FKBP-65 is a member of the immunophilin class of proteins consisting primarily of the cyclophilins and the FKBP's which bind the immunosuppressant drugs cyclosporin A and FK506, respectively. Immunophilins possess peptidylprolyl cis-trans isomerase (PPiase) activity which is inhibited upon binding of their respective macrolides. Specific cellular targets of most immunophilins and the role of PPiase activity in vivo remain largely unknown. FKBP-65 has been proposed as a molecular chaperone of tropoelastin (TE), the soluble precursor of elastin (Davis et al. 1998). TE contains 12% proline residues, many of which are found in VPGVG repeats. When P2 is in the trans conformation, these motifs form repeated type-II ~-turns and ~-spirals resulting in selfassociation of TE via an inverse temperature-dependent transition known as coacervation. Coacervation can be monitored by turbidity increases at 300 nm. We have used purified recombinant FKBP-65 in coacervation assays with chick aorta TE to show that FKBP65 specifically affects the coacervation characteristics of TE in a concentration-dependant manner. The overall extent of coacervation of TE could be increased by more than 2-fold over controls by inclusion of nM amounts of FKBP-65 in the assay. Also, FKBP-65 decreases the coacervation onset temperature of TE by 5-l 0°C. Structural evidence suggests that the influence of FKBP65 on tropoelastin coacervation may be due to its ability to increase the ~ structural content of tropoelastin. These results suggest that FKBP-65 may be a physiologically relevant, TE-specific molecular chaperone. / Thesis / Master of Science (MSc)

Molecular diffusion is an inherent feature of all fluid systems. The processes and interactions that characterize these systems are in some way dependent upon the mobility of the component molecules. Pulsed field-gradient spin-echo nuclear magnetic resonance (PGSE NMR) is a powerful tool for the study of molecular diffusion; for heterogeneous systems, such as those typically found in biology, this technique is unsurpassed in the diversity of systems that yield to its probing. The aim of the work presented in this thesis was to use an integrated NMR-based approach, in conjunction with computer modeling, for the study of molecular diffusion in compartmentalized and multicomponent biological systems. Erythrocyte suspensions provided an ideal experimental system for the study of compartmentalized diffusion in cells. Water exchanges rapidly between the intra- and extracellular regions and, as the major constituent of the cell, provides a strong and predominant proton NMR signal. In addition, the cells are known to align in the strong static magnetic field of the spectrometer. As a consequence of these two properties, the signal intensity from a suitably designed series of PGSE NMR experiments exhibits a series of maxima and minima when graphed as a function of the magnitude of the spatial wave number vector q. The apparently periodic phenomenon is mathematically analogous to optical diffraction and interference and is referred to here as diffusion-coherence. It is the characterization of this phenomenon, with the aid of computer-based models, which was the focus of a major section of the work described herein. Two quite distinct molecular systems formed the basis of the work in which I investigated diffusion in multicomponent systems. Both systems involved molecules that undergo self-association such that at equilibrium a population distribution of different oligomeric species is present. The first of these was tropoelastin, the monomeric subunit of elastin, which under certain conditions aggregates to form a coacervate. The second system was N-methylacetamide (NMA) which also undergoes extensive self-association. NMA oligomers have previously been studied as peptide analogues due to the presence in the monomer of a peptide linkage. In this work the aim was to use PGSE NMR diffusion measurements, in a manner that is in many ways analogous to analytical ultracentrifugation, to obtain estimates of hydrodynamic and thermodynamic parameters. Computer modeling was also used extensively in this section of work for the interpretation of the experimental data.

Molecular diffusion is an inherent feature of all fluid systems. The processes and interactions that characterize these systems are in some way dependent upon the mobility of the component molecules. Pulsed field-gradient spin-echo nuclear magnetic resonance (PGSE NMR) is a powerful tool for the study of molecular diffusion; for heterogeneous systems, such as those typically found in biology, this technique is unsurpassed in the diversity of systems that yield to its probing. The aim of the work presented in this thesis was to use an integrated NMR-based approach, in conjunction with computer modeling, for the study of molecular diffusion in compartmentalized and multicomponent biological systems. Erythrocyte suspensions provided an ideal experimental system for the study of compartmentalized diffusion in cells. Water exchanges rapidly between the intra- and extracellular regions and, as the major constituent of the cell, provides a strong and predominant proton NMR signal. In addition, the cells are known to align in the strong static magnetic field of the spectrometer. As a consequence of these two properties, the signal intensity from a suitably designed series of PGSE NMR experiments exhibits a series of maxima and minima when graphed as a function of the magnitude of the spatial wave number vector q. The apparently periodic phenomenon is mathematically analogous to optical diffraction and interference and is referred to here as diffusion-coherence. It is the characterization of this phenomenon, with the aid of computer-based models, which was the focus of a major section of the work described herein. Two quite distinct molecular systems formed the basis of the work in which I investigated diffusion in multicomponent systems. Both systems involved molecules that undergo self-association such that at equilibrium a population distribution of different oligomeric species is present. The first of these was tropoelastin, the monomeric subunit of elastin, which under certain conditions aggregates to form a coacervate. The second system was N-methylacetamide (NMA) which also undergoes extensive self-association. NMA oligomers have previously been studied as peptide analogues due to the presence in the monomer of a peptide linkage. In this work the aim was to use PGSE NMR diffusion measurements, in a manner that is in many ways analogous to analytical ultracentrifugation, to obtain estimates of hydrodynamic and thermodynamic parameters. Computer modeling was also used extensively in this section of work for the interpretation of the experimental data.

