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1
Javanbakht, Taraneh. „Optimization of Cdx Transcription Factors Characteristics“. Journal of Engineering Sciences 10, Nr. 2 (2023): E1—E7. http://dx.doi.org/10.21272/jes.2023.10(2).e1.
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This study presents a new application of TOPSIS for the optimization of transcription factors characteristics. This application is essential as it can help compare the characteristics of these proteins and determine the optimized output of their comparison with this decision-making method. The hypothesis in this article was that according to the previous study of the Cdx transcription factors, as the Cdx2 transcription factor showed more robust characteristics than Cdx1 and Cdx4, the TOPSIS method would show a better rank position of these first proteins in comparison with the two other ones. Moreover, the engrailed repressor domain EnRCdx1 used in the plasmid showed the reduction of the pax3 gene expression in comparison with the induced regulation of the gene expression with the production of the Cdx1, Cdx2, and Cdx4 transcription factors using the corresponding plasmids, the worst rank position with TOPSIS was expected for this repressor domain. The results obtained with this ranking method showed that the rank positions of the transcription factors and the repressor domain corresponded to their compared properties. Moreover, the change in the weight values of the candidates showed the modification of their distances from the best and worst alternatives and closeness coefficients. However, as expected, the candidates’ rank positions were unchanged, and the Cdx2 transcription factor was still the best candidate. The results of this article can be used in computer engineering to improve biological applications of these proteins.
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Denesyuk, Alexander I., Sergei E. Permyakov, Mark S. Johnson, Konstantin Denessiouk und Eugene A. Permyakov. „System Approach for Building of Calcium-Binding Sites in Proteins“. Biomolecules 10, Nr. 4 (11.04.2020): 588. http://dx.doi.org/10.3390/biom10040588.
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We introduce five new local metal cation (first of all, Ca2+) recognition units in proteins: Clampn,(n−2), Clampn,(n−1), Clampn,n, Clampn,(n+1) and Clampn,(n+2). In these units, the backbone oxygen atom of a residue in position “n” of an amino acid sequence and side-chain oxygen atom of a residue in position “n + i” (i = −2 to +2) directly interact with a metal cation. An analysis of the known “Ca2+-bound niches” in proteins has shown that a system approach based on the simultaneous use of the Clamp units and earlier proposed One-Residue (OR)/Three-Residue (TR) units significantly improves the results of constructing metal cation-binding sites in proteins.
3
Skvortsov, V. S., N. N. Alekseychuk, Yu V. Miroshnichenko und A. V. Rybina. „pIPredict Version 2: New Features and PTM Analysis“. Biomedical Chemistry: Research and Methods 1, Nr. 2 (2018): e00009. http://dx.doi.org/10.18097/bmcrm00009.
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pIPredict was created as a tool for prediction of the isoelectric point of peptides and proteins. It can also generate virtual 2D electrophoresis maps. The method of pI prediction is based on the Henderson-Hasselbach equation. In a new version the ProMoST and our new scales of pKa values were added. The other added features included: correction of electrophoretic shift by analyzing amino acid composition of proteins and prediction of pI values for proteins with a new set of posttranslational and other chemical modifications. Prediction of pI for proteins with PTM can be used to predict position of modified proteoforms on the virtual 2D electrophoresis map or as the tool of identifying which particular proteoform was observed in the experiment.The program also includes several widely used pKa scales, that can partially calculate values for proteins with some post-translational modifications. pIPredict is created as JAVA application and is freely available at http://www.ibmc.msk.ru/LPCIT/pIPredict.
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Zhang, Xiaoqi, Paul L. Bishop und Margaret J. Kupferle. „Measurement of polysaccharides and proteins in biofilm extracellular polymers“. Water Science and Technology 37, Nr. 4-5 (01.02.1998): 345–48. http://dx.doi.org/10.2166/wst.1998.0661.
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The structure of biofilm extracellular polymers (ECPs) was studied by measuring their polysaccharide and protein spatial distributions along biofilm depth. Biofilm was collected from two aerobic heterotrophic biofilm reactors, which were seeded with Sphingomonas sp. and Sphingomonas sp. plus mixed liquor, respectively, and operated under toxic organic (in this case, azo dye) degrading conditions. Complete mixing conditions in the two reactors were verified by measuring water content, and polysaccharide and protein quantities from three vertical sampling positions over time. Experimental results showed that: (1) the biofilm water content of either reactor did not change with sample position or biofilm age, with an average biofilm water content in both reactors of 97%; (2) polysaccharides and proteins in the ECPs did not change with sample position; (3) the profiles of polysaccharides and proteins along the biofilm depth showed a stratified biofilm structure, with their ratio (proteins/polysaccharides) being relatively stable over the depth. Oxygen and substrate transport and interactions among species were considered to be the main reasons for producing such a non-uniform biofilm structure; and (4) Sphingomonas sp. could not compete well with microorganisms derived from the mixed liquor of a wastewater treatment plant aeration basin.
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Miyaji, Akimitsu, Teppei Miyoshi, Ken Motokura und Toshihide Baba. „Discrimination of the prochiral hydrogens at the C-2 position of n-alkanes by the methane/ammonia monooxygenase family proteins“. Organic & Biomolecular Chemistry 13, Nr. 30 (2015): 8261–70. http://dx.doi.org/10.1039/c5ob00640f.
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The substrate binding site of AMO/pMMO family proteins can discriminate between the prochiral hydrogens at the C-2 position ofn-alkanes. We predict that at least one of the three amino acid residues at the di-copper site affects the discriminating ability of the family proteins.
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Moeller-Ehrlich, K., M. Ludlow, R. Beschorner, R. Meyermann, B. K. Rima, W. P. Duprex, S. Niewiesk und J. Schneider-Schaulies. „Two functionally linked amino acids in the stem 2 region of measles virus haemagglutinin determine infectivity and virulence in the rodent central nervous system“. Journal of General Virology 88, Nr. 11 (01.11.2007): 3112–20. http://dx.doi.org/10.1099/vir.0.83235-0.
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Rodent brain-adapted measles virus (MV) strains, such as CAM/RB and recombinant MVs based on the Edmonston strain containing the haemagglutinin (H) of CAM/RB, cause acute encephalitis after intracerebral infection of newborn rodents. We have demonstrated that rodent neurovirulence is modulated by two mutations at amino acid positions 195 and 200 in the H protein, one of these positions (200) being a potential glycosylation site. In order to analyse the effects of specific amino acids at these positions, we introduced a range of individual and combined mutations into the open reading frame of the H gene to generate a number of eukaryotic expression plasmids. The functionality of the mutant H proteins was assessed in transfected cells and by generating recombinant viruses. Interestingly, viruses caused acute encephalitis only if the amino acid Ser at position 200 was coupled with Gly at position 195, whereas viruses with single or combined mutations at these positions, including glycosylation at position 200, were attenuated. Neurovirulence was associated with virus spread and induction of neuronal apoptosis, whereas attenuated viruses failed to infect brain cells. Similar results were obtained by using primary brain-cell cultures. Our findings indicate that a structural alteration in the stem 2 region of the H protein at position 195 or 200 interferes with infectivity of rodent neurons, and suggest that the interaction of the viral attachment protein with cellular receptors on neurons is affected.
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Nufer, Oliver, Svend Guldbrandsen, Martin Degen, Felix Kappeler, Jean-Pierre Paccaud, Katsuko Tani und Hans-Peter Hauri. „Role of cytoplasmic C-terminal amino acids of membrane proteins in ER export“. Journal of Cell Science 115, Nr. 3 (01.02.2002): 619–28. http://dx.doi.org/10.1242/jcs.115.3.619.
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Export of membrane proteins from the ER is believed to be selective and require transport signals, but the identity of such signals has remained elusive. The recycling type I membrane protein ERGIC-53 carries a C-terminal diphenylalanine motif that is required for efficient ER export. Here we show that this motif can be functionally substituted by a single phenylalanine or tyrosine at position -2, two leucines or isoleucines at position -1 and -2 or a single valine at position -1. These motifs are common among mammalian type I membrane proteins. A single C-terminal valine, but none of the other motifs,accelerates transport of inefficiently exported reporter constructs and hence operates as an export signal. The valine signal is position, but not context,dependent. All transport motifs mediate COPII binding in vitro with distinct preferences for the COPII subunits Sec23p, Sec24Bp, Sec24Cp and p125. These results suggest that cytoplasmic C-terminal amino-acid motifs, either alone or in conjunction with other transport determinants, accelerate ER export of numerous type I and probably polytopic membrane proteins by mediating interaction with COPII coat components.
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Black, Katherine A., und Patricia C. Dos Santos. „Abbreviated Pathway for Biosynthesis of 2-Thiouridine in Bacillus subtilis“. Journal of Bacteriology 197, Nr. 11 (30.03.2015): 1952–62. http://dx.doi.org/10.1128/jb.02625-14.
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ABSTRACTThe 2-thiouridine (s2U) modification of the wobble position in glutamate, glutamine, and lysine tRNA molecules serves to stabilize the anticodon structure, improving ribosomal binding and overall efficiency of the translational process. Biosynthesis of s2U inEscherichia colirequires a cysteine desulfurase (IscS), a thiouridylase (MnmA), and five intermediate sulfur-relay enzymes (TusABCDE). TheE. coliMnmA adenylates and subsequently thiolates tRNA to form the s2U modification.Bacillus subtilislacks IscS and the intermediate sulfur relay proteins, yet its genome contains a cysteine desulfurase gene,yrvO, directly adjacent tomnmA. The genomic synteny ofyrvOandmnmAcombined with the absence of the Tus proteins indicated a potential functionality of these proteins in s2U formation. Here, we provide evidence that theB. subtilisYrvO and MnmA are sufficient for s2U biosynthesis. A conditionalB. subtilisknockout strain showed that s2U abundance correlates with MnmA expression, andin vivocomplementation studies inE. coliIscS- or MnmA-deficient strains revealed the competency of these proteins in s2U biosynthesis.In vitroexperiments demonstrated s2U formation by YrvO and MnmA, and kinetic analysis established a partnership between theB. subtilisproteins that is contingent upon the presence of ATP. Furthermore, we observed that the slow-growth phenotype ofE. coliΔiscSand ΔmnmAstrains associated with s2U depletion is recovered byB. subtilis yrvOandmnmA. These results support the proposal that the involvement of a devoted cysteine desulfurase, YrvO, in s2U synthesis bypasses the need for a complex biosynthetic pathway by direct sulfur transfer to MnmA.IMPORTANCEThe 2-thiouridine (s2U) modification of the wobble position in glutamate, glutamine, and lysine tRNA is conserved in all three domains of life and stabilizes the anticodon structure, thus guaranteeing fidelity in translation. The biosynthesis of s2U inEscherichia colirequires seven proteins: the cysteine desulfurase IscS, the thiouridylase MnmA, and five intermediate sulfur-relay enzymes (TusABCDE).Bacillus subtilisand most Gram-positive bacteria lack a complete set of biosynthetic components. Interestingly, themnmAcoding sequence is located adjacent toyrvO, encoding a cysteine desulfurase. In this work, we provide evidence that theB. subtilisYrvO is able to transfer sulfur directly to MnmA. Both proteins are sufficient for s2U biosynthesis in a pathway independent of the one used inE. coli.
