Kinetic traps in the folding/unfolding of procaspase-1 CARD domain

We have examined the folding and unfolding of the caspase recruitment domain of procaspase-1 (CP1-CARD), a member of the alpha-helical Greek key protein family. The equilibrium folding/unfolding of CP1-CARD is described by a two-state mechanism, and the results show CP1-CARD is marginally stable with a DeltaG(H2O) of 1.1 +/- 0.2 kcal/mole and an m-value of 0.65 +/- 0.06 kcal/mole/M (10 mM Tris-HCl at pH 8.0, 1 mM DTT, 25 degrees C). Consistent with the equilibrium folding data, CP1-CARD is a monomer in solution when examined by size exclusion chromatography. Single-mixing stopped-flow refolding and unfolding studies show that CP1-CARD folds and unfolds rapidly, with no detectable slow phases, and the reactions appear to reach equilibrium within 10 msec. However, double jump kinetic experiments demonstrate the presence of an unfolded-like intermediate during unfolding. The intermediate converts to the fully unfolded conformation with a half-time of 10 sec. Interrupted refolding studies demonstrate the presence of one or more nativelike intermediates during refolding, which convert to the native conformation with a half-time of about 60 sec. Overall, the data show that both unfolding and refolding processes are slow, and the pathways contain kinetically trapped species.     

Cloning, E. coli expression, refolding, and ATP-binding properties of a WD40-deleted Apaf-1 isoform

The apoptotic protease activating factor (Apaf-1) is central to the regulatory mechanism by which procaspase-9 is activated in the cytochrome c-mediated pathway of apoptosis. For a detailed biochemical and structural investigation of Apaf-1 function, we have cloned and expressed in Escherichia coli inclusion bodies the WD40-deleted protein (DeltaWD40 Apaf-1) from HepG2 cell. The construct contains an N-terminal His6 tag derived from the cloning vector so that the mass of the protein and the tag together is 51,594 Da, as determined by TOF/TOF mass spectrometric analysis. An optimized refolding protocol has allowed protein recovery in highly pure form. Basic fluorescence and CD probes indicate that the refolded protein retains secondary and tertiary structures, and unfolds in the presence of higher concentration of denaturant. The equilibrium ATP binding property of the protein has been measured by changes in fluorescence emission due to the fluorescent ATP analog, mant-ATP (2'(3')-O-(N-methylanthraniloyl) adenosine 5'-triphosphate). The results demonstrate a tight Apaf-1-ATP interaction, the binding affinity being 380 nM.     

环境高温促进猪睾丸Apaf-1和Caspase-9的表达

高温热应激条件下,凋亡蛋白表达量升高,生殖细胞凋亡增加。凋亡蛋白酶活化因子1（apoptosis protease activating factor 1,Apaf-1）和凋亡蛋白酶活化起始者含半胱氨酸的天冬氨酸蛋白水解酶9,(cysteine aspartic acid specific protease 9, Caspase-9）是细胞凋亡内源途径中的重要调节蛋白,热应激条件下猪睾丸Apaf-1和Caspase 9的表达未见报道。本研究发现,夏季畜舍高温使Apaf-1和Caspase-9表达量升高。qRT-PCR和Western印迹结果显示,与对照组（正常舍温20℃）相比,短时热应激组（40～42℃,1 h/d, 7 d）和长时热应激组（40～42℃,1 h/d, 42 d）,Apaf-1和Caspase-9 mRNA和蛋白的相对表达量均显著升高。免疫组织化学研究发现,Apaf-1在猪睾丸组织中免疫反应阳性物定位于间质细胞、支持细胞和各个发育阶段生精细胞。热应激处理导致精母细胞和精子细胞Apaf-1表达量升高。在各实验猪睾丸组织中,Caspase-9定位于间质细胞、支持细胞和各个发育阶段生精细胞的胞质中。与对照组相比,热应激处理导致减数分裂以后的生精细胞和支持细胞Caspase-9表达量升高。上述结果表明,高温热应激促进Apaf-1和Caspase-9的表达,提示Apaf-1和Caspase-9表达的变化可能与猪舍高温导致的猪精液品质下降存在关联。     

Solution structure of NOD1 CARD and mutational analysis of its interaction with the CARD of downstream kinase RICK

