Expansion and diversity of caspases in Mytilus coruscus contribute to larval metamorphosis and environmental adaptation.

BACKGROUND: Apoptosis is involved (directly and indirectly) in several physiological processes including tissue remodeling during the development, the turnover of immune cells, and a defense against harmful stimuli. The disordered apoptotic process participates in the pathogenesis of various diseases, such as neoplasms, and chronic inflammatory or systemic autoimmune diseases, which are associated with its inadequate regulation. Caspases are vital components of the apoptotic pathway that are involved in developmental and immune processes. However, genome-wide identification and functional analysis of caspase have not been conducted in Mytilus coruscus, which is an economically important bivalve. RESULTS: Here, 47 caspase genes were identified from the genomes of M. coruscus, and the expansion of caspase-2/9 and caspase-3/6/7 genes were observed. Tandem duplication acts as an essential driver of gene expansion. The expanded caspase genes were highly diverse in terms of sequence, domain structure, and spatiotemporal expression profiles, suggesting their functional differentiation. The high expression of the expanded caspase genes at the pediveliger larvae stage and the result of apoptosis location in the velum suggest that the apoptosis mediated by them plays a critical role in the metamorphosis of M. coruscus larvae. In gill, caspase genes respond differently to the challenge of different strains, and most caspase-2/9 and caspase-3/6/7 genes were induced by copper stress, whereas caspase-8/10 genes were suppressed. Additionally, most caspase genes were upregulated in the mantle under ocean acidification which could weaken the biomineralization capacity of the mantle tissue. CONCLUSIONS: These results provide a comprehensive overview of the evolution and function of the caspase family and enhanced the understanding of the biological function of caspases in M. coruscus larval development and response to biotic and abiotic challenges.

Exposure to high dose of polystyrene nanoplastics causes trophoblast cell apoptosis and induces miscarriage.

BACKGROUND: With rapid increase in the global use of various plastics, microplastics (MPs) and nanoplastics (NPs) pollution and their adverse health effects have attracted global attention. MPs have been detected out in human body and both MPs and NPs showed female reproductive toxicological effects in animal models. Miscarriage (abnormal early embryo loss), accounting for 15-25% pregnant women worldwide, greatly harms human reproduction. However, the adverse effects of NPs on miscarriage have never been explored. RESULTS: In this study, we identified that polystyrene (PS) plastics particles were present in women villous tissues. Their levels were higher in villous tissues of unexplained recurrent miscarriage (RM) patients vs. healthy control (HC) group. Furthermore, mouse assays further confirmed that exposure to polystyrene nanoplastics (PS-NPs, 50 nm in diameter, 50 or 100 mg/kg) indeed induced miscarriage. In mechanism, PS-NPs exposure (50, 100, 150, or 200 µg/mL) increased oxidative stress, decreased mitochondrial membrane potential, and increased apoptosis in human trophoblast cells by activating Bcl-2/Cleaved-caspase-2/Cleaved-caspase-3 signaling through mitochondrial pathway. The alteration in this signaling was consistent in placental tissues of PS-NPs-exposed mouse model and in villous tissues of unexplained RM patients. Supplement with Bcl-2 could efficiently suppress apoptosis in PS-NPs-exposed trophoblast cells and reduce apoptosis and alleviate miscarriage in PS-NPs-exposed pregnant mouse model. CONCLUSIONS: Exposure to PS-NPs activated Bcl-2/Cleaved-caspase-2/Cleaved-caspase-3, leading to excessive apoptosis in human trophoblast cells and in mice placental tissues, further inducing miscarriage.

Caspase-2 protects against ferroptotic cell death.

