qkat-quantitative-semi-automated-typing-killer-cell-immunoglobulin

qKAT: Quantitative Semi-automated Typing of Killer-cell Immunoglobulin-like Receptor Genes

Killer cell immunoglobulin-like receptors (KIRs) are a set of inhibitory and activating immune receptors, on natural killer (NK) and T cells, encoded by a ...

University of Cambridge

Royal Papworth Hospital

The chaperonin GroEL is a large complex composed of 14 identical 57-kDa subunits that requires ATP and GroES for some of its activities. We find that a monomeric polypeptide corresponding to residues 191 to 345 has the activity of the tetradecamer both in facilitating the refolding of rhodanese and cyclophilin A in the absence of ATP and in catalyzing the unfolding of native barnase. Its crystal structure, solved at 2.5 A resolution, shows a well-ordered domain with the same fold as in intact GroEL. We have thus isolated the active site of the complex allosteric molecular chaperone, which functions as a "minichaperone." This has mechanistic implications: the presence of a central cavity in the GroEL complex is not essential for those representative activities in vitro, and neither are the allosteric properties. The function of the allosteric behavior on the binding of GroES and ATP must be to regulate the affinity of the protein for its various substrates in vivo, where the cavity may also be required for special functions.

Chaperone activity and structure of monomeric polypeptide binding domains of GroEL.

1. We have characterized the human smooth muscle endothelin converting enzyme (ECE) present in the media of the endothelium-denuded human umbilical vein preparation. 2. Endothelin-1 (ET-1) and ET-2 were potent constrictors of umbilical vein with EC50 values of 9.2 nM and 29.6 nM, respectively. ET-1 was at least 30 times more potent than ET-3 suggesting the presence of constrictor ETA receptors. Little or no response was obtained to the ETB-selective agonist sarafotoxin 6c. These data suggest that endothelin-mediated vasoconstriction is via ETA receptors in this preparation. 3. Autoradiographical visualization of endothelin receptors with subtype selective ligands confirmed the predominance of the ETA receptor in the media of umbilical vein. High density of binding was obtained with the ETA selective [125I]-PD151242, with much lower levels detected with the ETB selective [125I]-BQ3020. 4. Big ET-1 (EC50 = 42.7 nM) and big ET-2(1-38) (EC50 = 99.0 nM) were less potent than ET-1 and ET-2, respectively. Big ET-2(1-38) was more potent than its isoform big ET-2(1-37) with concentration-response curves to big ET-2(1-37) incomplete at 300 nM. No response was obtained to big ET-3 at concentrations up to 700 nM. The C-terminal fragments, big ET-1(22-38) and big ET-2(22-38) were inactive. 5. Responses to ET-1 were unaffected by either the neutral endopeptidase (NEP) inhibitor thiorphan (10(-5) M) or by the dual NEP/ECE inhibitor phosphoramidon (10(-5) M). Big ET-1 was also unaffected by thiorphan but antagonized in a concentration-dependent manner by phosphoramidon (10(-5) M and 10(-4) M). 6. Addition of all four big endothelin peptides to human umbilical vein preparations resulted in detectable amounts of ET-IR in the bathing medium. Therefore, although big ET-3 was functionally inactive this reflects the low potency of ET-3 at the ETA receptor rather than the lack of ability of this smooth muscle ECE to convert big ET-3 to ET-3. 7. To conclude we have demonstrated the presence of a phosphoramidon-sensitive ECE on the smooth muscle layer of the human umbilical vein which can convert big ET-1, big ET-2(1-37), big ET-2(1-38) and big ET-3 to their mature biologically active forms. The precise subcellular localization of this enzyme and its physiological relevance remains to be determined.

Endothelin converting enzyme (ECE) activity in human vascular smooth muscle.

