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Huib Ovaa

Publications and source records attributed to Huib Ovaa.

At least 19 recordsLinked to original sources
PubMed2006

Two novel ubiquitin-fold modifier 1 (Ufm1)-specific proteases, UfSP1 and UfSP2.

Ubiquitin-fold modifier 1 (Ufm1) is a recently identified new ubiquitin-like protein, whose tertiary structure displays a striking resemblance to ubiquitin. Similar to ubiquitin, it has a Gly residue conserved across species at the C-terminal region with extensions of various amino acid sequences that need to be processed in vivo prior to conjugation to target proteins. Here we report the isolation, cloning, and characterization of two novel mouse Ufm1-specific proteases, named UfSP1 and UfSP2. UfSP1 and UfSP2 are composed of 217 and 461 amino acids, respectively, and they have no sequence homology with previously known proteases. UfSP2 is present in most, if not all, of multicellular organisms including plant, nematode, fly, and mammal, whereas UfSP1 could not be found in plant and nematode upon data base search. UfSP1 and UfSP2 cleaved the C-terminal extension of Ufm1 but not that of ubiquitin or other ubiquitin-like proteins, such as SUMO-1 and ISG15. Both were also capable of releasing Ufm1 from Ufm1-conjugated cellular proteins. They were sensitive to inhibition by sulfhydryl-blocking agents, such as N-ethylmaleimide, and their active site Cys could be labeled with Ufm1-vinylmethylester. Moreover, replacement of the conserved Cys residue by Ser resulted in a complete loss of the UfSP1 and UfSP2 activities. These results indicate that UfSP1 and UfSP2 are novel thiol proteases that specifically process the C terminus of Ufm1.

Amino Acid Substitution↗
PubMed2006

Comparative selectivity and specificity of the proteasome inhibitors BzLLLCOCHO, PS-341, and MG-132.

The 26S proteasome is a multicatalytic protease responsible for regulated intracellular protein degradation. Its function is mediated by three main catalytic activities: (a) chymotrypsin-like (CT-L), (b) trypsin-like, and (c) peptidylglutamyl peptide hydrolysing (PGPH). Proteasome inhibition is an emerging therapy for many cancers and is a novel treatment for multiple myeloma. Here, we profile the contributions of the three catalytic activities in multiple myeloma cell lines and compare the specificity and cytotoxicity of the novel proteasome inhibitor BzLLLCOCHO and inhibitors PS-341 (Velcade, bortezomib) and MG-132. Using fluorogenic substrates and an active site-directed probe specific for proteasome catalytic subunits, we show differential subunit specificity for each of the inhibitors. Addition of BzLLLCOCHO strongly inhibited all three catalytic activities, treatment with PS-341 completely inhibited CT-L and PGPH activities, and treatment with MG-132 resulted in weak inhibition of the CT-L and PGPH activities. Multiple myeloma cells were more sensitive to induction of apoptosis by PS-341 and MG-132 than BzLLLCOCHO. This study emphasizes the need for further investigation of the effects of these compounds on gene and protein expression in the cell to allow for the development of more specific and targeted inhibitors.

Apoptosis↗
PubMed2006

Design and use of conditional MHC class I ligands.

Major histocompatibility complex (MHC) class I molecules associate with a variety of peptide ligands during biosynthesis and present these ligands on the cell surface for recognition by cytotoxic T cells. We have designed conditional MHC ligands that form stable complexes with MHC molecules but degrade on command, by exposure to a defined photostimulus. 'Empty MHC molecules' generated in this manner can be loaded with arrays of peptide ligands to determine MHC binding properties and to monitor antigen-specific T-cell responses in a high-throughput manner. We document the value of this approach by identifying cytotoxic T-cell epitopes within the H5N1 influenza A/Vietnam/1194/04 genome.

Amino Acid Sequence↗
PubMed2006

Activity profiling of deubiquitinating enzymes in cervical carcinoma biopsies and cell lines.

Ubiquitin specific proteases (USPs) regulate the production and recycling of ubiquitin and are thereby critically involved in the control of cell growth, differentiation, and apoptosis. Increasing evidence implicates deregulation of USPs in malignant transformation but there is very little information on the overall and specific activity of USPs in normal and tumor tissues. We have used a chemistry-based functional proteomics approach to profile the activities of individual USPs in biopsies of human papillomavirus (HPV) carrying cervical carcinoma and adjacent normal tissue. To assess the contribution of HPV proteins, USP activity was also compared in HPV positive and negative cervical carcinoma cell lines and HPV E6/E7 immortalized human keratinocytes. The activity of the C-terminal hydrolases UCH-L3 and UCH37 was upregulated in the majority of tumor tissues compared to the adjacent normal tissues. UCH-L1 activity was lower in a significant proportion of the tumors but to a less extent in advanced tumors. In accordance with the relatively low UCH-L1 activity in tumor biopsies, UCH-L1 was detected only in one out of eight cervical carcinoma lines. UCH-L1, UCH-L3, USP7, and USP9X activity was upregulated following HPV E6/E7 immortalization of keratinocytes, suggesting a role of these enzymes in growth transformation.

