1997 — 1998 |
Schmidt, Walter K |
F32Activity Code Description: To provide postdoctoral research training to individuals to broaden their scientific background and extend their potential for research in specified health-related areas. |
Caax Processing Pathway Components @ Johns Hopkins University
methyltransferase; posttranslational modifications; molecular genetics; endopeptidases; enzyme activity; enzyme substrate; protein transport; immunoprecipitation; Saccharomyces cerevisiae;
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0.943 |
2005 |
Schmidt, Walter K |
R03Activity Code Description: To provide research support specifically limited in time and amount for studies in categorical program areas. Small grants provide flexibility for initiating studies which are generally for preliminary short-term projects and are non-renewable. |
Hts-Based Identification of Novel Rce1p Inhibitors(Rmi) @ University of Georgia (Uga)
DESCRIPTION (provided by applicant): Our long-term goal is to define the enzymatic properties of the Ras Converting Enzyme (Rce1p), a relatively uncharacterized protease that is required for CaaX protein-biosythensis. CaaX proteins are lipid-modified molecules that often function as signaling molecules in important cellular pathways. Ras and RhoB are key examples. Because of the role that CaaX proteins have in cellular transformation (e. g. activated forms of Ras are associated with 30% of all cancers), strategies that regulate the biosynthesis of these proteins are being explored as novel anti-cancer therapies. Rce1P is a new target in these strategies. Rce1p is an atypical protease, having multiple membrane spans and lacking a canonical protease motif. To date, the mechanism of Rce1p remains undefined, in part due to a lack of inhibitors that can be used as probes to investigate its catalytic properties. We have developed a coherent set of biochemical and genetic, chemical, and molecular assays that we are using for defining the active site and enzymatic properties of Rce1p and for identifying and characterizing novel Rcelp inhibitors. 1 of our assays is used to monitor the Rce1p-dependent cleavage of a quenched fluorescent peptide substrate. We have used this assay to evaluate the effect of novel inhibitors and mutations on the kinetic parameters of Rcelp. This assay is readily adaptable for HTS, and 1 of the aims of this proposal is fully assess the utility of this assay for HTS by optimizing the signal-to-noise ratio, determining reproducibility, and conducting a pilot screen for Rce1p inhibitors. As a second aim, we are proposing to integrate our other Rce1p assays into secondary screens that can be used to confirm HTS hits and to further assess the target specificity of candidate inhibitors. The ultimate goal of this project is the identification of novel Rce1p inhibitors that can serve as tools for providing insight into the mechanism of Rce1p; some of these compounds may also be of potential therapeutic value.
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0.987 |
2005 — 2009 |
Schmidt, Walter K |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Role of Rce1p in the Biogenesis of Caax Proteins @ University of Georgia (Uga)
DESCRIPTION (provided by applicant): Our goal is to define the enzymatic properties of the Ras Converting Enzyme (Rce1p), a protease that is required for CaaX protein biosynthesis. CaaX proteins are lipidated molecules that often function as signaling molecules in important cellular pathways. Ras and RhoB are key examples. Because of the role that CaaX proteins have in cellular transformation (e.g., activated forms of Ras are associated with 30% of all cancers), strategies that regulate the biosynthesis of these proteins are being explored as novel anticancer therapies. Rce1p is a new target in these strategies. Rce1p is an atypical protease, having multiple membrane spans and lacking a canonical protease motif. To date, the mechanism of Rce1p remains undefined. We hypothesize that the Rce1p active site is comprised of a subset of residues that are invariably conserved between Rce1p orthologs. In our preliminary studies, which take advantage of the S. cerevisiae system, we have 1) Identified 4 residues that are critically important for Rce1p function, 2) Discovered a novel compound that is potentially a specific and irreversible inhibitor of Rce1p, and 3) Utilized a dual genetic/ biochemical reporter and deletion analysis to assess the topology and importance of the Rce1p C-terminus. We have developed a coherent set of biochemical, genetic, chemical, and molecular approaches around our findings that will be used for defining the active site and enzymatic properties of Rce1p. Specifically, we will use a novel quantitative genetic assay to detail the importance of charge and position for residues deemed critical for enzymatic activity and to evaluate the substrate specificity of Rce1p mutants. We will use an in vitro assay that monitors cleavage of a quenched fluorescent peptide substrate to evaluate the effect of novel inhibitors and mutations on the kinetic parameters of Rce1p. mutants. Finally, we will use a dual genetic/biochemical topology reporter and deletion approaches to identify the functional domains of Rce1p. In sum, this proposal will clarify the enzymatic properties of Rce1p, a member of an emerging class of multi-span membrane-bound proteases having biomedical importance.
