Key points are not available for this paper at this time.
Human DJ-1 and Escherichia coli Hsp31 belong to ThiJ/PfpI family, whose members contain a conserved domain. DJ-1 is associated with autosomal recessive early onset parkinsonism and Hsp31 is a molecular chaperone. Structural comparisons between DJ-1, Hsp31, and an Archaea protease, a member of ThiJ/PfpI family, lead to the identification of the chaperone activity of DJ-1 and the proteolytic activity of Hsp31. Moreover, the comparisons provide insights into how the functional diversity is realized in proteins that share an evolutionarily conserved domain. On the basis of the chaperone activity the possible role of DJ-1 in the pathogenesis of Parkinson's disease is discussed. Human DJ-1 and Escherichia coli Hsp31 belong to ThiJ/PfpI family, whose members contain a conserved domain. DJ-1 is associated with autosomal recessive early onset parkinsonism and Hsp31 is a molecular chaperone. Structural comparisons between DJ-1, Hsp31, and an Archaea protease, a member of ThiJ/PfpI family, lead to the identification of the chaperone activity of DJ-1 and the proteolytic activity of Hsp31. Moreover, the comparisons provide insights into how the functional diversity is realized in proteins that share an evolutionarily conserved domain. On the basis of the chaperone activity the possible role of DJ-1 in the pathogenesis of Parkinson's disease is discussed. Completed or ongoing genome sequencing projects have added many members to protein families by finding out new proteins belonging to the families. ThiJ/PfpI family is such an expanding protein family whose members are evolutionarily distributed from Archaea to Eukarya. The family members share a domain (ThiJ domain) that is structurally related to type I glutamine amidotransferase domain (GAT 1The abbreviations used are: GAT, glutamine amidotransferase; AMC, 7-amido-4-methyl-coumarin; Hsp, Heat shock protein; CS, citrate synthase; BSA, bovine serum albumin; SeHsp31, selenomethionine-substituted Hsp31; SeDJ-1, selenomethionine-substituted DJ-1; Bicine, N,N-bis(2-hydroxyethyl)glycine; PD, Parkinson's Disease. domain) (1Horvath M.M. Grishin N.V. Proteins Struct. Funct. Genet. 2001; 42: 230-236Crossref PubMed Scopus (31) Google Scholar). GAT domains are found in a large group of biosynthetic enzymes that catalyze the transfer of water to free glutamine to release ammonia as a substrate for subsequent biosynthetic reactions at a contiguous synthase domain or subunit (2Zalkin H. Adv. Enzymol. Relat. Areas Mol. Biol. 1993; 66: 203-309PubMed Google Scholar). Unlike the GAT domain, however, the biological role of ThiJ domain is unclear. PFAM (3Bateman A. Birney E. Durbin R. Eddy S.R. Finn R.D. Sonnhammer E.L.L. Nucleic Acids Res. 1999; 27: 260-262Crossref PubMed Scopus (475) Google Scholar) shows that ThiJ/PfpI family contains many hypothetical proteins. Even the family members with known functions have distinct activities. For example, the domain has activities such as regulation of RNA-protein interaction, phosphorylation of hydroxymethylpyrimidine, and proteolysis. Thus, deciphering the molecular role of the domain and the evolutionary relationship among the family members is a great challenge. We have determined the crystal structures of Escherichia coli Hsp31 and human DJ-1, members of the ThiJ/PfpI family, as a start toward dissecting structural and functional relationships among the family members. Hsp31, the yedu gene product, is a molecular chaperone whose expression is induced by heat shock (4Blattner F.R. Plunkett G.R. Bloch C.A. Perna N.T. Burland V. Riley M. Collado-Vides J. Glasner J.D. Rode C.K. Mayhew G.F. Gregor J. Davis N.W. Kirkpatrick H.A. Goeden M.A. Rose D.J. Mau B. Shao Y. Science. 1997; 277: 1453-1474Crossref PubMed Scopus (6025) Google Scholar). Although the physiological role of Hsp31 has not been clearly established, the implication of Hsp31 in the protein quality control was discussed in the previous studies (5Malki A. Kern R. Abdallah J. Richarme G. Biochem. Biophys. Res. Commun. 