Date of Award
nucleobindin 1 (NUCB1), g proteins, Amyloid-Î² 42 (AÎ²42), calcium-binding proteins, amyloid fibrils
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 Î±-subunits. We show that dimeric Ca2+-free sNUCB1 binds to heterologously expressed GÎ±i1 and that Ca2+-binding inhibits this interaction. We further show that the binding of sNUCB1 to GÎ±i1 inhibits its basal rate of GDP release and slows its rate and extent of GTPÎ³S uptake. We conclude that sNUCB1 is a Ca2+-dependent guaninenucleotide dissociation inhibitor (GDI) for GÎ±i1. 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-Î² 42 (AÎ²42) peptides, as relevant to Type-2 Diabetes and Alzheimerâ€™s disease. In addition, we also show that Ca2+-free sNUCB1 can effectively dissociate amyloid fibrils formed by both hIAPP and AÎ²42. 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).
Kapoor, Neeraj, "Unzipping Amyloid Fibrils: How a Novel Calcium-Binding Protein, NUCB1, Prevents the Formation of Amyloid Fibrils" (2010). Student Theses and Dissertations. 74.