Copper-dependent Amine Oxidase Research Articles

Characterization of the Preprocessed Copper Site Equilibrium in Amine Oxidase and Assignment of the Reactive Copper Site in Topaquinone Biogenesis.

Copper-dependent amine oxidases produce their redox active cofactor, 2,4,5-trihydroxyphenylalanine quinone (TPQ), via the CuII-catalyzed oxygenation of an active site tyrosine. This study addresses possible mechanisms for this biogenesis process by presenting the geometric and electronic structure characterization of the CuII-bound, prebiogenesis (preprocessed) active site of the enzyme Arthrobacter globiformis amine oxidase (AGAO). CuII-loading into the preprocessed AGAO active site is slow ( kobs = 0.13 h-1), and is preceded by CuII binding in a separate kinetically favored site that is distinct from the active site. Preprocessed active site CuII is in a thermal equilibrium between two species, an entropically favored form with tyrosine protonated and unbound from the CuII site, and an enthalpically favored form with tyrosine bound deprotonated to the CuII active site. It is shown that the CuII-tyrosinate bound form is directly active in biogenesis. The electronic structure determined for the reactive form of the preprocessed CuII active site is inconsistent with a biogenesis pathway that proceeds through a CuI-tyrosyl radical intermediate, but consistent with a pathway that overcomes the spin forbidden reaction of 3O2 with the bound singlet substrate via a three-electron concerted charge-transfer mechanism.

MiR-210 promotes lung adenocarcinoma proliferation, migration, and invasion by targeting lysyl oxidase-like 4.

Accumulating evidence has revealed that various microRNAs are deregulated and involved in lung cancer development and metastasis. miR-210 is implicated in several cancer progression. However, the detailed biological function and role of miR-210 in lung adenocarcinoma remains unclear. Our current study was aimed to investigate the mechanism of miR-210 in lung adenocarcinoma progression. We observed that miR-210 was significantly upregulated in lung cancer cell lines (A549 and H1650) in comparison to BEAS-2B cells. In addition, we found that miR-210 was greatly elevated in lung adenocarcinoma tissues. Then, it was shown that overexpression of miR-210 was able to promote lung cancer cell proliferation and colony formation ability while inhibitors of miR-210 exhibited a reversed phenomenon. Subsequently, A549 and H1650 cell migration and invasion capacity were obviously restrained by miR-210 inhibition whereas induced by miR-210 mimics. Lysyl oxidase-like 4 (LOXL4), a member of the secreted copper-dependent amine oxidases has been found to be increased or decreased in different cancer types. Here, we confirmed that LOXL4 could serve as a downstream target of miR-210 and miR-210 promoted lung cancer progression via targeting LOXL4. In A549 and H1650 cells, knockdown of LOXL4 dramatically repressed lung cancer cell proliferation, migration, and invasion. In conclusion, our study implied that miR-210 might indicate a new perspective for lung cancer.

3D hydrogel breast cancer models for studying the effects of hypoxia on epithelial to mesenchymal transition.

Solid tumors are 3D assemblies of cancer cells, together with multiple stromal cell types within an extracellular matrix. Yet, the vast majority of cell-based studies to characterize oncogenesis and discovery of new anti-cancer drugs is conducted using conventional 2D monolayer culture systems, where cells are grown on plastic substratum under normoxic environments. In current study, we generated 3D breast cancer cell culture platform consists of photocrosslinkable hydrogels and encapsulated isogenic primary (21PT) and a metastatic (21MT-2) breast cancer cell lines derived from the primary tumor and pleural effusion from the same patient. We demonstrated that hypoxia decreased cellular assembly size and density, and promoted epithelial to mesenchymal transition (EMT) process, without affecting cell viability. Next, we showed hypoxia enhanced breast cancer cell migration, and expression and secretion of lysyl oxidase (LOX), which is copper-dependent amine oxidase and has the primary function to drive the crosslinking of collagen and elastin and is regulated by hypoxia. Furthermore, to recapitulate in vivo situation, we generated breast cancer and lung cells (derived from the same patient) contact model by stacking 3D hydrogel constructs with breast cancer cells onto lung mesenchymal cells (LMC) laden-hydrogel and then showed breast cancer cells migrated towards LMC during hypoxia. Lastly, as a validation of this model for future screen of therapeutic agents, we demonstrated that LOX inhibitor exhibited a significant decrease in breast cancer cell viability, migration, and EMT. Taken together, these results validate the use of hydrogels based models to examine hypoxia related EMT in breast cancer cells.

