Crystal structure of an intracellular protease from Pyrococcus horikoshii at 2-Å resolution

213

218

DJ-1 is a multifunctional protein that plays essential roles in tissues with higher order biological functions such as the testis and brain. DJ-1 is related to male fertility, and its level in sperm decreases in response to exposure to sperm toxicants. DJ-1 has also been identified as a hydroperoxide-responsive protein. Recently, a mutation of DJ-1 was found to be responsible for familial Parkinson's disease. Here, we present the crystal structure of DJ-1 refined to 1.95-Å resolution. DJ-1 forms a dimer in the crystal, and the monomer takes a flavodoxin-like Rossmann-fold. DJ-1 is structurally most similar to the monomer subunit of protease I, the intracellular cysteine protease from Pyrococcus horikoshii, and belongs to the Class I glutamine amidotransferaselike superfamily. However, DJ-1 contains an additional ␣-helix at the C-terminal region, which blocks the putative catalytic site of DJ-1 and appears to regulate the enzymatic activity. DJ-1 may induce conformational changes to acquire catalytic activity in response to oxidative stress.DJ-1 was initially identified as a novel oncogene product that transforms mouse NIH3T3 cells in cooperation with activated Ras. DJ-1 is an ϳ20-kDa protein comprising 189 amino acid residues ubiquitously expressed in various human tissues and with a particularly high level of expression in the testes (1). SP22 1 or CAP1, a rat homologue of human DJ-1, was subsequently identified as a key protein related to infertility in male rats exposed to sperm toxicants such as ornidazole and epichlorohydrin where DJ-1/CAP1/SP22 levels in the sperm and epididymis decreased with increased rat infertility (2-4). With the exception of DJ-1, no other protein decreased in response to exposure to sperm toxicants, supporting the close relationship between DJ-1 function and male fertility. Recently, Klinefelter et al. (5) revealed that DJ-1/CAP1/SP22 was located on the equatorial segment of the matured sperm head and anti-SP22

Section: Resultsmentioning

confidence: 99%

The Crystal Structure of DJ-1, a Protein Related to Male Fertility and Parkinson's Disease

Honbou

Suzuki

Horiuchi

et al. 2003

213

218

“…These data are consistent with recent x-ray diffraction data (17,18), which also demonstrate dimer formation. Different members of the DJ-1 superfamily of proteins form many different types of oligomeric species, and this data suggest that DJ-1 is more structurally similar to the bacterial Hsp31, which forms dimers (16) and less like PH1704, which forms hexamers (25). Full-length DJ-1 is necessary for its self-interaction, suggesting a contribution of both N and C termini to the proper folding or self-interactions of the protein.…”

Section: Discussionmentioning

confidence: 96%

L166P Mutant DJ-1, Causative for Recessive Parkinson's Disease, Is Degraded through the Ubiquitin-Proteasome System

Miller

Ahmad

Hague

et al. 2003

218

181

Mutations in a gene on chromosome 1, DJ-1, have been reported recently to be associated with recessive, earlyonset Parkinson's disease. While one mutation is a large deletion that is predicted to produce an effective knockout of the gene, the second is a point mutation, L166P, whose precise effects on protein function are unclear. In the present study, we show that L166P destabilizes DJ-1 protein and promotes its degradation through the ubiquitin-proteasome system. A double mutant (K130R, L166P) was more stable than L166P, suggesting that this lysine residue contributes to stability of the protein. Subcellular localization was broadly similar for both wild type and L166P forms of the protein, indicating that the effect of the mutation is predominantly on protein stability. These observations are reminiscent of other recessive gene mutations that produce an effective loss of function. The L166P mutation has the simple effect of promoting DJ-1 degradation, thereby reducing net DJ-1 protein within the cell.

“…The in-gel protease assay was carried out as described previously for determining the protease activity of the PfpI family of proteases (34,35). Purified wild-type or mutant DJ-1 protein (1-100 g) was electrophoresed onto 12% SDS-polyacrylamide gels containing 0.1% co-polymerized gelatin under non-reducing conditions.…”

Section: Dj-1 Expressionmentioning

confidence: 99%

“…Protease-Since DJ-1 also shares significant sequence and structural homology with the PfpI family of intracellular proteases (23,35), we sought to determine whether DJ-1 possesses intrinsic protease activity. We first assessed the proteolytic activity of DJ-1 by using the same in-gel protease assay that was used to demonstrate the protease activity of PfpI and PH1704 (34,35).…”

Section: Dj-1 Functions As a Cysteinementioning

confidence: 99%

“…We first assessed the proteolytic activity of DJ-1 by using the same in-gel protease assay that was used to demonstrate the protease activity of PfpI and PH1704 (34,35). No detectable proteolytic activity could be observed for DJ-1 protein despite the robust proteolytic activity exhibited by the positive control trypsin (data not shown).…”

Section: Dj-1 Functions As a Cysteinementioning

confidence: 99%

See 1 more Smart Citation

Familial Parkinson's Disease-associated L166P Mutation Disrupts DJ-1 Protein Folding and Function

Olzmann

Brown

Wilkinson

et al. 2004

262

282

Mutations in DJ-1, a protein of unknown function, were recently identified as the cause for an autosomal recessive, early onset form of familial Parkinson's disease. Here we report that DJ-1 is a dimeric protein that exhibits protease activity but no chaperone activity. The protease activity was abolished by mutation of Cys-106 to Ala, suggesting that DJ-1 functions as a cysteine protease. Our studies revealed that the Parkinson's disease-linked L166P mutation impaired the intrinsic folding propensity of DJ-1 protein, resulting in a spontaneously unfolded structure that was incapable of forming a homodimer with itself or a heterodimer with wild-type DJ-1. Correlating with the disruption of DJ-1 structure, the L166P mutation abolished the catalytic function of DJ-1. Furthermore, as a result of protein misfolding, the L166P mutant DJ-1 was selectively polyubiquitinated and rapidly degraded by the proteasome. Together these findings provide insights into the molecular mechanism by which loss-of-function mutations in DJ-1 lead to Parkinson's disease.