Caspase-2, one of the most evolutionarily conserved members of the caspase family, is an important regulator of the cellular response to oxidative stress. Given that ferroptosis is suppressed by antioxidant defense pathways, such as that involving selenoenzyme glutathione peroxidase 4 (GPX4), we hypothesized that caspase-2 may play a role in regulating ferroptosis. This study provides the first demonstration of an important and unprecedented function of caspase-2 in protecting cancer cells from undergoing ferroptotic cell death. Specifically, we show that depletion of caspase-2 leads to the downregulation of stress response genes including SESN2, HMOX1, SLC7A11, and sensitizes mutant-p53 cancer cells to cell death induced by various ferroptosis-inducing compounds. Importantly, the canonical catalytic activity of caspase-2 is not required for its role and suggests that caspase-2 regulates ferroptosis via non-proteolytic interaction with other proteins. Using an unbiased BioID proteomics screen, we identified novel caspase-2 interacting proteins (including heat shock proteins and co-chaperones) that regulate cellular responses to stress. Finally, we demonstrate that caspase-2 limits chaperone-mediated autophagic degradation of GPX4 to promote the survival of mutant-p53 cancer cells. In conclusion, we document a novel role for caspase-2 as a negative regulator of ferroptosis in cells with mutant p53. Our results provide evidence for a novel function of caspase-2 in cell death regulation and open potential new avenues to exploit ferroptosis in cancer therapy.

Bi-allelic truncating variants in CASP2 underlie a neurodevelopmental disorder with lissencephaly.

Lissencephaly (LIS) is a malformation of cortical development due to deficient neuronal migration and abnormal formation of cerebral convolutions or gyri. Thirty-one LIS-associated genes have been previously described. Recently, biallelic pathogenic variants in CRADD and PIDD1, have associated with LIS impacting the previously established role of the PIDDosome in activating caspase-2. In this report, we describe biallelic truncating variants in CASP2, another subunit of PIDDosome complex. Seven patients from five independent families presenting with a neurodevelopmental phenotype were identified through GeneMatcher-facilitated international collaborations. Exome sequencing analysis was carried out and revealed two distinct novel homozygous (NM_032982.4:c.1156delT (p.Tyr386ThrfsTer25), and c.1174 C > T (p.Gln392Ter)) and compound heterozygous variants (c.[130 C > T];[876 + 1 G > T] p.[Arg44Ter];[?]) in CASP2 segregating within the families in a manner compatible with an autosomal recessive pattern. RNA studies of the c.876 + 1 G > T variant indicated usage of two cryptic splice donor sites, each introducing a premature stop codon. All patients from whom brain MRIs were available had a typical fronto-temporal LIS and pachygyria, remarkably resembling the CRADD and PIDD1-related neuroimaging findings. Other findings included developmental delay, attention deficit hyperactivity disorder, hypotonia, seizure, poor social skills, and autistic traits. In summary, we present patients with CASP2-related ID, anterior-predominant LIS, and pachygyria similar to previously reported patients with CRADD and PIDD1-related disorders, expanding the genetic spectrum of LIS and lending support that each component of the PIDDosome complex is critical for normal development of the human cerebral cortex and brain function.

L-Arginine sulfate reduces irreversible protein binding in immobilized metal affinity chromatography.

Immobilized metal affinity chromatography (IMAC) is a powerful technique for capture and purification of relevant biopharmaceuticals in complex biological matrices. However, protein recovery can be drastically compromised due to surface induced spreading and unfolding of the analyte, leading to fouling of the stationary phase. Here, we report on the kinetics of irreversible adsorption of a protease on an IMAC resin in a time span ranging from minutes to several hours. This trend correlated with the thermal data measured by nano differential scanning calorimetry, and showed a time-dependent change in protein unfolding temperature. Our results highlight that 'soft' proteins show a strong time dependent increase in irreversible adsorption. Furthermore, commonly used co-solvents for preservation of the native protein conformation are tested for their ability to reduce fouling. Thermal data suggests that the amino acid l-arginine is beneficial in preventing unfolding, which was confirmed in batch adsorption experiments. The choice of counter-ions has to be considered when using this amino acid. These results show that l-arginine sulfate decelerates the irreversible adsorption kinetics of proteins on the IMAC stationary phase to a greater extent than l-arginine chloride.

ß-Lapachone Exerts Anticancer Effects by Downregulating p53, Lys-Acetylated Proteins, TrkA, p38 MAPK, SOD1, Caspase-2, CD44 and NPM in Oxaliplatin-Resistant HCT116 Colorectal Cancer Cells.

