Open-Bundle Structure as the Unfolding Intermediate of Cytochrome c′ Revealed by Small Angle Neutron Scattering

The dynamic structure changes, including the unfolding, dimerization, and transition from the compact to the open-bundle unfolding intermediate structure of Cyt c′, were detected by a small-angle neutron scattering experiment (SANS). The structure of Cyt c′ was changed into an unstructured random coil at pD = 1.7 (Rg = 25 Å for the Cyt c′ monomer). The four-α-helix bundle structure of Cyt c′ at neutral pH was transitioned to an open-bundle structure (at pD ~13), which is given by a numerical partial scattering function analysis as a joint-clubs model consisting of four clubs (α-helices) connected by short loops. The compactly folded structure of Cyt c′ (radius of gyration, Rg = 18 Å for the Cyt c′ dimer) at neutral or mildly alkaline pD transited to a remarkably larger open-bundle structure at pD ~13 (Rg = 25 Å for the Cyt c′ monomer). The open-bundle structure was also supported by ab initio modeling.

Characterization of oxidation of glutathione by cytochrome c

Cytochrome c is a member of the respiratory chain of the mitochondria. Non-membrane-bound (free) cytochrome c can be reduced by gluthatione as well as ascorbic acid. We investigated the effect of pH, Ca2+, Mg2+ and anionic phospholipids on the reduction of cytochrome c by glutathione.The reduction of cytochrome c by thiols was measured using photometry. Mitochondrial oxygen consumption was detected by use of oxygen electrode. Glutathione does not reduce cytochrome c at pH = 7.0 in the absence of Ca2+ and Mg2+. The reduction of cytochrome c by glutathione is inhibited by anionic lipids, especially cardiolipin. The typical conditions of apoptosis—elevated pH, Ca2+ level and Mg2+—increases the reduction of cytochrome c. Glutathione (5 mM) causes increased mitochondrial O2 consumption at pH = 8.0, in the presence of ADP either 1 mM Mg2+ or 1 mM Ca2+. Our results suggest that membrane bound cyt c does not oxidize glutathione. Free (not membrane bound) cytochrome c can oxidize glutathione. In mitochondria, O2 is depleted only in the presence of ADP, so the O2 depletion observed in the presence of glutathione can be related to the respiratory chain. Decreased glutathione levels play a role in apoptosis. Therefore, membrane unbound cyt c can contribute to apoptosis by oxidation of glutathione.

SIRT3 Protects Against Cognitive Dysfunction Induced by Sepsis-associated Encephalopathy Via JNK/p66Shc-Regulated Mitochondrial Apoptosis in Mice

Background: Sepsis-associated encephalopathy (SAE) is one of the severe central nervous system complications. Oxidative stress and synaptic dysfunction were involved in cognitive impairment induced by SAE. The mitochondrial nicotinamide adenine dinucleotide (NAD+) dependent deacetylase, sirtuin3 (SIRT3), plays a critical role in regulating mitochondrial function. The aim of this study was to evaluate the effect of SIRT3 in cognitive dysfunction induced by SAE.Methods: Mice were treated with lipopolysaccharide (LPS, 10 mg/kg, i.p.). Contextual and cue memory were evaluated by fear conditioning test in wild-type (WT) and SIRT3-deficient (SIRT3-/-) mice. Synapse-associated proteins and mitochondrial apoptosis-associated protein were examined by western blotting. In vitro studies, acetylation levels of cyclophilin D (CypD) were detected with different SIRT3 deacetylase activity in HT22 cells after LPS-induced microglia supernatant (Mi-sup) exposure. Oxidative stress was detected by reactive oxygen species (ROS) staining, and mitochondrial membrane potential (MMP) was detected by JC-1 staining, and mitochondrial membrane permeability transition pore (MPTP) opening was detected by Calcein and Co2+ staining. Furthermore, the phosphorylation levels of mitochondrial p66Shc and JNK were evaluated by western blotting.Results: SIRT3 expression was diminished in hippocampus of mice after LPS treatment. SIRT3-deficiency contributed to more severe contextual memory loss and synaptic dysfunction, decreased ratio of Bcl-2/Bax and increased Cyt C release to cytoplasm in hippocampus compared with wild-type controls. In HT22 cells, lysine acetylation levels of CypD were significantly increased after Mi-sup exposure and further enhanced with 3-TYP (SIRT3 deacetylation inhibitor) pretreatment, in association with the accumulation of ROS, declined MMP and increased MPTP opening, as well as the increased mitochondrial Cyt C release and phosphorylation levels of mitochondrial JNK and p66Shc-Ser36. SIRT3 overexpression restored CypD lysine acetylation levels and MPTP opening in HT22 cells after Mi-sup exposure and reduced mitochondrial JNK and p66Shc activation. Conclusions: Taken together, our results showed that SIRT3-mediated CypD deacetylation was involved in LPS-induced hippocampal synaptic dysfunction, via ROS accumulation, declined MMP, increased MPTP opening, mitochondrial Cyt C release and mitochondrial apoptosis of hippocampal neuron via JNK/p66Shc pathway. Our results revealed that SIRT3 may be a promising therapeutic and diagnostic target for cognitive dysfunction induced by SAE.

