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Apoptosis was associated with decreased sensory quality attributes of fish during postmortem storage. Based on cytochrome c (cyt-c) release plays a crucial role in apoptosis, the study aims to investigate the factors regulating cyt-c release and whether cyt-c acts as an endogenous pro-oxidant to trigger lipid oxidation. Within 12 h postmortem, dramatic changes in the intramuscular environment (glycogen from 1.57 mg/g to 0.65 mg/g; ATP reduced by 92.91%; pH value reaching the lowest (pH = 7.14)) and the mitochondrial environment (accumulation of mitochondrial ROS and Ca 2+ levels) are induced mitochondrial swelling and opening of the MPTP (increased 34.35% and 31.91%), leading to the release of cyt-c from the mitochondria into the cytoplasm and the activation of caspase-3. This leads to lipid oxidation and degradation of myofibrillar proteins, accelerating quality deterioration in color and texture. The results suggest that cyt-c is involved in lipid oxidation during postmortem through the apoptotic mitochondrial pathway., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Affinity-oriented online ligand screening with LC coupled to different detectors is widely popular to capture active compounds from herbal medicines (HMs). However, false-positive extensively occurs because insufficient information is recorded for the existence and stability of ligand-protein complex. Here, efforts were made to advance the hit confidences via configuring post-column infusion-LC-energy-resolved-affinity MS (PCI-LC-ER-AMS) to achieve "four-in-one" monitoring of: 1) response decrement of potential ligands; 2) response decrement of protein; 3) ions relating to ligand-protein complexes; and 4) ligand-protein binding strength. Ligand fishing for Cyt C from HMs was conducted as a proof-of-concept. For utility justification, a mimic sample containing twelve well-defined ligands and two negative controls underwent LC separation and met Cyt C prior to Qtof-MS measurements. Compared to Cyt C- or ligand-free assay, twelve ligands instead of negative controls showed response decrements that were consistent with twelve negative peaks observed at retention times corresponding to the ligands in Cyt C ion current chromatogram. Serial ions correlating to each ligand-Cyt C complex were observed. After recording breakdown graphs, optimal collision energy (OCE) corresponding to the non-covalent bond dissociation was positively correlated with binding strength. Two HMs including Scutellariae Radix (SR) and Aconiti Lateralis Radix Preparata were investigated. Consequently, 24 compounds were merely fished from SR, and particularly, flavonoid glycosides exhibited greater OCEs and also binding strengths over aglycones. Affinity assays and cellular evaluations consolidated the significant interactions between each captured compound and Cyt C. Overall, PCI-LC-ER-AMS is eligible for confidence-enhanced online ligand screening for Cyt C from HMs through "four-in-one" measurement., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Enzyme immobilization is an effective strategy for achieving efficient and sustainable enzyme catalysis. As a kind of promising enzyme-loading materials, the systematic research on zirconium based metal organic frameworks (Zr-MOFs) about immobilization performance at molecular level is still in its initial stage. In this work, UiO-66 was functionalized with various groups (-H, -NH 2 , -COOH, -OH, -2OH) for the immobilization of cytochrome c (Cyt c) and antioxidant enzyme catalase (CAT). Then the effects of surface-functionalized UiO-66 derivatives on the loading efficiency, enzyme stability and catalysis kinetics were systematically investigated. In addition, the affinity constants of Cyt c and CAT towards UiO-66-series MOFs carriers were also compared. The results have shown that hydroxyl group functionalized UiO-66 represents the highest enzyme loading capacity, enhanced activity and improved stability for Cyt c and CAT possibly due to high surface area and suitable microenvironments as well as enhanced affinity towards the enzymes provided by the introduction of a single hydroxyl group. Our research would foresee immense potential of MOFs in engineering biocatalysts., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The crosstalk between endoplasmic reticulum and mitochondria is of significance in apoptosis, in which cytochrome b 5 (Cyt b 5 ) is thought to be a major target for cytochrome c (Cyt c) upon its release from the mitochondria. In the absence of Cyt b 5 , the role of interactions of Cyt c with CYP-dependent monooxygenase system in apoptotic regulation was explored in this study. NADPH-dependent and Cyt c-induced formation of reactive oxygen species (ROS) and NADPH-independent Cyt c unfolding were revealed. With the aid of a CPR inhibitor and CYP antibodies, the interactions among Cyt c, cytochrome P450 reductase (CPR) and cytochrome P450 (CYP) are evidenced, which are found crucial for monooxygenase-derived ROS formation. The underlying structural basis of Cyt c-CYP complex was unveiled by molecular dynamics simulations. This study provides novel insights into how Cyt c regulates ROS formation through the interactions with CPR and CYP, and is implicated for a deeper understanding of the regulation mechanism in the mitochondria-endoplasmic reticulum apoptotic pathway., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Binding of cytochrome c (Cytc) to membranes containing cardiolipin (CL) is of considerable interest because of the importance of this interaction in the early stages of apoptosis. The molecular-level determinants of this interaction are still not well defined and there appear to be species-specific differences in Cytc affinity for CL-containing membranes. Many studies are carried out at low ionic strength far from the 100-150 mM ionic strength within mitochondria. Similarly, most binding studies are done at Cytc concentrations of 10 μM or less, much lower that the estimated range of 0.1 to 5 mM Cytc present in mitochondria. In this study, we evaluate binding of human and yeast Cytc to CL nanodiscs using size exclusion chromatography at 25 μM Cytc concentration and 100 mM ionic strength. We find that yeast Cytc affinity for CL nanodiscs is much stronger than that of human Cytc. Mutational analysis of the site A binding surface shows that lysines 86 and 87 are more important for yeast Cytc binding to CL nanodiscs than lysines 72 and 73, counter to results at lower ionic strength. Analysis of the electrostatic surface potential of human versus yeast Cytc shows that the positive potential due to lysines 86 and 87 and other nearby lysines (4, 5, 11, 89) is stronger than that due to lysines 72 and 73. In the case of human Cytc the positive potential around site A is less uniform and likely weakens electrostatic binding to CL membranes through site A., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Nanotexture on biocompatible surfaces promotes cell adhesion and proliferation. High aspect ratio nanoachitecture serves as an ideal interface between implant materials and host cells that is well-suited for localized therapeutic delivery. Despite this potential, nanotextured surfaces have not been widely applied for biomacromolecule delivery. Here, we employed a low-cost, industrially relevant nanoetching process to modify the surface of biocompatible stainless steel 316 (SS316L), creating nanotextured SS316L (NT-SS316L) as a material for intracellular biomacromolecule delivery. As biomacromolecule cargoes are adsorbed to the steel and ultimately would be used in protein-rich environments, we performed serum protein corona analysis on unmodified SS316L and NT-SS316L using tandem mass spectrometry. We observed an increase in proteins associated with cell adhesion on the surface of NT-SS316L compared to that of SS316L, supporting literature reports of enhanced adhesion on nanotextured materials. For delivery to adherent cells, a "hard corona" of model biomacromolecule cargoes including superfolder green fluorescent protein (sfGFP) charge variants, cytochrome c, and siRNA was adsorbed on NT-SS316L to assess delivery. Nanotextured surfaces enhanced cellular biomacromolecule uptake and delivered cytosolic-functional proteins and nucleic acids through energy-dependent endocytosis. Collectively, these findings indicate that NT-SS316L holds potential as a surface modification for implants to achieve localized drug delivery for a variety of biomedical applications.

Intracellular protein delivery has the potential to revolutionize cell-biological research and medicinal therapy, with broad applications in bioimaging, disease treatment, and genome editing. Herein, we demonstrate successful delivery of a functional protein, cytochrome c (CYC), by using a boron cluster anion as molecular carrier of the superchaotropic anion type (B 12 Br 11 OPr 2- ). CYC was delivered into lipid bilayer vesicles as well as living cells, with a cellular uptake ratio approaching 90%. Mechanistic studies showed that CYC was internalized into cells through a permeation pathway directly into the cytoplasm, bypassing endosomal entrapment. Upon carrier-assisted internalization, CYC retained its bioactivity, as reflected by an induced cell apoptosis rate of 25% at low dose (1 µM). This study furbishes a direct protein delivery method by a molecular carrier with high efficiency, confirming the potential of inorganic cluster ions as protein transport vehicles with an extensive range of future cell-biological or biomedical applications., Competing Interests: Competing interests statement:A.B.-B. and W.M.N. are co-inventors on a related patent (WO 2021/259668 A1).

