Caspase Substrates Research Articles

In Vitro Cleavage Assays using Purified Recombinant Drosophila Caspases for Substrate Screening.

Caspases are very specific cell death proteases that are involved in apoptotic and non-apoptotic processes. While the role of caspases during apoptosis has been very well defined and many apoptotic proteolytic substrates of caspases have been identified and characterized, the role of caspases for non-apoptotic processes is not well understood. In particular, few non-apoptotic substrates of caspases have been identified thus far. Here, in order to facilitate the identification and characterization of potential caspase substrates, a protocol that allows the testing of candidate substrates in caspase cleavage assays in vitro is described. This protocol includes the production and purification of recombinant caspase proteins, the production of the candidate substrates either recombinantly or in a cell-free expression system, and the actual in vitro cleavage reaction followed by SDS-PAGE and immunoblotting. This protocol is tailored for the Drosophila caspases Dronc and Drice but can easily be adapted for caspases from other organisms, including mammals.

GSDMD-mediated pyroptosis in retinal vascular inflammatory diseases: a review.

Pyroptosis is a newly discovered form of programmed pro-inflammatory cell death. The main signaling pathways include the classical scorch death pathway that depends on NLRP3 inflammatory vesicles and other activation caspase-1 and the non-classical scorch death pathway that depends on caspase-4 /5/11. The substrate of all inflammatory caspases is GSDMD; a large number of studies have confirmed that pyroptosis is associated with certain infectious diseases, atherosclerotic diseases, metabolic diseases, and aseptic inflammatory diseases of important organs. In recent years, pyroptosis has been studied partially in the ocular field. So, this article reviews the recent literature intending to help readers understand the main mechanisms of cellular scorch death and the progress of GSDMD-mediated cellular scorch death in retinal vascular inflammatory diseases. A detailed review of the literature related to pyroptosis and inflammatory diseases of the retinal vasculature is presented. The following 6 electronic databases were searched: CNKI, Wanfang, VIP, PubMed, The Cochrane Library, and Embase Databases, and the search period was from the database to May 2022. The main search keywords include "Pyroptosis," " GSDMD," "Retinal Vascular Inflammatory Disease," "Diabetic retinopathy," "Retinal vasculitis." The discovery of pyroptosis, the main molecular mechanisms, key proteins, and their pathogenesis and therapeutic prospects in retinal vasculitis diseases are extensively studied and summarized. The mechanisms of gasdermin D-mediated pyroptosis are elaborated and analyzed, with particular emphasis on their key role and potential in the pathogenesis and treatment of inflammatory retinal vascular lesions. Gasdermin D-mediated pyroptosis is a well-studied form of programmed pro-inflammatory cell death, which has a bidirectional regulatory effect on a variety of immune and inflammatory diseases. The literature reveals that pyroptosis is closely related to the pathogenesis of retinal vascular inflammatory diseases, and it may be an important therapeutic target for diabetic retinopathy and other retinal vasculitis eye diseases in the future.

GASDERMIN AND ITS ROLE IN PYROPTOSIS

The gasdermin family (GSDMs) contains a group of uncharacterized proteins that form membrane pores and serve as a major substrate for inflammatory caspases and the execution of pyroptosis, which is recognized as a novel type of programmed cell death [1]. Gasdermin (GSDM) family consists of Gasdermin A (GSDMA), Gasdermin B (GSDMB), Gasdermin C GSDMC), Gasdermin D (GSDMD), Gasdermin E(GSDME) and Pejvakin (PJVK). With the exception of PJVK, all GSDMs consist of two conserved domains: the C-terminal inhibitory domain (RD) and the N-terminal effector domain (PFD), where the N-terminal domain is cytotoxic, while the full-length structure It is not cytotoxic, indicating that the C-terminus of the GSDMs protein family (GSDMs-C) has auto-inhibitory and protective effects [2]. Because of the presence of the C-terminus, the GSDM protein does not cause cell death if it is not cleaved. Once RD is removed by hydrolysis, its PFD can combine with lipid components to form pores in the cell membrane [3]. Current studies have found that, except for PJVK, the N-terminal domains of almost all GSDMs have the ability to form pores in the plasma membrane. Among GSDMs, only the mechanism of GSDMD-induced pyroptosis is relatively clear.

