Dengue fever, a neglected emerging disease for which no vaccine or antiviral agents exist at present, is caused by dengue virus, a member of the Flavivirus genus, which includes several important human pathogens, such as yellow fever and West Nile viruses. The NS5 protein from dengue virus is bifunctional and contains 900 amino acids. The S-adenosyl methionine transferase activity resides within its N-terminal domain, and residues 270 to 900 form the RNA-dependent RNA polymerase (RdRp) catalytic domain. Viral replication begins with the synthesis of minus-strand RNA from the dengue virus positive-strand RNA genome, which is subsequently used as a template for synthesizing additional plus-strand RNA genomes. This essential function for the production of new viral particles is catalyzed by the NS5 RdRp. Here we present a high-throughput in vitro assay partly recapitulating this activity and the crystallographic structure of an enzymatically active fragment of the dengue virus RdRp refined at 1.85-Å resolution. The NS5 nuclear localization sequences, previously thought to fold into a separate domain, form an integral part of the polymerase subdomains. The structure also reveals the presence of two zinc ion binding motifs. In the absence of a template strand, a chain-terminating nucleoside analogue binds to the priming loop site. These results should inform and accelerate the structure-based design of antiviral compounds against dengue virus.Flaviviridae are enveloped viruses with positive-strand RNA genomes that have been grouped into three genera, Hepacivirus, Pestivirus, and Flavivirus (11,59). Several members of the Flavivirus genus, e.g., dengue virus (DENV), yellow fever virus (YFV), Japanese encephalitis virus (JEV), tick-borne encephalitis virus, and West Nile virus (WNV), are medically important arthropod-borne pathogens afflicting humans. DENV infects 50 to 100 million people each year, with ϳ500,000 patients developing the more severe disease dengue hemorrhagic fever, leading to hospitalizations and resulting in approximately 20,000 deaths, mainly in children (24,26,27,29). Based on serological studies, DENVs are further classified into four distinct serotypes, DENV 1 to 4, whose respective genomes share ϳ60% sequence identity, with ϳ90% sequence identity within a serotype (7, 26). The DENV RNA genome spans about 10.7 kb and contains a type I methyl guanosine cap structure at its 5Ј end but is devoid of a polyadenylate tail. The genomic RNA is translated into a single polyprotein (58), which is cleaved into three structural (C-prM-E) and seven nonstructural (NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5) proteins by both the viral and cellular proteases (28). The viral serine protease is within the N-terminal region of NS3, and recent structural studies show that part of its catalytic site is formed by the viral cofactor NS2B upon substrate binding (18). The C-terminal region of NS3 forms the RNA helicase domain, which is thought to either separate a double-stranded RNA template into individual strands or disrupt secon...

Background:The intrinsically disordered protein ␣-synuclein, a hallmark of Parkinson disease, is involved in mitochondrial dysfunction in neurodegeneration and directly interacts with mitochondria. Results: ␣-Synuclein regulates VDAC permeability; ␣-synuclein toxicity in yeast depends on VDAC. Conclusion: ␣-Synuclein both blocks VDAC and translocates via this channel across the mitochondrial outer membrane. Significance: (Patho)physiological roles of monomeric ␣-synuclein may originate from its interaction with VDAC.

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The presynaptic protein ␣-synuclein (␣-syn), particularly in its amyloid form, is widely recognized for its involvement in Parkinson disease (PD). Recent genetic studies reveal that mutations in the gene GBA are the most widespread genetic risk factor for parkinsonism identified to date. GBA encodes for glucocerebrosidase (GCase), the enzyme deficient in the lysosomal storage disorder, Gaucher disease (GD). In this work, we investigated the possibility of a physical linkage between ␣-syn and GCase, examining both wild type and the GD-related N370S mutant enzyme. Using fluorescence and nuclear magnetic resonance spectroscopy, we determined that ␣-syn and GCase interact selectively under lysosomal solution conditions (pH 5.5) and mapped the interaction site to the ␣-syn C-terminal residues, 118 -137. This ␣-syn-GCase complex does not form at pH 7.4 and is stabilized by electrostatics, with dissociation constants ranging from 1.2 to 22 M in the presence of 25 to 100 mM NaCl. Intriguingly, the N370S mutant form of GCase has a reduced affinity for ␣-syn, as does the inhibitor conduritol-␤-epoxidebound enzyme. Immunoprecipitation and immunofluorescence studies verified this interaction in human tissue and neuronal cell culture, respectively. Although our data do not preclude protein-protein interactions in other cellular milieux, we suggest that the ␣-syn-GCase association is favored in the lysosome, and that this noncovalent interaction provides the groundwork to explore molecular mechanisms linking PD with mutant GBA alleles.

