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• W2013025360 abstract "HslVU is an ATP-dependent protease in bacteria consisting of HslV dodecamer and HslU hexamer. Upon ATP binding, HslU ATPase allosterically activates the catalytic function of HslV protease by 1–2 orders of magnitude. However, relatively little is known about the role of HslV in the control of HslU function. Here we describe the involvement of the N-terminal Thr active sites (Thr-1) of HslV in the communication between HslV and HslU. Binding of proteasome inhibitors to Thr-1 led to a dramatic increase in the interaction between HslV and HslU with a marked increase in ATP hydrolysis by HslU. Moreover, carbobenzoxy-leucyl-leucyl-leucinal (MG132) could bind to Thr-1 of free HslV, and this binding induced a tight interaction between HslV and HslU with the activation of HslU ATPase, suggesting that substrate-bound HslV can allosterically regulate HslU function. Unexpectedly, the deletion of Thr-1 also caused a dramatic increase in the affinity between HslV and HslU even in the absence of ATP. Furthermore, the increase in the number of the Thr-1 deletion mutant subunit in place of HslV subunit in a dodecamer led to a proportional increase in the affinity between HslV and HslU with gradual activation of HslU ATPase. Although the molecular mechanism elucidating how the Thr-1 deletion influences the interaction between HslV and HslU remains unknown, these results suggest an additional allosteric mechanism for the control of HslU function by HslV. Taken together, our findings indicate a critical involvement of Thr-1 of HslV in the reciprocal control of HslU function and, thus, for their communication. HslVU is an ATP-dependent protease in bacteria consisting of HslV dodecamer and HslU hexamer. Upon ATP binding, HslU ATPase allosterically activates the catalytic function of HslV protease by 1–2 orders of magnitude. However, relatively little is known about the role of HslV in the control of HslU function. Here we describe the involvement of the N-terminal Thr active sites (Thr-1) of HslV in the communication between HslV and HslU. Binding of proteasome inhibitors to Thr-1 led to a dramatic increase in the interaction between HslV and HslU with a marked increase in ATP hydrolysis by HslU. Moreover, carbobenzoxy-leucyl-leucyl-leucinal (MG132) could bind to Thr-1 of free HslV, and this binding induced a tight interaction between HslV and HslU with the activation of HslU ATPase, suggesting that substrate-bound HslV can allosterically regulate HslU function. Unexpectedly, the deletion of Thr-1 also caused a dramatic increase in the affinity between HslV and HslU even in the absence of ATP. Furthermore, the increase in the number of the Thr-1 deletion mutant subunit in place of HslV subunit in a dodecamer led to a proportional increase in the affinity between HslV and HslU with gradual activation of HslU ATPase. Although the molecular mechanism elucidating how the Thr-1 deletion influences the interaction between HslV and HslU remains unknown, these results suggest an additional allosteric mechanism for the control of HslU function by HslV. Taken together, our findings indicate a critical involvement of Thr-1 of HslV in the reciprocal control of HslU function and, thus, for their communication. Binding of MG132 or deletion of the Thr active sites in HslV subunits increases the affinity of HslV protease for HslU ATPase and makes this interaction nucleotide-independent.Journal of Biological ChemistryVol. 