Expression and Purification of Human Granzyme B Fusion Protein to Induce Targeted Apoptosis in PSMA Positive Prostate Cancer Cells.

Granzyme B can induce apoptosis in target cells by direct and indirect activation of caspases and cleavage of central caspase substrates. Prostate-specific membrane antigen (PSMA) is a type II transmembrane glycoprotein and its expression increases following prostate cancer progression. In this study, we designed a fusion protein including mutant granzyme B, the influenza virus hemagglutinin HA-2 N-terminal, and PSMA ligand to construct GrB-HA-PSMA ligand fusion protein as a molecular agent for selective targeting of PSMA-positive (LNCaP) cells. The DNA sequence of our designed structure was synthesized and cloned into a pET28a expression vector. The recombinant protein was expressed in E. coli origami bacteria and then purified. The expression of the recombinant protein was verified by SDS PAGE and ELISA method. Furthermore, ELISA and flow cytometry assays were utilized to investigate the efficiency of binding and permeability of the recombinant protein into the LNCaP cells. Finally, cell proliferation and apoptosis rate were evaluated by MTT assay and flow cytometry assay, respectively. HeLa and PC3 cell lines were used as controls. The results showed that GrB-HA-PSMA ligand fusion protein could specifically bind and internalize into the PSMA-positive cells. Furthermore, treatment of the cells with GrB-HA-PSMA ligand fusion protein resulted in increased apoptotic cell death and decreased proliferation of LNCaP cells. Our findings indicate the specificity of GrB-HA-PSMA ligand fusion protein for PSMA-positive cells and suggest that this fusion protein is a potential candidate for prostate cancer targeted therapy.

Abstract 1403: ROS metabolism and autophagy as potential targets to improve head and neck cancer treatment

Abstract Introduction: Cancer cells exhibit a high degree of plasticity to adapt the rapidly changing tumor microenvironment (TME), in part by altering cellular metabolic pathways at a faster and more efficient pace than normal cells. Cancer cell plasticity, with genetic and epigenetic alterations, promote the diversity of cancer cells contributing to heterogeneity within the tumor. Head and neck cancer (HNC) is a notable example of this diversity; HNC tumors harbor cells with a wide genetic and metabolic heterogeneity, including cancer stem cells (CSC). Despite current treatment approaches, more than 40% of HNC patients have disease recurrence. Thus, we developed a multimodal approach to effectively prevent the development of radio/drug-resistance. Experimental Procedures: Nuclear-localized mkate2 expressing HNC cell numbers detected for every 6 hours by using IncuCyte S3 instrument. Apoptotic cells detected via NucView 488 Caspase-3 substrate and ROS levels determined using CellROX green simultaneously with cell numbers in the IncuCyte S3 instrument. Synergy scores calculated according to viability in SynergyFinder 2.0 web-application. Results: Our preliminary data profiling of metabolic regulators as radiosensitizer indicate that auranofin (AUR, thioredoxin reductase inhibitor) and buthionine sulfoximine (BSO, γ-glutamylcysteine synthetase inhibitor), given in combination with radiation (RT+AUR+BSO): 1) significantly inhibit proliferation in HNC cell lines, 2) increase ROS accumulation, and 3) reduce the surviving fraction of cells in colony forming assays compared to individual/dual agent applications. Furthermore, we added an autophagy inhibitor, SAR405, in our treatment regimen because of the crosstalk between ROS levels and autophagy. Human (FaDu and UM-SCC-47) and murine (MOC1 and MOC2) HNC cell were treated with +/-AUR+/-BSO+/-SAR405 and high-throughput combinatorial screening of cell numbers obtained over time. The combination of AUR+BSO+SAR405 registered a higher synergy score than the double combinations. Furthermore, as compared to single or double drug applications, the AUR+BSO+SAR405 group had a greater apoptotic cell rate and ROS levels. Conclusion: We present here a high-throughput method to screen multi-drug synergy. Multi-modal approach is important to prevent recurrence by providing a broader effect on heterogenous cancer cells in tumor. Moreover, multi-drug combinations allow usage of lower doses of drugs effectively, hence lowers the treatment-related adverse effects. This approach indicated the possibility of HNC treatment by increasing the ROS levels beyond the containable threshold which is consistent with the recent findings about the alterations in redox metabolism, particularly upregulation in thioredoxin and glutathione pathways, in many cancer types. In vivo experiments are ongoing to evaluate the potential of this combination for clinical studies. Citation Format: Zafer Gurel, Qianyun Luo, Michael S. Luy, Randall J. Kimple. ROS metabolism and autophagy as potential targets to improve head and neck cancer treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1403.

