Inhibitor Of DNase Research Articles

Novel high‐throughput DNase I assay

DNase I is the most active and abundant endonuclease in the body, mediates toxic injury of kidney cells induced by cisplatin. This presents a major obstacle for cancer chemotherapy. However, neither inhibitors of DNase I nor high‐throughput screening methods for screening of high‐volume chemical libraries for DNase I inhibitors are available. To overcome this problem, we have developed a high‐throughput DNase I assay. The assay is based on the increase of fluorescence intensity when fluorophore‐labeled oligonucleotides are degraded by a DNase. It is optimized for a 96‐well plate format. The assay is highly sensitive to DNase I compared to other endonucleases, reliable (Z′≥0.6), operationally simple, and has low intra‐ and inter‐assay variability. The assay was used to screen a chemical library and several potential DNase I inhibitors were identified. After comparison, one best inhibitor was selected and shown to protect against cisplatin‐induced kidney cell death in vitro. This assay will be suitable for identifying potential inhibitors of DNase I and other endonucleases. NIH R01 DK078908 and VA Merit Review grants to AGB.

Crystal Structure of a Voltage-gated K+ Channel Pore Module in a Closed State in Lipid Membranes

Voltage-gated K(+) channels underlie the electrical excitability of cells. Each subunit of the functional tetramer consists of the tandem fusion of two modules, an N-terminal voltage-sensor and a C-terminal pore. To investigate how sensor coupling to the pore generates voltage-dependent channel opening, we solved the crystal structure and characterized the function of a voltage-gated K(+) channel pore in a lipid membrane. The structure of a functional channel in a membrane environment at 3.1 Å resolution establishes an unprecedented connection between channel structure and function. The structure is unique in delineating an ion-occupied ready to conduct selectivity filter, a confined aqueous cavity, and a closed activation gate, embodying a dynamic entity trapped in an unstable closed state.

HMGB1 facilitates inflammasome-dependent IL-1β release (114.2)

Abstract HMGB1 is a potent mediator of late-phase shock, endotoxin toxicity and other inflammatory processes. HMGB1 is released passively from necrotic cells, and also following inflammasome activation in macrophages and dendritic cells. We hypothesized that caspase-1 activated nucleases liberate HMGB1 from the nucleus and allow for subsequent cellular release. To test this hypothesis, we measured inflammasome-mediated HMGB1 release in the presence of various nuclease inhibitors. Inhibitors of DNase γ blocked inflammasome-activated HMGB1 release. Surprisingly, DNase γ inhibitors also blocked IL-1β release, suggesting that nuclear proteins play a role in nonconventional cytokine secretion. To determine if HMGB1 regulates IL-1β release, we tested its intracellular association with IL-1β and the ability of HMGB1-deficient cells to secrete IL-1β. We found that HMGB1 co-immunoprecipitated with IL-1β in cells, indicating an interaction prior to IL-1β release. Furthermore, genetic disruption of HMGB1 in dendritic cells reduced their ability to secrete IL-1β in response to inflammasome activation. Taken together, these data suggest that HMGB1 and other nuclear proteins regulate IL-1β release through an inflammasome dependent process.

Low-Dose Spironolactone Prevents Apoptosis Repressor With Caspase Recruitment Domain Degradation During Myocardial Infarction

Low-dose mineralocorticoid receptor antagonists reduce morbidity and mortality in patients with heart failure and myocardial infarction, despite normal plasma aldosterone levels. Since apoptosis plays an important role in heart failure and postinfarction left ventricular remodeling, we examined whether low-dose mineralocorticoid receptor antagonists modulate cardiomyocyte death by regulating the apoptosis repressor protein apoptosis repressor with caspase recruitment domain to lessen the extent of apoptosis. Hearts from adult male Sprague-Dawley rats were subjected to regional ischemia followed by reperfusion ex vivo, with mineralocorticoid receptor antagonists added to perfusates before ischemia. Low-dose spironolactone (10 nmol/L) or eplerenone (100 nmol/L) significantly reduced infarct size. Spironolactone also prevented cleavage of the apoptotic chromatin condensation inducer in the nucleus and of the inhibitor of caspase-activated DNAse induced by ischemia-reperfusion, thereby abolishing chromatin condensation and internucleosomal cleavage. Ischemia-reperfusion-induced activation of caspases 2, 3, and 9, but not caspase 8, was prevented by spironolactone, suggesting targeted regulation of the intrinsic pathway. Low-dose spironolactone and eplerenone prevented loss of the apoptosis repressor with the caspase recruitment domain and reduced myocyte death. In H9c2 cells, mineralocorticoid receptor activation by aldosterone resulted in apoptosis repressor with caspase recruitment domain degradation and enhanced apoptosis; these actions were prevented by coadministration of spironolactone. Using a triple lysine mutant we identified that aldosterone enhances posttranscriptional degradation of the apoptosis repressor with a caspase recruitment domain via the ubiquitin-proteasomal pathway. Our data demonstrate that low-dose mineralocorticoid receptor antagonists reduce infarct size and apoptosis in the reperfused myocardium by preventing the apoptosis repressor with caspase recruitment domain degradation.

