An alkaloid initiates phosphodiesterase 3A-schlafen 12 dependent apoptosis without affecting the phosphodiesterase activity.
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ABSTRACT:
SUBMITTER: Ai Y
PROVIDER: S-EPMC7319972 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Cytotoxic molecules can kill cancer cells by disrupting critical cellular processes or by inducing novel activities. 6-(4-(Diethylamino)-3-nitrophenyl)-5-methyl-4,5-dihydropyridazin-3(2H)-one (DNMDP) is a small molecule that kills cancer cells by generation of novel activity. DNMDP induces complex formation between phosphodiesterase 3A (PDE3A) and schlafen family member 12 (SLFN12) and specifically kills cancer cells expressing elevated levels of these two proteins. Here, we examined the characteristics and covariates of the cancer cell response to DNMDP. On average, the sensitivity of human cancer cell lines to DNMDP is correlated with PDE3A expression levels. However, DNMDP could also bind the related protein, PDE3B, and PDE3B supported DNMDP sensitivity in the absence of PDE3A expression. Although inhibition of PDE3A catalytic activity did not account for DNMDP sensitivity, we found that expression of the catalytic domain of PDE3A in cancer cells lacking PDE3A is sufficient to confer sensitivity to DNMDP, and substitutions in the PDE3A active site abolish compound binding. Moreover, a genome-wide CRISPR screen identified the aryl hydrocarbon receptor-interacting protein (AIP), a co-chaperone protein, as required for response to DNMDP. We determined that AIP is also required for PDE3A-SLFN12 complex formation. Our results provide mechanistic insights into how DNMDP induces PDE3A-SLFN12 complex formation, thereby killing cancer cells with high levels of PDE3A and SLFN12 expression.
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2019-09-21 | GSE130569 | GEO
Project description:Platelet activation is critical for haemostasis, but if unregulated can lead to pathological thrombosis. Endogenous platelet inhibitory mechanisms are mediated by prostacyclin (PGI2)-stimulated cAMP signalling, which is regulated by phosphodiesterase 3A (PDE3A). However, spatiotemporal regulation of PDE3A activity in platelets is unknown. Here, we report that platelets possess multiple PDE3A isoforms with seemingly identical molecular weights (100 kDa). One isoform contained a unique N-terminal sequence that corresponded to PDE3A1 in nucleated cells but with negligible contribution to overall PDE3A activity. The predominant cytosolic PDE3A isoform did not possess the unique N-terminal sequence and accounted for >99% of basal PDE3A activity. PGI2 treatment induced a dose and time-dependent increase in PDE3A phosphorylation which was PKA-dependent and associated with an increase in phosphodiesterase enzymatic activity. The effects of PGI2 on PDE3A were modulated by A-kinase anchoring protein (AKAP) disruptor peptides, suggesting an AKAP-mediated PDE3A signalosome. We identified AKAP7, AKAP9, AKAP12, AKAP13, and moesin expressed in platelets but focussed on AKAP7 as a potential PDE3A binding partner. Using a combination of immunoprecipitation, proximity ligation techniques, and activity assays, we identified a novel PDE3A/PKA RII/AKAP7 signalosome in platelets that integrates propagation and termination of cAMP signalling through coupling of PKA and PDE3A.
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Project description:Acute respiratory virus infections, such as influenza and RSV, are predominant causes of asthma exacerbations. Eosinophils act as a double-edged sword in exacerbations in that they are activated by viral infections but also can capture and inactivate respiratory viruses. Phosphodiesterase type 4 (PDE4) is abundantly expressed by eosinophils and has been implicated in their activation. This exploratory study aims to determine whether these opposing roles of eosinophils activation of eosinophils upon interaction with virus can be modulated by selective PDE4 inhibitors and whether eosinophils from healthy, moderate and severe asthmatic subjects respond differently. Eosinophils were purified by negative selection from blood and subsequently exposed to RSV or influenza. Prior to exposure to virus, eosinophils were treated with vehicle or selective PDE4 inhibitors CHF6001 and GSK256066. After 18 hours of exposure, influenza, but not RSV, increased CD69 and CD63 expression by eosinophils from each group, which were inhibited by PDE4 inhibitors. ECP release, although not stimulated by virus, was also attenuated by PDE4 inhibitors. Eosinophils showed an increased Nox2 activity upon virus exposure, which was less pronounced in eosinophils derived from mild and severe asthmatics and was counteracted by PDE4 inhibitors. PDE4 inhibitors had no effect on binding of virus by eosinophils from each group. Our data indicate that PDE4 inhibitors can attenuate eosinophil activation, without affecting virus binding. By attenuating virus-induced responses, PDE4 inhibitors may mitigate virus-induced asthma exacerbations.
