Mycobacterium tuberculosis ClpC1: A POTENTIAL TARGET FOR TUBERCULOSIS DRUG DISCOVERY MELIANA RIWANTO NATIONAL UNIVERSITY OF SINGAPORE UNIVERSITY OF BASEL 2009 Mycobacterium tuberculosis ClpC1: A POTENTIAL TARGET FOR TUBERCULOSIS DRUG DISCOVERY MELIANA RIWANTO (B.Sc.(Pharm) (Hons.), National University of Singapore) A THESIS SUBMITTED FOR THE DEGREE OF MASTER OF SCIENCE IN INFECTIOUS DISEASES, VACCINOLOGY AND DRUG DISCOVERY DEPT OF MICROBIOLOGY, NATIONAL UNIVERSITY OF SINGAPORE BIOZENTRUM, UNIVERSITY OF BASEL 2009 Acknowledgements My deepest appreciation and gratitude goes to my thesis supervisor, Dr Luis Camacho Throughout this endeavor, he has provided me with tremendous guidance, insightful ideas and has always kept his door open, anytime I need help Without his ceaseless and selfless mentoring, this thesis would not have been possible He has been a great inspiration, a great teacher and a great friend My appreciation also goes to Dr Thomas Dick for the opportunity to work in the Tuberculosis Unit of the Novartis Institute for Tropical Diseases (NITD) I would especially like to extend my deep appreciation to the following people in the TB unit: Melvin for his support and guidance especially at the early stages of the project and for bestowing me with his protein purification knowledge; Elaine who has generously given her time and expertise to better my work especially her help in generating the knockout mutants; Bee Huat for his kind support and technical assistance; Srini for his critical and scientific advice I would also like to thank the rest of the members of the Tuberculosis Unit for their guidance, assistance and friendship I really appreciate David Beer for giving me the opportunity to conduct some of the experiments in the screening lab and for sharing some of his experience and knowledge I also thank members of the screening lab for their kind assistance, especially to Viral Patel for his meticulous and insightful inputs on the enzymatic assays I acknowledge the generous support from the committee of the Joint MSc programme, especially Dr Markus Wenk, Prof Marcel Tanner and Dr Thomas Keller for their great inspiration and ceaseless encouragement I especially thank Christine Mensch for her patience and efficiency in overseeing the administrative concerns My appreciation goes to Dr Veronique Dartois for being my academic supervisor and Prof Guy R Cornelis for being my Basel co-supervisor I I greatly appreciate the help from Kelly for reading and editing my thesis And my appreciation also goes to Patricia, for proof-reading my thesis and especially for her friendship, so full of energy and joy And I am especially grateful to Boon Zhi, Christina, Martin and Paul, who have provided me with great friendship, laughter, sanity and the occasional escape from science Many thanks to my family who have stood by me all these years I will not be where I am without their love and support Finally, I would like to thank Havard, whose care and encouragement have been my source of strength And I thank him for his company throughout the writing process, having painstakingly edited all the references in this thesis II Intellectual Property (IP) Statement In compliance with the IP policies of Novartis, we are unable to display the chemical structure of compound as well as the compound name used in this study Instead, we have replaced the name of the compound used in this study as Compound X (taken from Novartis compound library) III Table of Contents Chapter 1: INTRODUCTION 1.1 Tuberculosis 1.2 Overview of ATP-dependent protease 1.3 Clp proteolytic machinery 1.4 1.3.1 Clp ATPase 10 1.3.2 ClpP – the proteolytic component 11 1.3.3 Interplay of Clp/HSP100 