A germ line mutation in the death domain of DAPK-1 inactivates ERK-induced apoptosis

p53 is activated genetically by a set of kinases that are components of the calcium calmodulin kinase superfamily, including CHK2, AMP kinase, and DAPK-1. In dissecting the mechanism of DAPK-1 control, a novel mutation (N1347S) was identified in the death domain of DAPK-1. The N1347S mutation prevented the death domain module binding stably to ERK in vitro and in vivo. Gel filtration demonstrated that the N1347S mutation disrupted the higher order oligomeric nature of the purified recombinant death domain miniprotein. Accordingly, the N1347S death domain module is defective in vivo in the formation of high molecular weight oligomeric intermediates after cross-linking with ethylene glycol bis(succinimidylsuccinate). Full-length DAPK-1 protein harboring a N1347S mutation in the death domain was also defective in binding to ERK in cells and was defective in formation of an ethylene glycol bis(succinimidylsuccinate)-cross-linked intermediate in vivo. Full-length DAPK-1 encoding the N1347S mutation was attenuated in tumor necrosis factor receptor-induced apoptosis. However, the N1347S mutation strikingly prevented ERK:DAPK-1-dependent apoptosis as defined by poly(ADP-ribose) polymerase cleavage, Annexin V staining, and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling imaging. Significant penetrance of the N1347S allele was identified in normal genomic DNA indicating the mutation is germ line, not tumor derived. The frequency observed in genomic DNA was from 37 to 45% for homozygous wild-type, 41 to 47% for heterozygotes, and 12 to 15% for homozygous mutant. These data highlight a naturally occurring DAPK-1 mutation that alters the oligomeric structure of the death domain, de-stabilizes DAPK-1 binding to ERK, and prevents ERK:DAPK-1-dependent apoptosis.     

Deletion of the BH1 domain of Bcl-2 accelerates apoptosis by acting in a dominant negative fashion

To investigate the exact biochemical functions by which Bcl-2 regulates apoptosis, we established a stable human small cell lung carcinoma cell line, Ms-1, overexpressing wild-type human Bcl-2 or various deletion and point mutants thereof, and examined the effect of these Bcl-2 mutants on apoptosis induced by antitumor drugs such as camptothecin. Cytochrome c release, caspase-3-(-like) protease activation, and apoptosis induced by antitumor drugs were accelerated by overexpression of Bcl-2 lacking a Bcl-2 homology (BH) 1 domain (Bcl-2/ DeltaBH1), but not by that of BH2, BH3, or BH4 domain-deleted Bcl-2. A similar result was obtained upon the substitution of glycine 145 with alanine in the BH1 domain (Bcl-2/G145A), which failed to interact with either Bax or Bak. Pro-apoptotic Bax and Bak have been known to be activated in response to antitumor drugs, and Bcl-2/G145A as well as Bcl-2/DeltaBH1 also accelerated Bax- or Bak-induced apoptosis in HEK293T cells. These two mutants still retained the ability to interact with wild-type Bcl-2 and Bcl-xL, and abrogated the inhibitory effect of wild-type Bcl-2 or Bcl-xL on Bax- or Bak-induced apoptosis. In addition, immunoprecipitation studies revealed that Bcl-2/DeltaBH1 and Bcl-2/G145A interrupted the association between wild-type Bcl-2 and Bax/Bak. Taken together, our results demonstrate that Bcl-2/DeltaBH1 or Bcl-2/G145A acts as a dominant negative of endogenous anti-apoptotic proteins such as Bcl-2 and Bcl-xL, thereby enhancing antitumor drug-induced apoptosis, and that this dominant negative activity requires both a failure of interaction with Bax and Bak through the BH1 domain of Bcl-2 and retention of the ability to interact with Bcl-2 and Bcl-xL.     

A dominant negative inhibitor indicates that monocyte chemoattractant protein 1 functions as a dimer.

