Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.6.1.3 (ATPase)
65,361 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Apaf-1 plays a critical role in apoptosis by binding to and activating procaspase-9. We have identified a novel Apaf-1 cDNA encoding a protein of 1248 amino acids containing an insertion of 11 residues between the CARD and ATPase domains, and another 43 amino acid insertion creating an additional WD-40 repeat. The product of this Apaf-1 cDNA activated procaspase-9 in a cytochrome c and dATP/ATP-dependent manner. We used this Apaf-1 to show that Apaf-1 requires dATP/ATP hydrolysis to interact with cytochrome c, self-associate and bind to procaspase-9. A P-loop mutant (Apaf-1K160R) was unable to associate with Apaf-1 or bind to procaspase-9. Mutation of Met368 to Leu enabled Apaf-1 to self-associate and bind procaspase-9 independent of cytochrome c, though still requiring dATP/ATP for these activities. The Apaf-1M368L mutant exhibited greater ability to induce apoptosis compared with the wild-type Apaf-1. We also show that procaspase-9 can recruit procaspase-3 to the Apaf-1-procaspase-9 complex. Apaf-1(1-570), a mutant lacking the WD-40 repeats, associated with and activated procaspase-9, but failed to recruit procaspase-3 and induce apoptosis. These results suggest that the WD-40 repeats may be involved in procaspase-9-mediated procaspase-3 recruitment. These studies elucidate biochemical steps required for Apaf-1 to activate procaspase-9 and induce apoptosis.
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PMID:Role of cytochrome c and dATP/ATP hydrolysis in Apaf-1-mediated caspase-9 activation and apoptosis. 1039 75

Apaf-1 is an important apoptotic signaling molecule that can activate procaspase-9 in a cytochrome c/dATP-dependent fashion. Alternative splicing can create an NH(2)-terminal 11-amino acid insert between the caspase recruitment domain and ATPase domains or an additional COOH-terminal WD-40 repeat. Recently, several Apaf-1 isoforms have been identified in tumor cell lines, but their expression in tissues and ability to activate procaspase-9 remain poorly characterized. We performed analysis of normal tissue mRNAs to examine the relative expression of the Apaf-1 forms and identified Apaf-1XL, containing both the NH(2)-terminal and COOH-terminal inserts, as the major RNA form expressed in all tissues tested. We also identified another expressed isoform, Apaf-1LN, containing the NH(2)-terminal insert, but lacking the additional WD-40 repeat. Functional analysis of all identified Apaf-1 isoforms demonstrated that only those with the additional WD-40 repeat activated procaspase 9 in vitro in response to cytochrome c and dATP, while the NH(2)-terminal insert was not required for this activity. Consistent with this result, in vitro binding assays demonstrated that the additional WD-40 repeat was also required for binding of cytochrome c, subsequent Apaf-1 self-association, binding to procaspase-9, and formation of active Apaf-1 oligomers. These experiments demonstrate the expression of multiple Apaf-1 isoforms and show that only those containing the additional WD-40 repeat bind and activate procaspase-9 in response to cytochrome c and dATP.
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PMID:Expression and functional analysis of Apaf-1 isoforms. Extra Wd-40 repeat is required for cytochrome c binding and regulated activation of procaspase-9. 1072 81

Apaf-1 plays a crucial role in the cytochrome c/dATP-dependent activation of caspase-9 and -3. We found that the human myeloid leukemic K562 cells were more resistant to cytochrome c-induced activation of caspase-9 and -3 in a cell-free system compared with the human T-lymphoblastic subclone CEM/VLB(100) cells. Apaf-1 cDNA sequencing revealed an additional insert of 11 aa between the CARD and CED-4 (ATPase) domains in K562 cells, which was identical to the sequence of Apaf-1XL. Immunoprecipitation of Apaf-1 with caspase-9 after a cell-free reaction demonstrated that Apaf-1XL in the K562 cell line showed a lower binding ability to caspase-9 compared with Apaf-1L protein. The resistance of K562 cells to cytochrome c-dependent apoptosis may be partly due to this Apaf-1XL form. These results suggest that the additional insert between CARD and CED-4 domains might affect Apaf-1 recruitment of caspase-9 during apoptosis.
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PMID:Apaf-1XL is an inactive isoform compared with Apaf-1L. 1126 2

We have found a novel alternative splicing product of the apoptotic protease activating factor 1 (APAF-1), termed APAF-1-ALT, in the LNCaP human prostate cancer cell line. APAF-1-ALT harbors the caspase recruitment domain and an incomplete CED-4 like/ATPase domain, but lacks the WD-40 repeat units. The LNCaP cell expressed the full-length APAF-1 weakly and APAF-1-ALT rather abundantly, especially after mycoplasma infection. LNCaP underwent a retarded DNA damage-induced apoptosis, which was independent of caspase 9 activity. APAF-1-ALT functioned less effectively in inducing apoptosis than did APAF-1-XL, the full-length APAF-1, in transient transfection. Moreover, APAF-1-ALT interfered with APAF-1-XL's ability to induce apoptosis in transient double transfection experiment. These results indicate that APAF-1-ALT is a molecule that is deficient and impeded for mediating apoptosis and that it may contribute to the resistance to DNA damage-induced treatment observed in LNCaP.
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PMID:APAF-1-ALT, a novel alternative splicing form of APAF-1, potentially causes impeded ability of undergoing DNA damage-induced apoptosis in the LNCaP human prostate cancer cell line. 1280 98

