The Experts below are selected from a list of 294 Experts worldwide ranked by ideXlab platform
Woo Jin Park - One of the best experts on this subject based on the ideXlab platform.
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Determination of the substrate specificity of turnip mosaic virus NIa protease using a genetic method.
Journal of General Virology, 2001Co-Authors: Hara Kang, Yong Jae Lee, Jae Hwan Goo, Woo Jin ParkAbstract:The RNA genome of turnip mosaic potyvirus (TuMV) encodes a large polyprotein that is processed to mature proteins by virus-encoded proteases. The TuMV NIa protease is responsible for the cleavage of the polyprotein at seven different locations. These cleavage sites are defined by a conserved sequence motif Val-Xaa-His-Gln↓, with the Scissile Bond located after Gln. To determine the substrate specificity of the NIa protease, amino acid sequences cleaved by the NIa protease were obtained from randomized sequence libraries using a screening method referred to as GASP (genetic assay for site-specific proteolysis). Based on statistical analysis of the obtained sequences, a consensus substrate sequence was deduced: Yaa-Val-Arg-His-Gln↓Ser, with Yaa being an aliphatic amino acid and the Scissile Bond being located between Gln and Ser. This result is consistent with the conserved cleavage sequence motif, and should provide insight into the molecular activity of the NIa protease.
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Determination of the substrate specificity of turnip mosaic virus NIa protease using a genetic method.
The Journal of general virology, 2001Co-Authors: Hara Kang, Yong Jae Lee, Jae Hwan Goo, Woo Jin ParkAbstract:The RNA genome of turnip mosaic potyvirus (TuMV) encodes a large polyprotein that is processed to mature proteins by virus-encoded proteases. The TuMV NIa protease is responsible for the cleavage of the polyprotein at seven different locations. These cleavage sites are defined by a conserved sequence motif Val-Xaa-His-Gln decreased, with the Scissile Bond located after Gln. To determine the substrate specificity of the NIa protease, amino acid sequences cleaved by the NIa protease were obtained from randomized sequence libraries using a screening method referred to as GASP (genetic assay for site-specific proteolysis). Based on statistical analysis of the obtained sequences, a consensus substrate sequence was deduced: Yaa-Val-Arg-His-Gln decreased Ser, with Yaa being an aliphatic amino acid and the Scissile Bond being located between Gln and Ser. This result is consistent with the conserved cleavage sequence motif, and should provide insight into the molecular activity of the NIa protease.
Thomas Binz - One of the best experts on this subject based on the ideXlab platform.
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substrate recognition mechanism of vamp synaptobrevin cleaving clostridial neurotoxins
Journal of Biological Chemistry, 2008Co-Authors: Stefan Sikorra, Tina Henke, Thierry Galli, Thomas BinzAbstract:Botulinum neurotoxins (BoNTs) and tetanus neurotoxin (TeNT) inhibit neurotransmitter release by proteolyzing a single peptide Bond in one of the three soluble N-ethylmaleimide-sensitive factor attachment protein receptors SNAP-25, syntaxin, and vesicle-associated membrane protein (VAMP)/synaptobrevin. TeNT and BoNT/B, D, F, and G of the seven known BoNTs cleave the synaptic vesicle protein VAMP/synaptobrevin. Except for BoNT/B and TeNT, they cleave unique peptide Bonds, and prior work suggested that different substrate segments are required for the interaction of each toxin. Although the mode of SNAP-25 cleavage by BoNT/A and E has recently been studied in detail, the mechanism of VAMP/synaptobrevin proteolysis is fragmentary. Here, we report the determination of all substrate residues that are involved in the interaction with BoNT/B, D, and F and TeNT by means of systematic mutagenesis of VAMP/synaptobrevin. For each of the toxins, three or more residues clustered at an N-terminal site remote from the respective Scissile Bond are identified that affect solely substrate binding. These exosites exhibit different sizes and distances to the Scissile peptide Bonds for each neurotoxin. Substrate segments C-terminal of the cleavage site (P4-P4′) do not play a role in the catalytic process. Mutation of residues in the proximity of the Scissile Bond exclusively affects the turnover number; however, the importance of individual positions at the cleavage sites varied for each toxin. The data show that, similar to the SNAP-25 proteolyzing BoNT/A and E, VAMP/synaptobrevin-specific clostridial neurotoxins also initiate substrate interaction, employing an exosite located N-terminal of the Scissile peptide Bond.
