Selective nitration of Hsp90 acts as a metabolic switch promoting tumor cell proliferation.

Tumors develop in an oxidative environment characterized by peroxynitrite production and downstream protein tyrosine (Y) nitration. We showed that tyrosine nitration supports schwannoma cell proliferation and regulates cell metabolism in the inheritable tumor disorder NF2-related Schwannomatosis (NF2-SWN). Here, we identified the chaperone Heat shock protein 90 (Hsp90) as the first nitrated protein that acts as a metabolic switch to promote schwannoma cell proliferation. Doubling the endogenous levels of nitrated Hsp90 in schwannoma cells or supplementing nitrated Hsp90 into normal Schwann cells increased their proliferation. Metabolically, nitration on either Y33 or Y56 conferred Hsp90 distinct functions; nitration at Y33 (Hsp90NY33) down-regulated mitochondrial oxidative phosphorylation, while nitration at Y56 (Hsp90NY56) increased glycolysis by activating the purinergic receptor P2X7 in both schwannoma and normal Schwann cells. Hsp90NY33 and Hsp90NY56 showed differential subcellular and spatial distribution corresponding with their metabolic and proliferative functions in schwannoma three-dimensional cell culture models. Collectively, these results underscore the role of tyrosine nitration as a post-translational modification regulating critical cellular processes. Nitrated proteins, particularly nitrated Hsp90, emerge as a novel category of tumor-directed therapeutic targets.

Comprehensive Physicochemical Characterization, In Vitro Membrane Permeation, and In Vitro Human Skin Cell Culture of a Novel TOPK Inhibitor, HI-TOPK-032.

Nonmelanoma skin cancers (NMSC) are the most common skin cancers, and about 5.4 million people are diagnosed each year in the United States. A newly developed T-lymphokine-activated killer cell-originated protein kinase (TOPK) inhibitor, HI-TOPK-032, is effective in suppressing colon cancer cell growth, inducing the apoptosis of colon cancer cells and ultraviolet (UV) light-induced squamous cell carcinoma (SCC). This study aimed to investigate the physicochemical properties, permeation behavior, and cytotoxicity potential of HI-TOPK-032 prior to the development of a suitable topical formulation for targeted skin drug delivery. Techniques such as scanning electron microscopy (SEM), energy-dispersive X-ray (EDX) spectroscopy, differential scanning calorimetry (DSC), hot-stage microscopy (HSM), X-ray powder diffraction (XRPD), Karl Fisher (KF) coulometric titration, Raman spectrometry, confocal Raman microscopy (CRM), attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR), and Fourier transform infrared microscopy were used to characterize HI-TOPK-032. The dose effect of HI-TOPK-032 on in vitro cell viability was evaluated using a 2D cell culture of the human skin keratinocyte cell line (HaCaT) and primary normal human epidermal keratinocytes (NHEKs). Transepithelial electrical resistance (TEER) at the air-liquid interface as a function of dose and time was measured on the HaCAT human skin cell line. The membrane permeation behavior of HI-TOPK-032 was tested using the Strat-M® synthetic biomimetic membrane with an in vitro Franz cell diffusion system. The physicochemical evaluation results confirmed the amorphous nature of the drug and the homogeneity of the sample with all characteristic chemical peaks. The in vitro cell viability assay results confirmed 100% cell viability up to 10 µM of HI-TOPK-032. Further, a rapid, specific, precise, and validated reverse phase-high performance liquid chromatography (RP-HPLC) method for the quantitative estimation of HI-TOPK-032 was developed. This is the first systematic and comprehensive characterization of HI-TOPK-032 and a report of these findings.

Transmembrane protein TMEM184B is necessary for interleukin-31-induced itch.

ABSTRACT: Nociceptive and pruriceptive neurons in the dorsal root ganglia (DRG) convey sensations of pain and itch to the spinal cord, respectively. One subtype of mature DRG neurons, comprising 6% to 8% of neurons in the ganglia, is responsible for sensing mediators of acute itch and atopic dermatitis, including the cytokine IL-31. How itch-sensitive (pruriceptive) neurons are specified is unclear. Here, we show that transmembrane protein 184B (TMEM184B), a protein with roles in axon degeneration and nerve terminal maintenance, is required for the expression of a large cohort of itch receptors, including those for interleukin 31 (IL-31), leukotriene C4, and histamine. Male and female mice lacking TMEM184B show reduced responses to IL-31 but maintain normal responses to pain and mechanical force, indicating a specific behavioral defect in IL-31-induced pruriception. Calcium imaging experiments indicate that a reduction in IL-31-induced calcium entry is a likely contributor to this phenotype. We identified an early failure of proper Wnt-dependent transcriptional signatures and signaling components in Tmem184b mutant mice that may explain the improper DRG neuronal subtype specification. Accordingly, lentiviral re-expression of TMEM184B in mutant embryonic neurons restores Wnt signatures. Together, these data demonstrate that TMEM184B promotes adult somatosensation through developmental Wnt signaling and promotion of proper pruriceptive gene expression. Our data illuminate a new key regulatory step in the processes controlling the establishment of diversity in the somatosensory system.

Assessment of Persistent, Bioaccumulative and Toxic Organic Environmental Pollutants in Liver and Adipose Tissue of Alzheimer's Disease Patients and Age-matched Controls.