Inhibition of Lysyl Oxidase Activity Prevents Volume Overload Diastolic and Systolic Dysfunction

The cardiac extracellular matrix (ECM) is a structural framework which bridges cardiomyocytes to the cystoskeletal myofibril, preserving alignment, while providing tensile strength to the myocardium. Abnormalities in the architectural scaffolding of the ECM have been noted during various cardiac pathologies. The major structural components of the cardiac ECM are collagens, which are produced by fibroblasts. The relative expression of collagens and the degree of cross‐linking dictate the passive mechanical properties of the heart. Lysyl oxidase (LOX) is a copper‐dependent amine oxidase that plays a critical role in covalently cross‐linking fibrillar collagens, which determines the insolubility, stiffness and resistance to degradation of the resulting collagen fibrils. In our previous studies we have shown marked increases in LOX expression and activity in failing hearts relative to compensated and unstressed hearts. Our goal of this study was to determine whether LOX inhibition, initiated during the early stages of cardiac remodeling, could attenuate fibrosis and cardiac dysfunction.We used the aortocaval fistula (ACF) surgical model on Sprague‐Dawley rats to induce biventricular volume overload (VO). Two weeks after surgery, both sham‐operated and VO rats were treated with the LOX inhibitor, beta‐aminoproprionitrile (BAPN; 100 mg/kg/d), for 12 weeks. BAPN was administered via an osmotic pump surgically inserted into the abdominal cavity. After a total of 14 weeks of VO, left ventricular (LV) pressure‐volume catheterization was used to assess alterations in cardiac function. Western blot protein analyses were performed to assess alterations in LOX, collagens I and III, matrix metalloproteinases (MMPs), and their inhibitors (TIMPs). Further, LV strain and stiffness were calculated to determine alterations in diastolic function.Our data demonstrate that chronic VO caused a significant increase in LV wall stress at both diastole and systole, and produced significant LV hypertrophy (183%, 60%, and 92% increase, respectively vs. control; p<0.05). Further, VO induced significant increases in LV strain and stiffness (126% and 77% increase, respectively vs. control), as well as significant increases in the expression of fibrotic proteins including LOX, collagen I, collagen III, MMP2, and TIMP1 (135%, 58.4%, 26.7%, 39%, and 63% increase, respectively vs. control; p<0.05). Finally, VO also caused systolic dysfunction, as evident by decreases in cardiac contractility and ejection fraction (51% and 26% decrease, respectively vs. control; p<0.05). Further, our data highlight the cardioprotective role of LOX inhibition against adverse cardiac remodeling due to VO. LOX inhibition completely prevented VO‐induced increases in LV wall stress and ventricular hypertrophy. Further, LOX inhibition prevented VO‐induced fibrosis and diastolic dysfunction, as evident by normalized levels of collagens, LOX, MMPs, TIMPs, and LV stiffness. Finally, LOX inhibition prevented the VO‐induced decline in cardiac contractility. Overall, our data indicate that LOX inhibition prevented adverse ECM remodeling and cardiac dysfunction in the volume overloaded heart.Support or Funding InformationSupport for this project provided by the American Heart Association Southeast Affiliate and the NIH Heart, Lung and Blood Institute: Grant‐in‐Aid (#16GRNT30440008‐JDG), AHA Predoctoral Fellowship (# 16PRE29150010‐EEH), and F31 (#1F31HL134263‐ 01A1‐EEH).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Identification of Histidine 303 as the Catalytic Base of Lysyl Oxidase via Site-Directed Mutagenesis.

