Molecular chaperons stabilize protein folding and play a vital role in maintaining tissue homeostasis. To this intent, mitochondrial molecular chaperons may be involved in the regulation of oxidative phosphorylation and apoptosis during stress events such as infections. However, specific human infectious diseases relatable to defects in molecular chaperons have yet to be identified. To this end, we performed whole exome sequencing and functional immune assessment in a previously healthy Asian female, who experienced severe respiratory failure due to Pneumocystis jiroveci pneumonia and non-HIV-related CD4 lymphocytopenia. This revealed that a chaperon, the mitochondrial paralog of HSP90, TRAP1, may have been involved in the patient\'s susceptibility to an opportunistic infection. Two rare heterozygous variants in TRAP1, E93Q, and A64T were detected. The patient\'s peripheral blood mononuclear cells displayed diminished TRAP1 expression, but had increased active, cleaved caspase-3, caspase-7, and elevated IL-1β production. Transfection of A64T and E93Q variants in cell lines yielded decreased TRAP1 compared to transfected wildtype TRAP1 and re-capitulated the immunotypic phenotype of enhanced caspase-3 and caspase-7 activity. When infected with live P. jiroveci, the E93Q or A64T TRAP1 mutant expressing cells also exhibited reduced viability. Patient cells and cell lines transfected with the TRAP1 E93Q/A64T mutants had impaired respiration, glycolysis, and increased ROS production. Of note, co-expression of E93Q/A64T double mutants caused more functional aberration than either mutant singly. Taken together, our study uncovered a previously unrecognized role of TRAP1 in CD4+ lymphocytopenia, conferring susceptibility to opportunistic infections.

Aim: This study explores the cytotoxic and apoptotic effects of novel thiazolidinone-1,2,3-triazole hybrids on HT-1080, A-549, and MDA-MB-231 cancer cell lines.Methods & results: The synthesized compounds underwent comprehensive characterization (NMR and HRMS) to confirm their structures and purity. Subsequent anticancer activity screening across diverse cancer cell lines revealed promising antitumor potential notably, compounds 6f and 6g. Mechanistic investigations unveiled that compound 6f triggers apoptosis through the caspase-3/7 pathway. In terms of in silico studies, the compound 6f was identified as a potent inhibitor of caspase-3 and caspase-7.Conclusion: The present study underscores the therapeutic potential of thiazolidinone-1,2,3-triazole hybrids against certain cancer cells. These findings highlight a promising avenue for the development of cancer treatment strategies utilizing these (R)-Carvone-based derivatives.
[Box: see text].

Sterol-regulatory element binding proteins (SREBPs) are a conserved transcription factor family governing lipid metabolism. When cellular cholesterol level is low, SREBP2 is transported from the endoplasmic reticulum to the Golgi apparatus where it undergoes proteolytic activation to generate a soluble N-terminal fragment, which drives the expression of lipid biosynthetic genes. Malfunctional SREBP activation is associated with various metabolic abnormalities. In this study, we find that overexpression of the active nuclear form SREBP2 (nSREBP2) causes caspase-dependent lytic cell death in various types of cells. These cells display typical pyroptotic and necrotic signatures, including plasma membrane ballooning and release of cellular contents. However, this phenotype is independent of the gasdermin family proteins or mixed lineage kinase domain-like (MLKL). Transcriptomic analysis identifies that nSREBP2 induces expression of p73, which further activates caspases. Through whole-genome CRISPR-Cas9 screening, we find that Pannexin-1 (PANX1) acts downstream of caspases to promote membrane rupture. Caspase-3 or 7 cleaves PANX1 at the C-terminal tail and increases permeability. Inhibition of the pore-forming activity of PANX1 alleviates lytic cell death. PANX1 can mediate gasdermins and MLKL-independent cell lysis during TNF-induced or chemotherapeutic reagents (doxorubicin or cisplatin)-induced cell death. Together, this study uncovers a noncanonical function of SREBPs as a potentiator of programmed cell death and suggests that PANX1 can directly promote lytic cell death independent of gasdermins and MLKL.

