Transfer of Sulfur from IscS to IscU during Fe/S Cluster Assembly

The cysteine desulfurase enzymes NifS and IscS provide sulfur for the biosynthesis of Fe/S proteins. NifU and IscU have been proposed to serve as template or scaffold proteins in the initial Fe/S cluster assembly events, but the mechanism of sulfur transfer from NifS or IscS to NifU or IscU has not been elucidated. We have employed [35S]cysteine radiotracer studies to monitor sulfur transfer between IscS and IscU from Escherichia coli and have used direct binding measurements to investigate interactions between the proteins. IscS catalyzed transfer of 35S from [35S]cysteine to IscU in the absence of additional thiol reagents, suggesting that transfer can occur directly and without involvement of an intermediate carrier. Surface plasmon resonance studies and isothermal titration calorimetry measurements further revealed that IscU binds to IscS with high affinity (Kd ~2 µM) in support of a direct transfer mechanism. Transfer was inhibited by treatment of IscU with iodoacetamide, and 35S was released by reducing reagents, suggesting that transfer of persulfide sulfur occurs to cysteinyl groups of IscU. A deletion mutant of IscS lacking C-terminal residues 376-413 (IscSDelta 376-413) displayed cysteine desulfurase activity similar to the full-length protein but exhibited lower binding affinity for IscU, decreased ability to transfer 35S to IscU, and reduced activity in assays of Fe/S cluster assembly on IscU. The findings with IscSDelta 376-413 provide additional support for a mechanism of sulfur transfer involving a direct interaction between IscS and IscU and suggest that the C-terminal region of IscS may be important for binding IscU

A Twin Study of Early-Childhood Asthma in Puerto Ricans

Background:The relative contributions of genetics and environment to asthma in Hispanics or to asthma in children younger than 3 years are not well understood.Objective:To examine the relative contributions of genetics and environment to early-childhood asthma by performing a longitudinal twin study of asthma in Puerto Rican children ≤3 years old.Methods:678 twin infants from the Puerto Rico Neo-Natal Twin Registry were assessed for asthma at age 1 year, with follow-up data obtained for 624 twins at age 3 years. Zygosity was determined by DNA microsatellite profiling. Structural equation modeling was performed for three phenotypes at ages 1 and 3 years: physician-diagnosed asthma, asthma medication use in the past year, and ≥1 hospitalization for asthma in the past year. Models were additionally adjusted for early-life environmental tobacco smoke exposure, sex, and age.Results:The prevalences of physician-diagnosed asthma, asthma medication use, and hospitalization for asthma were 11.6%, 10.8%, 4.9% at age 1 year, and 34.1%, 40.1%, and 8.5% at 3 years, respectively. Shared environmental effects contributed to the majority of variance in susceptibility to physician-diagnosed asthma and asthma medication use in the first year of life (84%-86%), while genetic effects drove variance in all phenotypes (45%-65%) at age 3 years. Early-life environmental tobacco smoke, sex, and age contributed to variance in susceptibility.Conclusion:Our longitudinal study in Puerto Rican twins demonstrates a changing contribution of shared environmental effects to liability for physician-diagnosed asthma and asthma medication use between ages 1 and 3 years. Early-life environmental tobacco smoke reduction could markedly reduce asthma morbidity in young Puerto Rican children. © 2013 Bunyavanich et al

Hsc66 substrate specificity is directed toward a discrete region of the iron-sulfur cluster template protein IscU

Hsc66 and Hsc20 comprise a specialized chaperone system important for the assembly of iron-sulfur clusters in Escherchia coli. Only a single substrate, the Fe/S template protein IscU, has been identified for the Hsc66/Hsc20 system, but the mechanism by which Hsc66 selectively binds IscU is unknown. We have investigated Hsc66 substrate specificity using phage display and a peptide array of IscU. Screening of a heptameric peptide phage display library revealed that Hsc66 prefers peptides with a centrally located Pro-Pro motif. Using a cellulose-bound peptide array of IscU we determined that Hsc66 interacts specifically with a region (residues 99-103, LPPVK) that is invariant among all IscU family members. A synthetic peptide (ELPPVKIHC) corresponding to IscU residues 98-106 behaves in a similar manner to native IscU, stimulating the ATPase activity of Hsc66 with similar affinity as IscU, preventing Hsc66 suppression of bovine rhodanese aggregation, and interacting with the peptide-binding domain of Hsc66. Unlike native IscU, however, the synthetic peptide is not bound by Hsc20 and does not synergistically stimulate Hsc66 ATPase activity with Hsc20. Our results indicate that Hsc66 and Hsc20 recognize distinct regions of IscU and further suggest that Hsc66 will not bind LPPVK motifs with high affinity in vivo unless they are in the context of native IscU and can be directed to Hsc66 by Hsc20

Fluorescence Detection of a Protein-Bound 2Fe2S Cluster

A fluorescent biosensor is described for 2Fe2S clusters that is composed of green fluorescent protein (GFP) fused to glutaredoxin 2 (Grx2), as illustrated here. 2Fe2S detection is based on the reduction of GFP fluorescence upon the 2Fe2S-induced dimerization of GFP-Grx2. This assay is sufficiently sensitive to detect submicromolar changes in 2Fe2S levels, thus making it suitable for high-throughput measurements of metallocluster degradation and synthesis reactions