A combined miR-34a and arsenic trioxide nanodrug delivery system for synergistic inhibition of HCC progression after microwave ablation

Background Microwave ablation (MWA) has become an alternative treatment for unresectable hepatocellular carcinoma (HCC), but it does not eliminate the risk of recurrence and metastasis after treatment. Recent studies have demonstrated that miR-34a presents decreased gene expression in residual tumours after ablation therapy and can increase the therapeutic effect of arsenic trioxide against HCC, which brings new opportunities for HCC treatment. Methods A pH-sensitive charge inversion material was used to construct a nanotargeted delivery system based on the synergistic effects of miR-34a and As2O3. We established in vitro and in vivo models of HCC microwave ablation and performed in-depth research on the dual-drug system to inhibit the rapid progression and induce pyroptosis in HCC cells after microwave ablation. Results The antitumour effects were enhanced with the dual-drug nanoparticles relative to the single-drug formulations, and the therapeutic efficacy of the nanoparticles was more significant in a weakly acidic environment. The dual-drug nanoparticles increased the N-terminal portion of GSDME and decreased the expression of Cyt-c and c-met. Conclusions Dual-drug nanoparticles may improve the therapeutic efficacy of HCC treatment after insufficient ablation through Cyt-c and GSDME-N and decrease the expression levels of c-met. These nanoparticles are expected to provide new treatment methods for residual HCC after MWA, prolong the survival of patients and improve their quality of life.

Cytochrome c: Using Biological Insight toward Engineering an Optimized Anticancer Biodrug

The heme protein cytochrome c (Cyt c) plays pivotal roles in cellular life and death processes. In the respiratory chain of mitochondria, it serves as an electron transfer protein, contributing to the proliferation of healthy cells. In the cell cytoplasm, it activates intrinsic apoptosis to terminate damaged cells. Insight into these mechanisms and the associated physicochemical properties and biomolecular interactions of Cyt c informs on the anticancer therapeutic potential of the protein, especially in its ability to subvert the current limitations of small molecule-based chemotherapy. In this review, we explore the development of Cyt c as an anticancer drug by identifying cancer types that would be receptive to the cytotoxicity of the protein and factors that can be finetuned to enhance its apoptotic potency. To this end, some information is obtained by characterizing known drugs that operate, in part, by triggering Cyt c induced apoptosis. The application of different smart drug delivery systems is surveyed to highlight important features for maintaining Cyt c stability and activity and improving its specificity for cancer cells and high drug payload release while recognizing the continuing limitations. This work serves to elucidate on the optimization of the strategies to translate Cyt c to the clinical market.

Live-cell visualization of cytochrome c: a tool to explore apoptosis

Apoptosis dysfunction is associated with several malignancies, including cancer and autoimmune diseases. Apoptosis restoration could be an attractive therapeutic approach to those diseases. Mitochondrial outer membrane permeabilization is regarded as the point of no return in the ‘classical’ apoptosis triggering pathway. Cytoplasmic release of cytochrome c (cyt c), a mitochondrial electron transporter, is a prominent indicator of such critical step. Therefore, visualizing cyt c efflux in living cells is a convenient approach to address apoptosis triggering and monitor performance of apoptosis restoration strategies. Recent years have been prolific in the development of biosensors to visualize cyt c mitochondrial efflux in living cells, by fluorescence microscopy. These biosensors specifically detect endogenous, untagged cyt c, while showing efficient cellular uptake and reduced cell toxicity. A common aspect is their fluorescence quenching in the absence or presence of bound cyt c, resulting in two main biosensor types: ‘turn ON’ and ‘turn OFF’. In some of these systems, fluorescence intensity of fluorophore-bound aptamers is enhanced upon cyt c binding. In others, cyt c binding to quantum dots quenches their fluorescence. In the present minireview, I describe these biosensors and briefly introduce some hypotheses that could be addressed using these novel tools.

Hsp22 Inhibits Oxidative Stress-Induced Endplate Chondrocyte Apoptosis by Regulating Mitochondrial Pathway

Background: Facet joint degeneration (FJD), which is also called facet joint syndrome (FJS), has become one of the most commonly seen etiological factors for lumbago. Cartilage lesion triggered by lumbar facet joint (LFJ) degeneration might be related to mitochondrial impairment, but the its underlying mechanism remains unclear. Materials and methods: The endplate chondrocytes were induced by hydrogen peroxide (H2O2) to mimic the pathological conditions of oxidative stress. Enzyme linked immunosorbent assay (ELISA) were used for the evaluation of reactive oxygen species (ROS). Adenosine-triphosphate (ATP) level was assessed using ATP detection, along with the detection the expression of cytochrome C in mitochondria (mito-cyt c) and in cell cytoplasm (cyto-cyt c) and cleaved caspase 3 by Western blot analysis. TUNEL assay was conducted for the measurement of cell apoptosis in endplate chondrocytes. Reverse transcription-polymerase chain reaction (RT-qPCR) was used to verify the expression of heat shock protein 22 (HSP22) and the transfection efficiency of HSP22 interference plasmid. Results: It was found that H2O2 promoted the mitochondrial dysfunction, ROS generation and cell apoptosis in endplate chondrocytes. Moreover, HSP22 was down-regulated in H2O2-induced endplate chondrocytes, and interference of HSP22 decreased the ROS production, increased the ATP level and promoted the cell apoptosis, resulting in the enhanced impairment of endplate chondrocytes. Additionally, mitochondrial ROS inhibitor (Mito-TEMPO) ameliorated the injury effects of HSP22 silencing in the H2O2-induced endplate chondrocytes. Conclusion: In conclusion, HSP22 inhibits oxidative stress-induced endplate chondrocyte apoptosis by regulating mitochondrial pathway, possibly providing novel guidance direction for the treatment of LFJ degeneration.