Bioactive peptides and proteins in disease

Regulatory peptides and marker proteins are important to study in order to understand disease mechanisms. This applies of course also to our common diseases where all relationships are not yet known. Cancer and diabetes are two such complex diseases that affect hundreds of millions of people worldwide. This thesis addresses particular aspects of these two diseases, regarding one regulatory peptide (VIP, vasoactive intestinal polypeptide) that may be useful for tumor tracing and two proteins (apoCIII, apolipoprotein CIII, and TTR, transthyretin) that are altered in type 1 diabetes. VIP and functional VIP receptors are expressed in neuroblastomas, suggesting that the growth of these cells may be mediated in part by an autocrine action of VIP. VIP receptors are present in many epithelial cancers including breast, colon, non-small cell lung cancer, and pancreatic and prostate cancers. Due to the high density of VIP receptors on cancer cells, radiolabelled VIP may be used to image these tumours. It was therefore important for us to study in vivo distribution of the radiolabelled VIP prior to its usage as tumour tracer. We also studied the biological effects of VIP on tumours in an animal model, as there may be differences with respect to receptor expression between cultured tumour cells and tumour cells grown in vivo. Our studies could provide new insight into tumour imaging with respect to radiolabelled VIP. Type 1 diabetes serum was shown to increase intracellular Ca2+ and cause cell death. ApoCIII and TTR were isolated from sera of newly diagnosed type 1 diabetic patients based on a biological assay of increases of intracellular Ca2+. The exposure of the pancreatic beta-cell to apoCIII not only increases intracellular Ca2+, but also causes programmed cell death. Furthermore, the activity of apoCIII and type 1 diabetes serum was totally blocked when a polyclonal antibody against human apoCIII was added. TTR did not have any effect on cell death. When applying the patch clamp technique, both cells treated with apoCIII and those treated with TTR displayed larger Ca 2+ -channel currents than control cells. Research over the last 30 years has established that type 1 diabetes is an autoimmune disease, but the triggers of the initiation and progression of the disease are still not identified. Genetic, immunological and environmental factors are involved in the pathogenesis of type 1 diabetes and it is most likely that the events involved can differ between different patients. Further investigations are needed to elucidate all pathways and how they are related to the underlying autoimmunity, but our results show that there is at least a group of type 1 diabetes patients where apoCIII and TTR play a role

Conserved Structure and Function in the Granulysin and NK-Lysin Peptide Family

Granulysin and NK-lysin are homologous bactericidal proteins with a moderate residue identity (35%), both of which have antimycobacterial activity. Short loop peptides derived from the antimycobacterial domains of granulysin, NK-lysin, and a putative chicken NK-lysin were examined and shown to have comparable antimycobacterial but variable Escherichia coli activities. The known structure of the NK-lysin loop peptide was used to predict the structure of the equivalent peptides of granulysin and chicken NK-lysin by homology modeling. The last two adopted a secondary structure almost identical to that of NK-lysin. All three peptides form very similar three-dimensional (3-D) architectures in which the important basic residues assume the same positions in space. The basic residues in granulysin are arginine, while those in NK-lysin and chicken NK-lysin are a mixture of arginine and lysine. We altered the ratio of arginine to lysine in the granulysin fragment to examine the importance of basic residues for antimycobacterial activity. The alteration of the amino acids reduced the activity against E. coli to a larger extent than that against Mycobacterium smegmatis. In granulysin, the arginines in the loop structure are not crucial for antimycobacterial activity but are important for cytotoxicity. We suggest that the antibacterial domains of the related proteins granulysin, NK-lysin, and chicken NK-lysin have conserved their 3-D structure and their function against mycobacteria