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Authors: Wu, Mingxuan
Advisors: Fiedler, Dorothea
Contributors: Chemistry Department
Keywords: Cell Signaling
Diphosphoinositol Polyphosphate
Second Messengers
Subjects: Chemistry
Issue Date: 2015
Publisher: Princeton, NJ : Princeton University
Abstract: Diphosphoinositol polyphosphates (PP-IPs) represent a unique class of high energy messengers controlling a wide variety of cellular processes. It is proposed that these messengers transduce signals by protein binding or post-translational protein pyrophosphorylation. However, most details in PP-IP signalling have remained elusive due to a paucity of suitable tools. This thesis describes the synthesis of PP-IP bisphosphonate analogues (PCP-IPs) that are resistant to chemical and biochemical degradation. Evaluation of two regioisomers (1PCP-IP5 and 5PCP-IP5) in different biochemical assays highlights their utility for analysing PP-IP function. While both PCP-analogues inhibited Akt phosphorylation with similar potencies, 1PCP-IP5 was much more effective at inhibiting its cognate phosphatase hDIPP1. Furthermore, the PCP-analogues inhibit protein pyrophosphorylation because of their inability to transfer the phosphoryl group, and thus enable the distinction between PP-IP signalling mechanisms. Next, we chemically synthesized PCP-IP5 affinity reagents and applied in S. cerevisiae proteome. Using these reagents we isolated enzymes involved in diverse cellular processes including polyphosphate metabolism and nucleotide biosynthesis. Further investigation of Vtc4 revealed PP-IP5 promotes the inorganic polyphosphate polymerase activity of Vtc4 in vitro. Moreover, in the presence of Mg2+, a different set of proteins was enriched, some of which were confirmed in vitro as pyrophosphorylation substrates such as survival factor Svf1. As such, we have provided the first comprehensive data set on PP-IP interacting proteins, which included several known and many more novel pyrophosphorylation substrates. The PP-IPs affinity reagents hold a great promise for a variety of applications, and proteomic studies have expanded our understanding of PP-IP signaling.
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Type of Material: Academic dissertations (Ph.D.)
Language: en
Appears in Collections:Chemistry

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