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Seiberlich V.,University of Oldenburg | Goldbaum O.,University of Oldenburg | Zhukareva V.,LifeSensors Inc. | Richter-Landsberg C.,University of Oldenburg
Biochimica et Biophysica Acta - Molecular Cell Research | Year: 2012

A pathological hallmark of many neurodegenerative diseases is the aggregation of proteins. Protein aggregate formation may be linked to a failure of the ubiquitin proteasome system (UPS) and/or the autophagy pathway. The UPS involves the ubiquitination of proteins followed by proteasomal degradation. Deubiquitination of target proteins is performed by proteases called deubiquitinating proteins (DUBs). Inhibition of DUBs may lead to the dysregulation of homeostasis and have pathological consequences. To assess the effects of DUB-inhibition, we have used the oligodendroglial cell line, OLN-t40, stably expressing the longest human tau isoform. Cells were incubated with PR-619, a broad-range, reversible inhibitor of ubiquitin isopeptidases. Incubation with PR-619 led to morphological changes, the upregulation of heat shock proteins (HSP), including HSP70 and αB-crystallin, and to protein aggregates near the MTOC, containing ubiquitin, HSPs, and the ubiquitin binding protein p62, which may provide a link between the UPS and autophagy. Thus, inhibition of DUB activity caused stress responses and the formation of protein aggregates resembling pathological inclusions observed in aggregopathies. Furthermore, PR-619 led to the stabilization of the microtubule network, possibly through the modulation of tau phosphorylation, and small tau deposits assembled near the MTOC. Hence, organization and integrity of the cytoskeleton were affected, which is particularly important for the maintenance of the cellular architecture and intracellular transport processes, and essential for the functionality and survival of neural cells. Our data demonstrate that DUB inhibitors provide a useful tool to elucidate the manifold mechanisms of DUB functions in cells and their dysregulation in neurodegenerative diseases. This article is part of a Special Issue entitled: Ubiquitin Drug Discovery and Diagnostics. © 2012 Elsevier B.V. Source


Calixto A.,Columbia University | Calixto A.,Instituto Milenio Of Biologia Fundamentaly Aplicada | Calixto A.,University of Santiago de Chile | Chelur D.,Columbia University | And 5 more authors.
Nature Methods | Year: 2010

We expressed SID-1, a transmembrane protein from Caenorhabditis elegans that is required for systemic RNA interference (RNAi), in C. elegans neurons. This expression increased the response of neurons to double-stranded (ds)RNA delivered by feeding. Mutations in the lin-15b and lin-35 genes enhanced this effect. Worms expressing neuronal SID-1 showed RNAi phenotypes when fed with bacteria expressing dsRNA for known neuronal genes and for uncharacterized genes with no previously known neuronal phenotypes. Neuronal expression of sid-1 decreased nonneuronal RNAi, suggesting that neurons expressing transgenic sid-1(+) served as a sink for dsRNA. This effect, or a sid-1(-) background, can be used to uncover neuronal defects for lethal genes. Expression of sid-1(+) from cell-specific promoters in sid-1 mutants results in cell-specific feeding RNAi. We used these strains to identify a role for integrin signaling genes in mechanosensation. © 2010 Nature America, Inc. All rights reserved. Source


Balut C.M.,University of Pittsburgh | Loch C.M.,LifeSensors Inc. | Devor D.C.,University of Pittsburgh
FASEB Journal | Year: 2011

We recently demonstrated that plasma membrane KCa3.1 is rapidly endocytosed and targeted for lysosomal degradation via a Rab7-and ESCRT-dependent pathway. Herein, we assess the role of ubiquitylation in this process. Using a biotin ligase acceptor peptide (BLAP)-tagged KCa3.1, in combination with tandem ubiquitin binding entities (TUBEs), we demonstrate that KCa3.1 is polyubiquitylated following endocytosis. Hypertonic sucrose inhibited KCa3.1 endocytosis and resulted in a significant decrease in channel ubiquitylation. Inhibition of the ubiquitin-activating enzyme (E1) with UBEI-41 resulted in reduced KCa3.1 ubiquitylation and internalization. The general deubiquitylase (DUB) inhibitor, PR-619 attenuated KCa3.1 degradation, indicative of deubiquitylation being required for lysosomal delivery. Using the DUB Chip, a protein microarray containing 35 DUBs, we demonstrate a time-dependent association between KCa3.1 and USP8 following endocytosis, which was confirmed by coimmunoprecipitation. Further, overexpression of wild-type USP8 accelerates channel deubiquitylation, while either a catalytically inactive mutant USP8 or siRNA-mediated knockdown of USP8 enhanced accumulation of ubiquitylated KCa3.1, thereby inhibiting channel degradation. In summary, by combining BLAP-tagged KCa3.1 with TUBEs and DUB Chip methodologies, we demonstrate that polyubiquitylation mediates the targeting of membrane KCa3.1 to the lysosomes and also that USP8 regulates the rate of KCa3.1 degradation by deubiquitylating KCa3.1 prior to lysosomal delivery. © FASEB. Source


Grant
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 296.29K | Year: 2012

DESCRIPTION (provided by applicant): Identification, quantification, and isolation of low abundance proteins from complex mixtures is, at best, a difficult task. This is especially the case for proteins carrying a post-translational modification (PTM) that affects their half-life or regulatory properties. Examples of such PTMs include phosphorylation, glycosylation (especially O-GlcNAcylation), and ubiquitinylation. In many instances, one PTM can abrogate or enhance other PTMs on the same protein providingexquisite mechanisms for control of the protein's activity. Fishing a protein with one particular PTM out of the pool of possible modifie proteins becomes nearly impossible without selective tools. A further complication in the case of ubiquitinylation is the presence of multiple types of Ub-Ub linkages in polyubiquitin chains. Ubiquitin (Ub) is attached, via isopeptide bonds, to lysine residues in the target protein. These Ub-moieties can then serve as substrates for the conjugation of additional Ubs,again through the formation of isopeptide bonds between the C- terminus of one Ub and any of seven (7) lysines in the target Ub. The general consensus in the field is that chains with different linkages convey different meanings to the cell and hence, determine the ultimate fate of the protein, be it degradation, translocation, phosphorylation, etc. The precise information encoded in different chain linkages is largely unknown due to the lack of specific reagents that recognize different linkages. The goal of this proposal is to develop tools that allow the selective identification, quantification, and isolation of proteins modified by polyubiquitin chains containing different linkages. This will be accomplished using information encoded in the human genome that allows the cell to discriminate between different linkages, i.e. Ub-binding domains (UbDs). In Phase I, we will identify and characterize novel UbDs exhibiting, at least, partial selectivity. In Phase II we will use these UbDs to construct higher avidity reagents capable of linkage-specific discrimination. Both ubiquitinylation and de- ubiquitinylation have been linked to cancer, inflammation and neurological diseases; hence, the tools developed in this grant will have a major impact on our ability to dissect these disease processes. PUBLIC HEALTH RELEVANCE: Ubiquitinylation and de-ubiquitinylation are post-translational modifications that affect the function of many proteins and whose dysregulation has been implicated in many disease processes from cancer to inflammation to neurological degeneration. Study of these modifications is difficult due to their low abundance and the complexity of polyubiquitin chains. This grant proposes to generate specific reagents that will distinguish between different forms of polyubiquitin, enabling their detection and isolation from complex mixtures. Use of these tools will dramatically enhance our understanding of many diseases.


Patent
Progenra, Inc. and LifeSensors Inc. | Date: 2013-08-26

Methods for detection of the activity of proteolytic enzymes, particularly isopeptidases, are disclosed.

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