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Keith D Wilkinson

Publications and source records attributed to Keith D Wilkinson.

At least 19 recordsLinked to original sources
PubMed2006

Effects of ubiquitin system alterations on the formation and loss of a yeast prion.

The yeast prion [PSI+] is a self-propagating amyloidogenic isoform of the translation termination factor Sup35. Overproduction of the chaperone protein Hsp104 results in loss of [PSI+]. Here we demonstrate that this effect is decreased by deletion of either the gene coding for one of the major yeast ubiquitin-conjugating enzymes, Ubc4, or the gene coding for the ubiquitin-recycling enzyme, Ubp6. The effect of ubc4Delta on [PSI+] loss was increased by depletion of the Hsp70 chaperone Ssb but was not influenced by depletion of Ubp6. This indicates that Ubc4 affects [PSI+] loss via a pathway that is the same as the one affected by Ubp6 but not by Ssb. In the presence of Rnq1 protein, ubc4Delta also facilitates spontaneous de novo formation of [PSI+]. This stimulation is independent of [PIN+], the prion isoform of Rnq1. Numerous attempts failed to detect ubiquitinated Sup35 in the yeast extracts. While ubc4Delta and other alterations of ubiquitin system used in this work cause slight induction of some Hsps, these changes are insufficient to explain their effect on [PSI+]. However, ubc4Delta increases the proportion of the Hsp70 chaperone Ssa bound to Sup35, suggesting that misfolded Sup35 is either more abundant or more accessible to the chaperones in the absence of Ubc4. The proportion of [PSI+] cells containing large aggregated Sup35 structures is also increased by ubc4Delta. We propose that UPS alterations induce an adaptive response, resulting in accumulation of the large "aggresome"-like aggregates that promote de novo prion generation and prion recovery from the chaperone treatment.

Cloning, Molecular↗
PubMed2006

SUSP1 antagonizes formation of highly SUMO2/3-conjugated species.

Small ubiquitin-related modifier (SUMO) processing and deconjugation are mediated by sentrin-specific proteases/ubiquitin-like proteases (SENP/Ulps). We show that SUMO-specific protease 1 (SUSP1), a mammalian SENP/Ulp, localizes within the nucleoplasm. SUSP1 depletion within cell lines expressing enhanced green fluorescent protein (EGFP) fusions to individual SUMO paralogues caused redistribution of EGFP-SUMO2 and -SUMO3, particularly into promyelocytic leukemia (PML) bodies. Further analysis suggested that this change resulted primarily from a deficit of SUMO2/3-deconjugation activity. Under these circumstances, PML bodies became enlarged and increased in number. We did not observe a comparable redistribution of EGFP-SUMO1. We have investigated the specificity of SUSP1 using vinyl sulfone inhibitors and model substrates. We found that SUSP1 has a strong paralogue bias toward SUMO2/3 and that it acts preferentially on substrates containing three or more SUMO2/3 moieties. Together, our findings argue that SUSP1 may play a specialized role in dismantling highly conjugated SUMO2 and -3 species that is critical for PML body maintenance.

Cell Line, Tumor↗
PubMed2006

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.

Amino Acid Motifs↗
PubMed2006

Doa1 is a Cdc48 adapter that possesses a novel ubiquitin binding domain.

Cdc48 (p97/VCP) is an AAA-ATPase molecular chaperone whose cellular functions are facilitated by its interaction with ubiquitin binding cofactors (e.g., Npl4-Ufd1 and Shp1). Several studies have shown that Saccharomyces cerevisiae Doa1 (Ufd3/Zzz4) and its mammalian homologue, PLAA, interact with Cdc48. However, the function of this interaction has not been determined, nor has a physiological link between these proteins been demonstrated. Herein, we demonstrate that Cdc48 interacts directly with the C-terminal PUL domain of Doa1. We find that Doa1 possesses a novel ubiquitin binding domain (we propose the name PFU domain, for PLAA family ubiquitin binding domain), which appears to be necessary for Doa1 function. Our data suggest that the PUL and PFU domains of Doa1 promote the formation of a Doa1-Cdc48-ubiquitin ternary complex, potentially allowing for the recruitment of ubiquitinated proteins to Cdc48. DOA1 and CDC48 mutations are epistatic, suggesting that their interaction is physiologically relevant. Lastly, we provide evidence of functional conservation within the PLAA family by showing that a human-yeast chimera binds to ubiquitin and complements doa1Delta phenotypes in yeast. Combined, our data suggest that Doa1 plays a physiological role as a ubiquitin binding cofactor of Cdc48 and that human PLAA may play an analogous role via its interaction with p97/VCP.

