A Peptide Core Motif for Binding to Heterotrimeric G Protein α Subunits

Recently, in vitro selection using mRNA display was used to identify a novel peptide sequence that binds with high affinity to G{alpha}i1. The peptide was minimized to a 9-residue sequence (R6A-1) that retains high affinity and specificity for the GDP-bound state of G{alpha}i1 and acts as a guanine nucleotide dissociation inhibitor (GDI). Here we demonstrate that the R6A-1 peptide interacts with G{alpha} subunits representing all four G protein classes, acting as a core motif for G{alpha} interaction. This contrasts with the consensus G protein regulatory(GPR) sequence, a 28-mer peptide GDI derived from the GoLoco (G{alpha}i/0-Loco interaction)/GPR motif that shares no homology with R6A-1 and binds only to G{alpha}i1-3 in this assay. Binding of R6A-1 is generally specific to the GDP-bound state of the G{alpha} subunits and excludes association with G{beta}{gamma}. R6A-G{alpha}i1 complexes are resistant to trypsin digestion and exhibit distinct stability in the presence of Mg2+, suggesting that the R6A and GPR peptides exert their activities using different mechanisms. Studies using G{alpha}i1/G{alpha}s chimeras identify two regions of G{alpha}i1 (residues 1–35 and 57–88) as determinants for strong R6A-Gi{alpha}1 interaction. Residues flanking the R6A-1 peptide confer unique binding properties, indicating that the core motif could be used as a starting point for the development of peptides exhibiting novel activities and/or specificity for particular G protein subclasses or nucleotide-bound states

Model for eukaryotic tail-anchored protein binding based on the structure of Get3

The Get3 ATPase directs the delivery of tail-anchored (TA) proteins to the endoplasmic reticulum (ER). TA-proteins are characterized by having a single transmembrane helix (TM) at their extreme C terminus and include many essential proteins, such as SNAREs, apoptosis factors, and protein translocation components. These proteins cannot follow the SRP-dependent co-translational pathway that typifies most integral membrane proteins; instead, post-translationally, these proteins are recognized and bound by Get3 then delivered to the ER in the ATP dependent Get pathway. To elucidate a molecular mechanism for TA protein binding by Get3 we have determined three crystal structures in apo and ADP forms from Saccharomyces cerevisae (ScGet3-apo) and Aspergillus fumigatus (AfGet3-apo and AfGet3-ADP). Using structural information, we generated mutants to confirm important interfaces and essential residues. These results point to a model of how Get3 couples ATP hydrolysis to the binding and release of TA-proteins

Repositioning the Catalytic Triad Aspartic Acid of Haloalkane Dehalogenase: Effects on Stability, Kinetics, and Structure

Haloalkane dehalogenase (DhlA) catalyzes the hydrolysis of haloalkanes via an alkyl-enzyme intermediate. The covalent intermediate, which is formed by nucleophilic substitution with Asp124, is hydrolyzed by a water molecule that is activated by His289. The role of Asp260, which is the third member of the catalytic triad, was studied by site-directed mutagenesis. Mutation of Asp260 to asparagine resulted in a catalytically inactive D260N mutant, which demonstrates that the triad acid Asp260 is essential for dehalogenase activity. Furthermore, Asp260 has an important structural role, since the D260N enzyme accumulated mainly in inclusion bodies during expression, and neither substrate nor product could bind in the active-site cavity. Activity for brominated substrates was restored to D260N by replacing Asn148 with an aspartic or glutamic acid. Both double mutants D260N+N148D and D260N+N148E had a 10-fold reduced kcat and 40-fold higher Km values for 1,2-dibromoethane compared to the wild-type enzyme. Pre-steady-state kinetic analysis of the D260N+N148E double mutant showed that the decrease in kcat was mainly caused by a 220-fold reduction of the rate of carbon-bromine bond cleavage and a 10-fold decrease in the rate of hydrolysis of the alkyl-enzyme intermediate. On the other hand, bromide was released 12-fold faster and via a different pathway than in the wild-type enzyme. Molecular modeling of the mutant showed that Glu148 indeed could take over the interaction with His289 and that there was a change in charge distribution in the tunnel region that connects the active site with the solvent. On the basis of primary structure similarity between DhlA and other α/β-hydrolase fold dehalogenases, we propose that a conserved acidic residue at the equivalent position of Asn148 in DhlA is the third catalytic triad residue in the latter enzymes.