Non-Apoptotic Caspase Activity Preferentially Targets a Novel Consensus Sequence Associated With Cytoskeletal Proteins in the Developing Auditory Brainstem.

The auditory brainstem relies on precise circuitry to facilitate sound source localization. In the chick, the development of this specialized circuitry requires non-apoptotic activity of caspase-3, for which we previously identified several hundred proteolytic substrates. Here we tested whether the sequence of the caspase cleavage site differentially encodes proteolytic preference in apoptotic and non-apoptotic contexts. We constructed a consensus sequence for caspase activity in the non-apoptotic chick auditory brainstem comprising the four residues N-terminal to the cleavage site: IX(G/R)D↓ where X represents no significant enrichment and ↓ represents the cleavage site. We identified GO terms significantly enriched among caspase substrates containing motifs found in the above consensus sequence. (G/R)D↓ was associated with the term “Structural Constituent of Cytoskeleton” (SCoC), suggesting that SCoC proteins may be specifically targeted by caspase activity during non-apoptotic developmental processes. To ascertain whether this consensus sequence was specific to the non-apoptotic auditory brainstem at embryonic day (E) 10, we used protein mass spectrometry of brainstems harvested at a time when auditory brainstem neurons undergo apoptotic cell death (E13). The apoptotic motif VD was significantly enriched among E13 cleavage sites, indicating that motif preference at the P2 subsite had shifted toward the canonical caspase consensus sequence. Additionally, Monte Carlo simulations revealed that only the GD motif was associated with SCoC substrates in the apoptotic auditory brainstem, indicating that GD encodes specificity for SCoC proteins in both non-apoptotic and apoptotic contexts, despite not being preferred in the latter. Finally, to identify candidate human non-apoptotic consensus sequences, we used Monte Carlo analyses to determine motifs and motif pairs associated with SCoC caspase substrates in the Degrabase, a database of cleavage sites in human apoptotic cell lines. We found 11 motifs significantly associated with SCoC proteolysis, including IXXD and GD. We employed a stepwise method to select motif pairs that optimized SCoC specificity for a given coverage of SCoC cleavage events, yielding 11 motif pairs likely to be preferred in SCoC-directed human non-apoptotic caspase consensus sequences. GD + IXXD was among these motif pairs, suggesting a conservation of non-apoptotic consensus sites among vertebrates.

Investigation of Anticancer Effects of Apigenin on MOLT-3 Human Leukaemia Cell Line

Leukaemia is an aggressive blood malignancies characterized by abnormal accumulation and proliferation of WBCs. Among children younger than 15 years, leukaemia consider the most common type of cancer which accounting 30% of all diagnosed malignancies. The Saudi Cancer Registry (SCR) reported that leukaemia ranked as the 5th type of cancer among both male and female. Because of the undesirable side-effects of the chemotherapy commonly used for leukaemia treatment such as high toxicity and development of secondary cancer, the search for novel and new promising treatment with fewer side-effects and higher therapeutic efficiency is still a priority goal. Using bioactive compounds from natural sources was an effective treatment for a variety of blood cancer. Thus, the aim of this study was to use the bioactive compound Apigenin to treat human lymphoid leukaemia cell line MOLT-3 with three different doses (25, 50, 100 µM) for 24 h. The results of this study showed that cellular proliferation was significantly reduced with 25, 50 and 100 µM Apigenin. In addition, significant increase in apoptotic induction was demonstrated when assessed with Annexin V-FITC/ PI and Caspase-3 substrate based on flow cytometry. Cell cycle arrest was significantly reported at S phase within MOLT-3 cells in a dose response manner. Thus, the finding of this study suggested that Apigenin is a potent and efficient anticancer compound through its activity of blocked cell proliferation, apoptotic induction and cell cycle arrest. However, further studies are required to investigate if the dietary intake of Apigenin might interfere with chemotherapeutic treatment of leukaemia.