Abstract 1402: A xenograft pancreatic cancer mouse model to study the function of extracellular DNA in metastasis

Abstract Pancreatic cancer is one of the most lethal cancers with the lowest survival rate of all solid cancers. This is largely due to its highly aggressive metastasis, which almost always leads to death. Understanding the mechanisms and the process of pancreatic cancer metastasis is critical towards its prevention and treatments. Although the etiology of pancreatic cancer is not well understood, the relationship between inflammation and the development of this cancer has been well recognized. Extracellular DNA (exDNA) is a newly discovered component associated with inflammation. exDNA production can be induced by interleukin 8 (IL-8), one of the major inflammatory and angiogenic cytokine. IL-8 exhibits a significantly elevated level in pancreatic cancer cell lines MiaPaCa-2 and BxPC3 and has been shown to play a role in stimulating pancreatic tumorigenesis and metastasis. With this background, we initiated a study to examine the presence of exDNA associated with pancreatic cancer cells. We have discovered a large amount of extracellular DNA associated with cultured pancreatic cancer cells but not with the normal pancreatic cells. Here, we tested the hypothesis that pancreatic cancer cells produce extracellular DNA to promote their metastasis. Our in vitro study showed that removal of the exDNA by DNase I inhibited metastasis-associated characteristics of cancer cells including cell migration, invasion, and adhesion. To test our hypothesis in vivo, we generated the orthotopic xenograft pancreatic cancer mouse model. We used the pancreatic cancer cell line MiaPaCa-2/LucE expressing luciferase to induce tumors. We assessed the effect of DNase I treatment on pancreatic cancer metastasis from pancreases, the primary tumor site, to different organs. Tumors were examined directly in size and weight, and indirectly by the bioluminescent signal intensity that corresponds to the tumor size. Our preliminary data showed that continuous applications of a low dosage of DNase I reduced the tumor incidences and the tumor size measured by the chemiluminescence in all the organs examined. When mouse tumor tissues were stained with DAPI, a DNA-specific fluorescent dye and examined through immunohistochemistry (IHC) with DNA-specific antibody, high level of exDNA was found in the tumor tissues. More exDNA was found to be associated with the inflammatory area in the tumor tissues. We conclude that extracellular DNA play a novel role in metastatic behaviors of pancreatic cancer cells in vitro and facilitate metastasis in vivo. To our knowledge, this is the first report on the presence of exDNA associated with pancreatic cancer tissues and the first report on the inhibition of DNase I treatment on pancreatic cancer metastasis. Our results open new avenues for understanding the metastasis of pancreatic cancer, and facilitate the identification of new drug targets and development of new treatments. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1402. doi:1538-7445.AM2012-1402

Insulin-like Growth Factor Receptor-1 and Nuclear Factor κB Are Crucial Survival Signals That Regulate Caspase-3-mediated Lens Epithelial Cell Differentiation Initiation