Monocyte chemoattractant protein 1 (MCP-1) is a member of the chemokine family of proinflammatory cytokines, all of which share a high degree of amino acid sequence similarity. Aberrant expression of chemokines occurs in a variety of diseases that have an inflammatory component, such as atherosclerosis. Although structural analyses indicate that chemokines form homodimers, there is controversy about whether dimerization is necessary for activity. To address this question for MCP-1, we obtained evidence in four steps. First, coprecipitation experiments demonstrated that MCP-1 forms dimers at physiological concentrations. Second, chemically cross-linked MCP-1 dimers attract monocytes in vitro with a 50% effective concentration of 400 pM, identical to the activity of non-cross-linked MCP-1. Third, an N-terminal deletion variant of MCP-1 (called 7ND) that inhibits MCP-1-mediated monocyte chemotaxis specifically forms heterodimers with wild-type MCP-1. Finally, although 7ND inhibits wild-type MCP-1 activity, it has no effect on cross-linked MCP-1. These results indicate that 7ND is a dominant negative inhibitor, implying that MCP-1 activates its receptor as a dimer. In addition, chemical cross-linking restores activity to an inactive N-terminal insertional variant of MCP-1, further supporting the need for dimerization. Since the reported Kd for MCP-1 monomer dissociation is much higher than its 50% effective concentration or Kd for receptor binding, active dimer formation may require high local concentrations of MCP-1. Our data further suggest that the dimer interface can be a target for MCP-1 inhibitory drugs.     

Identification and analysis of dominant negative mutants of RIP1 DD that disrupt RIPoptosome core formation

The RIPoptosome, composed of RIP1 and caspase-8, plays an important role in the regulation of apoptosis and necroptosis; however, the mechanism of complex formation by oligomerization and how the caspase-activating process and necroptosis are mediated by the formation of the RIPoptosome is not well-understood. This study revealed that the assembly mechanism of the RIPoptosome core is dependent on salt concentration and not on pH and time. In addition, we demonstrated that three RIP1 mutations, E626K, M637K, and S657K, have dominant negative effects. These dominant negative mutations in RIP1 may have potential applications in therapeutic intervention.     

Identification of a functional domain in a GADD45-mediated G2/M checkpoint

Cell cycle checkpoints are essential for the maintenance of genomic stability in response to DNA damage. We demonstrated recently that GADD45, a DNA damage-inducible protein, activates a G(2)/M checkpoint induced by either UV radiation or alkylating agents. GADD45 can interact in vivo with the G(2) cell cycle-specific kinase, Cdc2, proliferating cell nuclear antigen (PCNA), and the cell cycle kinase inhibitor p21(waf1). The ability of GADD45 to induce a G(2)/M arrest may be caused in part by the inhibition of Cdc2 kinase activity. Here, we report the identification of a region of GADD45 that is involved in this G(2)/M checkpoint. Mutants of GADD45 that lacked either the first 35 or the last 80 residues still retained an ability to induce G(2)/M arrest. A mutant with a deletion of the central region (residues 50-76), which is conserved in the family members GADD45beta and GADD45gamma, lacked such activity. This mutant also lacked an ability to bind to Cdc2, PCNA, and p21(waf1) in vivo. Consistently, either GADD45beta or GADD45gamma bind to Cdc2 in vivo. However, unlike GADD45, neither GADD45beta nor GADD45gamma inhibited the Cdc2 kinase or induced G(2)/M arrest. The unique effect of GADD45 may be caused by the presence of a region containing DEDDDR residues. Alanine substitutions in the region abolished GADD45 induction of a G(2)/M arrest and its inactivation of the Cdc2 kinase but not its binding to Cdc2, PCNA, or p21(waf1). Therefore, the binding of GADD45 to Cdc2 was insufficient to induce a G(2)/M arrest, and additional activity contributed by the DEDDDR residues may be necessary to regulate the G(2)/M checkpoint.     

Identification of a UV-induced trans-acting protein that stimulates polyomavirus DNA replication.

Previous studies provided indirect evidence that the ability of a variety of DNA-damaging agents to induce asynchronous polyomavirus DNA replication in the H3 rat fibroblast cell line is mediated by a trans-acting factor. Using an erythrocyte insertion technique to introduce protein fractions from UV-irradiated cells into unirradiated H3 cells, we have now obtained evidence that this factor is a 60-kilodalton protein. These findings provide evidence that DNA damage in mammalian cells induces a factor that can alter the replication of a viral DNA.     

