Structure-Function Analysis of Endoproteolytic Cleavage: Site-Directed Mutagenesis Studies of the α-MSH Cleavage Site in Silurana tropicalis POMC

Author

Quinn Kun Chen, University of DenverFollow

Date of Award

1-1-2009

Document Type

Masters Thesis

Degree Name

M.S.

Organizational Unit

College of Natual Science and Mathematics

First Advisor

Robert M. Dores, Ph.D.

Second Advisor

Nancy Lorenzon

Third Advisor

Dwight Smith

Keywords

α-MSH, αTC1.9 cells, Endoproteolysis, Fusion protein, Joining peptide, Proopiomelanocortin

Abstract

The maturation process of many prohormone molecules typically requires endoproteolytic cleavage C-terminal of dibasic residues, such as the K¹⁴¹R¹⁴²↓ site N-terminal of the α-melanocyte stimulating hormone (α-MSH) sequence in Silurana tropicalis proopiomelanocortin (POMC). In order to determine the absolute requirement of basic amino acid residues in the cleavage process, site-directed mutagenesis was employed to substitute alanine for the wild-type residues in the frog POMC open reading frame. Specifically, the following underlined residues were individually targeted for alanine substitution: R ¹³⁷ Q ¹³⁸ E ¹³⁹ N ¹⁴⁰ K ¹⁴¹ R ¹⁴²↓. The mouse pancreatic αTC1.9 cell line, which expresses prohormone convertase 2 (PC2), was selected as the model system for transfection studies. The processing of wild-type frog POMC was correctly predicted to mimic that observed in the intermediate pituitaries of the frog. Moreover, biochemical analysis of mutant frog constructs in the α-cells revealed that alanine substitutions of K¹⁴¹ and R¹⁴², but not of R¹³⁷, disrupted endoproteolytic cleavage resulting in a JP-α-MSH mutant fusion protein. Quantitative real-time PCR analysis of the αTC1.9 cells detected the relative gene expression levels of mouse PC2, glucagon but not PC1/3; detection of S. tropicalis POMC was only evident in the transfected α-cells, which was consistent with immunocytochemical data.

Publication Statement

Copyright is held by the author. User is responsible for all copyright compliance.

Rights Holder

Quinn Kun Chen

Provenance

Received from ProQuest

File Format

application/pdf

Language

en

File Size

92 p.

Recommended Citation

Chen, Quinn Kun, "Structure-Function Analysis of Endoproteolytic Cleavage: Site-Directed Mutagenesis Studies of the α-MSH Cleavage Site in Silurana tropicalis POMC" (2009). Electronic Theses and Dissertations. 779.
https://digitalcommons.du.edu/etd/779

Copyright date

2009

Discipline

Endocrinology, Neurosciences