Background: Multiheme cytochromes c (MHC) provide prokaryotes with a broad metabolic versatility that contributes to their role in the biogeochemical cycling of the elements and in energy production in bioelectrochemical systems. However, MHC have only been isolated and studied in detail from a limited number of species. Among these, Desulfuromonadia spp. are particularly MHC-rich. To obtain a broad view of the diversity of MHC, we employed bioinformatic tools to study the cytochromome encoded in the genomes of the Desulfuromonadia class., Results: We found that the distribution of the MHC families follows a different pattern between the two orders of the Desulfuromonadia class and that there is great diversity in the number of heme-binding motifs in MHC. However, the vast majority of MHC have up to 12 heme-binding motifs. MHC predicted to be extracellular are the least conserved and show high diversity, whereas inner membrane MHC are well conserved and show lower diversity. Although the most prevalent MHC have homologues already characterized, nearly half of the MHC families in the Desulforomonadia class have no known characterized homologues. AlphaFold2 was employed to predict their 3D structures. This provides an atlas of novel MHC, including examples with high beta-sheet content and nanowire MHC with unprecedented high numbers of putative heme cofactors per polypeptide., Conclusions: This work illuminates for the first time the universe of experimentally uncharacterized cytochromes that are likely to contribute to the metabolic versatility and to the fitness of Desulfuromonadia in diverse environmental conditions and to drive biotechnological applications of these organisms., (© 2024. The Author(s).)

Increased intramitochondrial free iron is a key feature of various liver diseases, leading to oxidative stress, mitochondrial dysfunction, and liver damage. Polydatin is a polyphenol with a hepatoprotective effect, which has been attributed to its ability to enhance mitochondrial oxidative metabolism and antioxidant defenses, thereby inhibiting reactive oxygen species (ROS) dependent cellular damage processes and liver diseases. However, it has not been explored whether polydatin is able to exert its effects by protecting the phospholipid cardiolipin against damage from excess iron. Cardiolipin maintains the integrity and function of electron transport chain (ETC) complexes and keeps cytochrome c bound to mitochondria, avoiding uncontrolled apoptosis. Therefore, the effect of polydatin on oxidative lipid damage, ETC activity, cytochrome levels, and ROS production was explored in iron-exposed rat liver mitochondria. Fe 2+ increased lipid peroxidation, decreased cardiolipin and cytochromes c + c 1 and aa 3 levels, inhibited ETC complex activities, and dramatically increased ROS production. Preincubation with polydatin prevented all these effects to a variable degree. These results suggest that the hepatoprotective mechanism of polydatin involves the attenuation of free radical production by iron, which enhances cardiolipin levels by counteracting membrane lipid peroxidation. This prevents the loss of cytochromes, improves ETC function, and decreases mitochondrial ROS production.

Protein immobilization using water-soluble ionic metal-organic polyhedra (MOPs) acting as porous spacers has recently been demonstrated as a potent strategy for the preparation of biocatalysts. In this article, we describe a mixed-protein approach to achieve biocomposites with adjustable enzyme contents and excellent immobilization efficiencies, in a systematic and well-controlled manner. Self-assembly of either cationic or anionic MOPs with bovine serum albumin or egg white lysozyme combined with enzymes (alkaline phosphatase, laccase or cytochrome c) led to solid-state catalysts with a high retention of enzyme activity. Furthermore, for all these systems, the dilution of enzymes within the solid-state composite led to noticeably improved catalytic performances, with both higher specific activity and affinity for substrate.

This study aimed to assess the nephrotoxicity associated with VRP-034 (novel formulation of polymyxin B [PMB]) compared to marketed PMB in a three-dimensional (3D) kidney-on-a-chip model. To model the human kidney proximal tubule for analysis, tubular structures were established using 23 triple-channel chips seeded with RPTEC/hTERT1 cells. These cells were exposed to VRP-034 or PMB at seven concentrations (1-200 µM) over 12, 24, and 48 h. A suite of novel kidney injury biomarkers, cell health, and inflammatory markers were quantitatively assessed in the effluent. Additionally, caspase and cytochrome C levels were measured, and cell viability was evaluated using calcein AM and ethidium homodimer-1 (EthD-1). Exposure to marketed PMB resulted in significantly elevated levels ( P < 0.05) of four key biomarkers (KIM-1, cystatin C, clusterin, and OPN) compared to VRP-034, particularly at clinically relevant concentrations of ≥10 µM. At 25 µM, all biomarkers demonstrated a significant increase ( P < 0.05) with marketed PMB exposure compared to VRP-034. Inflammatory markers (interleukin-6 and interleukin-8) increased significantly ( P < 0.05) with marketed PMB at concentrations of ≥5 µM, relative to VRP-034. VRP-034 displayed superior cell health outcomes, exhibiting lower lactate dehydrogenase release, while ATP levels remained comparable. Morphological analysis revealed that marketed PMB induced more severe damage, disrupting tubular integrity. Both treatments activated cytochrome C, caspase-3, caspase-8, caspase-9, and caspase-12 in a concentration-dependent manner; however, caspase activation was significantly reduced ( P < 0.05) with VRP-034. This study demonstrates that VRP-034 significantly reduces nephrotoxicity compared to marketed PMB within a 3D microphysiological system, suggesting its potential to enable the use of full therapeutic doses of PMB with an improved safety profile, addressing the need for less nephrotoxic polymyxin antibiotics., Competing Interests: The authors of this study are employees of Venus Medicine Research Centre, which is the developer of the drug being investigated. This study was funded by Venus Medicine Research Centre. The authors declare no additional competing interests.

Cancer cell mitochondria are functionally different from those in normal cells and could be targeted to develop novel anticancer agents. The aryl-ureido fatty acid CTU (16({[4-chloro-3-(trifluoromethyl)phenyl]-carbamoyl}amino)hexadecanoic acid) is the prototype of a new class of targeted agents that enhance the production of reactive oxygen species (ROS) that disrupt the outer mitochondrial membrane (OMM) and kill cancer cells. However, the mechanism by which CTU disrupts the inner mitochondrial membrane (IMM) and activates apoptosis is not clear. Here, we show that CTU-mediated ROS selectively dysregulated the OMA1/OPA1 fusion regulatory system located in the IMM. The essential role of ROS was confirmed in experiments with the lipid peroxyl scavenger α-tocopherol, which prevented the dysregulation of OMA1/OPA1 and CTU-mediated MDA-MB-231 cell killing. The disruption of OMA1/OPA1 and IMM fusion by CTU-mediated ROS accounted for the release of cytochrome c from the mitochondria and the activation of apoptosis. Taken together, these findings demonstrate that CTU depolarises the mitochondrial membrane, activates ROS production, and disrupts both the IMM and OMM, which releases cytochrome c and activates apoptosis. Mitochondrial-targeting agents like CTU offer a novel approach to the development of new therapeutics with anticancer activity.

I., Background: Human induced pluripotent stem cell (hiPSC) derived cardiomyocytes (CMs) have been utilized in drug toxicity evaluation, drug discovery, and treating heart failure patients, showing substantial effects. Ensuring the quality, purity, and maturation of hiPSC-CMs during large-scale production is crucial. There is a growing demand for a novel method to characterize cell molecular profiles without labels and without causing damage. II., Methods: In this study, we employed label-free Raman microscopy to evaluate hiPSC-derived CMs. The study involved the characterization of cell molecular profiles without labels and without causing damage. The correlation between Raman spectroscopy of specific components, such as cytochrome c and myoglobin, and CM purity and maturation following hiPSC differentiation was investigated. Additionally, the validation of this correlation was performed by assessing mixtures of commercially available CMs (iCell cardiomyocytes2) and fibroblasts at various ratios as well as hiPSC-derived CMs with different efficiencies. Furthermore, CMs were matured using rapid pacing of traveling waves, and the Raman profiles of matured CMs were compared to those of immature ones. III., Results: Raman spectroscopy indicated that the cytochrome c and myoglobin showed correlation with the purity and maturation of CMs following differentiation of hiPSCs. This correlation was validated through experiments involving different CM-fibroblast mixtures and hiPSC-derived CMs with varying efficiencies. Moreover, matured CMs exhibited markedly different Raman profiles compared to immature ones, indicating the potential of Raman imaging as a tool for assessing CM maturation. IV., Conclusions: We discovered that Raman spectroscopy of certain components, such as cytochrome c and myoglobin, correlates with the CM purity and maturation following hiPSC differentiation. The findings of this study highlight the potential of label-free Raman microscopy as a nondestructive, high-content, and time-efficient method for quality control of hiPSC-derived CMs. This approach could significantly contribute to ensuring the quality and maturity of hiPSC-CMs for various applications in drug discovery and regenerative medicine.

