Refined structures of mouse P-glycoprotein.

The recently determined C. elegans P-glycoprotein (Pgp) structure revealed significant deviations compared to the original mouse Pgp structure, which suggested possible misinterpretations in the latter model. To address this concern, we generated an experimental electron density map from single-wavelength anomalous dispersion phasing of an original mouse Pgp dataset to 3.8 Šresolution. The map exhibited significantly more detail compared to the original MAD map and revealed several regions of the structure that required de novo model building. The improved drug-free structure was refined to 3.8 Šresolution with a 9.4 and 8.1% decrease in R(work) and R(free), respectively, (R(work) = 21.2%, R(free) = 26.6%) and a significant improvement in protein geometry. The improved mouse Pgp model contains ∼95% of residues in the favorable Ramachandran region compared to only 57% for the original model. The registry of six transmembrane helices was corrected, revealing amino acid residues involved in drug binding that were previously unrecognized. Registry shifts (rotations and translations) for three transmembrane (TM)4 and TM5 and the addition of three N-terminal residues were necessary, and were validated with new mercury labeling and anomalous Fourier density. The corrected position of TM4, which forms the frame of a portal for drug entry, had backbone atoms shifted >6 Šfrom their original positions. The drug translocation pathway of mouse Pgp is 96% identical to human Pgp and is enriched in aromatic residues that likely play a collective role in allowing a high degree of polyspecific substrate recognition.

Enhanced rat islet function and survival in vitro using a biomimetic self-assembled nanomatrix gel.

Peptide amphiphile (PA) is a peptide-based biomaterial that can self-assemble into a nanostructured gel-like scaffold, mimicking the chemical and biological complexity of natural extracellular matrix. To evaluate the capacity of the PA scaffold to improve islet function and survival in vitro, rat islets were cultured in three different groups--(1) bare group: isolated rat islets cultured in a 12-well nontissue culture-treated plate; (2) insert group: isolated rat islets cultured in modified insert chambers; (3) nanomatrix group: isolated rat islets encapsulated within the PA nanomatrix gel and cultured in modified insert chambers. Over 14 days, both the bare and insert groups showed a marked decrease in insulin secretion, whereas the nanomatrix group maintained glucose-stimulated insulin secretion. Moreover, entire islets in the nanomatrix gel stained positive for dithizone up to 14 days, indicating better maintained glucose-stimulated insulin production. Fluorescein diacetate/propidium iodide staining results also verified necrosis in the bare and insert groups after 7 days, whereas the PA nanomatrix gel maintained islet viability after 14 days. Thus, these results demonstrate the potential of PAs as an intermediary scaffold for increasing the efficacy of pancreatic islet transplantation.

AMP-activated protein kinase attenuates nitric oxide-induced beta-cell death.

During the initial autoimmune response in type 1 diabetes, islets are exposed to a damaging mix of pro-inflammatory molecules that stimulate the production of nitric oxide by beta-cells. Nitric oxide causes extensive but reversible cellular damage. In response to nitric oxide, the cell activates pathways for functional recovery and adaptation as well as pathways that direct beta-cell death. The molecular events that dictate cellular fate following nitric oxide-induced damage are currently unknown. In this study, we provide evidence that AMPK plays a primary role controlling the response of beta-cells to nitric oxide-induced damage. AMPK is transiently activated by nitric oxide in insulinoma cells and rat islets following IL-1 treatment or by the exogenous addition of nitric oxide. Active AMPK promotes the functional recovery of beta-cell oxidative metabolism and abrogates the induction of pathways that mediate cell death such as caspase-3 activation following exposure to nitric oxide. Overall, these data show that nitric oxide activates AMPK and that active AMPK suppresses apoptotic signaling allowing the beta-cell to recover from nitric oxide-mediated cellular stress.

CD4+ T cells target epitopes residing within the RNA-binding domain of the U1-70-kDa small nuclear ribonucleoprotein autoantigen and have restricted TCR diversity in an HLA-DR4-transgenic murine model of mixed connective tissue disease.

Mixed connective tissue disease (MCTD) is a systemic autoimmune disease with significant morbidity and premature mortality of unknown pathogenesis. In the present study, we characterized U1-70-kDa small nuclear ribonucleoprotein (70-kDa) autoantigen-specific T cells in a new murine model of MCTD. These studies defined 70-kDa-reactive T cell Ag fine specificities and TCR gene usage in this model. Similar to patients with MCTD, CD4(+) T cells can be readily identified from 70-kDa/U1-RNA-immunized HLA-DR4-transgenic mice. Using both freshly isolated CD4(+) T cells from spleen and lung, and T cell lines, we found that the majority of these T cells were directed against antigenic peptides residing within the RNA-binding domain of 70 kDa. We also found that TCR-beta (TRB) V usage was highly restricted among 70-kDa-reactive T cells, which selectively used TRBV subgroups 1, 2, 6, 8.1, 8.2, and 8.3, and that the TRB CDR3 had conserved sequence motifs which were shared across different TRBV subgroups. Finally, we found that the TRBV and CDR3 regions used by both murine and human 70-kDa-specific CD4(+) T cells were homologous. Thus, T cell recognition of the 70-kDa autoantigen by HLA-DR4-transgenic mice is focused on a limited number of T cell epitopes residing primarily within the RBD of the molecule, using a restricted number of TRBV and CDR3 motifs that are homologous to T cells isolated from MCTD patients.

