Completed Projects/COBRE Graduates

Proteomic Approaches to Target the PP6-mTORC2 Pathway in Glioblastoma

PI: Arminja Kettenbach, PhD
Associate Professor of Biochemistry and Cell Biology
Extramural Funding Awarded: NIH/R33, NIH/R35

Glioblastoma multiforme (GBM) is the most common and aggressive tumor of the central nervous system in adults and it is generally fatal within 12 months of diagnosis. We propose to identify targets in the newly uncovered and frequently overexpressed PP6-mTORC2 pathway for the development of new therapeutic strategies to fight this devastating disease.

Experimental strategy for the identification of mTORC1 and 2 substrates in Glioblastoma multiforme (GBM) cell lines and tumors using quantitative multiplex mass spectrometry-based phosphoproteomics.

Molecular Mechanisms of RSV F Activation and Inhibition 

PI: Jason McLellan, PhD
(Current) Associate Professor, Molecular Biosciences
The University of Texas at Austin
Extramural Funding Awarded: NIH/R01, Janssen Vaccines & Prevention B.V., MedImmune

Respiratory syncytial virus (RSV) causes respiratory tract infections that result in substantial morbidity and mortality in infants and the elderly. RSV entry into host cells is facilitated by an attachment protein (G) and a fusion protein (F) that in its active form adopts a metastable prefusion conformation. After attachment of RSV to cells, it is hypothesized that host-factors trigger the conformational rearrangement of F that results in fusion of the viral and cellular membranes. The prefusion conformation of F is therefore considered an ideal vaccine antigen, and antibodies and small molecules that disrupt its structure and function are actively being pursued. Development of effective therapeutics will be greatly enhanced by a molecular understanding of how the F glycoprotein interacts with host-cell factors to promote entry, and how neutralizing antibodies inhibit one or more steps in the entry process. For this project, we will test two hypotheses: first, that specific host-cell factors enhance the rate of RSV F triggering; and second, that the most potent neutralizing antibodies target receptor-binding sites and block conformational changes. We expect that the results from these studies will fill important gaps in our understanding of RSV entry and provide a molecular basis for RSV F activation and inhibition.

Schematic of RSV F-mediated membrane fusion. Host-cell factors promote entry, whereas neutralizing monoclonal antibodies block the function of RSV F.

Protein Design for Selective Interference with LPA Signaling in Colon Cancer

PI: Gevorg Grigoryan, Ph.D.
Associate Professor of Computer Science
Extramural Funding Awarded: NSF, NIH/R01

A computationally-designed inhibitor molecule (gray) interacts snuggly with its target domain (yellow) to prevent cancer-promoting activities.

Colorectal cancer was the third-most common cause of deaths from cancer in the United States in 2013. This disease claims over 50,000 lives and afflicts over 140,000 people annually in the US, underscoring a clear and urgent need for better control of this incurable malignancy. The goal of our study is to attenuate oncogenic activities in colon cancer cells by inhibiting a key proliferatory signaling pathway mediated by the powerful mitogen lysophosphatidic acid (LPA). Key towards this goal is to produce selective inhibitors—molecules that interact with and inhibit specific proteins in the cell, while avoiding any unintended side-interactions with other proteins. We are uniquely capable of providing such selectivity using computational technologies we have developed that enable us to target selectively a single member in a family of closely related protein domains.

Mechanisms of Increased Lung Disease Secondary to Polymorphisms in the ACE Gene

PI: Alix Ashare, M.D., Ph.D.
Associate Professor of Medicine and Microbiology & Immunology
Extramural Funding Awarded: NIH/R01, Cystic Fibrosis Foundation, American Asthma Foundation

Current treatments for chronic obstructive pulmonary disease (COPD) attempt to ameliorate symptoms and prevent exacerbations. Studies have identified an insertion (I)/deletion (D) polymorphism in an intron of the angiotensin converting enzyme (ACE) gene. D/D homozygotes (~25% of the population) exhibit more severe COPD but the mechanism is unclear. Our major goal is to investigate and target the role of ACE in the development of COPD. Patients with COPD are plagued by chronic inflammation, leading to increased lung compliance. Alveolar macrophages (AMs) are critical to the local inflammatory response in COPD. ACE is expressed on the surface of AMs and its abundance regulates levels of angiotensin II (ANGII), which in turn binds the ANGII type I (AT1) receptor on AMs. Inflammation in COPD causes bronchospasm, which can result in hypoxia. Hypoxia, in turn, causes an increase in ANGII, a key inflammatory mediator, creating a vicious cycle. Our preliminary data show that AMs generate an inflammatory response to hypoxia that is blocked by the AT1 receptor blocker, losartan. We also found increased pulmonary ACE and ANGII in subjects with the ACE D/D genotype. Our data suggest that the intronic sequence encodes microRNAs (miRNAs), small RNAs that can regulate protein abundance. Our overarching hypothesis is that the ACE D/D genotype increases translation of ACE protein, leading to an exaggerated ANGII response during hypoxia, increasing the severity of COPD. Aim 1 will test the hypothesis that the ACE D/D polymorphism is associated with elevated ACE activity and lung inflammation compared to I/I subjects. In parallel, we will determine if these levels predict differences in lung compliance, as quantified by magnetic resonance elastography (MRE). In Aim 2, we will test the hypothesis that hypoxia causes a dose-dependent increase in ACE and ANGII that is exaggerated in AMs from D/D versus I/I subjects. Our preliminary data suggest the expression of ACE-specific miRNAs from the intronic sequence. Aim 3 will test the hypothesis that reduced expression of these miRNAs in D/D subjects causes an increase in ACE translation compared to I/I subjects. This will foster discovery of previously unrecognized targets that could decrease ACE activity in the lung.