published_or_final_version / Dentistry / Doctoral / Doctor of Philosophy

published_or_final_version / Zoology / Doctoral / Doctor of Philosophy

The tissue inhibitors of metalloproteinases (TIMPs) are endogenous inhibitors of the matrix metalloproteinases (MMPs). Since unregulated MMP activities are linked to arthritis, cancer, and atherosclerosis, TIMP variants that are selective inhibitors of disease-related MMPs have potential therapeutic value. The structures of TIMP/MMP complexes reveal that most interactions with the MMP involve the N-terminal region of TIMP and the C-D B-strand connector which occupy the primed (right side of the active site) and unprimed (left side) regions of the active site. Substitutions for Thr2 of N-TIMP- 1 strongly influence MMP selectivity. In this study we found that Arg and Gly, which generally reduce MMP affinity, have less effect on binding to MMP-9. When the Arg mutation is added to the NTIMP-1 mutant with AB loop of TIMP-2, it produced a gelatinase-specific inhibitor with Ki values of 2.8 and 0.4 nM for MMP-2 and MMP-9, respectively. The Gly mutant has a Ki of 2.1 nM for MMP-9 and > 40 uM for MMP-2, indicating that engineered TIMPs can discriminate between MMPs in the same subfamily. In collaboration with Dr. Yingnan Zhang at Genentech, we have developed a protocol for the phage display of full-length human TIMP-2 to identify high-affinity selective inhibitors of human MMP-1, a protease that plays a role in cleaving extracellular matrix (ECM) components, connective tissue remodeling during development, angiogenesis, and apoptosis. We have generated a library containing 2x1010 variants of TIMP-2 randomized at residues 2-6 (L1), at residues 34-40 (L2) and 67-70 (L3). / The L1 library yielded a positive signal for MMP-1 binding. Clones from the L1 library, designated TM1, TM8, TM13, and TM14, were isolated after 5 rounds of selection on immobilized MMP-1 and MMP-3 and found to show a greater selectivity for MMP-1 relative to MMP-3. TM8, which has Ser2 to Asp and Ser4 to Ala substitutions, showed the greatest apparent selectivity of 10-fold toward MMP-1 compared to MMP-3. The various mutations identified by phage display were introduced into recombinant Nterminal TIMP-2 and the variants characterized as inhibitors of an array of MMP catalytic domains. The TM8-based mutant showed pronounced selectivity (> 1000-fold for MMP-1 vs. MMP-3) and may be a step towards the generation of MMP-1-specific inhibitors. Molecular modeling was used to rationalize the structural basis of MMP selectivity in the mutants. / by Harinathachari Bahudhanapati. / Thesis (Ph.D.)--Florida Atlantic University, 2009. / Includes bibliography. / Electronic reproduction. Boca Raton, Fla., 2009. Mode of access: World Wide Web.

Matrix metalloproteinases (MMPs) constitute the major class of enzymes capable of degrading all protein components of extracellular matrix (ECM) and have important roles in normal physiologic processes of maintaining tissue integrity and remodeling. However, excess MMP activities are associated with many diseases including rheumatoid arthritis and osteoarthritis, cardiomyopathy, and macular degeneration. The activity of MMPs is regulated by their endogenous protein inhibitors, the tissue inhibitors of metalloproteinases (TIMPs) which are avid broad-spectrum inhibitors of numerous human matrixins (MMPs and ADAMs). Uncontrolled matrix degradation occurs when the balance between TIMPs and MMPs is disrupted, resulting in serious diseases such as cancer, arthritis and chronic tissue ulcers. Thus, the engineering of TIMPs to produce highly selective and efficacious inhibitors of individual MMPs may be utilized for future treatment of diseases. Such engineering requires detailed analysis for the structural and biophysical information of MMP-TIMP interaction. Changes in the dynamics of proteins and solvent that accompany their associations with different binding partners, influence the specificity of binding through entropic effects. From the current studies it appears that the interactions of the inhibitory domains of TIMPs-1 and -2 (N-TIMPs) with MT1-MMP are driven by entropy increases that are partitioned between solvent and conformational entropy (ΔSsolv and ΔSconf), and a large conformational entropy penalty is responsible for the weak inhibition of MT1-MMP by NT1.We investigated how mutations that modify N-TIMP selectivity affect the thermodynamics of interactions with MMP1, MMP3 and MT1-MMP. The weak inhibition of MT1-MMP by N-TIMP-1 is enhanced by mutation of threonine 98, on the edge of the binding ridge, to leucine. This mutation increases the large ΔSconf cost for binding to MT1-MMP but this is offset by a greater increase in ΔSsolv. In contrast, this mutation enhances binding to MMP3 by increasing ΔSconf for the interaction. ΔSsolv and ΔSconf show mutual compensation for all interactions, with characteristic ranges for each MMP. Distinct electrostatic and dynamic features of MMPs are key factors in their selective inhibition. / Includes bibliography. / Dissertation (Ph.D.)--Florida Atlantic University, 2016. / FAU Electronic Theses and Dissertations Collection