Interferon-γ primes macrophages for pathogen ligand-induced killing via a caspase-8 and mitochondrial cell death pathway

Cell death plays an important role during pathogen infections. Here, we report that interferon-γ (IFNγ) sensitizes macrophages to Toll-like receptor (TLR)-induced death that requires macrophage-intrinsic death ligands and caspase-8 enzymatic activity, which trigger the mitochondrial apoptotic effectors, BAX and BAK. The pro-apoptotic caspase-8 substrate BID was dispensable for BAX and BAK activation. Instead, caspase-8 reduced pro-survival BCL-2 transcription and increased inducible nitric oxide synthase (iNOS), thus facilitating BAX and BAK signaling. IFNγ-primed, TLR-induced macrophage killing required iNOS, which licensed apoptotic caspase-8 activity and reduced the BAX and BAK inhibitors, A1 and MCL-1. The deletion of iNOS or caspase-8 limited SARS-CoV-2-induced disease in mice, while caspase-8 caused lethality independent of iNOS in a model of hemophagocytic lymphohistiocytosis. These findings reveal that iNOS selectively licenses programmed cell death, which may explain how nitric oxide impacts disease severity in SARS-CoV-2 infection and other iNOS-associated inflammatory conditions.

Inflammatory Caspases: Toward a Unified Model for Caspase Activation by Inflammasomes.

Inflammasomes are inflammatory signaling complexes that provide molecular platforms to activate the protease function of inflammatory caspases. Caspases-1, -4, -5, and -11 are inflammatory caspases activated by inflammasomes to drive lytic cell death and inflammatory mediator production, thereby activating host-protective and pathological immune responses. Here, we comprehensively review the mechanisms that govern the activity of inflammatory caspases. We discuss inflammatory caspase activation and deactivation mechanisms, alongside the physiological importance of caspase activity kinetics. We also examine mechanisms of caspase substrate selection and how inflammasome and cell identities influence caspase activity and resultant inflammatory and pyroptotic cellular programs. Understanding how inflammatory caspases are regulated may offer new strategies for treating infection and inflammasome-driven disease.

Cell Death Related Proteins Beyond Apoptosis in the CNS.

Cell death related (CDR) proteins are a diverse group of proteins whose original function was ascribed to apoptotic cell death signaling. Recently, descriptions of non-apoptotic functions for CDR proteins have increased. In this minireview, we comment on recent studies of CDR proteins outside the field of apoptosis in the CNS, encompassing areas such as the inflammasome and non-apoptotic cell death, cytoskeleton reorganization, synaptic plasticity, mitophagy, neurodegeneration and calcium signaling among others. Furthermore, we discuss the evolution of proteomic techniques used to predict caspase substrates that could potentially explain their non-apoptotic roles. Finally, we address new concepts in the field of non-apoptotic functions of CDR proteins that require further research such the effect of sexual dimorphism on non-apoptotic CDR protein function and the emergence of zymogen-specific caspase functions.

Determination of Caspase-Like Activities in Roots by the Use of Fluorogenic Substrates.

Activity of proteases in tissues can be influenced by various intrinsic and extrinsic factors. One of the activities that is regularly monitored in organisms ranging from prokaryotes to metazoans is the -aspase-like activity: activity of proteases, which cleave their substrates after the negatively charged amino acid residues, especially the aspartic acid. This activity is also known as the caspase-like activity, since the caspases, metazoan cysteine proteases, are one of the best characterized proteases with Asp-directed activities. Plants do not contain caspases; however, various plant proteases have been shown to exhibit caspase-like activity including saspases, phytaspases, and legumains (VPEs). The activity of these proteases can change in plants in response to stress. Here we present a simple method for monitoring of the caspase-like protease activity in roots, which have been treated with allelopathic extracts, using a set of commercially available caspase substrates. We show that activity towards some, but not all, caspase substrates is upregulated in treated but not control samples. The protocol can be used also for other plant tissues as well as for other stressors.

