INTRINSIC DISORDER WITHIN CYSTEINE-RICH, GRANULIN-B: IMPLICATIONS FOR MULTIFUNCTIONAL ROLES AND STRUCTURE

Abstract

Granulins (Grns) are a family of small, cysteine-rich proteins that are generated upon proteolytic cleavage of their precursor, progranulin. All seven Grns (A-G) contain twelve conserved cysteines that form six intramolecular disulfide bonds, rendering this family of proteins unique. Grns are known to play multi-functional roles, including wound healing, embryonic growth, and inflammation, and are also implicated in neurodegenerative diseases like frontotemporal dementia and Alzheimer disease. Despite their manifold functions, there exists a dearth of information regarding their structure-function relationship. Here, we sought to establish the role of disulfide bonds in promoting structure and function by studying the completely reduced GrnB (rGrnB). We have established that the monomeric rGrnB is an intrinsically disordered protein (IDP) at low concentrations and undergoes dimerization at higher concentrations to form a fuzzy complex without a net gain in the structure – a behavior increasingly identified as a hallmark of some IDPs. Interestingly, rGrnB also activates NF-B in human neuroblastoma cells in a concentration-dependent manner. This activation correlates with the observed monomer-dimer dynamics. We also show that only 10% of total GrnB expressed in E. coli is intramolecularly disulfide bonded protein, while rest forms intermoleularly bonded multimers. We are currently in the process of studying the biophysical and biochemical characteristics of the fully oxidized monomeric GrnB with intramolecular disulfide bonds along with serGrnB mutant, in which all the cysteines are mutated to serines. These data are presented and discussed.

Full text

INTRINSIC DISORDER WITHIN CYSTEINE-RICH, GRANULIN-B: IMPLICATIONS FOR
MULTIFUNCTIONAL ROLES AND STRUCTURE.
Gaurav Ghag1, Lauren M. Wolf2, Randi G. Reed1, Nicholas P. van der Munnik3, Claudius
Mundoma4, Melissa A. Moss2,3 and Vijayaraghavan Rangachari1.
1Department of Chemistry and Biochemistry, University of Southern Mississippi, Hattiesburg, MS
2Biomedical Engineering Program and 3Department of Chemical Engineering, University of South Carolina,
Columbia, SC
4Institute of Molecular Biophysics, Florida State University, Tallahassee, FL
Granulins (Grns) are a family of small, cysteine-rich proteins that are generated upon proteolytic cleavage
of their precursor, progranulin. All seven Grns (A-G) contain twelve conserved cysteines that form six
intramolecular disulfide bonds, rendering this family of proteins unique. Grns are known to play multi-
functional roles, including wound healing, embryonic growth, and inflammation, and are also implicated
in neurodegenerative diseases like frontotemporal dementia and Alzheimer disease. Despite their
manifold functions, there exists a dearth of information regarding their structure-function relationship.
Here, we sought to establish the role of disulfide bonds in promoting structure and function by studying
the completely reduced GrnB (rGrnB). We have established that the monomeric rGrnB is an intrinsically
disordered protein (IDP) at low concentrations and undergoes dimerization at higher concentrations to
form a fuzzy complex without a net gain in the structure – a behavior increasingly identified as a hallmark
of some IDPs. Interestingly, rGrnB also activates NF-B in human neuroblastoma cells in a
concentration-dependent manner. This activation correlates with the observed monomer-dimer dynamics.
We also show that only 10% of total GrnB expressed in E. coli is intramolecularly disulfide bonded
protein, while rest forms intermoleularly bonded multimers. We are currently in the process of studying
the biophysical and biochemical characteristics of the fully oxidized monomeric GrnB with
intramolecular disulfide bonds along with serGrnB mutant, in which all the cysteines are mutated to
serines. These data are presented and discussed.