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1 |
2008 |
Schmidt, Walter K |
R03Activity Code Description: To provide research support specifically limited in time and amount for studies in categorical program areas. Small grants provide flexibility for initiating studies which are generally for preliminary short-term projects and are non-renewable. |
Discovery of Specific Cell-Permeable Inhibitors of the Ras Converting Enzyme @ University of Georgia (Uga)
[unreadable] DESCRIPTION (provided by applicant): The RAS genes encode small GTPase Ras proteins that are important for controlling cell growth and differentiation. Activating mutations of Ras are associated with approximately 20-30% of all human cancers. All Ras proteins have a C-terminal CaaX motif that is extensively post-translationally modified. These modifications are prerequisite for localization of Ras proteins to the inner leaflet of the plasma membrane where they must be to function properly. It is therefore hypothesized that interfering with the post-translational modification of Ras could be an anti-cancer strategy. The CaaX motif is sequentially modified by the farnesyltransferase (FTase), the Ras converting enzyme (Rce1p), and the isoprenylcysteine carboxylmethyltransferase (ICMT), making them all promising anti-cancer targets. Our long-term goal is to identify agents that interfere with Rce1p. We hypothesize that pharmacological inhibition of Rce1p will lead to Ras mislocalization in vivo. Indeed, we have identified several compounds that inhibit Rce1p in vitro in a species-independent manner (i.e., human, yeast, and trypanosome) and disrupt Ras localization in vivo. This proposal aims to increase the number of similar acting compounds by identifying novel and potent Rce1p-specific inhibitors by high throughput screening (AIM 1). The primary screening of compounds will be performed in collaboration with the Emory HTS facility using a mix and measure fluorescence-based assay and Rce1p as the target enzyme. The specificity of primary hits will be determined using secondary screens to measure effects on two other enzymes, Ste24p and trypsin. Ste24p has a partially overlapping function with Rce1p in that it is required for the post-translational modification of certain CaaX motifs, but it is not specifically required for Ras modification. The expected hit rate using the proposed approach is 0.1% based on preliminary screening of a small compound library (i.e. NCI Diversity Set). The secondary hits will be further characterized by the applicant's laboratory (AIM 2). This analysis will include additional specificity assessments using a distinct reporter and assay system that is based on in vitro production of the yeast isoprenylated a-factor mating pheromone. In vitro approaches will be used to characterize compound in terms of their inhibition kinetics (e.g. KM, KI, vmax), potency (e.g. IC50), and biophysical properties. In vivo cell-based assays will be used to determine toxicity and cell permeability, the latter using a unique GFP-Ras2p localization assay that we have developed. We expect this study to yield several active compounds that will be valuable for understanding the enzymatic nature of the relatively uncharacterized Rce1p and for serving as lead compounds for anti-cancer therapeutics involving targeted inhibition of Rce1p. PUBLIC HEALTH RELEVANCE: The Ras converting enzyme (Rce1p) is an anti-cancer target. High throughput screening for Rce1p- specific inhibitors will be conducted in association with the Emory HTS facility using a direct mix and measure in vitro fluorescence-based primary assay and secondary assays to address target specificity. The identified compounds, representing lead compounds for anti-cancer therapeutics, will be evaluated for their toxicity profile and cell permeability using cell-based assays and will be further assessed for target specificity and kinetic parameters by in vitro methods. [unreadable] [unreadable] [unreadable]
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0.987 |
2016 — 2019 |
Schmidt, Walter K |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Role of Proteolysis in Regulating Caax Protein Function
PROJECT SUMMARY The goal of this study is to investigate how the properties of proteins are impacted by post-translational modifications (PTMs) associated with protein prenylation, specifically the proteolytic and carboxylmethylation events that typically follow isoprenylation. Most studies to date have investigated this issue using a select few reporters, including the Ras GTPases and the yeast a-factor mating pheromone. Our studies are bringing a new understanding to this issue by promoting a general view that the PTMs occurring to prenylated proteins are not necessarily coupled and are key regulators of protein function and localization. We have proposed two aims. One will investigate the coupling of PTMs using a set of prenylated protein reporters outside those previously studied. Our studies will challenge the conventional paradigm for how prenylated proteins are modified, which in turn will provide guidance to strategies aimed at interfering with protein prenylation and related downstream events as disease therapies. The second aim will investigate the target specificities of the Rce1p and Ste24p proteases that act on prenylated proteins. Our studies will demonstrate that these proteases have very distinct enzymatic profiles, which will provide additional guidance on the specific targeting of these enzymes in disease therapies. We bring to bear on our investigations an exceptionally strong set of preliminary findings, the complementary expertise of several research groups, and a comprehensive molecular toolbox for the study of prenylated proteins and the proteases involved.
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1 |
2020 — 2021 |
Kannan, Natarajan Schmidt, Walter K |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Determining the Scope of Prenylatable Protein Sequences
PROJECT SUMMARY This study investigates the specificity of farnesyl transferase (FTase) that isoprenylates CaaX proteins. Studies probing the in vivo activity of the FTase have historically used reporters (e.g. Ras GTPases) that undergo complex multi-step post-translational modification (PTM), involving initial farnesylation followed by CaaX proteolysis and carboxyl methylation. This study takes advantage of Hsp40 Ydj1, a farnesylation-only reporter for which we have developed a range of methods to monitor its PTM status. Its use reveals that farnesylation is not necessarily coupled to subsequent PTMs as has been generally accepted for CaaX proteins, and that FTase specificity is significantly more promiscuous than anticipated. These findings challenge the conventional paradigm for how farnesylated proteins are modified and which proteins are targeted by FTase. We will extend our studies to fully resolve the specificity of FTase using a combination of genetic, biochemical, bioinformatic, and biophysical studies. We bring to bear on our investigations an exceptionally strong set of preliminary findings, the complementary expertise of several research groups, and a comprehensive molecular toolbox for the study of farnesylated proteins and other enzymes associated with this post-translational modification pathway.
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1 |