Lysyl oxidase (LOX) is a copper-dependent amine oxidase enzyme that catalyzes the formation of crosslinkages of collagen and elastin in connective tissues by oxidative deamination of lysine. Using site-directed mutagenesis, Histidine 303 has been shown to be a key residue that acts as the necessary catalytic base for this enzyme to function properly. Histidine 303 was mutated to isoleucine to remove catalytic activity and to aspartate and glutamate, respectively, in order to provide alternate residues that could act as a general base that could maintain catalytic activity. Overexpression of the H303I mutant yielded 3.9mg of enzyme per liter of media, the H303D mutant yielded 3.3mg of enzyme per liter of media, and the H303E mutant yielded 3.0mg/L of media. Overexpression of wildtype LOX yielded 4.5mg/L of media, which is a slight improvement from previous yields. Total copper incorporation for H303I was calculated to be 68% and no copper was detected for the H303D and H303E mutants. As LOX requires the self-processed cofactor lysyl tyrosyl quinone (LTQ) for activity, total LTQ content was obtained by reacting the enzyme with phenylhydrazine and using the previously reported extinction coefficient of 15.4mM/cm. LTQ content for the wildtype enzyme was determined to be 92%, for H303I the total LTQ content was determined to be 36%, and no LTQ was detected for the H303D and H303E mutants. No catalytic activity was detected for any mutants when compared to the wildtype which has a previously reported activity of 0.11 U/mg. Comparison of excitation-emission matrices (EEM) of each of the mutants as compared to the wildtype indicate that all the mutations cause a change in the internal environment of the enzyme, albeit to varying degrees, as evidenced by the observed shifts.

Biochemical characterization and homology modeling of polyamine oxidase from cyanobacterium Synechocystis sp. PCC 6803

The intracellular polyamine contents are regulated not only by polyamine biosynthesis and transport but also by polyamine degradation catalyzed by copper-dependent amine oxidase (DAO) and FAD-dependent polyamine oxidase (PAO). The genome sequence of Synechocystis sp. PCC 6803 reveals the presence of at least one putative polyamine oxidase gene, slr5093. The open reading frame of slr5093 encoding Synechocystis polyamine oxidase (SynPAO, E.C. 1.5.3.17) was expressed in Escherichia coli. The purified recombinant enzyme had the characteristic absorption spectrum of a flavoprotein with absorbance peaks at 380 and 450 nm. The optimum pH and temperature for the oxidation of both spermidine and spermine are 8.5 and 30 °C, respectively. The enzyme catalyzed the conversion of spermine and spermidine to spermidine and putrescine, respectively, with higher catalytic efficiency when spermine served as substrate. These results suggest that SynPAO is a polyamine oxidase involved in a polyamine back-conversion pathway. Based on the structural analysis, Gln94, Tyr403 and Thr440 in SynPAO are predicted to be important residues in the active site.

Anabolic role of lysyl oxidase like-2 in cartilage of knee and temporomandibular joints with osteoarthritis

BackgroundLysyl oxidase like-2 (LOXL2) is a copper-dependent amine oxidase. Our previous studies showed that LOXL2 is elevated during mouse fracture healing. The goal of this study was to evaluate the potential of LOXL2 to act as an anabolic agent in cartilage affected by osteoarthritis (OA).MethodsLOXL2 was visualized in tissues from human knee and hip joints and temporomandibular joints (TMJ) by immunofluorescence. The activity of LOXL2 in human articular and TMJ chondrocytes was assessed by cell-based assays, microarray analysis, and RT-qPCR, and LOXL2-mediated activation of NF-κB and extracellular signal-related kinase (ERK) signaling pathways was measured by western blotting. To examine LOXL2-induced effect in vivo, we implanted Matrigel-imbedded human chondrocytes into nude mice and exposed them to exogenous LOXL2 for 6 weeks. Finally, LOXL2-induced effects on collagen type 2 α1 (COL2A1) and phospho-SMAD2/3 were evaluated by immunofluorescence analysis.ResultsLOXL2 staining was detected in damaged regions of human TMJ, hip and knee joints affected by OA. Stimulation with transforming growth factor (TGF)-β1 upregulated LOXL2 expression, while pro-inflammatory cytokines IL-1β and TNF-α downregulated LOXL2, in human chondrocytes. Viral transduction of LOXL2 in OA chondrocytes increased the mRNA levels of chondroitin sulfate proteoglycan (CSPG4), aggrecan (ACAN), sex determining region Y-box containing gene 9 (SOX9), and COL2A1 but reduced the levels of extracellular matrix (ECM)-degrading enzymes matrix metalloproteinase (MMP)1, MMP3, and MMP13. Further, forced expression of LOXL2 promoted chondrogenic lineage-specific gene expression, increased the expression of COL2A1 in the presence of TNF-α, and inhibited chondrocyte apoptosis. LOXL2 expression also inhibited IL-1β-induced phospho-NF-κB/p65 and TGF-β1-induced ERK1/2 phosphorylation. Matrigel constructs of human chondrocytes from the knee joint and TMJ implanted in nude mice showed anabolic responses after LOXL2 transduction, including increased expression of SOX9, ACAN, and COL2A1. Finally, immunofluorescence staining revealed co-localization of LOXL2 with SOX9 in the nuclei of cells in the implants, decreased phospho-SMAD2/3, and increased COL2A1 staining.ConclusionOur results suggest that although LOXL2 is upregulated in cartilage affected by OA, this may be a protective response that promotes anabolism while inhibiting specific catabolic responses in the pathophysiology of OA.

