All ETDs from UAB

Advisory Committee Chair

Chenbei Chang

Advisory Committee Members

Alecia K Gross

Jianbo Wang

James F Collawn

Matthew Alexander

Document Type

Dissertation

Date of Award

2020

Degree Name by School

Doctor of Philosophy (PhD) Heersink School of Medicine

Abstract

VAPB is one of three mammalian VAP proteins. It is a type-II ER transmembrane protein whose N-terminal major sperm protein domain (MSPd) is cleaved and secreted. Since the MSPd faces the cytosol, rather than the ER lumen, how it is cleaved and secreted is not yet known. In humans, P56S is a substitution mutation within the VAPB protein that segregates with cases of familial Amyotrophic Lateral Sclerosis (ALS) and prevents the secretion of VAPB MSPd. The work described in this thesis uses C. elegans to study how the N-terminal MSPd of VAPB is proteolytically processed, secreted, and regulated. C. elegans VPR-1 is the nematode homolog of VAPB. In Chapter 2 of this thesis, overexpression of VPR-1 with an N-terminal FLAG tag revealed that the N-terminal VPR-1 peptide is secreted from intestinal cells to bind the distal gonad. Genome-editing techniques were also used to tag the termini of endogenous vpr-1. Immunofluorescent imaging of endogenously tagged VPR-1 revealed a polar localization of VPR-1 termini in intestinal cells. In addition, western blots of these endogenously incorporated epitope tags revealed two stable VPR-1 products. Mass spectrometry determined that the smaller of the two products is the cleaved N-terminal peptide of VPR-1 that spans the MSPd and ends at a conserved leucine at the 156th amino acid position. This indicates that VPR-1 and other VAP homologs may be cleaved at L156 to release the MSPd. Chapter 3 describes an RNAi screen using C. elegans to identify genes required for MSPd cleavage or secretion. C. elegans null for vpr-1 are sterile and can be rescued by vpr-1 expression in the neurons, germline, or intestine. The brood size of vpr-1 null worms expressing vpr-1 in the intestine after RNAi knockdown was assessed for 422 genes. This screen identified a v-SNARE and several proteasome components as potential mediators of VPR-1 MSPd proteolysis and trafficking. Further analysis is needed in order to verify these candidates. In all, results from these two chapters advanced our understanding of MSPd cleavage and secretion and may pave the way to understanding ALS pathology.

Zein-Sabatto et al_G3_Table S1.xlsx (25 kB)
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