Progenra, Inc. and LifeSensors Inc. | Date: 2013-08-26
Methods for detection of the activity of proteolytic enzymes, particularly isopeptidases, are disclosed.
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.
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.
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase II | Award Amount: 945.45K | Year: 2012
DESCRIPTION (provided by applicant): In the last decade there has been an explosive growth in the field of ubiquitin research, with approximately 530 human genes predicted to encode enzymes involved in the conjugation and deconjugation of ubiquitin. Of these 95 encode for deubiquitylases (DUBs). In order understand the biology of these enzymes better, there is a need for better assays to measure the most physiologically relevant activity of the enzymes. All the currently available high throughput methods for measuring deubiquitylase activity rely on C-terminal amidohydrolase activity (involved in processing the precursors of ubiquitin) rather than the isopeptidase activity involved in ubiquitin deconjugation (important in regulating various cellular processes) of DUBs. The most widely used substrate, Ub-AMC has a small fluorophore attached to the C-terminus of ubiquitin via an amide bond. Hydrolysis of this C-terminal amide bond by a deubiquitylase leads to an increase in fluorescence. This assay format doesnot adequately mimic the more important physiological event - deconjugation of ubiquitin via isopeptidase activity. Furthermore, many DUBs do not possess C-terminal amidohydrolytic activity and hence are unable to cleave conjugates like Ub-AMC. Although itis possible to measure isopeptidase activity with physiological substrates such as commercially available ubiquitin chains by SDS-PAGE, western blotting or LC/MS, such options are viable only if a small number of samples are being tested. For screening small molecules or natural products for inhibitors of isopeptidases these methods are unacceptable. In Phase I we developed a novel fluorescent assay for measuring the actual isopeptidase activity of the DUBs with substrates that are more relevant to physiological conditions. This assay is amenable to high throughput screening and does not suffer from the limitations shared by current DUB assays. Briefly, we have created a series of diUb molecules in which one Ub chain is derivatized with a fluorescence quenching dye and the second Ub moiety carrys a fluorophore. The two Ubs are joined by an isopeptide bond linking the C-terminus of one Ub to either Lys48 (K48) or Lys63 (K63) of the second Ub. Following hydrolysis of this isopeptide bond by a DUB, FRET-quenching is released and the resulting increase in fluorescence is directly proportional to DUB activity. We have validated these substrates using DUBs which only cleave the K48- or K63-linkage. In the current grant application, we propose to extend this technology to the remaining 5 Lys's in ubiquitin and create a panel of substrates encompassing all of ubiquitin isopeptide linkages. These substrates will greatly enhance our understanding of DUB activity and selectivity and enable high throughput screening campaigns using physiologically relevant substrates. PUBLIC HEALTH RELEVANCE: Modification of proteins by ubiquitin plays important roles in many cellular processes. In the last decade there has been an explosive growth in the field of ubiquitin research. The enzymes that remove ubiquitin from target proteins are very important drug targets. There is a need for better assays to measure the activity of the enzymes, which are highly specific and physiologically relevant. The development of assays using substrates that are physiologically relevant, the topic of this proposal, represents a major advancement in the study of this important group of cellular enzymes.