Over the last 50 years resonance Raman spectroscopy has become an invaluable tool for the exploration of chromophores in biological macromolecules. Among them, heme proteins and metal complexes have attracted considerable attention. This interest results from the fact that resonance Raman spectroscopy probes the vibrational dynamics of these chromophores without direct interference from the surrounding. However, the indirect influence via through-bond and through-space chromophore-protein interactions can be conveniently probed and analyzed. This review article illustrates this point by focusing on class 1 cytochrome c, a comparatively simple heme protein generally known as electron carrier in mitochondria. The article demonstrates how through selective excitation of resonance Raman active modes information about the ligation, the redox state and the spin state of the heme iron can be obtained from band positions in the Raman spectra. The investigation of intensities and depolarization ratios emerged as tools for the analysis of in-plane and out-of-plane deformations of the heme macrocycle. The article further shows how resonance Raman spectroscopy was used to characterize partially unfolded states of oxidized cytochrome c. Finally, it describes its use for exploring structural changes due to the protein's binding to anionic surfaces like cardiolipin containing membranes., Competing Interests: Declaration of competing interest The author does not declare any conflict of interest., (Copyright © 2024 The Author. Published by Elsevier Inc. All rights reserved.)

Here, we revealed the reversibility of cabazitaxel (CBZ)-induced apoptosis in PC-3 castration resistant metastatic prostate cancer cells (mCRPC) through the hallmarks of apoptosis. The recovery of PC-3 cells from apoptosis upon removal of CBZ at different recovery periods was evaluated by Annexin V, DNA damage, oxidative damage, mitochondrial membrane depolarization, and caspase activation. Our results showed that the administration of CBZ caused apoptosis for 72 h in PC-3 cells. However, recovered cells exhibited decreased nuclear damage, plasma membrane disruption, ROS level, release cytochrome c level and caspase-3 activation with upregulation of Bcl-2 expression upon removal of especially 1 nM CBZ for 24 h recovery period in PC-3 cells. Our study indicates that CBZ treated PC-3 cells can recover after apoptotic cell death. However, advanced molecular analysis should elucidate the relationship between the molecular mechanisms of recovery and taxane response or resistance in PC-3 mCRPC cells., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2023. Published by Elsevier Ltd.)

The efficient delivery of therapeutic proteins to tumor sites is a promising cancer treatment modality. Hydrogen-bonded organic frameworks (HOFs) are successfully used for the protective encapsulation of proteins; however, easy precipitation and lack of controlled release of existing HOFs limit their further application for protein delivery in vivo. Here, a hypoxia-responsive HOF, self-assembled from azobenzenedicarboxylate/polyethylene glycol-conjugated azobenzenedicarboxylate and tetrakis(4-amidiniumphenyl)methane through charge-assisted hydrogen-bonding, is developed for systemic protein delivery to tumor cells. The newly generated HOF platform efficiently encapsulates representative cytochrome C, demonstrating good dispersibility under physiological conditions. Moreover, it can respond to overexpressed reductases in the cytoplasm under hypoxic conditions, inducing fast intracellular protein release to exert therapeutic effects. The strategy presented herein can be applied to other therapeutic proteins and can be expanded to encompass more intrinsic tumor microenvironment stimuli. This offers a novel avenue for utilizing HOFs in protein-based cancer therapy., (© 2024 Wiley‐VCH GmbH.)

Recent studies have established that cellular electrostatic interactions are more influential than assumed previously. Here, we use cryo-EM and perform steady-state kinetic studies to investigate electrostatic interactions between cytochrome (cyt.) c and the complex (C) III 2 -IV supercomplex from Saccharomyces cerevisiae at low salinity. The kinetic studies show a sharp transition with a Hill coefficient ≥2, which together with the cryo-EM data at 2.4 Å resolution indicate multiple cyt. c molecules bound along the supercomplex surface. Negatively charged loops of CIII 2 subunits Qcr6 and Qcr9 become structured to interact with cyt. c. In addition, the higher resolution allows us to identify water molecules in proton pathways of CIV and, to the best of our knowledge, previously unresolved cardiolipin molecules. In conclusion, the lowered electrostatic screening renders engagement of multiple cyt. c molecules that are directed by electrostatically structured CIII 2 loops to conduct electron transfer between CIII 2 and CIV., (© 2024. The Author(s).)

Background: Thiazole derivatives are gaining prominence in cancer research due to their potent anti-cancer effects and multifaceted biological activities. In leukemia research, these compounds are particularly studied for their ability to induce apoptosis, disrupt mitochondrial membrane potential (MMP), and modulate cell signaling pathways., Methods and Results: This study investigates the efficacy of 4-Methylthiazole in inducing apoptosis in HL-60 leukemia cells. Apoptosis was quantified via flow cytometry using FITC Annexin V and propidium iodide staining. Mitochondrial disruption was evaluated through alterations in mitochondrial membrane potential (MMP) as measured by the JC-1 assay. The compound significantly disrupted MMP, activated Caspase-3, and induced the release of Cytochrome C, all of which are critical markers of apoptosis (****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05). Additionally, treatment with 4-Methylthiazole markedly reduced CD45 and CD123 surface markers, indicating significant phenotypic alterations in leukemia cells (****p < 0.0001). High-dose treatment with 4-Methylthiazole significantly increased ROS levels, suggesting elevated oxidative stress and the presence of intracellular free radicals, contributing to its cytotoxic effects (*p < 0.05). A significant rise in TNF-α levels was observed post-treatment, indicating a pro-inflammatory response that may further inhibit leukemia cell viability. While IL-6 levels remained unchanged, a dose-dependent decrease in IL-10 levels was noted, suggesting a reduction in immunosuppressive conditions within the tumor microenvironment (*p < 0.05)., Conclusions: Overall, 4-Methylthiazole targets leukemia cells through multiple apoptotic mechanisms and modifies the immune landscape of the tumor microenvironment, enhancing its therapeutic potential. This study highlights the need for further clinical investigation to fully exploit the potential of thiazole derivatives in leukemia treatment., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Mitochondria-targeting agents, known as mitocans, are emerging as potent cancer therapeutics due to pronounced metabolic and apoptotic adaptations in the mitochondria of cancer cells. ONC212, an imipridone-family compound initially identified as a ClpP agonist, is currently under investigation as a potential mitocan with demonstrated preclinical efficacy against multiple malignancies. Despite this efficacy, the molecular mechanism underlying the cell death induced by ONC212 remains unclear. This study systematically investigates the mitochondrial involvement and signaling cascades associated with ONC212-induced cell death, utilizing HeLa and A549 cancer cells. Treated cancer cells exhibited characteristic apoptotic features, such as annexin-V positivity and caspase-3 activation; however, these occurred independently of typical mitochondrial events like membrane potential loss (ΔΨ m ) and cytochrome c release, as well as caspase-8 activation associated with the extrinsic pathway. Additionally, ONC212 treatment increased the expression of anti-apoptotic proteins Bcl-2 and Bcl-xL, which impeded apoptosis, as the overexpression of Bcl-2-GFP and Bcl-xL-GFP significantly reduced ONC212-mediated cell death. Furthermore, combining a sub-lethal dose of the Bcl-2/Bcl-xL inhibitor Navitoclax with ONC212 markedly augmented caspase-3 activation and cell death, still without any notable ΔΨ m loss or cytochrome c release. Moreover, inhibition of caspase-9 activity unexpectedly augmented, rather than attenuated, caspase-3 activation and the subsequent cell death. Collectively, our research identifies ONC212 as an atypical mitochondrial-independent, yet Bcl-2/Bcl-xL-inhibitable, caspase-3-mediated apoptotic cell death inducer, highlighting its potential for combination therapies in tumors with defective mitochondrial apoptotic signaling., (© 2024. The Author(s).)

Functional assays on intact tumor biopsies can complement genomics-based approaches for precision oncology, drug testing, and organs-on-chips cancer disease models by capturing key therapeutic response determinants, such as tissue architecture, tumor heterogeneity, and the tumor microenvironment. Most of these assays rely on fluorescent labeling, a semiquantitative method best suited for single-time-point assays or labor-intensive immunostaining analysis. Here, we report integrated aptamer electrochemical sensors for on-chip, real-time monitoring of cytochrome C, a cell death indicator, from intact microdissected tissues with high affinity and specificity. The platform features a multi-well sensor layout and a multiplexed electronic setup. The aptasensors measure increases in cytochrome C in the supernatant of mouse or human microdissected tumors after exposure to various drug treatments. Because of the sensor's high affinity, it primarily tracks rising concentrations of cytochrome C, capturing dynamic changes during apoptosis. This approach could help develop more advanced cancer disease models and apply to other complex in vitro disease models, such as organs-on-chips and organoids.

