PROFICS: A bacterial selection system for directed evolution of proteases.

Proteases serve as important tools in biotechnology and as valuable drugs or drug targets. Efficient protein engineering methods to study and modulate protease properties are thus of great interest for a plethora of applications. We established PROFICS (PRotease Optimization via Fusion-Inhibited Carbamoyltransferase-based Selection), a bacterial selection system, which enables the optimization of proteases for biotechnology, therapeutics or diagnosis in a simple overnight process. During the PROFICS process, proteases are selected for their ability to specifically cut a tag from a reporter enzyme and leave a native N-terminus. Precise and efficient cleavage after the recognition sequence reverses the phenotype of an Escherichia coli knockout strain deficient in an essential enzyme of pyrimidine synthesis. A toolbox was generated to select for proteases with different preferences for P1' residues (the residue immediately following the cleavage site). The functionality of PROFICS is demonstrated with viral proteases and human caspase-2. PROFICS improved caspase-2 activity up to 25-fold after only one round of mutation and selection. Additionally, we found a significantly improved tolerance for all P1' residues caused by a mutation in a substrate interaction site. We showed that this improved activity enables cells containing the new variant to outgrow cells containing all other mutants, facilitating its straightforward selection. Apart from optimizing enzymatic activity and P1' tolerance, PROFICS can be used to reprogram specificities, erase off-target activity, optimize expression via tags/codon usage, or even to screen for potential drug-resistance-conferring mutations in therapeutic targets such as viral proteases in an unbiased manner.

Exon 9 skipping of apoptotic caspase-2 pre-mRNA is promoted by SRSF3 through interaction with exon 8.

Alternative splicing plays an important role in gene expression by producing different proteins from a gene. Caspase-2 pre-mRNA produces anti-apoptotic Casp-2S and pro-apoptotic Casp-2L proteins through exon 9 inclusion or skipping. However, the molecular mechanisms of exon 9 splicing are not well understood. Here we show that knockdown of SRSF3 (also known as SRp20) with siRNA induced significant increase of endogenous exon 9 inclusion. In addition, overexpression of SRSF3 promoted exon 9 skipping. Thus we conclude that SRSF3 promotes exon 9 skipping. In order to understand the functional target of SRSF3 on caspase-2 pre-mRNA, we performed substitution and deletion mutagenesis on the potential SRSF3 binding sites that were predicted from previous reports. We demonstrate that substitution mutagenesis of the potential SRSF3 binding site on exon 8 severely disrupted the effects of SRSF3 on exon 9 skipping. Furthermore, with the approach of RNA pulldown and immunoblotting analysis we show that SRSF3 interacts with the potential SRSF3 binding RNA sequence on exon 8 but not with the mutant RNA sequence. In addition, we show that a deletion of 26nt RNA from 5' end of exon 8, a 33nt RNA from 3' end of exon 10 and a 2225nt RNA from intron 9 did not compromise the function of SRSF3 on exon 9 splicing. Therefore we conclude that SRSF3 promotes exon 9 skipping of caspase-2 pre-mRNA by interacting with exon 8. Our results reveal a novel mechanism of caspase-2 pre-mRNA splicing.

Oncogenic ras-induced down-regulation of pro-apoptotic protease caspase-2 is required for malignant transformation of intestinal epithelial cells.

Resistance of carcinoma cells to anoikis, apoptosis that is normally induced by loss of cell-to-extracellular matrix adhesion, is thought to be essential for the ability of these cells to form primary tumors, invade adjacent tissues, and metastasize to distant organs. Current knowledge about the mechanisms by which cancer cells evade anoikis is far from complete. In an effort to understand these mechanisms, we found that ras, a major oncogene, down-regulates protease caspase-2 (which initiates certain steps of the cellular apoptotic program) in malignant human and rat intestinal epithelial cells. This down-regulation could be reversed by inhibition of a protein kinase Mek, a mediator of Ras signaling. We also found that enforced down-regulation of caspase-2 in nonmalignant intestinal epithelial cells by RNA interference protected them from anoikis. Furthermore, the reversal of the effect of Ras on caspase-2 achieved by the expression of exogenous caspase-2 in detached ras-transformed intestinal epithelial cells promoted well established apoptotic events, such as the release of the pro-apoptotic mitochondrial factors cytochrome c and HtrA2/Omi into the cytoplasm of these cells, significantly enhanced their anoikis susceptibility, and blocked their long term growth in the absence of adhesion to the extracellular matrix. Finally, the blockade of the effect of Ras on caspase-2 substantially suppressed growth of tumors formed by the ras-transformed cells in mice. We conclude that ras-induced down-regulation of caspase-2 represents a novel mechanism by which oncogenic Ras protects malignant intestinal epithelial cells from anoikis, promotes their anchorage-independent growth, and allows them to form tumors in vivo.

