Recent studies support the association between metabolic syndrome (MetS), a cluster of cardiovascular risk factors, and diastolic dysfunction. Disproportionate collagen accumulation, particularly cross-linking of collagen, plays a key role in translating interstitial fibrosis into mechanical chamber stiffness and diastolic dysfunction. Characteristic changes in the expression and activity of myocardial lysyl oxidase (LOX), a matrix modifying enzyme that catalyzes cross-linked collagen, are unclear in MetS. We established a diet-induced MetS model to study diastolic dysfunction by treating male C57BL/6 mice a high-fat high-simple carbohydrate (HFHSC) diet for 6 months. Despite blunted gene expression of LOX isoforms, MetS mice demonstrated significant increase in the ratio of protein expression of mature to proenzyme LOX, enhanced LOX activity, and increased cardiac cross-linked collagen compared with controls. This fibrotic response coincided with marked increase in left ventricular end-diastolic pressure and stiffness and impaired diastolic filling pattern. Our data demonstrate that diet-induced MetS alters the remodeling enzyme LOX, thereby increasing the amount of crosslinking and inducing diastolic dysfunction.Furthermore we examined the role of T-lymphocytes in myocardial LOX regulation in diet-induced fibrotic hearts. Female SCID mice which are devoid of functional T-lymphocytes and C57BL/6 mice were treated with HFHSC diet for 12 months. Similar to male C67BL/6, female HFHSC-fed C57BL/6 mice demonstrated significant increase in maturation and catalytic activity of myocardial LOX, cross-linking, ventricular stiffness and diastolic dysfunction. Whereas induction of LOX protein was minimal in SCID mice compared with wild-type counterparts. Correspondingly fibrillar cross-linked collagen formation and diastolic dysfunction were less prominent in SCID mice. Our results suggest a potential role of T-lymphocytes in induction of myocardial stiffness and diastolic dysfunction through modulation of LOX-dependent collagen maturation.Moreover we studied the role of leptin, an adipokine over-produced in MetS with fibrotic effects in non-cardiac tissues, as a key mediator of profibrogenic responses in the heart by administrating leptin to C57BL/6 and leptin-deficient ob/ob mice. With exogenous leptin administration ob/ob mice displayed passive diastolic filling dysfunction that coincided with increase in myocardial collagen compared with ob/ob controls. Our findings suggest profibrotic effects of leptin in the heart, primarily through predominance of collagen synthesis over degradation.

Incretins are gastric hormones released by intestinal K-cells in response to food consumption and stimulate insulin secretion from pancreatic beta cells. One of these hormones, glucose-dependent insulinotropic peptide (GIP) is also anabolic in bone. Individuals with diabetes experience diminished bone quality caused by a low bone formation osteopenia. The present study seeks to identify a mechanism for diabetic osteopenia in which diabetes interferes with GIP-stimulated increases in the collagen cross-linking enzyme lysyl oxidase (LOX) in osteoblasts, leading to decreased collagen integrity and the trabecular abnormalities seen in diabetic bone. Micro-CT analysis and picrosirius red histology of long bones from LOX +/- and wild type mice made diabetic by low dose streptozotocin induction revealed a profound exacerbation of the decreased bone volume, impaired trabecular structure, and disorganized collagen matrix seen in diabetic mice when the mice were also haploinsufficient for LOX. Furthermore, qPCR of RNA isolated from diabetic long bones revealed a more than 20 fold decrease in LOX expression in diabetic bone from wild type mice. Treatment of wild type osteoblasts in culture with GIP results in a significant increase in LOX transcript and protein levels. Interestingly in our diabetic mice there is a decrease in osteoblast derived LOX and an abnormal increase in serum levels of the anti-incretin gut-derived dopamine, which is known to inhibit the effects of GIP in the pancreas. Therefore the ability of dopamine to inhibit GIP-stimulated signaling in osteoblasts was examined. Data indicate a strong dose-dependent inhibition of GIP-stimulated LOX expression when primary osteoblast cultures are pretreated with dopamine. Finally, pretreatment of primary osteoblasts with the dopamine receptor inhibitor amisulpride restored the impaired GIP stimulated increases in LOX expression in osteoblasts isolated from diabetic mice. This study defines a potential mechanism for diabetic bone disease and suggests that interference with dopamine signaling would likely restore bone health in diabetes.