NOD1 is a cytosolic signalling host pattern-recognition receptor composed of a caspase-activating and recruitment domain (CARD), a nucleotide-binding and oligomerization domain (NOD) and leucine-rich repeats. It plays a crucial role in innate immunity by activating the NF-kappaB pathway via its downstream effector the kinase RICK (RIP2) following the recognition of a specific bacterial ligand. RICK is recruited by NOD1 through interaction of their respective CARDs. Here we present the high resolution NMR structure of the NOD1 CARD. It is generally similar to other CARDs of known structure, consisting of six tightly packed helices, although the length and orientation of the last helix is unusual. Mutations in both the NOD1 and RICK CARD domains were assayed by immuno-precipitation of cell lysates and in vivo NF-kappaB activation in order to define residues important for CARD-CARD interaction and downstream signalling. The results show that the interaction is critically dependent on three acidic residues on NOD1 CARD and three basic residues on RICK CARD and thus is likely to have a strong electrostatic component, similar to other characterised CARD-CARD interactions.     

Apaf-1 and caspase-9 do not act as tumor suppressors in myc-induced lymphomagenesis or mouse embryo fibroblast transformation

Based on experiments with cultured fibroblasts, the apoptosis regulators caspase-9 and Apaf-1 are hypothesized to function as tumor suppressors. To investigate their in vivo role in lymphomagenesis, an IgH enhancer-driven c-myc transgene was crossed onto Apaf-1(-/-) and caspase-9(-/-) mice. Due to perinatal lethality, Emu-myc transgenic Apaf-1(-/-) or caspase-9(-/-) fetal liver cells were used to reconstitute lethally irradiated recipient mice. Surprisingly, no differences were seen in rate, incidence, or severity of lymphoma with loss of Apaf-1 or caspase-9, and Apaf-1 was not a critical determinant of anticancer drug sensitivity of c-myc-induced lymphomas. Moreover, loss of Apaf-1 did not promote oncogene-induced transformation of mouse embryo fibroblasts. Thus, Apaf-1 and caspase-9 do not suppress c-myc-induced lymphomagenesis and embryo fibroblast transformation.     

Interaction between NOD2 and CARD9 involves the NOD2 NACHT and the linker region between the NOD2 CARDs and NACHT domain

NOD2 activation by muramyl dipeptide causes a proinflammatory immune response in which the adaptor protein CARD9 works synergistically with NOD2 to drive p38 and c-Jun N-terminal kinase (JNK) signalling. To date the nature of the interaction between NOD2 and CARD9 remains undetermined. Here we show that this interaction is not mediated by the CARDs of NOD2 and CARD9 as previously suggested, but that NOD2 possesses two interaction sites for CARD9; one in the CARD–NACHT linker and one in the NACHT itself.     

Apaf-1 and caspase-9 accelerate apoptosis, but do not determine whether factor-deprived or drug-treated cells die

Apoptosis after growth factor withdrawal or drug treatment is associated with mitochondrial cytochrome c release and activation of Apaf-1 and caspase-9. To determine whether loss of Apaf-1, caspase-2, and caspase-9 prevented death of factor-starved cells, allowing them to proliferate when growth factor was returned, we generated IL-3-dependent myeloid lines from gene-deleted mice. Long after growth factor removal, cells lacking Apaf-1, caspase-9 or both caspase-9 and caspase-2 appeared healthy, retained intact plasma membranes, and did not expose phosphatidylserine. However, release of cytochrome c still occurred, and they failed to form clones when IL-3 was restored. Cells lacking caspase-2 alone had no survival advantage. Therefore, Apaf-1, caspase-2, and caspase-9 are not required for programmed cell death of factor-dependent cells, but merely affect its rate. In contrast, transfection with Bcl-2 provided long-term, clonogenic protection, and could act independently of the apoptosome. Unlike expression of Bcl-2, loss of Apaf-1, caspase-2, or caspase-9 would therefore be unlikely to enhance the survival of cancer cells.     

PAC-1 and isatin derivatives are weak matrix metalloproteinase inhibitors

Background

Dysregulation of apoptotic cell death is observed in a large number of pathological conditions. As caspases are central enzymes in the regulation of apoptosis, a large number of procaspase-activating compounds (PAC-1 derivatives) and inhibitors (isatin derivatives) have been developed. Matrix metalloproteinases (MMPs) have been shown to have a dual role in apoptosis. Hence compounds that either activate or inhibit caspases should ideally not affect MMPs. As many PAC-1 derivatives contain a zinc chelating ortho-hydroxy N-acyl hydrazone moiety and isatin derivatives has two carbonyl groups on the indole core, it was of interest to determine to which extent these compounds can inhibit MMPs.

Methods

Eight PAC-1 and five isatin derivatives were docked into MMP-9 and MMP-14. The same compounds were synthesized, characterized, purified and tested as inhibitors of MMP-9 and MMP-14, using fluorescence quenched peptide and biological substrates. Some of the compounds were also tested for fluorescence quenching.

