Diverse functions of cytochrome c in cell death and disease

Abstract

The redox-active protein cytochrome c is a highly positively charged hemoglobin that regulates cell fate decisions of life and death. Under normal physiological conditions, cytochrome c is localized in the mitochondrial intermembrane space, and its distribution can extend to the cytosol, nucleus, and extracellular space under specific pathological or stress-induced conditions. In the mitochondria, cytochrome c acts as an electron carrier in the electron transport chain, facilitating adenosine triphosphate synthesis, regulating cardiolipin peroxidation, and influencing reactive oxygen species dynamics. Upon cellular stress, it can be released into the cytosol, where it interacts with apoptotic peptidase activator 1 (APAF1) to form the apoptosome, initiating caspase-dependent apoptotic cell death. Additionally, following exposure to pro-apoptotic compounds, cytochrome c contributes to the survival of drug-tolerant persister cells. When translocated to the nucleus, it can induce chromatin condensation and disrupt nucleosome assembly. Upon its release into the extracellular space, cytochrome c may act as an immune mediator during cell death processes, highlighting its multifaceted role in cellular biology. In this review, we explore the diverse structural and functional aspects of cytochrome c in physiological and pathological responses. We summarize how posttranslational modifications of cytochrome c (e.g., phosphorylation, acetylation, tyrosine nitration, and oxidation), binding proteins (e.g., HIGD1A, CHCHD2, ITPR1, and nucleophosmin), and mutations (e.g., G41S, Y48H, and A51V) affect its function. Furthermore, we provide an overview of the latest advanced technologies utilized for detecting cytochrome c, along with potential therapeutic approaches related to this protein. These strategies hold tremendous promise in personalized health care, presenting opportunities for targeted interventions in a wide range of conditions, including neurodegenerative disorders, cardiovascular diseases, and cancer.

Fig. 1. Timeline in cytochrome c research.

The timeline of cytochrome c research highlights significant milestones that have contributed to our current understanding of its function and biology. Many discoveries couldn’t be included here due to space limitations. These milestones, among others, have paved the way for further investigations. 1884 MacMunn describes “respiratory pigments” (cytochromes) [1]. 1925 Keilin gives cytochromes their modern name [2]. 1989 Nomenclature is created for electron transfer proteins [3]. 1996 Cytochrome c is found to induce apoptosis in cell-free extract [212]. 1999 Antioxidant functions of cytochrome c are discovered [95]. 2000 CYCS global knockout mice are developed [10]. 2003 Cytochrome c is found to bind with ITPR1 (inositol 1,4,5-trisphosphate receptor type) [133]. 2004 Discovery of cytochrome c nuclear translocation [142]. 2005 Electron transfer from cytochrome c to p66Shc triggering mitochondrial apoptosis via reactive oxygen species [98]. 2005 Apoptotic functions of cytochrome c are ablated in neurons [21]. 2006 First discovery and characterization of mammalian cytochrome c phosphorylation site [63]. 2008 CYCS G41S-mutant is found to be associated with thrombocytopenia [150]. 2017 Architectures of human mitochondria respiratory megacomplex is defined through cryo-electron microscopy [49]. 2020 Pyroptosomes consisting of APAF1/cytochrome c/CASP4/11 are discovered [22]. 2022 Sublethal cytochrome c is found to generate drug-tolerant persister cells [31]. 2022 Translocation of cytochrome c to the nucleus can regulate the liquid-liquid phase separation within the nucleolus, resulting in the release of proteins sequestered by nucleophosmin [32]. 2023 Lysine 39 acetylation of cytochrome c enhances porcine skeletal muscle’s cellular respiration and resilience to ischemia-reperfusion injury [78].

Fig. 2. The structure of human cytochrome c.

The structure of human cytochrome c exhibits conserved amino acid sites across various species. It is a heme protein that undergoes alkaline transition and has specific binding sites for ATP, APAF1, cytochrome c₁, cytochrome c oxidase, and cardiolipin. In addition, cytochrome c can undergo modifications such as phosphorylation, acetylation, nitration, oxidation, and mutations associated with thrombocytopenia, including the variants G41S (PDB entry 3NWV), Y48H (PDB entry 5EXQ), and A51V (PDB entry 6DUJ). The 3D structure of cytochrome c, with highlighted residues, is displayed at the top (PDB entry 3ZCF).