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Hay, D. I., A. Bennick, D. H. Schlesinger, K. Minaguchi, G. Madapallimattam und S. K. Schluckebier. „The primary structures of six human salivary acidic proline-rich proteins (PRP-1, PRP-2, PRP-3, PRP-4, PIF-s and PIF-f)“. Biochemical Journal 255, Nr. 1 (01.10.1988): 15–21. http://dx.doi.org/10.1042/bj2550015.
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Human glandular salivary secretions contain several acidic proline-rich phosphoproteins (PRPs). These proteins have important biological functions related to providing a protective environment for the teeth, and appear to possess other activities associated with modulation of adhesion of bacteria to oral surfaces. These functions and activities depend on the primary structures of the PRPs. Previously determined amino acid sequences of two 150-residue molecules, PRP-1 and PRP-2, and two related 106-residue proteins, PRP-3 and PRP-4, indicated that residue 4 was Asn in PRP-1 and PRP-3, and Asp in PRP-2 and PRP-4, and position 50 was Asn in all four proteins. Recent data from cDNA sequence studies and further structural studies, however, showed that the previously proposed sequences cannot be completely correct. The present work has shown that the protein previously designated as PRP-1 actually consisted of two positional isomers, PIF-s, which has Asn and Asp at positions 4 and 50 respectively, and authentic PRP-1, which has the reverse arrangement. The same isomerism is present in the smaller proteins, PIF-f and PRP-3. Since the isomeric pairs have identical compositions and charges, their presence was not previously detected. Also, by using a more highly purified preparation, it has been found that position 50 in PRP-2 and PRP-4 is Asp, rather than Asn previously reported. These new findings for the six PRPs define their complete primary structures, which are now consistent with those proposed for PRP-1 and PIF-s from cDNA data, and are also consistent with the chromatographic and electrophoretic behaviours of the six PRPs and their derived peptides. These corrected structures are important for understanding the biological functions and activities of these unusual proteins.
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Boguszewska, A., R. Szyszka und N. Grankowski. „The phosphorylation sites of ribosomal P proteins from Saccharomyces cerevisiae cells by endogenous CK-2, PK60S and RAP protein kinases.“ Acta Biochimica Polonica 44, Nr. 2 (30.06.1997): 191–200. http://dx.doi.org/10.18388/abp.1997_4413.
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The phosphorylation sites of ribosomal acidic proteins (P proteins) from Saccharomyces cerevisiae were studied in vivo and in vitro by using CK-2, PK60S and RAP protein kinases. The three enzymes phosphorylate the last serine residues located in a highly conserved carboxyl end of the polypeptide chains. This was established by two-dimensional analysis of tryptic phosphopeptides from 32P-labelled proteins YP1 alpha, YP1 beta, YP2 alpha and YP2 beta, and by kinetic studies of the protein kinases with synthetic peptides corresponding to the fragments of endogenous ribosomal acidic polypeptides. In experiments with both endogenous P proteins and synthetic peptides as substrates protein kinase PK60S demonstrated unusual substrate specificity. In contrast to CK-2 and RAP protein kinases, PK60S phosphorylates predominantly two of the four P proteins, YP1 alpha and YP2 beta, with kinetic constants dependent on the primary structure of the N-terminal region of the polypeptide containing the target residue. The neutral amino acid, alanine, at position 3 in the peptide AAEESDDD (polypeptide fragments of YP1 beta and YP2 alpha) decreases the K(m) value more than 10-fold by comparison with the basic lysine residue at the same position in the peptide AKEESDDD (polypeptide fragments of YP1 alpha and YP2 beta).
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Songyang, Z., S. E. Shoelson, J. McGlade, P. Olivier, T. Pawson, X. R. Bustelo, M. Barbacid, H. Sabe, H. Hanafusa und T. Yi. „Specific motifs recognized by the SH2 domains of Csk, 3BP2, fps/fes, GRB-2, HCP, SHC, Syk, and Vav“. Molecular and Cellular Biology 14, Nr. 4 (April 1994): 2777–85. http://dx.doi.org/10.1128/mcb.14.4.2777-2785.1994.
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Src homology 2 (SH2) domains provide specificity to intracellular signaling by binding to specific phosphotyrosine (phospho-Tyr)-containing sequences. We recently developed a technique using a degenerate phosphopeptide library to predict the specificity of individual SH2 domains (src family members, Abl, Nck, Sem5, phospholipase C-gamma, p85 subunit of phosphatidylinositol-3-kinase, and SHPTP2 (Z. Songyang, S. E. Shoelson, M. Chaudhuri, G. Gish, T. Pawson, W. G. Haser, F. King, T. Roberts, S. Ratnofsky, R. J. Lechleider, B. G. Neel, R. B. Birge, J. E. Fajardo, M. M. Chou, H. Hanafusa, B. Schaffhausen, and L. C. Cantley, Cell 72:767-778, 1993). We report here the optimal recognition motifs for SH2 domains from GRB-2, Drk, Csk, Vav, fps/fes, SHC, Syk (carboxy-terminal SH2), 3BP2, and HCP (amino-terminal SH2 domain, also called PTP1C and SHPTP1). As predicted, SH2 domains from proteins that fall into group I on the basis of a Phe or Tyr at the beta D5 position (GRB-2, 3BP2, Csk, fps/fes, Syk C-terminal SH2) select phosphopeptides with the general motif phospho-Tyr-hydrophilic (residue)-hydrophilic (residue)-hydrophobic (residue). The SH2 domains of SHC and HCP (group III proteins with Ile, Leu, of Cys at the beta D5 position) selected the general motif phospho-Tyr-hydrophobic-Xxx-hydrophobic, also as predicted. Vav, which has a Thr at the beta D5 position, selected phospho-Tyr-Met-Glu-Pro as the optimal motif. Each SH2 domain selected a unique optimal motif distinct from motifs previously determined for other SH2 domains. These motifs are used to predict potential sites in signaling proteins for interaction with specific SH2 domain-containing proteins. The Syk SH2 domain is predicted to bind to Tyr-hydrophilic-hydrophilic-Leu/Ile motifs like those repeated at 10-residue intervals in T- and B-cell receptor-associated proteins. SHC is predicted to bind to a subgroup og these same motifs. A structural basis for the association of Csk with Src family members is also suggested from these studies.
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Songyang, Z., S. E. Shoelson, J. McGlade, P. Olivier, T. Pawson, X. R. Bustelo, M. Barbacid, H. Sabe, H. Hanafusa und T. Yi. „Specific motifs recognized by the SH2 domains of Csk, 3BP2, fps/fes, GRB-2, HCP, SHC, Syk, and Vav.“ Molecular and Cellular Biology 14, Nr. 4 (April 1994): 2777–85. http://dx.doi.org/10.1128/mcb.14.4.2777.
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Src homology 2 (SH2) domains provide specificity to intracellular signaling by binding to specific phosphotyrosine (phospho-Tyr)-containing sequences. We recently developed a technique using a degenerate phosphopeptide library to predict the specificity of individual SH2 domains (src family members, Abl, Nck, Sem5, phospholipase C-gamma, p85 subunit of phosphatidylinositol-3-kinase, and SHPTP2 (Z. Songyang, S. E. Shoelson, M. Chaudhuri, G. Gish, T. Pawson, W. G. Haser, F. King, T. Roberts, S. Ratnofsky, R. J. Lechleider, B. G. Neel, R. B. Birge, J. E. Fajardo, M. M. Chou, H. Hanafusa, B. Schaffhausen, and L. C. Cantley, Cell 72:767-778, 1993). We report here the optimal recognition motifs for SH2 domains from GRB-2, Drk, Csk, Vav, fps/fes, SHC, Syk (carboxy-terminal SH2), 3BP2, and HCP (amino-terminal SH2 domain, also called PTP1C and SHPTP1). As predicted, SH2 domains from proteins that fall into group I on the basis of a Phe or Tyr at the beta D5 position (GRB-2, 3BP2, Csk, fps/fes, Syk C-terminal SH2) select phosphopeptides with the general motif phospho-Tyr-hydrophilic (residue)-hydrophilic (residue)-hydrophobic (residue). The SH2 domains of SHC and HCP (group III proteins with Ile, Leu, of Cys at the beta D5 position) selected the general motif phospho-Tyr-hydrophobic-Xxx-hydrophobic, also as predicted. Vav, which has a Thr at the beta D5 position, selected phospho-Tyr-Met-Glu-Pro as the optimal motif. Each SH2 domain selected a unique optimal motif distinct from motifs previously determined for other SH2 domains. These motifs are used to predict potential sites in signaling proteins for interaction with specific SH2 domain-containing proteins. The Syk SH2 domain is predicted to bind to Tyr-hydrophilic-hydrophilic-Leu/Ile motifs like those repeated at 10-residue intervals in T- and B-cell receptor-associated proteins. SHC is predicted to bind to a subgroup og these same motifs. A structural basis for the association of Csk with Src family members is also suggested from these studies.
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Stasevych, Maryna, Viktor Zvarych, Olena Yaremkevych, Mykhaylo Vovk, Alla Vaskevych, Tetiana Halenova und Olexii Savchuk. „N-(9,10-Dioxo-9,10-dihydroanthracen-1(2)-yl)-2-(R-thio) Acetamides: Synthesis, Antioxidant and Antiplatelet Activity“. Acta Chimica Slovenica 69, Nr. 3 (26.09.2022): 584–95. http://dx.doi.org/10.17344/acsi.2022.7463.
