HS 1-associated protein X-1 is cleaved by caspase-3 during apoptosis

Caspase-3 (CASP3) plays a key role in apoptosis. In this study, HAX-1 was identified as a new substrate of CASP3 during apoptosis. HAX-1 was cleaved by CASP3 during etoposide-(ETO) induced apoptosis, and this event was inhibited by a CASP3-specific inhibitor. The cleavage site of HAX-1, at Asp(127), was located using N-terminal amino acid sequencing of in vitro cleavage products of recombinant HAX-1. Overexpression of HAX-1 inhibited ETO-induced apoptotic cell death. It also inhibited CASP3 activity. Together, these results suggest that HAX-1, a substrate of CASP3, inhibits the apoptotic process by inhibiting CASP3 activity.     

Human IL-21 and IL-4 bind to partially overlapping epitopes of common gamma-chain

Interleukin 21 (IL-21) is a recently identified novel cytokine that plays an important role in the regulation of B, T, and NK cell functions. Its effects depend on binding to and signaling through an IL-21 receptor complex consisting of the IL-21 receptor (IL-21R) and the common gamma-chain (gamma(c)). In this study using biosensor technique, the ligand-binding properties of IL-21R and gamma(c), which are presently poorly understood on a molecular level, were analyzed employing recombinant ectodomains of IL-21R and gamma(c). The formation of a binary complex between IL-21 and immobilized IL-21R (K(D) 70pM), gamma(c) and immobilized IL-21 (K(D) 160 microM) and a ternary complex between gamma(c) and IL-21 saturated immobilized IL-21R (K(D) 160nM) could be analyzed. The gamma(c) residues involved in IL-21 binding were defined by alanine-scanning mutational analysis. The epitope comprises gamma(c) residues N44, Y103, N128, L161, E162, and L208. It is not identical but partially overlapping with the previously established gamma(c) epitope for IL-4 binding. These results open the way to understand the molecular recognition mechanism in the IL-21 receptor system and also the promiscuous binding properties of gamma(c).     

Interleukin-1 receptor cluster: gene organization of IL1R2, IL1R1, IL1RL2 (IL-1Rrp2), IL1RL1 (T1/ST2), and IL18R1 (IL-1Rrp) on human chromosome 2q

The family of interleukin-1 receptor-like genes currently has six known members. We have constructed a contig of 10 overlapping human PAC clones that covers 530 kb and includes five of the six family members. The termini of the contig were mapped to the interval between D2S373 and D2S176 (chromosome 2q12) by radiation hybrid mapping. The contig contains the genes (cen --> tel), in the order given, for the type II interleukin-1 (IL-1) receptor (IL1R2), the type I IL-1 receptor (IL1R1), the IL-1 receptor-related protein 2 (IL1RL2), T1/ST2/fit-1 (IL1RL1), and the IL-1 receptor-related protein 1, which has recently been shown to be a component of the IL-18 receptor (IL18R1). We show that all the genes are transcribed in the same direction, with IL1R2 being transcribed toward the cluster. The only known family member that is absent from the human contig is the IL-1 receptor accessory protein gene (IL1RAP), which maps to 3q28.     

Caspase proteolysis of the cohesin component RAD21 promotes apoptosis

Caspases are a conserved family of proteases that play a critical role in the execution of apoptosis by cleaving key cellular proteins at Asp residues and modifying their function. Using an expression cloning strategy we recently developed, we isolated human RAD21/SCC1/MCD1 as a novel caspase substrate. RAD21 is a component of the cohesin complex that holds sister chromatids together during mitosis and repairs double-strand DNA breaks. Interestingly, RAD21 is cleaved by a caspase-like Esp1/separase at the onset of anaphase to trigger sister chromatid separation. Here, we demonstrate that human RAD21 is preferentially cleaved at Asp(279) by caspases-3 and -7 in vitro to generate two major proteolytic products of approximately 65 and 48 kDa. Moreover, we show that RAD21 is specifically proteolyzed by caspases into a similarly sized 65-kDa carboxyl-terminal product in cells undergoing apoptosis in response to diverse stimuli. We also demonstrate that caspase proteolysis of RAD21 precedes apoptotic chromatin condensation and has important functional consequences, viz. the partial removal of RAD21 from chromatin and the production of a proapoptotic carboxyl-terminal cleavage product that amplifies the cell death signal. Taken together, these findings point to an entirely novel function of RAD21 in the execution of apoptosis.     

