Publications by authors named "Sergey Savinov"

38 Publications

Method of Measuring High-LET Particles Dose.

Radiat Res 2021 Aug;196(2):192-196

Budker Institute of Nuclear Physics, iberian Branch of the Russian Academy of Sciences, Novosibirsk, Russia.

In boron neutron capture therapy, the total absorbed dose is the sum of four dose components with different relative biological effectiveness (RBE): boron dose, "nitrogen" dose, fast neutron dose and γ-ray dose. We present a new approach for measuring the first three doses. In this work, we provide the details of this method of dose measurement and results when this proposed method is employed.
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http://dx.doi.org/10.1667/RADE-21-00015.1DOI Listing
August 2021

Neutron Source Based on Vacuum Insulated Tandem Accelerator and Lithium Target.

Biology (Basel) 2021 Apr 21;10(5). Epub 2021 Apr 21.

Faculty of Physics, Novosibirsk State University, 2 Pirogov Str., 630090 Novosibirsk, Russia.

A compact accelerator-based neutron source has been proposed and created at the Budker Institute of Nuclear Physics in Novosibirsk, Russia. An original design tandem accelerator is used to provide a proton beam. The proton beam energy can be varied within a range of 0.6-2.3 MeV, keeping a high-energy stability of 0.1%. The beam current can also be varied in a wide range (from 0.3 mA to 10 mA) with high current stability (0.4%). In the device, neutron flux is generated as a result of the Li(p,n)Be threshold reaction. A beam-shaping assembly is applied to convert this flux into a beam of epithermal neutrons with characteristics suitable for BNCT. A lot of scientific research has been carried out at the facility, including the study of blistering and its effect on the neutron yield. The BNCT technique is being tested in in vitro and in vivo studies, and the methods of dosimetry are being developed. It is planned to certify the neutron source next year and conduct clinical trials on it. The neutron source served as a prototype for a facility created for a clinic in Xiamen (China).
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http://dx.doi.org/10.3390/biology10050350DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8143170PMC
April 2021

Design and synthesis of C-aryl angular luotonins via a one-pot aza-Nazarov-Friedlander sequence and their Topo-I inhibition studies along with C-aryl vasicinones and luotonins.

Bioorg Med Chem Lett 2021 06 30;41:127998. Epub 2021 Mar 30.

Department of Biochemistry and Molecular Biology, UMass Amherst, Amherst, MA 01003, USA.

A facile one-pot synthesis of C-ring substituted angular luotonins has been realized via a methanesulfonic acid mediated aza-Nazarov-Friedlander condensation sequence on quinazolinonyl enones. Topoisomerase I (topo-I) inhibition studies revealed that the angular luotonin library (7a-7l) and their regioisomeric analogs (linear luotonins, 8a-8l) are weak negative modulators, compared to camptothecin. These results would fare well for the design of topo-I-inert luotonins for non-oncological applications such as anti-fungal and insecticide lead developments. Surprisingly, the tricyclic vasicinones (9h, 9i, and 9j) showed better topo-I inhibition compared to pentacyclic C-aryl luotonins providing a novel pharmacophore for further explorations.
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http://dx.doi.org/10.1016/j.bmcl.2021.127998DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8113096PMC
June 2021

Similarity of angular distribution for THz radiation emitted by laser filament plasma channels of different lengths.

Opt Lett 2020 Jul;45(14):4009-4011

The influence of plasma channel length on an angular terahertz (THz) radiation distribution is experimentally studied for the channel formed under filamentation of an ultrashort laser pulse. It is shown that the angular distribution of the THz emission depends only on laser intensity in the filament and plasma density of the plasma channel and does not depend on the plasma channel length. A qualitative explanation of the THz emission screening by the filament plasma channel is proposed.
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http://dx.doi.org/10.1364/OL.394377DOI Listing
July 2020

Identification of Protein Recognition Elements within Heparin Chains Using Enzymatic Foot-Printing in Solution and Online SEC/MS.

Anal Chem 2020 06 13;92(11):7565-7573. Epub 2020 May 13.

Chemistry Department, University of Massachusetts-Amherst, 240 Thatcher Way, Amherst, Massachusetts 01003, United States.

Understanding molecular mechanisms governing interactions of glycosaminoglycans (such as heparin) with proteins remains challenging due to their enormous structural heterogeneity. Commonly accepted approaches seek to reduce the structural complexity by searching for "binding epitopes" within the limited subsets of short heparin oligomers produced either enzymatically or synthetically. A top-down approach presented in this work seeks to preserve the chemical diversity displayed by heparin by allowing the longer and structurally diverse chains to interact with the client protein. Enzymatic lysis of the protein-bound heparin chains followed by the product analysis using size exclusion chromatography with online mass spectrometry detection (SEC/MS) reveals the oligomers that are protected from lysis due to their tight association with the protein, and enables their characterization (both the oligomer length, and the number of incorporated sulfate and acetyl groups). When applied to a paradigmatic heparin/antithrombin system, the new method generates a series of oligomers with surprisingly distinct sulfation levels. The extent of sulfation of the minimal-length binder (hexamer) is relatively modest yet persistent, consistent with the notion of six sulfate groups being both essential and sufficient for antithrombin binding. However, the masses of longer surviving chains indicate complete sulfation of disaccharides beyond the hexasaccharide core. Molecular dynamics simulations confirm the existence of favorable electrostatic interactions between the high charge-density saccharide residues flanking the "canonical" antithrombin-binding hexasaccharide and the positive patch on the surface of the overall negatively charged protein. Furthermore, electrostatics may rescue the heparin/protein interaction in the absence of the canonical binding element.
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http://dx.doi.org/10.1021/acs.analchem.0c00115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8095033PMC
June 2020

A study of matrix and admixture elements in fluorine-rich ionic conductors by pulsed glow discharge mass spectrometry.

