An optimized protocol for expression and purification of monomeric full-length BAX protein for functional interrogations.

Diverse developmental signals and pro-death stresses converge on the regulation of the mitochondrial pathway of apoptosis. BAX, a proapoptotic BCL-2 effector, directly forms proteolipid pores in the outer mitochondrial membrane to activate the mitochondrial pathway of apoptosis. BAX is a viable pharmacological target for various human diseases, and increasing efforts have been made to study the molecular regulation of BAX while identifying small molecules selectively targeting BAX. However, generating large quantities of monomeric and functionally competent BAX has been challenging due to its aggregation-prone nature. Additionally, there is a lack of detailed and instructional protocols available for investigators who are not already familiar with recombinant BAX production. Here, we present a comprehensive protocol for expressing, purifying, and storing functional monomeric recombinant BAX protein. We use an intein-chitin binding domain-tagged BAX-expressing construct and employ a two-step chromatography strategy to capture and purify BAX. We also provide examples of standard assays to observe BAX activation, and highlight the best practices for handling and storing BAX to effectively preserve its quality, shelf life, and function.

An optimized high-yield protocol for expression and purification of monomeric full-length BAX protein.

Diverse developmental signals and pro-death stresses converge on regulation of the mitochondrial pathway of apoptosis. BAX, a pro-apoptotic BCL-2 effector, directly forms proteolipid pores in the outer mitochondrial member to activate the mitochondrial pathway of apoptosis. BAX is a viable pharmacological target for various human diseases, and increasing efforts have been made to study the molecular regulation of BAX and identify small molecules selectively targeting BAX. However, generating large quantities of monomeric and functionally-competent BAX has been challenging due to its aggregation-prone nature. Additionally, there is a lack of detailed and instructional protocols available for investigators who are not already familiar with recombinant BAX production. Here, we present a comprehensive high-yield protocol for expressing, purifying, and storing functional recombinant BAX protein. We utilize an intein-tagged BAX construct and employ a two-step chromatography strategy to capture and purify BAX, and provide example standard assays to observe BAX activation. We also highlight best practices for handling and storing BAX to effectively preserve its quality, shelf-life, and function.

The multifaceted roles of mitochondria.

Much more than the "powerhouse" of the cell, mitochondria have emerged as critical hubs involved in metabolism, cell death, inflammation, signaling, and stress responses. To open our mitochondria focus issue, we asked several scientists to share the unanswered questions, emerging themes, and topics of investigation that excite them.

IFN-γ+ cytotoxic CD4+ T lymphocytes are involved in the pathogenesis of colitis induced by IL-23 and the food colorant Red 40.

The food colorant Red 40 is an environmental risk factor for colitis development in mice with increased expression of interleukin (IL)-23. This immune response is mediated by CD4+ T cells, but mechanistic insights into how these CD4+ T cells trigger and perpetuate colitis have remained elusive. Here, using single-cell transcriptomic analysis, we found that several CD4+ T-cell subsets are present in the intestines of colitic mice, including an interferon (IFN)-γ-producing subset. In vivo challenge of primed mice with Red 40 promoted rapid activation of CD4+ T cells and caused marked intestinal epithelial cell (IEC) apoptosis that was attenuated by depletion of CD4+ cells and blockade of IFN-γ. Ex vivo experiments showed that intestinal CD4+ T cells from colitic mice directly promoted apoptosis of IECs and intestinal enteroids. CD4+ T cell-mediated cytotoxicity was contact-dependent and required FasL, which promoted caspase-dependent cell death in target IECs. Genetic ablation of IFN-γ constrained IL-23- and Red 40-induced colitis development, and blockade of IFN-γ inhibited epithelial cell death in vivo. These results advance the understanding of the mechanisms regulating colitis development caused by IL-23 and food colorants and identify IFN-γ+ cytotoxic CD4+ T cells as a new potential therapeutic target for colitis.

