Calcium-induced permeability transition in rat brain mitochondria is promoted by carbenoxolone through targeting connexin43

Carbenoxolone (Cbx), a substance from medicinal licorice, is used for antiinflammatory treatments. We investigated the mechanism of action of Cbx on Ca(2+)-induced permeability transition pore (PTP) opening in synaptic and nonsynaptic rat brain mitochondria (RBM), as well as in rat liver mitochondria (RLM), in an attempt to identify the molecular target of Cbx in mitochondria. Exposure to threshold Ca(2+) load induced PTP opening, as seen by sudden Ca(2+) efflux from the mitochondrial matrix and membrane potential collapse. In synaptic RBM, Cbx (1 μM) facilitated the Ca(2+)-induced, cyclosporine A-sensitive PTP opening, while in nonsynaptic mitochondria the Cbx threshold concentration was higher. A well-known molecular target of Cbx is the connexin (Cx) family, gap junction proteins. Moreover, Cx43 was previously found in heart mitochondria and attributed to the preconditioning mechanism of protection. Thus, we hypothesized that Cx43 might be a target for Cbx in brain mitochondria. For the first time, we detected Cx43 by Western blot in RBM, but Cx43 was absent in RLM. Interestingly, two anti-Cx43 antibodies, directed against amino acids 252 to 270 of rat Cx43, abolished the Cbx-induced enhancement of PTP opening in total RBM and in synaptic mitochondria, but not in RLM. In total RBM and in synaptic mitochondria, PTP caused dephosphorylation of Cx43 at serine 368. The phosphorylation level of serine 368 was decreased at threshold calcium concentration and additionally in the combined presence of Cbx in synaptic mitochondria. In conclusion, active mitochondrial Cx43 appears to counteract the Ca(2+)-induced PTP opening and thus might inhibit the PTP-ensuing mitochondrial demise and cell death. Consequently, we suggest that activity of Cx43 in brain mitochondria represents a novel molecular target for protection.     

The binding of precipitant ions in the tetragonal crystals of hen egg white lysozyme

Abstract

The bonds between lysozyme molecules and precipitant ions in single crystals grown with chlorides of several metals are analysed on the basis of crystal structure data. Crystals of tetragonal hen egg lysozyme (HEWL) were grown with chlorides of several alkali and transition metals (LiCl, NaCl, KCl, NiCl₂ and CuCl₂) as precipitants and the three-dimensional structures were determined at 1.35?Å resolution by X-ray diffraction method. The positions of metal and chloride ions attached to the protein were located, divided into three groups and analysed. Some of them, in accordance with the recently proposed and experimentally confirmed crystal growth model, provide connections in protein dimers and octamers that are precursor clusters in the crystallization lysozyme solution. The first group, including Cu⁺², Ni⁺² and Na⁺¹ cations, binds specifically to the protein molecule. The second group consists of metal and chloride ions bound inside the dimers and octamers. The third group of ions can participate in connections between the octamers that are suggested as building units during the crystal growth. The arrangement of chloride and metal ions associated with lysozyme molecule at all stages of the crystallization solution formation and crystal growth is discussed.

Communicated by Ramaswamy H. Sarma     

Expression of NLRP3 Inflammasomes in Neurogenic Niche Contributes to the Effect of Spatial Learning in Physiological Conditions but Not in Alzheimer’s Type Neurodegeneration

A common feature of neurodegenerative disorders, in particular Alzheimer's disease (AD), is a chronic neuroinflammation associated with aberrant neuroplasticity. Development of neuroinflammation affects efficacy of stem and progenitor cells proliferation, differentiation, migration, and integration of newborn cells into neural circuitry. However, precise mechanisms of neurogenesis alterations in neuroinflammation are not clear yet. It is well established that expression of NLRP3 inflammasomes in glial cells marks neuroinflammatory events, but less is known about contribution of NLRP3 to deregulation of neurogenesis within neurogenic niches and whether neural stem cells (NSCs), neural progenitor cells (NPCs) or immature neuroblasts may express inflammasomes in (patho)physiological conditions. Thus, we studied alterations of neurogenesis in rats with the AD model (intra-hippocampal injection of Aβ1-42). We found that in Aβ-affected brain, number of CD133+ cells was elevated after spatial training in the Morris water maze. The number of PSA-NCAM+ neuroblasts diminished by Aβ injection was completely restored by subsequent spatial learning. Spatial training leads to elevated expression of NLRP3 inflammasomes in the SGZ (subgranular zones): CD133+ and PSA-NCAM+ cells started to express NLRP3 in sham-operated, but not AD rats. Taken together, our data suggest that expression of NLRP3 inflammasomes in CD133+ and PSA-NCAM+ cells may contribute to stimulation of adult neurogenesis in physiological conditions, whereas Alzheimer’s type neurodegeneration abolishes stimuli-induced overexpression of NLRP3 within the SGZ neurogenic niche.