2003; 301: 430-436Crossref PubMed Scopus (41) Google Scholar, 6Sastry M.S.R. Korotkov K. Brodsky Y. Baneyx F. J. Biol. Chem. 2002; 277: 46026-46034Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). To control the protein quality, cells respond to a sudden increase of environmental temperature with the overexpression of heat shock proteins. During heat shock most of the heat shock proteins act either as molecular chaperones to assist unfolded proteins in folding or as proteases to degrade proteins that fail in refolding (7Parsell D.A. Lindquist S. Annu. Rev. Genet. 1993; 27: 437-496Crossref PubMed Scopus (1881) Google Scholar). DJ-1, which is preferentially expressed in the testis and moderately in other tissues, was first identified as a novel candidate of the oncogene product that transformed mouse NIH3T3 cells in cooperation with activated ras (8Nagakubo D. Taira T. Kitaura H. Ikeda M. Tamai K. Iguchi-Ariga S.M. Ariga H. Biochem. Biophys. Res. Commun. 1997; 231: 509-513Crossref PubMed Scopus (667) Google Scholar). After the first identification, various physiological roles of DJ-1 were successively revealed. DJ-1 was characterized as a protein that regulates an RNA-protein interaction (9Hod Y. Pentyala S.N. Whyard T.C. El-Maghrabi M.R. J. Cell. Biochem. 1999; 72: 435-444Crossref PubMed Scopus (173) Google Scholar). DJ-1 is also related to sperm fertilization (10Wagenfeld A. Yeung C.H. Shivaji S. Sundareswaran V.R. Ariga H. Cooper T.G. J. Androl. 2000; 21: 954-963PubMed Google Scholar) and positively modulates the androgen receptor by impairing the binding of PIAS (protein inhibitor of activated STAT xα) to the receptor (11Takahashi K. Taira T. Niki T. Seino C. Iguchi-Ariga S.M. Ariga H. J. Biol. Chem. 2002; 276: 37556-37563Abstract Full Text Full Text PDF Scopus (288) Google Scholar). Remarkably, the association of DJ-1 with autosomal recessive early onset parkinsonism was recently demonstrated (12Bonifati V. Rizzu P. van Baren M.J. Schaap O. Breedveld G.J. Krieger E. Dekker M.C.J. Squitieri F. Ibanez P. Joosse M. van Dongen J.W. Vanacore N. van Swieten J.C. Brice A. Meco G. van Duijn C.M. Oostra B.A. Heutink P. Science. 2003; 299: 256-259Crossref PubMed Scopus (2261) Google Scholar). In summary, DJ-1 has multiple functions whose correlation is presently obscure. Furthermore, it was reported that DJ-1 was expressed and that its pI was changed from 6.2 to 5.8 by treatment of cells with paraquat and endotoxin, which induce reactive oxygen species (13Mitsumoto A. Nakagawa Y. Free Radic. Res. 2001; 35: 885-893Crossref PubMed Scopus (251) Google Scholar, 14Mitsumoto A. Nakagawa Y. Takeuchi A. Okawa K. Iwamatsu A. Takanezawa Y. Free Radic. Res. 2001; 35: 301-310Crossref PubMed Scopus (238) Google Scholar), suggesting a function as an antioxidant protein. The transcription of YDR533C, a yeast DJ-1 homologue, is induced together with genes involved in the oxidative stress response (15de Nobel H. Lawrie L. Brul S. Klis F. Davis M. Alloush H. Coote P. Yeast. 2001; 18: 1413-1418Crossref PubMed Scopus (100) Google Scholar). Here, we describe the crystal structures and biochemical data to provide an important framework for the elucidation of the molecular mechanisms of DJ-1 and Hsp31 functions. Purification, Crystallization, and Structure Determination—Hsp31, C185A mutant of Hsp31 (referred to as C185A throughout the text), and SeHsp31 were purified as described (16Kim O.-G. Kim I.-K. Kim G.-H. Ko J. Park C. Suh P.-G. Kang S.-O. Lee H.-S. Cha S.