ß-lapachone (ß-Lap), a topoisomerase inhibitor, is a naturally occurring ortho-naphthoquinone phytochemical and is involved in drug resistance mechanisms. Oxaliplatin (OxPt) is a commonly used chemotherapeutic drug for metastatic colorectal cancer, and OxPt-induced drug resistance remains to be solved to increase chances of successful therapy. To reveal the novel role of ß-Lap associated with OxPt resistance, 5 µM OxPt-resistant HCT116 cells (HCT116-OxPt-R) were generated and characterized via hematoxylin staining, a CCK-8 assay and Western blot analysis. HCT116-OxPt-R cells were shown to have OxPt-specific resistance, increased aggresomes, upregulated p53 and downregulated caspase-9 and XIAP. Through signaling explorer antibody array, nucleophosmin (NPM), CD37, Nkx-2.5, SOD1, H2B, calreticulin, p38 MAPK, caspase-2, cadherin-9, MMP23B, ACOT2, Lys-acetylated proteins, COL3A1, TrkA, MPS-1, CD44, ITGA5, claudin-3, parkin and ACTG2 were identified as OxPt-R-related proteins due to a more than two-fold alteration in protein status. Gene ontology analysis suggested that TrkA, Nkx-2.5 and SOD1 were related to certain aggresomes produced in HCT116-OxPt-R cells. Moreover, ß-Lap exerted more cytotoxicity and morphological changes in HCT116-OxPt-R cells than in HCT116 cells through the downregulation of p53, Lys-acetylated proteins, TrkA, p38 MAPK, SOD1, caspase-2, CD44 and NPM. Our results indicate that ß-Lap could be used as an alternative drug to overcome the upregulated p53-containing OxPt-R caused by various OxPt-containing chemotherapies.

Caspase-2 mRNA levels are not elevated in mild cognitive impairment, Alzheimer's disease, Huntington's disease, or Lewy Body dementia.

Caspase-2 is a member of the caspase family that exhibits both apoptotic and non-apoptotic properties, and has been shown to mediate synaptic deficits in models of several neurological conditions, including Alzheimer's disease (AD), Huntington's disease (HD), and Lewy Body dementia (LBD). Our lab previously reported that caspase-2 protein levels are elevated in these diseases, leading us to hypothesize that elevated caspase-2 protein levels are due to increased transcription of caspase-2 mRNA. There are two major isoforms of caspase-2 mRNA, caspase-2L and caspase-2S. We tested our hypothesis by measuring the levels of these mRNA isoforms normalized to levels of RPL13 mRNA, a reference gene that showed no disease-associated changes. Here, we report no increases in caspase-2L mRNA levels in any of the three diseases studied, AD (with mild cognitive impairment (MCI)), HD and LBD, disproving our hypothesis. Caspase-2S mRNA showed a non-significant downward trend in AD. We also analyzed expression levels of SNAP25 and ßIII-tubulin mRNA. SNAP25 mRNA was significantly lower in AD and there were downward trends in MCI, LBD, and HD. ßIII-tubulin mRNA expression remained unchanged between disease groups and controls. These findings indicate that factors besides transcriptional regulation cause increases in caspase-2 protein levels. The reduction of SNAP25 mRNA expression suggests that presynaptic dysfunction contributes to cognitive deficits in neurodegeneration.

Unveiling caspase-2 regulation by non-coding RNAs.

Non-coding RNAs (ncRNAs) are a group of RNA molecules, such as small nucleolar RNAs, circular RNAs (circRNAs), microRNAs (miRNAs) and long-noncoding RNAs (ncRNAs), that do not encode proteins. Although their biofunctions are not well-understood, many regulatory ncRNAs appear to be highly involved in regulating the transcription and translation of several genes that have essential biological roles including cell differentiation, cell death, metabolism, tumorigenesis and so on. A growing number of studies have revealed the associations between dysregulated ncRNAs and caspases involved in cell death in numerous human diseases. As one of the initiator and executor caspases, caspase-2 is the most evolutionally conserved caspase in mammals, exerting both apoptotic and non-apoptotic functions. A great deal of studies has shown the involvement of caspase-2 as a tumor suppressor in multiple oncogene-driven cancers, and yet a comprehensive understanding of its biological roles remains largely unknown. In this review, we highlight a compilation of studies focused on the interaction between caspase-2 and miRNAs/lncRNAs in the context of different diseases in order to deepen our knowledge on the regulatory biofunctions of caspase-2 and, furthermore, provide more insight into understanding the role that ncRNAs/caspase-2 axis plays in the development of human diseases.