Rapid Commun Mass Spectrom 2020 Jun;34(11):e8786

Institute of Chemistry, St Petersburg State University, 199034, St Petersburg, Universitetskaya nab. 7/9, Russia.

Rationale: Dopants in ionic conductors play a crucial role in achieving the required electrochemical properties. A slight variation in their concentration considerably affects the conductivity of crystals and their applicability as ionic conductors and laser materials. To ensure the growth of high-quality fluoride crystals, adequate approaches for the quantification of matrix and admixture/dopant components are required.

Methods: A panel of SrF - and GdF -doped LaF single crystals was investigated. The electrical conductivity of the crystals was measured using impedance spectroscopy in the frequency range 100 Hz-1 MHz to control for crystal quality. Pulsed glow discharge mass spectrometry (GDMS) was used to simultaneously quantify fluorine, strontium, lanthanum, and gadolinium in the crystals. X-ray fluorescence, scanning electron microscopy-energy dispersive X-ray spectroscopy, and arc optical emission spectrometry were used for validation.

Results: Quasiperiodic intensity drifts under sputtering of the ionic conductors were observed and attributed to F redistribution on the sample surface, affecting surface conductivity and sputtering rate. Several sample preparation protocols were tested to address that effect. Full coating of the sample with a layer of silver several micrometers thick provided stable and effective sputtering. The parameters for the GDMS determination of F, Sr, La, and Gd were optimized. The elements' distribution was studied in different parts of the crystals.

Conclusions: An analytical approach to the direct multi-element analysis of fluoride-containing ionic conductors using pulsed GDMS with La Sr Gd F as an example was designed and tested. Instability effects of ionic conductivity were explained and coped with, providing effective and stable sputtering.
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http://dx.doi.org/10.1002/rcm.8786DOI Listing
June 2020

The genome of opportunistic fungal pathogen Fusarium oxysporum carries a unique set of lineage-specific chromosomes.

Commun Biol 2020 01 31;3(1):50. Epub 2020 Jan 31.

Department of Biochemistry and Molecular Biology, University of Massachusetts Amherst, Amherst, Massachusetts, 01003, USA.

Fusarium oxysporum is a cross-kingdom fungal pathogen that infects plants and humans. Horizontally transferred lineage-specific (LS) chromosomes were reported to determine host-specific pathogenicity among phytopathogenic F. oxysporum. However, the existence and functional importance of LS chromosomes among human pathogenic isolates are unknown. Here we report four unique LS chromosomes in a human pathogenic strain NRRL 32931, isolated from a leukemia patient. These LS chromosomes were devoid of housekeeping genes, but were significantly enriched in genes encoding metal ion transporters and cation transporters. Homologs of NRRL 32931 LS genes, including a homolog of ceruloplasmin and the genes that contribute to the expansion of the alkaline pH-responsive transcription factor PacC/Rim1p, were also present in the genome of NRRL 47514, a strain associated with Fusarium keratitis outbreak. This study provides the first evidence, to our knowledge, for genomic compartmentalization in two human pathogenic fungal genomes and suggests an important role of LS chromosomes in niche adaptation.
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http://dx.doi.org/10.1038/s42003-020-0770-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6994591PMC
January 2020

Synthesis of C-Ring-Substituted Vasicinones and Luotonins via Regioselective Aza-Nazarov Cyclization of Quinazolinonyl Enones.

Org Lett 2019 12 4;21(24):9824-9828. Epub 2019 Dec 4.

Department of Biochemistry and Molecular Biology , University of Massachusetts Amherst , Amherst , Massachusetts 01003 , United States.

A facile synthesis of C-ring substituted luotonins and vasicinones has been realized via a super-acid-mediated aza-Nazarov cyclization of quinazolinonyl enones. The regioselectivity of the cyclization is highly dependent on proton availability in the reaction medium.
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http://dx.doi.org/10.1021/acs.orglett.9b03586DOI Listing
December 2019

Mutations in TTC29, Encoding an Evolutionarily Conserved Axonemal Protein, Result in Asthenozoospermia and Male Infertility.

Am J Hum Genet 2019 12 14;105(6):1148-1167. Epub 2019 Nov 14.

Histologie Embryologie et Biologie de la Reproduction, Centre de Promotion des Sciences de la Reproduction, Polyclinique les Jasmins, Centre Urbain Nord, 1003 Tunis, Tunisia.

