Conflict of interest: HBG consults for and is on the advisory board of Aridis, a biotech company that has done preclinical research on novel delivery systems for rotavirus vaccines. He has provided educational lectures for Abbot, a pharmaceutical company that markets a licensed rotavirus vaccine in India.
Many RNA viruses replicate in cytoplasmic compartments (virus factories or viroplasms) composed of viral and cellular proteins, but the mechanisms required for their formation remain largely unknown. Rotavirus (RV) replication in viroplasms requires interactions between virus nonstructural proteins NSP2 and NSP5, which are associated with components of lipid droplets (LDs). We previously identified two forms of NSP2 in RV-infected cells, a cytoplasmically dispersed form (dNSP2) and a viroplasm-specific form (vNSP2), which interact with hypophosphorylated and hyperphosphorylated NSP5, respectively, indicating that a coordinated phosphorylation cascade controls viroplasm assembly. The cellular kinase CK1α phosphorylates NSP2 on serine 313, triggering the localization of vNSP2 to sites of viroplasm assembly and its association with hyperphosphorylated NSP5. Using reverse genetics, we generated a rotavirus with a phosphomimetic NSP2 (S313D) mutation to directly evaluate the role of CK1α NSP2 phosphorylation in viroplasm formation. Recombinant rotavirus NSP2 S313D (rRV NSP2 S313D) is significantly delayed in viroplasm formation and in virus replication and interferes with wild-type RV replication in coinfection. Taking advantage of the delay in viroplasm formation, the NSP2 phosphomimetic mutant was used as a tool to observe very early events in viroplasm assembly. We show that (i) viroplasm assembly correlates with NSP5 hyperphosphorylation and (ii) vNSP2 S313D colocalizes with RV-induced LDs without NSP5, suggesting that vNSP2 phospho-S313 is sufficient for interacting with LDs and may be the virus factor required for RV-induced LD formation. Further studies with the rRV NSP2 S313D virus are expected to reveal new aspects of viroplasm and LD initiation and assembly. IMPORTANCE Reverse genetics was used to generate a recombinant rotavirus with a single phosphomimetic mutation in nonstructural protein 2 (NSP2 S313D) that exhibits delayed viroplasm formation, delayed replication, and an interfering phenotype during coinfection with wild-type rotavirus, indicating the importance of this amino acid during virus replication. Exploiting the delay in viroplasm assembly, we found that viroplasm-associated NSP2 colocalizes with rotavirus-induced lipid droplets prior to the accumulation of other rotavirus proteins that are required for viroplasm formation and that NSP5 hyperphosphorylation is required for viroplasm assembly. These data suggest that NSP2 phospho-S313 is sufficient for interaction with lipid droplets and may be the virus factor that induces lipid droplet biogenesis in rotavirus-infected cells. Lipid droplets are cellular organelles critical for the replication of many viral and bacterial pathogens, and thus, understanding the mechanism of NSP2-mediated viroplasm/lipid droplet initiation and interaction will lead to new insights into this important host-pathogen interaction.
Prenylated indole alkaloids featuring spirooxindole rings possess a 3R or 3S carbon stereocenter, which determines the bioactivities of these compounds. Despite the stereoselective advantages of spirooxindole biosynthesis compared with those of organic synthesis, the biocatalytic mechanism for controlling the 3R or 3S-spirooxindole formation has been elusive. Here, we report an oxygenase/semipinacolase CtdE that specifies the 3S-spirooxindole construction in the biosynthesis of 21R-citrinadin A. High-resolution X-ray crystal structures of CtdE with the substrate and cofactor, together with site-directed mutagenesis and computational studies, illustrate the catalytic mechanisms for the possible β-face epoxidation followed by a regioselective collapse of the epoxide intermediate, which triggers semipinacol rearrangement to form the 3S-spirooxindole. Comparing CtdE with PhqK, which catalyzes the formation of the 3R-spirooxindole, we reveal an evolutionary branch of CtdE in specific 3S spirocyclization. Our study provides deeper insights into the stereoselective catalytic machinery, which is important for the biocatalysis design to synthesize spirooxindole pharmaceuticals.
Recent advances in mass spectrometry (MS) have provided means for large-scale phosphoproteomic profiling of specific tissues. Here, we report results from large-scale tandem MS [liquid chromatography (LC)-MS/MS]-based phosphoproteomic profiling of biochemically isolated membranes from the renal cortex, with focus on transporters and regulatory proteins. Data sets were filtered (by target-decoy analysis) to limit false-positive identifications to <2%. A total of 7,125 unique nonphosphorylated and 743 unique phosphorylated peptides were identified. Among the phosphopeptides identified were sites on transporter proteins, i.e., solute carrier (Slc, n = 63), ATP-binding cassette (Abc, n = 4), and aquaporin (Aqp, n = 3) family proteins. Database searches reveal that a majority of the phosphorylation sites identified in transporter proteins were previously unreported. Most of the Slc family proteins are apical or basolateral transporters expressed in proximal tubule cells, including proteins known to mediate transport of glucose, amino acids, organic ions, and inorganic ions. In addition, we identified potentially important phosphorylation sites for transport proteins from distal nephron segments, including the bumetanide-sensitive Na-K-2Cl cotransporter (Slc12a1 or NKCC2) at Ser(87), Thr(101), and Ser(126) and the thiazide-sensitive Na-Cl cotransporter (Slc12a3 or NCC) at Ser(71) and Ser(124). A subset of phosphorylation sites in regulatory proteins coincided with known functional motifs, suggesting specific regulatory roles. An online database from this study (http://dir.nhlbi.nih.gov/papers/lkem/rcmpd/) provides a resource for future studies of transporter regulation.