Induction of Apoptosis in Human Pancreatic Cancer Stem Cells by the Endoplasmic Reticulum-Targeted Alkylphospholipid Analog Edelfosine and Potentiation by Autophagy Inhibition.

Simple SummaryPancreatic cancer remains an incurable cancer with a gloomy prognosis and a median survival of months since the time of diagnosis. Disappointingly, no significant improvement in clinical outcomes has been achieved for the last fifty years. Cancer stem cells (CSCs) are suggested to be critical and responsible for tumor development, drug resistance and cancer relapse, therefore, an effective antitumor therapy against pancreatic cancer should deal with pancreatic CSCs. Here, we found that the alkylphospholipid analog edelfosine induces apoptosis in pancreatic CD44+CD24+EpCAM+ CSCs through its accumulation in the endoplasmic reticulum, leading to sustained endoplasmic reticulum stress and cell death. Edelfosine was effective against primary cultures from pancreatic cancer patients and their corresponding pancreatic CSCs. Autophagy inhibition potentiated the proapoptotic action of edelfosine in pancreatic CSCs. Thus, endoplasmic reticulum targeting and its combination with autophagy inhibitors could open a new framework in pancreatic cancer chemotherapy, directly involving pancreatic CSCs.Pancreatic cancer is one of the most lethal malignancies with a poor and gloomy prognosis and the highest mortality-to-incidence ratio. Pancreatic cancer remains an incurable malignancy, and current therapies are ineffective. We isolated cancer stem cells (CSCs) from the human PANC-1 pancreatic cancer cell line as CD44+CD24+EpCAM+ cells. These CSCs form pancreatic cancer spheres or spheroids and develop tumors in SCID mice after subcutaneous injection of as few as 100 cells per mouse. Here, we found that the alkylphospholipid analog edelfosine inhibited CSC pancreatic cancer spheroid formation and induced cell death, as assessed by an increase in the percentage of cells in the sub-G0/G1 region by means of flow cytometry, indicative of DNA breakdown and apoptosis. This correlated with an increase in caspase-3 activity and PARP breakdown, as a major substrate of caspase-3, following PANC-1 CSC treatment with edelfosine. The antitumor ether lipid edelfosine colocalized with the endoplasmic reticulum in both PANC-1 cells as well as PANC-1 CSCs by using a fluorescent edelfosine analog, and induced an endoplasmic reticulum stress response in both PANC-1 cells and PANC-1 CSCs, with a potent CHOP/GADD153 upregulation. Edelfosine elicited a strong autophagy response in both PANC-1 cells and PANC-1 CSCs, and preincubation of CSCs with autophagy inhibitors, chloroquine or bafilomycin A1, enhanced edelfosine-induced apoptosis. Primary cultures from pancreatic cancer patients were sensitive to edelfosine, as well as their respective isolated CSCs. Nontumorigenic pancreatic human cell line HPNE and normal human fibroblasts were largely spared. These data suggest that pancreatic CSCs isolated from established cell lines and pancreatic cancer patients are sensitive to edelfosine through its accumulation in the endoplasmic reticulum and induction of endoplasmic reticulum stress.

Antileukemic Natural Product Induced Both Apoptotic and Pyroptotic Programmed Cell Death and Differentiation Effect.

