The Experts below are selected from a list of 1545 Experts worldwide ranked by ideXlab platform
Jeremy C. Mottram - One of the best experts on this subject based on the ideXlab platform.
-
Substrate specificity and the effect of calcium on Trypanosoma brucei Metacaspase 2.
The FEBS journal, 2013Co-Authors: Maurício F.m. Machado, Jeremy C. Mottram, Marcelo F. Marcondes, Maria A. Juliano, Karen Mcluskey, Catherine X. Moss, Luiz Juliano, Vitor OliveiraAbstract:Metacaspases are cysteine peptidases that are distantly related to the mammalian caspases but found only in plants, fungi and protozoa [1]. Together with the caspases and paracaspases [1], they are endopeptidases which have been grouped into structural Family C14 in Clan CD of the MEROPS peptidase database [2]. In Trypanosoma brucei, five Metacaspase genes (TbMCA1-5) have been reported with three, TbMCA2, TbMCA3 and TbMCA5, predicted to code for active peptidases based on the conservation of an intact cysteine-histidine catalytic dyad [3, 4]. Interestingly, in TbMCA1 and TbMCA4 the catalytic cysteine is replaced by a serine, indicating that the coded proteins would not exhibit cysteine peptidase activity [3, 5]. Whilst plant Metacaspases have been shown to be involved in cell death pathways, trypanosomes appear to lack regulated cell death [6] and in these organisms Metacaspases have evolved alternative functions [5, 7]. Despite being related to the caspases, Metacaspases have been shown to be both structurally and functionally distinct. Metacaspases are known to cleave their substrates after Arg and Lys residues in the P1 position [8], as demonstrated for Metacaspases from T. brucei [9], T. cruzi [10], Leishmania major [11], plants [12-14] and yeast [13], while the caspases cleave their substrates after Asp [15]. Other functional differences between the two families of enzymes are that unlike the caspases, Metacaspases do not necessarily require processing or dimerization for activity and they are activated by calcium [9, 12-14, 16]. In addition, the recent crystallographic structures of TbMCA2 and the Saccharomyces cerevisiae Metacaspase (YCA1) [17, 18] provided a structural basis for comparing caspases and Metacaspases. The Metacaspase structures revealed a monomeric enzyme containing a core caspase/hemoglobinase fold [19] with an eight-stranded β-sheet, consisting of six parallel and two anti-parallel strands. This is unlike the caspases, which comprise a six-stranded β-sheet and a stable dimerisation interface, which forms the active enzyme. In addition, the recent structure of a paracaspase [20] shows that it is structurally very similar to the caspases and dimerizes in a similar manner. Conversely, the two extra strands in the β-sheet of the Metacaspases prevent such dimerisation, explaining its monomeric peptidase activity and distinguishing the Metacaspases structurally from other caspase-family members. The high-resolution crystal structure of TbMCA2 was determined using inactive mutants (TbMCA2C213A/G), as autoprocessing of the full-length recombinant enzyme in vitro was unfavourable in producing diffraction quality crystals [17, 18]. This revealed an unusual N-terminal region, which encircled the protein and crossed over the active site, blocking the S1 pocket. Consequently, the structure of TbMCA2 does not contain an active site substrate and the properties of its extended binding site have not yet been established. In this study, we undertook to examine the extended substrate specificity of recombinant and active TbMCA2 using a series of fluorescence resonance energy transfer (FRET) peptides. The specificity of TbMCA2 subsites S3 to S3′ were analysed by kinetic assays that monitored the hydrolysis of a FRET peptide series, where positions P3 to P3′ on the peptides are substituted by each of the natural amino acids. In addition, the effects of Ca2+ on the structure, activity and extended subsite specificity of TbMCA2 were investigated using a series of kinetic assays, intrinsic fluorescence analysis and circular dichroism (CD) spectroscopy.
