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1.

001-es BibID:BIBFORM020961
Első szerző:Austin, Brian P.
Cím:The substrate specificity of Metarhizium anisopliae and Bos taurus carboxypeptidases A : insights into their use as tools for the removal of affinity tags / Austin Brian P., Tözsér József, Bagossi Péter, Tropea Joseph E., Waugh David S.
Dátum:2011
ISSN:1046-5928
Megjegyzések:Carboxypeptidases may serve as tools for removal of C-terminal affinity tags. In the present study, we describe the expression and purification of an A-type carboxypeptidase from the fungal pathogen Metarhizium anisopliae (MeCPA) that has been genetically engineered to facilitate the removal of polyhistidine tags from the C-termini of recombinant proteins. A complete, systematic analysis of the specificity of MeCPA in comparison with that of bovine carboxypeptidase A (BoCPA) was carried out. Our results indicate that the specificity of the two enzymes is similar but not identical. Histidine residues are removed more efficiently by MeCPA. The very inefficient digestion of peptides with C-terminal lysine or arginine residues, along with the complete inability of the enzyme to remove a C-terminal proline, suggests a strategy for designing C-terminal affinity tags that can be trimmed by MeCPA (or BoCPA) to produce a digestion product with a homogeneous endpoint.
Tárgyszavak:Orvostudományok Elméleti orvostudományok idegen nyelvű folyóiratközlemény külföldi lapban
Megjelenés:Protein Expression And Purification. - 77 : 1 (2011), p. 53-61. -
További szerzők:Tőzsér József (1959-) (molekuláris biológus, biokémikus, vegyész) Bagossi Péter (1966-2011) (biokémikus, vegyész) Tropea, Joseph E. Waugh, David S.
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2.

001-es BibID:BIBFORM088520
035-os BibID:(cikkazonosító)E7686 (scopus)85092599352 (wos)000583012300001
Első szerző:Bozóki Beáta (molekuláris biológus)
Cím:Specificity Studies of the Venezuelan Equine Encephalitis Virus Non-Structural Protein 2 Protease Using Recombinant Fluorescent Substrates / Bozóki Beáta, Mótyán János András, Hoffka Gyula, Waugh David S., Tőzsér József
Dátum:2020
ISSN:1661-6596 1422-0067
Megjegyzések:The non-structural protein 2 (nsP2) of alphavirus Venezuelan equine encephalitis virus (VEEV) is a cysteine protease that is responsible for processing of the viral non-structural polyprotein and is an important drug target owing to the clinical relevance of VEEV. In this study we designed two recombinant VEEV nsP2 constructs to study the effects of an N-terminal extension on the protease activity and to investigate the specificity of the elongated enzyme in vitro. The N-terminal extension was found to have no substantial effect on the protease activity. The amino acid preferences of the VEEV nsP2 protease were investigated on substrates representing wild-type and P5, P4, P2, P1, P1·, and P2· variants of Semliki forest virus nsP1/nsP2 cleavage site, using a His6-MBP-mEYFP recombinant substrate-based protease assay which has been adapted for a 96-well plate-based format. The structural basis of enzyme specificity was also investigated in silico by analyzing a modeled structure of VEEV nsP2 complexed with oligopeptide substrate. To our knowledge, in vitro screening of P1· amino acid preferences of VEEV nsP2 protease remains undetermined to date, thus, our results may provide valuable information for studies and inhibitor design of different alphaviruses or other Group IV viruses.
Tárgyszavak:Orvostudományok Elméleti orvostudományok idegen nyelvű folyóiratközlemény külföldi lapban
folyóiratcikk
VEEV
Venezuelan equine encephalitis virus
nsp2
protease
alphavirus
alphaviral protease
non-structural protein
group IV virus
specificity
Megjelenés:International Journal Of Molecular Sciences. - 21 : 20 (2020), p. 1-26. -
További szerzők:Mótyán János András (1981-) (biokémikus, molekuláris biológus) Hoffka Gyula (1992-) (vegyész) Waugh, David S. Tőzsér József (1959-) (molekuláris biológus, biokémikus, vegyész)
Pályázati támogatás:NKFI-125238
Egyéb
GINOP-2.3.2-15-2016-00044
GINOP
TÁMOP 4.2.4B/2-11/1-2012-0001
Egyéb
NKFIH-1150-6/2019
Egyéb
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3.

