Abstract 1187: PDZ Domain Protein Arrays Define a Network of ABCA1 and SPLTC1 Protein-Protein Interactions that Regulate ABCA1 Trafficking and Efflux Activity

Protein microarrays are an emerging high throughput proteomic tool that can be used to study protein-protein interactions. Here we use protein arrays that have 123 PDZ domains spotted in duplicate from 77 PDZ proteins testing over 700 potential interactions and mapping a network of protein interactions that encompasses the ABCA1 transporter and SPLTC1. SPTLC1, through its associated serine palmitoyltransferase activity, catalyzes the initial step in the synthesis of sphingomyelin whose plasma level has been associated with an increased risk of cardiovascular disease. Conversely, ABCA1 effluxes cellular cholesterol to maintain plasma HDL levels which prevents cardiovascular disease. Using hierarchical clustering and networking algorithms analysis of our data set shows PDZ proteins are able to discriminate between closely related PDZ binding motifs contained in the C-termini of ABCA1 and SPTLC1. Mutation of the SPTLC1 PDZ motif disrupted SPTLC1’s ability to bind PDZ proteins and destabilized SPTLC1 protein expression demonstrating the functional importance of the motif. Co-expression of SPTLC1 with ABCA1 in 293 cells inhibited cholesterol efflux activity of the transporter by up to 80% (p < 0.01) as we previously reported and here we show this is through a mechanism that blocks the exit of the transporter from the endoplasmic reticulum. In contrast, treatment of primary human fibroblasts or mouse macrophages with myriocin, a specific serine palmitoyltransferase inhibitor, increased efflux by nearly 60% (p < 0.05). Our results demonstrate the utility using protein arrays to map protein-protein interactions that we hypothesis form a regulatory network that integrates the cholesterol efflux and sphingolipid synthesis pathways. That SPTLC1 activity negatively regulates ABCA1 efflux further suggests inhibition of SPTLC1 activity may represent a new therapeutic target to prevent cardiovascular disease.