Autosomal recessive juvenile parkinsonism (AR-JP), one of the most common familial forms of Parkinson disease, is characterized by selective dopaminergic neural cell death and the absence of the Lewy body, a cytoplasmic inclusion body consisting of aggregates of abnormally accumulated proteins. We previously cloned PARK2, mutations of which cause AR-JP (ref. 2), but the function of the gene product, parkin, remains unknown. We report here that parkin is involved in protein degradation as a ubiquitin-protein ligase collaborating with the ubiquitin-conjugating enzyme UbcH7, and that mutant parkins from AR-JP patients show loss of the ubiquitin-protein ligase activity. Our findings indicate that accumulation of proteins that have yet to be identified causes a selective neural cell death without formation of Lewy bodies. Our findings should enhance the exploration of the molecular mechanisms of neurodegeneration in Parkinson disease as well as in other neurodegenerative diseases that are characterized by involvement of abnormal protein ubiquitination, including Alzheimer disease, other tauopathies, CAG triplet repeat disorders and amyotrophic lateral sclerosis.

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N-glycosylation of proteins in the endoplasmic reticulum (ER) has a central role in protein quality control. Here we report that N-glycan serves as a signal for degradation by the Skp1-Cullin1-Fbx2-Roc1 (SCF(Fbx2)) ubiquitin ligase complex. The F-box protein Fbx2 (ref. 4) binds specifically to proteins attached to N-linked high-mannose oligosaccharides and subsequently contributes to ubiquitination of N-glycosylated proteins. Pre-integrin beta 1 is a target of Fbx2; these two proteins interact in the cytosol after inhibition of the proteasome. In addition, expression of the mutant Fbx2 Delta F, which lacks the F-box domain that is essential for forming the SCF complex, appreciably blocks degradation of typical substrates of the ER-associated degradation pathway. Our results indicate that SCF(Fbx2) ubiquitinates N-glycosylated proteins that are translocated from the ER to the cytosol by the quality control mechanism.

T.Kawakami and T.Chiba contributed equally to this workNEDD8/Rub1 is a ubiquitin (Ub)-like post-translational modi®er that is covalently linked to cullin (Cul)-family proteins in a manner analogous to ubiquitylation. NEDD8 is known to enhance the ubiquitylating activity of the SCF complex (composed of Skp1, Cul-1, ROC1 and F-box protein), but the mechanistic role is largely unknown. Using an in vitro reconstituted system, we report here that NEDD8 modi®cation of Cul-1 enhances recruitment of Ub-conjugating enzyme Ubc4 (E2) to the SCF complex (E3). This recruitment requires thioester linkage of Ub to Ubc4. Our ®ndings indicate that the NEDD8-modifying system accelerates the formation of the E2±E3 complex, which stimulates protein polyubiquitylation.

