Exploration of Natural and Artificial Sequence Spaces: Towards a Functional Remodeling of Membrane-bound Cytochrome P450

Abstract

Two complementary methods are described that associate in vitro and in vivo steps to generate sequence diversity by segment directed saturated mutagenesis and family shuffling. A high-throughput DNA chip-based procedure for the characterization and potentially the equalization of combinatorial libraries is also presented. Using these approaches, two combinatorial libraries of cytochrome P450 variants derived from the CYP1A subfamily were constructed and their sequence diversity characterized. The results of functional screening using high-throughput tools for the characterization of membrane P450-catalyzed activities, suggest that the 204–214 sequence segment of human CYP1A1 is not critical for polycyclic aromatic hydrocarbon recognition, as was hypothesized from previous data. Moreover, mutations in this segment do not alter the discrimination between alkoxyresorufins, which, for all tested mutants, remained similar to that of wild-type CYP1A1. In contrast, the constructed CYP1A1–CYP1A2 mosaic structures, containing multiple crossovers, exhibit a wide range of substrate preference and regioselectivity. These mosaic structures also discriminate between closely related alkoxyresorufin substrates. These results open the way to global high-throughput analysis of structure–function relationships using combinatorial libraries of enzymes together with libraries of structurally related substrates.     

Residential Selection across the Life Course: Adolescent Contextual and Individual Determinants of Neighborhood Disadvantage in Mid-Adulthood

Background

Numerous cross-sectional studies have examined neighborhood effects on health. Residential selection in adulthood has been stressed as an important cause of selection bias but has received little empirical attention, particularly its determinants from the earlier life course. The present study aims to examine whether neighborhood, family, school, health behaviors and health in adolescence are related to socioeconomic disadvantage of one''s neighborhood of residence in adulthood.

Methods

Based on the prospective Northern Swedish Cohort (analytical N = 971, 90.6% retention rate), information was collected at age 16 years concerning family circumstances, school adjustment, health behaviors and mental and physical health. Neighborhood register data was linked to the cohort and used to operationalize aggregated measures of neighborhood disadvantage (ND) at age 16 and 42. Data was analyzed with linear mixed models, with ND in adulthood regressed on adolescent predictors and neighborhood of residence in adolescence as the level-2 unit.

Results

Neighborhood disadvantage in adulthood was clustered by neighborhood of residence in adolescence (ICC = 8.6%). The clustering was completely explained by ND in adolescence. Of the adolescent predictors, ND (b = .14 (95% credible interval = .07–.22)), final school marks (b = −.18 (−.26–−.10)), socioeconomic disadvantage (b = .07 (.01–.14)), and, with borderline significance, school peer problems (b = .07 (−.00–.13)), were independently related to adulthood ND in the final adjusted model. In sex-stratified analyses, the most important predictors were school marks (b = −.21 (−.32–−.09)) in women, and neighborhood of residence (ICC = 15.5%) and ND (b = .20 (.09–.31)) in men.

Conclusions

These findings show that factors from adolescence – which also may impact on adult health – could influence the neighborhood context in which one will live in adulthood. This indicates that residential selection bias in neighborhood effects on health research may have its sources in early life.     

Vibrio cholerae Evades Neutrophil Extracellular Traps by the Activity of Two Extracellular Nucleases

The Gram negative bacterium Vibrio cholerae is the causative agent of the secretory diarrheal disease cholera, which has traditionally been classified as a noninflammatory disease. However, several recent reports suggest that a V. cholerae infection induces an inflammatory response in the gastrointestinal tract indicated by recruitment of innate immune cells and increase of inflammatory cytokines. In this study, we describe a colonization defect of a double extracellular nuclease V. cholerae mutant in immunocompetent mice, which is not evident in neutropenic mice. Intrigued by this observation, we investigated the impact of neutrophils, as a central part of the innate immune system, on the pathogen V. cholerae in more detail. Our results demonstrate that V. cholerae induces formation of neutrophil extracellular traps (NETs) upon contact with neutrophils, while V. cholerae in return induces the two extracellular nucleases upon presence of NETs. We show that the V. cholerae wild type rapidly degrades the DNA component of the NETs by the combined activity of the two extracellular nucleases Dns and Xds. In contrast, NETs exhibit prolonged stability in presence of the double nuclease mutant. Finally, we demonstrate that Dns and Xds mediate evasion of V. cholerae from NETs and lower the susceptibility for extracellular killing in the presence of NETs. This report provides a first comprehensive characterization of the interplay between neutrophils and V. cholerae along with new evidence that the innate immune response impacts the colonization of V. cholerae in vivo. A limitation of this study is an inability for technical and physiological reasons to visualize intact NETs in the intestinal lumen of infected mice, but we can hypothesize that extracellular nuclease production by V. cholerae may enhance survival fitness of the pathogen through NET degradation.     

