Production and Release of Proinflammatory Mediators by the Cockroach Allergen Bla g 1 via a Shared Membrane-Destabilization Mechanism.

The cockroach allergen Bla g 1 encloses an exceptionally large hydrophobic cavity, which allows it to bind and deliver unsaturated fatty acid ligands. Bla g 1-mediated delivery of naturally occurring (nMix) ligands has been shown to destabilize lipid membranes, contributing to its digestive/antiviral functions within the source organism. However, the consequences of this activity on Bla g 1 allergenicity following human exposure remain unknown. In this work, we show that Bla g 1-mediated membrane disruption can induce a proinflammatory immune response in mammalian cells via two complementary pathways. At high concentrations, the cytotoxic activity of Bla g 1 induces the release of proinflammatory cytosolic contents including damage-associated molecular patterns (DAMPs) such as heat-shock Protein-70 (HSP70) and the cytokine interleukin-1 (IL-1ß). Sublytic concentrations of Bla g 1 enhanced the ability of phospholipase A2 (PLA2) to extract and hydrolyze phospholipid substrates from cellular membranes, stimulating the production of free polyunsaturated fatty acids (PUFAs) and various downstream inflammatory lipid mediators. Both of these effects are dependent on the presence of Bla g 1's natural fatty-acid (nMix) ligands with CC50 values corresponding to the concentrations required for membrane destabilization reported in previous studies. Taken together, these results suggest that mechanisms through which Bla g 1-mediated lipid delivery and membrane destabilization could directly contribute to cockroach allergic sensitization.

Deep phenotyping of post-infectious myalgic encephalomyelitis/chronic fatigue syndrome.

Post-infectious myalgic encephalomyelitis/chronic fatigue syndrome (PI-ME/CFS) is a disabling disorder, yet the clinical phenotype is poorly defined, the pathophysiology is unknown, and no disease-modifying treatments are available. We used rigorous criteria to recruit PI-ME/CFS participants with matched controls to conduct deep phenotyping. Among the many physical and cognitive complaints, one defining feature of PI-ME/CFS was an alteration of effort preference, rather than physical or central fatigue, due to dysfunction of integrative brain regions potentially associated with central catechol pathway dysregulation, with consequences on autonomic functioning and physical conditioning. Immune profiling suggested chronic antigenic stimulation with increase in naïve and decrease in switched memory B-cells. Alterations in gene expression profiles of peripheral blood mononuclear cells and metabolic pathways were consistent with cellular phenotypic studies and demonstrated differences according to sex. Together these clinical abnormalities and biomarker differences provide unique insight into the underlying pathophysiology of PI-ME/CFS, which may guide future intervention.

Changes in metabolic landscapes shape divergent but distinct mutational signatures and cytotoxic consequences of redox stress.

Mutational signatures discerned in cancer genomes, in aging tissues and in cells exposed to toxic agents, reflect complex processes underlying transformation of cells from normal to dysfunctional. Due to its ubiquitous and chronic nature, redox stress contributions to cellular makeover remain equivocal. The deciphering of a new mutational signature of an environmentally-relevant oxidizing agent, potassium bromate, in yeast single strand DNA uncovered a surprising heterogeneity in the mutational signatures of oxidizing agents. NMR-based analysis of molecular outcomes of redox stress revealed profound dissimilarities in metabolic landscapes following exposure to hydrogen peroxide versus potassium bromate. The predominance of G to T substitutions in the mutational spectra distinguished potassium bromate from hydrogen peroxide and paraquat and mirrored the observed metabolic changes. We attributed these changes to the generation of uncommon oxidizing species in a reaction with thiol-containing antioxidants; a nearly total depletion of intracellular glutathione and a paradoxical augmentation of potassium bromate mutagenicity and toxicity by antioxidants. Our study provides the framework for understanding multidimensional processes triggered by agents collectively known as oxidants. Detection of increased mutational loads associated with potassium bromate-related mutational motifs in human tumors may be clinically relevant as a biomarker of this distinct type of redox stress.

Preventing oxidation of cellular XRCC1 affects PARP-mediated DNA damage responses.