Mutational dysfunction of PARKIN gene, which encodes a double RING finger protein and has ubiquitin ligase E3 activity, is the major cause of autosomal recessive juvenile Parkinsonism. Although many studies explored the functions of Parkin, its biochemical character is poorly understood. To address this issue, we established an E3 assay system using maltose-binding protein-fused Parkin purified from Escherichia coli. Using this recombinant Parkin, we found that not the front but the rear RING finger motif is responsible for the E3 activity of Parkin, and it catalyzes multiple monoubiquitylation. Intriguingly, for autosomal recessive juvenile Parkinsonismcausing mutations of Parkin, whereas there was loss of E3 activity in the rear RING domain, other pathogenic mutants still exhibited E3 activity equivalent to that of the wild-type Parkin. The evidence presented allows us to reconsider the function of Parkin-catalyzed ubiquitylation and to conclude that autosomal recessive juvenile Parkinsonism is not solely attributable to catalytic impairment of the E3 activity of Parkin.Recessive mutations in the human PARKIN gene are the most frequent cause of autosomal recessive juvenile parkinsonism, the common form of familial Parkinson disease (PD). 2 It has been shown that almost 50% of patients with familial autosomal recessive juvenile parkinsonism carry a series of exon rearrangements or point mutations in PARKIN. Moreover, recent findings of the haploinsufficiency of parkin and S-nitrosylation also imply its association in sporadic PD (1). The causal gene PARKIN encodes a double RING finger protein with ubiquitin ligase (E3) activity (2-5) and interestingly, missense mutations in the double RING finger motif resulted in an earlier onset of the disease than mutations in other function-unknown regions (6). To date, numerous biochemical studies have been performed to understand how mutations in Parkin lead to its dysfunction and to pathogenic outcome. However, because the biochemical characterization of E3 activity of Parkin has been difficult, it is still controversial whether the disease-relevant Parkin mutants lose their E3 activity or not. For example, one group of investigators implied that Parkin harboring K161N mutation loses its E3 activity (7), whereas another group suggested the same mutation dose not impair E3 activity (8). In the case of other PD mutations, the situation is even more complex (see supplemental Table 1). Thus, the mode of Parkin-catalyzed ubiquitylation remains poorly understood to date.Little is known about the reconstituted ubiquitylating experiment using recombinant Parkin. Almost all of the biochemical analyses reported so far have been performed using in vitro translated Parkin or immunoprecipitated Parkin. However, it is difficult to avoid trace contaminants of other proteins that could physically interact with Parkin. Indeed it has been reported that Parkin interacts with other E3s such as CHIP (9) and Nrdp1/FLRF (10), and thus the results of experiments using immunoprecipitated or in v...

A full-length cDNA encoding a SUMO-1-specific protease, named SUSP1, was identified and cloned for the first time from the human brain. Nucleotide sequence analysis of the cDNA containing an open reading frame of 3336 base pairs revealed that the protease consists of 1112 amino acids with a calculated molecular mass of 126,116 Da. Like yeast Ulp1, SUSP1 is a cysteine protease containing the well conserved His/Asp/Cys catalytic triad. SUSP1 expressed in Escherichia coli cells efficiently released SUMO-1 from SUMO-1⅐␤-galactosidase fusion but not from other ubiquitin-like protein fusions, including Smt3⅐␤-galactosidase, suggesting its role in the generation of matured SUMO-1 specifically from its precursors. Interestingly, reproductive organs, such as testis, ovary, and prostate, contained much higher amounts of SUSP1 mRNA than colon and peripheral blood leukocyte, whereas other tissues, such as heart and spleen, had little or none. In addition, confocal microscopy using green fluorescent protein⅐SUSP1 fusion showed that SUSP1 is exclusively localized to the cytoplasm of NIH3T3 and HeLa cells. These results suggest that SUSP1 may play a role in the regulation of SUMO-1-mediated cellular processes particularly related to reproduction.

We found previously that overexpression of an F-box protein ␤TrCP1 and the structurally related ␤TrCP2 augments ubiquitination of phosphorylated IB␣ (pIB␣) induced by tumor necrosis factor-␣ (TNF-␣), but the relationship of the two homologous ␤TrCP proteins remains unknown. Herein we reveal that deletion mutants of ␤TrCP1 and ␤TrCP2 lacking the F-box domain suppressed ubiquitination and destruction of pIB␣ as well as transcriptional activation of NF-B. The ectopically expressed ␤TrCP1 and ␤TrCP2 formed both homodimer and heterodimer complexes without displaying the trimer complex. Dimerization of ␤TrCP1 and/or ␤TrCP2 takes place at their conserved NH 2 -terminal regions, termed a "D-domain" (for dimerization domain), located upstream of the F-box domain. The D-domain was necessary and sufficient for the dimer formation. Intriguingly, the ␤TrCP homodimer, but not the heterodimer, was selectively recruited to pIB␣ induced by TNF-␣. These results indicate that not only ␤TrCP1 but also ␤TrCP2 participates in the ubiquitination-dependent destruction of IB␣ by forming SCF ␤TrCP1-␤TrCP1 and SCF ␤TrCP2-␤TrCP2 ubiquitin-ligase complexes.

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