ABSTRACT In 1995, Gary and Clarke identified a gene in E.coli (iadA) encoding a 41 kDa polypeptid... more ABSTRACT In 1995, Gary and Clarke identified a gene in E.coli (iadA) encoding a 41 kDa polypeptide that catalyzes the hydrolytic cleavage of L-isoaspartyl, or L-β-aspartyl, dipeptides [1]. Examination of the deduced amino acid sequence revealed no similarities to other peptidases or proteases. However, a marked similarity to dihydro-orotases and imidases was found, which are involved in the synthesis and the degradation of pyrimidines, reflecting the similarity in the structures of the substrates of these enzymes. L-aspartyl and asparaginyl residues are two of the most prominent sites for the spontaneous decomposition of proteins. These residues can undergo nonenzymatic intramolecular reactions resulting in the formation of deamidated, racemized, and isomerized derivatives. The major product is the L-isoaspartyl derivative in which the peptide bond from the aspartyl residue is made via the β-carboxyl of the side chain rather than through the α-carboxyl. This kink in the polypeptide chain, as well as the effect of the deamidation of the asparaginyl residue, can be detrimental to protein function. E. coli can convert L-isoaspartyl residues in proteins to normal L-aspartyl residues by the action of protein-L-isoaspartate O-methyltransferase [2]. E.coli lacking this transferase survive poorly in stationary phase and at elevated temperatures [3]. Additionally, the repair mechanism is limited by the accessibility of the modified residue on the protein surface and the affinity of the enzyme for different subsets of L-isoaspartyl containing proteins. These limitations suggest that additional mechanisms exist for the removal of isoaspartyl residues including catabolic pathways. Isoaspartyl dipeptides can arise from the degradation of damaged proteins because most proteases and peptidases don't recognize the β-peptide linkage connecting the isoaspartyl residue to its neighbor on the carboxyl side [4]. Without a specific dipeptidase, these dipeptides could accumulate inside the cell in stationary phase.
Proceedings of the National Academy of Sciences of the United States of America, Jan 31, 1998
Tumor necrosis factor-alpha (TNFalpha) is a cytokine that induces protective inflammatory reactio... more Tumor necrosis factor-alpha (TNFalpha) is a cytokine that induces protective inflammatory reactions and kills tumor cells but also causes severe damage when produced in excess, as in rheumatoid arthritis and septic shock. Soluble TNFalpha is released from its membrane-bound precursor by a membrane-anchored proteinase, recently identified as a multidomain metalloproteinase called TNFalpha-converting enzyme or TACE. We have cocrystallized the catalytic domain of TACE with a hydroxamic acid inhibitor and have solved its 2.0 A crystal structure. This structure reveals a polypeptide fold and a catalytic zinc environment resembling that of the snake venom metalloproteinases, identifying TACE as a member of the adamalysin/ADAM family. However, a number of large insertion loops generate unique surface features. The pro-TNFalpha cleavage site fits to the active site of TACE but seems also to be determined by its position relative to the base of the compact trimeric TNFalpha cone. The active-...
Tumor necrosis factor-α converting enzyme (TACE) is an ADAM ( isintegrin nd etalloproteinases) th... more Tumor necrosis factor-α converting enzyme (TACE) is an ADAM ( isintegrin nd etalloproteinases) that comprises an active catalytic domain and several C-terminal domains. We compare the binding affinity and association rate constants of the N-terminal ...
Homodimerization is an essential step for membrane type 1 matrix metalloproteinase (MT1-MMP) to a... more Homodimerization is an essential step for membrane type 1 matrix metalloproteinase (MT1-MMP) to activate proMMP-2 and to degrade collagen on the cell surface. To uncover the molecular basis of the hemopexin (Hpx) domain-driven dimerization of MT1-MMP, a crystal structure of the Hpx domain was solved at 1.7 Å resolution. Two interactions were identified as potential biological dimer interfaces in the crystal structure, and mutagenesis studies revealed that the biological dimer possesses a symmetrical interaction where blades II and III of molecule A interact with blades III and II of molecule B. The mutations of amino acids involved in the interaction weakened the dimer interaction of Hpx domains in solution, and incorporation of these mutations into the full-length enzyme significantly inhibited dimer-dependent functions on the cell surface, including proMMP-2 activation, collagen degradation, and invasion into the three-dimensional collagen matrix, whereas dimer-independent functions, including gelatin film degradation and two-dimensional cell migration, were not affected. These results shed light on the structural basis of MT1-MMP dimerization that is crucial to promote cellular invasion.
