Chilean salmon farming confronts persistent challenges, including climate change risks and pathogens, where the most prevalent diseases impacting Atlantic salmon are Caligidosis and Rickettsial Salmonid Septicemia (SRS). As a sustainable strategy, fish vaccines hold promise for preventing diseases and reducing the use of antibiotics. While most studies on Atlantic salmon responses to vaccines emphasize transcriptome profiling from tissues such as the liver, head kidney, and skin, blood cell transcriptomics to monitor immune response dynamics is emerging as a promising tool in salmon aquaculture. This study evaluated the Atlantic salmon blood cell transcriptome in response to vaccination and subsequent infection with the sea louse Caligus rogercresseyi and the intracellular bacterium Piscirickettsia salmonis. The vaccination trial included four groups: fish immunized with the recombinant IPath® vaccine and two commercial vaccines currently used in Chile for salmon production. (BlueGuard® and Alpha Ject LiVac® SRS), and the unvaccinated control group. The group vaccinated with IPath® showed a higher transcriptomic response than commercial vaccines. Additionally, all three groups significantly modulated genes associated with iron homeostasis and metabolism. Furthermore, the HIF-1 signaling pathway and ferroptosis were notably activated in the IPath® group, suggesting a potential role of IPath® in the hypoxia response and cell death. This research highlights the effectiveness of using Atlantic salmon blood cells to assess immune responses, offering valuable insights into the fish immune system without resorting to lethal sampling.

Caspases are critical regulators of cell death, development, innate immunity, host defense, and disease. Upon detection of pathogens, damage-associated molecular patterns, cytokines, or other homeostatic disruptions, innate immune sensors, such as NLRs, activate caspases to initiate distinct regulated cell death pathways, including non-lytic (apoptosis) and innate immune lytic (pyroptosis and PANoptosis) pathways. These cell death pathways are driven by specific caspases and distinguished by their unique molecular mechanisms, supramolecular complexes, and enzymatic properties. Traditionally, caspases are classified as either apoptotic (caspase-2, -3, -6, -7, -8, -9, and -10) or inflammatory (caspase-1, -4, -5, and -11). However, extensive data from the past decades have shown that apoptotic caspases can also drive lytic inflammatory cell death downstream of innate immune sensing and inflammatory responses, such as in the case of caspase-3, -6, -7, and -8. Therefore, more inclusive classification systems based on function, substrate specificity, or the presence of pro-domains have been proposed to better reflect the multifaceted roles of caspases. In this review, we categorize caspases into CARD-, DED-, and short/no pro-domain-containing groups and examine their critical functions in innate immunity and cell death, along with their structural and molecular mechanisms, including active site/exosite properties and substrates. Additionally, we highlight the emerging roles of caspases in cellular homeostasis and therapeutic targeting. Given the clinical relevance of caspases across multiple diseases, improved understanding of these proteins and their structure-function relationships is critical for developing effective treatment strategies.

In mammals, caspase 8 (CASP8) is a well-known initiator caspase of apoptosis. In invertebrates, the function of CASP8 is poorly understood. Herein, we examined the function of abalone Haliotis discus CASP8 (HdCASP8). Compared to mammalian CASP8, HdCASP8 possesses the conserved DED and CASc domains but also has an extra death domain (DD). HdCASP8 induced marked apoptosis of HEK293T cells without activating CASP3/6/7. Consistently, HdCASP8 did not cleave H. discus CASP3 (HdCASP3). HdCASP8 exhibited CASP3/6-like cleavage specificity and cleaved the apoptotic substrate DFF45. HdCASP3 is known to activate abalone pyroptosis by cleaving H. discus gasdermin E (HdGSDME) at two sites, DQVD and DEID. In the present work, HdCASP8 was found to interact with HdGSDME at its C-terminal region and induce pyroptosis by cleaving HdGSDME at DQVD but not at DEID. During bacterial infection, the expressions of HdCASP8 and HdGSDME were significantly upregulated in multiple tissues of abalone in a time-dependent manner. Together these results indicate that, most likely owing to its unique structural feature, HdCASP8 differs from the classical CASP8 by acting as an apoptosis/pyroptosis-regulating CASP3 and from the classical CASP3 in certain aspects of substrate specificity. These findings provide new insights into CASP8-mediated programmed cell death in invertebrates.

Background: Prostate cancer, as an androgen-dependent malignant tumor in older men, has attracted the attention of a wide range of clinicians. BCR remains a significant challenge following early prostate cancer treatment. Methods: The specific expression pattern of the Anoikis gene set in prostate cancer cells was first explored by single-cell and spatial transcriptomics analysis. Genes causally associated with prostate cancer were screened using Summary-data-based Mendelian Randomization (SMR). Subsequently, we explored the role and mechanism of CASP8 in prostate cancer cells and defined a new cell type: the CASP8 T cell. We constructed a prediction model that can better predict the BCR of prostate cancer, and explored the differences in various aspects of clinical subgroups, tumor microenvironments, immune checkpoints, drug sensitivities, and tumor-immune circulations between high- and low-risk groups. The results of SMR analysis indicated that CASP8 could increase the risk of prostate cancer. Based on the differential genes of CASP8-positive and -negative T cells, we constructed a four-gene prognostic model with a 5-year AUC of 0.713. Results: The results revealed that high-risk prostate cancer BCR patients had various characteristics such as higher tumor purity, higher BCR rate, downregulated SIRPA immune checkpoints, and unique drug sensitivity. Conclusions: In summary, CASP8 may be a potential biomarker for prostate cancer.

Caspases are a family of cysteine proteases that act as molecular scissors to cleave substrates and regulate biological processes such as programmed cell death and inflammation. Extensive efforts have been made to identify caspase substrates and to determine factors that dictate substrate specificity. We recently discovered that that the human inflammatory caspases (caspases-1, -4, and -5) cleave the cytokines IL-1β and IL-18 in a sequence-dependent manner. Here, we report the development of a new peptide-based probe and inhibitor based on the tetrapeptide sequence of IL-18 (LESD). We found that this inhibitor was most selective and potent at inhibiting caspase-8 activity (IC50 = 50 nM). We also discovered that our LESD-based inhibitor is more potent than the currently used z-IETD-FMK inhibitor that is thought to be the most selective and potent inhibitor of caspase-8. Accordingly, we demonstrate that the LESD based inhibitor prevents caspase-8 activation during Yersinia pseudotuberculosis infection in primary bone-marrow derived macrophages. Furthermore, we characterize the selectivity and potency of currently known substrates and inhibitors for the apoptotic and inflammatory caspases using the same activity units of each caspase. Our findings reveal that VX-765, a known caspase-1 inhibitor, also inhibits caspase-8 (IC50 = 1 μM) and even when specificities are shared, the caspases have different efficiencies and potencies for shared substrates and inhibitors. Altogether, we report the development of new tools that will facilitate the study of caspases and their roles in biology.

