MMP activation–associated aminopeptidase N reveals a bivalent 14-3-3 binding motif

Abstract

Aminopeptidase N (APN, CD13) is a transmembrane ectopeptidase involved in many crucial cellular functions. Besides its role as a peptidase, APN also mediates signal transduction and is involved in the activation of matrix metalloproteinases (MMPs). MMPs function in tissue remodeling within the extracellular space and are therefore involved in many human diseases, such as fibrosis, rheumatoid arthritis, tumor angiogenesis, and metastasis, as well as viral infections. However, the exact mechanism that leads to APN-driven MMP activation is unclear. It was previously shown that extracellular 14-3-3 adapter proteins bind to APN and thereby induce the transcription of MMPs. As a first step, we sought to identify potential 14-3-3–binding sites in the APN sequence. We constructed a set of phosphorylated peptides derived from APN to probe for interactions. We identified and characterized a canonical 14-3-3–binding site (site 1) within the flexible, structurally unresolved N-terminal APN region using direct binding fluorescence polarization assays and thermodynamic analysis. In addition, we identified a secondary, noncanonical binding site (site 2), which enhances the binding affinity in combination with site 1 by many orders of magnitude. Finally, we solved crystal structures of 14-3-3σ bound to mono- and bis-phosphorylated APN-derived peptides, which revealed atomic details of the binding mode of mono- and bivalent 14-3-3 interactions. Therefore, our findings shed some light on the first steps of APN-mediated MMP activation and open the field for further investigation of this important signaling pathway. Aminopeptidase N (APN, CD13) is a transmembrane ectopeptidase involved in many crucial cellular functions. Besides its role as a peptidase, APN also mediates signal transduction and is involved in the activation of matrix metalloproteinases (MMPs). MMPs function in tissue remodeling within the extracellular space and are therefore involved in many human diseases, such as fibrosis, rheumatoid arthritis, tumor angiogenesis, and metastasis, as well as viral infections. However, the exact mechanism that leads to APN-driven MMP activation is unclear. It was previously shown that extracellular 14-3-3 adapter proteins bind to APN and thereby induce the transcription of MMPs. As a first step, we sought to identify potential 14-3-3–binding sites in the APN sequence. We constructed a set of phosphorylated peptides derived from APN to probe for interactions. We identified and characterized a canonical 14-3-3–binding site (site 1) within the flexible, structurally unresolved N-terminal APN region using direct binding fluorescence polarization assays and thermodynamic analysis. In addition, we identified a secondary, noncanonical binding site (site 2), which enhances the binding affinity in combination with site 1 by many orders of magnitude. Finally, we solved crystal structures of 14-3-3σ bound to mono- and bis-phosphorylated APN-derived peptides, which revealed atomic details of the binding mode of mono- and bivalent 14-3-3 interactions. Therefore, our findings shed some light on the first steps of APN-mediated MMP activation and open the field for further investigation of this important signaling pathway. Aminopeptidase N (APN, CD13) is a zinc-dependent ectopeptidase of the M1 family. It is a type II integral membrane protein and is located on the surface of many mammalian cells like fibroblasts, epithelial and myeloid cells (1Luan Y. Xu W. The structure and main functions of aminopeptidase N.Curr. Med. Chem. 2007; 14 (17346152): 639-64710.2174/092986707780059571Crossref PubMed Scopus (155) Google Scholar, 2Wickström M. Larsson R. Nygren P. Gullbo J. Aminopeptidase