284Issue 25PreviewWe suggest that subscribers photocopy these corrections and insert the photocopies in the original publication at the location of the original article. Authors are urged to introduce these corrections into any reprints they distribute. Secondary (abstract) services are urged to carry notice of these corrections as prominently as they carried the original abstracts. Full-Text PDF Open Access HslVU is a two-component ATP-dependent protease in bacteria that comprises HslV protease and HslU ATPase (1Rohrwild M. Coux O. Huang H.C. Moerschell R.P. Yoo S.J. Seol J.H. Chung C.H. Goldberg A.L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5808-5813Crossref PubMed Scopus (213) Google Scholar, 2Yoo S.J. Seol J.H. Shin D.H. Rohrwild M. Kang M.S. Tanaka K. Goldberg A.L. Chung C.H. J. Biol. Chem. 1996; 271: 14035-14040Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 3Kessel M. Wu W. Gottesman S. Kocsis E. Steven A.C. Maurizi M.R. FEBS Lett. 1996; 398: 274-278Crossref PubMed Scopus (103) Google Scholar, 4Missiakas D. Schwager F. Betton J.M. Georgopoulos C. Raina S. EMBO J. 1996; 15: 6899-6909Crossref PubMed Scopus (154) Google Scholar, 5Rohrwild M. Pfeifer G. Santarius U. Muller S.A. Huang H.C. Engel A. Baumeister W. Goldberg A.L. Nat. Struct. Biol. 1997; 4: 133-139Crossref PubMed Scopus (175) Google Scholar). HslV, a homolog of the β-subunit of 20 S proteasome, is a self-compartmentalized protease that has two stacked hexameric rings of identical subunits, each of which has an N-terminal Thr (Thr-1) active site for proteolysis (6Yoo S.J. Shim Y.K. Seong I.S. Seol J.H. Kang M.S. Chung C.H. FEBS Lett. 1997; 412: 57-60Crossref PubMed Scopus (36) Google Scholar, 7Seemuller E. Lupas A. Baumeister W. Nature. 1996; 382: 468-471Crossref PubMed Scopus (183) Google Scholar, 8Bochtler M. Ditzel L. Groll M. Huber R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6070-6074Crossref PubMed Scopus (167) Google Scholar, 9Bochtler M. Hartmann C. Song H.K. Bourenkov G.P. Bartunik H.D. Huber R. Nature. 2000; 403: 800-805Crossref PubMed Scopus (378) Google Scholar, 10Sousa M.C. Trame C.B. Tsuruta H. Wilbanks S.M. Reddy V.S. McKay D.B. Cell. 2000; 103: 633-643Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar, 11Wang J. Song J.J. Franklin M.C. Kamtekar S. Im Y.J. Rho S.H. Seong I.S. Lee C.S. Chung C.H. Eom S.H. Structure. 2001; 9: 177-184Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar, 12Wang J. Song J.J. Seong I.S. Franklin M.C. Kamtekar S. Eom S.H. Chung C.H. Structure. 2001; 9: 1107-1116Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). The hexameric HslU ATPase, a member of AAA family (13Patel S. Latterich M. Trends Cell Biol. 1998; 8: 65-71Crossref PubMed Google Scholar, 14Neuwald A.F. Aravind L. Spouge J.L. Koonin E.V. Genome Res. 1999; 9: 27-43Crossref PubMed Google Scholar), binds to either one or both ends of an HslV dodecamer to form the HslVU complex. In the HslVU complex, the HslU and HslV central pores are aligned and the proteolytic active sites are sequestered in the internal chamber of HslV, with access to this chamber restricted to small axial pores (7Seemuller E. Lupas A. Baumeister W. Nature. 1996; 382: 468-471Crossref PubMed Scopus (183) Google Scholar, 8Bochtler M. Ditzel L. Groll M. Huber R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6070-6074Crossref PubMed Scopus (167) Google Scholar, 9Bochtler M. Hartmann C. Song H.K. Bourenkov G.P. Bartunik H.D. Huber R. Nature. 2000; 403: 800-805Crossref PubMed Scopus (378) Google Scholar, 10Sousa M.C. Trame C.B. Tsuruta H. Wilbanks S.M. Reddy V.S. McKay D.B. Cell. 2000; 103: 633-643Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar, 11Wang J. Song J.J. Franklin M.C. Kamtekar S. Im Y.J. Rho S.H. Seong I.S. Lee C.S. Chung C.H. Eom S.H. Structure. 