Expression of membrane-type matrix metalloproteinases (MT) and tissue inhibitors of matrix metalloproteinases (TIMP) was evaluated in sheep embryos and uterus during the pre- and peri-implantation periods. Embryos and uterine samples were surgically collected from ewes on days 9, 11, 13, and 15 of pregnancy (n=3 ewes/day) and of the estrous cycle (n=2 ewes/day). Total RNA was extracted and RT-PCR were performed using primers specifically designed from published human, mouse, and bovine complete cDNA sequences for MT-1, -2, -3, and -5, and TIMP-1, -2 and -3. Multiplex PCR were performed on uterine samples for each gene at optimal cycles and temperatures with 18S rRNA as the internal standard. For embryos, PCR were conducted for 40 cycles at optimal temperatures. MT-1, -2, -3, and -5 were observed in pregnant and nonpregnant uterus during all days of collection. No difference (P>0.10) was observed in MT-1 or -2 expression due to day of collection. However, pregnant uterus expressed more (P=0.096) MT-1 than nonpregnant uterus, whereas expression of MT-2 was greater (P<0.05) in nonpregnant compared to pregnant uterus. No differences (P>0.10) in MT-3 expression were observed due to pregnancy status, however Day 9 and 11 expressed more MT-3 than Day 15. Uterine MT-5 expression was not different (P>0.10) between pregnant and nonpregnant females, however Day 15 uterus expressed less (P<0.05) MT-5 then Day 11 and 13 uteri. TIMP-1 expression was greater (P<0.05) in pregnant compared to nonpregnant uterus, but did not differ (P>0.10) by day of collection. TIMP-2 did not differ (P>0.10) by pregnancy status or day of collection but the interaction was significant (P<0.05). TIMP-2 expression was greatest in Day 9 pregnant uterus and least in Day 9 nonpregnant uterus. No difference (P>0.10) was observed in expression of TIMP-3 due to day of collection or pregnancy status. Embryos expressed MT-3 and -5 during Days 9-15 of development, however, MT-1 and -2 were not detected. The presence of MT and TIMP in the endometrium suggests these proteins may play important roles in regulating extracellular matrix degradation and activating other matrix metalloproteinases for endometrial remodeling and preparation for implantation. Embryonic MT may participate in the processes of embryonic expansion, elongation and attachment. / Graduation date: 2002

In biotechnology and drug delivery silica materials are of interest but the controlled generation of silicic acid is difficult. To get more insight into the molecular mechanisms that control biosilification, it is important to study the proteins involved in this process. The sponge protein silicatein α synthesises part of the axial filament in the spicules which in situ polymerises silicic acid. It has been demonstrated that the polymerisation of siloxanes such as for example tetraethoxysilane (TEOS) can be carried out by both wild type and recombinant silicatein α. Unfortunately, it has not been possible yet to get reasonable amounts of wild type or recombinant silicatein α to perform biophysical studies. The human cysteine protease cathepsin L has almost 50 % identical residues with silicatein α. To get more insight into the mechanism of silica polymerisation, cathepsin L mutants were generated by our collaborators. Those mutants show sequence features and activity specific for silicatein α. The X-ray structure of one of those mutants (mutant 4SER) to 1.5 Å has allowed us to propose a new chemical mechanism for the catalysis of silicic acid polymerisation. ADP-β-D-glycero-D-mannoheptose and ADP-β-L-glycero-D-mannoheptose are interconverted by the SDR-enzyme ADP-β-L-glycero-D-mannoheptose 6-epimerase (AGME). This epimerisation reaction is the final reaction in the biosynthetic route of the precursor of heptose. Heptose is a part of the inner core of the lipopolysaccharide in Gram-negative bacteria. In mutants which do not have heptose, nonpolar compounds can penetrate more easily through the outer membrane. These mutants also show less pathogenicity. As a consequence the lipopolysaccharide biosynthetic pathway represents an interesting target for antimicrobial compounds. The crystal structure of AGME in complex with ADP-α-glucose has already been solved. From this structure a catalytic mechanism for this enzyme has been proposed with Tyr140 and Lys178 operating as acid/base residues. The disordered nature of the nucleotide sugar’s glucose moiety in the previous structure due to the wrong configuration of the sugar has hindered assignment of a mechanism. The determination of the X-ray structure of AGME Y140F in complex with a substrate in the β-manno configuration (ADP-β-mannose) to 2.4 Å resolution has given new insight into the mechanism of this SDR enzyme. A mechanism is proposed with only Tyr140 operating as catalytic acid/base. Initially it was thought that MMP-3 participates in the synovitis cascade. Glycoproteins, several parts of the ECM such as fibronectin and laminin and also collagens and procollagens are targets of this matrixin. Furthermore MMP-3 can undergo autocatalysis and is also able to cleave a range of other members of the matrixin family. Matrixins also play an important role in diseases such as cancer, rheumatoid arthritis and osteoporosis. This makes them targets for inhibitor design. Many structures of matrix metalloproteinases, such as stromelysin-1, in complex with various inhibitors have already been solved. The structures of the catalytic domain of MMP-3 in complex with two nonpeptide inhibitors are discussed.

