Role of lysyl oxidase propeptide in secretion and enzyme activity.
ABSTRACT: Lysyl oxidase (LOX) is secreted as a proenzyme (proLOX) that is proteolytically processed in the extracellular milieu to release the propeptide and mature, active LOX. LOX oxidizes lysyl residues of a number of protein substrates in the extracellular matrix and on the cell surface, which impacts several physiological and disease states. Although the LOX propeptide (LOX-PP) is glycosylated, little is known about the role of this modification in LOX secretion and activity. To gain insight into this issue, cells were transfected with native, full-length LOX cDNA (pre-pro-LOX), the N-glycosylation null pre-[N/Q]pro-LOX cDNA and the deletion mutant pre-LOX cDNA, referred to as secretory LOX, in which mature LOX is targeted to the secretory pathway without its N-terminal propeptide sequence. The results show that glycosylation of the LOX-PP is not required for secretion and extracellular processing of pro-LOX but it is required for optimal enzyme activity of the resulting mature LOX. Complete deletion of the propeptide sequence prevents mature LOX from exiting the endoplasmic reticulum (ER). Taken together, our study points out the requirement of the LOX-PP for pro-LOX exit from the ER and is the first to highlight the influence of LOX-PP glycosylation on LOX enzyme activity.
Project description:Lysyl oxidase enzyme activity is critical for the biosynthesis of mature and functional collagens and elastin. In addition, lysyl oxidase has tumor suppressor activity that has been shown to depend on the propeptide region (LOX-PP) derived from pro-lysyl oxidase (Pro-LOX) and not on lysyl oxidase enzyme activity. Pro-LOX is secreted as a 50 kDa proenzyme and then undergoes biosynthetic proteolytic processing to active approximately 30 kDa LOX enzyme and LOX-PP. The present study reports the efficient recombinant expression and purification of rat LOX-PP. Moreover, using enzymatic deglycosylation and DTT derivatization combined with mass spectrometry technologies, it is shown for the first time that rLOX-PP and naturally occurring LOX-PP contain both N- and O-linked carbohydrates. Structure predictions furthermore suggest that LOX-PP is a mostly disordered protein, which was experimentally confirmed in circular dichroism studies. Due to its high isoelectric point and its disordered structure, we propose that LOX-PP can associate with extracellular and intracellular binding partners to affect its known biological activities as a tumor suppressor and inhibitor of cell proliferation.
Project description:The lysyl oxidase propeptide (LOX-PP) is derived from pro-lysyl oxidase (Pro-LOX) by extracellular biosynthetic proteolysis. LOX-PP inhibits breast and prostate cancer xenograft tumor growth and has tumor suppressor activity. Although, several intracellular targets and molecular mechanisms of action of LOX-PP have been identified, LOX-PP uptake pathways have not been reported. Here we demonstrate that the major uptake pathway for recombinant LOX-PP (rLOX-PP) is PI3K-dependent macropinocytosis in PWR-1E, PC3, SCC9, MDA-MB-231 cell lines. A secondary pathway appears to be dynamin- and caveola dependent. The ionic properties of highly basic rLOX-PP provide buffering capacity at both high and low pHs. We suggest that the buffering capacity of rLOX-PP, which serves to limit endosomal acidification, sustains PI3K-dependent macropinocytosis in endosomes which in turn is likely to facilitate LOX-PP endosomal escape into the cytoplasm and its observed interactions with cytoplasmic targets and nuclear uptake.
Project description:Lysyl oxidase (LOX) plays a critical role in extracellular matrix maturation and limits VSMC proliferation and vascular remodeling. We have investigated whether this anti-proliferative effect relies on the extracellular catalytically active LOX or on its biologically active propeptide (LOX-PP). High expression levels of both LOX and LOX-PP were detected in the vascular wall from transgenic mice over-expressing the full-length human LOX cDNA under the control of SM22? promoter (TgLOX), which targets the transgene to VSMC without affecting the expression of mouse LOX isoenzymes. TgLOX VSMC also secrete high amounts of both mature LOX and LOX-PP. Wild-type (WT) mouse VSMC exposed to VSMC supernatants from transgenic animals showed reduced proliferative rates (low [3H]-thymidine uptake and expression of PCNA) than those incubated with conditioned media from WT cells, effect that was abrogated by ?-aminopropionitrile (BAPN), an inhibitor of LOX activity. Lentiviral over-expression of LOX, but not LOX-PP, decreased human VSMC proliferation, effect that was also prevented by BAPN. LOX transgenesis neither impacted local nor systemic inflammatory response induced by carotid artery ligation. Interestingly, in this model, BAPN normalized the reduced neointimal thickening observed in TgLOX mice. Therefore, extracellular enzymatically active LOX is required to limit both VSMC proliferation and vascular remodeling.
