Keith D. Wilkinson

The ubiquitin pathway in Parkinson's disease

Mutations of the α-synuclein gene, have been identified in some familial forms of Parkinsons disease, and α-synuclein protein has been shown to accumulate in the brains of patients with the disease. These findings suggest that Parkinsons disease may be caused by the abnormal aggregation of α-synuclein protein. Here we have identified in a German family with Parkinsons disease a missense mutation in the ubiquitin carboxy-terminal hydrolase L1 (UCH-L1) gene. We show that this mutation, Ile93Met, causes a partial loss of the catalytic activity of this thiol protease, which could lead to aberrations in the proteolytic pathway and aggregation of proteins.

Microarray identification of FMRP-associated brain mRNAs and altered mRNA translational profiles in fragile X syndrome.

Fragile X syndrome results from the absence of the RNA binding FMR protein. Here, mRNA was coimmunoprecipitated with the FMRP ribonucleoprotein complex and used to interrogate microarrays. We identified 432 associated mRNAs from mouse brain. Quantitative RT-PCR confirmed some to be >60-fold enriched in the immunoprecipitant. In parallel studies, mRNAs from polyribosomes of fragile X cells were used to probe microarrays. Despite equivalent cytoplasmic abundance, 251 mRNAs had an abnormal polyribosome profile in the absence of FMRP. Although this represents <2% of the total messages, 50% of the coimmunoprecipitated mRNAs with expressed human orthologs were found in this group. Nearly 70% of those transcripts found in both studies contain a G quartet structure, demonstrated as an in vitro FMRP target. We conclude that translational dysregulation of mRNAs normally associated with FMRP may be the proximal cause of fragile X syndrome, and we identify candidate genes relevant to this phenotype.

Regulation and Cellular Roles of Ubiquitin-Specific Deubiquitinating Enzymes

Deubiquitinating enzymes (DUBs) are proteases that process ubiquitin or ubiquitin-like gene products, reverse the modification of proteins by a single ubiquitin(-like) protein, and remodel polyubiquitin(-like) chains on target proteins. The human genome encodes nearly 100 DUBs with specificity for ubiquitin in five gene families. Most DUB activity is cryptic, and conformational rearrangements often occur during the binding of ubiquitin and/or scaffold proteins. DUBs with specificity for ubiquitin contain insertions and extensions modulating DUB substrate specificity, protein-protein interactions, and cellular localization. Binding partners and multiprotein complexes with which DUBs associate modulate DUB activity and substrate specificity. Quantitative studies of activity and protein-protein interactions, together with genetic studies and the advent of RNAi, have led to new insights into the function of yeast and human DUBs. This review discusses ubiquitin-specific DUBs, some of the generalizations emerging from recent studies of the regulation of DUB activity, and their roles in various cellular processes.

Regulation of ubiquitin-dependent processes by deubiquitinating enzymes.

An astounding number of important regulatory and structural proteins are subject to modification by the attachment of ubiquitin or ubiquitin‐like proteins. This modification acts as a targeting signal, delivering the modified protein to different locations in the cell and modifying its activity, macromolecular interactions, or half‐life. Deubiquitination, or the removal of this modification, is being recognized as an important regulatory strategy. This reaction is catalyzed by processing proteases known as deubiquitinating enzymes (DUBs). More than 60 DUBs are already known, although little is known about their biological roles. This review concentrates on recent findings and new insights into this fascinating class of enzymes.—Wilkinson, K. D. Regulation of ubiquitin‐dependent processes by deubiquitinating enzymes. FASEB J. 11, 1245–1256 (1997)

BAP1: a novel ubiquitin hydrolase which binds to the BRCA1 RING finger and enhances BRCA1-mediated cell growth suppression.

We have identified a novel protein, BAP1, which binds to the RING finger domain of the Breast/Ovarian Cancer Susceptibility Gene product, BRCA1. BAP1 is a nuclear-localized, ubiquitin carboxy-terminal hydrolase, suggesting that deubiquitinating enzymes may play a role in BRCA1 function. BAP1 binds to the wild-type BRCA1-RING finger, but not to germline mutants of the BRCA1-RING finger found in breast cancer kindreds. BAP1 and BRCA1 are temporally and spatially co-expressed during murine breast development and remodeling, and show overlapping patterns of subnuclear distribution. BAP1 resides on human chromosome 3p21.3; intragenic homozgyous rearrangements and deletions of BAP1 have been found in lung carcinoma cell lines. BAP1 enhances BRCA1-mediated inhibition of breast cancer cell growth and is the first nuclear-localized ubiquitin carboxy-terminal hydrolase to be identified. BAP1 may be a new tumor suppressor gene which functions in the BRCA1 growth control pathway.