Pro-angiogenic activity of isofuran.

Isofurans (IsoFs) are a series of novel discovered lipid peroxidation products. This study focused on the investigation of the angiogenic property of IsoF. MTT stain assay indicated that 1 µm IsoF had the most bioactivity in rat brain endothelial cells (RBECs). IsoF significantly promoted cellular proliferation and migration and remarkably decreased staurosporine-induced apoptosis by TUNEL assay in the RBECs. It successfully up-regulated rat aortic vascularization and choroid explant sprouting, extracellular regulated protein kinases (ERK)1/2, and triggered calcium release. RT-PCR examination indicated that IsoF up-regulated tumor necrosis factor (TNF)α, angiopoietin-1 receptor (Tie2), and vascular endothelial growth factor (VEGF)-A, but did not interfere with caspase 2 and VEGF-C in the RBECs. IsoF has pro-angiogenic activity. Calcium release and ERK1/2 phosphorylation may be involved in the signaling of the IsoF-induced up-regulation of TNFα, Tie2, and VEGF-A, which could be the molecular mechanism of the pro-angiogenic activity of the IsoF.

Tentatively Identified (UPLC/T-TOF-MS/MS) Compounds in the Extract of Saussurea costus Roots Exhibit In Vivo Hepatoprotection via Modulation of HNF-1α, Sirtuin-1, C/ebpα, miRNA-34a and miRNA-223.

Saussurea costus is a plant traditionally used for the treatment of several ailments. Our study accomplished the UPLC/T-TOF-MS/MS analysis of a methanol extract of Saussurea costus roots (MESC), in addition to lipoidal matter determination and assessment of its in vivo hepatoprotective activity. In this study, we were able to identify the major metabolites in MESC rather than the previously known isolated compounds, improving our knowledge of its chemical constituents. The flavones apigenin, acacetin, baicalein, luteolin, and diosmetin, and the flavonol aglycones quercetin, kaempferol, isorhamnetin, gossypetin, and myricetin and/or their glycosides and glucuronic derivatives were the major identified compounds. The hepatoprotective activity of MESC was evaluated by measuring catalase activity using UV spectrophotometry, inflammatory cytokines and apoptotic markers using ELISA techniques, and genetic markers using PCR. Paracetamol toxicity caused a significant increase in plasma caspase 2, cytokeratin 18 (CK18), liver tumor necrosis factor-α (TNF-α), interleukin 6 (IL-6), miRNA-34a, and miRNA-223, as well as a significant decrease in liver catalase (CAT) activity and in the levels of liver nuclear factor 1α (HNF-1α), sirtuin-1, and C/ebpα. Oral pretreatment with MESC (200 mg/kg) showed a significant decrease in caspase 2, CK18, TNF-α, IL-6 and a significant increase in liver CAT activity. MESC decreased the levels of liver miRNA-34a and miRNA-223 and induced HNF-1α, sirtuin-1, and C/ebpα gene expression. The histological examination showed a significant normalization in rats pretreated with MESC. Our findings showed that Saussurea costus may exert a potent hepatoprotective activity through the modulation of the expression of cellular cytokines, miRNA-34a, and miRNA-223.

The caspase-2 substrate p54nrb exhibits a multifaceted role in tumor cell death susceptibility via gene regulatory functions.