DNA stimulates Mec1-mediated phosphorylation of replication protein A

The cellular single-stranded DNA (ssDNA)-binding protein replication protein A (RPA) becomes phosphorylated periodically during the normal cell cycle and also in response to DNA damage. In Saccharomyces cerevisiae, RPA phosphorylation requires the checkpoint protein Mec1, a protein kinase homologous in structure and function to human ATR. We confirm here that immunocomplexes containing a tagged version of Mec1 catalyze phosphorylation of purified RPA, likely reflecting an RPA kinase activity intrinsic to Mec1. A significant stimulation of this activity is observed upon the addition of covalently closed ssDNA derived from the bacteriophage M13. This stimulation is not observed with mutant RPA deficient for DNA binding, indicating that DNA-bound RPA is a preferred substrate. Stimulation is also observed upon the addition of linear ssDNA homopolymers or hydrolyzed M13 ssDNA. In contrast to circular ssDNA, these DNA cofactors stimulate both wild type and mutant RPA phosphorylation. This finding suggests that linear ssDNA can also stimulate Mec1-mediated RPA phosphorylation by activating Mec1 or an associated protein. Although the Mec1-interacting protein Ddc2 is required for RPA phosphorylation in vivo, it is required for neither basal nor ssDNA-stimulated RPA phosphorylation in vitro. Therefore, activation of Mec1-mediated RPA phosphorylation by either circular or linear ssDNA does not operate through Ddc2. Our results provide insight into the mechanisms that function in vivo to specifically induce RPA phosphorylation upon initiation of DNA replication, repair, or recombination.     

Identification and functional analysis of two aromatic-ring-hydroxylating dioxygenases from a sphingomonas strain that degrades various polycyclic aromatic hydrocarbons

In this study, the enzymes involved in polycyclic aromatic hydrocarbon (PAH) degradation in the chrysene-degrading organism Sphingomonas sp. strain CHY-1 were investigated. [14C]chrysene mineralization experiments showed that PAH-grown bacteria produced high levels of chrysene-catabolic activity. One PAH-induced protein displayed similarity with a ring-hydroxylating dioxygenase beta subunit, and a second PAH-induced protein displayed similarity with an extradiol dioxygenase. The genes encoding these proteins were cloned, and sequence analysis revealed two distinct loci containing clustered catabolic genes with strong similarities to corresponding genes found in Novosphingobium aromaticivorans F199. In the first locus, two genes potentially encoding a terminal dioxygenase component, designated PhnI, were followed by a gene coding for an aryl alcohol dehydrogenase (phnB). The second locus contained five genes encoding an extradiol dioxygenase (phnC), a ferredoxin (phnA3), another oxygenase component (PhnII), and an isomerase (phnD). PhnI was found to be capable of converting several PAHs, including chrysene, to the corresponding dihydrodiols. The activity of PhnI was greatly enhanced upon coexpression of genes encoding a ferredoxin (phnA3) and a reductase (phnA4). Disruption of the phnA1a gene encoding the PhnI alpha subunit resulted in a mutant strain that had lost the ability to grow on PAHs. The recombinant PhnII enzyme overproduced in Escherichia coli functioned as a salicylate 1-hydroxylase. PhnII also used methylsalicylates and anthranilate as substrates. Our results indicated that a single enzyme (PhnI) was responsible for the initial attack of a range of PAHs, including chrysene, in strain CHY-1. Furthermore, the conversion of salicylate to catechol was catalyzed by a three-component oxygenase unrelated to known salicylate hydroxylases.     

DAPK-1 binding to a linear peptide motif in MAP1B stimulates autophagy and membrane blebbing

DAPK-1 (death-activated protein kinase) has wide ranging functions in cell growth control; however, DAPK-1 interacting proteins that mediate these effects are not well defined. Protein-protein interactions are driven in part by linear interaction motifs, and combinatorial peptide libraries were used to identify peptide interfaces for the kinase domain of DAPK-1. Peptides bound to DAPK-1core kinase domain fragments had homology to the N-terminal domain of the microtubule-associated protein MAP1B. Immunobinding assays demonstrated that DAPK-1 can bind to the full-length human MAP1B, a smaller N-terminal miniprotein containing amino acids 1-126 and the 12-amino acid polypeptides acquired by peptide selection. Amino acid starvation of cells induced a stable immune complex between MAP1B and DAPK-1, identifying a signal that forms the endogenous complex in cells. DAPK-1 and MAP1B co-expression form a synthetic lethal interaction as they cooperate to induce growth inhibition in a clonogenic assay. In cells, two co-localizing populations of DAPK-1 and MAP1B were observed using confocal microscopy; one pool co-localized with MAP1B plus tubulin, and a second pool co-localized with MAP1B plus cortical F-actin. Reduction of MAP1B protein using short interfering RNA attenuated DAPK-1-stimulated autophagy. Transfected MAP1B can synergize with DAPK-1 to stimulate membrane blebbing, whereas reduction of MAP1B using short interfering RNA attenuates DAPK-1 membrane blebbing activity. The autophagy inhibitor 3-methyladenine inhibits the DAPK-1 plus MAP1B-mediated membrane blebbing. These data highlight the utility of peptide aptamers to identify novel binding interfaces and highlight a role for MAP1B in DAPK-1-dependent signaling in autophagy and membrane blebbing.     