Protein ubiquitination plays a critical role in protein quality control in response to cellular stress. The excessive accumulation of ubiquitinated conjugates can be detrimental to cells and is recognized as a hallmark of multiple neurodegenerative diseases. However, an in-depth understanding of how the excessive ubiquitin chains are removed to maintain ubiquitin homeostasis post stress remains largely unclear. Here we found that caspase-2 (CASP2) accumulates in a ubiquitin and proteasome-positive biomolecular condensate, which we named ubstressome, following stress and functions as a deubiquitinase to remove overloaded ubiquitin chains on proteins prone to misfolding. Mechanistically, CASP2 binds to the poly-ubiquitinated conjugates through its allosteric ubiquitin-interacting motif-like region and decreases overloaded ubiquitin chains in a protease-dependent manner to promote substrate degradation. CASP2 deficiency in mice results in excessive accumulation of poly-ubiquitinated TAR DNA-binding protein 43, leading to motor defects. Our findings uncover a stress-evoked deubiquitinating activity of CASP2 in the maintenance of cellular ubiquitin homeostasis, which differs from the well-known roles of caspase in apoptosis and inflammation. These data also reveal unrecognized protein quality control functions of condensates in the removal of stress-induced ubiquitin chains.

Caspases are a family of protease enzymes that play a crucial role in apoptosis. Dysregulation of caspase activity has been implicated in various pathological conditions, making caspases an important focus of research in understanding cell death mechanisms and developing therapeutic strategies for diseases associated with abnormal apoptosis.

It is a comprehensive review of caspase inhibitors that have been comprising recently granted patents from 2016 to 2023. It includes peptide and non-peptide caspase inhibitors with their application for different diseases.

This review categorizes and analyses recently patented caspase inhibitors on various diseases. Diseases linked to caspase dysregulation, including neurodegenerative disorders, and autoimmune conditions, are highlighted to accentuate the therapeutic relevance of the patented caspase inhibitors. This paper serves as a valuable resource for researchers, clinicians, and pharmaceutical developers seeking an up-to-date understanding of recently patented caspase inhibitors. The integration of recent patented compounds, structural insights, and mechanistic details provides a holistic view of the progress in caspase inhibitor research and its potential impact on addressing various diseases.

Caspase-1 plays a central role in innate immunity, as its activation by inflammasomes induces the production of proinflammatory cytokines and pyroptosis. However, specific inhibition of the enzymatic activity of this protease is not effective in suppressing inflammation, owing to its enzyme-independent function. Herein, we identified a cyclohexenyl isothiocyanate compound (CIB-1476) that potently inhibited caspase-1 activity and suppressed the assembly and activation of the NLRP3 inflammasome and gasdermin-D-mediated pyroptosis. Mechanistically, CIB-1476 directly targeted pro-caspase-1 as an irreversible covalent inhibitor by binding to Cys285 and Cys397, resulting in more durable anti-inflammatory effects in the suppression of enzyme-dependent IL-1β production and enzyme-independent nuclear factor κB activation. Chemoproteomic profiling demonstrated the engagement of CIB-1476 with caspase-1. CIB-1476 showed potent therapeutic effects by suppressing inflammasome activation in mice, which was abolished in Casp1-/- mice. These results warrant further development of CIB-1476 along with its analogues as a novel strategy for caspase-1 inhibitors.

Caspases are a family of cysteine proteases that act as molecular scissors to cleave substrates and regulate biological processes such as programmed cell death and inflammation. Extensive efforts have been made to identify caspase substrates and to determine factors that dictate substrate specificity. Thousands of putative substrates have been identified for caspases that regulate an immunologically silent type of cell death known as apoptosis, but less is known about substrates of the inflammatory caspases that regulate an immunostimulatory type of cell death called pyroptosis. Furthermore, much of our understanding of caspase substrate specificities is derived from work done with peptide substrates, which do not often translate to native protein substrates. Our knowledge of inflammatory caspase biology and substrates has recently expanded and here, we discuss the recent advances in our understanding of caspase substrate specificities, with a focus on inflammatory caspases. We highlight new substrates that have been discovered and discuss the factors that engender specificity. Recent evidence suggests that inflammatory caspases likely utilize two binding interfaces to recognize and process substrates, the active site and a conserved exosite.

Caspase-1 is one of the main mediators of inflammatory caspases and has become a correspondent with inflammation, cell death, and several inflammatory diseases. In this review, we systematically summarize both original and recent advances in caspase-1 to provide references for a better understanding of the molecular mechanisms in its activation and functions. This study investigates and summarizes the published articles concerning caspase-1, inflammation, pyroptosis, apoptosis, and cell death by searching academic search systems, including the PubMed, Web of Science, and Google Scholar. Caspase-1 is one of the main mediators of inflammatory caspases and has become a correspondent with inflammation and cell death. In cell death, caspase-1 was originally found to cause apoptosis in fibroblasts. Importantly, caspase-1 was later reported to execute programmed cell death, including pyroptosis and apoptosis, in many immune cells in response to diverse stimuli. It is widely established that different pathways can activate caspase-1 and subsequently mediate cell death and inflammation. It has become increasingly clear that caspase-1 is responsible for the initiation and control of pyroptosis, apoptosis, and inflammation in addition to its well-known function in cleaving IL-1β. The significant advancement in the understanding of caspase-1-controlled cell death and novel substrates inspires new therapeutic approaches in the future.

Retinal diseases encompass various conditions associated with sight-threatening immune responses and are leading causes of blindness worldwide. These diseases include age-related macular degeneration, diabetic retinopathy, glaucoma and uveitis. Emerging evidence underscores the vital role of the innate immune response in retinal diseases, beyond the previously emphasized T-cell-driven processes of the adaptive immune system. In particular, pyroptosis, a newly discovered programmed cell death process involving inflammasome formation, has been implicated in the loss of membrane integrity and the release of inflammatory cytokines. Several disease-relevant animal models have provided evidence that the formation of inflammasomes and the induction of pyroptosis in innate immune cells contribute to inflammation in various retinal diseases. In this review article, we summarize current knowledge about the innate immune system and pyroptosis in retinal diseases. We also provide insights into translational targeting approaches, including novel drugs countering pyroptosis, to improve the diagnosis and treatment of retinal diseases.

