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1.
Biochim Biophys Acta Gene Regul Mech ; 1866(4): 194963, 2023 12.
Article in English | MEDLINE | ID: mdl-37499936

ABSTRACT

Transcriptional activity of the hypoxia inducible factor (HIF) relies on the formation of a heterodimer composed of an oxygen-regulated α-subunit and a stably expressed ß-subunit. Heterodimeric HIF activates expression by binding to RCGTG motifs within promoters of hypoxia-activated genes. Some hypoxia targets also possess an adjacent HIF ancillary sequence (HAS) reported to increase transcription but whose function remains obscure. Here, we investigate the contribution of the HAS element to the hypoxia response and its mechanism of action, using the HAS-containing prolyl 4-hydroxylase subunit α1 (P4HA1) as a gene model in NIH/3T3 mouse embryonic fibroblasts and HEK293 human embryonic kidney cells. Our HIF overexpression experiments demonstrate that the HAS motif is essential for full induction by hypoxia and that the presence of the tandem HAS/HIF, as opposed to HIF-only sequences, provides HIF proteins with the capacity to form complexes of stoichiometry beyond the classical heterodimer, likely tetramers, to cooperatively potentiate hypoxia-induced transcription. We also provide evidence of the crucial role played by the Fα helix of the PAS-B domain of the HIF1ß subunit to support the interaction between heterodimers. Functional analysis showed that human genes containing the HAS/HIF motifs are better responders to hypoxia, and their promoters are enriched for specific transcription factor binding sites. Gene ontology enrichment revealed a predominance of HAS/HIF in genes primarily related to tissue formation and development. Our findings add an extra level of regulation of the hypoxia/HIF signaling through multimerization of HIF proteins on regulatory elements containing the HAS/HIF motifs.


Subject(s)
DNA-Binding Proteins , Transcription Factors , Animals , Humans , Mice , Transcription Factors/metabolism , DNA-Binding Proteins/metabolism , HEK293 Cells , RNA, Messenger/metabolism , Fibroblasts/metabolism , Hypoxia
2.
Int J Mol Sci ; 23(6)2022 Mar 18.
Article in English | MEDLINE | ID: mdl-35328709

ABSTRACT

Members of the lysyl oxidase (LOX) family catalyze the oxidative deamination of lysine and hydroxylysine residues in collagen and elastin in the initiation step of the formation of covalent cross-links, an essential process for connective tissue maturation. Proteolysis has emerged as an important level of regulation of LOX enzymes with the cleavage of the LOX isoform by metalloproteinases of the BMP1 (bone morphogenetic protein 1) and ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) families as a model example. Lysyl oxidase-like 1 (LOXL1), an isoform associated with pelvic organ prolapse and pseudoexfoliation (PEX) glaucoma, has also been reported to be proteolytically processed by these proteases. However, precise molecular information on these proteolytic events is not available. In this study, using genetic cellular models, along with proteomic analyses, we describe that LOXL1 is processed by BMP1 and ADAMTS14 and identify the processing sites in the LOXL1 protein sequence. Our data show that BMP1 cleaves LOXL1 in a unique location within the pro-peptide region, whereas ADAMTS14 processes LOXL1 in at least three different sites located within the pro-peptide and in the first residues of the catalytic domain. Taken together, these results suggest a complex regulation of LOXL1 function by BMP1- and ADAMTS14-mediated proteolysis where LOXL1 enzymes retaining variable fragments of N-terminal region may display different capabilities.


Subject(s)
Exfoliation Syndrome , Protein-Lysine 6-Oxidase , ADAMTS Proteins/metabolism , Amino Acid Oxidoreductases/genetics , Amino Acid Oxidoreductases/metabolism , Bone Morphogenetic Protein 1/genetics , Bone Morphogenetic Protein 1/metabolism , Exfoliation Syndrome/genetics , Humans , Peptide Hydrolases/metabolism , Protein-Lysine 6-Oxidase/metabolism , Proteolysis , Proteomics
3.
Anal Biochem ; 639: 114508, 2022 02 15.
Article in English | MEDLINE | ID: mdl-34871563

ABSTRACT

The lysyl oxidase (LOX) family of enzymes catalyze the oxidative deamination of lysine and hydroxylysine residues in collagen and elastin in the initiation step of the formation of covalent cross-linkages, an essential process for extracellular matrix (ECM) maturation. Elevated LOX expression levels leading to increased LOX activity is associated with diverse pathologies including fibrosis, cancer, and cardiovascular diseases. Different protocols have been so far established to detect and quantify LOX activity from tissue samples and cultured cells, all of them showing advantages and drawbacks. This review article presents a critical overview of the main features of currently available methods as well as introduces some recent technologies called to revolutionize our approach to LOX catalysis.