ABSTRACT In 1995, Gary and Clarke identified a gene in E.coli (iadA) encoding a 41 kDa polypeptid... more ABSTRACT In 1995, Gary and Clarke identified a gene in E.coli (iadA) encoding a 41 kDa polypeptide that catalyzes the hydrolytic cleavage of L-isoaspartyl, or L-β-aspartyl, dipeptides [1]. Examination of the deduced amino acid sequence revealed no similarities to other peptidases or proteases. However, a marked similarity to dihydro-orotases and imidases was found, which are involved in the synthesis and the degradation of pyrimidines, reflecting the similarity in the structures of the substrates of these enzymes. L-aspartyl and asparaginyl residues are two of the most prominent sites for the spontaneous decomposition of proteins. These residues can undergo nonenzymatic intramolecular reactions resulting in the formation of deamidated, racemized, and isomerized derivatives. The major product is the L-isoaspartyl derivative in which the peptide bond from the aspartyl residue is made via the β-carboxyl of the side chain rather than through the α-carboxyl. This kink in the polypeptide chain, as well as the effect of the deamidation of the asparaginyl residue, can be detrimental to protein function. E. coli can convert L-isoaspartyl residues in proteins to normal L-aspartyl residues by the action of protein-L-isoaspartate O-methyltransferase [2]. E.coli lacking this transferase survive poorly in stationary phase and at elevated temperatures [3]. Additionally, the repair mechanism is limited by the accessibility of the modified residue on the protein surface and the affinity of the enzyme for different subsets of L-isoaspartyl containing proteins. These limitations suggest that additional mechanisms exist for the removal of isoaspartyl residues including catabolic pathways. Isoaspartyl dipeptides can arise from the degradation of damaged proteins because most proteases and peptidases don't recognize the β-peptide linkage connecting the isoaspartyl residue to its neighbor on the carboxyl side [4]. Without a specific dipeptidase, these dipeptides could accumulate inside the cell in stationary phase.
Proceedings of the National Academy of Sciences of the United States of America, Jan 31, 1998
Tumor necrosis factor-alpha (TNFalpha) is a cytokine that induces protective inflammatory reactio... more Tumor necrosis factor-alpha (TNFalpha) is a cytokine that induces protective inflammatory reactions and kills tumor cells but also causes severe damage when produced in excess, as in rheumatoid arthritis and septic shock. Soluble TNFalpha is released from its membrane-bound precursor by a membrane-anchored proteinase, recently identified as a multidomain metalloproteinase called TNFalpha-converting enzyme or TACE. We have cocrystallized the catalytic domain of TACE with a hydroxamic acid inhibitor and have solved its 2.0 A crystal structure. This structure reveals a polypeptide fold and a catalytic zinc environment resembling that of the snake venom metalloproteinases, identifying TACE as a member of the adamalysin/ADAM family. However, a number of large insertion loops generate unique surface features. The pro-TNFalpha cleavage site fits to the active site of TACE but seems also to be determined by its position relative to the base of the compact trimeric TNFalpha cone. The active-...
Tumor necrosis factor-α converting enzyme (TACE) is an ADAM ( isintegrin nd etalloproteinases) th... more Tumor necrosis factor-α converting enzyme (TACE) is an ADAM ( isintegrin nd etalloproteinases) that comprises an active catalytic domain and several C-terminal domains. We compare the binding affinity and association rate constants of the N-terminal ...
Homodimerization is an essential step for membrane type 1 matrix metalloproteinase (MT1-MMP) to a... more Homodimerization is an essential step for membrane type 1 matrix metalloproteinase (MT1-MMP) to activate proMMP-2 and to degrade collagen on the cell surface. To uncover the molecular basis of the hemopexin (Hpx) domain-driven dimerization of MT1-MMP, a crystal structure of the Hpx domain was solved at 1.7 Å resolution. Two interactions were identified as potential biological dimer interfaces in the crystal structure, and mutagenesis studies revealed that the biological dimer possesses a symmetrical interaction where blades II and III of molecule A interact with blades III and II of molecule B. The mutations of amino acids involved in the interaction weakened the dimer interaction of Hpx domains in solution, and incorporation of these mutations into the full-length enzyme significantly inhibited dimer-dependent functions on the cell surface, including proMMP-2 activation, collagen degradation, and invasion into the three-dimensional collagen matrix, whereas dimer-independent functions, including gelatin film degradation and two-dimensional cell migration, were not affected. These results shed light on the structural basis of MT1-MMP dimerization that is crucial to promote cellular invasion.
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Papers by K. Maskos