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The synthesis of new N-(9,10-dioxo-9,10-dihydroanthracen-1(2)-yl)-2-(R-thio) acetamides was carried out using reaction of 2-chloro-N-(9,10-dioxo-9,10-dihydroanthracene-1(2)-yl)acetamides with functionalized thiols in the presence of potassium carbonate in N,N-dimethylformamide (DMF) at room temperature. Evaluation of the synthesized compounds on such indicators of radical scavenging activity as lipid peroxidation (LP) and oxidative modification of proteins (OMP) in vitro in rat liver homogenate was carried out. It was determined that the compounds with a substituent in the first position of anthracedione core showed better antioxidant properties than their isomers with a substituent in the second position. The compounds 6 and 7 with the best indicators of radical-scavenging activity were determined. Antioxidant effect in OMP processes was also determined for compound 10. The antiplatelet activity study in vitro revealed compound 10 with the inhibited effect of ADP-induced aggregation.
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Kumar, Sandeep, und Manju Bansal. „Dissecting α-helices: Position-specific analysis of α-helices in globular proteins“. Proteins: Structure, Function, and Genetics 31, Nr. 4 (01.06.1998): 460–76. http://dx.doi.org/10.1002/(sici)1097-0134(19980601)31:4<460::aid-prot12>3.0.co;2-d.
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Phelan, M. L., R. Sadoul und M. S. Featherstone. „Functional differences between HOX proteins conferred by two residues in the homeodomain N-terminal arm“. Molecular and Cellular Biology 14, Nr. 8 (August 1994): 5066–75. http://dx.doi.org/10.1128/mcb.14.8.5066-5075.1994.
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Hox genes encode homeodomain-containing transcriptional regulators that function during development to specify positional identity along embryonic axes. The homeodomain is composed of a flexible N-terminal arm and three alpha helices, and it differentially binds DNA. A number of homeodomains recognize sites containing a TAAT core motif. The product of the murine Hoxd-4 (Hox-4.2) gene functions in a positive autoregulatory fashion in P19 cells that is dependent on two TAAT motifs in the Hoxd-4 promoter. This effect is specific in that murine HOXA-1 (HOX-1.6) is unable to activate transcription through the Hoxd-4 autoregulatory element. Here we show that this is due to an inability of the HOXA-1 homeodomain to bind a HOXD-4 recognition site effectively. We have produced chimeras between HOXD-4 and HOXA-1 to map specific residues responsible for this functional difference. When positions 2 and 3 in the N-terminal arm of HOXA-1 were converted to HOXD-4 identity, both strong DNA binding and transcriptional activation were rescued. This substitution appears to confer an increased DNA-binding ability on the HOXA-1 homeodomain, since we were unable to detect a high-affinity recognition sequence for HOXA-1 in a randomized pool of DNA probes. The contribution of position 3 to DNA binding has been implicated by structural studies, but this is the first report of the importance of position 2 in regulating homeodomain-DNA interactions. Additionally, specific homeodomain residues that confer major differences in DNA binding and transcriptional activation between Hox gene products have not been previously determined. Identity at these two positions is generally conserved among paralogs but varies between Hox gene subfamilies. As a result, these residues may be important for the regulation of target gene expression by specific Hox products.
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Phelan, M. L., R. Sadoul und M. S. Featherstone. „Functional differences between HOX proteins conferred by two residues in the homeodomain N-terminal arm.“ Molecular and Cellular Biology 14, Nr. 8 (August 1994): 5066–75. http://dx.doi.org/10.1128/mcb.14.8.5066.
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Hox genes encode homeodomain-containing transcriptional regulators that function during development to specify positional identity along embryonic axes. The homeodomain is composed of a flexible N-terminal arm and three alpha helices, and it differentially binds DNA. A number of homeodomains recognize sites containing a TAAT core motif. The product of the murine Hoxd-4 (Hox-4.2) gene functions in a positive autoregulatory fashion in P19 cells that is dependent on two TAAT motifs in the Hoxd-4 promoter. This effect is specific in that murine HOXA-1 (HOX-1.6) is unable to activate transcription through the Hoxd-4 autoregulatory element. Here we show that this is due to an inability of the HOXA-1 homeodomain to bind a HOXD-4 recognition site effectively. We have produced chimeras between HOXD-4 and HOXA-1 to map specific residues responsible for this functional difference. When positions 2 and 3 in the N-terminal arm of HOXA-1 were converted to HOXD-4 identity, both strong DNA binding and transcriptional activation were rescued. This substitution appears to confer an increased DNA-binding ability on the HOXA-1 homeodomain, since we were unable to detect a high-affinity recognition sequence for HOXA-1 in a randomized pool of DNA probes. The contribution of position 3 to DNA binding has been implicated by structural studies, but this is the first report of the importance of position 2 in regulating homeodomain-DNA interactions. Additionally, specific homeodomain residues that confer major differences in DNA binding and transcriptional activation between Hox gene products have not been previously determined. Identity at these two positions is generally conserved among paralogs but varies between Hox gene subfamilies. As a result, these residues may be important for the regulation of target gene expression by specific Hox products.
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Brandherm, Lukas, Antonio Mario Kobaš, Mara Klöhn, Yannick Brüggemann, Stephanie Pfaender, Joachim Rassow und Sebastian Kreimendahl. „Phosphorylation of SARS-CoV-2 Orf9b Regulates Its Targeting to Two Binding Sites in TOM70 and Recruitment of Hsp90“. International Journal of Molecular Sciences 22, Nr. 17 (26.08.2021): 9233. http://dx.doi.org/10.3390/ijms22179233.
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SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) is the causative agent of the COVID19 pandemic. The SARS-CoV-2 genome encodes for a small accessory protein termed Orf9b, which targets the mitochondrial outer membrane protein TOM70 in infected cells. TOM70 is involved in a signaling cascade that ultimately leads to the induction of type I interferons (IFN-I). This cascade depends on the recruitment of Hsp90-bound proteins to the N-terminal domain of TOM70. Binding of Orf9b to TOM70 decreases the expression of IFN-I; however, the underlying mechanism remains elusive. We show that the binding of Orf9b to TOM70 inhibits the recruitment of Hsp90 and chaperone-associated proteins. We characterized the binding site of Orf9b within the C-terminal domain of TOM70 and found that a serine in position 53 of Orf9b and a glutamate in position 477 of TOM70 are crucial for the association of both proteins. A phosphomimetic variant Orf9bS53E showed drastically reduced binding to TOM70 and did not inhibit Hsp90 recruitment, suggesting that Orf9b–TOM70 complex formation is regulated by phosphorylation. Eventually, we identified the N-terminal TPR domain of TOM70 as a second binding site for Orf9b, which indicates a so far unobserved contribution of chaperones in the mitochondrial targeting of the viral protein.
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Benned-Jensen, Tau, Christoffer Norn, Stephane Laurent, Christian M. Madsen, Hjalte M. Larsen, Kristine N. Arfelt, Romain M. Wolf, Thomas Frimurer, Andreas W. Sailer und Mette M. Rosenkilde. „Molecular Characterization of Oxysterol Binding to the Epstein-Barr Virus-induced Gene 2 (GPR183)“. Journal of Biological Chemistry 287, Nr. 42 (08.08.2012): 35470–83. http://dx.doi.org/10.1074/jbc.m112.387894.
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Oxysterols are oxygenated cholesterol derivates that are emerging as a physiologically important group of molecules. Although they regulate a range of cellular processes, only few oxysterol-binding effector proteins have been identified, and the knowledge of their binding mode is limited. Recently, the family of G protein-coupled seven transmembrane-spanning receptors (7TM receptors) was added to this group. Specifically, the Epstein-Barr virus-induced gene 2 (EBI2 or GPR183) was shown to be activated by several oxysterols, most potently by 7α,25-dihydroxycholesterol (7α,25-OHC). Nothing is known about the binding mode, however. Using mutational analysis, we identify here four key residues for 7α,25-OHC binding: Arg-87 in TM-II (position II:20/2.60), Tyr-112 and Tyr-116 (positions III:09/3.33 and III:13/3.37) in TM-III, and Tyr-260 in TM-VI (position VI:16/6.51). Substituting these residues with Ala and/or Phe results in a severe decrease in agonist binding and receptor activation. Docking simulations suggest that Tyr-116 interacts with the 3β-OH group in the agonist, Tyr-260 with the 7α-OH group, and Arg-87, either directly or indirectly, with the 25-OH group, although nearby residues likely also contribute. In addition, Tyr-112 is involved in 7α,25-OHC binding but via hydrophobic interactions. Finally, we show that II:20/2.60 constitutes an important residue for ligand binding in receptors carrying a positively charged residue at this position. This group is dominated by lipid- and nucleotide-activated receptors, here exemplified by the CysLTs, P2Y12, and P2Y14. In conclusion, we present the first molecular characterization of oxysterol binding to a 7TM receptor and identify position II:20/2.60 as a generally important residue for ligand binding in certain 7TM receptors.
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Söderberg, Jenny Johansson, Patrik Engström und Ulrich von Pawel-Rammingen. „The Intrinsic Immunoglobulin G Endopeptidase Activity of Streptococcal Mac-2 Proteins Implies a Unique Role for the Enzymatically Impaired Mac-2 Protein of M28 Serotype Strains“. Infection and Immunity 76, Nr. 5 (10.03.2008): 2183–88. http://dx.doi.org/10.1128/iai.01422-07.
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ABSTRACT IdeS, a secreted cysteine protease of the important human pathogen Streptococcus pyogenes, interferes with phagocytic killing by specifically cleaving the heavy chain of immunoglobulin G (IgG). Two allelic variants of the enzyme have been described, the IgG-specific endopeptidase, IdeS (or Mac-1) and Mac-2, a protein with only weak IgG endopeptidase activity, which has been suggested to interfere with opsonophagocytosis by blocking Fcγ receptors of phagocytic cells. However, despite the fact that Mac-2 proteins interact with Fcγ receptors, no inhibition of reactive oxygen species (ROS) production, opsonophagocytosis, or streptococcal killing by Mac-2 has been reported. In the present study, Mac-2 proteins are shown to contain IgG endopeptidase activity indistinguishable from the enzymatic activity exhibited by IdeS/Mac-1 proteins. The earlier reported weak IgG endopeptidase activity appears to be unique to Mac-2 of M28 serotype strains (Mac-2M28) and is most likely due to the formation of a disulfide bond between the catalytic site cysteine and a cysteine residue in position 257 of Mac-2M28. Furthermore, Mac-2 proteins are shown to inhibit ROS production ex vivo, independently of the IgG endopeptidase activity of the proteins. Inhibition of ROS generation per se, however, was not sufficient to mediate streptococcal survival in bactericidal assays. Thus, in contrast to earlier studies, implicating separate functions for IdeS and Mac-2 protein variants, the current study suggests that Mac-2 and IdeS are bifunctional proteins, combining Fcγ receptor binding and IgG endopeptidase activity. This finding implies a unique role for Mac-2 proteins of the M28 serotype, since this serotype has evolved and retained a Mac-2 protein lacking IgG endopeptidase activity.
