Influence of the heme distal pocket on nitrite binding orientation and reactivity in Sperm Whale myoglobin

Nitrite binding to recombinant wild-type Sperm Whale myoglobin (SWMb) was studied using a combination of spectroscopic methods including room-temperature magnetic circular dichroism. These revealed that the reactive species is free nitrous acid and the product of the reaction contains a nitrite ion bound to the ferric heme iron in the nitrito- (O-bound) orientation. This exists in a thermal equilibrium with a low-spin ground state and a high-spin excited state and is spectroscopically distinct from the purely low-spin nitro- (N-bound) species observed in the H64V SWMb variant. Substitution of the proximal heme ligand, histidine-93, with lysine yields a novel form of myoglobin (H93K) with enhanced reactivity towards nitrite. The nitrito-mode of binding to the ferric heme iron is retained in the H93K variant again as a thermal equilibrium of spin-states. This proximal substitution influences the heme distal pocket causing the pKa of the alkaline transition to be lowered relative to wild-type SWMb. This change in the environment of the distal pocket coupled with nitrito-binding is the most likely explanation for the 8-fold increase in the rate of nitrite reduction by H93K relative to WT SWMb

Microenvironmental Hypoxia Induces Dynamic Changes in Lung Cancer Synthesis and Secretion of Extracellular Vesicles

Extracellular vesicles (EVs) mediate critical intercellular communication within healthy tissues, but are also exploited by tumour cells to promote angiogenesis, metastasis, and host immunosuppression under hypoxic stress. We hypothesize that hypoxic tumours synthesize hypoxia-sensitive proteins for packing into EVs to modulate their microenvironment for cancer progression. In the current report, we employed a heavy isotope pulse/trace quantitative proteomic approach to study hypoxia sensitive proteins in tumour-derived EVs protein. The results revealed that hypoxia stimulated cells to synthesize EVs proteins involved in enhancing tumour cell proliferation (NRSN2, WISP2, SPRX1, LCK), metastasis (GOLM1, STC1, MGAT5B), stemness (STC1, TMEM59), angiogenesis (ANGPTL4), and suppressing host immunity (CD70). In addition, functional clustering analyses revealed that tumour hypoxia was strongly associated with rapid synthesis and EV loading of lysosome-related hydrolases and membrane-trafficking proteins to enhance EVs secretion. Moreover, lung cancer-derived EVs were also enriched in signalling molecules capable of inducing epithelial-mesenchymal transition in recipient cancer cells to promote their migration and invasion. Together, these data indicate that lung-cancer-derived EVs can act as paracrine/autocrine mediators of tumorigenesis and metastasis in hypoxic microenvironments. Tumour EVs may, therefore, offer novel opportunities for useful biomarkers discovery and therapeutic targeting of different cancer types and at different stages according to microenvironmental conditions

Hypoxia-induced tumor exosomes promote M2-like macrophage polarization of infiltrating myeloid cells and microRNA-mediated metabolic shift

Developing tumors rapidly outgrow their oxygen supply and are subject to hypoxia, which stimulates hypersecretion of tumor-derived exosomes that promote angiogenesis, metastasis, and immunosuppression, but the molecular mediators of these pathological effects remain poorly defined. Using quantitative proteomics, we identified that exosomes produced by hypoxic tumor cells are highly enriched in immunomodulatory proteins and chemokines including CSF-1, CCL2, FTH, FTL, and TGFβ. Modeling exosome effects on tumor-infiltrating immune cells, we observed a potent ability of these hypoxia-induced vesicles to influence macrophage recruitment and promote M2-like polarization both in vitro and in vivo. In addition, hypoxic, but not normoxic, tumor exosomes enhanced oxidative phosphorylation in bone marrow-derived macrophages via transfer of let-7a miRNA, resulting in suppression of the insulin-Akt-mTOR signaling pathway. Together, these data demonstrate that hypoxia promotes tumor secretion of biomolecule-loaded exosomes that can modify the immunometabolic profile of infiltrating monocyte-macrophages to better evade host immunity and enhance tumor progression.Ministry of Education (MOE)National Medical Research Council (NMRC)This work is in part supported by grants from the Singapore Ministry of Education (MOE2014-T2-2-043, MOE2016- T2-2-018, and MOE2016-T3-1-003) and the National Medical Research Council of Singapore (NMRC-OF-IRG-0003-2016)

Pulsed SILAC-based proteomic analysis unveils hypoxia- and serum starvation-induced de novo protein synthesis with PHD finger protein 14 (PHF14) as a hypoxia sensitive epigenetic regulator in cell cycle progression

Hypoxia is an environmental cue that is associated with multiple tumorigenic processes such as immunosuppression, angiogenesis, cancer invasion, metastasis, drug resistance, and poor clinical outcomes. When facing hypoxic stress, cells initiate several adaptive responses such as cell cycle arrest to reduce excessive oxygen consumption and co-activation of oncogenic factors. In order to identify the critical novel proteins for hypoxia responses, we used pulsed-SILAC method to trace the active cellular translation events in A431 cells. Proteomic discovery data and biochemical assays showed that cancer cells selectively activate key glycolytic enzymes and novel ER-stress markers, while protein synthesis is severely suppressed. Interestingly, deprivation of oxygen affected the expression of various epigenetic regulators such as histone demethylases and NuRD (nucleosome remodeling and deacetylase) complex in A431 cells. In addition, we identified PHF14 (the plant homeodomain finger-14) as a novel hypoxia-sensitive epigenetic regulator that plays a key role in cell cycle progress and protein synthesis. Hypoxia-mediated inhibition of PHF14 was associated with increase of key cell cycle inhibitors, p14ARF, p15INK4b, and p16INK4a, which are responsible for G1-S phase transition and decrease of AKT-mTOR-4E-BP1/pS6K signaling pathway, a master regulator of protein synthesis, in response to environmental cues. Analysis of TCGA colon cancer (n=461) and skin cancer (n=470) datasets revealed a positive correlation between PHF14 expression and protein translation initiation factors, eIF4E, eIF4B, and RPS6. Significance of PHF14 gene was further demonstrated by in vivo mouse xenograft model using PHF14 KD cell lines.Ministry of Education (MOE)National Medical Research Council (NMRC)Published versionThis study was supported by the Singapore Ministry of Education (MOE2014-T2-2-043, MOE2016-T2-2-018 and MOE2016-T3-1-003) and the National Medical Research Council of Singapore (NMRC-OF-IRG-0003-2016)

Microenvironmental Hypoxia Induces Dynamic Changes in Lung Cancer Synthesis and Secretion of Extracellular Vesicles

10.3390/cancers12102917CANCERS121

Internet marketing and the realty sector: some cross-national insights from Canada and the UK

Based on a sample of 135 recent Canadian and British property buyers, this paper explores the effects of increasing integration of Internet-related technologies on buying behaviour within the largely neglected, but important, realty sector. Its key findings include the increasing (but non-dominant) use of the medium in searching for realty-related information (in Canada to a greater extent); the continued relevance of gender and educational levels in predicting realty-related Internet usage; the importance of online service provision in lowering 'offline' expectations; and the positive association between online realty experience and property buyer-realtor relationships. The implications of these findings for managers of realty businesses in Canada, the UK and beyond are discussed