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Papers by Stephen Foundling
Advances in Experimental Medicine and Biology, 1991
All proteinases of avian and mammalian retroviruses belong to the family of aspartic proteinases,... more All proteinases of avian and mammalian retroviruses belong to the family of aspartic proteinases, are of similar size and of homologous primary structure; they all act catalytically in the form of highly symmetric molecular dimers.1 Detailed studies of retroviral proteinases were carried out on two almost identical proteinases of MAV2,3 and RS V4 (representing the group of avian retroviruses) and on the HIV proteinase.5,6 The knowledge of the 3D structure,2,4,5 catalytic activity and substrate specifity3,6 of the MAV and the HIV proteinase has changed the notion of their general similarity since several features that distinguish each proteinase from the other were revealed. The HIV-1 proteinase has a considerably higher activity3,6 which reflects the different conditions of the expression and action of this enzyme in vivo: 7 The “coding strategy” of MAV allows the expression of the proteinase from the first (gag) open reading frame and provides for the high (i.e. stoichiometrical) level of the relatively “weak” enzyme whereas the smaller amount of the more active HIV enzyme is a result of infrequent translational frameshift events that occur in the overlapping region of the gag and pol reading frames.8 The substrate specificities of retroviral proteinases seem complex and the requirement for a side chain in an individual subsite of a substrate is an outcome of the combination of residues occupying other closely located subsites.3 The two proteinases (MAV and HIV) show rather promiscuous substrate specificity, nevertheless several differences can be traced. We made an attempt to use protein engineering of the MAV proteinase to tackle directly problems of structural basis of these differences and, vice versa, to make more precise conclusions on the functional importance of the individual elements of its three dimensional structure. This article describes mutation of the MAV proteinase which resulted not only in an alteration of its substrate specificity but also in an increase of its enzymic activity — a rare case in protein engineering.
FEBS Letters, Aug 1, 1988
Journal of Biological Chemistry, Sep 1, 1993
Biochemistry, Oct 17, 1989
Proceedings of the National Academy of Sciences of the United States of America, Aug 1, 1995
Advances in Experimental Medicine and Biology, 1995
Biochemical Society Transactions, 1987
Acta Crystallographica Section A Foundations of Crystallography, 1984
Proceedings of the National Academy of Sciences, 1995
General transcription factor SIII, a heterotrimer composed of 110-kDa (p110), 18-kDa (p18), and 1... more General transcription factor SIII, a heterotrimer composed of 110-kDa (p110), 18-kDa (p18), and 15-kDa (p15) subunits, increases the catalytic rate of transcribing RNA polymerase II by suppressing transient pausing by polymerase at multiple sites on DNA templates. Here we report molecular cloning and biochemical characterization of the SIII p18 subunit, which is found to be a member of the ubiquitin homology (UbH) gene family and functions as a positive regulatory subunit of SIII. p18 is a 118-amino acid protein composed of an 84-residue N-terminal UbH domain fused to a 34-residue C-terminal tail. Mechanistic studies indicate that p18 activates SIII transcriptional activity above a basal level inherent in the SIII p110 and p15 subunits. Taken together, these findings establish a role for p18 in regulating the activity of the RNA polymerase II elongation complex, and they bring to light a function for a UbH domain protein in transcriptional regulation.
The low hydrolytic activity (k/sub cat/ < 0.001 sâ»Â¹) of chicken pepsin (CP) towards tri- an... more The low hydrolytic activity (k/sub cat/ < 0.001 sâ»Â¹) of chicken pepsin (CP) towards tri- and tetrapeptides is enhanced at least 100 times by modification of its single sulfhydryl group of Cys-115, with little effect on K/sub m/-values. Modification thus simulates the effect of secondary substrate binding on pepsin catalysis. The rate of Cys-115 modification is substantially decreased in the presence of some competitive inhibitors, suggesting its active site location. Experiments with CP alkylated at Cys-115 with Acrylodan as a fluorescent probe or with N-iodoacetyl-(4-fluoro)-aniline as a ¹â¹F-nmr probe suggest conformation change around Cys-115 to occur on substrate or substrate analog binding. The difference ¹H-nmr spectra (500 MHz) of unmodified free and inhibitor-complexed CP reveal chemical shifts almost exclusively in the aromatic region. The effects of Cu/sup + +/ on ¹â¹F- and ¹H-nmr spectra have been studied. Examination of a computer graphics model of CP based o...
Acta Crystallographica Section A Foundations of Crystallography, 2002
Molecular immunology, 1996
The 40 kDa secreted aspartyl proteinase (Saptl) of Candida tropicalis is a pepsin-like enzyme enc... more The 40 kDa secreted aspartyl proteinase (Saptl) of Candida tropicalis is a pepsin-like enzyme encoded by the SApTl gene. According to the deduced amino acid sequence, Saptl has a putative preproregion of 60 amino acids preceding the mature enzyme. Maturation and processing of Saptl was analysed in C. tropicalis and Sacchammyces cemvisiae strains expressing wildtype or mutated forms of SAPll. In s. cemvisiae, the glycosylated 46 kDa proenzyme was converted to the mature 40 kDa form of Saptl by K B 2 dependent proteolytic cleavage following the Lyssg-Argm sequence. The replacement of L y ~ ~ ~ A r g ~ by Lyss9-Gly60 revealed that the precursor can be processed by an autocatalytic cleavage. This alternative processing pathway to produce mature Saptl is less efficient than the Kex2-mediated pathway. Finally, it was shown that in C. fropicelis and S. cemvisiae the removal of the proregion was a prerequisite for the secretion of Saptl.