2001; 9: 177-184Abstract Full Text Full Text PDF PubMed Scopus (219) Google Scholar). Biochemical studies have shown that ATP binding and its subsequent hydrolysis by HslU play essential roles in controlling the proteolytic function of HslV and the interaction between HslU and HslV (10Sousa M.C. Trame C.B. Tsuruta H. Wilbanks S.M. Reddy V.S. McKay D.B. Cell. 2000; 103: 633-643Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar, 12Wang J. Song J.J. Seong I.S. Franklin M.C. Kamtekar S. Eom S.H. Chung C.H. Structure. 2001; 9: 1107-1116Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar, 15Huang H. Goldberg A.L. J. Biol. Chem. 1997; 272: 21364-21372Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 16Seol J.H. Yoo S.J. Shin D.H. Shim Y.K. Kang M.S. Goldberg A.L. Chung C.H. Eur. J. Biochem. 1997; 247: 1143-1150Crossref PubMed Scopus (51) Google Scholar, 17Yoo S.J. Seol J.H. Seong I.S. Kang M.S. Chung C.H. Biochem. Biophys. Res. Commun. 1997; 238: 581-585Crossref PubMed Scopus (37) Google Scholar). Hexamerization of HslU itself is largely favored by the nucleotide binding to the ATPase (17Yoo S.J. Seol J.H. Seong I.S. Kang M.S. Chung C.H. Biochem. Biophys. Res. Commun. 1997; 238: 581-585Crossref PubMed Scopus (37) Google Scholar). Moreover, HslV that by itself is a weak peptidase can be activated 1–2 orders of magnitude by ATP-bound HslU (15Huang H. Goldberg A.L. J. Biol. Chem. 1997; 272: 21364-21372Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 16Seol J.H. Yoo S.J. Shin D.H. Shim Y.K. Kang M.S. Goldberg A.L. Chung C.H. Eur. J. Biochem. 1997; 247: 1143-1150Crossref PubMed Scopus (51) Google Scholar). ATPγS, 4The abbreviations used are: ATPγS, adenosine 5′-O-(thiotriphosphate); MG132, carbobenzoxy-leucyl-leucyl-leucinal; NLVS, 4-hydroxy-5-iodo-3-nitrophenylacetyl-Leu-Leu-Leu-vinylsulfone; Z-GGL-AMC, carbobenzoxy-Gly-Gly-Leu-7-amido-4-methyl coumarin; CP, core particle; RP, regulatory particle; NTA, nitrilotriacetic acid. a nonhydrolyzable ATP analog, also supports HslV-mediated hydrolysis of small peptides but not that of native protein substrates, such as SulA, suggesting the role of ATP hydrolysis by HslU in unfolding of protein substrates for their access to and subsequent degradation at the inner proteolytic chamber of dodecameric HslV (18Seong I.S. Oh J.Y. Yoo S.J. Seol J.H. Chung C.H. FEBS Lett. 1999; 456: 211-214Crossref PubMed Scopus (64) Google Scholar). Importantly, chemical cross-linking analysis has shown that ATP-bound HslU interacts with HslV to form the HslVU complex, but ADP-bound HslU does not, implicating dynamic interaction between HslU and HslV during ATP hydrolysis cycles (17Yoo S.J. Seol J.H. Seong I.S. Kang M.S. Chung C.H. Biochem. Biophys. Res. Commun. 1997; 238: 581-585Crossref PubMed Scopus (37) Google Scholar). However, it was unknown how the HslVU complex is maintained during threading of unfolded polypeptide from HslU into the inner chamber of HslV and subsequent cleavage of peptide bonds at the Thr-1 active sites for the completion of a proteolytic cycle. Unlike eukaryotic 20 S proteasomes where substrate accessibility to proteolytic active sites is controlled by opening-and-closing the apical gates of α subunits (19Forster A. Whitby F.G. Hill C.P. EMBO J. 2003; 22: 4356-4364Crossref PubMed Scopus (96) Google Scholar, 20Groll M. Bajorek M. Kohler A. Moroder L. Rubin D.M. Huber R. Glickman M.H. Finley D. Nat. Struct. Biol. 2000; 7: 1062-1067Crossref PubMed Scopus (660) Google Scholar, 21Smith D.M. Kafri G. Cheng Y. Ng D. Walz T. Goldberg A.L. Mol. Cell. 2005; 20: 687-698Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar), HslVU has been shown to utilize an allosteric mechanism whereby the active sites of HslV are switched on-and-off through the nucleotide-dependent interaction of HslU with HslV (12Wang J. Song J.J. Seong I.S. Franklin M.C. Kamtekar S. Eom S.H. Chung C.H. Structure. 