Structural analysis of a designed inhibitor complexed with the hemagglutinin-neuraminidase of Newcastle disease virus

Viruses of the Paramyxoviridae family are the leading cause of respiratory disease in children. The human parainfluenza viruses (hPIV) are members of the Paramyxovirinae subfamily, which also includes mumps virus, Newcastle disease virus (NDV), Sendai virus (SV) and simian type 5 virus (SV5). On the surface of these viruses is the glycoprotein hemagglutinin-neuraminidase (HN), which is responsible for cell attachment, promotion of fusion and release of progeny virions. This multifunctional nature of HN makes it an attractive target for the development of inhibitors as a treatment for childhood respiratory diseases. Here we report the crystal structure of NDV HN in complex with a derivative of 2-deoxy-2,3-dehydro-N-acetylneuraminic acid, Neu5Ac2en, that has a functional group designed to occupy a large conserved binding pocket around the active site. The purpose of this study was to examine the effect of a bulky hydrophobic group at the O4 position of Neu5Ac2en, given the hydrophobic nature of the binding pocket. This derivative, with a benzyl group added to the O4 position of Neu5Ac2en, has an IC₅₀ of ∼10 μM in a neuraminidase assay against hPIV3 HN. The IC₅₀ value of the parent compound, Neu5Ac2en, in the same assay is ∼25 μM. These results highlight the striking difference between the influenza neuraminidase and paramyxovirus HN active sites, and provide a platform for the development of improved HN inhibitors.     

Single nucleotide RNA choreography

New structural analysis methods, and a tree formalism re-define and expand the RNA motif concept, unifying what previously appeared to be disparate groups of structures. We find RNA tetraloops at high frequencies, in new contexts, with unexpected lengths, and in novel topologies. The results, with broad implications for RNA structure in general, show that even at this most elementary level of organization, RNA tolerates astounding variation in conformation, length, sequence and context. However the variation is not random; it is well-described by four distinct modes, which are 3-2 switches (backbone topology variations), insertions, deletions and strand clips.     

The primary in vivo steroidal alkaloid glucosyltransferase from potato

To provide tools for breeders to control the steroidal glycoalkaloid (SGA) pathway in potato, we have investigated the steroidal alkaloid glycosyltransferase (Sgt) gene family. The committed step in the SGA pathway is the glycosylation of solanidine by either UDP-glucose or UDP-galactose leading to α-chaconine or α-solanine, respectively. The Sgt2 gene was identified by deduced protein sequence homology to the previously identified Sgt1 gene. SGT1 has glucosyltransferase activity in vitro, but in vivo serves as the UDP-galactose:solanidine galactosyltransferase. Two alleles of the Sgt2 gene were isolated and its function was established with antisense transgenic lines and in vitro assays of recombinant protein. In tubers of transgenic potato (Solanum tuberosum) cvs. Lenape and Desirée expressing an antisense Sgt2 gene construct, accumulation of α-solanine was increased and α-chaconine was reduced. Studies with recombinant SGT2 protein purified from yeast show that SGT2 glycosylation activity is highly specific for UDP-glucose as a sugar donor. This data establishes the function of the gene product (SGT2), as the primary UDP-glucose:solanidine glucosyltransferase in vivo.     

A pericellular collagenase directs the 3-dimensional development of white adipose tissue

White adipose tissue (WAT) serves as the primary energy depot in the body by storing fat. During development, fat cell precursors (i.e., preadipocytes) undergo a hypertrophic response as they mature into lipid-laden adipocytes. However, the mechanisms that regulate adipocyte size and mass remain undefined. Herein, we demonstrate that the membrane-anchored metalloproteinase, MT1-MMP, coordinates adipocyte differentiation in vivo. In the absence of the protease, WAT development is aborted, leaving tissues populated by mini-adipocytes which render null mice lipodystrophic. While MT1-MMP preadipocytes display a cell autonomous defect in vivo, null progenitors retain the ability to differentiate into functional adipocytes during 2-dimensional (2-D) culture. By contrast, within the context of the 3-dimensional (3-D) ECM, normal adipocyte maturation requires a burst in MT1-MMP-mediated proteolysis that modulates pericellular collagen rigidity in a fashion that controls adipogenesis. Hence, MT1-MMP acts as a 3-D-specific adipogenic factor that directs the dynamic adipocyte-ECM interactions critical to WAT development.     

Analysis of climatic and geographic factors affecting the presence of chytridiomycosis in Australia

Chytridiomycosis is an emerging fungal disease that has been implicated in the global decline of amphibian populations. Identifying climatic and geographic factors associated with its presence may be useful in control and prevention measures. Factors such as high altitude, cool temperature, and wet climate have been associated with chytridiomycosis outbreaks. Although some of these factors have been studied in a laboratory setting, there have been few studies in a natural setting. In this investigation, the relationship between altitude, average summer maximum temperature, or the amount of rainfall and the presence or absence of chytridiomycosis are statistically tested using data from 56 study sites in Australia. Currently, in Australia, 48 native species of wild amphibians have been found infected with chytridiomycosis. The 56 sites in the present study, extending along approximately 50% of the coastline of Australia, have been identified as either a chytrid site, where > or = 1 species are infected with chytridiomycosis, or a no-decline site, where none of the species present at the site are experiencing a decline or are known to be infected. The odds-ratio test and two-proportions test applied to this data indicate that the presence of chytridiomycosis in Australia is significantly related to temperature. In particular, the presence of chytridiomycosis is more likely at sites where the average summer maximum temperature is < 30 degrees C. The results of the analyses do not indicate a significant relationship between the presence of chytridiomycosis and altitude or rainfall.     

