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BBA - Proteins and Proteomics (v.1834, #2)
Enhancement of thermal stability of chondroitinase ABC I by site-directed mutagenesis: An insight from Ramachandran plot by Mahdieh Nazari-Robati; Khosro Khajeh; Mahdi Aminian; Nasrin Mollania; Abolfazl Golestani (pp. 479-486).
The application of chondroitinase ABC I (cABC I) in damaged nervous tissue is believed to prune glycosaminoglycan chains of proteoglycans, thereby facilitates axon regeneration. However, the utilization of cABC I as therapeutics is notably restricted due to its thermal instability. In the present study, we have explored the possibility of thermostabilization of cABC I through release of its conformational strain using Ramachandran plot information. In this regard, Gln140 with non-optimal φ and ψ values were replaced with Gly, Ala and Asn. The results indicated that Q140G and Q140A mutants were able to improve both activity and thermal stability of the enzyme while Q140N variant reduced the enzyme activity and destabilized it. Moreover, the two former variants displayed a remarkable resistance to trypsin degradation. Structural analysis of all mutants showed an increase in intrinsic fluorescence intensity and secondary structure content of Q140G and Q140A compared to the wild type which indicated more compact structure upon mutation. This investigation demonstrated that relief of conformational tension can be considered as a possible approach to increase the stability of the protein.► Thermal stability of cABC I and its mutants has been reported. ► Three mutations in a disallowed region of Ramachandran plot were designed. ► Q140G and Q140A mutations increased thermal stability of cABC I. ► Q140N mutation destabilized the enzyme. ► Structural analysis demonstrated that Q140N mutant became more flexible than the native enzyme.
Keywords: Abbreviations; cABC I; Chondroitinase ABC I; C6S; Chondroitin-6-sulfate; C4S; Chondroitin-4-sulfate; DS; Dermatan sulfate; GAGs; Glycosaminoglycans; CSPG; Chondroitin sulfate proteoglycan; SCI; Spinal cord injury; CD; Circular dichroism; PDB; Protein Data BankChondroitinase ABC I; Ramachandran plot; Site directed mutagenesis; Thermal stability
Utilization of protein intrinsic disorder knowledge in structural proteomics by Christopher J. Oldfield; Bin Xue; Ya-Yue Van; Eldon L. Ulrich; John L. Markley; A. Keith Dunker; Vladimir N. Uversky (pp. 487-498).
Intrinsically disordered proteins (IDPs) and proteins with long disordered regions are highly abundant in various proteomes. Despite their lack of well-defined ordered structure, these proteins and regions are frequently involved in crucial biological processes. Although in recent years these proteins have attracted the attention of many researchers, IDPs represent a significant challenge for structural characterization since these proteins can impact many of the processes in the structure determination pipeline. Here we investigate the effects of IDPs on the structure determination process and the utility of disorder prediction in selecting and improving proteins for structural characterization. Examination of the extent of intrinsic disorder in existing crystal structures found that relatively few protein crystal structures contain extensive regions of intrinsic disorder. Although intrinsic disorder is not the only cause of crystallization failures and many structured proteins cannot be crystallized, filtering out highly disordered proteins from structure-determination target lists is still likely to be cost effective. Therefore it is desirable to avoid highly disordered proteins from structure-determination target lists and we show that disorder prediction can be applied effectively to enrich structure determination pipelines with proteins more likely to yield crystal structures. For structural investigation of specific proteins, disorder prediction can be used to improve targets for structure determination. Finally, a framework for considering intrinsic disorder in the structure determination pipeline is proposed.► Intrinsically disordered proteins are common in various proteomes. ► These proteins are very dynamic and actively resist crystallization. ► Intrinsically disordered proteins can hinder structure determination pipelines. ► It is desirable to filter out highly disordered proteins from structural proteomics target lists. ► Protein intrinsic disorder knowledge can be utilized for prioritizing structural proteomics targets.
Keywords: Proteomics; Structural genomics; Structural proteomics; Intrinsically disordered protein
Microtubule-binding sites of the CH domain of EB1 and its autoinhibition revealed by NMR by Teppei Kanaba; Ryoko Maesaki; Tomoyuki Mori; Yutaka Ito; Toshio Hakoshima; Masaki Mishima (pp. 499-507).
