Question

6. Islet amyloid polypeptide (IAPP), often called amylin, is a peptide hormone that is co-secreted with insulin from pancreatic B-cells. In combination, insulin and amylin regulate blood glucose levels. In this signaling process, amylin participates in slowing gastric emptying and promoting satiety, helping to prevent spikes in blood glucose levels. Using your knowledge of protein sequencing and proteomics, answer the questions below related to amylin. A. Islet amyloid polypeptide (IAPP, amylin) is produced by processing of a precursor protein called pro-IAPP. The pro-IAPP precursor is processed by endogenous proteases in pancreatic cells, which remove signal peptide sequences and ultimately produce the functional IAPP hormone, which is 37 amino acids in length. The known processing pathway includes the proteolytic fragments in the left column of the table below. Recombinant pro- IAPP can also be digested in vitro with Lys-C, a bacterial protease which specifically cleaves amide bonds C- terminal to lysine residues. These fragments are in the right column below. Additionally, Edman degradation indicated that the N-terminal amino acid is methionine. Using this information, indicate the correct order of the fragments (from either set) that fully represents the pro-IAPP sequence from N-terminus to C-terminus. Note: the fragments are ordered by mass, not by primary sequence. You do not need to write out the primary sequence; just the order of one of two sets of fragments from N-terminus to C-terminus. (8 points) Pro-IAPP Processing Fragments Lys-C Fragments 1 KR 11 RK 2 SSTNVGSNTYG 2 MGILK 3 TPIESHQVEKR 3 RNAVEVLK 4 NAVEVLKREPLNYLPL 4 REPLNYLPL 5 MGILKLOVFLIVLSVALNHLKA 5 ATPIESHQVEK 6 KCNTATCATORLANFLVHSSNNFKAIL 6 AILSSTNVGSNTYGK 7 LOVFLIVLSVALNHLK 8 CNTATCATORLANFLVHSSNNFK B. Proteomics experiments have suggested that IAPP production in cells is subject to regulation by signaling processes which do not affect insulin production. For example, IAPP production has been hypothesized to be sensitive to the presence of tumor necrosis factor alpha (TNF-a), an inflammatory cytokine. Assuming that pancreatic B-cells can be cultured in vitro, propose a proteomics-based experiment to prove that IAPP production increases in the presence of TNF-a added to the cell culture media. You may assume that TNF-a can be easily obtained and that cell culture is routine (i.e. focus on quantitative proteomics). (7 points)

Answer 1

A)MGILKLQVFLIVLSUALNWLK

B)

Amylin, also known as islet amyloid polypeptide (IAPP), is a normal secretory protein mainly expressed by pancreatic beta cells. It plays important roles in energy homeostasis, bone growth and brain development. Amylin was discovered in 1987 as a major component of amyloid deposits in the pancreas of type 2 diabetes patients. Elevated circulating levels of amylin have been detected in patients with severe acute pancreatitis, pancreas transplantation, obesity and insulin resistance. Pancreatic amylin mRNA levels are also elevated in genetically obese, insulin-resistant rats. However, the mechanisms underlying amylin expression are not completely understood.

TNF-α is a critical player in the inflammatory response in acute pancreatitis and has been reported to be involved in pancreas graft dysfunction. Circulating TNF-α is increased in obesity and has been implicated as a causative factor in obesity-associated insulin resistance and the pathogenesis of type 2 diabetes. In this study, we used the cultured murine pancreatic beta cell line MIN6 and pancreatic islets, as well as human amylin promoter luciferase reporter constructs, to examine the effect of TNF-α on amylin expression and further explore the mechanisms involved.

MATERIAL-Pancreatic islet preparation and cell culture,RNA extraction and real-time PCR,TNFG-alpha,Western blotting,Plasmid construction and luciferase reporter assay,Electrophoretic mobility shift assay.

Result-

TNF-α induces murine amylin expression

To determine the effect of TNF-α on amylin gene expression, MIN6 cells were challenged with different concentrations of TNF-α for different lengths of time and the mRNA expression of amylin were detected by quantitative real-time PCR.MIN6 cells cultured in DMEM containing 5.6 mmol/l glucose expressed transcripts for amylin. This expression was significantly enhanced by TNF-α in time- and dose-dependent manners. The minimal concentration of TNF-α needed to significantly induce amylin gene expression was 0.57 nmol/l. Consistent with the results obtained from MIN6 cells, mRNA expression of amylin in murine pancreatic islets was significantly enhanced by TNF-α after 9 h of stimulation. Interestingly, the inductive effect of TNF-α on amylin mRNA was more potent in islets than in MIN6 cells, suggesting that amylin expression in response to TNF-α is more sensitive in islets than in transformed beta cells. As amylin and insulin are co-localised in beta cells and co-secreted in response to glucose, we then examined the effect of TNF-α on proinsulin expression in MIN6 cells. While TNF-α upregulated amylin mRNA levels in MIN6 cells and murine primary islets in a time-dependent manner, it had no effect on proinsulin 1 and proinsulin 2 mRNA expressions in MIN6 cells and islets, suggesting that TNF-α specifically induces amylin expression in beta cells. We checked the effect of TNF-α on cytokine expression in MIN6 cells and found that 3 to 15 h of TNF-α treatment at 2.87 nmol/l had no effect on Il1β (also known as Il1b), Il6 or Mcp-1 (also known as Ccl2) mRNA expression. These results suggest that the inductive effect of TNF-α on amylin expression was not indirectly mediated by cytokines induced by TNF-α.