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Escherichia coli Proteome Microarrays Identified the Substrates of ClpYQ Protease*

  • Chih-Hsuan Tsai
    Footnotes
    Affiliations
    Department of Agricultural Chemistry, National Taiwan University, No. 1, Sec. 4, Roosevelt Rd., Taipei 10617, Taiwan;
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  • Yu-Hsuan Ho
    Footnotes
    Affiliations
    Graduate Institute of Systems Biology and Bioinformatics, National Central University, No. 300, Jhongda Rd., Jhongli 32001, Taiwan;

    Department of Biomedical Sciences and Engineering, National Central University, No. 300, Jhongda Rd., Jhongli 32001, Taiwan
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  • Tzu-Cheng Sung
    Footnotes
    Affiliations
    Graduate Institute of Systems Biology and Bioinformatics, National Central University, No. 300, Jhongda Rd., Jhongli 32001, Taiwan;

    Department of Biomedical Sciences and Engineering, National Central University, No. 300, Jhongda Rd., Jhongli 32001, Taiwan
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  • Whei-Fen Wu
    Correspondence
    To whom correspondence should be addressed:Dept. of Agricultural Chemistry, National Taiwan University, No. 1, Sec. 4, Roosevelt Road, Taipei, Taiwan 106. Tel.:+886-2-33664818; E-mail: ;
    Affiliations
    Department of Agricultural Chemistry, National Taiwan University, No. 1, Sec. 4, Roosevelt Rd., Taipei 10617, Taiwan;
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  • Chien-Sheng Chen
    Correspondence
    Graduate Institute of Systems Biology and Bioinformatics, Dept. of Biomedical Sciences and Engineering, National Central University, No. 300, Jhongda Rd., Jhongli 32001, Taiwan. Tel.:+886-3-4227151 ext. 36103; Fax:+886-3-4273822; E-mail:.
    Affiliations
    Graduate Institute of Systems Biology and Bioinformatics, National Central University, No. 300, Jhongda Rd., Jhongli 32001, Taiwan;

    Department of Biomedical Sciences and Engineering, National Central University, No. 300, Jhongda Rd., Jhongli 32001, Taiwan
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  • Author Footnotes
    * This work was supported by Ministry of Science and Technology, Taiwan grants NSC-101-2313-B-002-065-MY3, MOST 103-2627-M-008-001, MOST 104-2320-B-002-038 and MOST 104-2320-B-008-002-MY3, The Aim for the Top University Project, National Central University and Landseed Hospital Join Research Program (NCU-LSH-103-A-001), National Central University and Cathay General Hospital Join Research Program (102NCU-CGH-02, 102NCU-CGH-07, 103CGH-NCU-A3), and National Health Research Institutes Career Development Grant (NHRI-EX103-10233SC).
    This article contains supplemental material.
    ** These authors contributed equally to this work.
Open AccessPublished:November 18, 2016DOI:https://doi.org/10.1074/mcp.M116.065482

