Identification of functionally relevant histidine residues in the apoptotic nuclease CAD

The caspase-activated DNase CAD (DFF40/CPAN) degrades chromosomal DNA during apoptosis. Chemical modification with DEPC inactivates the enzyme, suggesting that histidine residues play a decisive role in the catalytic mechanism of this nuclease. Sequence alignment of murine CAD with four homologous apoptotic nucleases reveals four completely (His242, His263, His304 and His308) and two partially (His127 and His313) conserved histidine residues in the catalytic domain of the enzyme. We have changed these residues to asparagine and characterised the variant enzymes with respect to their DNA cleavage activity, structural integrity and oligomeric state. All variants show a decrease in activity compared to the wild-type nuclease as measured by a plasmid DNA cleavage assay. H242N, H263N and H313N exhibit DNA cleavage activities below 5% and H308N displays a drastically altered DNA cleavage pattern compared to wild-type CAD. Whereas all variants but one have the same secondary structure composition and oligomeric state, H242N does not, suggesting that His242 has an important structural role. On the basis of these results, possible roles for His127, His263, His304, His308 and His313 in DNA binding and cleavage are discussed for murine CAD.     

Search for apoptotic nucleases in yeast: role of Tat-D nuclease in apoptotic DNA degradation

DNA fragmentation/degradation is an important step for apoptosis. However, in unicellular organisms such as yeast, this process has rarely been investigated. In the current study, we revealed eight apoptotic nuclease candidates in Saccharyomyces cerevisiae, analogous to the Caenorhabditis elegans apoptotic nucleases. One of them is Tat-D. Sequence comparison indicates that Tat-D is conserved across kingdoms, implicating that it is evolutionarily and functionally indispensable. In order to better understand the biochemical and biological functions of Tat-D, we have overexpressed, purified, and characterized the S. cerevisiae Tat-D (scTat-D). Our biochemical assays revealed that scTat-D is an endo-/exonuclease. It incises the double-stranded DNA without obvious specificity via its endonuclease activity and excises the DNA from the 3'- to 5'-end by its exonuclease activity. The enzyme activities are metal-dependent with Mg(2+) as an optimal metal ion and an optimal pH around 5. We have also identified three amino acid residues, His(185), Asp(325), and Glu(327), important for its catalysis. In addition, our study demonstrated that knock-out of TAT-D in S. cerevisiae increases the TUNEL-positive cells and cell survival in response to hydrogen hyperoxide treatment, whereas overexpression of Tat-D facilitates cell death. These results suggest a role of Tat-D in yeast apoptosis.     

Identification of the Xenopus DNA2 protein as a major nuclease for the 5'->3' strand-specific processing of DNA ends

The first step of homology-dependent DNA double-strand break (DSB) repair is the 5′ strand-specific processing of DNA ends to generate 3′ single-strand tails. Despite extensive effort, the nuclease(s) that is directly responsible for the resection of 5′ strands in eukaryotic cells remains elusive. Using nucleoplasmic extracts (NPE) derived from the eggs of Xenopus laevis as the model system, we have found that DNA processing consists of at least two steps: an ATP-dependent unwinding of ends and an ATP-independent 5′→3′ degradation of single-strand tails. The unwinding step is catalyzed by DNA helicases, the major one of which is the Xenopus Werner syndrome protein (xWRN), a member of the RecQ helicase family. In this study, we report the purification and identification of the Xenopus DNA2 (xDNA2) as one of the nucleases responsible for the 5′→3′ degradation of single-strand tails. Immunodepletion of xDNA2 resulted in a significant reduction in end processing and homology-dependent DSB repair. These results provide strong evidence that xDNA2 is a major nuclease for the resection of DNA ends for homology-dependent DSB repair in eukaryotes.     