2001; 9: 1107-1116Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). Specifically, the C-terminal tails of HslU show a dramatic movement in a nucleotide-dependent manner (i.e. they move toward HslV-HslV subunit interfaces from HslU-HslU subunit interfaces when ATP is bound) (10Sousa M.C. Trame C.B. Tsuruta H. Wilbanks S.M. Reddy V.S. McKay D.B. Cell. 2000; 103: 633-643Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar, 12Wang J. Song J.J. Seong I.S. Franklin M.C. Kamtekar S. Eom S.H. Chung C.H. Structure. 2001; 9: 1107-1116Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar, 22Sousa M.C. Kessler B.M. Overkleeft H.S. McKay D.B. J. Mol. Biol. 2002; 318: 779-785Crossref PubMed Scopus (63) Google Scholar). Moreover, a synthetic HslU tail peptide of 10 amino acids could replace HslU in the activation of HslV-mediated peptide hydrolysis (23Ramachandran R. Hartmann C. Song H.K. Huber R. Bochtler M. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 7396-7401Crossref PubMed Scopus (60) Google Scholar, 24Seong I.S. Kang M.S. Choi M.K. Lee J.W. Koh O.J. Wang J. Eom S.H. Chung C.H. J. Biol. Chem. 2002; 277: 25976-25982Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). Thus, it appears that HslU allosterically regulates the proteolytic function of HslV in a nucleotide-dependent fashion. On the other hand, relatively little is known about the role of HslV in the control of HslU function except for its ability to stimulate the ATPase activity several fold. In the present study we demonstrate that binding of proteasome inhibitors to the Thr-1 residues, which likely mimic the substrate-bound state of the active sites, dramatically increases the interaction between HslV and HslU. Significantly, MG132, unlike lactacystin or 4-hydroxy-5-iodo-3-nitrophenylacetyl-Leu-Leu-Leu-vinylsulfone (NLVS), could induce the interaction of HslV with HslU even in the absence of nucleotide or the sole presence of ADP. These findings provide a mechanism for the maintenance of stable HslVU complexes when substrates are bound to the Thr-1 active sites for the completion of a proteolytic cycle. Surprisingly, deletion of the Thr-1 residues was found to also cause a dramatic increase in the interaction between HslV and HslU in the absence of ATP. Collectively, our findings indicate that the N-terminal Thr active sites of HslV are involved in the communication between HslV and HslU in addition to its role in the catalysis of peptide bond cleavage. Materials—Enzymes for DNA cloning were purchased from Takara, New England Biolabs, and Stratagene. Carbobenzoxy-Gly-Gly-Leu-amidomethyl coumarin (Z-GGL-AMC) was purchased from Bachem. MG132, lactacystin, and NLVS were obtained from A. G. Scientific, Cayman Chemical, and Calbiochem, respectively. Other reagents were purchased from Sigma unless otherwise indicated. Strains and Cloning—BW25113 ΔhslVU::kan strain was generated from BW25113 strain by using λRed system (25Datsenko K.A. Wanner B.L. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6640-6645Crossref PubMed Scopus (11284) Google Scholar). Two primers (forward, gat gaa aat gat tga acg cga tta tag gat aaa acg gct cac tgg gct atc tgg aca agg; reverse, ccc cat cta taa ttg cat tat gcc ccg tac ttt tgt acg gcg tcc cgg aaa acg att ccg; the hslVU homologous regions are underlined) and pKD13 as a template were used to produce a PCR product for homologous recombination. The deletion of hslVU operon was confirmed by PCR and immunoblot analysis. pBR-PL was constructed from pBR322 by substituting the HindIII-NruI segment of the vector with a polylinker (aa gct tAC TAG TTA CCG CGG TCG ACA TCC ATG GAG CTC GGG CCC cga; the lowercase indicates vector sequences). pV-1 expressing only HslV was generated by deleting the NruI-BglII segment of hslU gene in pGEM-T/HslVU