End-binding protein 1 (EB1) is one of the best studied plus-end tracking proteins. It is known that EB1 specifically binds the plus ends of microtubules (MTs) and promotes MT growth. EB1 activity is thought to be autoinhibited by an intramolecular interaction. Recent cryo-EM analyses showed that the CH domain of Mal3p ( Schizosaccharomyces pombe EB1 homolog) binds to GMPCPP-MT (Sandblad, L. Cell 127 (2006) 1415-24), and strongly binds GTPγS-MT which is proposed to mimic MT plus ends better than GMPCPP-MT (Maurer S.P. et al. Cell 149 (2012) 371–82). Here, we report on the MT binding sites of the CH domain of EB1 as revealed by NMR using the transferred cross-saturation method. In this study, we used GMPCPP-MT and found that the MT binding sites are very similar to the binding site for GTPγS-MT as suggested by cryo-EM (Maurer S.P. et al. Cell 149 (2012) 371–82). Notably, the N-terminal tip of helix α6 of the CH domain did not make contact with GMPCPP-MT, in contrast to the cryo-EM study which showed that it is closely located to a putative switch region of β-tubulin in GTPγS-MT (Maurer S.P. et al. Cell 149 (2012) 371-82). Further, we found that the intramolecular interaction site of EB1 overlaps the MT binding sites, indicating that the MT binding sites are masked by interaction with the C-terminal domain. We propose a structural view of autoinhibition and its release mechanism through competition binding with binding partners such as adenomatous polyposis coli protein.► We report the microtubule binding sites of EB1 revealed by NMR. ► Microtubule binding sites are masked by an interaction between CH and C-terminal domain. ► A structural view of autoinhibition and its release mechanisms are proposed.
Keywords: Abbreviations; HSQC; heteronuclear single quantum correlation spectroscopy; TROSY; Transverse relaxation optimized spectroscopy; CSP; chemical shift perturbation; TCS; transferred cross-saturation; +; TIPs; plus-end tracking proteins; MT; microtubule; EB1; end-binding protein 1; CH domain; calponin homology domain; CAP-Gly domain; cytoskeleton-associated protein glycine-rich domain; CLIP; cytoplasmic linker protein; APC; adenomatous polyposis coliEB1; Microtubule; NMR; TCS; APC
A novel Aurora-A-mediated phosphorylation of p53 inhibits its interaction with MDM2 by Kai-Wei Hsueh; Shu-Ling Fu; Chirn-Bin Chang; Yu-Ling Chang; Chao-Hsiung Lin (pp. 508-515).
Purpose: Crosstalk between Aurora-A kinase and p53 has been proposed. While the genetic amplification of Aurora-A has been observed in many human cancers, how p53 is regulated by Aurora-A remains ambiguous. In this study, Aurora-A-mediated phosphorylation of p53 was analyzed by mass spectrometry in order to identify a new phosphorylation site. Subsequently, the functional consequences of such phosphorylation were examined. Experimental design: In vitro phosphorylation of p53 by Aurora-A was performed and the phosphorylated protein was then digested with trypsin and enriched for phosphopeptides by immobilized metal affinity chromatography. Subsequently, a combination of β-elimination and Michael addition was applied to the phosphopeptides in order to facilitate the identification of phosphorylation sites by MS. The functional consequences of the novel phosphorylation of p53 on the protein–protein interactions, protein stability and transactivation activity were then examined using co-immunoprecipitation, Western blotting and reporter assays. Results: Ser-106 of p53 was identified as a novel site phosphorylated by Aurora-A. A serine-to-alanine mutation at this site was found to attenuate Aurora-A-mediated phosphorylation in vitro. In addition, phosphate-sensitive Phos-tag SDS-PAGE was used to confirm that the Ser-106 of p53 is in vivo phosphorylated by Aurora-A. Finally, co-immunoprecipitation studies suggested that Ser-106 phosphorylation of p53 decreases its interaction with MDM2 and prolongs the half-life of p53. Conclusions: The inhibition of the interaction between p53 and MDM2 by a novel Aurora-A-mediated p53 phosphorylation was identified in this study and this provides important information for further investigations into the interaction between p53 and Aurora-A in terms of cancer biology.► Serine 106 of p53 is a novel phosphorylation site mediated by Aurora-A kinase. ► IMAC and chemical modifications facilitate identification of phosphorylated peptide. ► S106D mutant p53 showed the suppressed association with MDM2 than wild-type p53.
