Browse > Article

Nucleotide Triphosphates Inhibit the Degradation of Unfolded Proteins by HslV Peptidase  

Lee, Jung Wook (School of Biological Sciences, Seoul National University)
Park, Eunyong (School of Biological Sciences, Seoul National University)
Bang, Oksun (School of Biological Sciences, Seoul National University)
Eom, Soo-Hyun (Department of Biological Sciences, Gwangju Institute of Science and Technology)
Cheong, Gang-Won (Division of Applied Life Sciences and Environmental Biotechnology National Core Research Center, Gyeongsang National University)
Chung, Chin Ha (School of Biological Sciences, Seoul National University)
Seol, Jae Hong (School of Biological Sciences, Seoul National University)
Publication Information
Molecules and Cells / v.23, no.2, 2007 , pp. 252-257 More about this Journal
Abstract
Escherichia coli HslVU is an ATP-dependent protease consisting of two heat shock proteins, the HslU ATPase and HslV peptidase. In the reconstituted enzyme, HslU stimulates the proteolytic activity of HslV by one to two orders of magnitude, while HslV increases the rate of ATP hydrolysis by HslU several-fold. Here we show that HslV alone can efficiently degrade certain unfolded proteins, such as unfolded lactalbumin and lysozyme prepared by complete reduction of disulfide bonds, but not their native forms. Furthermore, HslV alone cleaved a lactalbumin fragment sandwiched by two thioredoxin molecules, indicating that it can hydrolyze the internal peptide bonds of lactalbumin. Surprisingly, ATP inhibited the degradation of unfolded proteins by HslV. This inhibitory effect of ATP was markedly diminished by substitution of the Arg86 residue located in the apical pore of HslV with Gly, suggesting that interaction of ATP with the Arg residue blocks access of unfolded proteins to the proteolytic chamber of HslV. These results suggest that uncomplexed HslV is inactive under normal conditions, but may can degrade unfolded proteins when the ATP level is low, as it is during carbon starvation.
Keywords
ATP-dependent Protease; ATPase; HslU; HslV; Lactalbumin; Unfolded Proteins;
Citations & Related Records
Times Cited By KSCI : 1  (Citation Analysis)
Times Cited By Web Of Science : 1  (Related Records In Web of Science)
연도 인용수 순위
1 Bochtler, M., Ditzel, L., Groll, M., and Huber, R. (1997) Crystal structure of heat shock locus V (HslV) from Escherichia coli. Proc. Natl. Acad. Sci. USA 94, 6070−6074
2 Bochtler, M., Hartmann, C., Song, H. K., Bourenkov, G. P., Bartunik, H. D., et al. (2000) The structures of HslU and the ATPdependent protease HslU-HslV. Nature 403, 800−805
3 Chung, C. H. (1993) Proteases in Escherichia coli. Science 262, 372−374   DOI
4 Gottesman, S. and Maurizi, M. R. (1992) Regulation by proteolysis: energy-dependent proteases and their targets. Microbiol. Rev. 56, 592−621
5 Hiraoka, Y., Segawa, T., Kuwajima, K., Sugai, S., and Murai, N. (1980) Alpha-Lactalbumin: a calcium metalloprotein. Biochem. Biophys. Res. Commun. 95, 1098−10104
6 Schagger, H. and von Jagow, G. (1987) Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal. Biochem. 166, 368−379
7 Seemuller, E., Lupas, A., Stock, D., Lowe, J., Huber, R., et al. (1995) Proteasome from Thermoplasma acidophilum: a threonine protease. Science 268, 579−582   DOI
8 Seong, I. S., Oh, J. Y., Yoo, S. J., Seol, J. H., and Chung, C. H. (1999) ATP-dependent degradation of SulA, a cell division inhibitor, by the HslVU protease in Escherichia coli. FEBS Lett. 456, 211−214
9 Wang, J., Hartling, J. A., and Flanagan, J. M. (1998) Crystal structure determination of Escherichia coli ClpP starting from an EM-derived mask. J. Struct. Biol. 124, 151−163
10 Wang, J., Song, J. J., Seong, I. S., Franklin, M. C., Kamtekar, S., et al. (2001b) Nucleotide-dependent conformational changes in a protease-associated ATPase HsIU. Structure 9, 1107−1116
11 Wenzel, T. and Baumeister, W. (1995) Conformational constraints in protein degradation by the 20S proteasome. Nat. Struct. Biol. 2, 199−204
12 Yoo, S. J., Shim, Y. K., Seong, I. S., Seol, J. H., Kang, M. S., et al. (1997a) Mutagenesis of two N-terminal Thr and five Ser residues in HslV, the proteolytic component of the ATP-dependent HslVU protease. FEBS Lett. 412, 57−60
13 Acharya, K. R., Ren, J. S., Stuart, D. I., Phillips, D. C., and Fenna, R. E. (1991) Crystal structure of human alpha-lactalbumin at 1.7 $\AA$ resolution. J. Mol. Biol. 221, 571−581
14 De Mot, R., Nagy, I., Walz, J., and Baumeister, W. (1999) Proteasomes and other self-compartmentalizing proteases in prokaryotes. Trends Microbiol. 7, 88−92
15 Gottesman, S. (2003) Proteolysis in bacterial regulatory circuits. Annu. Rev. Cell Dev. Biol. 19, 565−587
