Characterization of the human NLR protein NLRC5

Nucleotide-binding domain, leucine-rich repeat (NLR)-containing proteins play important roles in the innate immune system as intracellular pattern recognition receptors. The most prominent members, NOD1, NOD2 and NLRP3 have been extensively shown to trigger NF-kB activation or IL-1b/IL-18 processing upon pathogen infection, respectively. Nonetheless, other functions beyond pathogen recognition have also been reported for some NLR proteins. Here we report the first characterization of the human NLR protein NLRC5. NLRC5 lacks the typical N-terminal CARD or PYRIN domain of most NLR proteins, but harbours a death do-main fold effector domain with yet unknown function. Interestingly, NACHT and LRR domain alignments reveal close homology to the MHC class II transcriptional activator (CIITA), which is responsible for the transcriptional induction of MHC class II molecules, and moderate homology to NOD1 and NOD2. In the first part of this study, we addressed the expression and regulation of NLRC5 in different tissues and cell lines. We detected NLRC5 expression primarily in cells and tissues of the immune system, including CD4+ and CD8+ T cells and spleen, lymph node and bone marrow. Furthermore, we were able to induce a TLR3-dependent NLRC5 induction upon stimula-tion with the dsRNA-mimic poly(I:C) as well as an TLR3-independent NLRC5 induction using a Sendai Virus (SeV)-based infection model. In line with that, we revealed a role for NLRC5 in type I interferon (IFN) response against RNA viruses. Moreover, we adapted an infection model of primary human dermal fibroblasts (hFibr) with Sendai Virus (SeV), depicting a distinct role for NLRC5 in anti-viral immune processes. In the second part, we investigated the role of NLRC5 in MHC class I promoter activa-tion. Similar to MHC class II promoter activation by the non-DNA binding coactivator CIITA, we were able to obtain a clear role for NLRC5 in MHC class I expression and identified the domains, which are important for nuclear translocation and MHC class I promoter activation. We further analysed the involvement of a DNA-binding complex, the so-called enhanceosome, in NLRC5-dependent MHC class I expression, which is pivotal for CIITA-dependent MHC class II expression. Finally, we generated NLRC5-CIITA-chimeric proteins to decipher the NLRC5-dependent MHC class I and CIITA-dependent class II activation in more detail. Domain swapping of the N-terminal effector domains revealed, that the NLRC5 N-terminal effector domain fused to the C-terminus of CIITA is sufficient to activate both MHC class I and MHC class II expression. Taken together, in this study we identified a role for NLRC5 in anti-viral immune responses and further contributed to the understanding of NLRC5-mediated MHC class I expression

TAPBPR mediates peptide dissociation from MHC class I using a leucine lever.

Tapasin and TAPBPR are known to perform peptide editing on major histocompatibility complex class I (MHC I) molecules; however, the precise molecular mechanism(s) involved in this process remain largely enigmatic. Here, using immunopeptidomics in combination with novel cell-based assays that assess TAPBPR-mediated peptide exchange, we reveal a critical role for the K22-D35 loop of TAPBPR in mediating peptide exchange on MHC I. We identify a specific leucine within this loop that enables TAPBPR to facilitate peptide dissociation from MHC I. Moreover, we delineate the molecular features of the MHC I F pocket required for TAPBPR to promote peptide dissociation in a loop-dependent manner. These data reveal that chaperone-mediated peptide editing on MHC I can occur by different mechanisms dependent on the C-terminal residue that the MHC I accommodates in its F pocket and provide novel insights that may inform the therapeutic potential of TAPBPR manipulation to increase tumour immunogenicity.Wellcome PhD studentship (109076/Z/15/A). Wellcome Senior Research Fellowship (104647/Z/14/Z) South African Medical Research Council Bosch-Forschungsstiftung. Royal Society University Research Fellowship (UF100371)

TAPBPR bridges UDP-glucose : glycoprotein glucosyltransferase 1 onto MHC class I to provide quality control in the antigen presentation pathway

Funding Wellcome: Senior Research Fellowship 104647, Andreas Neerincx, Louise H Boyle Royal Society: University Research Fellowship, UF100371, Janet E Deane Cancer Research UK: Programme Grant, C7056A, Andy van Hateren, Tim Elliott Deutsche Forschungsgemeinschaft: SFB 685, Nico Trautwein, Stefan Stevanović Wellcome: PhD studentship, 089563, Clemens Hermann Wellcome: Strategic Award 100140, Robin Antrobus Wellcome: Programme grant, WT094847MA, Huan Cao Acknowledgements We are extremely grateful to Peter Cresswell and Najla Arshad (Yale University School of Medicine, New Haven, CT) for valuable advice, tapasin and TAP-specific antibody reagents, and the recombinant calreticulin proteins. We thank John Trowsdale (University of Cambridge, UK) for his mentorship and critical reading of this manuscript, and Jim Kaufman (University of Cambridge, UK) for useful discussions. We also thank Yi Cao (Cranfield University, UK) for MATLAB programming for densitometry analysis, and Mark Vickers and Sadie Henderson (Scottish National Blood Transfusion Services, UK) for permitting the use of and assistance with the Amersham WB system. The reagent ARP7099 FEC peptide pool was obtained from the Centre for AIDS Reagents, National Institute for Biological Standards and Control (NIBSC), and was donated by the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH.Peer reviewedPublisher PD

Utilizing TAPBPR to promote exogenous peptide loading onto cell surface MHC I molecules.