Abstract 73: Pre- and Post-Translational Regulation of Lysyl Oxidase/β1-Intergrin in Aortic Vascular Smooth Cell of Hypertension

Background: An increase in aortic stiffness is a fundamental component of hypertension . We previous showed that aortic vascular smooth muscle cells (VSMCs) play an causative role in aortic stiffening not only due to the increased intrinsic stiffness in VSMC itself but also due to its interaction with extracellular matrix (ECM). However, the molecular mechanism involved is unclear. Here, we test the hypothesis that the aortic VSMCs mediates pre- and post-translational regulation of Lysyl Oxidase (Lox)/β1intergrain pathway which may be one of the mechanisms of VSMCs-ECM remodeling in hypertension. Methods and Results: 4 months old male spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto (WKY) rats were studied. Aortic pressure and stiffness were measured with an invasive catheter and Doppler imaging echocardiography, respectively. Compared to WKY, SHR showed a significant higher aortic pressure (mean arterial pressure (MAP) 138.7±11.8 vs 102.7±7 mmHg, ( p <0.01) and a greater aorta stiffness reflected by 2.5-fold higher arterial stiffness index (ASI) in SHR vs WKY ( p <0.01). VSMCs were isolated from thoracic aorta, then cultured and measured at passages 2 to 4. Real time PCR and western blot were used to detect the gene and protein expression. Compared to WKY, VSMCs from SHR showed a significant increase in mRNA Lox, a copper-dependent amine oxidase mediating the crosslinking of collagens and elastin (2.7 folds vs WKY, p <0.01). The abundance of LOX protein was significant increased not only at pro-enzyme lox level, but also at the regulatory pro-peptide lox and matured active enzyme Lox in SHR VSMCs by 3.5 folds and 1.4 folds respectively ( p <0.01), indicating an increased post-translational regulation of LOX in SHR VSMCs. In addition, the glycosylated β1 integrin, a transmembrane receptor that facilitate cell-ECM adhesion, was found to be significant increase in SHR VSMCs vs WKY. Conclusion: The regulation of pre- and post-translational regulation of Lox/β1intergrain pathway in aortic VSMCs presents a novel mechanism of cell-ECM interaction, which may contribute to the increased aortic stiffness in SHR.

Identification of 4-(Aminomethyl)-6-(trifluoromethyl)-2-(phenoxy)pyridine Derivatives as Potent, Selective, and Orally Efficacious Inhibitors of the Copper-Dependent Amine Oxidase, Lysyl Oxidase-Like 2 (LOXL2).