Metastatic cancers are problematic because they spread throughout the body. A crucial step in cancer metastasis is the separation of the cancer cells from their surrounding normal cells. This occurs due to suppression or destruction of cell adhesion molecules such as E-cadherin, occludin, and various claudins. The Snail and Slug transcription factors play a direct role in suppressing these cell adhesion molecules through their SNAG repression domain. We explored the possibility of developing an ELISA diagnostics capable of detecting soluble E-cadherin, occludin, and claudin fragments in the serum of cancer patients. Using several bioinformatics tools, unique extracellular antigenic sequences were identified on claudins-1, 4, 16, occludin, and E-cadherin. These sequences were cloned as GST fusion proteins, expressed, and purified in large quantities to raise antibodies. In parallel, expression profiling of metastatic cancer cell lines was carried out to derive a correlation between Snail-Slug expression and suppression of cell adhesion molecules. / by Michael Irvine. / Thesis (M.S.)--Florida Atlantic University, 2009. / Includes bibliography. / Electronic reproduction. Boca Raton, Fla., 2009. Mode of access: World Wide Web.

Tissue inhibitors of metalloproteinases (TIMPs) comprise a family of four proteins in humans that modulate the turnover of the extracellular matrix by regulating the activities of endopeptidases that catalyze its degradation, especially the matrix metalloproteinases (MMP). In general, the four TIMPs are broad-spectrum tight binding inhibitors of MMPs with individual differences in specificity. In this study, we attempted to understand the basis of such variation by using membrane type-1 MMP (MT1-MMP) as a model, since it is inefficiently inhibited by TIMP-1 in contrast with the other TIMPs. We designed and engineered mutations in the N-domain of TIMP-1, based on current knowledge of TIMP interactions. By measuring inhibition levels of each mutant against several MMPs, including MT1-MMP, we were able to obtain a triple mutant with an vii improved affinity for MT1-MMP. / Our results, along with previous data, confirm that multiple residues in the critical interface segments between TIMPs and MMPs, namely at positions 2, 4, 5, 6, and 98, are key in determining the basic interaction between the two molecules. The second part of this work focused on naturally occurring mutations in TIMP-3 which cause an early form of macular degeneration called Sorsby's Fundus Dystrophy (SFD). The TIMP-3 mutants identified so far share certain features but the mechanism by which they result in macular disease is not yet understood. As an initial step, we expressed recombinant TIMP-3 carrying a truncation mutation, glutamic acid 139 to a stop codon (E139X), and assessed its activity towards representative MMPs and tumor necrosis factor-(Sa (Bconverting enzyme, another metalloproteinase normally inhibited by TIMP-3. Our results indicate that this mutation does not impair the inhibitory activity of TIMP-3. / Expression of this mutant in mammalian retinal cells revealed a difference in localization between wild-type and E139X mutant TIMP-3. Therefore, we concluded that the SFD mutations may actually influence the processing and/or binding properties of TIMP-3 in the retina. / by Asmaa Bilal Hamze. / Vita. / Thesis (Ph.D.)--Florida Atlantic University, 2008. / Includes bibliography. / Electronic reproduction. Boca Raton, Fla., 2008. Mode of access: World Wide Web.

Includes bibliographical references (leaves 292-352) Determines the role of matrix metalloproteinases, MMP-2 and MMP-9 in reperfusion injury following skeletal muscle ischaemia; and, whether inhibition of MMPs by doxycycline protects against tissue damage.

Includes bibliographical references (leaves 292-352) / xvi, 352 leaves : / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Determines the role of matrix metalloproteinases, MMP-2 and MMP-9 in reperfusion injury following skeletal muscle ischaemia; and, whether inhibition of MMPs by doxycycline protects against tissue damage. / Thesis (M.D.)--University of Adelaide, Dept. of Surgery, 2002