Inner mitochondrial translocase Tim50 interacts with 3β-hydroxysteroid dehydrogenase type 2 to regulate adrenal and gonadal steroidogenesis

In the adrenals, testes, and ovaries, 3β-hydroxysteroid dehydrogenase type 2 (3βHSD2) catalyzes the conversion of pregnenolone to progesterone and dehydroepiandrostenedione to androstenedione. Alterations in this pathway can have deleterious effects, including sexual development impairment, spontaneous abortion, and breast cancer. 3βHSD2, synthesized in the cytosol, is imported into the inner mitochondrial membrane (IMM) by translocases. Steroidogenesis requires that 3βHSD2 acts as both a dehydrogenase and isomerase. To achieve this dual functionality, 3βHSD2 must undergo a conformational change; however, what triggers that change remains unknown. We propose that 3βHSD2 associates with IMM or outer mitochondrial membrane translocases facing the intermembrane space (IMS) and that this interaction promotes the conformational change needed for full activity. Fractionation assays demonstrate that 3βHSD2 associated with the IMM but did not integrate into the membrane. Through mass spectrometry and Western blotting of mitochondrial complexes and density gradient ultracentrifugation, we show that that 3βHSD2 formed a transient association with the translocases Tim50 and Tom22 and with Tim23. This association occurred primarily through the interaction of Tim50 with the N terminus of 3βHSD2 and contributed to enzymatic activity. Tim50 knockdown inhibited catalysis of dehydroepiandrostenedione to androstenedione and pregnenolone to progesterone. Although Tim50 knockdown decreased 3βHSD2 expression, restoration of expression via proteasome and protease inhibition did not rescue activity. In addition, protein fingerprinting and CD spectroscopy reveal the flexibility of 3βHSD2, a necessary characteristic for forming multiple associations. In summary, Tim50 regulates 3βHSD2 expression and activity, representing a new role for translocases in steroidogenesis.     

The N terminus of the adhesion G protein-coupled receptor GPR56 controls receptor signaling activity

GPR56 is an adhesion G protein-coupled receptor that plays a key role in cortical development. Mutations to GPR56 in humans cause malformations of the cerebral cortex, but little is known about the normal function of the receptor. We found that the large N terminus (NT) of GPR56 is cleaved from the rest of the receptor during processing but remains non-covalently associated with the seven-transmembrane region of the receptor, as indicated by coimmunoprecipitation of the two GPR56 fragments from both transfected cells and native tissue. We also found that truncation of the GPR56 NT results in constitutive activation of receptor signaling, as revealed by increased GPR56-stimulated signaling upon transfection of HEK-293 cells with truncated GPR56, greatly enhanced binding of β-arrestins by truncated GPR56 relative to the full-length receptor, extensive ubiquitination of truncated GPR56, and cytotoxicity induced by truncated GPR56 that could be rescued by cotransfection of cells with β-arrestin 2. Furthermore, we found that the GPR56 NT is capable of homophilic trans-trans interactions that enhance receptor signaling activity. On the basis of these findings, we suggest a model of receptor activation in which the large N terminus of GPR56 constrains receptor activity but N-terminal interactions (GPR56 NT with an extracellular ligand and/or GPR56 NT homophilic trans-trans associations) can remove this inhibitory influence of the N terminus to activate receptor signaling.     

Protein kinase Cα promotes cell migration through a PDZ-dependent interaction with its novel substrate discs large homolog 1 (DLG1)

Protein scaffolds maintain precision in kinase signaling by coordinating kinases with components of specific signaling pathways. Such spatial segregation is particularly important in allowing specificity of signaling mediated by the 10-member family of protein kinase C (PKC) isozymes. Here we identified a novel interaction between PKCα and the Discs large homolog (DLG) family of scaffolds that is mediated by a class I C-terminal PDZ (PSD-95, disheveled, and ZO1) ligand unique to this PKC isozyme. Specifically, use of a proteomic array containing 96 purified PDZ domains identified the third PDZ domains of DLG1/SAP97 and DLG4/PSD95 as interaction partners for the PDZ binding motif of PKCα. Co-immunoprecipitation experiments verified that PKCα and DLG1 interact in cells by a mechanism dependent on an intact PDZ ligand. Functional assays revealed that the interaction of PKCα with DLG1 promotes wound healing; scratch assays using cells depleted of PKCα and/or DLG1 have impaired cellular migration that is no longer sensitive to PKC inhibition, and the ability of exogenous PKCα to rescue cellular migration is dependent on the presence of its PDZ ligand. Furthermore, we identified Thr-656 as a novel phosphorylation site in the SH3-Hook region of DLG1 that acts as a marker for PKCα activity at this scaffold. Increased phosphorylation of Thr-656 is correlated with increased invasiveness in non-small cell lung cancer lines from the NCI-60, consistent with this phosphorylation site serving as a marker of PKCα-mediated invasion. Taken together, these data establish the requirement of scaffolding to DLG1 for PKCα to promote cellular migration.     

