果蝇程序化死亡基因5(PDCD5)同源cDNA的克隆和序列分析

 为了解人类白血病细胞凋亡相关新基因 TFAR1 9(PDCD5,programmed cell death5)在不同种属间的序列同源性 ,利用 EST(expression sequence tag)拼接、RT- PCR、DNA序列测定技术及计算机分析技术 ,首次成功地进行了果蝇 PDCD5同源 c DNA编码区基因克隆和序列分析 .发现果蝇与小鼠及果蝇与人 PDCD5在核苷酸水平上分别有 57.5%和 57.1 %的同源性 ,在氨基酸水平上分别有 46.8%和 46.4%的同源性 .功能区分析发现 ,果蝇 PDCD5c DNA编码 1 33个氨基酸 ,计算机预测可能是一种核蛋白 ,含 5个可能的酪蛋白激酶 (casein kinase )磷酸化位点 ,2个可能的 PKC磷酸化位点 ,与人 PDCD5的功能区类似 .因而果蝇 PDCD5是与人 PDCD5同源的新基因 ,可能都与细胞程序化死亡相关 .     

Regulatory effects of programmed cell death 4 (PDCD4) protein in interferon (IFN)-stimulated gene expression and generation of type I IFN responses

The precise mechanisms by which the activation of interferon (IFN) receptors (IFNRs) ultimately controls mRNA translation of specific target genes to induce IFN-dependent biological responses remain ill defined. We provide evidence that IFN-α induces phosphorylation of programmed cell death 4 (PDCD4) protein on Ser67. This IFN-α-dependent phosphorylation is mediated by either the p70 S6 kinase (S6K) or the p90 ribosomal protein S6K (RSK) in a cell-type-specific manner. IFN-dependent phosphorylation of PDCD4 results in downregulation of PDCD4 protein levels as the phosphorylated form of PDCD4 interacts with the ubiquitin ligase β-TRCP (β-transducin repeat-containing protein) and undergoes degradation. This process facilitates IFN-induced eukaryotic translation initiation factor 4A (eIF4A) activity and binding to translation initiation factor eIF4G to promote mRNA translation. Our data establish that PDCD4 degradation ultimately facilitates expression of several ISG protein products that play important roles in the generation of IFN responses, including IFN-stimulated gene 15 (ISG15), p21(WAF1/CIP1), and Schlafen 5 (SLFN5). Moreover, engagement of the RSK/PDCD4 pathway by the type I IFNR is required for the suppressive effects of IFN-α on normal CD34(+) hematopoietic precursors and for antileukemic effects in vitro. Altogether, these findings provide evidence for a unique function of PDCD4 in the type I IFN system and indicate a key regulatory role for this protein in mRNA translation of ISGs and control of IFN responses.     

The 3C Protein of Enterovirus 71 Inhibits Retinoid Acid-Inducible Gene I-Mediated Interferon Regulatory Factor 3 Activation and Type I Interferon Responses

