Non-apoptotic Functions Research Articles

Death ligand receptor (DLR) signaling: Its non-apoptotic functions in cancer and the consequences of DLR-directed therapies.

Death ligands (DLs), particularly tumor necrosis factor alpha (TNF-α), FAS ligand (FASL), and TNF-related apoptosis-inducing ligand (TRAIL), collectively termed TFT, are pivotal members of the TNF superfamily. While traditionally linked to apoptosis, TFT proteins have emerged as key regulators of various non-apoptotic processes. This review summarizes the non-apoptotic functions of TFT in cancer and explores the intricate crosstalk signaling pathways and their impact on nuclear factor kappa B (NF-κB) signaling, inflammation, and pro-tumorigenic function. It also highlights the potential connections and hurdles that exist in translating synthetic lethality strategies involving DLs into clinical applications. Lastly, it discusses the challenges and opportunities associated with TFT-targeted therapeutic strategies for both malignant and non-malignant diseases.

MCL-1 regulates cellular transitions during oligodendrocyte development.

Oligodendrocytes are the myelinating cells of the central nervous system. Regulation of the early stages of oligodendrocyte development is critical to the function of the cell. Specifically, myelin sheath formation is an energetically demanding event that requires precision, as alterations may lead to dysmyelination. Recent work has established that fatty acid β-oxidation is required for the function of oligodendrocytes. We have shown that MCL-1, a well-characterized anti-apoptotic protein, is required for the development of oligodendrocytes in vivo. Further, it was recently uncovered that MCL-1 regulates long-chain fatty acid β-oxidation through its interaction with acyl-CoA synthetase long-chain family member 1 (ACSL1), an enzyme responsible for the conversion of long-chain fatty acids into acyl-CoA. Here, we introduce an in vitro system to isolate human stem cell-derived oligodendrocyte progenitor cells and investigate the involvement of MCL-1 during human oligodendrocyte development. Using this system, we pharmacologically inhibited MCL-1 in oligodendrocyte progenitor cells (OPCs) to elucidate the non-apoptotic function of the protein at this developmental stage. Additionally, we used a motor neuron co-culture system to investigate the downstream effects that MCL-1 inhibition has at later developmental stages when oligodendrocytes begin to contact axons and generate myelin basic protein. We demonstrate that the mitochondrial network changes in human oligodendrocyte development resemble those reported in vivo. Our findings point to MCL-1 as a critical factor essential at the OPC stage for proper oligodendrocyte morphogenesis.

Integrated application of target-based and ligand-based drug-designing approaches for the identification of novel caspase-6 inhibitors

Caspase-6 (CASP6) is an effector caspase that has been marked to possess various pathological attributes associated with neurodegeneration. It is widely expressed in the neurodegenerative brain and peripheral tissues. It plays a vital role in apoptotic cell death and also performs non-apoptotic functions like axon pruning which contribute to the degeneration of neurons. Increment in active CASP6 levels in the cerebrospinal fluid has been observed during inflammation and has been linked to the early onset of Alzheimer’s disease (AD). In the current study, a novel CASP6 inhibitor was identified with the help of integrated target-based and ligand-based drug-designing approaches. Various molecular features of US9 (PDB ID 8EG6) were used to generate models. The pharmacophore models were evaluated using the EF value, GH score, and percentage yield to select the best-suited model. The best model was used to screen the ZINC-15 database to obtain virtual hits. The undesirable compounds were eliminated using various nodes in KNIME workflow. The resulting compounds were further subjected to docking-based virtual screening (DBVS) to find the lead compounds. Further, the molecular docking studies were carried out in three stages, followed by pharmacokinetic property prediction and toxicity studies. The top two virtual hits, i.e. ZINC000012563650 and ZINC000069415222, were considered for molecular dynamics simulation studies. Compound ZINC000069415222 was found to possess better stability, drug-like properties, and lower toxicity under simulated conditions. Thus, ZINC000069415222 was identified as a potential CASP6 inhibitor that could be further explored experimentally as an anti-AD drug.