Dual apoptotic DNA fragmentation system in the fly: Drep2 is a novel nuclease of which activity is inhibited by Drep3

DNA fragmentation is the hallmark of apoptotic cells and mainly mediated by the DNA fragmentation factor DFF40(CAD)/DFF45(ICAD). DFF40 is a novel nuclease, whereas DFF45 is an inhibitor that can suppress the nuclease activity. Apoptotic DNA fragmentation in the fly is controlled by four DFF-related proteins, known as Drep1, 2, 3 and 4. However, the functions of Drep2 and Drep3 are totally unknown. Here, we found that Drep2 is a novel nuclease whose activity is inhibited by Drep3 through a tight interaction with the CIDE domain. Our results suggest that the fly has dual apoptotic DNA fragmentation systems: Drep1: Drep4 and Drep2: Drep3 complexes.Structured summary of protein interactionsDrep2 CIDE and Drep-3 CIDE bind by blue native page (View interaction)Drep2 CIDE and Drep-3 CIDE bind by molecular sieving (View interaction)     

A novel DNA nuclease is stimulated by association with the GINS complex

Chromosomal DNA replication requires the spatial and temporal coordination of the activities of several complexes that constitute the replisome. A previously uncharacterized protein, encoded by TK1252 in the archaeon Thermococcus kodakaraensis, was shown to stably interact with the archaeal GINS complex in vivo, a central component of the archaeal replisome. Here, we document that this protein (TK1252p) is a processive, single-strand DNA-specific exonuclease that degrades DNA in the 5' → 3' direction. TK1252p binds specifically to the GINS15 subunit of T. kodakaraensis GINS complex and this interaction stimulates the exonuclease activity in vitro. This novel archaeal nuclease, designated GINS-associated nuclease (GAN), also forms a complex in vivo with the euryarchaeal-specific DNA polymerase D. Roles for GAN in replisome assembly and DNA replication are discussed.     

Identification of novel regulators of apoptosis using a high-throughput cell-based screen

High-throughput subcellular imaging is a powerful tool for investigating the function of genes. In order to identify novel regulators of apoptosis we transiently transfected HeLa cells with 938 hypothetical genes of unknown function, and captured their nuclear images with an automated fluorescence microscope. We selected genes that induced greater than 3-fold increase in the percentage of apoptotic nuclei compared with vector-transfected cells. The full-length genes C10orf61, MGC 26717, and FLJ13855 were identified as candidate proapoptotic genes, and their apoptotic effects were confirmed by DNA fragmentation ELISAs and Western blotting for caspase-7 and PARP. We conclude that a subcellular image-based apoptotic screen is useful for identifying genes with proapoptotic activity.     

Identification of galectins as novel regulators of platelet signaling and function

Platelet activation at sites of vascular injury leads to the formation of a hemostatic plug. Activation of platelets is therefore crucial for normal hemostasis. However, uncontrolled platelet activation may also lead to the formation of occlusive thrombi that can cause ischemic events. Platelets can be activated by soluble molecules including thrombin, TXA2 , adenosine diphosphate (ADP), and serotonin or by adhesive extracellular matrix (ECM) proteins such as von Willebrand factor and collagen. In this article, we review recent advances on the role of galectins in platelet physiology. By acting in either soluble or immobilized form, these glycan-binding proteins trigger platelet activation through modulation of discrete signaling pathways. We also offer new hypotheses and some speculations about the role of platelet-galectin interactions not only in hemostasis and thrombosis but also in inflammation and related diseases such as atherosclerosis and cancer.     

DNA topoisomerase IIalpha interacts with CAD nuclease and is involved in chromatin condensation during apoptotic execution

Apoptotic execution is characterized by dramatic changes in nuclear structure accompanied by cleavage of nuclear proteins by caspases (reviewed in [1]). Cell-free extracts have proved useful for the identification and functional characterization of activities involved in apoptotic execution [2-4] and for the identification of proteins cleaved by caspases [5]. More recent studies have suggested that nuclear disassembly is driven largely by factors activated downstream of caspases [6]. One such factor, the caspase-activated DNase, CAD/CPAN/DFF40 [4,7,8] (CAD) can induce apoptotic chromatin condensation in isolated HeLa cell nuclei in the absence of other cytosolic factors [6,8]. As chromatin condensation occurs even when CAD activity is inhibited, however, CAD cannot be the sole morphogenetic factor triggered by caspases [6]. Here we show that DNA topoisomerase IIalpha (Topo IIalpha), which is essential for both condensation and segregation of daughter chromosomes in mitosis [9], also functions during apoptotic execution. Simultaneous inhibition of Topo IIalpha and caspases completely abolishes apoptotic chromatin condensation. In addition, we show that CAD binds to Topo IIalpha, and that their association enhances the decatenation activity of Topo IIalpha in vitro.     

