exploring-caspase-mutations-post-translational-modification-molecular

Exploring Caspase Mutations and Post-Translational Modification by Molecular Modeling Approaches

Apoptosis is a type of programmed cell death that eliminates damaged cells and controls the development and tissue homeostasis of multicellular organisms. ...

Lomonosov Moscow State University

Programmed cell death plays a central role in the regulation of homeostasis and development of multicellular organisms. Deregulation of programmed cell death is connected to a number of disorders, including cancer and autoimmune diseases. Initiation of cell death occurs in the multiprotein complexes or high molecular weight platforms. Composition, structure, and molecular interactions within these platforms influence the cellular decision toward life or death and, therefore, define the induction of a particular cell death program. Here, we discuss in detail the key cell-death complexes-including DISC, complex II, and TNFRI complex I/II, and the necrosome, RIPoptosome, apoptosome, and PIDDosome-that control apoptosis or necroptosis pathways as well as their regulation. The possibility of their pharmacological targeting leading to the development of new strategies of interference with cell death programs via control of the high molecular weight platforms will be discussed. 

Cell death controlling complexes and their potential therapeutic role.

Since their establishment in the early 1970s, the nuclear changes upon apoptosis induction, such as the condensation of chromatin, disassembly of nuclear scaffold proteins and degradation of DNA, were, and still are, considered as the essential steps and hallmarks of apoptosis. These are the characteristics of the execution phase of apoptotic cell death. In addition, accumulating data clearly show that some nuclear events can lead to the induction of apoptosis. In particular, if DNA lesions resulting from deregulation during the cell cycle or DNA damage induced by chemotherapeutic drugs or viral infection cannot be efficiently eliminated, apoptotic mechanisms, which enable cellular transformation to be avoided, are activated in the nucleus. The functional heterogeneity of the nuclear organization allows the tight regulation of these signaling events that involve the movement of various nuclear proteins to other intracellular compartments (and vice versa) to initiate and govern apoptosis. Here, we discuss how these events are coordinated to execute apoptotic cell death. 

Role of the nucleus in apoptosis: signaling and execution.

Apoptosis is a crucial program of cell death that controls development and homeostasis of multicellular organisms. The main initiators and executors of this process are the Cysteine-dependent ASPartate proteASES - caspases. A number of regulatory circuits tightly control caspase processing and activity. One of the most important, yet, at the same time still poorly understood control mechanisms of activation of caspases involves their post-translational modifications. The addition and/or removal of chemical groups drastically alters the catalytic activity of caspases or stimulates their nonapoptotic functions. In this review, we will describe and discuss the roles of key caspase modifications such as phosphorylation, ubiquitination, nitrosylation, glutathionylation, SUMOylation, and acetylation in the regulation of apoptotic cell death and cell survival.

Post-translational Modification of Caspases: The Other Side of Apoptosis Regulation.

The development of approaches that increase therapeutic effects of anti-cancer drugs is one of the most important tasks of oncology. Caloric restriction in vivo or serum deprivation (SD) in vitro has been shown to be an effective tool for sensitizing cancer cells to chemotherapeutic drugs. However, the detailed mechanisms underlying the enhancement of apoptosis in cancer cells by SD remain to be elucidated.

Modulation of Mcl-1 transcription by serum deprivation sensitizes cancer cells to cisplatin.

The role of caspase-2 in cell death regulation remains largely unknown. In this study we have analyzed the involvement of caspase-2 in RIPK1-regulated necrosis (necroptosis) in human ovarian carcinoma cells. We show that these cells undergo necroptosis upon treatment with the DNA damaging drug cisplatin in combination with the pan-caspase inhibitor zVAD-fmk. Downregulation of caspase-2 leads to an increase of necroptosis in CAOV-4 cells. Interestingly, an association of caspase-2 to the necrosome complex was not detected. Importantly, downregulation of caspase-2 with shRNA or CRISPR/Cas9 system led to an enhanced phosphorylation of RIPK1 and MLKL. Taken together, our data strongly indicate that caspase-2 negatively regulates necroptotic cell death, which might play an important role in further therapeutic applications.

Caspase-2 is a negative regulator of necroptosis.

To counteract oxidative stress, antioxidants including carotenoids are highly promising, yet their exploitation is drastically limited by the poor bioavailability and fast photodestruction, whereas current delivery systems are far from being efficient. Here we demonstrate that the recently discovered nanometer-sized water-soluble carotenoprotein from  sp. PCC 7120 (termed AnaCTDH) transiently interacts with liposomes to efficiently extract carotenoids via carotenoid-mediated homodimerization, yielding violet-purple protein samples. We characterize the spectroscopic properties of the obtained pigment-protein complexes and the thermodynamics of liposome-protein carotenoid transfer and demonstrate the delivery of carotenoid echinenone from AnaCTDH into liposomes with an efficiency of up to 70 ± 3%. Most importantly, we show efficient carotenoid delivery to membranes of mammalian cells, which provides protection from reactive oxygen species (ROS). Incubation of neuroblastoma cell line Tet21N in the presence of 1 μM AnaCTDH binding echinenone decreased antimycin A ROS production by 25% ( < 0.05). The described carotenoprotein may be considered as part of modular systems for the targeted antioxidant delivery.

Soluble Cyanobacterial Carotenoprotein as a Robust Antioxidant Nanocarrier and Delivery Module.