In humans, structural or functional defects of the sperm flagellum induce asthenozoospermia, which accounts for the main sperm defect encountered in infertile men. Herein we focused on morphological abnormalities of the sperm flagellum (MMAF), a phenotype also termed "short tails," which constitutes one of the most severe sperm morphological defects resulting in asthenozoospermia. In previous work based on whole-exome sequencing of a cohort of 167 MMAF-affected individuals, we identified bi-allelic loss-of-function mutations in more than 30% of the tested subjects. In this study, we further analyzed this cohort and identified five individuals with homozygous truncating variants in TTC29, a gene preferentially and highly expressed in the testis, and encoding a tetratricopeptide repeat-containing protein related to the intraflagellar transport (IFT). One individual carried a frameshift variant, another one carried a homozygous stop-gain variant, and three carried the same splicing variant affecting a consensus donor site. The deleterious effect of this last variant was confirmed on the corresponding transcript and protein product. In addition, we produced and analyzed TTC29 loss-of-function models in the flagellated protist T. brucei and in M. musculus. Both models confirmed the importance of TTC29 for flagellar beating. We showed that in T. brucei the TPR structural motifs, highly conserved between the studied orthologs, are critical for TTC29 axonemal localization and flagellar beating. Overall our work demonstrates that TTC29 is a conserved axonemal protein required for flagellar structure and beating and that TTC29 mutations are a cause of male sterility due to MMAF.
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http://dx.doi.org/10.1016/j.ajhg.2019.10.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6904810PMC
December 2019

Structural Heterogeneity in the Preamyloid Oligomers of β-2-Microglobulin.

J Mol Biol 2020 01 9;432(2):396-409. Epub 2019 Nov 9.

Department of Chemistry, University of Massachusetts, Amherst, MA 01003, United States. Electronic address:

In dialysis patients, the protein β2-microglobulin (β2m) forms amyloid fibrils in a condition known as dialysis-related amyloidosis. To understand the early stages of the amyloid assembly process, we have used native electrospray ionization (ESI) together with ion mobility mass spectrometry (IM-MS) to study soluble preamyloid oligomers. ESI-IM-MS reveals the presence of multiple conformers for the dimer, tetramer, and hexamer that precede the Cu(II)-induced amyloid assembly process, results which are distinct from β2m oligomers formed at low pH. Experimental and computational results indicate that the predominant dimer is a Cu(II)-bound structure with an antiparallel side-by-side configuration. In contrast, tetramers exist in solution in both Cu(II)-bound and Cu(II)-free forms. Selective depletion of Cu(II)-bound species results in two primary conformers-one that is compact and another that is more expanded. Molecular modeling and molecular dynamics simulations identify models for these two tetrameric conformers with unique interactions and interfaces that enthalpically compensate for the loss of Cu(II). Unlike with other amyloid systems in which conformational heterogeneity is often associated with different amyloid morphologies or off-pathway events, conformational heterogeneity in the tetramer seems to be a necessary aspect of Cu(II)-induced amyloid formation by β2m. Moreover, the Cu(II)-free models represent a new advance in our understanding of Cu(II) release in Cu(II)-induced amyloid formation, laying a foundation for further mechanistic studies as well as development of new inhibition strategies.
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http://dx.doi.org/10.1016/j.jmb.2019.10.030DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6995769PMC
January 2020

Ruthenium coordination preferences in imidazole-containing systems revealed by electrospray ionization mass spectrometry and molecular modeling: Possible cues for the surprising stability of the Ru (III)/tris (hydroxymethyl)-aminomethane/imidazole complexes.

J Mass Spectrom 2020 Feb 8;55(2):e4435. Epub 2019 Nov 8.

Department of Biochemistry and Molecular Biology, University of Massachusetts-Amherst, Amherst, Massachusetts, USA.

Ruthenium is a platinoid that exhibits a range of unique chemical properties in solution, which are exploited in a variety of applications, including luminescent probes, anticancer therapies, and artificial photosynthesis. This paper focuses on a recently demonstrated ability of this metal in its +3 oxidation state to form highly stable complexes with tris (hydroxymethyl)aminomethane (H NC(CH OH) , Tris-base or T) and imidazole (Im) ligands, where a single Ru cation is coordinated by two molecules of each T and Im. High-resolution electrospray ionization mass spectrometry (ESI MS) is used to characterize Ru complexes formed by placing a Ru complex [(NH ) Ru Cl]Cl in a Tris buffer under aerobic conditions. The most abundant ionic species in ESI MS represent mononuclear complexes containing an oxidized form of the metal, ie, [X Ru T - 2H] , where X could be an additional T (n = 1) or NH (n = 0-2). Di- and tri-metal complexes also give rise to a series of abundant ions, with the highest mass ion representing a metal complex with an empirical formula Ru C O N H (interpreted as cyclo(T RuO) , a cyclic oxo-bridged structure, where the coordination sphere of each metal is completed by two T ligands). The empirical formulae of the binuclear species are consistent with the structures representing acyclic fragments of cyclo(T RuO) with addition of various combinations of ammonia and dioxygen as ligands. Addition of histidine in large molar excess to this solution results in complete disassembly of poly-nuclear complexes and gives rise to a variety of ionic species in the ESI mass spectrum with a general formula [Ru His T (NH ) - 2H] , where k = 0 to 2, m = 0 to 3, and n = 0 to 4. Ammonia adducts are present for all observed combinations of k and m, except k = m = 2, suggesting that [His Ru T - 2H] represents a complex with a fully completed coordination sphere. The observed cornucopia of Ru complexes formed in the presence of histidine is in stark contrast to the previously reported selective reactivity of imidazole, which interacts with the metal by preserving the RuT core and giving rise to a single abundant ruthenium complex (represented by [Im Ru T - 2H] in ESI mass spectra). Surprisingly, the behavior of a hexa-histidine peptide (HHHHHH) is similar to that of a single imidazole, rather than a single histidine amino acid: The RuT core is preserved, with the following ionic species observed in ESI mass spectra: [HHHHHH·(Ru T ) - (3m-1)H] (m = 1-3). The remarkable selectivity of the imidazole interaction with the Ru T core is rationalized using energetic considerations at the quantum mechanical level of theory.
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http://dx.doi.org/10.1002/jms.4435DOI Listing
February 2020

Modulation of Amyloid-β42 Conformation by Small Molecules Through Nonspecific Binding.