-
crystal structure of a trypanosoma brucei Metacaspase
Proceedings of the National Academy of Sciences of the United States of America, 2012Co-Authors: Karen Mcluskey, Catherine X. Moss, Jana Rudolf, William R Proto, Neil W Isaacs, Graham H Coombs, Jeremy C. MottramAbstract:Metacaspases are distantly related caspase-family cysteine peptidases implicated in programmed cell death in plants and lower eukaryotes. They differ significantly from caspases because they are calcium-activated, arginine-specific peptidases that do not require processing or dimerization for activity. To elucidate the basis of these differences and to determine the impact they might have on the control of cell death pathways in lower eukaryotes, the previously undescribed crystal structure of a Metacaspase, an inactive mutant of Metacaspase 2 (MCA2) from Trypanosoma brucei, has been determined to a resolution of 1.4 A. The structure comprises a core caspase fold, but with an unusual eight-stranded β-sheet that stabilizes the protein as a monomer. Essential aspartic acid residues, in the predicted S1 binding pocket, delineate the arginine-specific substrate specificity. In addition, MCA2 possesses an unusual N terminus, which encircles the protein and traverses the catalytic dyad, with Y31 acting as a gatekeeper residue. The calcium-binding site is defined by samarium coordinated by four aspartic acid residues, whereas calcium binding itself induces an allosteric conformational change that could stabilize the active site in a fashion analogous to subunit processing in caspases. Collectively, these data give insights into the mechanistic basis of substrate specificity and mode of activation of MCA2 and provide a detailed framework for understanding the role of Metacaspases in cell death pathways of lower eukaryotes.
-
Trypanosoma brucei Metacaspase 4 is a pseudopeptidase and a virulence factor
The Journal of biological chemistry, 2011Co-Authors: William R Proto, Catherine X. Moss, Luiz Juliano, Graham H Coombs, Esther Castanys-munoz, Alana Black, Laurence Tetley, Jeremy C. MottramAbstract:Metacaspases are caspase family cysteine peptidases found in plants, fungi, and protozoa but not mammals. Trypanosoma brucei is unusual in having five Metacaspases (MCA1–MCA5), of which MCA1 and MCA4 have active site substitutions, making them possible non-enzymatic homologues. Here we demonstrate that recombinant MCA4 lacks detectable peptidase activity despite maintaining a functional peptidase structure. MCA4 is expressed primarily in the bloodstream form of the parasite and associates with the flagellar membrane via dual myristoylation/palmitoylation. Loss of function phenotyping revealed critical roles for MCA4; rapid depletion by RNAi caused lethal disruption to the parasite's cell cycle, yet the generation of MCA4 null mutant parasites (Δmca4) was possible. Δmca4 had normal growth in axenic culture but markedly reduced virulence in mice. Further analysis revealed that MCA4 is released from the parasite and is specifically processed by MCA3, the only Metacaspase that is both palmitoylated and enzymatically active. Accordingly, we have identified that the multiple Metacaspases in T. brucei form a membrane-associated proteolytic cascade to generate a pseudopeptidase virulence factor.
-
Metacaspase 2 of trypanosoma brucei is a calcium dependent cysteine peptidase active without processing
FEBS Letters, 2007Co-Authors: Catherine X. Moss, Luiz Juliano, Graham H Coombs, Gareth D. Westrop, Jeremy C. MottramAbstract:Metacaspases are cysteine peptidases that are distantly related to the caspases, for which proteolytic processing is central to their activation. Here, we show that recombinant Metacaspase 2 (MCA2) from Trypanosoma brucei has arginine/lysine-specific, Ca2+-dependent proteolytic activity. Autocatalytic processing of MCA2 occurred after Lys55 and Lys268; however, this was shown not to be required for the enzyme to be proteolytically active. The necessity of Ca2+, but not processing, for MCA2 enzymatic activity clearly distinguishes MCA2 from the caspases and would be consistent with different physiological roles.
-
Metacaspase 2 of Trypanosoma brucei is a calcium‐dependent cysteine peptidase active without processing
FEBS letters, 2007Co-Authors: Catherine X. Moss, Luiz Juliano, Graham H Coombs, Gareth D. Westrop, Jeremy C. MottramAbstract:Metacaspases are cysteine peptidases that are distantly related to the caspases, for which proteolytic processing is central to their activation. Here, we show that recombinant Metacaspase 2 (MCA2) from Trypanosoma brucei has arginine/lysine-specific, Ca2+-dependent proteolytic activity. Autocatalytic processing of MCA2 occurred after Lys55 and Lys268; however, this was shown not to be required for the enzyme to be proteolytically active. The necessity of Ca2+, but not processing, for MCA2 enzymatic activity clearly distinguishes MCA2 from the caspases and would be consistent with different physiological roles.
Maurício F.m. Machado - One of the best experts on this subject based on the ideXlab platform.