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Halada, Petr, Petr Man, Dana Grebeňová, Zbyněk Hrkal und Vladimír Havlíček. „Identification of HL60 Proteins Affected by 5-Aminolevulinic Acid-Based Photodynamic Therapy Using Mass Spectrometric Approach“. Collection of Czechoslovak Chemical Communications 66, Nr. 11 (2001): 1720–28. http://dx.doi.org/10.1135/cccc20011720.
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A combination of mass spectrometric techniques was used for identification of HL60 leukemia cell proteins affected by 5-aminolevulinic acid-based photodynamic therapy (ALA-PDT). We compared two-dimensional electrophoresis (2-DE) protein maps of ALA-treated non-irradiated and irradiated cells and found extensive changes in the proteome of HL60 cells. The silver-stained 2-DE pattern of HL60 proteins contained more than 1 350 spots. Matrix-assisted laser desorption/ionisation mass spectrometry and microcapillary liquid chromatography/tandem mass spectrometry have identified twelve proteins differing in their intensity or position following ALA-PDT. Several endoplasmic reticulum, mitochondrial, ribosomal and cytoplasmic proteins were determined showing the impact of ALA-PDT-mediated cytotoxicity on some cellular pathways.
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Angelova, Margarita T., Dilyana G. Dimitrova, Bruno Da Silva, Virginie Marchand, Caroline Jacquier, Cyrinne Achour, Mira Brazane et al. „tRNA 2′-O-methylation by a duo of TRM7/FTSJ1 proteins modulates small RNA silencing in Drosophila“. Nucleic Acids Research 48, Nr. 4 (16.01.2020): 2050–72. http://dx.doi.org/10.1093/nar/gkaa002.
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Abstract 2′-O-Methylation (Nm) represents one of the most common RNA modifications. Nm affects RNA structure and function with crucial roles in various RNA-mediated processes ranging from RNA silencing, translation, self versus non-self recognition to viral defense mechanisms. Here, we identify two Nm methyltransferases (Nm-MTases) in Drosophila melanogaster (CG7009 and CG5220) as functional orthologs of yeast TRM7 and human FTSJ1. Genetic knockout studies together with MALDI-TOF mass spectrometry and RiboMethSeq mapping revealed that CG7009 is responsible for methylating the wobble position in tRNAPhe, tRNATrp and tRNALeu, while CG5220 methylates position C32 in the same tRNAs and also targets additional tRNAs. CG7009 or CG5220 mutant animals were viable and fertile but exhibited various phenotypes such as lifespan reduction, small RNA pathways dysfunction and increased sensitivity to RNA virus infections. Our results provide the first detailed characterization of two TRM7 family members in Drosophila and uncover a molecular link between enzymes catalyzing Nm at specific tRNAs and small RNA-induced gene silencing pathways.
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Ramjeesingh, Mohabir, Canhui Li, Yi-Min She und Christine E. Bear. „Evaluation of the membrane-spanning domain of ClC-2“. Biochemical Journal 396, Nr. 3 (29.05.2006): 449–60. http://dx.doi.org/10.1042/bj20060043.
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The ClC family of chloride channels and transporters includes several members in which mutations have been associated with human disease. An understanding of the structure–function relationships of these proteins is essential for defining the molecular mechanisms underlying pathogenesis. To date, the X-ray crystal structures of prokaryotic ClC transporter proteins have been used to model the membrane domains of eukaryotic ClC channel-forming proteins. Clearly, the fidelity of these models must be evaluated empirically. In the present study, biochemical tools were used to define the membrane domain boundaries of the eukaryotic protein, ClC-2, a chloride channel mutated in cases of idiopathic epilepsy. The membrane domain boundaries of purified ClC-2 and accessible cysteine residues were determined after its functional reconstitution into proteoliposomes, labelling using a thiol reagent and proteolytic digestion. Subsequently, the lipid-embedded and soluble fragments generated by trypsin-mediated proteolysis were studied by MS and coverage of approx. 71% of the full-length protein was determined. Analysis of these results revealed that the membrane-delimited boundaries of the N- and C-termini of ClC-2 and the position of several extramembrane loops determined by these methods are largely similar to those predicted on the basis of the prokaryotic protein [ecClC (Escherichia coli ClC)] structures. These studies provide direct biochemical evidence supporting the relevance of the prokaryotic ClC protein structures towards understanding the structure of mammalian ClC channel-forming proteins.
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Schneider, Gisbert, Oliver Grammel, Tilman Todt und Paul Wrede. „Patterns of Amino Acid Properties Around Mitochondrial Signal Peptidase I Cleavage-Sites“. Protein & Peptide Letters 2, Nr. 2 (Oktober 1995): 327–32. http://dx.doi.org/10.2174/092986650201220524123740.
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Abstract: Target sequences of ntitochondrial signal peptidase I from Neurospora crassa and human nuclear encoded matrix precursor proteins were analyzed for characteristic motifs. The distributions of positively charged and hydrophobic residues reveal predominant patterns. Differences between human and Neurospora crassa cleavage-sites seem to exist for position -2 and around position -5. Based on property histograms putative cleavage-site sequences were constructed de novo.
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Tzortzakakis, Emmanuel, Vivian Blok, Mohamed Adam und Mark Phillips. „Characterisation of mjap genes encoding novel secreted proteins from the root-knot nematode, Meloidogyne javanica“. Nematology 11, Nr. 2 (2009): 253–65. http://dx.doi.org/10.1163/156854109x429583.
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AbstractTwo homologues of map-1, which encodes a putative avirulence factor, were found in a single egg mass line of the root-knot nematode Meloidogyne javanica that was virulent to the Mi-resistant gene. The main difference between the two encoded proteins of these homologues, MJ-MAP-1 and MJ-MAP-2, was the position of a 13 amino acid repeat region. Genes encoding two related but novel proteins, MJAP-1 and MJAP-2 (290 and 283 amino acids, respectively, including a potential signal secretion peptide), were also isolated from M. javanica. They have high similarity to MAP at the C-terminus. MJAP-1 and MJAP-2 differ from each other in the number and position of a seven-amino-acid repeat and in five other amino acids. The mjap genes are expressed in the subventral pharyngeal glands of second-stage juveniles of M. javanica, and transcription analysis in different developmental stages showed expression in the juvenile stage but not in eggs or adult females. Both mjap-1 and mjap-2 were expressed in both Mi-virulent and avirulent lines of M. javanica.
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Wayne, Laura L., Daniel J. Gachotte, Paul R. Graupner, Yelena Adelfinskaya, David G. McCaskill, James G. Metz, Ross Zirkle und Terence A. Walsh. „Plant and algal lysophosphatidic acid acyltransferases increase docosahexaenoic acid accumulation at the sn-2 position of triacylglycerol in transgenic Arabidopsis seed oil“. PLOS ONE 16, Nr. 8 (25.08.2021): e0256625. http://dx.doi.org/10.1371/journal.pone.0256625.
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Although docosahexaenoic acid (DHA), an important dietary omega-3 polyunsaturated fatty acid (PUFA), is at present primarily sourced from marine fish, bioengineered crops producing DHA may offer a more sustainable and cost-effective source. DHA has been produced in transgenic oilseed crops, however, DHA in seed oil primarily occupies the sn-1/3 positions of triacylglycerol (TAG) with relatively low amounts of DHA in the sn-2 position. To increase the amount of DHA in the sn-2 position of TAG and in seed oil, putative lysophosphatidic acid acyltransferases (LPAATs) were identified and characterized from the DHA-producing alga Schizochytrium sp. and from soybean (Glycine max). The affinity-purified proteins were confirmed to have LPAAT activity. Expression of the Schizochytrium or soybean LPAATs in DHA-producing Arabidopsis expressing the Schizochytrium PUFA synthase system significantly increased the total amount of DHA in seed oil. A novel sensitive band-selective heteronuclear single quantum coherence (HSQC) NMR method was developed to quantify DHA at the sn-2 position of glycerolipids. More than two-fold increases in sn-2 DHA were observed for Arabidopsis lines expressing Schizochytrium or soybean LPAATs, with one Schizochytrium LPAAT driving DHA accumulation in the sn-2 position to 61% of the total DHA. Furthermore, expression of a soybean LPAAT led to a redistribution of DHA-containing TAG species, with two new TAG species identified. Our results demonstrate that transgenic expression of Schizochytrium or soybean LPAATs can increase the proportion of DHA at the sn-2 position of TAG and the total amount of DHA in the seed oil of a DHA-accumulating oilseed plant. Additionally, the band-selective HSQC NMR method that we developed provides a sensitive and robust method for determining the regiochemistry of DHA in glycerolipids. These findings will benefit the advancement of sustainable sources of DHA via transgenic crops such as canola and soybean.
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Sumarningsih, Sumarningsih, Simson Tarigan, H. Farhid und Jagoda Ignjatovic. „Characterisation of M2e Antigenicity using anti-M2 Monoclonal Antibody and anti-M2e Polyclonal Antibodies“. Jurnal Ilmu Ternak dan Veteriner 24, Nr. 3 (24.09.2019): 122. http://dx.doi.org/10.14334/jitv.v24i3.1987.
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Matrix 2 ectodomain (M2e) protein is a potential antigen for detection of influenza A virus infection in vaccinated poultry (DIVA test). However the M2e antigenicity and immune response it induces in either humans or animals are poorly understood. Seventeen M2e peptides and sixteen recombinant M2e (rM2e) proteins with amino acid (aa) changes introduced at position 10, 11, 12, 13 14, 16, 18 and 20 were compared by western blot (WB) and enzyme-linked immunosorbent assay (ELISA) using mouse anti-M2 monoclonal antibody (mAb) 14C2, and anti-M2e peptide chicken and rabbit polyclonal antibody (pAb). The mAb 14C had the best discriminating power and indicated that all six positions contributed to the M2e antigenicity. Position 11 was the important immunodominant and affected Mab14C binding to a greatest degree. Changes in the adjacent position 14, 16 and 18 also influenced the binding, and it detected regardless of the method (WB or ELISA), or the antigen used (M2e peptide or rM2e). For chicken pAb and rabbit pAb, the immunodominant aa was position 10 and the antibody reaction was not affected by aa change at 11. The binding of rabbit pAb was also affected by changes at 14 and 16, which confirm the contribution of these positions to the M2e antigenicity. Position 10 was the only important position for the binding of chicken pAb to M2e. Overall, the study showed that the M2e antigenic sites are located between residues 10 – 18 and that aa changes at position 10, 11, 12, 14, 16 and 18 may all affect the antibody binding within the M2e protein.
