Student Theses and Dissertations
Date of Award
2010
Document Type
Thesis
RU Laboratory
Sakmar Laboratory
Keywords
nucleobindin 1 (NUCB1), g proteins, Amyloid-β 42 (Aβ42), calcium-binding proteins, amyloid fibrils
Abstract
Nucleobindin 1 (NUCB1) is a widely expressed multi-domain Ca2+-binding protein whose precise physiological and biochemical functions are not well understood. We engineered and heterologously expressed a soluble form of NUCB1 (sNUCB1) and characterized its biophysical and biochemical properties. We show that sNUCB1 exists as a dimer in solution and that each monomer binds two divalent Ca2+ cations. Ca2+-binding causes conformational changes in sNUCB1 as judged by circular dichroism and fluorescence spectroscopy experiments. Earlier reports suggested that NUCB1 might interact with heterotrimeric G protein α-subunits. We show that dimeric Ca2+-free sNUCB1 binds to heterologously expressed Gαi1 and that Ca2+-binding inhibits this interaction. We further show that the binding of sNUCB1 to Gαi1 inhibits its basal rate of GDP release and slows its rate and extent of GTPγS uptake. We conclude that sNUCB1 is a Ca2+-dependent guaninenucleotide dissociation inhibitor (GDI) for Gαi1. To our knowledge sNUCB1 is the first example of a calcium-dependent GDI for heterotrimeric G proteins. In addition, we also established novel and unique anti-amyloidogenic functional ability of sNUCB1. We show that Ca2+-free sNUCB1 can inhibit fibril formation by highly amyloidogenic human Islet Amyloid PolyPeptide (hIAPP) and Amyloid-β 42 (Aβ42) peptides, as relevant to Type-2 Diabetes and Alzheimer’s disease. In addition, we also show that Ca2+-free sNUCB1 can effectively dissociate amyloid fibrils formed by both hIAPP and Aβ42. Interestingly, Ca2+ disrupts this unique functional ability of sNUCB1. In order to circumvent this, we designed and purified a Ca2+-insentive mutant namely sNUCB1(tetramutant) that is functionally similar to sNUCB1 even in the presence of excess Ca2+ in the reaction mixture. Mechanistically, we show that sNUCB1 inhibits fibril formation by capping the ends of high molecular mass prefibrillar species. Subsequently, we also isolated a 39-amino-acid long C-terminal (CT) peptide fragment of NUCB1 namely NUCB1(381-419) which, similar to sNUCB1, inhibits and disaggregates hIAPP fibrillization. Finally we have also extended this work to other amyloidogenic proteins namely tau K19(C322S), a synthetic construct comprising microtubule binding domain of human tau protein. Specifically, we show that Ca2+-free sNUCB1 effectively inhibits amyloid fibril formation by tauK19(C322S).
Permanent URL
http://hdl.handle.net/10209/356
Recommended Citation
Kapoor, Neeraj, "Unzipping Amyloid Fibrils: How a Novel Calcium-Binding Protein, NUCB1, Prevents the Formation of Amyloid Fibrils" (2010). Student Theses and Dissertations. 74.
https://digitalcommons.rockefeller.edu/student_theses_and_dissertations/74
Comments
A thesis presented to the faculty of The Rockefeller University in partial fulfillment of the requirements for the degree of Doctor of Philosophy.