A kinetic fluorescence polarization ligand assay for monitoring BAX early activation.

Developmental, homeostatic, and pharmacological pro-apoptotic signals converge by activating the BCL-2 family member BAX. Studies investigating molecular regulation of BAX are commonly limited to methodologies measuring endpoint phenotypes and do not assess activation of monomeric BAX. Here, we present FLAMBE, a fluorescence polarization ligand assay for monitoring BAX early activation, that measures activation-induced release of a peptide probe in real time. Using complementary parallel and tandem biochemical techniques, we validate, corroborate, and apply FLAMBE to a contemporary repertoire of BAX modulators, characterizing their contributions within the early steps of BAX activation. Additionally, we use FLAMBE to reveal that historically "dead" BAX mutants remain responsive to activation as quasi-functional monomers. We also identify data metrics for comparative analyses and demonstrate that FLAMBE data align with downstream functional observations. Collectively, FLAMBE advances our understanding of BAX activation and fills a methodological void for studying BAX with broad applications in cell biology and therapeutic development. MOTIVATION In vitro BAX activation studies are invaluable platforms for studying cellular and pharmacological modulators of apoptosis. The gold standard for studying BAX function relies on membrane permeabilization assays, which assess the pore-forming activity of oligomeric BAX. However, there are currently no rapid or kinetic assays to interrogate real-time activation of monomeric BAX in solution, thereby limiting any molecular insights that occur upstream of mitochondrial permeabilization. Furthermore, available methods to observe the activation of monomeric BAX suffer from low throughput and static observations. To address this methodological gap, we developed FLAMBE, a kinetic fluorescence polarization-based assay to measure monomeric BAX activation in solution via concomitant displacement of a labeled peptide. This approach maintains the benefits of rapid kinetic data generation in a low-cost microplate format without requiring specialized equipment or large quantities of protein. FLAMBE compliments available experimental strategies and expands the accessibility of investigators to monitor early steps within the BAX activation continuum.

FLAMBE: A kinetic fluorescence polarization assay to study activation of monomeric BAX.

BAX activation techniques are crucial to studying the intrinsic pathway of apoptosis- thousands of pro-apoptotic signals converge on BAX activation. Current methodologies are predominantly limited to membrane permeabilization studies, which assess endpoint functionality of oligomeric BAX, but overlook early activation steps of cytosolic BAX. Here we detail FLAMBE: a fluorescence polarization ligand assay for monitoring BAX early-activation in solution. We also describe a dual-metric parameterization strategy for distillation of kinetic data and comparative analyses when studying candidate ligands. For complete details on the use and execution of this protocol, please refer to Gelles et al. (2022).

T cell-derived tumor necrosis factor induces cytotoxicity by activating RIPK1-dependent target cell death.

TNF ligation of TNF receptor 1 (TNFR1) promotes either inflammation and cell survival by (a) inhibiting RIPK1's death-signaling function and activating NF-κB or (b) causing RIPK1 to associate with the death-inducing signaling complex to initiate apoptosis or necroptosis. The cellular source of TNF that results in RIPK1-dependent cell death remains unclear. To address this, we employed in vitro systems and murine models of T cell-dependent transplant or tumor rejection in which target cell susceptibility to RIPK1-dependent cell death could be genetically altered. We show that TNF released by T cells is necessary and sufficient to activate RIPK1-dependent cell death in target cells and thereby mediate target cell cytolysis independently of T cell frequency. Activation of the RIPK1-dependent cell death program in target cells by T cell-derived TNF accelerates murine cardiac allograft rejection and synergizes with anti-PD1 administration to destroy checkpoint blockade-resistant murine melanoma. Together, the findings uncover a distinct immunological role for TNF released by cytotoxic effector T cells following cognate interactions with their antigenic targets. Manipulating T cell TNF and/or target cell susceptibility to RIPK1-dependent cell death can be exploited to either mitigate or augment T cell-dependent destruction of allografts and malignancies to improve outcomes.

