Streamline Proteomic Approach for Characterizing Protein−Protein Interaction Network in a RAD52 Protein Complex

Large-scale identification of protein-protein interactions (PPIs) in functional complexes represents an efficient route to elucidate the regulatory rules of cellular functions. While many methods have been developed to identify the PPIs associated with particular target/bait protein in complexes, little information is available about the interaction relationships among all components in a complex. Here, we have established a strategy of integrating proteomic identification of complex components with mammalian two-hybrid screening of their binary relationships to achieve information content of both breadth (i.e., identifying all potential interacting partners of the protein of interest) and depth (i.e., detailed mapping of the physical interactions of a subset of the identified and functionally related proteins) in characterizing protein complexes. In the initial phase of quantitative proteomic analysis of this streamline, the proteins that specifically complex with the target/bait protein were pulled down by immunoprecipitation and identified by mass spectrometry (MS)-based “dual-tagging” quantitative proteomic approach. In the second phase of in-depth characterizations of binary relationships, the physical interactions of a subset of functionally closely related complex components are mapped by mammalian two-hybrid assay. The screening for binary relationships of complex components not only serves as a validation of the first phase of proteomic identification, but also further deepens the understanding of the protein complex of interest. With this streamlined approach, we studied the protein complexes that are associated with a DNA recombination protein RAD52. In the initial phase, multiple proteins both known and unknown to interact with RAD52 were identified by the “dual-tagging” proteomic method. In the second phase, a complex protein-protein interaction network, which may play important roles in coordinating the activity of DNA repair with that of cell division, was defined by the mammalian two-hybrid assay

Comparison of Efficiencies of Non-invasive Prenatal Testing, Karyotyping, and Chromosomal Micro-Array for Diagnosing Fetal Chromosomal Anomalies in the Second and Third Trimesters

In this study, we aimed to compare the efficiency of non-invasive prenatal testing (NIPT), karyotyping, and chromosomal micro-array (CMA) for the diagnosis of fetal chromosomal anomalies in the second and third trimesters. Pregnant women, who underwent amniocenteses for prenatal genetic diagnoses during their middle and late trimesters, were recruited at the Prenatal Diagnosis Center of Taizhou City. Maternal blood was separated for NIPT, and amniotic fluid cells were cultured for karyotyping and CMA. The diagnostic efficiency of NIPT for detecting fetal imbalanced anomalies was compared with karyotyping and CMA. A total of 69 fetal chromosomal imbalances were confirmed by CMA, 37 were diagnosed by NIPT and 35 were found by karyotyping. The sensitivities of NIPT and karyotyping for diagnosing aneuploidy were 96.3% and 100% respectively. Only one mosaic sexual chromosome monosomy was misdiagnosed by NIPT, whereas the sensitivity of NIPT and karyotyping was 70% and 30%, respectively, for detecting pathogenic deletions and duplications sized from 5–20 Mb. Taken together, our results suggest that the efficiency of NIPT was similar to the formula karyotyping for detecting chromosome imbalance in the second and third trimesters

Acceptability and feasibility of smartphone-assisted 24 h recalls in the Chinese population

Abstract Objective To examine the acceptability and feasibility of using smartphone technology to assess beverage intake and evaluate whether the feasibility of smartphone use is greater among key sub-populations. Design An acceptability and feasibility study of recording the video dietary record, the acceptability of the ecological momentary assessment (EMA), wearing smartphones and whether the videos helped participants recall intake after a cross-over validation study. Setting Rural and urban area in Shanghai, China. Subjects Healthy adults ( n 110) aged 20–40 years old. Results Most participants reported that the phone was acceptable in most aspects, including that videos were easy to use (70 %), helped with recalls (77 %), EMA reminders helped them record intake (75 %) and apps were easy to understand (85 %). However, 49 % of the participants reported that they had trouble remembering to take videos of the beverages before consumption or 46 % felt embarrassed taking videos in front of others. Moreover, 72 % reported that the EMA reminders affected their consumption. When assessing overall acceptability of using smartphones, 72 % of the participants were favourable responders. There were no statistically significant differences in overall acceptability for overweight v. normal-weight participants or for rural v. urban residents. However, we did find that the overall acceptability was higher for males (81 %) than females (61 %, P =0·017). Conclusions Our study did not find smartphone technology helped with dietary assessments in a Chinese population. However, simpler approaches, such as using photographs instead of videos, may be more feasible for enhancing 24 h dietary recalls

