Novel and Engineered Type II CRISPR Systems from Uncultivated Microbes with Broad Genome Editing Capability.

Type II Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 nucleases have been extensively used in biotechnology and therapeutics. However, many applications are not possible owing to the size, targetability, and potential off-target effects associated with currently known systems. In this study, we identified thousands of CRISPR type II effectors by mining an extensive, genome-resolved metagenomics database encompassing hundreds of thousands of microbial genomes.

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

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).

Proteasomal degradation of NOD2 by NLRP12 in monocytes promotes bacterial tolerance and colonization by enteropathogens.

Mutations in the nucleotide-binding oligomerization domain protein 12 (NLRP12) cause recurrent episodes of serosal inflammation. Here we show that NLRP12 efficiently sequesters HSP90 and promotes K48-linked ubiquitination and degradation of NOD2 in response to bacterial muramyl dipeptide (MDP). This interaction is mediated by the linker-region proximal to the nucleotide-binding domain of NLRP12.

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.

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.

Preferential interaction of MHC class I with TAPBPR in the absence of glycosylation.

We recently discovered that TAPBPR promotes reglucosylation of the N-linked glycan on MHC class I molecules, a modification that restores their recognition by calreticulin and reincorporation into the peptide-loading complex. We wondered whether TAPBPR displayed some degree of glycan specificity, as is known to be the case for tapasin via its interaction with calreticulin & ERp57, or whether its interaction with MHC class I was glycan independent. Here, we explored this by comparing the ability of TAPBPR to bind to MHC class I containing either an intact or disrupted NxS/T glycosylation consensus sequence.

Properties of the tapasin homologue TAPBPR.

The presentation of antigenic peptides by MHC class I molecules plays a vital role in generating T cell responses against infection and cancer. Over the last two decades the central role of tapasin as a peptide editor that influences the loading and optimisation of peptides onto MHC class I molecules has been extensively characterised. Recently, it has become evident that the tapasin-related protein, TAPBPR, functions as a second peptide editor which influences the peptides displayed by MHC class I molecules.

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

Recently, we revealed that TAPBPR is a peptide exchange catalyst that is important for optimal peptide selection by MHC class I molecules. Here, we asked whether any other co-factors associate with TAPBPR, which would explain its effect on peptide selection. We identify an interaction between TAPBPR and UDP-glucose:glycoprotein glucosyltransferase 1 (UGT1), a folding sensor in the calnexin/calreticulin quality control cycle that is known to regenerate the GlcManGlcNAc moiety on glycoproteins.

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.

NLRC5 interacts with RIG-I to induce a robust antiviral response against influenza virus infection.

The NLR protein, NLRC5 is an important regulator of MHC class I gene expression, however, the role of NLRC5 in other innate immune responses is less well defined. In the present study, we report that NLRC5 binds RIG-I and that this interaction is critical for robust antiviral responses against influenza virus. Overexpression of NLRC5 in the human lung epithelial cell line, A549, and normal human bronchial epithelial cells resulted in impaired replication of influenza virus A/Puerto Rico/8/34 virus (PR8) and enhanced IFN-β expression.

The N-terminal domain of NLRC5 confers transcriptional activity for MHC class I and II gene expression.

Ag presentation to CD4(+) and CD8(+) T cells depends on MHC class II and MHC class I molecules, respectively. One important regulatory factor of this process is the transcriptional regulation of MHC gene expression. It is well established that MHC class II transcription relies on the NLR protein CIITA.

NLRC5, at the Heart of Antigen Presentation.

Nucleotide-binding domain and leucine-rich repeat containing receptors (NLRs) are intracellular proteins mainly involved in pathogen recognition, inflammatory responses, and cell death. Until recently, the function of the family member NLR caspase recruitment domain (CARD) containing 5 (NLRC5) has been a matter of debate. It is now clear that NLRC5 acts as a transcriptional regulator of the major-histocompatibility complex class I.

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.

NLRC5 controls basal MHC class I gene expression in an MHC enhanceosome-dependent manner.

Nucleotide-binding domain and leucine-rich repeat (NLR) proteins play important roles in innate immune responses as pattern-recognition receptors. Although most NLR proteins act in cell autonomous immune pathways, some do not function as classical pattern-recognition receptors. One such NLR protein is the MHC class II transactivator, the master regulator of MHC class II gene transcription.

A role for the human nucleotide-binding domain, leucine-rich repeat-containing family member NLRC5 in antiviral responses.

Proteins of the nucleotide-binding domain, leucine-rich repeat (NLR)-containing family recently gained attention as important components of the innate immune system. Although over 20 of these proteins are present in humans, only a few members including the cytosolic pattern recognition receptors NOD1, NOD2, and NLRP3 have been analyzed extensively. These NLRs were shown to be pivotal for mounting innate immune response toward microbial invasion.