BACKGROUND: Lifetime exposure to environmental (neuro) toxicants may contribute to the pathogenesis of Alzheimer's Disease (AD). Since many contaminants do not cross the blood-brain barrier, brain tissue alone cannot serve to assess the spectrum of environmental exposures. METHODS: We used liquid and gas chromatography tandem mass spectrometry to monitor, in postmortem liver and adipose tissues of AD patients and age-matched controls, the occurrence and concentrations of 11 environmental contaminants. RESULTS: Seven toxicants were detected at 100% frequency: p,p'-DDE, dieldrin, triclosan, methylparaben, bisphenol A, fipronil and tetrabromobisphenol A (TBBPA). Intra-individual, tissuedependent differences were detected for triclosan, methylparaben, fipronil and TBBPA. High concentrations of p,p'-DDE and dieldrin were observed in adipose tissue when compared to liver values for both AD cases and controls. CONCLUSION: This study provides vital data on organ-specific human body burdens to select analytes and demonstrate the feasibility of analyzing small sample quantities for toxicants suspected to constitute AD risk factors.

Role of environmental contaminants in the etiology of Alzheimer's disease: a review.

Alzheimer's dis ease (AD) is a leading cause of mortality in the developed world with 70% risk attributable to genetics. The remaining 30% of AD risk is hypothesized to include environmental factors and human lifestyle patterns. Environmental factors possibly include inorganic and organic hazards, exposure to toxic metals (aluminium, copper), pesticides (organochlorine and organophosphate insecticides), industrial chemicals (flame retardants) and air pollutants (particulate matter). Long term exposures to these environmental contaminants together with bioaccumulation over an individual's life-time are speculated to induce neuroinflammation and neuropathology paving the way for developing AD. Epidemiologic associations between environmental contaminant exposures and AD are still limited. However, many in vitro and animal studies have identified toxic effects of environmental contaminants at the cellular level, revealing alterations of pathways and metabolisms associated with AD that warrant further investigations. This review provides an overview of in vitro, animal and epidemiological studies on the etiology of AD, highlighting available data supportive of the long hypothesized link between toxic environmental exposures and development of AD pathology.

Metabolic proteomics of the liver and mammary gland during lactation.

The liver and the mammary gland have complementary metabolic roles during lactation. Glucose synthesized by the liver is released into the circulation and is taken up by the mammary gland where major metabolic products of glucose include milk sugar (lactose) and the glycerol backbone of milk fat (triglycerides). Hepatic synthesis of glucose is often accompanied by ß-oxidation in that organ to provide energy for glucose synthesis, while mammary gland synthesizes rather than oxidizes fat during lactation. We have therefore compared enzyme abundances between the liver and mammary gland of lactating Friesian cows where metabolic output is well established. Quantitative differences in protein amount were assessed using two-dimensional differential in-gel electrophoresis. As predicted, the abundances of enzymes catalysing gluconeogenesis and ß-oxidation were greatest in the liver, and enzyme abundances in mammary tissue were consistent with fat synthesis rather than ß-oxidation.

Proteomic changes at 8 weeks after infection are associated with chronic liver pathology in experimental schistosomiasis.

Chronic Schistosoma mansoni infection can present as a moderate or severe disease, termed intestinal or hepatosplenic schistosomiasis, respectively. Similarly, either moderate splenomegaly or hypersplenomegaly syndrome develops in CBA/J mice by 20weeks of infection and is similar to intestinal or hepatosplenic schistosomiasis respectively. Using this mouse model and two-dimensional differential in gel electrophoresis, the liver proteomic signatures of uninfected mice and mice infected for 6, 8, 12, or 20weeks were compared, and significant protein spots identified using mass spectrometry. We found the greatest number of changes at 12weeks suggesting that this period represents the peak time of change. Pathway analysis identified specific proteins and pathways that correlated to the pathological changes indicative of severe disease, and these pathways were involved as early as 8weeks after infection. These findings provide insight into the development of severe liver pathology in schistosomiasis and may aid in developing biomarkers for hepatosplenic schistosomiasis.

Identification of cytokeratin 18 as a biomarker of mouse and human hepatosplenic schistosomiasis.

Previously, we demonstrated unique protein expression patterns in 20-week-Schistosoma mansoni-infected CBA/J mice with moderate splenomegaly syndrome (MSS) or hypersplemomegaly syndrome (HSS). To better understand the development of severe pathology, we compared the two-dimensional differential in-gel electrophoresis (2D-DIGE) proteomic signatures of livers from uninfected mice and mice infected for 6, 8, 12, or 20 weeks and found significant changes in collagen isoforms, interleukin-2 (IL-2), cytokeratin 18, hydroxyproline, S. mansoni phosphoenolpyruvate carboxykinase, major urinary protein isoforms, and peroxiredoxin 6. Cytokeratin 18, hydroxyproline, and connective tissue growth factor (CTGF) were chosen for analysis in mouse and human sera using targeted biochemical assays. Consistent with the liver analysis, cytokeratin 18, CTGF, and hydroxyproline were significantly elevated in sera from mice with HSS compared to those from uninfected mice or mice with MSS. Moreover, cytokeratin 18 and CTGF were found to be markers for subjects with hepatosplenic and intestinal schistosomiasis, respectively, while serum hydroxyproline was a strong indicator of fibrosis for severe HS. These findings indicate that schistosome-associated changes to the liver can be detected in the serum and reveal the potential for cytokeratin 18 to be used as a diagnostic marker for early detection of hepatosplenic schistosomiasis.