Keywords: Abbreviations; IMAC; immobilized metal affinity chromatography; GST; glutathione S-transferase; SDS-PAGE; sodium dodecyl sulfate polyacrylamide gel electrophoresis; RIPA buffer; radioimmune precipitation assay buffer; DTT; 1,4-dithiothreitol; TFA; trifluoroacetic acid; AET; 2-aminoethanethiol; CHCA; α-cyano-4-hydroxycinnamic acid; DHB; 2,5-dihydroxybenzoic acidPhosphorylation; p53; Aurora-A; Phosphopeptide; IMAC
Insights into the function of RifI2: Structural and biochemical investigation of a new shikimate dehydrogenase family protein by James Peek; Christel Garcia; John Lee; Dinesh Christendat (pp. 516-523).
The shikimate dehydrogenase (SDH) family consists of enzymes with diverse roles in secondary metabolism. The two most widespread members of the family, AroE and YdiB, function in amino acid biosynthesis and quinate catabolism, respectively. Here, we have determined the crystal structure of an SDH homolog belonging to the RifI class, a group of enzymes with proposed roles in antibiotic biosynthesis. The structure of RifI2 from Pseudomonas putida exhibits a number of distinctive features, including a substantial C-terminal truncation and an atypical mode of oligomerization. The active site of the enzyme contains substrate- and cofactor-binding motifs that are significantly different from those of any previously characterized member of the SDH family. These features are reflected in the novel kinetic properties of the enzyme. RifI2 exhibits much lower activity using shikimate as a substrate than AroE, and a strong preference for NAD+ instead of NADP+ as a cofactor. Moreover, the enzyme has only trace activity using quinate, unlike YdiB. Cocrystallization of RifI2 with NAD+ provided the opportunity to determine the mode of cofactor selectivity employed by the enzyme. We complemented this analysis by probing the role of a strictly conserved residue in the cofactor-binding domain, Asn193, by site directed mutagenesis. This study presents the first crystal structure and formal kinetic characterization of a new NAD+-dependent member of the SDH family.► The crystal structure of Pseudomonas putida RifI2 was solved in complex with NAD+. ► The structure has a novel C-terminal truncation and an unusual dimerization mode. ► A unique collection of substrate- and cofactor-binding motifs was identified. ► These features are reflected in the distinctive kinetic properties of the enzyme.
Keywords: Abbreviations; SDH; shikimate dehydrogenase; SdhL; shikimate dehydrogenase-like; Ael1; AroE-like1; AHBA; 3-amino-5-hydroxybenzoic acid; IPTG; isopropyl ß-; d; -thiogalactopyranoside; Ni-NTA; nickel-nitriloacetic acid; R.M.S.D.; root mean square deviationShikimate dehydrogenase; Pseudomonas putida; Crystal structure; Enzyme kinetics; Site-directed mutagenesis
Unusual binding of Grb2 protein to a bivalent polyproline-ligand immobilized on a SPR sensor: Intermolecular bivalent binding by Nico J. de Mol; John A.W. Kruijtzer; Ed E. Moret; Isabelle Broutin; Rob M.J. Liskamp (pp. 524-535).