Functional Dissection of the Drosophila melanogaster Condensin Subunit Cap-G Reveals Its Exclusive Association with Condensin I

The heteropentameric condensin complexes have been shown to participate in mitotic chromosome condensation and to be required for unperturbed chromatid segregation in nuclear divisions. Vertebrates have two condensin complexes, condensin I and condensin II, which contain the same structural maintenance of chromosomes (SMC) subunits SMC2 and SMC4, but differ in their composition of non–SMC subunits. While a clear biochemical and functional distinction between condensin I and condensin II has been established in vertebrates, the situation in Drosophila melanogaster is less defined. Since Drosophila lacks a clear homolog for the condensin II–specific subunit Cap-G2, the condensin I subunit Cap-G has been hypothesized to be part of both complexes. In vivo microscopy revealed that a functional Cap-G-EGFP variant shows a distinct nuclear enrichment during interphase, which is reminiscent of condensin II localization in vertebrates and contrasts with the cytoplasmic enrichment observed for the other EGFP-fused condensin I subunits. However, we show that this nuclear localization is dispensable for Cap-G chromatin association, for its assembly into the condensin I complex and, importantly, for development into a viable and fertile adult animal. Immunoprecipitation analyses and complex formation studies provide evidence that Cap-G does not associate with condensin II–specific subunits, while it can be readily detected in complexes with condensin I–specific proteins in vitro and in vivo. Mass-spectrometric analyses of proteins associated with the condensin II–specific subunit Cap-H2 not only fail to identify Cap-G but also the other known condensin II–specific homolog Cap-D3. As condensin II–specific subunits are also not found associated with SMC2, our results question the existence of a soluble condensin II complex in Drosophila.     

Proteoglycans Act as Cellular Hepatitis Delta Virus Attachment Receptors

The hepatitis delta virus (HDV) is a small, defective RNA virus that requires the presence of the hepatitis B virus (HBV) for its life cycle. Worldwide more than 15 million people are co-infected with HBV and HDV. Although much effort has been made, the early steps of the HBV/HDV entry process, including hepatocyte attachment and receptor interaction are still not fully understood. Numerous possible cellular HBV/HDV binding partners have been described over the last years; however, so far only heparan sulfate proteoglycans have been functionally confirmed as cell-associated HBV attachment factors. Recently, it has been suggested that ionotrophic purinergic receptors (P2XR) participate as receptors in HBV/HDV entry. Using the HBV/HDV susceptible HepaRG cell line and primary human hepatocytes (PHH), we here demonstrate that HDV entry into hepatocytes depends on the interaction with the glycosaminoglycan (GAG) side chains of cellular heparan sulfate proteoglycans. We furthermore provide evidence that P2XR are not involved in HBV/HDV entry and that effects observed with inhibitors for these receptors are a consequence of their negative charge. HDV infection was abrogated by soluble GAGs and other highly sulfated compounds. Enzymatic removal of defined carbohydrate structures from the cell surface using heparinase III or the obstruction of GAG synthesis by sodium chlorate inhibited HDV infection of HepaRG cells. Highly sulfated P2XR antagonists blocked HBV/HDV infection of HepaRG cells and PHH. In contrast, no effect on HBV/HDV infection was found when uncharged P2XR antagonists or agonists were applied. In summary, HDV infection, comparable to HBV infection, requires binding to the carbohydrate side chains of hepatocyte-associated heparan sulfate proteoglycans as attachment receptors, while P2XR are not actively involved.     