J Chem Theory Comput 2019 Oct 4;15(10):5169-5174. Epub 2019 Sep 4.

Department of Biochemistry and Molecular Biology , University of Massachusetts Amherst , Amherst , Massachusetts 01003 , United States.

Aggregation of amyloid-β (Aβ) peptides is a crucial step in the progression of Alzheimer's disease (AD). Identifying aggregation inhibitors against AD has been a great challenge. We report an atomistic simulation study of the inhibition mechanism of two small molecules, homotaurine and -inositol, which are AD drug candidates currently under investigation. We show that both small molecules promote a conformational change of the Aβ42 monomer toward a more collapsed phase through a nonspecific binding mechanism. This finding provides atomistic-level insights into designing potential drug candidates for future AD treatments.
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http://dx.doi.org/10.1021/acs.jctc.9b00599DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6783347PMC
October 2019

Drug Screening for Discovery of Broad-spectrum Agents for Soil-transmitted Nematodes.

Sci Rep 2019 08 26;9(1):12347. Epub 2019 Aug 26.

Program in Molecular Medicine, University of Massachusetts Medical School Worcester, Worcester, USA.

Soil-transmitted nematodes (STNs), namely hookworms, whipworms, and ascarids, are extremely common parasites, infecting 1-2 billion of the poorest people worldwide. Two benzimidazoles, albendazole and mebendazole, are currently used in STN mass drug administration, with many instances of low/reduced activity reported. New drugs against STNs are urgently needed. We tested various models for STN drug screening with the aim of identifying the most effective tactics for the discovery of potent, safe and broad-spectrum agents. We screened a 1280-compound library of approved drugs to completion against late larval/adult stages and egg/larval stages of both the human hookworm parasite Ancylostoma ceylanicum and the free-living nematode Caenorhabditis elegans, which is often used as a surrogate for STNs in screens. The quality of positives was further evaluated based on cheminformatics/data mining analyses and activity against evolutionarily distant Trichuris muris whipworm adults. From these data, two pairs of positives, sulconazole/econazole and pararosaniline/cetylpyridinium, predicted to target nematode CYP-450 and HSP-90 respectively, were prioritized for in vivo evaluation against A. ceylanicum infections in hamsters. One of these positives, pararosaniline, showed a significant impact on hookworm fecundity in vivo. Taken together, our results suggest that anthelmintic screening with A. ceylanicum larval stages is superior to C. elegans based on both reduced false negative rate and superior overall quality of actives. Our results also highlight two potentially important targets for the discovery of broad-spectrum human STN drugs.
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http://dx.doi.org/10.1038/s41598-019-48720-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6710243PMC
August 2019

Chemoproteomics Using Nucleotide Acyl Phosphates Reveals an ATP Binding Site at the Dimer Interface of Procaspase-6.

Biochemistry 2019 12 24;58(52):5320-5328. Epub 2019 May 24.

ActivX Biosciences, Inc. , La Jolla , California 92037 , United States.

Acyl phosphates of ATP (ATPAc) and related nucleotides have proven to be useful for the interrogation of known nucleotide binding sites via specific acylation of conserved lysines (K). In addition, occasional K acylations are identified in proteins without such known sites. Here we present a robust and specific acylation of procaspase-6 by ATPAc at K133 in Jurkat cell lysates. The K133 acylation is dependent on π-π stacking interactions between the adenine moiety of ATPAc and a conserved Y198-Y198 site formed at the homodimeric interface of procaspase-6. Significantly, the Y198A mutation in procaspase-6 abolishes K133 acylation but has no effect on the proteolytic activity of the mature, active caspase-6 Y198A variant. Additional studies show that ATP can inhibit the autoproteolytic activation of procaspase-6. These observations suggest that ATP, and possibly other nucleotides, may serve as the endogenous ligands for the allosteric site at the procaspase-6 dimer interface, a site that has persisted in its "orphan" status for more than a decade.
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http://dx.doi.org/10.1021/acs.biochem.9b00290DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6874883PMC
December 2019

Mass Spectrometry Reveals a Multifaceted Role of Glycosaminoglycan Chains in Factor Xa Inactivation by Antithrombin.

Biochemistry 2018 08 25;57(32):4880-4890. Epub 2018 Jul 25.

Factor Xa (fXa) inhibition by antithrombin (AT) enabled by heparin or heparan sulfate is critical for controlling blood coagulation. AT activation by heparin has been investigated extensively, while interaction of heparin with trapped AT/fXa intermediates has received relatively little attention. We use native electrospray ionization mass spectrometry to study the role of heparin chains of varying length [hexa-, octa-, deca-, and eicosasaccharides (dp6, dp8, dp10, and dp20, respectively)] in AT/fXa complex assembly. Despite being critical promoters of AT/Xa binding, shorter heparin chains are excluded from the final products (trapped intermediates). However, replacement of short heparin segments with dp20 gives rise to a prominent ionic signal of ternary complexes. These species are also observed when the trapped intermediate is initially prepared in the presence of a short oligoheparin (dp6), followed by addition of a longer heparin chain (dp20), indicating that binding of heparin to AT/fXa complexes takes place after the inhibition event. The importance of the heparin chain length for its ability to associate with the trapped intermediate suggests that the binding likely occurs in a bidentate fashion (where two distinct segments of oligoheparin make contacts with the protein components, while the part of the chain separating these two segments is extended into solution to minimize electrostatic repulsion). This model is corroborated by both molecular dynamics simulations with an explicit solvent and ion mobility measurements in the gas phase. The observed post-inhibition binding of heparin to the trapped AT/fXa intermediates hints at the likely role played by heparan sulfate in their catabolism.
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http://dx.doi.org/10.1021/acs.biochem.8b00199DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6445383PMC
August 2018

Rare human Caspase-6-R65W and Caspase-6-G66R variants identify a novel regulatory region of Caspase-6 activity.