Acute myeloid leukemia (AML) is one of the most common forms of leukemia. Despite advances in the management of such malignancies and the progress of novel therapies, unmet medical needs still exist in AML because of several factors, including poor response to chemotherapy and high relapse rates. Ardisianone, a plant-derived natural component with an alkyl benzoquinone structure, induced apoptosis in leukemic HL-60 cells. The determination of dozens of apoptosis-related proteins showed that ardisianone upregulated death receptors and downregulated the inhibitor of apoptosis protein (IAPs). Western blotting showed that ardisianone induced a dramatic increase in tumor necrosis factor receptor 2 (TNFR2) protein expression. Ardisianone also induced downstream signaling by activating caspase-8 and -3 and degradation in Bid, a caspase-8 substrate. Furthermore, ardisianone induced degradation in DNA fragmentation factor 45 kDa (DFF45), a subunit of inhibitors of caspase-activated DNase (ICAD). Q-VD-OPh (a broad-spectrum caspase inhibitor) significantly diminished ardisianone-induced apoptosis, confirming the involvement of caspase-dependent apoptosis. Moreover, ardisianone induced pyroptosis. Using transmission electron microscopic examination and Western blot analysis, key markers including gasdermin D, high mobility group box1 (HMGB1), and caspase-1 and -5 were detected. Notably, ardisianone induced the differentiation of the remaining survival cells, which were characterized by an increase in the expression of CD11b and CD68, two markers of macrophages and monocytes. Wright–Giemsa staining also showed the differentiation of cells into monocyte and macrophage morphology. In conclusion, the data suggested that ardisianone induced the apoptosis and pyroptosis of leukemic cells through downregulation of IAPs and activation of caspase pathways that caused gasdermin D cleavage and DNA double-stranded breaks and ultimately led to programmed cell death. Ardisianone also induced the differentiation of leukemic cells into monocyte-like and macrophage-like cells. The data suggested the potential of ardisianone for further antileukemic development.

TRPA1 promotes cisplatin-induced nephrotoxicity through inflammation mediated by the MAPK/NF-κB signaling pathway.

BackgroundThe nephrotoxicity induced by cisplatin (DDP) has been a severe obstacle for its clinical use in anticancer treatment. The apoptosis and inflammation induced by DDP are the main causes of the nephrotoxicity. Transient receptor potential ankyrin 1 (TRPA1) is a non-selective cation ligand-gated channel that is involved in the inflammation progress.MethodsThe apoptosis, inflammation, MAPK/NF-κB signaling pathway, and TRPA1 expression were assessed after HEK293 cells had been induced by DDP, and the role of TRPA1 in apoptosis and inflammation of DDP-induced HEK293 cells treated with TRPA1 antagonist HC-030031 was also evaluated by quantitative reverse transcription-polymerase chain reaction (qRT-PCR), flow cytometry, and western blot assays.ResultsThe cell viability was reduced by DDP in both a time-dependent and dose-dependent manner with a minimal cytotoxic concentration of 10 μM. Moreover, DDP induced an enhancement of the apoptosis and inflammation in a dose-dependent manner, as indicated by the increase of the relative protein level of cleaved-caspase3 (cleaved-cas3), the cleavage product of caspase-3 substrate poly-ADP-ribose polymerase (cleaved-PARP) and inducible nitric oxide synthase (iNOS), and the messenger RNA (mRNA) expression level of interleukin (IL)-1β, IL-6, tumor necrosis factor-α (TNF-α), and interferon-γ (INF-γ). Additionally, DDP treatment increased the protein phosphorylation expression of IKKβ, JNK, ERK, and p38 in a dose-dependent manner, which was antagonized by the treatment of NF-κB-specific inhibitor BAY 11-7082 and pan-MAPK inhibitor U0126. It was also found that DDP upregulated the expression of TRPA1 at both the mRNA and protein levels in a dose-dependent manner. Besides, block of TRPA1 with HC-030031 relieved the apoptosis, diminished the level of IL-1β, IL-6, TNF-α, and INF-γ, reduced the level of cleaved-cas3, cleaved-PARP, and iNOS, decreased the p-IKKβ, p-JNK, p-ERK, and p-p38 expression, and enhanced the expression of IκBα.ConclusionsTaken together, these results indicate that TRPA1 regulates DDP-induced nephrotoxicity via inflammation mediated by the MAPK/NF-κB signaling pathway in HEK293 cells.