-S. Acta Crystallogr. Sect. D. 2002; 58: 1217-1219Crossref PubMed Scopus (2) Google Scholar). DJ-1 gene (encoding residues 1–189) was amplified by polymerase chain reaction from a human kidney cDNA library. The gene was inserted downstream of the T7 promoter on the expression plasmid pET-21a, and the plasmid was introduced in E. coli strain B834 (DE3), a methionine auxotroph strain. Cells were grown to an A 600 of ∼0.6 in minimal media containing selenomethionine and 0.1 mg/ml ampicillin at 37 °C, and the expression of SeDJ-1 was induced by 0.5 mm isopropyl-d-thiogalactoside. After a 4-h induction at 37 °C, cells were harvested and resuspended in a 20 mm Tris buffer (pH 7.5) and disrupted by sonication. After centrifugation supernatants were loaded on Q-Sepharose Fast Flow column (Amersham Biosciences). The unbound fraction was consecutively loaded on SP-Sepharose Fast Flow column (Amersham Biosciences). The eluted fractions containing SeDJ-1 were collected and used for crystallization. Because SeDJ-1 was obtained in large we used SeDJ-1 for the studies The reported of Hsp31 (16Kim O.-G. Kim I.-K. Kim G.-H. Ko J. Park C. Suh P.-G. Kang S.-O. Lee H.-S. Cha S.-S. Acta Crystallogr. Sect. D. 2002; 58: 1217-1219Crossref PubMed Scopus (2) Google Scholar) were not we found a new Hsp31 was at with of and 0.1 (pH and to group with a to Hsp31 DJ-1 a a in were grown at by of protein and of containing citrate and 0.1 (pH For data were at a were in a containing in the A data of DJ-1 was collected with a on a data and data of Hsp31 were collected with a at of were and with and Enzymol. 1997; 276: PubMed Scopus Google Scholar). of SeHsp31 were and were the The subsequent by to an DJ-1 was determined by molecular with as a of in is for the in is for the is the of the of related of of of of and is the protein from the free was with of the in and are the from The in is for the is the of the of related of and is the protein from the free was with of the in and are the from in a new of DJ-1, we added 0.5 mm to the protein the of DJ-1 were obtained with the that was used for the crystal of and was with in were with The of SeHsp31 was on an by Although the was we were to the of SeHsp31 for residues with the After the of a of SeHsp31, the were and a and a were successively out the the data was by and of and were The in the were to other throughout the The was to and free of and was to the SeDJ-1 by the from molecular SeDJ-1 has at the with The to the was in the first by the molecular the was After to DJ-1 the was to a of The to water were added by the of The subsequent and of the and free to and The of the was by J. Crystallogr. 1993; Google Scholar). The and of residues are in the most and and are in the and of by DJ-1, Hsp31, and C185A were by The buffer of mm mm mm and 20 mm Tris (pH To the of the reaction buffer we used 20 mm for 20 mm for or 20 mm for activities were by the of from the and of Hsp31 was for an with substrate in mm Tris (pH mm mm and at at was the reaction was by the of of activity was by the of the proteins on the of and at The of and DJ-1 in the mm (pH 7.5) buffer with 0.5 mm was for For the was into mm (pH 7.5) at in the or of the and the reaction was for After the the of from the or was on a with a temperature was to and the were to and The was on the basis of DJ-1 DJ-1 contains of DJ-1 and structures of SeDJ-1 DJ-1 structures were reported was in the L. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Y. H. L. H. H. 2003; PubMed Scopus Google Scholar). Although the was described insights data on the chaperone activity of DJ-1 were and Hsp31 the of the of Hsp31 was reported by group Korotkov K. Baneyx F. S. A. 2003; PubMed Scopus Google Scholar). not the binding in Hsp31. Furthermore, to the proteolytic activity of Hsp31. that are structurally and of the structures are in and Hsp31 was reported as a (5Malki A. Kern R. Abdallah J. Richarme G. Biochem. Biophys. Res. Commun. 