Caspases are a highly conserved family of cysteine-aspartyl proteases known for their essential roles in regulating apoptosis, inflammation, cell differentiation, and proliferation. Complementary to genetic approaches, small-molecule probes have emerged as useful tools for modulating caspase activity. However, due to the high sequence and structure homology of all 12 human caspases, achieving selectivity remains a central challenge for caspase-directed small-molecule inhibitor development efforts. Here, using mass spectrometry-based chemoproteomics, we first identify a highly reactive noncatalytic cysteine that is unique to caspase-2. By combining both gel-based activity-based protein profiling (ABPP) and a tobacco etch virus (TEV) protease activation assay, we then identify covalent lead compounds that react preferentially with this cysteine and afford a complete blockade of caspase-2 activity. Inhibitory activity is restricted to the zymogen or precursor form of monomeric caspase-2. Focused analogue synthesis combined with chemoproteomic target engagement analysis in cellular lysates and in cells yielded both pan-caspase-reactive molecules and caspase-2 selective lead compounds together with a structurally matched inactive control. Application of this focused set of tool compounds to stratify the functions of the zymogen and partially processed (p32) forms of caspase-2 provide evidence to support that caspase-2-mediated response to DNA damage is largely driven by the partially processed p32 form of the enzyme. More broadly, our study highlights future opportunities for the development of proteoform-selective caspase inhibitors that target nonconserved and noncatalytic cysteine residues.

The cyclic-oligonucleotide-based anti-phage signalling system (CBASS) is a type of innate prokaryotic immune system. Composed of a cyclic GMP-AMP synthase (cGAS) and CBASS-associated proteins, CBASS uses cyclic oligonucleotides to activate antiviral immunity. One major class of CBASS contains a homologue of eukaryotic ubiquitin-conjugating enzymes, which is either an E1-E2 fusion or a single E2. However, the functions of single E2s in CBASS remain elusive. Here, using biochemical, genetic, cryo-electron microscopy and mass spectrometry investigations, we discover that the E2 enzyme from Serratia marcescens regulates cGAS by imitating the ubiquitination cascade. This includes the processing of the cGAS C terminus, conjugation of cGAS to a cysteine residue, ligation of cGAS to a lysine residue, cleavage of the isopeptide bond and poly-cGASylation. The poly-cGASylation activates cGAS to produce cGAMP, which acts as an antiviral signal and leads to cell death. Thus, our findings reveal a unique regulatory role of E2 in CBASS.

Our epithelium represents a battle ground against a variety of insults including pathogens and danger signals. It encodes multiple sensors that detect and respond to such insults, playing an essential role in maintaining and defending tissue homeostasis. One key set of defense mechanisms is our inflammasomes which drive innate immune responses including, sensing and responding to pathogen attack, through the secretion of pro-inflammatory cytokines and cell death. Identification of physiologically relevant triggers for inflammasomes has greatly influenced our ability to decipher the mechanisms behind inflammasome activation. Furthermore, identification of patient mutations within inflammasome components implicates their involvement in a range of epithelial diseases. This review will focus on exploring the roles of inflammasomes in epithelial immunity and cover: the diversity and differential expression of inflammasome sensors amongst our epithelial barriers, their ability to sense local infection and damage and the contribution of the inflammasomes to epithelial homeostasis and disease.

The protein scaffold that includes the caspases is ancient and found in all domains of life. However, the stringent specificity that defines the caspase biologic function is relatively recent and found only in multicellular animals. During the radiation of the Chordata, members of the caspase family adopted roles in immunity, events coinciding with the development of substrates that define the modern innate immune response. This review focuses on the switch from the non-inflammatory cellular demise of apoptosis to the highly inflammatory innate response driven by distinct members of the caspase family, and the interplay between these two regulated cell death pathways.

Caspase-5 is a protease that induces inflammation in response to lipopolysaccharide (LPS), a component of the cell envelope of Gram-negative bacteria. The expression level of the CASP5 gene is very low in the basal state, but strongly increases in the presence of LPS. Intracellular LPS binds to the caspase activation and recruitment domain (CARD) of caspase-5, leading to the formation of a non-canonical inflammasome. Subsequently, the catalytic domain of caspase-5 cleaves gasdermin D and thereby facilitates the formation of cell membrane pores through which pro-inflammatory cytokines of the interleukin-1 family are released. Caspase-4 is also able to form a non-canonical inflammasome upon binding to LPS, but its expression is less dependent on LPS than the expression of caspase-5. Caspase-4 and caspase-5 have evolved via the duplication of a single ancestral gene in a subclade of primates, including humans. Notably, the main biomedical model species, the mouse, has only one ortholog, namely caspase-11. Here, we review the structural features and the mechanisms of regulation that are important for the pro-inflammatory roles of caspase-5. We summarize the interspecies differences and the evolution of pro-inflammatory caspases in mammals and discuss the potential roles of caspase-5 in the defense against Gram-negative bacteria and in sepsis.

Caspase (CASP) is a protease family that plays a vital role in apoptosis, development, and immune response. Herein, we reported the identification and characterization of two CASPs, AjCASPX1 and AjCASPX2, from the sea cucumber Apostichopus japonicus, an important aquaculture species. AjCASPX1/2 share similar domain organizations with the vertebrate initiator caspases CASP2/9, including the CARD domain and the p20/p10 subunits with conserved functional motifs. However, compared with human CASP2/9, AjCASPX1/2 possess unique structural features in the linker region between p20 and p10. AjCASPX1, but not AjCASPX2, induced marked apoptosis of human cells by activating CASP3/7. The recombinant proteins of AjCASPX2 and the CARD domain of AjCASPX2 were able to bind to a wide range of bacteria, as well as bacterial cell wall components, and inhibit bacterial growth. AjCASPX1, when expressed in Escherichia coli, was able to kill the host bacteria. Under normal conditions, AjCASPX1 and AjCASPX2 expressions were most abundant in sea cucumber muscle and coelomocytes, respectively. After bacterial infection, both AjCASPX1 and AjCASPX2 expressions were significantly upregulated in sea cucumber tissues and cells. Together, these results indicated that AjCASPX1 and AjCASPX2 were initiator caspases with antimicrobial activity and likely functioned in apoptosis and immune defense against pathogen infection.

