The Nucleotide Exchange Factor Ric-8A is a Chaperone for the Conformationally Dynamic Nucleotide-Free State of G Alpha I1

Heterotrimeric G protein alpha subunits are activated upon exchange of GDP for GTP at the nucleotide binding site of G alpha, catalyzed by guanine nucleotide exchange factors (GEFs). In addition to transmembrane G protein-coupled receptors (GPCRs), which act on G protein heterotrimers, members of the family cytosolic proteins typified by mammalian Ric-8A are GEFs for Gi/q/12/13-class G alpha subunits. Ric-8A binds to G alpha.GDP, resulting in the release of GDP. The Ric-8A complex with nucleotide-free G alpha i1 is stable, but dissociates upon binding of GTP to G alpha i1. To gain insight into the mechanism of Ric-8A-catalyzed GDP release from G alpha i1, experiments were conducted to characterize the physical state of nucleotide-free G alpha i1 (hereafter referred to as G alpha i1[]) in solution, both as a monomeric species, and in the complex with Ric-8A. We found that Ric-8A-bound, nucleotide-free G alpha i1 is more accessible to trypsinolysis than G alpha i1.GDP, but less so than G alpha i1[] alone. The TROSY-HSQC spectrum of [N-15]G alpha i1[] bound to Ric-8A shows considerable loss of peak intensity relative to that of [N-15]G alpha i1.GDP. Hydrogen-deuterium exchange in G alpha i1[] bound to Ric-8A is 1.5-fold more extensive than in G alpha i1.GDP. Differential scanning calorimetry shows that both Ric-8A and G alpha i1.GDP undergo cooperative, irreversible unfolding transitions at 47 degrees and 52 degrees, respectively, while nucleotide-free G alpha i1 shows a broad, weak transition near 35 degrees. The unfolding transition for Ric-8A: G alpha i1[] is complex, with a broad transition that peaks at 50 degrees, suggesting that both Ric-8A and G alpha i1[] are stabilized within the complex, relative to their respective free states. The C-terminus of G alpha i1 is shown to be a critical binding element for Ric-8A, as is also the case for GPCRs, suggesting that the two types of GEF might promote nucleotide exchange by similar mechanisms, by acting as chaperones for the unstable and dynamic nucleotide-free state of G alpha

Group A Streptococcus Secreted Esterase Hydrolyzes Platelet-Activating Factor to Impede Neutrophil Recruitment and Facilitate Innate Immune Evasion

The innate immune system is the first line of host defense against invading organisms. Thus, pathogens have developed virulence mechanisms to evade the innate immune system. Here, we report a novel means for inhibition of neutrophil recruitment by Group A Streptococcus (GAS). Deletion of the secreted esterase gene (designated sse) in M1T1 GAS strains with (MGAS5005) and without (MGAS2221) a null covS mutation enhances neutrophil ingress to infection sites in the skin of mice. In trans expression of SsE in MGAS2221 reduces neutrophil recruitment and enhances skin invasion. The sse deletion mutant of MGAS5005 (ΔsseMGAS5005) is more efficiently cleared from skin than the parent strain. SsE hydrolyzes the sn-2 ester bond of platelet-activating factor (PAF), converting biologically active PAF into inactive lyso-PAF. KM and kcat of SsE for hydrolysis of 2-thio-PAF were similar to those of the human plasma PAF acetylhydrolase. Treatment of PAF with SsE abolishes the capacity of PAF to induce activation and chemotaxis of human neutrophils. More importantly, PAF receptor-deficient mice significantly reduce neutrophil infiltration to the site of ΔsseMGAS5005 infection. These findings identify the first secreted PAF acetylhydrolase of bacterial pathogens and support a novel GAS evasion mechanism that reduces phagocyte recruitment to sites of infection by inactivating PAF, providing a new paradigm for bacterial evasion of neutrophil responses

Demonstration of Protein-Based Human Identification Using the Hair Shaft Proteome

