Mccormickmckenzie3174

Conversely, the DAD2D166A variant could not interact with PhMAX2A in the presence of SL, but its interaction with PhD53A remained unaffected. Structural analyses of DAD2N242I and DAD2D166A revealed only small differences compared with the structure of the wild-type receptor. Results of molecular dynamics simulations of the DAD2N242I structure suggested that increased flexibility is a likely cause for its SL-independent interaction with PhMAX2A. Our results suggest that PhMAX2A and PhD53A have distinct binding sites on the SL receptor and that its flexibility is a major determinant of its interactions with these two downstream regulators. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.The ileal apical sodium-dependent bile acid transporter (ASBT) is crucial for the enterohepatic circulation of bile acids. ASBT function is rapidly regulated by several post-translational modifications (PTMs). One reversible PTM is S-acylation, involving the covalent attachment of fatty acids (FAs) to cysteine residues in proteins. However, whether S-acylation affects ASBT function and membrane expression has not been determined. selleck chemicals Using the acyl resin-assisted capture (acyl-RAC) method, here we found that the majority of ASBT (~80%) was S-acylated in ileal brush border membrane vesicles from human organ donors, as well as in HEK293 cells stably transfected with ASBT (2BT cells). Metabolic labeling with alkyne-palmitic acid (100 μM for 15 h) also showed that ASBT is S-acylated in 2BT cells. Incubation with the acyltransferase inhibitor 2-bromopalmitate (25 μM for 15 h) significantly reduced ASBT S-acylation, function, and levels on the plasma membrane. Treatment of 2BT cells with saturated palmitic acid (100 μM for 15 h) increased ASBT function, whereas treatment with unsaturated oleic acid significantly reduced ASBT function. Metabolic labeling with alkyne-oleic acid (100 μM for 15 h) revealed that oleic acid attaches to ASBT, suggesting that unsaturated fatty acids may decrease ASBT's function via a direct covalent interaction with ASBT. We also identified Cys-314 as a potential S-acylation site. In conclusion, these results provide evidence that S-acylation is involved in the modulation of ASBT function. These findings underscore the potential for unsaturated FAs to reduce ASBT function, which may be useful in disorders in which bile acid toxicity is implicated. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.Endothelial cells (ECs) lining the vasculature of vertebrates respond to low oxygen (hypoxia) by maintaining vascular homeostasis and initiating adaptive growth of new vasculature through angiogenesis. Previous studies have uncovered the molecular underpinnings of the hypoxic response in ECs; however, there is a need for comprehensive temporal analysis of the transcriptome during hypoxia. Here, we sought to investigate the early transcriptional programs of hypoxic ECs by using RNA-Seq of primary cultured human umbilical vein ECs (HUVECs) exposed to progressively increasing severity and duration of hypoxia. We observed that hypoxia modulates the expression levels of about one third of the EC transcriptome. Intriguingly, expression of the gene encoding the developmental transcription factor SRY-box transcription factor 7 (SOX7) rapidly and transiently increased during hypoxia. Transcriptomic and functional analyses of ECs following SOX7 depletion established its critical role in regulating hypoxia-induced angiogenesis. We also observed that depletion of the hypoxia-inducible factor (HIF) genes, HIF1A (encoding HIF-1α) and endothelial PAS domain protein 1 (EPAS1 encoding HIF-2α) inhibited both distinct and overlapping transcriptional programs. Our results indicated a role for HIF1A in down-regulating mitochondrial metabolism, while concomitantly up-regulating glycolytic genes, whereas EPAS1 primarily up-regulated the angiogenesis transcriptional program. These results identify the concentration and time dependence of the endothelial transcriptomic response to hypoxia and an early key role for SOX7 in mediating angiogenesis. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.Protein phosphatase 2A (PP2A) critically regulates cell signaling and is a human tumor suppressor. PP2A complexes are modulated by proteins such as cancerous inhibitor of protein phosphatase 2A (CIP2A), protein phosphatase methylesterase 1 (PME-1), and SET nuclear proto-oncogene (SET) that often are deregulated in cancers. However, how they impact cellular phosphorylation and how redundant they are in cellular regulation is poorly understood. Here, we conducted a systematic phosphoproteomics screen for phospho-targets modulated by siRNA-mediated depletion of CIP2A, PME-1, and SET (to reactivate PP2A) or the scaffolding A-subunit of PP2A (PPP2R1A)(to inhibit PP2A) in HeLa cells. We identified PP2A-modulated targets in diverse cellular pathways, including kinase signaling, cytoskeleton, RNA splicing, DNA repair, and nuclear lamina. The results indicate non-redundancy among CIP2A, PME-1, and SET in phospho-target regulation. Notably, PP2A inhibition or reactivation affected largely distinct phosphopeptides, introducing a concept of non-overlapping phosphatase inhibition and activation responsive sites (PIRS and PARS, respectively). This phenomenon is explained by the PPP2R1A inhibition impacting primarily dephosphorylated threonines, whereas PP2A reactivation results in dephosphorylation of clustered and acidophilic sites. Using comprehensive drug-sensitivity screening in PP2A-modulated cells to evaluate the functional impact of PP2A across diverse cellular pathways targeted by these drugs, we found that consistent with global phosphoproteome effects, PP2A modulations broadly affect responses to more than 200 drugs inhibiting a broad spectrum of cancer-relevant targets. These findings advance our understanding of the phosphoproteins, pharmacological responses, and cellular processes regulated by PP2A modulation and may enable the development of combination therapies. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.