O microambiente tumoral é composto por células, como fibroblastos, células do sistema imune, células endoteliais e pericitos, envoltas por uma matriz extracelular, além de possuir fatores solúveis que participam da comunicação celular. Nas últimas décadas, têm-se entendido cada vez melhor seu papel na iniciação e progressão dos tumores. É de fundamental importância, portanto, entender a biologia dos seus componentes e como podem agir em favor do desenvolvimento tumoral. Diversos trabalhos demonstram que há uma associação entre a presença dos pericitos nos vasos tumorais com a agressividade e prognóstico de alguns tipos de câncer. Uma vez ativadas, além do papel estrutural, essas células modulam as atividades das células endoteliais durante a formação de novos vasos, além de adquirirem propriedades como proliferação e migração. Neste contexto, os pericitos passam a secretar fatores importantes na comunicação célula-a-célula e liberam enzimas moduladoras na matriz extracelular. A lisil oxidase (LOX) é uma das principais enzimas que atuam sobre a matriz extracelular. Já está bem descrito que, quando superexpressa em células tumorais, a LOX pode alterar a migração e invasão dessas células, promovendo a geração de metástases. Entretanto, pouco se sabe a respeito da atuação dessa enzima sobre os demais componentes celulares do estroma tumoral, como os pericitos. Sendo assim, o presente trabalho teve como objetivo principal verificar se enzima LOX é relevante para a ativação de propriedades dos pericitos que possam contribuir para suas funções pró-tumorigênicas, como migração, proliferação e formação de vasos. Os resultados foram gerados avaliando essas atividades dos pericitos após pré-tratamento de 24 horas com β-aminopropionitrile (βAPN), um inibidor irreversível da LOX. Foram utilizadas duas linhagens de pericitos derivados de tecido normal (adiposo e muscular) e duas linhagens de pericitos provenientes de tecido tumores do sistema nervoso central (neuroblastoma e ependimoma). Este composto foi capaz de diminuir a capacidade de migração das células de todas as linhagens testadas e, de maneira geral, tornou o processo de formação de estruturas tubulares in vitro menos eficiente. Entretanto, não foram observadas alterações na proliferação celular. Os dados indicam, portanto, que a enzima LOX pode ser importante para a ativação dos pericitos e, possivelmente, influenciem no seu comportamento no microambiente tumoral / The tumor microenvironment is composed of non-cancer cells, such as fibroblasts, immune cells, endothelial cells and pericytes, surrounded by an extracellular matrix, in addition to soluble factors involved in cellular crosstalk. In the last decades, it has been better understood its role in the initiation and progression of tumors. It is critical, therefore, to understand the biology of its components and how they can act in favor of tumor development. Several studies show an association between the presence of pericytes in tumor vessels with aggressiveness and prognosis of some cancers. Once activated, these cells modulate the activities of endothelial cells during the new vessels formation, and acquire properties as proliferation and migration. In this context, pericytes triggers the secretion of important factors in cell-to-cell communication and release modulating enzymes of extracellular matrix. The lysyl oxidase (LOX) is one of the main enzymes that act on the extracelular matrix. It is well described that when overexpressed in tumor cells, LOX can alter the migration and invasion of these cells, promoting the generation of metastases. However, little is known about the role of this enzyme over other cellular components of the tumor stroma, such as pericytes. Therefore, the aim of this study was to verify whether LOX enzyme is relevant to the activation of properties of the pericytes that could contribute to its pro-tumorigenic functions such as migration, proliferation and vessel formation. All the results were generated by evaluation of the activities of these pericytes after 24 hours pretreatment with β-aminopropionitrile (βAPN), an irreversible inhibitor of LOX. This study used two cell lines of pericytes derived from normal tissue (fat and muscle) and two isolated from tissue of the central nervous system. The βAPN was able to reduce the migration of cells of all tested cell lines and, in general, alter the tubular formation in vitro. However, changes in cell proliferation weren′t observed. The data showed, that the LOX family may be important for the activation of pericytes and possibly influence on their behavior in the tumor microenvironment