Biopsy↗
PubMed2006

A fluorescent broad-spectrum proteasome inhibitor for labeling proteasomes in vitro and in vivo.

The proteasome is an essential evolutionary conserved protease involved in many regulatory systems. Here, we describe the synthesis and characterization of the activity-based, fluorescent, and cell-permeable inhibitor Bodipy TMR-Ahx(3)L(3)VS (MV151), which specifically targets all active subunits of the proteasome and immunoproteasome in living cells, allowing for rapid and sensitive in-gel detection. The inhibition profile of a panel of commonly used proteasome inhibitors could be readily determined by MV151 labeling. Administration of MV151 to mice allowed for in vivo labeling of proteasomes, which correlated with inhibition of proteasomal degradation in the affected tissues. This probe can be used for many applications ranging from clinical profiling of proteasome activity, to biochemical analysis of subunit specificity of inhibitors, and to cell biological analysis of the proteasome function and dynamics in living cells.

Animals↗
PubMed2006

Crystal structure of the boronic acid-based proteasome inhibitor bortezomib in complex with the yeast 20S proteasome.

The dipeptide boronic acid bortezomib, also termed VELCADE, is a proteasome inhibitor now in use for the treatment of multiple myeloma, and its use for the treatment of other malignancies is being explored. We determined the crystal structure of the yeast 20S proteasome in complex with bortezomib to establish the specificity and binding mode of bortezomib to the proteasome's different catalytically active sites. This structure should enable the rational design of new boronic acid derivatives with improved affinities and specificities for individual active subunits.

Animals↗
PubMed2006

Chemical proteomics profiling of proteasome activity.

Proteolysis is a key mechanism for protein homeostasis in living cells. This process is effected by different classes of proteases. The proteasome is one of the most abundant and versatile proteases, bearing three different proteolytic active sites. The proteasome plays an important role in essential biological pathways such as antigen presentation, signal transduction, and cell-cycle control feedback loops. The aim of this work is to design novel chemical strategies for capturing, detection, identification, and quantification--in one word, profiling--the active protease fractions of interest, in cells of different phenotypes. Here, a set of chemistry-based functional proteomics techniques is demonstrated by profiling the multi-catalytic protease activities of the proteasome. Importantly, functional profiling is complementary to expression level profiling and is an indispensable parameter for better understanding of mechanisms underlying biological processes.

Blotting, Western↗
PubMed2006

Ubiquitin proteasome system as a pharmacological target in neurodegeneration.

Ubiquitinated protein aggregates are observed in the brains of Alzheimer's, Parkinson's and Huntington's disease patients and in other neurodegenerative disorders. These aggregates indicate that the ubiquitin proteasome system may be impaired in these diseases. To date no therapy is available that specifically targets this system, although preventing aggregate formation or stimulating the degradation of already formed aggregates by targeting components of the ubiquitin proteasome system is an attractive therapeutic approach. Here, we review the role of the ubiquitin proteasome system in aggregate formation with respect to neurodegenerative diseases, discussing the unfolded protein response, endoplasmic reticulum-associated degradation, aggresome formation and accumulation as well as aggregation and neurotoxicity of proteins involved in neurodegeneration. The potential of pharmacological intervention within this system in patients suffering from neurodegenerative diseases will be evaluated.

Animals↗
PubMed2005

Loss of HAUSP-mediated deubiquitination contributes to DNA damage-induced destabilization of Hdmx and Hdm2.

The p53 tumor suppressor protein has a major role in protecting the integrity of the genome. In unstressed cells, p53 is maintained at low levels by the ubiquitin-proteasome pathway. A balance between ubiquitin ligase activity (Hdm2, COP1, and Pirh2) and the ubiquitin protease activity of the Herpes virus-associated ubiquitin-specific protease (HAUSP) determines the half-life of p53. HAUSP also modulates p53 stability indirectly by deubiquitination and stabilization of Hdm2. The Hdmx protein affects p53 stability as well through its interaction with and regulation of Hdm2. Vice versa, Hdmx is a target for Hdm2-mediated ubiquitination and degradation. Here, we show that HAUSP also interacts with Hdmx, resulting in its direct deubiquitination and stabilization. HAUSP activity is required to maintain normal Hdmx protein levels. Therefore, the balance between HAUSP and Hdm2 activity determines Hdmx protein stability. Importantly, impaired deubiquitination of Hdmx/Hdm2 by HAUSP contributes to the DNA damage-induced degradation of Hdmx and transient instability of Hdm2.