16 Maurizi, M. R. (1992) Proteases and protein degradation in Escherichia coli. Experientia 48, 178−201   DOI   ScienceOn
17 Seong, I. S., Kang, M. S., Choi, M. K., Lee, J. W., Koh, O. J., et al. (2002) The C-terminal tails of HslU ATPase act as a molecular switch for activation of HslV peptidase. J. Biol. Chem. 277, 25976−25982
18 Groll, M., Bajorek, M., Kohler, A., Moroder, L., Rubin, D. M., et al. (2000) A gated channel into the proteasome core particle. Nat. Struct. Biol. 7, 1062−1067
19 Park, S. C., Jia, B., Yang, J. K., Van, D. L., Shao, Y. G., et al. (2006) Oligomeric structure of the ATP-dependent protease La (Lon) of Escherichia coli. Mol. Cells 21, 129−134
20 Rohrwild, M., Coux, O., Huang, H. C., Moerschell, R. P., Yoo, S. J., et al. (1996) HslV-HslU: A novel ATP-dependent protease complex in Escherichia coli related to the eukaryotic proteasome. Proc. Natl. Acad. Sci. USA 93, 5808−5813
21 Sousa, M. C., Trame, C. B., Tsuruta, H., Wilbanks, S. M., Reddy, V. S., et al. (2000) Crystal and solution structures of an HslUV protease-chaperone complex. Cell 103, 633−643
22 Yoo, S. J., Seol, J. H., Shin, D. H., Rohrwild, M., Kang, M. S., et al. (1996) Purification and characterization of the heat shock proteins HslV and HslU that form a new ATP-de-pendent protease in Escherichia coli. J. Biol. Chem. 271, 14035−14040
23 Bradford, M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248−254
24 Chung, C. H., Yoo, S. J., Seol, J. H., and Kang, M. S. (1997) Characterization of energy-dependent proteases in bacteria. Biochem. Biophys. Res. Commun. 241, 613−616
25 Ewbank, J. J. and Creighton, T. E. (1993) Pathway of disulfidecoupled unfolding and refolding of bovine alpha-lactalbumin. Biochemistry, 32, 3677−3693
26 Goldber, A. L. and St. John, A. C. (1976) Intracellular protein degradation in mammalian and bacterial cells: part 2. Annu. Rev. Biochem. 45, 747−803
27 Wang, J., Song, J. J., Franklin, M. C., Kamtekar, S., Im, Y. J., et al. (2001a) Crystal structures of the HslVU peptidase-ATPase complex reveal an ATP-dependent proteolysis mechanism. Structure 9, 177−184
28 Whitby, F. G., Masters, E. I., Kramer, L., Knowlton, J. R., Yao, Y., et al. (2000) Structural basis for the activation of 20S proteasomes by 11S regulators. Nature 408, 115−120   DOI
29 Goldberg, A. L. (1992) The mechanism and functions of ATPdependent proteases in bacterial and animal cells. Eur. J. Biochem. 203, 9−23
30 Yoo, S. J., Seol, J. H., Seong, I. S., Kang, M. S., and Chung, C. H. (1997b) ATP binding, but not its hydrolysis, is required for assembly and proteolytic activity of the HslVU protease in Escherichia coli. Biochem. Biophys. Res. Commun. 238, 581−585
31 Gaal, T., Bartlett, M. S., Ross, W., Turnbough, C. L. Jr., and Gourse, R. L. (1997) Transcription regulation by initiating NTP concentration: rRNA synthesis in bacteria. Science 278, 2092−2097
32 Seong, I. S., Oh, J. Y., Lee, J. W., Tanaka, K., and Chung, C. H. (2000) The HslU ATPase acts as a molecular chaperone in prevention of aggregation of SulA, an inhibitor of cell division in Escherichia coli. FEBS Lett. 477, 224−229   DOI   ScienceOn
33 Chuang, S. E., Burland, V., Plunkett, G., 3rd, Daniels, D. L., and Blattner, F. R. (1993) Sequence analysis of four new heatshock genes constituting the hslTS/ibpAB and hslVU operons in Escherichia coli. Gene 134, 1−6
34 Liu, C. W., Corboy, M. J., DeMartino, G. N., and Thomas, P. J. (2003) Endoproteolytic activity of the proteasome. Science 299, 408−411   DOI   ScienceOn
35 Park, E., Rho, Y. M., Koh, O., Ahn, S. W., Seong, I. S., et al. (2005) Role of the GYVG pore motif of HslU ATPase in protein unfolding and translocation for degradation by HslV peptidase J. Biol. Chem. 280, 22892−22898
36 Gottesman, S. (1996) Proteases and their targets in Escherichia coli. Annu. Rev. Genet. 30, 465−506
37 Holt, C. and Sawyer, L. (1988) Primary and predicted secondary structures of the caseins in relation to their biological functions. Protein Eng. 2, 251−259
38 St. John, A. C. and Goldberg, A. L. (1978) Effects of reduced energy production on protein degradation, guanosine tetraphosphate, and RNA synthesis in Escherichia coli. J. Biol. Chem. 253, 2705−2711
39 Syme, C. D., Blanch, E. W., Holt, C., Jakes, R., Goedert, M., et al. (2002) A raman optical activity study of rheomorphism in caseins, synucleins and tau. New insight into the structure and behavior of natively unfolded proteins. Eur. J. Biochem. 269, 148−156
40 Seol, J. H., Yoo, S. J., Shin, D. H., Shim, Y. K., Kang, M. S., et al. (1997) The heat-shock protein HslVU from Escherichia coli is a protein-activated ATPase as well as an ATP-de-pendent proteinase. Eur. J. Biochem. 247, 1143−1150