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1.

001-es BibID:BIBFORM040648
Első szerző:Boross Péter (biokémikus, vegyész)
Cím:Effect of substrate residues on the P2' preference of retroviral proteinases / Boross Peter, Bagossi Peter, Copeland Terry D., Oroszlan Stephen, Louis John M., Tozser Jozsef
Dátum:1999
ISSN:0014-2956
Megjegyzések:The substrate sequence requirements for preference toward P2' Glu residue by human immunodeficiency virus type 1 (HIV-1) proteinase were studied in both the matrix protein/ capsid protein (MA/CA) and CA/p2 cleavage site sequence contexts. These sequences represent typical type 1 (-aromatic*Pro-) and type 2 (-hydrophobic* hydrophobic-) cleavage site sequences, respectively. While in the type 1 sequence context, the preference for P2' Glu over Ile or Gln was found to be strongly dependent on the ionic strength and the residues being outside the P2-P2' region of the substrate, it remained preferable in the type 2 substrates when typical type 1 substrate sequence residues were substituted into the outside regions. The pH profile of the specificity constants suggested a lower pH optimum for substrates having P2' Glu in contrast to those having uncharged residues, in both sequence contexts. The very low frequency of P2' Glu in naturally occurring retroviral cleavage sites of various retroviruses including equine infectious anemia virus (EIAV) and murine leukemia virus (MuLV) suggests that such a residue may not have a general regulatory role in the retroviral life cycle. In fact, unlike HIV-1 and HIV-2, EIAV and MuLV proteinases do not favor P2' Glu in either the MA/CA or CA/p2 sequence contexts.
Tárgyszavak:Orvostudományok Elméleti orvostudományok idegen nyelvű folyóiratközlemény külföldi lapban
Megjelenés:European Journal Of Biochemistry. - 264 : 3 (1999), p. 921-929. -
További szerzők:Bagossi Péter (1966-2011) (biokémikus, vegyész) Copeland, Terry D. Oroszlan, Stephen Louis, John M. Tőzsér József (1959-) (molekuláris biológus, biokémikus, vegyész)
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2.

001-es BibID:BIBFORM072540
Első szerző:Bozóki Beáta (molekuláris biológus)
Cím:A recombinant fusion protein-based, fluorescent protease assay for high throughput-compatible substrate screening / Beáta Bozóki, Lívia Gazda, Ferenc Tóth, Márió Miczi, János András Mótyán, József Tőzsér
Dátum:2018
ISSN:0003-2697
Megjegyzések:In connection with the intensive investigation of proteases, several methods have been developed for analysis of the substrate specificity. Due to the great number of proteases and the expected target molecules to be analyzed, time- and cost-efficient high-throughput screening (HTS) methods are preferred. Here we describe the development and application of a separation-based HTS-compatible fluorescent protease assay, which is based on the use of recombinant fusion proteins as substrates of proteases. The protein substrates used in this assay consists of N-terminal (hexahistidine and maltose binding protein) fusion tags, cleavage sequences of the tobacco etch virus (TEV) and HIV-1 proteases, and a C-terminal fluorescent protein (mApple or mTurquoise2). The assay is based on the fluorimetric detection of the fluorescent proteins, which are released from the magnetic bead-attached substrates by the proteolytic cleavage. The protease assay has been applied for activity measurements of TEV and HIV-1 proteases to test the suitability of the system for enzyme kinetic measurements, inhibition studies, and determination of pH optimum. We also found that denatured fluorescent proteins can be renatured after SDS-PAGE of denaturing conditions, but showed differences in their renaturation abilities. After in-gel renaturation both substrates and cleavage products can be identified by in-gel UV detection.
Tárgyszavak:Természettudományok Biológiai tudományok idegen nyelvű folyóiratközlemény külföldi lapban
Megjelenés:Analytical Biochemistry. - 540-541 (2018), p. 52-63. -
További szerzők:Gazda Lívia Diána (1989-) Tóth Ferenc (1980-) (molekuláris biológus) Miczi Márió Mótyán János András (1981-) (biokémikus, molekuláris biológus) Tőzsér József (1959-) (molekuláris biológus, biokémikus, vegyész)
Pályázati támogatás:GINOP-2.3.2-15-2016-00044
GINOP
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3.

