The rockefeller university

Proteomic identifications hinge on the measurement of both parent and fragment masses and matching these to amino acid sequences via database search engines. The correctness of the identifications is assessed by statistical means. Here we present an experimental approach to test identifications. Chemical modification of all peptides in a sample leads to shifts in masses depending on the chemical properties of each peptide. The identification of a native peptide sequence and its perturbed version with a different parent mass and fragment ion masses provides valuable information. Labeling all peptides using reductive alkylation with formaldehyde is one such perturbation where the ensemble of peptides shifts mass depending on the number of reactive amine groups. Matching covalently perturbed fragmentation patterns from the same underlying peptide sequence increases confidence in the assignments and can salvage low scoring post-translationally modified peptides. Applying this strategy to bovine alpha-crystallin, we identify 9 lysine acetylation sites, 4 O-GlcNAc sites and 13 phosphorylation sites.

In vivo protection by antimicrobial neutralizing Abs can require the contribution of effector functions mediated by Fe-Fc gamma receptor (Fc-Fc gamma R) interactions for optimal efficacy. In influenza, broadly neutralizing anti-hemagglutinin (anti-HA) stalk mAbs require Fc-Fc gamma R interactions to mediate in vivo protection, but strain-specific anti-HA head mAbs do not. Whether this rule applies only to anti-stalk Abs or is applicable to any broadly neutralizing Ab (bNAb) against influenza is unknown. Here, we characterized the contribution of Fc-Fc gamma R interactions during in vivo protection for a panel of 13 anti-HA mAbs, including bNAbs and non-neutralizing Abs, against both the stalk and head domains. All classes of broadly binding anti-HA mAbs required Fc-Fc gamma R interactions to provide protection in vivo, including those mAbs that bind the HA head and those that do not neutralize virus in vitro. Further, abroadly neutralizing anti-neuraminidase (anti-NA) mAb also required Fc gamma Rs to provide protection in vivo, but a strain-specific anti-NA mAb did not. Thus, these findings suggest that the breadth of reactivity of anti-influenza Abs, regardless of their epitope, necessitates interactions with Fc gamma Rs on effector cell populations to mediate in vivo protection. These findings will guide the design of antiviral Ab therapeutics and inform vaccine design to elicit Abs with optimal binding properties and effector functions.

The agr locus encodes a quorum-sensing (QS) circuit required for the virulence of a spectrum of Gram-positive pathogens and is, therefore, regarded as an important target for the development of chemotherapeutics. In recent years, many of the biochemical events in the Staphylococcus aureus agr circuit have been reconstituted and subject to quantitative analysis in vitro. This work, in conjunction with structural studies on several key players in the signaling circuit, has furnished mechanistic insights into the regulation and evolution of the agr QS system. Here, we review this progress and discuss the remaining open questions in the area. We also highlight advances in the discovery of small-molecule agr modulators and how the newly available biochemical and structural information might be leveraged for the design of next-generation therapeutics targeting the agr system.

Clostridium difficile is a Gram-positive bacillus and is the leading cause of toxin-mediated nosocomial diarrhea following antibiotic use. C. difficile flagella play a role in colonization, adherence, biofilm formation, and toxin production, which might contribute to the overall virulence of certain strains. Human and animal studies indicate that anti-flagella immune responses may play a role in protection against colonization by C. difficile and subsequent disease outcome. Here we report that recombinant C. difficile flagellin (FliC) is immunogenic and protective in a murine model of C. difficile infection (CDI) against a clinical C. difficile strain, UK1. Passive protection experiments using anti-FliC polyclonal serum in mice suggest this protection to be antibody-mediated. FliC immunization also was able to afford partial protection against CDI and death in hamsters following challenge with C. difficile 630 Delta erm. Additionally, immunization against FliC does not have an adverse effect on the normal gut flora of vaccinated hamsters as evidenced by comparing the fecal microbiome of vaccinated and control hamsters. Therefore, the use of FliC as a vaccine candidate against CDI warrants further testing.

Ubiquitylation of histone H2B at lysine 120 (H2B-Ub), a post-translational modification first discovered in 1980, plays a critical role in diverse nuclear processes including the regulation of transcription and DNA damage repair. Herein, we use a suite of protein chemistry methods to explore how H2B-Ub stimulates hDot1L-mediated methylation of histone H3 on lysine 79 (H3K79me). By using semisynthetic 'designer' chromatin containing H2B-Ub bearing a site-specifically installed photocrosslinker, here we report an interaction between a functional hotspot on ubiquitin and the N-terminus of histone H2A. Our biochemical studies indicate that this interaction is required for stimulation of hDot1L activity and leads to a repositioning of hDot1L on the nucleosomal surface, which likely places the active site of the enzyme proximal to H3K79. Collectively, our data converge on a possible mechanism for hDot1L stimulation in which H2B-Ub physically 'corrals' the enzyme into a productive binding orientation.