Macroscopic quantum phenomena in high-Tc superconducting material

The observation of macroscopic quantum phenomena in a high-Tc superconducting material [YBa2Cu3O9-y (Y-Bu-Cu-O), Tc=93.4 K] is reported. Relationships between current, voltage, and magnetic fields of Sn–Y-Ba-Cu-O and Y-Ba-Cu-O–Y-Ba-Cu-O point contracts were measured. In both cases the critical current is periodic in the magnetic field, which is typical for double point contacts (dc superconducting quantum interference devices). In the Sn–Y-Ba-Cu-O contacts the oscillations were observed below the Tc of tin. In the Y-Ba-Cu-O–Y-Ba-Cu-O contacts the oscillations were observed at temperatures up to 66 K, clearly demonstrating macroscopic quantum phemonena in this high-Tc material

Identification of a G(iα) binding site on type V adenylyl cyclase

The stimulatory G protein α subunit G(sα) binds within a cleft in adenylyl cyclase formed by the α1-α2 and α3-β4 loops of the C2 domain. The pseudosymmetry of the C1 and C2 domains of adenylyl cyclase suggests that the homologous inhibitory α subunit G(iα) could bind to the analogous cleft within C1. We demonstrate that myristoylated guanosine 5\u27-3-O- (thio)triphosphate-G(iα1) forms a stable complex with the C1 (but not the C2) domain of type V adenylyl cyclase. Mutagenesis of the membrane-bound enzyme identified residues whose alteration either increased or substantially decreased the IC50 for inhibition by G(iα1). These mutations suggest binding of G(iα) within the cleft formed by the α2 and α3 helices of C1, analogous to the G(sα) binding site in C2. Adenylyl cyclase activity reconstituted by mixture of the C1 and C2 domains of type V adenylyl cyclase was also inhibited by G(iα). The C(1b) domain of the type V enzyme contributed to affinity for G(iα), but the source of C2 had little effect. Mutations in this soluble system faithfully reflected the phenotypes observed with the membrane-bound enzyme. The pseudosymmetrical structure of adenylyl cyclase permits bidirectional regulation of activity by homologous G protein α subunits

A peptide core motif for binding to heterotrimeric G protein alpha subunits

Recently, in vitro selection using mRNA display was used to identify a novel peptide sequence that binds with high affinity to Galpha(i1). The peptide was minimized to a 9-residue sequence (R6A-1) that retains high affinity and specificity for the GDP-bound state of Galpha(i1) and acts as a guanine nucleotide dissociation inhibitor (GDI). Here we demonstrate that the R6A-1 peptide interacts with Galpha subunits representing all four G protein classes, acting as a core motif for Galpha interaction. This contrasts with the consensus G protein regulatory(GPR) sequence, a 28-mer peptide GDI derived from the GoLoco (Galpha(i/0)-Loco interaction)/GPR motif that shares no homology with R6A-1 and binds only to Galpha(i1-3) in this assay. Binding of R6A-1 is generally specific to the GDP-bound state of the Galpha subunits and excludes association with Gbetagamma. R6A-Galpha(i1) complexes are resistant to trypsin digestion and exhibit distinct stability in the presence of Mg(2+), suggesting that the R6A and GPR peptides exert their activities using different mechanisms. Studies using Galpha(i1)/Galpha(s) chimeras identify two regions of Galpha(i1) (residues 1-35 and 57-88) as determinants for strong R6A-G(ialpha1) interaction. Residues flanking the R6A-1 peptide confer unique binding properties, indicating that the core motif could be used as a starting point for the development of peptides exhibiting novel activities and/or specificity for particular G protein subclasses or nucleotide-bound states

Consideration of the bioavailability of metal/metalloid species in freshwaters: experiences regarding the implementation of biotic ligand model-based approaches in risk assessment frameworks