Caspase-2 represents an evolutionary conserved caspase, which plays a role in genotoxic stress-induced apoptosis, ageing-related metabolic changes, and in deleting aneuploid cells in tumors. Genetic deletion of caspase-2 leads to increased tumor susceptibility in vivo. The exact downstream signaling mechanism by which caspase-2 accomplishes its specific tumor suppressor functions is not clear. Caspase-2, uniquely among caspases, resides in the nucleus and other cellular compartments. In this study, we identify a nuclear caspase-2 specific substrate, p54nrb, which is selectively cleaved by caspase-2 at D422, leading to disruption of the C-terminal site, the putative DNA binding region of the protein. P54nrb is an RNA and DNA binding protein, which plays a role in RNA editing, transport, and transcriptional regulation of genes. Overexpression of p54nrb is observed in several human tumor types, such as cervix adenocarcinoma, melanoma, and colon carcinoma. In contrast, the loss of p54nrb in tumor cell lines leads to increased cell death susceptibility and striking decrease in tumorigenic potential. By employing high resolution quantitative proteomics, we demonstrate that the loss/cleavage of p54nrb results in altered expression of oncogenic genes, among which the downregulation of the tumorigenic protease cathepsin-Z and the anti-apoptotic gelsolin can be detected universally across three tumor cell types, including adenocarcinoma, melanoma and colon carcinoma. Finally, we demonstrate that p54nrb interacts with cathepsin-Z and gelsolin DNA, but not RNA. Taken together, this study uncovers a so far not understood mechanism of caspase-2 tumor suppressor function in human tumor cells.

miR-150-3p enhances neuroprotective effects of neural stem cell exosomes after hypoxic-ischemic brain injury by targeting CASP2.

Brains are vulnerable to ischemic/hypoxic damage, which are directly caused by stroke, hypoxic-ischemic encephalopathy and other cerebral diseases. Currently, therapeutic strategies against cerebral ischemia and hypoxia are extremely limited. Recent studies have indicated that stem cell-derived exosomes play a neuroprotective role in hypoxic-ischemic brain injury. However, the treatment mechanism remains unclear. In this study, we cultured neural stem cells (NSCs) in vitro successfully. Exosomes isolated from NSCs (NSCs-Ex) inhibited the apoptosis while promoting the proliferation of SH-SY5Y cells both in normal and oxygen-glucose deprivation (OGD) culture conditions. Moreover, in vivo studies demonstrated that NSCs-Ex significantly reduced the infarction area in the middle cerebral artery occlusion (MCAO) model and suppressed the apoptosis of neurons. Furthermore, miR-150-3p was identified as the most abundantly expressed miRNA in exosomes compared to their parent NSCs. The miR-150-3p mimic displayed neuroprotective effects while miR-150-3p inhibitor exacerbated nerve injury both in vivo and in vitro. We further identified CASP2 as a miR-150-3p target. Thus, our data indicate that NSC-Ex facilitate the neuroprotective effects via transfer of miR-150-3p which targets CASP2, thus suppressing neuronal apoptosis after brain injury. Our results suggest that NSCs-Ex prevent cerebral injury by transferring miR-150-3p which promotes neurons proliferation by inhibiting CASP2 signaling pathway.

Non-apoptotic activation of Drosophila caspase-2/9 modulates JNK signaling, the tumor microenvironment, and growth of wound-like tumors.

Resistance to apoptosis due to caspase deregulation is considered one of the main hallmarks of cancer. However, the discovery of novel non-apoptotic caspase functions has revealed unknown intricacies about the interplay between these enzymes and tumor progression. To investigate this biological problem, we capitalized on a Drosophila tumor model with human relevance based on the simultaneous overactivation of the EGFR and the JAK/STAT signaling pathways. Our data indicate that widespread non-apoptotic activation of initiator caspases limits JNK signaling and facilitates cell fate commitment in these tumors, thus preventing the overgrowth and exacerbation of malignant features of transformed cells. Intriguingly, caspase activity also reduces the presence of macrophage-like cells with tumor-promoting properties in the tumor microenvironment. These findings assign tumor-suppressing activities to caspases independent of apoptosis, while providing molecular details to better understand the contribution of these enzymes to tumor progression.

Use of the ß-Glucan-Producing Lactic Acid Bacteria Strains Levilactobacillus brevis and Pediococcus claussenii for Sourdough Fermentation-Chemical Characterization and Chemopreventive Potential of In Situ-Enriched Wheat and Rye Sourdoughs and Breads.