Identification and purification of a protein that stimulates the helicase activity of the Escherichia coli Rep protein

A polypeptide (Mr = 15,000) has been purified from Escherichia coli cell extracts that significantly stimulates the duplex DNA unwinding reaction catalyzed by E. coli Rep protein. The Rep helicase unwinding reaction was stimulated by as much as 20-fold, upon addition of the stimulatory protein, using either a 71-base pair or a 343-base pair partial duplex DNA molecule as a substrate. The purified Rep helicase stimulatory protein (RHSP) had no intrinsic helicase activity or ATP hydrolysis activity and did not stimulate the single-stranded DNA-dependent ATP hydrolysis reaction catalyzed by Rep protein. It is likely that RHSP stimulates the Rep helicase unwinding reaction by stoichiometric binding to single-stranded DNA. However, a specific interaction between Rep protein and RHSP cannot be ruled out, since RHSP did not stimulate the duplex DNA unwinding reactions catalyzed by E. coli helicase I or the recently discovered 75-kDa helicase. RHSP did stimulate the duplex DNA unwinding reaction catalyzed by E. coli helicase II. The identification and subsequent purification of RHSP from cell extracts demonstrates the feasibility of using direct helicase assays to purify stimulatory proteins.     

RAD51AP1 is a structure-specific DNA binding protein that stimulates joint molecule formation during RAD51-mediated homologous recombination

Homologous recombination is essential for preserving genome integrity. Joining of homologous DNA molecules through strand exchange, a pivotal step in recombination, is mediated by RAD51. Here, we identify RAD51AP1 as a RAD51 accessory protein that specifically stimulates joint molecule formation through the combination of structure-specific DNA binding and physical contact with RAD51. At the cellular level, we show that RAD51AP1 is required to protect cells from the adverse effects of DNA double-strand break-inducing agents. At the biochemical level, we show that RAD51AP1 has a selective affinity for branched-DNA structures that are obligatory intermediates during joint molecule formation. Our results highlight the importance of structural transitions in DNA as control points in recombination. The affinity of RAD51AP1 for the central protein and DNA intermediates of recombination confers on it the ability to control the preservation of genome integrity at a number of critical mechanistic steps.     

Identification of a conserved anti-apoptotic protein that modulates the mitochondrial apoptosis pathway

Here we identified an evolutionarily highly conserved and ubiquitously expressed protein (C9orf82) that shows structural similarities to the death effector domain of apoptosis-related proteins. RNAi knockdown of C9orf82 induced apoptosis in A-549 and MCF7/casp3-10b lung and breast carcinoma cells, respectively, but not in cells lacking caspase-3, caspase-10 or both. Apoptosis was associated with activated caspases-3, -8, -9 and -10, and inactivation of caspases 10 or 3 was sufficient to block apoptosis in this pathway. Apoptosis upon knockdown of C9orf82 was associated with increased caspase-10 expression and activation, which was required for the generation of an 11 kDa tBid fragment and activation of Caspase-9. These data suggest that C9orf82 functions as an anti-apoptotic protein that modulates a caspase-10 dependent mitochondrial caspase-3/9 feedback amplification loop. We designate this ubiquitously expressed and evolutionarily conserved anti-apoptotic protein Conserved Anti-Apoptotic Protein (CAAP). We also demonstrated that treatment of MCF7/casp3-10b cells with staurosporine and etoposides induced apoptosis and knockdown of CAAP expression. This implies that the CAAP protein could be a target for chemotherapeutic agents.     

Identification of a domain that mediates vesicle aggregation reveals functional diversity of annexin repeats.

Annexins are structurally related proteins that bind phospholipids in a Ca2(+)-dependent manner and possess at least four conserved 70-amino acid repeat domains. The ability of certain annexins to promote contact between vesicle membranes in vitro has prompted the suggestion that these proteins regulate membrane traffic in exocytosis. We have previously found that annexins I and II promote contact between vesicles whereas annexin V does not. In order to understand the mechanism of annexin I-mediated vesicle-vesicle contact, we prepared a monoclonal antibody that specifically inhibits annexin I-mediated vesicle aggregation. We identified the domain of annexin I recognized by this monoclonal antibody by using it to screen an expression library containing random fragments of annexin I cDNA. The antibody identified a fragment encoding amino acids 41-118 (the first repeat plus 8 residues of the amino-terminal tail). We constructed a chimeric protein containing these amino acids of annexin I fused to the second, third, and fourth repeats of annexin V. Transfer of this domain conferred the ability to promote vesicle aggregation, confirming that this domain participates directly in mediating contact between vesicle membranes.     