BACKGROUND: There is a need for novel treatments for neuroblastoma, despite the emergence of new biological and immune treatments, since refractory pediatric neuroblastoma is still a medical challenge. Phyto cannabinoids and their hemisynthetic derivatives have shown evidence supporting their anticancer potential. The aim of this research was to examine Phytocannabinoids or hemisynthetic cannabinoids, which reduce the SHSY-5Y, neuroblastoma cell line\'s viability.
METHODS: Hexane and acetyl acetate extracts were produced starting with Cannabis sativa L. as raw material, then, 9-tetrahidrocannabinol, its acid counterpart and CBN were isolated. In addition, acetylated derivatives of THC and CBN were synthesized. The identification and purity of the chemicals was determined by High Performance Liquid Chromatography and 1H y 13C Magnetic Nuclear Resonance. Then, the capacity to affect the viability of SHSY-5Y, a neuroblastoma cell line, was examined using the resazurin method. Finally, to gain insight into the mechanism of action of the extracts, phytocannabinoids and acetylated derivatives on the examined cells, a caspase 3/7 determination was performed on cells exposed to these compounds.
RESULTS: The structure and purity of the isolated compounds was demonstrated. The extracts, the phytocannabinoids and their acetylated counterparts inhibited the viability of the SHSY 5Y cells, being CBN the most potent of all the tested molecules with an inhibitory concentration of 50 percent of 9.5 µM.
CONCLUSIONS: Each of the evaluated molecules exhibited the capacity to activate caspases 3/7, indicating that at least in part, the cytotoxicity of the tested phytocannabinoids and their hemi-synthetic derivatives is mediated by apoptosis.

Dengue virus (DENV) is a major global health concern, causing millions of infections annually. Understanding the cellular response to DENV infection is crucial for developing effective therapies. This study provides an in-depth analysis of the cellular response to Dengue virus (DENV) infection, with a specific focus on the interplay between microRNAs (miRNAs), apoptosis, and viral load across different DENV serotypes. Utilizing a variety of cell lines infected with four DENV serotypes, the research methodically quantifies viral load, and the expression levels of miRNA-15, miRNA-16, and BCL2 protein, alongside measuring apoptosis markers. Methodologically, the study employs quantitative PCR for viral load and miRNA expression analysis, and Western blot for apoptosis and BCL2 detection, with a statistical framework that includes ANOVA and correlation analysis to discern significant differences and relationships. The findings reveal that despite similar viral loads across DENV serotypes, DENV-2 exhibits a marginally higher load. A notable upregulation of miRNA-15 and miRNA-16 correlates positively with increased viral load, suggesting their potential role in modulating viral replication. Concurrently, a marked activation of caspases 3 and 7, along with changes in BCL2 protein levels, underscores the role of apoptosis in the cellular response to DENV infection. Conclusively, the study enhances the understanding of miRNA involvement in DENV pathogenesis, highlighting miRNA-15 and miRNA-16 as potential regulatory agents in viral replication and apoptosis. These findings pave the way for further exploration into miRNA-based therapeutic strategies against DENV infection.

Allergic conjunctivitis (AC) is the most common form of allergic eye disease and an increasingly prevalent condition. Topical eye drop treatments are the usual approach for managing AC, although their impact on the ocular surface is not frequently investigated. The aim of this study was to perform a comparative physicochemical characterization, and in vitro biological evaluations in primary conjunctival and corneal epithelial cells of the new multidose preservative-free bilastine 0.6% and main commercially available eye drops. MTT assay was used to measure cell viability; oxidative stress was analyzed with a ROS-sensitive probe; and apoptosis was evaluated monitoring caspase 3/7 activation. Differences in pH value, osmolarity, viscosity and phosphate levels were identified. Among all formulations, bilastine exhibited pH, osmolarity and viscosity values closer to tear film (7.4, 300 mOsm/l and ~ 1.5-10 mPa·s, respectively), and was the only phosphates-free solution. Single-dose ketotifen did not induce ROS production, and single-dose azelastine and bilastine only induced a mild increase. Bilastine and single-dose ketotifen and azelastine showed high survival rates attributable to the absence of preservative in its formulation, not inducing caspase-3/7-mediated apoptosis after 24 h. Our findings support the use of the new bilastine 0.6% for treating patients with AC to preserve and maintain the integrity of the ocular surface.