The Grb2 adapter protein is involved in the activation of the Ras signaling pathway. It recruits the Sos protein by binding of its two SH3 domains to Sos polyproline sequences. We observed that the binding of Grb2 to a bivalent ligand, containing two Sos-derived polyproline-sequences immobilized on a SPR sensor, shows unusual kinetic behavior. SPR-kinetic analysis and supporting data from other techniques show major contributions of an intermolecular bivalent binding mode. Each of the two Grb2 SH3 domains binds to one polyproline-sequence of two different ligand molecules, facilitating binding of a second Grb2 molecule to the two remaining free polyproline binding sites. A molecular model based on the X-ray structure of the Grb2 dimer shows that Grb2 is flexible enough to allow this binding mode. The results fit with a role of Grb2 in protein aggregation, achieving specificity by multivalent interactions, despite the relatively low affinity of single SH3 interactions.Display Omitted► Kinetic analysis of Grb2 binding to monovalent and bivalent polyproline surfaces. ► Intermolecular rather than intramolecular bivalent binding. ► Intermolecular binding maybe due to high flexibility of Grb2 SH3-SH2 linkers. ► This binding mode fits well in non-linear networks of protein aggregation.
Keywords: Abbreviations; PP; polyproline; RTK; receptor tyrosine kinase; K; LM; apparent Langmuir binding constant; K; S; binding constant in solution derived from SPR competition experimentsGrb2; Polyproline; SH3-domain; Sos-protein; Surface plasmon resonance (SPR); Divalent binding
X-ray crystallography and QM/MM investigation on the oligosaccharide synthesis mechanism of rice BGlu1 glycosynthases by Jinhu Wang; Salila Pengthaisong; James R. Ketudat Cairns; Yongjun Liu (pp. 536-545).
Nucleophile mutants of retaining β-glycosidase can act as glycosynthases to efficiently catalyze the synthesis of oligosaccharides. Previous studies proved that rice BGlu1 mutants E386G, E386S and E386A catalyze the oligosaccharide synthesis with different rates. The E386G mutant gave the fastest transglucosylation rate, which was approximately 3- and 19-fold faster than those of E386S and E386A. To account for the differences of their activities, in this paper, the X-ray crystal structures of BGlu1 mutants E386S and E386A were solved and compared with that of E386G mutant. However, they show quite similar active sites, which implies that their activities cannot be elucidated from the crystal structures alone. Therefore, a combined quantum mechanical/molecular mechanical (QM/MM) calculations were further performed. Our calculations reveal that the catalytic reaction follows a single-step mechanism, i.e., the extraction of proton by the acid/base, E176, and the formation of glycosidic bond are concerted. The energy barriers are calculated to be 19.9, 21.5 and 21.9kcal/mol for the mutants of E386G, E386S and E386A, respectively, which is consistent with the order of their experimental relative activities. But based on the calculated activation energies, 1.1kcal/mol energy difference may translate to nearly 100 fold rate difference. Although the rate limiting step in these mutants has not been established, considering the size of the product and the nature of the active site, it is likely that the product release, rather than chemistry, is rate limiting in these oligosaccharides synthesis catalyzed by BGlu1 mutants.Display Omitted► The mechanism of glycosynthase was explored by X-ray crystallographic and QM/MM methods. ► The catalytic reaction proceeds via a single-step mechanism. ► The energy barriers are sensitive to hindered interactions of the mutated residues. ► The incoming crystal water Wat1 was important to the reaction.
Keywords: Abbreviations; QM/MM; quantum mechanical/molecular mechanical; α-GlcF; alpha-glucosyl fluoride; p; NPC2; p; NP-cellobioside; RMSD; root-mean-square deviation; MD; molecular dynamics; HB; hydrogen bond; R; reactant; TS; transition state; P; productOligosaccharide synthesis; Rice BGlu1 E386 mutant; QM/MM; Energy barrier; rice BGlu1 E386G E386A, E386S
Monodisperse and LPS-free Aggregatibacter actinomycetemcomitans leukotoxin: Interactions with human β2 integrins and erythrocytes by Jesper Reinholdt; Knud Poulsen; Christel R. Brinkmann; Søren V. Hoffmann; Romualdas Stapulionis; Jan J. Enghild; Uffe B. Jensen; Thomas Boesen; Thomas Vorup-Jensen (pp. 546-558).