The Most Popular Smartphone Apps for Weight Loss: A Quality Assessment

Background: Advancements in mobile phone technology have led to the development of smartphones with the capability to run apps. The availability of a plethora of health- and fitness-related smartphone apps has the potential, both on a clinical and public health level, to facilitate healthy behavior change and weight management. However, current top-rated apps in this area have not been extensively evaluated in terms of scientific quality and behavioral theory evidence base. Objective: The purpose of this study was to evaluate the quality of the most popular dietary weight-loss smartphone apps on the commercial market using comprehensive quality assessment criteria, and to quantify the behavior change techniques (BCTs) incorporated. Methods: The top 200-rated Health & Fitness category apps from the free and paid sections of Google Play and iTunes App Store in Australia (n=800) were screened in August 2014. To be included in further analysis, an app had to focus on weight management, include a facility to record diet intake (self-monitoring), and be in English. One researcher downloaded and used the eligible apps thoroughly for 5 days and assessed the apps against quality assessment criteria which included the following domains: accountability, scientific coverage and content accuracy of information relevant to weight management, technology-enhanced features, usability, and incorporation of BCTs. For inter-rater reliability purposes, a second assessor provided ratings on 30% of the apps. The accuracy of app energy intake calculations was further investigated by comparison with results from a 3-day weighed food record (WFR). Results: Across the eligible apps reviewed (n=28), only 1 app (4%) received full marks for accountability. Overall, apps included an average of 5.1 (SD 2.3) out of 14 technology-enhanced features, and received a mean score of 13.5 (SD 3.7) out of 20 for usability. The majority of apps provided estimated energy requirements (24/28, 86%) and used a food database to calculate energy intake (21/28, 75%). When compared against the WFR, the mean absolute energy difference of apps which featured energy intake calculations (23/28, 82%) was 127 kJ (95% CI -45 to 299). An average of 6.3 (SD 3.7) of 26 BCTs were included. Conclusions: Overall, the most popular commercial apps for weight management are suboptimal in quality, given the inadequate scientific coverage and accuracy of weight-related information, and the relative absence of BCTs across the apps reviewed. With the limited regulatory oversight around the quality of these types of apps, this evaluation provides clinicians and consumers an informed view of the highest-quality apps in the current popular app pool appropriate for recommendation and uptake. Further research is necessary to assess the effectiveness of apps for weight management

Genetic influences on thought problems in 7-year-olds: A twin-study of genetic, environmental and rater effects.

The Thought-Problem scale (TP) of the CBCL assesses symptoms such as hallucinations and strange thoughts/behaviors and has been associated with other behavioral disorders. This study uses parental reports to examine the etiology of variation in TP, about which relatively little is known, in 7-year-old twins. Parental ratings on TP were collected in 8,962 7-year-old twin pairs. Because the distribution of TP scores was highly skewed scores were categorized into 3 classes. The data were analyzed under a threshold liability model with genetic structural equation modeling. Ratings from both parents were simultaneously analyzed to determine the rater agreement phenotype (or common phenotype [TPc]) and the rater specific phenotype [TPs] that represents rater disagreement caused by rater bias, measurement error and/or a unique view of the parents on the child's behavior. Scores on the TP-scale varied as a function of rater (fathers rated fewer problems), sex (boys scored higher) and zygosity (DZ twins scored higher). The TPc explained 67% of the total variance in the parental ratings. Variation in TPc was influenced mainly by the children's genotype (76%). Variance in TPs also showed a contribution of genetic factors (maternal reports: 61%, paternal reports: 65%), indicating that TPs does not only represent rater bias. Shared environmental influences were only found in the TPs. No sex differences in genetic architecture were observed. These results indicate an important contribution of genetic factors to thought problems in children as young as 7 years

In Vivo Fluorescent Detection of Fe-S Clusters Coordinated by Human GRX2

A major challenge to studying Fe-S cluster biosynthesis in higher eukaryotes is the lack of simple tools for imaging metallocluster binding to proteins. We describe the first fluorescent approach for in vivo detection of 2Fe2S clusters that is based upon the complementation of Venus fluorescent protein fragments via human glutaredoxin 2 (GRX2) coordination of a 2Fe2S cluster. We show that Escherichia coli and mammalian cells expressing Venus fragments fused to GRX2 exhibit greater fluorescence than cells expressing fragments fused to a C37A mutant that cannot coordinate a metallocluster. In addition, we find that maximal fluorescence in the cytosol of mammalian cells requires the iron-sulfur cluster assembly proteins ISCU and NFS1. These findings provide evidence that glutaredoxins can dimerize within mammalian cells through coordination of a 2Fe2S cluster as observed with purified recombinant proteins

A Genetically Encoded AND Gate for Cell-Targeted Metabolic Labeling of Proteins

We describe a genetic AND gate for cell-targeted metabolic labeling and proteomic analysis in complex cellular systems. The centerpiece of the AND gate is a bisected methionyl-tRNA synthetase (MetRS) that charges the Met surrogate azidonorleucine (Anl) to tRNAMet. Cellular protein labeling occurs only upon activation of two different promoters that drive expression of the N- and C-terminal fragments of the bisected MetRS. Anl-labeled proteins can be tagged with fluorescent dyes or affinity reagents via either copper-catalyzed or strain-promoted azide–alkyne cycloaddition. Protein labeling is apparent within 5 min after addition of Anl to bacterial cells in which the AND gate has been activated. This method allows spatial and temporal control of proteomic labeling and identification of proteins made in specific cellular subpopulations. The approach is demonstrated by selective labeling of proteins in bacterial cells immobilized in the center of a laminar-flow microfluidic channel, where they are exposed to overlapping, opposed gradients of inducers of the N- and C-terminal MetRS fragments. The observed labeling profile is predicted accurately from the strengths of the individual input signals