Question

In part A, yes you should reconstitute the full-length protein sequence from the fragments. However, you do not need to write out the amino acid sequence - you can just provide the order of the fragment numbers (from either set of fragments). For example (and this is not the answer), you could say: "Using the Pro-IAPP fragment set, starting from the N-terminus, the fragment order is '5-4-6-3-2-1' " and that would fully address the question.

In part B, the hint is to focus on quantitative proteomics.

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. As it starts with methionine at N terminal, it will start with the sequence MGILKLQVFLIVSVALNHLKA and the sequence will continue in order 5-3-1-6-2-4

B. IAPP activates macrophages and induces islet inflammation . Inflammasome activation is the critical event in the initiation of islet inflammation by IAPP, as IAPP induced IL-1β but has no effect on IL-6 or TNF secretion from mmLDL-primed dendritic cells in vitro. The significance of the inflammasome/IL-1 pathway to IAPP-induced islet inflammation in vitro demonstrated by the finding that TNF secretion from macrophages and chemokine secretion from isolated islets induced by long-term stimulation with IAPP was strongly suppressed by IL-Ra. In vivo, IL-1Ra treatment improved glucose tolerance in recipients of IAPP-Tg islets and conferred nearly complete protection from macrophage recruitment to the transplanted hIAPP-Tg islets. IL-1Ra also improved islet function without affecting insulin sensitivity in IAPP-Tg KKAy mice.