N (CD13) as a target for cancer chemotherapy.Cancer Sci. 2011; 102 (21205077): 501-50810.1111/j.1349-7006.2010.01826.xCrossref PubMed Scopus (225) Google Scholar). APN consists of 967 amino acids (aa), which can be divided into three regions. A short N-terminal region is located in the cytoplasm (aa 1–9), followed by a single-helix transmembrane domain (aa 10–27) and a large extracellular region (aa 28–967) (3Wong A.H.M. Zhou D. Rini J.M. The x-ray crystal structure of human aminopeptidase N reveals a novel dimer and the basis for peptide processing.J. Biol. Chem. 2012; 287 (22932899): 36804-3681310.1074/jbc.M112.398842Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). APN is involved in multiple processes. It is most widely known for its protease activity in the renin-angiotensin system, where it proteolytically converts angiotensin III to IV (4Danziger R.S. Aminopeptidase N in arterial hypertension.Heart Fail. Rev. 2008; 13 (18008160): 293-29810.1007/s10741-007-9061-yCrossref PubMed Scopus (44) Google Scholar). In addition to its enzymatic activity, it functions as a receptor for coronaviruses and has been proposed to participate in the endocytosis of cholesterol (5Nomura R. Kiyota A. Suzaki E. Kataoka K. Ohe Y. Miyamoto K. Senda T. Fujimoto T. Human coronavirus 229E binds to CD13 in rafts and enters the cell through caveolae.J. Virol. 2004; 78 (15280478): 8701-870810.1128/JVI.78.16.8701-8708.2004Crossref PubMed Scopus (120) Google Scholar, 6Mina-Osorio P. The moonlighting enzyme CD13: old and new functions to target.Trends Mol. Med. 2008; 14 (18603472): 361-37110.1016/j.molmed.2008.06.003Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar, 7Kolb A.F. Hegyi A. Maile J. Heister A. Hagemann M. Siddell S.G. Molecular analysis of the coronavirus-receptor function of aminopeptidase N.Adv. Exp. Med. Biol. 1998; 440 (9782265): 61-6710.1007/978-1-4615-5331-1_8Crossref PubMed Scopus (28) Google Scholar). Some of the functions of APN are mediated by protein-protein interactions. Binding of extracellular 14-3-3 proteins, for instance, was shown to induce transcription of various matrix-metalloproteinases (MMPs) via p38 MAPK signaling (8Ghahary A. Marcoux Y. Karimi-Busheri F. Li Y. Tredget E.E. Kilani R.T. Lam E. Weinfeld M. Differentiated keratinocyte-releasable stratifin (14-3-3 Sigma) stimulates MMP-1 expression in dermal fibroblasts.J. Invest. Dermatol. 2005; 124 (15654971): 170-17710.1111/j.0022-202X.2004.23521.xAbstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar, 9Asdaghi N. Kilani R.T. Hosseini-Tabatabaei A. Odemuyiwa S.O. Hackett T.L. Knight D.A. Ghahary A. Moqbel R. Extracellular 14-3-3 from human lung epithelial cells enhances MMP-1 expression.Mol. Cell. Biochem. 2012; 360 (21948273): 261-27010.1007/s11010-011-1065-1Crossref PubMed Scopus (16) Google Scholar, 10Ghahary A. Karimi-Busheri F. Marcoux Y. Li Y. Tredget E.E. Kilani R.T. Li L. Zheng J. Karami A. Keller B.O. Weinfeld M. Keratinocyte-releasable stratifin functions as a potent collagenase-stimulating factor in fibroblasts.J. Invest. Dermatol. 2004; 122 (15140222): 1188-119710.1111/j.0022-202X.2004.22519.xAbstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 11Ghaffari A. Li Y. Kilani R.T. Ghahary A. 14-3-3σ associates with cell surface aminopeptidase N in the regulation of matrix metalloproteinase-1.J. Cell Sci. 2010; 123 (20699358): 2996-300510.1242/jcs.069484Crossref PubMed Scopus (26) Google Scholar, 12Eun K.L. Youn S.L. Lee H. Cheol Y.C. Seok H.P. 14-3-3ε protein increases matrix metalloproteinase-2 gene expression via p38 MAPK signaling in NIH3T3 fibroblast cells.Exp. Mol. Med. 2009; 41 (19322035): 453-46110.3858/emm.2009.41.7.050Crossref PubMed Scopus (11) Google Scholar). MMPs act in tissue remodeling by rearranging the extracellular matrix (13Birkedal-Hansen H. Moore W.G.I. Bodden M.K. Windsor L.J. Birkedal-Hansen B. DeCarlo A. Engler J.A. Matrix metalloproteinases: a review.Crit. Rev. Oral Biol. Med. 1993; 4 (8435466): 197-25010.1177/10454411930040020401Crossref PubMed Scopus (2577) Google Scholar). They are involved in several human diseases, such as fibrosis and rheumatoid arthritis (14Maksymowych W.P. van der Heijde D. Allaart C.F. Landewé R. Boire G. Tak P.P. Gui Y. Ghahary A. Kilani R. A. is a novel with the of rheumatoid arthritis and PubMed Scopus Google Scholar, A. Li Y. Karami A. M. Tredget E.E. Ghahary A. extracellular matrix gene expression in to keratinocyte-releasable Cell. Biochem. PubMed Scopus Google Scholar). MMPs also important in of by and M. functions for the matrix metalloproteinases in cancer Rev. PubMed Scopus Google Scholar, Matrix metalloproteinases in a target for Invest. PubMed Scopus Google Scholar). The of 14-3-3 proteins are adapter proteins, which are involved in several protein-protein and therefore a of cellular functions. are in human and with a of A. van T. G. J. 14-3-3 a of Biochem. Sci. Full Text PDF PubMed Scopus Google Scholar). 14-3-3 consists of and via its N-terminal region and in 14-3-3 proteins and the PubMed Scopus Google Scholar, G. J. 14-3-3 new to PubMed Scopus Google Scholar). of the binding to with 14-3-3 binding in a in which a of the target protein is phosphorylated and to bind to a within 14-3-3 J. B. H. binds a phosphorylated peptide and peptide via its Biol. Chem. 1998; Full Text Full Text PDF PubMed Scopus Google Scholar, K. A. H. The basis for binding Full Text Full Text PDF PubMed Scopus Google Scholar). to its 14-3-3 dimer of binding and several shown bivalent binding 14-3-3 and D. A. M. D. P. T. Human and is for 14-3-3 PubMed Scopus Google Scholar, R. of by PubMed Scopus (16) Google Scholar, B. A. of 14-3-3 binding site within 2009; PubMed Scopus Google Scholar, L. and 14-3-3 J. PubMed Scopus Google Scholar). the its some 14-3-3 known as stratifin and shown to be and are also in the extracellular to extracellular 14-3-3 be to in expression rheumatoid arthritis as factor A. Karimi-Busheri F. Marcoux Y. Li Y. Tredget E.E. Kilani R.T. Li L. Zheng J. Karami A. Keller B.O. Weinfeld M. Keratinocyte-releasable stratifin functions as a potent collagenase-stimulating factor in fibroblasts.J. Invest. Dermatol. 2004; 122 (15140222): 1188-119710.1111/j.0022-202X.2004.22519.xAbstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, W.P. van der Heijde D. Allaart C.F. Landewé R. Boire G. Tak P.P. Gui Y. Ghahary A. Kilani R. A. is a novel with the of rheumatoid arthritis and PubMed Scopus Google Scholar, A. Li Y. Karami A. M. Tredget E.E. Ghahary A. extracellular matrix gene expression in to keratinocyte-releasable Cell. Biochem. PubMed Scopus Google Scholar, R. A. L. A. P. N. E. A. Ghahary A. type in dermal fibroblasts.J. Cell. Biochem. 2012; PubMed Scopus Google Scholar, E. R. R. N. Ghahary A. of a that the of stratifin and and PubMed Scopus Google Scholar). In to the signal transduction that MMPs via the p38 MAPK the extracellular of APN by 14-3-3 is Extracellular 14-3-3 binds to APN in a A. Li Y. Kilani R.T. Ghahary A. 14-3-3σ associates with cell surface aminopeptidase N in the regulation of matrix metalloproteinase-1.J. Cell Sci. 2010; 123 (20699358): 2996-300510.1242/jcs.069484Crossref PubMed Scopus (26) Google Scholar, M. L. G. G. F. The 14-3-3ε is a of CD13 in Cell Sci. PubMed Scopus Google Scholar). can be by the 14-3-3 binding by P. L. N. and novel of 14-3-3 PubMed Scopus Google Scholar, A. D. N. K. L. of peptides that protein-protein Med. Chem. PubMed Scopus (26) Google Scholar). APN-derived peptide a phosphorylated was shown to the MMP M. L. G. G. F. The 14-3-3ε is a of CD13 in Cell Sci. PubMed Scopus Google