Mutations in GBA, the gene encoding glucocerebrosidase, the lysosomal enzyme deficient in Gaucher disease increase the risk for developing Parkinson disease. Recent research suggests a relationship between glucocerebrosidase and the Parkinson disease-related amyloid-forming protein, α-synuclein; however, the specific molecular mechanisms responsible for association remain elusive. Previously, we showed that α-synuclein and glucocerebrosidase interact selectively under lysosomal conditions, and proposed that this newly identified interaction might influence cellular levels of α-synuclein by either promoting protein degradation and/or preventing aggregation. Here, we demonstrate that membrane-bound α-synuclein interacts with glucocerebrosidase, and that this complex formation inhibits enzyme function. Using site-specific fluorescence and Förster energy transfer probes, we mapped the protein-enzyme interacting regions on unilamellar vesicles. Our data suggest that on the membrane surface, the glucocerebrosidase-α-synuclein interaction involves a larger α-synuclein region compared to that found in solution. In addition, α-synuclein acts as a mixed inhibitor with an apparent IC50 in the submicromolar range. Importantly, the membrane-bound, α-helical form of α-synuclein is necessary for inhibition. This glucocerebrosidase interaction and inhibition likely contribute to the mechanism underlying GBA-associated parkinsonism.

The Flavivirus NS5 protein possesses both (guanine-N7)-methyltransferase and nucleoside-2 -O methyltransferase activities required for sequential methylation of the cap structure present at the 5 end of the Flavivirus RNA genome. Seventeen mutations were introduced into the dengue virus type 2 NS5 methyltransferase domain, targeting amino acids either predicted to be directly involved in S-adenosyl-L-methionine binding or important for NS5 conformation and/or charged interactions. The effects of the mutations on (i) (guanine-N7)-methyltransferase and nucleoside-2 -O methyltransferase activities using biochemical assays based on a bacterially expressed NS5 methyltransferase domain and (ii) viral replication using a dengue virus type 2 infectious cDNA clone were examined. Clustered mutations targeting the S-adenosyl-L-methionine binding pocket or an active site residue abolished both methyltransferase activities and viral replication, demonstrating that both methyltransferase activities utilize a single S-adenosyl-L-methionine binding pocket. Substitutions to single amino acids binding S-adenosyl-L-methionine decreased both methyltransferase activities by varying amounts. However, viruses that replicated at wild type levels could be recovered with mutations that reduced both activities by >75%, suggesting that only a threshold level of methyltransferase activity was required for virus replication in vivo. Mutation of residues outside of regions directly involved in S-adenosyl-L-methionine binding or catalysis also affected methyltransferase activity and virus replication. The recovery of viruses containing compensatory second site mutations in the NS5 and NS3 proteins identified regions of the methyltransferase domain important for overall stability of the protein or likely to play a role in virus replication distinct from that of cap methylation.Cellular and many viral mRNAs contain a modified 5Ј-terminal guanosine "cap" structure covalently linked to the 5Ј end of the mRNA. The study of mRNA cap formation using viral, eukaryotic, and protozoan systems has shown that the formation of the 5Ј-RNA cap structure requires three sequential enzymatic reactions. First, the 5Ј-terminal triphosphate of the nascent RNA is hydrolyzed to a diphosphate by the enzyme RNA triphosphatase. Second, the RNA is capped with GMP in a 5Ј-5Ј triphosphate linkage by mRNA guanyltransferase, and third, the guanosine is methylated at the N 7 position by a (guanine-N7)-methyltransferase (N7 MTase) 2 using S-adenosyl-Lmethionine (AdoMet) as a methyl donor to form a type 0 ( 7Me G 5Ј -ppp 5Ј N) cap structure (1, 2). Nucleotides adjacent to the cap structure may be further methylated by nucleoside-2Ј-O methyltransferases (2Ј-O MTase) to give type I ( 7Me G 5Ј -ppp 5Ј N Me ) or type II cap structures ( 7Me G 5Ј -ppp 5Ј N Me N Me ). In the simplest case, such as for yeast, each of the enzymatic activities required for RNA capping resides in an individual protein. However, studies on viral capping systems have revealed many interesting variations o...

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Background: A specific interaction exists between ␣-synuclein and glucocerebrosidase on the lipid membrane, resulting in enzyme inhibition. Results: Binding glucocerebrosidase has a profound effect on ␣-synuclein, moving roughly half of its embedded helical region above the membrane plane. Conclusion: A model is proposed with structural insights into glucocerebrosidase inhibition by ␣-synuclein. Significance: ␣-Synuclein-glucocerebrosidase interaction provides a molecular connection between Parkinson and Gaucher diseases.