Positive feedback favors invasion by a submersed freshwater plant

The submersed macrophyte Utricularia inflata has invaded lakes in northern New York State, thereby threatening native isoetids such as Eriocaulon aquaticum. Isoetids often dominate and modify softwater lakes due to their capacity to oxidize sediment and thus influence solute mobilization. Greenhouse experiments tested the hypotheses that U. inflata invasion could result in higher porewater iron (Fe) concentrations and greater ammonium (NH₄ ⁺) and Fe release from the sediment into the water column, and that this mobilization would stimulate further U. inflata growth. In the first experiment, three levels of U. inflata impact on E. aquaticum were imposed using sediment cores overlain by lake water: E. aquaticum alone, E. aquaticum with a cover of U. inflata, and bare sediment—the latter to simulate local extirpation of the isoetid by the invasive. After 16 weeks, sediment porewater NH₄ ⁺ and total dissolved Fe concentrations were significantly higher (P < 0.05) for the U. inflata and bare sediment treatments. Water column concentrations of these solutes were five-fold higher (P < 0.05) for the bare sediment treatment than E. aquaticum alone, indicating that isoetid extirpation by U. inflata can compromise water quality. A second experiment demonstrated that U. inflata grew faster over bare sediment than over sediment with E. aquaticum (P < 0.05), likely due to greater solute mobilization in the absence of E. aquaticum. Where U. inflata causes a decline of native isoetids in Adirondack Mountain lakes, changes to lake sediment and water chemistry can create a positive feedback loop further escalating the impact of this invasive species.     

STRONG SELECTION BARRIERS EXPLAIN MICROGEOGRAPHIC ADAPTATION IN WILD SALAMANDER POPULATIONS

Microgeographic adaptation occurs when populations evolve divergent fitness advantages across the spatial scales at which focal organisms regularly disperse. Although an increasing number of studies find evidence for microgeographic adaptation, the underlying causes often remain unknown. Adaptive divergence requires some combination of limited gene flow and strong divergent natural selection among populations. In this study, we estimated the relative influence of selection, gene flow, and the spatial arrangement of populations in shaping patterns of adaptive divergence in natural populations of the spotted salamander (Ambystoma maculatum). Within the study region, A. maculatum co‐occur with the predatory marbled salamander (Ambystoma opacum) in some ponds, and past studies have established a link between predation risk and adaptive trait variation in A. maculatum. Using 14 microsatellite loci, we found a significant pattern of genetic divergence among A. maculatum populations corresponding to levels of A. opacum predation risk. Additionally, A. maculatum foraging rate was strongly associated with predation risk, genetic divergence, and the spatial relationship of ponds on the landscape. Our results indicate the sorting of adaptive genotypes by selection regime and strongly suggest that substantial selective barriers operate against gene flow. This outcome suggests that microgeographic adaptation in A. maculatum is possible because strong antagonistic selection quickly eliminates maladapted phenotypes despite ongoing and substantial immigration. Increasing evidence for microgeographic adaptation suggests a strong role for selective barriers in counteracting the homogenizing influence of gene flow.     

Temperature and Carbon Assimilation Regulate the Chlorosome Biogenesis in Green Sulfur Bacteria

Green photosynthetic bacteria adjust the structure and functionality of the chlorosome—the light-absorbing antenna complex—in response to environmental stress factors. The chlorosome is a natural self-assembled aggregate of bacteriochlorophyll (BChl) molecules. In this study, we report the regulation of the biogenesis of the Chlorobaculum tepidum chlorosome by carbon assimilation in conjunction with temperature changes. Our studies indicate that the carbon source and thermal stress culture of C. tepidum grows slower and incorporates fewer BChl c in the chlorosome. Compared with the chlorosome from other cultural conditions we investigated, the chlorosome from the carbon source and thermal stress culture displays (a) smaller cross-sectional radius and overall size, (b) simplified BChl c homologs with smaller side chains, (c) blue-shifted Q_y absorption maxima, and (d) a sigmoid-shaped circular dichroism spectra. Using a theoretical model, we analyze how the observed spectral modifications can be associated with structural changes of BChl aggregates inside the chlorosome. Our report suggests a mechanism of metabolic regulation for chlorosome biogenesis.     

Context-dependent role of ATG4B as target for autophagy inhibition in prostate cancer therapy

ATG4B belongs to the autophagin family of cysteine proteases required for autophagy, an emerging target of cancer therapy. Developing pharmacological ATG4B inhibitors is a very active area of research. However, detailed studies on the role of ATG4B during anticancer therapy are lacking. By analyzing PC-3 and C4-2 prostate cancer cells overexpressing dominant negative ATG4B^C74Ain vitro and in vivo, we show that the effects of ATG4B^C74A are cell type, treatment, and context-dependent. ATG4B^C74A expression can either amplify the effects of cytotoxic therapies or contribute to treatment resistance. Thus, the successful clinical application of ATG4B inhibitors will depend on finding predictive markers of response.