p38 mitogen-activated protein kinase is a key regulator of 5-phenylselenyl- and 5-methylselenyl-methyl-2'-deoxyuridine-induced apoptosis in human HL-60 cells.

Two novel, modified thymidine nucleosides, 5-phenylselenyl-methyl-2'-deoxyuridine (PhSe-T) and 5-methylselenyl-methyl-2'-deoxyuridine (MeSe-T), trigger reactive oxygen species (ROS) generation and DNA damage and thereby induce caspase-mediated apoptosis in human HL-60 cells; however, the mechanism leading to caspase activation and apoptotic cell death remains unclear. Therefore, we investigated the signaling molecules involved in nucleoside derivative-induced caspase activation and apoptosis in HL-60 cells. PhSe-T/MeSe-T treatment activated two mitogen-activated protein kinases (MAPKs), extracellular-receptor kinase (ERK) and p38, and induced the phosphorylation of two downstream targets of p38, ATF-2 and MAPKAPK2. In addition, the selective p38 inhibitor SB203580 suppressed PhSe-T/MeSe-T-induced apoptosis and activation of caspase-3, -9, -8, and -2, whereas the jun amino-terminal kinase (JNK) inhibitor SP600125 and the ERK inhibitor PD98059 had no effect. SB203580 and an ROS scavenger, tiron, inhibited PhSe-T/MeSe-T-induced histone H2AX phosphorylation, which is a DNA damage marker. Moreover, tiron inhibited PhSe-T/MeSe-T-induced phosphorylation of p38 and enhanced p38 MAP kinase activity, indicating a role for ROS in PhSe-T/MeSe-T-induced p38 activation. Taken together, our results suggest that PhSe-T/MeSe-T-induced apoptosis is mediated by the p38 pathway and that p38 serves as a link between ROS generation and DNA damage/caspase activation in HL-60 cells.

Quercetin-induced apoptosis of HL-60 cells by reducing PI3K/Akt.

To explore the effect and mechanism of quercetin on proliferation and apoptosis of leukemia cells, and provide a theoretical basis for its clinical application. HL-60 leukemia cell lines was treated with different dose quercetin, the proliferation activity of leukemia cells was assessed by MTT method; the morphological changes of apoptosis of HL-60 cells, including nuclear condensation and DNA fragmentation, were observed by Hoechst 33258 fluorescence staining, the apoptosis rate and caspase 2,3 activation were assessed by flow cytometry, and the cell signal pathway including phosphatidylinositol 3-kinase (PI3K), phosphorylated protein kinase B (pAkt), Bcl-2, Bax were detected by western blotting. Quercetin could significantly decrease the proliferation activity of HL-60 cells through the blockade of G(0)/G(1) phase, and induce the apoptosis of HL-60 cells in a time- and dose-dependent manner. Quercetin caused leukemia cells apoptosis by decreasing the protein expression of PI3K and Bax, the inhibitory phosphorylation of Akt, the decreased levels of Bcl-2 protein and increased activations of caspase-2 and -3, and increased poly(ADP-ribose) polymerase cleavage. Our results indicate that the apoptotic processes caused by quercetin are mediated by the decrease of pAkt and Bcl-2 levels, the increase of Bax level, and the activation of caspase families in HL-60 cells.

Apoptosis of bladder cancer by sodium butyrate and cisplatin.