001-es BibID:BIBFORM040655
Első szerző:Fehér Anita
Cím:Effect of sequence polymorphism and drug resistance on two HIV-1 Gag processing sites / Fehér Anita, Weber Irene T., Bagossi Péter, Boross Péter, Mahalingam Bhuvaneshwari, Louis John M., Copeland Terry D., Torshin Ivan Y., Harrison Robert W., Tözsér József
Dátum:2002
ISSN:0014-2956
Megjegyzések:The HIV-1 proteinase (PR) has proved to be a good target for antiretroviral therapy of AIDS, and various PR inhibitors are now in clinical use. However, there is a rapid selection of viral variants bearing mutations in the proteinase that are resistant to clinical inhibitors. Drug resistance also involves mutations of the nucleocapsid/p1 and p1/p6 cleavage sites of Gag, both in vitro and in vivo. Cleavages at these sites have been shown to be rate limiting steps for polyprotein processing and viral maturation. Furthermore, these sites show significant sequence polymorphism, which also may have an impact on virion infectivity. We have studied the hydrolysis of oligopeptides representing these cleavage sites with representative mutations found as natural variations or that arise as resistant mutations. Wild-type and five drug resistant PRs with mutations within or outside the substrate binding site were tested. While the natural variations showed either increased or decreased susceptibility of peptides toward the proteinases, the resistant mutations always had a beneficial effect on catalytic efficiency. Comparison of the specificity changes obtained for the various substrates suggested that the maximization of the van der Waals contacts between substrate and PR is the major determinant of specificity: the same effect is crucial for inhibitor potency. The natural nucleocapsid/p1 and p1/p6 sites do not appear to be optimized for rapid hydrolysis. Hence, mutation of these rate limiting cleavage sites can partly compensate for the reduced catalytic activity of drug resistant mutant HIV-1 proteinases.
Tárgyszavak:Orvostudományok Elméleti orvostudományok idegen nyelvű folyóiratközlemény külföldi lapban
Megjelenés:European Journal Of Biochemistry. - 269 : 16 (2002), p. 4114-4120. -
További szerzők:Weber, Irene T. Bagossi Péter (1966-2011) (biokémikus, vegyész) Boross Péter (1972-) (biokémikus, vegyész) Mahalingam, Bhuvaneshwari Louis, John M. Copeland, Terry D. Torshin, Ivan Y. Harrison, Robert W. Tőzsér József (1959-) (molekuláris biológus, biokémikus, vegyész)
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4.