-
processing of Metacaspase 2 from trypanosoma brucei tbmca2 broadens its substrate specificity
Biochimica et Biophysica Acta, 2017Co-Authors: Joyce Meire Gilio, Maurício F.m. Machado, Marcelo F. Marcondes, Maria A. Juliano, Luiz Juliano, Vitor Oliveira, Debora FerrariAbstract:Metacaspases are members of the cysteine peptidase family and may be implicated in programmed cell death in plants and lower eukaryotes. These proteases exhibit calcium-dependent activity and specificity for arginine residues at P1. In contrast to caspases, they do not require processing or dimerization for activity. Indeed, unprocessed Metacaspase-2 of Trypanosoma brucei (TbMCA2) is active; however, it has been shown that cleavages at Lys55 and Lys268 increase TbMCA2 hydrolytic activity on synthetic substrates. The processed TbMCA2 comprises 3 polypeptide chains that remain attached by non-covalent bonds. Replacement of Lys55 and Lys268 with Gly via site-directed mutagenesis results in non-processed but enzymatically active mutant, TbMCA2 K55/268G. To investigate the importance of this processing for the activity and specificity of TbMCA2, we performed activity assays comparing the non-processed mutant (TbMCA2 K55/268G) with the processed TbMCA2 form. Significant differences between TbMCA2 WT (processed form) and TbMCA2 K55/268G (non-processed form) were observed. Specifically, we verified that although non-processed TbMCA2 is active when assayed with small synthetic substrates, the TbMCA2 form does not exhibit hydrolytic activity on large substrates such as azocasein, while processed TbMCA2 is able to readily digest this protein. Such differences can be relevant for understanding the physiological regulation and function of TbMCA2.
-
processing of Metacaspase 2 from trypanosoma brucei tbmca2 broadens its substrate specificity proteins and proteomics
Biochimica et Biophysica Acta, 2017Co-Authors: Joyce Meire Gilio, Marcelo F. Marcondes, Maria A. Juliano, Luiz Juliano, Vitor Oliveira, Debora Ferrari, Maurício F.m. MachadoAbstract:Metacaspases are members of the cysteine peptidase family and may be implicated in programmed cell death in plants and lower eukaryotes. These proteases exhibit calcium-dependent activity and specificity for arginine residues at P1. In contrast to caspases, they do not require processing or dimerization for activity. Indeed, unprocessed Metacaspase-2 of Trypanosoma brucei (TbMCA2) is active; however, it has been shown that cleavages at Lys⁵⁵ and Lys²⁶⁸ increase TbMCA2 hydrolytic activity on synthetic substrates. The processed TbMCA2 comprises 3 polypeptide chains that remain attached by non-covalent bonds. Replacement of Lys⁵⁵ and Lys²⁶⁸ with Gly via site-directed mutagenesis results in non-processed but enzymatically active mutant, TbMCA2 K55/268G. To investigate the importance of this processing for the activity and specificity of TbMCA2, we performed activity assays comparing the non-processed mutant (TbMCA2 K55/268G) with the processed TbMCA2 form. Significant differences between TbMCA2 WT (processed form) and TbMCA2 K55/268G (non-processed form) were observed. Specifically, we verified that although non-processed TbMCA2 is active when assayed with small synthetic substrates, the TbMCA2 form does not exhibit hydrolytic activity on large substrates such as azocasein, while processed TbMCA2 is able to readily digest this protein. Such differences can be relevant for understanding the physiological regulation and function of TbMCA2.
-
Substrate specificity and the effect of calcium on Trypanosoma brucei Metacaspase 2.