Citrus sinensis (L.) Osbeck (Rutaceae), commonly known as the sweet orange, is a popular and widely consumed fruit with several medicinal properties. The present study aimed to perform the in silico screening of 18 flavonoids and eight volatile components from the peel of C. sinensis against apoptotic and inflammatory proteins, metalloprotease, and tumor suppressor markers. Flavonoids obtained higher probabilities than volatile components against selected anti-cancer drug targets. Hence, the data from the binding energies against the essential apoptotic and cell proliferation proteins substantiate that they may be promising compounds in developing effective candidates to block cell growth, proliferation, and induced cell death by activating the apoptotic pathway. Further, the binding stability of the selected targets and the corresponding molecules were analyzed by 100 ns molecular dynamics (MD) simulations. Chlorogenic acid has the most binding affinity against the important anti-cancer targets iNOS, MMP-9, and p53. The congruent binding mode to different drug targets focused on cancer shown by chlorogenic acid suggests that it may be a compound with significant therapeutic potential. Moreover, the binding energy predictions indicated that the compound had stable electrostatic and van der Waal energies. Thus, our data reinforce the medicinal importance of flavonoids from C. sinensis and expand the need for more studies, seeking to optimize results and amplify the impacts of further in vitro and in vivo studies. Communicated by Ramaswamy H. Sarma.

We describe bedside-to-bench immunological and genetic elucidation of defective pyroptosis attributable to novel caspase 4 defect mediating pathogen-triggered inflammatory programmed cell death, in the setting of severe pneumonia and abscess-forming melioidosis in an overtly healthy host failing to clear Burkholderia pseudomallei infection, and how targeted adjunctive biological therapy led to a successful outcome.

Peptides and peptide aptamers have emerged as promising molecules for a wide range of biomedical applications due to their unique properties and versatile functionalities. The screening strategies for identifying peptides and peptide aptamers with desired properties are discussed, including high-throughput screening, display screening technology, and in silico design approaches. The synthesis methods for the efficient production of peptides and peptide aptamers, such as solid-phase peptide synthesis and biosynthesis technology, are described, along with their advantages and limitations. Moreover, various modification techniques are explored to enhance the stability, specificity, and pharmacokinetic properties of peptides and peptide aptamers. This includes chemical modifications, enzymatic modifications, biomodifications, genetic engineering modifications, and physical modifications. Furthermore, the review highlights the diverse biomedical applications of peptides and peptide aptamers, including targeted drug delivery, diagnostics, and therapeutic. This review provides valuable insights into the advancements in screening, synthesis, modification, and biomedical applications of peptides and peptide aptamers. A comprehensive understanding of these aspects will aid researchers in the development of novel peptide-based therapeutics and diagnostic tools for various biomedical challenges.

Caspases are a highly conserved family of cysteine-aspartyl proteases known for their essential roles in regulating apoptosis, inflammation, cell differentiation, and proliferation. Complementary to genetic approaches, small-molecule probes have emerged as useful tools for modulating caspase activity. However, due to the high sequence and structure homology of all twelve human caspases, achieving selectivity remains a central challenge for caspase-directed small-molecule inhibitor development efforts. Here, using mass spectrometry-based chemoproteomics, we first identify a highly reactive non-catalytic cysteine that is unique to caspase-2. By combining both gel-based activity-based protein profiling (ABPP) and a tobacco etch virus (TEV) protease activation assay, we then identify covalent lead compounds that react preferentially with this cysteine and afford a complete blockade of caspase-2 activity. Inhibitory activity is restricted to the zymogen or precursor form of monomeric caspase-2. Focused analogue synthesis combined with chemoproteomic target engagement analysis in cellular lysates and in cells yielded both pan-caspase reactive molecules and caspase-2 selective lead compounds together with a structurally matched inactive control. Application of this focused set of tool compounds to stratify caspase contributions to initiation of intrinsic apoptosis, supports compensatory caspase-9 activity in the context of caspase-2 inactivation. More broadly, our study highlights future opportunities for the development of proteoform-selective caspase inhibitors that target non-conserved and non-catalytic cysteine residues.

The noncanonical inflammasome is a signalling complex critical for cell defence against cytosolic Gram-negative bacteria. A key step in the human noncanonical inflammasome pathway involves unleashing the proteolytic activity of caspase-4 within this complex. Caspase-4 induces inflammatory responses by cleaving gasdermin-D (GSDMD) to initiate pyroptosis; however, the molecular mechanisms that activate caspase-4 and govern its capacity to cleave substrates remain poorly defined. Caspase-11, the murine counterpart of caspase-4, acquires protease activity within the noncanonical inflammasome by forming a dimer that self-cleaves at D285 to cleave GSDMD. These cleavage events trigger signalling via the NLRP3-ASC-caspase-1 axis, leading to downstream cleavage of the pro-IL-1β cytokine precursor. Here, we show that caspase-4 first dimerises then self-cleaves at two sites-D270 and D289-in the interdomain linker to acquire full proteolytic activity, cleave GSDMD, and induce cell death. Surprisingly, caspase-4 dimerisation and self-cleavage at D289 generate a caspase-4 p34/p9 protease species that directly cleaves pro-IL-1β, resulting in its maturation and secretion independently of the NLRP3 inflammasome in primary human myeloid and epithelial cells. Our study thus elucidates the key molecular events that underpin signalling by the caspase-4 inflammasome and identifies IL-1β as a natural substrate of caspase-4.

Ischemic heart disease remains a leading cause of human mortality worldwide. One form of ischemic heart disease is ischemia-reperfusion injury caused by the reintroduction of blood supply to ischemic cardiac muscle. The short and long-term damage that occurs due to ischemia-reperfusion injury is partly due to the proteolysis of diverse protein substrates inside and outside of cardiomyocytes. Ischemia-reperfusion activates several diverse intracellular proteases, including, but not limited to, matrix metalloproteinases, calpains, cathepsins, and caspases. This review will focus on the biological roles, intracellular localization, proteolytic targets, and inhibitors of these proteases in cardiomyocytes following ischemia-reperfusion injury. Recognition of the intracellular function of each of these proteases includes defining their activation, proteolytic targets, and their inhibitors during myocardial ischemia-reperfusion injury. This review is a step toward a better understanding of protease activation and involvement in ischemic heart disease and developing new therapeutic strategies for its treatment.

Many eukaryotic receptors and enzymes rely on glycosylphosphatidylinositol (GPI) anchors for membrane localization and function. The transmembrane complex GPI-T recognizes diverse proproteins at a signal peptide region that lacks consensus sequence and replaces it with GPI via a transamidation reaction. How GPI-T maintains broad specificity while preventing unintentional cleavage is unclear. Here, substrates- and products-bound human GPI-T structures identify subsite features that enable broad proprotein specificity, inform catalytic mechanism, and reveal a multilevel safeguard mechanism against its promiscuity. In the absence of proproteins, the catalytic site is invaded by a locally stabilized loop. Activation requires energetically unfavorable rearrangements that transform the autoinhibitory loop into crucial catalytic cleft elements. Enzyme-proprotein binding in the transmembrane and luminal domains respectively powers the conformational rearrangement and induces a competent cleft. GPI-T thus integrates various weak specificity regions to form strong selectivity and prevent accidental activation. These findings provide important mechanistic insights into GPI-anchored protein biogenesis.

