Dissertation/Thesis Abstract

Secretory Protein Biogenesis: From Membrane Translocation to Vitamin K-Dependent Disulfide Bridge Formation
by Schulman, Sol, Ph.D., Harvard University, 2010, 156; 3435574
Abstract (Summary)

The eukaryotic endoplasmic reticulum (ER) and prokaryotic plasma membrane are the gateway to the secretory pathway. After insertion into or translocation across these membranes, the nascent proteins are embraced by a specialized folding environment rich in oxidative folding catalysts. This thesis will address three fundamental questions underlying secretory protein biogenesis: (1) How are proteins translocated selectively while maintaining a membrane seal? (2) How do bacterial homologs of mammalian vitamin K epoxide reductase (VKOR) cooperate with a thioredoxin (Tnc)-like redox partner to catalyze disulfide bridge formation? (3) What are the Trx-like redox partners of human VKOR?

Secretory proteins are directed to the SecY complex by N-terminal signal sequences. When we deleted a short a-helical plug at the center of E. coli SecY these mutants were functional and even translocated proteins with defective signal sequences. Structures of equivalent plug deletions in M. jannaschii demonstrated these mutants were viable because new plugs had formed These plugs lacked many interactions that normally stabilize the closed channel, explaining how a membrane seal is ordinarily maintained and why mutant channels open for proteins with signal sequence mutations.

Homologs of VKOR catalyze disulfide bridge formation in many bacteria. We determined the crystal structure of a VKOR homolog from Synechococcus sp. in an arrested state of electron transfer with its periplasmic Trx-like redox partner. We propose a pathway explaining how VKOR uses electrons from the reduced cysteines of secretory proteins to reduce a quinone, a mechanism confirmed by in vitro reconstitution of vitamin K-dependent disulfide bridge formation. The structure helps to understand how human VKOR mutations cause resistance to warfarin, the most commonly used oral anticoagulant.

Finally, we identified physiological redox partners of human VKOR by screening mammalian Trx-like ER proteins for their ability to form a key intermediate with human VKOR. VKOR interacts most strongly with TMX, a membrane-anchored Trx-like protein with a unique CPAC active site. This interaction is dependent on the same conserved extracellular loop cysteines required in Synechococcus sp. These results demonstrate that human VKOR employs the same electron transfer pathway as its bacterial homologs, and that VKORs generally prefer membrane-bound Inc-like redox partners.

Indexing (document details)
Advisor: Rapoport, Tom A.
Commitee:
School: Harvard University
School Location: United States -- Massachusetts
Source: DAI-B 72/01, Dissertation Abstracts International
Source Type: DISSERTATION
Subjects: Cellular biology, Biochemistry, Medicine
Keywords: Disulfide bond formation, Membrane translocation, Secretory proteins, Thioredoxin, Vitamin K, Warfarin
Publication Number: 3435574
ISBN: 9781124340463
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