Caspase-2 is a unique and conservative cysteine protease which plays an important role in several cellular processes including apoptotic cell death. Although the molecular mechanisms of its activation remain largely unclear, a major role belongs to the architecture of the caspase-2 active center. We demonstrate that the substitution of the putative phosphorylation site of caspase-2, Serine-384 to Alanine, blocks caspase-2 processing and decreases its enzymatic activity. Strikingly, in silico analysis using molecular dynamics simulations has shown that Serine-384 is crucially involved in interactions within the caspase-2 active center. It stabilizes Arginine-378, which forms a crucial hydrogen bond with the aspartate residue of a substrate. Hence, Serine-384 is essential for supporting a proper architecture of the active center of caspase-2. Moreover, molecular modeling strongly proved steric inaccessibility of Ser-384 to be phosphorylated. Importantly, a multiple alignment has demonstrated that both Serine-384 and Arg-378 residues are highly conservative across all members of caspase family, which allows us to suggest that this diade is indispensable for caspase processing and activity. Spontaneous mutations in this diade might influence oncosuppressive function of caspases, in particular of caspase-2. Likewise, the mutation of Ser-384 is associated with the development of lung squamous cell carcinoma and adenocarcinoma. Taken together, we have uncovered a central feature of the caspase-2 activation mechanism which is crucial for the regulation of its signaling network.

Requirement for Serine-384 in Caspase-2 processing and activity.

An emerging role of long non-coding RNAs (lncRNAs) in tumor progression has been revealed in the last decade. Through interactions with nucleic acids and proteins, lncRNAs could act as enhancers, scaffolds or decoys for a number of oncoproteins and tumor suppressors. The aberrant lncRNA expression or mutations are often associated with changes in a variety of cellular processes, including proliferation, stress response and cell death. Here, we will focus on the tumor-associated lncRNAs in ovarian cancer according to their contribution to cancer hallmarks, such as intense proliferation, cell death resistance, altered energy metabolism, invasion and metastasis, and immune evasion. Moreover, the potential clinical implications of lncRNAs and their significance for the diagnosis, prognosis and therapy of ovarian cancer will be discussed.

Long non-coding RNAs: A view to kill ovarian cancer.

BH3 mimetics represent a promising tool in cancer treatment. Recently, the drugs targeting the Mcl-1 protein progressed into clinical trials, and numerous studies are focused on the investigation of their activity in various preclinical models. We investigated two BH3 mimetics to Mcl-1, A1210477 and S63845, and found their different efficacies in on-target doses, despite the fact that both agents interacted with the target. Thus, S63845 induced apoptosis more effectively through a Bak-dependent mechanism. There was an increase in the level of Bcl-xL protein in cells with acquired resistance to Mcl-1 inhibition. Cell lines sensitive to S63845 demonstrated low expression of Bcl-xL. Tumor tissues from patients with lung adenocarcinoma were characterized by decreased Bcl-xL and increased Bak levels of both mRNA and proteins. Concomitant inhibition of Bcl-xL and Mcl-1 demonstrated dramatic cytotoxicity in six of seven studied cell lines. We proposed that co-targeting Bcl-xL and Mcl-1 might lead to a release of Bak, which cannot be neutralized by other anti-apoptotic proteins. Surprisingly, in Bak-knockout cells, inhibition of Mcl-1 and Bcl-xL still resulted in pronounced cell death, arguing against a sole role of Bak in the studied phenomenon. We demonstrate that Bak and Bcl-xL are co-factors for, respectively, sensitivity and resistance to Mcl-1 inhibition.

Bak and Bcl-xL Participate in Regulating Sensitivity of Solid Tumor Derived Cell Lines to Mcl-1 Inhibitors.

Mitotic catastrophe is a defensive mechanism that promotes elimination of cells with aberrant mitosis by triggering the cell-death pathways and/or cellular senescence. Nowadays, it is known that apoptosis, autophagic cell death, and necrosis could be consequences of mitotic catastrophe. Here, we demonstrate the ability of a DNA-damaging agent, doxorubicin, at 600 nM concentration to stimulate mitotic catastrophe. We observe that the inhibition of caspase activity leads to accumulation of cells with mitotic catastrophe hallmarks in which RIP1-dependent necroptotic cell death is triggered. The suppression of autophagy by a chemical inhibitor or  knockout upregulates RIP1 phosphorylation and promotes necroptotic cell death. Thus, in certain conditions mitotic catastrophe, in addition to apoptosis and autophagy, can precede necroptosis.

Article	Year
Cell death controlling complexes and their potential therapeutic role. Cellular and molecular life sciences : CMLS\| PubMed ID: 25323133	2015
Role of the nucleus in apoptosis: signaling and execution. Cellular and molecular life sciences : CMLS\| PubMed ID: 26346492	2015
Post-translational Modification of Caspases: The Other Side of Apoptosis Regulation. Trends in cell biology\| PubMed ID: 28188028	2017
Modulation of Mcl-1 transcription by serum deprivation sensitizes cancer cells to cisplatin. Biochimica et biophysica acta. General subjects\| PubMed ID: 29203282	2018
Caspase-2 is a negative regulator of necroptosis. The international journal of biochemistry & cell biology\| PubMed ID: 30025878	2018
Soluble Cyanobacterial Carotenoprotein as a Robust Antioxidant Nanocarrier and Delivery Module. Antioxidants (Basel, Switzerland)\| PubMed ID: 32942578	2020
Requirement for Serine-384 in Caspase-2 processing and activity. Cell death & disease\| PubMed ID: 33011746	2020
Long non-coding RNAs: A view to kill ovarian cancer. Biochimica et biophysica acta. Reviews on cancer\| PubMed ID: 34157315	2021
Bak and Bcl-xL Participate in Regulating Sensitivity of Solid Tumor Derived Cell Lines to Mcl-1 Inhibitors. Cancers\| PubMed ID: 35008345	2021
Necroptosis as a Novel Facet of Mitotic Catastrophe. International journal of molecular sciences\| PubMed ID: 35409093	2022

Alexey V. Zamaraev

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