Cell-free plasma DNA (cfDNA) is circulating extracellular DNA arising from cell death mechanisms (apoptosis, necrosis, etc.). It is commonly existent in healthy individuals, but its ranks increase in diverse clinical circumstances, such as kidney disease, sepsis, myocardial infarction, trauma and cancer. In patients with advanced chronic kidney disease, cfDNA is connected to inflammation, and it has been associated with higher mortality. Caspase-3 plays a dominant role in apoptosis, a mechanism of programmed cell death involved in the pathogenesis and progression of chronic kidney disease (CKD). The aim of this pilot study was the evaluation of cfDNA levels and caspase-3 concentrations in patients with chronic kidney disease, in order to investigate the potential role of these molecules, deriving from inflammatory and apoptotic mechanisms, in the progression of renal damage.

We compared cfDNA and caspase-3 levels in 25 CKD patients and in 10 healthy subjects, evaluating their levels based on CKD stage. We also explored correlations between cfDNA and caspase-3 levels in CKD patients and between cfDNA and caspase-3 levels and serum creatinine and urea in this population.

We observed that cfDNA and caspase-3 levels were higher in patients with CKD compared to healthy subjects, in particular in patients with advanced renal disease (CKD stage 5). A positive correlation between cfDNA and caspase-3 levels and between cfDNA and caspase-3 and creatinine and urea were also noticed.

Patients with chronic kidney disease show higher levels of cfDNA and caspase-3 levels compared to the control group. Based on these preliminary results, we speculated that the worsening of renal damage and the increase in uremic toxin concentration could be associated with higher levels of cfDNA and caspase-3 levels, thus reflecting the potential role of inflammation and apoptosis in the progression of CKD. Future studies should focus on the validation of these promising preliminary results.

Breast cancer exhibits the highest global incidence among all tumor types. Regardless of the type of breast cancer, metastasis is a crucial cause of poor prognosis. Anoikis, a form of apoptosis initiated by cell detachment from the native environment, is an outside-in process commencing with the disruption of cytosolic connectors such as integrin-ECM and cadherin-cell. This disruption subsequently leads to intracellular cytoskeletal and signaling pathway alterations, ultimately activating caspases and initiating programmed cell death. Development of an anoikis-resistant phenotype is a critical initial step in tumor metastasis. Breast cancer employs a series of stromal alterations to suppress anoikis in cancer cells. Comprehensive investigation of anoikis resistance mechanisms can inform strategies for preventing and regressing metastatic breast cancer. The present review first outlines the physiological mechanisms of anoikis, elucidating the alterations in signaling pathways, cytoskeleton, and protein targets that transpire from the outside in upon adhesion loss in normal breast cells. The specific anoikis resistance mechanisms induced by pathological changes in various spatial structures during breast cancer development are also discussed. Additionally, the genetic loci of targets altered in the development of anoikis resistance in breast cancer, are summarized. Finally, the micro-RNAs and targeted drugs reported in the literature concerning anoikis are compiled, with keratocin being the most functionally comprehensive. Video Abstract.

Caspase-7 (C7), a cysteine protease involved in apoptosis, is a valuable drug target for its role in human diseases (e. g., Parkinson's, Alzheimer's, sepsis). The C7 allosteric site has great potential for small-molecule targeting, but numerous drug discovery efforts have identified precious few allosteric inhibitors. Here we present the first selective, drug-like inhibitor of C7 along with several other improved inhibitors based on our previous fragment hit. We also provide a rational basis for the impact of allosteric binding on the C7 catalytic cycle by using an integrated approach including X-ray crystallography, stopped-flow kinetics, and molecular dynamics simulations. Our findings suggest allosteric binding disrupts C7 pre-acylation by neutralization of the catalytic dyad, displacement of substrate from the oxyanion hole, and altered dynamics of substrate binding loops. This work advances drug targeting efforts and bolsters our understanding of allosteric structure-activity relationships (ASARs).

Cigarette smoke (CS) poses a significant risk factor for respiratory, vascular, and organ diseases owing to its high content of harmful chemicals and reactive oxygen species (ROS). These substances are known to induce oxidative stress, inflammation, apoptosis, and senescence due to their exposure to environmental pollutants and the presence of oxidative enzymes. The lung is particularly susceptible to oxidative stress. Persistent oxidative stress caused by chronic exposure to CS can lead to respiratory diseases such as chronic obstructive pulmonary disease (COPD), pulmonary fibrosis (PF), and lung cancer. Avoiding exposure to environmental pollutants, like cigarette smoke and air pollution, can help mitigate oxidative stress. A comprehensive understanding of oxidative stress and its impact on the lungs requires future research. This includes identifying strategies for preventing and treating lung diseases as well as investigating the underlying mechanisms behind oxidative stress. Thus, this review aims to investigate the cellular processes induced by CS, specifically inflammation, apoptosis, senescence, and their associated biomarkers. Furthermore, this review will delve into the alveolar response provoked by CS, emphasizing the roles of potential therapeutic target markers and strategies in inflammation and oxidative stress.

Targeting amyloid-β plaques and tau tangles has failed to provide effective treatments for Alzheimer's disease and related dementias (ADRD). A more fruitful pathway to ADRD therapeutics may be the development of therapies that target common signaling pathways that disrupt synaptic connections and impede communication between neurons. In this review, we present our characterization of a signaling pathway common to several neurological diseases featuring dementia including Alzheimer's disease, frontotemporal dementia, Lewy body dementia, and Huntington's disease. This signaling pathway features the cleavage of tau by caspase-2 (Casp2) yielding Δtau314 (Casp2/tau/Δtau314). Through a not yet fully delineated mechanism, Δtau314 catalyzes the mislocalization and accumulation of tau to dendritic spines leading to the internalization of AMPA receptors and the concomitant weakening of synaptic transmission. Here, we review the accumulated evidence supporting Casp2 as a druggable target and its importance in ADRD. Additionally, we provide a brief overview of our initial medicinal chemistry explorations aimed at the preparation of novel, brain penetrant Casp2 inhibitors. We anticipate that this review will spark broader interest in Casp2 as a target for restoring synaptic dysfunction in ADRD.