Sci Rep 2018 03 13;8(1):4428. Epub 2018 Mar 13.

Bloomfield Center for Research in Aging, Lady Davis Institute for Medical Research, Jewish General Hospital, 3755 Ch. Cote Ste-Catherine, Montreal, QC, H3T 1E2, Canada.

The cysteine protease Caspase-6 (Casp6) is a potential therapeutic target of Alzheimer Disease (AD) and age-dependent cognitive impairment. To assess if Casp6 is essential to human health, we investigated the effect of CASP6 variants sequenced from healthy humans on Casp6 activity. Here, we report the effects of two rare Casp6 amino acid polymorphisms, R65W and G66R, on the catalytic function and structure of Casp6. The G66R substitution eliminated and R65W substitution significantly reduced Casp6 catalytic activity through impaired substrate binding. In contrast to wild-type Casp6, both Casp6 variants were unstable and inactive in transfected mammalian cells. In addition, Casp6-G66R acted as a dominant negative inhibitor of wild-type Casp6. The R65W and G66R substitutions caused perturbations in substrate recognition and active site organization as revealed by molecular dynamics simulations. Our results suggest that full Casp6 activity may not be essential for healthy humans and support the use of Casp6 inhibitors against Casp6-dependent neurodegeneration in age-dependent cognitive impairment and AD. Furthermore, this work illustrates that studying natural single amino acid polymorphisms of enzyme drug targets is a promising approach to uncover previously uncharacterized regulatory sites important for enzyme activity.
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http://dx.doi.org/10.1038/s41598-018-22283-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5849602PMC
March 2018

Homozygous missense mutation L673P in adenylate kinase 7 (AK7) leads to primary male infertility and multiple morphological anomalies of the flagella but not to primary ciliary dyskinesia.

Hum Mol Genet 2018 04;27(7):1196-1211

INSERM U1016, Institut Cochin, Paris 75014, France.

Motile cilia and sperm flagella share an extremely conserved microtubule-based cytoskeleton, called the axoneme, which sustains beating and motility of both organelles. Ultra-structural and/or functional defects of this axoneme are well-known to cause primary ciliary dyskinesia (PCD), a disorder characterized by recurrent respiratory tract infections, chronic otitis media, situs inversus, male infertility and in most severe cases, hydrocephalus. Only recently, mutations in genes encoding axonemal proteins with preferential expression in the testis were identified in isolated male infertility; in those cases, individuals displayed severe asthenozoospermia due to Multiple Morphological Abnormalities of the sperm Flagella (MMAF) but not PCD features. In this study, we performed genetic investigation of two siblings presenting MMAF without any respiratory PCD features, and we report the identification of the c.2018T > G (p.Leu673Pro) transversion in AK7, encoding an adenylate kinase, expressed in ciliated tissues and testis. By performing transcript and protein analyses of biological samples from individual carrying the transversion, we demonstrate that this mutation leads to the loss of AK7 protein in sperm cells but not in respiratory ciliated cells, although both cell types carry the mutated transcript and no tissue-specific isoforms were detected. This work therefore, supports the notion that proteins shared by both cilia and sperm flagella may have specific properties and/or function in each organelle, in line with the differences in their mode of assembly and organization. Overall, this work identifies a novel genetic cause of asthenozoospermia due to MMAF and suggests that in humans, more deleterious mutations of AK7 might induce PCD.
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http://dx.doi.org/10.1093/hmg/ddy034DOI Listing
April 2018

Interaction of Cholesterol with Perfringolysin O: What Have We Learned from Functional Analysis?

Toxins (Basel) 2017 11 23;9(12). Epub 2017 Nov 23.

Department of Biochemistry and Molecular Biology, University of Massachusetts, Amherst, MA 01003, USA.

Cholesterol-dependent cytolysins (CDCs) constitute a family of pore-forming toxins secreted by Gram-positive bacteria. These toxins form transmembrane pores by inserting a large β-barrel into cholesterol-containing membranes. Cholesterol is absolutely required for pore-formation. For most CDCs, binding to cholesterol triggers conformational changes that lead to oligomerization and end in pore-formation. Perfringolysin O (PFO), secreted by , is the prototype for the CDCs. The molecular mechanisms by which cholesterol regulates the cytolytic activity of the CDCs are not fully understood. In particular, the location of the binding site for cholesterol has remained elusive. We have summarized here the current body of knowledge on the CDCs-cholesterol interaction, with focus on PFO. We have employed sterols in aqueous solution to identify structural elements in the cholesterol molecule that are critical for its interaction with PFO. In the absence of high-resolution structural information, site-directed mutagenesis data combined with binding studies performed with different sterols, and molecular modeling are beginning to shed light on this interaction.
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http://dx.doi.org/10.3390/toxins9120381DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5744101PMC
November 2017

Caspase-6 Undergoes a Distinct Helix-Strand Interconversion upon Substrate Binding.

J Biol Chem 2017 03 2;292(12):4885-4897. Epub 2017 Feb 2.