Deorphanizing Caspase-3 and Caspase-9 Substrates In and Out of Apoptosis with Deep Substrate Profiling.

Caspases are a family of enzymes that regulate biological processes such as inflammation and programmed cell death, through proteolysis. For example, in the intrinsic pathway of apoptosis, cell death signaling involves cytochrome c release from the mitochondria, which leads to the activation of caspase-9 and eventually the executioners caspase-3 and -7. One key step in our understanding of these proteases is to identify their respective protein substrates. Although hundreds of substrates have been linked to caspase-3, only a small handful of substrates have been reported for caspase-9. Employing deep profiling by subtiligase N-terminomics, we present here an unbiased analysis of caspase-3 and caspase-9 substrates in native cell lysates. We identified 906 putative protein substrates associated with caspase-3 and 124 protein substrates for caspase-9. This is the most comprehensive list of caspase substrates reported for each of these proteases, revealing a pool of new substrates that could not have been discovered using other approaches. Over half of the caspase-9 substrates were also cleaved by caspase-3, but often at unique sites, suggesting an evolved functional redundancy for these two proteases. Correspondingly, nearly half of the caspase-9 cleavage sites were not recognized by caspase-3. Our results suggest that in addition to its important role in activating the executioners, the role of caspase-9 is likely broader and more complex than previously appreciated, which includes proteolysis of key apoptotic substrates other than just caspase-3 and -7 and involvement in non-apoptotic pathways. Our results are well poised to aid the discovery of new biological functions for these two caspases.

A switch-on molecular biosensor for detection of caspase-3 and imaging of apoptosis of cells

Apoptosis is a form of programmed cell death that is essential for maintaining internal environmental stability. Disordered apoptosis can cause a variety of diseases; therefore, sensing apoptosis can provide help in study of mechanism of the relevant diseases and drug development. It is known that caspase-3 is a key enzyme involved in apoptosis and the expression of its activity is an indication of apoptosis. Here, we present a genetically encoded switch-on mNeonGreen2-based molecular biosensor. mNeonGreen2 is the brightest monomeric green fluorescent protein. The substrate of caspase-3, DEVD amino acid residues, is inserted in it, while cyclized by insertion of Nostoc punctiforme DnaE intein to abolish the fluorescence (inactive state). Caspase-3-catalyzed cleavage of DEVD linearizes mNeonGreen2 and rebuilds the natural barrel structure to restore the fluorescence (activated state). The characterization exhibited that the Caspase-3 biosensor has shortened response time, higher sensitivity, and prolonged functional shelf life in detection of caspase-3 amongst the existing counterparts. We also used the Caspase-3 biosensor to evaluate the effect of several drugs on the induction of apoptosis of HeLa and MCF-7 tumor cells and inhibition of Zika virus invasion.Supporting InformationThe supporting information is available online at 10.1007/s11427-021-1986-7. The supporting materials are published as submitted, without typesetting or editing. The responsibility for scientific accuracy and content remains entirely with the authors.

Kinases leave their mark on caspase substrates.

Apoptosis is a cell death program that is executed by the caspases, a family of cysteine proteases that typically cleave after aspartate residues during a proteolytic cascade that systematically dismantles the dying cell. Extensive signaling crosstalk occurs between caspase-mediated proteolysis and kinase-mediated phosphorylation, enabling integration of signals from multiple pathways into the decision to commit to apoptosis. A new study from Maluch et al. examines how phosphorylation within caspase cleavage sites impacts the efficiency of substrate cleavage. The results demonstrate that while phosphorylation in close proximity to the scissile bond is generally inhibitory, it does not necessarily abrogate substrate cleavage, but instead attenuates the rate. In some cases, this inhibition can be overcome by additional favorable substrate features. These findings suggest potential nuanced physiological roles for phosphorylation of caspase substrates with exciting implications for targeting caspases with chemical probes and therapeutics.

Treatment with ascorbic acid normalizes the aerobic capacity, antioxidant defence, and cell death pathways in thermally stressed Mytilus galloprovincialis.