Sub-nanosecond photolysis studies of Fe protoporphyrin IX solubilized in neat dimethyl sulfoxide

The present study examines the optical properties of the sub-nanosecond photolytic transient of Fe(II)protoporphyrin IX (Fe(II)PPIX) in neat dimethyl sulfoxide (DMSO). Previous nanosecond studies have revealed that photolysis of the (DMSO)₂Fe(II)PPIX complex in neat DMSO results in the formation of a five-coordinate high-spin (DMSO)Fe(II)PPIX complex within ∼100 ns which decayed with a pseudo-first order rate constant of 2 × 10⁶ s⁻¹ (Larsen et al. (1995) [19]). The results presented here demonstrate that the five-coordinate (DMSO)Fe(II)PPIX species is generated in <100 ps and that no significant changes occur in the kinetic difference between 100 ps and ∼100 ns. The 100 ps transient spectrum of the (DMSO)Fe(II)PPIX complex was also constructed from the kinetic difference spectrum and the equilibrium spectrum of the (DMSO)₂Fe(II)PPIX. The 100 ps transient spectrum exhibits a Soret maximum at ∼432 nm close to that of deoxyMb (435 nm, imidazole coordination) consistent with S-bonded DMSO occupying the fifth coordination site. Neither base elimination is detected on time scales down to 100 ps nor is there evidence for transient O-bonded DMSO followed by linkage isomerization to the equilibrium S-bonded form. The unusually slow rate of DMSO recombination is attributed to electrostatic interactions between DMSO and the five-coordinate heme iron as well as intermolecular interactions between solvent molecules in the bulk, as has been previously proposed.     

Focus Issue on Roots: Duplicate and Conquer: Multiple Homologs of PHOSPHORUS-STARVATION TOLERANCE1 Enhance Phosphorus Acquisition and Sorghum Performance on Low-Phosphorus Soils

