Structural studies and cytotoxicity assays of "aggregation-prone" IAPP(8-16) and its non-amyloidogenic variants suggest its important role in fibrillogenesis and cytotoxicity of human amylin.

Amyloid deposits to the islets of Langerhans are responsible for the gradual loss of pancreatic ß-cells leading to type II diabetes mellitus. Human mature islet amyloid polypeptide (hIAPP), a 37-residue pancreatic hormone, has been identified as the primary component of amyloid fibrils forming these deposits. Several individual segments along the entire sequence length of hIAPP have been nominated as regions with increased amyloidogenic potential, such as regions 8-20, 20-29, and 30-37. A smaller fragment of the 8-20 region, spanning residues 8-16 of hIAPP has been associated with the formation of early transient α-helical dimers that promote fibrillogenesis and also as a core part of hIAPP amyloid fibrils. Utilizing our aggregation propensity prediction tools AmylPred and AmylPred2, we have identified the high aggregation propensity of the 8-16 segment of hIAPP. A peptide analog corresponding to this segment was chemically synthesized and its amyloidogenic properties were validated using electron microscopy, X-ray fiber diffraction, ATR FT-IR spectroscopy, and polarized microscopy. Additionally, two peptides introducing point mutations L12R and L12P, respectively, to the 8-16 segment, were chemically synthesized. Both mutations disrupt the α-helical properties of the 8-16 region and lower its amyloidogenic potential, which was confirmed experimentally. Finally, cytotoxicity assays indicate that the 8-16 segment of hIAPP shows enhanced cytotoxicity, which is relieved by the L12R mutation but not by the L12P mutation. Our results indicate that the chameleon properties and the high aggregation propensity of the 8-16 region may significantly contribute to the formation of amyloid fibrils and the overall cytotoxic effect of hIAPP.

Structure-activity studies of lGnRH-III through rational amino acid substitution and NMR conformational studies.

Lamprey gonadotropin-releasing hormone type III (lGnRH-III) is an isoform of GnRH isolated from the sea lamprey (Petromyzon marinus) with negligible endocrine activity in mammalian systems. Data concerning the superior direct anticancer activity of lGnRH-III have been published, raising questions on the structure-activity relationship. We synthesized 21 lGnRH-III analogs with rational amino acid substitutions and studied their effect on PC3 and LNCaP prostate cancer cell proliferation. Our results question the importance of the acidic charge of Asp6 for the antiproliferative activity and indicate the significance of the stereochemistry of Trp in positions 3 and 7. Furthermore, conjugation of an acetyl-group to the side chain of Lys8 or side chain cyclization of amino acids 1-8 increased the antiproliferative activity of lGnRH-III demonstrating that the proposed salt bridge between Asp6 and Lys8 is not crucial. Conformational studies of lGnRH-III were performed through NMR spectroscopy, and the solution structure of GnRH-I was solved. In solution, lGnRH-III adopts an extended backbone conformation in contrast to the well-defined ß-turn conformation of GnRH-I.

Enzymatic stability, solution structure, and antiproliferative effect on prostate cancer cells of leuprolide and new gonadotropin-releasing hormone peptide analogs.

Analogs of GnRH, including [DLeu6, desGly1o]-GnRH-NHEt (leuprolide, commercial product), have been widely used in oncology to induce reversible chemical castration. Several studies have provided evidence that, besides their pituitary effects, GnRH analogs may exert direct antiproliferative effects on tumor cells. To study the effect of modifications in positions 4 and 6 of leuprolide on prostate cancer cell proliferation, we synthesized 12 new leuprolide analogs. All GnRH analogs lacked the carboxy-terminal Gly10-amide of GnRH, and an ethylamide residue was added to Pro9. Gly6 was substituted by DLys, Nepsilon-modified DLys, Glu, and DGlu. To improve the enzymatic stability, NMeSer was incorporated in position 4, and the rate of hydrolysis by alpha-chymotrypsin and subtilisin was investigated. Our results demonstrate that this incorporation increases enzymatic stability in all analogs of GnRH, whereas the antiproliferative effect on PC3 and LNCaP prostate cancer cells is similar to that of leuprolide. Conformational studies were performed to elucidate structural changes occurring on substitution of native residues and to study structure-activity relationship for these analogs. The solution models of [DLeu6, desGly10]-GnRH-NHEt (leuprolide), [NMeSer4, DGlu6, desGly10]-GnRH-NHEt, [Glu6, desGly10]-GnRH-NHEt, and [DGIu6, desGly10]-GnRH-NHEt peptides were determined through two-dimensional nuclear magnetic resonance spectroscopy in dimethylsulfoxide. Nuclear magnetic resonance data provide experimental evidence for the U-turn-like structure appeared in all four analogs, which could be characterized as beta-hairpin conformation. The most stable analog [NMeSer4, DGlu6, desGly10]-GnRH-NHEt against proteolytic cleavage forms a second extra backbone turn observed for residues 1-4.

Helicobacter pylori neutrophil-activating protein activates neutrophils by its C-terminal region even without dodecamer formation, which is a prerequisite for DNA protection--novel approaches against Helicobacter pylori inflammation.

Helicobacter pylori neutrophil-activating protein (HP-NAP) protects DNA from free radicals as a dodecamer through its ferroxidase activity without, however, directly binding to it. The retardation that was observed at pH 7.5 could be easily attributed to an iron effect, as it was revealed by experiments in the absence of HP-NAP. A total loss of ferroxidase activity, dodecamer formation and DNA protection in environments rich in free radicals was observed after replacement of His25, His37, Asp52 and Lys134, which are located within the ferroxidase site, with Ala. Molecular dynamics simulations revealed that dimer formation is highly unlikely following mutation of the above amino acids, as the Fe(2+) is no longer attracted with equal strength by both subunits. These findings probably indicate that iron plays an important role in the conformation of HP-NAP by initiating the formation of stable dimers that are indispensable for the ensuing dodecamer structure. Very surprisingly, neutrophil activation appeared to be stimulated by structural elements that are localized within the C-terminal region of both mutant HP-NAP and wild-type dodecamer HP-NAP. In particular, the dodecamer conformation does not seem to be necessary for activation, and helices H3 (Leu69-Leu75) and H4 (Lys89-Leu114) or the linking coils (His63-Thr68 and Thr76-Ser88) are probably critical in stimulating neutrophil activation.