vector. Site-directed mutagenesis (QuikChange, Stratagene) was performed to insert the His6 tag at the C terminus of HslV (SYKAHHHHHH; the HslV sequence is underlined), resulting in pVH-1 vector. Mutations in start codons and Thr-1 deletion were also generated by site-directed mutagenesis. All mutations were confirmed by DNA sequencing. Vectors for the production of mixed dodecamers consisting of HslV and Thr-1-deletion mutant (T1Δ) subunits (Table 1) were constructed by sequential insertions of hslV and t1Δ genes (restriction fragments of pV-1 or pVH-1) into the polylinker site of pBR-PL.TABLE 1Vector constructs used for in vivo generation of HslV mixed dodecamers The start codons used in the constructs were GTG in hslv, ATG in hslVa, TTG in hslVb, and GTG in t1Δ (see Fig. 5B).ConstructsGene 1Gene 2Gene 3ahslVahslVat1ΔbhslVat1ΔnonechslVat1Δnonedt1ΔhslVnoneet1Δt1ΔhslVft1Δt1ΔhslVb Open table in a new tab Protein Expression and Purification—HslU and HslV were purified as described previously (2Yoo S.J. Seol J.H. Shin D.H. Rohrwild M. Kang M.S. Tanaka K. Goldberg A.L. Chung C.H. J. Biol. Chem. 1996; 271: 14035-14040Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 16Seol J.H. Yoo S.J. Shin D.H. Shim Y.K. Kang M.S. Goldberg A.L. Chung C.H. Eur. J. Biochem. 1997; 247: 1143-1150Crossref PubMed Scopus (51) Google Scholar). pETDuet-1 vectors (Novagen) were used for co-expression of HslU and His-tagged HslV proteins. BL21 (DE3) ΔhslVU cells transformed with the vectors were cultured at 37 °C to an optical density of 0.5–0.6 at 600 nm and then treated with 0.1 mm isopropyl 1-thio-β-d-galactopyranoside for 30 min for protein induction. To express HslV mixed dodecamers, BW25113 ΔhslVU::kan cells harboring appropriate vectors were grown overnight at 37 °C in Luria broth supplemented with ampicillin. Proteins were purified by using Ni2+-nitrilotriacetic acid (NTA)-agarose columns according to the manufacturer's instruction (Qiagen) with some modifications; i.e. imidazole was used at 50–60 mm for washing and at 450 mm for elution. Purified proteins were dialyzed against 20 mm Tris-HCl buffer (pH 7.8) containing 100 mm NaCl, 0.5 mm EDTA, 1 mm dithiothreitol, and 10% glycerol and stored at -70 °C for further uses. Protein concentration was measured by the Bradford method using bovine serum albumin as a standard. NTA Pulldown Analysis—Reaction mixtures (0.5 ml) containing HslU (150 nm) and His-HslV (75 nm) in 50 mm HEPES buffer (pH 8) containing 150 mm NaCl, 5% glycerol, and 0.04% Triton X-100 were incubated at 4 °C for 1 h in the absence or presence of 2 mm adenine nucleotides and 5 mm MgCl2. After incubation, the mixtures were supplemented with 10 μl of 1 m imidazole and 20 μl of Ni2+-NTA resins and rocked at 4 °C for 1 h. The resins were washed 4 times with 0.5 ml of 50 mm HEPES buffer (pH 8) containing 300 mm NaCl, 5 mm MgCl2, 60 mm imidazole, 5% glycerol, 0.04% Triton X-100, and 2 mm adenine nucleotides. Proteins bound to NTA resins were eluted by SDS sampling buffer, subjected to SDS-PAGE, and stained with Coomassie Blue R-250. Assays—ATP hydrolysis was measured using an enzyme-coupled assay (26Norby J.G. Methods Enzymol. 1988; 156: 116-119Crossref PubMed Scopus (254) Google Scholar). HslU (0.2 μm) and HslV (0.2 μm) in 100 mm Tris-HCl buffer (pH 8) containing 150 mm NaCl, 2 mm KCl, 5 mm MgCl2, and 0.5 mm EDTA were incubated at 37 °C with 2 mm ATP, 3 mm phosphoenolpyruvate, 0.5 mm NADH, 20 units/ml of pyruvate kinase, and 20 units/ml of lactic dehydrogenase. Absorbance at 340 nm was continuously recorded using a spectrophotometer (Ultrospec2000, GE Healthcare) equipped with a temperature controller. The rate of ATP hydrolysis was calculated from the slope within a linear range, based on the extinction