Chaseellison8732

Published under license by The American Society for Biochemistry and Molecular Biology, Inc.Fatty acid transport protein 2 (FATP2) is highly expressed in the liver, small intestine, and kidney where it functions in both the transport of exogenous long-chain fatty acids and the activation of very-long-chain fatty acids. Here, using  a murine model, we investigated the phenotypic impacts of deleting FATP2, followed by a transcriptomic analysis using unbiased  RNA-Seq to identify concomitant changes in the liver transcriptome. Selleck BTK inhibitor Wildtype and FATP2-null (Fatp2-/-) mice (5 weeks) were maintained on a standard chow diet for 6 weeks. The Fatp2-/- mice had reduced weight gain, lowered serum triglyceride and increased serum cholesterol levels, and attenuated dietary fatty acid absorption. Transcriptomic analysis of the liver revealed 258 differentially expressed genes in male Fatp2-/- mice and a total of 91 in female Fatp2-/- mice. These genes mapped to the following gene ontology categories fatty acid degradation, peroxisome biogenesis, fatty acid synthesis, and retinol and arachidonic acid metabolism. Targeted RT-qPCR verified the altered expression of selected genes. Of note, most of the genes with increased expression were known to be regulated by peroxisome proliferator-activated receptor alpha (PPARα), suggesting that FATP2 activity is linked to a PPARα-specific proximal ligand. Targeted metabolomic experiments in the Fatp2-/- liver revealed increases of total C160, C161, and C181 fatty acids; increases in lipoxinA4 and prostaglandinJ2; and a decrease in 20-hydroxyeicosatetraenoic acid. We conclude that the expression of FATP2 in the liver broadly affects the metabolic landscape through PPARα, indicating that FATP2 provides an important role in liver lipid metabolism  through its transport or activation activities. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.The protein tyrosine phosphatase SHP2 is an allosteric enzyme critical for cellular events downstream of growth factor receptors.Mutations in the SHP2gene have beenlinked to many different types of human diseases, including developmental disorders, leukemia and solid tumors.Unlike most SHP2-activating mutations, the T507K substitution in SHP2 is unique in that it exhibits oncogenic Ras-like transforming activity. However, the biochemical basis of how the SHP2/T507K variant elicits transformation remains unclear. By combining kinetic and biophysical methods, X-ray crystallography and molecular modeling, as well as using cell biology approaches, here we uncovered that the T507K substitution alters both SHP2 substrate specificity and its allosteric regulatory mechanism. We found that although SHP2/T507K exists in the closed, autoinhibited conformation similar to the wild-type enzyme, the interactions between its N-SH2 and PTP domains are weakened such that SHP2/T507K possesses a higher affinity for the scaffolding protein Grb2-associated binding protein 1 (Gab1). We also discovered that the T507K substitution alters the structure of the SHP2 active site, resulting in a change in SHP2 substrate preference for Sprouty1, a known negative regulator of Ras signaling and potential tumor suppressor. Our results suggest that SHP2/T507K's shift in substrate specificity coupled with its preferential association of SHP2/T507K with Gab1 enables the mutant SHP2 to more efficiently dephosphorylate Sprouty1 at pTyr53. This dephosphorylation hyperactivates Ras signaling, which is likely responsible for SHP2/T507K's Ras-like transforming activity. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.T-type (Cav3) Ca2+ channels are important regulators of excitability and rhythmic activity of excitable cells. Among other voltage-gated Ca2+ channels, Cav3 channels are uniquely sensitive to oxidation and zinc. Using recombinant protein expression in HEK293 cells, patch-clamp electrophysiology, site-directed mutagenesis, and homology modeling, we report here that modulation of Cav3.2 by redox agents and zinc is mediated by a unique extracellular module containing i) a high-affinity metal-binding site formed by the extracellular IS1-IS2 and IS3-IS4 loops of domain I, and ii) a cluster of extracellular cysteines in the IS1-IS2 loop. Patch clamp recording of recombinant Cav3.2 currents revealed that two cysteine-modifying agents, sodium (2-sulfonatoethyl) methanethiosulfonate (MTSES) and N-ethylmaleimide (NEM), as well as a reactive oxygen species-producing neuropeptide, substance P (SP), inhibit Cav3.2 current to similar degrees and that this inhibition is reversed by a reducing agent and a zinc chelator. Pre-application of MTSES prevented further SP-mediated current inhibition. Substitution of the zinc-binding residue His-191 in Cav3.2 reduced the channel's sensitivity to MTSES, and introduction of the corresponding histidine into Cav3.1 sensitized it to MTSES. Removal of extracellular cysteines from the IS1-IS2 loop of Cav3.2 reduced its sensitivity to both MTSES and SP. We hypothesize that oxidative modification of IS1-IS2 loop cysteines induces allosteric changes in the zinc-binding site of Cav3.2, such that it become sensitive to ambient zinc. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.Mutations  in retinaldehyde binding protein 1 (RLBP1), encoding the visual cycle protein cellular retinaldehyde-binding protein (CRALBP), cause an autosomal recessive form of retinal degeneration. By binding to 11-cis-retinoid, CRALBP augments the isomerase activity of retinoid isomerohydrolase RPE65 (RPE65) and facilitates 11-cis-retinol oxidation to 11-cis-retinal. CRALBP also maintains the 11-cis configuration and protects against unwanted retinaldehyde activity. Studying a sibling pair that are compound heterozygous for mutations in RLBP1/CRALBP, here we expand the phenotype of affected individuals, elucidate a previously unreported phenotype in RLBP1/CRALBP carriers, and demonstrate consistencies between the affected individuals and Rlbp/Cralbp-/- mice. In the RLBP1/CRALBP-affected individuals, non-recordable rod-specific electroretinogram traces recovered after prolonged dark adaptation. In ultrawide-field fundus images, we observed radially arranged puncta typical of RLBP1/CRALBP-associated disease. Spectral domain optical coherence tomography (SD-OCT) revealed hyperreflective aberrations within photoreceptor-associated bands.