The FEBS journal, 2013Co-Authors: Maurício F.m. Machado, Jeremy C. Mottram, Marcelo F. Marcondes, Maria A. Juliano, Karen Mcluskey, Catherine X. Moss, Luiz Juliano, Vitor OliveiraAbstract:Metacaspases are cysteine peptidases that are distantly related to the mammalian caspases but found only in plants, fungi and protozoa [1]. Together with the caspases and paracaspases [1], they are endopeptidases which have been grouped into structural Family C14 in Clan CD of the MEROPS peptidase database [2]. In Trypanosoma brucei, five Metacaspase genes (TbMCA1-5) have been reported with three, TbMCA2, TbMCA3 and TbMCA5, predicted to code for active peptidases based on the conservation of an intact cysteine-histidine catalytic dyad [3, 4]. Interestingly, in TbMCA1 and TbMCA4 the catalytic cysteine is replaced by a serine, indicating that the coded proteins would not exhibit cysteine peptidase activity [3, 5]. Whilst plant Metacaspases have been shown to be involved in cell death pathways, trypanosomes appear to lack regulated cell death [6] and in these organisms Metacaspases have evolved alternative functions [5, 7]. Despite being related to the caspases, Metacaspases have been shown to be both structurally and functionally distinct. Metacaspases are known to cleave their substrates after Arg and Lys residues in the P1 position [8], as demonstrated for Metacaspases from T. brucei [9], T. cruzi [10], Leishmania major [11], plants [12-14] and yeast [13], while the caspases cleave their substrates after Asp [15]. Other functional differences between the two families of enzymes are that unlike the caspases, Metacaspases do not necessarily require processing or dimerization for activity and they are activated by calcium [9, 12-14, 16]. In addition, the recent crystallographic structures of TbMCA2 and the Saccharomyces cerevisiae Metacaspase (YCA1) [17, 18] provided a structural basis for comparing caspases and Metacaspases. The Metacaspase structures revealed a monomeric enzyme containing a core caspase/hemoglobinase fold [19] with an eight-stranded β-sheet, consisting of six parallel and two anti-parallel strands. This is unlike the caspases, which comprise a six-stranded β-sheet and a stable dimerisation interface, which forms the active enzyme. In addition, the recent structure of a paracaspase [20] shows that it is structurally very similar to the caspases and dimerizes in a similar manner. Conversely, the two extra strands in the β-sheet of the Metacaspases prevent such dimerisation, explaining its monomeric peptidase activity and distinguishing the Metacaspases structurally from other caspase-family members. The high-resolution crystal structure of TbMCA2 was determined using inactive mutants (TbMCA2C213A/G), as autoprocessing of the full-length recombinant enzyme in vitro was unfavourable in producing diffraction quality crystals [17, 18]. This revealed an unusual N-terminal region, which encircled the protein and crossed over the active site, blocking the S1 pocket. Consequently, the structure of TbMCA2 does not contain an active site substrate and the properties of its extended binding site have not yet been established. In this study, we undertook to examine the extended substrate specificity of recombinant and active TbMCA2 using a series of fluorescence resonance energy transfer (FRET) peptides. The specificity of TbMCA2 subsites S3 to S3′ were analysed by kinetic assays that monitored the hydrolysis of a FRET peptide series, where positions P3 to P3′ on the peptides are substituted by each of the natural amino acids. In addition, the effects of Ca2+ on the structure, activity and extended subsite specificity of TbMCA2 were investigated using a series of kinetic assays, intrinsic fluorescence analysis and circular dichroism (CD) spectroscopy.
Luiz Juliano - One of the best experts on this subject based on the ideXlab platform.
-
processing of Metacaspase 2 from trypanosoma brucei tbmca2 broadens its substrate specificity
Biochimica et Biophysica Acta, 2017Co-Authors: Joyce Meire Gilio, Maurício F.m. Machado, Marcelo F. Marcondes, Maria A. Juliano, Luiz Juliano, Vitor Oliveira, Debora FerrariAbstract:Metacaspases are members of the cysteine peptidase family and may be implicated in programmed cell death in plants and lower eukaryotes. These proteases exhibit calcium-dependent activity and specificity for arginine residues at P1. In contrast to caspases, they do not require processing or dimerization for activity. Indeed, unprocessed Metacaspase-2 of Trypanosoma brucei (TbMCA2) is active; however, it has been shown that cleavages at Lys55 and Lys268 increase TbMCA2 hydrolytic activity on synthetic substrates. The processed TbMCA2 comprises 3 polypeptide chains that remain attached by non-covalent bonds. Replacement of Lys55 and Lys268 with Gly via site-directed mutagenesis results in non-processed but enzymatically active mutant, TbMCA2 K55/268G. To investigate the importance of this processing for the activity and specificity of TbMCA2, we performed activity assays comparing the non-processed mutant (TbMCA2 K55/268G) with the processed TbMCA2 form. Significant differences between TbMCA2 WT (processed form) and TbMCA2 K55/268G (non-processed form) were observed. Specifically, we verified that although non-processed TbMCA2 is active when assayed with small synthetic substrates, the TbMCA2 form does not exhibit hydrolytic activity on large substrates such as azocasein, while processed TbMCA2 is able to readily digest this protein. Such differences can be relevant for understanding the physiological regulation and function of TbMCA2.