YesHuman identification from biological material is largely dependent on the ability to characterize genetic polymorphisms in DNA. Unfortunately, DNA can degrade in the environment, sometimes below the level at which it can be amplified by PCR. Protein however is chemically more robust than DNA and can persist for longer periods. Protein also contains genetic variation in the form of single amino acid polymorphisms. These can be used to infer the status of non-synonymous single nucleotide polymorphism alleles. To demonstrate this, we used mass spectrometry-based shotgun proteomics to characterize hair shaft proteins in 66 European-American subjects. A total of 596 single nucleotide polymorphism alleles were correctly imputed in 32 loci from 22 genes of subjects’ DNA and directly validated using Sanger sequencing. Estimates of the probability of resulting individual non-synonymous single nucleotide polymorphism allelic profiles in the European population, using the product rule, resulted in a maximum power of discrimination of 1 in 12,500. Imputed non-synonymous single nucleotide polymorphism profiles from European–American subjects were considerably less frequent in the African population (maximum likelihood ratio = 11,000). The converse was true for hair shafts collected from an additional 10 subjects with African ancestry, where some profiles were more frequent in the African population. Genetically variant peptides were also identified in hair shaft datasets from six archaeological skeletal remains (up to 260 years old). This study demonstrates that quantifiable measures of identity discrimination and biogeographic background can be obtained from detecting genetically variant peptides in hair shaft protein, including hair from bioarchaeological contexts.The Technology Commercialization Innovation Program (Contracts #121668, #132043) of the Utah Governors Office of Commercial Development, the Scholarship Activitie

Mass Spectrometry Imaging of Chlorhexidine and Bacteria in a Model Wound

The ability to generate two-dimensional images of a wound that contains information about the distribution of bacteria overlaid with the distribution of drugs and metabolites could enhance our understanding of wound healing processes. Advances in technology are leading to a rapid expansion in mass spectrometry-based imaging. When combined with the ability of matrix assisted laser desorption ionization to ionize a wide range of molecules, imaging mass spectrometry is a powerful biomedical research tool. However, this technique has yet to be used to investigate bacterial colonization of wounds or the distribution of antimicrobial agents on tissue. To address this, distribution and persistence of the antimicrobial agent chlorhexidine on a model human tissue was investigated. The ability to detect and localize Staphylococcus aureus on the same tissue model was also addressed. Sub-millimeter resolution ion images from these experiments show the promise of using mass spectrometry imaging to investigate the growth and treatment of bacteria on skin. This methodology will be of value in the development of wound dressings with improved antimicrobial properties and a more careful analysis of the concentration of antimicrobial agents required to prevent biofilm formation and persistence

The nucleotide exchange factor Ric-8A is a chaperone for the conformationally dynamic nucleotide-free state of Gαi1.

Heterotrimeric G protein α subunits are activated upon exchange of GDP for GTP at the nucleotide binding site of Gα, catalyzed by guanine nucleotide exchange factors (GEFs). In addition to transmembrane G protein-coupled receptors (GPCRs), which act on G protein heterotrimers, members of the family cytosolic proteins typified by mammalian Ric-8A are GEFs for Gi/q/12/13-class Gα subunits. Ric-8A binds to Gα•GDP, resulting in the release of GDP. The Ric-8A complex with nucleotide-free Gαi1 is stable, but dissociates upon binding of GTP to Gαi1. To gain insight into the mechanism of Ric-8A-catalyzed GDP release from Gαi1, experiments were conducted to characterize the physical state of nucleotide-free Gαi1 (hereafter referred to as Gαi1[ ]) in solution, both as a monomeric species, and in the complex with Ric-8A. We found that Ric-8A-bound, nucleotide-free Gαi1 is more accessible to trypsinolysis than Gαi1•GDP, but less so than Gαi1[ ] alone. The TROSY-HSQC spectrum of [(15)N]Gαi1[ ] bound to Ric-8A shows considerable loss of peak intensity relative to that of [(15)N]Gαi1•GDP. Hydrogen-deuterium exchange in Gαi1[ ] bound to Ric-8A is 1.5-fold more extensive than in Gαi1•GDP. Differential scanning calorimetry shows that both Ric-8A and Gαi1•GDP undergo cooperative, irreversible unfolding transitions at 47° and 52°, respectively, while nucleotide-free Gαi1 shows a broad, weak transition near 35°. The unfolding transition for Ric-8A:Gαi1[ ] is complex, with a broad transition that peaks at 50°, suggesting that both Ric-8A and Gαi1[ ] are stabilized within the complex, relative to their respective free states. The C-terminus of Gαi1 is shown to be a critical binding element for Ric-8A, as is also the case for GPCRs, suggesting that the two types of GEF might promote nucleotide exchange by similar mechanisms, by acting as chaperones for the unstable and dynamic nucleotide-free state of Gα