Animals↗
PubMed2005

Activity probe for in vivo profiling of the specificity of proteasome inhibitor bortezomib.

Proteasome inhibitors, such as the dipeptide boronic acid bortezomib, are emerging as important tools in the treatment of the fatal hematologic malignancy multiple myeloma. Despite the recent US Food and Drug Administration approval of bortezomib (PS341, Velcade) for the treatment of refractory multiple myeloma, many of the basic pharmacologic parameters of bortezomib and its mode of action on myeloma cells remain to be determined. We describe the synthesis and use of a cell-permeant active site-directed probe, which allows profiling of proteasomal activities in living cells. When we compared proteasome activity patterns in cultured cells and crude cell extracts with this probe, we observed substantial differences, stressing the importance for bioassays compatible with live cells to ensure accuracy of such measurements. Using this probe, we investigated the in vivo subunit specificities of bortezomib and another inhibitor, MG132.

Animals↗
PubMed2005

A novel orally active proteasome inhibitor induces apoptosis in multiple myeloma cells with mechanisms distinct from Bortezomib.

Bortezomib therapy has proven successful for the treatment of relapsed and/or refractory multiple myeloma (MM); however, prolonged treatment is associated with toxicity and development of drug resistance. Here, we show that the novel proteasome inhibitor NPI-0052 induces apoptosis in MM cells resistant to conventional and Bortezomib therapies. NPI-0052 is distinct from Bortezomib in its chemical structure, effects on proteasome activities, mechanisms of action, and toxicity profile against normal cells. Moreover, NPI-0052 is orally bioactive. In animal tumor model studies, NPI-0052 is well tolerated and prolongs survival, with significantly reduced tumor recurrence. Combining NPI-0052 and Bortezomib induces synergistic anti-MM activity. Our study therefore provides the rationale for clinical protocols evaluating NPI-0052, alone and together with Bortezomib, to improve patient outcome in MM.

Administration, Oral↗
PubMed2005

Phosphoproteomic analysis with a solid-phase capture-release-tag approach.

A comprehensive study of global phosphorylation events in biological systems is critical. We report a chemistry-based capture-release-tag method for isolation of complex phospho-Ser/Thr-containing peptides by liquid beta-elimination combined with solid-phase Michael addition. The free thiol groups of 6-(mercapto-acetylamino)-hexanoic acid functionalized resin are used as immobilized Michael donors to capture dehydro-serine/threonine peptides. After an acid-mediated release step, phospho-peptides are labeled with a 6-(2-mercapto-acetylamine)-hexanoic amide tag at phosphorylated sites. We applied this method to analyze the phosphorylation status of microtubule-associated proteins. We find that a CDK5 substrate microtubule-associated protein 2 (MAP2) is phosphorylated on residues that are within a homologous region of Tau. The chemical method corroborates previous results and suggests that Tau and MAP2 may contain a CDK5 phosphorylation motif.

Amino Acid Sequence↗
PubMed2005

Immunotherapeutic potential for ceramide-based activators of iNKT cells.

Invariant natural killer T (iNKT) cells are a subpopulation of T cells that are reactive with glycolipids that are bound by CD1d antigen-presenting molecules. alpha-Galactosylceramide (alpha-GalCer) is a synthetic glycolipid that is a potential treatment for several autoimmune diseases, infectious diseases and cancer. Its therapeutic effect can be traced back to its ability to bind CD1d and activate iNKT cells, which results in the production of T helper 1 (Th1) and Th2 cytokines. However, the effectiveness of alpha-GalCer therapy is limited by the opposing actions of Th1 and Th2 cytokines. The alpha-GalCer analogs OCH and the recently designed and synthesized C-glycosidic alpha-GalCer selectively activate one of two types of cytokine profiles, possibly because either reduced or enhanced overall stability of the CD1d-glycolipid-T-cell-receptor complex leads to a higher potency in vivo, compared with the parent compound. These discoveries boost the potential of glycolipid-based therapies.

Animals↗
PubMed2005

Mechanism-based proteomics tools based on ubiquitin and ubiquitin-like proteins: crystallography, activity profiling, and protease identification.