Subject(s)
Enzyme Assays/methods , Protein-Lysine 6-Oxidase/metabolism , Animals , Biosensing Techniques/instrumentation , Biosensing Techniques/methods , Cardiovascular Diseases/enzymology , Enzyme Assays/instrumentation , Humans , Neoplasms/enzymology , Optical Imaging/instrumentation , Optical Imaging/methods , Protein-Lysine 6-Oxidase/analysis
4.
Methods Mol Biol ; 2299: 115-122, 2021.
Article in English | MEDLINE | ID: mdl-34028738

ABSTRACT

Synthesis, deposition, and cross-linking of collagen are hallmarks of fibroblast to myofibroblast differentiation. Standard methods for determining collagen from tissue samples are not directly applicable to cell culture conditions, where the overall synthesis and deposition of collagen is clearly unfavorable, mainly due to quantity limitations and dilution of required extracellular remodeling factors. In this chapter, we describe the methods we have established to analyze collagen production and deposition into the extracellular matrix by cultured myo/fibroblasts, as well as to determine lysyl oxidase (LOX) activity in cell supernatants as an index of the capacity of the cell to cross-link collagen in vitro.


Subject(s)
Collagen/metabolism , Fibroblasts/cytology , Myofibroblasts/cytology , Protein-Lysine 6-Oxidase/metabolism , Animals , Cell Differentiation , Cell Line , Cross-Linking Reagents/pharmacology , Extracellular Matrix/metabolism , Fibroblasts/metabolism , Humans , Mice , Myofibroblasts/metabolism , NIH 3T3 Cells
5.
Methods Cell Biol ; 156: 259-270, 2020.
Article in English | MEDLINE | ID: mdl-32222222

ABSTRACT

This book chapter describes the use of exogenous application of lysyl oxidase (LOX) and bone morphogenetic protein-1 (BMP1) to enhance collagen synthesis and deposition from fibroblasts in culture. The protocol includes the generation of human embryonic kidney (HEK) 293 cell lines overexpressing human LOX and BMP1 constructs in order to obtain supernatants enriched in these factors. Incubation of fibroblast monolayers with these conditioned media strongly increases the capacity of these cells to deposit collagen onto the insoluble extracellular matrix. We also describe the use of these decellularized fibroblast-derived matrices as a substrate for the growth and differentiation of mesenchymal stem cells.


Subject(s)
Bone Morphogenetic Protein 1/metabolism , Collagen/metabolism , Protein-Lysine 6-Oxidase/metabolism , Cell Differentiation , Extracellular Matrix/metabolism , Fibroblasts/metabolism , HEK293 Cells , Humans , Mesenchymal Stem Cells/cytology , Mesenchymal Stem Cells/metabolism , Osteogenesis
6.
J Biol Chem ; 294(39): 14308-14318, 2019 09 27.
Article in English | MEDLINE | ID: mdl-31391253

ABSTRACT

Collagens are extracellular matrix (ECM) proteins that support the structural and biomechanical integrity of many tissues. Procollagen-lysine, 2-oxoglutarate 5-dioxygenase 2 (PLOD2) encodes the only lysyl hydroxylase (LH) isoform that specifically hydroxylates lysine residues in collagen telopeptides, a post-translational modification required for the formation of stabilized cross-links. PLOD2 expression is induced by hypoxia and transforming growth factor-ß1 (TGF-ß1), well-known stimuli for the formation of a fibrotic ECM, which can lead to pathological fibrosis underlying several diseases. Here, using human and murine fibroblasts, we studied the molecular determinants underlying hypoxia- and TGF-ß1-induced PLOD2 expression and its impact on collagen biosynthesis. Deletion mapping and mutagenesis analysis identified specific binding sites for hypoxia-inducible factors (HIF) and TGF-ß1-activated SMAD proteins on the human PLOD2 gene promoter that were required for these stimuli to induce PLOD2 expression. Interestingly, our experiments also revealed that HIF signaling plays a preponderant role in the SMAD pathway, as intact HIF sites were absolutely required for TGF-ß1 to exert its effect on SMAD-binding sites. We also found that silencing PLOD2 expression did not alter soluble collagen accumulation in the extracellular medium, but it effectively abolished the deposition into the insoluble collagen matrix. Taken together, our findings reveal the existence of a hierarchical relationship between the HIF and SMAD signaling pathways for hypoxia- and TGF-ß1-mediated regulation of PLOD2 expression, a key event in the deposition of collagen into the ECM.