Scholar). identify the region of direct APN and we the analysis of potential binding phosphorylated peptides, we fluorescence polarization and characterized potential APN phosphorylated and 14-3-3–binding We that a noncanonical 14-3-3–binding site increases the affinity and therefore a bivalent we solved the crystal structure of mono- and bis-phosphorylated APN in with Therefore, we a analysis of a bivalent 14-3-3 and the of secondary, noncanonical 14-3-3–binding a analysis of APN binding to we for the of its exact binding As phosphorylated and are in 14-3-3–binding sites T. basis of 14-3-3 protein Cell Biol. 2011; PubMed Scopus Google Scholar, of 14-3-3 with signaling proteins is mediated by the of Full Text Full Text PDF PubMed Scopus Google Scholar, P. 14-3-3 2005; PubMed Scopus Google Scholar, M.K. the of Cell Sci. 2004; PubMed Scopus Google we analysis of extracellular and of APN using three we the potential of for 14-3-3 binding F. M. G. E. M. R. to PubMed Scopus Google the three we that to be in potential 14-3-3–binding within APN we that the be surface N. and the for protein PubMed Scopus Google which the of to potential 14-3-3–binding of and are located in the structurally N-terminal region of APN (3Wong A.H.M. Zhou D. Rini J.M. The x-ray crystal structure of human aminopeptidase N reveals a novel dimer and the basis for peptide processing.J. Biol. Chem. 2012; 287 (22932899): 36804-3681310.1074/jbc.M112.398842Abstract Full Text Full Text PDF PubMed Scopus (98) Google Finally, the potential binding to be located in structure of proteins via E. R. G. A of for 2011; PubMed Scopus Google Scholar, W. of protein of and PubMed Scopus Google Scholar, G. A and for 2005; Scopus Google which in potential 14-3-3–binding sites and of potential 14-3-3–binding within APN revealed potential we peptides via peptide peptide was of N-terminal and three amino acids to the and N-terminal via a The 14-3-3–binding of was as a and peptides for direct binding 14-3-3σ in and 1 was identified as the In addition, is to be phosphorylated and N. and of protein Mol. Biol. PubMed Scopus Google Scholar, this peptide a in the to and the of our the APN peptide was and in our In to binding 14-3-3σ a for the amino acids the the of the peptides 1 and a probe As from our direct was to with In 1 was to with as the peptide which that APN-derived 1 is a direct 14-3-3σ As some of 14-3-3 are to be also located in the extracellular space A. M. Extracellular functions of 14-3-3 PubMed Scopus Google we to 1 is bound by a of 14-3-3 We using human 14-3-3 and and to the previously characterized binding with and the and the of peptide 1 with 14-3-3 proteins in we a this we peptides 1 and on and of The proteins via of the a the of with the for the peptide The and in to the P. The moonlighting enzyme CD13: old and new functions to target.Trends Mol. Med. 2008; 14 (18603472): 361-37110.1016/j.molmed.2008.06.003Abstract Full Text Full Text PDF PubMed Scopus (247) Google peptide 1 binds to of are peptide 1 was to 14-3-3 proteins the and the and the of the binding of peptide 1 to 14-3-3 in we peptide 1 and 14-3-3σ (aa The set was to a of and the structure was solved in space by P. A. of the and Biol. 2011; PubMed Scopus Google Scholar, 2007; PubMed Scopus Google using a previously solved 14-3-3σ structure as A B. M. R. of a Biol. 2010; PubMed Scopus Google of and P. B. K. and of Biol. 2010; PubMed Scopus Google Scholar, D. G. B. structure using and in Biol. PubMed Scopus Google to the the exact and of peptide 1 within the binding of 14-3-3 As the structure revealed 14-3-3 the dimer is via the binding of the dimer with peptide of structure and Cell of in a new for APN be into the A of 14-3-3 structures revealed that peptide 1 was bound to the site with the to the exact of APN bound to we