Identification of a novel putative Ran-binding protein and its close homologue

In the process of cloning genes at the breakpoint of t(5;14) (q34;q11), a recurring translocation in acute lymphoblastic leukemia, we isolated and characterized a novel gene at 5q34, and a close human homologue (66% amino acid identity) located at 8p11-12. The presence of an importin-beta N-terminal domain at their N-terminus, their size of approximately 110 kD, their nuclear localization and the identity of the homologue to a gene of a recently submitted RanGTP binding protein (RanBP16), suggest that its protein is a novel member of the importin-beta superfamily of nuclear transport receptors, therefore called RanBP17. Northern blot analysis of human tissues revealed a ubiquitous expression pattern of the RanBP16 gene and a very restricted expression pattern of the RanBP17 gene, showing high expression in testis and pancreas. Both genes are evolutionary conserved and show a high (99 and 94%) amino acid conservation with their murine counterparts and a striking similarity (40%) to a protein product of Caenorhabditis elegans (C35A5.8).     

利用两种不相容质粒在大肠杆菌中共表达DFF45和DFF40

DNA断裂因子（DNA fragmentation factor,DFF）是细胞凋亡过程中起重要作用的蛋白质之一,它由分子量为45kD和40kD的两个亚基构成,分别称为DFF45和DFF40。利用RT－PCR技术从人宫颈癌细胞系HeLa的总RNA中扩增了DFF45和DFF40的cDNA,分别克隆到到卡那霉素抗性表达载体pET-28a( )中,构建了pET28a-DFF45和pET28a-DFF40。用它们单独转化大肠杆菌BL21（DE3）后,经IPTG诱导都可获得高效表达。表达的重组蛋白质各自约占菌体总蛋白质的56％和22％。再将DFF45的cDNA克隆到氨苄青霉素抗性表达载体pET-21a( )中,得到了pET21a-DFF45。利用二者的不同抗性,将pET28a-DFF40和pET21a-DFF45共同转化大肠杆菌BL21（DE3）,工程菌经IPTG诱导后实现了DFF45和DFF40的共表达,表达产物各约占菌体总蛋白质的30％和17％。为了研究这两种不相容质粒在细菌中共存的稳定性,我们将共转化子在同时含有卡那霉素和氨苄青霉素的液体培养基中连续培养14h,发现此时仍有75％以上的细菌可同时耐受两种抗生素,即同时含有pET28a-DFF45和pET28a-DFF40,说明利用两个具有不同抗性的不相容载体进行蛋白质共表达的方法是可行的。     

Identification of novel autophagy regulators by a luciferase-based assay for the kinetics of autophagic flux

Macroautophagy (hereafter referred to as autophagy) has recently emerged as an attractive target for the treatment of various degenerative diseases and cancer. The discovery of effective pharmaceutical regulators of autophagy has, however, been hindered by a lack of feasible assay systems for autophagic flux. Here, we present a luciferase-based reporter assay that measures autophagic flux in real time in living cells and demonstrate that this assay system is apt for the detection of dose- and stimulus-dependent differences in autophagy kinetics. Furthermore, by screening a small molecule kinase inhibitor library containing 80 compounds we identified 12 compounds as inducers of autophagic flux. Importantly, six inhibitors of the class I phosphoinositide 3-kinase – protein kinase B – mammalian target of rapamycin complex 1 axis, the central signaling pathway repressing autophagy, scored as autophagy inducers adequately validating the screen. We conclude that the assay system presented here allows easy and rapid monitoring of autophagy kinetics and is suitable for screening of small molecule libraries.     

Cleavage preferences of the apoptotic endonuclease DFF40 (caspase-activated DNase or nuclease) on naked DNA and chromatin substrates

Here we report the co-factor requirements for DNA fragmentation factor (DFF) endonuclease and characterize its cleavage sites on naked DNA and chromatin substrates. The endonuclease exhibits a pH optimum of 7.5, requires Mg(2+), not Ca(2+), and is inhibited by Zn(2+). The enzyme generates blunt ends or ends with 1-base 5'-overhangs possessing 5'-phosphate and 3'-hydroxyl groups and is specific for double- and not single-stranded DNA or RNA. DFF endonuclease has a moderately greater sequence preference than micrococcal nuclease or DNase I, and the sites attacked possess a dyad axis of symmetry with respect to purine and pyrimidine content. Using HeLa cell nuclei or chromatin reconstituted on a 5 S rRNA gene tandem array, we prove that the enzyme attacks chromatin in the internucleosomal linker, generating oligonucleosomal DNA ladders sharper than those created by micrococcal nuclease. Histone H1, high mobility group-1, and topoisomerase II activate DFF endonuclease activity on naked DNA substrates but much less so on chromatin substrates. We conclude that DFF is a useful reagent for chromatin research.     