001-es BibID:BIBFORM056377
Első szerző:Louis, John M.
Cím:Enhanced stability of monomer fold correlates with extreme drug resistance of HIV-1 protease / John M. Louis, József Tözsér, Julien Roche, Krisztina Matúz, Annie Aniana, Jane M. Sayer
Dátum:2013
ISSN:0006-2960
Megjegyzések:During treatment, mutations in HIV-1 protease (PR) are selected rapidly that confer resistance by decreasing affinity to clinical protease inhibitors (PIs). As these unique drug resistance mutations can compromise the fitness of the virus to replicate, mutations that restore conformational stability and activity while retaining drug resistance are selected on further evolution. Here we identify several compensating mechanisms by which an extreme drug-resistant mutant bearing 20 mutations (PR20) with >5-fold increased Kd and >4000-fold decreased affinity to the PI darunavir functions. (1) PR20 cleaves, albeit poorly, Gag polyprotein substrates essential for viral maturation. (2) PR20 dimer, which exhibits distinctly enhanced thermal stability, has highly attenuated autoproteolysis, thus likely prolonging its lifetime in vivo. (3) The enhanced stability of PR20 results from stabilization of the monomer fold. Both monomeric PR20(T26A) and dimeric PR20 exhibit Tm values 6-7.5 ?C higher than those for their PR counterparts. Two specific mutations in PR20, L33F and L63P at sites of autoproteolysis, increase the Tm of monomeric PR(T26A) by ~8 ?C, similar to PR20(T26A). However, without other compensatory mutations as seen in PR20, L33F and L63P substitutions, together, neither restrict autoproteolysis nor significantly reduce binding affinity to darunavir. To determine whether dimer stability contributes to binding affinity for inhibitors, we examined single-chain dimers of PR and PR(D25N) in which the corresponding identical monomer units were covalently linked by GGSSG sequence. Linking of the subunits did not appreciably change the ?Tm on inhibitor binding; thus stabilization by tethering appears to have little direct effect on enhancing inhibitor affinity.
Tárgyszavak:Orvostudományok Elméleti orvostudományok idegen nyelvű folyóiratközlemény külföldi lapban
HIV-1
Megjelenés:Biochemistry. - 52 : 43 (2013), p. 7678-7688. -
További szerzők:Tőzsér József (1959-) (molekuláris biológus, biokémikus, vegyész) Roche, Julien Matúz Krisztina (1980-) (vegyész, biokémikus) Aniana, Annie Sayer, Jane M.
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5.

001-es BibID:BIBFORM040668
Első szerző:Mahalingam, Bhuvaneshwari
Cím:Crystal structures of HIV protease V82A and L90M mutants reveal changes in the indinavir-binding site / Mahalingam Bhuvaneshwari, Wang Yuan-Fang, Boross Peter I., Tozser Jozsef, Louis John M., Harrison Robert W., Weber Irene T.
Dátum:2004
ISSN:0014-2956
Megjegyzések:The crystal structures of the wild-type HIV-1 protease (PR) and the two resistant variants, PR(V82A) and PR(L90M), have been determined in complex with the antiviral drug, indinavir, to gain insight into the molecular basis of drug resistance. V82A and L90M correspond to an active site mutation and nonactive site mutation, respectively. The inhibition (K(i)) of PR(V82A) and PR(L90M) was 3.3- and 0.16-fold, respectively, relative to the value for PR. They showed only a modest decrease, of 10-15%, in their k(cat)/K(m) values relative to PR. The crystal structures were refined to resolutions of 1.25-1.4 A to reveal critical features associated with inhibitor resistance. PR(V82A) showed local changes in residues 81-82 at the site of the mutation, while PR(L90M) showed local changes near Met90 and an additional interaction with indinavir. These structural differences concur with the kinetic data.
Tárgyszavak:Orvostudományok Elméleti orvostudományok idegen nyelvű folyóiratközlemény külföldi lapban
Megjelenés:European Journal Of Biochemistry. - 271 : 8 (2004), p. 1516-1524. -
További szerzők:Wang, Yuan-Fang Boross Péter (1972-) (biokémikus, vegyész) Tőzsér József (1959-) (molekuláris biológus, biokémikus, vegyész) Louis, John M. Harrison, Robert W. Weber, Irene T.
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6.

001-es BibID:BIBFORM042832
Első szerző:Raran-Kurussi, Sreejith
Cím:Differential temperature dependence of tobacco etch virus and rhinovirus 3C proteases / Sreejith Raran-Kurussi, József Tözsér, Scott Cherry, Joseph E. Tropea, David S. Waugh
Dátum:2013
ISSN:0003-2697
Tárgyszavak:Orvostudományok Elméleti orvostudományok idegen nyelvű folyóiratközlemény külföldi lapban
Megjelenés:Analytical Biochemistry 436 : 2 (2013), p. 142-144. -
További szerzők:Tőzsér József (1959-) (molekuláris biológus, biokémikus, vegyész) Cherry, Scott Tropea, Joseph E. Waugh, David S.
Pályázati támogatás:K101591
OTKA
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7.