Methylation of CpG dinucleotides is the major epigenetic modification of mammalian genomes, critical for regulating chromatin structure and gene activity. The mixed-lineage leukaemia (MLL) CXXC domain selectively binds nonmethyl-CpG DNA, and is required for transformation by MLL fusion proteins that commonly arise from recurrent chromosomal translocations in infant and secondary treatment-related acute leukaemias. To elucidate the molecular basis of nonmethyl-CpG DNA recognition, we determined the structure of the human MLL CXXC domain by multidimensional NMR spectroscopy. The CXXC domain has a novel fold in which two zinc ions are each coordinated tetrahedrally by four conserved cysteine ligands provided by two CGXCXXC motifs and two distal cysteine residues. We have identified the CXXC domain DNA binding interface by means of chemical shift perturbation analysis, cross-saturation transfer and site-directed mutagenesis. In particular, we have shown that residues in an extended surface loop are in close contact with the DNA. These data provide a template for the design of specifically targeted therapeutics for poor prognosis MLL-associated leukaemias.

Solution structure of the nonmethyl-CpG-binding CXXC domain of the leukaemia-associated MLL histone methyltransferase.

We report results of a nonsynonymous SNP scan for ulcerative colitis and identify a previously unknown susceptibility locus at ECM1. We also show that several risk loci are common to ulcerative colitis and Crohn's disease (IL23R, IL12B, HLA, NKX2-3 and MST1), whereas autophagy genes ATG16L1 and IRGM, along with NOD2 (also known as CARD15), are specific for Crohn's disease. These data provide the first detailed illustration of the genetic relationship between these common inflammatory bowel diseases.

Genetic determinants of ulcerative colitis include the ECM1 locus and five loci implicated in Crohn's disease.

Mutations affecting NPM1 (nucleophosmin) are the most common genetic lesions found in acute myeloid leukemia (AML). NPM1 is one of the most abundant proteins found in the nucleolus and has links to the MDM2/p53 tumor suppressor pathway. A distinctive feature of NPM1 mutants in AML is their aberrant localization to the cytoplasm of leukemic cells. This mutant phenotype is the result of the substitution of several C-terminal residues, including one or two conserved tryptophan residues, with a leucine-rich nuclear export signal. The exact molecular mechanism underlying the loss of nucleolar retention, and the role of the tryptophans, remains unknown. In this study we have determined the structure of an independently folded globular domain in the C terminus of NPM1 using NMR spectroscopy, and we report that the conserved tryptophans are critical for structure. This domain is necessary for the nucleolar targeting of NPM1 and is disrupted by mutations in AML with cytoplasmic NPM1. Furthermore, we identify conserved surface-exposed lysine residues that are functionally rather than structurally important for nucleolar localization. This study provides new focus for efforts to understand the pathogenesis of AML with cytoplasmic NPM1 and may be used to aid the design of small molecules that target the C-terminal domain of NPM1 to act as novel anti-proliferative and anti-leukemia therapeutics.

Structural consequences of nucleophosmin mutations in acute myeloid leukemia.

Presenilin-mediated endoproteolysis of transmembrane proteins plays a key role in physiological signaling and in the pathogenesis of Alzheimer disease and some cancers. Numerous inhibitors have been found via library screens, but their structural mechanisms remain unknown. We used several biophysical techniques to investigate the structure of human presenilin complexes and the effects of peptidomimetic γ-secretase inhibitors. The complexes are bilobed. The head contains nicastrin ectodomain. The membrane-embedded base has a central channel and a lateral cleft, which may represent the initial substrate docking site. Inhibitor binding induces widespread structural changes, including rotation of the head and closure of the lateral cleft. These changes block substrate access to the catalytic pocket and inhibit the enzyme. Intriguingly, peptide substrate docking has reciprocal effects on the inhibitor binding site. Similar reciprocal shifts may underlie the mechanisms of other inhibitors and of the "lateral gate" through which substrates access to the catalytic site.

Structural interactions between inhibitor and substrate docking sites give insight into mechanisms of human PS1 complexes.