Agency: Department of Health and Human Services | Branch: National Institutes of Health | Program: SBIR | Phase: Phase I | Award Amount: 224.66K | Year: 2016
Linkage specific ubiquitylation patterns as highly sensitive markers for neurodegenerative disease Abstract Despite an intensive effort by the government pharmaceutical companies and academic groups the diagnosis and treatment of Alzheimerandapos s disease remains elusive The incidence of Alzheimerandapos s disease is predicted to increase reaching more than million cases in USA by Thus the development of quantitative simple and reproducible diagnostic markers is extremely important Current methods for diagnosis of Alzheimerandapos s disease are dependent on clinical and neuropsychological assessment cerebrospinal fluid CSF analysis and brain imaging procedures all of which have significant cost and access to care barriers Thus the need for simple blood based biomarkers has never been greater We hypothesize that dysfunction in the ubiquitin proteasome pathway occurs decades before the clinical manifestation of Alzheimerandapos s disease symptoms Neurons generally do not divide and their survival depends heavily on the removal of misfolded proteins The first step in neurodegenerative disease begins with a diminished response to unfolded protein removal and proteasomal dysfunction Therefore neuronal diseases are marked by the appearance or change in pattern of ubiquitylated proteins the main hypothesis of this proposal which describes a unique combination of affinity purification of ubiquitylated proteins and mass spectroscopy approaches to detect ubiquitylated proteins and also determine the nature of the poly ubiquitin chain linkage Although the roles of the ubiquitin pathway in cell physiology and pathology have been recognized for the last three decades surprisingly there are no reliable and sensitive methods available to monitor patterns of ubiquitylation The development of this technology will enable clinicians to make early diagnoses of Alzheimerandapos s disease facilitate the discovery of disease modifying drugs and open doors for cell biologists to rapidly identify patterns of poly ubiquitylated proteins in tissues cells or body fluids PUBLIC HEALTH SIGNIFICANCE The proposed studies are based on the hypothesis that changes in the pattern of ubiquitylation of certain proteins both in extent and in the structure of the polyubiquitin chains appear early on in the onset of neurodegenerative diseases especially Alzheimers Disease AD Identification of these changes by a combination of linkage specific polyubiquitin affinity based purification matrices and high resolution quantitative mass spectrometry should provide lead markers for the development of diagnostic tests for the early detection of AD and new therapeutic approaches
Agency: Department of Health and Human Services | Branch: National Institutes of Health | Program: SBIR | Phase: Phase I | Award Amount: 224.67K | Year: 2016
Abstract Cell based assays more closely mimic biology than direct inhibition studies Therefore drug discovery companies are increasingly utilizing cellular screening assays Current reporter systems are unable to reliably measure in cell transient protein protein interactions Furthermore many are based upon split enzyme systems which require long incubation steps are not real time and are typically low throughput In order to improve on these existing technologies we will develop a split SUMOstar system engineered to contain a tetracysteine recognition motif for a biarsenical based fluorophores There are several advantages for using split a SUMOstar fluorescent reporter system including relatively small fusions Kda to the target proteins of interest lower non specific interaction with native proteins and the ability to take real time measurements in high throughput applications Furthermore using structural based mutagenesis we can engineer the kinetics and binding affinity of the split SUMO interaction allowing us to fine tune the reporter assay to measure transient protein protein interactions Transient protein protein interactions are key to understanding cellular processes in neurological disease states Currently these interactions are poorly understood in a cellular environment We propose to build a detection system for monitoring transient protein protein interactions in cells This will allow high throughput drug screening against new targets and enable novel research of neurological disease state pathways
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 225.00K | Year: 2014
DESCRIPTION (provided by applicant): In the last decade there has been an explosive growth in the field of ubiquitin research, with approximately 530 human genes predicted to encode enzymes involved in the conjugation and deconjugation of ubiquitin. Of these 95 encode deubiquitylases (DUBs) and these enzymes have become the target of many on-going drug discovery efforts. All DUBs carry-out the enzymatic removal of ubiquitin (Ub) from target proteins, whether that protein is a normal cellular protein or is Ub itself. Deconjugation requires the cleavage of an isopeptide bond formed between the C-terminus of Ub and the -NH2-group of a Lys residue in the target protein. With the exception of the JAMM-family of DUBs, all DUBs fall into the peptidase C19 familyand have an activated cysteine residue in the active site. As a consequence, most screening programs have a high hit rate of cys-reactive compounds that show low selectivity and potency. The most potent DUB inhibitors are generally produced by incorpo
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 223.71K | Year: 2014