Aggregatibacter actinomycetemcomitans is a gram-negative, facultatively anaerobic cocco-bacillus and a frequent member of the human oral flora. It produces a leukotoxin, LtxA, belonging to the repeats-in-toxin (RTX) family of bacterial cytotoxins. LtxA efficiently kills neutrophils and mononuclear phagocytes. The known receptor for LtxA on leukocytes is integrin αLβ2 (LFA-1 or CD11a/CD18). However, the molecular mechanisms involved in LtxA-mediated cytotoxicity are poorly understood, partly because LtxA has proven difficult to prepare for experiments as free of contaminants and with its native structure. Here, we describe a protocol for the purification of LtxA from bacterial culture supernatant, which does not involve denaturing procedures. The purified LtxA was monodisperse, well folded as judged by the combined use of synchrotron radiation circular dichroism spectroscopy (SRCD) and in silico prediction of the secondary structure content, and free of bacterial lipopolysaccharide. The analysis by SRCD and similarity to a lipase from Pseudomonas with a known three dimensional structure supports the presence of a so-called beta-ladder domain in the C-terminal part of LtxA. LtxA rapidly killed K562 target cells transfected to express β2 integrin. Cells expressing αMβ2 (CD11b/CD18) or αXβ2 (CD11c/CD18) were killed as efficiently as cells expressing αLβ2. Erythrocytes, which do not express β2 integrins, were lysed more slowly. In ligand blotting experiments, LtxA bound only to the β2 chain (CD18). These data support a previous suggestion that CD18 harbors the major binding site for LtxA as well as identifies integrins αMβ2 and αXβ2 as novel receptors for LtxA.Display Omitted► We describe a protocol for purifying LPS-free LtxA from A. actinomycetemcomitans. ► We characterize the structural order of LtxA by the use of SRCD. ► LtxA-mediated killing of cells through integrin αLβ2 as well as αMβ2 and αXβ2.
Keywords: Bacterial toxin; Leukotoxin; Integrin; Synchrotron radiation circular dichroism spectroscopy
The major mRNP protein YB-1: Structural and association properties in solution by Sergey G. Guryanov; Vladimir V. Filimonov; Alexander A. Timchenko; Bogdan S. Melnik; Hiroshi Kihara; Victor P. Kutyshenko; Lev P. Ovchinnikov; Gennady V. Semisotnov (pp. 559-567).
YB-1 is a major mRNP protein participating in the regulation of transcription and translation of a wide range of eukaryotic genes in many organisms probably due to its influence on mRNA packing into mRNPs. While the functional properties of YB-1 are extensively studied, little is known about its structural properties. In the present work we focused on studying its secondary structure, rigidity of its tertiary structure, compactness, and oligomerization in vitro by using far UV-CD, DSC, one-dimensional1H NMR, SAXS, sedimentation and FPLC. It was shown that only the cold shock domain within the entire YB-1 chain has a well-packed tertiary structure undergoing cooperative heat and cold denaturation transitions. In contrast, the rest of the YB-1 molecule is not rigidly packed and consists of PP II-like helical secondary structure elements and coil-like regions. At the same time, the overall dimension of the protein molecule is unexpectedly small. The polypeptide chains of YB-1 have a high tendency to form oligomers at neutral pH, while the extent and structural organization of the oligomers depend on protein concentration and ionic strength varying from compact monomeric units up to high molecular weight oligomers. These oligomers in solution are unstable and dissociate upon protein concentration decrease.► YB-1 is a multifunctional protein regulating transcription and translation. ► The extent of YB-1 oligomerization correlates with ionic strength. ► In solution YB-1 is mainly unfolded but compact protein rich of polypro II helices. ► Cold shock domain of YB-1 is folded and undergoes heat- and cold-induced unfolding.
Keywords: Abbreviations; UV; ultraviolet; CD; circular dichroism; DSC; differential scanning calorimetry; NMR; nuclear magnetic resonance; SAXS; small-angle X-ray scattering; FPLC; fast protein liquid chromatography; CSD; cold shock domain; GuHCl; guanidine hydrochloride; BSA; bovine serum albumin; PPII; poly(L)proline type II helixCold shock domain; Heat and cold denaturation; Disordered protein; Polyproline type II helix; Compactness; Oligomerization
Influenza A virus protein PB1-F2 from different strains shows distinct structural signatures by Sara M.Ø. Solbak; Alok Sharma; Karsten Bruns; Roder René Röder; David Mitzner; Friedrich Hahn; Rebekka Niebert; Anni Vedeler; Petra Henklein; Peter Henklein; Ulrich Schubert; Victor Wray; Torgils Fossen (pp. 568-582).