Fig. 3. The subcellular-dependent functions of cytochrome c.

Cytochrome c exhibit distinct functions depending on its subcellular localization. In mitochondria, cytochrome c serves as a vital component of the electron transport chain, facilitating electron transfer between complexes III and IV in the mitochondrial intermembrane space. The interaction between cytochrome c and HIGD1A (hypoxia-inducible domain family member 1A) is pivotal in regulating mitochondrial oxidative phosphorylation and the cellular stress response. Cytochrome c also plays a role in scavenging reactive oxygen species (ROS), particularly through the detoxification of hydrogen peroxide (H₂O₂). Moreover, when cytochrome c interacts with cardiolipin, it demonstrates peroxidase activity, playing a role in the initiation of apoptosis. It can further amplify ROS generation and trigger apoptosis via its interaction with p66Shc. Cytochrome c also interacts with other proteins, such as the growth hormone-inducible transmembrane protein (GHITM, also known as MICS1). Alongside GHITM, coiled-coil-helix-coiled-coil-helix domain containing 2 (CHCHD2) binds to cytochrome c. These interactions influence mitochondrial morphology or the release of cytochrome c. Members of the BCL-2 family, including BCL2 and BCL2L1, can inhibit the release of cytochrome c. A Under cellular stress, BH3-only proteins, such as Bim and Bid, become activated, bind and inactivate the anti-apoptotic proteins, such as BCL2 and BCL2L1, whose primary function is to restrain the effectors Bax and Bak. Once the anti-apoptotic BCL2 proteins are neutralized by the BH3-only proteins, Bax and Bak undergo spontaneous activation, homo-oligomerize, and form pores on the mitochondrial outer membrane, allowing the escape of cytochrome c and other apoptogeneic factors from the intermembrane space. After cytochrome c is translocated to the cytosol, it interacts with APAF1, initiating apoptosome assembly and subsequently activating CASP9 and CASP3. B It can interact with the inositol 1,4,5-trisphosphate receptor type 1 (ITPR1) on the endoplasmic reticulum membranes, triggering calcium ion (Ca²⁺) release. In addition, cytochrome c can bind to 14-3-3epsilon and block 14-3-3epsilon–mediated inhibition of APAF1, thus acting as an indirect activator of CASP9/3. Heat shock proteins have been identified as inhibitors of cytochrome c release or apoptosome formation. C Mitochondrial permeability transition (MPT) induces the assembly of the pyroptosome, which consists of APAF1, cytochrome c, and CASP4. This complex cleaves CASP3 and triggers the activation of gasdermin E (GSDME), leading to pyroptosis. D In the presence of cardiolipin, O₂, and H₂O₂, cytochrome c oxidizes the plasmalogen vinyl ether linkage, facilitating its hydrolytic cleavage leading to lipid peroxidation. E BH3 mimetics can induce sublethal cytochrome c release, which activates heme-regulated inhibitor kinase (HRI or EIF2AK1) and enables the translation of activating transcription factor 4 (ATF4), resulting in a persister phenotype. Furthermore, cytochrome c can enter the nucleus, where it directly binds and inhibits histone chaperone SET/TAF-Iβ during DNA damage, and thus hinders SET/TAF-Iβ nucleosome assembly activity. Cytochrome c can directly bind to nucleolar nucleophosmin (NPM) by triggering a conformational change, driving alternative reading frame (ARF) release followed by the activation of p53 pathway in response to DNA damage. NPM engages in liquid-liquid phase separation. Finally, cytochrome c can be released into the extracellular space from damaged cells and taken up by astrocytes or immune cells in a toll-like receptor 4 (TLR4)-dependent manner. This uptake leads to the secretion of cytokines such as IL-1β, IL-12, and GM-CSF that trigger immune responses.

Fig. 4. The functions of cytochrome c in apoptosis, pyroptosis, and the persister phenotype.

