Date of Award

4-13-2017

Publication Type

Doctoral Thesis

Degree Name

Ph.D.

Department

Chemistry and Biochemistry

Supervisor

Vacratsis, Panayiotis

Rights

info:eu-repo/semantics/openAccess

Abstract

Human YVH1 (hYVH1, also known as DUSP12) is a poorly-characterized atypical dual-specificity phosphatase widely expressed in human tissues. The yvh1 gene is evolutionarily conserved and all orthologues possess an N-terminal dual specificity phosphatase domain and a novel C-terminal zinc-binding domain (ZBD). We developed a low-pH thiol-labeling and enrichment strategy coupled to mass spectrometry for characterizing thiol oxidation by mass spectrometry. Employing this method, we provide further evidence that hYVH1 utilizes a disulfide exchange mechanism to regulate its activity under oxidative environment. Moreover, we have identified hYVH1 to be associated with various ribonucleoprotein particles using affinity chromatography coupled to mass spectrometry employing ion mobility separation. Of particular importance, hYVH1 was confirmed to associate with mRNP particles containing Y-box-binding protein 1 (YB-1) and fragile X mental retardation protein (FMRP), proteins that function in translational repression and stress granule regulation. Follow-up microscopy studies demonstrated that hYVH1 overexpression reduces stress granule size, while knocking down hYVH1 expression attenuated stress granule breakdown during recovery from arsenite stress, implicating hYVH1 as a novel stress granule disassembly factor. Structural mass spectrometry experiments were conducted to gain insight into the hYVH1 topology landscape as a first step towards mapping regions mediating RNP association. Pro272, Trp275 and Met276 within the ZBD were predicted to be solvent exposed by in silico prediction modeling and hydrogen/deuterium exchange mass spectrometry (HDX-MS) analysis. Site-directed mutagenesis revealed that these three highly conserved amino acids moderately affect stress granule dynamics. In addition, HDX-MS results showed that residues 160-189 located in the linker region are highly solvent accessible. Furthermore, limited proteolysis experiments demonstrated this region to be inaccessible to proteolysis only under oxidative conditions suggesting that this region undergoes dynamic conformational changes during hYVH1 oxidation. Taken together, a variety of mass spectrometry based techniques have advanced the understanding of hYVH1 in RNP dynamics and discovered putative regions of structural importance for how hYVH1 functions in cellular regulation.

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