Abstract Lysyl hydroxylase (EC1.14.11.4, LH) catalyzes post-translationally the hydroxylation of lysyl residues in collagens and other proteins with collagenous domains. Hydroxylysyl residues may also be glycosylated by hydroxylysyl galactosyltransferase (EC 2.4.1.50, GT) or galactosylhydroxylysyl glucosyltransferase (EC 2.4.1.66, GGT) to form galactosylhydroxylysyl or glucosylgalactosylhydroxylysyl residues, structures unique to collagen. Three LH isoenzymes (LH1, LH2a/2b, LH3) have been characterized so far. We analyzed mRNA levels of these isoforms, as well as the mRNAs of the main collagen types (I, III, IV, V) and the α subunit of PH-4 in different human cell lines. Large variations were found in mRNA expression of LH1 and LH2 but not LH3. The mRNA levels of LH1, LH2, and the α subunit of PH-4 showed significant correlation with each other whereas LH3 correlated with none. No correlation was observed between the LH isoforms and individual collagen types. Three human LH isoforms were expressed in different expression systems. The purified recombinant protein produced by LH3 cDNA was found to be the only one possessing LH, GT and GGT activities. The molecular weight of the partially purified LH3 expressed in Sf9 or Cos-7 cells corresponded to about 85 kDa whereas that in E.coli cells was about 81 kDa probably due to a deficiency of glycosylation in bacterial cells. The recombinant protein of C. elegans LH cDNA was expressed in a cell-free translation system and in E.coli cells. The data indicated that the glycosyltransferase activities, GT and GGT, were also associated with this gene product. The sequence alignment of LH isoforms from different species revealed that there are 29 amino acids conserved between human LH3, mouse LH3 and C. elegans LH sequences and scattered evenly in the molecule, but differing from those of LH1 and LH2. In vitro mutagenesis data showed that the amino acids important for the glycosyltransferase activities were located at the amino-terminal part of the molecule, being separate from the LH active site. Mutation of a conserved LH3 specific, non-disulfide linked cysteine to isoleucine caused a dramatic reduction in GT and GGT activity but had no effect on LH activity. Mutations of the amino-terminal DxD motif (D187-191) characteristic of many glycosyltransferases eliminated both GT and GGT activities, showing the importance of this motif for collagen glycosyltransferases and suggesting that it might serve as the Mn2+ binding site in the molecule.

Abstract Lysyl hydroxylase (LH, EC 1.14.11.4) catalyzes the post-translational hydroxylation of lysyl residues in collagens and other proteins with collagenous domains. The hydroxylysyl residues participate in the formation of collagen cross-links, and some of the hydroxylysyl residues are further glycosylated. Three lysyl hydroxylase isoforms LH1, LH2 and LH3, encoded by three individual genes have been characterized and one isoform, LH3 is a multifunctional enzyme containing lysyl hydroxylase, collagen galactosyltransferase (GT, E.C. 2.4.1.50) and glucosyltransferase (GGT, E.C. 2.4.1.66) activities in vitro. In this thesis the genes for the mouse lysyl hydroxylases were each mapped to a different chromosome. In addition, the roles of the lysyl hydroxylase isoforms were characterized in mice by studying their expression during development and the distribution of LH2 and LH3 in adult mice. The results revealed a widespread expression of the mouse lysyl hydroxylases during embryonic development whereas LH2 and LH3 showed tissue- or cell-specific expression patterns in the adult. Alternative splicing of the gene for LH2 also showed developmental and tissue-specific regulation. The different functions of LH3 were studied in vivo by generating three different LH3 manipulated mouse lines. Analysis of the mouse lines revealed that LH3 has lysyl hydroxylase and glucosyltransferase activities in vivo, and that, in particular, the glucosyltransferase activity of LH3 is essential for normal development. The loss of glucosyltransferase activity caused disruption of basement membranes leading to embryonic lethality while the absence of lysyl hydroxylase activity led to ultrastructural alterations in muscle and basement membranes and disorganization of collagen fibrils. The disruption of basement membrane was due to an intracellular accumulation of unglycosylated type IV collagen, whereas the ultrastructural alterations were related to the abnormal aggregation and distribution of underglycosylated type VI collagen. The results demonstrate that hydroxylysine-linked glycosylations are critical for the secretion of type IV collagen and its assembly into basement membranes, and for the assembly and distribution of type VI collagen.

No description available.

No description available.

