Proteasomal Degradation of Proinsulin Requires Derlin-2, HRD1 and p97.

Patients with type 1 diabetes (T1D) suffer from beta-cell destruction by CD8+ T-cells that have preproinsulin as an important target autoantigen. It is of great importance to understand the molecular mechanism underlying the processing of preproinsulin into these CD8+ T-cell epitopes. We therefore studied a pathway that may contribute to the production of these antigenic peptides: degradation of proinsulin via ER associated protein degradation (ERAD). Analysis of the MHC class I peptide ligandome confirmed the presentation of the most relevant MHC class I-restricted diabetogenic epitopes in our cells: the signal peptide-derived sequence A15-A25 and the insulin B-chain epitopes H29-A38 and H34-V42. We demonstrate that specific silencing of Derlin-2, p97 and HRD1 by shRNAs increases steady state levels of proinsulin. This indicates that these ERAD constituents are critically involved in proinsulin degradation and may therefore also play a role in subsequent antigen generation. These ERAD proteins therefore represent interesting targets for novel therapies aiming at the reduction and possibly also prevention of beta-cell directed auto-immune reactions in T1D.

Anti-tumor activity of human IgG1 anti-gp75 TA99 mAb against B16F10 melanoma in human FcgammaRI transgenic mice.

Patients suffering from advanced melanoma have a very poor prognosis. Despite recent advances in the understanding of oncogenic mechanisms and therapeutic interventions, the median survival of patients with metastatic disease is less than 12 months. Immunotherapy of melanoma has been intensely investigated and holds great promises. Tyrosinase-related protein-1 or gp75 (TYRP-1/gp75) antigen is a melanosomal polypeptide. It is the most abundant glycoprotein synthesized by pigmented melanocytes and melanomas. It is specific for melanocytes and both primary and metastatic melanomas. In mice, administration of the mouse mAb anti-gp75 TA99 prevents outgrowth of B16F10 melanoma metastases. The activity of TA99 is dependent on the presence and activity of the IgG specific, Fc receptors. TA99 cross-reacts with human gp75, and is currently being used for diagnosis of patients. Here, we sequenced mIgG2a TA99 and found that the locus harboring the endogenous light chain of the fusion partner in the TA99 hybridoma cells is not inactivated, resulting in the production of a mixed pool of mAbs that mitigates binding to gp75. Since human IgG1 (hIgG1) is the most frequently used mAb format in clinical studies, we produced a recombinant hIgG1 TA99 molecule. Whereas it is known that hIgG1 can functionally interact with mouse Fc receptors, we found that hIgG1 TA99 did not exhibit in vivo activity against B16F10 melanoma in wild type C57BL/6 mice. However, results obtained in this study demonstrated anti-tumor activity of hIgG1 TA99 in FcγRIIB knockout mice and in human FcγRI transgenic mice. These results emphasize the need for testing hIgG mAb in mice with functional human FcγRs.

Swarming of Pseudomonas aeruginosa is controlled by a broad spectrum of transcriptional regulators, including MetR.

Pseudomonas aeruginosa exhibits swarming motility on semisolid surfaces (0.5 to 0.7% agar). Swarming is a more than just a form of locomotion and represents a complex adaptation resulting in changes in virulence gene expression and antibiotic resistance. In this study, we used a comprehensive P. aeruginosa PA14 transposon mutant library to investigate how the complex swarming adaptation process is regulated. A total of 233 P. aeruginosa PA14 transposon mutants were verified to have alterations in swarming motility. The swarming-associated genes functioned not only in flagellar or type IV pilus biosynthesis but also in processes as diverse as transport, secretion, and metabolism. Thirty-three swarming-deficient and two hyperswarming mutants had transposon insertions in transcriptional regulator genes, including genes encoding two-component sensors and response regulators; 27 of these insertions were newly identified. Of the 25 regulatory mutants whose swarming motility was highly impaired (79 to 97%), only 1 (a PA1458 mutant) had a major defect in swimming, suggesting that this regulator might influence flagellar synthesis or function. Twitching motility, which requires type IV pili, was strongly affected in only two regulatory mutants (pilH and PA2571 mutants) and was moderately affected in three other mutants (algR, ntrB, and nosR mutants). Microarray analyses were performed to compare the gene expression profile of a swarming-deficient PA3587 mutant to that of the wild-type PA14 strain under swarming conditions. PA3587 showed 63% homology to metR, which encodes a regulator of methionine biosynthesis in Escherichia coli. The observed dysregulation in the metR mutant of nine different genes required for swarming motility provided a possible explanation for the swarming-deficient phenotype of this mutant.

Human host defense peptide LL-37 prevents bacterial biofilm formation.

The ability to form biofilms is a critical factor in chronic infections by Pseudomonas aeruginosa and has made this bacterium a model organism with respect to biofilm formation. This study describes a new, previously unrecognized role for the human cationic host defense peptide LL-37. In addition to its key role in modulating the innate immune response and weak antimicrobial activity, LL-37 potently inhibited the formation of bacterial biofilms in vitro. This occurred at the very low and physiologically meaningful concentration of 0.5 microg/ml, far below that required to kill or inhibit growth (MIC = 64 microg/ml). LL-37 also affected existing, pregrown P. aeruginosa biofilms. Similar results were obtained using the bovine neutrophil peptide indolicidin, but no inhibitory effect on biofilm formation was detected using subinhibitory concentrations of the mouse peptide CRAMP, which shares 67% identity with LL-37, polymyxin B, or the bovine bactenecin homolog Bac2A. Using microarrays and follow-up studies, we were able to demonstrate that LL-37 affected biofilm formation by decreasing the attachment of bacterial cells, stimulating twitching motility, and influencing two major quorum sensing systems (Las and Rhl), leading to the downregulation of genes essential for biofilm development.