Xeroderma pigmentosum protein XPD controls caspase-mediated stress responses

Caspases regulate and execute a spectrum of functions including cell deaths, non-apoptotic developmental functions, and stress responses. Despite these disparate roles, the same core cell-death machinery is required to enzymatically activate caspase proteolytic activities. Thus, it remains enigmatic how distinct caspase functions are differentially regulated. In this study, we show that Xeroderma pigmentosum protein XPD has a conserved function in activating the expression of stress-responsive caspases in C. elegans and human cells without triggering cell death. Using C. elegans, we show XPD-1-dependent activation of CED-3 caspase promotes survival upon genotoxic UV irradiation and inversely suppresses responses to non-genotoxic insults such as ER and osmotic stressors. Unlike the TFDP ortholog DPL-1 which is required for developmental apoptosis in C. elegans, XPD-1 only activates stress-responsive functions of caspase. This tradeoff balancing responses to genotoxic and non-genotoxic stress may explain the seemingly contradictory nature of caspase-mediated stress resilience versus sensitivity under different stressors.

Inhibition of caspase-8 cascade restrains the osteoclastogenic fate of bone marrow cells.

Osteoclasts are multinucleated cells of hematopoietic origin, with a pivotal role in bone development and remodeling. Failure in osteoclast differentiation and activation leads to various bone disorders; thus, attention has focused on a search of molecules involved in osteoclast regulatory pathways. Caspase-8 appears to be an interesting candidate for further exploration, due to its potential function in bone development and homeostasis. Mouse bone marrow cells were differentiated into osteoclasts by RANKL stimulation. Increased activation of caspase-8 and its downstream executioner caspases (caspase-3 and caspase-6) was found during osteoclastogenesis. Subsequent inhibition of caspase-8, caspase-3, or caspase-6, respectively, during osteoclast differentiation showed distinct changes in the formation of TRAP-positive multinucleated cells and reduced expression of osteoclast markers including Acp5, Ctsk, Dcstamp, and Mmp9. Analysis of bone matrix resorption confirmed significantly reduced osteoclast function after caspase inhibition. The results clearly showed the role of caspases in the proper development of osteoclasts and contributed new knowledge about non-apoptotic function of caspases.

FasL impacts Tgfb signaling in osteoblastic cells

Fas ligand (FasL, CD178) belongs to classical apoptotic molecules, however, recent evidence expands the spectrum of FasL functions into non-apoptotic processes which also applies for the bone. Tgfb subfamily members (Tgfb1, Tgfb2, Tgfb3) represent major components in osteogenic pathways and extracellular matrix. Their possible association with FasL has not yet been investigated but can be postulated. To test such a hypothesis, FasL deficient (gld) calvaria-derived cells were examined with a focus on the expression of Tgfb receptor ligands. The qPCR analysis revealed significantly increased expression of Tgfb1, Tgfb2 and Tgfb3 in gld cells. To check the vice versa effect, the gld cells were stimulated by soluble FasL. As a consequence, a dramatic decrease in expression levels of all three ligands was observed. This phenomenon was also confirmed in IDG-SW3 (osteoblastic cells of endochondral origin).TFLink gateway identified Fosl2 as an exclusive candidate of FasL capable to impact expression of all three Tgfb ligands. However, Fosl2 siRNA did not cause any significant changes in expression of Tgfb ligands. Therefore, the upregulation of the three ligands is likely to occur separately. In this respect, we tested the only exclusive candidate transcription factor for Tgfb3, Prrx1. Additionally, an overlapping candidate for Tgfb1 and Tgfb2, Mef2c capable to modulate expression of sclerostin, was examined. Prrx1 as well as Mef2c were found upregulated in gld samples and their expression decreased after addition of FasL. The same effect of FasL treatment was observed in the IDG-SW3 model.Taken together, FasL deficiency causes an increase in the expression of Tgfb ligands and stimulation by FasL reduces Tgfb expression in osteoblastic cells. The candidates mediating the effect comprise Prrx1 for Tgfb3 and Mef2c for Tgfb1/2. These results indicate FasL as a novel cytokine interfering with Tgfb signaling and thus the complex osteogenic network. The emerging non-apoptotic functions of FasL in bone development and maintenance should also be considered in treatment strategies such as the anti-osteoporotic factor.

Targeting Myeloid Leukemia-1 in Cancer Therapy: Advances and Directions.

