Role of methyl groups in dynamics and evolution of biomolecules

Recent studies have discovered strong differences between the dynamics of nucleic acids (RNA and DNA) and proteins, especially at low hydration and low temperatures. This difference is caused primarily by dynamics of methyl groups that are abundant in proteins, but are absent or very rare in RNA and DNA. In this paper, we present a hypothesis regarding the role of methyl groups as intrinsic plasticizers in proteins and their evolutionary selection to facilitate protein dynamics and activity. We demonstrate the profound effect methyl groups have on protein dynamics relative to nucleic acid dynamics, and note the apparent correlation of methyl group content in protein classes and their need for molecular flexibility. Moreover, we note the fastest methyl groups of some enzymes appear around dynamical centers such as hinges or active sites. Methyl groups are also of tremendous importance from a hydrophobicity/folding/entropy perspective. These significant roles, however, complement our hypothesis rather than preclude the recognition of methyl groups in the dynamics and evolution of biomolecules.     

Chlamydia trachomatis Infection of Endocervical Epithelial Cells Enhances Early HIV Transmission Events

Chlamydia trachomatis causes a predominantly asymptomatic, but generally inflammatory, genital infection that is associated with an increased risk for HIV acquisition. Endocervical epithelial cells provide the major niche for this obligate intracellular bacterium in women, and the endocervix is also a tissue in which HIV transmission can occur. The mechanism by which CT infection enhances HIV susceptibility at this site, however, is not well understood. Utilizing the A2EN immortalized endocervical epithelial cell line grown on cell culture inserts, we evaluated the direct role that CT-infected epithelial cells play in facilitating HIV transmission events. We determined that CT infection significantly enhanced the apical-to-basolateral migration of cell-associated, but not cell-free, HIV_BaL, a CCR5-tropic strain of virus, across the endocervical epithelial barrier. We also established that basolateral supernatants from CT-infected A2EN cells significantly enhanced HIV replication in peripheral mononuclear cells and a CCR5+ T cell line. These results suggest that CT infection of endocervical epithelial cells could facilitate both HIV crossing the mucosal barrier and subsequent infection or replication in underlying target cells. Our studies provide a mechanism by which this common STI could potentially promote the establishment of founder virus populations and the maintenance of local HIV reservoirs in the endocervix. Development of an HIV/STI co-infection model also provides a tool to further explore the role of other sexually transmitted infections in enhancing HIV acquisition.     

Validation of signature polarlipid fatty acid biomarkers for alkane-utilizing bacteria in soils and subsurface aquifer materials

Abstract Extractable cell membrane-derived polarlipid ester-linked fatty acids (PLFA) obtained from aerated soils gassed with methane or propane and from methane- and propane-oxidizing bacteria isolated from the soils were analyzed by capillary gas chromatography/mass spectrometry. Exposure of aerated soils to methane resulted in the formation of a high proportion of an unusual 18-carbon mono-unsaturated PLFA, 18:lw8c. High proportions of this fatty acid biomarker are found in monocultures from this soil grown in minimal media with methane. This PLFA has been previously established as associated with authentic type II methane-oxidizing bacteria. The microbiota in aerated soils exposed to hydrocarbons containing propane, formed a suite of PLFA characterized by high proportions of a 16-carbon mono-unsaturated acid, 16:lw6c, and an 18-carbon saturated fatty acid with an additional methyl branch at the 10 position, 10 Me 18:0. This PLFA pattern has been detected in several monocultures enriched from the soil with propane-amended minimal media. The correspondence of high proportions of these unusual mono-unsaturated PLFA in the isolated monocultures and in situ in the soils after stimulation with the appropriate hydrocarbon is a strong validation of the utility of these biomarkers in defining the community structure of the surface soil microbial community.     