and adaptor proteins 13 1.3.4 Substrate recognition 14 1.3.5 Modes of substrate recognition, unfolding and proteolysis 17 1.3.6 Self-compartmentalized bacterial proteases and pathogenesis 19 1.3.7 Mycobacterium tuberculosis and its protein degradation machinery 21 Preliminary data 23 1.4.1 Isolation and characterization of Compound X 23 1.4.2 Frequency of spontaneous Compound X-resistant mutants in mycobacteria 26 1.4.3 Compound X bind to ClpC1 27 AIMS OF THESIS 29 Chapter 2: MATERIALS AND METHODS 31 2.1 Bacterial strains, Growth conditions 32 2.2 Antibacterial activity of Compound X 32 2.3 Construction of M bovis BCG clpC1 deletion mutant 33 IV 2.3.1 Construction of vectors 33 2.3.2 Isolation of single cross-over strain (clpC1-sco strain) 34 2.3.3 Isolation of double cross-over strains from clpC1-sco strain 34 2.3.4 Preparation of electrocompetent of M bovis BCG and M smegmatis 35 2.3.5 Transformation into electro-competent M bovis BCG and M smegmatis cells 35 2.4 Isolation and characterization of Clp proteins as putative target of Compound X 36 2.4.1 Sequence alignment of clpC1, clpP1 and clpP2 36 2.4.2 Expression and purification of ClpC1, ClpX, ClpP1 ClpP2 and GFP-ssrA 36 2.4.2.1 Sub-cloning and creation of expression vectors 36 2.4.2.2 Transformation into chemical-competent E coli Top10 cells using heat-shock treatment 38 2.4.3 2.4.2.3 Small scale protein expression and purification 38 2.4.2.4 Large scale protein expression and purification 39 2.4.2.5 SDS PAGE 40 Biochemical assays to assess the activity of Clp proteins 41 2.4.3.1 ATPase activity of M tuberculosis ClpC1 and ClpX 41 2.4.3.2 Peptidase activity of M tuberculosis ClpP1 and ClpP2 42 2.4.3.3 Proteolytic activity of ClpC1-ClpP complex with β-casein as substrate 42 2.5 Effect of Compound X on the degradation of GFP-ssrA tag in M smegmatis 42 2.5.1 Construction of GFP-ssrA tag plasmids 42 2.5.2 Dose-dependent tetracycline regulation of pMind_GFP, pMind_GFP_SsrA/LAA and pMind_GFP_SsrA/LDD in M smegmatis 44 2.5.3 Effect of Compound X on the degradation of GFP-ssrA tag into M smegmatis 45 V 2.6 In vitro proteolytic degradation of GFP carrying SsrA tag 45 2.7 Overexpression of M tuberculosis ClpP1 and ClpP2 in M smegmatis 46 2.7.1 Construction of overexpressed mutants 46 2.7.2 Western Blot analysis of overexpressed ClpP1 and ClpP2 strains 47 2.7.3 Cell and colony morphology of M smegmatis overexpressing M tuberculosis ClpP1 and ClpP2 48 2.8 Primers and strains used in this study 48 Chapter 3: RESULTS 52 3.1 Antibacterial activity of Compound X 53 3.2 Essentiality of ClpC1 gene in M bovis BCG 53 3.3 Sub-cloning and expression of ClpC1 55 3.4 ClpC1 Displays Basal ATPase activity 57 3.5 Effect of Compound X on the ATPase activity of ClpC1 60 3.6 Compound X confer increased peptidase activity to ClpP via ClpC1 63 3.7 Compound X confer degradation of GFP-ssrA in vivo 68 3.8 In vitro proteolysis of GFP-SsrA by M tuberculosis ClpC1P complex 72 3.9 Overexpression of ClpP1 and ClpP2 73 Chapter 4: DISCUSSION 77 Discussion 78 Future perspectives 83 Chapter 5: CONCLUSION 85 Conclusion 86 VI BIBLIOGRAPHY 87 SUPPLEMENTARY DATA 96 VII Abstract A whole cell-based screen of natural product for new anti-TB agents at Novartis identified a Compound X displaying potent activity against M tuberculosis This compound is active against multidrug resistant clinical isolates, implying a new target In a pull-down experiment with immobilized Compound X and BCG lysate, ClpC1 was identified as the potential target (Schmitt E et al unpublished data) The ClpC form of Clp/HSP100 family is an essential, highly conserved protein that interacts with ClpP peptidase to degrade specific substrates, and yet little is known