001-es BibID:BIBFORM108790
035-os BibID:(Scopus)85148381582 (WoS)000944102100001
Első szerző:Hoffka Gyula (vegyész)
Cím:Self-inhibited state of Venezuelan equine encephalitis virus (VEEV) nsP2 cysteine protease : a crystallographic and molecular dynamics analysis / Gyula Hoffka, George T. Lountos, Danielle Needle, Alexander Wlodawer, David S. Waugh, József Tőzsér, János András Mótyán
Dátum:2023
ISSN:0022-2836
Megjegyzések:The Venezuelan equine encephalitis virus (VEEV) belongs to the Togaviridae family and is pathogenic to both humans and equines. The VEEV non-structural protein 2 (nsP2) is a cysteine protease (nsP2pro) that processes the polyprotein and thus it is a drug target for inhibitor discovery. The atomic structure of the VEEV nsP2 catalytic domain was previously characterized by both X-ray crystallography and computational studies. A modified nsP2pro harboring a N475A mutation in the N terminus was observed to exhibit an unexpected conformation: the N-terminal residues bind to the active site, mimicking binding of a substrate. The large conformational change of the N terminus was assumed to be induced by the N475A mutation, as N475 has an important role in stabilization of the N terminus and the active site. This conformation was first observed in the N475A mutant, but we also found it while determining a crystal structure of the catalytically active nsP2pro containing the wild-type N475 active site residue and K741A/K767A surface entropy reduction mutations. This suggests that the N475A mutation is not a prerequisite for self-inhibition. Here, we describe a high resolution (1.46 ?A) crystal structure of a truncated nsP2pro (residues 463-785, K741A/K767A) and analyze the structure further by molecular dynamics to study the active and self-inhibited conformations of nsP2pro and its N475A mutant. A comparison of the different conformations of the N-terminal residues sheds a light on the interactions that play an important role in the stabilization of the enzyme.
Tárgyszavak:Orvostudományok Elméleti orvostudományok idegen nyelvű folyóiratközlemény külföldi lapban
folyóiratcikk
Venezuelan equine encephalitis virus
protease
alphavirus
crystallography
molecular dynamics
Megjelenés:Journal Of Molecular Biology. - 435 : 6 (2023), p. 1-20. -
További szerzők:Lountos, George T. Needle, Danielle Wlodawer, Alexander Waugh, David S. Tőzsér József (1959-) (molekuláris biológus, biokémikus, vegyész) Mótyán János András (1981-) (biokémikus, molekuláris biológus)
Pályázati támogatás:TKP2021-EGA-20 (Biotechnology)
Egyéb
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4.

001-es BibID:BIBFORM040661
Első szerző:Kapust, Rachel B.
Cím:The P1' specificity of tobacco etch virus protease / Kapust Rachel B., Tözsér József, Copeland Terry D., Waugh David S.
Dátum:2002
ISSN:0006-291X
Megjegyzések:Affinity tags have become indispensable tools for protein expression and purification. Yet, because they have the potential to interfere with structural and functional studies, it is usually desirable to remove them from the target protein. The stringent sequence specificity of the tobacco etch virus (TEV) protease has made it a useful reagent for this purpose. However, a potential limitation of TEV protease is that it is believed to require a Gly or Ser residue in the P1' position of its substrates to process them with reasonable efficiency. Consequently, after an N-terminal affinity tag is removed by TEV protease, the target protein will usually retain a non-native Ser or Gly residue on its N-terminus, and in some cases this may affect its biological activity. To investigate the stringency of the requirement for Gly or Ser in the P1' position of a TEV protease recognition site, we constructed 20 variants of a fusion protein substrate with an otherwise optimal recognition site, each containing a different amino acid in the P1' position. The efficiency with which these fusion proteins were processed by TEV protease was compared both in vivo and in vitro. Additionally, the kinetic parameters K(M) and k(cat) were determined for a representative set of peptide substrates with amino acid substitutions in the P1' position. The results indicate that many side-chains can be accommodated in the P1' position of a TEV protease recognition site with little impact on the efficiency of processing.
Tárgyszavak:Orvostudományok Elméleti orvostudományok idegen nyelvű folyóiratközlemény külföldi lapban
Megjelenés:Biochemical And Biophysical Research Communications. - 294 : 5 (2002), p. 949-955. -
További szerzők:Tőzsér József (1959-) (molekuláris biológus, biokémikus, vegyész) Copeland, Terry D. Waugh, David S.
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5.