LOXL2 catalyzes the oxidative deamination of ε-amines of lysine and hydroxylysine residues within collagen and elastin, generating reactive aldehydes (allysine). Condensation with other allysines or lysines drives the formation of inter- and intramolecular cross-linkages, a process critical for the remodeling of the ECM. Dysregulation of this process can lead to fibrosis, and LOXL2 is known to be upregulated in fibrotic tissue. Small-molecules that directly inhibit LOXL2 catalytic activity represent a useful option for the treatment of fibrosis. Herein, we describe optimization of an initial hit 2, resulting in identification of racemic-trans-(3-((4-(aminomethyl)-6-(trifluoromethyl)pyridin-2-yl)oxy)phenyl)(3-fluoro-4-hydroxypyrrolidin-1-yl)methanone 28, a potent irreversible inhibitor of LOXL2 that is highly selective over LOX and other amine oxidases. Oral administration of 28 significantly reduced fibrosis in a 14-day mouse lung bleomycin model. The (R,R)-enantiomer 43 (PAT-1251) was selected as the clinical compound which has progressed into healthy volunteer Phase 1 trials, making it the "first-in-class" small-molecule LOXL2 inhibitor to enter clinical development.

LOXL3-sv2, a novel variant of human lysyl oxidase-like 3 (LOXL3), functions as an amine oxidase.

Human lysyl oxidase-like 3(LOXL3) functions as a copper-dependent amine oxidase toward collagen and elastin. The LOXL3protein contains four scavenger receptor cysteine-rich(SRCR) domains in the N-terminus in addition to the C-terminal characteristic domains of the lysyl oxidase(LOX) family, such as a copper-binding domain, a cytokine receptor‑like domain and residues for the lysyl-tyrosyl quinone cofactor. Using BLASTNsearches, we identified a novel variant of LOXL3(termed LOXL3-sv2), which lacked the sequences corresponding to exons4 and5 of LOXL3. The LOXL3-sv2mRNA is at least 2,398bp in length, encoding a 608amino acid-long polypeptide with a calculated molecular mass of 67.4kDa. The deletion of exons4 and5 do not change the open-reading frame of LOXL3 but results in deletion of the SRCR domain2. The recombinant LOXL3-sv2protein showed a β-aminopropionitrile-inhibitable amine oxidase activity toward collagen typeI. In RT-PCR analysis, LOXL3-sv2 was detected in all human tissues tested, along with LOXL3 and LOXL3-sv1, a previously identified variant of LOXL3. These findings indicate that the human LOXL3gene encodes at least three variants, LOXL3, LOXL3-sv1 andLOXL3-sv2, all of which function as amine oxidases.

Lysyl Oxidase Is a Strong Determinant of Tumor Cell Colonization in Bone.

Lysyl oxidase (LOX) is a secreted copper-dependent amine oxidase whose primary function is to drive collagen crosslinking and extracellular matrix stiffness. LOX in colorectal cancer synergizes with hypoxia-inducible factor-1 (HIF-1) to promote tumor progression. Here we investigated whether LOX/HIF1 endows colorectal cancer cells with full competence for aggressive colonization in bone. We show that a high LOX expression in primary tumors from patients with colorectal cancer was associated with poor clinical outcome, irrespective of HIF-1 In addition, LOX was expressed by tumor cells in the bone marrow from colorectal cancer patients with bone metastases. In vivo experimental studies show that LOX overexpression in colorectal cancer cells or systemic delivery of the conditioned medium from LOX-overexpressing colorectal cancer cells promoted tumor cell dissemination in the bone marrow and enhanced osteolytic lesion formation, irrespective of HIF-1 Conversely, silencing or pharmacologic inhibition of LOX activity blocked dissemination of colorectal cancer cells in the bone marrow and tumor-driven osteolytic lesion formation. In vitro, tumor-secreted LOX supported the attachment and survival of colorectal cancer cells to and in the bone matrix, and inhibited osteoblast differentiation. LOX overexpression in colorectal cancer cells also induced a robust production of IL6. In turn, both LOX and IL6 were acting in concert to promote RANKL-dependent osteoclast differentiation, thereby creating an imbalance between bone resorption and bone formation. Collectively, our findings show that LOX supports colorectal cancer cell dissemination in the bone marrow and they reveal a novel mechanism through which LOX-driven IL6 production by colorectal cancer cells impairs bone homeostasis. Cancer Res; 77(2); 268-78. ©2016 AACR.

