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Containing Helicase Cleaved During Apoptosis, Accelerates DNA Degradation

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Containing Helicase Cleaved During Apoptosis, Accelerates DNA Degradation View metadata, citation and similar papers at core.ac.uk brought to you by CORE Current Biology, Vol. 12, 838–843, May 14, 2002, 2002 Elsevier Science Ltd. All rights reserved. PII S0960-9822(02)00842-4provided by Elsevier - Publisher Connector Overexpression of Helicard, a CARD- Containing Helicase Cleaved during Apoptosis, Accelerates DNA Degradation Magdalena Kovacsovics,1 Fabio Martinon,1 human cells may express more than 50 different heli- Olivier Micheau,1 Jean-Luc Bodmer,1 cases [8]. These estimates are largely based on the Kay Hofmann,2 and Ju¨ rg Tschopp1,3 presence of seven highly conserved motifs identified in 1Institute of Biochemistry most known helicases (see below). Recent studies have University of Lausanne shown that helicases, via DNA twisting, have also an Chemin des Boveresses 155 effect on chromatin remodeling [9]. This activity can CH-1066 Epalinges alter the position of nucleosomes along DNA, transfer Switzerland histone octamers from one DNA fragment to another, 2 MEMOREC Stoffel GmbH and thus modulates the access of transcription factors Stoeckheimer Weg 1 to DNA. D-50829 Koeln In proapoptotic signaling pathways, the interaction Germany between the different initiator units, such as the death receptor Fas, the various adaptor proteins, and cas- pases is primarily mediated by three structurally related Summary protein-protein domains called death domain (DD), death effector domain (DED), and caspase-recruitment Apoptotic cell death is characterized by several morpho- domain (CARD) [10]. Therefore, to identify novel candi- logical nuclear changes, such as chromatin condensa- date proteins that are implicated in proapoptotic signal- tion and extensive fragmentation of chromosomal DNA ing pathways, we screened public databases with a [1]. These alterations are primarily triggered through generalized profile of CARD motifs from all currently the activation of caspases, which subsequently cleave known CARD sequences [11]. Several hitherto noniden- nuclear substrates. Caspase-3 induces processing of tified CARD-containing proteins were found, one of Acinus, which leads to chromatin condensation [2]. which particularly attracted our attention, since, in addi- DNA fragmentation is dependent on the DNase CAD, tion to the CARD domain, it also contained a predicted which is released from its inhibitor, ICAD, upon cleav- helicase domain. The corresponding full-length cDNA age by caspase-3 [3]. DNA degradation is also induced was cloned via a combination of available EST clones by AIF [4] and endonuclease G [5, 6], which are both and RT-PCR. The predicted murine protein has 1025 released from mitochondria upon death stimuli but amino acids and contains at the amino-terminal region do not require prior processing by caspases for their two CARD domains (Figures 1A and 1B) followed by a DNase activity. Here we report the identification of a region showing high homology to helicases (Figure 1C). widely expressed helicase designated Helicard, which We refer to this novel protein as Helicard. Most of the contains two N-terminal CARD domains and a C-ter- helicases contain seven characteristic motifs in a region minal helicase domain. Upon apoptotic stimuli, Heli- of 300–500 residues. The DECH sequence found in motif card is cleaved by caspases, thereby separating the II of Helicard indicates that it is a member of the DExH- CARD domains from the helicase domain. While Heli- box subfamily of RNA and DNA helicases. The corre- card localizes in the cytoplasm, the helicase-con- sponding human homolog shows extensive sequence taining fragment is found in the nucleus. Helicard ac- homology (84% identical aa, data not shown). Database celerates Fas ligand-mediated DNA degradation, searches revealed that proteins with a similar overall whereas a noncleavable or a helicase-dead Helicard structure exist in human (RIG1), pig (RIG1), and C. ele- mutant does not, implicating Helicard in the nuclear gans (F15B10.2), indicating that Helicards are evolution- remodeling occurring during apoptosis. ary highly conserved. The expression of RIG1 is upregu- lated by retoinic acids, but its function is not known [12]. Given the homology of Helicard with helicases, we Results and Discussion tested Helicard for its helicase activity using an indirect method, based on the ATPase activity of helicases [13]. DNA and RNA helicases are a ubiquitous and diverse We expressed the helicase domain of Helicard (aa 252– group of enzymes defined by their capacity to catalyze 1025) and found that the recombinant protein exhibited the unwinding of duplex nucleic acid molecules [7]. They an ATPase activity which was triggered by RNA and to participate