regarding its specific activity as a molecular chaperone in M tuberculosis To address this and to confirm the target of Compound X, ClpC1 from M tuberculosis was purified using an E coli-based overexpression system We have found that recombinant ClpC1 display basal ATPase activity, similar to that of other types of HSP100 proteins but without the need of other chaperones or adaptor proteins Most significantly, we demonstrate that ClpC1 basal ATPase activity was enhanced in the presence of Compound X and was specific to this ATPase Consistent with this observation, we show that only structural derivatives of Compounds X with potent whole cell activity had stimulating ClpC1 ATPase activity Of further interest is the finding that binding of Compounds X to ClpC1 also resulted in enhanced proteolytic efficiency of ClpC1P complex Such activation leads to inhibition of bacterial growth and suggest that ClpC1 is involved in key processes of importance for the multiplication of M tuberculosis VIII Chapter Five: Conclusion 85 Conclusion In conclusion, the identification of ClpC1 as a 'target' for antibacterial compounds broadens our perspective of how to kill mycobacteria Virtually all clinically useful antibacterial compounds inhibit essential bacterial functions—but Compounds X hyperactivates an essential function, inducing widespread protein degradation Brötz-Oesterhelt et al (2005) already shows that a class of molecules, acyldepsipeptides acts by removing the stringent controls on a bacterial protease, unleashing the enzyme to degrade 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Paal, K., Cusack, S., Bukau, B., and Dougan, D.A (2002) Structural analysis of the adaptor protein ClpS in complex with the N-terminal domain of ClpA Nat Struct Biol 9, 906-911 Zhou, Y., Gottesman, S., Hoskins, J.R., Maurizi, M.R., and Wickner, S (2001) The RssB response regulator directly targets sigma(S) for degradation by ClpXP Genes Dev 15, 627-637 Zwickl, P., Baumeister, W., and Steven, A (2000) Dis-assembly lines: the proteasome and related ATPase-assisted proteases Curr Opin Struct Biol 10, 242-250 95 Supplementary Data Supplementary data Sequence alignment of ClpC1 homologues corresponding to the Tuberculist entry for M tuberculosis (Rv3596c), M bovis BCG (Mb3629c) and the TIGR entry for M smegmatis (6091 MC2) by CLUSTAL 2.0.10 Residues marked with the asterisks correspo1d to residues or nucleotides identical in all sequences ":" means that conserved substitutions have been observed "." means that semi-conserved substitutions are observed M_tuberculosis M_bovis M_smegmatis MFERFTDRARRVVVLAQEEARMLNHNYIGTEHILLGLIHEGEGVAAKSLE 50 MFERFTDRARRVVVLAQEEARMLNHNYIGTEHILLGLIHEGEGVAAKSLE 50 MFERFTDRARRVVVLAQEEARMLNHNYIGTEHILLGLIHEGEGVAAKSLE 50 ************************************************** M_tuberculosis M_bovis M_smegmatis SLGISLEGVRSQVEEIIGQGQQAPSGHIPFTPRAKKVLELSLREALQLGH 100 SLGISLEGVRSQVEEIIGQGQQAPSGHIPFTPRAKKVLELSLREALQLGH 100 SLGISLEGVRSQVEEIIGQGQQAPSGHIPFTPRAKKVLELSLREALQLGH 100 ************************************************** M_tuberculosis M_bovis M_smegmatis NYIGTEHILLGLIREGEGVAAQVLVKLGAELTRVRQQVIQLLSGYQGKEA 150 NYIGTEHILLGLIREGEGVAAQVLVKLGAELTRVRQQVIQLLSGYQGKEA 150 NYIGTEHILLGLIREGEGVAAQVLVKLGAELTRVRQQVIQLLSGYQGKEA 150 ************************************************** M_tuberculosis M_bovis M_smegmatis AEAGTGGRGGESGSPSTSLVLDQFGRNLTAAAMEGKLDPVIGREKEIERV 200 AEAGTGGRGGESGSPSTSLVLDQFGRNLTAAAMEGKLDPVIGREKEIERV 200 AEAGTGGRGGESGNPSTSLVLDQFGRNLTAAAMEGKLDPVIGREKEIERV 200 *************.