Enteroaggregative Escherichia coli (EAEC) is a significant cause of acute and chronic diarrhea, foodborne outbreaks, infections of the immunocompromised, and growth stunting in children in developing nations. There is no vaccine and resistance to antibiotics is rising. Unlike related E. coli pathotypes that are often associated with acute bouts of infection, EAEC is associated with persistent diarrhea and subclinical long-term colonization. Several secreted virulence factors have been associated with EAEC pathogenesis and linked to disease in humans, less certain are the molecular drivers of adherence to the intestinal mucosa. We previously established human intestinal enteroids (HIEs) as a model system to study host-EAEC interactions and aggregative adherence fimbriae A (AafA) as a major driver of EAEC adherence to HIEs. Here, we report a large-scale assessment of the host response to EAEC adherence from all four segments of the intestine across at least three donor lines for five E. coli pathotypes. The data demonstrate that the host response in the duodenum is driven largely by the infecting pathotype, whereas the response in the colon diverges in a patient-specific manner. Major pathways altered in gene expression in each of the four enteroid segments differed dramatically, with responses observed for inflammation, apoptosis and an overwhelming response to different mucin genes. In particular, EAEC both associated with large mucus droplets and specific mucins at the epithelial surface, binding
Profiling using high-throughput MS has discovered an overwhelming number of novel protein phosphorylation sites (“phosphosites”). However, the functional relevance of these sites is not always clear. In light of recent studies on the evolutionary mechanism of phosphorylation, we have developed CPhos, a Java program that can assess the conservation of phosphosites among species using an information theory-based approach. The degree of conservation established using CPhos can be used to assess the functional significance of phosphosites. CPhos has a user friendly graphical user interface and is available both as a web service and as a standalone Java application to assist phosphoproteomic researchers in analyzing and prioritizing lists of phosphosites for further experimental validation. CPhos can be accessed or downloaded at http://helixweb.nih.gov/CPhos/.
Human noroviruses (NoVs) are the main cause of epidemic and sporadic gastroenteritis. Phylogenetically, noroviruses are divided into seven genogroups, with each divided into multiple genotypes. NoVs belonging to genogroup II and genotype 4 (GII.4) are globally most prevalent. Genetic diversity among the NoVs and the periodic emergence of novel strains present a challenge for the development of vaccines and antivirals to treat NoV infection. NoV protease is essential for viral replication and is an attractive target for the development of antivirals. The available structure of GI.1 protease provided a basis for the design of inhibitors targeting the active site of the protease. These inhibitors, although potent against the GI proteases, poorly inhibit the GII proteases, for which structural information is lacking. To elucidate the structural basis for this difference in the inhibitor efficiency, we determined the crystal structure of a GII.4 protease. The structure revealed significant changes in the S2 substratebinding pocket, making it noticeably smaller, and in the active site, with the catalytic triad residues showing conformational changes. Furthermore, a conserved arginine is found inserted into the active site, interacting with the catalytic histidine and restricting substrate/inhibitor access to the S2 pocket. This interaction alters the relationships between the catalytic residues and may allow for a pHdependent regulation of protease activity. The changes we observed in the GII.4 protease structure may explain the reduced potency of the GI-specific inhibitors against the GII protease and therefore must be taken into account when designing broadly cross-reactive antivirals against NoVs. IMPORTANCE Human noroviruses (NoVs) cause sporadic and epidemic gastroenteritis worldwide. They are divided into seven genogroups (GI to GVII), with each genogroup further divided into several genotypes. Human NoVs belonging to genogroup II and genotype 4 (GII.4) are the most prevalent. Currently, there are no vaccines or antiviral drugs available for NoV infection. The protease encoded by NoV is considered a valuable target because of its essential role in replication. NoV protease structures have only been determined for the GI genogroup. We show here that the structure of the GII.4 protease exhibits several significant changes from GI proteases, including a unique pairing of an arginine with the catalytic histidine that makes the proteolytic activity of GII.4 protease pH sensitive. A comparative analysis of NoV protease structures may provide a rational framework for structure-based drug design of broadly cross-reactive inhibitors targeting NoVs.
Marine extremophiles produce cold-adapted and/or salt-tolerant enzymes to survive in harsh conditions. These enzymes are naturally evolved with unique structural features that confer a high level of flexibility, solubility and substrate-binding ability compared to mesophilic and thermostable homologs. Here, we identified and characterized an amylase, SdG5A, from the marine bacterium Saccharophagus degradans 2-40T. We expressed the protein in Bacillus subtilis and found that the purified SdG5A enabled highly specific production of maltopentaose, an important health-promoting food and nutrition component. Notably, SdG5A exhibited outstanding cold adaptation and salt tolerance, retaining approximately 30 and 70% of its maximum activity at 4°C and in 3 M NaCl, respectively. It converted 68 and 83% of starch into maltooligosaccharides at 4 and 25°C, respectively, within 24 h, with 79% of the yield being the maltopentaose. By analyzing the structure of SdG5A, we found that the C-terminal carbohydrate-binding module (CBM) coupled with an extended linker, displayed a relatively high negative charge density and superior conformational flexibility compared to the whole protein and the catalytic domain. Consistent with our bioinformatics analysis, truncation of the linker-CBM region resulted in a significant loss in activities at low temperature and high salt concentration. This highlights the linker-CBM acting as the critical component for the protein to carry out its activity in biologically unfavorable condition. Together, our study indicated that these unique properties of SdG5A have great potential for both basic research and industrial applications in food, biology, and medical and pharmaceutical fields.
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