      Abstract

      Proteolysis is a vital mechanism to regulate the cellular proteome in all kingdoms of life, and ATP-dependent proteases play a crucial role within this process. In Escherichia coli, ClpYQ is one of the primary ATP-dependent proteases. In addition to function with removals of abnormal peptides in the cells, ClpYQ degrades regulatory proteins if necessary and thus let cells adjust to various environmental conditions. In E. coli, SulA, RcsA, RpoH and TraJ as well as RNase R, have been identified as natural protein substrates of ClpYQ. ClpYQ contains ClpY and ClpQ. The ATPase ClpY is responsible for protein recognition, unfolding, and translocation into the catalytic core of ClpQ. In this study, we use an indirect identification strategy to screen possible ClpY targets with E. coli K12 proteome chips. The chip assay results showed that YbaB strongly bound to ClpY. We used yeast two-hybrid assay to confirm the interactions between ClpY and YbaB protein and determined the Kd between ClpY and YbaB by quartz crystal microbalance. Furthermore, we validated that YbaB was successfully degraded by ClpYQ protease activity using ClpYQ in vitro and in vivo degradation assay. These findings demonstrated the YbaB is a novel substrate of ClpYQ protease. This work also successfully demonstrated that with the use of recognition element of a protease can successfully screen its substrates by indirect proteome chip screening assay.
      Protease is responsible for protein degradation that can digest long protein chains into shorter fragments by splitting the peptide bonds (
      • Neurath H.
      • Walsh K.A.
      Role of proteolytic enzymes in biological regulation (a review).
      ). Protease exists in a wide range of organisms. One of the most important functions for protease is to degrade the misfolding or functionless proteins. Misfolding or functionless proteins usually lead to severe diseases, such as Alzheimer's disease, bovine spongiform encephalopathy, cardiovascular disease, cancer, osteoporosis, neurological disorders, and type II diabetes (
      • Harper J.D.
      • Lansbury P.T.
      Models of amyloid seeding in Alzheimer's disease and scrapie:mechanistic truths and physiological consequences of the time-dependent solubility of amyloid proteins.
      ,
      • Kahn S.E.
      • Andrikopoulos S.
      • Verchere C.B.
      Islet amyloid: a long-recognized but underappreciated pathological feature of type 2 diabetes.
      ,
      • Turk B.
      Targeting proteases: successes, failures and future prospects.
      ). In addition, the protease-induced protein regulation is very important in the wide-range organism. Protease-induced protein regulation in bacterial cells is related to the antibiotic resistance, environmental resilience, and infectivity of bacteria (
      • Gottesman S.
      Proteases and their targets in Escherichia coli.
      ). Altering the growth environments of bacteria (e.g. adjusting temperature, osmotic pressure, nutrient deficiency, ultraviolet irradiation, addition of antibiotics or other chemicals) can induce abnormal protein synthesis and accumulation in bacterial cells. Under such conditions, external environmental stimuli can activate the response systems of bacteria, including heat-shock and SOS responses, which can trigger the biosynthesis of the corresponding proteases, potentially aiding bacteria in overcoming unfavorable growth conditions (
      • Wickner S.
      • Maurizi M.R.
      • Gottesman S.
      Posttranslational quality control: folding, refolding, and degrading proteins.
      ,
      • Zwickl P.
      • Baumeister W.
      • Steven A.
      Dis-assembly lines: the proteasome and related ATPase-assisted proteases.
      ). The ATP-dependent protease of E. coli provides us an excellent example to understand the importance of protein degradation role in cell physiology. The homologs of these energy (ATP or GTP)-dependent proteases were also found in eukaryotes indicating the similar regulation cascades between prokaryotes and eukaryotes (
      • Gottesman S.
      Proteases and their targets in Escherichia coli.
      ). There are four important ATP-dependent proteases in prokaryotes, Lon, FtsH, ClpAP/XP, and ClpYQ (
      • Gottesman S.
      Proteolysis in bacterial regulatory circuits.