Characterization of the Archaeal Thermophile Sulfolobus Turreted Icosahedral Virus Validates an Evolutionary Link among Double-Stranded DNA Viruses from All Domains of Life

Icosahedral nontailed double-stranded DNA (dsDNA) viruses are present in all three domains of life, leading to speculation about a common viral ancestor that predates the divergence of Eukarya, Bacteria, and Archaea. This suggestion is supported by the shared general architecture of this group of viruses and the common fold of their major capsid protein. However, limited information on the diversity and replication of archaeal viruses, in general, has hampered further analysis. Sulfolobus turreted icosahedral virus (STIV), isolated from a hot spring in Yellowstone National Park, was the first icosahedral virus with an archaeal host to be described. Here we present a detailed characterization of the components forming this unusual virus. Using a proteomics-based approach, we identified nine viral and two host proteins from purified STIV particles. Interestingly, one of the viral proteins originates from a reading frame lacking a consensus start site. The major capsid protein (B345) was found to be glycosylated, implying a strong similarity to proteins from other dsDNA viruses. Sequence analysis and structural predication of virion-associated viral proteins suggest that they may have roles in DNA packaging, penton formation, and protein-protein interaction. The presence of an internal lipid layer containing acidic tetraether lipids has also been confirmed. The previously presented structural models in conjunction with the protein, lipid, and carbohydrate information reported here reveal that STIV is strikingly similar to viruses associated with the Bacteria and Eukarya domains of life, further strengthening the hypothesis for a common ancestor of this group of dsDNA viruses from all domains of life

Ultrafast Excited-State Deactivation of the Bacterial Pigment Violacein

The photophysical properties of the natural pigment violacein extracted from an Antarctic organism adapted to high exposure levels of UV radiation were measured in a combined steady-state and time-resolved spectroscopic study for the first time. In the low-viscosity solvents methanol and acetone, violacein exhibits low fluorescence quantum yields on the order of 1 × 10^–4, and femtosecond transient absorption measurements reveal excited-state lifetimes of 3.2 ± 0.2 and 4.5 ± 0.2 ps in methanol and acetone, respectively. As solvent viscosity is increased, both the fluorescence quantum yield and excited-state lifetime of this intensely colored pigment increase dramatically, and stimulated emission decays 30-fold more slowly in glycerol than in methanol at room temperature. Excited-state deactivation is suggested to occur via a molecular-rotor mechanism in which torsion about an interring bond leads to a conical intersection with the ground state

Effects of anticholinergic drugs on cognitive function in older Austalians: Results from the AIBL study

The nature and extent of adverse cognitive effects due to the prescription of anticholinergic drugs in older people with and without dementia is unclear. Methods: We calculated the anticholinergic load (ACL) of medications taken by participants of the Australian Imaging, Biomarkers and Lifestyle (AIBL) study of ageing, a cohort of 211 Alzheimer’s disease (AD) patients, 133 mild cognitive impairment (MCI) patients and 768 healthy controls (HC) all aged over 60 years. The association between ACL and cognitive function was examined for each diagnostic group (HC, MCI, AD). Results: A high ACL within the HC group was associated with significantly slower response speeds for the Stroop color and incongruent trials. No other significant relationships between ACL and cognition were noted. Conclusion: In this large cohort, prescribed anticholinergic drugs appeared to have modest effects upon psychomotor speed and executive function, but not on other areas of cognition in healthy older adults