Mechanistic connections between mitochondrial biology and regulated cell death.

The ancient, dynamic, and multifaceted functions of the mitochondrial network are essential for organismal homeostasis and contribute to numerous human diseases. As central hubs for metabolism, ion transport, and multiple macromolecular synthesis pathways, mitochondria establish and control extensive signaling networks to ensure cellular survival. In this review, we explore how these same mitochondrial functions also participate in the control of regulated cell death (RCD). We discuss the complementary essential mitochondrial functions as compartments that participate in the production and presentation of key molecules and platforms that actively enable, initiate, and execute RCD.

Why not add some SPARKL to your life (and death)!?

Quantifying cytostatic and cytotoxic outcomes of cells responding to perturbagens is an essential component of mechanism-based studies and pharmacological screening approaches. We recently described an easy and versatile method for single-cell and population-level analyses using real-time kinetic labeling (SPARKL). This technology enables zero-handling, non-disruptive protocols for integrating proliferation profiles with cell death mechanisms, along with advanced mathematics for robust analyses.

Complex I and MDM2: hit me baby one more time.

MDM2 (mouse double minute 2) functions as both a tumor suppressor and oncogene, yet little is known if MDM2 regulates cancer cell biology by altering cellular metabolism. We recently found that MDM2 binds NDUFS1 (NADH:ubiquinone oxidoreductase 75 kDa Fe-S protein 1), a key protein involved in Complex I assembly, function, and efficiency. The MDM2⋅NDUFS1 interaction promotes reactive oxygen species production, DNA damage, and apoptosis.

MDM2 Integrates Cellular Respiration and Apoptotic Signaling through NDUFS1 and the Mitochondrial Network.

Signaling diversity and subsequent complexity in higher eukaryotes is partially explained by one gene encoding a polypeptide with multiple biochemical functions in different cellular contexts. For example, mouse double minute 2 (MDM2) is functionally characterized as both an oncogene and a tumor suppressor, yet this dual classification confounds the cell biology and clinical literatures. Identified via complementary biochemical, organellar, and cellular approaches, we report that MDM2 negatively regulates NADH:ubiquinone oxidoreductase 75 kDa Fe-S protein 1 (NDUFS1), leading to decreased mitochondrial respiration, marked oxidative stress, and commitment to the mitochondrial pathway of apoptosis. MDM2 directly binds and sequesters NDUFS1, preventing its mitochondrial localization and ultimately causing complex I and supercomplex destabilization and inefficiency of oxidative phosphorylation. The MDM2 amino-terminal region is sufficient to bind NDUFS1, alter supercomplex assembly, and induce apoptosis. Finally, this pathway is independent of p53, and several mitochondrial phenotypes are observed in Drosophila and murine models expressing transgenic Mdm2.

Think We Understand the Role of DRP1 in Mitochondrial Biology? Zinc Again!

In this issue of Molecular Cell, Cho et al. (2019) identify a mechanism by which the mitochondrial division machinery provides selective pressure to identify dysfunctional organelles through the coordinated action of DRP1, Zip1, and Zn2+ transport into mitochondria.

MDM2 and mitochondrial function: One complex intersection.

Decades of research reveal that MDM2 participates in cellular processes ranging from macro-molecular metabolism to cancer signaling mechanisms. Two recent studies uncovered a new role for MDM2 in mitochondrial bioenergetics. Through the negative regulation of NDUFS1 (NADH:ubiquinone oxidoreductase 75 kDa Fe-S protein 1) and MT-ND6 (NADH dehydrogenase 6), MDM2 decreases the function and efficiency of Complex I (CI). These observations propose several important questions: (1) Where does MDM2 affect CI activity? (2) What are the cellular consequences of MDM2-mediated regulation of CI? (3) What are the physiological implications of these interactions? Here, we will address these questions and position these observations within the MDM2 literature.