In this work, the adsorption behavior of cytochrome c (Cyt-c) on five different self-assembled monolayers (SAMs) (i.e., CH3-SAM, OH-SAM, NH2-SAM, COOH-SAM, and OSO3--SAM) was studied by combined parallel tempering Monte Carlo and molecular dynamics simulations. The results show that Cyt-c binds to the CH3-SAM through a hydrophobic patch (especially Ile81) and undergoes a slight reorientation, while the adsorption on the OH-SAM is relatively weak. Cyt-c cannot stably bind to the lower surface charge density (SCD, 7% protonation) NH2-SAM even under a relatively high ionic strength condition, while a higher SCD of 25% protonation promotes Cyt-c adsorption on the NH2-SAM. The preferred adsorption orientations of Cyt-c on the negatively-charged surfaces are very similar, regardless of the surface chemistry and the SCD. As the SCD increases, more counterions are attracted to the charged surfaces, forming distinct counterion layers. The secondary structure of Cyt-c is well kept when adsorbed on these SAMs except the OSO3--SAM surface. The deactivation of redox properties for Cyt-c adsorbed on the highly negatively-charged surface is due to the confinement of heme reorientation and the farther position of the central iron to the surfaces, as well as the relatively larger conformation change of Cyt-c adsorbed on the OSO3--SAM surface. This work may provide insightful guidance for the design of Cyt-c-based bioelectronic devices and controlled enzyme immobilization., (2024 Published under an exclusive license by the AVS.)

Purpose: This study investigated the potential of 1,25-dihydroxyvitamin D 3 (1,25(OH) 2 VD 3 ) to mitigate bisphenol A (BPA)-induced apoptosis in human ovarian granulosa KGN cells with the aim of establishing a theoretical foundation for understanding of how vitamin D improved ovarian function in patients with polycystic ovary syndrome (PCOS)., Methods: The impact of varying concentrations of BPA and 1,25(OH) 2 VD 3 on KGN cell viability was elucidated. It was established that BPA-induced apoptosis in KGN cells. Subsequently, KGN cells underwent pretreatment with 1,25(OH) 2 VD 3 , followed by exposure to BPA. The apoptosis rate, reactive oxygen species (ROS) levels, and mitochondrial function of the cells were meticulously assessed, along with the expression levels of genes associated with apoptosis as well as antioxidant and mitochondrial biogenesis., Results: BPA induced a notable increase in apoptosis (P < 0.001) and oxidative stress (P < 0.001) in KGN cells, accompanied by a significant reduction in mitochondrial membrane potential (P < 0.001) and severe impairment of mitochondrial function. Following pretreatment of KGN cells with 1,25(OH) 2 VD 3 , there was a significant decrease in the apoptosis rate (P = 0.004), coupled with a reduction in ROS production (P = 0.002). Concomitantly, the upregulation of PGC-1α (P = 0.009) and SOD (P = 0.018) was observed, while mRNA expression of BAX (P = 0.011), Cyt c (P = 0.001), Apaf-1 (P = 0.012), caspase-9 (P < 0.001), and caspase-3 (P = 0.011) was downregulated. Notably, the mitigation of mitochondrial damage was evident through restored mitochondrial membrane potential (P < 0.001), as corroborated by electron microscope results., Conclusions: 1,25(OH) 2 VD 3 mitigated BPA-induced damage and apoptosis in KGN cells by upregulating the expression of PGC-1α and impeding the mitochondrial cytochrome c (Cyt c) apoptotic pathway. This study established a novel theoretical foundation for utilizing vitamin D in the treatment of PCOS patients., (© 2024. The Author(s).)

Hexokinases (HKs) control the first step of glucose catabolism. A switch of expression from liver HK (glucokinase, GCK) to the tumor isoenzyme HK2 is observed in hepatocellular carcinoma progression. Our prior work revealed that HK isoenzyme switch in hepatocytes not only regulates hepatic metabolic functions but also modulates innate immunity and sensitivity to Natural Killer (NK) cell cytotoxicity. This study investigates the impact of HK2 expression and its mitochondrial binding on the resistance of human liver cancer cells to NK-cell-induced cytolysis. We have shown that HK2 expression induces resistance to NK cell cytotoxicity in a process requiring mitochondrial binding of HK2. Neither HK2 nor GCK expression affects target cells' ability to activate NK cells. In contrast, mitochondrial binding of HK2 reduces effector caspase 3/7 activity both at baseline and upon NK-cell activation. Furthermore, HK2 tethering to mitochondria enhances their resistance to cytochrome c release triggered by tBID. These findings indicate that HK2 mitochondrial binding in liver cancer cells is an intrinsic resistance factor to cytolysis and an escape mechanism from immune surveillance., (© 2024 The Author(s). European Journal of Immunology published by Wiley‐VCH GmbH.)

This study aimed to investigate the mechanism of the apoptosis of erythroblasts in rat bone marrow after the exposure to hypobaric hypoxia. Male SD rats were randomly divided into three groups. The hypoxic group was kept in a hypobaric hypoxia chamber at a simulated altitude of 5000 m for 7 and 28 days, respectively. The control group was kept at an altitude of 2260 m. We found that myeloid: erythroid (M:E) ratio was significantly lower after hypoxia exposure and the proportions of polychromatic erythroblasts and orthochromatic erythroblasts significantly increased compared to control group, along with significant increase in the proportion of CD71+ cells and apoptosis rate. The expression levels of caspase-3, Bax, and Cyt-C in CD71+ cells were higher after hypoxia exposure than those in control group, while there was no significant difference in the expression levels of TNFR and Fas. In conclusion, after exposure to hypobaric hypoxia the proliferation of peripheral blood and bone marrow erythroblasts in rats increased, and apoptosis also increased, indicating that bone marrow erythroblasts in rats is regulated by both proliferation and apoptosis, and the mitochondrial pathway is one of the important pathways for apoptosis., Competing Interests: Declaration of competing interest All authors declare no conflict of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