001-es BibID:BIBFORM040684
Első szerző:Tőzsér József (molekuláris biológus, biokémikus, vegyész)
Cím:Kinetic and modeling studies of S3-S3' subsites of HIV proteinases / Tözsér J., Weber I. T., Gustchina A., Bláha I., Copeland T. D., Louis J. M., Oroszlan S.
Dátum:1992
ISSN:0006-2960
Megjegyzések:Kinetic analysis and modeling studies of HIV-1 and HIV-2 proteinases were carried out using the oligopeptide substrate [formula: see text] and its analogs containing single amino acid substitutions in P3-P3' positions. The two proteinases acted similarly on the substrates except those having certain hydrophobic amino acids at P2, P1, P2', and P3' positions (Ala, Leu, Met, Phe). Various amino acids seemed to be acceptable at P3 and P3' positions, while the P2 and P2' positions seemed to be more restrictive. Polar uncharged residues resulted in relatively good binding at P3 and P2 positions, while at P2' and P3' positions they gave very high Km values, indicating substantial differences in the respective S and S' subsites of the enzyme. Lys prevented substrate hydrolysis at any of the P2-P2' positions. The large differences for subsite preference at P2 and P2' positions seem to be at least partially due to the different internal interactions of P2 residue with P1', and P2' residue with P1. As expected on the basis of amino acid frequency in the naturally occurring cleavage sites, hydrophobic residues at P1 position resulted in cleavable peptides, while polar and beta-branched amino acids prevented hydrolysis. On the other hand, changing the P1' Pro to other amino acids prevented substrate hydrolysis, even if the substituted amino acid had produced a good substrate in other oligopeptides representing naturally occurring cleavage sites. The results suggest that the subsite specificity of the HIV proteinases may strongly depend on the sequence context of the substrate.
Tárgyszavak:Orvostudományok Elméleti orvostudományok idegen nyelvű folyóiratközlemény külföldi lapban
Megjelenés:Biochemistry. - 31 : 20 (1992), p. 4793-4800. -
További szerzők:Weber, Irene T. Gustchina, Alla Bláha, Ivo Copeland, Terry D. Louis, John M. Oroszlan, Stephen
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8.

001-es BibID:BIBFORM040686
Első szerző:Tőzsér József (molekuláris biológus, biokémikus, vegyész)
Cím:Studies on the substrate specificity of the proteinase of equine infectious anemia virus using oligopeptide substrates / Tozser Jozsef, Friedman Deirdre, Weber Irene T., Blaha Ivo, Oroszlan Stephen
Dátum:1993
ISSN:0006-2960
Megjegyzések:The proteinase of the equine infectious anemia virus (EIAV), a lentivirus closely related to human immunodeficiency virus (HIV), was purified from concentrated virus. The specificity of the enzyme was characterized using oligopeptides representing naturally occurring cleavage sites in the Gag and Gag-Pol polyproteins. The length of the substrate binding pocket was found to be 1-2 residues longer than that of HIV proteinases. Although the EIAV and HIV proteinases cleaved most of the peptides at the same bond, some were hydrolyzed by only the EIAV enzyme. Oligopeptides representing cleavage sites in the nucleocapsid protein were also found to be substrates of the EIAV proteinase. However, these peptides were not hydrolyzed by the HIV proteinases. While peptides representing the corresponding sequences in the first cysteine arrays of the nucleocapsid proteins of HIV-1 and HIV-2 were substrates of the proteinases, peptides representing the homologous sequences in the second Cys arrays were resistant against the proteolytic attack. A three-dimensional model of the EIAV proteinase built on the basis of homology with HIV-1 proteinase was used to interpret the differences. In addition to the oligopeptides representing cleavage sites in the Gag and Gag-Pol polyproteins, the EIAV proteinase was also able to cleave an oligopeptide mimicking a cleavage site in the transmembrane protein. Our results suggest that the specificity of lentiviral proteinases share common characteristics, although substantial differences may exist in hydrolysis of some peptides.
Tárgyszavak:Orvostudományok Elméleti orvostudományok idegen nyelvű folyóiratközlemény külföldi lapban
Megjelenés:Biochemistry. - 32 : 13 (1993), p. 3347-3353. -
További szerzők:Friedman, Deirdre Weber, Irene T. Bláha, Ivo Oroszlan, Stephen
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9.