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Substrate recognition mechanism of VAMP/synaptobrevin-cleaving clostridial neurotoxins.
The Journal of biological chemistry, 2008Co-Authors: Stefan Sikorra, Tina Henke, Thierry Galli, Thomas BinzAbstract:Botulinum neurotoxins (BoNTs) and tetanus neurotoxin (TeNT) inhibit neurotransmitter release by proteolyzing a single peptide Bond in one of the three soluble N-ethylmaleimide-sensitive factor attachment protein receptors SNAP-25, syntaxin, and vesicle-associated membrane protein (VAMP)/synaptobrevin. TeNT and BoNT/B, D, F, and G of the seven known BoNTs cleave the synaptic vesicle protein VAMP/synaptobrevin. Except for BoNT/B and TeNT, they cleave unique peptide Bonds, and prior work suggested that different substrate segments are required for the interaction of each toxin. Although the mode of SNAP-25 cleavage by BoNT/A and E has recently been studied in detail, the mechanism of VAMP/synaptobrevin proteolysis is fragmentary. Here, we report the determination of all substrate residues that are involved in the interaction with BoNT/B, D, and F and TeNT by means of systematic mutagenesis of VAMP/synaptobrevin. For each of the toxins, three or more residues clustered at an N-terminal site remote from the respective Scissile Bond are identified that affect solely substrate binding. These exosites exhibit different sizes and distances to the Scissile peptide Bonds for each neurotoxin. Substrate segments C-terminal of the cleavage site (P4-P4') do not play a role in the catalytic process. Mutation of residues in the proximity of the Scissile Bond exclusively affects the turnover number; however, the importance of individual positions at the cleavage sites varied for each toxin. The data show that, similar to the SNAP-25 proteolyzing BoNT/A and E, VAMP/synaptobrevin-specific clostridial neurotoxins also initiate substrate interaction, employing an exosite located N-terminal of the Scissile peptide Bond.
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Substrate recognition mechanism of VAMP/synaptobrevin cleaving clostridial neurotoxins.
Journal of Biological Chemistry, 2008Co-Authors: Stefan Sikorra, Tina Henke, Thierry Galli, Thomas BinzAbstract:Botulinum neurotoxins (BoNTs) and tetanus neurotoxin (TeNT) inhibit neurotransmitter release by proteolyzing a single peptide Bond in one of the three soluble N-ethylmaleimide sensitive factor attachment protein receptors SNAP-25, syntaxin, and vesicle associated membrane protein (VAMP)/synaptobrevin. TeNT and BoNT/B, D, F and G of the seven known BoNTs cleave the synaptic vesicle protein VAMP/synaptobrevin. Except for BoNT/B and TeNT they cleave unique peptide Bonds and prior work suggested that different substrate segments are required for the interaction of each toxin. Whereas the mode of SNAP-25 cleavage by BoNT/A and E has recently been studied in detail, the mechanism of VAMP/synaptobrevin proteolysis is fragmentary. Here, we report the determination of all substrate residues that are involved in the interaction with BoNT/B, D, F, and TeNT by means of systematic mutagenesis of VAMP/synaptobrevin. For each of the toxins three or more residues clustered at an N terminal site remote to the respective Scissile Bond are identified that affect solely substrate binding. These exosites exhibit different sizes and distances to the Scissile peptide Bonds for each neurotoxin. Substrate segments C-terminal of the cleavage site (P4-P4') do not play a role in the catalytic process. Mutation of residues in the proximity of the Scissile Bond exclusively affects the turnover number, however, the importance of individual positions at the cleavage sites varied for each toxin. The data evidence that similar to the SNAP-25 proteolyzing BoNT/A and E, VAMP/synaptobrevin specific CNTs also initiate substrate interaction employing an exosite located N terminal of the Scissile peptide Bond.
David A. Lane - One of the best experts on this subject based on the ideXlab platform.
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A common mechanism by which type 2A von Willebrand disease mutations enhance ADAMTS13 proteolysis revealed with a von Willebrand factor A2 domain FRET construct.