Suppression of RhoA activity by focal adhesion kinase-induced activation of p190RhoGAP: role in regulation of endothelial permeability

The interaction of endothelial cells with extracellular matrix proteins at focal adhesions sites contributes to the integrity of vascular endothelial barrier. Although focal adhesion kinase (FAK) activation is required for the recovery of the barrier function after increased endothelial junctional permeability, the basis for the recovery remains unclear. We tested the hypothesis that FAK activates p190RhoGAP and, thus, negatively regulates RhoA activity and promotes endothelial barrier restoration in response to the permeability-increasing mediator thrombin. We observed that thrombin caused a transient activation of RhoA but a more prolonged FAK activation temporally coupled to the recovery of barrier function. Thrombin also induced tyrosine phosphorylation of p190RhoGAP, which coincided with decrease in RhoA activity. We further showed that FAK was associated with p190RhoGAP, and importantly, recombinant FAK phosphorylated p190RhoGAP in vitro. Inhibition of FAK by adenoviral expression of FRNK (a dominant negative FAK construct) in monolayers prevented p190RhoGAP phosphorylation, increased RhoA activity, induced actin stress fiber formation, and produced an irreversible increase in endothelial permeability in response to thrombin. We also observed that p190RhoGAP was unable to attenuate RhoA activation in the absence of FAK activation induced by FRNK. The inhibition of RhoA by the C3 toxin (Clostridium botulinum toxin) restored endothelial barrier function in the FRNK-expressing cells. These findings in endothelial cells were recapitulated in the lung microcirculation in which FRNK expression in microvessel endothelia increased vascular permeability. Our studies demonstrate that FAK-induced down-modulation of RhoA activity via p190RhoGAP is a crucial step in signaling endothelial barrier restoration after increased endothelial permeability.     

Rapid quantitative characterization of protein interactions by composition gradient static light scattering

Two new applications of the recently developed technique of composition gradient static light scattering (CG-SLS) are presented. 1), The method is demonstrated to be capable of detecting and quantitatively characterizing reversible association of chymotrypsin and bovine pancreatic trypsin inhibitor in a solution mixture and simultaneously occurring reversible self-association of chymotrypsin at low pH in the same mixture. The values of equilibrium constants for both self- and heteroassociation may be determined with reasonable precision from the analysis of data obtained from a single experiment requiring <15 min and <1 mg of each protein. 2), Analysis of the results of a single CG-SLS experiment carried out on Ftsz, a protein that self-associates to form linear oligomers of indefinite size in the presence of guanosine diphosphate, yields the dependence of the equilibrium constant for monomer addition upon oligomer size.     

The p75(NTR) tumor suppressor induces cell cycle arrest facilitating caspase mediated apoptosis in prostate tumor cells

The p75 neurotrophin receptor (p75(NTR)) is a death receptor which belongs to the tumor necrosis factor receptor super-family of membrane proteins. This study shows that p75(NTR) retarded cell cycle progression by induced accumulation of cells in G0/G1 and a reduction in the S phase of the cell cycle. The rescue of tumor cells from cell cycle progression by a death domain deleted (DeltaDD) dominant-negative antagonist of p75(NTR) showed that the death domain transduced anti-proliferative activity in a ligand-independent manner. Conversely, addition of NGF ligand rescued retardation of cell cycle progression with commensurate changes in components of the cyclin/cdk holoenzyme complex. In the absence of ligand, p75(NTR)-dependent cell cycle arrest facilitated an increase in apoptotic nuclear fragmentation of the prostate cancer cells. Apoptosis of p75(NTR) expressing cells occurred via the intrinsic mitochondrial pathway leading to a sequential caspase-9 and -7 cascade. Since the death domain deleted dominant-negative antagonist of p75(NTR) rescued intrinsic caspase associated apoptosis in PC-3 cells, this shows p75(NTR) was integral to ligand independent induction of apoptosis. Moreover, the ability of ligand to ameliorate the p75(NTR)-dependent intrinsic apoptotic cascade indicates that NGF functioned as a survival factor for p75(NTR) expressing prostate cancer cells.     

Structure and activity analyses of Escherichia coli K-12 NagD provide insight into the evolution of biochemical function in the haloalkanoic acid dehalogenase superfamily

The HAD superfamily is a large superfamily of proteins which share a conserved core domain that provides those active site residues responsible for the chemistry common to all family members. The superfamily is further divided into the four subfamilies I, IIA, IIB, and III, based on the topology and insertion site of a cap domain that provides substrate specificity. This structural and functional division implies that members of a given HAD structural subclass may target substrates that have similar structural characteristics. To understand the structure/function relationships in all of the subfamilies, a type IIA subfamily member, NagD from Escherichia coli K-12, was selected (type I, IIB, and III members have been more extensively studied). The structure of the NagD protein was solved to 1.80 A with R(work) = 19.8% and R(free) = 21.8%. Substrate screening and kinetic analysis showed NagD to have high specificity for nucleotide monophosphates with k(cat)/K(m) = 3.12 x 10(4) and 1.28 x 10(4) microM(-)(1) s(-)(1) for UMP and GMP, respectively. This specificity is consistent with the presence of analogues of NagD that exist as fusion proteins with a nucleotide pyrophosphatase from the Nudix family. Docking of the nucleoside substrate in the active site brings it in contact with conserved residues from the cap domain that can act as a substrate specificity loop (NagD residues 144-149) in the type IIA subfamily. NagD and other subfamily IIA and IIB members show the common trait that substrate specificity and catalytic efficiencies (k(cat)/K(m)) are low (1 x 10(4) M(-)(1) s(-)(1)) and the boundaries defining physiological substrates are somewhat overlapping. The ability to catabolize other related secondary metabolites indicates that there is regulation at the genetic level.