Juneja, P. | Search bioRxiv

bioRxiv2020

Allosteric regulation of Thioesterase Superfamily Member 1 by free fatty acids and lysophosphatidylcholine

Non-shivering thermogenesis occurs in brown adipose tissue to generate heat in response to cold temperatures. Thioesterase superfamily member 1 (Them1) is transcriptionally upregulated in brown adipose tissue upon cold exposure and suppresses thermogenesis to conserve energy reserves. Them1 hydrolyzes long-chain fatty acyl-CoAs, preventing their use as fuel for thermogenesis. Them1 contains a C-terminal StAR-related lipid transfer domain (StarD) with unknown ligand or function. By complementary biophysical approaches, we show that StarD binds to long-chain fatty acids, products of Them1s enzymatic reaction, as well lysophosphatidylcholine (LPC), which activate thermogenesis in brown adipocytes. Certain fatty acids stabilize the StarD and allosterically enhance Them1 catalysis of acyl-CoA, whereas 18:1 LPC destabilizes and inhibits activity, which we verify in cell culture. Additionally, we demonstrate that the StarD functions to localize Them1 near lipid droplets. These findings define the role of the StarD as a lipid sensor that allosterically regulates Them1 activity and localization.

biochemistry↗

bioRxiv2019

Biochemical and structural analysis reveals the Neurofibromin (NF1) protein forms a high-affinity dimer

Neurofibromin is the protein product of the NF1 gene which is mutated in the Rasopathy disease Neurofibromatosis Type I. Defects in NF1 lead to aberrant signaling through the RAS-MAPK pathway due to disruption of the Neurofibromin GTPase-activating function on RAS family small GTPases. Very little is known about the function of the majority of Neurofibromin--to date, biochemical and structural data exist only for the GAP domain and the region containing a Sec-PH motif. To better understand the role of this large protein, we carried out a series of biochemical and biophysical studies which demonstrate that full length Neurofibromin forms a high-affinity dimer. Neurofibromin dimerization also occurs in cells, and likely has biological and clinical implications. Analysis of purified full-length and truncated variants of Neurofibromin by negative stain electron microscopy reveals the overall architecture of the dimer and predicts the potential interactions which contribute to the dimer interface. Structures resembling high-affinity full-length dimers could be reconstituted by mixing N- and C-terminal protein domains in vitro. Taken together these data suggest how Neurofibromin dimers might form and be stabilized within the cell.

biochemistry↗