Chemoresistance in human ovarian cancer: the role of apoptotic regulators

Ovarian cancer is among the most lethal of all malignancies in women. While chemotherapy is the preferred treatment modality, chemoresistance severely limits treatment success. Recent evidence suggests that deregulation of key pro- and anti-apoptotic pathways is a key factor in the onset and maintenance of chemoresistance. Furthermore, the discovery of novel interactions between these pathways suggests that chemoresistance may be multi-factorial. Ultimately, the decision of the cancer cell to live or die in response to a chemotherapeutic agent is a consequence of the overall apoptotic capacity of that cell. In this review, we discuss the biochemical pathways believed to promote cell survival and how they modulate chemosensitivity. We then conclude with some new research directions by which the fundamental mechanisms of chemoresistance can be elucidated.     

Intracellullar peptides as putative natural regulators of protein interactions

Extralysosomal proteolysis by multicatalytic complexes such as the 26S proteasome produces large amounts of peptides in the cytosol, mitochondria and nuclei of eukaryotic cells, and there is increasing evidence that the resulting free intracellular peptides can modulate specific protein interactions. The demonstration that free peptides added to the intracellular milieu can regulate cellular functions mediated by protein interactions suggests new putative roles for these molecules in gene regulation, metabolism, cell signaling and protein targeting. Such interactions frequently involve specific consensus amino acid sequences that can be predicted based on similarities in domain composition. We have recently developed a new strategy for identifying novel natural peptides, the sequences of which correspond to fragments of intracellular proteins and contain putative post-translational modification sites. In this review, we examine the evidence that intracellular peptides released by proteasomes may be involved in regulating protein interactions. In particular, the role of endopeptidase 24.15 (thimet oligopeptidase; EC 3.4.24.15) is discussed in detail as this enzyme has been implicated in intracellular peptide metabolism in vivo in concert with the 26S proteasome.     

Identification of a novel member of the T1R family of putative taste receptors

In the gustatory system, the recognition of sugars, amino acids and bitter-tasting compounds is the function of specialized G protein-coupled receptors. Recently, two members of novel subfamily of G protein-coupled receptors were proposed to function as taste receptors based on their specific expression in taste receptor cells. Here, we report the identification of a third member, T1R3, of this family of receptors. T1R3 maps near the telomere of mouse chromosome 4 rendering it a candidate for the Sac locus, a primary determinant of sweet preference in mice. Consistent with its candidacy for the Sac locus, T1R3 displays taste receptor cell-specific expression. In addition, taster and non-taster strains of mouse harbor different alleles of T1R3.     

A novel inhibitor that protects apoptotic DNA fragmentation catalyzed by DNase gamma

The internucleosomal cleavage of genomic DNA is the biochemical hallmark of apoptosis. DNase gamma, a Ca(2+)/Mg(2+)-dependent endonuclease, has been suggested to be one of the apoptotic endonucleases. We identified here 4-(4,6-dichloro-[1,3,5]-triazin-2-ylamino)-2-(6-hydroxy-3-oxo-3H-xanthen-9-yl)-benzoic acid (DR396) as a novel and potent DNase gamma inhibitor using stable HeLa S3 transfectants of DNase gamma (HeLa-gamma cells). DR396 inhibited apoptotic DNA fragmentation in HeLa-gamma cells induced by staurosporine (STS) and in rat splenocytes exposed to gamma-ray irradiation in a dose-dependent manner. This compound potently and selectively inhibited DNase gamma activity with an IC(50) value of 3.2 microM. DR396 did not delay the apoptotic processes as judged by the morphological changes and the cleavage of a death substrate, poly(ADP-ribose) polymerase (PARP). Furthermore, the compound did not prevent apoptotic DNA fragmentation in Jurkat cells induced by anti-Fas antibody (Ab), which is catalyzed by caspase-activated DNase (CAD). These findings clearly indicate that DR396 exerts chemical knockdown effect of DNase gamma on cells, suggesting that the compound could be an attractive tool for understanding of the physiological significance of DNase gamma.