Technology Development of Multiplex Substrate Profiling by Mass Spectrometry for Kinases and Application to the HIV-1 Tat - P-TEFb Interaction

University Of California, San Francisco, San Francisco CA

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Abstract

? DESCRIPTION (provided by applicant): Human immunodeficiency virus (HIV) has a small genome encoding only 15 proteins and is therefore highly dependent upon host cellular machinery for replication and proliferation. Modulation of host factors by HIV is accomplished in a variety of mechanisms, and post-translational modifications (PTM's) have been increasingly recognized as an essential and poorly understood aspect in controlling viral pathogenesis. The physical interactions between each of the viral proteins and various host proteins were determined using an unbiased affinity tagging and purification mass spectrometry-based approach. Ten percent of the 497 high-confidence HIV-host protein-protein interactions identified involve host PTM enzymes. I aim to characterize one such interaction - the interaction between HIV-1 Tat and the host kinase positive transcription elongation factor b (P-TEFb). In order to carry out the proposed studies I am developing a mass spectrometry-based method to profile kinases that will be a useful technology for other researchers aiming to characterize a kinase. Transcription of the integrated viral genome is carried out by RNA polymerase II (RNAP II). This transcription is regulated by the binding of two negative elongation factors (NELF and DSIF) as well as by differential phosphorylation of the RNAP II C-terminal domain. In HIV-infected cells, Tat induces transcriptional activation by indirectly stimulating RNAP II. Tat recruits the host kinase P-TEFb and several other host transcription factors to the site of transcription. This complex, consisting of Tat, P-TEFb, and several host transcription factors is known as the Super Elongation Complex (SEC). Tat mediates its transactivation activity by coordinating P-TEFb phosphorylation of the RNAP II C-terminal domain and the two negative elongation factors. These phosphorylation events allow for increased transcription of the viral genome. There are still unanswered questions regarding Tat's transactivation through P-TEFb and the role of the other factors in the Super Elongation Complex. It is unclear what effect Tat has on the substrate specificity and catalytic efficiency of P-TEFb. It is also unclear how the presence of each individual factor of the SEC alters P-TEFb kinase activity. I aim to determine what effects Tat and factors of the SEC have on P-TEFb substrate specificity and catalytic efficiency. By doing this, I will be able to decipher what protein-protein interactions are integral to Tat's transactivation activity. These key protein-protein interactions may present novel druggable targets, the effective inhibition of which could halt viral pathogenesis. Targeting a protein-proten interaction involving a host protein will present an advantage over targeting a viral factor since host proteins are less prone to rapid mutagenesis.