001-es BibID:BIBFORM040689
Első szerző:Tőzsér József (molekuláris biológus, biokémikus, vegyész)
Cím:Activity of Tethered Human Immunodeficiency Virus 1 Protease Containing Mutations in the Flap Region of One Subunit / Tozser Jozsef, Yin Fay H., Cheng Yin-Shyun E., Bagossi Peter, Weber Irene T., Harrison Robert W., Oroszlan Stephen
Dátum:1997
ISSN:0014-2956
Megjegyzések:The tethered-dimer protease of human immunodeficiency virus 1 (HIV-1) [Cheng Y.-S. E., Yin, F.H., Foundling, S., Blomstrom, D. & Kettner, C. A. (1990) Proc. Natl Acad. Sci. USA 87, 9660-9664] and its mutants containing amino acid substitutions or deletions or both in only one flap region were expressed in Escherichia coli. These mutant enzymes showed various degrees of self-processing and significantly reduced catalytic activity toward oligopeptide substrates compared with the wild type. Kinetic parameters determined for one of the oligopeptide substrates showed a dramatic increase in K(m) and decrease in Kcat values. Unexpectedly, the substrate cleavage was more efficient in low salt concentration for a mutant containing a shortened hydrophilic flap. Assays with oligopeptides representing naturally occurring cleavage sites or oligopeptides containing single amino acid substitutions at the P2 and P2' substrate positions showed only moderate changes in the substrate specificity of the mutant proteases. Predicted structures for the mutants were constructed by molecular modeling and used to interpret the results of kinetic measurements. In general, the data suggest that the mutated part of the flaps does not have a major role in determining substrate specificity; rather, it provides the hydrophobic environment and hydrogen-bond interactions with the conserved water that are necessary for efficient substrate binding and catalysis.
Tárgyszavak:Orvostudományok Elméleti orvostudományok idegen nyelvű folyóiratközlemény külföldi lapban
Megjelenés:European Journal Of Biochemistry 244 : 1 (1997), p. 235-241. -
További szerzők:Yin, Fay H. Cheng, Yin-Shyun E. Bagossi Péter (1966-2011) (biokémikus, vegyész) Weber, Irene T. Harrison, Robert W. Oroszlan, Stephen
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10.

001-es BibID:BIBFORM040690
Első szerző:Tőzsér József (molekuláris biológus, biokémikus, vegyész)
Cím:Effect of serine and tyrosine phosphorylation on retroviral proteinase substrates / Tozser Jozsef, Bagossi Peter, Boross Peter, Louis John M., Majerova Eva, Oroszlan Stephen, Copeland Terry D.
Dátum:1999
ISSN:0014-2956
Megjegyzések:Vimentin, a cellular substrate of HIV type 1 (HIV-1) proteinase, contains a protein kinase C (PKC) phosphorylation site at one of its cleavage sites. Peptides representing this site were synthesized in P2 Ser-phosphorylated and nonphosphorylated forms. While the nonphosphorylated peptide was a fairly good substrate of the enzyme, phosphorylation prevented hydrolysis. Phosphorylation of human recombinant vimentin by PKC prevented its processing within the head domain, where the phosphorylation occurred. Oligopeptides representing naturally occurring cleavage sites at the C-terminus of the Rous sarcoma virus integrase were assayed as substrates of the avian proteinase. Unlike the nonphosphorylated peptides, a Ser-phosphorylated peptide was not hydrolyzed by the enzyme at the Ser-Pro bond, suggesting the role of previously established phosphorylation in processing at this site. Ser-phosphorylated and Tyr-phosphorylated forms of model substrates were also tested as substrates of the HIV-1 and the avian retroviral proteinases. In contrast to the moderate effect of P4 Ser phosphorylation, phosphorylation of P1 Tyr prevented substrate hydrolysis by HIV-1 proteinase. Substrate phosphorylation had substantially smaller effects on the hydrolysis by the avian retroviral proteinase. As the active retroviral proteinase as well as various protein kinases are incorporated into mature virions, substrate phosphorylation resulting in attenuation or prevention of proteolytic processing may have important consequences in the regulation of the retroviral life cycle as well as in virus-host cell interactions.
Tárgyszavak:Orvostudományok Elméleti orvostudományok idegen nyelvű folyóiratközlemény külföldi lapban
Megjelenés:European Journal Of Biochemistry. - 265 : 1 (1999), p. 423-429. -
További szerzők:Bagossi Péter (1966-2011) (biokémikus, vegyész) Boross Péter (1972-) (biokémikus, vegyész) Louis, John M. Majerova, Eva Oroszlan, Stephen Copeland, Terry D.
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11.