The repertoire of peptides displayed at the cell surface by MHC I molecules is shaped by two intracellular peptide editors, tapasin and TAPBPR. While cell-free assays have proven extremely useful in identifying the function of both of these proteins, here we explored whether a more physiological system could be developed to assess TAPBPR-mediated peptide editing on MHC I. We reveal that membrane-associated TAPBPR targeted to the plasma membrane retains its ability to function as a peptide editor and efficiently catalyzes peptide exchange on surface-expressed MHC I molecules. Additionally, we show that soluble TAPBPR, consisting of the luminal domain alone, added to intact cells, also functions as an effective peptide editor on surface MHC I molecules. Thus, we have established two systems in which TAPBPR-mediated peptide exchange on MHC class I can be interrogated. Furthermore, we could use both plasma membrane-targeted and exogenous soluble TAPBPR to display immunogenic peptides on surface MHC I molecules and consequently induce T cell receptor engagement, IFN-γ secretion, and T cell-mediated killing of target cells. Thus, we have developed an efficient way to by-pass the natural antigen presentation pathway of cells and load immunogenic peptides of choice onto cells. Our findings highlight a potential therapeutic use for TAPBPR in increasing the immunogenicity of tumors in the future

TAPBPR alters MHC class I peptide presentation by functioning as a peptide exchange catalyst.

Our understanding of the antigen presentation pathway has recently been enhanced with the identification that the tapasin-related protein TAPBPR is a second major histocompatibility complex (MHC) class I-specific chaperone. We sought to determine whether, like tapasin, TAPBPR can also influence MHC class I peptide selection by functioning as a peptide exchange catalyst. We show that TAPBPR can catalyse the dissociation of peptides from peptide-MHC I complexes, enhance the loading of peptide-receptive MHC I molecules, and discriminate between peptides based on affinity in vitro. In cells, the depletion of TAPBPR increased the diversity of peptides presented on MHC I molecules, suggesting that TAPBPR is involved in restricting peptide presentation. Our results suggest TAPBPR binds to MHC I in a peptide-receptive state and, like tapasin, works to enhance peptide optimisation. It is now clear there are two MHC class I specific peptide editors, tapasin and TAPBPR, intimately involved in controlling peptide presentation to the immune system.This is the final version of the article. It first appeared from eLife via http://dx.doi.org/10.755

Sialic acid-binding immunoglobulin-like lectin (Sigelac)-15 is a rapidly internalised cell-surface antigen expressed by acute myeloid leukaemia cells.

Funder: )Sialic acid-binding immunoglobulin-like lectin (Siglec)-15 has recently been identified as a critical tumour checkpoint, augmenting the expression and function of programmed death-ligand 1. We raised a monoclonal antibody, A9E8, specific for Siglec-15 using phage display. A9E8 stained myeloid leukaemia cell lines and peripheral cluster of differentiation (CD)33+ blasts and CD34+ leukaemia stem cells from patients with acute myeloid leukaemia (AML). By contrast, there was minimal expression on healthy donor leucocytes or CD34+ stem cells from non-AML donors, suggesting targeting Siglec-15 may have significant therapeutic advantages over its fellow Siglec CD33. After binding, A9E8 was rapidly internalised (half-life of 180 s) into K562 cells. Antibodies to Siglec-15 therefore hold therapeutic potential for AML treatment

TRIM27 Negatively Regulates NOD2 by Ubiquitination and Proteasomal Degradation

NOD2, the nucleotide-binding domain and leucine-rich repeat containing gene family (NLR) member 2 is involved in mediating antimicrobial responses. Dysfunctional NOD2 activity can lead to severe inflammatory disorders, but the regulation of NOD2 is still poorly understood. Recently, proteins of the tripartite motif (TRIM) protein family have emerged as regulators of innate immune responses by acting as E3 ubiquitin ligases. We identified TRIM27 as a new specific binding partner for NOD2. We show that NOD2 physically interacts with TRIM27 via the nucleotide-binding domain, and that NOD2 activation enhances this interaction. Dependent on functional TRIM27, ectopically expressed NOD2 is ubiquitinated with K48-linked ubiquitin chains followed by proteasomal degradation. Accordingly, TRIM27 affects NOD2-mediated pro-inflammatory responses. NOD2 mutations are linked to susceptibility to Crohns disease. We found that TRIM27 expression is increased in Crohns disease patients, underscoring a physiological role of TRIM27 in regulating NOD2 signaling. In HeLa cells, TRIM27 is partially localized in the nucleus. We revealed that ectopically expressed NOD2 can shuttle to the nucleus in a Walker A dependent manner, suggesting that NOD2 and TRIM27 might functionally cooperate in the nucleus. We conclude that TRIM27 negatively regulates NOD2-mediated signaling by degradation of NOD2 and suggest that TRIM27 could be a new target for therapeutic intervention in NOD2-associated diseases.Funding Agencies|German Research Foundation (DFG)|SFB670-NG01|Swedish Society of Medicine||Regional Research Council of South-East Sweden (FORSS)||Swedish Research Council division of Medicine||Gustav V 90th anniversary foundation||Italian Telethon Foundation||DFG|SE 1122/2-1|</p

Solve-RD: systematic pan-European data sharing and collaborative analysis to solve rare diseases.