Results

Molecular docking suggested that the different compounds can bind to the MMP active sites. However, kinetic studies showed that neither of these compounds was a strong MMP inhibitor. IC₅₀ values over 100 μM were obtained after the enzyme activities were corrected for quenching. These IC₅₀ values are far above the concentrations needed to activate or inhibit the caspases.

Conclusion

The use of PAC-1 and isatin derivatives against caspases should have little or no effect on the activity of MMPs.

General significance

Activators and inhibitors of caspases are important potential therapeutic agents for several diseases such as cancer, diabetes and neurodegenerative disorders.     

Apoptosis repressor with caspase recruitment domain (ARC) is expressed in cancer cells and localizes to nuclei

Apoptosis repressor with caspase recruitment domain is expressed at high levels in brain and myogenic tissues, consistent with a role to inhibit apoptosis in the terminally differentiated cells. Expression of ARC in cancers is not known. In this study, we reported that ARC was highly expressed in various non-myogenic and non-neurogenic human and rat cancer cell lines. Unexpectedly, ARC was localized almost exclusively to the nuclei of cancer cells, which was unlike the cytoplasmic localization of ARC in non-cancer cells. Furthermore, nuclear ARC in cancer cells did not co-localize with nucleolus protein of 30 kDa, an alternatively spliced ARC isoform. These findings indicate that ARC is distributed differently in cancer cells than non-cancer cells and thus might play a role in neoplastic transformation.     

Decreased expression of Apaf‐1 with progression of melanoma

Defects in apoptotic system may contribute in the pathogenesis and resistance of malignant melanoma cells to chemotherapy. Apoptotic protease‐activating factor‐1 (Apaf‐1) is a cell death effector that acts with cytochrome c and caspase‐9 to mediate apoptosis. Recently it was shown that metastatic melanomas often lose Apaf‐1 and are concomitantly resistant to apoptosis. It is not known, however, whether Apaf‐1 protein is lost during melanoma progression from localized to metastatic tumor. To this end, we evaluated Apaf‐1 protein expression by immunohistochemistry in 10 cases of human nevi, 11 melanomas in situ, 26 primary melanomas and 15 metastases. Significant decreases in Apaf‐1 expression was observed when comparing nevi and melanomas (chi‐square = 33.719; P < 0.0001). Moreover, primary melanomas with greater tumor thickness showed lesser expression of Apaf‐1 (chi‐square = 16.182; P < 0.003). Intriguingly, we were unable to detect Apaf‐1 expression in lesions of metastatic melanomas. These data demonstrated that there is an inverse correlation between Apaf‐1 expression and pathologic stage of melanoma. This suggests that the decreased expression of Apaf‐1 seen in correlation with melanoma progression renders melanoma more resistant to chemotherapy.     

Evidence that CED-9/Bcl2 and CED-4/Apaf-1 localization is not consistent with the current model for C. elegans apoptosis induction

In C. elegans, the BH3-only domain protein EGL-1, the Apaf-1 homolog CED-4 and the CED-3 caspase are required for apoptosis induction, whereas the Bcl-2 homolog CED-9 prevents apoptosis. Mammalian B-cell lymphoma 2 (Bcl-2) inhibits apoptosis by preventing the release of the Apaf-1 (apoptotic protease-activating factor 1) activator cytochrome c from mitochondria. In contrast, C. elegans CED-9 is thought to inhibit CED-4 by sequestering it at the outer mitochondrial membrane by direct binding. We show that CED-9 associates with the outer mitochondrial membrane within distinct foci that do not overlap with CED-4, which is predominantly perinuclear and does not localize to mitochondria. CED-4 further accumulates in the perinuclear space in response to proapoptotic stimuli such as ionizing radiation. This increased accumulation depends on EGL-1 and is abrogated in ced-9 gain-of-function mutants. CED-4 accumulation is not sufficient to trigger apoptosis execution, even though it may prime cells for apoptosis. Our results suggest that the cell death protection conferred by CED-9 cannot be solely explained by a direct interaction with CED-4.     