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Project description:About 30% of medulloblastomas (MBs), a tumor of the cerebellum, arise from cerebellar granule cell precursors (GCPs) undergoing transformation following activation of the Sonic hedgehog (Shh) pathway. To study this process, we generated a new MB model by crossing Patched1 heterozygous (Ptch1 +/-) mice, which develop spontaneous Shh-type MBs, with mice lacking B-cell translocation gene 1 (Btg1), a regulator of cerebellar development. In MBs developing in Ptch1 +/- mice, deletion of Btg1 does not alter tumor and lesion frequencies, nor affect the proliferation of neoplastic precursor cells. However, in both tumors and lesions arising in Ptch1 +/- mice, ablation of Btg1 increases by about 25% the apoptotic neoplastic precursor cells, as judged by positivity to activated caspase-3. Moreover, although Btg1 ablation in early postnatal GCPs, developing in the external granule cell layer, leads to a significant increase of proliferation, and decrease of differentiation, relative to wild-type, no synergy occurs with the Ptch1 +/- mutation. However, Btg1 deletion greatly increases apoptosis in postnatal GCPs, with strong synergy between Btg1-null and Ptch1 +/- mutations. That pronounced increase of apoptosis observed in Ptch1 +/- /Btg1 knockout young or neoplastic GCPs may be responsible for the lack of effect of Btg1 ablation on tumorigenesis. This increased apoptosis may be a consequence of increased expression of protein arginine methyltransferase 1 (Prmt1) protein that we observe in Btg1 knockout/Ptch1 +/- MBs. In fact, apoptotic genes, such as BAD, are targets of Prmt1. Moreover, in Btg1-null MBs, we observed a two-fold increase of cells positive to CD15, which labels tumor stem cells, raising the possibility of activation of quiescent tumor cells, known for their role in long-term resistance to treatment and relapses. Thus, Btg1 appears to play a role in cerebellar tumorigenesis by regulating the balance between apoptosis and proliferation during MB development, also influencing the number of tumor stem cells.
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Project description:Latency is a major barrier towards virus elimination in HIV-1-infected individuals. Yet, the mechanisms that contribute to the maintenance of HIV-1 latency are incompletely understood. Here we describe the Schlafen 12 protein (SLFN12) as an HIV-1 restriction factor that establishes a post-transcriptional block in HIV-1-infected cells and thereby inhibits HIV-1 replication and virus reactivation from latently infected cells. The inhibitory activity is dependent on the HIV-1 codon usage and on the SLFN12 RNase active sites. Within HIV-1-infected individuals, SLFN12 expression in PBMCs correlated with HIV-1 plasma viral loads and proviral loads suggesting a link with the general activation of the immune system. Using an RNA FISH-Flow HIV-1 reactivation assay, we demonstrate that SLFN12 expression is enriched in infected cells positive for HIV-1 transcripts but negative for HIV-1 proteins. Thus, codon-usage dependent translation inhibition of HIV-1 proteins participates in HIV-1 latency and can restrict the amount of virus release after latency reversal.