Project description:Enhanced RAS signaling and decreased androgen dependence of prostate cancer cells accompany poor clinical outcomes. Elevated autocrine fibroblast growth factors 2 (FGF-2) signaling promotes prostate cancer cell growth and survival. Expression of lysyl oxidase (LOX) inhibits RAS transforming activity. LOX is secreted as 50 kDa pro-LOX protein and then undergoes extracellular proteolytic processing to form approximately 30 kDa LOX enzyme and approximately 18 kDa propeptide (LOX-PP). We have previously shown that LOX-PP inhibits breast cancer cell transformation and tumor formation, but mechanisms of action of LOX-PP have not been fully elucidated. Here we report that LOX expression is reduced in prostate cancer cell lines and that recombinant LOX-PP protein inhibits serum-stimulated DNA synthesis and MEK/ERK and PI3K/AKT pathways in DU 145 and PC-3 androgen-independent cell lines. In DU 145 cells, treatment with a pharmacologic FGF-receptor inhibitor or a neutralizing anti-FGFR1 antibody mimicked LOX-PP inhibition of serum-stimulated DNA synthesis. FGF-2-stimulated DNA synthesis, ERK1/2, AKT and FRS2alpha activation were found all to be inhibited by LOX-PP in DU 145 cells. LOX-PP reduced specific binding of FGF-2 to DU 145 cells, suggesting that LOX-PP targets FGF signaling at the receptor. Interestingly, PC-3 cells did not respond to FGF-2, consistent with previous reports. We conclude that LOX-PP inhibits proliferation of DU 145 cells by interfering with FGFR(s) binding and signaling, and that LOX-PP has other mechanisms of action in PC-3 cells.
Project description:The lysyl oxidase (LOX) gene encodes an enzyme (LOX) critical for extracellular matrix maturation. The LOX gene has also been shown to inhibit the transforming activity of Ras oncogene signaling. In particular, the pro-peptide domain (LOX-PP) released from the secreted precursor protein (Pro-LOX) was found to inhibit the transformed phenotype of breast, lung, and pancreatic cancer cells. However, the mechanisms of action of LOX-PP remained to be determined. Here, the ability of LOX-PP to attenuate the integrin signaling pathway, which leads to phosphorylation of focal adhesion kinase (FAK), and the activation of its downstream target p130Cas, was determined. In NF639 breast cancer cells driven by Her-2/neu, which signals via Ras, ectopic Pro-LOX and LOX-PP expression inhibited fibronectin-stimulated protein tyrosine phosphorylation. Importantly, phosphorylation of FAK on Tyr-397 and Tyr-576, and p130Cas were substantially reduced. The amount of endogenous p130Cas in the Triton X-100-insoluble protein fraction, and fibronectin-activated haptotaxis were decreased. Interestingly, expression of mature LOX enzyme enhanced fibronectin-stimulated integrin signaling. Of note, treatment with recombinant LOX-PP selectively reduced fibronectin-mediated haptotaxis of NF639, MDA-MB-231, and Hs578T breast cancer cells. Thus, evidence is provided that one mechanism of action of LOX-PP tumor suppression is to block fibronectin-stimulated signaling and cell migration.