001-es BibID:BIBFORM040662
Első szerző:Kapust, Rachel B.
Cím:Tobacco etch virus protease : mechanism of autolysis and rational design of stable mutants with wild-type catalytic proficiency / Kapust, R. B., Tozser, J., Fox, J. D., Anderson, D. E., Cherry, S., Copeland, T. D., Waugh, D. S.
Dátum:2001
ISSN:1741-0126
Megjegyzések:Because of its stringent sequence specificity, the catalytic domain of the nuclear inclusion protease from tobacco etch virus (TEV) is a useful reagent for cleaving genetically engineered fusion proteins. However, a serious drawback of TEV protease is that it readily cleaves itself at a specific site to generate a truncated enzyme with greatly diminished activity. The rate of autoinactivation is proportional to the concentration of TEV protease, implying a bimolecular reaction mechanism. Yet, a catalytically active protease was unable to convert a catalytically inactive protease into the truncated form. Adding increasing concentrations of the catalytically inactive protease to a fixed amount of the wild-type enzyme accelerated its rate of autoinactivation. Taken together, these results suggest that autoinactivation of TEV protease may be an intramolecular reaction that is facilitated by an allosteric interaction between protease molecules. In an effort to create a more stable protease, we made amino acid substitutions in the P2 and P1' positions of the internal cleavage site and assessed their impact on the enzyme's stability and catalytic activity. One of the P1' mutants, S219V, was not only far more stable than the wild-type protease (approximately 100-fold), but also a more efficient catalyst.
Tárgyszavak:Orvostudományok Elméleti orvostudományok idegen nyelvű folyóiratközlemény külföldi lapban
Megjelenés:Protein Engineering Design & Selection. - 14 : 12 (2001), p. 993-1000. -
További szerzők:Tőzsér József (1959-) (molekuláris biológus, biokémikus, vegyész) Fox, Jeffrey D. Anderson, D. Eric Cherry, Scott Copeland, Terry D. Waugh, David S.
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6.

001-es BibID:BIBFORM040675
Első szerző:Nallamsetty, Sreedevi
Cím:Efficient site-specific processing of fusion proteins by tobacco vein mottling virus protease in vivo and in vitro / Nallamsetty Sreedevi, Kapust Rachel B., Tözsér József, Cherry Scott, Tropea Joseph E., Copeland Terry D., Waugh David S.
Dátum:2004
ISSN:1046-5928
Megjegyzések:Affinity tags are widely used as vehicles for the production of recombinant proteins. Yet, because of concerns about their potential to interfere with the activity or structure of proteins, it is almost always desirable to remove them from the target protein. The proteases that are most often used to cleave fusion proteins are factor Xa, enterokinase, and thrombin, yet the literature is replete with reports of fusion proteins that were cleaved by these proteases at locations other than the designed site. It is becoming increasingly evident that certain viral proteases have more stringent sequence specificity. These proteases adopt a trypsin-like fold but possess an unconventional catalytic triad in which Cys replaces Ser. The tobacco etch virus (TEV) protease is the best-characterized enzyme of this type. TEV protease cleaves the sequence ENLYFQG/S between QG or QS with high specificity. The tobacco vein mottling virus (TVMV) protease is a close relative of TEV protease with a distinct sequence specificity (ETVRFQG/S). We show that, like TEV protease, TVMV protease can be used to cleave fusion proteins with high specificity in vitro and in vivo. We compared the catalytic activity of the two enzymes as a function of temperature and ionic strength, using an MBP-NusG fusion protein as a model substrate. The behavior of TVMV protease was very similar to that of TEV protease. Its catalytic activity was greatest in the absence of NaCl, but diminished only threefold with increasing salt up to 200 mM. We found that the optimum temperatures of the two enzymes are nearly the same and that they differ only two-fold in catalytic efficiency, both at room temperature and 4 degrees C. Hence, TVMV protease may be a useful alternative to TEV protease when a recombinant protein happens to contain a sequence that is similar to a TEV protease recognition site or for protein expression strategies that involve the use of more than one protease.
Tárgyszavak:Orvostudományok Elméleti orvostudományok idegen nyelvű folyóiratközlemény külföldi lapban
Megjelenés:Protein Expression And Purification. - 38 : 1 (2004), p. 108-115. -
További szerzők:Kapust, Rachel B. Tőzsér József (1959-) (molekuláris biológus, biokémikus, vegyész) Cherry, Scott Tropea, Joseph E. Copeland, Terry D. Waugh, David S.
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7.

