2003 — 2004 |
Hartson, Steven D |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Regulation of the Hsp90 Co-Chaperone Network @ Oklahoma State University Stillwater
DESCRIPTION (provided by applicant): Hsp90 senses proteotoxic stress and provides obligatory support to a diverse family of normal and mutant signal transduction proteins. To fulfill these roles, Hsp90 associates with networks of non-client co-chaperone partners to generate an unknown number of discrete Hsp90 "machines." These networks appear to regulate or target Hsp90 function. Due to Hsp90's pivotal role in normal and aberrant cell physiology, Hsp90 inhibitors are in clinical trials to treat human disease, and the expanding network of known co-chaperones includes several proto-oncoproteins and several noteworthy pharmacological targets. Our long-term goal is to define the roles of Hsp90 and its co-chaperones, and to elucidate the mechanisms underlying their functions. The evolving field of proteomics now provides the tools needed to examine the composition, organization, and dynamic regulation of the Hsp90 co-chaperone network. Thus, this R21 application proposes to initiate the discovery novel Hsp90 co-chaperones, to confirm their interactions with Hsp90, and to determine if individual Hsp90-binding proteins behave as co-chaperones versus clients. Additionally, we propose to test the hypothesis that the Hsp90 co-chaperone network is regulated by proteotoxic cell stresses such as hyperthermia, heavy metal, and ethanol. These Aims will be pursued by isolating the Hsp90 co-chaperone network via one-step purification, separating and quantifying Hsp90-associated proteins via 1-D and 2-D electrophoresis, and by using digital imaging and data-basing software to compare Hsp90 co-chaperone networks isolated from stressed versus unstressed tissues. Individual Hsp90-associated proteins will be identified by trypsinolytic fingerprinting and MALDI-based mass spectrometry. Associations with Hsp90 will be confirmed via reciprocal immunoadsorptions. Authentic co-chaperones will be differentiated from Hsp90 clients on the basis of the prototypical effects of Hsp90 antagonists. Potential regulatory mechanisms will be examined via biochemical characterizations of dynamically responsive co-chaperones. The research proposed will elucidate the composition and dynamic regulation of a discrete protein network that supports human development, and will characterize how environmental stress may dysregulate this network.
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1 |
2007 — 2010 |
Massiah, Michael Mort, Andrew Hartson, Steven Soulages, Jose (co-PI) [⬀] Burnap, Robert (co-PI) [⬀] Picking, William |
N/AActivity Code Description: No activity code was retrieved: click on the grant title for more information |
Mri: Acquisition of An Ltq Mass Spectrometer @ Oklahoma State University
This award is for the acquisition of an ion trap mass spectrometer (MS) to support a diverse portfolio of basic research activities. These activities are united by their strong need for the MS/MS fragmentation capabilities, the superior sensitivity, and the ability to resolve and analyze peptide mixes using upstream in-line liquid chromatography. The MS will provide crucial insights into the structure, function, and regulation of five fundamentally important biomolecules, including the Midline-1 phosphoprotein, the lipid-droplet-associated protein-1, and apolipoprotein A-I. The MS will provide molecular insights into cellular differentiation and lipid transport processes. The MS will illuminate limitations to photosynthetic productivity, and it will contribute to the use of pectins in foodstuffs and as bio-industrial precursors. The MS will also be used to identify proteins governing plant-insect and plant-microbe relationships, protein chaperone networks, plant responses to oxidative stress, and plant meiosis. Because the most advanced MS on campus has no fragmentation, Liquid Chromatography, or Electro-Spray Ionization capabilities, the new MS will be valuable for many of the university's current research activities.
