Abalones reveal unique taste after processing,mainly because of their abundant free amino acids(FAAs)and nucleotides.FAAs are nutrition components that can contribute to the unique taste.However,which factor(s)is resp...Abalones reveal unique taste after processing,mainly because of their abundant free amino acids(FAAs)and nucleotides.FAAs are nutrition components that can contribute to the unique taste.However,which factor(s)is responsible for the accumulation of FAAs still need further studies.To analyze the production of FAAs,we studied the variation of FAAs during 7 days of storage at 4℃.The content of taste-active amino acids,including Asp,Glu,Ser,and Gly increased by 1.7-fold,2.0-fold,3.0-fold,and 8.4-fold,respectively.The relative activity of cathepsin L and aminopeptidase(AP)increased significantly during the cold storage period.To identify AP in abalone and its function in mediating the production of FAAs,an aminopeptidase with wide substrate specificity was then extracted and purified from abalone muscle to homogeneity.Purified AP with a molecular mass of 100 kDa exhibited its maximum activity at 30℃,pH 7.5,and was further confirmed by LC-MS.Bestatin specifically inhibited the activity of AP,and metalloproteinase inhibitors EDTA,EGTA and 1,10-phenanthroline also suppressed its activity to different degrees.Based on its highest activity to substrate Leu-MCA and its peptide sequences,the purified enzyme was identified as leucine aminopeptidase(LAP).Our present study indicated the essential role of AP for FAAs accumulation during cold storage of abalone.展开更多
A cDNA encoding aminopeptidase N was cloned by degenerated PCR combined with RACE technique in this paper. The full-length of APN-Harm is 3 043 bp. Open reading frame is 2 856 bp in length, encoding 951 amino acid res...A cDNA encoding aminopeptidase N was cloned by degenerated PCR combined with RACE technique in this paper. The full-length of APN-Harm is 3 043 bp. Open reading frame is 2 856 bp in length, encoding 951 amino acid residues. Its predicted molecular weight and isoelectric point are 108. 3 kDa and 5.29, respectively. This deduced amino acid sequence shares some common structural features with aminopeptidase N from several moth species, including the consensus zinc-binding motif HEXXHX18E and the GAMEN motif common to gluzincin aminopeptidases. The first 20 amino acid residues at N-termini is hydrophobic transmembrane helix. The sequence of APN-Harm was deposited in GenBank and the accession number is AY181026.展开更多
Aminopeptidase N(APN/CD13), a Zn<sup>2+</sup>-dependent ectopeptidase localized on the cell surface, is widely considered to influence the invasion of tumor cells. We found that boroleucine and dino-leucin...Aminopeptidase N(APN/CD13), a Zn<sup>2+</sup>-dependent ectopeptidase localized on the cell surface, is widely considered to influence the invasion of tumor cells. We found that boroleucine and dino-leucine borate exhibited a strong inhibitory effect on the enzyme activity of aminopeptidase N. The tested assay indicated that both compounds had an anti-proliferative effect on triple-negative breast cancer cells. Wound healing assay, migration test and matrigel-coated transwell assay showed that both boroleucine and dino-leucine borate inhibited the migration and invasion of breast cancer cells. Immunoblot analysis showed that both compounds down-regulated the expression of matrix metalloproteinase-2/9. In the capillary tube formation assay of human umbilical vein endothelial cells (HUVECs), dino-leucine borate showed better antiangiogenic activity than ubenimex even at a low concentration (10 μM). Moreover, compared with ubenimex, the anti-metastatic activity of dino-leucine borate in vivo was similar to or even better than that of ubenimex in the H22 pulmonary metastasis mouse model. In this paper, we found the novel APN inhibitors to markedly suppress the enzyme activity of APN and inhibit the migration and invasion of tumor cells in vitro and in vivo.展开更多
Human endoplasmic reticulum aminopeptidase 1 (ERAP1) is one of two ER luminal aminopeptidases that participate in the final processing of peptide precursors and generates the N-termini of the MHC class I-restricted ep...Human endoplasmic reticulum aminopeptidase 1 (ERAP1) is one of two ER luminal aminopeptidases that participate in the final processing of peptide precursors and generates the N-termini of the MHC class I-restricted epitopes. In order to investigate the interactions of its binding site with substrate peptides, X-ray crystallographic analyses have been carried out to study structures of ERAP1 regulatory (ERAP1_R) domain in complex with antigenic peptides. Single-chain bimodular constructs with various antigenic peptides linked to the C-terminal end of ERAP1_R domain are designed to facilitate crystallization process of these complexes. These recombinant proteins have been purified and crystalized, and x-ray diffraction data of one crystal have been processed to a resolution of 2.8 . The crystal belongs to the space group P21, with unit cell parameters a =64.2, b = 66.8, c = 66.3 , β = 110.2°. A Refmac-refined omit map reveals a clear density for the antigenic peptide’s carboxylate-end that is in contact with the ERAP1 regulatory domain of neighboring molecule. Thus the single-chain bimodular constructs have provided an expedited approach to study sequence-specific interactions between the ERAP1 regulatory domain and antigen peptide’s C-terminal ends.展开更多
