Salvaging Pyrococcus furiosus Protein Targets at SECSG

Proteins derived from the coding regions of Pyrococcus furiosus are targets for three-dimensional X-ray and NMR structure determination by the Southeast Collaboratory for Structural Genomics (SECSG). Of the 2200 open reading frames (ORFs) in this organism, 220 protein targets were cloned and expressed in a high-throughput (HT) recombinant system for crystallographic studies. However, only 96 of the expressed proteins could be crystallized and, of these, only 15 have led to structures. To address this issue, SECSG has recently developed a two-tier approach to protein production and crystallization. In this approach, tier-1 efforts are focused on producing protein for new Pfu(italics?) targets using a high-throughput approach. Tier-2 protein production efforts support tier-1 activities by (1) producing additional protein for further crystallization trials, (2) producing modified protein (further purification, methylation, tag removal, selenium labeling, etc) as required and (3) serving as a salvaging pathway for failed tier-1 proteins. In a recent study using this two-tiered approach, nine structures were determined from a set of 50 Pfu proteins, which failed to produce crystals suitable for X-ray diffraction analysis. These results validate this approach and suggest that it has application to other HT crystal structure determination applications.     

Discrimination against deoxyribonucleotide substrates by bacterial RNA polymerase

Nucleic acid polymerases have evolved elaborate mechanisms that prevent incorporation of the non-cognate substrates, which are distinguished by both the base and the sugar moieties. While the mechanisms of substrate selection have been studied in single-subunit DNA and RNA polymerases (DNAPs and RNAPs, respectively), the determinants of substrate binding in the multisubunit RNAPs are not yet known. Molecular modeling of Thermus thermophilus RNAP-substrate NTP complex identified a conserved beta' subunit Asn(737) residue in the active site that could play an essential role in selection of the substrate ribose. We utilized the Escherichia coli RNAP model system to assess this prediction. Functional in vitro analysis demonstrates that the substitutions of the corresponding beta' Asn(458) residue lead to the loss of discrimination between ribo- and deoxyribonucleotide substrates as well as to defects in RNA chain extension. Thus, in contrast to the mechanism utilized by the single-subunit T7 RNAP where substrate selection commences in the inactive pre-insertion site prior to its delivery to the catalytic center, the bacterial RNAPs likely recognize the sugar moiety in the active (insertion) site.     

Switching of membrane organelles between cytoskeletal transport systems is determined by regulation of the microtubule-based transport

Intracellular transport of membrane organelles occurs along microtubules (MTs) and actin filaments (AFs). Although transport along each type of the cytoskeletal tracks is well characterized, the switching between the two types of transport is poorly understood because it cannot be observed directly in living cells. To gain insight into the regulation of the switching of membrane organelles between the two major transport systems, we developed a novel approach that combines live cell imaging with computational modeling. Using this approach, we measured the parameters that determine how fast membrane organelles switch back and forth between MTs and AFs (the switching rate constants) and compared these parameters during different signaling states. We show that regulation involves a major change in a single parameter: the transferring rate from AFs onto MTs. This result suggests that MT transport is the defining factor whose regulation determines the choice of the cytoskeletal tracks during the transport of membrane organelles.     

VEGFR2 and Src kinase inhibitors suppress Andes virus-induced endothelial cell permeability