-
processing of Metacaspase 2 from trypanosoma brucei tbmca2 broadens its substrate specificity proteins and proteomics
Biochimica et Biophysica Acta, 2017Co-Authors: Joyce Meire Gilio, Marcelo F. Marcondes, Maria A. Juliano, Luiz Juliano, Vitor Oliveira, Debora Ferrari, Maurício F.m. MachadoAbstract:Metacaspases are members of the cysteine peptidase family and may be implicated in programmed cell death in plants and lower eukaryotes. These proteases exhibit calcium-dependent activity and specificity for arginine residues at P1. In contrast to caspases, they do not require processing or dimerization for activity. Indeed, unprocessed Metacaspase-2 of Trypanosoma brucei (TbMCA2) is active; however, it has been shown that cleavages at Lys⁵⁵ and Lys²⁶⁸ increase TbMCA2 hydrolytic activity on synthetic substrates. The processed TbMCA2 comprises 3 polypeptide chains that remain attached by non-covalent bonds. Replacement of Lys⁵⁵ and Lys²⁶⁸ with Gly via site-directed mutagenesis results in non-processed but enzymatically active mutant, TbMCA2 K55/268G. To investigate the importance of this processing for the activity and specificity of TbMCA2, we performed activity assays comparing the non-processed mutant (TbMCA2 K55/268G) with the processed TbMCA2 form. Significant differences between TbMCA2 WT (processed form) and TbMCA2 K55/268G (non-processed form) were observed. Specifically, we verified that although non-processed TbMCA2 is active when assayed with small synthetic substrates, the TbMCA2 form does not exhibit hydrolytic activity on large substrates such as azocasein, while processed TbMCA2 is able to readily digest this protein. Such differences can be relevant for understanding the physiological regulation and function of TbMCA2.
-
Substrate specificity and the effect of calcium on Trypanosoma brucei Metacaspase 2.
The FEBS journal, 2013Co-Authors: Maurício F.m. Machado, Jeremy C. Mottram, Marcelo F. Marcondes, Maria A. Juliano, Karen Mcluskey, Catherine X. Moss, Luiz Juliano, Vitor OliveiraAbstract:Metacaspases are cysteine peptidases that are distantly related to the mammalian caspases but found only in plants, fungi and protozoa [1]. Together with the caspases and paracaspases [1], they are endopeptidases which have been grouped into structural Family C14 in Clan CD of the MEROPS peptidase database [2]. In Trypanosoma brucei, five Metacaspase genes (TbMCA1-5) have been reported with three, TbMCA2, TbMCA3 and TbMCA5, predicted to code for active peptidases based on the conservation of an intact cysteine-histidine catalytic dyad [3, 4]. Interestingly, in TbMCA1 and TbMCA4 the catalytic cysteine is replaced by a serine, indicating that the coded proteins would not exhibit cysteine peptidase activity [3, 5]. Whilst plant Metacaspases have been shown to be involved in cell death pathways, trypanosomes appear to lack regulated cell death [6] and in these organisms Metacaspases have evolved alternative functions [5, 7]. Despite being related to the caspases, Metacaspases have been shown to be both structurally and functionally distinct. Metacaspases are known to cleave their substrates after Arg and Lys residues in the P1 position [8], as demonstrated for Metacaspases from T. brucei [9], T. cruzi [10], Leishmania major [11], plants [12-14] and yeast [13], while the caspases cleave their substrates after Asp [15]. Other functional differences between the two families of enzymes are that unlike the caspases, Metacaspases do not necessarily require processing or dimerization for activity and they are activated by calcium [9, 12-14, 16]. In addition, the recent crystallographic structures of TbMCA2 and the Saccharomyces cerevisiae Metacaspase (YCA1) [17, 18] provided a structural basis for comparing caspases and Metacaspases. The Metacaspase structures revealed a monomeric enzyme containing a core caspase/hemoglobinase fold [19] with an eight-stranded β-sheet, consisting of six parallel and two anti-parallel strands. This is unlike the caspases, which comprise a six-stranded β-sheet and a stable dimerisation interface, which forms the active enzyme. In addition, the recent structure of a paracaspase [20] shows that it is structurally very similar to the caspases and dimerizes in a similar manner. Conversely, the two extra strands in the β-sheet of the Metacaspases prevent such dimerisation, explaining its monomeric peptidase activity and distinguishing the Metacaspases structurally from other caspase-family members. The high-resolution crystal structure of TbMCA2 was determined using inactive mutants (TbMCA2C213A/G), as autoprocessing of the full-length recombinant enzyme in vitro was unfavourable in producing diffraction quality crystals [17, 18]. This revealed an unusual N-terminal region, which encircled the protein and crossed over the active site, blocking the S1 pocket. Consequently, the structure of TbMCA2 does not contain an active site substrate and the properties of its extended binding site have not yet been established. In this study, we undertook to examine the extended substrate specificity of recombinant and active TbMCA2 using a series of fluorescence resonance energy transfer (FRET) peptides. The specificity of TbMCA2 subsites S3 to S3′ were analysed by kinetic assays that monitored the hydrolysis of a FRET peptide series, where positions P3 to P3′ on the peptides are substituted by each of the natural amino acids. In addition, the effects of Ca2+ on the structure, activity and extended subsite specificity of TbMCA2 were investigated using a series of kinetic assays, intrinsic fluorescence analysis and circular dichroism (CD) spectroscopy.