2003; 301: 430-436Crossref PubMed Scopus (41) Google Scholar, 6Sastry M.S.R. Korotkov K. Brodsky Y. Baneyx F. J. Biol. Chem. 2002; 277: 46026-46034Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar), and DJ-1 was also found to a by and not were in the protein In DJ-1 DJ-1 of the molecular of The in DJ-1 are between and from and between from The between from an A was found in the of Hsp31. residues on and on from the and The of a water into the a which clearly shows that the residues a not a water The is to the of in Hsp31. To the of the on the we added mm to the purified Hsp31 and the buffer the The of on the of Hsp31 not Furthermore, the residues are not conserved to the among the Hsp31 in other M.S.R. Korotkov K. Brodsky Y. Baneyx F. J. Biol. Chem. 2002; 277: 46026-46034Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). Thus, the not to for the of Hsp31. of a with structures The and the are by The residues are as between The the domain. The of ThiJ domain is The into the is by The of the is to of Hsp31 is to A. The residues and residues involved in the binding are in The the of the binding The the of of the Hsp31 with the and are and The is to B. The the of the and at and the binding of chaperone activity of Hsp31 (5Malki A. Kern R. Abdallah J. Richarme G. Biochem. Biophys. Res. Commun. 2003; 301: 430-436Crossref PubMed Scopus (41) Google Scholar, 6Sastry M.S.R. Korotkov K. Brodsky Y. Baneyx F. J. Biol. Chem. 2002; 277: 46026-46034Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar) and the structural between Hsp31 and DJ-1 to the chaperone activity of Because of the of molecular chaperone function is an to the of we DJ-1 the of CS, a chaperone A in was To DJ-1 substrate is to or we DJ-1 activity with chaperone CS, and at J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). DJ-1 was also to the of in a The of a protein with chaperone activity was demonstrated not to the of and M.S.R. Korotkov K. Brodsky Y. Baneyx F. J. Biol. Chem. 2002; 277: 46026-46034Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, M. J.C. Cell. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). the of the that DJ-1 contains a chaperone The DJ-1 activity was not by the of or not of DJ-1 and structural in chaperones is the of on the with substrate proteins the In to the the with unfolded proteins have The on the are for the with proteins. The in molecular chaperones are in the E. Structure 2002; Full Text Full Text PDF PubMed Scopus Google Scholar, Lee Struct. Biol. 2001; PubMed Scopus Google Scholar, J.D. K. Struct. Biol. 2000; PubMed Scopus Google Scholar). The of DJ-1 and Hsp31 structures that contain and for the with proteins. The of DJ-1 is in the at the molecular Hsp31 contains a in The of DJ-1 is by and that with other and A of residues and from is in the In to DJ-1, the and of Hsp31 is important in the of the that of of residues and are on the of Hsp31 The residues on the in Hsp31 and DJ-1 are conserved or by other with in the in other not is the in DJ-1 is that the chain of is To the function of the we a mutant of DJ-1 of and an mutant of Hsp31 of and Because the of proteins are not in the of the of ThiJ domain of and the structures of the are to conserved with the of the that the are not The in the of the DJ-1 mutant with that in the of the of type DJ-1 not The Hsp31 mutant was not to the refolding of in to type Hsp31 not to the of the in the of the functional in the proteins. of the that and ThiJ domain of E. coli among ThiJ/PfpI family members were structurally J. N. J. C. J. Structure Full Text Full Text PDF Scopus (100) Google Scholar, Kim R. J. H. Kim S. A. 2000; PubMed Scopus Google Scholar). The structural among members Hsp31, and an of ThiJ domain and The of a and either of the and The to large the of residues in Hsp31 not the discussed in the to is to associated with the functional diversity of ThiJ domain. Remarkably, a in the and The is a that was first in In an the of a either a or a a in the of the in PFAM (3Bateman A. Birney E. Durbin R. Eddy S.R. Finn R.D. Sonnhammer E.L.L. Nucleic Acids Res. 1999; 27: 260-262Crossref PubMed Scopus (475) Google Scholar) shows