HLA-DM (DM) catalyzes CLIP release, stabilizes MHC class II molecules and edits the peptide repertoire presented by class II. Impaired DM function may have profound effects on antigen presentation events in the thymus and periphery that are critical for maintenance of self-tolerance. The associations of the HLA-DQ2 (DQ2) allele with celiac disease and type 1 diabetes mellitus have been appreciated for a long time. The explanation for these associations, however, remains unknown. We previously found that DQ2 is a poor substrate for DM. Here, to further characterize DQ2-DM interaction, we introduced point mutations into DQ2 on the proposed DQ2/DM interface in order to restore the sensitivity of DQ2 to DM. The effects of mutations were investigated by measuring the peptide dissociation and exchange rate in vitro, CLIP and DQ2 expression on the cell surface and the presentation of α-II-gliadin epitope (residues 62-70) to murine, DQ2-restricted T cell hybridomas. We found that the three α chain mutations (α+53G, α+53R or αY22F) decreased the intrinsic stability of peptide-class II complex. More interestingly, the α+53G mutant restored DQ2 sensitivity to DM, likely due to improved interaction with DM. Our data also suggest that α-II-gliadin 62-70 is a DM-suppressed epitope. The DQ2 resistance to DM changes the fate of this peptide from a cryptic to an immunodominant epitope. Our findings elucidate the structural basis for reduced DQ2-DM interaction and have implications for mechanisms underlying disease associations of DQ2.

IntroductionFollicular lymphoma (FL) is a slowly progressive and largely incurable human B-cell malignancy. Transformation to a more aggressive lymphoma, such as diffuse large B-cell lymphoma, is common and strongly associated with an increase in morbidity and mortality. A chromosomal translocation t(14:18) is the hallmark of this disease, and it is found in 85%-90% of cases. It results in the juxtaposition of the BCL2 proto-oncogene with the immunoglobulin (Ig) heavy chain gene, IGH, leading to deregulated overexpression of Bcl-2 protein, a major inhibitor of apoptosis. However, the t(14:18) translocation is insufficient to cause malignancy as it is detectable in rare B cells from healthy persons. 1-3 Thus, FL pathogenesis requires additional signals beyond that imparted by the deregulation of BCL2. The observation that FL cells isolated from patients fail to survive in vitro and undergo spontaneous apoptosis supports the hypothesis that extrinsic microenvironmental factors are required for maintenance and expansion of FL. 4 Phenotypically, FL tumor cells resemble antigen-experienced germinal center B cells. Their Ig genes, which are rearranged to produce a functional B-cell receptor (BCR), have numerous point mutations compared with their germline counterparts, and this process of somatic hypermutation (SHM) is ongoing as the malignant clone expands and diversifies. Thus, individual tumor cells can each have slightly different Ig variable region sequences. 5 Random mutations should eventually result in stop codons and loss of BCR protein expression. However, FL tumors maintain a surface BCR, indicating a selective force favoring retention of a functional BCR. Furthermore, therapy with anti-idiotype antibodies directed against the BCR did not select for the outgrowth of BCR-negative variants. Rather, this therapy selected for the outgrowth of cells that had amino acid substitutions in the targeted V region sequence, making them unrecognizable by the anti-idiotype antibody. 6 Other in vitro studies with malignant B-cell lines have shown that experimental knockdowns of the BCR and members of its signaling pathway result in growth arrest, implicating their importance in tumor cell survival. 7 The BCR can transmit a tonic survival signal, but this is greatly augmented on its binding to a cognate antigen. 8 There is indirect evidence to suggest that antigen recognition plays a role in the pathogenesis of FL. SHM can introduce silent or replacement mutations, the latter leading to an amino acid substitution. In a normal immune response, B cells with mutations resulting in higher binding affinity for the inciting antigen preferentially survive. This selective pressure leads to enrichment of replacement mutations in the complementarity determining regions (CDRs) of the BCR, and an under representation of replacement mutations in the framework regions (FWRs). 9 This same distribution of replacement and silent mutations has been reported for the BCRs of FL cells, 5 and the intraclonal diversity resulting from ongoing SHM...

The β 2 adrenergic receptor (β 2 AR) signals through both G s and G i in cardiac myocytes, and the G i pathway counteracts the G s pathway. However, G i coupling is much less efficient than G s coupling in most cell-based and biochemical assays, making it difficult to study β 2 AR−G i interactions. Here we investigate the role of phospholipid composition on G s and G i coupling. While negatively charged phospholipids are known to enhance agonist affinity and stabilize an active state of the β 2 AR, we find that they impair coupling to G i3 and facilitate coupling to G s . Positively charged Ca 2+ and Mg 2+ , known to interact with the negative charge on phospholipids, facilitates G i3 coupling. Mutational analysis suggests that Ca 2+ coordinates an interaction between phospholipid and the negatively charged EDGE motif on the amino terminal helix of G i3 . Taken together, our observations suggest that local membrane charge modulates the interaction between β 2 AR and competing G protein subtypes.

The aim for deterministic control of the interactions between macroions in aqueous media has motivated widespread experimental and theoretical work. Although it has been well established that like-charged macromolecules can aggregate under the influence of oppositely charged condensing agents, the specific conditions for the stability of such aggregates can only be determined empirically. We examine these conditions, which involve an interplay of electrostatic and osmotic effects, by using a well defined model system composed of F-actin, an anionic rod-like polyelectrolyte, and lysozyme, a cationic globular protein with a charge that can be genetically modified. The structure and stability of actin-lysozyme complexes for different lysozyme charge mutants and salt concentrations are examined by using synchrotron x-ray scattering and molecular dynamics simulations. We provide evidence that supports a structural transition from columnar arrangements of F-actin held together by arrays of lysozyme at the threefold interstitial sites of the actin sublattice to marginally stable complexes in which lysozyme resides at twofold bridging sites between actin. The reduced stability arises from strongly reduced partitioning of salt between the complex and the surrounding solution. Changes in the stability of actin-lysozyme complexes are of biomedical interest because their formation has been reported to contribute to the persistence of airway infections in cystic fibrosis by sequestering antimicrobials such as lysozyme. We present x-ray microscopy results that argue for the existence of actin-lysozyme complexes in cystic fibrosis sputum and demonstrate that, for a wide range of salt conditions, charge-reduced lysozyme is not sequestered in ordered complexes while retaining its bacterial killing activity.antimicrobial ͉ cystic fibrosis ͉ self-assembly ͉ small-angle x-ray scattering ͉ x-ray microscopy

The peptide-exchange catalyst, HLA-DM, and its inhibitor, HLA-DO control endosomal generation of peptide/class II major histocompatibility protein (MHC-II) complexes; these complexes traffic to the cell surface for inspection by CD4+ T cells. Some evidence suggests that pH influences DO regulation of DM function, but pH also affects the stability of polymorphic MHC-II proteins, spontaneous peptide loading, DM/MHC-II interactions and DM catalytic activity, imposing challenges on approaches to determine pH effects on DM-DO function and their mechanistic basis. Using optimized biochemical methods, we dissected pH-dependence of spontaneous and DM-DO-mediated class II peptide exchange and identified an MHC-II allele-independent relationship between pH, DO/DM ratio and efficient peptide exchange. We demonstrate that active, free DM is generated from DM-DO complexes at late endosomal/lysosomal pH due to irreversible, acid-promoted DO destruction rather than DO/DM molecular dissociation. Any soluble DM that remains in complex with DO stays inert. pH-exposure of DM-DO in cell lysates corroborates such a pH-regulated mechanism, suggesting acid-activated generation of functional DM in DO-expressing cells.