Genetic incorporation of unnatural amino acids for the synthesis of proteins with defined modifications

Residue-specific incorporation and site-specific incorporation methods are widely used to incorporate an increasing number of novel unnatural amino acids (UAAs) into proteins with great potential to facilitate their structure-function studies. This dissertation presents my work on the genetic incorporation of several UAAs by using these two methods into proteins and their applications in the synthesis of proteins with defined modifications, which are important for understanding their role in cellular events. These two methods together with other protein synthesis and modification techniques are introduced in Chapter 1 of this thesis. In chapter 2, a residue-specific incorporation method was used to incorporate azidonorleucine (ANL) and azidonorvaline (ANV) into proteins via methionine auxotrophic strain. The genetically incorporated ANL served as the orthogonal lysine precursor in the protein, onto which one ligatable auxiliary group was installed for native chemical ubiquitination. The semisynthesis of diubiquitin and its characterization was demonstrated. To further expand the utility of our method, ubiquitylated H2A was also synthesized efficiently in a similar way, which was used to study the behavior of several deubiquitinases towards it and the crosstalk between H2A ubiquitination and H3K36 methylation. In this chapter, we also attempted to develop one new method to prepare arginine methylated protein using genetically incorporated ANV. One mutant MetRS that can efficiently activate ANV was found. Several guanidinylating reagents were synthesized and tested for the site-specific guanidinylate ion at the protein level. The pyrrolysyl-tRNA synthetase (PylRS)/PylT pair has been wildly used to incorporate site-specifically UAAs into proteins in E. coli. In chapter 3, several pyrrolysine analogs were shown to be incorporated into proteins via orthogonal PylRS/PylT pair. One genetically incorporated α-hydroxyl amino acid was used to synthesize protein α-thioester by utilization of cysteinyl prolyl ester (CPE) auto-activating motif on protein. The proposed formation of diketopiperazine thioester via an intramolecular N–S acyl shift reaction was not successful possibly due to the steric hindrance of the α-hydroxyl amino acid located in our CPE motif. One genetically incorporated Cbz-protected homocysteine was used to synthesize histones with two different modifications by orthogonal cysteine-based chemistry. One model H3 protein was shown to be installed efficiently with a dimethylated lysine mimic at K27. The attempt to install the second modification into H3 failed due to the poor efficiency of the deprotection of Cbz-protected homocysteine using silver acetate or iodine in the acetic acid buffer. In the last part of this chapter, to incorporate more new UAAs into protein using PylRS/tRNA pair, the two plasmids based selection system was established in our lab. The construction and functional test of two plasmids used in the positive and negative selections were presented. Two libraries of mutant PylRS was constructed and successfully used to screen the mutant that can recognize the pyrrolysine analogs.​Doctor of Philosophy (SBS

Diosmetin ameliorates glucose metabolism in KK-Ay diabetic mice through regulation of Corynebacterium glutamicum via IRS/PI3K/ AKT signaling pathway

Background and Purpose: Diosmetin (Dios), a flavonoid compound with multiple pharmacological activities. However, fewer studies have reported its effects on diabetes. Here, we address the effect of Dios on glucose metabolism and gut microbiota in KK-Ay diabetic mice. Experimental Approach: Wild type C57BL/6J mice or diabetic KK-Ay mice were treated with vehicle or Dios for one month. The liver RNA-Seq was used to reveal the key signaling pathway interfered with Dios. The liver 16S rRNA gene sequencing was used to reveal the effects of Dios on gut microbiota. The experiment of C. glu transplantation was used to reveal the relationship between Dios and C. glu on glucose metabolism. Key Results: Dios treatment significantly decreased blood glucose and increased serum insulin concentrations. Transcriptome sequencing analysis found that the underlining mechanism of diosmetin on T2DM by regulating signal pathways of glucose metabolism, which was proved by up-regulating IRS/PI3K/AKT signaling pathway to promote glycogen synthesis and GLUT4 translocation. Besides, Dios treatment reshaped the unbalanced gut microbiota by suppressing the ratio of Firmicutes/Bacteroidetes and markedly increasing the richness of C. glu. Moreover, Treatment with C. glu and Dios together can markedly ameliorate glucose metabolism by up-regulating IRS/PI3K/AKT signaling pathway to promoting glycogen synthesis and GLUT4 translocation. Conclusions and Implications: Dios treatment remarkably ameliorated glucose metabolism in KK-Ay diabetic mice by the regulation of C. glu on IRS/PI3K/AKT signaling pathway and reshaped the unbalanced gut microbiota. Our study provided evidence for the application of Dios to the treatment of T2DM.</jats:p