Abstract 2727: LOX is a novel mitotic spindle-associated protein essential for mitosis

Abstract Lysyl oxidase (LOX) is a copper-dependent amine oxidase that plays a critical role in the biogenesis of connective tissue matrices. LOX is highly expressed in aggressive cancers; has been associated with a shorter survival and regulates cancer progression in a variety of human malignancies. Here, we report a new function of LOX in mitosis in anaplastic thyroid cancer cell line (THJ-16T), breast cancer cell line (MDA-MB231) and HeLa cells. We show that LOX co-localizes to mitotic spindles from metaphase to telophase, and p-H3(Ser10)-positive cells harbor strong LOX staining. Further, purification of mitotic spindles from synchronized cells show that LOX fails to bind to microtubules in the presence of nocodazole, whereas paclitaxel treated samples showed enrichment in LOX expression suggesting that LOX binds to stabilized microtubules only. Functional studies of LOX depletion resulted in decreased cellular proliferation, G2/M cell cycle arrest, and formation of gigantic nuclei. Further, LOX knockdown led to reduced p-H3(Ser10), cyclin B1, CDK1, and Aurora B expression. Lastly, LOX knockdown significantly increased sensitivity of cancer cells to chemotherapeutic agents that target microtubules and this effect was synergistic. Our results show identify a novel function of LOX in cancer cell mitosis, and that loss of LOX expression in cancer cells can enhanced effects of anti-microtubules agents used in cancer therapy. Citation Format: Myriem Boufraqech, Darmood Wei, Urbain Weyemi, Lisa Zhang, Martha Quezado, Petr Kalab, Electron Kebebew. LOX is a novel mitotic spindle-associated protein essential for mitosis. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2727.

Lysyl oxidase promotes epithelial-to-mesenchymal transition during paraquat-induced pulmonary fibrosis.

Lysyl oxidase (LOX) is a copper-dependent amine oxidase that plays a critical role in pulmonary fibrosis. Our previous study demonstrated that epithelial-to-mesenchymal transition (EMT) was strongly associated with paraquat (PQ) induced pulmonary fibrosis. This present study was aimed to evaluate the potential involvement of LOX on EMT in the process of pulmonary fibrosis induced by PQ. We established an in vivo rat model and an in vitro cell model induced by PQ treatment and found that LOX protein expression was significantly up-regulated and collagen deposition was enhanced in rats. The EMT process was strongly found in A549 and RLE-6TN cells after PQ exposure. After inactivating LOX with an inhibitor, pulmonary fibrosis was significantly reduced and EMT was also suppressed. Additionally, small interfering RNA (siRNA) targeting LOX was used to silence LOX expression to observe EMT in A549 cells. As a result, LOX could promote the progress of EMT, and inactivating LOX alleviated the EMT process in PQ-induced pulmonary fibrosis and mesenchymal-to-epithelial transition (MET) occurred after inactivating LOX in vitro and in vivo. In conclusion, LOX could promote the progress of EMT and inactivating LOX alleviated EMT in PQ-induced pulmonary fibrosis. Therefore, LOX could potentially be a new candidate therapeutic target for pulmonary fibrosis induced by PQ by regulating the balance between EMT and MET.

LOXL2 Highly-Expressed Induce the Transition of Stromal Cells into Cancer-Associated Fibroblasts Which Maybe Involve in Myeloproliferative Neoplasms Progession