It is now known that the function of the caspase family of proteases is not restricted to effectors of programmed cell death. For example, there is a significant non-apoptotic role for caspase-3 in cell differentiation. Our own studies in the developing lens show that caspase-3 is activated downstream of the canonical mitochondrial death pathway to act as a molecular switch in signaling lens cell differentiation. Importantly, for this function, caspase-3 is activated at levels far below those that induce apoptosis. We now have provided evidence that regulation of caspase-3 for its role in differentiation induction is dependent on the insulin-like growth factor-1 receptor (IGF-1R) survival-signaling pathway. IGF-1R executed this regulation of caspase-3 by controlling the expression of molecules in the Bcl-2 and inhibitor of apoptosis protein (IAP) families. This effect of IGF-1R was mediated through NFκB, demonstrated here to function as a crucial downstream effector of IGF-1R. Inhibition of expression or activation of NFκB blocked expression of survival proteins in the Bcl-2 and IAP families and removed controls on the activation state of caspase-3. The high level of caspase-3 activation that resulted from inhibiting this IGF-1R/NFκB signaling pathway redirected cell fate from differentiation toward apoptosis. These results provided the first evidence that the IGF-1R/NFκB cell survival signal is a crucial regulator of the level of caspase-3 activation for its non-apoptotic function in signaling cell differentiation.

Prolonged mitotic arrest triggers partial activation of apoptosis, resulting in DNA damage and p53 induction

Mitotic arrest induced by antimitotic drugs can cause apoptosis or p53-dependent cell cycle arrest. It can also cause DNA damage, but the relationship between these events has been unclear. Live, single-cell imaging in human cancer cells responding to an antimitotic kinesin-5 inhibitor and additional antimitotic drugs revealed strong induction of p53 after cells slipped from prolonged mitotic arrest into G1. We investigated the cause of this induction. We detected DNA damage late in mitotic arrest and also after slippage. This damage was inhibited by treatment with caspase inhibitors and by stable expression of mutant, noncleavable inhibitor of caspase-activated DNase, which prevents activation of the apoptosis-associated nuclease caspase-activated DNase (CAD). These treatments also inhibited induction of p53 after slippage from prolonged arrest. DNA damage was not due to full apoptosis, since most cytochrome C was still sequestered in mitochondria when damage occurred. We conclude that prolonged mitotic arrest partially activates the apoptotic pathway. This partly activates CAD, causing limited DNA damage and p53 induction after slippage. Increased DNA damage via caspases and CAD may be an important aspect of antimitotic drug action. More speculatively, partial activation of CAD may explain the DNA-damaging effects of diverse cellular stresses that do not immediately trigger apoptosis.

Simple Generation of Human Induced Pluripotent Stem Cells Using Poly-β-amino Esters As the Non-viral Gene Delivery System

Reprogramming of somatic cells to induced pluripotent stem (iPS) cells can be achieved by the delivery of a combination of transcription factors, including Oct4, Sox2, Klf4, and c-Myc. Retroviral and lentiviral vectors are commonly used to express these four reprogramming factors separately and obtain reprogrammed iPS cells. Although efficient and reproducible, these approaches involve the time-consuming and labor-intensive production of retroviral or lentiviral particles together with a high risk of working with potentially harmful viruses overexpressing potent oncogenes, such as c-Myc. Here, we describe a simple method to produce bona fide iPS cells from human fibroblasts using poly-β-amino esters as the transfection reagent for the delivery of a single CAG-driven polycistronic plasmid expressing Oct4, Sox2, Klf4, c-Myc, and a GFP reporter gene (OSKMG). We demonstrate for the first time that poly-β-amino esters can be used to deliver a single polycistronic reprogramming vector into human fibroblasts, achieving significantly higher transfection efficiency than with conventional transfection reagents. After a protocol of serial transfections using poly-β-amino esters, we report a simple methodology to generate human iPS cells from human fibroblasts avoiding the use of viral vectors.

DR396, an apoptotic DNase γ inhibitor, attenuates high mobility group box 1 release from apoptotic cells