As a tripartite cell death switch, B-cell lymphoma protein 2 (Bcl-2) family members precisely regulate the endogenous apoptosis pathway in response to various cell signal stresses through protein-protein interactions. Myeloid leukemia-1 (Mcl-1), a key anti-apoptotic Bcl-2 family member, is positioned downstream in the endogenous apoptotic pathway and plays a central role in regulating mitochondrial function. Mcl-1 is highly expressed in a variety of hematological malignancies and solid tumors, contributing to tumorigenesis, poor prognosis, and chemoresistance, making it an attractive target for cancer treatment. This Perspective aims to discuss the mechanism by which Mcl-1 regulates apoptosis and non-apoptotic functions in cancer cells and to outline the discovery and optimization process of potent Mcl-1 modulators. In addition, we summarize the structural characteristics of potent inhibitors that bind to Mcl-1 through multiple co-crystal structures and analyze the cardiotoxicity caused by current Mcl-1 inhibitors, providing prospects for rational targeting of Mcl-1.

Interaction of SMAC with a survivin-derived peptide alters essential cancer hallmarks: Tumor growth, inflammation, and immunosuppression

SMAC/Diablo is known as a pro-apoptotic mitochondrial protein released into the cytosol in response to apoptotic signals. We recently reported SMAC overexpression in cancers as essential for cell proliferation and tumor growth due to non-apoptotic functions including phospholipid synthesis regulation. These functions may be associated with its interactions with partner proteins. Using a peptide array with 768 peptides derived from 11 selected SMAC-interacting proteins, we identified SMAC-interacting sequences. These SMAC-binding sequences were produced as cell penetrating peptides targeted to the cytosol, mitochondria, or nucleus, inhibiting cell proliferation and inducing apoptosis in several cell lines. For in-vivo study, a survivin/BIRC5-derived peptide was selected due to its overexpression in many cancers, and involvement in mitosis, apoptosis, autophagy, cell proliferation, inflammation, and immune responses, as a target for cancer therapy. Specifically, a SMAC-targeting survivin/BIRC5-derived peptide, given intratumorally or intravenously, strongly inhibited lung tumor growth, cell proliferation, angiogenesis, and inflammation, induced apoptosis, and remodeled the tumor microenvironment. The peptide promoted tumor infiltration of CD8+ cells and increased cell-intrinsic PD-1/PD-L1 expression, resulting in cancer cell self-destruction and increased tumor cell death, preserving immune cells. Thus, targeting the interaction between the multifunctional proteins SMAC and survivin represents a innovative therapeutic cancer paradigm.

The effect of cytochrome c on Na,K-ATPase.

Na,K-ATPase is a crucial enzyme responsible for maintaining Na+, K+-gradients across the cell membrane, which is essential for numerous physiological processes within various organs and tissues. Due to its significance in cellular physiology, inhibiting Na,K-ATPase can have profound physiological consequences. This characteristic makes it a target for various pharmacological applications, and drugs that modulate the pump's activity are thus used in the treatment of several medical conditions. Cytochrome c (Cytc) is a protein with dual functions in the cell. In the mitochondria, it is essential for ATP synthesis and energy production. However, in response to apoptotic stimuli, it is released into the cytosol, where it triggers programmed cell death through the intrinsic apoptosis pathway. Aside from its role in canonical intrinsic apoptosis, Cytc also plays additional roles. For instance, Cytc participates in certain non-apoptotic functions -those which are less well-understood in comparison to its role in apoptosis. Within this in vitro study, we have shown the impact of Cytc on Na,K-ATPase for the first time. Cytc has a biphasic action on Na,K-ATPase, with activation at low concentrations (0.06ng/ml; 6ng/ml) and inhibition at high concentration (120ng/ml). Cytc moreover displays isoform/subunit specificity and regulates the Na+ form of the enzyme, while having no effect on the activity or kinetic parameters of the K+-dependent form of the enzyme. Changing the affinity of p-chloromercuribenzoic acid (PCMB) by Cytc is therefore both a required and sufficient condition for confirming that PCMB and Cytc share the same target, namely the thiol groups of cysteine in Na,K-ATPase.

Caspase-9 Is a Positive Regulator of Osteoblastic Cell Migration Identified by diaPASEF Proteomics.