coefficient of NADH (ϵ340 nm = 6.22 × 103). Peptide hydrolysis was assayed by incubation of HslU (10 nm) and HslV (5 nm) in 100 mm Tris-HCl buffer (pH 8) containing 5 mm MgCl2, 0.5 mm EDTA, and 2 mm ATP with 0.1 mm Z-GGL-AMC at 37 °C (27Park 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. J. Biol. Chem. 2005; 280: 22892-22898Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). Fluorescence (λexcitation = 355 nm, λemission = 460 nm) of released AMC was continuously measured using a fluorometer (FluoStar, BMG) equipped with a temperature controller. The rate of peptide hydrolysis was then calculated from the slope within a linear range. Gel Filtration by Spin-column—Spin-columns were prepared by packing Sephadex G-25 (0.2 ml) in 0.5-ml Eppendorf tubes that have a hole at the tip of their bottoms. After equilibration with 20 mm Tris-HCl buffer (pH 7.8) containing 100 mm NaCl and 5% glycerol, they were loaded with HslV incubated with proteasome inhibitors, put in 1.5-ml Eppendorf tubes, and spun for 10 s. The samples eluted from the columns were then used for further analysis. Effects of Proteasome Inhibitors on the Interaction between HslV and HslU—To facilitate the purification of HslV and to assay the interaction between HslV and HslU by NTA pulldown analysis, poly-His (His6) was tagged to the C-terminal end of HslV. The resulting protein (referred to as HslV-His) was purified to apparent homogeneity and assayed for its ability to cleave Z-GGL-AMC in the presence of HslU. We also examined the ability of HslV-His to promote ATP hydrolysis by HslU. HslV-His cleaved the peptide and stimulated the ATP hydrolysis by HslU as well as HslV (Fig. 1A). In the presence of HslU and ATP, HslV-His could also degrade protein substrates, including α-casein and MBP-SulA as well as HslV (data not shown). These results indicate that the C-terminal His tag does not interfere with the interaction between HslV and HslU. HslV and HslU interact with each other (i.e. form the HslVU complex) in the presence of ATP, and this interaction is required for their mutual activation (1Rohrwild M. Coux O. Huang H.C. Moerschell R.P. Yoo S.J. Seol J.H. Chung C.H. Goldberg A.L. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5808-5813Crossref PubMed Scopus (213) Google Scholar, 2Yoo S.J. Seol J.H. Shin D.H. Rohrwild M. Kang M.S. Tanaka K. Goldberg A.L. Chung C.H. J. Biol. Chem. 1996; 271: 14035-14040Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, 3Kessel M. Wu W. Gottesman S. Kocsis E. Steven A.C. Maurizi M.R. FEBS Lett. 1996; 398: 274-278Crossref PubMed Scopus (103) Google Scholar, 17Yoo S.J. Seol J.H. Seong I.S. Kang M.S. Chung C.H. Biochem. Biophys. Res. Commun. 1997; 238: 581-585Crossref PubMed Scopus (37) Google Scholar). In an attempt to determine how HslV allosterically activates HslU ATPase, we first monitored the interaction between HslV and HslU in the presence of each of three well known proteasome inhibitors: MG132, lactacystin, and NLVS. MG132 is known to reversibly react with the N-terminal Thr active sites of the 20 S proteasome β-subunits, whereas lactacystin and NLVS irreversibly modify them (22Sousa M.C. Kessler B.M. Overkleeft H.S. McKay D.B. J. Mol. Biol. 2002; 318: 779-785Crossref PubMed Scopus (63) Google Scholar, 28Lee D.H. Goldberg A.L. J. Biol. Chem. 1996; 271: 27280-27284Abstract Full Text Full Text PDF PubMed Scopus (344) Google Scholar, 29Bogyo M. McMaster J.S. Gaczynska M. Tortorella D. Goldberg A.L. Ploegh H. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6629-6634Crossref PubMed Scopus (408) Google Scholar, 30Kisselev A.F. Goldberg A.L. Chem. Biol. 2001; 8: 739-758Abstract Full Text Full Text PDF PubMed Scopus (1001) Google Scholar). HslV-His was incubated with each of the inhibitors in the absence or presence of adenine nucleotides. The samples were then subjected to NTA pulldown analysis. In the presence of ATP, all three inhibitors caused a dramatic increase in the amount of HslU co-precipitated with His-HslV as compared with DMSO that was used as a control (Fig. 1B, upper panel). Under the same experimental conditions, these inhibitors abolished the peptidase activity of HslV, indicating that they efficiently block the Thr-1 active sites of HslV (Fig. 1C, upper panel). These results demonstrate that the binding of proteasome inhibitors to the Thr-1 active sites leads to a marked increase in the interaction between HslV and HslU. Unexpectedly, MG132, but not lactacystin or NLVS, could increase the interaction of HslV-His with HslU even in the presence of ADP or the absence of any nucleotide. Thus, it appears that MG132 can bind to free HslV (i.e. HslV uncomplexed with HslU), and this binding induces tight interaction between HslV and HslU even in the absence of ATP. On the other hand, lactacystin and NLVS appear to bind only to HslV that is complexed with ATP-bound HslU, resulting in further stabilization of the HslVU complex. Taken together, these results suggest that the Thr-1 active sites of HslV protease are involved in the interaction between HslV and HslU. To determine whether MG132, unlike lactacystin and NLVS, could indeed bind to free HslV, each of the inhibitors was subjected to incubation with HslV-His alone followed by gel filtration to remove unbound inhibitors by using Sephadex G-25-filled spin-columns. Eluted HslV proteins were then subjected to incubation with HslU in the presence of ATP followed by NTA pulldown analysis. Gel filtration abrogated the stimulatory effect of lactacystin or NLVS on the interaction between HslV and HslU but showed little or no influence on that of MG132 (Fig. 1B, lower panel). Consistently, gel filtration abolished the inhibitory effects of lactacystin and NLVS on the peptidase activity of HslV but not that of MG132 (Fig. 1C, lower panel). These results indicate that MG132 binding to the Thr-1 active sites of free HslV is responsible for the induction of tight interaction between HslV and HslU in the absence of ATP or the presence of ADP. However, it remains unclear how MG132, unlike lactacystin and NLVS, can bind to free HslV. Although both MG132 and NLVS have the same tri-leucine peptide backbone, they have different N-terminal capping groups and C-terminal reactive groups. Therefore, one possibility is that, due to different steric or chemical properties of reactive groups, MG132, but not NLVS, is capable of reacting with or stably binding to the Thr-1 active sites of free HslV. However, this differential interaction of MG132 is not due to the reactive aldehyde group itself because N-acetyl-DEVD-aldehyde, a caspase-3 inhibitor, did not show any of the properties exhibited by MG132 (data not shown). Because lactacystin or NLVS promoted the interaction between HslV and HslU only when ATP was present, we examined whether the presence of ATP might be persistently required for maintaining the stable interaction of HslU with the inhibitor-bound HslV. HslV-His and HslU were incubated with ATP in the absence or presence of lactacystin or NLVS, and the HslVU complexes formed were pulled down by NTA resins. Precipitates were then extensively washed with buffers containing ATP, ADP, or none of the nucleotides followed by SDS-PAGE. In the absence of the inhibitors, HslU was dissociated from HslVU complexes by washing with buffers containing ADP or no nucleotide (Fig. 1D). In their presence, however, HslU remained stably associated with HslV under all washing conditions tested, indicating that the stability of inhibitor-bound