From the Departments of Chemistry and

Caspases are cysteine aspartate proteases that are major players in key cellular processes, including apoptosis and inflammation. Specifically, caspase-6 has also been implicated in playing a unique and critical role in neurodegeneration; however, structural similarities between caspase-6 and other caspase active sites have hampered precise targeting of caspase-6. All caspases can exist in a canonical conformation, in which the substrate binds atop a β-strand platform in the 130's region. This caspase-6 region can also adopt a helical conformation that has not been seen in any other caspases. Understanding the dynamics and interconversion between the helical and strand conformations in caspase-6 is critical to fully assess its unique function and regulation. Here, hydrogen/deuterium exchange mass spectrometry indicated that caspase-6 is inherently and dramatically more conformationally dynamic than closely related caspase-7. In contrast to caspase-7, which rests constitutively in the strand conformation before and after substrate binding, the hydrogen/deuterium exchange data in the L2' and 130's regions suggested that before substrate binding, caspase-6 exists in a dynamic equilibrium between the helix and strand conformations. Caspase-6 transitions exclusively to the canonical strand conformation only upon substrate binding. Glu-135, which showed noticeably different calculated pK values in the helix and strand conformations, appears to play a key role in the interconversion between the helix and strand conformations. Because caspase-6 has roles in several neurodegenerative diseases, exploiting the unique structural features and conformational changes identified here may provide new avenues for regulating specific caspase-6 functions for therapeutic purposes.
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http://dx.doi.org/10.1074/jbc.M116.773499DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5377803PMC
March 2017

Terahertz continuous wave nonlinear-optical detection without phase-locking between a source and the detector.

Opt Lett 2016 Sep;41(17):4075-8

We demonstrate the possibility of nonlinear-optical detection of terahertz (THz) wave radiation without phase-locking between a source and a detector. Spectrally resolved room-temperature incoherent nonlinear-optical detection is demonstrated for 0.22-THz continuous wave (CW) radiation by upconversion using a 100-mW CW laser in a 15-mm-long Mg:LiNbO3 crystal.
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http://dx.doi.org/10.1364/OL.41.004075DOI Listing
September 2016

Interactions of Haptoglobin with Monomeric Globin Species: Insights from Molecular Modeling and Native Electrospray Ionization Mass Spectrometry.

Biochemistry 2016 Mar 17;55(12):1918-28. Epub 2016 Mar 17.

Department of Chemistry and ‡Department of Biochemistry and Molecular Biology, University of Massachusetts Amherst , Amherst, Massachusetts 01003, United States.

Haptoglobin (Hp) binds free hemoglobin (Hb) dimers to prevent negative consequences of Hb circulation in the extracellular environment. Although both monomeric Hb and myoglobin (Mb) species also present potential risks, their interactions with Hp have not been extensively studied. Mb is homologous to both the α- and β-chains of Hb and shares many conserved Hb/Hp interface residues, yet whether Hp binds Mb remains unclear. To address this, computational biology tools were used to predict the interactions required for Hp to bind monomeric globins, and the predicted association was tested using native electrospray ionization mass spectrometry (ESI-MS). The Hb/Hp crystal structure was used as the template to create molecular models of two Mb molecules bound to an Hp heterodimer (Mb2/Hp). Molecular modeling suggests that Mb can bind at the Hp α-chain binding site, where 73% of the globin/Hp interactions are conserved. By contrast, several ionic β-chain residues involved in complementary electrostatic interactions with Hp correspond to residues with the opposite charge in Mb, suggesting unfavorable electrostatic Hp/Mb interactions at the β-chain binding site. As shown by native ESI-MS, isolated monomeric Hbα subunits can form 2:1 complexes with Hp heterotetramers in the absence of Hb β-chains. Native ESI-MS also confirmed that Mb can bind to Hp heterotetramers in solution with stoichiometries of 1:1 and 2:1 at physiological pH and ionic strength. The affinity of Hp for Mb appears to be diminished relative to that of Hb α-chains. Our in silico experiments rationalize this change and demonstrate that molecular modeling of protein/protein interactions is a valuable aid for MS experiments.
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http://dx.doi.org/10.1021/acs.biochem.5b00807DOI Listing
March 2016

Homozygous mutation of PLCZ1 leads to defective human oocyte activation and infertility that is not rescued by the WW-binding protein PAWP.

Hum Mol Genet 2016 Mar 31;25(5):878-91. Epub 2015 Dec 31.

Université Grenoble Alpes, Grenoble, F-38000, Grenoble, France, Institut Albert Bonniot, INSERM U823, La Tronche F-38700, France,

In mammals, sperm-oocyte fusion initiates Ca(2+) oscillations leading to a series of events called oocyte activation, which is the first stage of embryo development. Ca(2+) signaling is elicited by the delivery of an oocyte-activating factor by the sperm. A sperm-specific phospholipase C (PLCZ1) has emerged as the likely candidate to induce oocyte activation. Recently, PAWP, a sperm-born tryptophan domain-binding protein coded by WBP2NL, was proposed to serve the same purpose. Here, we studied two infertile brothers exhibiting normal sperm morphology but complete fertilization failure after intracytoplasmic sperm injection. Whole exomic sequencing evidenced a missense homozygous mutation in PLCZ1, c.1465A>T; p.Ile489Phe, converting Ile 489 into Phe. We showed the mutation is deleterious, leading to the absence of the protein in sperm, mislocalization of the protein when injected in mouse GV and MII oocytes, highly abnormal Ca(2+) transients and early embryonic arrest. Altogether these alterations are consistent with our patients' sperm inability to induce oocyte activation and initiate embryo development. In contrast, no deleterious variants were identified in WBP2NL and PAWP presented normal expression and localization. Overall we demonstrate in humans, the absence of PLCZ1 alone is sufficient to prevent oocyte activation irrespective of the presence of PAWP. Additionally, it is the first mutation located in the C2 domain of PLCZ1, a domain involved in targeting proteins to cell membranes. This opens the door to structure-function studies to identify the conserved amino acids of the C2 domain that regulate the targeting of PLCZ1 and its selectivity for its lipid substrate(s).
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http://dx.doi.org/10.1093/hmg/ddv617DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4754041PMC
March 2016

