Microfluidic deep mutational scanning of the human executioner caspases reveals differences in structure and regulation

The human caspase family comprises 12 cysteine proteases that are centrally involved in cell death and inflammation responses. The members of this family have conserved sequences and structures, highly similar enzymatic activities and substrate preferences, and overlapping physiological roles. In this paper, we present a deep mutational scan of the executioner caspases CASP3 and CASP7 to dissect differences in their structure, function, and regulation. Our approach leverages high-throughput microfluidic screening to analyze hundreds of thousands of caspase variants in tightly controlled in vitro reactions. The resulting data provides a large-scale and unbiased view of the impact of amino acid substitutions on the proteolytic activity of CASP3 and CASP7. We use this data to pinpoint key functional differences between CASP3 and CASP7, including a secondary internal cleavage site, CASP7 Q196 that is not present in CASP3. Our results will open avenues for inquiry in caspase function and regulation that could potentially inform the development of future caspase-specific therapeutics.

Intrinsic apoptosis is evolutionary divergent among metazoans

Apoptosis is characterised by an analogous set of morphological features1 that depend on a proteolytic multigenic family, the caspases. Each apoptotic signalling pathway involves a specific initiator caspase, upstream of the pathway regulation, which finally converges to common executioner caspases. Intrinsic apoptosis, previously known as the mitochondrial apoptotic pathway, is often considered as ancestral and evolutionary conserved among animals. First identified in the nematode Caenorhabditis elegans, intrinsic apoptosis was next characterised in fruit fly Drosophila melanogaster and mammals. Intrinsic apoptosis depends on the key initiator caspase-9 (named Ced-3 and Dronc in Caenorhabditis and Drosophila, respectively), the activator Apaf-1 and the Bcl-2 multigenic family. Many functional studies have led to a deep characterisation of intrinsic apoptosis based on those classical models. Nevertheless, the biochemical role of mitochondria, the pivotal function of cytochrome c and the modality of caspases activation remain highly heterogeneous and hide profound molecular divergences among apoptotic pathways in animals. Independent of functional approaches, the phylogenetic history of the signal transduction actors, mostly the caspase family, is the Rosetta Stone to shed light on intrinsic apoptosis evolution. Here, after exhaustive research on CARD-caspases, we demonstrate by phylogenetic analysis that the caspase-9, the fundamental key of intrinsic apoptosis, is deuterostomes-specific, while it is the caspase-2 which is ancestral and common to bilaterians. Our analysis of Bcl-2 family and Apaf-1 confirm the high heterogeneity in apoptotic pathways elaboration in animals. Taken together, our results support convergent emergence of distinct intrinsic apoptotic pathways during metazoan evolution.

Genuine Selective Caspase-2 Inhibition with new Irreversible Small Peptidomimetics

Caspase-2 (Casp2) is a promising therapeutic target in several human diseases including nonalcoholic steatohepatitis (NASH) and Alzheimer's disease (AD). However, the design of active-site-directed inhibitor selective to individual caspase family members is challenging because caspases have extremely similar active sites. Here we present new peptidomimetics derived from the VDVAD pentapeptide structure, harboring non-natural modifications at the P2 position and an irreversible warhead. Enzyme kinetics shows that these new compounds, such as LJ2 or its specific isomer LJ2a, and LJ3a, strongly and irreversibly inhibit Casp2 with genuine selectivity. According to Casp2 role in cellular stress responses, LJ2 inhibits cell death induced by microtubule destabilization or hydroxamic acid-based deacetylase inhibition. The most potent peptidomimetic, LJ2a, inhibits human Casp2 with a remarkably high inactivation rate (k3/Ki ~ 5 500 000 M-1s-1) and the most selective inhibitor, LJ3a, has a near to 1000 times higher inactivation rate on Casp2 as compared to Casp3. Structural analysis of LJ3a shows that spatial configuration of C[alpha]; at the P2 position determines inhibitor efficacy. In transfected human cell lines overexpressing site-1 protease (S1P), sterol regulatory element-binding protein 2 (SREBP2) and Casp2, LJ2a and LJ3a fully inhibit SREBP2 activation, suggesting a potential to prevent NASH development. Furthermore, in primary hippocampal neurons treated with [beta]-amyloid oligomers, submicromolar concentrations of LJ2a and of LJ3a prevent synapse loss, indicating a potential for further investigations in AD treatment.