Le virus de l’immunodéficience humaine de type 1 (VIH-1), est un rétrovirus dont le génome est composé de deux molécules d’ARN simple brin. La transcriptase inverse codée par le VIH-1 utilise l’ARNt3Lys de la cellule hôte pour amorcer la réplication de son génome ARN en ADN proviral. L’ARNt3Lys est encapsidé dans les virions lors de l’assemblage; la lysyl-ARNt synthétase (LysRS) cellulaire est impliquée dans ce mécanisme et sert de co-transporteur à l’ARNt3Lys.Chez l’homme, il existe deux formes de LysRS, une forme cytoplasmique (cLysRS) et une forme mitochondriale (pmLysRS) qui donnera la forme mature (mLysRS) après translocation dans la mitochondrie. Les deux LysRS sont issues d’un même gène par épissage alternatif. Il a été démontré que seule la forme mitochondriale est présente dans les particules virales.Nous avons établi un modèle des interactions protéine-protéine impliquées dans la formation du complexe d’encapsidation de l’ARNt3Lys. En recherchant les interactions des précurseurs Gag et GagPol avec les LysRS et leurs domaines, nous avons démontré que seul le domaine Pol du précurseur GagPol a la capacité de s’associer à la LysRS. Ce sont les sous-domaines transframe TF et intégrase IN du domaine Pol qui permettent l’association entre LysRS et GagPol. Cette association se fait via le domaine catalytique de l’enzyme. La sélectivité de l'encapsidation de la forme mitochondriale de LysRS aux dépens de sa forme cytoplasmique pourrait résider dans la stricte compartimentation cellulaire de ces deux formes enzymatiques. Nous avons voulu établir à quel stade l’encapsidation de la LysRS mitochondriale a lieu, soit avant sa translocation mitochondriale sous forme de précurseur pmLysRS, soit après sous forme mLysRS maturée. Nous avons déterminé le site de maturation du précurseur pmLysRS puis caractérisé les deux formes mitochondriales de la LysRS, en déterminant leurs paramètres cinétiques et leur affinité pour l’ARNt3Lys. Alors que la forme pmLysRS ne forme pas de complexe stable avec l’ARNt, la forme maturée mLysRS est la plus apte à interagir avec l’ARNt3Lys. Ce serait donc la mLysRS qui serait impliquée dans le transport de l’ARNt3Lys dans les particules virales lors du bourgeonnement.Comme l'interaction GagPol:LysRS n'est pas spécifique in vitro de la forme mLysRS qui est la seule espèce de LysRS encapsidée, nous avons recherché si d’autres protéines virales pouvaient intervenir dans la formation du complexe d’encapsidation et conférer la spécificité pour la seule mLysRS. Nous avons montré que les protéines auxiliaires Rev et Vpr ont la capacité à s’associer à la LysRS sans distinction d'origine, mais ne peuvent interagir dans le contexte du complexe d'encapsidation GagPol:mLysRS:ARNt3Lys. Les différentes formes de LysRS pourraient ainsi réguler l'activité de Vpr et Rev à d'autres étapes du cycle viral. / The Human immunodeficiency virus type 1 (HIV-1) is a retrovirus with a genome composed of two molecules of single stranded RNA. The reverse transcriptase encoded by HIV-1 uses the cellular tRNA3Lys to prime the replication of its RNA genome into a proviral DNA. The tRNA3Lys is packaged into the viral particles during their assembly; the cellular lysyl-tRNA synthetase (LysRS) is involved in this mechanism as a co-carrier of tRNA3Lys.In human, there are two forms of LysRS, a cytoplasmic form (cLysRS) and a mitochondrial form (pmLysRS) that will be maturated into mLysRS after translocation into the mitochondrion. Both LysRS arise from the same gene by alternative splicing. It was demonstrated that only the mitochondrial species is present in the viral particles.We established a model of the protein-protein interactions which are implied in the formation of the packaging complex of tRNA3Lys. By searching for interactions of the viral precursors Gag and GagPol with the LysRS species and their domains, we demonstrated that only the Pol domain of the GagPol precursor has the capacity to interact with LysRS. The transframe (TF) and integrase (IN) domains of the Pol region of the polyprotein GagPol are required for association of LysRS with GagPol. This association is mediated by the catalytic domain of the enzyme. The selectivity of the packaging of the mitochondrial species of LysRS but not of its cytoplasmic species would rest on the cellular compartmentalization of these two enzyme forms. To establish at which step the mitochondrial LysRS is packaged, either as the pmLysRS precursor before its mitochondrial translocation, or after as the mature mLysRS, we determined the site of maturation of the pmLysRS precursor, then we characterized both mitochondrial forms of LysRS, by determining their kinetic parameters and their affinity for tRNA3Lys. Whereas the pmLysRS species did not form a stable complex with tRNA, the mature pmLysRS species did. Thus, mLysRS is the only LysRS species which could be implied in the transport of tRNA3Lys into the viral particles during the budding step. In vitro, the interaction GagPol:LysRS is not specific for the mLysRS species, but only the mitochondrial LysRS is packaged into the viral particles. We determined if another viral protein could impact the specificity of mLysRS packaging. We showed that the auxiliary proteins Rev and Vpr have the capacity to interact with LysRS but this intercation is not recovered in the context of the GagPol:mLysRS:tRNA3Lys packaging complex. These data suggest that the different forms of LysRS might regulate the activity of Vpr and Rev at other steps of the viral cycle.