Background Depending on the epidemiological setting, a variable proportion of leprosy patients will suffer from excessive pro-inflammatory responses, termed type-1 reactions (T1R). The LRRK2 gene encodes a multi-functional protein that has been shown to modulate pro-inflammatory responses. Variants near the LRRK2 gene have been associated with leprosy in some but not in other studies. We hypothesized that LRRK2 was a T1R susceptibility gene and that inconsistent association results might reflect different proportions of patients with T1R in the different sample settings. Hence, we evaluated the association of LRRK2 variants with T1R susceptibility. Methodology An association scan of the LRRK2 locus was performed using 156 single-nucleotide poly-morphisms (SNPs). Evidence of association was evaluated in two family-based samples: A set of T1R-affected and a second set of T1R-free families. Only SNPs significant for T1R-affected families with significant evidence of heterogeneity relative to T1R-free families were considered T1R-specific. An expression quantitative trait locus (eQTL) analysis was applied to evaluate the impact of T1R-specific SNPs on LRRK2 gene transcriptional levels. Principal Findings A total of 18 T1R-specific variants organized in four bins were detected. The core SNP capturing the T1R association was the LRRK2 missense variant M2397T (rs3761863) that affects LRRK2 protein turnover. Additionally, a bin of nine SNPs associated with T1R were eQTLs for LRRK2 in unstimulated whole blood cells but not after exposure to Mycobacterium leprae antigen. Significance The results support a preferential association of LRRK2 variants with T1R. LRRK2 involvement in T1R is likely due to a pathological pro-inflammatory loop modulated by LRRK2 availability. Interestingly, the M2397T variant was reported in association with Crohn's disease with the same risk allele as in T1R suggesting common inflammatory mechanism in these two distinct diseases.

A search is presented for the production of both first- and second-generation scalar leptoquarks with a final state of either two electrons and one jet or two muons and one jet. The search is based on a data sample of proton-proton collisions at center-of-mass energy root s = 8 TeV recorded with the CMS detector and corresponding to an integrated luminosity of 19.6 fb(-1). Upper limits are set on both the first- and second-generation leptoquark production cross sections as functions of the leptoquark mass and the leptoquark couplings to a lepton and a quark. Results are compared with theoretical predictions to obtain lower limits on the leptoquark mass. At 95% confidence level, single production of first- generation leptoquarks with a coupling and branching fraction of 1.0 is excluded for masses below 1730 GeV, and second-generation leptoquarks with a coupling and branching fraction of 1.0 is excluded for masses below 530 GeV. These are the best overall limits on the production of first-generation leptoquarks to date.

Prevalent HSV-2 infection increases the risk of HIV acquisition both in men and women even in asymptomatic subjects. Understanding the impact of HSV-2 on the mucosal micro-environment may help to identify determinants of susceptibility to HIV. Vaginal HSV-2 infection increases the frequency of cells highly susceptible to HIV in the vaginal tissue of women and macaques and this correlates with increased susceptibility to vaginal SHIV infection in macaques. However, the effect of rectal HSV-2 infection on HIV acquisition remains understudied. We developed a model of rectal HSV-2 infection in macaques in combination with rectal SIVmac239 Delta nef (SIV Delta nef) vaccination and our results suggest that rectal HSV-2 infection may increase the susceptibility of macaques to rectal SIVmac239 wild-type (wt) infection even in SIV Delta nef-infected animals. Rectal SIV Delta nef infection/vaccination protected 7 out of 7 SIV Delta nef-infected macaques from SIVmac239wt rectal infection (vs 12 out of 16 SIV Delta nef-negative macaques), while 1 out of 3 animals co-infected with SIV Delta nef and HSV-2 acquired SIVmac239wt infection. HSV-2/SIVmac239wt co-infected animals had increased concentrations of inflammatory factors in their plasma and rectal fluids and a tendency toward higher acute SIVmac239wt plasma viral load. However, they had higher blood CD4 counts and reduced depletion of CCR5+ CD4+ T cells compared to SIV-mac239wt-only infected animals. Thus, rectal HSV-2 infection generates a pro-inflammatory environment that may increase susceptibility to rectal SIV infection and may impact immunological and virological parameters during acute SIV infection. Studies with larger number of animals are needed to confirm these findings.

While searching for new therapeutics against malaria, Lanzavecchia and colleagues discovered that antibodies can be diversified by DNA sequences encoded outside of antibody genes.

Protein fatty-acylation in eukaryotes has been associated with many fundamental biological processes. However, the diversity, abundance and regulatory mechanisms of protein fatty-acylation in vivo remain to be explored. Herein, we review the proteomic analysis of fatty-acylated proteins, with a focus on N-myristoylation and S-palmitoylation. We then highlight major challenges and emerging methods for direct site identification, quantitation, and lipid structure characterization to understand the functions and regulatory mechanisms of fatty-acylated proteins in physiology and disease.