Adaptor Proteins, Signal Transducing↗
PubMed2005

The discovery of ubiquitin-dependent proteolysis.

In early 1980, Irwin A. Rose, Avram Hershko, and Aaron Ciechanover published two papers in PNAS that reported the astounding observation that energy-dependent intracellular proteolysis was far more complicated than the previously accepted models of lysosomal proteolysis or the action of ATP-dependent proteases such as bacterial lon. In fact, it has turned out to be even more complicated than they could have suspected. The general model of covalently attaching a small protein as a targeting signal has proved to be every bit as important to eukaryotic cells as the better understood modifications such as phosphorylation or acetylation. The key player in this modification, a small protein called ubiquitin (APF-1 in these papers), is the founding member of a large family of proteins containing the beta-grasp fold and is used as a posttranslational targeting signal to modify the structure, function, and/or localization of other proteins. The story of this discovery is a textbook example of the confluence of intellectual curiosity, unselfish collaboration, chance, luck, and preparation.

ATP-Dependent Proteases↗
PubMed2005

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.

Amino Acid Sequence↗
PubMed2005

Ubiquitin-proteasome degradation of KLF5 transcription factor in cancer and untransformed epithelial cells.

Ubiquitin-mediated proteolysis plays a central role in controlling intracellular levels of essential regulatory molecules such as p53, cyclins, myc, BRCA1, HIF-1alpha, etc. The Kruppel-like factor 5 (KLF5) transcription factor regulates biological processes involved in carcinogenesis, angiogenesis, and smooth muscle cell differentiation. In carcinogenesis, KLF5's role has been indicated by frequent genetic deletion as well as functional studies. Here we show that KLF5 is an unstable protein with a short half-life. Destruction of KLF5 was prevented by each of the proteasome-specific inhibitors tested but not by an inhibitor for trypsin-like proteases and cysteine proteases or by a lysosome inhibitor in epithelial cells. Furthermore, KLF5 underwent ubiquitination, and deletion of a 56-amino-acid sequence adjacent to a known transactivation domain of KLF5 significantly reduced its ubiquitination and degradation. Interestingly, cancer cells appeared to be more active in KLF5 degradation than untransformed epithelial cells, yet their proteasome activity was not higher. These results suggest that KLF5 protein is degraded at least in part through ubiquitination-proteasome pathway, which may have become hyperactive for KLF5 in cancer cells.

Blotting, Western↗
PubMed2005

Structure of a complex between Nedd8 and the Ulp/Senp protease family member Den1.

The Nedd8 conjugation pathway is conserved from yeast to humans and is essential in many organisms. Nedd8 is conjugated to cullin proteins in a process that alters SCF E3 ubiquitin ligase activity, and it is presumed that Nedd8 deconjugation would reverse these effects. We now report the X-ray structures of the human Nedd8-specific protease, Den1, in a complex with the inhibitor Nedd8 aldehyde, thus revealing a model for the tetrahedral transition state intermediate generated during proteolysis. Although Den1 is closely related to the SUMO-specific protease family (Ulp/Senp family), structural analysis of the interface suggests determinants involved in Nedd8 selectivity by Den1 over other ubiquitin-like family members and suggests how the Ulp/Senp architecture has been modified to interact with different ubiquitin-like modifiers.

Amino Acid Sequence↗
PubMed2005

Derivitization of the C-terminus of ubiquitin and ubiquitin-like proteins using intein chemistry: methods and uses.