Identification of peptides that antagonize formyl peptide receptor-like 1-mediated signaling

Formyl peptide receptor-like 1 (FPRL1) is an important classical chemoattractant receptor that is expressed in phagocytic cells in the peripheral blood and brain. Recently, various novel agonists have been identified from several origins, such as host-derived molecules. Activation of FPRL1 is closely related to inflammatory responses in the host defense mechanism and neurodegenerative disorders. In the present study we identified several novel peptides by screening hexapeptide libraries that inhibit the binding of one of FPRL1's agonists (Trp-Lys-Tyr-Met-Val-D-Met-CONH(2) (WKYMVm)) to its specific receptor, FPRL1, in RBL-2H3 cells. Among the novel peptides, Trp-Arg-Trp-Trp-Trp-Trp-CONH(2) (WRWWWW (WRW(4))) showed the most potent activity in terms of inhibiting WKYMVm binding to FPRL1. We also found that WRW(4) inhibited the activation of FPRL1 by WKYMVm, resulting in the complete inhibition of the intracellular calcium increase, extracellular signal-regulated kinase activation, and chemotactic migration of cells toward WKYMVm. For the receptor specificity of WRW(4) to the FPR family, we observed that WRW(4) specifically inhibit the increase in intracellular calcium by the FPRL1 agonists MMK-1, amyloid beta42 (Abeta42) peptide, and F peptide, but not by the FPR agonist, fMLF. To investigate the effect of WRW(4) on endogenous FPRL1 ligand-induced cellular responses, we examined its effect on Abeta42 peptide in human neutrophils. Abeta42 peptide-induced superoxide generation and chemotactic migration of neutrophils were inhibited by WRW(4), which also completely inhibited the internalization of Abeta42 peptide in human macrophages. WRW(4) is the first specific FPRL1 antagonist and is expected to be useful in the study of FPRL1 signaling and in the development of drugs against FPRL1-related diseases.     

Identification of a domain in guanylyl cyclase-activating protein 1 that interacts with a complex of guanylyl cyclase and tubulin in photoreceptors

The membrane-bound guanylyl cyclase in rod photoreceptors is activated by guanylyl cyclase-activating protein 1 (GCAP-1) at low free [Ca2+]. GCAP-1 is a Ca2+-binding protein and belongs to the superfamily of EF-hand proteins. We created an oligopeptide library of overlapping peptides that encompass the entire amino acid sequence of GCAP-1. Peptides were used in competitive screening assays to identify interaction regions in GCAP-1 that directly bind the guanylyl cyclase in bovine photoreceptor cells. We found four regions in GCAP-1 that participate in regulating guanylyl cyclase. A 15-amino acid peptide located adjacent to the second EF-hand motif (Phe73-Lys87) was identified as the main interaction domain. Inhibition of GCAP-1-stimulated guanylyl cyclase activity by the peptide Phe73-Lys87 was completely relieved when an excess amount of GCAP-1 was added. An affinity column made from this peptide was able to bind a complex of photoreceptor guanylyl cyclase and tubulin. Using an anti-GCAP-1 antibody, we coimmunoprecipitated GCAP-1 with guanylyl cyclase and tubulin. Complex formation between GCAP-1 and guanylyl cyclase was observed independent of [Ca2+]. Our experiments suggest that there exists a tight association of guanylyl cyclase and tubulin in rod outer segments.     

Identification and functional characterization of methyl-CpG binding domain protein from Tribolium castaneum

Methyl-CpG binding domain proteins (MBD) can specifically bind to methylated CpG sites and play important roles in epigenetic gene regulation. Here, we identified and functionally characterized the MBD protein in Tribolium castaneum. T. castaneum genome encodes only one MBD protein: TcMBD2/3. RNA interference targeting this gene at different developmental stages caused lethal phenotypes including metamorphosis deficiency in larvae and pupae, gastrointestinal system problems and fecundity deficiency in adult. Moreover, Tcmbd2/3 knockdown adult showed progressive reduced locomoter activity, a typical neurodegeneration phenotype. This is a common feature of DNA methylation in mammals and has not been found in other insects. However, band shift assays demonstrated that TcMBD2/3 could not bind to methylated DNA, indicating the essential roles of TcMBD2/3 is independent of DNA methylation. Our study provides Tcmbd2/3 plays important roles in T. castaneum and gives new insights into the potential mechanism of action of MBD proteins in insect.