      ). Lon protease is a major cytosol protease in E. coli, which is a DNA heat shock protein responsible for degradation of misfolding proteins (
      • Parsell D.A.
      • Sauer R.T.
      Induction of a heat shock-like response by unfolded protein in Escherichia coli: dependence on protein level not protein degradation.
      ,
      • Goff S.A.
      • Goldberg A.L.
      Production of abnormal proteins in E. coli stimulates transcription of lon and other heat shock genes.
      ). FtsH is a membrane-bound essential protease, which degrades the heat-shock transcription factor sigma 32 (
      • Tomoyasu T.
      • Gamer J.
      • Bukau B.
      • Kanemori M.
      • Mori H.
      • Rutman A.J.
      • Oppenheim A.B.
      • Yura T.
      • Yamanaka K.
      • Niki H.
      Escherichia coli FtsH is a membrane-bound, ATP-dependent protease which degrades the heat-shock transcription factor sigma 32.
      ). The ClpAP/XP and ClpYQ belong to the Clp protease family. The clp stands for caseinolytic protease, which can degrade casein in vitro (
      • Katayama-Fujimura Y.
      • Gottesman S.
      • Maurizi M.R.
      A multiple-component, ATP-dependent protease from Escherichia coli.
      ,
      • Gottesman S.
      • Roche E.
      • Zhou Y.
      • Sauer R.T.
      The ClpXP and ClpAP proteases degrade proteins with carboxy-terminal peptide tails added by the SsrA-tagging system.
      ). ATP-dependent protease ClpYQ is a type of protease complex. The translational products of clpQ+Y+ operon are ClpQ (HslV), the peptidase responsible for proteolysis, and ClpY (HslU), an ATPase with unfolding activity (
      • Chuang S.E.
      • Burland V.
      • Plunkett 3rd, G.
      • Daniels D.L.
      • Blattner F.R.
      Sequence analysis of four new heat-shock genes constituting the hslTS/ibpABhslVU operons in Escherichia coli.
      ). ClpYQ was suggested as an alternative protease to partially restore the function of Lon protease (
      • Wu W.F.
      • Zhou Y.
      • Gottesman S.
      Redundant in vivo proteolytic activities of Escherichia coli Lon and the ClpYQ (HslUV) protease.
      ,
      • Kuo M.S.
      • Chen K.P.
      • Wu W.F.
      Regulation of RcsA by the ClpYQ (HslUV) protease in Escherichia coli.
      ). Bochtler et al. (
      • Bochtler M.
      • Hartmann C.
      • Song H.K.
      • Bourenkov G.P.
      • Bartunik H.D.
      • Huber R.
      The structures of HslU and the ATP-dependent protease HslU-HslV.
      ) reported the protein crystal structure of ClpYQ, which is composed of ClpY and ClpQ hexamers (ClpY6 and ClpQ6). The centers and both ends of two ClpQ hexamers are each connected to a ClpY hexamer, thereby forming a ClpYQ complex (
      • Kessel M.
      • Wu W.F.
      • Gottesman S.
      • Kocsis E.
      • Steven A.C.
      • Maurizi M.R.
      Six-fold rotational symmetry of ClpQ, the E. coli homolog of the 20S proteasome, and its ATP-dependent activator, ClpY.
      ,
      • Missiakas D.
      • Schwager F.
      • Betton J.M.
      • Georgopoulos C.
      • Raina S.
      Identification and characterization of HsIV HsIU (ClpQ ClpY) proteins involved in overall proteolysis of misfolded proteins in Escherichia coli.
      ,
      • Rohrwild M.
      • Coux O.
      • Huang H.C.
      • Moerschell R.P.
      • Yoo S.J.
      • Seol J.H.
      • Chung C.H.
      • Goldberg A.L.
      HslV-HslU: A novel ATP-dependent protease complex in Escherichia coli related to the eukaryotic proteasome.
      ). Previous studies (
      • Bochtler M.
      • Hartmann C.
      • Song H.K.
      • Bourenkov G.P.
      • Bartunik H.D.
      • Huber R.
      The structures of HslU and the ATP-dependent protease HslU-HslV.
      ,
      • Rohrwild M.
      • Coux O.
      • Huang H.C.
      • Moerschell R.P.
      • Yoo S.J.
      • Seol J.H.
      • Chung C.H.
      • Goldberg A.L.
      HslV-HslU: A novel ATP-dependent protease complex in Escherichia coli related to the eukaryotic proteasome.
      ) have verified that ClpY can only form a ring complex under the presence of nucleotides such as of ATP, ADP, and nonhydrolysable ATP analogs (e.g. AMP-PNP or ATP-γ-S). By contrast, ClpQ can spontaneously form a six-membered ring. Therefore, synthesis of a complete ClpYQ complex must involve nucleotides including ATP (
      • Rohrwild M.
      • Pfeifer G.
      • Santarius U.