-
Trypanosoma brucei Metacaspase 4 is a pseudopeptidase and a virulence factor
The Journal of biological chemistry, 2011Co-Authors: William R Proto, Catherine X. Moss, Luiz Juliano, Graham H Coombs, Esther Castanys-munoz, Alana Black, Laurence Tetley, Jeremy C. MottramAbstract:Metacaspases are caspase family cysteine peptidases found in plants, fungi, and protozoa but not mammals. Trypanosoma brucei is unusual in having five Metacaspases (MCA1–MCA5), of which MCA1 and MCA4 have active site substitutions, making them possible non-enzymatic homologues. Here we demonstrate that recombinant MCA4 lacks detectable peptidase activity despite maintaining a functional peptidase structure. MCA4 is expressed primarily in the bloodstream form of the parasite and associates with the flagellar membrane via dual myristoylation/palmitoylation. Loss of function phenotyping revealed critical roles for MCA4; rapid depletion by RNAi caused lethal disruption to the parasite's cell cycle, yet the generation of MCA4 null mutant parasites (Δmca4) was possible. Δmca4 had normal growth in axenic culture but markedly reduced virulence in mice. Further analysis revealed that MCA4 is released from the parasite and is specifically processed by MCA3, the only Metacaspase that is both palmitoylated and enzymatically active. Accordingly, we have identified that the multiple Metacaspases in T. brucei form a membrane-associated proteolytic cascade to generate a pseudopeptidase virulence factor.
-
Metacaspase 2 of trypanosoma brucei is a calcium dependent cysteine peptidase active without processing
FEBS Letters, 2007Co-Authors: Catherine X. Moss, Luiz Juliano, Graham H Coombs, Gareth D. Westrop, Jeremy C. MottramAbstract:Metacaspases are cysteine peptidases that are distantly related to the caspases, for which proteolytic processing is central to their activation. Here, we show that recombinant Metacaspase 2 (MCA2) from Trypanosoma brucei has arginine/lysine-specific, Ca2+-dependent proteolytic activity. Autocatalytic processing of MCA2 occurred after Lys55 and Lys268; however, this was shown not to be required for the enzyme to be proteolytically active. The necessity of Ca2+, but not processing, for MCA2 enzymatic activity clearly distinguishes MCA2 from the caspases and would be consistent with different physiological roles.
Zhensheng Kang - One of the best experts on this subject based on the ideXlab platform.
-
tamca4 a novel wheat Metacaspase gene functions in programmed cell death induced by the fungal pathogen puccinia striiformis f sp tritici
Molecular Plant-microbe Interactions, 2012Co-Authors: Xiaodong Wang, Xiaojie Wang, Hao Feng, Chunlei Tang, Lili Huang, Zhensheng KangAbstract:Programmed cell death (PCD) is a physiological process to remove redundant or harmful cells, for the development of multicellular organisms, or for restricting the spread of pathogens (hypersensitive response). Metacaspases are cysteine-dependent proteases which play an essential role in PCD. Triticum aestivum Metacaspase 4 (TaMCA4) is a type II Metacaspase gene cloned from ‘Suwon11’ wheat, with typical structural features such as peptidase C14 caspase domain and a long linker sequence between the two subunits. Transient expression of TaMCA4 in tobacco leaves failed to induce PCD directly but enhanced cell death triggered by a mouse Bax gene or a candidate effector gene from Puccinia striiformis f. sp. tritici. Enhancement of PCD was also observed in wheat leaves co-bombarded with TaMCA4. When challenged with the avirulent race of P. striiformis f. sp. tritici, the expression level of TaMCA4 in wheat leaves was sharply upregulated, whereas the transcript level was not significantly induced by the virulent...