001-es BibID:BIBFORM040682
Első szerző:Tőzsér József (molekuláris biológus, biokémikus, vegyész)
Cím:In vitro processing of HIV-1 nucleocapsid protein by the viral proteinase : effects of amino acid substitutions at the scissile bond in the proximal zinc finger sequence / Tözsér József, Shulenin Sergey, Louis John M., Copeland Terry D., Oroszlan Stephen
Dátum:2004
ISSN:0006-2960
Megjegyzések:The human immunodeficiency virus type 1 (HIV-1) nucleocapsid protein flanked by Gag sequences (r-preNC) was expressed in Escherichia coli and purified. HIV-1 proteinase cleaved r-preNC to the "mature" NCp7 form, which is comprised of 55 residues. Further incubation resulted in cleavages of NCp7 itself between Phe16 and Asn17 of the proximal zinc finger domain and between Cys49 and Thr50 in the C-terminal part. Kinetic parameters determined for the cleavage of oligopeptides corresponding to the cleavage sites in r-preNC correlated well with the sequential processing of r-preNC. Mutations of Asn17 were introduced to alter the susceptibility of NC protein to HIV-1 proteinase. While mutating Asn17 to Ala resulted in a protein which was processed in a manner similar to that of the wild type, mutating it to Phe or Leu resulted in proteins which were processed at a substantially higher rate at this site than the wild type. Mutation of Asn17 to Lys or Gly resulted in proteins which were very poorly cleaved at this site. Oligopeptides containing the same amino acid substitutions at the cleavage site of the proximal zinc finger domain were also tested as substrates of the proteinase, and the kinetic parameters agreed well with the semiquantitative results obtained with the protein substrates.
Tárgyszavak:Orvostudományok Elméleti orvostudományok idegen nyelvű folyóiratközlemény külföldi lapban
Megjelenés:Biochemistry. - 43 : 14 (2004), p. 4304-4312. -
További szerzők:Shulenin, Sergey Louis, John M. Copeland, Terry D. Oroszlan, Stephen
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12.

001-es BibID:BIBFORM040126
Első szerző:Tőzsér József (molekuláris biológus, biokémikus, vegyész)
Cím:Comparison of the substrate specificity of the human T-cell leukemia virus and human immunodeficiency virus proteinases / József Tőzsér, Gábor Zahuczky, Péter Bagossi, John M. Louis, Terry D. Copeland, Stephen Oroszlan, Robert W. Harrison, Irene T. Weber
Dátum:2000
Tárgyszavak:Természettudományok Biológiai tudományok idegen nyelvű folyóiratközlemény külföldi lapban
Megjelenés:European Journal of Biochemistry. - 267 : 20 (2000), p. 6287-6295. -
További szerzők:Zahuczky Gábor (1975-) (molekuláris biológus, biokémikus, vegyész) Bagossi Péter (1966-2011) (biokémikus, vegyész) Louis, John M. Copeland, Terry D. Oroszlan, Stephen Harrison, Robert W. Weber, Irene T.
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