An optimized live bacterial delivery vehicle safely and efficaciously delivers bacterially transcribed therapeutic nucleic acids

There is an unmet need for delivery platforms that realize the full potential of next-generation nucleic acid therapeutics. The in vivo usefulness of current delivery systems is limited by numerous weaknesses, including poor targeting specificity, inefficient access to target cell cytoplasm, immune activation, off-target effects, small therapeutic windows, limited genetic encoding and cargo capacity, and manufacturing challenges. Here we characterize the safety and efficacy of a delivery platform comprising engineered live, tissue-targeting, non-pathogenic bacteria (Escherichia coli SVC1) for intracellular cargo delivery. SVC1 bacteria are engineered to specifically bind to epithelial cells via a surface-expressed targeting ligand, to allow escape of their cargo from the phagosome, and to have minimal immunogenicity. We describe SVC1's ability to deliver short hairpin RNA (shRNA), localized SVC1 administration to various tissues, and its minimal immunogenicity. To validate the therapeutic potential of SVC1, we used it to deliver influenza-targeting antiviral shRNAs to respiratory tissues in vivo. These data are the first to establish the safety and efficacy of this bacteria-based delivery platform for use in multiple tissue types and as an antiviral in the mammalian respiratory tract. We expect that this optimized delivery platform will enable a variety of advanced therapeutic approaches.     

Phosphatidylinositol 4-kinase from Saccharomyces cerevisiae. Kinetic analysis using Triton X-100/phosphatidylinositol-mixed micelles.

Phosphatidylinositol 4-kinase (ATP:phosphatidylinositol 4-phosphotransferase, EC 2.7.1.67) was purified from Saccharomyces cerevisiae by an improved procedure over that previously reported (Belunis, C.J., Bae-Lee, M., Kelley, M.J., and Carman, G.M. (1988) J. Biol. Chem. 263, 18897-18903) for the enzyme. The molecular mass of the enzyme was 45 kDa. The 35-kDa protein previously identified as PI 4-kinase was a proteolysis product of the 45-kDa protein. A detailed kinetic analysis of the purified enzyme was performed with Triton X-100/phosphatidylinositol-mixed micelles according to the "surface dilution" (Deems, R.A., Eaton, B.R., and Dennis, E.A. (1975) J. Biol. Chem. 250, 9013-9020) and "dual phospholipid" (Hendrickson, H.S., and Dennis, E.A. (1984) J. Biol. Chem. 259, 5734-5739) kinetic models. Phosphatidylinositol 4-kinase activity followed saturation kinetics with respect to the bulk and surface concentrations of phosphatidylinositol at concentrations of phosphatidylinositol below 0.1 mM. Above 0.1 mM activity was only dependent on the surface concentration of phosphatidylinositol. The enzyme more closely followed the dual phospholipid model where the enzyme associated with Triton X-100 micelles when phosphatidylinositol was present. The interfacial Michaelis constant (KmB) for phosphatidylinositol was 0.0036 mol fraction and the dissociation constant (KsA) for phosphatidylinositol in the micelle surface was 0.26 mM. The results of glycerol gradient centrifugation studies showed that the enzyme was physically associated with Triton X-100/phosphatidylinositol micelles.     

Distinctive Profile of IsomiR Expression and Novel MicroRNAs in Rat Heart Left Ventricle

MicroRNAs (miRNAs) are single-stranded non-coding RNAs that negatively regulate target gene expression through mRNA cleavage or translational repression. There is mounting evidence that they play critical roles in heart disease. The expression of known miRNAs in the heart has been studied at length by microarray and quantitative PCR but it is becoming evident that microRNA isoforms (isomiRs) are potentially physiologically important. It is well known that left ventricular (patho)physiology is influenced by transmural heterogeneity of cardiomyocyte phenotype, and this likely reflects underlying heterogeneity of gene expression. Given the significant role of miRNAs in regulating gene expression, knowledge of how the miRNA profile varies across the ventricular wall will be crucial to better understand the mechanisms governing transmural physiological heterogeneity. To determinine miRNA/isomiR expression profiles in the rat heart we investigated tissue from different locations across the left ventricular wall using deep sequencing. We detected significant quantities of 145 known rat miRNAs and 68 potential novel orthologs of known miRNAs, in mature, mature* and isomiR formation. Many isomiRs were detected at a higher frequency than their canonical sequence in miRBase and have different predicted targets. The most common miR-133a isomiR was more effective at targeting a construct containing a sequence from the gelsolin gene than was canonical miR-133a, as determined by dual-fluorescence assay. We identified a novel rat miR-1 homolog from a second miR-1 gene; and a novel rat miRNA similar to miR-676. We also cloned and sequenced the rat miR-486 gene which is not in miRBase (v18). Signalling pathways predicted to be targeted by the most highly detected miRNAs include Ubiquitin-mediated Proteolysis, Mitogen-Activated Protein Kinase, Regulation of Actin Cytoskeleton, Wnt signalling, Calcium Signalling, Gap junctions and Arrhythmogenic Right Ventricular Cardiomyopathy. Most miRNAs are not expressed in a gradient across the ventricular wall, with exceptions including miR-10b, miR-21, miR-99b and miR-486.     