The effects of sodium butyrate (SB), a histone deacetylase inhibitor, in combination with cisplatin (CDDP), for inhibition of cell growth and induction of apoptosis were investigated in bladder cancer cell lines in vitro. Bladder cancer cell lines T24, 253J, and UMUC3 were treated with different concentrations of CDDP or SB. Cell proliferation was studied by XTT assay. Cell-cycle analysis and induction of apoptosis were analyzed by laser scanning cytometry (LSC). Western blot analysis was used to determine expression of p21, p27, TRADD, FADD, caspase-2, and caspase-7. We observed that SB in combination with CDDP induced significant inhibition of cell growth in a dose-dependent manner through G1 arrest and apoptosis, as determined by LSC. When bladder cancer cell lines were treated with SB plus CDDP, Western blotting showed increased expression of p21 but not p27 in T24 cells, whereas both p21 and p27 increased in 253J and UMUC3 cells. All cell lines exhibited a moderate increase in TRADD and decrease in procaspase-2 but no significant change in FADD and procaspase-7. The results showed the synergistic anticancer effect of SB in combination with CDDP, their potential for treatment of bladder cancer, and their mechanism of action in terms of cell signal transduction and induction of apoptosis.

TAp73beta and DNp73beta activate the expression of the pro-survival caspase-2S.

p73, the p53 homologue, exists as a transactivation-domain-proficient TAp73 or deficient deltaN(DN)p73 form. Expectedly, the oncogenic DNp73 that is capable of inactivating both TAp73 and p53 function, is over-expressed in cancers. However, the role of TAp73, which exhibits tumour-suppressive properties in gain or loss of function models, in human cancers where it is hyper-expressed is unclear. We demonstrate here that both TAp73 and DNp73 are able to specifically transactivate the expression of the anti-apoptotic member of the caspase family, caspase-2(S). Neither p53 nor TAp63 has this property, and only the p73beta form, but not the p73alpha form, has this competency. Caspase-2 promoter analysis revealed that a non-canonical, 18 bp GC-rich Sp-1-binding site-containing region is essential for p73beta-mediated activation. However, mutating the Sp-1-binding site or silencing Sp-1 expression did not affect p73beta's transactivation ability. In vitro DNA binding and in vivo chromatin immunoprecipitation assays indicated that p73beta is capable of directly binding to this region, and consistently, DNA binding p73 mutant was unable to transactivate caspase-2(S). Finally, DNp73beta over-expression in neuroblastoma cells led to resistance to cell death, and concomitantly to elevated levels of caspase-2(S.) Silencing p73 expression in these cells led to reduction of caspase-2(S) expression and increased cell death. Together, the data identifies caspase-2(S) as a novel transcriptional target common to both TAp73 and DNp73, and raises the possibility that TAp73 may be over-expressed in cancers to promote survival.

Production of Circularly Permuted Caspase-2 for Affinity Fusion-Tag Removal: Cloning, Expression in Escherichia coli, Purification, and Characterization.

Caspase-2 is the most specific protease of all caspases and therefore highly suitable as tag removal enzyme creating an authentic N-terminus of overexpressed tagged proteins of interest. The wild type human caspase-2 is a dimer of heterodimers generated by autocatalytic processing which is required for its enzymatic activity. We designed a circularly permuted caspase-2 (cpCasp2) to overcome the drawback of complex recombinant expression, purification and activation, cpCasp2 was constitutively active and expressed as a single chain protein. A 22 amino acid solubility tag and an optimized fermentation strategy realized with a model-based control algorithm further improved expression in Escherichia coli and 5.3 g/L of cpCasp2 in soluble form were obtained. The generated protease cleaved peptide and protein substrates, regardless of N-terminal amino acid with high activity and specificity. Edman degradation confirmed the correct N-terminal amino acid after tag removal, using Ubiquitin-conjugating enzyme E2 L3 as model substrate. Moreover, the generated enzyme is highly stable at -20 °C for one year and can undergo 25 freeze/thaw cycles without loss of enzyme activity. The generated cpCasp2 possesses all biophysical and biochemical properties required for efficient and economic tag removal and is ready for a platform fusion protein process.