Backgroud and objective: Myeloproliferative neoplasms (MPNs) are malignant disorders by proliferation of one of the myeloid lineages and characteristically show an increase in bone marrow reticulin reticulin-fibrosis.Lysyl oxidases like-2(LOXL2) is copper-dependent amine oxidases that play a critical role in the biogenesis of connective tissue by crosslinking extracellular matrix proteins, collagen and elastin,and Cancer associated-fibroblasts (CAFs) are major mediators in tumor microenvironment. Studies found that loxl2 stimulate CAFs grouth solid tumor,and the expression of LOXL2 is increased in MPN patients,espessionly in PMF patients.Here, we want to evaluate whether the expression of higher LOXL2 associated to CAFs during various MPN progression.Patients and methods: We compared normal bone marrows and those from patients with chronic myeloid leukemia(CML)(include CML-CP n=20,CML-BC n=13),polycythemia vera(PV)(n=18), essential thrombocythemia(ET) (n=23), and primary myelofibrosis (PMF) (n=8). We detected α-smooth actin and reticulin protein by immunohistochemical staining, examined LOXL2 expression by western blot in bone marrow and ELIZA kit in serum.Results: LOXL2 was not detected in normal bone marrows and serum.The level of LOXL2 gene is over expressed in PMF (p<0.01) and CML-BC (p=0.02). In other MPNs a differential pattern of expression were statistically significant (P< 0.010).The level of LOXL2 expression associated with reticulin protein expression in bone marrow, especially if reticulin protein expressed higher than 2+(p=0.01).We detected α-smooth actin positive stromal cells in CML-BC and PMF patients,and the level of LOXL2 expression is related to α-smooth actin positive stromal cells(p<0.05).we also detected α-smooth actin after co-cultured mesenchymal stem cell(MSCs) with sLOXL2 for 96 hour.Conclusion: Higher level of LOXL2 could be promote MPN progression by modulating several functions of surrounding stromal cells which acquire features of cancer-associated fibroblasts involved in the pathogenesis of MPN. These findings maybe used as the basis for future targeted therapy directed against MPN progression. DisclosuresNo relevant conflicts of interest to declare.

Estradiol plays a role in regulating the expression of lysyl oxidase family genes in mouse urogenital tissues and human Ishikawa cells.

The lysyl oxidase (LOX) family encodes the copper-dependent amine oxidases that play a key role in determining the tensile strength and structural integrity of connective tissues by catalyzing the crosslinking of elastin or collagen. Estrogen may upregulate the expression of LOX and lysyl oxidase-like 1 (LOXL1) in the vagina. The objective of this study was to determine the effect of estrogen on the expression of all LOX family genes in the urogenital tissues of accelerated ovarian aging mice and human Ishikawa cells. Mice and Ishikawa cells treated with estradiol (E2) showed increased expression of LOX family genes and transforming growth factor β1 (TGF-β1). Ishikawa cells treated with TGF-β1 also showed increased expression of LOX family genes. The Ishikawa cells were then treated with either E2 plus the TGF-β receptor (TGFBR) inhibitor SB431542 or E2 alone. The expression of LOX family genes induced by E2 was reduced in the Ishikawa cells treated with TGFBR inhibitor. Our results showed that E2 increased the expression of the LOX family genes, and suggest that this induction may be mediated by the TGF-β signal pathway. E2 may play a role in regulating the expression of LOX family genes.

Domain mobility as probed by small-angle X-ray scattering may account for substrate access to the active site of two copper-dependent amine oxidases.

Amine oxidases are a family of dimeric enzymes that contain one copper(II) ion and one 2,4,5-trihydroxyphenyalanine quinone per subunit. Here, the low-resolution structures of two Cu/TPQ amine oxidases from lentil (Lens esculenta) seedlings and from Euphorbia characias latex have been determined in solution by small-angle X-ray scattering. The active site of these enzymes is highly buried and requires a conformational change to allow substrate access. The study suggests that the funnel-shaped cavity located between the D3 and D4 domains is narrower within the crystal structure, whereas in solution the D3 domain could undergo movement resulting in a protein conformational change that is likely to lead to easier substrate access.

A molecular model of human Lysyl Oxidase (LOX) with optimal copper orientation in the catalytic cavity for induced fit docking studies with potential modulators.

Lysyl oxidase (LOX) is a copper dependent amine oxidase which catalyses the cross linking of collagen and elastin towards the maturation of extracellular matrix. The expression and activity of LOX is known to vary under pathological conditions such as tumorigenesis, hyperhomocysteinemia, copper deficiency diseases, pseudoexfoliation syndrome and proliferative diabetic retinopathy. Despite the implication of LOX in many diseases, there is inadequate information about its structure. Therefore, we describe a molecular model of Human Lysyl Oxidase (LOX) with optimal copper orientation in the catalytic cavity for induced fit docking studies with potential modulators. The predicted model was found to be highly plausible as per the stereochemistry checks. Further, Molecular Dynamics (MD) studies also inferred the stability of the predicted structure. We performed Induced Fit Docking (IFD) of LOX modulators to the predicted structure and also validated the molecular interactions in implicit solvent model by calculating Molecular Mechanics Generalized Born Surface Area (MMGBSA). The IFD results strongly reveal that aspartic acid residues in the catalytic cavity as the key players in establishing interactions with small molecules. The insights from this study will aid in better exploration of the structure-function relationship of LOX.