Isopeptidases that specifically remove ubiquitin or ubiquitin-like molecules from polypeptide adducts are emerging as key regulatory enzymes in a multitude of biochemical pathways. We have developed a set of tools that covalently target the active site of ubiquitin or ubiquitin-like deconjugating enzymes. We have used epitope-tagged ubiquitin and ubiquitin-like derivatives in immunoprecipitation assays to identify active proteases by mass spectrometry (MS/MS). The epitope tag confers the ability to conduct an immunoblot-based profiling assay for active isopeptidases in cell extracts. We have applied a ubiquitin-based probe in the structural analysis of the ubiquitin hydrolase UCH-L3 in its ligand-bound state. We describe the use of these electrophilic derivatives of ubiquitin and ubiquitin-like molecules in the identification, activity profiling, and structural analysis of these proteases. These tools can be used to rapidly profile activity of multiple Ub/UBL-specific proteases in parallel in cell extracts. We also show that in vitro these probes can be conjugated onto parts of the Ub/UBL conjugating machinery.

Crystallography↗
PubMed2005

Mechanism-based proteomics tools based on ubiquitin and ubiquitin-like proteins: synthesis of active site-directed probes.

The families of ubiquitin and ubiquitin-like modifiers are involved in the regulation of many biochemical pathways. The steady-state level of polypeptide modification with these molecules depends on the opposing activity of conjugating and deconjugating enzymes. Here we describe the generation of mechanism-dependent active site-directed probes that target the large family of ubiquitin/ubiquitin-like isopeptidases. To maintain substrate specificity for these enzymes, we have based the development of these probes on full-length sequences of ubiquitin and several ubiquitin-like molecules. For their construction, this approach necessitates the use of a combination of organic synthesis and expressed protein ligation. These probes have been used in the isolation and identification of active isopeptidases from crude cell extracts and have been instrumental in the discovery of novel ubiquitin and ubiquitin-like deconjugating enzymes. These probes may be generated with or without an epitope tag that enables activity profiling of proteases from cell extracts. In addition, we have used a ubiquitin-based probe in the structural analysis of the ligand-bound form of the enzyme UCH-L3 by x-ray crystallography. Together, these probes greatly facilitate the study of ubiquitin and ubiquitin-like proteases.

Binding Sites↗
PubMed2004

Structure of the ubiquitin hydrolase UCH-L3 complexed with a suicide substrate.

Ubiquitin C-terminal hydrolases (UCHs) comprise a family of small ubiquitin-specific proteases of uncertain function. Although no cellular substrates have been identified for UCHs, their highly tissue-specific expression patterns and the association of UCH-L1 mutations with human disease strongly suggest a critical role. The structure of the yeast UCH Yuh1-ubiquitin aldehyde complex identified an active site crossover loop predicted to limit the size of suitable substrates. We report the 1.45 A resolution crystal structure of human UCH-L3 in complex with the inhibitor ubiquitin vinylmethylester, an inhibitor that forms a covalent adduct with the active site cysteine of ubiquitin-specific proteases. This structure confirms the predicted mechanism of the inhibitor and allows the direct comparison of a UCH family enzyme in the free and ligand-bound state. We also show the efficient hydrolysis by human UCH-L3 of a 13-residue peptide in isopeptide linkage with ubiquitin, consistent with considerable flexibility in UCH substrate size. We propose a model for the catalytic cycle of UCH family members which accounts for the hydrolysis of larger ubiquitin conjugates.

Amino Acid Sequence↗
PubMed2004

Differential dependence of CD4+CD25+ regulatory and natural killer-like T cells on signals leading to NF-kappaB activation.

Natural killer-like (NK) T, regulatory T (TR), and memory type T cells display surface phenotypes reminiscent of activated T cells. Previously, we reported that the generation of TR cells and, to a lesser extent, of memory type T cells, depends on IkappaB kinase 2. Here, we show that T cell-specific ablation of IkappaB kinase 2, in addition, completely precludes NKT cell development. T cell antigen receptor (TCR)-induced signals to activate NF-kappaB are essential for mature T cell activation, leading us to hypothesize that this pathway could play an important role in the generation of the antigen-driven T cell subsets comprising TR, memory type T, and NKT cells. TCR-mediated NF-kappaB activation critically depends on Bcl10 and PKCtheta. By using mice deficient for these proteins, we demonstrate that the generation of TR and, to a lesser extent, of memory type T cells, depends on Bcl10 and PKCtheta, and therefore, most likely on NF-kappaB activation initiated by TCR engagement. NKT cells, on the other hand, require PKCtheta for thymic development, whereas absence of Bcl10 leads primarily to the reduction of peripheral NKT cell numbers.

Adaptor Proteins, Signal Transducing↗