Low soil phosphorus (P) availability is a major constraint for crop production in tropical regions. The rice (Oryza sativa) protein kinase, PHOSPHORUS-STARVATION TOLERANCE1 (OsPSTOL1), was previously shown to enhance P acquisition and grain yield in rice under P deficiency. We investigated the role of homologs of OsPSTOL1 in sorghum (Sorghum bicolor) performance under low P. Association mapping was undertaken in two sorghum association panels phenotyped for P uptake, root system morphology and architecture in hydroponics and grain yield and biomass accumulation under low-P conditions, in Brazil and/or in Mali. Root length and root surface area were positively correlated with grain yield under low P in the soil, emphasizing the importance of P acquisition efficiency in sorghum adaptation to low-P availability. SbPSTOL1 alleles reducing root diameter were associated with enhanced P uptake under low P in hydroponics, whereas Sb03g006765 and Sb03g0031680 alleles increasing root surface area also increased grain yield in a low-P soil. SbPSTOL1 genes colocalized with quantitative trait loci for traits underlying root morphology and dry weight accumulation under low P via linkage mapping. Consistent allelic effects for enhanced sorghum performance under low P between association panels, including enhanced grain yield under low P in the soil in Brazil, point toward a relatively stable role for Sb03g006765 across genetic backgrounds and environmental conditions. This study indicates that multiple SbPSTOL1 genes have a more general role in the root system, not only enhancing root morphology traits but also changing root system architecture, which leads to grain yield gain under low-P availability in the soil.Increasing food production is one of the major global challenges in dealing with continuously growing population and food consumption (Godfray et al., 2010). One of the major obstacles to improve crop production in tropical regions is phosphorus (P) deficiency caused by P fixation in the soil clays. P is one of the most important plant nutrients, contributing approximately 0.2% of a plant’s dry weight, and is a component of key organic molecules such as nucleic acids, phospholipids, and ATP (Schachtman et al., 1998). On tropical soils, even when the total amount of soil P is high, its bioavailability is low due to P fixation by aluminum and iron oxides in clay minerals (Marschner, 1995) and immobilization into organic forms (Schachtman et al., 1998). Approximately half of the world’s agricultural lands occurs on low-P soils (Lynch, 2011); hence, crop adaptation to P insufficiency should be a major breeding target to enable sustainable agricultural production worldwide. In addition, because phosphate rock fertilizer is a nonrenewable resource that is being depleted by agricultural demands, increasing fertilizer prices negatively impact agriculture, particularly for small-holder farmers in developing countries in the tropics and subtropics (Cordell et al., 2009; Sattari et al., 2012). In sorghum (Sorghum bicolor), breeding strategies for low-P adaptation have been developed based on multienvironment trials in West Africa, indicating the importance of undertaking selection in low-P soil conditions (Leiser et al., 2012a, 2012b). Therefore, developing crops with greater ability to grow and maintain satisfactory yields on soils with reduced P availability is expected to substantially improve food security worldwide.Tolerance to P deficiency in plants can be achieved by mechanisms underlying both P acquisition and P internal utilization efficiency (Parentoni and Souza Junior, 2008). One of the major mechanisms that plants have evolved to overcome low-P availability is to maximize the ability of the roots to acquire and absorb P from the soil. Plants can mobilize P through the exudation of organic acids, acid phosphatases, and ribonucleases, resulting in enhanced P availability and uptake (Hinsinger, 2001; Ryan et al., 2001; Dakora and Phillips, 2002; Hammond and White, 2008; Ma et al., 2009; Pang et al., 2009). Another strategy to cope with low-P availability is to increase the soil volume accessed by root systems by forming mycorrhizal symbioses (Li et al., 2012; Smith and Smith, 2012; Rai et al., 2013). Due to low-P mobility on tropical soils, changes in root architecture and morphology enhance P uptake by facilitating soil exploration (Williamson et al., 2001; Ho et al., 2005; Walk et al., 2006; Svistoonoff et al., 2007; Lynch, 2011; Ingram et al., 2012; Niu et al., 2013). Root structural changes leading to higher P uptake include increased root hair growth (Yan et al., 2004; Haling et al., 2013; Lan et al., 2013) and length and enhancing lateral root over primary root growth (Williamson et al., 2001; Wang et al., 2013). In addition, increased root surface area is achieved by a combination of reduced root diameter and enhanced elongation of relatively thinner roots (Fitter et al., 2002). There is both intraspecific and interspecific genetic variation for P deficiency tolerance in crop species (Lynch and Brown, 2001, 2012; Mudge et al., 2002; Paszkowski et al., 2002; Rausch and Bucher, 2002; Huang et al., 2011; Zhang et al., 2011; Leiser et al., 2014a) that can be explored to develop P-efficient cultivars.In rice (Oryza sativa), Phosphorus uptake1 (Pup1), a major quantitative trait locus (QTL) for P deficiency tolerance donated by an aus-type Indian variety, Kasalath, was mapped to the long arm of chromosome 12 (Ni et al., 1998; Wissuwa et al., 1998, 2002; Heuer et al., 2009). Near-isogenic lines bearing the Kasalath allele at Pup1 showed 3-fold higher P uptake and grain yield in low-P trials compared with the recurrent parent, cv Nipponbare, which is intolerant to P starvation (Wissuwa and Ae, 2001). Following high-resolution mapping of Pup1, comparative sequence analyses of homologous bacterial artificial chromosomes showed that a Kasalath genomic fragment contained several genes not present in cv Nipponbare, highlighting an approximately 90-kb deletion in the cv Nipponbare reference genome that encompassed the Pup1 locus (Heuer et al., 2009). Within this insertion/deletion, OsPupK46-2, a gene encoding a Ser/Thr kinase of the Receptor-like Protein Kinase LRK10L-2 subfamily, was found to enhance grain yield and P uptake in rice lines overexpressing this gene, indicating that this protein kinase underlies the Pup1 locus (Gamuyao et al., 2012). OsPupK46-2, which is now designated PHOSPHORUS-STARVATION TOLERANCE1 (OsPSTOL1), was found to be up-regulated in the root tissues of tolerant near-isogenic lines under P-deficient conditions and was shown to increase P uptake by a physiological mechanism based on the enhancement of early root growth and development. Furthermore, lines overexpressing OsPupK46-2 showed an approximately 30% grain yield increase in comparison with the null lines, suggesting that PSTOL1 has potential for molecular breeding applications to improve crop performance under low-P conditions. Consistent with the proposed physiological mechanism underlying OsPSTOL1, the superior performance of the transgenic lines was related to enhanced root dry weight, root length, and root surface area (Gamuyao et al., 2012).Sorghum is the world’s fifth most important cereal crop and is a staple food for more than half a billion people. It is widely adapted to harsh environmental conditions, and more specifically, to arid and semiarid regions of the world (Doumbia et al., 1993, 1998). It has been estimated that rice diverged from its most recent common ancestor with sorghum and maize (Zea mays) approximately 50 million years ago (Kellogg, 1998; Paterson et al., 2000, 2004; Paterson, 2008). About 60% of the genes in the sorghum genome are located in syntenic regions to rice (Paterson et al., 2009), emphasizing the potential for using comparative genomics for cross-species identification of genes underlying abiotic stress tolerance in the grass family. Here, we applied association analysis to specifically study the role of sorghum homologs of rice OsPSTOL1 in tolerance to P starvation in sorghum. Single-nucleotide polymorphisms (SNPs) within PSTOL1 homologs in sorghum, collectively designated SbPSTOL1, were significantly associated with grain yield under low-P conditions and also root morphology and root system architecture (RSA) traits phenotyped from hydroponically grown plants. Under low P, SbPSTOL1 genes increased biomass accumulation and P content in the African landrace panel and grain yield in the sorghum association panel phenotyped in a low-P Brazilian soil. This suggests a stable effect across environments and sorghum genotypes that potentially can be used for molecular breeding applications. QTL mapping with a large sorghum recombinant inbred line population was used to validate the association results, indicating that SbPSTOL1 homologs colocalize with QTLs related to root morphology and performance under low P. Our results indicate that SbPSTOL1 homologs have the ability to enhance P uptake and sorghum performance in low-P soils by a mechanism related not only to early root growth enhancement, as was the case for rice OsPSTOL1, but also by modulating RSA.     

