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.     

The (in)dependence of alternative splicing and gene duplication

Alternative splicing (AS) and gene duplication (GD) both are processes that diversify the protein repertoire. Recent examples have shown that sequence changes introduced by AS may be comparable to those introduced by GD. In addition, the two processes are inversely correlated at the genomic scale: large gene families are depleted in splice variants and vice versa. All together, these data strongly suggest that both phenomena result in interchangeability between their effects. Here, we tested the extent to which this applies with respect to various protein characteristics. The amounts of AS and GD per gene are anticorrelated even when accounting for different gene functions or degrees of sequence divergence. In contrast, the two processes appear to be independent in their influence on variation in mRNA expression. Further, we conducted a detailed comparison of the effect of sequence changes in both alternative splice variants and gene duplicates on protein structure, in particular the size, location, and types of sequence substitutions and insertions/deletions. We find that, in general, alternative splicing affects protein sequence and structure in a more drastic way than gene duplication and subsequent divergence. Our results reveal an interesting paradox between the anticorrelation of AS and GD at the genomic level, and their impact at the protein level, which shows little or no equivalence in terms of effects on protein sequence, structure, and function. We discuss possible explanations that relate to the order of appearance of AS and GD in a gene family, and to the selection pressure imposed by the environment.     

Electrophysiological characterization of Tityus obscurus β toxin 1 (To1) on Na+-channel isoforms

To1, previously named Tc49b, is a peptide neurotoxin isolated from venom of the scorpion Tityus obscurus that is responsible for lethal human poisoning cases in the Brazilian Amazonian region. Previously, To1 was shown to be lethal to mice and to change Na⁺ permeation in cerebellum granular neurons from rat brain. In addition, To1 did not affect Shaker B K⁺ channels. Based on sequence similarities, To1 was described as a β-toxin. In the present work, To1 was purified from T. obscurus venom and submitted to an electrophysiological characterization in human and invertebrate Na_V channels. The analysis of the electrophysiological experiments reveal that To1 enhances the open probability at more negative potentials of human Na_V 1.3 and 1.6, of the insect channel BgNa_V1 and of arachnid VdNa_V1 channel. In addition, To1 reduces the peak of Na⁺ currents in some of the Na_Vs tested. These results support the classification of the To1 as a β-toxin. A structure and functional comparison to other β-toxins that share sequence similarity to To1 is also presented.     

Cats under cover: Habitat models indicate a high dependency on woodlands by Atlantic Forest felids

Four Neotropical small and medium felids—the ocelot, jaguarundi, margay, and southern tiger cat—have overlapping geographic distributions in the endangered Atlantic Forest. Local studies show that these felids avoid areas with high human impact, but the three smaller ones use human‐modified areas more frequently than do ocelots. To understand how landscape changes affect the habitat distribution of these four felids in the Atlantic Forest of Argentina, we used maximum entropy models to analyze the effect of environmental and anthropogenic factors. We estimated niche breadth and overlap among these felids. The conversion of the native forest to land uses without trees was the most important variable that determined the habitat distribution of the four species. For all four species, the optimal habitat covered about 1/3 of the study area and corresponds mainly to the native forest areas. Nearly 50% of these areas had some level of protection. The niche width was higher for the small felids than for ocelots. Niche overlap was high for all species pairs, but higher among the small felids and lower for each of these with the ocelot. The four felids were negatively affected by native forest loss, with ocelots being more sensitive than the smaller felids. The conversion of unprotected forest areas to other types of land uses would imply a greater habitat loss for these felids. The protection of current remnants of Atlantic Forest in Argentina is important for the long‐term conservation of the four felids. Abstract in Spanish is available with online material.     