L’angiogenèses par bourgeonnement est associée à une réorganisation majeure de la matrice extracellulaire (MEC). Nous avons déjà démontré que la lysyl oxydase-like 2 (LOXL2), une enzyme responsable du crosslinking de la MEC, régule la formation de vaisseaux intersomitiques dans les embryons de poisson zèbre et de capillaires en hydrogels 3D. Dans ce manuscrit, nous avons examiné les mécanismes impliqués dans cette régulation. Nous avons constaté l’association intracellulaire de LOXL2 avec la fibronectine et le collagène IV, avant d’être incorporée dans des structures fibrillaires dès l’exocytose. De plus, l’inhibition de l’expression de LOXL2 entraine des défauts de déposition de la MEC et diminue sa rigidité, inhibant secondairement la maturation des structures d'adhésion cellulaire. Alors que LOXL2 n‘est pas nécessaire pour la formation de capillaires dans un modèle 2D sur MEC de fibroblastes, les défauts de déposition de MEC sont corrélés à l'inhibition de formation des capillaires en hydrogel 3D. Ni l’addition de LOXL2 exogène, ni l’augmentation de la rigidité des hydrogels ne compense la perte d’expression de LOXL2. Enfin, nous avons pu montrer que ni l'activité catalytique ni le domaine catalytique de LOXL2 ne sont essentiels pour la formation de capillaire dans le poisson zèbre et dans les hydrogels et pour l’assemblage du collagène IV par des cellules endothéliales. L’ensemble de ces données suggère donc que les domaines SRCR de LOXL2 exprimés par des cellules endothéliales régulent l’échafaudage de fibronectine et de collagène IV dans la MEC qui est nécessaire à la formation de capillaires. / Sprouting angiogenesis is associated with major extracellular matrix (ECM) remodelling, consisting in both degradation of the microenvironment and generation of a new basement membrane. We have previously reported that lysyl oxidase-like 2 (LOXL2), an enzyme responsible for ECM crosslinking, regulates formation of intersomitic vessels (ISV) of zebrafish embryos and of capillaries in 3D hydrogels. In this manuscript we investigated the mechanisms involved. We found that LOXL2 associates with fibronectin and collagen IV intracellulary before direct incorporation in fibrillar structures of the ECM upon exocytosis. In addition, silencing LOXL2 demonstrated its involvement in ECM deposition as both composition and stiffness of the ECM were affected, which subsequently altered maturation of cell adhesion structures. Whereas LOXL2 is not required for formation of capillaries on top of a fibroblast monolayer, in a 2D assay, ECM defaults were associated with altered formation of capillaries in 3D hydrogels.Neither addition of exogenous LOXL2, nor increasing the stiffness of hydrogels could restore capillary formation. Moreover, we could show that neither the catalytic activity nor the catalytic domain were required for capillary formation in vivo and in 3D hydrogels, and for collagen IV deposition by endothelial cells. Altogether, these data suggest that the SRCR domains of LOXL2 expressed by endothelial cells regulate 3D capillary morphogenesis through scaffolding of fibronectin and collagen IV in the ECM.

Lubica Dudakova Doctoral Thesis ABSTRACT Keratoconus (KC) is a non-inflammatory disease of the cornea, in which ectasia and thinning occur probably due to defects in the collagen fibers binding. It is one of the most common indications for corneal transplantation. KC is a complex disorder with the involvement of both genetic and environmental factors; however the exact pathogenic mechanisms leading to the disease development have not been elucidated. The main aim of our work was to compare the presence and enzyme activity of cross- linking enzymes lysyl oxidases (LOX and LOX-like enzymes), in control human cornea samples and explanted cornea gained from patients with KC. We also focused on diseases previously described to be associated with KC with the aim to identify common signs among them. Furthermore, we replicated association of single nucleotide polymorphisms (SNPs) in LOX and hepatocyte growth factor (HGF) with KC risk. We attempted to link all pathophysiological disturbances observed in KC into one common pathway. We have used a wide spectrum of methods (cell culturing, immunohisto- and immunocytochemistry, microscopy, fluorimetric enzyme activity measurement, genotyping and direct sequencing, statistical analysis). We demonstrated the presence of entire family of LOX enzymes in control and in KC...