Upregulation of miR-34a by Inhibition of IRE1α Has Protective Effect against Aß-Induced Injury in SH-SY5Y Cells by Targeting Caspase-2.

BACKGROUND: Neurotoxicity induced by the amyloid-ß (Aß) peptide is one of the most important pathological mechanisms of Alzheimer's disease (AD). Based on accumulating evidence in AD research, both endoplasmic reticulum stress (ER stress) and alterations in the microRNA (miRNA) network contribute to the pathogenesis of the disease, making them potential therapeutic targets for AD. The present study was performed to investigate whether miR-34a and the inositol-requiring enzyme 1 (IRE1) are involved in the regulation of Aß-induced cytotoxicity. METHODS: Human neuroblastoma SH-SY5Y cells were treated with Aß1-40. Cell viability was assessed by the MTT assay. The integrity of the plasma membrane was assessed by LDH release. The expression levels of XBP1s, IRE1α, p-IRE1α, and Caspase-2 were detected by Western blot analysis. Spliced-XBP1 mRNA and miR-34a were detected by reverse transcription- (RT-) PCR and quantitative real-time PCR, respectively. Caspase-2 activity was measured using the Caspase-2 cellular activity assay kit. The IRE1 inhibitor (STF-083010) was used to determine the role of IRE1α on miR-34a expression. SH-SY5Y cells were transfected with miR-34a mimics to assess the role of miR-34a on the activation of Caspase-2 and the viability of Aß-exposed SH-SY5Y cells. RESULTS: We showed that Aß caused concentration- and duration-dependent death of SH-SY5Y cells. The expression levels of XBP1s, p-IRE1α, and Caspase-2 were increased, along with a corresponding decrease in the miR-34a levels in Aß-exposed SH-SY5Y cells. The IRE1 inhibitor (STF-083010) upregulated the expression of miR-34a and suppressed the activation of Caspase-2, effectively alleviating the Aß-induced death of SH-SY5Y cells. Transfection studies show that miR-34a mimics inhibit the expression of Caspase-2 and restore the viability of Aß-exposed SH-SY5Y cells. CONCLUSION: Aß peptide induced downregulation of miR-34a through the activation of IRE1α, which may induce cytotoxicity by targeting Caspase-2. Upregulation of miR-34a by inhibition of IRE1α has protective effects against Aß-induced injury in SH-SY5Y cells.

Reproductive tissue regression: involvement of caspases, inducible nitric oxide synthase and nitric oxide during moulting in White Leghorn hens.

Moulting is a natural physiological process where the reproductive system of birds undergoes complete remodeling in preparation for the next laying cycle. In domestic chickens, moulting is artificially induced by feed withdrawal to recycle the old laying flock for best profit margins. This has received severe criticism from animal welfare organizations, forcing several countries to stop this practice. Several alternative methods to feed withdrawal methods were developed but were found to produce inconsistent results. Understanding the actual mechanism of moulting would help in designing a new animal welfare friendly method. The present investigation attempted to study the molecular mechanism of moulting in White Leghorn hens. Eighty-four layers (75 weeks) were divided into two groups. The birds in the first group were subjected to moulting by feed withdrawal (FW) while the other group received high dietary Zn (ZnF) treatment for 10 days. Six birds from each group were sacrificed on 0, 1-4, 6 and 10 days of moulting and mRNA expression of caspases-1, -2 and iNOS, along with the apoptotic ladder pattern and nitric oxide (NO) in the ovary and oviduct, was investigated. The mRNA expression of iNOS was upregulated with a corresponding increase in NO levels. Caspases-1 and -2 were differentially upregulated in the ovary and oviduct of moulted birds. A constant decline in serum estradiol and progesterone levels was also observed. It can be concluded that the pattern of reproductive regression during moulting by the two methods is different, as the expression of genes studied in the present investigation is different.

Prognostic Value of CD95, Active Caspase-3, and Bcl-2 Expression in Adult Patients with De Novo Acute Lymphoblastic Leukemia.

