The etiology of many human diseases involves structural changes in multi-component protein complexes caused by aberrant posttranslational modifications (PTM). We propose to develop a novel in-cell NMR-based technology for mapping the STructural changes that accompany protein-protein INTeractions in the cell (STINT-NMR) and use it to analyze molecular interactions of biological significance. STINT-NMR analysis greatly reduces the time required to obtain atomic resolution information on protein complexes compared to traditional in vitro NMR and x-ray crystallography. The resulting data define structural details of the interacting surfaces at atomic resolution. By sequentially expressing enzymatic activities that modify protein-protein interactions, STINT-NMR also allows us to perform "biochemistry inside the cell", where we can monitor the structural consequences of post-translational modifications. The broad objective of this proposal is to develop a novel in vivo atomic resolution technique to understand on structural level how posttranslational modifications regulate protein complexes involved in important biological processes. To fulfill this objective we have three specific aims: 1) Develop STINT-NMR methodology to structurally characterize multiprotein interactions in bacterial cells. As a biological system, we will use the interactions between Ubiquitin and unmodified or posttranslationally modified endocytic proteins Hrs, STAM2, Eps15. For this aim we will create STINT-NMR compatible plasmid constructs capable of overexpressing endocytic proteins, protein kinases, and monoubiquitination machinery in bacteria. We will perform series of STINT-NMR experiments using different combinations of unmodified and modified endocytic proteins to assess the structural changes resulting from PTM's. We will supplement our in- cell NMR experiments with in vitro studies of the modified protein complexes. 2) Develop STINT-NMR for eukaryotic cells to test the influence of intracellular structures on protein-protein interactions. For this aim we will optimize overexpression of Ubiquitin and STAM2 in yeast cells to perform STINT-NMR experiments. 3) Extend STINT-NMR for high-throughput screening of small molecules capable of interfering with the complexes formed between viral proteins required for budding and host endocytic proteins. We will create STINT-NMR compatible plasmid constructs capable of overexpressing viral HIV p6 domain, endocytic proteins TSG101 and Hrs, protein kinase MEK1 and ERK2, and monoubiquitination machinery in bacteria. We will perform a series of STINT-NMR experiments using different combinations of unmodified and modified proteins to establish structural changes associated with complex formation. As a positive control of STINT-NMR HTS methodology, we will screen a small library of known antagonists against TSG101-directed HIV-1 budding. Later, a library of small drug-like molecules (NCI Discover set) will be screened against the p6-TSG101-Hrs complexes using STINT-NMR to identify possible classes of the compounds capable of interfering with viral budding.

Public Health Relevance

Many human diseases are caused by aberrant post-translational modifications which, it turn, result in changes in protein-protein interactions. We developed a new technology, STINT-NMR, which allows us to study STructural INTeractions between post- translationally modified proteins inside the cell with atomic resolution by using Nuclear Magnetic Resonance (NMR) spectroscopy. We will apply this technology to study aberrant structures and the molecular processes that may lead to disease states. We will also develop this technology to search for small drug-like molecules that may regulate these molecular processes.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM085006-04
Application #
8499358
Study Section
Enabling Bioanalytical and Biophysical Technologies Study Section (EBT)
Program Officer
Wehrle, Janna P
Project Start
2010-07-01
Project End
2015-06-30
Budget Start
2013-07-01
Budget End
2014-06-30
Support Year
4
Fiscal Year
2013
Total Cost
$217,729
Indirect Cost
$68,694
Name
State University of New York at Albany
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
152652822
City
Albany
State
NY
Country
United States
Zip Code
12222
Xue, Jing; Ray, Rashmi; Singer, David et al. (2014) The receptor for advanced glycation end products (RAGE) specifically recognizes methylglyoxal-derived AGEs. Biochemistry 53:3327-35
Bharat, Somireddy Venkata; Shekhtman, Alexander; Pande, Jayanti (2014) The cataract-associated V41M mutant of human ?S-crystallin shows specific structural changes that directly enhance local surface hydrophobicity. Biochem Biophys Res Commun 443:110-4
Karlsson, Amelia B; Washington, Jacqueline; Dimitrova, Valentina et al. (2014) The role of spartin and its novel ubiquitin binding region in DALIS occurrence. Mol Biol Cell 25:1355-65
Majumder, Subhabrata; DeMott, Christopher M; Burz, David S et al. (2014) Using singular value decomposition to characterize protein-protein interactions by in-cell NMR spectroscopy. Chembiochem 15:929-33
Maldonado, Andres Y; Burz, David S; Reverdatto, Sergey et al. (2013) Fate of pup inside the Mycobacterium proteasome studied by in-cell NMR. PLoS One 8:e74576
Jagadish, Krishnappa; Borra, Radhika; Lacey, Vanessa et al. (2013) Expression of fluorescent cyclotides using protein trans-splicing for easy monitoring of cyclotide-protein interactions. Angew Chem Int Ed Engl 52:3126-31
Ji, Yanbin; Majumder, Subhabrata; Millard, Melissa et al. (2013) In vivo activation of the p53 tumor suppressor pathway by an engineered cyclotide. J Am Chem Soc 135:11623-33
Reverdatto, Sergey; Rai, Vivek; Xue, Jing et al. (2013) Combinatorial library of improved peptide aptamers, CLIPs to inhibit RAGE signal transduction in mammalian cells. PLoS One 8:e65180
Aboye, Teshome L; Li, Yilong; Majumder, Subhabrata et al. (2012) Efficient one-pot cyclization/folding of Rhesus ?-defensin-1 (RTD-1). Bioorg Med Chem Lett 22:2823-6
Gould, Andrew; Li, Yilong; Majumder, Subhabrata et al. (2012) Recombinant production of rhesus ?-defensin-1 (RTD-1) using a bacterial expression system. Mol Biosyst 8:1359-65

Showing the most recent 10 out of 18 publications