Targeting and Understanding the Function of Laminin Receptor

C. Samuel Umbaugh, Purdue University

Abstract

Laminin receptor (67 LR) is a 67 kDa protein derived from a 37 kDa precursor protein (37 LR) and the derivation process most likely involves post-translational modification of 37 LR, thus laminin receptor is referred to as 37/67 LR. Found in both the membrane and cytosolic compartments, 37/67 LR normally binds laminin and pathologically is associated with cancer progression, metastasis, and invasion. We used an in silico drug discovery approach to target the interaction interface between the potent anti-angiogenic protein pigment epithelium derived factor (PEDF) and 37 LR using a Maybridge HitFinder library. Following cell based counter screening and binding validation, we characterized a hit compound’s anti-viability, activation of PEDF signaling-related genes, anti-wound healing, and anti-cancer signaling properties in a PC-3 prostate cancer cell model. Our hit compound, HTS07944, which we nicknamed C3, was able to bind the laminin interacting domain of 37 LR (Peptide G), inhibited PC-3 cell viability at low micromolar concentrations, elicited PEDF-like gene expression and inhibited angiogenic tube formation and wound healing in a scratch assay. Proteomic analysis revealed that C3’s mechanism of action may be through inhibition of chromosomal maintenance proteins (MCM family and ubiquitin E2 ligases) and pathway analysis suggested that C3 would inactive the oncogene MYC. C3 inhibited MYC binding in a luciferase promoter assay. This hit compound has potential for future development as a lead compound for treating tumor growth and inhibiting angiogenesis. To better understand function of 37 LR, we designed a series of constructs lacking critical lysines in putative small ubiquitin-like modifier (SUMO) motifs of 37 LR (K11, K42, K212) and tested the hypothesis that SUMOylation generated the 67 LR species. SUMOylation is a ligase driven, lysine directed conjugation of approximately 12 kDa proteins to client proteins that alters protein-protein interactions, cellular location/trafficking, and general function. After failure to detect 67 LR, we examined the role putative SUMO motifs played in the general fate and function of 37 LR. Using phylogenetic analysis, we showed that a common consensus SUMO motif arose in mammals and via SUMO-1 pulldown and confocal analysis using anti-SUMO-2/3 antibody, we demonstrated that 37 LR associates with SUMO. Mutations to putative SUMO motifs altered half-life of 37 LR, with a marked enhancement to stability in the case of K42R mutation as shown via cyclohexamide chase assay. K42R also failed to accumulate after MG-132 treatment, suggesting that 37 LR may possess either a direct ubiquitin (Ub) site or a mixed SUMO/Ub site that regulates proteasomal degradation. Functionally, mutations to putative SUMO motifs in 37 LR significantly altered steady state levels of early pre-rRNA including 47S and 45S. Taken together, we have highlighted a role for SUMO in the function of 37 LR.

Degree

Ph.D.

Advisors

Figueiredo, Purdue University.

Subject Area

Molecular biology|Chemistry

COinS