Insights into the structure and dynamics of lysyl oxidase propeptide, a flexible protein with numerous partners

Abstract

Lysyl oxidase (LOX) catalyzes the oxidative deamination of lysine and hydroxylysine residues in collagens and elastin, which is the first step of the cross-linking of these extracellular matrix proteins. It is secreted as a proenzyme activated by bone morphogenetic protein-1, which releases the LOX catalytic domain and its bioactive N-terminal propeptide. We characterized the recombinant human propeptide by circular dichroism, dynamic light scattering, and small-angle X-ray scattering (SAXS), and showed that it is elongated, monomeric, disordered and flexible (Dmax: 11.7 nm, Rg: 3.7 nm). We generated 3D models of the propeptide by coarse-grained molecular dynamics simulations restrained by SAXS data, which were used for docking experiments. Furthermore, we have identified 17 new binding partners of the propeptide by label-free assays. They include four glycosaminoglycans (hyaluronan, chondroitin, dermatan and heparan sulfate), collagen I, cross-linking and proteolytic enzymes (lysyl oxidase-like 2, transglutaminase-2, matrix metalloproteinase-2), a proteoglycan (fibromodulin), one growth factor (Epidermal Growth Factor, EGF), and one membrane protein (tumor endothelial marker-8). This suggests new roles for the propeptide in EGF signaling pathway.

Intracellular retention of mutant lysyl oxidase leads to aortic dilation in response to increased hemodynamic stress

“…An intriguing possibility is that the mutant proteins contribute to disease pathogenesis through mechanisms that do not involve their amine oxidase activity. Noncatalytic functions for LOX are emerging (5,36,37), and much remains to be learned about how mutations in this multifunctional enzyme lead to aortic disease.…”

Computational and experimental characterization of the novel ECM glycoprotein SNED1 and prediction of its interactome

“…For large-scale expression of fl-SNED1 and the N-terminal fragment of SNED1, cells were cultured in HYPERFlasks™ in DMEM (Sigma-Aldrich, D5796) supplemented with 50 μg/ml of gentamicin (Sigma-Aldrich, G1272) as previously described (58). Culture media were harvested every 48 h for up to 18 days.…”

“…Fl-SNED1 and the N-terminal fragment were purified by affinity chromatography on an anti-FLAG resin (Sigma-Aldrich, A2220) as previously described (58) in presence of 150 mM NaCl. In brief, FLAGtagged proteins were purified at 4°C on the anti-FLAG resin at a flow rate of 20 ml/h with a P1 pump (GE Healthcare), and eluted by competition with a FLAG peptide solution at 200 μg/ml in 10 mM Hepes, 150 mM NaCl, pH 7.4 (HBS).…”

“…Conditioned media from 293T cells stably expressing FLAG-tagged fl-SNED1 and the N-terminal fragment of SNED1 were incubated with PNGase F as previously described (58), or with heparinase III and chondroitinase ABC (2 mU per 40 μL of CM) as previously described (59). Proteins were separated by SDS-PAGE, and transferred onto nitrocellulose membranes.…”

Functional genomic analyses highlights a shift inGpr17-regulated cellular processes in oligodendrocyte progenitor cells (OPC) and underlying myelin dysregulation in the aged forebrain

“…Notably, neuronal numbers are largely unaltered in the aging human brain (Fabricius et al, 2013, Pelvig et al, 2008. In comparison, there is evidence of gradual losses in oligodendrocytes and myelin in aging and that these changes are key factors in cognitive decline and to decreased capacity for repair following pathology (Vanzulli et al, 2020, Bartzokis, 2004, Vallet et al, 2018. Sustaining myelin and oligodendrocytes throughout life is therefore critical and is the function of a reservoir of oligodendrocyte progenitor cells (OPCs) Butt, 2019, Reich et al, 2018).…”