-
TaMCA4, a novel wheat Metacaspase gene functions in programmed cell death induced by the fungal pathogen Puccinia striiformis f. sp. tritici.
Molecular plant-microbe interactions : MPMI, 2012Co-Authors: Xiaodong Wang, Xiaojie Wang, Hao Feng, Chunlei Tang, Lili Huang, Pengfei Bai, Guorong Wei, Zhensheng KangAbstract:Programmed cell death (PCD) is a physiological process to remove redundant or harmful cells, for the development of multicellular organisms, or for restricting the spread of pathogens (hypersensitive response). Metacaspases are cysteine-dependent proteases which play an essential role in PCD. Triticum aestivum Metacaspase 4 (TaMCA4) is a type II Metacaspase gene cloned from ‘Suwon11’ wheat, with typical structural features such as peptidase C14 caspase domain and a long linker sequence between the two subunits. Transient expression of TaMCA4 in tobacco leaves failed to induce PCD directly but enhanced cell death triggered by a mouse Bax gene or a candidate effector gene from Puccinia striiformis f. sp. tritici. Enhancement of PCD was also observed in wheat leaves co-bombarded with TaMCA4. When challenged with the avirulent race of P. striiformis f. sp. tritici, the expression level of TaMCA4 in wheat leaves was sharply upregulated, whereas the transcript level was not significantly induced by the virulent...
Vitor Oliveira - One of the best experts on this subject based on the ideXlab platform.
-
processing of Metacaspase 2 from trypanosoma brucei tbmca2 broadens its substrate specificity
Biochimica et Biophysica Acta, 2017Co-Authors: Joyce Meire Gilio, Maurício F.m. Machado, Marcelo F. Marcondes, Maria A. Juliano, Luiz Juliano, Vitor Oliveira, Debora FerrariAbstract:Metacaspases are members of the cysteine peptidase family and may be implicated in programmed cell death in plants and lower eukaryotes. These proteases exhibit calcium-dependent activity and specificity for arginine residues at P1. In contrast to caspases, they do not require processing or dimerization for activity. Indeed, unprocessed Metacaspase-2 of Trypanosoma brucei (TbMCA2) is active; however, it has been shown that cleavages at Lys55 and Lys268 increase TbMCA2 hydrolytic activity on synthetic substrates. The processed TbMCA2 comprises 3 polypeptide chains that remain attached by non-covalent bonds. Replacement of Lys55 and Lys268 with Gly via site-directed mutagenesis results in non-processed but enzymatically active mutant, TbMCA2 K55/268G. To investigate the importance of this processing for the activity and specificity of TbMCA2, we performed activity assays comparing the non-processed mutant (TbMCA2 K55/268G) with the processed TbMCA2 form. Significant differences between TbMCA2 WT (processed form) and TbMCA2 K55/268G (non-processed form) were observed. Specifically, we verified that although non-processed TbMCA2 is active when assayed with small synthetic substrates, the TbMCA2 form does not exhibit hydrolytic activity on large substrates such as azocasein, while processed TbMCA2 is able to readily digest this protein. Such differences can be relevant for understanding the physiological regulation and function of TbMCA2.
-
processing of Metacaspase 2 from trypanosoma brucei tbmca2 broadens its substrate specificity proteins and proteomics
Biochimica et Biophysica Acta, 2017Co-Authors: Joyce Meire Gilio, Marcelo F. Marcondes, Maria A. Juliano, Luiz Juliano, Vitor Oliveira, Debora Ferrari, Maurício F.m. MachadoAbstract:Metacaspases are members of the cysteine peptidase family and may be implicated in programmed cell death in plants and lower eukaryotes. These proteases exhibit calcium-dependent activity and specificity for arginine residues at P1. In contrast to caspases, they do not require processing or dimerization for activity. Indeed, unprocessed Metacaspase-2 of Trypanosoma brucei (TbMCA2) is active; however, it has been shown that cleavages at Lys⁵⁵ and Lys²⁶⁸ increase TbMCA2 hydrolytic activity on synthetic substrates. The processed TbMCA2 comprises 3 polypeptide chains that remain attached by non-covalent bonds. Replacement of Lys⁵⁵ and Lys²⁶⁸ with Gly via site-directed mutagenesis results in non-processed but enzymatically active mutant, TbMCA2 K55/268G. To investigate the importance of this processing for the activity and specificity of TbMCA2, we performed activity assays comparing the non-processed mutant (TbMCA2 K55/268G) with the processed TbMCA2 form. Significant differences between TbMCA2 WT (processed form) and TbMCA2 K55/268G (non-processed form) were observed. Specifically, we verified that although non-processed TbMCA2 is active when assayed with small synthetic substrates, the TbMCA2 form does not exhibit hydrolytic activity on large substrates such as azocasein, while processed TbMCA2 is able to readily digest this protein. Such differences can be relevant for understanding the physiological regulation and function of TbMCA2.