This article describes the molecular mechanism by which tetraalkylammonium chloride ([R 4 N]Cl: R- = methyl (Me), ethyl (Et), propyl (Pr),butyl (Bu)) modulates the stability, folding, and dynamics of cytochrome c (Cyt c). Analysis of [R 4 N]Cl effects on thermal/chemical denaturations, millisecond refolding/unfolding kinetics, and slow CO-association kinetics of Cyt c without and with denaturant providing some significant results: (i) [R 4 N]Cl decreasing the unfolding free energy estimated by thermodynamic and kinetic analysis of thermal/chemical denaturation curves and kinetic chevrons (Log k obs -[GdmCl]) of Cyt c, respectively (ii) hydrophobicity of R-group of [R 4 N]Cl, preferential inclusion of [R 4 N]Cl at the protein surface, and destabilizing enthalpic attractive interactions of [Me 4 N]Cl and steric entropic interactions of [Et 4 N]Cl,[Pr 4 N]Cl and [Bu 4 N]Cl with protein contribute to [R 4 N]Cl-induced decrease thermodynamic stability of Cyt c (iii) [R 4 N]Cl exhibits an additive effect with denaturant to decrease thermodynamic stability and refolding rates of Cyt c (iv) low concentrations of [R 4 N]Cl (≤ 0.5 M) constrain the motional dynamics while the higher concentrations (>0.75 M [R 4 N]Cl) enhance the structural-fluctuations that denture protein (v) hydrophobicity of R-group of [R 4 N]Cl alters the [denaturant]-dependent conformational stability, refolding-unfolding kinetics, and CO-association kinetics of Cyt c. Furthermore, the MD simulations depicted that the addition of 1.0 M of [R 4 N]Cl increased the conformational fluctuations in Cyt c leading to decreased structural stability in the order [Me 4 N]Cl < [Et 4 N]Cl < [Pr 4 N]Cl < [Bu 4 N]Cl consistent with the experimental results., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Ethnopharmacological Relevance: Ischemic stroke is currently a major public health hazard.Yangyin Tongnao Granules (YYTN), a traditional Chinese medicinal prescription, exerts potential therapeutic effects on subsequent cerebral ischemia-reperfusion injury (CIRI) after ischemic stroke. However, further studies are required to comprehend the underlying mechanism of YYTN for treating CIRI and the associated spectrum-effect mechanisms., Aim of the Study: To investigate the coordinated correlation between the fingerprint and the pharmacodynamic indexes of the effective components (total flavonoids, total saponins, total alkaloids, and total phenolic acids) in YYTN for treating CIRI in rats., Methods: The fingerprints of five specific components (ligustrazine, puerarin, ferulic acid, calycosin, and formononetin) of YYTN in rats with middle cerebral artery occlusion (MCAO) were established using high-performance liquid chromatography (HPLC), and their peak areas were quantified in plasma samples. The pharmacodynamic indexes of tumor necrosis factor-alpha (TNF-α), cytochrome c (Cyt-C), and total superoxide dismutase (T-SOD) were integrated using the Criteria Importance Through Intercriteria Correlation (CRITIC) method to create a comprehensive evaluation index. Spectrum-effect correlation was analyzed by performing gray relation analysis (GRA), correlation analysis (CA), and partial least squares regression (PLSR). The Borda method was then applied to integrate the obtained results., Results: In MCAO rats, the effective components of YYTN reduced TNF-α and Cyt-C and increased T-SOD, which indicates their anti-inflammatory, antiapoptotic, and antioxidant effects. Spectrum-effect CA revealed certain associations between the chromatographic peaks of the five main components and the comprehensive pharmacodynamic evaluation index. Of these components, formononetin displayed the highest correlation, whereas ferulic acid exhibited the lowest correlation. All components showed a positive correlation. Using the Borda method, the components were ranked as follows based on correlation: formononetin > calycosin > ligustrazine > puerarin > ferulic acid., Conclusions: The effective components of YYTN exhibited synergistic effects in the treatment of MCAO rats, which could potentially be attributed to their multitarget and multipathway mechanisms. The Borda method-based spectrum-effect correlation analysis provides a coordinated approach to investigate the relationship between fingerprint and pharmacodynamics of traditional Chinese medicine (TCM)., Competing Interests: Declaration of competing interest We hereby affirm that there are no known conflicts of interest associated with this publication, and no significant financial support has been received for this work that could have influenced its outcome., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Mitochondria are essential for cell metabolism and survival as they produce the majority of cellular ATP through oxidative phosphorylation as well as regulate critical processes such as cell proliferation and apoptosis. NIPSNAP family of proteins are predominantly mitochondrial matrix proteins. However, the molecular and cellular functions of the NIPSNAPs, particularly NIPSNAP3A, have remained elusive. Here, we demonstrated that NIPSNAP3A knockdown in HeLa cells inhibited their proliferation and migration and attenuated apoptosis induced by Actinomycin D (Act-D). These findings suggested a complex relationship between cellular processes and mitochondrial functions, mediated by NIPSNAP3A. Further investigations revealed that NIPSNAP3A knockdown not only inhibited mitochondrial fission through reduction of DRP1-S616, but also suppressed cytochrome c release in apoptosis. Collectively, our findings highlight the critical role of NIPSNAP3A in coordinating cellular processes, likely through its influence on mitochondrial dynamics., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The design of synthetic analogs of cytochrome c oxidase (CcO) is a formidable task due to its intricate structure encompassing multiple metal prosthetic sites and protein subunits. In recent years, artificial enzymes based on alloy nanomaterials have garnered significant attention due to the alloy design approach holds promise for the effective tuning of the properties of metal catalysts. In this study, we present copper-cerium alloy nanozymes (Cu-Ce-a NEs), where Cu mimics the active site of CcO, while Ce endows the alloy phase and enhances the capacity to catalyze the oxidation to cytochrome c (Cyt c). Cu-Ce-a NEs functionally mimics CcO, the terminal enzyme in the respiratory electron transport chain (ETC), by catalyzing the four-electron reduction of dioxygen to water. Utilizing the CcO-like properties of Cu-Ce-a NEs, we successfully implemented the electrochemical detection of Cyt c. The Cu-Ce-a NEs based electrochemical sensor revealed a favorable linear range spanning from 2 to 20 μM Cyt c, with a detection limit (LOD) of 2 μM. This method demonstrates high accuracy in Cyt c quantitation in pharmaceuticals, with results closely aligning with the actual concentrations. This finding not only offers new perspectives in the design of enzyme analogs, but also underscores the potential of this method for clinical Cyt c detection, highlighting its significance in biomedical research and diagnostics., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Post-translational modifications (PTMs) of proteins are ubiquitous processes present in all life kingdoms, involved in the regulation of protein stability, subcellular location and activity. In this context, cytochrome c (Cc) is an excellent case study to analyze the structural and functional changes induced by PTMS as Cc is a small, moonlighting protein playing different roles in different cell compartments at different cell-cycle stages. Cc is actually a key component of the mitochondrial electron transport chain (ETC) under homeostatic conditions but is translocated to the cytoplasm and even the nucleus under apoptotic conditions and/or DNA damage. Phosphorylation does specifically alter the Cc redox activity in the mitochondria and the Cc non-redox interaction with apoptosis-related targets in the cytoplasm. However, little is known on how phosphorylation alters the interaction of Cc with histone chaperones in the nucleus. Here, we report the effect of Cc Tyr48 phosphorylation by examining the protein interaction with SET/TAF-Iβ in the nuclear compartment using a combination of molecular dynamics simulations, biophysical and structural approaches such as isothermal titration calorimetry (ITC) and nuclear magnetic resonance (NMR) and in cell proximity ligation assays. From these experiments, we infer that Tyr48 phosphorylation allows a fine-tuning of the Cc-mediated inhibition of SET/TAF-Iβ histone chaperone activity in vitro. Our findings likewise reveal that phosphorylation impacts the nuclear, stress-responsive functions of Cc, and provide an experimental framework to explore novel aspects of Cc post-translational regulation in the nucleus., (© 2024 The Author(s). Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.)

Inflammation is an intrinsic protective mechanism against various forms of cellular injuries in humans; however, its undesired activation results in tissue damage and cell death. The onset of chronic inflammation and oxidative stress are the key characteristics of autoimmune inflammatory diseases such as rheumatoid arthritis (RA), for which an effective treatment is yet to be developed. Therefore, in this study, we investigated the protective effects and molecular mechanisms of a novel herbal preparation, Jing-Si herbal tea (JS), against H 2 O 2 -induced inflammation and cellular damage in HIG-82 synoviocytes. We found that JS did not show any significant alterations in cell viability at <188 μg/mL; however, a cytotoxic effect was observed at 188-1883 μg/mL concentrations tested. We found that expressions of inflammation associated extracellular matrix (ECM)-degrading proteases MMP-13, ADAMTS-2, -8, and -17 were abnormally enhanced under H 2 O 2 -induced pathological oxidative stress (ROS) in HIG-82 cells. Interestingly, JS treatment not only reduced the ROS levels but also significantly repressed the protein expressions of collagen degrading proteases in a dose-dependent manner. Treatment with JS showed enhanced cell viability against H 2 O 2 -induced toxic ROS levels. The expressions of cell protective aggrecan, Collagen II, and Bcl-2 were increased, whereas MMP-13, ADAMTS-2, Cytochrome C, and cleaved Caspase 3 were decreased by JS under inflammatory agents H 2 O 2 , MIA, LPS, and TNF-α treatment, respectively, in HIG-82 cells. Interestingly, the cytoprotective effect of JS treatment was attributed to a decreased mitochondrial localization of Bax and a reduction of Cytochrome C release into the cytoplasm of H 2 O 2 -treated HIG-82 cells. Collectively, our results suggest a novel protective mechanism of JS for RA treatment, which could be potentially applied as a complementary treatment or as an alternative therapeutic approach to mitigate inflammatory diseases., (© 2024 Wiley Periodicals LLC.)

