Abstract 1723: The Sphingolipid Synthesis Regulator SPTLC1 is Bound by PARD3 through a Type II PDZ Protein-Protein Interaction
Elevated sphingolipids in humans associate with an increased risk of cardiovascular disease. Conversely, in mice, inhibition of de novo sphingolipid synthesis, which is initiated by serine palmitoyltransferase (SPT), an enzyme composed of the SPTLC1 & 2 subunits, protects against atherosclerosis. Here we describe a novel protein-protein interaction between SPTLC1 and the PDZ protein named Partitioning Defective Protein 3 (PARD3). Analysis of the SPTLC1 open reading frame indicated it has a highly conserved C-terminus that conforms to a type II PDZ protein interaction domain. Thus, PDZ domain protein arrays were probed with a SPTLC1 C-terminal peptide, which indicated SPTLC1 has the capacity to directly interact with the 3rd PDZ domain of PARD3. Overlay assays confirmed that the interaction of full length PARD3 with SPTLC1 depended upon the PDZ motif encoded within the last three amino acids in the SPTLC1 C-terminus. In human THP-1 macrophages the SPTLC1-PARD3 complex was detected at endogenous expression levels of the interacting proteins as assessed by co-immunoprecipitations demonstrating the physiologic existence of the complex. Increased PARD3 expression increased SPTLC1 expression, while mutation of the SPTLC1 PDZ interaction motif destabilized SPTLC1 expression and induced a diffuse cellular distribution of SPTLC1 as assessed by confocal microscopy and biochemical fractionation assays. Present experiments are focused on assessing the functional role of the SPTLC1-PARD3 complex in THP-1 macrophages using siRNA lentiviral vectors to suppress endogenous PARD3 expression. In conclusion, we have used protein arrays to identify a novel SPTLC1-PARD3 complex that effects the cellular localization and stability of this critical regulator of sphingolipid synthesis. Given the proatherosclerotic activity of SPTLC1, disruption of the SPTLC1-PARD3 complex may be of therapeutic use to block SPT activity and inhibit atherosclerotic vascular disease.