In vivo clonal expansion and phenotypes of hypocretin-specific CD4(+) T cells in narcolepsy patients and controls

Individuals with narcolepsy suffer from abnormal sleep patterns due to loss of neurons that uniquely supply hypocretin (HCRT). Previous studies found associations of narcolepsy with the human leukocyte antigen (HLA)-DQ6 allele and T-cell receptor alpha (TRA) J24 gene segment and also suggested that in vitro-stimulated T cells can target HCRT. Here, we present evidence of in vivo expansion of DQ6-HCRT tetramer(+)/TRAJ24(+)/CD4(+) T cells in DQ6(+) individuals with and without narcolepsy. We identify related TRAJ24(+) TCRalphabeta clonotypes encoded by identical alpha/beta gene regions from two patients and two controls. TRAJ24-G allele(+) clonotypes only expand in the two patients, whereas a TRAJ24-C allele(+) clonotype expands in a control. A representative tetramer(+)/G-allele(+) TCR shows signaling reactivity to the epitope HCRT87-97. Clonally expanded G-allele(+) T cells exhibit an unconventional effector phenotype. Our analysis of in vivo expansion of HCRT-reactive TRAJ24(+) cells opens an avenue for further investigation of the autoimmune contribution to narcolepsy development

Inactivating mutations and X-ray crystal structure of the tumor suppressor OPCML reveal cancer-associated functions

OPCML, a tumor suppressor gene, is frequently silenced epigenetically in ovarian and other cancers. Here we report, by analysis of databases of tumor sequences, the observation of OPCML somatic missense mutations from various tumor types and the impact of these mutations on OPCML function, by solving the X-ray crystal structure of this glycoprotein to 2.65 A resolution. OPCML consists of an extended arrangement of three immunoglobulin-like domains and homodimerizes via a network of contacts between membrane-distal domains. We report the generation of a panel of OPCML variants with representative clinical mutations and demonstrate clear phenotypic effects in vitro and in vivo including changes to anchorage-independent growth, interaction with activated cognate receptor tyrosine kinases, cellular migration, invasion in vitro and tumor growth in vivo. Our results suggest that clinically occurring somatic missense mutations in OPCML have the potential to contribute to tumorigenesis in a variety of cancers

SepA Enhances Shigella Invasion of Epithelial Cells by Degrading Alpha-1 Antitrypsin and Producing a Neutrophil Chemoattractant

Shigella spp. are highly adapted pathogens that cause bacillary dysentery in human and nonhuman primates. An unusual feature of Shigella pathogenesis is that this organism invades the colonic epithelia from the basolateral pole. Therefore, it has evolved the ability to disrupt the intestinal epithelial barrier to reach the basolateral surface. We have shown previously that the secreted serine protease A (SepA), which belongs to the family of serine protease autotransporters of Enterobacteriaceae, is responsible for the initial destabilization of the intestinal epithelial barrier that facilitates Shigella invasion. However, the mechanisms used by SepA to regulate this process remain unknown. To investigate the protein targets cleaved by SepA in the intestinal epithelium, we incubated a sample of homogenized human colon with purified SepA or with a catalytically inactive mutant of this protease. We discovered that SepA targets an array of 18 different proteins, including alpha-1 antitrypsin (AAT), a major circulating serine proteinase inhibitor in humans. In contrast to other serine proteases, SepA cleaved AAT without forming an inhibiting complex, which resulted in the generation of a neutrophil chemoattractant. We demonstrated that the products of the AAT-SepA reaction induce a mild but significant increase in neutrophil transepithelial migration in vitro. Moreover, the presence of AAT during Shigella infection stimulated neutrophil migration and dramatically enhanced the number of bacteria invading the intestinal epithelium in a SepA-dependent manner. We conclude that by cleaving AAT, SepA releases a chemoattractant that promotes neutrophil migration, which in turn disrupts the intestinal epithelial barrier to enable Shigella invasion. IMPORTANCE Shigella is the second leading cause of diarrheal death globally. In this study, we identified the host protein targets of SepA, Shigella\u27s major protein secreted in culture. We demonstrated that by cleaving AAT, a serine protease inhibitor important to protect surrounding tissue at inflammatory sites, SepA releases a neutrophil chemoattractant that enhances Shigella invasion. Moreover, SepA degraded AAT without becoming inhibited by the cleaved product, and SepA catalytic activity was enhanced at higher concentrations of AAT. Activation of SepA by an excess of AAT may be physiologically relevant at the early stages of Shigella infection, when the amount of synthesized SepA is very low compared to the concentration of AAT in the intestinal lumen. This observation may also help to explain the adeptness of Shigella infectivity at low dose, despite the requirement of reaching the basolateral side to invade and colonize the colonic epithelium

Structural insights into the transcriptional and translational roles of Ebp1.

