Novel mutant P277 peptide VP to ameliorate atherogenic side-effects and to preserve anti-diabetic effects in NOD mice.

P277 is a 24 amino-acids peptide, residues 437-460 of human heat shock protein 60 (HSP60). P277 or sequence repeated 6 × P277 was previously found showing potency preventive and therapeutic anti-diabetes functions in NOD mice, but aroused atherosclerosis due to the induction of anti-HSP65 autoantibodies as reported. To determine the intrinsic B epitope sequence, we screened P277 with pepscan method and then proved by detection of sera IgG from peptide fragments vaccinated mouse and rabbits. Results indicated HSP60 443-448 (ALLRCI) is potential intrinsic B epitope sequence of P277. We modified P277 by deleting the former three amino acids of ALLRCI (VP) or replacing these six with alanine (AP). The detection of serum lipid parameter in NOD mice and aorta endothelial damage levels in high-cholesterol diets fed rabbits demonstrated that VP induced higher anti-diabetes efficacy and caused less arteriosclerosis-liked diseases separately. With less TLR2/4 activation of dendritic cells and macrophages, VP treatment reduced Th1 related P277 specific pro-inflammatory cytokines production and increased regulatory immune responses both in vivo and in vitro. These results indicated that optimized VP peptide might serve as a promising candidate for mouse type 1 diabetes therapy.

Aligator: A computational tool for optimizing total chemical synthesis of large proteins.

The scope of chemical protein synthesis (CPS) continues to expand, driven primarily by advances in chemical ligation tools (e.g., reversible solubilizing groups and novel ligation chemistries). However, the design of an optimal synthesis route can be an arduous and fickle task due to the large number of theoretically possible, and in many cases problematic, synthetic strategies. In this perspective, we highlight recent CPS tool advances and then introduce a new and easy-to-use program, Aligator (Automated Ligator), for analyzing and designing the most efficient strategies for constructing large targets using CPS. As a model set, we selected the E. coli ribosomal proteins and associated factors for computational analysis. Aligator systematically scores and ranks all feasible synthetic strategies for a particular CPS target. The Aligator script methodically evaluates potential peptide segments for a target using a scoring function that includes solubility, ligation site quality, segment lengths, and number of ligations to provide a ranked list of potential synthetic strategies. We demonstrate the utility of Aligator by analyzing three recent CPS projects from our lab: TNFα (157 aa), GroES (97 aa), and DapA (312 aa). As the limits of CPS are extended, we expect that computational tools will play an increasingly important role in the efficient execution of ambitious CPS projects such as production of a mirror-image ribosome.

Access to the inaccessible sequence of cpn 60.1 (195-217) by temporary oxazolidine protection of selected amide bonds.

The solid-phase peptide synthesis of a reportedly inaccessible peptide sequence of chaperonin 60.1 (195-219) is described using oxazolidine containing dipeptide building blocks ('pseudo-proline' dipeptide units). Two attempts at the synthesis of the chaperonin 60.1 sequence are outlined using one and two pseudo-proline units, respectively, and these results are compared with the outcome of an ordinary stepwise (double) coupling procedure. The only successful synthesis is that combining two pseudo-proline building blocks.

Tetradecameric chaperonin 60 can be assembled in vitro from monomers in a process that is ATP independent.

The present work shows that monomers of cpn60 (groEL) formed at 2.5 M urea could be assembled to tetradecamers in a process that was independent of ATP. Reassembled cpn60 was able to assist the folding of urea unfolded rhodanese. When cpn60 was incubated at urea concentrations higher than 2.75 M, assembly of tetradecameric cpn60 did not occur after dialysis, and the presence of ATP did not stimulate the assembly process. The cpn60 used here did not display the previously reported ATP-dependent self-assembly of cpn60 monomers that required a higher urea concentration (4 M) for formation (Lissen et al. (1990) Nature 348, 339-342). Assembly and disassembly of cpn60 tetradecamers were followed as a function of the urea concentration by ultracentrifugation and gel electrophoresis in the presence of urea. The electrophoresis results demonstrate that there is rapid assembly of tetradecamers following preincubation and rapid removal of urea at concentrations lower than 2.5 M. Thus, previous methods monitored irreversible dissociation of cpn60, and the present results indicate that the cpn60 assembly requirements for ATP are dependent on pretreatment conditions.