BACKGROUND: Lung cancer, a chronic and heterogeneous disease, is the leading cause of cancer-related death on a global scale. Presently, despite a variety of available treatments, their effectiveness is limited, often resulting in considerable toxicity and adverse effects. Additionally, the development of chemoresistance in cancer cells poses a challenge. Trilobolide-6-O-isobutyrate (TBB), a natural sesquiterpene lactone extracted from Sphagneticola trilobata, has exhibited antitumor effects. Its pharmacological properties in NSCLC lung cancer, however, have not been explored.
OBJECTIVE: This study evaluated the impact of TBB on the A549 and NCI-H460 tumor cell lines in vitro, examining its antiproliferative properties and initial mechanisms of cell death.
METHODS: TBB, obtained at 98 % purity from S. trilobata leaves, was characterized using chromatographic techniques. Subsequently, its impact on inhibiting tumor cell proliferation in vitro, TBB-induced cytotoxicity in LLC-MK2, THP-1, AMJ2-C11 cells, as well as its effects on sheep erythrocytes, and the underlying mechanisms of cell death, were assessed.
RESULTS: In silico predictions have shown promising drug-likeness potential for TBB, indicating high oral bioavailability and intestinal absorption. Treatment of A549 and NCI-H460 human tumor cells with TBB demonstrated a direct impact, inducing significant morphological and structural alterations. TBB also reduced migratory capacity without causing toxicity at lower concentrations to LLC-MK2, THP-1 and AMJ2-C11 cell lines. This antiproliferative effect correlated with elevated oxidative stress, characterized by increased levels of ROS, superoxide anion radicals and NO, accompanied by a decrease in antioxidant markers: SOD and GSH. TBB-stress-induced led to changes in cell metabolism, fostering the accumulation of lipid droplets and autophagic vacuoles. Stress also resulted in compromised mitochondrial integrity, a crucial aspect of cellular function. Additionally, TBB prompted apoptosis-like cell death through activation of caspase 3/7 stressors.
CONCLUSIONS: These findings underscore the potential of TBB as a promising candidate for future studies and suggest its viability as an additional component in the development of novel anticancer drugs prototypes.

Caspase (CASP) is a family of proteases involved in cleavage and activation of gasdermin, the executor of pyroptosis. In humans, CASP3 and CASP7 recognize the same consensus motif DxxD, which is present in gasdermin E (GSDME). However, human GSDME is cleaved by CASP3 but not by CASP7. The underlying mechanism of this observation is unclear. In this study, we identified a pyroptotic pufferfish GSDME that was cleaved by both pufferfish CASP3/7 and human CASP3/7. Domain swapping between pufferfish and human CASP and GSDME showed that the GSDME C-terminus and the CASP7 p10 subunit determined the cleavability of GSDME by CASP7. p10 contains a key residue that governs CASP7 substrate discrimination. This key residue is highly conserved in vertebrate CASP3 and in most vertebrate (except mammalian) CASP7. In mammals, the key residue is conserved in non-primates (e.g., mouse) but not in primates. However, mouse CASP7 cleaved human GSDME but not mouse GSDME. These findings revealed the molecular mechanism of CASP7 substrate discrimination and the divergence of CASP3/7-mediated GSDME activation in vertebrate. These results also suggested that mutation-mediated functional alteration of CASP probably enabled the divergence and specialization of different CASP members in the regulation of complex cellular activities in mammals.
Cell death is essential for an organism to develop and survive as it plays key roles in processes such as embryo development and tissue regeneration. Cell death is also an important form of defence during an infection. A form of programmed cell death known as pyroptosis can be induced in infected cells, which helps to kill the infectious agent as well as alert the immune system to the infection. Pyroptosis is driven by Gasdermin E, a protein made up of two domains. At one end of the protein, the ‘N-terminal’ domain punctures holes in cell membranes, which can lead to cell death. At the other end, the ‘C-terminal’ domain inhibits the activity of the N-terminal domain. A family of proteins called caspases activate Gasdermin E by cleaving it, which releases the N-terminal domain from the inhibitory C-terminal domain. In humans, two caspases known as CASP3 and CASP7 recognize a specific sequence of amino acids – the building blocks of proteins – in Gasdermin E. However, only CASP3 is able to cleave the protein. After discovering that, unlike in humans, pufferfish Gasdermin E can be cleaved by both CASP3 and CASP7, Xu et al. wanted to investigate the underlying mechanisms behind this difference. Swapping the domains of human and pufferfish Gasdermin E and creating different versions of CASP7 revealed that the C-terminal domain of Gasdermin E and a single amino acid in CASP7 determine whether cleavage is possible. Interestingly, the key amino acid sequence required for cleavage by CASP7 is present in most vertebrate CASP3 and CASP7 proteins. However, it is absent in most mammalian CASP7. The findings of Xu et al. suggest that the different activity of human CASP7 and CASP3 is driven by a single amino acid mutation. This change likely played an important role in the process of different CASP proteins evolving to regulate different cellular activities in mammalian cells. This knowledge will be useful for future studies on the evolution and specialization of other closely related proteins.