Combined Metabolomics and Network Toxicology to Explore the Molecular Mechanism of Phytolacca acinose Roxb-Induced Hepatotoxicity in Zebrafish Larvae in Vivo

Phytolacca acinosa Roxb (PAR), a traditional Chinese medicine, has been widely used as a diuretic drug for a long period of time for the treatment edema, swelling, and sores. However, it has been reported that PAR might induce hepatotoxicity, while the mechanisms of its toxic effect are still unclear. In this study, network toxicology and metabolomic technique were applied to explore PAR-induced hepatotoxicity on zebrafish larvae. We evaluated the effect of PAR on the ultrastructure and the function of the liver, predictive targets, and pathways in network toxicology, apoptosis of liver cells by PCR and western blot, and metabolic profile by GC-MS. PAR causes liver injury, abnormal liver function, and apoptosis in zebrafish. The level of arachidonic acid in endogenous metabolites treated with PAR was significantly increased, leading to oxidative stress in vivo. Excessive ROS further activated the p53 signal pathway and caspase family, which were obtained from KEGG enrichment analysis of network toxicology. The gene levels of caspase-3, caspase-8, and caspase-9 were significantly increased by RT-PCR, and the level of Caps3 protein was also significantly up-regulated through western blot. PAR exposure results in the liver function abnormal amino acid metabolism disturbance and motivates hepatocyte apoptosis, furthermore leading to liver injury.

Idarubicin-induced oxidative stress and apoptosis in cardiomyocytes: An in vitro molecular approach

Idarubicin (IDA) is an anthracycline antibiotic, frequently used for the treatment of various human cancers. In vivo rodent model studies have identified a variety of possible adverse outcomes from IDA including heart effects like increased heart weights, myocardial histopathological injury, electrocardiogram abnormalities, and cardiac dysfunction. Despite significant investigations, the molecular mechanisms responsible for the cardiotoxicity of IDA have not been fully clarified. The aim of the current study was to investigate the effects of IDA on the HL-1 cardiac muscle cell. Different concentrations of IDA (10−6, 10−5, 10−4, and 10−3 M) were used at different time (6, 12, 24, and 48 h) periods, and the Cell Counting Kit-8 (CCK-8); 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) probe method; and enzyme-linked immunosorbent assay (ELISA) were used to detect the oxidative stress level. In addition, we used network analysis to predict IDA-induced cardiotoxicity. The TUNEL assay, qRT-PCR, ELISA assay, and Western blotting detection of related apoptotic factors including caspase family, Bax, and Bcl-2. Overall, we found that IDA was generally more toxic at high concentrations or extended durations of exposure. At the same time, IDA can increase the content of reactive oxygen species (ROS), malondialdehyde (MDA), and decrease the level of superoxide dismutase (SOD), catalase (CAT), and glutathione (GSH) in cells, and increase the content of lactate dehydrogenase (LDH) and nitric oxide synthase (NOS) in the medium. Network analysis showed that the apoptosis signaling pathway was activated; specifically, the caspase family was involved in the signal pathway. The results of the TUNEL assay, qRT-PCR, ELISA, and Western blot found that IDA can activate apoptotic factors. The mechanism may be related to the activation of apoptosis signaling pathway. These results indicate that the cardiotoxic effects of IDA are most likely associated with oxidative stress and ROS formation, which finally ends in apoptotic factors’ activation and induction of cell apoptosis.

Regulated Cell Death in Urinary Malignancies

Urinary malignancies refer to a series of malignant tumors that occur in the urinary system and mainly include kidney, bladder, and prostate cancers. Although local or systemic radiotherapy and chemotherapy, immunotherapy, castration therapy and other methods have been applied to treat these diseases, their high recurrence and metastasis rate remain problems for patients. With in-depth research on the pathogenesis of urinary malignant tumors, this work suggests that regulatory cell death (RCD) plays an important role in their occurrence and development. These RCD pathways are stimulated by various internal and external environmental factors and can induce cell death or permit cell survival under the control of various signal molecules, thereby affecting tumor progression or therapeutic efficacy. Among the previously reported RCD methods, necroptosis, pyroptosis, ferroptosis, and neutrophil extracellular traps (NETs) have attracted research attention. These modes transmit death signals through signal molecules, such as cysteine-aspartic proteases (caspase) family and tumor necrosis factor-α (TNF-α) that have a wide and profound influence on tumor proliferation or death and even change the sensitivity of tumor cells to therapy. This review discussed the effects of necroptosis, pyroptosis, ferroptosis, and NETs on kidney, bladder and prostate cancer and summarized the latest research and achievements in these fields. Future directions and possibility of improving the denouement of urinary system tumors treatment by targeting RCD therapy were also explored.