The proapoptotic influenza A virus PB1-F2 protein contributes to viral pathogenicity and is present in most human and avian influenza isolates. The structures of full-length PB1-F2 of the influenza strains Pandemic flu 2009 H1N1, 1918 Spanish flu H1N1, Bird flu H5N1 and H1N1 PR8, have been characterized by NMR and CD spectroscopy. The study was conducted using chemically synthesized full-length PB1-F2 protein and fragments thereof. The amino acid residues 30–70 of PR8 PB1-F2 were found to be responsible for amyloid formation of the protein, which could be assigned to formation of β-sheet structures, although α-helices were the only structural features detected under conditions that mimic a membranous environment. At membranous conditions, in which the proteins are found in their most structured state, significant differences become apparent between the PB1-F2 variants investigated. In contrast to Pandemic flu 2009 H1N1 and PR8 PB1-F2, which exhibit a continuous extensive C-terminal α-helix, both Spanish flu H1N1 and Bird flu H5N1 PB1-F2 contain a loop region with residues 66–71 that divides the C-terminus into two shorter helices. The observed structural differences are located to the C-terminal ends of the proteins to which most of the known functions of these proteins have been assigned. A C-terminal helix–loop–helix motif might be a structural signature for PB1-F2 of the highly pathogenic influenza viruses as observed for 1918 Spanish flu H1N1 and Bird flu H5N1 PB1-F2. This signature could indicate the pathological nature of viruses emerging in the future and thus aid in the recognition of these viruses.Display Omitted► PB1-F2 derived from different IAV strains show distinct secondary structures. ► A divided C-terminal helix may be a signature of the most lethal influenza viruses. ► The central region of PR8 PB1-F2 was responsible for amyloid formation. ► The main secondary structure of PB1-F2 was α-helical.
Keywords: Abbreviations; ACN; acetonitrile; CD; circular dichroism; COSY; correlation spectroscopy; CSI; chemical shift index; IAV; influenza A virus; MTS; mitochondrial targeting sequence; NMR; nuclear magnetic resonance; NOESY; Nuclear Overhauser effect spectroscopy; RMSD; root mean square deviations; SDS; sodium dodecyl sulfate; TFE; trifluoroethanol; TOCSY; total correlation spectroscopyInfluenza; PB1-F2; 2009 Pandemic flu; 1918 Spanish flu; Bird flu; PR8
High throughput characterization of structural differences between closely related proteins in solution by Alexander Zaslavsky; Pedro Madeira; Leonid Breydo; Vladimir N. Uversky; Arnon Chait; Boris Zaslavsky (pp. 583-592).
Partitioning of a protein in an aqueous two-phase system (ATPS) is governed by interactions of the protein with aqueous media in the two phases. Here we describe how partitioning of proteins in a set of ATPS of different compositions can be used to quantify differences between 3D structures of closely related proteins. We also provide perspective on practical applications of the technology when comparative analysis of the higher-order structure of proteins is desired.Display Omitted► Distinguishing protein isoforms without detailed structural analysis is often needed. ► Analytical tool exploiting isoform differences in solvent interaction is introduced. ► This technique is based on the protein partition in aqueous two-phase systems (ATPS). ► ATPS generate very crowded environment. ► Macromolecular crowding conditions of ATPS amplify structural differences of isoforms.
Keywords: Partitioning; Protein structure; Aqueous two-phase system; Structural change; Solvent interaction analysis
Interaction of reduced lysozyme with surfactants by Weiying Zhu; Timothy A. Keiderling (pp. 593-600).