CED-4, a pro-apoptotic factor in Caenorhabditis elegans, activates the cell death protease CED-3. CED-9 directly binds to CED-4 and represses this. However, it has remained unclear whether a mammalian CED-9 homologue, Bcl-XL, inhibits the function of the mammalian CED-4 homologue, Apaf-1, by direct binding. To analyze the interaction, we adopted a yeast two-hybrid system. Since Bcl-XL and the CED-4-like portion of Apaf-1 failed to exhibit a positive result in the assay, we prepared "fragment libraries" of bcl-XL or apaf-1 cDNA. By screening of the apaf-1 "fragment library," we obtained nine clones interacting with Bcl-XL, all containing the same region within the ATPase domain, designated BBR: the Bcl-XL binding region. Binding of BBR to Bcl-XL was also confirmed by immunoprecipitation assays. Bcl-2, Bcl-w, A1/Bfl-1, and Boo/Diva failed to show the same capacity for binding to BBR as Bcl-XL. These results indicate that Bcl-XL directly binds to a specific region in Apaf-1.
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PMID:Identification of a Bcl-XL binding region within the ATPase domain of Apaf-1. 1296 20

Apoptotic protease activating factor-1 (Apaf-1) is a critical regulator of apoptosis and a crucial part of the apoptosome that is assembled in response to several cellular stresses like hypoxia. We have previously shown that hypoxia results in increased influx of nuclear Ca(2+) and increased expression of nuclear apoptotic proteins. The present study investigates that Apaf-1 is expressed during hypoxia in the cerebral cortex of newborn piglets and that administration of clonidine prevents the hypoxia induced increase expression of Apaf-1. Studies were conducted in 19 newborn piglets, 6 normoxic (Nx), 7 hypoxic (Hx FiO(2) of 0.05-0.07 for 1h) and 6 clonidine-treated hypoxic (Hx-Clo) piglets. Tissue hypoxia was confirmed biochemically by determining the levels of high energy phosphates ATP and phosphocreatine (PCr). Neuronal nuclei, mitochondrial membranes and cytosolic fractions were isolated and separated by 12% SDS-PAGE and probed with specific antibodies to Apaf-1. The expression of Apaf-1 in neuronal nuclei was 48.86+/-5.27 in Nx, 108.43+/-6.37 in Hx and 78.53+/-7.00 in Hx-Clo. The Apaf-1 expression of in mitochondrial fraction was 72.73+/-11.76 in Nx, 132.27+/-16.15 in Hx and 85.17+/-5.64 in Hx-Clo. Similarly, the expression of Apaf-1 in cytosolic fraction was 86.79+/-6.97 in Nx, 193.95+/-15.41 in Hx and 111.07+/-7.91 in Hx-Clo. In summary, the results show that hypoxia results in increased expression of Apaf-1 proteins in neuronal nuclear, mitochondrial and cytosolic fractions. Administration of a high affinity Ca(2+)-ATPase, prevented the hypoxia induced increased expression of Apaf-1 protein, suggesting that the hypoxia-induced increased expression of Apaf-1 proteins is nuclear Ca(2+)-influx mediated. We conclude that cerebral hypoxia-induced increase in Apaf-1 protein will lead to increased activation of procaspase-9 to caspase-9 in the cytosolic compartment leading to a cascade of hypoxic neuronal death.
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PMID:Mechanisms of expression of apoptotic protease activating factor-1 (Apaf-1) in nuclear, mitochondrial and cytosolic fractions of the cerebral cortex of newborn piglets. 1727 90

Three dimensional models of NB-ARC domains in five different proteins were constructed based on the recently published crystal structure of the apoptotic protease activating factor 1, of which two are for tomato species, one each for flax, Arabidopsis, and nematode. Standard multiple sequence alignment was performed for chosen members of the NB-ARC domains, very divergent from each other in protein sequence, followed by homology model building and structure refinement. In this alignment, amino acid insertions and deletions between members generally fall in loop regions or at ends of alpha helices. Despite the presence of sequence divergence between the species, it is argued that the NB-ARC domains carry out the similar biological functions in the various species, highlighting the ATP binding and ATPase activity. By our comparative study of these models, it is predicted that NB-ARC domains should bind ADP/ATP rather than GDP/GTP. Both natural and induced mutants of Arabidopsis within the RPS2 locus and their phenotypes for disease reaction against Pseudomonas syringae are rationalized from the protein model. Apaf-1 Thr263 and Arg265 positions conserved totally within the NB-ARC domains are predicted to take active part in the catalytic activity of kinase-3 motif, the arginine known as the sensor I motif in AAA+ proteins. This was later verified for the Ced-4 crystal structure in complex with Ced-9. Our model of Ced-4 based on Apaf-1 was also compared with its crystal structure in the Ced-4-Ced-9 complex; the 3 layered alpha/beta domain superposes quite well, helical domain I is shifted by about 5 A but the winged helix domain is rotated away to a new position. Since Apaf-1 was crystallized with ADP and Ced-4-Ced9 with magnesium-ATP, this rotation signifies a change in structure of these NB-ARC domains between the two forms. Further, we hypothesize that certain mutants in the plant R proteins called 'constitutive gain-of-function' or 'autocatalytic' dispose their winged helix domains permanently like the magnesium-ATP form as observed for Ced-4, avoiding the closed ADP conformation. The models are also validated with mutagenesis data for a related tomato protein I-2, tomato prf and flax, including loss of function, wild type and autocatalytic phenotypes, and compared with similar data for potato and tobacco proteins, for which models were not built. These three dimensional models would help us to understand the spatial arrangement, function of R proteins and their conserved motifs.
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PMID:Three-dimensional models of NB-ARC domains of disease resistance proteins in tomato, Arabidopsis, and flax. 1809 30