High mobility group box1 (HMGB1) is a non-histone chromatin chromosomal protein playing an important role in chromatin architecture and transcriptional regulation. Recently, HMGB1 has been shown to be secreted into extracellular milieu in necrosis and apoptosis, and involved in inflammatory responses. However, the mechanism by which apoptotic cells release HMGB1 is unclear. In this study, to investigate the mechanism of HMGB1 release, we searched inhibitors of HMGB1 release from apoptotic cells. As a result, three compounds, 4-(4,6-dichloro-[1,3,5]-triazin-2-ylamino)-2-(6-hydroxy-3-oxo-3H-xanthen-9-yl)-benzoic acid (DR396), Pontacyl Violet 6R (PV6R), and Fmoc-D-Cha-OH (FDCO) in our in-house chemical library were found to inhibit HMGB1 release from staurosporine (STS)-induced apoptotic HeLa S3 cells. Interestingly, these three compounds have been previously categorized into apoptotic DNase γ inhibitors. Therefore, we examined whether apoptotic nucleosomal DNA fragmentation is involved in the release of HMGB1 during apoptosis. Expectedly, DR396, which is the most potent and specific inhibitor of DNase γ, was found to almost completely inhibit both HMGB1 release and internucleosomal DNA cleavage in HeLa S3 cells transfected with DNase γ expression vector and stably expressing DNase γ (HeLa S3/γ cells). These results clearly suggest that nucleosomal DNA fragmentation catalyzed by DNase γ is critical in the release of HMGB1 from apoptotic cells.

Interference of Real-Time PCR Quantification of Vibrio vulnificus by a Novel DNase from the Eastern Oyster (Crassostrea virginica)

A novel DNase from oyster tissue with an optimum temperature at 65°C was found to interfere with the real-time PCR (Rti-PCR) quantification of Vibrio vulnificus. The DNase activity of oyster extracts at 65°C was 7.5-fold higher than that at 35°C after 30 min of DNA digestion and was linear at 50°C, 55°C, 60°C and 65°C for at least 80 min. The oyster DNase activity was strongly inhibited by EDTA, suggesting that EDTA sequestered the enzyme cofactor Mg++. The Ct values from Rti-PCR amplification of DNA from V. vulnificus cells heated at 65°C or 70°C in the presence of oyster tissue were larger than the Ct values from DNA derived from cells heated at these temperatures in the absence of oyster tissue, which suggested that oyster DNase is a potential PCR inhibitor. These observations suggest that Rti-PCR inhibition was most probably due to the DNase entering the cells at 65°C and 70°C. Activated carbon coated with benonite was used to remove oyster DNase and other PCR inhibitors, resulting in a minimum detection level of 10 CFU of V. vulnificus per gram of oyster tissue, which was a 10-fold higher level of detection sensitivity than the 100 CFU/g obtained with the Wizard DNA purification system.

Mutation of Actin Tyr-53 Alters the Conformations of the DNase I-binding Loop and the Nucleotide-binding Cleft

All but 11 of the 323 known actin sequences have Tyr at position 53, and the 11 exceptions have the conservative substitution Phe, which raises the following questions. What is the critical role(s) of Tyr-53, and, if it can be replaced by Phe, why has this happened so infrequently? We compared the properties of purified endogenous Dictyostelium actin and mutant constructs with Tyr-53 replaced by Phe, Ala, Glu, Trp, and Leu. The Y53F mutant did not differ significantly from endogenous actin in any of the properties assayed, but the Y53A and Y53E mutants differed substantially; affinity for DNase I was reduced, the rate of nucleotide exchange was increased, the critical concentration for polymerization was increased, filament elongation was inhibited, and polymerized actin was in the form of small oligomers and imperfect filaments. Growth and/or development of cells expressing these actin mutants were also inhibited. The Trp and Leu mutations had lesser but still significant effects on cell phenotype and the biochemical properties of the purified actins. We conclude that either Tyr or Phe is required to maintain the functional conformations of the DNase I-binding loop (D-loop) in both G- and F-actin, and that the conformation of the D-loop affects not only the properties that directly involve the D-loop (binding to DNase I and polymerization) but also allosterically modifies the conformation of the nucleotide-binding cleft, thus increasing the rate of nucleotide exchange. The apparent evolutionary "preference" for Tyr at position 53 may be the result of Tyr allowing dynamic modification of the D-loop conformation by phosphorylation (Baek, K., Liu, X., Ferron, F., Shu, S., Korn, E. D., and Dominguez, R. (2008) Proc. Natl. Acad. Sci. U.S.A. 105, 11748-11753) with effects similar, but not identical, to those of the Ala and Glu mutations.