Objective: Previous study demonstrated the involvement of basic aminopeptidase (APB) activity in the development of collagen-induced arthritis (CIA). Two zinc dependent metalloenzymes (EC 3.4.11.6 and EC 3.3.2.6) are ...Objective: Previous study demonstrated the involvement of basic aminopeptidase (APB) activity in the development of collagen-induced arthritis (CIA). Two zinc dependent metalloenzymes (EC 3.4.11.6 and EC 3.3.2.6) are known to exhibit concomitantly APB and leukotriene-A4-hydrolase (LT-A4-H) activities. Influence of the interrelationship between both activities on arthritic processes, however, is presently uncertain. This study aimed to compare these activities in CIA. Methods: CIA was induced in rats and arthritis was assessed macroscopically. Ultracentrifugation was used to separate soluble (S) and solubilized membrane-bound (M) fractions from peripheral blood mononuclear cells (PBMCs) and synovial tissue (ST). Enzyme immunoassay was used to measure LT-A4-H activity, and Real Time Polymerase Chain Reaction was used for evaluating EC 3.4.11.6 and EC 3.3.2.6 gene expressions. Results: The existence of genes for EC 3.3.2.6 and EC 3.4.11.6 was demonstrated in the ST. Compared with control, LT-A4-H activity increased in synovial fluid (SF) and in S-PBMCs of CIA-arthritic and CIA-resistant and in M-ST of CIA-resistant, while it decreased in M-PBMCs of CIA-arthritic and CIA-resistant. In all these locations APB activity remained unchanged or inversely correlated with LT-A4-H activity. Conclusions: LT-A4-H and APB activities in joint-related samples are associated, for the first time, with EC 3.3.2.6 and EC 3.4.11.6 genes, exhibiting a compartment-dependent differential modulation of their specificity, efficiency and/or affinity or an inverse concurrent pattern. Changes in LT-A4-H activity have implications for development or resistance to arthritis in CIA model with a potential to be a diagnostic tool.展开更多
Aim: This study aimed to decipher the molecular mechanism underlying the synergistic effect of inhibitors of the mevalonate-cholesterol pathway (i.e., statins) and aminopeptidase inhibitors (APis) on APi-sensitive and...Aim: This study aimed to decipher the molecular mechanism underlying the synergistic effect of inhibitors of the mevalonate-cholesterol pathway (i.e., statins) and aminopeptidase inhibitors (APis) on APi-sensitive and -resistant acute myeloid leukemia (AML) cells.Methods: U937 cells and their sublines with low and high levels of acquired resistance to (6S)-[(R)-2-((S)-Hydroxy-hydroxycarbamoyl-methoxy-methyl)-4-methyl-pentanoylamino]-3,3 dimethyl-butyric acid cyclopentyl ester (CHR2863), an APi prodrug, served as main AML cell line models. Drug combination effects were assessed with CHR2863 and in vitro non-toxic concentrations of various statins upon cell growth inhibition, cell cycle effects, and apoptosis induction. Mechanistic studies involved analysis of Rheb prenylation required for mTOR activation.Results: A strong synergy of CHR2863 with the statins simvastatin, fluvastatin, lovastatin, and pravastatin was demonstrated in U937 cells and two CHR2863-resistant sublines. This potent synergy between simvastatin and CHR2863 was also observed with a series of other human AML cell lines (e.g., THP1, MV4-11, and KG1), but not with acute lymphocytic leukemia or multiple solid tumor cell lines. This synergistic activity was: (i) specific for APis (e.g., CHR2863 and Bestatin), rather than for other cytotoxic agents;and (ii) corroborated by enhanced induction of apoptosis and cell cycle arrest which increased the sub-G1 fraction. Consistently, statin potentiation of CHR2863 activity was abrogated by co-administration of mevalonate and/or farnesyl pyrophosphate, suggesting the involvement of protein prenylation;this was experimentally confirmed by impaired Rheb prenylation by simvastatin.Conclusion: These novel findings suggest that the combined inhibitory effect of impaired Rheb prenylation and CHR2863-dependent mTOR inhibition instigates a potent synergistic inhibition of statins and APis on human AML cells.展开更多
基金supported by the National Key R&D Program of China(No.2018YFD0901004)the National Natural Science Foundation of China(No.31772049).