p21(WAF1/CIP1) inhibits initiator caspase cleavage by TRAIL death receptor DR4

Death receptors of the Tumor Necrosis Factor (TNF) family form membrane-bound self-activating signaling complexes that initiate apoptosis through cleavage of proximal caspases including CASP8 and 10. Here we show that overexpression of the cytoplasmic domain (CD) of the DR4 TRAIL receptor (TNFRSF10A, TRAIL R1) in human breast, lung, and colon cancer cell lines, using an adenovirus vector (Ad-DR4-CD), leads to p53-independent apoptotic cell death involving cleavage of CASP8 and 10 proximally and CASP3, 6, and 7 distally. DR4-CD overexpression also leads to cleavage of poly(ADP-ribose) polymerase (PARP) and the DNA fragmentation factor (DFF45; ICAD). Importantly, normal lung fibroblasts are resistant to DR4-CD overexpression and show no evidence of PARP-, CASP8- or CASP3-cleavage despite similar levels of adenovirus-delivered DR4-CD protein as the cancer cells. These results suggest that DR4 may signal death through known caspases and that further studies are required to evaluate Ad-DR4-CD as a novel anti-cancer agent. Finally, we show that overexpression of the cyclin-dependent kinase inhibitor p21(WAF1/CIP1) (CDKN1A), or its N-terminal 91 amino acids containing cell cycle-inhibitory activity, inhibits DR4-CD-dependent proximal caspase cleavage. The blockage of initiator caspase activation provides a novel insight into how p21 may suppress apoptosis and enhance cell survival.     

Are genetic variations in IL-21–IL-23R–IL-17A cytokine axis involved in a pathogenic pathway of rheumatoid arthritis? Bayesian hierarchical meta-analysis

Inflammatory cytokines have been established to be involved in the pathogenesis of rheumatoid arthritis (RA). The genetic polymorphisms in the interleukin (IL) 23 receptor (IL23R), IL21, and IL17 have been associated with RA risk. However, there is no conclusive understanding of the genes encoding the immunoinflammatory IL-21–IL-23R–IL-17A pathway in RA aetiopathogenesis. This meta-analysis was conducted to attain this goal. A comprehensive literature search was conducted in Scopus and PubMed to look for the relevant case–control studies up until 2018. A Bayesian hierarchical meta-analysis was carried out to assess the association between the polymorphisms and the risk of RA. The association was estimated by calculating the logarithm of odds ratio (Log OR) and 95% credible interval (95% CI). In this meta-analysis, 37 case–control studies comprising 23,506 RA patients and 25,984 healthy individuals were found for analyzing the IL23R, IL21, and IL1A gene polymorphism and risk of RA. In the IL23R gene rs1343151 SNP, the minor A allele significantly increased the risk of RA (Log OR = 0.085, 95% CI = 0.008, 0.156). Moreover, the minor AA genotype was significantly associated with increased RA risk (Log OR = 0.176, 95% CI = 0.028, 0.321). In addition, the C allele of the IL23R gene rs2201841 SNP significantly decreased the disease risk (Log OR = −0.544, 95% CI = −1.0, −0.065). Since Bayesian meta-analysis is a powerful strategy to pool the data, it can be mentioned that genetic polymorphisms of IL23R, but not IL21 and IL17A, are involved in susceptibility to RA.     

Assignment of the genes encoding human interleukin-8 receptor types 1 and 2 and an interleukin-8 receptor pseudogene to chromosome 2q35.

Two human cDNA clones that encode different interleukin-8 (IL8) receptors have recently been isolated. The interleukin-8 receptor type 1 (IL8R1) binds IL8 only, whereas the interleukin-8 receptor type 2 (IL8R2) (previously designated IL8RA) also binds growth regulated gene (GRO), and neutrophil activating protein-2 (NAP-2) with high affinity. In the process of screening a genomic library with these cDNAs to obtain large clones for use in chromosomal localization studies, we isolated an interleukin-8 receptor pseudogene (IL8RP) that bears greatest similarity to IL8R2. Using Southern hybridization analysis of human x rodent somatic cell hybrid DNAs with cDNA probes for IL8R1 and IL8R2 and probes from the IL8RP locus, we assigned the three loci to chromosome 2; fluorescence in situ hybridization (FISH) to metaphase chromosome preparations using genomic clones from each locus refined this localization to chromosome 2, band q35, for all three. By virtue of their chromosomal location, IL8R1 and IL8R2 may be considered candidate genes for several human disorders in which the involved locus has been mapped to distal 2q or that are associated with structural abnormalities of this segment, including van der Woude syndrome and the neoplastic diseases rhabdomyosarcoma and uterine leiomyomata. In addition, because this region of chromosome 2q is homologous to proximal mouse chromosome 1 in the segment containing the Lsh-Ity-Bcg locus involved in mediating host resistance to infection with intracellular pathogens, examination for abnormalities of the murine homologues of the IL8R genes should be considered in mice affected by mutations of this locus.     