Trophic niche changes associated with habitat fragmentation in a Neotropical bat species

Habitat fragmentation could alter ecological traits including species trophic habits. Here, we used carbon and nitrogen stable isotope ratios to establish differences in isotopic niche width and food resource use between forest fragments and the continuous forest for the phyllostomid frugivorous bat Artibeus lituratus. Using mist nests, we captured bats from two forest fragments and two sites in continuous forest, and sampled from each individual captured three body tissues with contrasting turnover rates (skin, muscle, and liver). Samples were collected between February and March (austral summer) and between August and September (austral winter). In addition, in each sampling site and season we collected potential food resources (fruits and insects) consumed by our A. lituratus. Our findings indicate that A. lituratus had a predominantly omnivorous diet, with high consumption of insects during summer in forest fragments. The increasing consumption of insects in these fragments seems to have led to a wider isotopic niche, in relation to the continuous forest. Because A. lituratus is typically a seed disperser, changes in trophic habits in the forest fragments from frugivory to insectivory may diminish their role in forest regeneration. Abstract in Portuguese is available with online material.     

Molecular dynamics simulation and binding free energy studies of novel leads belonging to the benzofuran class inhibitors of Mycobacterium tuberculosis Polyketide Synthase 13

In this work, the binding mechanism of new Polyketide Synthase 13 (Pks13) inhibitors has been studied through molecular dynamics simulation and free energy calculations. The drug Tam1 and its analogs, belonging to the benzofuran class, were submitted to 100 ns simulations, and according to the results obtained for root mean square deviation, all the simulations converged from approximately 30 ns. For the analysis of backbone flotation, the root mean square fluctuations were plotted for the Cα atoms; analysis revealed that the greatest fluctuation occurred in the residues that are part of the protein lid domain. The binding free energy value (ΔG_bind) obtained for the Tam16 lead molecule was of ?51.43 kcal/mol. When comparing this result with the ΔG_bind values for the remaining analogs, the drug Tam16 was found to be the highest ranked: this result is in agreement with the experimental results obtained by Aggarwal and collaborators, where it was verified that the IC₅₀ for Tam16 is the smallest necessary to inhibit the Pks13 (IC₅₀ = 0.19 μM). The energy decomposition analysis suggested that the residues which most interact with inhibitors are: Ser1636, Tyr1637, Asn1640, Ala1667, Phe1670, and Tyr1674, from which the greatest energy contribution to Phe1670 was particularly notable. For the lead molecule Tam16, a hydrogen bond with the hydroxyl of the phenol not observed in the other analogs induced a more stable molecular structure. Aggarwal and colleagues reported this hydrogen bonding as being responsible for the stability of the molecule, optimizing its physic-chemical, toxicological, and pharmacokinetic properties.     

Sperm protein (sp50) binds to acromosome and plasma membranes in a Ca2+-dependent manner: Possible role in acrosome reaction

Annexins are a family of Ca²⁺-binding proteins involved in the exocytotic process. The presence and the role of annexins in mammalian spermatozoa have not been well established. Two annexin-like proteins were obtained from guinea pig testis, a doublet of Mr 31–33 kD (p31/33) and a protein of Mr 50 kD (p50). Both proteins were able to bind to erythrocyte ghosts in a Ca²⁺-dependent fashion. Polyclonal antibodies against p31/33 reacted with two major proteins, Mrs 50 kD (sp50) and 42 kD (sp42), from mature and immature guinea pig spermatozoa. p50 and sp50 are likely the native proteins from testis and spermatozoa, respectively, and they are seemingly related. By immunofluorescence, sp50 was only found in the apical acrosome region of immature and capacitated and noncapacitated spermatozoa, and its location was intracellular. In spermatozoa undergoing acrosome reaction, sp50 was detected in the whole acrosome, while in spermatozoa that had undergone acrosome reaction sp50 was not detected. However, in the protein pattern of acrosome reaction vesicles, anti-p31/33 antibody revealed diffuse bands of Mr 35–38 kD. sp50 was able to bind to plasma membrane fragments and acrosome outer membrane from demembranated sperm in a Ca²⁺-dependent fashion. The presence of sp50 in the acrosome region, its distribution throughout the acrosome membrane just before the acrosome reaction, and its ability to bind both plasma and outer acrosome membranes in a Ca²⁺-dependent manner suggest that sp50 may participate in the acrosome reaction mechanism in guinea pig spermatozoa. © 1996 Wiley-Liss, Inc.