Magnolol induces apoptosis via activation of both mitochondrial and death receptor pathways in A375-S2 cells

Magnolol inhibited proliferation of human malignant melanoma A375-S2 cells. The drug induced oligonucleosomal fragmentation of DNA in A375-S2 cells and increased caspase-3, 8, 9 activities followed by the degradation of caspase-3 substrates, inhibitor of caspase dependent DNase (ICAD) and poly-(ADP-ribose) polymerase (PARP). Pan-caspase inhibitor (z-VADfmk), caspase-3 inhibitor (z-DEVD-fmk), capase-8 inhibitor (z-IETD-fmk), caspase-9 inhibitor (z-LEHD-fmk) and caspase-10 inhibitor (z-AEVD-fmk) inhibited magnolol-induced A375-S2 cell apoptosis. The level of anti-apoptotic mitochondrial protein Bcl-2 was up-regulated while the level of pro-apoptotic protein Bax was down-regulated. Taken together, our results indicate that magnolol induces apoptosis by activation of both mitochondrial and death receptor pathways in A375-S2 cells.

Proteome-wide Substrate Analysis Indicates Substrate Exclusion as a Mechanism to Generate Caspase-7 Versus Caspase-3 Specificity

Caspase-3 and -7 are considered functionally redundant proteases with similar proteolytic specificities. We performed a proteome-wide screen on a mouse macrophage lysate using the N-terminal combined fractional diagonal chromatography technology and identified 46 shared, three caspase-3-specific, and six caspase-7-specific cleavage sites. Further analysis of these cleavage sites and substitution mutation experiments revealed that for certain cleavage sites a lysine at the P5 position contributes to the discrimination between caspase-7 and -3 specificity. One of the caspase-7-specific substrates, the 40 S ribosomal protein S18, was studied in detail. The RPS18-derived P6-P5' undecapeptide retained complete specificity for caspase-7. The corresponding P6-P1 hexapeptide still displayed caspase-7 preference but lost strict specificity, suggesting that P' residues are additionally required for caspase-7-specific cleavage. Analysis of truncated peptide mutants revealed that in the case of RPS18 the P4-P1 residues constitute the core cleavage site but that P6, P5, P2', and P3' residues critically contribute to caspase-7 specificity. Interestingly, specific cleavage by caspase-7 relies on excluding recognition by caspase-3 and not on increasing binding for caspase-7.

Deoxyribonuclease I is Essential for DNA Fragmentation Induced by Gamma Radiation in Mice

Gamma radiation is known to induce cell death in several organs. This damage is associated with endonuclease-mediated DNA fragmentation; however, the enzyme that produces the latter and is likely to cause cell death is unknown. To determine whether the most abundant cytotoxic endonuclease DNase I mediates gamma-radiation-induced tissue injury, we used DNase I knockout mice and zinc chelate of 3,5-diisopropylsalicylic acid (Zn-DIPS), which, as we show, has DNase I inhibiting activity in vitro. The study demonstrated for the first time that inactivation or inhibition of DNase I ameliorates radiation injury to the white pulp of spleen, intestine villi and bone marrow as measured using a quantitative TUNEL assay. The spleen and intestine of DNase I knockout mice were additionally protected from radiation by Zn-DIPS, perhaps due to the broad radioprotective effect of the zinc ions. Surprisingly, the main DNase I-producing tissues such as the salivary glands, pancreas and kidney showed no effect of DNase I inactivation. Another unexpected observation was that even without irradiation, DNA fragmentation and cell death were significantly lower in the intestine of DNase I knockout mice than in wild-type mice. This points to the physiological role of DNase I in normal cell death in the intestinal epithelium. In conclusion, our results suggested that DNase I-mediated mechanism of DNA damage and subsequent tissue injury are essential in gamma-radiation-induced cell death in radiosensitive organs.