X-ray Structural and Functional Studies of the Three Tandemly Linked Domains of Non-structural Protein 3 (nsp3) from Murine Hepatitis Virus Reveal Conserved Functions.

J Biol Chem 2015 Oct 19;290(42):25293-306. Epub 2015 Aug 19.

From the Department of Biological Sciences, the Center for Cancer Research, and the Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907 and

Murine hepatitis virus (MHV) has long served as a model system for the study of coronaviruses. Non-structural protein 3 (nsp3) is the largest nsp in the coronavirus genome, and it contains multiple functional domains that are required for coronavirus replication. Despite the numerous functional studies on MHV and its nsp3 domain, the structure of only one domain in nsp3, the small ubiquitin-like domain 1 (Ubl1), has been determined. We report here the x-ray structure of three tandemly linked domains of MHV nsp3, including the papain-like protease 2 (PLP2) catalytic domain, the ubiquitin-like domain 2 (Ubl2), and a third domain that we call the DPUP (domain preceding Ubl2 and PLP2) domain. DPUP has close structural similarity to the severe acute respiratory syndrome coronavirus unique domain C (SUD-C), suggesting that this domain may not be unique to the severe acute respiratory syndrome coronavirus. The PLP2 catalytic domain was found to have both deubiquitinating and deISGylating isopeptidase activities in addition to proteolytic activity. A computationally derived model of MHV PLP2 bound to ubiquitin was generated, and the potential interactions between ubiquitin and PLP2 were probed by site-directed mutagenesis. These studies extend substantially our structural knowledge of MHV nsp3, providing a platform for further investigation of the role of nsp3 domains in MHV viral replication.
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http://dx.doi.org/10.1074/jbc.M115.662130DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4646180PMC
October 2015

Evaluation of AaDOP2 receptor antagonists reveals antidepressants and antipsychotics as novel lead molecules for control of the yellow fever mosquito, Aedes aegypti.

J Pharmacol Exp Ther 2015 Jan 20;352(1):53-60. Epub 2014 Oct 20.

Department of Medicinal Chemistry and Molecular Pharmacology (J.M.C., T.B.D., V.J.W.), Department of Entomology (J.M.M., A.B.N., C.A.H.), and Bindley Bioscience Center, Discovery Park (S.N.S.), Purdue University, West Lafayette, Indiana

The yellow fever mosquito, Aedes aegypti, vectors disease-causing agents that adversely affect human health, most notably the viruses causing dengue and yellow fever. The efficacy of current mosquito control programs is challenged by the emergence of insecticide-resistant mosquito populations, suggesting an urgent need for the development of chemical insecticides with new mechanisms of action. One recently identified potential insecticide target is the A. aegypti D1-like dopamine receptor, AaDOP2. The focus of the present study was to evaluate AaDOP2 antagonism both in vitro and in vivo using assay technologies with increased throughput. The in vitro assays revealed AaDOP2 antagonism by four distinct chemical scaffolds from tricyclic antidepressant or antipsychotic chemical classes, and elucidated several structure-activity relationship trends that contributed to enhanced antagonist potency, including lipophilicity, halide substitution on the tricyclic core, and conformational rigidity. Six compounds displayed previously unparalleled potency for in vitro AaDOP2 antagonism, and among these, asenapine, methiothepin, and cis-(Z)-flupenthixol displayed subnanomolar IC50 values and caused rapid toxicity to A. aegypti larvae and/or adults in vivo. Our study revealed a significant correlation between in vitro potency for AaDOP2 antagonism and in vivo toxicity, suggesting viability of AaDOP2 as an insecticidal target. Taken together, this study expanded the repertoire of known AaDOP2 antagonists, enhanced our understanding of AaDOP2 pharmacology, provided further support for rational targeting of AaDOP2, and demonstrated the utility of efficiency-enhancing in vitro and in vivo assay technologies within our genome-to-lead pipeline for the discovery of next-generation insecticides.
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http://dx.doi.org/10.1124/jpet.114.219717DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4279103PMC
January 2015

The Cdk/cDc14 module controls activation of the Yen1 holliday junction resolvase to promote genome stability.

Mol Cell 2014 Apr 13;54(1):80-93. Epub 2014 Mar 13.