Innate immunity represents an important system with a variety of vital processes at the core of many diseases. In recent years, the central role of the Nod-like receptor (NLR) protein family became increasingly appreciated in innate immune responses. NLRs are classified as part of the signal transduction ATPases with numerous domains (STAND) clade within the AAA+ ATPase family. They typically feature an N-terminal effector domain, a central nucleotide-binding domain (NACHT) and a C-terminal ligand-binding region that is composed of several leucine-rich repeats (LRRs). NLRs are believed to initiate or regulate host defense pathways through formation of signaling platforms that subsequently trigger the activation of inflammatory caspases and NF-kB. Despite their fundamental role in orchestrating key pathways in innate immunity, their mode of action in molecular terms remains largely unknown. Here we present the first comprehensive sequence and structure modeling analysis of NLR proteins, revealing that NLRs possess a domain architecture similar to the apoptotic initiator protein Apaf-1. Apaf-1 performs its cellular function by the formation of a heptameric platform, dubbed apoptosome, ultimately triggering the controlled demise of the affected cell. The mechanism of apoptosome formation by Apaf-1 potentially offers insight into the activation mechanisms of NLR proteins. Multiple sequence alignment analysis and homology modeling revealed Apaf-1-like structural features in most members of the NLR family, suggesting a similar biochemical behaviour in catalytic activity and oligomerization. Evolutionary tree comparisons substantiate the conservation of characteristic functional regions within the NLR family and are in good agreement with domain distributions found in distinct NLRs. Importantly, the analysis of LRR domains reveals surprisingly low conservation levels among putative ligand-binding motifs. The same is true for the effector domains exhibiting distinct interfaces ensuring specific interactions with downstream target proteins. All together these factors suggest specific biological functions for individual NLRs.
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PMID:The Nod-like receptor (NLR) family: a tale of similarities and differences. 1844 35

Cdc6 is the bifunctional AAA+ ATPase that assembles prereplicative complexes on origins of replication and activates p21(CIP1)- or p27(KIP1)-bound Cdk2. During the G(1)-S transition, the Cdc6 gene essential for chromosomal replication is activated by the E2F transcriptional factor. Paradoxically, Apaf-1 encoding the central component of the apoptosome is also activated at the same time and by E2F. Consequently, genes for antipodal life and death are regulated in the same manner by the same transcriptional factor. Here we report a striking solution to this paradox. Besides performing prereplicative complex assembly and Cdk2 activation, Cdc6 obstructed apoptosome assembly by forming stable complexes very likely with a monomer of cytochrome c-activated Apaf-1 molecules. This function depended on its own ATPase domain but not on the cyclin-binding motif. In proliferating rodent fibroblasts, Cdc6 continued to block apoptosome assembly induced by a non-cytochrome c or some other mechanism, suppressing seemingly unintended apoptosis when promoting cell proliferation. Thus, Cdc6 is an AAA+ ATPase with three functions, all working for life.
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PMID:Cdc6 protein obstructs apoptosome assembly and consequent cell death by forming stable complexes with activated Apaf-1 molecules. 2568 11

Cdc6 is the AAA+ ATPase that assembles prereplicative complexes on replication origins in eukaryotic chromosomes. Recently, the same Cdc6 protein was found to exert two more functions in mammalian cells to promote cell proliferation and survival: ATP-dependent activation of p21(CIP1)- or p27(KIP1)-bound Cdk2-cyclin A/E complexes and obstruction of apoptosome assembly and consequent cell death by forming stable complexes with activated Apaf-1 molecules. These findings not only redefined the biological role of mammalian Cdc6 but also led the discovery of entirely new mechanisms controlling Cdk2 activity and apoptosis. This review focuses on this multi-functional AAA+ ATPase and the newly discovered mechanisms by which it controls the G(1)-S transition and cell survival during proliferation.
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PMID:Cdc6: a trifunctional AAA+ ATPase that plays a central role in controlling the G(1)-S transition and cell survival. 2290 36


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