Distributions of Salmonella Subtypes Differ between Two U.S. Produce-Growing Regions

Salmonella accounts for approximately 50% of produce-associated outbreaks in the United States, several of which have been traced back to contamination in the produce production environment. To quantify Salmonella diversity and aid in identification of Salmonella contamination sources, we characterized Salmonella isolates from two geographically diverse produce-growing regions in the United States. Initially, we characterized the Salmonella serotype and subtype diversity associated with 1,677 samples collected from 33 produce farms in New York State (NYS). Among these 1,677 samples, 74 were Salmonella positive, yielding 80 unique isolates (from 147 total isolates), which represented 14 serovars and 23 different pulsed-field gel electrophoresis (PFGE) types. To explore regional Salmonella diversity associated with production environments, we collected a smaller set of samples (n = 65) from South Florida (SFL) production environments and compared the Salmonella diversity associated with these samples with the diversity found among NYS production environments. Among these 65 samples, 23 were Salmonella positive, yielding 32 unique isolates (from 81 total isolates), which represented 11 serovars and 17 different PFGE types. The most common serovars isolated in NYS were Salmonella enterica serovars Newport, Cerro, and Thompson, while common serovars isolated in SFL were Salmonella serovars Saphra and Newport and S. enterica subsp. diarizonae serovar 50:r:z. High PFGE type diversity (Simpson''s diversity index, 0.90 ± 0.02) was observed among Salmonella isolates across both regions; only three PFGE types were shared between the two regions. The probability of three or fewer shared PFGE types was <0.000001; therefore, Salmonella isolates were considerably different between the two sampled regions. These findings suggest the potential for PFGE-based source tracking of Salmonella in production environments.     