The reformation of secondary structure for unfolded, disulfide reduced hen egg white lysozyme (HEWL) upon interaction with surfactants was studied using CD, fluorescence and IR (infrared) techniques. Equilibrium CD studies showed that reduced HEWL when mixed with negatively charged surfactants, such as SDS (sodium dodecyl sulfate), gradually regains average helical structure to a level equivalent to that obtained for the oxidized form also in SDS, but both forms lose tertiary structure in such environments. This non-native structure recovery process begins with monomer surfactant interaction but at higher concentrations is in part dependent on micelle formation, with the helical fraction reaching its maximum value with each surfactant only above the CMC. Fluorescence changes were more complex, evidencing an intermediate state at lower surfactant concentration. With positively charged surfactants the degree of helicity recovered was less, and the intermediate state in fluorescence was not seen. Stopped flow dynamics studies showed the CD kinetics fit to two exponentials as did the fluorescence. The faster steps in CD and fluorescence detected kinetics appear to be correlated which suggests formation of an intermediate on rapid interaction of the micelle and protein. The second step then reflected attainment of a stable surfactant solvated state which attains maximum helicity and moves the Trps to a more hydrophobic environment, which may occur in independent steps, as the slower kinetics are not well correlated.► Charge, CMC, hydrophobicity of surfactant micelles affect refolding reduced HEWL. ► Reduced lysozyme helicity, enhanced with negatively charged surfactant micelles. ► Kinetics with CD and fluorescence suggest separate mechanistic steps. ► Reduced and native lysozyme mechanisms differ, have similar molten globule state.
Keywords: Disulfide bond; Reduction; Surfactant; Micelle; Secondary structure; Membrane
Proteomic identification of differentially expressed genes during differentiation of cynomolgus monkey ( Macaca fascicularis) embryonic stem cells to astrocyte progenitor cells in vitro by Kuniko Akama; Tomoe Horikoshi; Takashi Nakayama; Masahiro Otsu; Noriaki Imaizumi; Megumi Nakamura; Tosifusa Toda; Michiko Inuma; Hisashi Hirano; Yasushi Kondo; Yutaka Suzuki; Nobuo Inoue (pp. 601-610).
Understanding astrocytogenesis is valuable for the treatment of nervous system disorders, as astrocytes provide structural, metabolic and defense support to neurons, and regulate neurons actively. However, there is limited information about the molecular events associated with the differentiation from primate ES cells to astrocytes. We therefore investigated the differentially expressed proteins in early astrocytogenesis, from cynomolgus monkey ES cells (CMK6 cell line) into astrocyte progenitor (AstP) cells via the formation of primitive neural stem spheres (Day 4), mature neural stem spheres (NSS), and neural stem (NS) cells in vitro, using two-dimensional gel electrophoresis (2-DE) and liquid chromatography-tandem mass spectrometry (LC-MS-MS). We identified 66 differentially expressed proteins involved in these five differentiation stages. Together with the results of Western blotting, RT-PCR, and a search of metabolic pathways related to the identified proteins, these results indicated that collapsin response mediator protein 2 (CRMP2), its phosphorylated forms, and cellular retinoic acid binding protein 1 (CRABP1) were upregulated from ES cells to Day 4 and NSS cells, to which differentiation stages apoptosis-associated proteins such as caspases were possibly related; Phosphorylated CRMP2s were further upregulated but CRABP1 was downregulated from NSS cells to NS cells, during which differentiation stage considerable axon guidance proteins for development of growth cones, axon attraction, and repulsion were possibly readied; Nonphosphorylated CRMP2 was downregulated but CRABP1 was re-upregulated from NS cells to AstP cells, in which differentiation stage reorganization of actin cytoskeleton linked to focal adhesion was possibly accompanied. These results provide insight into the molecular basis of early astrocytogenesis in monkey.► We investigated differentiation of monkey ES cells to astrocyte progenitor (AstP) cells. ► CRMP2 and CRABP1 were upregulated from ES cells to neural stem sphere (NSS). ► CRMP2 was further upregulated but CRABP1 was downregulated from NSS to NS cells. ► CRMP2 was downregulated and CRABP1 was re-upregulated from NS cells to AstP cells.
