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No description available.

Cells in embryonic tendon deposit parallel arrays of narrow-diameter collagen fibrils that form a template parallel arrangement of the fibrils is established during mid-embryonic development when the tendon anlagen is formed by cadherin-11-influenced condensation of pre-tendon mesenchyme. It is widely accepted that the assembly, parallelism and packing collagen fibrils is cell mediated however the molecular and cellular mechanisms are unknown. Recent evidence from the Kadler group supports a new model for the orchestration of collagen fibril assembly and organisation during late embryonic development.

Collagenolysis, the catabolism of triple helical collagen, is essential for the physiological remodeling of connective tissues during growth and development. Aberrant collagen degradation is a feature of both inflammatory diseases and cancer cell invasion. Matrix metalloproteinase-1 (MMP-1) is a known collagenase and previous studies have implicated its hemopexin (HPX) domain in binding and possibly destabilising collagen in preparation for hydrolysis by the catalytic (CAT) domain. More recently, conformational freedom and domain separation of the CAT and HPX domains has been proposed to play a role in collagen degradation. This study aims to explore HPX mediated collagen recognition and the postulated flexible state of MMP-1, in order to enhance our understanding of the collagenolytic mechanism. Here, biophysical methods have been used to study the complex formed between the MMP-1 HPX domain and a synthetic triple helical peptide (THP) that encompasses the MMP-1 cleavage site of the collagen α1(I) chain. A programme of site-directed mutagenesis was used to produce an extensive library of recombinant proteins. Surface plasmon resonance (SPR) has been used to characterise a previously unknown collagen binding site (exosite) located in blade 1 of the HPX domain and small angle x-ray scattering has been used to probe the conformational freedom and transient domain separation in mutant forms of both zymogen and mature MMP-1 enzymes. Significant reductions in THP affinity were observed on mutation of either Phe301, Val319 and Asp338, residues forming part of a “ball and socket” joint in the CAT-HPX interface. Disruption of the CAT-HPX interface by mutagenesis, of any of these three residues, severely impacts collagen recognition. Conversely, the reduced collagen binding activity of a "stapled" mutant (in which the CAT and HPX domains are constrained with a disulphide bridge) indicates that the ability of the domains to transiently dislocate is also important. Thus, a balanced equilibrium between these compact and dislocated states is an essential feature of MMP-1 collagenolytic activity.

Oxygen dependent prolyl-4-hydroxylation of the alpha-subunit of the hypoxia inducible transcription factor (HIF-alpha) plays an essential role in the hypoxic response. Hydroxylation of proline residues in the N- or C-terminal oxygen dependent degradation domains (NODD or CODD) increases the affinity of HIF-alpha to the von Hippel-Lindau protein (pVHL) by approx. 1000 fold so signalling for HIF-alpha degradation. With limiting oxygen, HIF-alpha hydroxylation slows, it dimerises with HIF-beta and activates the transcription of a gene array. Prolyl-4-hydroxylation also stabilises the triple helix structure of collagen, the most abundant human protein. Both the collagen and the HIF prolyl hydroxylases (PHDs) are Fe(II) and 2-oxoglutarate (2OG) dependent oxygenases. Crystal structures of PHD2 in complex with CODD were determined in the current study. Together with biochemical analyses, the results demonstrate that catalysis involves a mobile region of PHD2 that encloses the hydroxylation site and stabilises the PHD2.Fe(II).2OG complex. When bound to PHD2 the pyrrolidine ring of the non-hydroxylated proline-residue adopts a C⁴-endo conformation. Evidence is provided that 4R-hydroxylation enables a stereoelectronic effect that changes the proline conformation to the C⁴-exo state, as observed when hydroxylated HIF-alpha is bound to pVHL and in collagen. The results help to rationalise NODD/CODD selectivity data for PHD isoforms and the effects of clinically observed mutations on PHD2 catalysis. Analyses on the interaction of nitric oxide with PHD2 are described and discussed with respect to regulation of the hypoxic response by nitric oxide.

Le collagène de type I osseux subit une série de modifications post-traductionnelles au cours du processus de maturation. Un certain nombre de ces modifications sont quantifiables par dosage : c’est le cas des molécules de pontage enzymatiques pyridinoline (PYD) et désoxypyridinoline (DPD), de la pentosidine (PEN) qui est un produit de glycation, et de la forme native (α) et isomérisée (β) des C-télopeptides (α et β CTX) du collagène de type I. A l’aide d’un modèle de maturation in vitro d’os bovin foetal, nous avons montré que le taux de solubilisation du collagène osseux par la cathepsine K augmente avec la durée d’incubation des os à 37°C. Nous avons également montré que cette augmentation est corrélée au taux des modifications post-traductionnelles du collagène mesurées. Lors d’une étude précédente utilisant ce même modèle de maturation d’os foetal, nous avions déterminé que les modifications post-traductionnelles du collagène osseux influençaient les propriétés mécaniques de l’os. Dans le cadre de ce travail de thèse et pour compléter cette étude, nous avons mis au point un modèle pour étudier isolément l’influence des molécules de pontage PYD et DPD sur les propriétés mécaniques de l’os. Ce modèle utilise de l’os cortical bovin traité aux U.V. puis soumis à des tests de flexion trois points. Dans la même optique, nous avons contribué à la mise au point d’un dosage en chromatographie en phase liquide à haute performance qui permet de quantifier à la fois les formes matures et immatures des molécules de pontage pyridinoliques (PYD, DPD, HLNL et DHLNL) sur le même chromatogramme / Type I bone collagen undergoes a series of posttranslational modifications during maturation process. Some of them are quantifiable by assays: the enzymatic cross-links pyridinoline (PYD) and deoxypyridinoline (DPD), the advanced glycation end product pentosidine (PEN), and the native (α) and isomerized (β) forms of the type I collagen C-telopeptides (α and β CTX). With an in vitro model of bovine fetal bone maturation, we showed that bone collagen solubilization by cathepsin K increases with the duration of bone incubation at 37°C. We also showed a correlation between this increase of solubilization and the level of measured collagen posttranslational modifications. In a previous study, using the same fetal bovine bone maturation, our results had suggested a link between bone collagen posttranslational modifications and bone mechanical properties. In the aim to complete this study, we developed a model to focus on PYD and DPD influence in bone mechanical properties. This model uses bovine cortical bone subjected to ultraviolet light before three points binding tests. In the same purpose, we contributed to develop a High-Performance Liquid Chromatography essay, quantifying mature and immature forms of pyridinium crosslinks (PYD, DPD, HLNL and DHLNL) in an unique chromatogram