in multiple cellular processes, including DNA a lesser extent by DNA (Figure 2D). replication, DNA repair, recombination, transcription, By Northern blot analysis, Helicard was found to be transcriptional regulation, RNA processing, and transla- widely expressed as a 4.4 kb transcript, with prominent tion. In fact, in every process involving the manipulation expression in lung, liver, kidney, heart, and spleen (Fig- of nucleic acids, helicases play important roles. The ure 2E). Using a polyclonal antibody raised against the diversity of functions in which helicases operate is re- CARD domains of Helicard, the expression pattern was 140ف flected in the number of different helicases present in confirmed at the protein level. In addition to the prokaryotic and eukaryotic cells. Escherichia coli con- kDa full-length protein, most tissues examined also con- .kDa 45ف tains at least 12 biochemically distinct helicases, and tained a reactive band migrating at around Although we cannot rule out the presence of an alterna- 3 Correspondence: [email protected] tively spliced form of Helicard, it is more likely that the Brief Communication 839 Figure 1. Characterization and Expression of Helicard (A) Predicted amino acid sequence and schematic representation of murine Helicard, which contains two CARD motifs and a helicase domain .amino acids 100ف linked by an intermediate region of (B) Alignment of the two CARD domains of human and murine Helicard with CARD motifs present in the Helicard related proteins RIG1 and C. elegans F15B10.2 and in various other proteins implicated in apoptosis. Black boxes indicate a greater than 50% amino acid sequence identity, and gray boxes indicate greater than 50% amino acid similarity through conservative amino acid substitution. (C) Alignment of the helicase domains of human and murine Helicard with the helicase domains of human and pig RIG1, C. elegans F15B10.2, Current Biology 840 Figure 2. Helicard Is Cleaved in Cells Undergoing Apoptosis (A) Cleavage of Helicard upon FasL induced apoptosis. (Left panel) 293T cells were transfected with cDNA encoding the Flag-tagged full- length Helicard and stimulated for the indicated time with FasL (200 ng/ml). Cell lysates were subsequently analyzed by Western blot analysis using an anti-Flag-antibody. (Middle panel) Cleavage of Helicard upon TRAIL stimulation. 293T cells were transfected and stimulated for 3 hr with TRAIL (100 ng/ml). (Right panel) Apoptosis was induced with staurosporine (Stau, 1 ␮g/ml). Note the presence of a fragment (F) of 45 kDa, even in the absence of proapoptotic stimuli. (B) Cleavage of endogenous Helicard in murine A20 B lymphomas undergoing Fas-mediated apoptosis. A20 cells, either sensitive (S) or resistant (R) to FasL, were treated with 200 ng/ml of FasL and harvested at the indicated times. Cell extracts were analyzed by Western blot analysis using either an anti-Helicard antibody or a monoclonal antibody recognizing the cleaved form of PARP as an indicator of apoptosis. (C) Caspase inhibition prevents FasL-induced cleavage of Helicard. 293 T cells were transfected with full-length Helicard cDNA. Thirty min before FasL stimulation, the indicated caspase inhibitors were added at given concentrations. Cells were then incubated with FasL (200 ng/ml) for 3 hr and analyzed as in (A). (D) Identification of Helicard cleavage sites. 293T cells were transfected with either the wild-type, ⌬200-221, D208, or the D251 mutants of Helicard and stimulated for 3 hr with FasL (200 ng/ml). Werner helicase, Xeroderma Pigmentosum B and D helicase, Bloom helicase, and Cockayne helicase. The seven motifs that are characteristic for helicases are indicated. Numbers between motifs refer to aa separating the motifs. The highly conserved DExH box required for ATP binding is indicated. (D) ATPase activity of helicase activity. ATPase activity of 293T supernatants containing recombinant Helicard (helicase-containing fragment) was measured in the presence of murine DNA or RNA or with no nucleic acid. For control purposes, supernatants of nontransfected cells were assayed. Oxidation of NADH, which is directly proportional to the rate of ATP hydrolysis, was continuously monitored by measuring the absorbance at 338 nm. Means Ϯ standard errors of a represenative experiment are shown. The experiment has been repeated three times. (E) Expression of Helicard RNA in various murine tissues. A Northern blot of various mouse tissues (Clontech) was probed with a 32P-labeled antisense RNA fragment covering the CARD-coding region of Helicard. The blot was subsequently hybridized with a ␤-actin probe. (F) Western blot analysis of various murine tissues. A blot containing 50 ␮g of protein extracted from the indicated cells was probed with an anti-Helicard antibody, directed toward the N-terminal portion of the protein. The blot was reprobed with anti-tubulin
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