Evidence for contemporary and historical gene flow between guppy populations in different watersheds,with a test for associations with adaptive traits

In dendritic river systems, gene flow is expected to occur primarily within watersheds. Yet, rare cross‐watershed transfers can also occur, whether mediated by (often historical) geological events or (often contemporary) human activities. We explored these events and their potential evolutionary consequences by analyzing patterns of neutral genetic variation (microsatellites) and adaptive phenotypic variation (male color) in wild guppies (Poecilia reticulata) distributed across two watersheds in northern Trinidad. We found the expected signatures of within‐watershed gene flow; yet we also inferred at least two instances of cross‐watershed gene flow—one in the upstream reaches and one further downstream. The upstream cross‐watershed event appears to be very recent (41 ± 13 years), suggesting dispersal via recent flooding or undocumented human‐mediated transport. The downstream cross‐watershed event appears to be considerably older (577 ± 265 years), suggesting a role for rare geological or climatological events. Alongside these strong signatures of both contemporary and historical gene flow, we found little evidence of impacts on presumably adaptive phenotypic differentiation, except perhaps in the one instance of very recent cross‐watershed gene flow. Selection in this system seems to overpower gene flow—at least on the spatiotemporal scales investigated here.     

Salmonella SPI1 effector SipA persists after entry and cooperates with a SPI2 effector to regulate phagosome maturation and intracellular replication

Salmonellae employ two type III secretion systems (T3SSs), SPI1 and SPI2, to deliver virulence effectors into mammalian cells. SPI1 effectors, including actin-binding SipA, trigger initial bacterial uptake, whereas SPI2 effectors promote subsequent replication within customized Salmonella-containing vacuoles (SCVs). SCVs sequester actin filaments and subvert microtubule-dependent motors to migrate to the perinuclear region. We demonstrate that SipA delivery continues after Salmonella internalization, with dosage being restricted by host-mediated degradation. SipA is exposed on the cytoplasmic face of the SCV, from where it stimulates bacterial replication in both nonphagocytic cells and macrophages. Although SipA is sufficient to target and redistribute late endosomes, during infection it cooperates with the SPI2 effector SifA to modulate SCV morphology and ensure perinuclear positioning. Our findings define an unexpected additional function for SipA postentry and reveal precise intracellular communication between effectors deployed by distinct T3SSs underlying SCV biogenesis.     

Cofactor requirements and reconstitution of microcin B17 synthetase: a multienzyme complex that catalyzes the formation of oxazoles and thiazoles in the antibiotic microcin B17

In the maturation of the Escherichia coli antibiotic Microcin B17 (MccB17), the McbA prepro-antibiotic is modified post-translationally by the multimeric microcin synthetase complex (composed of the McbB, -C, and -D proteins), which cyclizes four cysteines and four serines to thiazoles and oxazoles, respectively. Herein, we report the purification of individual subunits of MccB17 synthetase as fusions to maltose binding protein (MBP), and the in vitro reconstitution of heterocyclization activity. Preliminary characterization of each subunit reveals McbB to be a zinc-containing protein that may catalyze the initial cyclodehydration step, and McbC to contain flavin, consistent with an anticipated role for a dehydrogenase. We have previously demonstrated that McbD is a regulated ATPase/GTPase that may function as a conformational switch. Photolabeling experiments with the McbA propeptide now identify McbD as the initial site of substrate recognition. Heterocyclization activity was reconstituted only by combining all three subunits, demonstrating that each protein is required for heterocycle formation. Titration assays indicate that the subunits bind to each other with at least micromolar affinities, although McbD affords activity only after the MBP tag is proteolytically removed. Subunit competition assays with an McbDD147A mutant, which yields a catalytically deficient synthetase in vivo, show it to be defective in complex formation, whereas the McbBC181A/C184A double mutant, which is also inactive, competitively inhibits reconstitution by native McbB. Addition of the HtpG chaperone (originally shown to copurify with MccB17 synthetase), does not stimulate synthetase reconstitution or heterocyclization activity in vitro. A model for synthetase activity is proposed.