Date of Award

Spring 5-2017

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Chemistry and Biochemistry

School

Mathematics and Natural Sciences

Committee Chair

Vijay Rangachari

Committee Chair Department

Chemistry and Biochemistry

Committee Member 2

Douglas Masterson

Committee Member 2 Department

Chemistry and Biochemistry

Committee Member 3

Faqing Huang

Committee Member 3 Department

Chemistry and Biochemistry

Committee Member 4

Sarah Morgan

Committee Member 4 Department

Polymers and High Performance Materials

Committee Member 5

Philip Bates

Committee Member 5 Department

Chemistry and Biochemistry

Abstract

Granulins (GRNs) are a family of small, cysteine-rich proteins that are generated upon proteolytic cleavage of their precursor, progranulin (PGRN) during inflammation. All seven GRNs (1 – 7 or A – G) contain twelve conserved cysteines that form six intramolecular disulfide bonds, rendering this family of proteins unique. GRNs play multiple roles and are involved in a myriad of physiological as well as pathological processes. They are known to a play role in growth and embryonic development, wound healing, and signaling cascades as well as in tumorigenesis. They are also implicated in neurodegenerative diseases like frontotemporal dementia (FTD), Alzheimer disease (AD), and amyotrophic lateral sclerosis (ALS). Despite their manifold functions, there is a paucity in the information about the structure-function relationship of these proteins, especially, with the role of the twelve conserved cysteines and the disulfide linkages in determining their structure and the functions. In this study, the role of disulfide bonds is probed by comparing the structures of the fully reduced GRN-3 (rGRN-3) and native GRN-3. We report that monomeric rGRN-3 is an intrinsically disordered protein (IDP) at low concentrations and undergoes dimerization at higher concentrations to form a fuzzy complex. Interestingly, we show that rGRN-3 is also able to activate NF-kappaB in human neuroblastoma cells in a concentration-dependent manner. We also show that both E. coli and mammalian HEK cells are inefficient in forming correct disulfide linkages and are incapable of generating monomeric native GRN-3 (GRN-3) exclusively, thus, predominately generating multimeric GRN-3 (mGRN-3) with scrambled inter-molecular bonds. We establish that GRN-3 has a more ordered structure as compared to that of mGRN-3 or rGRN-3, stabilized exclusively by the disulfide bonds which form a fulcrum imparting order to an otherwise disordered protein. We determined the potential involvement of GRN-3 in AD pathogenesis by showing that GRN-3 augments A-beta, the protein implicated in AD, aggregation in a concentration- and time-dependent manner and it interacts both with A-beta monomers and oligomers thereby providing a proof of concept for neuroinflammation triggered neurodegeneration.

ORCID ID

0000-0002-0941-2680

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