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Soonsanga, Sumarin, Jin-Won Lee und John D. Helmann. „Conversion of Bacillus subtilis OhrR from a 1-Cys to a 2-Cys Peroxide Sensor“. Journal of Bacteriology 190, Nr. 17 (27.06.2008): 5738–45. http://dx.doi.org/10.1128/jb.00576-08.
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ABSTRACTOhrR proteins can be divided into two groups based on their inactivation mechanism: 1-Cys (represented byBacillus subtilisOhrR) and 2-Cys (represented byXanthomonas campestrisOhrR). A conserved cysteine residue near the amino terminus is present in both groups of proteins and is initially oxidized to the sulfenic acid. TheB. subtilis1-Cys OhrR protein is subsequently inactivated by formation of a mixed-disulfide bond with low-molecular-weight thiols or by cysteine overoxidation to sulfinic and sulfonic acids. In contrast, theX. campestris2-Cys OhrR is inactivated when the initially oxidized cysteine sulfenate forms an intersubunit disulfide bond with a second Cys residue from the other subunit of the protein dimer. Here, we demonstrate that the 1-CysB. subtilisOhrR can be converted into a 2-Cys OhrR by introducing another cysteine residue in either position 120 or position 124. Like theX. campestrisOhrR protein, these mutants (G120C and Q124C) are inactivated by intermolecular disulfide bond formation. Analysis of oxidized 2-Cys variants both in vivo and in vitro indicates that intersubunit disulfide bond formation can occur simultaneously at both active sites in the protein dimer. Rapid formation of intersubunit disulfide bonds protects OhrR against irreversible overoxidation in the presence of strong oxidants much more efficiently than do the endogenous low-molecular-weight thiols.
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Bonventre, J. V. „Phospholipase A2 and signal transduction.“ Journal of the American Society of Nephrology 3, Nr. 2 (August 1992): 128–50. http://dx.doi.org/10.1681/asn.v32128.
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Phospholipases A2 (PLA2) comprise a family of enzymes that hydrolyze the acyl bond at the sn-2 position of phospholipids to generate free fatty acids and lysophospholipids. Different forms of PLA2 are involved in digestion, inflammation, and intercellular and intracellular signal transduction. The sn-2 position of phospholipids in mammalian cells is enriched in arachidonic acid, the precursor of eicosanoids, which have diverse physiologic and pathophysiologic effects on the kidney and other organs. Thus, the regulation of PLA2 activity has important implications for kidney function. PLA2 regulation involves: calcium, pH, protein kinases, GTP-binding proteins, inhibitory and activating proteins, metabolic product inhibition, and transcriptional control. The various roles of arachidonic acid and cyclooxygenase, lipoxygenase, and cytochrome P450 mono-oxygenase products of arachidonic acid metabolism, as intracellular messengers, in the regulation of membrane channel activities, intracellular enzyme activities, cellular calcium homeostasis, mitogenesis, differentiation, cytokine and early response gene expression are discussed.
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Tiedemann, Michael T., Naomi Muryoi, David E. Heinrichs und Martin J. Stillman. „Characterization of IsdH (NEAT domain 3) and IsdB (NEAT domain 2) in Staphylococcus aureus by magnetic circular dichroism spectroscopy and electrospray ionization mass spectrometry“. Journal of Porphyrins and Phthalocyanines 13, Nr. 10 (Oktober 2009): 1006–16. http://dx.doi.org/10.1142/s1088424609001352.
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Absorption and magnetic circular dichroism (MCD) spectra, together with electrospray ionization mass spectral (ESI-MS) data are reported for the first two proteins in the Isd sequence of proteins in Staphylococcus aureus. IsdH-NEAT domain 3 (IsdH-N3) and IsdB-NEAT domain 2 (IsdB-N2) are considered to be involved in heme transport following heme scavenging from the hemoglobin of the host. The ESI-MS data show that a single heme binds to each of these NEAT domains. The charge states of the native proteins indicate that there is minimal change in conformation when heme binds to the heme-free native protein. Acid denaturation releases the bound heme and results in protein that exhibits significantly higher charge states, which we associate with unfolding of the protein structure. MCD spectra of the heme-bound native proteins show that the heme-iron is in a high-spin state, which is similar to that in IsdC-N. Addition of cyanide results in a spectral envelope characteristic of low-spin ferric hemes. The lack of complete binding for IsdH-N3 suggests that there is considerable congestion in the heme-binding site region. Unusually, reduction to the ferrous heme results in spectral characteristics of six coordination of the ferrous heme. CO is shown to bind strongly to both heme bound proteins, resulting in six-coordinate bound hemes. The spectra following reduction most closely resemble spectra recorded for heme with histidine in the fifth position and methionine in the sixth position. We report a theoretical model calculated from the X-ray structure coordinates of IsdH-N3, in which the heme is coordinated to nearby histidine and methionine. We propose that this structure accounts for the spectroscopic properties of the protein with the ferrous heme.
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Fernandes, L., C. Rodrigues-Pousada und K. Struhl. „Yap, a novel family of eight bZIP proteins in Saccharomyces cerevisiae with distinct biological functions.“ Molecular and Cellular Biology 17, Nr. 12 (Dezember 1997): 6982–93. http://dx.doi.org/10.1128/mcb.17.12.6982.
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Saccharomyces cerevisiae contains eight members of a novel and fungus-specific family of bZIP proteins that is defined by four atypical residues on the DNA-binding surface. Two of these proteins, Yap1 and Yap2, are transcriptional activators involved in pleiotropic drug resistance. Although initially described as AP-1 factors, at least four Yap proteins bind most efficiently to TTACTAA, a sequence that differs at position +/-2 from the optimal AP-1 site (TGACTCA); further, a Yap-like derivative of the AP-1 factor Gcn4 (A239Q S242F) binds efficiently to the Yap recognition sequence. Molecular modeling suggests that the Yap-specific residues make novel contacts and cause physical constraints at the +/-2 position that may account for the distinct DNA-binding specificities of Yap and AP-1 proteins. To various extents, Yap1, Yap2, Yap3, and Yap5 activate transcription from a promoter containing a Yap recognition site. Yap-dependent transcription is abolished in strains containing high levels of protein kinase A; in contrast, Gcn4 transcriptional activity is stimulated by protein kinase A. Interestingly, Yap1 transcriptional activity is stimulated by hydrogen peroxide, whereas Yap2 activity is stimulated by aminotriazole and cadmium. In addition, unlike other yap mutations tested, yap4 (cin5) mutations affect chromosome stability, and they suppress the cold-sensitive phenotype of yap1 mutant strains. Thus, members of the Yap family carry out overlapping but distinct biological functions.
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Yi, Jennifer, Vivian Kellner, Hyun Joo, Nathaniel Chien, Shivarni Patel, Zaina Chaban und Jerry Tsai. „Characterizing the consensus residue specificity and surface of BCL-2 binding to BH3 ligands using the Knob-Socket model“. PLOS ONE 18, Nr. 2 (16.02.2023): e0281463. http://dx.doi.org/10.1371/journal.pone.0281463.
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Cancer cells bypass cell death by changing the expression of the BCL-2 family of proteins, which are apoptotic pathway regulators. Upregulation of pro-survival BCL-2 proteins or downregulation of cell death effectors BAX and BAK interferes with the initiation of the intrinsic apoptotic pathway. In normal cells, apoptosis can occur through pro-apoptotic BH3-only proteins interacting and inhibiting pro-survival BCL-2 proteins. When cancer cells over-express pro-survival BCL-2 proteins, a potential remedy is the sequestration of these pro-survival proteins through a class of anti-cancer drugs called BH3 mimetics that bind in the hydrophobic groove of pro-survival BCL-2 proteins. To improve the design of these BH3 mimetics, the packing interface between BH3 domain ligands and pro-survival BCL-2 proteins was analyzed using the Knob-Socket model to identify the amino acid residues responsible for interaction affinity and specificity. A Knob-Socket analysis organizes all the residues in a binding interface into simple 4 residue units: 3-residue sockets defining surfaces on a protein that pack a 4th residue knob from the other protein. In this way, the position and composition of the knobs packing into sockets across the BH3/BCL-2 interface can be classified. A Knob-Socket analysis of 19 BCL-2 protein and BH3 helix co-crystals reveal multiple conserved binding patterns across protein paralogs. Conserved knob residues such as a Gly, Leu, Ala and Glu most likely define binding specificity in the BH3/BCL-2 interface, whereas other residues such as Asp, Asn, and Val are important for forming surface sockets that bind these knobs. These findings can be used to inform the design of BH3 mimetics that are specific to pro-survival BCL-2 proteins for cancer therapeutics.
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Bakker, Arjen H. F., Edward F. Rehberg, Keith R. Marotti und Jan H. Verheijen. „The position of the structurally autonomous kringle 2 domain influences the functional features of tissue-type plasminogen activator“. "Protein Engineering, Design and Selection" 8, Nr. 3 (1995): 293–300. http://dx.doi.org/10.1093/protein/8.3.293.
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Ferguson, C., A. Lakey, A. Hutchings, G. W. Butcher, K. R. Leonard und B. Bullard. „Cytoskeletal proteins of insect muscle: location of zeelins in Lethocerus flight and leg muscle“. Journal of Cell Science 107, Nr. 5 (01.05.1994): 1115–29. http://dx.doi.org/10.1242/jcs.107.5.1115.