In plants, proteolysis is emerging as an important field of study due to a growing understanding of the critical involvement of proteases in plant cell death, disease and development. Because proteases irreversibly modify the structure and function of their target substrates, proteolytic activities are stringently regulated at multiple levels. Most proteases are produced as dormant isoforms and only activated in specific conditions such as altered ion fluxes or by post-translational modifications. Some of the regulatory mechanisms initiating and modulating proteolytic activities are restricted in time and space, thereby ensuring precision activity, and minimizing unwanted side effects. Currently, the activation mechanisms and the substrates of only a few plant proteases have been studied in detail. Most studies focus on the role of proteases in pathogen perception and subsequent modulation of the plant reactions, including the hypersensitive response (HR). Proteases are also required for the maturation of coexpressed peptide hormones that lead essential processes within the immune response and development. Here, we review the known mechanisms for the activation of plant proteases, including post-translational modifications, together with the effects of proteinaceous inhibitors.

Caspases are cysteine proteases in charge of breaking a peptide bond next to an aspartate residue. Caspases constitute an important family of enzymes involved in cell death and inflammatory processes. A plethora of diseases, including neurological and metabolic diseases and cancer, are associated with the poor regulation of caspase-mediated cell death and inflammation. Human caspase-1 in particular carries out the transformation of the pro-inflammatory cytokine pro-interleukin-1β into its active form, a key process in the inflammatory response and then in many diseases, such as Alzheimer's disease. Despite its importance, the reaction mechanism of caspases has remained elusive. The standard mechanistic proposal valid for other cysteine proteases and that involves the formation of an ion pair in the catalytic dyad is not supported by experimental evidence. Using a combination of classical and hybrid DFT/MM simulations, we propose a reaction mechanism for the human caspase-1 that explains experimental observations, including mutagenesis, kinetic, and structural data. In our mechanistic proposal, the catalytic cysteine, Cys285, is activated after a proton transfer to the amide group of the scissile peptide bond, a process facilitated by hydrogen-bond interactions with Ser339 and His237. The catalytic histidine does not directly participate in any proton transfer during the reaction. After formation of the acylenzyme intermediate, the deacylation step takes place through the activation of a water molecule by the terminal amino group of the peptide fragment formed during the acylation step. The overall activation free energy obtained from our DFT/MM simulations is in excellent agreement with the value derived from the experimental rate constant, 18.7 vs 17.9 kcal·mol^-1, respectively. Simulations of the H237A mutant support our conclusions and agree with the reported reduced activity observed for this caspase-1 variant. We propose that this mechanism can explain the reactivity of all cysteine proteases belonging to the CD clan and that differences with respect to other clans could be related to the larger preference showed by enzymes of the CD clan for charged residues at position P1. This mechanism would avoid the free energy penalty associated with the formation of an ion pair. Finally, our structural description of the reaction process can be useful to assist in the design of inhibitors of caspase-1, a target in the treatment of several human diseases.

The actin cytoskeleton impacts practically every function of a eukaryotic cell. Historically, the best-characterized cytoskeletal activities are in cell morphogenesis, motility, and division. The structural and dynamic properties of the actin cytoskeleton are also crucial for establishing, maintaining, and changing the organization of membrane-bound organelles and other intracellular structures. Such activities are important in nearly all animal cells and tissues, although distinct anatomical regions and physiological systems rely on different regulatory factors. Recent work indicates that the Arp2/3 complex, a broadly expressed actin nucleator, drives actin assembly during several intracellular stress response pathways. These newly described Arp2/3-mediated cytoskeletal rearrangements are coordinated by members of the Wiskott-Aldrich Syndrome Protein (WASP) family of actin nucleation-promoting factors. Thus, the Arp2/3 complex and WASP-family proteins are emerging as crucial players in cytoplasmic and nuclear activities including autophagy, apoptosis, chromatin dynamics, and DNA repair. Characterizations of the functions of the actin assembly machinery in such stress response mechanisms are advancing our understanding of both normal and pathogenic processes, and hold great promise for providing insights into organismal development and interventions for disease.

The caspase-4/11-GSDMD pyroptosis axis recognizes cytosolic lipopolysaccharide for antibacterial defenses. Shigella flexneri employs an OspC3 effector to block pyroptosis by catalyzing NAD⁺-dependent arginine ADP-riboxanation of caspase-4/11. Here, we identify Ca²⁺-free calmodulin (CaM) that binds and stimulates OspC3 ADP-riboxanase activity. Crystal structures of OspC3-CaM and OspC3-caspase-4 binary complexes reveal unique CaM binding to an OspC3 N-terminal domain featuring an ADP-ribosyltransferase-like fold and specific recognition of caspase-4 by an OspC3 ankryin repeat domain, respectively. CaM-OspC3-caspase-4 ternary complex structures show that NAD⁺ binding reorganizes the catalytic pocket, in which D231 and D177 activate the substrate arginine for initial ADP-ribosylation and ribosyl 2'-OH in the ADP-ribosylated arginine, respectively, for subsequent deamination. We also determine structures of unmodified and OspC3-ADP-riboxanated caspase-4. Mechanisms derived from this series of structures covering the entire process of OspC3 action are supported by biochemical analyses in vitro and functional validation in S. flexneri-infected mice.

Cover cropping is commonly acknowledged to promote soil health in agriculture. However, contradictory findings on the benefits of cover crops for soil health, crop productivity, economic and ecological factors, as well as the influence of inherent soil parameters on such benefits exist in the scientific literature. Here, we critically assessed evidence of cover crop benefits through a systematic review of the published literature. To access relevant papers, we searched the literature for cover crops and soil health indicators using Scopus (1996-2020), ScienceDirect (1996-2020) and Google scholar (1970-1996) with specific keywords and combinations. Only English research papers including experimental plots and control groups were considered. We analyzed 102 unique peer-reviewed papers and 1494 corresponding unique plots encompassing various cover crops, soil textures, climates, management systems and experimental duration (1-3 years, 4-6 years, 7-10 years and over 10 years). Strong evidence suggests that cover crops can enhance soil structure and promote soil health by improving soil physical and chemical properties, including saturated hydraulic conductivity (mean net change of 105.6 %), total organic carbon (10.1 %), and total nitrogen (20.2 %). On the other hand, cover crops exhibit weak effects on properties like bulk density and microporosity with fairly low values of net change. In most cases, cover crops increase the soil carbon content, including microbial biomass carbon (19.5 %) and particulate organic carbon (49.5 %). In this systematic review, we found limited studies on the effect of cover crops on soil health as influenced by soil texture, regional climate, rainfall and duration of the cover crop practices. The paucity of long-term regional systematic research of soil physics, chemistry and biology makes it difficult to forecast future implications of cover crops on soil health indicators.