Objectives: Diabetes is closely linked to hearing loss, yet the exact mechanisms remain unclear. Cochlear stria vascularis and pericytes (PCs) are crucial for hearing. This study investigates whether high glucose induces apoptosis in the cochlear stria vascularis and pericytes via elevated ROS levels due to oxidative stress, impacting hearing loss., Methods: We established a type II diabetes model in C57BL/6J mice and used auditory brainstem response (ABR), Evans blue staining, HE staining, immunohistochemistry, and immunofluorescence to observe changes in hearing, blood-labyrinth barrier (BLB) permeability, stria vascularis morphology, and apoptosis protein expression. Primary cultured stria vascularis pericytes were subjected to high glucose, and apoptosis levels were assessed using flow cytometry, Annexin V-FITC, Hoechst 33342 staining, Western blot, Mitosox, and JC-1 probes., Results: Diabetic mice showed decreased hearing thresholds, reduced stria vascularis density, increased oxidative stress, cell apoptosis, and decreased antioxidant levels. High glucose exposure increased apoptosis and ROS content in pericytes, while mitochondrial membrane potential decreased, with AIF and cytochrome C (CytC) released from mitochondria to the cytoplasm. Adding oxidative scavengers reduced AIF and CytC release, decreasing pericyte apoptosis., Discussion: Hyperglycemia may induce mitochondrial apoptosis of cochlear stria vascularis pericytes through oxidative stress.

The pattern of the activity of proteases is related to distinct physiological states of living organisms. Often activity changes of a certain protease can be assigned to a specific disease. Hence, they are useful biomarkers and a simple and fast determination method of their activity could be a valuable tool for the efficient monitoring of numerous diseases. Here, two different methods for the qualitative and quantitative determination of protease activity are demonstrated using the model system of proteinase K. The first test system is based on a protein-modified and colored 3D silica structure that changes color when exposed to the enzyme. This method has also been used for the detection of matrix metallo-protease 2 (MMP2) with gelatine as protease substrate on the plates. The second detection system uses the decrease in the voltammetric signal of a cytochrome c/DNA multilayer electrode after incubation with a protease to quantitatively determine its proteolytic activity. While activities down to 0.15 U/ml can be detected with the first method, the second one provides detection limits of about 0.03U/ml (for proteinase K.) The functionality of both systems can be demonstrated and ways for further enhancement of sensitivity have been elucidated., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

The hydrogen/deuterium (H/D) exchange rate is an optimal measure for studying the structures and dynamics of hydrogen bonding systems, as it reflects the molecular contact environment and the strength of the hydrogen bonds. A method for rapid measurement of the H/D exchange reaction rates is required to examine the intermolecular environments of molecules in solutions. We developed a droplet collision atmospheric pressure infrared laser ablation mass spectrometry technique for this purpose. We obtained the H/D exchange reaction rate of cytochrome c in a methanol/H 2 O·D 2 O solution. We revealed that the first hydration shell of the cytochrome c molecule hinders the penetration of D 2 O to the surface of the molecule from the rates, which provides a novel method to investigate solution structures by a mass-spectrometric method. The droplet-collision mass spectrometry method developed in the present study can be extended to research on the molecular interactions in solutions, such as the mutual interactions of protein molecules, which are of importance in living cells.

Nanopore sensing is a label-free single-molecule technique that enables the study of the dynamical structural properties of proteins. Here, we detect the translocation of cytochrome c (Cyt c ) through an asymmetric thin nanopore with photothermal heating to evaluate the influence of temperature on Cyt c conformation during its translocation in an electric field. Before Cyt c translocates through an asymmetric thin SiN x nanopore, ∼1 ms trapping events occur due to electric field-induced denaturation. These trapping events were corroborated by a control analysis with a transmission electron microscopy-drilled pore and denaturant buffer. Cyt c translocation events exhibited markedly greater broad current blockade when the pores were photothermally heated. Collectively, our molecular dynamics simulation predicted that an increased temperature facilitates denaturation of the α-helical structure of Cyt c , resulting in greater blockade current during Cyt c trapping. Our photothermal heating method can be used to study the influence of temperature on protein conformation at the single-molecule level in a label-free manner.

Oxidized light-density lipoprotein (ox-LDL) causes endothelial dysfunction, which is an important determinant of atherogenesis, and subsequently leads to apoptosis. Atherosclerosis is one of the most significant cardiovascular diseases (CVDs) threatening human health and causes death worldwide. Recently, long noncoding RNAs (lncRNAs) have been suggested to involved in vascular biology. Ox-LDL activates nuclear factor kappa-B (NF-κB), and NF-κB interacting lncRNA (NKILA) inhibits NF-κB signaling. In this study, the hypothesis is that NKILA may regulate endothelial cell (EC) apoptosis and, therefore, play a role in the pathogenesis of atherosclerosis. This hypothesis is based on the knowledge that EC apoptosis contributes to atherosclerosis development and that NKILA has become a prominent lncRNA in CVDs. The expression of Bcl-2-associated X protein (BAX), caspase 9 (CASP9), cytochrome c (Cyt c, CYCS), apoptotic protease activating factor 1 (APAF1), and B-cell lymphoma 2 (BCL-2) genes in human umbilical vein endothelial cells (HUVEC) treated with ox-LDL and transfected with NKILA siRNA was analyzed using quantitative reverse transcription polymerase chain reaction (RT-qPCR). BAX, CASP9, CYCS, APAF1, and BCL-2 gene expression was downregulated in ox-LDL and NKILA siRNA-treated HUVEC. In addition, when threshold/quantification cycle (Cq) values of NKILA gene expression increased, Cq values of BAX, CASP9, APAF1, and BCL-2 gene expression increased statistics significantly. The expression detection of all these genes, resulting from NKILA gene silencing, may provide guidance for epigenetic studies on EC apoptosis in atherosclerosis., Competing Interests: The authors declare there are no competing interests., (©2024 Bayyurt et al.)

Cytochrome c (CytC) is conjugated with a small molecule TG6 to give TG6-CytC, which is directly delivered into cytosol, triggering the release of endogenous CytC from mitochondria, and inducing a caspase-3-dependent apoptosis with an IC 50 down to 2.4 nM. This work shows an efficient strategy for intracellular protein delivery.

Extracellular ATP is a dynamic signaling molecule that modulates myriad of cellular functions through P2 purinergic receptors activation and is cytotoxic to a variety of cells at high concentration. But the mechanism of this extracellular ATP/ATP analogs- elicited cytotoxicity is not fully understood. In this study we aim to investigate whether there is differential sensitivity towards induction of apoptosis by ATP analogs (2'-Me ATP and 3'-Me ATP) and its effect on receptor mediated or extrinsic and mitochondria mediated or intrinsic apoptotic signaling pathways. Our findings demonstrated that the IC50 values for 2'-Me ATP and 3'-Me ATP were 3mM and 2mM, respectively, in Hep2, and SiHa cells. The downregulation of anti-apoptotic proteins Bcl-2 and Bcl-xL, along with a significant increase in the expression of the pro-apoptotic protein Bax (p<0.05), indicated the involvement of both pro- and anti-apoptotic factors in HeP2 cells, whereas in SiHa cells, a downregulation of anti-apoptotic proteins Bcl-2 and Bcl-xL was observed, whereas the expression level of the pro-apoptotic protein Bax remained unaffected. Furthermore, an upregulation of p53 and apoptosis-inducing factor (AIF) was observed in HeP2 cells (p<0.05) whereas, an upregulation of p53 was observed while no change was seen on the level of apoptosis inducing factor (AIF) was observed in SiHa cells. Additionally, there was a notable rise in caspase-3 and -9 activities, PARP cleavage, and the release of cytochrome c (p<0.05) from the mitochondria to the cytosol in both cells. Collectively, our study suggests that 3'-Me ATP induces apoptosis in Hep2 and SiHa cells through the intrinsic mitochondrial pathway.