Here we describe a general method to synthesize and use a panel of reagents with selectivity for deubiquitinating enzymes exhibiting specificity for each ubiquitin-like protein. A substrate (Ubl-AMC), a reversible inhibitor (Ubl-aldehyde), and an active-site-directed irreversible inhibitor (Ubl-vinylsulfone) are described for each Ubl. Because of space constraints, we give details for only the Nedd8 derivatives, but these methods have been used in our laboratory to produce these derivatives of Ubiquitin, Nedd8, Sumo-1, Sumo-2, and Isg15. These reagents are useful in defining the specificity of DUBs, as well as in purifying and identifying these important proteins. The reagents are selective and useful in crude extracts. The reactivity of these reagents reveals differences in both the S1 and S1' sites of deubiquitinating enzymes. Only active enzymes are efficiently detected with these reagents. Published results indicate that specificity is not strictly defined by the evolutionary relationships of these DUBs.

Base Sequence↗
PubMed2005

Deubiquitinating enzyme purification, assay inhibitors, and characterization.

Despite the identification of numerous deubiquitinating enzymes (DUBs) in recent years, the large majority of this class of enzymes has not been well characterized. This chapter describes biochemical methods that can be used to characterize the function and substrate specificity of DUBs. Methods described will include: fluorescence assay using ubiquitin-amidomethylcoumarin (AMC); a high-performance liquid chromatography assay using ubiquitin ethyl ester or ubiquitin fusion peptides as model substrates to monitor DUB activity; and the purification of a recombinant human DUB, isopeptidase T, in E. coli using low-temperature expression as well as ion-exchange and affinity chromatography.

Endopeptidases↗
PubMed2004

Hsp70 chaperones as modulators of prion life cycle: novel effects of Ssa and Ssb on the Saccharomyces cerevisiae prion [PSI+].

[PSI(+)] is a prion isoform of the yeast release factor Sup35. In some assays, the cytosolic chaperones Ssa1 and Ssb1/2 of the Hsp70 family were previously shown to exhibit "pro-[PSI(+)]" and "anti-[PSI(+)]" effects, respectively. Here, it is demonstrated for the first time that excess Ssa1 increases de novo formation of [PSI(+)] and that pro-[PSI(+)] effects of Ssa1 are shared by all other Ssa proteins. Experiments with chimeric constructs show that the peptide-binding domain is a major determinant of differences in the effects of Ssa and Ssb proteins on [PSI(+)]. Surprisingly, overproduction of either chaperone increases loss of [PSI(+)] when Sup35 is simultaneously overproduced. Excess Ssa increases both the average size of prion polymers and the proportion of monomeric Sup35 protein. Both in vivo and in vitro experiments uncover direct physical interactions between Sup35 and Hsp70 proteins. The proposed model postulates that Ssa stimulates prion formation and polymer growth by stabilizing misfolded proteins, which serve as substrates for prion conversion. In the case of very large prion aggregates, further increase in size may lead to the loss of prion activity. In contrast, Ssb either stimulates refolding into nonprion conformation or targets misfolded proteins for degradation, in this way counteracting prion formation and propagation.

Adenosine Triphosphatases↗
PubMed2004

Identification of a novel 29-linked polyubiquitin binding protein, Ufd3, using polyubiquitin chain analogues.

Lysine 48-linked polyubiquitin chains are the best understood form of polyubiquitin and are necessary for the function of the ubiquitin-proteasome system. However, other forms of polyubiquitin (e.g., K29- and K63-linked chains) are also present in vivo. Less is known about the functional roles of these linkages or the proteins specifically interacting with these forms of polyubiquitin. Use of native polyubiquitin chains to identify binding proteins is complicated by the difficulties of synthesis and stability. Here, we report the synthesis of a nonhydrolyzable analogue of 29-linked polyubiquitin chains on an affinity support and its use in identifying proteins that bind 29-linked polyubiquitin chains. The 29-linked Ub4 resin was stable and tightly bound recombinant human Isopeptidase T (USP5), a deubiquitinating enzyme known to bind the 29-linked polyubiquitin chains. Two high affinity interactors of the 29-linked polyubiquitin analogues were identified from Saccharomyces cerevisiae lysates. They were identified as Ubp14, the yeast ortholog of Isopeptidase T, and Ufd3, a member of the ubiquitin-fusion degradation pathway with unknown function. Purified recombinant Ufd3 bound to the resin as well, confirming that Ufd3 is a novel binding partner of polyubiquitin. These results demonstrate the efficacy of using polyubiquitin analogue affinity supports to identify novel binding partners of specifically linked polyubiquitin chains. Identification of these proteins will lead to a greater understanding of the physiological relevance of different polyubiquitin linkages.