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Project description:Background/aimsHuman enterocytic differentiation is altered during development, fasting, adaptation, and bariatric surgery, but its intracellular control remains unclear. We hypothesized that Schlafen 12 (SLFN12) regulates enterocyte differentiation.MethodsWe used laser capture dissection of epithelium, qRT-PCR, and immunohistochemistry to evaluate SLFN12 expression in biopsies of control and fasting human duodenal mucosa, and viral overexpression and siRNA to trace the SLFN12 pathway in human Caco-2 and HIEC6 intestinal epithelial cells.ResultsFasting human duodenal mucosa expressed less SLFN12 mRNA and protein, accompanied by decreases in enterocytic markers like sucrase-isomaltase. SLFN12 overexpression increased Caco-2 sucrase-isomaltase promoter activity, mRNA, and protein independently of proliferation, and activated the SLFN12 putative promoter. SLFN12 coprecipitated Serpin B12 (SERPB12). An inactivating SLFN12 point mutation prevented both SERPB12 binding and sucrase-isomaltase induction. SERPB12 overexpression also induced sucrase-isomaltase, while reducing SERPB12 prevented the SLFN12 effect on sucrase-isomaltase. Sucrase-isomaltase induction by both SLFN12 and SERPB12 was attenuated by reducing UCHL5 or USP14, and blocked by reducing both. SERPB12 stimulated USP14 but not UCHL5 activity. SERPB12 coprecipitated USP14 but not UCHL5. Moreover, SLFN12 increased protein levels of the sucrase-isomaltase-promoter-binding transcription factor cdx2 without altering Cdx2 mRNA. This was prevented by reducing UCHL5 and USP14. We further validated this pathway in vitro and in vivo. SLFN12 or SERPB12 overexpression induced sucrase-isomaltase in human non-malignant HIEC-6 enterocytes.ConclusionsSLFN12 regulates human enterocytic differentiation by a pathway involving SERPB12, the deubiquitylases, and Cdx2. This pathway may be targeted to manipulate human enterocytic differentiation in mucosal atrophy, short gut or obesity.
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Project description:Using DNA microarray and clustering of expressed genes we have analyzed the mechanism of inhibition of wild-type p53-induced apoptosis by the cytokine interleukin 6 (IL-6) and the calcium mobilizer thapsigargin (TG). Clustering analysis of 1,786 genes, the expression level of which changed after activation of wild-type p53 in the absence or presence of IL-6 or TG, showed that these compounds did not cause a general inhibition of the ability of p53 to up-regulate or down-regulate gene expression. Expression of various p53 targets implicated as mediators of p53-induced apoptosis was also not affected by IL-6 or TG. These compounds thus can bypass the effect of wild-type p53 on gene expression and inhibit apoptosis. IL-6 and TG activated different p53-independent pathways of gene expression that include up-regulation of antiapoptotic genes. IL-6 and TG also activated different differentiation-associated genes. The ability of compounds such as cytokines and calcium mobilizers to inhibit p53-mediated apoptosis without generally inhibiting gene expression regulated by p53 can facilitate tumor development and tumor resistance to radiation and chemotherapy in cells that retain wild-type p53.
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Project description:The association between the phosphodiesterase 3A (PDE3A) rs7134375 single nucleotide polymorphism (SNP) and serum lipid levels are not well understood in the general population. The present study was performed in order to detect the association between the rs7134375 SNP and serum lipid levels in the Guangxi Mulao and Han populations. The genotypes of the PDE3A rs7134375 SNP in 761 subjects of the Mulao population and 774 subjects of the Han Chinese population were determined by polymerase chain reaction and restriction fragment length polymorphism combined with gel electrophoresis, and then confirmed by direct sequencing. It was observed that serum low-density lipoprotein cholesterol and apolipoprotein B levels were higher in the Mulao population than in the Han population (P<0.05 for each). The frequencies of the C and A alleles were 72.14 and 27.86% in the Mulao population, and 78.55 and 21.45% in the Han population (P<0.01), respectively. The frequencies of the CC, CA and AA genotypes were 52.04, 40.21 and 7.75% in the Mulao population, and 61.50, 34.11 and 4.39% in the Han population (P<0.01), respectively. The frequencies of the C and A alleles were 74.89 and 25.11% in Mulao females, and 68.08 and 31.92% in Mulao males (P<0.01), respectively. The serum triglyceride (TG) levels were different among the genotypes in the Mulao population; however, not in the Han population (P<0.01), and the A allele carriers exhibited lower TG levels than the A allele noncarriers. The serum lipid parameters were also correlated with several environmental factors in the two ethnic groups (P<0.05-0.001). It was concluded that the genotypic and allelic frequencies of the rs7134375 SNP are different between the Mulao and Han populations. In addition, the PDE3A rs7134375 SNP is associated with serum TG levels in the Mulao population, however, not in the Han population.
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