Project description:Ewing sarcoma is the second most common bone malignancy in children and young adults. It is driven by oncogenic fusion proteins (i.e. EWS/FLI1) acting as aberrant transcription factors that upregulate and downregulate target genes, leading to cellular transformation. Thus, identificating these target genes and understanding their contribution to Ewing sarcoma tumorigenesis are key for the development of new therapeutic strategies. In this study we show that lysyl oxidase (LOX), an enzyme involved in maintaining structural integrity of the extracellular matrix, is downregulated by the EWS/FLI1 oncoprotein and in consequence it is not expressed in Ewing sarcoma cells and primary tumors. Using a doxycycline inducible system to restore LOX expression in an Ewing sarcoma derived cell line, we showed that LOX displays tumor suppressor activities. Interestingly, we showed that the tumor suppressor activity resides in the propeptide domain of LOX (LOX-PP), an N-terminal domain produced by proteolytic cleavage during the physiological processing of LOX. Expression of LOX-PP reduced cell proliferation, cell migration, anchorage-independent growth in soft agar and formation of tumors in immunodeficient mice. By contrast, the C-terminal domain of LOX, which contains the enzymatic activity, had the opposite effects, corroborating that the tumor suppressor activity of LOX is mediated exclusively by its propeptide domain. Finally, we showed that LOX-PP inhibits ERK/MAPK signalling pathway, and that many pathways involved in cell cycle progression were significantly deregulated by LOX-PP, providing a mechanistic explanation to the cell proliferation inhibition observed upon LOX-PP expression. In summary, our observations indicate that deregulation of the LOX gene participates in Ewing sarcoma development and identify LOX-PP as a new therapeutic target for one of the most aggressive paediatric malignancies. These findings suggest that therapeutic strategies based on the administration of LOX propeptide or functional analogues could be useful for the treatment of this devastating paediatric cancer.
Project description:Lysyl oxidase (LOX) catalyzes the oxidative deamination of lysine residues in collagen and elastin, key components of connective tissue. LOX is synthesized as an inactive 50 kD pre-proenzyme, and secreted to the extracellular matrix where it is cleaved into an active 32 kD LOX, and an 18kD free propeptide (LOX-PP), purportedly an inhibitor of fibroblast growth factor-2 (FGF-2) signaling. Given that adipocytes are distributed inside the connective tissue, it is likely that LOX-PP has an important regulatory role in adipogenesis, which has not been studied. Using NIH 3T3-L1 cells, we observed that FGF-2 inhibited adipogenesis, and LOX-PP promoted adipogenesis of 3T3-L1 cells in the presence of FGF-2; the expression of peroxisome proliferator-activated receptor (PPAR) ? and CCAAT-enhancer binding protein (C/EBP) ?, two markers of adipogenesis, were enhanced in the presence of LOX-PP. We further observed that LOX-PP down-regulated AKT and ERK1/2, two proliferative signaling proteins down-stream of FGF-2 signaling. Similarly, inhibition of FGF-2 receptor signaling by canofin, a competitive inhibitor of FGF-2 receptor, promoted adipogenesis albeit less effective compared to LOX-PP. To further explore whether LOX-PP promoted adipogenesis through inhibition of FGF-2 signaling, site directed mutagenesis of LOX-PP, resulting in an Arg158 to Gln158 mutation which abolishes the inhibitory activity of LOX-PP to FGF-2 receptor, attenuated the adipogenic promoting properties of LOX-PP. In summary, for the first time, our data show that LOX-PP enhances adipogenesis at least partially through inhibition of FGF-2 receptor signaling. Our data suggest that LOX-PP may serve as a bona fide therapeutic target for regulating adipogenesis and adipose tissue development.
Project description:In this study we show that lysyl oxidase (LOX), an enzyme involved in maintaining structural integrity of the extracellular matrix, is expressed at low levels in Ewing sarcoma cells and primary tumors and is downregulated by the EWS/FLI1 oncoprotein characteristic of these tumors. Using a doxycycline inducible system to restore LOX expression in an Ewing sarcoma derived cell line, we show that LOX displays tumor suppressor activities. Interestingly, we show that the tumor suppressor activity resides in the propeptide domain of LOX (LOX-PP), an N-terminal domain produced by proteolytic cleavage during the physiological processing of LOX. Finally, we show that LOX-PP inhibits ERK/MAPK signalling pathway, and that many pathways involved in cell cycle progression were significant deregulated by LOX-PP, providing a mechanistic explanation to the cell proliferation inhibition observed upon LOX-PP expression. In summary, our observations indicate that deregulation of the LOX gene participates in Ewing sarcoma development and identify LOX-PP as a new therapeutic target for one of the most aggressive paediatric malignancies. These findings suggest that therapeutic strategies based in the administration of LOX propeptide or functional analogues could be useful in the treatment of this devastating paediatric cancer. A673 cells derived from Ewing sarcoma were genetically enginereed to express LOX-PP upon doxycycline stimulation (72 hours). Three independent experiments from control cells and three independent experiments from A673 cells expressing LOX-PP were done. Gene expression profile in A673 cells expressing LOX-PP vs control cells were compared.
