Whitlatch, Kindra
(2018)
PROGRESS TOWARDS THE STRUCTURAL BASIS OF TEC-FAMILY KINASE ACTIVATION BY HIV-1 NEF.
Master's Thesis, University of Pittsburgh.
(Unpublished)
Abstract
HIV-1 Nef is a viral accessory factor that is essential for virus infectivity, host immune evasion and AIDS progression. Nef lacks intrinsic catalytic activity and functions instead via interactions with multiple classes of host cell proteins involved in signal transduction and endocytic trafficking. Nef interacts with the Src-family tyrosine kinases Hck and Lyn through their SH3 domains, resulting in constitutive kinase activity. Nef also binds to select members of the Tec-family of tyrosine kinases, including Btk, Bmx, and Itk, all of which are expressed in HIV-1 target cells. Of particular interest is Itk, which is expressed in CD4+ T cells and is activated by Nef. Selective Itk inhibitors block Nef-dependent enhancement of HIV-1 infectivity and replication, suggesting an important role in the viral life cycle. While the interaction between Itk and Nef has been demonstrated at the plasma membrane in cell-based fluorescence complementation assays, the structural basis of this interaction has not been reported. Like Src-family kinases, Itk has a core region consisting of sequential SH3, SH2 and kinase domains. In addition, Itk has an N-terminal pleckstrin homology (PH) domain important for membrane targeting as well as a Tec homology(TH) region involved in kinase regulation. To explore the structure of the Nef:Tec-family kinase (TFK) complexes, I have created a panel of bacterial expression constructs for the Itk and Btk regulatory region. These include the entire PH-TH-SH3-SH2 region, the SH3-SH2 region, and the isolated SH3 domain, all of which have yielded mg amounts of soluble protein. I have also produced recombinant, N-terminally myristoylated (Myr) Nef in bacteria, a post-translational modification essential for Nef membrane localization in cells. Preliminary Surface Plasmon Resonance (SPR) studies show that Myr-Nef binds membrane bilayers with low µM affinity in a Myr-dependent manner. These proteins will provide the foundation for future structural determination of Nef-TFK complexes by X-ray crystallography as well as the nature of this interaction in lipid bilayers, the physiological site of interaction in HIV-infected cells.
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| Item Type: |
University of Pittsburgh ETD
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| Status: |
Unpublished |
| Creators/Authors: |
|
| ETD Committee: |
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| Date: |
4 September 2018 |
| Date Type: |
Publication |
| Defense Date: |
17 August 2018 |
| Approval Date: |
4 September 2018 |
| Submission Date: |
3 September 2018 |
| Access Restriction: |
No restriction; Release the ETD for access worldwide immediately. |
| Number of Pages: |
72 |
| Institution: |
University of Pittsburgh |
| Schools and Programs: |
School of Medicine > Molecular Biophysics and Structural Biology |
| Degree: |
MS - Master of Science |
| Thesis Type: |
Master's Thesis |
| Refereed: |
Yes |
| Uncontrolled Keywords: |
HIV-1, Nef, Tec family kinase, kinase, Itk, Btk |
| Date Deposited: |
04 Sep 2018 18:30 |
| Last Modified: |
04 Sep 2018 18:30 |
| URI: |
http://d-scholarship.pitt.edu/id/eprint/35293 |
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