Considering temperature's upcoming increase due to climate change, combined with the fact that Mediterranean mussels Mytilus galloprovincialis (Lamarck, 1819) live at their upper limits [critical temperatures (Tc) beyond 25°C], we cannot be sure of this species' sustainable future in the Mediterranean Sea. Deviation from optimum temperatures leads to cellular damage due to oxidative stress. Although ascorbic acid (AA) is a major scavenger of reactive oxygen species (ROS), its capacity to minimize oxidative stress effects is scarcely studied in aquatic organisms. Thus, treatment with 5mM and 10mM AA of thermally stressed molluscs had been employed in order to examine its antioxidant capacity. While 5mM had no effect, 10mM normalized COX1 and ND2 relative mRNA levels, and superoxide dismutase (SOD), catalase, and glutathione reductase (GR) enzymatic activity levels in both examined tissues: posterior adductor muscle (PAM) and mantle. ATP levels, probably providing the adequate energy for antioxidant defence in thermally stressed mussels, is also normalized under 10mM AA treatment. Moreover, autophagic indicators such as LC3 II/I and SQSTM1/p62 levels are normalized, indicating autophagy amelioration. Apoptosis also seems to be inhibited since both Bax/Bcl-2 and cleaved caspase substrate levels decrease with 10mM AA treatment. Therefore, treatment of mussels with AA seems to produce threshold effects, although the precise underlying mechanisms must be elucidated in future studies. These findings show that treatment of mussels with effective antioxidants can be useful as a strategic approach for the reduction of the deleterious effects on mussels' summer mortality in aquaculture zones.

Great balls of fire: activation and signalling of inflammatory caspases.

Innate immune responses are tightly regulated by various pathways to control infections and maintain homeostasis. One of these pathways, the inflammasome pathway, activates a family of cysteine proteases called inflammatory caspases. They orchestrate an immune response by cleaving specific cellular substrates. Canonical inflammasomes activate caspase-1, whereas non-canonical inflammasomes activate caspase-4 and -5 in humans and caspase-11 in mice. Caspases are highly specific enzymes that select their substrates through diverse mechanisms. During inflammation, caspase activity is responsible for the secretion of inflammatory cytokines and the execution of a form of lytic and inflammatory cell death called pyroptosis. This review aims to bring together our current knowledge of the biochemical processes behind inflammatory caspase activation, substrate specificity, and substrate signalling.

GSDMD contributes to host defence against Staphylococcus aureus skin infection by suppressing the Cxcl1\u2013Cxcr2 axis

Gasdermin D (GSDMD), a member of the gasdermin protein family, is a caspase substrate, and its cleavage is required for pyroptosis and IL-1β secretion. To date, the role and regulatory mechanism of GSDMD during cutaneous microbial infection remain unclear. Here, we showed that GSDMD protected against Staphylococcus aureus skin infection by suppressing Cxcl1–Cxcr2 signalling. GSDMD deficiency resulted in larger abscesses, more bacterial colonization, exacerbated skin damage, and increased inflammatory cell infiltration. Although GSDMD deficiency resulted in defective IL-1β production, the critical role of IL-1β was counteracted by the fact that Caspase-1/11 deficiency also resulted in less IL-1β production but did not aggravate disease severity during S. aureus skin infection. Interestingly, GSDMD-deficient mice had increased Cxcl1 secretion accompanied by increased recruitment of neutrophils, whereas Caspase-1/11-deficient mice presented similar levels of Cxcl1 and neutrophils as wild-type mice. Moreover, the absence of GSDMD promoted Cxcl1 secretion in bone marrow-derived macrophages induced by live, dead, or different strains of S. aureus. Corresponding to higher transcription and secretion of Cxcl1, enhanced NF-κB activation was shown in vitro and in vivo in the absence of GSDMD. Importantly, inhibiting the Cxcl1–Cxcr2 axis with a Cxcr2 inhibitor or anti-Cxcl1 blocking antibody rescued host defence defects in the GSDMD-deficient mice. Hence, these results revealed an important role of GSDMD in suppressing the Cxcl1–Cxcr2 axis to facilitate pathogen control and prevent tissue damage during cutaneous S. aureus infection.