Department of Biochemistry, Purdue University, West Lafayette, IN 47907, USA; Center for Cancer Research, Purdue University, West Lafayette, IN 47907, USA. Electronic address:

Faithful genome transmission during cell division requires precise, coordinated action of DNA metabolic enzymes, including proteins responsible for DNA damage detection and repair. Dynamic phosphorylation plays an important role in controlling repair enzymes during the DNA damage response (DDR). Cdc14 phosphatases oppose cyclin-dependent kinase (Cdk) phosphorylation and have been implicated in the DDR in several model systems. Here, we have refined the substrate specificity of budding yeast Cdc14 and, using this insight, identified the Holliday junction resolvase Yen1 as a DNA repair target of Cdc14. Cdc14 activation at anaphase triggers nuclear accumulation and enzymatic activation of Yen1, likely to resolve persistent recombinational repair intermediates. Consistent with this, expression of a phosphomimetic Yen1 mutant increased sister chromatid nondisjunction. In contrast, lack of Cdk phosphorylation resulted in constitutive activity and elevated crossover-associated repair. The precise timing of Yen1 activation, governed by core cell-cycle regulators, helps coordinate DNA repair with chromosome segregation and safeguards against genome destabilization.
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http://dx.doi.org/10.1016/j.molcel.2014.02.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3988236PMC
April 2014

Chemical and biological mechanisms of phytochemical activation of Nrf2 and importance in disease prevention.

Recent Adv Phytochem 2013;43:121-155. Epub 2013 Dec 3.

Purdue University Center for Cancer Research, West Lafayette, Indiana, 47907.

Plants are an incredibly rich source of compounds that activate the Nrf2 transcription factor, leading to upregulation of a battery of cytoprotective genes. This perspective surveys established and proposed molecular mechanisms of Nrf2 activation by phytochemicals with a special emphasis on a common chemical property of Nrf2 activators: the ability as "soft" electrophiles to modify cellular thiols, either directly or as oxidized biotransformants. In addition, the role of reactive oxygen/nitrogen species as secondary messengers in Nrf2 activation is discussed. While the uniquely reactive C151 of Keap1, an Nrf2 repressor protein, is highlighted as a key target of cytoprotective phytochemicals, also reviewed are other stress-responsive proteins, including kinases, which play non-redundant roles in the activation of Nrf2 by plant-derived agents. Finally, the perspective presents two key factors accounting for the enhanced therapeutic windows of effective phytochemical activators of the Keap1-Nrf2 axis: enhanced selectivity toward sensor cysteines and reversibility of addition to thiolate molecules.
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http://dx.doi.org/10.1007/978-3-319-00581-2_7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4739799PMC
December 2013

Intracellular detection and evolution of site-specific proteases using a genetic selection system.

Appl Biochem Biotechnol 2012 Mar 21;166(5):1340-54. Epub 2012 Jan 21.

Department of Chemistry, Purdue University, West Lafayette, IN 47907, USA.

Development of endoproteases, programmed to promote degradation of peptides or proteins responsible for pathogenic states, represents an attractive therapeutic strategy, since such biocatalytic agents could be directed against a potentially unlimited repertoire of extracellular proteinaceous targets. Difficulties associated with engineering enzymes with tailor-made substrate specificities have, however, hindered the discovery of proteases possessing both the efficiency and selectivity to act as therapeutics. Here, we disclose a genetic system, designed to report on site-specific proteolysis through the survival of a bacterial host, and the implementation of this method in the directed evolution of proteases with a non-native substrate preference. The high sensitivity potential of this system was established by monitoring the activity of the Tobacco Etch Virus protease (TEV-Pr) against co-expressed substrates of various recognition level and corroborated by both intracellular and cell-free assays. The genetic selection system was then used in an iterative mode with a library of TEV-Pr mutants to direct the emergence of proteases favoring a nominally poor substrate of the stringently selective protease. The retrieval of mutant enzymes displaying enhanced proteolytic properties against the non-native sequence combined with reduced recognition of the cognate hexapeptide substrate demonstrates the potential of this system for evolving proteases with improved or completely unprecedented properties.
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http://dx.doi.org/10.1007/s12010-011-9522-6DOI Listing
March 2012

Functional profiling of p53-binding sites in Hdm2 and Hdmx using a genetic selection system.

Bioorg Med Chem 2010 Aug 22;18(16):6099-108. Epub 2010 Jun 22.

Department of Chemistry, Purdue University, West Lafayette, IN 47907, USA.

Upregulation of structurally homologous oncoproteins Hdm2 and Hdmx has been linked to the depletion or inactivation of their common regulation target the tumor suppressor p53 protein leading to the progression of cancer. The restoration of the p53 function, rendered suppressed or dormant by these negative regulators, establishes, therefore, a unique opportunity for a targeted induction of apoptosis in cancers that retain wild-type p53. While several small molecules have been reported to rescue the tumor suppressor by antagonizing the Hdm2-p53 interaction, these agents displayed limited application scope by being ineffective in tumors enriched with active Hdmx. Here, we describe the use of a genetic selection system and encoded library of conformationally pre-organized peptides to perform functional profiling of each regulator revealing specific recognition features that guide the antagonism of Hdm2-p53 and Hdmx-p53 interactions. Structure-activity relationship analysis of the most effective leads identified functional and structural elements mediating selective recognition of the two structurally related regulators, while providing convenient starting points for further activity optimization.
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http://dx.doi.org/10.1016/j.bmc.2010.06.053DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2922946PMC
August 2010

Direct evaluation of cellular internalization rates using chromogenic disulfides.

Mol Biosyst 2010 Jul 20;6(7):1176-9. Epub 2010 Apr 20.

Department of Chemistry, Purdue University, West Lafayette, IN 47909, USA.

A practical assay, which exploits the maintenance of the cytoplasmic redox balance in live cells, has been developed to report in real time on relative cellular internalization rates of molecules derivatized as chromogenic disulfides.
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http://dx.doi.org/10.1039/c003969aDOI Listing
July 2010
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