     

Assembly and maintenance of nodes of ranvier rely on distinct sources of proteins and targeting mechanisms

We have investigated the source(s) and targeting of components to PNS nodes of Ranvier. We show adhesion molecules are freely diffusible within the axon membrane and accumulate at forming nodes from local sources, whereas ion channels and cytoskeletal components are largely immobile and require transport to the node. We further characterize targeting of NF186, an adhesion molecule that pioneers node formation. NF186 redistributes to nascent nodes from a mobile, surface pool. Its initial accumulation and clearance from the internode require extracellular interactions, whereas targeting to mature nodes, i.e., those flanked by paranodal junctions, requires intracellular interactions. After incorporation into the node, NF186 is immobile, stable, and promotes node integrity. Thus, nodes assemble from two sources: adhesion molecules, which initiate assembly, accumulate by diffusion trapping via interactions with Schwann cells, whereas ion channels and cytoskeletal components accumulate via subsequent transport. In mature nodes, components turnover slowly and are replenished via transport. VIDEO ABSTRACT:     

Heparanase uptake is mediated by cell membrane heparan sulfate proteoglycans

Heparanase is a mammalian endoglycosidase that degrades heparan sulfate (HS) at specific intrachain sites, an activity that is strongly implicated in cell dissemination associated with metastasis and inflammation. In addition to its structural role in extracellular matrix assembly and integrity, HS sequesters a multitude of polypeptides that reside in the extracellular matrix as a reservoir. A variety of growth factors, cytokines, chemokines, and enzymes can be released by heparanase activity and profoundly affect cell and tissue function. Thus, heparanase bioavailability, accessibility, and activity should be kept tightly regulated. We provide evidence that HS is not only a substrate for, but also a regulator of, heparanase. Addition of heparin or xylosides to cell cultures resulted in a pronounced accumulation of, heparanase in the culture medium, whereas sodium chlorate had no such effect. Moreover, cellular uptake of heparanase was markedly reduced in HS-deficient CHO-745 mutant cells, heparan sulfate proteoglycan-deficient HT-29 colon cancer cells, and heparinase-treated cells. We also studied the heparanase biosynthetic route and found that the half-life of the active enzyme is approximately 30 h. This and previous localization studies suggest that heparanase resides in the endosomal/lysosomal compartment for a relatively long period of time and is likely to play a role in the normal turnover of HS. Co-localization studies and cell fractionation following heparanase addition have identified syndecan family members as candidate molecules responsible for heparanase uptake, providing an efficient mechanism that limits extracellular accumulation and function of heparanase.     

Preparation and Properties of Recombinant Rat and Human Procholecystokinin57 –95

Recombinant human progastrin_6–80 binds two ferric ions with an apparent dissociation constant of 2.2 ± 0.1 μM [Baldwin (2004) Protein J 23:65–70]. The aims of the present study were to express fragments of recombinant procholecystokinin and to determine whether or not they bound ferric ions. Recombinant rat and human procholecystokinin_57–95 were expressed as glutathione S-transferase fusion proteins in E. coli. The fusion proteins were bound to glutathione-agarose, cleaved with thrombin, and purified by reverse phase HPLC. Recombinant procholecystokinin_57–95 did not bind to either the CCK1 or CCK2 receptor with high affinity. No change in absorption spectrum was observed on addition of ferric ions, and analysis of the quenching of tryptophan fluorescence observed in the presence of ferric ions indicated that binding to procholecystokinin_57–95 was at least 40–fold weaker than the binding of ferric ions to progastrin_6–80.     

Genetic re-engineering of polyunsaturated phospholipid profile of Saccharomyces cerevisiae identifies a novel role for Cld1 in mitigating the effects of cardiolipin peroxidation

Cardiolipin (CL) is a unique phospholipid localized almost exclusively within the mitochondrial membranes where it is synthesized. Newly synthesized CL undergoes acyl remodeling to produce CL species enriched with unsaturated acyl groups. Cld1 is the only identified CL-specific phospholipase in yeast and is required to initiate the CL remodeling pathway. In higher eukaryotes, peroxidation of CL, yielding CL_OX, has been implicated in the cellular signaling events that initiate apoptosis. CL_OX can undergo enzymatic hydrolysis, resulting in the release of lipid mediators with signaling properties. Our previous findings suggested that CLD1 expression is upregulated in response to oxidative stress, and that one of the physiological roles of CL remodeling is to remove peroxidized CL. To exploit the powerful yeast model to study functions of CLD1 in CL peroxidation, we expressed the H. brasiliensis Δ¹²-desaturase gene in yeast, which then synthesized poly unsaturated fatty acids(PUFAs) that are incorporated into CL species. Using LC-MS based redox phospholipidomics, we identified and quantified the molecular species of CL and other phospholipids in cld1Δ vs. WT cells. Loss of CLD1 led to a dramatic decrease in chronological lifespan, mitochondrial membrane potential, and respiratory capacity; it also resulted in increased levels of mono-hydroperoxy-CLs, particularly among the highly unsaturated CL species, including tetralinoleoyl-CL. In addition, purified Cld1 exhibited a higher affinity for CL_OX, and treatment of cells with H₂O₂ increased CLD1 expression in the logarithmic growth phase. These data suggest that CLD1 expression is required to mitigate oxidative stress. The findings from this study contribute to our overall understanding of CL remodeling and its role in mitigating oxidative stress.     

Oestrogen receptors interact with the α-catalytic subunit of AMP-activated protein kinase

Normal and pathological stressors engage the AMP-activated protein kinase (AMPK) signalling axis to protect the cell from energetic pressures. Sex steroid hormones also play a critical role in energy metabolism and significantly modify pathological progression of cardiac disease, diabetes/obesity and cancer. AMPK is targeted by 17β-oestradiol (E2), the main circulating oestrogen, but the mechanism by which E2 activates AMPK is currently unknown. Using an oestrogen receptor α/β (ERα/β) positive (T47D) breast cancer cell line, we validated E2-dependent activation of AMPK that was mediated through ERα (not ERβ) by using three experimental strategies. A series of co-immunoprecipitation experiments showed that both ERs associated with AMPK in cancer and striated (skeletal and cardiac) muscle cells. We further demonstrated direct binding of ERs to the α-catalytic subunit of AMPK within the βγ-subunit-binding domain. Finally, both ERs interacted with the upstream liver kinase B 1 (LKB1) kinase complex, which is required for E2-dependent activation of AMPK. We conclude that E2 activates AMPK through ERα by direct interaction with the βγ-binding domain of AMPKα.