Targeting of plasmid DNA‐lipoplexes to cells with molecules anchored via a metal chelator lipid

The ability to deliver plasmid DNA (pDNA) to specific cells in vivo is crucial for achieving efficient targeted transfection with nonviral vectors. We previously used stealth liposomes containing the chelator lipid 3(nitrilotriacetic acid)-ditetradecylamine (NTA(3)-DTDA) to target delivery of antigen and cytokines to immune cells in vivo. In the present study, we utilized liposomes containing NTA(3)-DTDA and the ionizable aminolipid 1,2-dioleoyl-3-dimethyl-ammonium-propane (DODAP) to incorporate pDNA into complexes for targeting to cells. Liposomes containing DODAP, NTA(3)-DTDA and helper lipids were acidified (pH 5.5) and mixed with pDNA to form complexes. These lipoplexes were neutralized and engrafted with His-tagged molecules for targeting to extracellular receptors. Targeted transfection efficiency was assessed using the enhanced green fluorescent protein reporter gene. Initial transfections of HEK-293 cells using a His-tagged peptide (T2) related to the Arg-rich motif of HIV-1 TAT protein resulted in a low transfection efficiency (<2.5%). Optimization of the lipid formulation and use of an endosome-destabilizing peptide and inhibitor of DNase II, increased transfection approximately 20-fold. These lipoplexes are approximately 250 nm in diameter, and transfection efficiencies were: approximately 50% for HEK-293 cells targeted with lipoplexes containing pEGFP-N1 and engrafted with T2, and 30-40% for HepG2 cells targeted with lipoplexes engrafted with a peptide specific for the VEGF receptor Flt-1. The results show that DODAP-containing lipoplexes incorporating NTA(3)-DTDA enable the engraftment of targeting molecules and the effective targeting of pDNA to cells in serum-containing media, resulting in efficient transgene expression. The strategy may provide a convenient approach for targeting pDNA to cells in vivo in therapeutic applications.

Characterization of Biofilm Formation by the Bioleaching Acidophilic Bacterium &lt;i&gt;Acidithiobacillus Ferrooxidans&lt;/i&gt; by a Microarray Transcriptome Analysis

Bioleaching is the extraction of metals, such as copper or gold, from ore by microorganisms. Bacterial attachment increases leaching activities due to the formation of a "reaction space" between the metal sulfide surface and the cell. This process depends on abiotic characteristics such as purity and degree of crystallization of the metal sulfide, as well as biotic ones such as the capacity of the bacteria for detecting favourable attachment sites and synthesizing a suitable cell envelope (EPS), for adhesion. Planktonic and sessile cells should differ significantly in their metabolic activities and therefore in their gene expression patterns. To help to understand At. ferrooxidans biofilm formation, microarray transcript profiling was carried out to compare planktonic and sessile cells. The high contents of EPS and ferric iron of the biofilms are interfering with RNA extraction, causing inhibition of DNAse, reverse transcriptase and/or polymerase activities required to get labelled target cDNA. In order to have sufficient high quality RNA suitable for transcriptomic analysis, we have optimized the biofilm formation of At. ferrooxidans on pyrite (FeS2) and the RNA extraction from the sessile cell population. DNA microarrays have been hybridized with labelled cDNAs from sessile and planktonic cells and preliminary data suggest that some genes are differently expressed between these two subpopulations. The understanding of these differences may help us to shift populations of leaching bacteria from the planktonic state towards the sessile state in order to influence bioleaching.

Radioprotection by inactivation of deoxyribonuclease I

Gamma radiation‐induced cell death is associated with and mediated by enzymatic DNA fragmentation. A nuclease that produces this fragmentation of DNA is unknown. To determine whether deoxyribonuclease I (DNase I), a major cytotoxic/apoptotic nuclease expressed in all cells and tissues, can mediate gamma radiation‐induced tissue injury, we used DNase I knockout (KO) mice and a zinc chelate of 3,5‐diisopropylsalicylic acid (Zn‐DIPS) that, as we show, has DNase I inhibiting activity in vitro. Our study demonstrated that inactivation or inhibition of DNase I strongly suppressed radiation injury (8 Gy, LD50/30) to white pulp of spleen, intestine villi, and bone marrow as measured using quantitative TUNEL assay. Spleen and intestine of DNase I KO mice were additionally protected from radiation by Zn‐DIPS due to the broad radioprotective efficacy of zinc. It is interesting that main DNase I‐producing tissues such as salivary gland, pancreas, and kidney showed no effect of DNase I inactivation. These results suggest that DNase I‐mediated mechanism of DNA damage and tissue injury has important regulatory role in radiation‐mediated cell death in radiosensitive organs. The study was supported by NIH/NIDDK and VA Merit Review grants to A.G.B.