Caspase-9 is traditionally considered the initiator caspase of the intrinsic apoptotic pathway. In the past decade, however, other functions beyond initiation/execution of cell death have been described including cell type-dependent regulation of proliferation, differentiation/maturation, mitochondrial, and endosomal/lysosomal homeostasis. As previous studies revealed nonapoptotic functions of caspases in osteogenesis and bone homeostasis, this study was performed to identify proteins and pathways deregulated by knockout of caspase-9 in mouse MC3T3-E1 osteoblasts. Data-independent acquisition-parallel accumulation serial fragmentation (diaPASEF) proteomics was used to compare protein profiles of control and caspase-9 knockout cells. A total of 7669 protein groups were quantified, and 283 upregulated/141 downregulated protein groups were associated with the caspase-9 knockout phenotype. The deregulated proteins were mainly enriched for those associated with cell migration and motility and DNA replication/repair. Altered migration was confirmed in MC3T3-E1 cells with the genetic and pharmacological inhibition of caspase-9. ABHD2, an established regulator of cell migration, was identified as a possible substrate of caspase-9. We conclude that caspase-9 acts as a modulator of osteoblastic MC3T3-E1 cell migration and, therefore, may be involved in bone remodeling and fracture repair.

Synthesis of N-Glycosylated Soluble Fas Ligand.

Controlled cell death is essential for the regulation of the immune system and plays a role in pathogen defense. It is often altered in pathogenic conditions such as cancer, viral infections and autoimmune diseases. The Fas receptor and its corresponding membrane-bound ligand (FasL) are part of the extrinsic apoptosis pathway activated in these cases. A soluble form of FasL (sFasL), produced by ectodomain shedding, displays a diverse but still elusive set of non-apoptotic functions and sometimes even serves as a pro-survival factor. To gather more knowledge about the characteristics of this protein and the impact N-glycosylations may have, access to homogeneous posttranslationally modified variants of sFasL is needed. Therefore, we developed a flexible strategy to obtain such homogeneously N-glycosylated variants of sFasL by applying chemical protein synthesis. This strategy can be flexibly combined with enzymatic methods to introduce more complex, site selective glycosylations.

Is death a living being?

The death process begins with the loss of function of one or more of the three classic vital organs: the heart, the brain, and/or the lungs. Failure to resuscitate the function of the affected primary organ leads to the cessation of the function of other organs. The cell carries death factors or elements such as caspases and some mitochondrial secretions - At the same time the mitochondria represent the basic source of energy and life in the cell. Death occurs only when activating the molecules and factors of cell annihilation. Apoptosis is a type of cell death mechanism, controlled by interactions between several molecules - and is responsible for removing unwanted cells from the body. Apoptosis can be induced by signals from inside the cell or by death signals coming from outside the cell via cosmic energy waves. Caspases are cysteine aspartate-specific proteases that are essential for the initiation and execution of apoptosis. As one of the initiator and executor caspases, caspase-2 is the most evolutionally conserved caspase, exerting both apoptotic and non-apoptotic functions. Caspases also have a role in inflammation, whereby they directly process proinflammatory cytokines such as pro-IL1β. These are signaling molecules that allow recruitment of immune cells to an infected cell or tissue. In addition to apoptosis, caspases play a role in necroptosis, pyroptosis, and autophagy, which are non-apoptotic cell death processes. Mitochondria coordinate cell stress responses, such as autophagy, and control nonapoptotic cell death routines, such as regulated necrosis. Mitochondrial permeabilization leads to release of these proteins, which then interfere with the IAP-inhibition of caspases, resulting in potentiation of cell death. Initiators of mitochondrial-driven inflammation include the release of mitochondrial dsRNA and mt DNA thereby initiating type I interferons. It is concluded that mitochondria not only power the metabolic cell activities with energy but also power the cell death with both energy and signaling molecules denoting that cell death is an active living being.

How does caspases regulation play role in cell decisions? apoptosis and beyond.

Caspases are a family of cysteine proteases, and the key factors behind the cellular events which occur during apoptosis and inflammation. However, increasing evidence shows the non-conventional pro-survival action of apoptotic caspases in crucial processes. These cellular events include cell proliferation, differentiation, and migration, which may appear in the form of metastasis, and chemotherapy resistance in cancerous situations. Therefore, there should be a precise and strict control of caspases activity, perhaps through maintaining the threshold below the required levels for apoptosis. Thus, understanding the regulators of caspase activities that render apoptotic caspases as non-apoptotic is of paramount importance both mechanistically and clinically. Furthermore, the functions of apoptotic caspases are affected by numerous post-translational modifications. In the present mini-review, we highlight the various mechanisms that directly impact caspases with respect to their anti- or non-apoptotic functions. In this regard, post-translational modifications (PTMs), isoforms, subcellular localization, transient activity, substrate availability, substrate selection, and interaction-mediated regulations are discussed.