************************************ M_tuberculosis M_bovis M_smegmatis MQVLSRRTKNNPVLIGEPGVGKTAVVEGLAQAIVHGEVPETLKDKQLYTL 250 MQVLSRRTKNNPVLIGEPGVGKTAVVEGLAQAIVHGEVPETLKDKQLYTL 250 MQVLSRRTKNNPVLIGEPGVGKTAVVEGLAQAIVHGEVPETLKDKQLYTL 250 ************************************************** M_tuberculosis M_bovis M_smegmatis DLGSLVAGSRYRGDFEERLKKVLKEINTRGDIILFIDELHTLVGAGAAEG 300 DLGSLVAGSRYRGDFEERLKKVLKEINTRGDIILFIDELHTLVGAGAAEG 300 DLGSLVAGSRYRGDFEERLKKVLKEINTRGDIILFIDELHTLVGAGAAEG 300 ************************************************** M_tuberculosis M_bovis M_smegmatis AIDAASILKPKLARGELQTIGATTLDEYRKYIEKDAALERRFQPVQVGEP 350 AIDAASILKPKLARGELQTIGATTLDEYRKYIEKDAALERRFQPVQVGEP 350 AIDAASILKPKLARGELQTIGATTLDEYRKYIEKDAALERRFQPVQVGEP 350 ************************************************** M_tuberculosis M_bovis M_smegmatis TVEHTIEILKGLRDRYEAHHRVSITDAAMVAAATLADRYINDRFLPDKAI 400 TVEHTIEILKGLRDRYEAHHRVSITDAAMVAAATLADRYINDRFLPDKAI 400 TVEHTIEILKGLRDRYEAHHRVSITDSAMVAAATLADRYINDRFLPDKAI 400 **************************:*********************** M_tuberculosis M_bovis M_smegmatis DLIDEAGARMRIRRMTAPPDLREFDEKIAEARREKESAIDAQDFEKAASL 450 DLIDEAGARMRIRRMTAPPDLREFDEKIAEARREKESAIDAQDFEKAASL 450 DLIDEAGARMRIRRMTAPPDLREFDEKIADARREKESAIDAQDFEKAAAL 450 *****************************:******************:* M_tuberculosis M_bovis M_smegmatis RDREKTLVAQRAEREKQWRSGDLDVVAEVDDEQIAEVLGNWTGIPVFKLT 500 RDREKTLVAQRAEREKQWRSGDLDVVAEVDDEQIAEVLGNWTGIPVFKLT 500 RDKEKQLVAQRAEREKQWRSGDLDVVAEVDDEQIAEVLGNWTGIPVFKLT 500 **:** ******************************************** M_tuberculosis EAETTRLLRMEEELHKRIIGQEDAVKAVSKAIRRTRAGLKDPKRPSGSFI 550 96 M_bovis M_smegmatis EAETTRLLRMEEELHKRIIGQEDAVKAVSKAIRRTRAGLKDPKRPSGSFI 550 EEETTRLLRMEEELHKRIIGQEDAVKAVSKAIRRTRAGLKDPKRPSGSFI 550 * ************************************************ M_tuberculosis M_bovis M_smegmatis FAGPSGVGKTELSKALANFLFGDDDALIQIDMGEFHDRFTASRLFGAPPG 600 FAGPSGVGKTELSKALANFLFGDDDALIQIDMGEFHDRFTASRLFGAPPG 600 FAGPSGVGKTELSKALANFLFGDDDALIQIDMGEFHDRFTASRLFGAPPG 600 ************************************************** M_tuberculosis M_bovis M_smegmatis YVGYEEGGQLTEKVRRKPFSVVLFDEIEKAHQEIYNSLLQVLEDGRLTDG 650 YVGYEEGGQLTEKVRRKPFSVVLFDEIEKAHQEIYNSLLQVLEDGRLTDG 650 YVGYEEGGQLTEKVRRKPFSVVLFDEIEKAHQEIYNSLLQVLEDGRLTDG 650 ************************************************** M_tuberculosis M_bovis M_smegmatis QGRTVDFKNTVLIFTSNLGTSDISKPVGLGFSKGGGENDYERMKQKVNDE 700 QGRTVDFKNTVLIFTSNLGTSDISKPVGLGFSKGGGENDYERMKQKVNDE 700 QGRTVDFKNTVLIFTSNLGTSDISKAVGLGFSQGGSENNYERMKQKVHDE 700 *************************.******:**.**:********:** M_tuberculosis M_bovis M_smegmatis LKKHFRPEFLNRIDDIIVFHQLTREEIIRMVDLMISRVAGQLKSKDMALV 750 LKKHFRPEFLNRIDDIIVFHQLTREEIIRMVDLMISRVAGQLKSKDMALV 750 LKKHFRPEFLNRIDDIIVFHQLTQDEIIQMVDLMIGRVSNQLKTKDMALE 750 ***********************::***:******.**:.***:***** M_tuberculosis M_bovis M_smegmatis LTDAAKALLAKRGFDPVLGARPLRRTIQREIEDQLSEKILFEEVGPGQVV 800 LTDAAKALLAKRGFDPVLGARPLRRTIQREIEDQLSEKILFEEVGPGQVV 800 LSDKAKALLAKRGFDPVLGARPLRRTIQREIEDQLSEKILFEEIGPGQLV 800 *:* ***************************************:****:* M_tuberculosis M_bovis M_smegmatis TVDVDNWDGEGPGEDAVFTFTG TRKPPAEPDLAKAGAHSAGGPEPAAR 848 TVDVDNWDGEGPGEDAVFTFTG TRKPPAEPDLAKAGAHSAGGPEPAAR 848 TVDVEGWDGEGQGEDAKFTFSGGPKRAETAEPDLAGAG AAGAPTAGTE 848 ****:.***** **** ***:* * ****** ** :**.* : SeqA Name Len(aa) SeqB Name Len(aa) Score =================================================================================== M_tuberculosis_Rv3596c 848 M_bovis_Mb3627c 848 100 M_tuberculosis_Rv3596c 848 M_smegmatis_6091 848 94 M_bovis_Mb3627c 848 M_smegmatis_6091 848 94 =================================================================================== 97 Supplementary Data Sequence alignment of ClpP1 homologues corresponding to the Tuberculist entry for M tuberculosis (Rv2461c), and the TIGR entry for M smegmatis (4673 MC2) Residues marked with the asterisks correspo1d to residues or nucleotides identical in all sequences ":" means that conserved substitutions have been observed "." means that semi-conserved substitutions are observed M_tuberculosis M_smegmatis MSQ -VTDMRSNSQGLSLTDSVYERLLSERIIFLGSEVNDEIA 41 MYQDVVESRYPVVTDMRGTGQGLNLVDSVYERLLAERIIFLGSQVDDDIA 50 * * ***** ***.