For the first time in Europe hundreds of rare disease (RD) experts team up to actively share and jointly analyse existing patient's data. Solve-RD is a Horizon 2020-supported EU flagship project bringing together >300 clinicians, scientists, and patient representatives of 51 sites from 15 countries. Solve-RD is built upon a core group of four European Reference Networks (ERNs; ERN-ITHACA, ERN-RND, ERN-Euro NMD, ERN-GENTURIS) which annually see more than 270,000 RD patients with respective pathologies. The main ambition is to solve unsolved rare diseases for which a molecular cause is not yet known. This is achieved through an innovative clinical research environment that introduces novel ways to organise expertise and data. Two major approaches are being pursued (i) massive data re-analysis of >19,000 unsolved rare disease patients and (ii) novel combined -omics approaches. The minimum requirement to be eligible for the analysis activities is an inconclusive exome that can be shared with controlled access. The first preliminary data re-analysis has already diagnosed 255 cases form 8393 exomes/genome datasets. This unprecedented degree of collaboration focused on sharing of data and expertise shall identify many new disease genes and enable diagnosis of many so far undiagnosed patients from all over Europe

Twist exome capture allows for lower average sequence coverage in clinical exome sequencing

Background Exome and genome sequencing are the predominant techniques in the diagnosis and research of genetic disorders. Sufficient, uniform and reproducible/consistent sequence coverage is a main determinant for the sensitivity to detect single-nucleotide (SNVs) and copy number variants (CNVs). Here we compared the ability to obtain comprehensive exome coverage for recent exome capture kits and genome sequencing techniques. Results We compared three different widely used enrichment kits (Agilent SureSelect Human All Exon V5, Agilent SureSelect Human All Exon V7 and Twist Bioscience) as well as short-read and long-read WGS. We show that the Twist exome capture significantly improves complete coverage and coverage uniformity across coding regions compared to other exome capture kits. Twist performance is comparable to that of both short- and long-read whole genome sequencing. Additionally, we show that even at a reduced average coverage of 70× there is only minimal loss in sensitivity for SNV and CNV detection. Conclusion We conclude that exome sequencing with Twist represents a significant improvement and could be performed at lower sequence coverage compared to other exome capture techniques

Solving patients with rare diseases through programmatic reanalysis of genome-phenome data.

Funder: EC | EC Seventh Framework Programm | FP7 Health (FP7-HEALTH - Specific Programme "Cooperation": Health); doi: https://doi.org/10.13039/100011272; Grant(s): 305444, 305444Funder: Ministerio de Economía y Competitividad (Ministry of Economy and Competitiveness); doi: https://doi.org/10.13039/501100003329Funder: Generalitat de Catalunya (Government of Catalonia); doi: https://doi.org/10.13039/501100002809Funder: EC | European Regional Development Fund (Europski Fond za Regionalni Razvoj); doi: https://doi.org/10.13039/501100008530Funder: Instituto Nacional de Bioinformática ELIXIR Implementation Studies Centro de Excelencia Severo OchoaFunder: EC | EC Seventh Framework Programm | FP7 Health (FP7-HEALTH - Specific Programme "Cooperation": Health)Reanalysis of inconclusive exome/genome sequencing data increases the diagnosis yield of patients with rare diseases. However, the cost and efforts required for reanalysis prevent its routine implementation in research and clinical environments. The Solve-RD project aims to reveal the molecular causes underlying undiagnosed rare diseases. One of the goals is to implement innovative approaches to reanalyse the exomes and genomes from thousands of well-studied undiagnosed cases. The raw genomic data is submitted to Solve-RD through the RD-Connect Genome-Phenome Analysis Platform (GPAP) together with standardised phenotypic and pedigree data. We have developed a programmatic workflow to reanalyse genome-phenome data. It uses the RD-Connect GPAP's Application Programming Interface (API) and relies on the big-data technologies upon which the system is built. We have applied the workflow to prioritise rare known pathogenic variants from 4411 undiagnosed cases. The queries returned an average of 1.45 variants per case, which first were evaluated in bulk by a panel of disease experts and afterwards specifically by the submitter of each case. A total of 120 index cases (21.2% of prioritised cases, 2.7% of all exome/genome-negative samples) have already been solved, with others being under investigation. The implementation of solutions as the one described here provide the technical framework to enable periodic case-level data re-evaluation in clinical settings, as recommended by the American College of Medical Genetics