Caspase-8 and Tyrosine Kinases: A Dangerous Liaison in Cancer

Caspase-8 is a cysteine-aspartic acid protease that has been identified as an initiator caspase that plays an essential role in the extrinsic apoptotic pathway. Evasion of apoptosis is a hallmark of cancer and Caspase-8 expression is silenced in some tumors, consistent with its central role in apoptosis. However, in the past years, several studies reported an increased expression of Caspase-8 levels in many tumors and consistently identified novel “non-canonical” non-apoptotic functions of Caspase-8 that overall promote cancer progression and sustain therapy resistance. These reports point to the ability of cancer cells to rewire Caspase-8 function in cancer and raise the question of which are the signaling pathways aberrantly activated in cancer that may contribute to the hijack of Caspase-8 activity. In this regard, tyrosine kinases are among the first oncogenes ever identified and genomic, transcriptomic and proteomic studies indeed show that they represent a class of signaling molecules constitutively activated in most of the tumors. Here, we aim to review and discuss the role of Caspase-8 in cancer and its interplay with Src and other tyrosine kinases.

Activity-dependent regulation of the BAX/BCL-2 pathway protects cortical neurons from apoptotic death during early development

During early brain development, homeostatic removal of cortical neurons is crucial and requires multiple control mechanisms. We investigated in the cerebral cortex of mice whether the BAX/BCL-2 pathway, an important regulator of apoptosis, is part of this machinery and how electrical activity might serve as a set point of regulation. Activity is known to be a pro-survival factor; however, how this effect is translated into enhanced survival chances on a neuronal level is not fully understood. In this study, we show that caspase activity is highest at the neonatal stage, while developmental cell death peaks at the end of the first postnatal week. During the first postnatal week, upregulation of BAX is accompanied by downregulation of BCL-2 protein, resulting in a high BAX/BCL-2 ratio when neuronal death rates are high. In cultured neurons, pharmacological blockade of activity leads to an acute upregulation of Bax, while elevated activity results in a lasting increase of BCL-2 expression. Spontaneously active neurons not only exhibit lower Bax levels than inactive neurons but also show almost exclusively BCL-2 expression. Disinhibition of network activity prevents the death of neurons overexpressing activated CASP3. This neuroprotective effect is not the result of reduced caspase activity but is associated with a downregulation of the BAX/BCL-2 ratio. Notably, increasing neuronal activity has a similar, non-additive effect as the blockade of BAX. Conclusively, high electrical activity modulates BAX/BCL-2 expression and leads to higher tolerance to CASP3 activity, increases survival, and presumably promotes non-apoptotic CASP3 functions in developing neurons.

Something new under the sun: caspases promote cell survival under moderate heat stress.

Since the discovery of caspases in 1993 (Yuan etal, 1993), they have largely been regarded as essential Cys proteases in apoptosis. However, more recently, it has become evident that caspases also have non-apoptotic functions in the so-called caspase-dependent non-lethal processes (CDPs) including cell proliferation and cell differentiation (Aram etal, 2017). While most of the non-apoptotic functions were characterized under normal developmental conditions, a new study by Galasso etal took this examination a step further-to the analysis of moderate stress conditions. The authors now report that caspases have a non-apoptotic prosurvival function in Drosophila ovarian somatic cells exposed to moderate heat stress by controlling Hedgehog signaling and autophagy (Galasso etal, 2023).

Non-lethal roles of the initiator caspase Dronc in Drosophila

The role of caspases, or cysteine-aspartic proteases, in apoptosis has been well-studied across multiple organisms. These apoptotic caspases can be divided into initiator and effector caspases, with the former cleaving and activating the latter to trigger cell death. However, emerging evidence is supporting non-lethal roles of caspases in development, tissue homeostasis and disease. In comparison to effector caspases, less is known about the non-apoptotic functions of initiator caspases because of their more restricted activities and fewer known substrates. This review focuses on some recent findings in Drosophila on non-lethal roles of the initiator caspase Dronc. We discuss their biological importance, underlying regulatory mechanisms, and implications for our understanding of their mammalian counterparts. Deciphering the non-apoptotic functions of Dronc will provide valuable insights into the multifaceted functions of caspases during development and in diseases including cancer.