*.********:********:*:*:** M_tuberculosis M_smegmatis NRLCAQILLLAAEDASKDISLYINSPGGSISAGMAIYDTMVLAPCDIATY 91 NRLCAQILLLSAEDPTKDIHLYINSPGGSISAGMAIYDTMVLAPCDIATY 100 **********:***.:*** ****************************** M_tuberculosis M_smegmatis AMGMAASMGEFLLAAGTKGKRYALPHARILMHQPLGGVTGSAADIAIQAE 141 AMGMAASMGEFLLAAGTKGKRYALPHARILMHQPLGGVTGSAADIAIQAE 150 ************************************************** M_tuberculosis M_smegmati QFAVIKKEMFRLNAEFTGQPIERIEADSDRDRWFTAAEALEYGFVDHIIT 191 QFAVIKKEMFRLNAEFTGQPIERIEADSDRDRWFTAQEALEYGFVDHIIT 200 ************************************ ************* M_tuberculosis M_smegmatis RAHVNGEAQ 200 SASVNGEGPGAGLDK 215 * **** SeqA Name Len(aa) SeqB Name Len(aa) Score =================================================================================== M_tuberculosis_Rv2461c 200 M_smegmatis_MSMEG_4673 215 89 =================================================================================== 98 Supplementary Data Sequence alignment of ClpP2 homologues corresponding to the Tuberculist entry for M tuberculosis (Rv2460c), and the TIGR entry for M smegmatis (4672 MC2) Residues marked with the asterisks correspond to residues or nucleotides identical in all sequences ":" means that conserved substitutions have been observed "." means that semi-conserved substitutions are observed M_tuberculosis M_smegmatis MN SQNSQIQPQARYILPSFIEHSSFGVKESNPYNKLFEERIIFLGV 46 MSNIHPSLDARLQPQARYILPSFIEHSSFGVKESNPYNKLFEERIIFLGV 50 * * ::::************************************** M_tuberculosis M_smegmatis QVDDASANDIMAQLLVLESLDPDRDITMYINSPGGGFTSLMAIYDTMQYV 96 QVDDASANDIMAQLLVLESLDPDRDITMYINSPGGSFTSLMAIYDTMQYV 100 ***********************************.************** M_tuberculosis M_smegmatis RADIQTVCLGQAASAAAVLLAAGTPGKRMALPNARVLIHQPSLSGVIQGQ 146 RADIQTVCLGQAASAAAVLLAAGTPGKRLALPNARVLIHQPALSGVIQGQ 150 ****************************:************:******** M_tuberculosis M_smegmatis FSDLEIQAAEIERMRTLMETTLARHTGKDAGVIRKDTDRDKILTAEEAKD 196 FSDLEIQAAEIERMRTLMETTLARHTGKDPAQIRKDTDRDKILTAEEAKE 200 ***************************** *****************: M_tuberculosis M_smegmatis YGIIDTVLEYRKLSAQTA 214 YGIIDTVLQYRKLSAQTS 218 ********:********: SeqA Name Len(aa) SeqB Name Len(aa) Score =================================================================================== M_tuberculosis_Rv2460c 214 M_smegmatis_MSMEG_4672 218 92 =================================================================================== 99 [...]... ClpA has a subunit Mr of 83,000 and possesses an intrinsic ATPase activity that is increased in the presence of ClpP and substrates (Hwang et al., 1988; Katayama et al., 1988) ClpA is purified as a monomer-dimer mixture, but addition of magnesium, ATP or analogs of ATP promotes association of ClpA to a hexamer with Mr 450,000 to 500,000 (Maurizi, 1991) ClpA has a basal ATPase activity that is activated... A site of the ribosome causing the release of the truncated mRNA lacking a STOP codon The SsrA RNA also adds 11 amino acid tag (AANDENYALAA) to the incomplete protein Proteins tagged with the SsrA peptide are targeted for degradation The C-terminal Ala-Ala residues are critical for SsrA recognition by ClpX, or other proteases (Gottesman et al., 1998; Keiler and Shapiro, 2003) When the two C-terminal... AAA+ superfamily of ATPases AAA+ proteins are generally modulated by a group of otherwise unrelated proteins termed adaptor proteins An adaptor protein serves as an accessory component