Sarcococca saligna is a valuable source of bioactive secondary metabolites exhibiting antioxidant, anti-inflammatory and acetylcholinesterase inhibitory activities. The study was intended to explore the therapeutic pursuits of S. saligna in amelioration of cognitive and motor dysfunctions induced by D-galactose and linked mechanistic pathways. Alzheimer's disease model was prepared by administration of D-galactose subcutaneous injection100 mg/kg and it was treated with rivastigmine (100 mg/kg, orally) and plant extract for 42 days. Cognitive and motor functions were evaluated by behavioral tasks and oxidative stress biomarkers. Level of acetylcholinesterase, reduced level of glutathione, protein and nitrite level, and brain neurotransmitters were analyzed in brain homogenate. The level of apoptosis regulator Bcl-2, Caspases 3 and heat shock protein HSP-70 in brain homogenates were analyzed by ELISA and colorimetric method, respectively. AChE, IL-1β, TNF-α, IL-1α and β secretase expressions were analyzed by RT-PCR. S. saligna dose dependently suppressed the neurodegenerative effects of D-galactose induced behavioral and biochemical impairments through modulation of antioxidant enzymes and acetylcholinesterase inhibition. S. saligna markedly (P < 0.05) ameliorated the level of brain neurotransmitters, Bcl-2, HSP-70 and Caspases-3 level. S. saligna at 500-1000 mg/kg considerably recovered the mRNA expression of neurodegenerative and neuro-inflammatory biomarkers, also evident from histopathological analysis. These findings suggest that S. saligna could be applicable in cure of Alzheimer's disease.

Caspases (cysteine-aspartic proteases) play critical roles in inflammation and the programming of cell death in the form of necroptosis, apoptosis, and pyroptosis. The name of these enzymes has been chosen in accordance with their cysteine protease activity. They act as cysteines in nucleophilically active sites to attack and cleave target proteins in the aspartic acid and amino acid C-terminal. Based on the substrate's structure and the specificity, the physiological activity of caspases is divided. However, in apoptosis, the division of caspases into initiating caspases (caspase 2, 8, 9, and 10) and executive caspases (caspase 3, 6, and 7) is essential. The present study aimed to perform Proteochemometrics Modeling to generalize the data on caspases, which could predict ligand and protein interactions. In this study, we employed protein and ligand descriptors. Moreover, protein descriptors were computed using the Protr R package, while PADEL-Descriptor was employed for the computation of ligand descriptors. In addition, NCA (Neighborhood Component Analyses) was used for descriptor selection, and SVR, decision tree, and ensemble methods were utilized for the proteochemometrics modeling. This study shows that the ensemble model demonstrates superior performance compared with other models in terms of R², Q², and RMSE criteria.

Inflammasomes are supramolecular platforms that organize in response to various damage-associated molecular patterns and pathogen-associated molecular patterns. Upon activation, inflammasome sensors (with or without the help of ASC) activate caspase-1 and other inflammatory caspases that cleave gasdermin D and pro-IL-1β/pro-IL-18, leading to pyroptosis and mature cytokine secretion. Pyroptosis enables intracellular pathogen niche disruption and intracellular content release at the cost of cell death, inducing pro-inflammatory responses in the neighboring cells. IL-1β is a potent pro-inflammatory regulator for neutrophil recruitment, macrophage activation, and T-cell expansion. Thus, pyroptosis and cytokine secretion are the two main mechanisms that occur downstream of inflammasome signaling; they maintain homeostasis, drive the innate immune response, and shape adaptive immunity. This review aims to discuss the possible mechanisms, timing, consequences, and significance of the two uncoupling preferences downstream of inflammasome signaling. While pyroptosis and cytokine secretion may be usually coupled, pyroptosis-predominant and cytokine-predominant uncoupling are also observed in a stimulus-, cell type-, or context-dependent manner, contributing to the pathogenesis and development of numerous pathological conditions such as cryopyrin-associated periodic syndromes, LPS-induced sepsis, and Salmonella enterica serovar Typhimurium infection. Hyperactive cells consistently release IL-1β without LDH leakage and pyroptotic death, thereby leading to prolonged inflammation, expanding the lifespans of pyroptosis-resistant neutrophils, and hyperactivating stimuli-challenged macrophages, dendritic cells, monocytes, and specific nonimmune cells. Death inflammasome activation also induces GSDMD-mediated pyroptosis with no IL-1β secretion, which may increase lethality in vivo. The sublytic GSDMD pore formation associated with lower expressions of pyroptotic components, GSDMD-mediated extracellular vesicles, or other GSDMD-independent pathways that involve unconventional secretion could contribute to the cytokine-predominant uncoupling; the regulation of caspase-1 dynamics, which may generate various active species with different activities in terms of GSDMD or pro-IL-1β, could lead to pyroptosis-predominant uncoupling. These uncoupling preferences enable precise reactions to different stimuli of different intensities under specific conditions at the single-cell level, promoting cooperative cell and host fate decisions and participating in the pathogen "game". Appropriate decisions in terms of coupling and uncoupling are required to heal tissues and eliminate threats, and further studies exploring the inflammasome tilt toward pyroptosis or cytokine secretion may be helpful.

The biggest challenge to immune control of HIV infection is the rapid within-host viral evolution, which allows selection of viral variants that escape from T cell and antibody recognition. Thus, it is impossible to clear HIV infection without targeting "immutable" components of the virus. Unlike the adaptive immune system that recognizes cognate epitopes, the CARD8 inflammasome senses the essential enzymatic activity of the HIV-1 protease, which is immutable for the virus. Hence, all subtypes of HIV clinical isolates can be recognized by CARD8. In HIV-infected cells, the viral protease is expressed as a subunit of the viral Gag-Pol polyprotein and remains functionally inactive prior to viral budding. A class of anti-HIV drugs, the non-nucleoside reverse transcriptase inhibitors (NNRTIs), can promote Gag-pol dimerization and subsequent premature intracellular activation of the viral protease. NNRTI treatment triggers CARD8 inflammasome activation, which leads to pyroptosis of HIV-infected CD4⁺ T cells and macrophages. Targeting the CARD8 inflammasome can be a potent and broadly effective strategy for HIV eradication.