MCF-7 breast carcinoma cells do not express caspase-3

Apoptosis, a fundamental process essential for normal tissue homeostasis and development, is closely associated with the activation of a class of aspartate-specific cysteinedependent proteases, called caspases, that lead to the demise of the cell via limited proteolysis of a multitude of cellular substrates [1, 2]. Caspases are expressed as inactive zymogens that become activated upon cleavage by other caspases in a so-called caspase activation cascade, or by mere oligomerization instigated by the formation of large multi-protein complexes such as the death-inducing signaling complex (DISC) or the apoptosome [3, 4]. Whereas DISC formation occurs via the so-called extrinsic death pathway that is instigated by activation of members of the death receptor family such as CD95, tumor necrosis factor receptor (TNF-R) or the receptors of the TNF-Rrelated apoptosis-inducing ligand (TRAIL), the apoptosome is formed following activation of mitochondria and is hence termed the intrinsic or mitochondrial death pathway. Based on their order in cell death pathways, caspases can be divided into initiator (caspase-2, -8, -9, and -10) and effector (caspase-3, -6, and -7) caspases. Among them, caspase-3, a member of the latter group, is absolutely crucial for apoptosis induction, as this enzyme is not only activated downstream of both the extrinsic and intrinsic death pathway, it is also responsible for the cleavage of the majority of substrates known so far [1, 5]. More importantly, with the proteolysis of discrete substrates, caspase-3 evokes some of the typical morphological and biochemical alterations associated with apoptosis. For instance, whereas the caspase-3-mediated cleavages of a-fodrin, gelsolin, rho-associated kinase-1 (ROCK-1) and p21-activated kinase 2 (PAK2) contribute to membrane blebbing, cleavage of the inhibitor of the caspase-activated DNase (ICAD) leads to the typical DNA fragmentation pattern observed in apoptosis [1]. Furthermore, with the cleavagemediated activation of the calcium-independent phospholipase A2 and subsequent production of the chemotactic phospholipid lysophosphatidylcholine, caspase-3 appears to be also responsible for the generation of so-called ‘‘eatme’’ signals that induce migration of phagocytes to the site of apoptotic cell death [6]. Thus, caspase-3 not only instigates and pursues the demise of a cell, but, in addition, makes sure that the corpse is properly disposed, a function crucial for avoiding inflammatory processes. Hence, determination of the processing and activation of caspase-3 are common means to assess apoptotic signal transduction pathways in numerous cell lines of varying origin. Curiously, several reports still claim the presence of caspase-3 in the breast carcinoma cell line MCF-7 in which this enzyme is supposed to contribute to apoptosis signaling [7–15]. This was not only demonstrated indirectly via fluorometric assay systems measuring caspase-3-like activities—that might be also elicited by the closely related caspase-7—but by Western blotting analyses demonstrating directly the presence of this protease in MCF-7 cells. However, in addition to the lack of caspase-10 [16], an initiator caspase in the extrinsic death pathway, MCF-7 cells do also not express caspase-3 [17]. Ten years ago, we demonstrated unambiguously that the lack of caspase-3 in these cells is caused by a 47-base pair deletion within exon 3 of the CASP3 gene resulting in the skipping of this exon during premRNA splicing and introduction of a premature stop codon at position 42 that completely abrogates translation of the CASP-3 mRNA [17]. Although caspase-3-deficient MCF-7 R. U. Janicke (&) Laboratory of Molecular Radiooncology, Clinic and Policlinic for Radiation Therapy and Radiooncology, University of Dusseldorf, Universitatsstrasse 1, 40225 Dusseldorf, Germany e-mail: Janicke@uni-duesseldorf.de

Neuroprotective Actions of PIKE-L by Inhibition of SET Proteolytic Degradation by Asparagine Endopeptidase

Ischemia and seizure cause excessive neuronal excitation that is associated with brain acidosis and neuronal cell death. However, the molecular mechanism of acidification-triggered neuronal injury is incompletely understood. Here, we show that asparagine endopeptidase (AEP) is activated under acidic condition, cuts SET, an inhibitor of DNase, and triggers DNA damage in brain, which is inhibited by PIKE-L. SET, a substrate of caspases, was cleaved by acidic cytosolic extract independent of caspase activation. Fractionation of the acidic cellular extract yielded AEP that is required for SET cleavage. We found that kainate provoked AEP activation and SET cleavage at N175, triggering DNA nicking in wild-type, but not AEP null, mice. PIKE-L strongly bound SET and prevented its degradation by AEP, leading to resistance of neuronal cell death. Moreover, AEP also mediated stroke-provoked SET cleavage and cell death in brain. Thus, AEP might be one of the proteinases activated by acidosis triggering neuronal injury during neuroexcitotoxicity or ischemia.