Project description:The lysyl oxidase gene (LOX) inhibits Ras signaling in transformed fibroblasts and breast cancer cells. Its activity was mapped to the 162-amino-acid propeptide domain (LOX-PP) of the lysyl oxidase precursor protein. LOX-PP inhibits Erk signaling, motility, and tumor formation in a breast cancer xenograft model; however, its mechanism of action is largely unknown. Here, a copurification-mass spectrometry approach was taken using ectopically expressed LOX-PP in HEK293T cells and the heat shock/chaperone protein Hsp70 identified. Hsp70 interaction with LOX-PP was confirmed using coimmunoprecipitation of intracellularly and bacterially expressed and endogenous proteins. The interaction was mapped to the Hsp70 peptide-binding domain and to LOX-PP amino acids 26 to 100. LOX-PP association reduced Hsp70 chaperone activities of protein refolding and survival after heat shock. LOX-PP interacted with the Hsp70 chaperoned protein c-Raf. With the use of ectopic expression of LOX-PP wild-type and deletion proteins, small interfering RNA (siRNA) knockdown, and Lox(-/-) mouse embryo fibroblasts, LOX-PP interaction with c-Raf was shown to decrease downstream activation of MEK and NF-?B, migration, and anchorage-independent growth and reduce its mitochondrial localization. Thus, the interaction of LOX-PP with Hsp70 and c-Raf inhibits a critical intermediate in Ras-induced MEK signaling and plays an important role in the function of this tumor suppressor.
Project description:The lysyl oxidase gene inhibits Ras signaling in transformed fibroblasts and breast cancer cells. Its activity was mapped to the 162 amino acid propeptide domain (LOX-PP) of the lysyl oxidase precursor protein. LOX-PP inhibited the Her-2/Ras signaling axis in breast cancer cells, and reduced the Her-2-driven breast tumor burden in a xenograft model. Since its mechanism of action is largely unknown, co-affinity-purification/mass spectrometry was performed and the "Cbl-interacting protein of 85-kDa" (CIN85) identified as an associating protein. CIN85 is an SH3-containing adapter protein that is overexpressed in invasive breast cancers. The CIN85 SH3 domains interact with c-Cbl, an E3 ubiquitin ligase, via an unconventional PxxxPR ligand sequence, with the highest affinity displayed by the SH3-B domain. Interaction with CIN85 recruits c-Cbl to the AMAP1 complex where its ubiquitination activity is necessary for cancer cells to develop an invasive phenotype and to degrade the matrix. Direct interaction of LOX-PP with CIN85 was confirmed using co-immunoprecipitation analysis of lysates from breast cancer cells and of purified expressed proteins. CIN85 interaction with c-Cbl was reduced by LOX-PP. Domain specific CIN85 regions and deletion mutants of LOX-PP were prepared and used to map the sites of interaction to the SH3-B domain of CIN85 and to an epitope encompassing amino acids 111 to 116 of LOX-PP. Specific LOX-PP point mutant proteins P111A and R116A failed to interact with CIN85 or to compete for CIN85 binding with c-Cbl. Structural modeling identified a new atypical PxpxxRh SH3-binding motif in this region of LOX-PP. The LOX-PP interaction with CIN85 was shown to reduce the invasive phenotype of breast cancer cells, including their ability to degrade the surrounding extracellular matrix and for Matrigel outgrowth. Thus, LOX-PP interacts with CIN85 via a novel SH3-binding motif and this association reduces CIN85-promoted invasion by breast cancer cells.