Fipronil induces CYP isoforms and cytotoxicity in human hepatocytes

Recent studies have demonstrated the potential of pesticides to either inhibit or induce xenobiotic metabolizing enzymes in humans. Exposure of human hepatocytes to doses of fipronil (5-amino-1-[2,6-dichloro-4-(trifluoromethyl)phenyl]-4-[(trifluoromethyl) sulfinyl]-1H-pyrazole-3-carbonitrile) ranging from 0.1 to 25 microM resulted in a dose dependent increase in CYP1A1 mRNA expression (3.5 to approximately 55-fold) as measured by the branched DNA assay. In a similar manner, CYP3A4 mRNA expression was also induced (10-30-fold), although at the higher doses induction returned to near control levels. CYP2B6 and 3A5 were also induced by fipronil, although at lower levels (2-3-fold). Confirmation of bDNA results were sought through western blotting and/or enzyme activity assays. Western blots using CYP3A4 antibody demonstrated a dose responsive increase from 0.5 to 1 microM followed by decreasing responses at higher concentrations. Similar increases and decreases were observed in CYP3A4-specific activity levels as measured using 6beta-hydroxytestosterone formation following incubation with testosterone. Likewise, activity levels for a CYP1A1-specific substrate, luciferin CEE, demonstrated that CYP1A1 enzyme activities were maximally induced by 1 microM fipronil followed by dramatically declining activity measurements at 10 and 25 microM. Cytotoxic effects of fipronil and fipronil sulfone were examined using the adenylate kinase and the trypan blue exclusion assays in HepG2 cells and human hepatocytes. The results indicate both that HepG2 cells and primary human hepatocytes are sensitive to the cytotoxic effects of fipronil. The maximum induction of adenylate kinase was ca. 3-fold greater than the respective controls in HepG2 and 6-10-fold in the case of primary hepatocytes. A significant time- and dose-dependent induction of adenylate kinase activity in HepG2 cells was noted from 0.1 to 12.5 microM fipronil followed by decreasing activities at 25 and 50 microM. For fipronil sulfone, cytotoxic effects increased throughout the dose range. The trypan blue assay indicated that cytotoxic effects contributing to an increase of greater than 10% of control values was indicated at doses above 12.5 microM. However, fipronil sulfone induced cytotoxic effects at lower doses. The possibility that cytotoxic effects were due to apoptosis was indicated by significant time- and dose-dependent induction of caspase-3/7 activity in both HepG2 cells and human hepatocytes. Fipronil mediated activation of caspase-3/7 in concurrence with compromised ATP production and viability are attributed to apoptotic cell death.     

Protease signalling in cell death: caspases versus cysteine cathepsins

Proteases were, for a long time, mainly considered as protein degrading enzymes. However, in the last decade this view has changed dramatically, and the focus is now on proteases as signalling molecules. One of the best examples is apoptosis, the major mechanism used by eukaryotes to remove superfluous, damaged and potentially dangerous cells, in which a number of proteases have been found to play a central role. Of these the caspases have been considered to be the major players. However, more recently, other proteases have been increasingly suggested as being important in apoptosis, in particular the cysteine cathepsins. In this review the roles of caspases and cysteine cathepsins in apoptosis signalling are compared and discussed.     

喉鳞癌Apaf-1基因表达及启动子区甲基化研究

喉鳞癌细胞存在多种癌基因和抑癌基因异常,Apaf-1(apoptotic protease activating factor-1)基因是诱导细胞凋亡的肿瘤抑制基因。为探讨Apaf-1基因在喉鳞癌发生中的作用,应用半定量PCR方法分析Apaf-1的表达,用比较基因组杂交(Comparative Genomic Hybrodization,CGH)和杂合性丢失(Loss of Heterozygosity,LOH)分析方法对喉鳞癌病人Apaf-1基因所在的12q22—23区域缺失情况进行研究．并用甲基化特异PCR对该基因启动子区甲基化情况进行了分析。结果表明：11例喉鳞癌组织出现Apaf-1 mRNA表达明显下调,占40．7％(11／27),而6例良性喉肿瘤未发现缺失或下调;CGH分析发现,18例喉鳞癌仅发现2例存在12q22-23区域缺失,未发现扩增;LOH分析发现,72例喉癌组织Apaf-1基因的5个多态位点LOH发生频率低,分别为18．2％(D12S346)、13．9％(D12S1706)、18．2％(D12S327)、22．2％(D12S1657)和16．6％(D12S393),11例出现Apaf-1 mRNA下调的喉癌组织均检测到启动子区甲基化,而16例Apaf-1 mRNA表达未下调者仅1例有甲基化,两者有显著差异(x^2检验,P=0．0001)。通过以上结果,首次证实Apaf-1基因与喉鳞癌相关,在喉鳞癌中Apaf-1基因缺失发生率低．提示启动子区甲基化是该基因失活的首要机制。     