the amino and of the We in the region the membrane and the domain of APN (aa space for 14-3-3 Therefore, we a of our 14-3-3 and the APN crystal structure (3Wong A.H.M. Zhou D. Rini J.M. The x-ray crystal structure of human aminopeptidase N reveals a novel dimer and the basis for peptide processing.J. Biol. Chem. 2012; 287 (22932899): 36804-3681310.1074/jbc.M112.398842Abstract Full Text Full Text PDF PubMed Scopus (98) Google and the on the of amino acids within the region we the the domain and the membrane to be space for a 14-3-3 protein dimer of to bind The 14-3-3 dimer can bind to a APN via the site to the APN via We the of N-terminal and of the APN region noncanonical binding In this 1) was identified as a potential canonical 14-3-3–binding site in our analysis affinity We to this and potential sites in combination with to bis-phosphorylated peptides in binding affinity peptides and for affinity and in The peptide a affinity the peptide 1 and a affinity to peptides in binding affinity and We a and binding the site was located within the region of with in the three noncanonical binding the peptides (14Maksymowych W.P. van der Heijde D. Allaart C.F. Landewé R. Boire G. Tak P.P. Gui Y. Ghahary A. Kilani R. A. is a novel with the of rheumatoid arthritis and PubMed Scopus Google Scholar, A. Li Y. Karami A. M. Tredget E.E. Ghahary A. extracellular matrix gene expression in to keratinocyte-releasable Cell. Biochem. PubMed Scopus Google Scholar, M. functions for the matrix metalloproteinases in cancer Rev. PubMed Scopus Google and in our for affinity three noncanonical binding sites in the and be to be 14-3-3 as the combination of a canonical binding site 1 with a noncanonical binding site in binding of bis-phosphorylated We of with and to the thermodynamic of the binding 4 and and in that our 14-3-3 as dimer in we affinity of bis-phosphorylated and a affinity of with 1 and which is in with our via be in our affinity and thermodynamic be is in with the affinity of peptide As we a for the peptide to the surface of The to binding of peptide and 1 are which in binding for the region of In addition, we for to a In the of in to Therefore, the binding a on In with a and as binding (site peptide 1 and it is important to the of in which the in a the binding we 1 and which that peptide binds to 14-3-3 peptides bind to 14-3-3 peptide a a bivalent binding mode in which peptide binds to 14-3-3 the bis-phosphorylated peptide bound to we with 14-3-3σ (aa using the in a The set was to a of The structure was solved in space with 14-3-3 using as As the dimer is by of the the of peptide (site 1 and site is within the binding site 1 into the binding of a to the the was the site was the of the the bis-phosphorylated peptide binds in a within the crystal we crystal and solved the set in space Molecular in analysis of the revealed as that the binding of the 14-3-3 dimer APN site 1 and site with of The bis-phosphorylated peptide binds with to the 14-3-3 and the structure in to a We to the structure in space 1) and a of APN peptide with amino acids to the phosphorylated and N-terminal be to peptide of amino acids from the phosphorylated the exact of peptide from the a that sites 1 and in the 14-3-3–binding to to the this we that sites bind in the within the 14-3-3–binding and this leads to of the bis-phosphorylated peptide bound to the 14-3-3 dimer and The of APN by extracellular 14-3-3 is the first in the p38 signaling that the of MMPs for tissue remodeling A. Li Y. Kilani R.T. Ghahary A. 14-3-3σ associates with cell surface aminopeptidase N in the regulation of matrix metalloproteinase-1.J. Cell Sci. 2010; 123 (20699358): 2996-300510.1242/jcs.069484Crossref PubMed Scopus (26) Google Scholar). As we are details of the step, we in the and of a potential binding region for 14-3-3 in We identified potential 14-3-3–binding in the extracellular region of by F. M. G. E. M. R. to PubMed Scopus Google and located in flexible, regions. The potential 1) and in to binding this is known be extracellular and involved in binding to APN A. Li Y. Kilani R.T. Ghahary A. 14-3-3σ associates with cell surface aminopeptidase N in the regulation of matrix metalloproteinase-1.J. Cell Sci. 2010; 123 (20699358): 2996-300510.1242/jcs.069484Crossref PubMed Scopus (26) Google Scholar). 