Hepatocellular carcinoma remains a challenging contributor to the global cancer and related mortality, and claims approximately 800,000 deaths each year. Dysregulation or loss of function mutations involving the tumor suppressor gene, phosphatase and tensin homolog deleted on chromosome ten (PTEN), has been well-characterized in various cancers to elicit anomalous cell proliferation and oncogenic transformation. However, the delivery and bioavailability of genes/drugs of interest to carcinomas remains a serious bottleneck behind the success of any anti-cancer formulation. In this study, we have engineered nanoliposomes containing PTEN plasmids, plumbagin, and antioxidant cerium oxide nanoparticles (Lipo-PTEN-Plum) to restore the PTEN expression and inhibit the AKT/PI3K pathway. The Lipo-PTEN-Plum was quasi-spherical in shape with ∼110 nm diameter and ∼64% plumbagin loading efficiency. The Lipo-PTEN-Plum was successfully internalized HepG2 cells, restore PTEN expression and inhibit PI3K/AKT pathway to induce death in cells grown in monolayer and in form of spheroids. Mechanistically, the formulation showed G2/M cell cycle arrest, DNA damage and apoptosis in hepatic cancer cells. Other cellular events such as Caspase-7 overexpression and PI3K (phosphoinositide 3-kinase), AKT (a serine/threonine protein kinase), PARP [Poly (ADP-ribose) polymerases], and mTOR (Mammalian target of rapamycin) inhibition led to the apoptosis in hepatic cancer cells. The mRNA expression profile of PTEN, PI3K, AKT3, Caspase-7, PARP and mTOR proteins, primarily controlling the cancer cell proliferation and apoptosis, suggest that exogenous supply of PTEN could regulate the expression of oncogenic proteins and thus cancer progression.

GSDMB is associated with several inflammatory diseases, such as asthma, sepsis and colitis. GZMA is released by cytotoxic lymphocytes and cleaves GSDMB at the K244 site and to induce GSDMB N-terminus dependent pyroptosis. This cleavage of GSDMB is noncell autonomous. In this study, we demonstrated that the GSDMB-N domain (1-91 aa) was important for a novel cell-autonomous function and that GSDMB could bind caspase-4 and promote noncanonical pyroptosis. Furthermore, activated caspase-7 cleaved GSDMB at the D91 site to block GSDMB-mediated promotion of noncanonical pyroptosis during apoptosis. Mechanistically, the cleaved GSDMB-C-terminus (92-417 aa) binds to the GSDMB-N-terminus (1-91 aa) to block the function of GSDMB. During E. coli and S. Typhimurium infection, inhibition of the caspase-7/GSDMB axis resulted in more pyroptotic cells. Furthermore, in a septic mouse model, caspase-7 inhibition or deficiency in GSDMB-transgenic mice led to more severe disease phenotypes. Overall, we demonstrate that apoptotic caspase-7 activation inhibits non-canonical pyroptosis by cleaving GSDMB and provide new targets for sepsis therapy.