Handbook of Proteolytic Enzymes, 2013
Handbook of Proteolytic Enzymes, 2004
Handbook of Proteolytic Enzymes, 2004
Advances in Experimental Medicine and Biology, 1991
All proteinases of avian and mammalian retroviruses belong to the family of aspartic proteinases,... more All proteinases of avian and mammalian retroviruses belong to the family of aspartic proteinases, are of similar size and of homologous primary structure; they all act catalytically in the form of highly symmetric molecular dimers.1 Detailed studies of retroviral proteinases were carried out on two almost identical proteinases of MAV2,3 and RS V4 (representing the group of avian retroviruses) and on the HIV proteinase.5,6 The knowledge of the 3D structure,2,4,5 catalytic activity and substrate specifity3,6 of the MAV and the HIV proteinase has changed the notion of their general similarity since several features that distinguish each proteinase from the other were revealed. The HIV-1 proteinase has a considerably higher activity3,6 which reflects the different conditions of the expression and action of this enzyme in vivo: 7 The “coding strategy” of MAV allows the expression of the proteinase from the first (gag) open reading frame and provides for the high (i.e. stoichiometrical) level of the relatively “weak” enzyme whereas the smaller amount of the more active HIV enzyme is a result of infrequent translational frameshift events that occur in the overlapping region of the gag and pol reading frames.8 The substrate specificities of retroviral proteinases seem complex and the requirement for a side chain in an individual subsite of a substrate is an outcome of the combination of residues occupying other closely located subsites.3 The two proteinases (MAV and HIV) show rather promiscuous substrate specificity, nevertheless several differences can be traced. We made an attempt to use protein engineering of the MAV proteinase to tackle directly problems of structural basis of these differences and, vice versa, to make more precise conclusions on the functional importance of the individual elements of its three dimensional structure. This article describes mutation of the MAV proteinase which resulted not only in an alteration of its substrate specificity but also in an increase of its enzymic activity — a rare case in protein engineering.
FEBS Letters, Aug 1, 1988
Journal of Biological Chemistry, Sep 1, 1993
Biochemistry, Oct 17, 1989
Proceedings of the National Academy of Sciences of the United States of America, Aug 1, 1995
Advances in Experimental Medicine and Biology, 1995
Biochemical Society Transactions, 1987
Acta Crystallographica Section A Foundations of Crystallography, 1984
Proceedings of the National Academy of Sciences, 1995
General transcription factor SIII, a heterotrimer composed of 110-kDa (p110), 18-kDa (p18), and 1... more General transcription factor SIII, a heterotrimer composed of 110-kDa (p110), 18-kDa (p18), and 15-kDa (p15) subunits, increases the catalytic rate of transcribing RNA polymerase II by suppressing transient pausing by polymerase at multiple sites on DNA templates. Here we report molecular cloning and biochemical characterization of the SIII p18 subunit, which is found to be a member of the ubiquitin homology (UbH) gene family and functions as a positive regulatory subunit of SIII. p18 is a 118-amino acid protein composed of an 84-residue N-terminal UbH domain fused to a 34-residue C-terminal tail. Mechanistic studies indicate that p18 activates SIII transcriptional activity above a basal level inherent in the SIII p110 and p15 subunits. Taken together, these findings establish a role for p18 in regulating the activity of the RNA polymerase II elongation complex, and they bring to light a function for a UbH domain protein in transcriptional regulation.
The low hydrolytic activity (k/sub cat/ < 0.001 sâ»Â¹) of chicken pepsin (CP) towards tri- an... more The low hydrolytic activity (k/sub cat/ < 0.001 sâ»Â¹) of chicken pepsin (CP) towards tri- and tetrapeptides is enhanced at least 100 times by modification of its single sulfhydryl group of Cys-115, with little effect on K/sub m/-values. Modification thus simulates the effect of secondary substrate binding on pepsin catalysis. The rate of Cys-115 modification is substantially decreased in the presence of some competitive inhibitors, suggesting its active site location. Experiments with CP alkylated at Cys-115 with Acrylodan as a fluorescent probe or with N-iodoacetyl-(4-fluoro)-aniline as a ¹â¹F-nmr probe suggest conformation change around Cys-115 to occur on substrate or substrate analog binding. The difference ¹H-nmr spectra (500 MHz) of unmodified free and inhibitor-complexed CP reveal chemical shifts almost exclusively in the aromatic region. The effects of Cu/sup + +/ on ¹â¹F- and ¹H-nmr spectra have been studied. Examination of a computer graphics model of CP based o...
Acta Crystallographica Section A Foundations of Crystallography, 2002
Molecular immunology, 1996
The 40 kDa secreted aspartyl proteinase (Saptl) of Candida tropicalis is a pepsin-like enzyme enc... more The 40 kDa secreted aspartyl proteinase (Saptl) of Candida tropicalis is a pepsin-like enzyme encoded by the SApTl gene. According to the deduced amino acid sequence, Saptl has a putative preproregion of 60 amino acids preceding the mature enzyme. Maturation and processing of Saptl was analysed in C. tropicalis and Sacchammyces cemvisiae strains expressing wildtype or mutated forms of SAPll. In s. cemvisiae, the glycosylated 46 kDa proenzyme was converted to the mature 40 kDa form of Saptl by K B 2 dependent proteolytic cleavage following the Lyssg-Argm sequence. The replacement of L y ~ ~ ~ A r g ~ by Lyss9-Gly60 revealed that the precursor can be processed by an autocatalytic cleavage. This alternative processing pathway to produce mature Saptl is less efficient than the Kex2-mediated pathway. Finally, it was shown that in C. fropicelis and S. cemvisiae the removal of the proregion was a prerequisite for the secretion of Saptl.
Handbook of Proteolytic Enzymes, 2013
Handbook of Proteolytic Enzymes, 2004
Handbook of Proteolytic Enzymes, 2004