SLX4 binds to three nucleases (XPF-ERCC1, MUS81-EME1, and SLX1), and its deficiency leads to genomic instability, sensitivity to DNA crosslinking agents, and Fanconi anemia. However, it is not understood how SLX4 and its associated nucleases act in DNA crosslink repair. Here, we uncover consequences of mouse Slx4 deficiency and reveal its function in DNA crosslink repair. Slx4-deficient mice develop epithelial cancers and have a contracted hematopoietic stem cell pool. The N-terminal domain of SLX4 (mini-SLX4) that only binds to XPF-ERCC1 is sufficient to confer resistance to DNA crosslinking agents. Recombinant mini-SLX4 enhances XPF-ERCC1 nuclease activity up to 100-fold, directing specificity toward DNA forks. Mini-SLX4-XPF-ERCC1 also vigorously stimulates dual incisions around a DNA crosslink embedded in a synthetic replication fork, an essential step in the repair of this lesion. These observations define vertebrate SLX4 as a tumor suppressor, which activates XPF-ERCC1 nuclease specificity in DNA crosslink repair. 

Mouse SLX4 is a tumor suppressor that stimulates the activity of the nuclease XPF-ERCC1 in DNA crosslink repair.

Identifying the site of vascular bleeding in fatal cases of traumatic basal subarachnoid hemorrhage (TBSAH) is important, but can be very difficult to achieve when there is extensive blood clot in the posterior cranial fossa. Post mortem angiography in these circumstances has been reported previously but with mixed results, and is rarely used in current practice within the United Kingdom. We have developed a simple and effective post mortem angiography method, using fluoroscopy and clear modern contrast medium, suitable for use in the autopsy room. Contrast medium was injected through an angiographic catheter positioned (with water filled balloon) in the extracranial vertebral artery (ECVA) just behind the clavicle, whilst the base of the skull and upper cervical spine area was visualized by fluoroscopy following digital subtraction of the bony images. The procedure was developed over a series of 8 TBSAH cases and using unfixed cadaveric specimens. A clear leak point was identified in 3 cases and the likely site indicated in a further fatality (all with tears subsequently confirmed by histology). In 4 cases, the bleeding point was not identified by angiography. In 7 deaths, the anatomy of the upper cervical loops and intracranial course of the vertebral arteries were well delineated by angiography and several small loop segments were documented. In 1 case, with severe degenerative vascular disease, extracranial vessel wall trauma and luminal thrombosis, angiography was unsuccessful above the level of the first cervical (C1) vertebra. Injecting contrast through the internal carotid artery, just above the bifurcation, was also shown to visualize the anterior cerebral circulation well. This method is likely to identify the main site of vascular tearing in about 50% of cases of TBSAH and to delineate the important anatomy of the vessels, prior to dissection of the upper neck and posterior fossa, thus minimizing handling artefacts and guiding the histological sampling of the vessels.

A simple post mortem room angiography method for the investigation of traumatic basal subarachnoid hemorrhage.

Biodiversity is essential to human well-being, but people have been reducing biodiversity throughout human history. Loss of species and degradation of ecosystems are likely to further accelerate in the coming years. Our understanding of this crisis is now clear, and world leaders have pledged to avert it. Nonetheless, global goals to reduce the rate of biodiversity loss have mostly not been achieved. However, many examples of conservation success show that losses can be halted and even reversed. Building on these lessons to turn the tide of biodiversity loss will require bold and innovative action to transform historical relationships between human populations and nature.

Biodiversity losses and conservation responses in the Anthropocene.

Ciliopathies are a group of genetic disorders caused by a failure to form functional cilia. Due to a lack of structural information, it is currently poorly understood how ciliopathic mutations affect protein functionality to give rise to the underlying disease. Using X-ray crystallography, we show that the ciliopathy-associated centriolar protein CEP120 contains three C2 domains. The point mutations V194A and A199P, which cause Joubert syndrome (JS) and Jeune asphyxiating thoracic dystrophy (JATD), respectively, both reduce the thermostability of the second C2 domain by targeting residues that point toward its hydrophobic core. Genome-engineered cells homozygous for these mutations have largely normal centriole numbers but show reduced CEP120 levels, compromised recruitment of distal centriole markers, and deficient cilia formation. Our results provide insight into the disease mechanism of two ciliopathic mutations in CEP120, identify putative binding partners of CEP120 C2B, and suggest a complex genotype-phenotype relation of the CEP120 ciliopathy alleles.

