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roles of substrate sequence and thermal stability in the collagenolytic action of matrix metalloproteinases

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Date Issued:
2006
Summary:
Matrix metalloproteinases are implicated in diseases accompanied by pathological destruction of tissues. Collagen is an important physiological barrier between tissues and therefore the proteases that can cleave intact collagen are of great interest as possible therapeutic targets. The collagenolytic activity of MMPs varies greatly and within the MMP family distinct preferences for collagen types are seen. Subtle variations in sequence, triple-helix local stability, and Gly register were hypothesized to be among the determinants that may guide these specificities. In order to develop collagenolysis-based inhibitors, we need to examine the interactions between MMPs and collagen on a molecular level. We utilized FRET triple-helical substrates to compare the triple-helical peptidase activities and effects of hypothesized specificity determinants on triple-helical peptidase activities of collagenolytic MMPs. Kinetic parameters of triple-helical peptide hydrolysis were determined for MMP-1, -8, -13, MT1-MMP, and MT2-MMP, and activation energies for this process were calculated. It was determined that MMPs possess differential abilities to process more thermally stable triple-helices. MMP-13 was least sensitive and MMP-1 and MT1-MMP were most sensitive to the enhanced thermal stability of substrates. In agreement with kinetic parameters, hydrolysis of more thermally stable substrates required higher activation energies. MMP collagen specificity also has been examined using a triple-helical substrate library. The consensus type I-III collagen sequence was modified in positions P1' and P2. Single substitution of Gln by Orn in P2 position was beneficial for MMP-13 and MT2 MMP while detrimental for MMP-1, MMP-2, MMP-8, and MT1-MMP activities. Single substitution of Leu by Cys(Mob) in P1' position was disfavored by MMP-1, -2, and -9 and favored by MMP-8, MMP-13, MT1-MMP, and MT2-MMP. Assays of MMP reactions with a sequence containing substitutions in both positions revealed that these sites are not independent from each other, which might indicate an enzyme "induced fit". The significance of triple-helicity for MMP substrate specificity was evaluated by using a library of peptides containing interruptions of Gly register in the vicinity of the scissile bond. Interruption of the Gly register led to the decreased triple-helicity of substrates and subsequently to the inability of membrane type MMPs to cleave such substrates within the interrupted region.
Title: The roles of substrate sequence and thermal stability in the collagenolytic action of matrix metalloproteinases.
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Name(s): Minond, Dmitriy
Florida Atlantic University, Degree Grantor
Charles E. Schmidt College of Science
Department of Chemistry and Biochemistry
Type of Resource: text
Genre: Electronic Thesis Or Dissertation
Issuance: monographic
Date Issued: 2006
Publisher: Florida Atlantic University
Place of Publication: Boca Raton, Fla.
Physical Form: application/pdf
Extent: 200 p.
Language(s): English
Summary: Matrix metalloproteinases are implicated in diseases accompanied by pathological destruction of tissues. Collagen is an important physiological barrier between tissues and therefore the proteases that can cleave intact collagen are of great interest as possible therapeutic targets. The collagenolytic activity of MMPs varies greatly and within the MMP family distinct preferences for collagen types are seen. Subtle variations in sequence, triple-helix local stability, and Gly register were hypothesized to be among the determinants that may guide these specificities. In order to develop collagenolysis-based inhibitors, we need to examine the interactions between MMPs and collagen on a molecular level. We utilized FRET triple-helical substrates to compare the triple-helical peptidase activities and effects of hypothesized specificity determinants on triple-helical peptidase activities of collagenolytic MMPs. Kinetic parameters of triple-helical peptide hydrolysis were determined for MMP-1, -8, -13, MT1-MMP, and MT2-MMP, and activation energies for this process were calculated. It was determined that MMPs possess differential abilities to process more thermally stable triple-helices. MMP-13 was least sensitive and MMP-1 and MT1-MMP were most sensitive to the enhanced thermal stability of substrates. In agreement with kinetic parameters, hydrolysis of more thermally stable substrates required higher activation energies. MMP collagen specificity also has been examined using a triple-helical substrate library. The consensus type I-III collagen sequence was modified in positions P1' and P2. Single substitution of Gln by Orn in P2 position was beneficial for MMP-13 and MT2 MMP while detrimental for MMP-1, MMP-2, MMP-8, and MT1-MMP activities. Single substitution of Leu by Cys(Mob) in P1' position was disfavored by MMP-1, -2, and -9 and favored by MMP-8, MMP-13, MT1-MMP, and MT2-MMP. Assays of MMP reactions with a sequence containing substitutions in both positions revealed that these sites are not independent from each other, which might indicate an enzyme "induced fit". The significance of triple-helicity for MMP substrate specificity was evaluated by using a library of peptides containing interruptions of Gly register in the vicinity of the scissile bond. Interruption of the Gly register led to the decreased triple-helicity of substrates and subsequently to the inability of membrane type MMPs to cleave such substrates within the interrupted region.
Identifier: 9780542675331 (isbn), 12212 (digitool), FADT12212 (IID), fau:9119 (fedora)
Collection: FAU Electronic Theses and Dissertations Collection
Note(s): Adviser: Gregg B. Fields.
Thesis (Ph.D.)--Florida Atlantic University, 2006.
Subject(s): Chemistry, Biochemistry
Held by: Florida Atlantic University Libraries
Persistent Link to This Record: http://purl.flvc.org/fcla/dt/12212
Sublocation: Digital Library
Use and Reproduction: Copyright © is held by the author, with permission granted to Florida Atlantic University to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
Use and Reproduction: http://rightsstatements.org/vocab/InC/1.0/
Host Institution: FAU
Is Part of Series: Florida Atlantic University Digital Library Collections.