Cytochrome C (cyt C), the protein involved in oxidative phosphorylation, plays several other crucial roles necessary for both cell life and death. Studying natural variants of cyt C offers the possibility to better characterize the structure-to-function relationship that modulates the different activities of this protein. Naturally mutations in human cyt C (G41S and Y48H) occur in the protein central Ω-loop and cause thrombocytopenia 4. In this study, we have investigated the binding of such variants and of wild type (wt) cyt C to synthetic cardiolipin-containing vesicles. The mutants have a lower propensity in membrane binding, displaying higher dissociation constants with respect to the wt protein. Compressibility measurements reveal that both variants are more flexible than the wt, suggesting that the native central Ω-loop is important for the interaction with membranes. Such hypothesis is supported by molecular dynamics simulations. A minimal distance analysis indicates that in the presence of cardiolipin the central Ω-loop of the mutants is no more in contact with the membrane, as it happens instead in the case of wt cyt C. Such finding might provide a hint for the reduced membrane binding capacity of the variants and their enhanced peroxidase activity in vivo., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

The urgent global health problem of antimicrobial resistance (AMR) calls for the discovery of new antibiotics with innovative modes of action while considering the low toxicity to mammalian cells. This paper proposes a novel strategy for designing antibiotics with selective bacterial toxicity by exploiting the positional differences of electron transport chains (ETC) in bacterial and mammalian cells. The focus is on cytochrome c (cyt C) and its maturation system in E. coli. The catalytic oxidative activity of metallophthalocyanine (MPc), which have a distinctive M-N4 structure, is being investigated. Unlike previous applications based on light-activated reactive oxygen species (ROS) generation, this study exploits the ability of MPcs to oxidize Fe 2+ to Fe 3+ in cyt C and catalyze the formation of disulfide bonds between cysteine residues to interfere with cyt C maturation, disrupt the bacterial respiratory chain and selectively kills bacteria. In contrast, in mammalian cells, these MPcs are located in the lysosomes and cannot access the ETC in the mitochondria, thus achieving selective bacterial toxicity. Two MPcs that showed effective antibacterial activity in a wound infection model were identified. This study provides a valuable reference for the design of novel antibiotics based on M-N4-based metal complex molecules., Competing Interests: Declaration of competing interest The authors declare that there is no conflict of interest in this submission and responsible for the authenticity, completeness and accuracy of the submitted literature. The content of the literature does not infringe on the rights and interests of any individual or organization., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Free interconversion of cytochrome C (CytC) between native ferrous (Cyt-Fe II ) and oxidized ferric (CytC-Fe III ) states is necessary to maintain the respiratory function of mitochondria. Disturbances in CytC-Fe III to total CytC ratio may indicate mitochondrial dysfunction and apoptosis. Thus, tracking CytC oxidation state delivers important information about cellular physiology. In this work, we propose a novel methodology based on resonance Raman (rR) imaging optimized uniquely to track and qualitatively analyze the transition of Cyt-Fe II to CytC-Fe III within live cells without affecting their morphology. None of the commonly used excitation lines allows such clear-cut differentiation, contrary to the 405 nm applied in this work. The presented methodology provides a novel pathway in the label-free detection of ferrous and ferric heme proteins., (© 2024 Federation of European Biochemical Societies.)

Cytochrome c (CytC), a one-electron carrier, transfers electrons from complex bc 1 to cytochrome c oxidase (CcO) in the electron-transport chain. Electrostatic interaction with the partners, complex bc 1 and CcO, is ensured by a lysine cluster near the heme forming the Universal Binding Site (UBS). We constructed three mutant variants of mitochondrial CytC with one (2Mut), four (5Mut), and five (8Mut) Lys->Glu substitutions in the UBS and some compensating Glu->Lys substitutions at the periphery of the UBS for charge compensation. All mutants showed a 4-6 times increased peroxidase activity and accelerated binding of cyanide to the ferric heme of CytC. In contrast, decomposition of the cyanide complex with ferrous CytC, as monitored by magnetic circular dichroism spectroscopy, was slower in mutants compared to WT. Molecular dynamic simulations revealed the increase in the fluctuations of C α atoms of individual residues of mutant CytC compared to WT, especially in the Ω-loop (70-85), which can cause destabilization of the Fe…S(Met80) coordination link, facilitation of the binding of exogenous ligands cyanide and peroxide, and an increase in peroxidase activity. It was found that only one substitution K72E is enough to induce all these changes, indicating the significance of K72 and the Ω-loop (70-85) for the structure and physiology of mitochondrial CytC. In this work, we also propose using a ferro-ferricyanide buffer as a substrate to monitor the peroxidase activity of CytC. This new approach allows us to determine the rate of peroxidase activity at moderate (200 µM) concentrations of H 2 O 2 and avoid complications of radical formation during the reaction.

In the present study, the apoptosis-inducing potential of a chloroform fraction from an alcoholic extract of Vallaris solanacea aerial parts (VS) was examined using human promyelocytic leukemia HL-60 cells. We discovered a concentration and time-dependent decrease in cell growth using MTT assay. Scanning electron micrographs and fluorescence microscopy were used to observe several well-documented morphological and nuclear alterations, such as reduction in cell size, chromatin condensation, fragmentation, and the creation of cell surface blebs. A considerable rise in the Sub-G 0 population was revealed by cell cycle analysis. Additionally, a dose-dependent rise in cells positive for Annexin V was observed. DCFH-DA test on VS-treated HL-60 cells showed an increase in endogenous ROS generation of up to 4.3 fold. Additionally, suppression in Bcl-2 levels and increased mitochondrial membrane depolarization in treated cells were also associated with a rise in cytosolic cytochrome-c levels that was consequently followed by the activation of the caspase cascade. Further, the DNA fragmentation assay exhibited a typical ladder formation at 25 μg/ml, which became prominent in a concentration-dependent manner. Our study revealed that VS has apoptosis-inducing potential towards HL-60 cells in vitro and is an effective candidate for further anti-cancer studies., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

NADPH-oxidase (NOX) is a multi-subunit enzyme complex. The upregulation of NOX causes massive production of superoxide (O 2 ¯ ), which avidly reacts with nitric oxide (NO) and increases cellular reactive oxygen/nitrogen species (ROS/RNS). Increased ROS/RNS plays pivotal role in the sporadic Alzheimer's disease (sAD) development and brain damage following impaired insulin signaling. Hence, this study aimed to examine early-time course of changes in NOX and NOS expression, and apoptotic proteins in the rats hippocampi following insulin signaling impairment [induced by STZ injection; intraperitoneal (IP) or in cerebral ventricles (ICV)]. Early effects (1, 3, or 6 weeks) on the NOX activity, translocation of NOX subunits from cytosol to the membrane, NO-synthases [neuronal-, inducible- and endothelial-NOS; nNOS, iNOS and eNOS], The Rac-1 protein expression, levels of NO and O 2 ¯ , cytochrome c release, caspase-3 and 9 activations (cleavage) were studied. STZ injection (in both models) increased NOX activity, O 2 ¯ production, and enhanced cytosolic subunits translocation into membrane. The iNOS but not nNOS and eNOS expression and NO levels were increased in STZ treated rats. Finally, STZ injection increased cytochrome c release, caspase-3 and 9 activations in a manner that was significantly associated with levels of O 2 ¯ and NO in the hippocampus. ICV-STZ administration resulted in significant profound changes over the IP route. In conclusion, impairment in insulin function induces early changes in ROS/RNS contents through NOX and iNOS upregulation and neuronal apoptosis in the hippocampus. Our results could mechanistically explain the role of impaired insulin function in the development of sAD., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

This study investigated the effects of ergosterol peroxide(EP) on the proliferation and apoptosis of MCF-7 breast cancer cells, explored its possible mechanisms of action, and verified the effects and mechanisms by in vitro experiments. Network pharmaco-logy was used to screen the target proteins of EP and construct target networks and protein-protein interaction(PPI) networks to predict the potential target proteins and related pathways involved in EP anti-breast cancer effects. The MTT assay was performed to measure the inhibitory effect of EP on MCF-7 cell proliferation, and the colony formation assay was used to assess the cell cloning ability. Flow cytometry and laser confocal microscopy were employed to evaluate cell apoptosis, mitochondrial membrane potential and reactive oxygen species(ROS) levels. Western blot analysis was conducted to examine the expression levels of B-cell lymphoma 2(Bcl-2), Bcl-2-associated X protein(Bax), cytochrome C(Cyt C), caspase-7, cleaved caspase-7, phosphatidylinositol 3-kinase(PI3K), and se-rine/threonine kinase B(AKT) in MCF-7 cells treated with EP. The results of network pharmacology prediction yielded 173 common targets between EP and breast cancer; the results of Kyoto Encyclopedia of Genes and Genomes(KEGG) enrichment analysis showed that EP treatment for breast cancer mainly affected the signaling pathways such as cancer pathway, PI3K-AKT signaling pathway, cellular senescence signaling pathway, and viral carcinogenesis pathway; and the MTT assay results showed that the viability of MCF-7 cells in the EP group was significantly lower than that in the control group, exhibiting a time-and concentration-dependent trend, and EP can inhibit colony formation of MCF-7 breast cancer cells. Treatment with 10, 20, and 40 μmol·L~(-1) EP for 24 h resulted in a significant increase in the total apoptosis rate of MCF-7 cells, a significant decrease in mitochondrial membrane potential, and a significant increase in ROS levels. In addition, treatment with EP led to an upregulation of Cyt C, Bax, and cleaved caspase-7 protein expression, and a downregulation of p-PI3K, p-AKT, and Bcl-2 protein expression in MCF-7 cells. Studies have shown that EP inhibits MCF-7 breast cancer cell proliferation and reduces colony formation by a mechanism that may be related to the PI3K-AKT pathway mediating the mitochondrial apoptotic pathway.