BACKGROUND: Acute lymphoblastic leukemia is an aggressive malignant disease with high mortality rates in adults. AIM OF THE STUDY: The expression levels of CD95, active caspase-3, and Bcl-2 were determined in 111 patients with de novo acute lymphoblastic leukemia (ALL) and correlated with overall survival (OS) and disease-free survival (DFS). METHODS: The immunophenotyped ok leukemia and the expression of CD95, active caspase-3, and Bcl-2, were determined by flow cytometry. Apoptotic variables were correlated by Spearman test, and survival by Kaplan-Meier method. Log-rank test was used to compare survival curves. RESULTS: From a total of 111 patients, 56 cases were B-ALL, 16 T-ALL, 16 B-ALL/CD33+, and 23 ambiguous lineage-AL (AmbLin-AL). The median expression of CD95 (61.5%) and active-caspase-3 (19.4%) was higher in T-ALL (p <0.05), whereas Bcl-2 was lower in T-ALL (p <0.038). There was a highly significant correlation in B-ALL, B-ALL/CD33+ and AmbLin-AL between CD95 and Bcl-2, CD95-Active caspase-3, and Bcl-2-Active caspase-3; while in T-ALL, there was only a correlation between CD95-Active caspase-3, and Bcl-2-Active caspase-3. OS and DFS were better for T-ALL than the other groups, especially in patients having higher values of CD95 and active caspase 3, and lower values of Bcl-2. The worse survival rates were observed in patients with B-ALL/CD33+, and AmbLin-AL. CONCLUSIONS: The prognosis of ALL in adults is influenced by the expression levels of Bcl-2, active-caspase-3, and CD95.

Inhibition of microRNA-383 has tumor suppressive effect in human epithelial ovarian cancer through the action on caspase-2 gene.

BACKGROUND: MicroRNAs are important cancer regulators. In this work, we examined the expression pattern and mechanistic implications of microRNA-383 (miR-383) in human epithelial ovarian cancer (EOC). METHODS: Gene expression level of miR-383 was compared by qRT-PCR between EOC cell lines and normal ovarian epithelial cell line, and between clinical EOC tumors and adjacent non-tumor ovarian epithelial tissues. Endogenous miR-383 was downregulated through lentiviral infection. Its effects on regulating EOC proliferation, cell cycle, invasion and in vivo explant development were assessed. Possible downstream target of miR-383 in EOC, human caspase-2 gene (CASP2), was evaluated by luciferase assay and qRT-PCR. CASP2 was then genetically knocked down by siRNA to assess its functional relationship with miR-383 in regulating EOC development both in vitro and in vivo. RESULTS: MiR-383 was overexpressed in both immortal EOC cell lines and human EOC tumors. In stably miR-383-downregulated EOC cell lines, cancer proliferation, cell cycle progression, invasion and in vivo explant development were significantly suppressed. CASP2 was confirmed to be downstream of miR-383 in EOC. SiRNA-mediated CASP2 downregulation had reverse relationship with miR-383 downregulation in regulating EOC development both in vitro and in vivo. CONCLUSION: Inhibition of miR-383 has profound tumor suppressing effect on EOC development. And the functional regulation of miR-383 in EOC is very likely inversely associated with CASP2 gene.

Knockdown of S100P by lentiviral-mediated RNAi promotes apoptosis and suppresses the colony-formation ability of gastric cancer cells.

S100P is a putative candidate oncogene in several types of human tumors. However, expression of S100P, its potential role and its clinical significance in gastric cancer remain unclear. In the present study, S100P expression was examined by immunohistochemistry using a tissue microarray. Positive staining for S100P was noted in 77.1% of the cases while 22.9% were negative. In two gastric cancer cell lines, MGC-803 and SGC-7901, S100P expression was knocked down by a lentiviral short hairpin delivery system. The RNA interference-mediated downregulation of S100P expression markedly promoted cell apoptosis and inhibited cell colony-formation ability of the gastric cancer cells. In addition, knockdown of S100P significantly regulated the expression of 12 apoptosis-associated genes with a >1.5-fold change compared with the negative control. Among them, FOS, DDIT3 and FN1 were significantly upregulated, while FASLG, DAPK1, CTNNB1 and CASP2 were notably downregulated following S100P silencing. These results suggest that S100P acts as an oncogenic factor in gastric cancer and is a potential molecular target for gastric cancer gene therapy.