PloS one, 2017Co-Authors: Christopher J. Lynch, Adam D. Cawte, Carolyn M. Millar, David Rueda, David A. LaneAbstract:Rheological forces in the blood trigger the unfolding of von Willebrand factor (VWF) and its A2 domain, exposing the Scissile Bond for proteolysis by ADAMTS13. Under quiescent conditions, the Scissile Bond is hidden by the folded structure due to the stabilisation provided by the structural specialisations of the VWF A2 domain, a vicinal disulphide Bond, a calcium binding site and a N1574-glycan.The reduced circulating high MW multimers of VWF in patients with type 2A von Willebrand disease (VWD) may be associated with mutations within the VWF A2 domain and this is attributed to enhanced ADAMTS13 proteolysis. We investigated 11 VWF A2 domain variants identified in patients with type 2A VWD. In recombinant full-length VWF, enhanced ADAMTS13 proteolysis was detected for all of the expressed variants in the presence of urea-induced denaturation. A subset of the FLVWF variants displayed enhanced proteolysis in the absence of urea. The mechanism of enhancement was investigated using a novel VWF A2 domain FRET construct. In the absence of induced unfolding, 7/8 of the expressed mutants exhibited a disrupted domain fold, causing spatial separation of the N- and C- termini. Three of the type 2A mutants were not secreted when studied within the VWF A2 domain FRET construct. Urea denaturation revealed for all 8 secreted mutants reduced unfolding cooperativity and stability of the VWF A2 domain. As folding stability was progressively disrupted, proteolysis by ADAMTS13 increased. Due to the range of folding stabilities and wide distribution of VWF A2 domain mutations studied, we conclude that these mutations disrupt regulated folding of the VWF A2 domain. They enhance unfolding by inducing separation of N- and C-termini, thereby promoting a more open conformation that reveals its binding sites for ADAMTS13 and the Scissile Bond.
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N-linked glycan stabilization of the VWF A2 domain
Blood, 2016Co-Authors: Christopher J. Lynch, David A. LaneAbstract:Shear forces in the blood trigger a conformational transition in the VWF A2 domain, from its native folded to an unfolded state, in which the cryptic Scissile Bond (Y1505-M1606) is exposed and can then be proteolysed by ADAMTS13. The conformational transition depends upon a Ca 2+ binding site and a vicinal cysteine disulphide Bond. Glycosylation at N1574 has previously been suggested to modulate VWF A2 domain interaction with ADAMTS13 through steric hindrance by the bulky carbohydrate structure. We investigated how the N-linked glycans of the VWF A2 domain affect thermostability and regulate both the exposure of the ADAMTS13 binding sites and the Scissile Bond. We show by differential scanning fluorimetry that the N-linked glycans thermodynamically stabilise the VWF A2 domain. The essential component of the glycan structure is the first sugar residue (GlcNAc) at the N1574 attachment site. From its crystal structures, N1574-GlcNAc is predicted to form stabilising intradomain interactions with Y1544 and nearby residues. Substitution of the surface exposed Y1544 to aspartic acid is able to stabilise the domain in the absence of glycosylation and to protect against ADAMTS13 proteolysis in both the VWF A2 domain and FLVWF. Glycan stabilisation of the VWF A2 domain acts together with the Ca 2+ binding site and vicinal cysteine disulphide Bond to control unfolding and ADAMTS13 proteolysis.
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Control of VWF A2 domain stability and ADAMTS13 access to the Scissile Bond of full length VWF
Blood, 2014Co-Authors: Christopher J. Lynch, David A. Lane, Brenda M. LukenAbstract:Rheological shear forces in the blood trigger von Willebrand factor (VWF) unfolding which exposes the Y1605-M1606 Scissile Bond within the VWF A2 domain for cleavage by ADAMTS13. The VWF A2 domain contains 2 structural features that provide it with stability: a vicinal disulphide Bond and a Ca2+-binding site (CBS). We investigated how these 2 structural features interplay to determine stability and regulate the exposure of the Scissile Bond in full-length VWF. We have used differential scanning fluorimetry together with site-directed mutagenesis of residues involved in both the vicinal disulphide Bond and the CBS to demonstrate that both of these sites contribute to stability against thermal unfolding of the isolated VWF A2 domain. Moreover, we show that the combination of site mutations can result in increased susceptibility of FL-VWF to proteolysis by ADAMTS13, even in the absence of an agent (such as urea) required to induce unfolding. These studies demonstrate that VWF A2 domain stability provided by its 2 structural elements (vicinal disulphide Bond and CBS) is a key protective determinant against FL-VWF cleavage by ADAMTS13. They suggest a 2-step mechanism for VWF A2 domain unfolding.