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Asynchronous insect flight muscles produce oscillatory contractions and can contract at high frequency because they are activated by stretch as well as by Ca2+. Stretch activation depends on the high stiffness of the fibres and the regular structure of the filament lattice. Cytoskeletal proteins may be important in stabilising the lattice. Two proteins, zeelin 1 (35 kDa) and zeelin 2 (23 kDa), have been isolated from the cytoskeletal fraction of Lethocerus flight muscle. Both zeelins have multiple isoforms of the same molecular mass and different charge. Zeelin 1 forms micelles and zeelin 2 forms filaments when renatured in low ionic strength solutions. Filaments of zeelin 2 are ribbons 10 nm wide and 3 nm thick. The position of zeelins in fibres from Lethocerus flight and leg muscle was determined by immunofluorescence and immunoelectron microscopy. Zeelin 1 is found in flight and leg fibres and zeelin 2 only in flight fibres. In flight myofibrils, both zeelins are in discrete regions of the A-band in each half sarcomere. Zeelin 1 is across the whole A-band in leg myofibrils. Zeelins are not in the Z-disc, as was thought previously, but migrate to the Z-disc in glycerinated fibres. Zeelins are associated with thick filaments and analysis of oblique sections showed that zeelin 1 is closer to the filament shaft than zeelin 2. The antibody labelling pattern is consistent with zeelin molecules associated with myosin near the end of the rod region. Alternatively, the position of zeelins may be determined by other A-band proteins. There are about 2.0 to 2.5 moles of myosin per mole of each zeelin. The function of these cytoskeletal proteins may be to maintain the ordered structure of the thick filament.
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Goh, Chul Jun, und Yoonsoo Hahn. „Analysis of proteolytic processing sites in potyvirus polyproteins revealed differential amino acid preferences of NIa-Pro protease in each of seven cleavage sites“. PLOS ONE 16, Nr. 1 (25.01.2021): e0245853. http://dx.doi.org/10.1371/journal.pone.0245853.
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Potyviruses encode a large polyprotein that undergoes proteolytic processing, producing 10 mature proteins: P1, HC-Pro, P3, 6K1, CI, 6K2, VPg, NIa-Pro, NIb-RdRp, and CP. While P1/HC-Pro and HC-Pro/P3 junctions are cleaved by P1 and HC-Pro, respectively, the remaining seven are processed by NIa-Pro. In this study, we analyzed 135 polyprotein sequences from approved potyvirus species and deduced the consensus amino acid residues at five positions (from −4 to +1, where a protease cleaves between −1 and +1) in each of nine cleavage sites. In general, the newly deduced consensus sequences were consistent with the previous ones. However, seven NIa-Pro cleavage sites showed distinct amino acid preferences despite being processed by the same protease. At position −2, histidine was the dominant amino acid residue in most cleavage sites (57.8–60.7% of analyzed sequences), except for the NIa-Pro/NIb-RdRp junction where it was absent. At position −1, glutamine was highly dominant in most sites (88.2–97.8%), except for the VPg/NIa-Pro junction where glutamic acid was found in all the analyzed proteins (100%). At position +1, serine was the most abundant residue (47.4–86.7%) in five out of seven sites, while alanine (52.6%) and glycine (82.2%) were the most abundant in the P3/6K1 and 6K2/VPg junctions, respectively. These findings suggest that each NIa-Pro cleavage site is finely tuned for differential characteristics of proteolytic reactions. The newly deduced consensus sequences may be useful resources for the development of models and methods to accurately predict potyvirus polyprotein processing sites.
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Chu, Hongkang, und Taigang Liu. „Comprehensive Research on Druggable Proteins: From PSSM to Pre-Trained Language Models“. International Journal of Molecular Sciences 25, Nr. 8 (19.04.2024): 4507. http://dx.doi.org/10.3390/ijms25084507.
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Identification of druggable proteins can greatly reduce the cost of discovering new potential drugs. Traditional experimental approaches to exploring these proteins are often costly, slow, and labor-intensive, making them impractical for large-scale research. In response, recent decades have seen a rise in computational methods. These alternatives support drug discovery by creating advanced predictive models. In this study, we proposed a fast and precise classifier for the identification of druggable proteins using a protein language model (PLM) with fine-tuned evolutionary scale modeling 2 (ESM-2) embeddings, achieving 95.11% accuracy on the benchmark dataset. Furthermore, we made a careful comparison to examine the predictive abilities of ESM-2 embeddings and position-specific scoring matrix (PSSM) features by using the same classifiers. The results suggest that ESM-2 embeddings outperformed PSSM features in terms of accuracy and efficiency. Recognizing the potential of language models, we also developed an end-to-end model based on the generative pre-trained transformers 2 (GPT-2) with modifications. To our knowledge, this is the first time a large language model (LLM) GPT-2 has been deployed for the recognition of druggable proteins. Additionally, a more up-to-date dataset, known as Pharos, was adopted to further validate the performance of the proposed model.
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Miyake, Ariko, Mikako Fujita, Haruna Fujino, Ryoko Koga, Sogo Kawamura, Masami Otsuka, Hirotaka Ode et al. „Poly-proline motif in HIV-2 Vpx is critical for its efficient translation“. Journal of General Virology 95, Nr. 1 (01.01.2014): 179–89. http://dx.doi.org/10.1099/vir.0.057364-0.
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Human immunodeficiency virus type 2 (HIV-2) carries an accessory protein Vpx that is important for viral replication in natural target cells. In its C-terminal region, there is a highly conserved poly-proline motif (PPM) consisting of seven consecutive prolines, encoded in a poly-pyrimidine tract. We have previously shown that PPM is critical for Vpx expression and viral infectivity. To elucidate the molecular basis underlying this observation, we analysed the expression of Vpx proteins with various PPM mutations by in vivo and in vitro systems. We found that the number and position of consecutive prolines in PPM are important for Vpx expression, and demonstrated that PPM is essential for efficient Vpx translation. Furthermore, mutational analysis to synonymously disrupt the poly-pyrimidine tract suggested that the context of PPM amino acid sequences is required for efficient translation of Vpx. We similarly analysed HIV-1 and HIV-2 Vpr proteins structurally related to HIV-2 Vpx. Expression level of the two Vpr proteins lacking PPM was shown to be much lower relative to that of Vpx, and not meaningfully enhanced by introduction of PPM at the C terminus. Finally, we examined the Vpx of simian immunodeficiency virus from rhesus monkeys (SIVmac), which also has seven consecutive prolines, for PPM-dependent expression. A multi-substitution mutation in the PPM markedly reduced the expression level of SIVmac Vpx. Taken together, it can be concluded that the notable PPM sequence enhances the expression of Vpx proteins from viruses of the HIV-2/SIVmac group at the translational level.
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Borrego, Francisco, Matthias Ulbrecht, Elisabeth H. Weiss, John E. Coligan und Andrew G. Brooks. „Recognition of Human Histocompatibility Leukocyte Antigen (HLA)-E Complexed with HLA Class I Signal Sequence–derived Peptides by CD94/NKG2 Confers Protection from Natural Killer Cell–mediated Lysis“. Journal of Experimental Medicine 187, Nr. 5 (02.03.1998): 813–18. http://dx.doi.org/10.1084/jem.187.5.813.
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Human histocompatibility leukocyte antigen (HLA)-E is a nonclassical HLA class I molecule, the gene for which is transcribed in most tissues. It has recently been reported that this molecule binds peptides derived from the signal sequence of HLA class I proteins; however, no function for HLA-E has yet been described. We show that natural killer (NK) cells can recognize target cells expressing HLA-E molecules on the cell surface and this interaction results in inhibition of the lytic process. Furthermore, HLA-E recognition is mediated primarily through the CD94/NKG2-A heterodimer, as CD94-specific, but not killer cell inhibitory receptor (KIR)–specific mAbs block HLA-E–mediated protection of target cells. Cell surface HLA-E could be increased by incubation with synthetic peptides corresponding to residues 3–11 from the signal sequences of a number of HLA class I molecules; however, only peptides which contained a Met at position 2 were capable of conferring resistance to NK-mediated lysis, whereas those having Thr at position 2 had no effect. Interestingly, HLA class I molecules previously correlated with CD94/NKG2 recognition all have Met at residue 4 of the signal sequence (position 2 of the HLA-E binding peptide), whereas those which have been reported not to interact with CD94/NKG2 have Thr at this position. Thus, these data show a function for HLA-E and suggest an alternative explanation for the apparent broad reactivity of CD94/NKG2 with HLA class I molecules; that CD94/NKG2 interacts with HLA-E complexed with signal sequence peptides derived from “protective” HLA class I alleles rather than directly interacting with classical HLA class I proteins.
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May, H. D., D. R. Dean und W. E. Newton. „Altered nitrogenase MoFe proteins from Azotobacter vinelandii. Analysis of MoFe proteins having amino acid substitutions for the conserved cysteine residues within the β-subunit“. Biochemical Journal 277, Nr. 2 (15.07.1991): 457–64. http://dx.doi.org/10.1042/bj2770457.
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The regions surrounding the three strictly conserved cysteine residues (positions 70, 95 and 153) in the beta-subunit of the Azotobacter vinelandii nitrogenase MoFe protein have been proposed to provide P-cluster environments [Dean, Setterquist, Brigle, Scott, Laird & Newton (1990) Mol. Microbiol. 4, 1505-1512]. In the present study, each of these cysteine residues was individually substituted by either serine or alanine by site-directed mutagenesis of the nifK gene, which encodes the MoFe protein beta-subunit. A mutant strain for which the codon for Cys-153 is removed was also isolated. Significant structural or functional roles are indicated for the cysteine residues at positions 70 and 95, where substitution by either serine or alanine eliminates diazotrophic growth of the resulting strains and abolishes or markedly decreases both MoFe-protein acetylene-reduction activity and the intensity of the whole-cell S = 3/2 e.p.r. signal. Changes introduced at position 153 have various effects on the functional properties of the enzyme. The strains produced either by deletion of the Cys-153 residue or its substitution by serine exhibit only a moderate decrease in diazotrophic growth and MoFe-protein activity and no loss of the whole-cell e.p.r.-signal intensity. In contrast, substitution by alanine eliminates diazotrophic growth and very markedly decreases both MoFe-protein activity and e.p.r.-signal intensity. These results are interpreted in terms of a metallocluster-driven protein rearrangement. After purification of the altered MoFe protein, in which serine replaces Cys-153, an investigation of its catalytic and spectroscopic properties confirms that neither the FeMo cofactor, i.e. the substrate-reduction site, nor the component-protein interaction site has been affected. Instead, these data indicate a disruption in electron transfer within the MoFe protein, which is consistent with a role for this residue (and region) at the P clusters.