Crystal structure of interleukin-21 receptor (IL-21R) bound to IL-21 reveals that sugar chain interacting with WSXWS motif is integral part of IL-21R

IL-21 is a class I cytokine that exerts pleiotropic effects on both innate and adaptive immune responses. It signals through a heterodimeric receptor complex consisting of the IL-21 receptor (IL-21R) and the common γ-chain. A hallmark of the class I cytokine receptors is the class I cytokine receptor signature motif (WSXWS). The exact role of this motif has not been determined yet; however, it has been implicated in diverse functions, including ligand binding, receptor internalization, proper folding, and export, as well as signal transduction. Furthermore, the WXXW motif is known to be a consensus sequence for C-mannosylation. Here, we present the crystal structure of IL-21 bound to IL-21R and reveal that the WSXWS motif of IL-21R is C-mannosylated at the first tryptophan. We furthermore demonstrate that a sugar chain bridges the two fibronectin domains that constitute the extracellular domain of IL-21R and anchors at the WSXWS motif through an extensive hydrogen bonding network, including mannosylation. The glycan thus transforms the V-shaped receptor into an A-frame. This finding offers a novel structural explanation of the role of the class I cytokine signature motif.     

Crystal structures of human caspase 6 reveal a new mechanism for intramolecular cleavage self-activation

Dimeric effectors caspase 3 and caspase 7 are activated by initiator caspase processing. In this study, we report the crystal structures of effector caspase 6 (CASP6) zymogen and N-Acetyl-Val-Glu-Ile-Asp-al-inhibited CASP6. Both of these forms of CASP6 have a dimeric structure, and in CASP6 zymogen the intersubunit cleavage site (190)TEVD(193) is well structured and inserts into the active site. This positions residue Asp 193 to be easily attacked by the catalytic residue Cys 163. We demonstrate biochemically that intramolecular cleavage at Asp 193 is a prerequisite for CASP6 self-activation and that this activation mechanism is dependent on the length of the L2 loop. Our results indicate that CASP6 can be activated and regulated through intramolecular self-cleavage.     

TRIM-directed selective autophagy regulates immune activation

Selectivity of autophagy is achieved by target recognition; however, the number of autophagy receptors identified so far is limited. In this study we demonstrate that a subset of tripartite motif (TRIM) proteins mediate selective autophagy of key regulators of inflammatory signaling. MEFV/TRIM20, and TRIM21 act as autophagic receptors recognizing their cognate targets and delivering them for autophagic degradation. MEFV recognizes the inflammasome components NLRP3, CASP1 and NLRP1, whereas TRIM21 specifically recognizes the activated, dimeric from of IRF3 inducing type I interferon gene expression. MEFV and TRIM21 have a second activity, whereby they act not only as receptors but also recruit and organize key components of autophagic machinery consisting of ULK1, BECN1, ATG16L1, and mammalian homologs of Atg8, with a preference for GABARAP. MEFV capacity to organize the autophagy apparatus is affected by common mutations causing familial Mediterranean fever. These findings reveal a general mode of action of TRIMs as autophagic receptor-regulators performing a highly-selective type of autophagy (precision autophagy), with MEFV specializing in the suppression of inflammasome and CASP1 activation engendering IL1B/interleukin-1β production and implicated in the form of cell death termed pyroptosis, whereas TRIM21 dampens type I interferon responses.     

Development of a high-throughput screen targeting caspase-8-mediated cleavage of the amyloid precursor protein

Caspases, effectors of apoptosis, are key mediators of neuronal death in several neurodegenerative diseases. Caspase-8 and caspase-6 have been implicated in the pathogenesis of amyotrophic lateral sclerosis, multiple sclerosis, Parkinson’s disease, and Alzheimer’s disease (AD). ß-Amyloid precursor protein (APP) is cleaved at Asp664 in its intracellular domain by caspase-8. We and other laboratories recently showed that obliteration of the caspase cleavage site on APP alleviates functional AD-like deficits in a mouse model. Therefore, caspase cleavage of APP constitutes a potential novel target for therapeutic intervention. To identify chemical inhibitors of caspase-8 cleavage, we screened a subset of the chemical library at the Harvard NeuroDiscovery Center’s Laboratory for Drug Discovery in Neurodegeneration. We show that caspase-8, but not caspase-1, -3, or -9, cleaves a biotinylated peptide derived from APP at Asp664, and we report the development of a sensitive high-throughput assay for caspase-8 cleavage of APP and the use of that assay for the identification of specific small molecule “hit” compounds that potently inhibit Asp664 cleavage of APP. Furthermore, we demonstrate that one of these compounds (LDN-0021835) inhibits the cleavage of APP at Asp664 in cell-based assays.     