Ipomoeassin F Binds Sec61α to Inhibit Protein Translocation

Funding Information: We thank the Arkansas Nano & Bio Materials Characterization Facility at the Institute for Nano Sciences & Engineering for our imaging studies, and Prof Yoshito Kishi (Harvard University) for the kind gift of synthetic mycolactone A/B used by S.H. and R.S. W.S. is supported by Grant No. R15GM116032 from the National Institute of General Medical Sciences of the National Institutes of Health (NIH) and startup funds from the University of Arkansas. This work was also supported in part by Grant No. P30 GM103450 from the National Institute of General Medical Sciences of the NIH and by seed money from the Arkansas Biosciences Institute (ABI). S.O’K. is the recipient of a Biotechnology and Biological Sciences Research Council (BBSRC) Doctoral Training Programme Award (BB/J014478/ 1), and S.H. holds a Welcome Trust Investigator Award in Science (204957/Z/16/Z). The alpha-1 antitrypsin work was supported by the Alpha-1 Foundation (J.I. and M.J.I.). J.I. and M.J.H. were supported by the intramural program of NCATS, National Institutes of Health, projects 1ZIATR000048-03 (J.I.) and ZIATR000063-04 (M.J.H.). R.S. holds a Welcome Trust Investigator Award in Science (202843/Z/16/Z). C.D. received funding from the Institut Pasteur, the Institut National de la Santé et de la Recherche Med́ icale, and the Fondation Raoul Follereau. N.B.’s synthesis and chemical biology studies of mycolactone were supported by CNRS, Université de Strasbourg, Fondations Potier et Follereau, and the Investisse-ment d’Avenir (Idex Unistra). V.O.P. is supported by the Academy of Finland (Grants 289737 and 314672) and the Sigrid Juselius Foundation. Funding Information: We thank the Arkansas Nano & Bio Materials Characterization Facility at the Institute for Nano Sciences & Engineering for our imaging studies, and Prof Yoshito Kishi (Harvard University) for the kind gift of synthetic mycolactone A/B used by S.H. and R.S. W.S. is supported by Grant No. R15GM116032 from the National Institute of General Medical Sciences of the National Institutes of Health (NIH) and startup funds from the University of Arkansas. This work was also supported in part by Grant No. P30 GM103450 from the National Institute of General Medical Sciences of the NIH and by seed money from the Arkansas Biosciences Institute (ABI). S.O'K. is the recipient of a Biotechnology and Biological Sciences Research Council (BBSRC) Doctoral Training Programme Award (BB/J014478/1), and S.H. holds a Welcome Trust Investigator Award in Science (204957/Z/16/Z). The alpha-1 antitrypsin work was supported by the Alpha-1 Foundation (J.I. and M.J.I.). J.I. and M.J.H. were supported by the intramural program of NCATS, National Institutes of Health, projects 1ZIATR000048-03 (J.I.) and ZIATR000063-04 (M.J.H.). R.S. holds a Welcome Trust Investigator Award in Science (202843/Z/16/Z). C.D. received funding from the Institut Pasteur, the Institut National de la Sante et de la Recherche Medicale, and the Fondation Raoul Follereau. N.B.'s synthesis and chemical biology studies of mycolactone were supported by CNRS, Universite de Strasbourg, Fondations Potier et Follereau and the Investissement d'Avenir (Idex Unistra). V.O.P. is supported by the Academy of Finland (Grants 289737 and 314672) and the Sigrid Juselius Foundation. Publisher Copyright: © 2019 American Chemical Society.Ipomoeassin F is a potent natural cytotoxin that inhibits growth of many tumor cell lines with single-digit nanomolar potency. However, its biological and pharmacological properties have remained largely unexplored. Building upon our earlier achievements in total synthesis and medicinal chemistry, we used chemical proteomics to identify Sec61 alpha (protein transport protein Sec61 subunit alpha isoform 1), the pore-forming subunit of the Sec61 protein translocon, as a direct binding partner of ipomoeassin F in living cells. The interaction is specific and strong enough to survive lysis conditions, enabling a biotin analogue of ipomoeassin F to pull down Sec61 alpha from live cells, yet it is also reversible, as judged by several experiments including fluorescent streptavidin staining, delayed competition in affinity pulldown, and inhibition of TNF biogenesis after washout. Sec61 alpha forms the central subunit of the ER protein translocation complex, and the binding of ipomoeassin F results in a substantial, yet selective, inhibition of protein translocation in vitro and a broad ranging inhibition of protein secretion in live cells. Lastly, the unique resistance profile demonstrated by specific amino acid single-point mutations in Sec61 alpha provides compelling evidence that Sec61 alpha is the primary molecular target of ipomoeassin F and strongly suggests that the binding of this natural product to Sec61 alpha is distinctive. Therefore, ipomoeassin F represents the first plant-derived, carbohydrate-based member of a novel structural class that offers new opportunities to explore Sec61 alpha function and to further investigate its potential as a therapeutic target for drug discovery.Peer reviewe