The aim of the present study was to examine ß-glucan production and the potential prebiotic and chemopreventive effects of wheat and rye sourdoughs and breads generated with wild-type and non-ß-glucan-forming isogenic mutant strains of Levilactobacillus brevis and Pediococcus claussenii. Sourdough and bread samples were subjected to in vitro digestion and fermentation. Fermentation supernatants (FS) and pellets (FP) were analyzed (pH values, short-chain fatty acids (SCFA), ammonia, bacterial taxa) and the effects of FS on LT97 colon adenoma cell growth, viability, caspase-2 and -3 activity, genotoxic and antigenotoxic effects and on gene and protein expression of p21, cyclin D2, catalase and superoxide dismutase 2 (SOD2) were examined. Concentrations of SCFA were increased and concentrations of ammonia were partly reduced in the FS. The relative abundance of Bifidobacteriaceae was increased in all FPs. Treatment with FS reduced the growth and viability of LT97 cells and significantly increased caspase-2 and -3 activities without exhibiting genotoxic or antigenotoxic effects. The p21 mRNA and protein levels were increased while that of cyclin D2 was reduced. Catalase and SOD2 mRNA and protein expression were marginally induced. The presented results indicate a comparable chemopreventive potential of wheat and rye sourdoughs and breads without an additional effect of the formed ß-glucan.

PIDD1 in cell cycle control, sterile inflammation and cell death.

The death fold domain-containing protein PIDD1 has recently attracted renewed attention as a regulator of the orphan cell death-related protease, Caspase-2. Caspase-2 can activate p53 to promote cell cycle arrest in response to centrosome aberrations, and its activation requires formation of the PIDDosome multi-protein complex containing multimers of PIDD1 and the adapter RAIDD/CRADD at its core. However, PIDD1 appears to be able to engage with multiple client proteins to promote an even broader range of biological responses, such as NF-κB activation, translesion DNA synthesis or cell death. PIDD1 shows features of inteins, a class of self-cleaving proteins, to create different polypeptides from a common precursor protein that allow it to serve these diverse functions. This review summarizes structural information and molecular features as well as recent experimental advances that highlight the potential pathophysiological roles of this unique death fold protein to highlight its drug-target potential.

lncRNA GMDS­AS1 upregulates IL­6, TNF­α and IL­1ß, and induces apoptosis in human monocytic THP­1 cells via miR­96­5p/caspase 2 signaling.

Long non­coding RNA (lncRNA) is considered a crucial modulator of the initiation and progression of several diseases. However, the roles of lncRNA in sepsis have yet to be fully elucidated. Thus, the aim of the present study was to investigate the effects of the lncRNA GDP­mannose 4,6­dehydratase antisense 1 (GMDS­AS1) and its target in order to understand its role in the pathogenesis of sepsis. An in vitro sepsis model was established by lipopolysaccharide (LPS) induction. Reverse transcription­quantitative PCR analysis was applied to detect the expression of inflammatory cytokines and the levels of GMDS­AS1, microRNA (miR)­96­5p and caspase­2 (CASP2). Flow cytometry was used to quantify the rate of apoptosis. In addition, the interaction between miR­96­5p and CASP2 was verified using a luciferase reporter assay. Western blot analysis was performed to assess the protein levels of CASP2 following alterations in GMDS­AS1 and miR­96­5p expression using transfection. The levels of interleukin (IL)­6, tumor necrosis factor­α and IL­1ß were increased by LPS treatment in THP­1 cells, whereas miR­96­5p expression was downregulated. miR­96­5p overexpression inhibited LPS­induced inflammatory responses and apoptosis. In addition, GMDS­AS1 expression increased, and upregulation of GMDS­AS1 inhibited, the expression of miR­96­5p in the in vitro sepsis model. Moreover, CASP2 was confirmed to be a direct target of miR­96­5p. Therefore, the lncRNA GMDS­AS1 regulated inflammatory responses and apoptosis by modulating CASP2 and sponging miR­96­5p in LPS­induced THP­1 cells. In summary, the findings of the present study demonstrated that lncRNA GMDS­AS1 could promote the development of sepsis by targeting miR­96­5p/CASP2, indicating that the GMDS­AS1/miR­96­5p/CASP2 axis may be a new therapeutic target and potential research direction for sepsis therapy.