Apoptotic proteins with non-apoptotic activity: expression and function in cancer.

Apoptosis is a process of programmed cell death in which a cell commits suicide while maintaining the integrity and architecture of the tissue as a whole. Apoptosis involves activation of one of two major pathways: the extrinsic pathway, where extracellular pro-apoptotic signals, transduced through plasma membrane death receptors, activate a caspase cascade leading to apoptosis. The second, the intrinsic apoptotic pathway, where damaged DNA, oxidative stress, or chemicals, induce the release of pro-apoptotic proteins from the mitochondria, leading to the activation of caspase-dependent and independent apoptosis. However, it has recently become apparent that proteins involved in apoptosis also exhibit non-cell death-related physiological functions that are related to the cell cycle, differentiation, metabolism, inflammation or immunity. Such non-conventional activities were predominantly reported in non-cancer cells although, recently, such a dual function for pro-apoptotic proteins has also been reported in cancers where they are overexpressed. Interestingly, some apoptotic proteins translocate to the nucleus in order to perform a non-apoptotic function. In this review, we summarize the unconventional roles of the apoptotic proteins from a functional perspective, while focusing on two mitochondrial proteins: VDAC1 and SMAC/Diablo. Despite having pro-apoptotic functions, these proteins are overexpressed in cancers and this apparent paradox and the associated pathophysiological implications will be discussed. We will also present possible mechanisms underlying the switch from apoptotic to non-apoptotic activities although a deeper investigation into the process awaits further study.

MiR-155-5p-mediated increase in p53 content induced by dacarbazine in melanoma cells

BACKGROUND: Cellular senescence is a stress response, triggered by various stimuli such as chemotherapy treatment and causes G0/G1 cell cycle arrest followed by the production of a senescence associated secretory phenotype. p53 considered to be a modulator of these events although the precise mechanisms of it remains not clear.
 AIMS: To determine the non-apoptotic functions of the p53 protein the formation of the senescence associated secretory phenotype phenotype of melanoma cells under the treatment of the cytostatic agent dacarbazine.
 MATERIALS AND METHODS: The study was conducted on BRO and SK-MEL-2 skin melanoma cell lines. Melanoma cells were were treated by cytostatic agent dacarbazine. Then immunocytochemical study was performed to determine the proportion of G0-positive cells and the expression of the tumor suppressor protein p53. A bioinformatic analysis was accomplished to identify for p53 regulators with determining of miR-155-5p levels in exosomes released by dacarbazine-treated melanoma cells.
 RESULTS: The cytostatic drug dacarbazine increases the proportion of cells residing in the G0 phase of the cell cycle. Onco-microRNA miR-155-5p was expressed in the exosomes of the two studied cell lines BRO and SK-MEL-2 of skin melanoma. Changes in the expression level of p53 correlate with changes in miR-155-5p microRNA expression. The absence of changes in p53 expression in BRO melanoma cells may be due to the absence of changes in miR-155-5p expression levels. In the BRO cell line, no changes in the expression of the oncosuppressor p53 were observed with an increased percentage of G0-positive cells, which may be associated with the activation of other mechanisms of cell cycle arrest in the G0/G1 phase.
 CONCLUSIONS: Heterogeneous effect of the cytostatic agent dacarbazine on melanoma cells was revealed. For the SK-MEL-2 cell line, dacarbazine induces the release of senescence associated secretory phenotype by inhibiting exosomal production of miR-155-5p, which activates the p53 oncosuppressor, which was not observed in the BRO line.

Yca1 metacaspase: diverse functions determine how yeast live and let die.

The Yca1 metacaspase was discovered due to its role in the regulation of apoptosis in Saccharomyces cerevisiae. However, the mechanisms that drive apoptosis in yeast remain poorly understood. Additionally, Yca1 and other metacaspase proteins have recently been recognized for their involvement in other cellular processes, including cellular proteostasis and cell cycle regulation. In this minireview, we outline recent findings on Yca1 that will enable the further study of metacaspase multifunctionality and novel apoptosis pathways in yeast and other nonmetazoans. In addition, we discuss advancements in high-throughput screening technologies that can be applied to answer complex questions surrounding the apoptotic and nonapoptotic functions of metacaspase proteins across a diverse range of species.