Inhibition of diverse opportunistic viruses by structurally optimized retrograde trafficking inhibitors

Opportunistic viruses are a major problem for immunosuppressed individuals, particularly following organ or stem cell transplantation. Current treatments are non-existent or suffer from problems such as high toxicity or development of resistant strains. We previously published that a trafficking inhibitor that targets a host protein greatly reduces the replication of human cytomegalovirus. This inhibitor was also shown to be moderately effective against polyomaviruses, another family of opportunistic viruses. We have developed a panel of analogues for this inhibitor and have shown that these analogues maintain their high efficacy against HCMV, while substantially lowering the concentration required to inhibit polyomavirus replication. By targeting a host protein these compounds are able to inhibit the replication of two very different viruses. These observations open up the possibility of pan-viral inhibitors for immunosuppressed individuals that are effective against multiple, diverse opportunistic viruses.     

Functional analysis tools for post‐translational modification: a post‐translational modification database for analysis of proteins and metabolic pathways

Post‐translational modifications (PTMs) are critical regulators of protein function, and nearly 200 different types of PTM have been identified. Advances in high‐resolution mass spectrometry have led to the identification of an unprecedented number of PTM sites in numerous organisms, potentially facilitating a more complete understanding of how PTMs regulate cellular behavior. While databases have been created to house the resulting data, most of these resources focus on individual types of PTM, do not consider quantitative PTM analyses or do not provide tools for the visualization and analysis of PTM data. Here, we describe the Functional Analysis Tools for Post‐Translational Modifications (FAT‐PTM) database ( https://bioinformatics.cse.unr.edu/fat-ptm/ ), which currently supports eight different types of PTM and over 49 000 PTM sites identified in large‐scale proteomic surveys of the model organism Arabidopsis thaliana. The FAT‐PTM database currently supports tools to visualize protein‐centric PTM networks, quantitative phosphorylation site data from over 10 different quantitative phosphoproteomic studies, PTM information displayed in protein‐centric metabolic pathways and groups of proteins that are co‐modified by multiple PTMs. Overall, the FAT‐PTM database provides users with a robust platform to share and visualize experimentally supported PTM data, develop hypotheses related to target proteins or identify emergent patterns in PTM data for signaling and metabolic pathways.     

Impact of grazing,resource availability and light on prokaryotic growth and diversity in the oligotrophic surface global ocean

The impact of grazing, resource competition and light on prokaryotic growth and taxonomic composition in subtropical and tropical surface waters were studied through 10 microcosm experiments conducted between 30°N and 30°S in the Atlantic, Pacific and Indian oceans. Under natural sunlight conditions, significant changes in taxonomic composition were only observed after the reduction of grazing by sample filtration in combination with a decrease in resource competition by sample dilution. Sunlight exposure significantly reduced prokaryote growth (11 ± 6%) and community richness (14 ± 4%) compared to continuous darkness but did not significantly change community composition. The largest growth inhibition after sunlight exposure occurred at locations showing deep mixed layers. The reduction of grazing had an expected and significant positive effect on growth, but caused a significant decrease in community richness (16 ± 6%), suggesting that the coexistence of many different OTUs is partly promoted by the presence of predators. Dilution of the grazer-free prokaryotic community significantly enhanced growth at the level of community, but consistently and sharply reduced the abundance of Prochlorococcus and SAR11 populations. The decline of these oligotrophic bacterial taxa following an increase in resource availability is consistent with their high specialization for exploiting the limited resources available in the oligotrophic warm ocean.     

Role of electrostatic interactions in amyloid beta-protein (A beta) oligomer formation: a discrete molecular dynamics study

Pathological folding and oligomer formation of the amyloid beta-protein (A beta) are widely perceived as central to Alzheimer's disease. Experimental approaches to study A beta self-assembly provide limited information because most relevant aggregates are quasi-stable and inhomogeneous. We apply a discrete molecular dynamics approach combined with a four-bead protein model to study oligomer formation of A beta. We address the differences between the two most common A beta alloforms, A beta 40 and A beta 42, which oligomerize differently in vitro. Our previous study showed that, despite simplifications, our discrete molecular dynamics approach accounts for the experimentally observed differences between A beta 40 and A beta 42 and yields structural predictions amenable to in vitro testing. Here we study how the presence of electrostatic interactions (EIs) between pairs of charged amino acids affects A beta 40 and A beta 42 oligomer formation. Our results indicate that EIs promote formation of larger oligomers in both A beta 40 and A beta 42. Both A beta 40 and A beta 42 display a peak at trimers/tetramers, but A beta 42 displays additional peaks at nonamers and tetradecamers. EIs thus shift the oligomer size distributions to larger oligomers. Nonetheless, the A beta 40 size distribution remains unimodal, whereas the A beta 42 distribution is trimodal, as observed experimentally. We show that structural differences between A beta 40 and A beta 42 that already appear in the monomer folding, are not affected by EIs. A beta 42 folded structure is characterized by a turn in the C-terminus that is not present in A beta 40. We show that the same C-terminal region is also responsible for the strongest intermolecular contacts in A beta 42 pentamers and larger oligomers. Our results suggest that this C-terminal region plays a key role in the formation of A beta 42 oligomers and the relative importance of this region increases in the presence of EIs. These results suggest that inhibitors targeting the C-terminal region of A beta 42 oligomers may be able to prevent oligomer formation or structurally modify the assemblies to reduce their toxicity.     