1 to be a 14-3-3 and was further by a of APN has been by and some 14-3-3 are known to be located in the extracellular we the binding affinity of 1 of the human of a binding affinity with and the and are involved in of MMPs to 14-3-3σ A. M. Extracellular functions of 14-3-3 PubMed Scopus Google Scholar). A from cellular and a thermodynamic analysis using and further the affinity of peptide It a canonical 14-3-3 mode II binding site (site 1) 14-3-3 proteins and the PubMed Scopus Google Scholar). the 14-3-3–binding K. A. H. The basis for binding Full Text Full Text PDF PubMed Scopus Google are to be flexible, for 14-3-3 such as in and in and a in R. and of J. 2010; PubMed Scopus Google Scholar). The analysis of 1 bound to 14-3-3σ the and of the peptide within the protein A and and the of the 14-3-3 dimer bound to the extracellular domain of APN was to a binding mode of 14-3-3σ to APN is In addition to the site we identify as a noncanonical binding site (site 4 and was with site a was in the affinity of and We that the three the binding affinity A. Karimi-Busheri F. Marcoux Y. Li Y. Tredget E.E. Kilani R.T. Li L. Zheng J. Karami A. Keller B.O. Weinfeld M. Keratinocyte-releasable stratifin functions as a potent collagenase-stimulating factor in fibroblasts.J. Invest. Dermatol. 2004; 122 (15140222): 1188-119710.1111/j.0022-202X.2004.22519.xAbstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 11Ghaffari A. Li Y. Kilani R.T. Ghahary A. 14-3-3σ associates with cell surface aminopeptidase N in the regulation of matrix metalloproteinase-1.J. Cell Sci. 2010; 123 (20699358): 2996-300510.1242/jcs.069484Crossref PubMed Scopus (26) Google Scholar, 12Eun K.L. Youn S.L. Lee H. Cheol Y.C. Seok H.P. 14-3-3ε protein increases matrix metalloproteinase-2 gene expression via p38 MAPK signaling in NIH3T3 fibroblast cells.Exp. Mol. Med. 2009; 41 (19322035): 453-46110.3858/emm.2009.41.7.050Crossref PubMed Scopus (11) Google the from the of peptides the and the phosphorylated and the which to the that also noncanonical 14-3-3–binding sites a The of peptides from canonical 14-3-3–binding by the in and the in the 14 and in as in peptide the is It is has been that noncanonical as which enhances the affinity in in the of a orders of D. A. M. D. P. T. Human and is for 14-3-3 PubMed Scopus Google Scholar, R. of by PubMed Scopus (16) Google Scholar, B. A. of 14-3-3 binding site within 2009; PubMed Scopus Google Scholar, L. and 14-3-3 J. PubMed Scopus Google Scholar, M. binding of the phosphorylated and sites of to Mol. Biol. 2012; PubMed Scopus Google Scholar, Lam L. and of the binding 14-3-3 and the domain of Sci. A. PubMed Scopus Google Scholar). In our thermodynamic we peptides and which the and therefore a in the binding sites for it is binding which is in the The affinity for peptide is via to a binding (site 1 and site We the binding of (14-3-3 and with a in which APN bind to 14-3-3 dimer via site our a mechanism in which 14-3-3 dimer binds to APN via site 1 and site the APN bis-phosphorylated peptide with we that of peptide in the 14-3-3 dimer are within the crystal and thereby and We therefore a protein in APN to function as bivalent to we identified as a binding site 1 and noncanonical binding site which in a affinity of structure analysis in 14-3-3 structures