Transcriptomic analysis unveils correlations between regulative apoptotic caspases and genes of cholesterol homeostasis in human brain.

Regulative circuits controlling expression of genes involved in the same biological processes are frequently interconnected. These circuits operate to coordinate the expression of multiple genes and also to compensate dysfunctions in specific elements of the network. Caspases are cysteine-proteases with key roles in the execution phase of apoptosis. Silencing of caspase-2 expression in cultured glioblastoma cells allows the up-regulation of a limited number of genes, among which some are related to cholesterol homeostasis. Lysosomal Acid Lipase A (LIPA) was up-regulated in two different cell lines in response to caspase-2 down-regulation and cells silenced for caspase-2 exhibit reduced cholesterol staining in the lipid droplets. We expanded this observation by large-scale analysis of mRNA expression. All caspases were analyzed in terms of co-expression in comparison with 166 genes involved in cholesterol homeostasis. In the brain, hierarchical clustering has revealed that the expression of regulative apoptotic caspases (CASP2, CASP8 CASP9, CASP10) and of the inflammatory CASP1 is linked to several genes involved in cholesterol homeostasis. These correlations resulted in altered GBM (Glioblastoma Multiforme), in particular for CASP1. We have also demonstrated that these correlations are tissue specific being reduced (CASP9 and CASP10) or different (CASP2) in the liver. For some caspases (CASP1, CASP6 and CASP7) these correlations could be related to brain aging.

ARC is highly expressed in nasopharyngeal carcinoma and confers X-radiation and cisplatin resistance.

Apoptosis repressor with caspase recruitment domain (ARC), an inhibitor of apoptosis, is primarily expressed in terminally differentiated tissues. Recent studies have revealed that ARC is highly expressed in a variety of human cancer cell lines and epithelial-derived cancers, which suggests that ARC plays an important role in the process of carcinogenesis. However, whether ARC is involved in the development of nasopharyngeal carcinoma (NPC) and the various roles it plays in NPC remain unclear. In the present study, we examined the expression of ARC in NPC cell lines and NPC tissues and the relationship between its subcellular expression and clinicopathological grade; moreover, we explored the effect of this protein on radiation resistance and chemoresistance in NPC cells. We found that cytoplasmic ARC was expressed at high levels in NPC tissues, at moderate levels in severe atypical hyperplasia and at low levels in benign nasopharyngeal tissues. High expression of cytoplasmic and nuclear ARC was correlated with advanced local invasion. However, only a small amount of nuclear ARC was expressed in NPC in contrast to cytoplasmic ARC. We also found that attenuation of ARC expression by miRNA resulted in decreased X-radiation and cisplatin resistance in NPC CNE-2 cells. In contrast, overexpression of ARC resulted in increased X-radiation and cisplatin resistance in NPC 6-10B cells. Furthermore, we demonstrated that ARC appears to be critical for blocking the activation of casapse-8 and casapse-2 in NPC cells subjected to X-radiation or cisplatin. These results suggest that high expression of ARC plays an important role in the pathogenesis of NPC and leads to X-radiation and cisplatin resistance in NPC.

Ocular neuroprotection by siRNA targeting caspase-2.

Retinal ganglion cell (RGC) loss after optic nerve damage is a hallmark of certain human ophthalmic diseases including ischemic optic neuropathy (ION) and glaucoma. In a rat model of optic nerve transection, in which 80% of RGCs are eliminated within 14 days, caspase-2 was found to be expressed and cleaved (activated) predominantly in RGC. Inhibition of caspase-2 expression by a chemically modified synthetic short interfering ribonucleic acid (siRNA) delivered by intravitreal administration significantly enhanced RGC survival over a period of at least 30 days. This exogenously delivered siRNA could be found in RGC and other types of retinal cells, persisted inside the retina for at least 1 month and mediated sequence-specific RNA interference without inducing an interferon response. Our results indicate that RGC apoptosis induced by optic nerve injury involves activation of caspase-2, and that synthetic siRNAs designed to inhibit expression of caspase-2 represent potential neuroprotective agents for intervention in human diseases involving RGC loss.