After the scientific development of Biotic Ligand Models (BLMs) in recent decades these models are now considered suitable for implementation in regulatory risk assessment of metals in freshwater bodies. The approach has been developed over several years and has been described in many peer-reviewed publications. The original complex BLMs have been applied in prospective risk assessment reports for metals and metal compounds and are also recommended as suitable concepts for the evaluation of monitoring data in the context of the European Water Framework Directive. Currently, several user-friendly BLM-based bioavailability software tools are available for assessing the aquatic toxicity of a limited number of metals (mainly copper, nickel, and zinc). These tools need only a basic set of water parameters as input (e.g., pH, hardness, dissolved organic matter and dissolved metal concentration). Such tools seem appropriate to foster the implementation in routine water quality assessments. This work aims to review the existing bioavailability-based regulatory approaches and the application of available BLM-based bioavailability tools for this purpose. Advantages and possible drawbacks of these tools (e.g., feasibility, boundaries of validity) are discussed, and recommendations for further implementation are given

Evidence-Informed Guidelines for Pediatric Pandemic Planning and Response

From the executive summary: Pandemic events are unpredictable and inevitable. When they occur, the impact is both all-encompassing and asymmetrical; each pandemic targets specific, vulnerable populations, but ultimately impacts individuals, families and communities throughout the world. Regardless of origin or circumstances, the next pandemic will certainly count infants, children, and adolescents among its most vulnerable targets. As evidenced by the 2009 H1N1 influenza pandemic, children may be at higher risk than populations more typically seen as susceptible to pandemic illness (the elderly, those with weakened immune systems, etc.). Children also can function as disease vectors, spreading the virus through their ubiquitous presence in settings where they live, attend school, and play. This document is the result of a two-year international, mixed-methods study of the physical, social, and mental health effects of pandemic on children and families – particularly the impact of quarantine and hospital isolation during these events. This project also examined the psychosocial effects of pandemic disaster on professionals who care for children before, during, and after pandemic. Based on the empirical findings of this study, researchers developed a set of evidence-informed, child-focused, best practice guidelines for use by stakeholders during future pandemics across a variety of relevant fields. In addition, data gathered and analyzed for the project have been used to create a set of Kentucky-specific recommendations that respond to the state’s unique geographic and population needs

Weighing poor immunometabolic health in relatives for severity of affective symptoms:A study of patients with depressive and anxiety disorders and their siblings

BACKGROUND: Affective (i.e. depressive and anxiety) disorders often co-occur with immunometabolic diseases and related biological pathways. Although many large population-based and meta-analytic studies have confirmed this link in community and clinical samples, studies in at-risk samples of siblings of persons with affective disorders are lacking. Furthermore, this somatic-mental co-occurrence may be partially explained by familial clustering of the conditions. First, we examined whether the association between a wide range of immunometabolic diseases and related biomarker based risk-profiles with psychological symptoms replicates in at-risk siblings of probands with affective disorders. Second, leveraging on a sibling-pair design, we disentangled and quantified the effect of probands' immunometabolic health on siblings' psychological symptoms and on the association between immunometabolic health and these symptoms in siblings.METHODS: The sample included 636 participants (M age = 49.7; 62.4% female) from 256 families, each including a proband with lifetime depressive and/or anxiety disorders and at least one of their sibling(s) (N = 380 proband-sibling pairs). Immunometabolic health included cardiometabolic and inflammatory diseases, body mass index (BMI), and composite metabolic (based on the five metabolic syndrome components) and inflammatory (based on interleukin-6 and C-reactive protein) biomarker indices. Overall affective symptoms and specific atypical, energy-related depressive symptoms were derived from self-report questionnaires. Mixed-effects analyses were used to model familial clustering. RESULTS: In siblings, inflammatory disease (γ = 0.25, p = 0.013), higher BMI (γ = 0.10, p = 0.033) and metabolic index (γ = 0.28, p &lt; 0.001) were associated with higher affective symptoms, with stronger associations for atypical, energy-related depressive symptoms (additionally associated with cardiometabolic disease; γ = 0.56, p = 0.048). Immunometabolic health in probands was not independently associated with psychological symptoms in siblings nor did it moderate the association between immunometabolic health and psychological symptoms estimated in siblings.CONCLUSIONS: Our findings demonstrate that the link between later life immunometabolic health and psychological symptoms is consistently present also in adult siblings at high risk for affective disorders. Familial clustering did not appear to have a substantial impact on this association. Instead, individual lifestyle, rather than familial factors, may have a relatively higher impact in the clustering of later life immunometabolic conditions with psychological symptoms in at-risk adult individuals. Furthermore, results highlighted the importance of focusing on specific depression profiles when investigating the overlap with immunometabolic health.</p