Hantaviruses predominantly infect human endothelial cells and, in the absence of cell lysis, cause two diseases resulting from increased vascular permeability. Andes virus (ANDV) causes a highly lethal acute pulmonary edema termed hantavirus pulmonary syndrome (HPS). ANDV infection enhances the permeability of endothelial cells in response to vascular endothelial growth factor (VEGF) by increasing signaling responses directed by the VEGFR2-Src-VE-cadherin pathway, which directs adherens junction (AJ) disassembly. Here we demonstrate that inhibiting pathway-specific VEGFR2 and Src family kinases (SFKs) blocks ANDV-induced endothelial cell permeability. Small interfering RNA (siRNA) knockdown of Src within ANDV-infected endothelial cells resulted in an ～70% decrease in endothelial cell permeability compared to that for siRNA controls. This finding suggested that existing FDA-approved small-molecule kinase inhibitors might similarly block ANDV-induced permeability. The VEGFR2 kinase inhibitor pazopanib as well as SFK inhibitors dasatinib, PP1, bosutinib, and Src inhibitor 1 dramatically inhibited ANDV-induced endothelial cell permeability. Consistent with their kinase-inhibitory concentrations, dasatinib, PP1, and pazopanib inhibited ANDV-induced permeability at 1, 10, and 100 nanomolar 50% inhibitory concentrations (IC(50)s), respectively. We further demonstrated that dasatinib and pazopanib blocked VE-cadherin dissociation from the AJs of ANDV-infected endothelial cells by >90%. These findings indicate that VEGFR2 and Src kinases are potential targets for therapeutically reducing ANDV-induced endothelial cell permeability and, as a result, capillary permeability during HPS. Since the functions of VEGFR2 and SFK inhibitors are already well defined and FDA approved for clinical use, these findings rationalize their therapeutic evaluation for efficacy in reducing HPS disease. Endothelial cell barrier functions are disrupted by a number of viruses that cause hemorrhagic, edematous, or neurologic disease, and as a result, our findings suggest that VEGFR2 and SFK inhibitors should be considered for regulating endothelial cell barrier functions altered by additional viral pathogens.     

Interleukin-6 enhances insulin secretion by increasing glucagon-like peptide-1 secretion from L cells and alpha cells

Exercise, obesity and type 2 diabetes are associated with elevated plasma concentrations of interleukin-6 (IL-6). Glucagon-like peptide-1 (GLP-1) is a hormone that induces insulin secretion. Here we show that administration of IL-6 or elevated IL-6 concentrations in response to exercise stimulate GLP-1 secretion from intestinal L cells and pancreatic alpha cells, improving insulin secretion and glycemia. IL-6 increased GLP-1 production from alpha cells through increased proglucagon (which is encoded by GCG) and prohormone convertase 1/3 expression. In models of type 2 diabetes, the beneficial effects of IL-6 were maintained, and IL-6 neutralization resulted in further elevation of glycemia and reduced pancreatic GLP-1. Hence, IL-6 mediates crosstalk between insulin-sensitive tissues, intestinal L cells and pancreatic islets to adapt to changes in insulin demand. This previously unidentified endocrine loop implicates IL-6 in the regulation of insulin secretion and suggests that drugs modulating this loop may be useful in type 2 diabetes.     

gamma-synuclein has a dynamic intracellular localization

gamma-Synuclein is a member of the synuclein family consisting of three proteins. Within the last several years increasing attention has focused on these proteins because of their role in human diseases. alpha-Synuclein relevance to Parkinson's disease is based on mutations found in familial cases of the disease and its presence in filaments and inclusion bodies in sporadic cases. gamma-Synuclein is implicated in some forms of cancer and ocular diseases, while beta-synuclein may antagonize their pathological functions. In this paper we present data on the localization and properties of gamma-synuclein in several neuronal and nonneuronal cell cultures. We show that contrary to the current opinion, gamma-synuclein is not an exclusively cytoplasmic protein, but has a dynamic localization and can associate with subcellular structures. It is present in the perinuclear area and may be associated to centrosomes. On late steps of mitosis gamma-synuclein is not found in the centrosomes, and redistributes to the midbody in telophase. Under stress conditions a translocation of gamma-synuclein from the perinuclear area to the nucleus occurs exhibiting nucleocytoplasmic shuttling. gamma-Synuclein overexpression reduces neurite outgrowth in a greater extent then alpha-synuclein overexpression. These data support the view that gamma-synuclein may change its intracellular localization and associate with subcellular structures in response to intracellular signaling or stress.     