Lysyl oxidase genetic variants affect gene expression in cervical cancer.

Lysyl oxidase (LOX) is a copper-dependent amine oxidase that plays important roles in the homeostasis of tumors. The aim of this study was to investigate the association between LOX polymorphisms and cervical cancer, and the effect of these polymorphisms on gene expression. We evaluated two polymorphisms of LOX, rs1800449G/A (G473A) and rs2278226C/G, in 262 cervical cancer cases and 298 healthy controls in the Chinese population. Results showed that the prevalence of rs1800449AA genotype was significantly increased in cases than in controls (p=0.004). Individuals who carried the rs1800449A allele had a 1.56-fold increased risk for cervical cancer than those with the rs1800449G allele (p=0.003). The rs2278226CG genotype also revealed a significantly higher proportion in cases (20.6%) than in controls (7.7%, p<0.001). Interestingly, when analyzing these two polymorphisms with the serum level of LOX, we identified that cervical cancer patients carrying the rs2278226CG genotype had a significantly elevated level of LOX than those with rs2278226CC wild type, whereas the same phenomenon was not observed in controls. The rs1800449 polymorphism did not affect the LOX serum level in either controls or patients. These results suggest that the polymorphisms in the LOX gene may be involved in the development of cervical cancer through various mechanisms.

PXS-4681A, a Potent and Selective Mechanism-Based Inhibitor of SSAO/VAP-1 with Anti-Inflammatory Effects In Vivo

Semicarbazide-sensitive amine oxidase (SSAO), also known as vascular adhesion protein-1 (VAP-1), is a member of the copper-dependent amine oxidase family that is associated with various forms of inflammation and fibrosis. To investigate the therapeutic potential of SSAO/VAP-1 inhibition, potent and selective inhibitors with drug-like properties are required. PXS-4681A [(Z)-4-(2-(aminomethyl)-3-fluoroallyloxy)benzenesulfonamide hydrochloride] is a mechanism-based inhibitor of enzyme function with a pharmacokinetic and pharmacodynamic profile that ensures complete, long-lasting inhibition of the enzyme after a single low dose in vivo. PXS-4681A irreversibly inhibits the enzyme with an apparent Ki of 37 nM and a kinact of 0.26 min(-1) with no observed turnover in vitro. It is highly selective for SSAO/VAP-1 when profiled against related amine oxidases, ion channels, and seven-transmembrane domain receptors, and is superior to previously reported inhibitors. In mouse models of lung inflammation and localized inflammation, dosing of this molecule at 2 mg/kg attenuates neutrophil migration, tumor necrosis factor-α, and interleukin-6 levels. These results demonstrate the drug-like properties of PXS-4681A and its potential use in the treatment of inflammation.

Lysyl oxidase genetic variants and the prognosis of glioma

Lysyl oxidase (LOX) is a copper-dependent amine oxidase that plays important roles in the development and homeostasis of primary brain tumors such as glioma. The aim of this study was to investigate whether polymorphisms in the LOX gene were associated with susceptibility to glioma. We tested two functional polymorphisms of LOX, -22G/C and 473G/A, and compared them between 466 glioma cases and 502 healthy controls in the Chinese population. Results showed that the prevalence of 473AA genotype was significantly increased in cases than in controls (p = 0.001). Individuals who carried 473A allele had a 1.44-fold of increased risk for glioma than those with 473G allele (p = 0.002). In addition, when analyzing the survival time of glioma patients with LOX 473G/A polymorphism, cases with AA genotype had significantly shorter survival time compared to the patients carrying G allele (25.0 months vs 43.0 months, p = 0.0009). These results suggested that polymorphism in LOX gene was associated with increased susceptibility to glioma and could be used as prognostic factor for this malignancy.