WSB-1, a novel IL-21 receptor binding molecule, enhances the maturation of IL-21 receptor

Interleukin-21 (IL-21) is a pleiotropic cytokine that regulates T-cell, B-cell, NK-cell, and myeloid-cell functions. IL-21 binds with its cognate receptor complex, which consists of the IL-21 receptor (IL-21R) and the common gamma chain. We identified a novel IL-21R-binding molecule, WSB-1, which contains WD-40 repeats and a SOCS-box domain. WSB-1 associates with the middle part of intracytoplasmic region of IL-21R and enhances the maturation of IL-21R from N-linked glycosylated form to fully glycosylated mature form. Furthermore, WSB-1 moderates IL-21R degradation. Taken together, our present study suggests that WSB-1 has a role in the tuning of the maturation and degradation of IL-21R.     

Inhibition of cathepsin S attenuates myocardial ischemia/reperfusion injury by suppressing inflammation and apoptosis

Myocardial ischemia/reperfusion (I/R) injury leads to high mortality and morbidity due to the incomplete understanding of the underlying mechanism and the consequent lack of effective therapy. The present study revealed and validated key candidate genes in relation to inflammation and apoptosis pathways underlying myocardial I/R injury. Cathepsin S was identified as the top hub protein based on the protein–protein interaction analysis, and, thus, its role during myocardial I/R injury was further investigated. Myocardial I/R in mice resulted in significantly increased levels of myocardial injury biomarkers (cardiac troponin I, lactic dehydrogenase, and creatinine kinase‐MB) and inflammatory cytokines (interleukin‐1β [IL‐1β], IL‐6, and tumor necrosis factor‐α), elevated apoptosis rate, and upregulated protein expression of cleaved caspase‐8, cleaved caspase‐3, and cleaved poly ADP‐ribose polymerase. These abovementioned changes were blocked by two different selective cathepsin S inhibitors, LY3000328 or MIV‐247. Moreover, Kaplan–Meier survival plot showed that cathepsin S inhibition improved 21‐day survival rate following myocardial I/R injury. This study demonstrated that the inhibition of cathepsin S alleviated myocardial I/R‐induced injury by suppressing inflammation and apoptosis, which may be used in clinical applications of cardioprotection.     

Serum IL-1beta,sIL-2R,IL-6, IL-8 and TNF-alpha in schizophrenic patients,relation with symptomatology and responsiveness to risperidone treatment.

Activation of the inflammatory response system and varied levels of cytokines in acute schizophrenia have been suggested by recent studies. Psychopharmacologic agents can differentially effect cytokine production, which suggests that therapeutic function of neuroleptics may involve immunomodulation. The present study was carried out to examine: (i) serum concentrations of interleukin (IL)-1beta, soluble interleukin-2 receptor (sIL-2R), IL-6, IL-8 and tumour necrosis factor (TNF)-alpha in schizophrenic patients; (ii) their relation with psychopathological assessment; and (iii) the relation of the initial cytokine levels with responsiveness to risperidone therapy. Thirty-four drug-free schizophrenic patients with acute exacerbation and 23 age- and gender-matched healthy controls were recruited for this study. Psychopathological assessments at admission and throughout risperidone treatment for 60 days were recorded. Serum cytokine concentrations were determined with chemilumunescence assays. According to our results, serum IL-1beta, sIL-2R, IL-6, IL-8 and TNF-alpha concentrations adjusted for age, gender, body mass index and smoking were no different in patients with schizophrenia and controls and among subtypes of schizophrenia. However, the initial TNF-alpha concentrations had a significant effect on Brief Psychiatric Rating Scale and Scale Assessment of Positive Symptoms scores. The initial cytokine concentrations of the patients responsive to risperidone were not significantly different from those of non-responsive patients. The present study demonstrates that plasma levels of IL-1beta, sIL-2R, IL-6, IL-8 and TNF-alpha adjusted for confounding factors are not altered in drug-free schizophrenic patients at acute exacerbation. We suggest that, if cytokine production is altered in schizophrenia, these alterations may not be detectable in systemic circulation. According to our results, the therapeutic effect of risperidone is not related to basal levels of the aforementioned cytokines. However, serum TNF-alpha may contribute to symptomatology in schizophrenia     

Characterization of a caspase-3-substrate kinome using an N- and C-terminally tagged protein kinase library produced by a cell-free system

