Characterization of a UBXD1 Polymorphic Variant and Identification of UBXD1 Interacting Proteins

Abstract

p97, a member of the AAA ATPase (ATPases Associated with diverse cellular Activities) family of proteins, exists as a hexamer with two centrally located ATPase domains D1 and D2. The ATPase function of p97 is the means by which mechanical force is applied to substrates, consequently changing their conformation. As a highly abundant protein within cells, p97 has been shown to function in multiple pathways whereby specificity is directed via adaptor proteins. The largest family of bona fide p97 adaptors is the `Ubiquitin regulatory X' (UBX) domain containing family of adaptors. In addition to the UBX domain containing family of adaptors, proteins containing a PUB domain have been implicated in binding to p97. The p97 adaptor protein UBXD1 contains both a UBX and PUB domain, however the UBX domain does not participate in binding p97 due to absence of the conserved motif required for binding. Recently, a highly conserved region within the first 150 amino acids of the UBXD1 N-terminus has been shown to participate in p97 binding (Kern et al., 2009). The cellular function of UBXD1 remains largely unknown. One of the focuses of the Haines laboratory is to elucidate the function of UBXD1. Unpublished results from within the laboratory suggest UBXD1 to be defective at interacting with p97 mutants found in Inclusion Body Myopathy associated with Paget's Disease of Bone and Frontotemporal Dementia (IBMPFD) and Amyotrophic Lateral Sclerosis (ALS) disease, leading to a disruption of the autophagy pathway. These results suggest a role for UBXD1 as a human disease relevant protein. Search of a Single Nucleotide Polymorphism (SNP) database for polymorphisms within conserved regions of UBXD1 was carried out. Interestingly, the SNP database revealed a polymorphic variant within a conserved region of the PUB domain. The SNP was found within the asparagine residue of the evolutionarily conserved surface patch, resulting in an asparagine(N) to serine(S) substitution, termed N184S. The two main objectives of my master's thesis project are 1) to characterize this UBXD1 polymorphic variant in terms of p97 binding capabilities and determine the prevalence of the N184S PUB domain SNP within the population and 2) identification/verification of UBXD1 interacting proteins that may provide a clue for UBXD1 function in autophagy and define domains required for association. As a result of interaction-based studies, I have been able to show a severe loss-of-binding phenotype by the UBXD1 N184S polymorphic variant. While the N184S PUB domain SNP could not be validated within my sample population, the interaction data led to the discovery of potential UBXD1 interacting proteins. Additionally, proteomics data generated by Dr. Dale Haines in the lab of Dr. Raymond Deshaies revealed ERGIC-53 to be a novel binding partner of UBXD1. Through interaction-based studies, I have been able to determine the regions required for the interaction between UBXD1 and ERGIC-53 as well as propose a possible role for p97 UBXD1 complexes in autophagy.