DESCRIPTION (provided by applicant): Ubiquitin (Ub), a 76 amino acid protein, is implicated in protein degradation and a myriad of cell signaling pathways, including DNA damage response, protein trafficking, cell-cycle progression, inflammation, immune response and regulation of apoptosis. Ubiquitylation occurs through formation of an isopeptide bond between the C-terminus of Ub and the ?-amino group of a lysine (Lys) residue on target proteins. Ub itself has seven Lys residues (K6, K11, K27, K29, K33, K48,and K63), each of which can be further ubiquitylated, generating poly-Ub chains ranging from 2 to 7 Ubs in length. The ability of Ub to form polymers through various lysines appears to be central to the versatility of this system in regulating cell processes. Research in the ubiquitin field is hampered by a lack of antibodies or other tools that selectively recognize poly-ubiquitylated proteins. To address this issue we have combined our experience of generating isopeptide selective poly-Ubs and yeast
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 234.97K | Year: 2010
DESCRIPTION (provided by applicant): As the prominence of the ubiquitin and ubiquitin-like pathways increases the need for assays to measure the activity of the enzymes involved in these pathways grows. Currently the only high throughput methods for measuring ubiquitin and ubiquitin-like isopeptidase activity rely on non-physiological ubiquitin conjugates. The most widely used is Ub-AMC. In this assay the C- terminus of ubiquitin is fused to a small fluorophore. Upon cleavage by an ubiquitin isopeptidase there is an increase in fluorescence. This assay format does not represent a physiological event which may explain why many isopeptidases are unable to cleave this conjugate. Although it is possible to measure isopeptidase activity with physiological substrates such as commercially available ubiquitin chains by western blotting this is only a viable option if a small number of samples are being tested. For instance in order to screen small molecules or natural products for inhibitors of isopeptidases SDS-PAGE and western blotting are unacceptable methods. For these reasons we propose to develop a novel assay for measuring ubiquitin isopeptidase activity with physiological substrates. This assay will be amenable to high throughput screening and will not suffer from the limitations shared by current ubiquitin isopeptidase assays. Briefly, C-terminus of wild type ubiquitin is conjugated to two different ubiquitins, which contain only lysine 48 or lysine 63, available for isopeptide bond formation and fluorescent labeling. In vitro conjugation wild type ubiquitin, via an isopeptide bond to another ubiquitin results in di-ubiquitin that will contain optimized internally quenched fluorescent pairs. We will establish physiological role of the novel di-ubiquitin substrates by demonstrating cleavage with lysine 48 or 63 isopeptide bond selective de-ubiquitylases. The generation of high throughput assays for quantifying ubiquitin isopeptidase activity using physiological substrates represents a major advancement in the study of these crucial cellular enzymes. PUBLIC HEALTH RELEVANCE: Modification of proteins by ubiquitin plays important roles in many cellular processes. In the last decade there has been an explosive growth in the field of ubiquitin research. The enzymes that remove ubiquitin from target proteins are very important drug targets. There is a need for better assays to measure the activity of the enzymes, which are highly specific and physiologically relevant. The development of assays using physiological substrates represents a major advancement in the study of this important group of cellular enzymes and the topic of this proposal.
Agency: Department of Health and Human Services | Branch: | Program: SBIR | Phase: Phase I | Award Amount: 278.22K | Year: 2010
DESCRIPTION (provided by applicant): The network of enzymes involved in the ubiquitination (ligases) and deubiquitination (deubiquitinases or DUbs) of cellular proteins is one of the largest in the human proteome and plays a major role in the regulation of cellular homeostasis. This network interacts with other networks in the cell, especially the kinome, in regulating transcriptional activation and protein degradation among other functions. Finally, dysregulation of this network has been linked to a variety of diseases including neurodegenerative diseases, cancer, and arthritis and inflammation. One of the most powerful tools for determining the specificity of proteases has been the use of peptide libraries to define substrate preferences and determine the influence of amino acids flanking the scissile bond on cleavage efficiency. These studies also provide insights into the nature of side chains interacting with binding subsites within the enzyme's active site. Because deubiquitinases hydrolyze isopeptide bonds between ubiquitin (Ub) and the protein substrate, this type of analysis cannot be performed with such linear peptide libraries which do not mimic the geometry of an isopeptide linkage. In this application, we propose to construct a peptide library linked to Ub through a physiologically relevant isopeptide bond between the carboxyl of the C-terminal Gly of Ub and the 5-amine of a lysine residue in the peptide. A microarray of the library will be constructed on a glass substrate to allow facile analysis of the activity of individual DUbs against each Ub-isopeptide in the library. The data generated in this system will allow us to begin to define the substrate selectivity of different DUbs and the influence of different amino acids flanking the isopeptide lysine on catalytic efficiency of the DUbs. These results will help refine the pattern of interactions in the ubiquitin network and their role in regulating cellular physiology. PUBLIC HEALTH RELEVANCE: LifeSensors proposes to develop an innovative series of new tools that will impact the characterization of the ubiquitination/deubiquitination network. This network is intimately involved - both directly and indirectly through its interactions with other regulatory networks - in the maintenance of cellular homeostasis. Dysregulation of specific members of this network has been implicated in the etiology of a variety of human pathologies including neurodegenerative diseases, cancer, and arthritis and inflammation. The tools developed under this grant will provide new methods for understanding the functioning of ubiquitin pathway enzymes and new approaches to the development of therapeutic agents targeting these enzymes.