to main proteins in a signal transduction pathway Several adaptor proteins for bacterial Clp/HSP100 proteins have been identified and characterized They exhibit a great diversity in sequence and structure and vary in size,... Introduction ATPases are responsible for the recognition, unfolding and translocation of substrates into the ClpP degradation chamber (Sauer et al., 2004) ATP hydrolysis is not required for association between ClpA and ClpP; nonhydrolyzable analogs such as AMPPNP and adenosine-5'thiotriphosphate (ATP-γ-S) promote self-association of ClpA and formation of the ClpA-ClpP complex (Maurizi, 1991) A B C Figure 1.2 A. .. the FtsH/AAA family This family of proteases are anchored to membranes but contain cytoplasmic domains with ATPase activity and Zn2+ metalloprotease active sites (Tomoyasu et al., 1995; Tomoyasu et al., 1993) Similar to Lon, it forms a homo- 8 Introduction oligomer with both ATPase and protease active site in a single polypeptide (Tomoyasu et al., 1995; Tomoyasu et al., 1993) Meanwhile, the heat shock... protein degradation by these proteases As a result of this structure, proteins must first be unfolded and threaded into the protease chamber for degradation Thus, the second common feature of these proteases is the requirement of ATP hydrolysis, usually by ATPases of the AAA or AAA+ superfamily (ATPases associated with various cellular activities) (Neuwald et al., 1999) These ATPase chaperones or chaperonins... BCG vaccination and exposure to non-tuberculous environmental bacteria (Nahid et al., 2006; Pai, 2005) To circumvent this problem, an IFNγ-based assay was developed using RD1 antigens such as namely 6-kDa early secreted antigenic target (ESAT-6) and 10-kDa culture filtrate protein (CFP-10) (Dheda et al., 2005; Lalvani, 2003; Nahid et al., 2006; Pai, 2005; Pai et al., 2004) Studies have shown that this... its name suggests, degrades casein and other proteins only in the presence of ATP (Katayama et al., 1986) ClpP alone can rapidly cleave short (3- to 6-amino-acid) peptides and will also cleave longer unstructured polypeptides, such as oxidized insulin B chain (Gottesman and Maurizi, 1992).It is composed of two heptameric rings stacked back-to-back forming a tetradecamer with a central pore that contains... we summarize a handful of these studies, not necessarily confined to the antimycobacterial research We aim to highlight the importance of bacterial protease as potential drug target and extrapolate the results of previous studies in other bacteria to our need in antituberculous research 1.2 Overview of ATP-dependent protease Bacterial protease has been implicated in the regulation of intracellular protein... promote degradation of casein when complexed with the latter (Hwang et al., 1988; Katayama et al., 1988; Maurizi et al., 199 0a; Maurizi et al., 1990b) ClpX was later identified as an alternative chaperone to ClpA (Gottesman et al., 1993) Alone, ClpP is able to degrade only small peptides Degradation of large or folded proteins require that they first be unfolded through ATP hydrolysis by the chaperone ... of ATP hydrolysis, usually by ATPases of the AAA or AAA+ superfamily (ATPases associated with various cellular activities) (Neuwald et al., 1999) These ATPase chaperones or chaperonins unfold and... trans-translation, in which an alanyl-tmRNA enters the empty A site of the ribosome causing the release of the truncated mRNA lacking a STOP codon The SsrA RNA also adds 11 amino acid tag (AANDENYALAA)... the same group demonstrated that mpa and pafA mutants are severely attenuated in a mouse model of infection (Darwin et al., 2005) Mpa is an ATPase that forms hexamers like the Clp ATPase Meanwhile,