文摘Abalones reveal unique taste after processing,mainly because of their abundant free amino acids(FAAs)and nucleotides.FAAs are nutrition components that can contribute to the unique taste.However,which factor(s)is responsible for the accumulation of FAAs still need further studies.To analyze the production of FAAs,we studied the variation of FAAs during 7 days of storage at 4℃.The content of taste-active amino acids,including Asp,Glu,Ser,and Gly increased by 1.7-fold,2.0-fold,3.0-fold,and 8.4-fold,respectively.The relative activity of cathepsin L and aminopeptidase(AP)increased significantly during the cold storage period.To identify AP in abalone and its function in mediating the production of FAAs,an aminopeptidase with wide substrate specificity was then extracted and purified from abalone muscle to homogeneity.Purified AP with a molecular mass of 100 kDa exhibited its maximum activity at 30℃,pH 7.5,and was further confirmed by LC-MS.Bestatin specifically inhibited the activity of AP,and metalloproteinase inhibitors EDTA,EGTA and 1,10-phenanthroline also suppressed its activity to different degrees.Based on its highest activity to substrate Leu-MCA and its peptide sequences,the purified enzyme was identified as leucine aminopeptidase(LAP).Our present study indicated the essential role of AP for FAAs accumulation during cold storage of abalone.
文摘A cDNA encoding aminopeptidase N was cloned by degenerated PCR combined with RACE technique in this paper. The full-length of APN-Harm is 3 043 bp. Open reading frame is 2 856 bp in length, encoding 951 amino acid residues. Its predicted molecular weight and isoelectric point are 108. 3 kDa and 5.29, respectively. This deduced amino acid sequence shares some common structural features with aminopeptidase N from several moth species, including the consensus zinc-binding motif HEXXHX18E and the GAMEN motif common to gluzincin aminopeptidases. The first 20 amino acid residues at N-termini is hydrophobic transmembrane helix. The sequence of APN-Harm was deposited in GenBank and the accession number is AY181026.
文摘Aminopeptidase N(APN/CD13), a Zn<sup>2+</sup>-dependent ectopeptidase localized on the cell surface, is widely considered to influence the invasion of tumor cells. We found that boroleucine and dino-leucine borate exhibited a strong inhibitory effect on the enzyme activity of aminopeptidase N. The tested assay indicated that both compounds had an anti-proliferative effect on triple-negative breast cancer cells. Wound healing assay, migration test and matrigel-coated transwell assay showed that both boroleucine and dino-leucine borate inhibited the migration and invasion of breast cancer cells. Immunoblot analysis showed that both compounds down-regulated the expression of matrix metalloproteinase-2/9. In the capillary tube formation assay of human umbilical vein endothelial cells (HUVECs), dino-leucine borate showed better antiangiogenic activity than ubenimex even at a low concentration (10 μM). Moreover, compared with ubenimex, the anti-metastatic activity of dino-leucine borate in vivo was similar to or even better than that of ubenimex in the H22 pulmonary metastasis mouse model. In this paper, we found the novel APN inhibitors to markedly suppress the enzyme activity of APN and inhibit the migration and invasion of tumor cells in vitro and in vivo.
文摘Human endoplasmic reticulum aminopeptidase 1 (ERAP1) is one of two ER luminal aminopeptidases that participate in the final processing of peptide precursors and generates the N-termini of the MHC class I-restricted epitopes. In order to investigate the interactions of its binding site with substrate peptides, X-ray crystallographic analyses have been carried out to study structures of ERAP1 regulatory (ERAP1_R) domain in complex with antigenic peptides. Single-chain bimodular constructs with various antigenic peptides linked to the C-terminal end of ERAP1_R domain are designed to facilitate crystallization process of these complexes. These recombinant proteins have been purified and crystalized, and x-ray diffraction data of one crystal have been processed to a resolution of 2.8 . The crystal belongs to the space group P21, with unit cell parameters a =64.2, b = 66.8, c = 66.3 , β = 110.2°. A Refmac-refined omit map reveals a clear density for the antigenic peptide’s carboxylate-end that is in contact with the ERAP1 regulatory domain of neighboring molecule. Thus the single-chain bimodular constructs have provided an expedited approach to study sequence-specific interactions between the ERAP1 regulatory domain and antigen peptide’s C-terminal ends.