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unraveling the Scissile Bond how adamts13 recognizes and cleaves von willebrand factor
Blood, 2011Co-Authors: James T B Crawley, Brenda M. Luken, Rens De Groot, Yaozu Xiang, David A. LaneAbstract:von Willebrand factor (VWF) is a large adhesive glycoprotein with established functions in hemostasis. It serves as a carrier for factor VIII and acts as a vascular damage sensor by attracting platelets to sites of vessel injury. VWF size is important for this latter function, with larger multimers being more hemostatically active. Functional imbalance in multimer size can variously cause microvascular thrombosis or bleeding. The regulation of VWF multimeric size and platelet-tethering function is carried out by ADAMTS13, a plasma metalloprotease that is constitutively active. Unusually, protease activity of ADAMTS13 is controlled not by natural inhibitors but by conformational changes in its substrate, which are induced when VWF is subject to elevated rheologic shear forces. This transforms VWF from a globular to an elongated protein. This conformational transformation unfolds the VWF A2 domain and reveals cryptic exosites as well as the Scissile Bond. To enable VWF proteolysis, ADAMTS13 makes multiple interactions that bring the protease to the substrate and position it to engage with the cleavage site as this becomes exposed by shear. This article reviews recent literature on the interaction between these 2 multidomain proteins and provides a summary model to explain proteolytic regulation of VWF by ADAMTS13.
Hara Kang - One of the best experts on this subject based on the ideXlab platform.
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Determination of the substrate specificity of turnip mosaic virus NIa protease using a genetic method.
Journal of General Virology, 2001Co-Authors: Hara Kang, Yong Jae Lee, Jae Hwan Goo, Woo Jin ParkAbstract:The RNA genome of turnip mosaic potyvirus (TuMV) encodes a large polyprotein that is processed to mature proteins by virus-encoded proteases. The TuMV NIa protease is responsible for the cleavage of the polyprotein at seven different locations. These cleavage sites are defined by a conserved sequence motif Val-Xaa-His-Gln↓, with the Scissile Bond located after Gln. To determine the substrate specificity of the NIa protease, amino acid sequences cleaved by the NIa protease were obtained from randomized sequence libraries using a screening method referred to as GASP (genetic assay for site-specific proteolysis). Based on statistical analysis of the obtained sequences, a consensus substrate sequence was deduced: Yaa-Val-Arg-His-Gln↓Ser, with Yaa being an aliphatic amino acid and the Scissile Bond being located between Gln and Ser. This result is consistent with the conserved cleavage sequence motif, and should provide insight into the molecular activity of the NIa protease.
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Determination of the substrate specificity of turnip mosaic virus NIa protease using a genetic method.
The Journal of general virology, 2001Co-Authors: Hara Kang, Yong Jae Lee, Jae Hwan Goo, Woo Jin ParkAbstract:The RNA genome of turnip mosaic potyvirus (TuMV) encodes a large polyprotein that is processed to mature proteins by virus-encoded proteases. The TuMV NIa protease is responsible for the cleavage of the polyprotein at seven different locations. These cleavage sites are defined by a conserved sequence motif Val-Xaa-His-Gln decreased, with the Scissile Bond located after Gln. To determine the substrate specificity of the NIa protease, amino acid sequences cleaved by the NIa protease were obtained from randomized sequence libraries using a screening method referred to as GASP (genetic assay for site-specific proteolysis). Based on statistical analysis of the obtained sequences, a consensus substrate sequence was deduced: Yaa-Val-Arg-His-Gln decreased Ser, with Yaa being an aliphatic amino acid and the Scissile Bond being located between Gln and Ser. This result is consistent with the conserved cleavage sequence motif, and should provide insight into the molecular activity of the NIa protease.
Neil Osheroff - One of the best experts on this subject based on the ideXlab platform.