Poly(ADP-ribose) polymerase-1 (PARP-1) binds intermediates of base excision repair (BER) and becomes activated for poly(ADP-ribose) (PAR) synthesis. PAR mediates recruitment and functions of the key BER factors XRCC1 and DNA polymerase ß (pol ß) that in turn regulate PAR. Yet, the molecular mechanism and implications of coordination between XRCC1 and pol ß in regulating the level of PAR are poorly understood. A complex of PARP-1, XRCC1 and pol ß is found in vivo, and it is known that pol ß and XRCC1 interact through a redox-sensitive binding interface in the N-terminal domain of XRCC1. We confirmed here that both oxidized and reduced forms of XRCC1 are present in mouse fibroblasts. To further understand the importance of the C12-C20 oxidized form of XRCC1 and the interaction with pol ß, we characterized cell lines representing stable transfectants in Xrcc1(-/-) mouse fibroblasts of wild-type XRCC1 and two mutants of XRCC1, a novel reduced form with the C12-C20 disulfide bond blocked (C12A) and a reference mutant that is unable to bind pol ß (V88R). XRCC1-deficient mouse fibroblasts are extremely hypersensitive to methyl methanesulfonate (MMS), and transfected wild-type and C12A mutant XRCC1 proteins similarly reversed MMS hypersensitivity. However, after MMS exposure the cellular PAR level was found to increase to a much greater extent in cells expressing the C12A mutant than in cells expressing wild-type XRCC1. PARP inhibition resulted in very strong MMS sensitization in cells expressing wild-type XRCC1, but this sensitization was much less in cells expressing the C12A mutant. The results suggest a role for the oxidized form of XRCC1 in the interaction with pol ß in (1) controlling the PAR level after MMS exposure and (2) enabling the extreme cytotoxicity of PARP inhibition during the MMS DNA damage response.

Lactate dehydrogenase C and energy metabolism in mouse sperm.

We demonstrated previously that disruption of the germ cell-specific lactate dehydrogenase C gene (Ldhc) led to male infertility due to defects in sperm function, including a rapid decline in sperm ATP levels, a decrease in progressive motility, and a failure to develop hyperactivated motility. We hypothesized that lack of LDHC disrupts glycolysis by feedback inhibition, either by causing a defect in renewal of the NAD(+) cofactor essential for activity of glyceraldehyde 3-phosphate dehydrogenase, sperm (GAPDHS), or an accumulation of pyruvate. To test these hypotheses, nuclear magnetic resonance analysis was used to follow the utilization of labeled substrates in real time. We found that in sperm lacking LDHC, glucose consumption was disrupted, but the NAD:NADH ratio and pyruvate levels were unchanged, and pyruvate was rapidly metabolized to lactate. Moreover, the metabolic disorder induced by treatment with the lactate dehydrogenase (LDH) inhibitor sodium oxamate was different from that caused by lack of LDHC. This supported our earlier conclusion that LDHA, an LDH isozyme present in the principal piece of the flagellum, is responsible for the residual LDH activity in sperm lacking LDHC, but suggested that LDHC has an additional role in the maintenance of energy metabolism in sperm. By coimmunoprecipitation coupled with mass spectrometry, we identified 27 proteins associated with LDHC. A majority of these proteins are implicated in ATP synthesis, utilization, transport, and/or sequestration. This led us to hypothesize that in addition to its role in glycolysis, LDHC is part of a complex involved in ATP homeostasis that is disrupted in sperm lacking LDHC.

Estrogen receptor beta mediates gender differences in ischemia/reperfusion injury.

Under hypercontractile conditions associated with increased intracellular calcium, male hearts show enhanced ischemia/reperfusion injury compared to female hearts. Our aim in this study was to identify the specific estrogen receptor involved in this gender difference. Following brief treatment with isoproterenol, isolated mouse hearts were subjected to ischemia and reperfusion. Postischemic contractile function and infarct size were measured in wild-type (WT) male and female hearts, and female hearts lacking functional alpha estrogen receptor (alpha ERKO), or the beta estrogen receptor (beta ERKO). WT male hearts exhibited significantly less functional recovery and more necrosis than WT females. alpha ERKO female hearts exhibited ischemia/reperfusion injury similar to that observed in WT females, whereas beta ERKO females exhibited significantly less functional recovery than WT females and were similar to WT males. These data suggest that estrogen, through the beta-estrogen receptor, plays a role in the protection observed in the female heart. Furthermore, we identified genes that were differentially expressed in beta ERKO female hearts compared to alpha ERKO and WT female hearts, and found altered expression of a number of metabolism genes, which may be important in ischemic injury. We further showed that WT female hearts have increased ratio of carbohydrate to fatty acid metabolism relative to WT males.