Removing allosteric feedback inhibition of tomato 4-coumarate:CoA ligase by directed evolution.

Plant secondary metabolites, such as those derived from the phenylpropanoid pathway, have a beneficial effect on human health. Manipulation of metabolic flux in the phenylpropanoid pathway is important for achieving enhanced production of compounds such as anthocyanins, flavonoids and isoflavonoids. Here, we describe the development of a high-throughput molecular evolution approach that can be used for catalytic improvement of at least four key phenylpropanoid pathway enzymes, within the context of the metabolic pathway. This method uses yeast cells that express plant phenylpropanoid pathway enzymes, leading to formation of a colored intermediate that can be used as a readout in high-throughput screening. Here we report the identification of improved tomato peel 4-coumarate:CoA ligase variants using this approach. We found that the wild-type enzyme is strongly allosterically inhibited by naringenin, a downstream product of the pathway. Surprisingly, at least two of the improved variants are completely insensitive to feedback inhibition by naringenin. We suggest that this inhibition is exerted through a unique and previously unrecognized allosteric domain.

Elimination of negative feedback control mechanisms along the insulin signaling pathway improves beta-cell function under stress.

OBJECTIVE: Cellular stress and proinflammatory cytokines induce phosphorylation of insulin receptor substrate (IRS) proteins at Ser sites that inhibit insulin and IGF-1 signaling. Here, we examined the role of Ser phosphorylation of IRS-2 in mediating the inhibitory effects of proinflammatory cytokines and cellular stress on beta-cell function. RESEARCH DESIGN AND METHODS: Five potential inhibitory Ser sites located proximally to the P-Tyr binding domain of IRS-2 were mutated to Ala. These IRS-2 mutants, denoted IRS-2(5A), and their wild-type controls (IRS-2(WT)) were introduced into adenoviral constructs that were infected into Min6 cells or into cultured murine islets. RESULTS: When expressed in cultured mouse islets, IRS-2(5A) was better than IRS-2(WT) in protecting beta-cells from apoptosis induced by a combination of IL-1beta, IFN-gamma, TNF-alpha, and Fas ligand. Cytokine-treated islets expressing IRS2(5A) secreted significantly more insulin in response to glucose than did islets expressing IRS-2(WT). This could be attributed to the higher transcription of Pdx1 in cytokine-treated islets that expressed IRS-2(5A). Accordingly, transplantation of 200 islets expressing IRS2(5A) into STZ-induced diabetic mice restored their ability to respond to a glucose load similar to naïve mice. In contrast, mice transplanted with islets expressing IRS2(WT) maintained sustained hyperglycemia 3 days after transplantation. CONCLUSIONS: Elimination of a physiological negative feedback control mechanism along the insulin-signaling pathway that involves Ser/Thr phosphorylation of IRS-2 affords protection against the adverse effects of proinflammatory cytokines and improves beta-cell function under stress. Genetic approaches that promote IRS2(5A) expression in pancreatic beta-cells, therefore, could be considered a rational treatment against beta-cell failure after islet transplantation.

Regulation of transcription of the RNA splicing factor hSlu7 by Elk-1 and Sp1 affects alternative splicing.

Alternative splicing plays a major role in transcriptome diversity and plasticity, but it is largely unknown how tissue-specific and embryogenesis-specific alternative splicing is regulated. The highly conserved splicing factor Slu7 is involved in 3' splice site selection and also regulates alternative splicing. We show that Slu7 has a unique spatial pattern of expression among human and mouse embryonic and adult tissues. We identified several functional Ets binding sites and GC-boxes in the human Slu7 (hSlu7) promoter region. The Ets and GC-box binding transcription factors, Elk-1 and Sp1, respectively, exerted opposite effects on hSlu7 transcription: Sp1 protein enhances and Elk-1 protein represses transcription in a dose-dependent manner. Sp1 protein bound to the hSlu7 promoter in vivo, and depletion of Sp1 by RNA interference (RNAi) repressed hSlu7 expression. Elk-1 protein bound to the hSlu7 promoter in vivo, and depletion of Elk-1 by RNAi caused an increase in the endogenous level of hSlu7 mRNA. Further, depletion of either Sp1 or Elk-1 affected alternative splicing. Our results provide indications of a complex transcription regulation mechanism that controls the spatial and temporal expression of Slu7, presumably allowing regulation of tissue-specific alternative splicing events.

Stress alters the subcellular distribution of hSlu7 and thus modulates alternative splicing.

During pre-mRNA splicing, introns are removed and exons are ligated to form an mRNA. Exon choice is determined by different nuclear protein concentrations varying among tissues and cell types or by developmental stage. These can be altered by different cellular circumstances such as physiological stimuli, environmental effects and phosphorylation state. The splicing factor hSlu7 plays an important role in 3' splice site selection during the second step of splicing in vitro and has been suggested to affect alternative splicing in vivo. Our results indicate that an ultraviolet-C (UV-C) stress stimulus triggers changes in the alternative splicing patterns of cellular genes by decreasing the nuclear concentration of hSlu7 through the modulation of its nucleus-to-cytoplasm transport. This shift is mostly dependent on the Jun N-terminal kinase (JNK) cascade. Although we found by RNAi knockdown that hSlu7 is not essential for cell viability, its nuclear concentration effects exon choice and inclusion:skipping ratio of alternative splicing. A possible spatial and temporal regulatory mechanism by which hSlu7 protein levels are regulated within the nucleus is suggested, thus implying a broad effect of hSlu7 on alternative splicing.