Differential tissue targeting of autoimmunity manifestations by autoantigen-associated Y RNAs.

OBJECTIVE: To assess the Y RNAs, a family of homologous RNAs that bind to the Ro autoantigen, for the ability to contribute to autoimmune disease by activating RNA-responsive Toll-like receptors (TLRs). METHODS: Using cell lines expressing or stably transfected with TLR-3, TLR-7, or TLR-8, we determined the patterns of RNA-specific TLR activation by in vitro transcripts of all of the known murine and human Y RNAs. Next, 8-10-week-old female mice were exposed to a single 50-microg subcutaneous injection of mouse Y1 or mouse Y3 RNA, and the effects were observed. RESULTS: Y RNA family members differed in their TLR reactivities. Both human and mouse Y3 RNAs, but not other human or mouse Y RNAs, prominently induced TLR-3 activation. Although most human and mouse Y RNAs activated TLR-7 efficiently, mouse Y3 RNA and human Y5 RNA did not. Single subcutaneous injections of mice with either mouse Y1 RNA or mouse Y3 RNA induced or inhibited lymphoid infiltrates in different target organs based on the Y RNA and TLR status of the mouse used. Mouse Y1 RNA induced kidney lesions in TLR-3-intact mice but not in TLR-3-knockout mice. In contrast, mouse Y3 RNA treatment was associated with nephritis in TLR-3-knockout mice but not in TLR-3-intact mice. Sialoadenitis developed in untreated TLR-3-/- mice and in TLR-3-/- mice treated with mouse Y3 RNA, but sialoadenitis was not present in TLR--/-) mice treated with mouse Y1 RNA. CONCLUSION: Y RNAs can induce innate immune responses and influence clinical manifestations of autoimmunity, suggesting that they are relevant to syndromes of anti-Ro autoimmunity. Distinct patterns of tissue targeting can be seen after exposure to different Y RNAs, in a pattern that correlates with the innate immune signals they induce. Thus, the balance of innate immune signals induced by exposure to endogenous Y RNAs may help determine the nature of the clinical syndrome in anti-Ro autoimmunity.

A murine model of mixed connective tissue disease induced with U1 small nuclear RNP autoantigen.

OBJECTIVE: To test whether immunizing mice with autoantigens closely linked to mixed connective tissue disease (MCTD) could induce an MCTD-like clinical syndrome distinguishable from systemic lupus erythematosus (SLE). METHODS: Transgenic and knockout C57BL/6-derived mice were immunized subcutaneously at age 8-12 weeks with U1-70-kd small nuclear RNP (70K) fusion protein along with either Freund's complete adjuvant (CFA) or U1 RNA. After 2 months, mice were killed and analyzed histologically and serologically. RESULTS: Immunization of C57BL/6-derived mice transgenic for human HLA-DR4 with 70K and either CFA or U1 RNA led to anti-70K antibodies in 62% of mice (21 of 34), and diversified anti-RNP immune responses. MCTD-like lung disease also developed in 50% of immunized mice (17 of 34), and anti-70K antibodies were strongly correlated with lung disease. CFA and U1 RNA were comparably able to induce this syndrome. Mice deficient in Toll-like receptor 3 (TLR-3) also developed this same syndrome when immunized with 70K and CFA. However, TLR-3(-/-) mice failed to develop MCTD-like lung disease when treated with 70K and U1 RNA. Rather, TLR-3(-/-) mice immunized with 70K and U1 RNA developed an autoimmune syndrome characterized by glomerulonephritis typical of SLE. CONCLUSION: Exposure to 70K in an appropriate context is sufficient to induce autoimmunity and target organ injury consistent with MCTD. This system represents a new model of autoimmune interstitial lung disease, and establishes a closer link between anti-70K immunity and MCTD-like lung disease. Of note, changes in innate immune signaling can cause the same trigger to lead to the development of SLE-like nephritis rather than MCTD-like lung disease.

Human T cell clones specific for heterogeneous nuclear ribonucleoprotein A2 autoantigen from connective tissue disease patients assist in autoantibody production.

OBJECTIVE: To identify and characterize human T cells reactive with heterogeneous nuclear RNP A2 (hnRNP A2) antigen, and to determine the ability of hnRNP-reactive T cells to assist in the production of human autoantibodies. METHODS: T cells from patients with high serum levels of anti-hnRNP IgG autoantibody were stimulated with an hnRNP recombinant fusion protein, and the cells were cloned by limiting dilution. The surface phenotype and cytokine profiles of the T cells were examined by flow cytometry and enzyme-linked immunosorbent assay (ELISA), respectively. T cell clones were cultured with highly purified autologous B cells, and the ability of T cells to enhance autoantibody production under a variety of conditions was measured by ELISA. RESULTS: Human T cells reactive with hnRNP antigen were cloned from 2 patients with systemic lupus erythematosus (SLE) and 1 patient with mixed connective tissue disease (MCTD). The T cells were CD4+ and had a Th1-like functional phenotype. In coculture in vitro with autologous B cells, T cell clones augmented anti-hnRNP autoantibody production and did so without the need for direct T cell-B cell contact. CONCLUSION: This study provides direct evidence for a role of anti-hnRNP-reactive T cells in autoantibody production in SLE and MCTD. These findings support the notion that hnRNP-reactive T cells play a role in the pathogenesis of these diseases.