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  • Organ fibrosis is thought to be initiated by repeated

    2021-04-30

    Organ fibrosis is thought to be initiated by repeated or chronic epithelial injury. The current belief is that damaged epithelial TAME induce an aberrant and unresolved wound repair process by activating fibroblasts via various profibrotic cues. Upon injury, epithelial cells activate the profibrotic cytokine transforming growth factor β (TGF-β) from latent complexes in the extracellular matrix (ECM) [[4], [5], [6]] and release TGF-β-containing exosomes [7]. TGF-β is well-established as a central driver of fibrogenesis through induction of expression and deposition of major ECM components like type I collagen and fibronectin by fibroblasts [1,[8], [9], [10]]. Furthermore, fibrotic ECM as synthesized by IPF fibroblasts provides profibrotic cues resulting in even more ECM synthesis, i.e. a positive feedback loop that may underlie the highly progressive character of the disease [[11], [12], [13], [14], [15]]. However, to the best of our knowledge, no study has taken an unbiased, global proteomic approach to comprehensively quantify the changes in ECM proteins and their modifications synthesized by IPF fibroblasts upon TGF-β1 stimulation. Constituting between 30% and 70% of ECM protein in all tissue types, collagen clearly is one of the main components of the ECM [16]. Collagen biosynthesis and deposition is highly upregulated in fibrotic disease, leading to impaired ECM homeostasis and scar formation [[17], [18], [19]]. Importantly, all collagens are subject to various intracellular post-translational modifications (PTMs) as well as extracellular maturation steps [20,21]. Evidence has emerged that strongly argues for an important impact of collagen PTMs on protein-protein or protein-cell interactions [[22], [23], [24], [25], [26], [27], [28]], which can be highly relevant for disease. For instance, collagen prolyl-3-hydroxylation, a comparatively rare collagen PTM, has been shown to affect binding to the proteoglycan decorin, an extracellular regulator of TGF-β activity in the context of osteogenesis imperfecta [27]. Causal associations have been established for prolyl-3-hydroxylase 1 (P3H1, LEPRE1) gene mutations with osteogenesis imperfecta [29,30], for prolyl-3-hydroxylase 2 (P3H2, LEPREL1) gene mutations with myopia and several other eye defects [31,32], and for prolyl-3-hydroxylase 3 (P3H3, LEPREL2) gene mutations with Ehlers-Dalnos syndrome type VIA [33]. Although collagen PTMs have been studied by targeted mass spectrometry as well as computational prediction methods [34,35], they have neither been described in depth, nor in the context of lung fibrosis [36,37]. Therefore, strategies enabling global assessment and characterization of collagen PTMs are likely to provide important insight about their biological function.
    Results and discussion
    Experimental procedures
    Introduction Prolyl 4-hydroxylase (P4H) is a key enzyme of the collagen biosynthesis catalyzing the hydroxylation of proline residues in -Xaa-Pro-Gly-sequences typical of proteins with collagen domains (for review, see Kivirikko et al., 1989, Kivirikko and Myllyharju, 1998, Myllyharju, 2003). The synthesis of 4-hydroxyproline residues is essential to TAME the folding of the newly synthetized chains into the triple helices of collagens. In vertebrates, this enzyme is a α2β2 tetramer, where the α subunits contain the catalytic active site for the proline hydroxylation, while the β subunits, that are identical to protein disulfide-isomerase (PDI), act to maintain the α subunits in a soluble form and to retain the enzyme in the ER lumen (Kivirikko et al., 1989). The pivotal role of this enzyme in collagen metabolism makes it a potential specific target for pharmacological regulation of fibrotic diseases and, for this reason, several studies have been carried out in order to elucidate the details of the hydroxylation mechanism (Myllyharju and Kivirikko, 1997, Lamberg et al., 1995). Attempts to assemble an active P4H tetramer in vitro from its individual α and β subunits have been unsuccessful (Koivu and Myllylä, 1986, Nietfeld et al., 1981). Human P4H was produced in recombinant form in insect cells by baculovirus vectors (Vuori et al., 1992) and more recently in E. coli (Kersteen et al., 2004). The recombinant production of a stable P4H tetramer proves to be of great significance not only to perform accurate structure/function analyses but also to promote further studies on the enzymatic activity mechanism by site-specific mutagenesis. Last but not least, this protein is considered a fundamental biotechnological tool for the production of recombinant proteins needing prolyl-hydroxylation in their post-translational modifications as collagen.