The insulin polymorphism -23Hph increases the risk for type 1 diabetes mellitus in the Romanian population

The insulin -23Hph and IGF2 Apa polymorphisms were genotyped in Romanian patients with T1DM (n = 204), T2DM (n = 215) or obesity (n = 200) and normoponderal healthy subjects (n = 750). The genotypes of both polymorphisms were distributed in concordance with Hardy-Weinberg equilibrium in all groups. The -23Hph AA genotype increased the risk for T1DM (OR: 3.22, 95%CI: 2.09-4.98, p < 0,0001), especially in patients without macroalbuminuria (OR: 4.32, 95%CI: 2.54-7.45, p < 0,0001). No other significant association between the alleles or genotypes of insulin -23Hph and IGF2 Apa and diabetes or obesity was identified.     

Life at the border: Adaptation of proteins to anisotropic membrane environment

This review discusses main features of transmembrane (TM) proteins which distinguish them from water‐soluble proteins and allow their adaptation to the anisotropic membrane environment. We overview the structural limitations on membrane protein architecture, spatial arrangement of proteins in membranes and their intrinsic hydrophobic thickness, co‐translational and post‐translational folding and insertion into lipid bilayers, topogenesis, high propensity to form oligomers, and large‐scale conformational transitions during membrane insertion and transport function. Special attention is paid to the polarity of TM protein surfaces described by profiles of dipolarity/polarizability and hydrogen‐bonding capacity parameters that match polarity of the lipid environment. Analysis of distributions of Trp resides on surfaces of TM proteins from different biological membranes indicates that interfacial membrane regions with preferential accumulation of Trp indole rings correspond to the outer part of the lipid acyl chain region—between double bonds and carbonyl groups of lipids. These “midpolar” regions are not always symmetric in proteins from natural membranes. We also examined the hydrophobic effect that drives insertion of proteins into lipid bilayer and different free energy contributions to TM protein stability, including attractive van der Waals forces and hydrogen bonds, side‐chain conformational entropy, the hydrophobic mismatch, membrane deformations, and specific protein–lipid binding.     

Predictive Dynamics of Human Pain Perception

While the static magnitude of thermal pain perception has been shown to follow a power-law function of the temperature, its dynamical features have been largely overlooked. Due to the slow temporal experience of pain, multiple studies now show that the time evolution of its magnitude can be captured with continuous online ratings. Here we use such ratings to model quantitatively the temporal dynamics of thermal pain perception. We show that a differential equation captures the details of the temporal evolution in pain ratings in individual subjects for different stimulus pattern complexities, and also demonstrates strong predictive power to infer pain ratings, including readouts based only on brain functional images.     

Syntheses and crystal structures of cyano-bridged cluster-metal layered coordination polymers [Cu(dien)]3[W4Te4(CN)12] · 9H2O and [Ni(en)(NH3)]3[W4Se4(CN)12] · 7.5H2O

Two new tetrahedral tungsten cyanide cluster compounds, [Cu(dien)]₃[W₄Te₄(CN)₁₂] · 9H₂O (1) (dien=diethylenetriamine) and [Ni(en)(NH₃)]₃[W₄Se₄(CN)₁₂] · 7.5H₂O (2) (en=ethylenediamine), were synthesized by treating aqueous solutions of the saltlike cluster compound K₆[W₄Te₄(CN)₁₂] · 5H₂O/K₆[W₄Se₄(CN)₁₂] · 6H₂O with copper(II)/nickel(II) chloride in aqueous ammonia containing dien/en. The cyano-bridged layered coordination polymeric compounds were characterized by single-crystal X-ray diffraction analysis: monoclinic, space group P2₁ for 1; trigonal, space group for 2. Structures of 1 and 2 consist of infinite neutral layers of cluster components {W₄Te₄(CN)₁₂}/{W₄Se₄(CN)₁₂} connected, one another by {Cu(dien)} or {Ni(en)(NH₃)} fragments, respectively.