The rockefeller university

Inteins are autoprocessing domains that cut themselves out of host proteins in a traceless manner. This process, known as protein splicing, involves multiple chemical steps that must be coordinated to ensure fidelity in the process. The committed step in splicing involves attack of a conserved Asn side-chain amide on the adjacent backbone amide, leading to an intein-succinimide product and scission of that peptide bond. This cleavage reaction is stimulated by formation of a branched intermediate in the splicing process. The mechanism by which the Asn side-chain becomes activated as a nucleophile is not understood. Here we solve the crystal structure of an intein trapped in the branched intermediate step in protein splicing. Guided by this structure, we use protein-engineering approaches to show that intein-succinimide formation is critically dependent on a backbone-to-side-chain hydrogen-bond. We propose that this interaction serves to both position the side-chain amide for attack and to activate its nitrogen as a nucleophile. Collectively, these data provide an unprecedented view of an intein poised to carry out the rate-limiting step in protein splicing, shedding light on how a nominally nonnucleophilic group, a primary amide, can become activated in a protein active site.

CRISPR-Cas encodes an adaptive immune system that defends prokaryotes against infectious viruses and plasmids. Immunity is mediated by Cas nucleases, which use small RNA guides (the crRNAs) to specify a cleavage site within the genome of invading nucleic acids. In type II CRISPR-Cas systems, the DNA-cleaving activity is performed by a single enzyme Cas9 guided by an RNA duplex. Using synthetic single RNA guides, Cas9 can be reprogrammed to create specific double-stranded DNA breaks in the genomes of a variety of organisms, ranging from human cells to bacteria, and thus constitutes a powerful tool for genetic engineering. Here we describe recent advancements in our understanding of type II CRISPR-Cas immunity and how these studies led to revolutionary genome editing applications.

During the Mobile Genetic Elements and Genome Evolution Keystone Symposium in March 2014, the Editors of Mobile DNA caught up with a panel of conference speakers to select key advances in the field, and hear their thoughts on where mobile DNA research is going.

Substance P (SP) is a neuropeptide that mediates numerous physiological responses, including transmission of pain and inflammation through the neurokinin-1 (NK1) receptor, a G protein-coupled receptor. Previous mutagenesis studies and photoaffinity labeling using ligand analogues suggested that the binding site for SP includes multiple domains in the N-terminal (Nt) segment and the second extracellular loop (ECLII) of NK1. To map precisely the NK1 residues that interact with SP, we applied a novel receptor-based targeted photocross-linking approach. We used amber codon suppression to introduce the photoreactive unnatural amino acid p-benzoyl-L-phenylalanine (BzF) at 11 selected individual positions in the Nt tail (residues 11-21) and 23 positions in the ECLII (residues 170(C-10)-193(C+13)) of NK1. The 34 NK1 variants were expressed in mammalian HEK293 cells and retained the ability to interact with a fluorescently labeled SP analog. Notably, 10 of the receptor variants with BzF in the Nt tail and 4 of those with BzF in ECLII cross-linked efficiently to SP, indicating that these 14 sites are juxtaposed to SP in the ligand-bound receptor. These results show that two distinct regions of the NK1 receptor possess multiple determinants for SP binding and demonstrate the utility of genetically encoded photocross-linking to map complex multitopic binding sites on G protein-coupled receptors in a cell-based assay format.

Measurements are presented of the t-channel single-top-quark production cross section in proton-proton collisions at root s = 8 TeV. The results are based on a data sample corresponding to an integrated luminosity of 19.7 fb(-1) recorded with the CMS detector at the LHC. The cross section is measured inclusively, as well as separately for top (t) and antitop (t) over bar, in final states with a muon or an electron. The measured inclusive t-channel cross section is sigma(t-ch.) = 83.6 +/- 2.3 (stat.) +/- 7.4 (syst.) pb. The single t and (t) cross sections are measured to be sigma(t-ch.)(t) = 53.8 +/- 1.5 (stat.) +/- 4.4 (syst.) pb and sigma(t-ch.)(t) over bar = 27.6 +/- 1.3 (stat.) +/- 3.7 (syst.) pb, respectively. The measured ratio of cross sections is Rt-ch. = sigma(t-ch.)(t)/sigma(t-ch.)(t) over bar = 1.95 +/- 0.10 (stat.) +/- 0.19 (syst.), in agreement with the standard model prediction. The modulus of the Cabibbo-Kobayashi-Maskawa matrix element V-tb is extracted and, in combination with a previous CMS result at root s = 7 TeV, a value vertical bar V-tb vertical bar = 0.998 +/- 0.038 (exp.) +/- 0.016 (theo.) is obtained.