The role of caspase family in acute brain injury: the potential therapeutic targets in the future

: The caspase family is commonly involved in the pathophysiology of acute brain injury (ABI) through complex apoptotic, pyroptotic, and inflammatory pathways. Current translational strategies for caspase modulation in ABI primarily focus on caspase inhibitors. Because there are no caspase-inhibiting drugs approved for clinical use on the market, the development of caspase inhibitors remains an attractive challenge for researchers and clinicians. Therefore, we conducted the present review with the aim of providing a comprehensive introduction of caspases in ABI. In this review, we summarized the available evidence and potential mechanisms regarding the biological function of caspases. We also reviewed the therapeutic effects of caspase inhibitors on ABI and its subsequent complications. However, various important issues remain unclear, prompting further verification of the efficacy and safety regarding clinical application of caspase inhibitors. We believe that our work will be helpful to further understand the critical role of the caspase family, and will provide novel therapeutic potential for ABI treatment.

Evolution of the folding landscape of effector caspases

Caspases are a family of cysteinyl proteases that control programmed cell death and maintain homeostasis in multicellular organisms. The caspase family is an excellent model to study protein evolution because all caspases are produced as zymogens (procaspases) that must be activated to gain full activity; the protein structures are conserved through hundreds of millions of years of evolution; and some allosteric features arose with the early ancestor while others are more recent evolutionary events. The apoptotic caspases evolved from a common ancestor into two distinct subfamilies: monomers (initiator caspases) or dimers (effector caspases). Differences in activation mechanisms of the two subfamilies, and their oligomeric forms, play a central role in the regulation of apoptosis. Here, we examine changes in the folding landscape by characterizing human effector caspases and their common ancestor. The results show that the effector caspases unfold by a minimum three-state equilibrium model at pH 7.5, where the native dimer is in equilibrium with a partially folded monomeric (procaspase-7, common ancestor) or dimeric (procaspase-6) intermediate. In comparison, the unfolding pathway of procaspase-3 contains both oligomeric forms of the intermediate. Overall, the data show that the folding landscape was first established with the common ancestor and was then retained for >650 million years. Partially folded monomeric or dimeric intermediates in the ancestral ensemble provide mechanisms for evolutionary changes that affect stability of extant caspases. The conserved folding landscape allows for the fine-tuning of enzyme stability in a species-dependent manner while retaining the overall caspase-hemoglobinase fold.

Anticancer activity study of paeoniflorin Ag(I) complexes against human hepatoma cells 3B cells

A new paeoniflorin Ag(I) complex was synthesized using the hydrothermal method, which was characterized by some spectral techniques like elemental analysis, thermal analysis, 1H nuclear magnetic resonance (1H NMR), Fourier-transform infrared spectroscopy (FTIR), electrospray ionization mass spectrometry (ESI-MS) and geometry optimization. Cell Counting Kit-8 (CCK-8) was used to test the in vitro cytotoxicity of the paeoniflorin Ag(I) complexes in various cells (such as A549, Caco2, HepG2, Hep3B, LO2, H9C2 and PC12 cells). It is shown that the paeoniflorin Ag(I) complexes possessed a remarkable cytotoxicity against the Hep3B cell line, but a low cytotoxicity against the LO2 cell line. Cell cycle progression and membrane potential of Hep3B cells were studied using flow cytometry, and the cell examination of apoptosis was made by Annexin V-FITC/PI staining. Moreover, expressions levels of caspase family proteins and Bcl-2 were also investigated. Our findings demonstrated that the paeoniflorin Ag(I) complexes induced apoptosis of Hep3B cells via the mitochondrial membrane potential access by regulating Bcl-2 and caspase family proteins.

Caspases in the Developing Central Nervous System: Apoptosis and Beyond

The caspase family of cysteine proteases represents the executioners of programmed cell death (PCD) type I or apoptosis. For years, caspases have been known for their critical roles in shaping embryonic structures, including the development of the central nervous system (CNS). Interestingly, recent findings have suggested that aside from their roles in eliminating unnecessary neural cells, caspases are also implicated in other neurodevelopmental processes such as axon guidance, synapse formation, axon pruning, and synaptic functions. These results raise the question as to how neurons regulate this decision-making, leading either to cell death or to proper development and differentiation. This review highlights current knowledge on apoptotic and non-apoptotic functions of caspases in the developing CNS. We also discuss the molecular factors involved in the regulation of caspase-mediated roles, emphasizing the mitochondrial pathway of cell death.