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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8.

001-es BibID:BIBFORM012850
Első szerző:Sun, Ping
Cím:Structural determinants of tobacco vein mottling virus protease substrate specificity / Sun, P., Austin, B.P., Tözsér, J., Waugh, D. S.
Dátum:2010
Tárgyszavak:Természettudományok Biológiai tudományok idegen nyelvű folyóiratközlemény külföldi lapban
Megjelenés:Protein Science. - 19 : 11 (2010), p. 2240-2251. -
További szerzők:Austin, Brian P. Tőzsér József (1959-) (molekuláris biológus, biokémikus, vegyész) Waugh, David S.
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9.

001-es BibID:BIBFORM040692
Első szerző:Tőzsér József (molekuláris biológus, biokémikus, vegyész)
Cím:Comparison of the substrate specificity of two potyvirus proteases / Tözsér József, Tropea Joseph E., Cherry Scott, Bagossi Peter, Copeland Terry D., Wlodawer Alexander, Waugh David S.
Dátum:2005
ISSN:1742-464X
Megjegyzések:The substrate specificity of the nuclear inclusion protein a (NIa) proteolytic enzymes from two potyviruses, the tobacco etch virus (TEV) and tobacco vein mottling virus (TVMV), was compared using oligopeptide substrates. Mutations were introduced into TEV protease in an effort to identify key determinants of substrate specificity. The specificity of the mutant enzymes was assessed by using peptides with complementary substitutions. The crystal structure of TEV protease and a homology model of TVMV protease were used to interpret the kinetic data. A comparison of the two structures and the experimental data suggested that the differences in the specificity of the two enzymes may be mainly due to the variation in their S4 and S3 binding subsites. Two key residues predicted to be important for these differences were replaced in TEV protease with the corresponding residues of TVMV protease. Kinetic analyses of the mutants confirmed that these residues play a role in the specificity of the two enzymes. Additional residues in the substrate-binding subsites of TEV protease were also mutated in an effort to alter the specificity of the enzyme.
Tárgyszavak:Orvostudományok Elméleti orvostudományok idegen nyelvű folyóiratközlemény külföldi lapban
Megjelenés:Febs Journal. - 272 : 2 (2005), p. 514-523. -
További szerzők:Tropea, Joseph E. Cherry, Scott Bagossi Péter (1966-2011) (biokémikus, vegyész) Copeland, Terry D. Wlodawer, Alexander Waugh, David S.
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10.

001-es BibID:BIBFORM020959
Első szerző:Zhang, Di
Cím:Molecular cloning, overproduction, purification and biochemical characterization of the p39 nsp2 protease domains encoded by three alphaviruses / Zhang D., Tözsér J., Waugh D. S.
Dátum:2009
ISSN:1046-5928
Megjegyzések:Alphaviruses cause serious diseases that pose a potential health threat to both humans and livestock. The nonstructural protein 2 (nsp2) encoded by alphaviruses is a multifunctional enzyme that is essential for viral replication and maturation. Its 39-kDa C-terminal domain (nsp2pro) is a cysteine protease that is responsible for cleaving a viral polyprotein at three sites to generate nonstructural proteins 1, 2, 3 and 4. In the present study, we evaluated nsp2pro domains from the following three sources as reagents for site-specific cleavage of fusion proteins: Venezuelan Equine Encephalitis Virus (VEEV), Semliki Forest Virus (SFV) and Sindbis Virus (SIN). All three alphavirus proteases cleaved model fusion protein substrates with high specificity but they were much less efficient enzymes than potyviral proteases from tobacco etch virus (TEV) and tobacco vein mottling virus (TVMV). Oligopeptide substrates were also cleaved with very low efficiency by the alphavirus proteases. We conclude that, in general, alphavirus nsp2pro proteases are not very useful tools for the removal of affinity tags from recombinant proteins although they do remain promising therapeutic targets for the treatment of a variety of diseases
Tárgyszavak:Orvostudományok Elméleti orvostudományok idegen nyelvű folyóiratközlemény külföldi lapban
Megjelenés:Protein Expression And Purification. - 64 : 1 (2009), p. 89-97. -
További szerzők:Tőzsér József (1959-) (molekuláris biológus, biokémikus, vegyész) Waugh, David S.
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