a and a bis-phosphorylated APN to the field of bivalent 14-3-3 and the of analysis of 14-3-3 we shed some light on the first steps of APN signal is to the cellular of sites in of phosphorylated binding was by using the of the human aminopeptidase N F. M. G. E. M. R. to PubMed Scopus Google The potential binding matrix further for surface (3Wong A.H.M. Zhou D. Rini J.M. The x-ray crystal structure of human aminopeptidase N reveals a novel dimer and the basis for peptide processing.J. Biol. Chem. 2012; 287 (22932899): 36804-3681310.1074/jbc.M112.398842Abstract Full Text Full Text PDF PubMed Scopus (98) Google using N. and the for protein PubMed Scopus Google Scholar). the within structure was using E. R. G. A of for 2011; PubMed Scopus Google Scholar, W. of protein of and PubMed Scopus Google Scholar, G. A and for 2005; Scopus Google Scholar). amino acids that and a surface of and in a a into further peptides using on peptide of the was in using 1 of amino with of for was using for of of the was using in for of amino acids was by first using 4 of amino 4 of 4 of and of in for and in a 4 of amino 4 of 4 of and of in for of was using in for and the peptides first with a amino was by using 4 of the and of in for peptides, a of was to the peptide of the from was with a of and for 1 of the by peptides with in and of peptides was using with a using A and with a of of peptides was using with a and the A and of peptides was by using the in a a by using the peptides using via and a peptide 14-3-3 proteins with E. was with the gene and in was expression was using the was 4 and the cell was in 1 the steps 4 cell and and cells using a The was via and the was on a with The was with of 1 and protein was with 4 of 1 The protein was using a and was using on with via as to the protein was in and protein (14-3-3 was and as of the protein from the protein was using 1 and proteolytically using protease in The protein was a for 1 was using on system, from with via as to the protein was in and the the 14-3-3 proteins to a in of peptides in to a of the of in of a A with of protein was and of peptide was to well for a of The was for was in a analysis was with and via a of with The fluorescence polarization was by the 14-3-3σ protein to with and peptide of was in of a and a with of peptide 1 was of the of 14-3-3 protein and peptide was to the to a of The was for and the was using a analysis was with and via a of with E. cell was by E. cells via in and cell by 4 of with and with of peptide for on the three with the for in the cell three with and in for direct for with with of peptide 1 for and three with of on a H. 1 PubMed Scopus Google and for was for in a of and and for in of the proteins in the was in a of a 1 of for was by in with a was on the of 14-3-3σ (aa with peptide 1 by protein in a with of the was with of and as in 4 of 14-3-3σ (aa and peptide in the as was for into and in was the the of was using W. D. Biol. 2010; PubMed Scopus Google Scholar). Molecular was with from the P. A. of the and Biol. 2011; PubMed Scopus Google by using B. M. R. of a Biol. 2010; PubMed Scopus Google as the and was using P. B. K. and of Biol. 2010; PubMed Scopus Google and D. G. B. structure using and in Biol. PubMed Scopus Google P. F. for the of crystal Biol. 2011; PubMed Scopus Google for The on 14-3-3 proteins for a to was on a system, peptides and a of protein was to a of peptide in steps in 4 peptide a of protein was to a of peptide with the The was set to and the was set to was using analysis structures in this been in the and can be with the and are within the and the We the of the for with aminopeptidase N amino acids structure of proteins fluorescence polarization protein matrix metalloproteinases peptide

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