Doddcollins9830

We identified TmAAE1 and TmAAE5 as the most efficient enzymes for the activation of butyric acid (taxol D side chain), TmAAE13 as the best candidate for generating a CoA ester of tiglic acid (taxol C side chain), TmAAE3 and TmAAE13 as suitable for the activation of 4-methylbutyric acid (N-debenzoyl-N-(2-methylbutyryl)taxol side chain), TmAAE15 as a highly efficient candidate for hexanoic acid activation (taxol B side chain), and TmAAE4 as suitable candidate for esterification of benzoic acid with CoA (taxol side chain). This study lays important groundwork for metabolic engineering efforts aimed at improving taxol production in cell cultures. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.The small GTPases H, K, and NRAS are molecular switches indispensable for proper regulation of cellular proliferation and growth. Several mutations in the genes encoding members of this protein family are associated with cancer and result in aberrant activation of signaling processes caused by a deregulated recruitment of downstream effector proteins. In this study, we engineered variants of the Ras-binding domain (RBD) of C-Raf proto-oncogene, Ser/Thr kinase (CRAF). These variants bound with high affinity to the effector-binding site of Ras in an active conformation. Structural characterization disclosed how the newly identified RBD mutations cooperate and thereby enhance affinity to the effector-binding site in Ras compared with WT RBD. The engineered RBD variants closely mimicked the interaction mode of naturally occurring Ras effectors and acted as dominant-negative affinity reagents that block Ras signal transduction. Tanespimycin research buy Experiments with cancer cells showed that expression of these RBD variants inhibits Ras signaling, reducing cell growth and inducing apoptosis. Using these optimized RBD variants, we stratified patient-derived colorectal cancer organoids with known Ras mutational status according to their response to Ras inhibition. These results revealed that the presence of Ras mutations was insufficient to predict sensitivity to Ras inhibition, suggesting that not all of these tumors required Ras signaling for proliferation. In summary, by engineering the Ras/Raf interface of the CRAF-RBD, we identified potent and selective inhibitors of Ras in its active conformation that outcompete binding of Ras-signaling effectors. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.Heart muscle contractility and performance are controlled by posttranslational modifications of sarcomeric proteins. Although myosin regulatory light chain (RLC) phosphorylation has been extensively studied both in vitro and in vivo, the precise role of cardiac myosin light chain kinase (cMLCK), the primary kinase acting upon RLC, in the regulation of cardiomyocyte contractility remains poorly understood. In the current study, using recombinantly expressed and purified proteins, various analytical methods, in vitro and in situ kinase assays, and mechanical measurements in isolated ventricular trabeculae, we demonstrate that human cMLCK is not a dedicated kinase for RLC, but can phosphorylate other sarcomeric proteins with well-characterized regulatory functions. We show that cMLCK specifically mono-phosphorylates Ser-23 of human cardiac troponin I (cTnI) both in isolation and in the trimeric troponin complex in vitro and in situ in the native environment of the muscle myofilament lattice. Moreover, we observed that human cMLCK phosphorylates rodent cTnI to a much smaller extent both in vitro and in situ, suggesting a species-specific adaptation of cMLCK. Although cMLCK treatment of ventricular trabeculae exchanged with either rat or human troponin increased their cross-bridge kinetics, the increase in sensitivity of myofilaments to calcium was significantly blunted by human TnI, suggesting that human cTnI phosphorylation by cMLCK modifies the functional consequences of RLC phosphorylation. We propose that cMLCK-mediated phosphorylation of TnI is functionally significant and represents a critical signaling pathway that coordinates the regulatory states of thick and thin filaments in both physiological and potentially pathophysiological conditions of the heart. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.Vascular endothelial growth factor (VEGF) contributes to blood-retinal barrier (BRB) dysfunction in several blinding eye diseases, including diabetic retinopathy. Signaling via the secreted protein norrin through the frizzled class receptor 4 (FZD4)/LDL receptor-related protein 5-6 (LRP5-6)/tetraspanin 12 (TSPAN12) receptor complex is required for developmental vascularization and BRB formation. Here, we tested the hypothesis that norrin restores BRB properties after VEGF-induced vascular permeability in diabetic rats or in animals intravitreally injected with cytokines. Intravitreal co-injection of norrin with VEGF completely ablated VEGF-induced BRB permeability to Evans blue-albumin. Likewise, 5-month diabetic rats exhibited increased permeability of FITC-albumin, and a single norrin injection restored BRB properties. These results were corroborated in vitro, where co-stimulation of norrin with VEGF or stimulation of norrin after VEGF exposure, restored barrier properties, indicated by electrical resistance or 70 kDa RITC-dextran permeability in primary endothelial cell culture. Interestingly, VEGF promoted norrin signaling by increasing the FZD4 co-receptor TSPAN12 at cell membranes in a MAPK/ERK kinase (MEK)/ERK-dependent manner. Norrin signaling through β-catenin was required for BRB restoration, but glycogen synthase kinase 3 α/β (GSK-3α/β) inhibition did not restore BRB properties. Moreover, levels of the tight junction protein claudin-5 were increased with norrin and VEGF or VEGF alone, but both norrin and VEGF were required for enriched claudin-5 localization at the tight junction. These results suggest that VEGF simultaneously induces vascular permeability and promotes responsiveness to norrin. Norrin, in turn, restores tight junction complex organization and BRB properties in a β-catenin-dependent manner. Published under license by The American Society for Biochemistry and Molecular Biology, Inc.