that of the ThiJ/PfpI family members DJ-1, Hsp31, and have a conserved The structures of DJ-1, Hsp31, and the of the conserved in DJ-1, in Hsp31, and in in the of the The structures also that the for the conserved in an in the is the of the in a The of containing a an activity of ThiJ domain. contains a proteolytic activity Kim R. J. H. Kim S. A. 2000; PubMed Scopus Google Scholar). activity of has been reported the conserved is with a in the structures we that Hsp31 contains a that is structurally with the of on we are of the proteolytic activity of Hsp31, and as Hsp31 that was reported to proteolytic activity (5Malki A. Kern R. Abdallah J. Richarme G. Biochem. Biophys. Res. Commun. 2003; 301: 430-436Crossref PubMed Scopus (41) Google Scholar, 6Sastry M.S.R. Korotkov K. Brodsky Y. Baneyx F. J. Biol. Chem. 2002; 277: 46026-46034Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar) a protein In DJ-1 that has at a of from the conserved an not degrade to the The proteolytic activity to the of the in the conserved in the not the activity of ThiJ domain. For the activity the Thus, we that the ThiJ/PfpI family members with the conserved contain The proteolytic activity of Hsp31 toward is at 37 to of a of to of The of the in Hsp31 an on the activity toward a protein The of Hsp31 is a with of The of the is The to the substrate of Hsp31 to protein Thus, we the activity of Hsp31 with Hsp31 activity a with at and Hsp31 was not to however, that Hsp31 has a for Hsp31 is to a with as and Hsp31 degrade protein of by of at various of that the not by C185A by In the of the of to the of Hsp31 The of at various of that the not by C185A by In the of the of to the of Hsp31 in a new The of the of the protein have In of a proteolytic have been protease, and and J. D. B. P. R. Science. PubMed Scopus Google Scholar). in E. a with a of residues D. P. Annu. Rev. Biochem. 1999; PubMed Scopus Google Scholar), and to of residues N. F. T. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). product to release T. N. R. F. Science. PubMed Scopus Google Scholar). heat the proteolytic to degrade proteins that fail to by and that into are in large In Hsp31 a role to to that are in the as of the of in on the and the proteolytic activity that is to the of to a To the activity of we a C185A The mutant not as as and suggesting that is the of Hsp31. Structure of DJ-1 is to a with a pI in response to oxidative (13Mitsumoto A. Nakagawa Y. Free Radic. Res. 2001; 35: 885-893Crossref PubMed Scopus (251) Google Scholar, 14Mitsumoto A. Nakagawa Y. Takeuchi A. Okawa K. Iwamatsu A. Takanezawa Y. Free Radic. Res. 2001; 35: 301-310Crossref PubMed Scopus (238) Google Scholar), we determined the crystal of DJ-1 to the of the The of the DJ-1 is to the the of In the we in the of in the A is into the A to and to and J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar). Because the of and are the of the the pI of proteins C.M. PubMed Scopus Google Scholar). Thus, the reported of DJ-1 pI in is to the of DJ-1 and Parkinson's is the protein to identified as involved in PD, the other and the role of DJ-1 in in with and is a of which is a of J. P. Rev. 2002; PubMed Scopus Google Scholar). is an that is in the protein H. N. S. Y. S. S. N. K. T. K. T. Genet. 2000; PubMed Scopus Google Scholar, Y. J. H. S. A. 2000; PubMed Scopus Google Scholar). of the in and is the of proteins. A of is that a a of protein that to the in type J. P. Rev. 2002; PubMed Scopus Google Scholar). in lead to in the of proteins that are to M.R. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). In PD, the and the of are with the of proteins 2001; Full Text Full Text PDF PubMed Scopus Google Scholar). is that the mechanisms the of proteins are in the of Because the chaperone is the the of proteins in the chaperone activity of DJ-1 that DJ-1 a role in the pathogenesis of Remarkably, the of a chaperone activity in the of the chaperone function in for Lee Science. 