Crystal structure of VC1805, a conserved hypothetical protein from a Vibrio cholerae pathogenicity island, reveals homology to human p32

The severe diarrhoeal disease cholera is caused by the gram-negative bacterium Vibrio cholerae and continues to be a major cause of morbidity and mortality. Like many Vibrio species V. cholerae inhabits an aquatic ecosystem, and most V. cholerae isolates do not possess the ability to cause cholera. Of more than 200 known serotypes, only O1 and O139 serogroups are highly pathogenic and acknowledged to cause epidemic disease.1 The O1 serogroup can be divided into two groups: classical and El Tor, with the first cholera pandemic, beginning in Asia in 1817, and the subsequent five pandemics probably caused by the classical biotype. The seventh and present pandemic began in 1961 caused by the El Tor biotype.2 In 1992 a novel O-serogroup, O-139, emerged to cause epidemic cholera.3 All V. cholerae O1 and O139 serogroups encode the major virulence factors cholera toxin and toxin coregulated pilus, the latter being encoded on a pathogenicity island, named Vibrio Pathogenicity Island-I (VPI-1).4 Several other genomic regions have been identified that occur mainly among epidemic O1 and O139 serogroup isolates, including the so-called Vibrio seventh pandemic island-I (VSP-I) encoding ORFs VC0175 to VC0185, VSP-II encoding ORFs VC0490 to VC0516 and VPI-2 encoding ORFs VC1758 to VC1809.5–7 A microarray analysis of V. cholerae El Tor isolates was used to identify VSP-I and VSP-II encompassing genes that are possibly responsible for the unique characteristics of the seventh pandemic (El Tor) strains.5 An evolutionary genetic analysis of clinical and environmental isolates suggested that pandemic strains arose from a common O1 serogroup progenitor through the successive acquisition of new virulence regions.8 VPI-2 is a 57.3 kb region which consists of 52 ORFs present in all toxigenic O1 and O139 serogroup isolates, but lacking in non-O1 and non-O139 nontoxigenic isolates.6 VPI-2 encodes a type-I restriction modification system, a nan-nag region of genes involved in sialic acid metabolism that may play a nutritional role,9 a sialidase/neuraminidase known to convert intestinal higher-order gangliosides to GM1,10 and a region with homology to Mu phage. In addition, VPI-2 contains several genes that code for hypothetical proteins. One of these proteins, VC1805, is a 148 amino acid protein with no function revealed so far through sequence analysis. VC1805 is also encoded in the reduced 20 kb VPI-2 region present among most O139 serogroup isolates that are missing ORFs VC1761 to VC1788.6 A paralogue of VC1805 exists within VSP-II, VC0508 a 147 residue protein that shares 59% amino acid sequence identity with VC1805. A search of the sequence database using PSI-BLAST reveals homologues of VC1805 in several Vibrio species: V. vulnificus, V. splendidus, V. alginolyticus and V. fischeri, and orthologues in Altermonas macleodii, Aeromonas hydrophilia, and some Shewanella species. In addition, PSI-BLAST reveals that the adjacent hypothetical protein VC1804, with 104 residues, is a homologue of VC1805 sharing 26% sequence identity. Similarly the hypothetical protein VC0509 is a homologue of the adjacent protein VC0508. The four proteins VC0508, VC0509, VC1804, and VC1805 are therefore likely to share the same protein fold and be functionally related. As part of a structural genomics approach to understanding the function of hypothetical proteins within V. cholerae genomic islands we have determined the crystal structure of VC1805 to a resolution of 2.1 A using heavy atom isomorphous replacement. The structure reveals a similarity to the human mitochondrial protein p32 that is known to have several binding partners, including the human complement system protein C1q. This study shows that VC1805 does bind C1q, suggesting potential biological roles for the protein and its homologues.