Plant-Pathogenic Oomycetes,Escherichia coli Strains,and Salmonella spp. Frequently Found in Surface Water Used for Irrigation of Fruit and Vegetable Crops in New York State

In the United States, surface water is commonly used to irrigate a variety of produce crops and can harbor pathogens responsible for food-borne illnesses and plant diseases. Understanding when pathogens infest water sources is valuable information for produce growers to improve the food safety and production of these crops. In this study, prevalence data along with regression tree analyses were used to correlate water quality parameters (pH, temperature, turbidity), irrigation site properties (source, the presence of livestock or fowl nearby), and precipitation data to the presence and concentrations of Escherichia coli, Salmonella spp., and hymexazol-insensitive (HIS) oomycetes (Phytophthora and Pythium spp.) in New York State surface waters. A total of 123 samples from 18 sites across New York State were tested for E. coli and Salmonella spp., of which 33% and 43% were positive, respectively. Additionally, 210 samples from 38 sites were tested for HIS oomycetes, and 88% were found to be positive, with 10 species of Phytophthora and 11 species of Pythium being identified from the samples. Regression analysis found no strong correlations between water quality parameters, site factors, or precipitation to the presence or concentration of E. coli in irrigation sources. For Salmonella, precipitation (≤0.64 cm) 3 days before sampling was correlated to both presence and the highest counts. Analyses for oomycetes found creeks to have higher average counts than ponds, and higher turbidity levels were associated with higher oomycete counts. Overall, information gathered from this study can be used to better understand the food safety and plant pathogen risks of using surface water for irrigation.     

Comparative Host Feeding Patterns of the Asian Tiger Mosquito,Aedes albopictus,in Urban and Suburban Northeastern USA and Implications for Disease Transmission

Background

Aedes albopictus is an invasive species which continues expanding its geographic range and involvement in mosquito-borne diseases such as chikungunya and dengue. Host selection patterns by invasive mosquitoes are critically important because they increase endemic disease transmission and drive outbreaks of exotic pathogens. Traditionally, Ae. albopictus has been characterized as an opportunistic feeder, primarily feeding on mammalian hosts but occasionally acquiring blood from avian sources as well. However, limited information is available on their feeding patterns in temperate regions of their expanded range. Because of the increasing expansion and abundance of Ae. albopictus and the escalating diagnoses of exotic pathogens in travelers returning from endemic areas, we investigated the host feeding patterns of this species in newly invaded areas to further shed light on its role in disease ecology and assess the public health threat of an exotic arbovirus outbreak.

Methodology/Principal Findings

We identified the vertebrate source of 165 blood meals in Ae. albopictus collected between 2008 and 2011 from urban and suburban areas in northeastern USA. We used a network of Biogents Sentinel traps, which enhance Ae. albopictus capture counts, to conduct our collections of blooded mosquitoes. We also analyzed blooded Culex mosquitoes collected alongside Ae. albopictus in order to examine the composition of the community of blood sources. We found no evidence of bias since as expected Culex blood meals were predominantly from birds (n = 149, 93.7%) with only a small proportion feeding on mammals (n = 10, 6.3%). In contrast, Aedes albopictus fed exclusively on mammalian hosts with over 90% of their blood meals derived from humans (n = 96, 58.2%) and domesticated pets (n = 38, 23.0% cats; and n = 24, 14.6% dogs). Aedes albopictus fed from humans significantly more often in suburban than in urban areas (χ², p = 0.004) and cat-derived blood meals were greater in urban habitats (χ², p = 0.022). Avian-derived blood meals were not detected in any of the Ae. albopictus tested.

Conclusions/Significance

The high mammalian affinity of Ae. albopictus suggests that this species will be an efficient vector of mammal- and human-driven zoonoses such as La Crosse, dengue, and chikungunya viruses. The lack of blood meals obtained from birds by Ae. albopictus suggest that this species may have limited exposure to endemic avian zoonoses such as St. Louis encephalitis and West Nile virus, which already circulate in the USA. However, growing populations of Ae. albopictus in major metropolitan urban and suburban centers, make a large autochthonous outbreak of an arbovirus such as chikungunya or dengue viruses a clear and present danger. Given the difficulties of Ae. albopictus suppression, we recommend that public health practitioners and policy makers install proactive measures for the imminent mitigation of an exotic pathogen outbreak.