Hepatocellular carcinoma is a primary liver cancer, characterised by diverse etiology, late diagnoses, and poor prognosis. Hepatocellular carcinoma is mostly resistant to current treatment options, therefore, identification of more effective druggable therapeutic targets is needed. We found microRNA miR-20a-5p is upregulated during mouse liver tumor progression and in human hepatocellular carcinoma patients. In this study, we elucidated the therapeutic potential of targeting oncogenic miR-20a-5p, in vivo, in a xenograft model and in two transgenic hepatocellular carcinoma mouse models via adeno-associated virus-mediated miR-20a-Tough-Decoy treatment. In vivo knockdown of miR-20a-5p attenuates tumor burden and prolongs survival in the two independent hepatocellular carcinoma mouse models. We identified and validated cytochrome c as a novel target of miR-20a-5p. Cytochrome c plays a key role in initiation of the apoptotic cascade and in the electron transport chain. We show for the first time, that miR-20a modulation affects both these key functions of cytochrome c during HCC development. Our study thus demonstrates the promising 'two birds with one stone' approach of therapeutic in vivo targeting of an oncogenic miRNA, whereby more than one key deregulated cellular process is affected, and unequivocally leads to more effective attenuation of HCC progression and significantly longer overall survival., (© 2024. The Author(s).)

Lys-ligated cytochromes make up an emerging family of heme proteins. Density functional theory calculations on the amine/imidazole-ligated c -type ferric heme were employed to develop force-field parameters for molecular dynamics (MD) simulations of structural and dynamic features of these proteins. The new force-field parameters were applied to the alkaline form of yeast iso -1 cytochrome c to rationalize discrepancies resulting from distinct experimental conditions in prior structural studies and to provide insights into the mechanisms of the alkaline transition. Our simulations have revealed the dynamic nature of Ω-loop C in the Lys-ligated protein and its unfolding in the Lys-ligated conformer having this loop in the same position as in the native Met-ligated protein. The proximity of Tyr67 or Tyr74 to the Lys ligand of ferric heme iron suggests a possible mechanism of the backward alkaline transition where a proton donor Tyr assists in Lys dissociation. The developed force-field parameters will be useful in structural and dynamic characterization of other native or engineered Lys-ligated heme proteins.

Background: The concentration of cytochrome C is demonstrated to be an effective indicator of the microbial corrosion strength of metals. Traditional cytochrome C sensor can detect cytochrome C with a low detection limit, but their use is limited by their high cost, cumbersome operation, and susceptibility to malignant environments. In addition, studies on the monitoring of cytochrome C in the field of microbial corrosion has still not been carried out. Therefore, there is a need for a highly sensitive, selective, low-cost, anti-interference, and stable cytochrome C sensor with online monitoring and remote sensing capabilities for in-situ measurement of microbial corrosion strength., Results: This paper proposed a highly sensitive label-free fiber-optic sensor based on Mach-Zehnder interferometer (MZI) for in-situ measurement of the microbial corrosion marker cytochrome C. Two-dimensional Ti 2 C-MXene material is uniformly immobilized onto the surface of the sensing area to improve the sensitivity, hydrophilicity, and specific surface area of the sensing area, as well as to facilitate the immobilization of specific sensitive materials. The cytochrome C antibody is modified on the surface of Ti 2 C-MXene to specifically recognize cytochrome C, whose concentration variation can be measured by monitoring the spectral shift of MZI sensor. Results demonstrate a measurement sensitivity of 1.428 nm/μM for cytochrome C concentrations ranging from 0 to 7.04 μM. The detection limit of the sensor is calculated to be 0.392 μM with remarkable performance, including selectivity, stability, and reliability. Besides, the measurement result of the proposed sensor in real microbial corrosive environment is consistent with that of the ideal environment., Significance and Novelty: This is the first instance of achieving in-situ and label-free measurement of cytochrome C by using a fiber-optic MZI sensor, which undoubtedly provides a feasible solution for the effective monitoring of microbial metal corrosion in the environment., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Montmorillonite (MM) crystal nanoplates acquire anticancer properties when coated with the mitochondrial protein cytochrome c (cytC) due to the cancer cells' capability to phagocytize cytC-MM colloid particles. The introduced exogenous cytC initiates apoptosis: an irreversible cascade of biochemical reactions leading to cell death. In the present research, we investigate the organization of the cytC layer on the MM surface by employing physicochemical and computer methods-microelectrophoresis, static, and electric light scattering-to study cytC adsorption on the MM surface, and protein electrostatics and docking to calculate the local electric potential and Gibbs free energy of interacting protein globules. The found protein concentration dependence of the adsorbed cytC quantity is nonlinear, manifesting a positive cooperative effect that emerges when the adsorbed cytC globules occupy more than one-third of the MM surface. Computer analysis reveals that the cooperative effect is caused by the formation of protein associates in which the cytC globules are oriented with oppositely charged surfaces. The formation of dimers and trimers is accompanied by a strong reduction in the electrostatic component of the Gibbs free energy of protein association, while the van der Waals component plays a secondary role.

Cytochrome c (Cyt c ) is important for both mitochondrial respiration and apoptosis, both of which are altered in cancer cells that switch to Warburg metabolism and manage to evade apoptosis. We earlier reported that lysine 53 (K53) of Cyt c is acetylated in prostate cancer. K53 is conserved in mammals that is known to be essential for binding to cytochrome c oxidase and apoptosis protease activating factor-1 (Apaf-1). Here we report the effects of this acetylation on the main functions of cytochrome c by expressing acetylmimetic K53Q in cytochrome c double knockout cells. Other cytochrome c variants analyzed were wild-type, K53R as a control that maintains the positive charge, and K53I, which is present in some non-mammalian species. Intact cells expressing K53Q cytochrome c showed 49% decreased mitochondrial respiration and a concomitant increase in glycolytic activity (Warburg effect). Furthermore, mitochondrial membrane potential was decreased, correlating with notably reduced basal mitochondrial superoxide levels and decreased cell death upon challenge with H 2 O 2 or staurosporine. To test for markers of cancer aggressiveness and invasiveness, cells were grown in 3D spheroid culture. K53Q cytochrome c -expressing cells showed profoundly increased protrusions compared to WT, suggesting increased invasiveness. We propose that K53 acetylation of cytochrome c is an adaptive response that mediates prostate cancer metabolic reprogramming and evasion of apoptosis, which are two hallmarks of cancer, to better promote tumor survival and metastasis.

This study aims to investigate the effect of ergosterol peroxide(EP) on the apoptosis of human hepatocellular carcinoma and its mechanism of action. The cell viability of HepG2 and SK-Hep-1 cells with 0(blank control), 2.5, 5, 10, 20, 40, and 80 μmol·L~(-1) of EP after 24, 48, and 72 h of action was detected by using CCK-8 assay, and the half inhibitory concentrations(IC&#95;(50)) at 24, 48, and 72 h were calculated. Formal experiments were performed to detect the effect of EP on intracellular reactive oxygen species(ROS) using DCFH-DA staining, the effect of EP on intracellular mitochondrial membrane potential using JC-1 staining, the number of apoptotic cells using Annexin V-FITC/PI double-staining after HepG2 cells were co-cultured with 0(blank control), 10, 20, 40 μmol·L~(-1) EP for 48 h. The effects of EP at different concentrations on apoptotic morphology were detected using AO/EB staining. The effects of different concentrations of EP on the protein expression of mitochondrial apoptosis pathway-related proteins B cell lymphoma 2(Bcl-2), cytochrome C(Cyt-C), Bcl-2-related X protein(Bax), caspase-3, cleaved caspase-3, caspase-9, and cleaved caspase-9 were examined by using Western blot. The results showed that different concentrations of EP could inhibit the proliferation of hepatocellular carcinoma with concentration-and time-dependent trends. Compared with the blank control group, the ROS level in the EP-treated group increased significantly(P&lt;0.05). The mitochondrial membrane potential decreased significantly(P&lt;0.05). The total apoptosis rate increased significantly(P&lt;0.05). The expression of Bcl-2 protein was significantly down-regulated, and the expression of Cyt-C, Bax, cleaved caspase-9, and cleaved caspase-3 were significantly up-regulated(P&lt;0.05). In summary, EP may inhibit the proliferation of hepatocellular carcinoma by modulating the mitochondria-mediated apoptosis pathway and induce apoptosis.