<p> FKBP65 is a 65 kDa FK-506 binding protein containing 4 putative peptidyl prolyl isomerase (PPiase) domains, whose expression level parallels that oftropoelastin, the soluble precursor ofelastin. Studies from other laboratories have established that FKBP65 associates with tropoelastin (TE) in the endoplasmic reticulum (ER) and dissociates from TE before reaching the Golgi apparatus (Patterson et al., 2000). TE contains 12% proline residues, which are often found in VPGVG repeats, and it has been suggested that these repeats formp-turns and subsequently P-spirals (Urry et al., 1992). The formation ofthe P-spiral is thought to be essential to endow the elastic properties of the elastin fibers. In order to form a P-turn, the proline residue at position 2 ofthe VPGVG sequence must be in trans conformation (Urry et al., 1995). Therefore, it was hypothesized by Davis and coworkers (Davis et al., 1998) that FKBP65, as a PPiase, may play an important role in the folding oftropoelastin by enhancing the formation ofP-turns in the ER, and thus elastic fiber formation. In the present study we have studied the coacervation (a reversible, temperature-dependent, self association process) ofTE and recombinant elastin model polypeptide, EP4, in the absence or presence ofrecombinant FKBP65 (rFKBP65). rFKBP65 was shown to enhance the coacervation process of TE, by lowering the coacervation temperature (T c) and increasing the overall extent of coacervation. In the kinetic study ofcoacervation ofTE at a constant temperature, rFKBP65 increased both the initial rate ofthe coacervation process and the overall extent ofcoacervation. These effects are specific to rFKBP65, as FKBP12 has no effect on the coacervation process. Rapamycin, an inhibitor ofthe PPiase activity ofFK-506 binding proteins, did not alter rFKBP65's effect on TE coacervation. </p> <p>In contrast to TE, rFKBP65 affected the coacervation process ofEP4 by increasing the T c, and by enhancing the dissociation of coacervates when temperature is decreased. Once again, these effects are specific to rFKBP65, as FKBP12 and BSA were shown to have no effect on the coacervation ofEP4. The effect of small pH changes on rFKBP65 was also investigated, and it was found that lowering the pH from 7.5 to 6.0 had no effect on rFKBP65's secondary structure or coacervation-altering activity. </p> <p> In summary, this study, along with an earlier study from this laboratory, has shown that FKBP65 affects the coacervation process ofTE. In addition, the coacervation pro·cess of an elastin model polypeptide, EP4, is also modulated by FKBP65. However, the mechanism ofthese effects remains unclear. Nevertheless, along with the data established by other laboratories, FKBP65 does appear to be a strong candidate as a molecular chaperone for tropoelastin, and may play an important role in the elastogenesis process. </p> / Thesis / Master of Science (MSc)

<p>"Smart" polymers that sense stimuli in aqueous environments and that respond with a pronounced change in their solvation are of great utility in biotechnology and medicine. Currently, however, only few peptide polymers are known to display this behavior. Here, we uncover the syntax -- defined as the arrangement of amino acids (letters) into repeat units (words) that have a functional behavior of interest -- of a novel and extensive family of genetically encoded "smart" peptide polymers, termed syntactomers, that dictates their ability to undergo a soluble to insoluble phase transition at temperatures above a lower critical solution temperature (LCST) or below an upper critical solution temperature (UCST). We show that this syntax ranges from phase transition polymers composed of simple repeats of a few amino acids to polymers whose syntax resembles the complex sequence of peptide drugs and protein domains that exhibit dual levels of function, as seen by their stimulus responsiveness and biological activity. This seamless fusion of materials and protein design embodied by syntactomers promises, we hope, a new generation of designer polymers with multiple levels of embedded functionality that should lead to new functional materials of broad interest</p> / Dissertation

An increase in coronary disease prevalence and mortality highlights the growing need for therapies to treat atherosclerotic vessels. While current bypass procedures utilize autologous vessels for small caliber grafts, there is a big push towards the use of engineered tissues to bypass diseased portions of arteries. Cardiovascular tissue engineering is the emerging discipline that aims to create a functional substitute. Ideally, a tissue engineered blood vessel would possess the relevant cells and matrix proteins that interact in a physiologic manner and will respond to the environmental cues of the host. A particular obstacle to achieving appropriate vessel structure is the inclusion of elastin in a tissue engineered media equivalent. Rat arterial smooth muscle cells that were retrovirally mediated to overexpress versican V3 have been shown to have an enhanced expression of tropoelastin in vitro as well as in injury models. The unique tropoelastin expression by these adult cells was studied in the context of tissue engineered media equivalents. Changes to the extracellular matrix architecture and composition, stimulation with medium additives, and cyclic distension, were shown to increase tropoelastin synthesis in V3 versican overexpressing cells. This study not only expanded the characterization of V3 versican overexpressing smooth muscle cells, it also explored the novel use of these cells as a tropoelastin source in a tissue engineered media equivalent.