Identification of the ADPR binding pocket in the NUDT9 homology domain of TRPM2

Activation of the transient receptor potential melastatin 2 (TRPM2) channel occurs during the response to oxidative stress under physiological conditions as well as in pathological processes such as ischemia and diabetes. Accumulating evidence indicates that adenosine diphosphate ribose (ADPR) is the most important endogenous ligand of TRPM2. However, although it is known that ADPR binds to the NUDT9 homology (NUDT9-H) domain in the intracellular C-terminal region, the molecular mechanism underlying ADPR binding and activation of TRPM2 remains unknown. In this study, we generate a structural model of the NUDT9-H domain and identify the binding pocket for ADPR using induced docking and molecular dynamics simulation. We find a subset of 11 residues—H1346, T1347, T1349, L1379, G1389, S1391, E1409, D1431, R1433, L1484, and H1488—that are most likely to directly interact with ADPR. Results from mutagenesis and electrophysiology approaches support the predicted binding mechanism, indicating that ADPR binds tightly to the NUDT9-H domain, and suggest that the most significant interactions are the van der Waals forces with S1391 and L1484, polar solvation interaction with E1409, and electronic interactions (including π–π interactions) with H1346, T1347, Y1349, D1431, and H1488. These findings not only clarify the roles of a range of newly identified residues involved in ADPR binding in the TRPM2 channel, but also reveal the binding pocket for ADPR in the NUDT9-H domain, which should facilitate structure-based drug design for the TRPM2 channel

Inactivation of TRPM2 channels by extracellular divalent copper

Cu2+ is an essential metal ion that plays a critical role in the regulation of a number of ion channels and receptors in addition to acting as a cofactor in a variety of enzymes. Here, we showed that human melastatin transient receptor potential 2 (hTRPM2) channel is sensitive to inhibition by extracellular Cu2+. Cu2 + at concentrations as low as 3 μM inhibited the hTRPM2 channel completely and irreversibly upon washing or using Cu2+ chelators, suggesting channel inactivation. The Cu2+-induced inactivation was similar when the channels conducted inward or outward currents, indicating the permeating ions had little effect on Cu2+-induced inactivation. Furthermore, Cu2+ had no effect on singe channel conductance. Alanine substitution by site-directed mutagenesis of His995 in the pore-forming region strongly attenuated Cu2+-induced channel inactivation, and mutation of several other pore residues to alanine altered the kinetics of channel inactivation by Cu2+. In addition, while introduction of the P1018L mutation is known to result in channel inactivation, exposure to Cu2+ accelerated the inactivation of this mutant channel. In contrast with the hTRPM2, the mouse TRPM2 (mTRPM2) channel, which contains glutamine at the position equivalent to His995, was insensitive to Cu2+. Replacement of His995 with glutamine in the hTRPM2 conferred loss of Cu2+-induced channel inactivation. Taken together, these results suggest that Cu2+ inactivates the hTRPM2 channel by interacting with the outer pore region. Our results also indicate that the amino acid residue difference in this region gives rise to species-dependent effect by Cu2+ on the human and mouse TRPM2 channels

CEO succession and the CEO’s commitment to the status quo

Chief executive officer (CEO) commitment to the status quo (CSQ) is expected to play an important role in any firm’s strategic adaptation. CSQ is used often as an explanation for strategic change occurring after CEO succession: new CEOs are expected to reveal a lower CSQ than established CEOs. Although widely accepted in the literature, this relationship remains imputed but unobserved. We address this research gap and analyze whether new CEOs reveal lower CSQ than established CEOs. By analyzing the letters to the shareholders of German HDAX firms, we find empirical support for our hypothesis of a lower CSQ of newly appointed CEOs compared to established CEOs. However, our detailed analyses provide a differentiated picture. We find support for a lower CSQ of successors after a forced CEO turnover compared to successors after a voluntary turnover, which indicates an influence of the mandate for change on the CEO’s CSQ. However, against the widespread assumption, we do not find support for a lower CSQ of outside successors compared to inside successors, which calls for deeper analyses of the insiderness of new CEOs. Further, our supplementary analyses propose a revised tenure effect: the widely assumed relationship of an increase in CSQ when CEO tenure increases might be driven mainly by the event of CEO succession and may not universally and continuously increase over time, pointing to a “window of opportunity” to initiate strategic change shortly after the succession event. By analyzing the relationship between CEO succession and CEO CSQ, our results contribute to the CSQ literature and provide fruitful impulses for the CEO succession literature