Mitochondrial Isolation and Real-Time Monitoring of MOMP.

Isolated model systems have proven to be the standard in the apoptosis field to deconstruct MOMP into individual steps and to study the behavior of a subset of MOMP regulators. Here we describe the method to isolate, JC-1-label, and purify mouse liver mitochondria and subsequently describe how to utilize the JC-1-labeled mitochondria for real-time MOMP measurements.

Dual suppression of inner and outer mitochondrial membrane functions augments apoptotic responses to oncogenic MAPK inhibition.

Mitogen-activated protein kinase (MAPK) pathway inhibitors show promise in treating melanoma, but are unsuccessful in achieving long-term remission. Concordant with clinical data, BRAFV600E melanoma cells eliminate glycolysis upon inhibition of BRAFV600E or MEK with the targeted therapies Vemurafenib or Trametinib, respectively. Consequently, exposure to these therapies reprograms cellular metabolism to increase mitochondrial respiration and restrain cell death commitment. As the inner mitochondrial membrane (IMM) is sub-organellar site of oxidative phosphorylation (OXPHOS), and the outer mitochondrial membrane (OMM) is the major site of anti-apoptotic BCL-2 protein function, we hypothesized that suppressing these critical mitochondrial membrane functions would be a rational approach to maximize the pro-apoptotic effect of MAPK inhibition. Here, we demonstrate that disruption of OXPHOS with the mitochondria-specific protonophore BAM15 promotes the mitochondrial pathway of apoptosis only when oncogenic MAPK signaling is inhibited. Based on RNA-sequencing analyses of nevi and primary melanoma samples, increased pro-apoptotic BCL-2 family expression positively correlates with high-risk disease suggesting a highly active anti-apoptotic BCL-2 protein repertoire likely contributes to worse outcome. Indeed, combined inhibition of the anti-apoptotic BCL-2 repertoire with BH3-mimetics, OXPHOS, and oncogenic MAPK signaling induces fulminant apoptosis and eliminates clonogenic survival. Altogether, these data suggest that dual suppression of IMM and OMM functions may unleash the normally inadequate pro-apoptotic effects of oncogenic MAPK inhibition to eradicate cancer cells, thus preventing the development of resistant disease, and ultimately, supporting long-term remission.

The peroxisomes strike BAK: Regulation of peroxisome integrity by the Bcl-2 family.

Within the mitochondrial pathway of apoptosis, VDAC2 controls both the localization and proapoptotic activity of BAK. In this issue, Hosoi et al. (2017. J. Cell Biol. https://doi.org/10.1083/jcb.201605002) find that loss of VDAC2 diverts BAK into peroxisome membranes, revealing the ability of BAK to control peroxisome membrane integrity and the release of soluble peroxisomal matrix proteins.

Mitochondrial dynamics as regulators of cancer biology.

Mitochondria are dynamic organelles that supply energy required to drive key cellular processes, such as survival, proliferation, and migration. Critical to all of these processes are changes in mitochondrial architecture, a mechanical mechanism encompassing both fusion and fragmentation (fission) of the mitochondrial network. Changes to mitochondrial shape, size, and localization occur in a regulated manner to maintain energy and metabolic homeostasis, while deregulation of mitochondrial dynamics is associated with the onset of metabolic dysfunction and disease. In cancers, oncogenic signals that drive excessive proliferation, increase intracellular stress, and limit nutrient supply are all able to alter the bioenergetic and biosynthetic requirements of cancer cells. Consequently, mitochondrial function and shape rapidly adapt to these hostile conditions to support cancer cell proliferation and evade activation of cell death programs. In this review, we will discuss the molecular mechanisms governing mitochondrial dynamics and integrate recent insights into how changes in mitochondrial shape affect cellular migration, differentiation, apoptosis, and opportunities for the development of novel targeted cancer therapies.