Adaptor Proteins, Signal Transducing↗
PubMed2004

Modulating huntingtin half-life alters polyglutamine-dependent aggregate formation and cell toxicity.

A common finding among the expanded polyglutamine disorders is intracellular protein aggregates. Although the precise role of these aggregates in the disease process is unclear, they are generally ubiquitinated, implicating the ubiquitin-proteasome pathway in neuronal degeneration. To investigate the mechanism of aggregate formation, we have developed a cell culture model to express huntingtin designed to have an altered degradation rate through the ubiquitin-dependent N-end rule pathway. We fused the first 171 amino acids of huntingtin, containing either a pathogenic or normal polyglutamine tract, to the enhanced green fluorescent protein (EGFP). The half-life of soluble huntingtin-EGFP was dependent on the degradation signal and the polyglutamine tract length. However, once huntingtin-EGFP with a pathogenic tract had aggregated, the protein was extremely stable. Huntingtin-EGFP with a pathogenic glutamine tract and a shorter half-life displayed a delayed onset of aggregate formation and was more toxic to transfected cells. These data suggest that rapid clearance through the ubiquitin-proteasome pathway slows aggregate formation, yet increases cellular toxicity. Polyglutamine-induced neurotoxicity may therefore be triggered by non-aggregated protein, and aggregate formation itself may be a cellular defense mechanism.

Cell Line↗
PubMed2004

Specific and covalent targeting of conjugating and deconjugating enzymes of ubiquitin-like proteins.

Modification of proteins by ubiquitin (Ub)-like proteins (UBLs) plays an important role in many cellular processes, including cell cycle progression, nuclear transport, and autophagy. Protein modification occurs via UBL-conjugating and -deconjugating enzymes, which presumably exert a regulatory function by determining the conjugation status of the substrate proteins. To target and identify UBL-modifying enzymes, we produced Nedd8, ISG15, and SUMO-1 in Escherichia coli and equipped them with a C-terminal electrophilic trap (vinyl sulfone [VS]) via an intein-based method. These C-terminally modified UBL probes reacted with purified UBL-activating (E1), -conjugating (E2), and -deconjugating enzymes in a covalent fashion. Modified UBLs were radioiodinated and incubated with cell lysates prepared from mouse cell lines and tissues to allow visualization of polypeptides reactive with individual UBL probes. The cell type- and tissue-specific labeling patterns observed for the UBL probes reflect distinct expression profiles of active enzymes, indicating tissue-specific functions of UBLs. We identify Ub C-terminal hydrolase L1 (UCH-L1) and DEN1/NEDP1/SENP8, in addition to UCH-L3, as proteases with specificity for Nedd8. The Ub-specific protease isopeptidase T/USP5 is shown to react with ISG15-VS. Furthermore, we demonstrate that the desumoylation enzyme SuPr-1 can be modified by SUMO-1-VS, a modification that is dependent on the SuPr-1 active-site cysteine. The UBL probes described here will be valuable tools for the further characterization of the enzymatic pathways that govern modification by UBLs.

Animals↗
PubMed2004

Quantitative analysis of protein-protein interactions.

Numerous authors, including contributors to this volume, have described methods to detect protein-protein interactions. Many of these approaches are now accessible to the inexperienced investigator thanks to core facilities and/or affordable instrumentation. This chapter discusses some common design considerations that are necessary to obtain valid measurements, as well as the assumptions and analytical methods that are relevant to the quantitation of these interactions.

Kinetics↗