Caspase-3 (CASP3) cleaves many proteins including protein kinases (PKs). Understanding the relationship(s) between CASP3 and its PK substrates is necessary to delineate the apoptosis signaling cascades that are controlled by CASP3 activity. We report herein the characterization of a CASP3-substrate kinome using a simple cell-free system to synthesize a library that contained 304 PKs tagged at their N- and C-termini (NCtagged PKs) and a luminescence assay to report CASP3 cleavage events. Forty-three PKs, including 30 newly identified PKs, were found to be CASP3 substrates, and 28 cleavage sites in 23 PKs were determined. Interestingly, 16 out of the 23 PKs have cleavage sites within 60 residues of their N- or C-termini. Furthermore, 29 of the PKs were cleaved in apoptotic cells, including five that were cleaved near their termini in vitro. In total, approximately 14% of the PKs tested were CASP3 substrates, suggesting that CASP3 cleavage of PKs may be a signature event in apoptotic-signaling cascades. This proteolytic assay method would identify other protease substrates.     

Association of twelve candidate gene polymorphisms and response to challenge with Salmonella enteritidis in poultry

Breeding for disease resistance to Salmonella enteritidis (SE) could be an effective approach to control Salmonella in poultry. The candidate gene approach is a useful method to investigate genes that are involved in genetic resistance. In this study, 12 candidate genes that are involved in the pathogenesis of Salmonella infection were investigated using five different genetic groups of meat-type chicken. The genes were natural resistance associated macrophage protein 1 (SLC11A1, previously known as NRAMP1), inhibitor of apoptosis protein 1 (IAP1), prosaposin (PSAP), Caspase-1 (CASP1), inducible nitric oxide production (iNOS), interferon-gamma (IFNG), interleukin-2 (IL2), immunoglobulin light chain (IGL), ZOV3, and transforming growth factors B2, B3 and B4 (TGFB2, B3 and B4). In total, 117 birds of all groups were challenged with SE at the age of 3 weeks. In all birds at 7-day post-infection SE load in caecum content, spleen and liver were quantified. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assays were used to genotype all animals for each gene. Overall we found the most significant associations with caecum content, nine of 12 genes showed a significant association (SLC11A1, IAP1, PSAP, CASP1, iNOS, IL2, IGL, TGFB2 and TGFB4). For liver, five genes (SLC11A1, CASP1, IL2, IGL, and TGFB4) and for spleen, only one gene (TGFB3) showed a significant association with SE load. By showing associations of 12 PCR-RFLP assays with SE load after a pathogen challenge, this study confirmed the polygenic nature of disease resistance to SE.     

Structure and chromosome localization of the human CASP8 gene

The human CASP8 gene, whose product is also known as caspase 8 and FLICE, encodes an interleukin-1β converting enzyme (ICE)-related cysteine protease that is activated by the engagement of several different death receptors. Caspase 8 is immediately recruited to the Fas receptor once it oligomerizes, and its protease activity is crucial for the apoptotic response generated by the resulting death-inducing signaling complex (DISC). We report here that the CASP8 gene contains at least 11 exons spanning ∼30 kb on human chromosome band 2q33–34. This region of human chromosome 2 was previously reported as the location of the CASP10 gene, whose product is closely related to caspase 8. Chromosome 2 band q33–34 is also involved in tumorigenesis, with loss of heterogeneity (LOH) being reported in a number of tumors. We also report EcoRI and HindIII polymorphisms that may prove to be useful in disease analysis. Both caspases 8 and 10 contain long pro-domains with duplicated death effector domains (DEDs), as well as their corresponding cysteine protease catalytic domains. Thus, it appears that CASP8 and CASP10 have evolved by tandem gene duplication, much like the CASP1, CASP4 and CASP5 gene cluster on human chromosome 11q22.2–22.3.     

Crystal structure of the IL-22/IL-22R1 complex and its implications for the IL-22 signaling mechanism

Interleukin-22 (IL-22) is a member of the interleukin-10 cytokine family, which is involved in anti-microbial defenses, tissue damage protection and repair, and acute phase responses. Its signaling mechanism involves the sequential binding of IL-22 to interleukin-22 receptor 1 (IL-22R1), and of this dimer to interleukin-10 receptor 2 (IL-10R2) extracellular domain. We report a 1.9A crystal structure of the IL-22/IL-22R1 complex, revealing crucial interacting residues at the IL-22/IL-22R1 interface. Functional importance of key residues was confirmed by site-directed mutagenesis and functional studies. Based on the X-ray structure of the binary complex, we discuss a molecular basis of the IL-22/IL-22R1 recognition by IL-10R2. STRUCTURED SUMMARY: