Phosphorylation of bid by casein kinases I and II regulates its cleavage by caspase 8

Bid plays an essential role in Fas-mediated apoptosis of the so-called type II cells. In these cells, following cleavage by caspase 8, the C-terminal fragment of Bid translocates to mitochondria and triggers the release of apoptogenic factors, thereby inducing cell death. Here we report that Bid is phosphorylated by casein kinase I (CKI) and casein kinase II (CKII). Inhibition of CKI and CKII accelerated Fas-mediated apoptosis and Bid cleavage, whereas hyperactivity of the kinases delayed apoptosis. When phosphorylated, Bid was insensitive to caspase 8 cleavage in vitro. Moreover, a mutant of Bid that cannot be phosphorylated was found to be more toxic than wild-type Bid. Together, these data indicate that phosphorylation of Bid represents a new mechanism whereby cells control apoptosis.     

Inhibition of tau polymerization by its carboxy-terminal caspase cleavage fragment

Abnormal aggregation of the microtubule-associated protein, tau, occurs in many neurodegenerative diseases, making it important to understand the mechanisms of tau polymerization. Previous work has indicated that the C-terminal region of tau inhibits polymerization in vitro, and a growing body of evidence implicates caspase cleavage of tau at Asp 421 in the C-terminus as an important inducer of tau polymerization in Alzheimer's disease. In the present study, we provide evidence that the C-terminal peptide fragment produced by caspase cleavage inhibits tau polymerization, suggesting that caspase cleavage of tau enhances its polymerization by removing the inhibitory control element. Moreover, we provide evidence that the peptide assumes an alpha-helical configuration and inhibits tau assembly by interacting with residues 321-375 in the microtubule binding repeat region. These findings indicate that formation of the fibrillar pathologies during the course of Alzheimer's disease may be driven or sustained by apoptotic events leading to caspase activation.     

FADD cleavage by NK cell granzyme M enhances its self-association to facilitate procaspase-8 recruitment for auto-processing leading to caspase cascade

Granzyme M (GzmM), an orphan Gzm, is constitutively and abundantly expressed in innate effector natural killer cells. We previously demonstrated that GzmM induces caspase (casp)-dependent apoptosis and cytochrome c release from mitochondria. We also resolved the crystal structure for GzmM and generated its specific inhibitor. However, how GzmM causes casp activation has not been defined. Here we found that casp-8 is an initiator caspase in GzmM-induced casp cascade, which causes other casp activation and Bid cleavage. GzmM does not directly cleave procaspase-3 and Bid, whose processing is casp dependent. Casp-8 knockdown or deficient cells attenuate or abolish GzmM-induced proteolysis of procaspase-3 and Bid. Extrinsic death receptor pathway adaptor Fas-associated protein with death domain (FADD) contributes to GzmM-induced casp-8 activation. GzmM specifically cleaves FADD after Met 196 to generate truncated FADD (tFADD) that enhances its self-association for oligomerization. The oligomerized tFADD facilitates procaspase-8 recruitment to promote its auto-processing leading to casp activation cascade. FADD-deficient cells abrogate GzmM-induced activation of casp-8 and apoptosis as well as significantly inhibit lymphokine-activated killer cell-mediated cytotoxicity. FADD processing by GzmM can potentiate killing efficacy against tumor cells and intracellular pathogens.     

Cytoskeletal confinement of CX3CL1 limits its susceptibility to proteolytic cleavage by ADAM10

CX₃CL1 is a unique chemokine that acts both as a transmembrane endothelial adhesion molecule and, upon proteolytic cleavage, a soluble chemoattractant for circulating leukocytes. The constitutive release of soluble CX₃CL1 requires the interaction of its transmembrane species with the integral membrane metalloprotease ADAM10, yet the mechanisms governing this process remain elusive. Using single-particle tracking and subdiffraction imaging, we studied how ADAM10 interacts with CX₃CL1. We observed that the majority of cell surface CX₃CL1 diffused within restricted confinement regions structured by the cortical actin cytoskeleton. These confinement regions sequestered CX₃CL1 from ADAM10, precluding their association. Disruption of the actin cytoskeleton reduced CX₃CL1 confinement and increased CX₃CL1–ADAM10 interactions, promoting the release of soluble chemokine. Our results demonstrate a novel role for the cytoskeleton in limiting membrane protein proteolysis, thereby regulating both cell surface levels and the release of soluble ligand.     

Cloning of the MspI modification enzyme. The site of modification and its effects on cleavage by MspI and HpaII

The gene for the MspI modification enzyme from Moraxella was cloned in Escherichia coli using the plasmid vector pBR322. Selection of transformants carrying the gene was based on the resistance of the modified plasmid encoding the enzyme to cleavage by MspI. Both chromosomal and plasmid DNA were modified in the selected clones. None of the clones obtained produced the cognate restriction enzyme which suggests that in this system the genes for the restriction enzyme and methylase are not closely linked. Crude cell extracts prepared from the recombinant strains, but not the host (E. coli HB101), contain an S-adenosylmethionine-dependent methyltransferase specific for the MspI recognition site, CCGG. Production of the enzyme is 3-4-fold greater in the transformants than in the original Moraxella strain. 5-Methylcytosine was identified as the product of the reaction chromatographically. The outer cytosine of the recognition sequence, *CCGG, was shown to be the site of methylation by DNA-sequencing methods. This modification blocks cleavage by both MspI and its isoschizomer HpaII. HpaII, but not MspI, is able to cleave the unmethylated strand of a hemimethylated substrate. The relevance of these results to the use of MspI and HpaII to analyze patterns of methylation in genomic DNA is discussed.     

Genetic modification of alternative respiration has differential effects on antimycin A-induced versus salicylic acid-induced resistance to Tobacco mosaic virus

Salicylic acid (SA), a natural defensive signal chemical, and antimycin A, a cytochrome pathway inhibitor, induce resistance to Tobacco mosaic virus (TMV). Pharmacological evidence suggested signaling during resistance induction by both chemicals involved alternative oxidase (AOX), sole component of the alternative respiratory pathway (AP). Roles of the AP include regulation of intramitochondrial reactive oxygen species and maintenance of metabolic homeostasis. Transgenic tobacco (Nicotiana tabacum) with modified AP capacities (2- to 3-fold increased or decreased) showed no alteration in phenotype with respect to basal susceptibility to TMV or the ability to display SA-induced resistance to systemic viral disease. However, in directly inoculated tissue, antimycin A-induced TMV resistance was inhibited in plants with increased AP capacities, whereas SA and antimycin A-induced resistance was transiently enhanced in plant lines with decreased AP capacities. We conclude that SA-induced TMV resistance results from activation of multiple mechanisms, a subset of which are inducible by antimycin A and influenced by AOX. Other antiviral factors, potentially including the SA-inducible RNA-dependent RNA polymerase, are regulated by AOX-independent mechanisms.     

Suppression of the caspase cleavage of beta-amyloid precursor protein by its cytoplasmic phosphorylation

beta-Amyloid precursor protein (APP) is a type I transmembrane protein. Its cleavages by beta- and gamma-secretases yield beta-amyloid, which is the main constituent of senile plaques in Alzheimer's disease (AD). In apoptotic cells and AD brains, APP is alternatively cleaved by caspases in the cytoplasmic region after the Asp664 residue (with respect to the numbering conversion for the APP695 isoform). Caspase-cleaved fragments of APP are cytotoxic and have been implicated in AD pathogenesis; however, the mechanisms regulating the cleavage have not been studied. APP is constitutively phosphorylated at Thr668 in brain. In the present study, we demonstrate that APP phosphorylated at Thr668 is less vulnerable to cytoplasmic cleavage by caspase-3 and caspase-8. This suggests that APP phosphorylation suppresses the generation of caspase-cleaved fragments of APP in the brain and that perturbation of this phosphorylation may be involved in APP-mediated neurotoxicity.     

Post-translational modification of TRAIL receptor type 1 on various tumor cells and the susceptibility of tumors to TRAIL-induced apoptosis

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and its receptors (TRAIL-R1 and TRAIL-R2) are promising targets for tumor therapy. However, their clinical use is limited because some tumors show resistance to TRAIL-treatment. Here, we analyzed epitopes of nine TRAIL-R1-specific human monoclonal antibodies and demonstrated at least five tentative epitopes on human TRAIL-R1. We found that some of the five were post-translationally modified on some tumor cell lines. Interestingly, one of them, an epitope of TR1-272 antibody (TR1-272-epitope) disappeared on the tumor cells that are more susceptible to TRAIL-induced apoptosis compared to TR1-272-epitope positive cells. Treatment of TR1-272-epitope negative cells with TRAIL induced large cluster formation of TRAIL-R1, while treatment of TR1-272-epiope positive cells with TRAIL did not. These results suggest that TR1-272-antibody might distinguish the TRAIL-R1 conformation that could deliver stronger death signals. Further analysis of epitope-appearance and sensitivity to TRAIL should clarify the mechanisms of TRAIL-induced apoptosis of tumor cells and would provide useful information about tumor therapy using TRAIL and TRAIL-R signaling.     

Caspase-8-mediated cleavage inhibits IRF-3 protein by facilitating its proteasome-mediated degradation

Interferon regulatory factor 3 (IRF-3) plays a central role in inducing the expression of cellular antiviral genes, including the interferon-β gene, in response to Pattern Recognition Receptors. IRF-3 is targeted for proteasome-mediated degradation, which modulates the strength and duration of the innate immune responses that depend on it. We have found that caspase-8, which is activated by cytosolic RIG-I-dependent signaling, catalyzes an essential intermediate step in the ubiquitination and proteasome-mediated degradation of IRF-3. Mutation of a consensus cleavage site within IRF-3 generates a form that is not cleaved by caspase-8 and that is protected from ubiquitination and degradation. An in vitro assay confirms the direct action of caspase-8 cleavage on IRF-3. We also show that caspase-8-mediated cleavage of IRF-3 helps to modulate dsRNA-dependent gene induction.     

Inhibition of human initiator caspase 8 and effector caspase 3 by cross-class inhibitory bovSERPINA3-1 and A3-3

Serpins are a superfamily of structurally conserved proteins. Inhibitory serpins use a suicide substrate-like mechanism. Some are able to inhibit cysteine proteases in cross-class inhibition. Here, we demonstrate for the first time the strong inhibition of initiator and effector caspases 3 and 8 by two purified bovine SERPINA3s. SERPINA 3-1 (uniprotkb:Q9TTE1) binds tighly to human CASP3 (uniprotkb:P42574) and CASP8 (uniprotkb:Q14790) with k_ass of 4.2 × 10⁵ and 1.4 × 10⁶ M⁻¹ s⁻¹, respectively. A wholly similar inhibition of human CASP3 and CASP8 by SERPINA3-3 (uniprotkb:Q3ZEJ6) was also observed with k_ass of 1.5 × 10⁵ and 2.7 × 10⁶ M⁻¹ s⁻¹, respectively and form SDS-stable complexes with both caspases. By site-directed mutagenesis of bovSERPINA3-3, we identified Asp³⁷¹ as the potential P1 residue for caspases. The ability of other members of this family to inhibit trypsin and caspases was analysed and discussed.

Structured summary

MINT-7234656: CASP8 (uniprotkb:Q14790) and SERPINA3-1 (uniprotkb:Q9TTE1) bind (MI:0407) by biochemical (MI:0401)MINT-7234634: SERPINA3-3 (uniprotkb:Q3ZEJ6) and CASP3 (uniprotkb:P42574) bind (MI:0407) by biochemical (MI:0401)MINT-7234663: CASP8 (uniprotkb:Q14790) and SERPINA3-3 (uniprotkb:Q3ZEJ6) bind (MI:0407) by biochemical (MI:0401)MINT-7234625: SERPINA3-1 (uniprotkb:Q9TTE1) and CASP3 (uniprotkb:P42574) bind (MI:0407) by biochemical (MI:0401)     

The herpes simplex virus-1 Us3 protein kinase blocks CD8T cell lysis by preventing the cleavage of Bid by granzyme B

The Us3 kinase is part of the antiapoptotic arsenal that salvages herpes simplex virus (HSV)-1-infected cells from damage caused by different stimuli. We demonstrate that Us3 protects HSV-1-infected cells from lysis by MHC class I-restricted CD8T cells without affecting antigen presentation. Expression of Us3 was associated with inhibition of caspase activation and reduced cleavage of the proapoptotic protein Bid. Recombinant granzyme B (GrB) failed to cleave Bid in cytosolic extracts from Us3 positive cells, while recombinant Bid served as substrate for Us3 phosphorylation, suggesting that modification of Bid by Us3 blocks its processing by GrB. Our data illustrate a new strategy of viral escape, where modification of a cellular proapoptotic substrate may prevent lysis of the infected cells without affecting other T-cell functions.     

组蛋白翻译后修饰与DNA损伤响应蛋白招募的调控

组蛋白翻译后修饰是细胞DNA损伤早期应答反应的重要内涵,一方面是松弛、开放染色质结构的必要分子调节事件,以便DNA损伤响应蛋白能接近DNA损伤位点;另一方面直接参与DNA损伤修复蛋白招募过程的调控。综述了在DNA损伤信号激发下,发生的组蛋白主要修饰类型,异组蛋白H2AX、H2A.Z在DNA损伤部位与组蛋白置换,及其对DNA损伤响应蛋白招募的调节作用和机制。     

Influence of subterminal viral DNA nucleotides on differential susceptibility to cleavage by human immunodeficiency virus type 1 and visna virus integrases.

A comparison of the extents of site-specific cleavage of U5 and U3 viral DNA termini by the integrases of human immunodeficiency virus type 1 and visna virus guided the quantitative testing of oligonucleotide substrates containing specific base substitutions. The simultaneous exchange of positions 5 and 6 between U3 substrates switched the patterns of differential susceptibility to the two integrases. The activity of visna virus integrase was more dependent on the identity of position 5 adjacent to the invariant CA bases than on position 6, whereas human immunodeficiency virus type 1 integrase appeared to interact even more critically with position 6. Although the paired natural substrates of most lentiviral integrases match at positions 7 and 8, these bases were not important for susceptibility of U5 substrates. In fact, the final six U5 positions contained all of the sequence information necessary for susceptibility. These results suggest that constraints other than integration influence the terminal inverted repeats of retroviral DNA.     

Post-translational modification of the RhoGTPase activating protein 21, ARHGAP21, by SUMO2/3

ARHGAP21 is a 217 kDa RhoGAP protein shown to modulate cell migration through the control of Cdc42 and FAK activities. In the present work a 250 kDa-ARHGAP21 was identified by mass spectrometry. This modified form is differentially expressed among cell lines and human primary cells. Co-immunoprecipitations and in vitro SUMOylation confirmed ARHGAP21 specific modification by SUMO2/3 and mapped the SUMOylation site to ARHGAP21 lysine K1443. Immunofluorescence staining revealed that ARHGAP21 co-localizes with SUMO2/3 in the cytoplasm and membrane compartments. Interestingly, our results suggest that ARHGAP21 SUMOylation may be related to cell proliferation. Therefore, SUMOylation of ARHGAP21 may represent a way of guiding its function.     

HIV-1 protease processes procaspase 8 to cause mitochondrial release of cytochrome c,caspase cleavage and nuclear fragmentation

Infection of T cells with HIV-1 induces apoptosis and modulates apoptosis regulatory molecules. Similar effects occur following treatment of cells with individual HIV-1 encoded proteins. While HIV-1 protease is known to be cytotoxic, little is known of its effect on apoptosis and apoptosis regulatory molecules. The ability of HIV-1 protease to kill cells, coupled with the degenerate substrate specificity of HIV-1 protease, suggests that HIV-1 protease may activate cellular factor(s) which, in turn, induce apoptosis. We demonstrate that HIV-1 protease directly cleaves and activates procaspase 8 in T cells which is associated with cleavage of BID, mitochondrial release of cytochrome c, activation of the downstream caspases 9 and 3, cleavage of DFF and PARP and, eventually, to nuclear condensation and DNA fragmentation that are characteristic of apoptosis. The effect of HIV-1 protease is not seen in T cell extracts which have undetectable levels of procaspase 8, indicating a specificity and requirement for procaspase 8.     

Pseudohyperphosphorylation has differential effects on polymerization and function of tau isoforms

The microtubule-associated protein tau exists as six isoforms created through the splicing of the second, third, and tenth exons. The isoforms are classified by their number of N-terminal exons (0N, 1N, or 2N) and by their number of microtubule-binding repeat regions (3R or 4R). Hyperphosphorylated isoforms accumulate in insoluble aggregates in Alzheimer's disease and other tauopathies. These neurodegenerative diseases can be categorized based on the isoform content of the aggregates they contain. Hyperphosphorylated tau has the general characteristics of an upward electrophoretic shift, decreased microtubule binding, and an association with aggregation. Previously we have shown that a combination of seven pseudophosphorylation mutations at sites phosphorylated by GSK-3β, referred to as 7-Phos, induced several of these characteristics in full-length 2N4R tau and led to the formation of fewer but longer filaments. We sought to determine whether the same phosphorylation pattern could cause differential effects in the other tau isoforms, possibly through varied conformational effects. Using in vitro techniques, we examined the electrophoretic mobility, aggregation properties, and microtubule stabilization of all isoforms and their pseudophosphorylated counterparts. We found that pseudophosphorylation affected each isoform, but in several cases certain isoforms were affected more than others. These results suggest that hyperphosphorylation of tau isoforms could play a major role in determining the isoform composition of tau aggregates in disease.