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Lê, Hương Giang, Jung-Mi Kang, Tuấn Cường Võ, Won Gi Yoo, Kon Ho Lee und Byoung-Kuk Na. „Biochemical Properties of Two Plasmodium malariae Cysteine Proteases, Malapain-2 and Malapain-4“. Microorganisms 10, Nr. 1 (16.01.2022): 193. http://dx.doi.org/10.3390/microorganisms10010193.
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Cysteine proteases belonging to the falcipain (FP) family play a pivotal role in the biology of malaria parasites and have been extensively investigated as potential antimalarial drug targets. Three paralogous FP-family cysteine proteases of Plasmodium malariae, termed malapains 2–4 (MP2–4), were identified in PlasmoDB. The three MPs share similar structural properties with the FP-2/FP-3 subfamily enzymes and exhibit a close phylogenetic lineage with vivapains (VXs) and knowpains (KPs), FP orthologues of P. vivax and P. knowlesi. Recombinant MP-2 and MP-4 were produced in a bacterial expression system, and their biochemical properties were characterized. Both recombinant MP-2 and MP-4 showed enzyme activity across a broad range of pH values with an optimum activity at pH 5.0 and relative stability at neutral pHs. Similar to the FP-2/FP-3 subfamily enzymes in other Plasmodium species, recombinant MP-2 and MP-4 effectively hydrolyzed hemoglobin at acidic pHs. They also degraded erythrocyte cytoskeletal proteins, such as spectrin and band 3, at a neutral pH. These results imply that MP-2 and MP-4 are redundant hemoglobinases of P. malariae and may also participate in merozoite egression by degrading erythrocyte cytoskeletal proteins. However, compared with other FP-2/FP-3 enzymes, MP-2 showed a strong preference for arginine at the P2 position. Meanwhile, MP-4 showed a primary preference for leucine at the P2 position but a partial preference for phenylalanine. These different substrate preferences of MPs underscore careful consideration in the design of optimized inhibitors targeting the FP-family cysteine proteases of human malaria parasites.
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Xin, Li Li, und Gregory S. Chirikjian. „Mechanics of Interactions of Helices in Proteins“. Key Engineering Materials 326-328 (Dezember 2006): 823–26. http://dx.doi.org/10.4028/www.scientific.net/kem.326-328.823.
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This paper concerns a mechanics of interactions of helical structures in proteins. Helices are the most important secondary structures of proteins and contribute the formation of a more complex 3-D structure, and so the analysis of interactions of helices is quite critical. We examine 1290 protein structures that have 2.0 Å or better resolutions and less than 20 percent of their sequences in common. Interactions between helices are represented by two parameters: the distance and angle. Assuming that helices are slender rigid rods with finite length, we define three different mechanisms of interactions: (1) line-on-line contact; (2) endpoint-to-line contact; and (3) endpointto- endpoint contact. In this paper, interactions for the first case are expressed with the 3-D relative rigid-body motion (position and orientation) and the unique volume element for correctly integrating over rigid-body motions are determined using six parameters. The results are extremely useful for the correct analysis of interactions in terms of distance and angle without the statistical biases inherent in the three data sets.
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Thủy, Vì Thị Xuân, Lò Thị Mai Thu, Hồ Mạnh Tường, Lê Văn Sơn, Nguyễn Vũ Thanh Thanh und Chu Hoàng Mậu. „Characteristics of defensin1 gene and designing structure to create resistant transgenic corn lines to weevils“. Vietnam Journal of Biotechnology 14, Nr. 2 (30.06.2016): 279–86. http://dx.doi.org/10.15625/1811-4989/14/2/9353.
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Plant defensins are multifunctional proteins, inhibiting the growth of fungal, anti-bacterial, altering membrane channels, inhibiting activity of trypsin and α-amylase. Plant defensin consists of 18 groups in which the group 1 includes defensins to inhibit either α-amylase enzyme or trypsin. Defensins bind to the active site of α-amylase in the weevil gut, thus inhibit starch digestion in weevils. In this report, we present the results of cloning and determining the ZmDEF1 gene sequence isolated from mRNA and DNA of Sonla province local maize and LVN99 hybrid maize cultivar. The coding region of ZmDEF1 gene isolated from some maize samples had the size of 243 nucleotides, encoding 80 amino acids. Gen ZmDEF1 isolated from DNA had the size 345bp consists of two exons and one in tron (102 bp). The nucleotide sequences of ZmDEF1 gene (DNA) of the samples have 6 positions nucleotide difference, on exon 1 has two points difference (position 43, 53), on intron has a difference (position 150), on exon 2 has 3 nucleotide site difference (203, 263 and 297 position). Deduced amino acid sequences of defensin of the Sonla local maize sample has 8 cysteines to make 4 disulfide bridges, while LVN99 hybrid maize has 7 cysteines, which can formed only 3 disulfide bridges. Transformation vector pBetaPhaso-ZmDEF1 has been designed successfully, in which ZmDEF1 is controlled by seed specific Phasoline promoter. The correct insertion and expression of ZmDEF1 was examinated in transgenic tobacco plants throught PCR and RT-PCR, respectively. These results provide an firm evident for using the designed transformation vector to produce transgenic maỉze lines with an improved resistant ability to weevils.
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Zhang, Nan, Yuxin Zuo, Yu Peng und Lielian Zuo. „Function of N6-Methyladenosine Modification in Tumors“. Journal of Oncology 2021 (23.11.2021): 1–10. http://dx.doi.org/10.1155/2021/6461552.
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N6-Methyladenosine (m6A) modification is a dynamic and reversible methylation modification at the N6-position of adenosine. As one of the most prevalent posttranscriptional methylation modifications of RNA, m6A modification participates in several mRNA processes, including nuclear export, splicing, translation, and degradation. Some proteins, such as METTL3, METTL14, WTAP, ALKBH5, FTO, and YTHDF1/2/3, are involved in methylation. These proteins are subdivided into writers (METTL3, METTL14, WTAP), erasers (ALKBH5, FTO), and readers (YTHDF1/2/3) according to their functions in m6A modification. Several studies have shown that abnormal m6A modification occurs in tumors, including colorectal cancer, liver cancer, breast cancer, nasopharyngeal carcinoma, and gastric cancer. The proteins for m6A modification are involved in tumor proliferation, angiogenesis, metastasis, immunity, and other processes. Herein, the roles of m6A modification in cancer are discussed, which will improve the understanding of tumorigenesis, as well as the diagnosis, treatment, and prognosis of tumors.
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Zhang, L., A. Jhingan und FJ Castellino. „Role of individual gamma-carboxyglutamic acid residues of activated human protein C in defining its in vitro anticoagulant activity“. Blood 80, Nr. 4 (15.08.1992): 942–52. http://dx.doi.org/10.1182/blood.v80.4.942.942.
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Abstract To evaluate the contributions of individual gamma-carboxyglutamic acid (gla) residues to the overall Ca(2+)-dependent anticoagulant activity of activated human protein C (APC), we used recombinant (r) DNA technology to generate protein C (PC) variants in which each of the gla precursor glutamic acid (E) residues (positions 6, 7, 14, 16, 19, 20, 25, 26, and 29) was separately altered to aspartic acid (D). In one case, a gla26V mutation ([gla26V]r-PC) was constructed because a patient with this particular substitution in coagulation factor IX had been previously identified. Two additional r-PC mutants were generated, viz, an r-PC variant containing a substitution at arginine (R) 15 ([R15]r-PC), because this particular R residue is conserved in all gla- containing blood coagulation proteins, as well as a variant r-PC with substitution of an E at position 32 ([F31L, Q32E]r-PC), because gla residues are found in other proteins at this sequence location. This latter protein did undergo gamma-carboxylation at the newly inserted E32 position. For each of the 11 recombinant variants, a subpopulation of PC molecules that were gamma-carboxylated at all nonmutated gla- precursor E residues has been purified by anion exchange chromatography and, where necessary, affinity chromatography on an antihuman PC column. The r-PC muteins were converted to their respective r-APC forms and assayed for their amidolytic activities and Ca(2+)-dependent anticoagulant properties. While no significant differences were found between wild-type (wt) r-APC and r-APC mutants in the amidolytic assays, lack of a single gla residue at any of the following locations, viz, 7, 16, 20, or 26, led to virtual complete disappearance of the Ca(2+)-dependent anticoagulant activity of the relevant r-APC mutant, as compared with its wt counterpart. On the other hand, single eliminations of any of the gla residues located at positions 6, 14, or 19 of r-APC resulted in variant recombinant molecules with substantial anticoagulant activity (80% to 92%), relative to wtr-APC. Mutation of gla residues at positions 25 and 29 resulted in r-APC variants with significant but low (24% and 9% of wtr-APC, respectively) levels of anticoagulant activity. The variant, [R15L]r-APC, possessed only 19% of the anticoagulant activity of wrt-APC, while inclusion of gla at position 32 in the variant, [F31L, Q32gla]r-APC, resulted in a recombinant enzyme with an anticoagulant activity equivalent to that of wtr-APC.
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Zhang, L., A. Jhingan und FJ Castellino. „Role of individual gamma-carboxyglutamic acid residues of activated human protein C in defining its in vitro anticoagulant activity“. Blood 80, Nr. 4 (15.08.1992): 942–52. http://dx.doi.org/10.1182/blood.v80.4.942.bloodjournal804942.
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To evaluate the contributions of individual gamma-carboxyglutamic acid (gla) residues to the overall Ca(2+)-dependent anticoagulant activity of activated human protein C (APC), we used recombinant (r) DNA technology to generate protein C (PC) variants in which each of the gla precursor glutamic acid (E) residues (positions 6, 7, 14, 16, 19, 20, 25, 26, and 29) was separately altered to aspartic acid (D). In one case, a gla26V mutation ([gla26V]r-PC) was constructed because a patient with this particular substitution in coagulation factor IX had been previously identified. Two additional r-PC mutants were generated, viz, an r-PC variant containing a substitution at arginine (R) 15 ([R15]r-PC), because this particular R residue is conserved in all gla- containing blood coagulation proteins, as well as a variant r-PC with substitution of an E at position 32 ([F31L, Q32E]r-PC), because gla residues are found in other proteins at this sequence location. This latter protein did undergo gamma-carboxylation at the newly inserted E32 position. For each of the 11 recombinant variants, a subpopulation of PC molecules that were gamma-carboxylated at all nonmutated gla- precursor E residues has been purified by anion exchange chromatography and, where necessary, affinity chromatography on an antihuman PC column. The r-PC muteins were converted to their respective r-APC forms and assayed for their amidolytic activities and Ca(2+)-dependent anticoagulant properties. While no significant differences were found between wild-type (wt) r-APC and r-APC mutants in the amidolytic assays, lack of a single gla residue at any of the following locations, viz, 7, 16, 20, or 26, led to virtual complete disappearance of the Ca(2+)-dependent anticoagulant activity of the relevant r-APC mutant, as compared with its wt counterpart. On the other hand, single eliminations of any of the gla residues located at positions 6, 14, or 19 of r-APC resulted in variant recombinant molecules with substantial anticoagulant activity (80% to 92%), relative to wtr-APC. Mutation of gla residues at positions 25 and 29 resulted in r-APC variants with significant but low (24% and 9% of wtr-APC, respectively) levels of anticoagulant activity. The variant, [R15L]r-APC, possessed only 19% of the anticoagulant activity of wrt-APC, while inclusion of gla at position 32 in the variant, [F31L, Q32gla]r-APC, resulted in a recombinant enzyme with an anticoagulant activity equivalent to that of wtr-APC.