Decreased pCO(2) accumulation by eliminating bicarbonate addition to high cell-density cultures

High-density perfusion cultivation of mammalian cells can result in elevated bioreactor CO(2) partial pressure (pCO(2)), a condition that can negatively influence growth, metabolism, productivity, and protein glycosylation. For BHK cells in a perfusion culture at 20 x 10(6) cells/mL, the bioreactor pCO(2) exceeded 225 mm Hg with approximate contributions of 25% from cellular respiration, 35% from medium NaHCO(3), and 40% from NaHCO(3) added for pH control. Recognizing the limitations to the practicality of gas sparging for CO(2) removal in perfusion systems, a strategy based on CO(2) reduction at the source was investigated. The NaHCO(3) in the medium was replaced with a MOPS-Histidine buffer, while Na(2)CO(3) replaced NaHCO(3) for pH control. These changes resulted in 63-70% pCO(2) reductions in multiple 15 L perfusion bioreactors, and were reproducible at the manufacturing-scale. Bioreactor pCO(2) values after these modifications were in the 68-85 mm Hg range, pCO(2) reductions consistent with those theoretically expected. Low bioreactor pCO(2) was accompanied by both 68-123% increased growth rates and 58-92% increased specific productivity. Bioreactor pCO(2) reduction and the resulting positive implications for cell growth and productivity were brought about by process changes that were readily implemented and robust. This philosophy of pCO(2) reduction at the source through medium and base modification should be readily applicable to large-scale fed-batch cultivation of mammalian cells.     

The ATPase activity of the DNA transporter TrwB is modulated by protein TrwA: implications for a common assembly mechanism of DNA translocating motors

Conjugative systems contain an essential integral membrane protein involved in DNA transport called the Type IV coupling protein (T4CP). The T4CP of conjugative plasmid R388 is TrwB, a DNA-dependent ATPase. Biochemical and structural data suggest that TrwB uses energy released from ATP hydrolysis to pump DNA through its central channel by a mechanism similar to that used by F1-ATPase or ring helicases. For DNA transport, TrwB couples the relaxosome (a DNA-protein complex) to the secretion channel. In this work we show that TrwA, a tetrameric oriT DNA-binding protein and a component of the R388 relaxosome, stimulates TrwBDeltaN70 ATPase activity, revealing a specific interaction between the two proteins. This interaction occurs via the TrwA C-terminal domain. A 68-kDa complex between TrwBDeltaN70 and TrwA C-terminal domain was observed by gel filtration chromatography, consistent with a 1:1 stoichiometry. Additionally, electron microscopy revealed the formation of oligomeric TrwB complexes in the presence, but not in the absence, of TrwA protein. TrwBDeltaN70 ATPase activity in the presence of TrwA was further enhanced by DNA. Interestingly, maximal ATPase rates were achieved with TrwA and different types of dsDNA substrates. This is consistent with a role of TrwA in facilitating the interaction between TrwB and DNA. Our findings provide a new insight into the mechanism by which TrwB recruits the relaxosome for DNA transport. The process resembles the mechanism used by other DNA-dependent molecular motors, such as the RuvA/RuvB system, to be targeted to the DNA followed by hexamer assembly.     

Inflammation: a way to understanding the evolution of portal hypertension

Background

Portal hypertension is a clinical syndrome that manifests as ascites, portosystemic encephalopathy and variceal hemorrhage, and these alterations often lead to death.

Hypothesis

Splanchnic and/or systemic responses to portal hypertension could have pathophysiological mechanisms similar to those involved in the post-traumatic inflammatory response. The splanchnic and systemic impairments produced throughout the evolution of experimental prehepatic portal hypertension could be considered to have an inflammatory origin. In portal vein ligated rats, portal hypertensive enteropathy, hepatic steatosis and portal hypertensive encephalopathy show phenotypes during their development that can be considered inflammatory, such as: ischemia-reperfusion (vasodilatory response), infiltration by inflammatory cells (mast cells) and bacteria (intestinal translocation of endotoxins and bacteria) and lastly, angiogenesis. Similar inflammatory phenotypes, worsened by chronic liver disease (with anti-oxidant and anti-enzymatic ability reduction) characterize the evolution of portal hypertension and its complications (hepatorenal syndrome, ascites and esophageal variceal hemorrhage) in humans.

Conclusion

Low-grade inflammation, related to prehepatic portal hypertension, switches to high-grade inflammation with the development of severe and life-threatening complications when associated with chronic liver disease.     

Xeroderma pigmentosum complementation group A protein is driven to nucleotide excision repair sites by the electrostatic potential of distorted DNA

The presumed DNA-binding cleft of xeroderma pigmentosum group A (XPA) protein, a key regulatory subunit of the eukaryotic nucleotide excision repair complex, displays a distinctive array of 6 positively charged amino acid side chains. Here, the molecular function of these closely spaced electropositive residues has been tested by systematic site-directed mutagenesis. After the introduction of single amino acid substitutions, the mutants were probed for protein-DNA interactions in electrophoretic mobility shift and photochemical crosslinking assays. This analysis led to the identification of a critical hot-spot for DNA substrate recognition composed of two neighboring lysines at codons 141 and 179 of the human XPA sequence. The replacement of other basic side chains in the DNA interaction domain conferred more moderate defects of substrate binding. When the function of XPA was tested as a fusion product with either mCherry or green-fluorescent protein, a glutamate substitution of one of the positively charged residues at positions 141 and 179 was sufficient to decrease DNA repair activity in human fibroblasts. Thus, the removal of a single cationic side chain abolished DNA-binding activity and significant excision repair defects could be induced by single charge inversions on the XPA surface, indicating that this molecular sensor participates in substrate recognition by monitoring the electrostatic potential of distorted DNA repair sites.