      • Muller S.A.
      • Huang H.C.
      • Engel A.
      • Baumeister W.
      • Goldberg A.L.
      The ATP-dependent HslVU protease from Escherichia coli is a four-ring structure resembling the proteasome.
      ). A previous study (
      • Bochtler M.
      • Hartmann C.
      • Song H.K.
      • Bourenkov G.P.
      • Bartunik H.D.
      • Huber R.
      The structures of HslU and the ATP-dependent protease HslU-HslV.
      ) divided the structure of ClpY into three domains: the N-terminal domain (N domain, Ser2-Lys109, Ile244-Leu332), intermediate domain (I domain, Met110-Ala243), and C-terminal domain (C domain, Gln333-Leu443). The N domain comprises ATP binding sites. The I domain is comparatively further away from ClpQ than the N domain is and contains a double-loop structure for recognizing and tethering substrates. The C domain involves aggregating the atoms in the ClpY and ClpQ complexes (
      • Lien H.Y.
      • Shy R.S.
      • Peng S.S.
      • Wu Y.L.
      • Weng Y.T.
      • Chen H.H.
      • Su P.C.
      • Ng W.F.
      • Chen Y.C.
      • Chang P.Y.
      • Wu W.F.
      Characterization of the Escherichia coli ClpY (HslU) substrate recognition site in the ClpYQ (HslUV) protease using the yeast two-hybrid system.
      ,
      • Lee Y.Y.
      • Chang C.F.
      • Kuo C.L.
      • Chen M.C.
      • Yu C.H.
      • Lin P.I.
      • Wu W.F.
      Subunit oligomerization and substrate recognition of the Escherichia coli ClpYQ (HslUV) protease implicated by in vivo protein-protein interactions in the yeast two-hybrid system.
      ,
      • Song H.K.
      • Hartmann C.
      • Ramachandran R.
      • Bochtler M.
      • Behrendt R.
      • Moroder L.
      • Huber R.
      Mutational studies on HslU and its docking mode with HslV.
      ). Previous studies on how ClpY recognizes substrates have revealed that the G90Y91V92G93 pore motif (pore 1 site) located at the center pore of ClpY is conservative and essential for unfolding and transporting substrates (
      • Sundar S.
      • Baker T.A.
      • Sauer R.T.
      The I domain of the AAA+ HslUV protease coordinates substrate binding, ATP hydrolysis, and protein degradation.
      ,
      • Park E.
      • Rho Y.M.
      • Koh O.J.
      • Ahn S.W.
      • Seong I.S.
      • Song J.J.
      • Bang O.
      • Seol J.H.
      • Wang J.
      • Eom S.H.
      • Chung C.H.
      Role of the GYVG pore motif of HslU ATPase in protein unfolding and translocation for degradation by HslV peptidase.
      ,
      • Hsieh F.C.
      • Chen C.T.
      • Weng Y.T.
      • Peng S.S.
      • Chen Y.C.
      • Huang L.-Y.
      • Hu H.T.
      • Wu Y.L.
      • Lin N.C.
      • Wu W.F.
      Stepwise activity of ClpY (HslU) mutants in the processive degradation of Escherichia coli ClpYQ (HslUV) protease substrates.
      ).
      Currently, few substrates have been identified for the ClpYQ protease in comparison with other ATP-dependent proteases (
      • Sundar S.
      • Baker T.A.
      • Sauer R.T.
      The I domain of the AAA+ HslUV protease coordinates substrate binding, ATP hydrolysis, and protein degradation.
      ,
      • Park E.
      • Rho Y.M.
      • Koh O.J.
      • Ahn S.W.
      • Seong I.S.
      • Song J.J.
      • Bang O.
      • Seol J.H.
      • Wang J.
      • Eom S.H.
      • Chung C.H.
      Role of the GYVG pore motif of HslU ATPase in protein unfolding and translocation for degradation by HslV peptidase.
      ,
      • Hsieh F.C.
      • Chen C.T.
      • Weng Y.T.
      • Peng S.S.
      • Chen Y.C.
      • Huang L.-Y.
      • Hu H.T.
      • Wu Y.L.
      • Lin N.C.
      • Wu W.F.
      Stepwise activity of ClpY (HslU) mutants in the processive degradation of Escherichia coli ClpYQ (HslUV) protease substrates.
      ). Because the substrate of a protease can reflect its role in a bacterial cell, identifying potential enzyme substrates is essential in protease research. Using substrate-enzyme interactions as indicators for screening enzyme substrates in large batches has become a prevalent approach in recent studies on ATP-dependent protease substrates (
      • Westphal K.
      • Langklotz S.
      • Thomanek N.
      • Narberhaus F.
      A Trapping approach reveals novel substrates and physiological functions of the essential protease FtsH in Escherichia coli.
      ,

      Flynn, J. M., Neher, S. B., Kim, Y.-I., Sauer, R. T., and Baker, T. A., Proteomic discovery of cellular substrates of the ClpXP protease reveals five classes of ClpX-recognition signals. Mol. Cell 11, 671–683,

      ). Because ClpY is responsible for substrate recognition, in this study, we used E. coli proteome microarrays to identify ClpY recognition proteins and the results showed that the ClpY exhibited strong interactions with the YbaB. We also used a yeast two-hybrid assay system to validate the interactions between ClpY and YbaB. Additionally, a quartz crystal microbalance (QCM)
      The abbreviations used are: QCM, quartz crystal microbalance.
      1The abbreviations used are: QCM, quartz crystal microbalance.
      was used to measure the Kd values of YbaB- ClpY interactions. The in vitro and in vivo degradation tests were conducted to confirm the degradation of YabB by ClpYQ. These results proposed the YabB is a novel target of ATP-dependent protease ClpYQ.

      DISCUSSION

      This is the first study to globally screen protease substrates using protein microarrays. Because the protein substrates on a protein chip would be degraded by the protease probe, it is very challenging to identify a protease substrate by using a regular proteome microarray assay directly. In this study, we chose an indirect strategy to first identity the protease-binding proteins. We used ClpY only, which is the recognition unit for ClpYQ protease, to screen for ClpY-binding proteins with E. coli proteome microarrays. After identifying the protein recognized by ClpY, we validated the protease activity by in vitro and in vivo degradation assay. By using this indirect strategy, we identified that the YbaB is the substrate of ClpYQ.
      When E. coli suffers in heat, the misfolding proteins will accumulate. Thus, the heat shock proteins are induced (
      • Rosen R.
      • Biran D.
      • Gur E.
      • Becher D.
      • Hecker M.
      • Ron E.Z.
      Protein aggregation in Escherichia coli: role of proteases.
      ). However; in the normal physiological state, overexpression of heat shock proteins will be toxic to the cell. Some of antibiotics, such as puromycin, induced heat shock regulation protein expression. Missiaka et al. (
      • Missiakas D.
      • Schwager F.
      • Betton J.M.
      • Georgopoulos C.
      • Raina S.
      Identification and characterization of HsIV HsIU (ClpQ ClpY) proteins involved in overall proteolysis of misfolded proteins in Escherichia coli.
      ) founds ClpYQ is able to inhibit the constitutive heat shock regulation induced by puromycin. They reported the ClpYQ can degrade abnormal “puromycyl polypeptides”. Thus, it is helping cell to lower the toxic effect of the abnormal heat shock response. The SOS response protein SulA (
      • Bi E.
      • Lutkenhaus J.
      Analysis of ftsZ mutations that confer resistance to the cell division inhibitor SulA (SfiA).
      ) is a known substrate of ClpYQ (
      • Wu W.F.
      • Zhou Y.
      • Gottesman S.
      Redundant in vivo proteolytic activities of Escherichia coli Lon and the ClpYQ (HslUV) protease.
      ). SulA inhibited the cell division when SOS response activated (
      • Bi E.
      • Lutkenhaus J.
      Analysis of ftsZ mutations that confer resistance to the cell division inhibitor SulA (SfiA).
      ). The other knowing targets of ClpYQ are RcsA, RpoH, TraJ and RNaseR (
      • Wu W.F.
      • Zhou Y.
      • Gottesman S.
      Redundant in vivo proteolytic activities of Escherichia coli Lon and the ClpYQ (HslUV) protease.
      ,
      • Kuo M.S.
      • Chen K.P.
      • Wu W.F.
      Regulation of RcsA by the ClpYQ (HslUV) protease in Escherichia coli.
      ,
      • Lau-Wong I.C.
      • Locke T.
      • Ellison M.J.
      • Raivio T.L.
      • Frost L.S.