-
Substrate specificity and the effect of calcium on Trypanosoma brucei Metacaspase 2.
The FEBS journal, 2013Co-Authors: Maurício F.m. Machado, Jeremy C. Mottram, Marcelo F. Marcondes, Maria A. Juliano, Karen Mcluskey, Catherine X. Moss, Luiz Juliano, Vitor OliveiraAbstract:Metacaspases are cysteine peptidases that are distantly related to the mammalian caspases but found only in plants, fungi and protozoa [1]. Together with the caspases and paracaspases [1], they are endopeptidases which have been grouped into structural Family C14 in Clan CD of the MEROPS peptidase database [2]. In Trypanosoma brucei, five Metacaspase genes (TbMCA1-5) have been reported with three, TbMCA2, TbMCA3 and TbMCA5, predicted to code for active peptidases based on the conservation of an intact cysteine-histidine catalytic dyad [3, 4]. Interestingly, in TbMCA1 and TbMCA4 the catalytic cysteine is replaced by a serine, indicating that the coded proteins would not exhibit cysteine peptidase activity [3, 5]. Whilst plant Metacaspases have been shown to be involved in cell death pathways, trypanosomes appear to lack regulated cell death [6] and in these organisms Metacaspases have evolved alternative functions [5, 7]. Despite being related to the caspases, Metacaspases have been shown to be both structurally and functionally distinct. Metacaspases are known to cleave their substrates after Arg and Lys residues in the P1 position [8], as demonstrated for Metacaspases from T. brucei [9], T. cruzi [10], Leishmania major [11], plants [12-14] and yeast [13], while the caspases cleave their substrates after Asp [15]. Other functional differences between the two families of enzymes are that unlike the caspases, Metacaspases do not necessarily require processing or dimerization for activity and they are activated by calcium [9, 12-14, 16]. In addition, the recent crystallographic structures of TbMCA2 and the Saccharomyces cerevisiae Metacaspase (YCA1) [17, 18] provided a structural basis for comparing caspases and Metacaspases. The Metacaspase structures revealed a monomeric enzyme containing a core caspase/hemoglobinase fold [19] with an eight-stranded β-sheet, consisting of six parallel and two anti-parallel strands. This is unlike the caspases, which comprise a six-stranded β-sheet and a stable dimerisation interface, which forms the active enzyme. In addition, the recent structure of a paracaspase [20] shows that it is structurally very similar to the caspases and dimerizes in a similar manner. Conversely, the two extra strands in the β-sheet of the Metacaspases prevent such dimerisation, explaining its monomeric peptidase activity and distinguishing the Metacaspases structurally from other caspase-family members. The high-resolution crystal structure of TbMCA2 was determined using inactive mutants (TbMCA2C213A/G), as autoprocessing of the full-length recombinant enzyme in vitro was unfavourable in producing diffraction quality crystals [17, 18]. This revealed an unusual N-terminal region, which encircled the protein and crossed over the active site, blocking the S1 pocket. Consequently, the structure of TbMCA2 does not contain an active site substrate and the properties of its extended binding site have not yet been established. In this study, we undertook to examine the extended substrate specificity of recombinant and active TbMCA2 using a series of fluorescence resonance energy transfer (FRET) peptides. The specificity of TbMCA2 subsites S3 to S3′ were analysed by kinetic assays that monitored the hydrolysis of a FRET peptide series, where positions P3 to P3′ on the peptides are substituted by each of the natural amino acids. In addition, the effects of Ca2+ on the structure, activity and extended subsite specificity of TbMCA2 were investigated using a series of kinetic assays, intrinsic fluorescence analysis and circular dichroism (CD) spectroscopy.