Keywords: Abbreviations; ACM; astrocyte-conditioned medium; AstP; astrocyte progenitor; CRABP1; cellular retinoic acid binding protein 1; CRMP2; collapsin response mediator protein 2; Day 4; primitive neural stem spheres; 2-DE; two-dimensional gel electrophoresis; EGF; epidermal growth factor; ES; embryonic stem; FABP7; fatty acid binding protein 7; FGF; fibroblast growth factor; GFAP; glial fibrillary acidic protein; IEF; isoelectric focusing; IPG; immobilized pH gradient; LC-MS-MS; liquid chromatography-tandem mass spectrometry; MS; mass spectrometry; NS; neural stem; NSS; neural stem spheres; PMF; peptide mass fingerprinting; T-TBS; TBS containing 0.05% TweenEmbryonic stem cell; Neural stem cell; Astrocyte progenitor cell; Cellular retinoic acid binding protein 1; Collapsin response mediator protein 2
Dimer exchange and cleavage specificity of the DNA damage response protein UmuD by Jaylene N. Ollivierre; Jacquelyn L. Sikora; Penny J. Beuning (pp. 611-620).
The cellular response to DNA damage in Escherichia coli is controlled in part by the activity of the umuD gene products. The full-length dimeric UmuD2 is the initial product that is expressed shortly after the induction of the SOS response and inhibits bacterial mutagenesis, allowing for error-free repair to occur. Over time, the slow auto-cleavage of UmuD2 to UmuD′2 promotes mutagenesis to ensure cell survival. The intracellular levels of UmuD2 and UmuD′2 are further regulated by degradation in vivo, returning the cell to a non-mutagenic state. To further understand the dynamic regulatory roles of the umuD gene products, we monitored the kinetics of exchange and cleavage of the UmuD2 and UmuD′2 homodimers as well as of the UmuDD′ heterodimer under equilibrium conditions. We found that the heterodimer is the preferred but not exclusive protein form, and that both the heterodimer and homodimers exhibit slow exchange kinetics which is further inhibited in the presence of interacting partner DinB. In addition, the heterodimer efficiently cleaves to form UmuD′2. Together, this work reveals an intricate UmuD lifecycle that involves dimer exchange and cleavage in the regulation of the DNA damage response.Display Omitted► UmuD homodimers and the heterodimer exhibit slow exchange kinetics. ► The UmuDD′ heterodimer preferentially forms over the homodimers. ► The UmuDD′ heterodimer preferentially cleaves in trans to form UmuD′.
Keywords: SOS mutagenesis; DNA damage; Heterodimer; Heterodimer cleavage
Structural alteration of Escherichia coli Hsp31 by thermal unfolding increases chaperone activity by Dongwook Choi; Kyoung-Seok Ryu; Chankyu Park (pp. 621-628).
Escherichia coli Hsp31, encoded by hchA, is a heat-inducible molecular chaperone. We found that Hsp31 undergoes a conformational change via temperature-induced unfolding, generating a high molecular weight (HMW) form with enhanced chaperone activity. Although it has previously been reported that some subunits of the Hsp31 crystal structure show structural heterogeneity with increased hydrophobic surfaces, Hsp31 basically forms a dimer. We found that a C-terminal deletion (CΔ19) of Hsp31 exhibited structurally and functionally similar characteristics to that of the HMW form. Both the CΔ19 and HMW forms achieved a structure with considerably more β-sheets and less α-helices than the native dimeric form, exposing a portion of its hydrophobic surfaces. The structural alterations were determined from its spectral changes in circular dichroism, intrinsic fluorescence of tryptophan residues, and fluorescence of bis-ANS binding to a hydrophobic surface. Interestingly, during thermal transition, the dimeric Hsp31 undergoes a conformational change to the HMW species via the CΔ19 structure, as monitored with near-UV CD spectrum, implying that the CΔ19 resembles an intermediate state between the dimer and the HMW form. From these results, we propose that Hsp31 transforms itself into a fully functional chaperone by altering its tertiary and quaternary structures.► E. coli Hsp31 undergoes a conformational transition from dimer to oligomer. ► The oligomer differs in secondary structures from that of dimer. ► The oligomer lacks enzyme activity and exhibits a chaperone activity. ► C-terminal deletion of Hsp31 mimics the structure and function of the oligomer.
Keywords: Hsp31; Chaperone; Conformational change; Oligomerization; Glyoxalase