      Activation of the Cpx regulon destabilizes the F plasmid transfer activator, TraJ, via the HslVU protease in Escherichia coli.
      ,
      • Kanemori M.
      • Nishihara K.
      • Yanagi H.
      • Yura T.
      Synergistic roles of HslVU and other ATP-dependent proteases in controlling in vivo turnover of sigma32 and abnormal proteins in Escherichia coli.
      ,
      • Liang W.
      • Deutscher M.P.
      Transfer-messenger RNA-SmpB protein regulates ribonuclease R turnover by promoting binding of HslUV and Lon proteases.
      ). RpoH is a heat shock transcription factor (
      • Kuo M.S.
      • Chen K.P.
      • Wu W.F.
      Regulation of RcsA by the ClpYQ (HslUV) protease in Escherichia coli.
      ). RcsA is a capsule synthesis activation protein and also a substrate of Lon (
      • Gottesman S.
      • Trisler P.
      • Torres-Cabassa A.
      Regulation of capsular polysaccharide synthesis in Escherichia coli K-12: characterization of three regulatory genes.
      ). TraJ is responsible for F plasmid conjugation activation (
      • Lau-Wong I.C.
      • Locke T.
      • Ellison M.J.
      • Raivio T.L.
      • Frost L.S.
      Activation of the Cpx regulon destabilizes the F plasmid transfer activator, TraJ, via the HslVU protease in Escherichia coli.
      ). RNaseR is an important exoribonuclease responsible for degradation of structured RNAs in E. coli (
      • Liang W.
      • Deutscher M.P.
      Transfer-messenger RNA-SmpB protein regulates ribonuclease R turnover by promoting binding of HslUV and Lon proteases.
      ). These findings suggested the ClpYQ participate in important cell physiological functions. In this study, we showed that YbaB is a novel target of ClpYQ. YbaB is a DNA binding protein with a probable histone-like activity in bacterial cells that facilitates packing the genetic materials of prokaryotes by forming nucleoids (
      • Jutras B.L.
      • Bowman A.
      • Brissette C.A.
      • Adams C.A.
      • Verma A.
      • Chenail A.M.
      • Stevenson B.
      EbfC (YbaB) is a new type of bacterial nucleoid-associated protein and a global regulator of gene expression in the lyme disease spirochete.
      ,
      • Dillon S.C.
      • Dorman C.J.
      Bacterial nucleoid-associated proteins, nucleoid structure and gene expression.
      ). However, the actual function of YbaB remains unclear (
      • Jutras B.L.
      • Bowman A.
      • Brissette C.A.
      • Adams C.A.
      • Verma A.
      • Chenail A.M.
      • Stevenson B.
      EbfC (YbaB) is a new type of bacterial nucleoid-associated protein and a global regulator of gene expression in the lyme disease spirochete.
      ). Because YbaB is a histone-like protein, it may mediate gene-silencing and antisilencing activities through regulating the nucleoid structure (
      • Dillon S.C.
      • Dorman C.J.
      Bacterial nucleoid-associated proteins, nucleoid structure and gene expression.
      ). This suggested that ClpYQ protease participated in the YbaB's regulations functions and other gene regulation cascade. Also, YbaB shares homology with many pathogens, such as Haemophilus influenzae, E. coli, Vibrio cholerae, Pseudomonas putida, Rickettsia rickettsiae, Neisseria gonorrhoeae, Bdellovibrio bacteriovorus, Clostridium perfringens, Bacillus subtilis, Enterococcus faecalis, Streptococcus pneumoniae, Mycobacterium tuberculosis, Bacteroides capillosus, and Borrelia burgdorferi (
      • Cooley A.E.
      • Riley S.P.
      • Kral K.
      • Miller M.C.
      • DeMoll E.
      • Fried M.G.
      • Stevenson B.
      DNA-binding by Haemophilus influenzaeEscherichia coli YbaB, members of a widely-distributed bacterial protein family.
      ). Their hosts include human, plants, and animals. This suggests that the regulation of YbaB function through digestion by ClpYQ is a very important mechanism in bacteria.

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