Accepted versio

Lessons from mouse chimaera experiments with a reiterated transgene marker:revised marker criteria and a review of chimaera markers

Recent reports of a new generation of ubiquitous transgenic chimaera markers prompted us to consider the criteria used to evaluate new chimaera markers and develop more objective assessment methods. To investigate this experimentally we used several series of fetal and adult chimaeras, carrying an older, multi-copy transgenic marker. We used two additional independent markers and objective, quantitative criteria for cell selection and cell mixing to investigate quantitative and spatial aspects of developmental neutrality. We also suggest how the quantitative analysis we used could be simplified for future use with other markers. As a result, we recommend a five-step procedure for investigators to evaluate new chimaera markers based partly on criteria proposed previously but with a greater emphasis on examining the developmental neutrality of prospective new markers. These five steps comprise (1) review of published information, (2) evaluation of marker detection, (3) genetic crosses to check for effects on viability and growth, (4) comparisons of chimaeras with and without the marker and (5) analysis of chimaeras with both cell populations labelled. Finally, we review a number of different chimaera markers and evaluate them using the extended set of criteria. These comparisons indicate that, although the new generation of ubiquitous fluorescent markers are the best of those currently available and fulfil most of the criteria required of a chimaera marker, further work is required to determine whether they are developmentally neutral. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s11248-015-9883-7) contains supplementary material, which is available to authorized users

Structural and Mutagenic Analysis of Foot-and-Mouth Disease Virus 3C Protease Reveals the Role of the β-Ribbon in Proteolysis

The 3C protease (3C(pro)) from foot-and-mouth disease virus (FMDV), the causative agent of a widespread and economically devastating disease of domestic livestock, is a potential target for antiviral drug design. We have determined the structure of a new crystal form of FMDV 3C(pro), a chymotrypsin-like cysteine protease, which reveals features that are important for catalytic activity. In particular, we show that a surface loop which was disordered in previous structures adopts a β-ribbon structure that is conformationally similar to equivalent regions on other picornaviral 3C proteases and some serine proteases. This β-ribbon folds over the peptide binding cleft and clearly contributes to substrate recognition. Replacement of Cys142 at the tip of the β-ribbon with different amino acids has a significant impact on enzyme activity and shows that higher activity is obtained with more hydrophobic side chains. Comparison of the structure of FMDV 3C(pro) with homologous enzyme-peptide complexes suggests that this correlation arises because the side chain of Cys142 contacts the hydrophobic portions of the P2 and P4 residues in the peptide substrate. Collectively, these findings provide compelling evidence for the role of the β-ribbon in catalytic activity and provide valuable insights for the design of FMDV 3C(pro) inhibitors

The Crystal Structure of Human Endoplasmic Reticulum Aminopeptidase 2 Reveals the Atomic Basis for Distinct Roles in Antigen Processing

Endoplasmic reticulum aminopeptidases ERAP1 and ERAP2 cooperate to trim a vast variety of antigenic peptide precursors to generate mature epitopes for binding to major histocompatibility class I molecules. We report here the first structure of ERAP2 determined at 3.08 Å by X-ray crystallography. On the basis of residual electron density, a lysine residue has been modeled in the active site of the enzyme; thus, the structure corresponds to an enzyme–product complex. The overall domain organization is highly similar to that of the recently determined structure of ERAP1 in its closed conformation. A large internal cavity adjacent to the catalytic site can accommodate large peptide substrates. The ERAP2 structure provides a structural explanation for the different peptide N-terminal specificities between ERAP1 and ERAP2 and suggests that such differences extend throughout the whole peptide sequence. A noncrystallographic dimer observed may constitute a model for a proposed ERAP1–ERAP2 heterodimer. Overall, the structure helps explain how two homologous aminopeptidases cooperate to process a large variety of sequences, a key property of their biological role

The Crystal Structure of Human Endoplasmic Reticulum Aminopeptidase 2 Reveals the Atomic Basis for Distinct Roles in Antigen Processing

Endoplasmic reticulum aminopeptidases ERAP1 and ERAP2 cooperate to trim a vast variety of antigenic peptide precursors to generate mature epitopes for binding to major histocompatibility class I molecules. We report here the first structure of ERAP2 determined at 3.08 Å by X-ray crystallography. On the basis of residual electron density, a lysine residue has been modeled in the active site of the enzyme; thus, the structure corresponds to an enzyme–product complex. The overall domain organization is highly similar to that of the recently determined structure of ERAP1 in its closed conformation. A large internal cavity adjacent to the catalytic site can accommodate large peptide substrates. The ERAP2 structure provides a structural explanation for the different peptide N-terminal specificities between ERAP1 and ERAP2 and suggests that such differences extend throughout the whole peptide sequence. A noncrystallographic dimer observed may constitute a model for a proposed ERAP1–ERAP2 heterodimer. Overall, the structure helps explain how two homologous aminopeptidases cooperate to process a large variety of sequences, a key property of their biological role