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Begic, Sanela, und Elizabeth A. Worobec. „Site-directed mutagenesis studies to probe the role of specific residues in the external loop (L3) of OmpF and OmpC porins in susceptibility of Serratia marcescens to antibiotics“. Canadian Journal of Microbiology 53, Nr. 6 (Juni 2007): 710–19. http://dx.doi.org/10.1139/w07-018.
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Serratia marcescens is a nosocomial bacterium with natural resistance to a broad spectrum of antibiotics, making treatment challenging. One factor contributing to this natural antibiotic resistance is reduced outer membrane permeability, controlled in part by OmpF and OmpC porin proteins. To investigate the direct role of these porins in the diffusion of antibiotics across the outer membrane, we have created an ompF–ompC porin-deficient strain of S. marcescens. A considerable similarity between the S. marcescens porins and those from other members of Enterobacteriaceae was detected by sequence alignment, with the exception of a change in a conserved region of the third external loop (L3) of the S. marcescens OmpC protein. Serratia marcescens OmpC has aspartic acid instead of glycine in position 112, methionine instead of aspartic acid in position 114, and glutamine in position 124, while in S. marcescens OmpF this is a glycine at position 124. To investigate the role of amino acid positions 112, 114, and 124 and how the observed changes within OmpC porin may play a part in pore permeability, 2 OmpC sites were altered in the Enterobacteriaceae consensus (D112G and M114D) through site-directed mutagenesis. Also, Q124G in OmpC, G124Q in OmpF, and double mutants of these amino acid residues were constructed. Antibiotic accumulation assays and minimal inhibitory concentrations of the strains harboring the mutated porins were performed, while liposome swelling experiments were performed on purified porins. Our results demonstrate that the amino acid at position 114 is not responsible for either antibiotic size or ionic selection, the amino acid at position 112 is responsible for size selection only, and position 124 is involved in both size and ionic selection.
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Hatem, C. L., N. R. Gough und D. M. Fambrough. „Multiple mRNAs encode the avian lysosomal membrane protein LAMP-2, resulting in alternative transmembrane and cytoplasmic domains“. Journal of Cell Science 108, Nr. 5 (01.05.1995): 2093–100. http://dx.doi.org/10.1242/jcs.108.5.2093.
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Lysosomal membranes are enriched in extensively glycosylated transmembrane proteins, LAMP-1 and LAMP-2. LAMP-1 proteins have been characterized from several mammalian species and from chickens, but no non-mammalian homologues of LAMP-2 have been described, and no splice variants of either protein have been reported. Here we report the characterization of three cDNA clones encoding chicken LAMP-2. The nucleotide sequences of the cDNAs diverge at their 3′ ends within the open reading frame, resulting in sequences that code for three different transmembrane and cytoplasmic domains. Southern analysis suggests that a single gene encodes the common region of chicken LAMP-2. The position of the divergence and the identity of the common sequence are consistent with alternative splicing of 3′ exons. Analysis of the mRNAs present in adult chicken tissues suggests tissue-specific expression of the three chicken LAMP-2 variants, with LAMP-2b expressed primarily in the brain. The cytoplasmic domain of LAMP-type proteins contains the targeting signal for directing these molecules to the lysosome. Using chimeras consisting of the lumenal domain of chicken LEP100 (a LAMP-1) and the transmembrane and cytoplasmic domains of the LAMP-2 variants, we demonstrate in transfected mouse L cells that all three LAMP-2 carboxyl-terminal regions are capable of targeting the chimeric proteins to lysosomes. Levels of expression, subcellular distribution, and glycosylation of the LAMP proteins have all been shown to change with differentiation in mammalian cells and to be correlated with metastatic potential in certain tumor cell lines. Alternative splicing of the LAMP-2 transcript may play a role in these changes.
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Perry, David J., Kathy J. Austin und Thomas R. Hansen. „Cloning of Interferon-Stimulated Gene 17: The Promoter and Nuclear Proteins That Regulate Transcription“. Molecular Endocrinology 13, Nr. 7 (01.07.1999): 1197–206. http://dx.doi.org/10.1210/mend.13.7.0294.
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Abstract A member of the interferon-stimulated gene (ISG) family encodes a 17-kDa ubiquitin homolog called ISG17 that is induced in the bovine uterine endometrium by interferon-τ (IFN-τ) during early pregnancy. The bovine (b) ISG17 cDNA shares 30% identity with a tandem ubiquitin repeat and 70% identity with human (h) ISG15. The present experiments were designed to sequence the bISG17 gene, compare general structure with the hISG15 gene, and to identify transcription factors that were induced by IFN-τ in bovine endometrial (BEND) cells. The promoter of the bISG17 gene was similar to the hISG15 gene in placement of a tandem IFN-stimulatory response element (ISRE) at position −90, but unique in the presence of three additional ISREs at positions −123, −332, and −525. IFN-τ (25 nm) induced nuclear proteins in BEND cells that interacted with a tandem bISG17 ISRE in electrophoretic mobility shift assay (EMSA). IFN-regulatory factor-1 (IRF-1) bound to this ISRE based upon supershift EMSA using antiserum against IRF-1. IFN-τ activated STAT-1 (signal transducer and activator of transcription-1) and -2 by 0.5 h, and IRF-1 by 2 h in BEND cells. It is concluded that the bISG17 gene is similar to the hISG15 gene, retains an ISRE that interacts with IRF-1, and is possibly induced initially by the STATs and later by IRF-1 in response to IFN-τ during early pregnancy.
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Severson, Aaron F., und Bruce Bowerman. „Myosin and the PAR proteins polarize microfilament-dependent forces that shape and position mitotic spindles in Caenorhabditis elegans“. Journal of Cell Biology 161, Nr. 1 (14.04.2003): 21–26. http://dx.doi.org/10.1083/jcb.200210171.
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In Caenorhabditis elegans, the partitioning proteins (PARs), microfilaments (MFs), dynein, dynactin, and a nonmuscle myosin II all localize to the cortex of early embryonic cells. Both the PARs and the actomyosin cytoskeleton are required to polarize the anterior-posterior (a-p) body axis in one-cell zygotes, but it remains unknown how MFs influence embryonic polarity. Here we show that MFs are required for the cortical localization of PAR-2 and PAR-3. Furthermore, we show that PAR polarity regulates MF-dependent cortical forces applied to astral microtubules (MTs). These forces, which appear to be mediated by dynein and dynactin, produce changes in the shape and orientation of mitotic spindles. Unlike MFs, dynein, and dynactin, myosin II is not required for the production of these forces. Instead, myosin influences embryonic polarity by limiting PAR-3 to the anterior cortex. This in turn produces asymmetry in the forces applied to MTs at each pole and allows PAR-2 to accumulate in the posterior cortex of a one-cell zygote and maintain asymmetry.
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Cadwell, Ken, und Laurent Coscoy. „The Specificities of Kaposi's Sarcoma-Associated Herpesvirus-Encoded E3 Ubiquitin Ligases Are Determined by the Positions of Lysine or Cysteine Residues within the Intracytoplasmic Domains of Their Targets“. Journal of Virology 82, Nr. 8 (13.02.2008): 4184–89. http://dx.doi.org/10.1128/jvi.02264-07.
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ABSTRACT Kaposi's sarcoma-associated herpesvirus encodes two homologous E3 ligases, MIR1 and MIR2, that mediate the ubiquitination and subsequent downregulation of several cell surface proteins, and in particular major histocompatibility complex class I (MHC-I) molecules. We have previously shown that, in addition to lysine ubiquitination, MIR1 has the unique ability of transferring ubiquitin onto MHC-I molecules lacking available lysine residues, in a cysteine-dependent manner. Here we report that MIR1 activity is maximal when either a lysine or cysteine residue is placed approximately 15 amino acids away from the transmembrane domain, whereas MIR2 preferentially targets residues, including cysteines, that are closer to the transmembrane domain. Thus MIR1 and -2 can distinguish their substrates based on the position of the lysine or cysteine residues, suggesting that these proteins have evolved to target different sets of surface molecules. These results indicate that the position of target residues within a substrate is an essential determinant of E3 ubiquitin ligase specificity.
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Najarian, D., M. E. Dihanich, N. C. Martin und A. K. Hopper. „DNA sequence and transcript mapping of MOD5: features of the 5' region which suggest two translational starts“. Molecular and Cellular Biology 7, Nr. 1 (Januar 1987): 185–91. http://dx.doi.org/10.1128/mcb.7.1.185-191.1987.
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A mutation in the yeast nuclear gene MOD5 drastically reduces the biosynthesis of the modified base isopentenyladenosine in tRNAs located in different cellular compartments: the mitochondria and the nucleus or cytoplasm. Several lines of evidence strongly suggest that MOD5 is the structural gene encoding the tRNA-modifying enzyme delta 2-isopentenyl pyrophosphate:tRNA isopentenyl transferase. DNA sequence analysis of MOD5 reveals an open reading frame of 428 amino acids. A set of mRNAs heterogeneous at both the 5' and 3' termini are transcribed from this gene. Although all of these transcripts initiate upstream of the first AUG codon of the open reading frame, a subset has an extremely short (greater than or equal to 1 base) 5' leader. Furthermore, in positions important for efficient initiation of translation and generally occupied by purines, this first AUG codon is flanked by a U (position -3) and a C (position +4). It is possible that two proteins, one with an amino-terminal extension of basic charge, could be generated from the MOD5 gene via differential translational starts.