Disease-Associated Mutations in CEP120 Destabilize the Protein and Impair Ciliogenesis.

British Thoracic Society Guideline for bronchiectasis in adults.

p53 is frequently mutated in human cancers. Its levels are tightly regulated by the E3 ubiquitin ligase MDM2. The complex between MDM2 and p53 is largely formed by the interaction between the N-terminal domain of MDM2 and the N-terminal transactivation (TA) domain of p53 (residues 15-29). We investigated the kinetic and thermodynamic basis of the MDM2/p53 interaction by using wild-type and mutant variants of the TA domain. We focus on the effects of phosphorylation at positions Thr18 and Ser20 including their substitution with phosphomimetics. Conformational propensities of the isolated peptides were investigated using in silico methods and experimentally by circular dichroism and H-NMR in aqueous solution. Both experimental and computational analyses indicate that the p53 peptides are mainly disordered in aqueous solution, with evidence of nascent helix around the Ser20-Leu25 region. Both phosphorylation and the phosphomimetics at Thr18 result in a decrease in the binding affinity by ten- to twenty-fold when compared to the wild-type. Phosphorylation and phosphomimetics at Ser20 result in a smaller decrease in the affinity. Mutation of Lys24 and Leu25 also disrupts the interaction. Our results may be useful for further development of peptide-based drugs targeting the MDM2/p53 interaction.

Kinetic and thermodynamic effects of phosphorylation on p53 binding to MDM2.

The full British Thoracic Society Guideline for Bronchiectasis in Adults is published in . The following is a summary of the recommendations and good practice points. The sections referred to in the summary refer to the full guideline. The appendices are available in the full guideline.

Article	Year
Chaperone activity and structure of monomeric polypeptide binding domains of GroEL. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 8986757	1996
Endothelin converting enzyme (ECE) activity in human vascular smooth muscle. British journal of pharmacology\| PubMed ID: 9422810	1997
Solution structure of the nonmethyl-CpG-binding CXXC domain of the leukaemia-associated MLL histone methyltransferase. The EMBO journal\| PubMed ID: 16990798	2006
Genetic determinants of ulcerative colitis include the ECM1 locus and five loci implicated in Crohn's disease. Nature genetics\| PubMed ID: 18438406	2008
Structural consequences of nucleophosmin mutations in acute myeloid leukemia. The Journal of biological chemistry\| PubMed ID: 18511415	2008
Structural interactions between inhibitor and substrate docking sites give insight into mechanisms of human PS1 complexes. Structure (London, England : 1993)\| PubMed ID: 24210759	2014
Mouse SLX4 is a tumor suppressor that stimulates the activity of the nuclease XPF-ERCC1 in DNA crosslink repair. Molecular cell\| PubMed ID: 24726326	2014
A simple post mortem room angiography method for the investigation of traumatic basal subarachnoid hemorrhage. Forensic science, medicine, and pathology\| PubMed ID: 28093693	2017
Biodiversity losses and conservation responses in the Anthropocene. Science (New York, N.Y.)\| PubMed ID: 28428393	2017
Disease-Associated Mutations in CEP120 Destabilize the Protein and Impair Ciliogenesis. Cell reports\| PubMed ID: 29847808	2018
British Thoracic Society Guideline for bronchiectasis in adults. Thorax\| PubMed ID: 30545985	2019
Kinetic and thermodynamic effects of phosphorylation on p53 binding to MDM2. Scientific reports\| PubMed ID: 30679555	2019
British Thoracic Society guideline for bronchiectasis in adults. BMJ open respiratory research\| PubMed ID: 30687502	2018

Christopher Johnson

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