2002; PubMed Scopus Google Scholar). that a chaperone have on the of of oxidative stress were in studies of H. Lee Free Radic. Biol. 2002; PubMed Scopus (251) Google Scholar, M.M. 2002; 58: PubMed Google Scholar). DJ-1 is an oxidative protein (13Mitsumoto A. Nakagawa Y. Free Radic. Res. 2001; 35: 885-893Crossref PubMed Scopus (251) Google Scholar, 14Mitsumoto A. Nakagawa Y. Takeuchi A. Okawa K. Iwamatsu A. Takanezawa Y. Free Radic. Res. 2001; 35: 301-310Crossref PubMed Scopus (238) Google Scholar), and the and the chaperone activity of DJ-1 are in the of and it is that DJ-1 a role in the of to the overexpression of DJ-1 the of in or in A in DJ-1 was in (12Bonifati V. Rizzu P. van Baren M.J. Schaap O. Breedveld G.J. Krieger E. Dekker M.C.J. Squitieri F. Ibanez P. Joosse M. van Dongen J.W. Vanacore N. van Swieten J.C. Brice A. Meco G. van Duijn C.M. Oostra B.A. Heutink P. Science. 2003; 299: 256-259Crossref PubMed Scopus (2261) Google Scholar). is in which is a of the (12Bonifati V. Rizzu P. van Baren M.J. Schaap O. Breedveld G.J. Krieger E. Dekker M.C.J. Squitieri F. Ibanez P. Joosse M. van Dongen J.W. Vanacore N. van Swieten J.C. Brice A. Meco G. van Duijn C.M. Oostra B.A. Heutink P. Science. 2003; 299: 256-259Crossref PubMed Scopus (2261) Google Scholar) the the The of is to with the between and Because and roles in the the of the between the is to the and Thus, in of the chaperone is to a in Proteins ThiJ ThiJ/PfpI family is not a ThiJ domain contains other biochemical activities the proteolytic activity and the chaperone For example, proteins with ThiJ domain are known to involved in the and of synthase I from Moreover, the identification of functions of hypothetical proteins that a large of ThiJ/PfpI family novel activities of ThiJ domain. is the of the functional diversity of ThiJ domain the of The structural of DJ-1, Hsp31, and on the of the activities of ThiJ domain. of the structural of the proteins is the distinct the a Kim R. J. H. Kim S. A. 2000; PubMed Scopus Google Scholar). The of DJ-1 is also to that of Hsp31 and The induced by in ThiJ domain, which are by the in the and of ThiJ domains in the are for the of The biochemical activities of and DJ-1 are with of the in is its on the and the with a from an at the molecular Kim R. J. H. Kim S. A. 2000; PubMed Scopus Google Scholar) The in DJ-1 a at the molecular of a which DJ-1 as a chaperone. In the of Hsp31, the to of the of the functional domain on of ThiJ domain is to the of the functional domain the by on the and the is also on of the domain The shows that Hsp31 contains residues DJ-1 and domain is of and and and that are by the residues and In summary, the functional of DJ-1, Hsp31, and in the molecular or in the domain, not in the ThiJ domain the and the of a new domain are for the functional diversity of ThiJ domain. the first of the chaperone activity of DJ-1 that has roles in various biological the pathogenesis of The roles of the chaperone in the pathogenesis of Lee Science. 2002; PubMed Scopus Google Scholar, S. A. 2002; PubMed Scopus Google Scholar, P. B. P. E. J. M. A. J. 2000; PubMed Scopus Google Scholar, P. S. Science. 2000; PubMed Scopus Google Scholar, Y. A. M. M. M. K. G. J. Biol. Chem. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar, E. K. N. Lindquist S. S. A. 2000; PubMed Scopus Google Scholar, Y. Genet. 1999; PubMed Scopus Google Scholar) that the of DJ-1 to protein a in the pathogenesis of We also describe the proteolytic activity of Hsp31 molecular that was reported to contain proteolytic The and the of C185A on the proteolytic activity that Hsp31 is a The of the proteolytic activity of Hsp31 a of a protein that is a chaperone and a the structures of DJ-1, Hsp31, and that the or the of a new domain is to the various biochemical activities of ThiJ/PfpI family members. We for a of
Lee et al. (Sat,) studied this question.