Intracellular IL-15 controls mast cell survival

The regulation of mast cell activities and survival is a central issue in inflammatory immune responses. Here, we have investigated the role of mouse interleukin-15, a pro-inflammatory and pleiotropic cytokine, in the control of mast cell survival and homeostasis. We report that aged IL-15−/− mice show a reduced number of peritoneal mast cells compared to WT mice. Furthermore, IL-15 deficiency in bone marrow derived mouse mast cells (BMMCs) results in increased susceptibility to apoptosis mediated by growth factor deprivation and A-SMase-treatment. IL-15−/− BMMCs show a constitutive stronger mRNA and protein expression as well as enzymatic activity of the members of the mitochondrial apoptotic pathways including acidic lysosomal aspartate protease cathepsin D (CTSD), endogenous acid sphingomyelinase (A-SMase), caspase-3 and -7 compared to wild type (WT) BMMCs. Furthermore, IL-15−/− BMMCs constitutively generate more A-SMase-derived ceramide than WT controls and display a decreased expression of pro-survival sphingosin-1-phosphate (SPP) both in cytosol and membrane cell fractions. Furthermore, pre-treatment of mast cells with imipramine or pepstatin A, inhibitors of the intracellular acid sphingomyelinase and cathepsin D pathways respectively, increases survival in IL-15−/− BMMCs. These findings suggest that intracellular IL-15 is a key regulator of pathways controlling primary mouse mast cell homeostasis.     

Relationship between propeptide pH unfolding and inhibitory ability during ProDer p 1 activation mechanism

The major allergen Der p 1 of the house dust mite Dermatophagoides pteronyssinus is a papain-like cysteine protease (CA1) produced as an inactive precursor and associated with allergic diseases. The propeptide of Der p 1 exhibits a specific fold that makes it unique in the CA1 propeptide family. In this study, we investigated the activation steps involved in the maturation of the recombinant protease Der p 1 expressed in Pichia pastoris and the interaction of the full-length and truncated soluble propeptides with their parent enzyme in terms of activity inhibition and BIAcore interaction analysis. According to our results, the activation of protease Der p 1 is a multistep mechanism that is characterized by at least two intermediates. The propeptide strongly inhibits unglycosylated and glycosylated recombinant Der p 1 (K_D = 7 nM) at neutral pH. This inhibition is pH dependent. It decreases from pH 7 to pH 4 and can be related to conformational changes of the propeptide characterized by an increase of its flexibility and formation of a molten globule state. Our results indicate that activation of the zymogen at pH 4 is a compromise between activity preservation and propeptide unfolding.     

Polyglutamine expansion disturbs the endoplasmic reticulum formation,leading to caspase-7 activation through Bax

The endoplasmic reticulum (ER) plays a pivotal role in cellular functions such as the ER stress response. However, the effect of the ER membrane on caspase activation remains unclear. This study reveals that polyglutamine oligomers augmented at ER induce insertion of Bax into the ER membrane, thereby activating caspase-7. In line with the role of ER in cell death induced by polyglutamine expansion, the ER membrane was found to be disrupted and dilated in the brain of a murine model of Huntington’s disease. We can conclude that polyglutamine expansion may drive caspase-7 activation by disrupting the ER membrane.     

Mechanisms of NLRP1-Mediated Autoinflammatory Disease in Humans and Mice

NLRP1 was the first NOD-like receptor described to form an inflammasome, recruiting ASC to activate caspase-1, which processes interleukin-1β and interleukin-18 to their active form. A wealth of new genetic information has now redefined our understanding of this innate immune sensor. Specifically, rare loss-of-function variants in the N-terminal pyrin domain indicate that this part of NLRP1 is autoinhibitory and normally acts to prevent a familial autoinflammatory skin disease associated with cancer. In the absence of a ligand to trigger human NLRP1, these mutations have now confirmed the requirement of NLRP1 autolytic cleavage within the FIIND domain, which had previously been implicated in NLRP1 activation. Autolytic cleavage generates a C-terminal fragment of NLRP1 containing the CARD domain which then forms an ASC-dependent inflammasome. The CARD domain as an inflammasome linker is consistent with the observation that under some conditions, particularly for mouse NLRP1, caspase-1 can be engaged directly, and although it is no longer processed, it is still capable of producing mature IL-1β. Additional rare variants in a linker region between the LRR and FIIND domains of NLRP1 also cause autoinflammatory disease in both humans and mice. This new genetic information is likely to provide for more mechanistic insight in the years to come, contributing to our understanding of how NLRP1 functions as an innate immune sensor of infection and predisposes to autoimmune or autoinflammatory diseases.