Cytochrome c plays a critical role in various cellular processes, including apoptosis, pyroptosis, and the development of a persister phenotype. a In apoptosis, stressful stimuli such as chemotherapy or low doses of bile acids trigger MOMP. This leads to the release of cytochrome c into the cytosol, where it interacts with APAF1, initiating apoptosome assembly. Activated caspase-9 and caspase-3 are then recruited, resulting in apoptosis. b Under specific stimuli like bile acids, calcium overload, or activators of ANT, mitochondrial MPT promotes the assembly of a pyroptosome. The pyroptosome consists of APAF1, cytochrome c, and CASP4/11 (a general sensor of MPT) but not CASP9. This assembly leads to the cleavage of CASP3 and GSDME, inducing pyroptosis. c In the context of BH3-mimetic treatment, a sublethal release of cytochrome c enables cancer cells to acquire a persister phenotype. This phenotype is driven by the cytochrome c-EIF2AK1/HRI-ATF4 pathway. It allows cancer cells to persist despite treatment and contributes to treatment resistance. ANT adenine nucleotide translocator, MOMP mitochondrial outer membrane permeabilization, MPT mitochondrial permeability transition, ATF4 activating transcription factor 4, HRI heme-regulated inhibitor/EIF2AK1, eukaryotic translation initiation factor 2 alpha kinase 1.

Fig. 5. Cytochrome c-associated human diseases, detection, and therapeutic strategies for engineering precision nanoparticles (NPs).

A Cytochrome c has been implicated in various human diseases, including neurodegenerative diseases, cardiovascular diseases, inflammatory diseases, and cancer. It is also relevant to treatments such as anti-retroviral therapy and cancer treatment. Furthermore, methodologies have been developed for detecting cytochrome c in serum, aiding in disease diagnosis and monitoring. Traditional techniques such as enzyme-linked immunosorbent assays (ELISAs), western blots, high-performance liquid chromatography (HPLC), immunocytochemistry (IC), flow cytometry (FCM), surface plasmon resonance (SPR) and biosensors could potentially be used to detect and analyze cytochrome c. B Biosensors offer a valuable tool for monitoring mitochondria damage or therapeutic interventions that induce cell death in tumors, resulting in increased levels of cytochrome c release that can be found in the bloodstream. These biosensors consist of three main components: (a) Bioreceptor: The bioreceptor employs capture probes such as aptamers, enzymes, or antibodies specifically designed for sensing cytochrome c. The electrode surfaces are modified with gold nanoparticles (AuNPs), carbon nanotubes (CNTs), quantum dots, trypsin, enzymes (e.g., cytochrome c oxidase [CcO] and cytochrome c reductase [CcR]), antibodies, or aptamers. (b) Detector Element or Transducer System: This component of the biosensor utilizes various techniques such as square wave voltammetry, electrochemistry, fluorescence, differential pulse voltammetry, interferometric reflectance spectroscopy, or surface-enhanced Raman scattering (SERS) to detect and measure the presence of cytochrome c. (c) Reader Device: The biosensor is accompanied by a reader device that enables the analysis and interpretation of the detected signals. The specifications of these biosensors include high sensitivity, rapid response times, low cost, multiplexing capability (i.e., simultaneous detection of multiple targets), and miniaturization for portability and ease of use. By incorporating these components and meeting the specified requirements, cytochrome c biosensors provide a valuable analytical tool for sensitive, rapid, and cost-effective detection and quantification of cytochrome c in serum. C The strategies involve specific property designs to enhance targeted delivery and tailor the platform for cytochrome c delivery and include: (a) Incorporation of targeting molecules on the NP surface: Antibodies (e.g., HER-2 antibody), toxins, peptides, aptamers, and ligands (e.g., transferrin) can be attached to the NP surface, facilitating specific and efficient NP uptake by cancer cells. (b) Utilization of therapeutic delivery systems: NP-based delivery systems coated with ligands that recognize cancer cells through surface markers. cytochrome c can be encapsulated inside the NP/nanogel or bound to its surface. (c) Implementation of linkers: A linker is employed to connect the cytochrome c-conjugated protein or peptide to the NP. For example, a redox-sensitive linker containing a disulfide bond can be used, as the disulfide bonds are cleaved within the reductive chemical environment of the cancer cell cytoplasm, leading to the release of cytochrome c. (d) Enhancement of cytochrome c stability: Methods such as PEGylation, ionic liquids, B-DNA, or conjugation with proteins or peptides can be employed to improve the stability of cytochrome c. PEGylation involves the conjugation of poly(ethylene glycol) (PEG) to drugs or nanoparticles, increasing their circulation time and reducing unwanted host responses. By incorporating these strategies, precision NPs can be developed to effectively deliver cytochrome c to cancer cells, enabling targeted therapeutic interventions.