Subject(s)
Basic Helix-Loop-Helix Transcription Factors/metabolism , Hypoxia-Inducible Factor 1/metabolism , Oxygen/metabolism , Procollagen-Lysine, 2-Oxoglutarate 5-Dioxygenase/genetics , Smad Proteins/metabolism , 3T3 Cells , Animals , Cell Hypoxia , Cell Line, Tumor , Extracellular Matrix/metabolism , Fibroblasts/drug effects , Fibroblasts/metabolism , Humans , Mice , Procollagen-Lysine, 2-Oxoglutarate 5-Dioxygenase/metabolism , Promoter Regions, Genetic , Transforming Growth Factor beta1/pharmacology
7.
J Biol Chem ; 294(29): 11087-11100, 2019 07 19.
Article in English | MEDLINE | ID: mdl-31152061

ABSTRACT

Collagens are the main structural component of the extracellular matrix and provide biomechanical properties to connective tissues. A critical step in collagen fibril formation is the proteolytic removal of N- and C-terminal propeptides from procollagens by metalloproteinases of the ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) and BMP1 (bone morphogenetic protein 1)/Tolloid-like families, respectively. BMP1 also cleaves and activates the lysyl oxidase (LOX) precursor, the enzyme catalyzing the initial step in the formation of covalent collagen cross-links, an essential process for fibril stabilization. In this study, using murine skin fibroblasts and HEK293 cells, along with immunoprecipitation, LOX enzymatic activity, solid-phase binding assays, and proteomics analyses, we report that the LOX precursor is proteolytically processed by the procollagen N-proteinases ADAMTS2 and ADAMTS14 between Asp-218 and Tyr-219, 50 amino acids downstream of the BMP1 cleavage site. We noted that the LOX sequence between the BMP1- and ADAMTS-processing sites contains several conserved tyrosine residues, of which some are post-translationally modified by tyrosine O-sulfation and contribute to binding to collagen. Taken together, these findings unravel an additional level of regulation in the formation of collagen fibrils. They point to a mechanism that controls the binding of LOX to collagen and is based on differential BMP1- and ADAMTS2/14-mediated cleavage of a tyrosine-sulfated domain.


Subject(s)
ADAMTS Proteins/metabolism , Bone Morphogenetic Protein 1/metabolism , Collagen/metabolism , Protein-Lysine 6-Oxidase/metabolism , Tyrosine/analogs & derivatives , Animals , Binding Sites , Cattle , Cells, Cultured , HEK293 Cells , Humans , Mice , Protein Binding , Protein Processing, Post-Translational , Protein-Lysine 6-Oxidase/chemistry , Proteolysis , Tyrosine/metabolism
8.
J Glaucoma ; 27 Suppl 1: S15-S19, 2018 07.
Article in English | MEDLINE | ID: mdl-29419646

ABSTRACT

Lysyl oxidases (LOX) are copper-dependent enzymes that oxidize lysyl and hydroxylysyl residues in collagen and elastin, as a first step in the stabilization of these extracellular matrix proteins through the formation of covalent cross-linkages, an essential process for connective tissue maturation. Five different LOX enzymes have been identified in mammals, LOX and LOX-like (LOXL) 1 to 4, being genetically different protein products with a high degree of homology in the catalytic carboxy terminal end and a more variable amino terminal proregion. Intensive investigation in the last years has delineated the main biological functions of these enzymes and their involvement in several pathologies including fibrosis, cancer, and ocular disorders. This review article summarizes the major findings on the role of LOX isoforms, with particular focus on their contribution to the development and progression of human disorders.


Subject(s)
Exfoliation Syndrome/enzymology , Glaucoma, Open-Angle/enzymology , Protein-Lysine 6-Oxidase/physiology , Animals , Bone Diseases/enzymology , Cardiovascular Diseases/enzymology , Humans , Isoenzymes/physiology , Neoplasms/enzymology
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