Les peptides élastiques (ELP, Elastin-like peptide) sont d'excellents exemples de polymères biomimétiques récemment proposés en médecine régénérative, en particulier dans le domaine de l'ingénierie tissulaire des tissus mous (peau, vaisseaux sanguins, poumons…) pour lesquels la modélisation est complexe car l'instruction correcte des cellules nécessite une élasticité fonctionnelle. L'ajustement précis de la structure primaire des ELP peut moduler voire améliorer les propriétés physico-chimiques, structurales et fonctionnelles de la protéine native. De plus, la capacité des ELP à ajuster leurs caractéristiques physico-chimiques en réponse à des stimuli externes (température, pH), les définit comme des polymères intelligents. Ces polymères bioactifs offrent ainsi une large gamme d'applications très prometteuses encore très peu explorées dans les technologies d'ingénierie tissulaire et les systèmes d'administration de médicaments. Dans ce travail de thèse, nous avons développé, caractérisé et évalué les potentiels thérapeutiques d'une protéine élastique synthétique, Elactiv', inspirée de la structure unique de la tropoélastine humaine, précurseur soluble de l'élastine. Elactiv' conserve les caractéristiques physico-chimiques (comportement thermosensible, propriétés d'autoassemblage) et les fonctions biologiques de la protéine native (prolifération, différenciation et survie des fibroblastes dermiques et kératinocytes humains, sensibilité à la dégradation enzymatique). De plus, Elactiv' possède la particularité in vitro de s'incorporer dans les fibres élastiques néo-synthétisées par des fibroblastes dermiques sains, et d'induire la synthèse de tropoélastine fibrillaire par des fibroblastes pathologiques, syndrome de Williams-Beuren, qui ne synthétisent pas ou très peu de fibres élastiques. Un hydrogel formé exclusivement d'Elactiv' a permis d'accéder aux propriétés mécaniques de l'ensemble et de vérifier sa biocompatibilité in vitro et son innocuité et sa résorption in vivo. Enfin, l'association de la protéine Elactiv' aux dendrigrafts de poly(L-lysine), polymères synthétiques hautement fonctionnalisables, a permis de faire évoluer l'architecture de l'hydrogel vers un biomatériau hybride dans le but d'augmenter ses propriétés mécaniques et biologiques. Ainsi, les potentiels biomimétiques et thérapeutiques de la protéine Elactiv' en font un candidat prometteur pour la régénération des tissus mous / Elastin-like peptides are excellent examples of biomimetic polymers recently proposed in regenerative medicine, particularly for soft tissue engineering (skin, blood vessels, lung ...) for which modeling is a complex task requiring functional elasticity to insctruct cells properelly. Fine-tuning of ELP’s primary structure can modulate or improve physicochemical, structural and functional properties of the native protein. In addition, the adjustment of ELP physicochemical characteristics through external stimuli (temperature, pH) defined them as intelligent polymers. These bioactive polymers thus provide a wide range of very promising applications in tissue engineering and drug delivery, although this has been under-explored until then. In this thesis, we have developed, characterized and evaluated therapeutic potentials of Elactiv', a synthetic elastic protein inspired by the unique structure of the human tropoelastin, the soluble precursor of elastin. Elactiv’ retains physicochemical characteristics (thermoresponsive behavior, self-assembly properties) and biological functions of the native protein (proliferation, differentiation and survival of human keratinocytes and dermal fibroblasts, susceptibility to enzymatic degradation). Besides, Elactiv’ is able to incorporate into neosynthesized elastic fibers by healthy dermal fibroblasts, and to induce fibrillar tropoelastin synthesis by pathological fibroblasts, Williams-Beuren syndrome, which do not synthesize or very few elastic fibres. A hydrogel formed exclusively of Elactiv’ allowed to access to mechanical properties of the scaffold and to verify its biocompatibility in vitro and its safety and resorption in vivo. Finally, the association of Elactiv' protein to poly(L-lysine) dendrigrafts, highly functionalizable synthetic polymers, enabled to evolve the hydrogel's architecture to a hybrid biomaterial in order to increase its mechanical and biological properties for skin tissue engineering. Taken together, biomimetic and therapeutic potentials of Elactiv' protein make it a promising candidate for soft tissue regeneration