HslVU complex is no longer influenced by the presence or absence of any adenine nucleotide. These results also indicate that ATP-bound HslU is required for the initial step where the inhibitors bind to the Thr-1 residue of HslV, probably through a covalent modification of the hydroxyl group of the Thr-1 residue. This notion is consistent with previous reports that HslV requires association with ATP-bound HslU to allosterically activate the Thr-1 residue of HslV (15Huang H. Goldberg A.L. J. Biol. Chem. 1997; 272: 21364-21372Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 22Sousa M.C. Kessler B.M. Overkleeft H.S. McKay D.B. J. Mol. Biol. 2002; 318: 779-785Crossref PubMed Scopus (63) Google Scholar, 31Kwon A.R. Kessler B.M. Overkleeft H.S. McKay D.B. J. Mol. Biol. 2003; 330: 185-195Crossref PubMed Scopus (40) Google Scholar). Because HslV is known to stimulate the ATPase activity of HslU by 2–3-folds (Refs. 2Yoo S.J. Seol J.H. Shin D.H. Rohrwild M. Kang M.S. Tanaka K. Goldberg A.L. Chung C.H. J. Biol. Chem. 1996; 271: 14035-14040Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar and 16Seol J.H. Yoo S.J. Shin D.H. Shim Y.K. Kang M.S. Goldberg A.L. Chung C.H. Eur. J. Biochem. 1997; 247: 1143-1150Crossref PubMed Scopus (51) Google Scholar; see Fig. 1B) and because all three tested proteasome inhibitors markedly increase the interaction between HslV and HslU when ATP is present, we examined whether this increased interaction leads to a further enhancement of HslV-stimulated ATPase activity of HslU. All of the inhibitors dramatically enhanced the HslV-stimulated ATPase activity of HslU, although to different extents (Fig. 1E, left panel). In addition, removal of unbound inhibitors by gel filtration on spin-columns abolished the stimulatory effect of lactacystin or NLVS, not that of MG132, on ATP hydrolysis by HslU (Fig. 1E, right panel). These results again reveal that MG132 can bind to free HslV, and this binding induces tight association of HslV with HslU, resulting in a dramatic activation of HslU ATPase. Collectively, these results implicate a role of the Thr-1 active sites of HslV in the interaction between HslV and HslU and thereby in the control of HslU function. Effect of the Deletion of N-terminal Thr on the Interaction between HslV and HslU—To clarify the involvement of Thr-1 active sites in the interaction between HslV and HslU, we generated a HslV mutant lacking the Thr-1 residue, tagged poly-His to its C terminus, and purified by using NTA-agarose columns. Unexpectedly, the Thr-1-deletion mutant (referred to as T1Δ-His), unlike HslV-His, was co-purified with HslU from the NTA-agarose column (Fig. 2A) despite the buffer used for the affinity chromatography not being supplemented with ATP. These results indicate that, like MG132-bound HslV, T1Δ can form stable complexes with HslU even in the absence of ATP. To confirm this finding, T1Δ-His was purified to apparent homogeneity (i.e. separated from HslU) and then incubated with HslU in the absence or presence of ADP or ATP. NTA pulldown analysis reveals that HslU co-precipitates with T1Δ-His under all conditions tested (Fig. 2B), indicating that T1Δ interacts with HslU regardless of the binding of either adenine nucleotide to HslU. Moreover, the amount of HslU co-precipitated with T1Δ-His was much greater than that with HslV-His, indicating that the deletion of Thr-1 leads to a marked increase in the i" @default.
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• W2013025360 title "Binding of MG132 or Deletion of the Thr Active Sites in HslV Subunits Increases the Affinity of HslV Protease for HslU ATPase and Makes This Interaction Nucleotide-independent" @default.
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