The production of a Z boson, decaying into two leptons and produced in association with one or more b jets, is studied using proton-proton collisions delivered by the LHC at a centre-of-mass energy of 7 TeV. The data were recorded in 2011 with the CMS detector and correspond to an integrated luminosity of 5 fb(-1). The Z(ll) + b-jets cross sections (where ll = mu mu or ee) are measured separately for a Z boson produced with exactly one b jet and with at least two b jets. In addition, a cross section ratio is extracted for a Z boson produced with at least one b jet, relative to a Z boson produced with at least one jet. The measured cross sections are compared to various theoretical predictions, and the data favour the predictions in the five-flavour scheme, where b quarks are assumed massless. The kinematic properties of the reconstructed particles are compared with the predictions from the MadGraph event generator using the pythia parton shower simulation.

Soil microbiomes are a rich source of uncharacterized natural product biosynthetic gene clusters. Here we use short conserved biosynthetic gene sequences (natural product sequence tags) amplified from soil microbiomes as phylogenetic markers to correlate genotype to chemotype and target the discovery of novel bioactive pentangular polyphenols from the environment. The heterologous expression of an environmental DNA-derived gene cluster (the ARX cluster), whose ketosynthase beta (KS beta) sequence tag was phylogenetically distinct from any known KS beta sequence, led to the discovery of the arixanthomycins. Arixanthomycin A (1) exhibits potent antiproliferative activity against human cancer cell lines.

mRNA export factor 1 (Rae1) and nucleoporin 98 (Nup98) are host cell targets for the matrix (M) protein of vesicular stomatitis virus (VSV). How Rae1 functions in mRNA export and how M protein targets both Rae1 and Nup98 are not understood at the molecular level. To obtain structural insights, we assembled a 1: 1: 1 complex of M center dot Rae1 center dot Nup98 and established a crystal structure at 3.15-angstrom resolution. We found that the M protein contacts the Rae1_ Nup98 heterodimer principally by two protrusions projecting from the globular domain of M like a finger and thumb. Both projections clamp to the side of the beta-propeller of Rae1, with the finger also contacting Nup98. The most prominent feature of the finger is highly conserved Methionine 51 (Met51) with upstream and downstream acidic residues. The complementary surface on Rae1 displays a deep hydrophobic pocket, into which Met51 fastens like a bolt, and a groove of basic residues on either side, which bond to the acidic residues of the finger. Notably, the M protein competed for in vitro binding of various oligonucleotides to Rae1 center dot Nup98. We localized this competing activity of M to its finger using a synthetic peptide. Collectively, our data suggest that Rae1 serves as a binding protein for the phosphate backbone of any nucleic acid and that the finger of M mimics this ligand. In the context of mRNA export, we propose that a given mRNA segment, after having been deproteinated by helicase, is transiently reproteinated by Nup98-tethered Rae1. We suggest that such repetitive cycles provide cytoplasmic stopover sites required for ratcheting mRNA across the nuclear pore.

Background: Atopic dermatitis (AD) is the most common inflammatory disease. Evolving disease models link changes in epidermal growth and differentiation to T(H)2/T(H)22 cytokine activation. However, these models have not been tested by in vivo suppression of T-cell cytokines. Cyclosporine (CsA) is an immunosuppressant that is highly effective for severe disease, but its mechanism in AD skin lesions has not been studied. Objective: We sought to establish the ability of a systemic immunosuppressant to modulate immune and epidermal alterations that form the pathogenic disease phenotype and to correlate changes with clinical improvement. Methods: CsA's effects on AD skin pathology were evaluated by using gene expression and immunohistochemistry studies in baseline, week 2, and week 12 lesional and nonlesional biopsy specimens from 19 patients treated with 5 mg/kg/d CsA for 12 weeks. Results: After 2 and 12 weeks of treatment, we observed significant reductions of 51% and 72%, respectively, in SCORAD scores. Clinical improvements were associated with significant gene expression changes in lesional but also nonlesional skin, particularly reductions in levels of T(H)2-, T(H)22-, and some T(H)17-related molecules (ie, IL-13, IL-22, CCL17, S100As, and elafin/peptidase inhibitor 3), and modulation of epidermal hyperplasia and differentiation measures. Conclusions: This is the first study that establishes a relationship between cytokine activation and molecular epidermal alterations, as well as correlations between disease biomarkers in the skin and clinical improvement. The reversal of the molecular phenotype with CsA and the associated biomarkers can serve as a reference for the successful modulation of tissue inflammation with specific immune antagonists in future studies, contributing to the understanding of the specific cytokines involved in epidermal pathology.