Molecular discrimination of structurally equivalent Lys 63-linked and linear polyubiquitin chains

At least eight types of ubiquitin chain exist, and individual linkages affect distinct cellular processes. The only distinguishing feature of differently linked ubiquitin chains is their structure, as polymers of the same unit are chemically identical. Here, we have crystallized Lys 63‐linked and linear ubiquitin dimers, revealing that both adopt equivalent open conformations, forming no contacts between ubiquitin molecules and thereby differing significantly from Lys 48‐linked ubiquitin chains. We also examined the specificity of various deubiquitinases (DUBs) and ubiquitin‐binding domains (UBDs). All analysed DUBs, except CYLD, cleave linear chains less efficiently compared with other chain types, or not at all. Likewise, UBDs can show chain specificity, and are able to select distinct linkages from a ubiquitin chain mixture. We found that the UBAN (ubiquitin binding in ABIN and NEMO) motif of NEMO (NF‐κB essential modifier) binds to linear chains exclusively, whereas the NZF (Npl4 zinc finger) domain of TAB2 (TAK1 binding protein 2) is Lys 63 specific. Our results highlight remarkable specificity determinants within the ubiquitin system.

Chemistry-based functional proteomics reveals novel members of the deubiquitinating enzyme family.

The ubiquitin (Ub)-proteasome system includes a large family of deubiquitinating enzymes (DUBs). Many members are assigned to this enzyme class by sequence similarity but without evidence for biological activity. A panel of novel DUB-specific probes was generated by a chemical ligation method. These probes allowed identification of DUBs and associated components by tandem mass spectrometry, as well as rapid demonstration of enzymatic activity for gene products whose functions were inferred from primary structure. We identified 23 active DUBs in EL4 cells, including the tumor suppressor CYLD1. At least two DUBs tightly interact with the proteasome 19S regulatory complex. An OTU domain-containing protein, with no sequence homology to any known DUBs, was isolated. We show that this polypeptide reacts with the C terminus of Ub, thus demonstrating DUB-like enzymatic activity for this novel superfamily of proteases.

A novel active site-directed probe specific for deubiquitylating enzymes reveals proteasome association of USP14

A C‐terminally modified ubiquitin (Ub) derivative, ubiquitin vinyl sulfone (UbVS), was synthesized as an active site‐directed probe that irreversibly modifies a subset of Ub C‐terminal hydrolases (UCHs) and Ub‐specific processing proteases (UBPs). Specificity of UbVS for deubiquitylating enzymes (DUBs) is demonstrated not only by inhibition of [125I]UbVS labeling with N‐ethylmaleimide and Ub aldehyde, but also by genetic analysis. [125I]UbVS modifies six of the 17 known and putative yeast deubiquitylating enzymes (Yuh1p, Ubp1p, Ubp2p, Ubp6p, Ubp12p and Ubp15p), as revealed by analysis of corresponding mutant strains. In mammalian cells, greater numbers of polypeptides are labeled, most of which are likely to be DUBs. Using [125I]UbVS as a probe, we report the association of an additional DUB with the mammalian 26S proteasome. In addition to the 37 kDa enzyme reported to be part of the 19S cap, we identified USP14, a mammalian homolog of yeast Ubp6p, as being bound to the proteasome. Remarkably, labeling of 26S‐associated USP14 with [125I]UbVS is increased when proteasome function is impaired, suggesting functional coupling between the activities of USP14 and the proteasome.

Structure and mechanisms of the proteasome-associated deubiquitinating enzyme USP14.

The ubiquitin‐specific processing protease (UBP) family of deubiquitinating enzymes plays an essential role in numerous cellular processes. Mammalian USP14 (Ubp6 in yeast) is unique among known UBP enzymes in that it is activated catalytically upon specific association with the 26S proteasome. Here, we report the crystal structures of the 45‐kDa catalytic domain of USP14 in isolation and in a complex with ubiquitin aldehyde, which reveal distinct structural features. In the absence of ubiquitin binding, the catalytic cleft leading to the active site of USP14 is blocked by two surface loops. Binding by ubiquitin induces a significant conformational change that translocates the two surface loops thereby allowing access of the ubiquitin C‐terminus to the active site. These structural observations, in conjunction with biochemical characterization, identify important regulatory mechanisms for USP14.

The Ubiquitin Binding Domain ZnF UBP Recognizes the C-Terminal Diglycine Motif of Unanchored Ubiquitin

Ubiquitin binding proteins regulate the stability, function, and/or localization of ubiquitinated proteins. Here we report the crystal structures of the zinc-finger ubiquitin binding domain (ZnF UBP) from the deubiquitinating enzyme isopeptidase T (IsoT, or USP5) alone and in complex with ubiquitin. Unlike other ubiquitin binding domains, this domain contains a deep binding pocket where the C-terminal diglycine motif of ubiquitin is inserted, thus explaining the specificity of IsoT for an unmodified C terminus on the proximal subunit of polyubiquitin. Mutations in the domain demonstrate that it is required for optimal catalytic activation of IsoT. This domain is present in several other protein families, and the ZnF UBP domain from an E3 ligase also requires the C terminus of ubiquitin for binding. These data suggest that binding the ubiquitin C terminus may be necessary for the function of other proteins.