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Coordinating the Two Protomer Active Sites of Human Topoisomerase IIα: Nicks as Topoisomerase II Poisons†
Biochemistry, 2009Co-Authors: Joseph E. Deweese, Neil OsheroffAbstract:Topoisomerase II modulates DNA topology by generating double-stranded breaks in DNA. Results of the current study indicate that the presence of a nick at one Scissile Bond dramatically increases the rate of cleavage by human topoisomerase IIα at the Scissile Bond on the opposite strand. We propose that this enhanced activity at the second strand coordinates the two protomer subunits of topoisomerase II and allows the enzyme to create double-stranded breaks. Finally, the presence of a nick on one strand induces cleavage on the opposite strand. Thus, nicks are topoisomerase II poisons that generate novel sites of DNA cleavage.
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using 3 bridging phosphorothiolates to isolate the forward dna cleavage reaction of human topoisomerase iiα
Biochemistry, 2008Co-Authors: Joseph E. Deweese, Alex B Burgin, Neil OsheroffAbstract:The ability to cleave DNA is critical to the cellular and pharmacological functions of human type II topoisomerases. However, the low level of cleavage at equilibrium and the tight coupling of the cleavage and ligation reactions make it difficult to characterize the mechanism by which these enzymes cut DNA. Therefore, to establish a system that isolates topoisomerase II-mediated DNA scission from ligation, oligonucleotide substrates were developed that contained a 3'-bridging phosphorothiolate at the Scissile Bond. Scission of these substrates generates a 3'-terminal -SH moiety that is a poor nucleophile relative to the normal 3'-terminal -OH group. Consequently, topoisomerase II cannot efficiently ligate phosphorothiolate substrates once they are cleaved. The characteristics of topoisomerase IIalpha-mediated cleavage of phosphorothiolate oligonucleotides were identical to those seen with wild-type substrates, except that no ligation was observed. This unidirectional accumulation of cleavage complexes provided critical information regarding coordination of the protomer subunits of topoisomerase IIalpha and the mechanism of action of topoisomerase II poisons. Results indicate that the two enzyme subunits are partially coordinated and that cleavage at one Scissile Bond increases the degree of cleavage at the other. Furthermore, anticancer drugs such as etoposide and amsacrine that strongly inhibit topoisomerase II-mediated DNA ligation have little effect on the forward scission reaction. In contrast, abasic sites that increase levels of cleavage complexes without affecting ligation stimulate the forward rate of scission. Phosphorothiolate substrates provide significant advantages over traditional "suicide substrates" and should be valuable for future studies on DNA scission and the topoisomerase II-DNA cleavage complex.
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Quinolone Action against Human Topoisomerase IIα: Stimulation of Enzyme-Mediated Double-Stranded DNA Cleavage†
Biochemistry, 2003Co-Authors: Kenneth D. Bromberg, And Alex B. Burgin, Neil OsheroffAbstract:Several important antineoplastic drugs kill cells by increasing levels of topoisomerase II-mediated DNA breaks. These compounds act by two distinct mechanisms. Agents such as etoposide inhibit the ability of topoisomerase II to ligate enzyme-linked DNA breaks. Conversely, compounds such as quinolones have little effect on ligation and are believed to stimulate the forward rate of topoisomerase II-mediated DNA cleavage. The fact that there are two Scissile Bonds per double-stranded DNA break implies that there are two sites for drug action in every enzyme-DNA cleavage complex. However, since agents in the latter group are believed to act by locally perturbing DNA structure, it is possible that quinolone interactions at a single Scissile Bond are sufficient to distort both strands of the double helix and generate an enzyme-mediated double-stranded DNA break. Therefore, an oligonucleotide system was established to further define the actions of topoisomerase II-targeted drugs that stimulate the forward rate of DNA cleavage. Results indicate that the presence of the quinolone CP-115,953 at one Scissile Bond increased the extent of enzyme-mediated scission at the opposite Scissile Bond and was sufficient to stimulate the formation of a double-stranded DNA break by human topoisomerase IIalpha. These findings stand in marked contrast to those for etoposide, which must be present at both Scissile Bonds to stabilize a double-stranded DNA break [Bromberg, K. D., et al. (2003) J. Biol. Chem. 278, 7406-7412]. Moreover, they underscore important mechanistic differences between drugs that enhance DNA cleavage and those that inhibit ligation.