In addition to the scientific benefit, the MS will be useful for the investigators' undergraduates, graduate students, and postdoctoral trainees. All of these students and trainees will analyze data from the MS, and about a third of them will be trained to use this sophisticated instrument directly. The MS will enhance the existing semi-annual workshops on ""Mass Spectrometry and Proteomics,? and it will be integrated into the university's undergraduate biochemistry laboratory curricula to expose students to modern analytical approaches. The MS will be featured in outreach efforts that will include presentations on-campus and off-campus, as well as other novel outreach efforts. Approximately half of this outreach will be directed toward: minority programs, the minority institute Langston University, two of the State's non-Ph.D. institutes, and students who do not currently consider themselves research-bound.
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0.915 |
2017 |
Deng, Junpeng (co-PI) [⬀] Hartson, Steven D Matts, Robert L. [⬀] |
R15Activity Code Description: Supports small-scale research projects at educational institutions that provide baccalaureate or advanced degrees for a significant number of the Nation’s research scientists but that have not been major recipients of NIH support. The goals of the program are to (1) support meritorious research, (2) expose students to research, and (3) strengthen the research environment of the institution. Awards provide limited Direct Costs, plus applicable F&A costs, for periods not to exceed 36 months. This activity code uses multi-year funding authority; however, OER approval is NOT needed prior to an IC using this activity code. |
Structure/Function Studies of Anti-Cancer Hsp90 Inhibitors That Target the C-Terminal Domain @ Oklahoma State University Stillwater
The Hsp90 family of molecular chaperones is required for the maturation, activation, and/or stability of diverse proteins that play central roles in malignant progression. As a result of this diversity, Hsp90 inhibitors simultaneously antagonize a wide variety of oncogenic pathways and processes. Thus, Hsp90 inhibitors are widely envisioned to have great potential as anti-cancer drugs. The Hsp90 inhibitors that have progressed to clinical trials share a common mechanism of action, namely binding to the ATP binding site in Hsp90's N- terminal domain and inhibiting Hsp90 function in vivo. Unfortunately, N-terminal Hsp90 inhibitors also have the untoward effect of inducing the pro-survival heat shock response, which is acknowledged to undermine the clinical efficacies of N-terminal Hsp90 inhibitors. Small molecules that bind to Hsp90's C-terminus have also been identified that inhibit Hsp90 function in vitro, and show cytostatic/cytotoxic activity in cultured cancer cell lines, but do not appear to induce the heat shock response. Thus, targeting the C-terminal domain of Hsp90 may be a superior strategy for anti-cancer therapies based on the inhibition of Hsp90. However, the progression of C-terminal Hsp90 inhibitors as clinical agents is hindered by two significant knowledge gaps: (i) The structural basis of their binding to Hsp90 is unknown, but must be determined for the rational design and optimization of novel inhibitory compounds; and (ii) the physiological basis of their anti-proliferative / cytotoxic activities is poorly understood. To further the development of C-terminal Hsp90 inhibitors with clinical potential, we propose to: 1) determine the structure of Hsp90 complexed to C-terminal Hsp90 inhibitors; and 2) compare and contrast the impacts of C-terminal vs. N-terminal inhibitors on the proteomes of breast cancer cells cultured as 3D spheroids. The structure of a N-terminally truncated Hsp90 construct bound to Hsp90 C- terminal inhibitors will be determined by X-ray crystallography. The impact of N- and C-terminal Hsp90 inhibitors on breast cancer cell proteomes will be characterized by Stable Isotope Labeling of Amino Acids in Cell Culture (SILAC), in conjunction with multi-dimensional liquid chromatography and a state-of-the-art Fusion Tribrid mass spectrometer. The proposed studies will provide critical structural information regarding the binding of C-terminal inhibitors to Hsp90, culminating in the rational design of C-terminal inhibitors with improved affinities for Hsp90 and clinical potential. The mechanistic insights into the selective tumoricidal activities of N- and C-terminal Hsp90 inhibitors generated by the proteomic studies will also reveal tumor- specific features that might guide chemotherapeutic decisions, tumor cell responses that might be monitored during clinical treatment, and promising modalities for combinatorial treatments with both families of Hsp90 inhibitors.
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