Caspase-2 regulates S-phase cell cycle events to protect from DNA damage accumulation independent of apoptosis.

In addition to its classical role in apoptosis, accumulating evidence suggests that caspase-2 has non-apoptotic functions, including regulation of cell division. Loss of caspase-2 is known to increase proliferation rates but how caspase-2 is regulating this process is currently unclear. We show that caspase-2 is activated in dividing cells in G1-phase of the cell cycle. In the absence of caspase-2, cells exhibit numerous S-phase defects including delayed exit from S-phase, defects in repair of chromosomal aberrations during S-phase, and increased DNA damage following S-phase arrest. In addition, caspase-2-deficient cells have a higher frequency of stalled replication forks, decreased DNA fiber length, and impeded progression of DNA replication tracts. This indicates that caspase-2 protects from replication stress and promotes replication fork protection to maintain genomic stability. These functions are independent of the pro-apoptotic function of caspase-2 because blocking caspase-2-induced cell death had no effect on cell division, DNA damage-induced cell cycle arrest, or DNA damage. Thus, our data supports a model where caspase-2 regulates cell cycle and DNA repair events to protect from the accumulation of DNA damage independently of its pro-apoptotic function.

miR-2 contributes to WSSV infection by targeting Caspase 2 in mud crab (Scylla paramamosain).

Caspase 2 is widely studied for its function in the regulation of apoptosis in mammals. Despite the fundamental role of apoptosis during the anti-viral immune response, the relationship between Caspase 2 and virus infection has not been extensively explored in invertebrates. Also, whether or not miRNAs involve this process remains unclear. To address this issue, the miRNA-mediated regulation of Caspase 2 in mud crab (Scylla paramamosain) (Sp-Caspase 2) was characterized in this study. Sp-Caspase 2 contains an open reading frame (ORF) of 969 bp encoding 322 deduced amino acids and possesses a conserved CASc domain. The results suggested that Sp-Caspase 2 could suppress white spot syndrome virus infection via apoptosis induction. The further data showed that Sp-Caspase 2 was directly targeted by miR-2 in mud crab. Silencing or overexpression of miR-2 could affect apoptosis and WSSV replication through the regulation of Sp-Caspase 2 expression. Taken together, these results demonstrated the crucial role of the miR-2-Caspase 2 pathway in the innate immunity of mud crabs and revealed a novel mechanism in the anti-viral immune response in marine invertebrates.

Programmed Cell Death in Health and Disease.

Programmed cell death is a conserved evolutionary process of cell suicide that is central to the development and integrity of eukaryotic organisms [...].

PROFICS: A bacterial selection system for directed evolution of proteases.

Proteases serve as important tools in biotechnology and as valuable drugs or drug targets. Efficient protein engineering methods to study and modulate protease properties are thus of great interest for a plethora of applications. We established PROFICS (PRotease Optimization via Fusion-Inhibited Carbamoyltransferase-based Selection), a bacterial selection system, which enables the optimization of proteases for biotechnology, therapeutics or diagnosis in a simple overnight process. During the PROFICS process, proteases are selected for their ability to specifically cut a tag from a reporter enzyme and leave a native N-terminus. Precise and efficient cleavage after the recognition sequence reverses the phenotype of an Escherichia coli knockout strain deficient in an essential enzyme of pyrimidine synthesis. A toolbox was generated to select for proteases with different preferences for P1' residues (the residue immediately following the cleavage site). The functionality of PROFICS is demonstrated with viral proteases and human caspase-2. PROFICS improved caspase-2 activity up to 25-fold after only one round of mutation and selection. Additionally, we found a significantly improved tolerance for all P1' residues caused by a mutation in a substrate interaction site. We showed that this improved activity enables cells containing the new variant to outgrow cells containing all other mutants, facilitating its straightforward selection. Apart from optimizing enzymatic activity and P1' tolerance, PROFICS can be used to reprogram specificities, erase off-target activity, optimize expression via tags/codon usage, or even to screen for potential drug-resistance-conferring mutations in therapeutic targets such as viral proteases in an unbiased manner.