptidase Clp/química , Endopeptidase Clp/metabolismo , Disgenesia Gonadal 46 XX/metabolismo , Perda Auditiva Neurossensorial/metabolismo , Humanos , Mitocôndrias/genética , Mitocôndrias/metabolismo , Modelos Moleculares , Mutação , Conformação Proteica
20.
Curr Biol ; 13(8): R326-37, 2003 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-12699647

RESUMO

Apart from a handful of proteins encoded by the mitochondrial genome, most proteins residing in this organelle are nuclear-encoded and synthesised in the cytosol. Thus, delivery of proteins to their final destination depends on a network of specialised import components that form at least four main translocation complexes. The import machinery ensures that proteins earmarked for the mitochondrion are recognised and delivered to the organelle, transported across membranes, sorted to the correct compartment and assisted in overcoming energetic barriers.


Assuntos
Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Modelos Biológicos , Transporte Proteico/fisiologia , Transdução de Sinais/fisiologia , Proteínas de Transporte/metabolismo , Citosol/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Mitocôndrias/fisiologia , Proteínas do Complexo de Importação de Proteína Precursora Mitocondrial , Chaperonas Moleculares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo
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