基金This study was financially supported by a Research Grant 09/17613-0 from FAPESP(Fundacao de Amparoà Pes-quisa do Estado de Sao Paulo,Brazil)P.F.S was recipient of a Productivity Grant 302533/2011-7 from CNPq(Conselho Nacional de Desenvolvimento Cientifico e Tecnologico,Brazil)M.T.M.was recipient of a CAPES(Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior,Brasil)fellowship.
文摘Objective: Previous study demonstrated the involvement of basic aminopeptidase (APB) activity in the development of collagen-induced arthritis (CIA). Two zinc dependent metalloenzymes (EC 3.4.11.6 and EC 3.3.2.6) are known to exhibit concomitantly APB and leukotriene-A4-hydrolase (LT-A4-H) activities. Influence of the interrelationship between both activities on arthritic processes, however, is presently uncertain. This study aimed to compare these activities in CIA. Methods: CIA was induced in rats and arthritis was assessed macroscopically. Ultracentrifugation was used to separate soluble (S) and solubilized membrane-bound (M) fractions from peripheral blood mononuclear cells (PBMCs) and synovial tissue (ST). Enzyme immunoassay was used to measure LT-A4-H activity, and Real Time Polymerase Chain Reaction was used for evaluating EC 3.4.11.6 and EC 3.3.2.6 gene expressions. Results: The existence of genes for EC 3.3.2.6 and EC 3.4.11.6 was demonstrated in the ST. Compared with control, LT-A4-H activity increased in synovial fluid (SF) and in S-PBMCs of CIA-arthritic and CIA-resistant and in M-ST of CIA-resistant, while it decreased in M-PBMCs of CIA-arthritic and CIA-resistant. In all these locations APB activity remained unchanged or inversely correlated with LT-A4-H activity. Conclusions: LT-A4-H and APB activities in joint-related samples are associated, for the first time, with EC 3.3.2.6 and EC 3.4.11.6 genes, exhibiting a compartment-dependent differential modulation of their specificity, efficiency and/or affinity or an inverse concurrent pattern. Changes in LT-A4-H activity have implications for development or resistance to arthritis in CIA model with a potential to be a diagnostic tool.
基金This study was supported by Cancer Center Amsterdam grants 07/36 and 2012-1-08.
文摘Aim: This study aimed to decipher the molecular mechanism underlying the synergistic effect of inhibitors of the mevalonate-cholesterol pathway (i.e., statins) and aminopeptidase inhibitors (APis) on APi-sensitive and -resistant acute myeloid leukemia (AML) cells.Methods: U937 cells and their sublines with low and high levels of acquired resistance to (6S)-[(R)-2-((S)-Hydroxy-hydroxycarbamoyl-methoxy-methyl)-4-methyl-pentanoylamino]-3,3 dimethyl-butyric acid cyclopentyl ester (CHR2863), an APi prodrug, served as main AML cell line models. Drug combination effects were assessed with CHR2863 and in vitro non-toxic concentrations of various statins upon cell growth inhibition, cell cycle effects, and apoptosis induction. Mechanistic studies involved analysis of Rheb prenylation required for mTOR activation.Results: A strong synergy of CHR2863 with the statins simvastatin, fluvastatin, lovastatin, and pravastatin was demonstrated in U937 cells and two CHR2863-resistant sublines. This potent synergy between simvastatin and CHR2863 was also observed with a series of other human AML cell lines (e.g., THP1, MV4-11, and KG1), but not with acute lymphocytic leukemia or multiple solid tumor cell lines. This synergistic activity was: (i) specific for APis (e.g., CHR2863 and Bestatin), rather than for other cytotoxic agents;and (ii) corroborated by enhanced induction of apoptosis and cell cycle arrest which increased the sub-G1 fraction. Consistently, statin potentiation of CHR2863 activity was abrogated by co-administration of mevalonate and/or farnesyl pyrophosphate, suggesting the involvement of protein prenylation;this was experimentally confirmed by impaired Rheb prenylation by simvastatin.Conclusion: These novel findings suggest that the combined inhibitory effect of impaired Rheb prenylation and CHR2863-dependent mTOR inhibition instigates a potent synergistic inhibition of statins and APis on human AML cells.