Pyroptosis is an active and ordered form of programmed cell death. The signaling pathways of pyroptosis are mainly divided into canonical pathways mediated by caspase-1 and noncanonical pathways mediated by caspase-11. Cell pyroptosis is characterized by the activation of inflammatory caspases (mainly caspase-1, 4, 5, 11) and cleavage of various members of the Gasdermin family to form membrane perforation components, leading to cell membrane rupture, inflammatory mediators release, and cell death. Moderate pyroptosis is an innate immune response that fights against infection and plays an important role in the occurrence and development of the normal function of the immune system. However, excessive pyroptosis occurs and leads to immune disorders in many pathological conditions. Based on canonical pathways, research on pyroptosis regulation has demonstrated several pyroptotic inhibitors, including small-molecule drugs, natural products, and formulations of traditional Chinese medicines. In this paper, we review the characteristics and molecular mechanisms of pyroptosis, summarize inhibitors of pyroptosis, and propound that herbal medicines should be a focus on the research and development for pyroptosis blockers.

Primary biliary cholangitis (PBC) is an autoimmune disease that involves chronic inflammation and injury to biliary epithelial cells. To identify critical genetic factor(s) in PBC patients, we performed whole-exome sequencing of five female siblings, including one unaffected and four affected sisters, in a multi-PBC family, and identified 61 rare heterozygote variants that segregated only within the affected sisters. Among them, we were particularly interested in caspase-10, for although several caspases are involved in cell death, inflammation and autoimmunity, caspase-10 is little known from this perspective. We generated caspase-10 knockout macrophages, and then investigated the obtained phenotypes in comparison to those of its structurally similar protein, caspase-8. Unlike caspase-8, caspase-10 does not play a role during differentiation into macrophages, but after differentiation, it regulates the process of inflammatory cell deaths such as necroptosis and pyroptosis more strongly. Interestingly, caspase-10 displays better protease activity than caspase-8 in the process of RIPK1 cleavage, and an enhanced ability to form a complex with RIPK1 and FADD in human macrophages. Higher inflammatory cell death affected the fibrotic response of hepatic stellate cells; this effect could be recovered by treatment with UDCA and OCA, which are currently approved for PBC patients. Our findings strongly indicate that the defective roles of caspase-10 in macrophages contribute to the pathogenesis of PBC, thereby suggesting a new therapeutic strategy for PBC treatment.

Cysteinyl aspartate specific proteinase-3 (Caspase-3) is an important protein involved in the apoptosis and gasdermin E (GSDME)-mediated cell pyroptosis pathways in vertebrates. A Caspase-3 homologue (designated as CgCaspase-3) was previously identified as an immune receptor specific for lipopolysaccharide (LPS) to regulate apoptosis in the Pacific oyster Crassostrea gigas. In the present study, the binding activity of CgCaspase-3 to different pathogen associated molecular patterns (PAMPs) and its effects on CgGSDME translocation in haemocytes were further investigated in C. gigas. The mRNA expression of CgCaspase-3 could be detected in all the tested tissues, including hepatopancreas, labial palp, adductor muscle, gonad, gill, mantle and haemocytes, and it was highly expressed in labial palp, gonad, haemocytes, and adductor muscle. The mRNA expression of CgCaspase-3 in haemocytes increased significantly at 3, 24, 48 and 72 h after LPS stimulation, and it increased significantly at 6, 12, 24 and 48 h after Vibrio splendidus stimulation. The recombinant CgCaspase-3 displayed binding activity towards LPS, mannose (MAN), peptidoglycan (PGN), and polyinosinic-polycytidylic acid potassium salt (Poly (I:C)). The positive signals of CgGSDME on haemocyte membrane became stronger at 3 h after V. splendidus stimulation, compared with that of Seawater group, and the co-localization of CgCaspase-3 and CgGSDME was observed in the haemocyte membrane. After the injection of dsCgCaspase-3, the positive signals of CgGSDME on haemocyte membrane became weaker compared with that of EGFP-RNAi group at 24 h after V. splendidus stimulation. The results suggested that CgCaspase-3 was able to bind diverse PAMPs and activate the translocation of CgGSDME in haemocytes of oyster response against pathogen invasion.

Rapid progress in nano-scales and nanostructure extremely altered the way of diagnosing or preventing numerous diseases. One of the most important nano-medicines used in cancer treatment and diagnosis is silver nanoparticles (AgNPs). Regardless of their extensive utilization, their prospective neurotoxicity wasn't studied yet. Herein, male Swiss Albino mice were intoxicated via two Nano-scales of AgNPs; (20 nm and 100 nm) for one month (100 mg/kg) then treated by leaves extracts of both Casimiroa edulis (C. edulis) and Glycosmis pentaphylla (G. pentaphylla), in addition to, mucilage and protein, the separated compounds from C. edulis fruits and seeds respectively in a dose of (500 mg/kg). Molecular, Biochemical and histopathological examinations were then conducted. Data recorded showed a significant elevation in hydrogen peroxide (H2O2) level and reduction in glutathione peroxidase (GPX) level post AgNPs intoxication. The oxidative stress occurred was modulated upon treatment regimens. Protein expression of C-reactive protein (CRP) showed a significant elevation and Molecular analysis recorded a significant up-regulation in the expression of both Bax and caspace-3 genes upon AgNPs intoxication in both particles size. On the contrary, both Bcl2 and P53 gene expression were shown to be significantly reduced. Treatment by C. edulis, G. pentaphylla, protein and mucilage extracts revealed modulation in apoptotic and pro-apoptotic biomarkers. Histopathological examination confirmed the obtained results. AgNPs exposure could induce neurotoxicity, genetic alternation and oxidative stress; the targeted extracts could be considered as a promising candidate in modulating apoptosis and neurotoxicity induced by AgNPs.

Legumain is a lysosomal cysteine protease with strict specificity for cleaving after asparagine residues. By sequence comparison, legumain belongs to MEROPS clan CD of the cysteine proteases, which indicates its structural and mechanistic relation to caspases. Contrasting caspases, legumain harbors a pH-dependent ligase activity in addition to the protease activity. Although we already have a significant body of knowledge on the catalytic activities of legumain, many mechanistic details are still elusive. In this study, we provide evidence that extended active site residues and substrate conformation are steering legumain activities. Biochemical experiments and bioinformatics analysis showed that the catalytic Cys189 and His148 residues are regulated by sterically close Glu190, Ser215 and Asn42 residues. While Glu190 serves as an activity brake, Ser215 and Asn42 have a favorable effect on legumain protease activity. Mutagenesis studies using caspase-9 as model enzyme additionally showed that a similar Glu190 activity brake is also implemented in the caspases. Furthermore, we show that the substrate's conformational flexibility determines whether it will be hydrolyzed or ligated by legumain. The functional understanding of the extended active site residues and of substrate prerequisites will allow us to engineer proteases with increased enzymatic activity and better ligase substrates, with relevance for biotechnological applications.