Searching for Novel Regulators of the Mevalonate Pathway

The mevalonate pathway is an important metabolic pathway that provides cells with vital bioactive molecules. The pathway converts mevalonate into various sterol and nonsterol isoprenoids required for cellular maintenance and health, including, cholesterol, ubiquinione, dolichol, heme A, farnesyl, and gernaylgeranyl protein anchors. However, these molecules are energetically expensive to synthesize and toxic in excess, so tight regulation of the pathway is necessary at multiple steps. In order to identify novel proteins that help regulate the mevalonate pathway, we performed unbiased proteomic screens to detect protein-protein interactions in Huh7 cells, a human hepatocellular carcinoma cell line. Proximity labeling with a promiscuous biotin ligase (BioID), and affinity purification were performed using multiple enzymes from the pathway as bait (MVK, MVD, FDFT1, FDPS, SQLE, and LSS). Several proteins were detected using both techniques at statistically significant levels compared to control. An extensive literature review revealed that two hits, NAB1 (NGFI-A-binding protein 1) and KHDRBS1 (KH RNA Binding Domain Containing, Signal Transduction Associated 1), could have relevant and understudied interactions with mevalonate kinase (MVK). Therefore, to determine if NAB1 or KHDRBS1 regulate MVK, they were independently knocked down using siRNAs in vitro . Knockdown of NAB1 led to decreased mRNA expression and protein levels of MVK under sterol depleted conditions, and decreased expression of high-density lipoprotein (HDL) associated genes ( ABCA1 , ApoA1 , etc). Conversely, when MVK was knocked down under sterol replete conditions, NAB1 protein levels were similarly reduced. Thus, these two proteins undergo a previously unknown interaction which warrants further study. NAB1-MVK interaction might help explain the association between MVK single nucleotide polymorphisms (SNP) and circulating HDL levels in humans, and the increase of circulating HDL in NAB1 knock out (KO) mice.