Enterovirus 71 (EV71) is a human pathogen that induces hand, foot, and mouth disease and fatal neurological diseases. Immature or impaired immunity is thought to associate with increased morbidity and mortality. In a murine model, EV71 does not facilitate the production of type I interferon (IFN) that plays a critical role in the first-line defense against viral infection. Administration of a neutralizing antibody to IFN-α/β exacerbates the virus-induced disease. However, the molecular events governing this process remain elusive. Here, we report that EV71 suppresses the induction of antiviral immunity by targeting the cytosolic receptor retinoid acid-inducible gene I (RIG-I). In infected cells, EV71 inhibits the expression of IFN-β, IFN-stimulated gene 54 (ISG54), ISG56, and tumor necrosis factor alpha. Among structural and nonstructural proteins encoded by EV71, the 3C protein is capable of inhibiting IFN-β activation by virus and RIG-I. Nevertheless, EV71 3C exhibits no inhibitory activity on MDA5. Remarkably, when expressed in mammalian cells, EV71 3C associates with RIG-I via the caspase recruitment domain. This precludes the recruitment of an adaptor IPS-1 by RIG-I and subsequent nuclear translocation of interferon regulatory factor 3. An R84Q or V154S substitution in the RNA binding motifs has no effect. An H40D substitution is detrimental, but the protease activity associated with 3C is dispensable. Together, these results suggest that inhibition of RIG-I-mediated type I IFN responses by the 3C protein may contribute to the pathogenesis of EV71 infection.Enterovirus 71 (EV71) is a single-stranded, positive-sense RNA virus belonging to the Picornaviridae family. The viral genome is approximately 7,500 nucleotides in length, with a single open reading frame that encodes a large precursor protein. Upon infection, this protein precursor is processed into four structural (VP1, VP2, VP3, and VP4) and seven nonstructural (2A, 2B, 2C, 3A, 3B, 3C, and 3D) proteins (32). EV71 infection manifests most frequently as the childhood exanthema known as hand, foot, and mouth disease (HFMD). Additionally, EV71 infection may cause neurological diseases, which include aseptic meningitis, brain stem and/or cerebellar encephalitis, and acute flaccid paralysis (32). Young children and infants are especially susceptible to EV71 infection. Since the initial recognition of EV71 in the United States, outbreaks have been reported in Southeast Asia, Europe, and Australia (1-3, 11, 14, 24, 30-32). Recently, large epidemics of HFMD occurred in the mainland of China (26, 42, 52).The mechanism of EV71 pathogenesis remains obscure. It is believed that immature or impaired immunity, upon EV71 infection, is associated with increased morbidity and mortality (7, 14, 17). In a murine infection model, lymphocyte as well as antibody responses reduce tissue viral loads and EV71 lethality (28). Notably, EV71 induces skin rash at the early stage and hind limb paralysis or death at the late stage. Oral infection leads to initial replication in the intestine and subsequent spread to various organs such as the spinal cord and the brain stem (8). Intriguingly, EV71 does not facilitate the production of type I interferon (IFN), a family of cytokines involved in first-line defense against virus infection. Indeed, administration of neutralizing antibody to IFN-α/β increases tissue viral loads and exacerbates the virus-induced disease (29).Type I IFN is produced in response to viral infections (22). For example, Toll-like receptor 3 (TLR3) in the endosome recognizes double-stranded RNA (dsRNA), where it recruits the adaptor Toll/interleukin-1 receptor (TIR) domain-containing adaptor inducing IFN-β (TRIF) (22). TRIF, together with tumor necrosis factor (TNF) receptor-associated factor 3 (TRAF3), then activates the two IKK-related kinases, TANK-binding kinase 1 (TBK1) and inducible IκB kinase (IKKi), both of which phosphorylate interferon regulatory factor 3/7 (IRF3/7) (10, 13, 36, 45). IRF3 or IRF7, in turn, stimulates the expression of target genes, such as IFN-α/β (33, 37, 39, 51). In parallel, TRIF also induces NF-κB activation via TRAF6 (18, 19). In addition, alternative mechanisms exist in host cells to detect cytosolic nucleic acids. Two RNA helicases, retinoid acid-inducible gene I (RIG-I) and melanoma differentiation-associated gene 5 (MDA5), recognize viral RNA present in the cytoplasm and subsequently recruit the adaptor IFN promoter-stimulating factor 1 (IPS-1; also called Cardif, MAVS, and VISA) (22, 23, 54). The interaction of IPS-1, TRAF3, and TBK1/IKKi activates IRF3/IRF7 and induces the expression of IFN-α/β while the interaction of IPS-1 with the Fas-associated protein-containing death domain (FADD) leads to NF-κB activation. It has been shown that MDA5 recognizes long double-stranded RNAs, such as in cells infected with picornaviruses, whereas RIG-I senses 5′ triphosphate single-stranded RNA with poly(U/A) motifs and short dsRNA in cells infected with a variety of RNA viruses (16, 20, 40, 43).The objective of this study was to investigate the interaction of EV71 with the type I IFN system. We demonstrate that, unlike Sendai virus or double-stranded RNA, EV71 does not stimulate the expression of antiviral genes in mammalian cells. Among structural and nonstructural proteins encoded by EV71, the 3C protein is able to inhibit virus-induced activation of the IFN-β promoter. We provide evidence that when expressed in mammalian cells, the 3C protein suppresses RIG-I signaling by disruption of the RIG-I-IPS-1 complex and IRF3 nuclear translocation. While H40, KFRDI, and VGK motifs are involved, the protease and RNA binding activities are dispensable. Collectively, these results suggest that control of RIG-I by the 3C protein impairs type I IFN responses, which may contribute to the pathogenesis of EV71 infection.     

动物细胞培养过程中的细胞凋亡

细胞培养过程中的细胞凋亡是细胞受环境因素的影响而发生的现象。随着对细胞凋亡的分子生物学和细胞生物学了解的深入,显示了有效地控制动物细胞培养中细胞凋亡的巨大潜力。包括采用ＤＮＡ重组技术把抗细胞凋亡的基因导入细胞和在培基中加入具有抗细胞凋亡的生存因子或化合物等手段已用于控制细胞培养过程中的细胞凋亡。这些技术将大大延长细胞达到饱和密度后的培养时间,提高细胞培养系统的生产效率。     

Programmed Cell Death 6 (PDCD6) Protein Interacts with Death-Associated Protein Kinase 1 (DAPk1): Additive Effect on Apoptosis via Caspase-3 Dependent Pathway

Programmed cell death 6 (PDCD6) protein is a 22 kDa EF-hand type Ca²⁺-binding protein involved in apoptosis. To define the regulating mechanism of PDCD6 activity in the apoptotic pathway, we searched a human ovary cDNA library for a novel PDCD6 binding protein using a yeast two-hybrid system. The selected protein was the human death-associated protein kinase 1 (DAPk1), another protein that functions as a positive mediator of apoptosis. Co-transfection of PDCD6 and DAPk1 cDNA into a tumor cell line accelerated apoptosis via caspase-3 dependent pathway.J.H. Lee and S.B. Rho contributed equally to this workRevisions requested 4 March 2005; Revisions received 10 May 2005     

Enhanced Arginine Methylation of Programmed Cell Death 4 Protein during Nutrient Deprivation Promotes Tumor Cell Viability

The role of programmed cell death 4 (PDCD4) in tumor biology is context-dependent. PDCD4 is described as a tumor suppressor, but its coexpression with protein arginine methyltransferase 5 (PRMT5) promotes accelerated tumor growth. Here, we report that PDCD4 is methylated during nutrient deprivation. Methylation occurs because of increased stability of PDCD4 protein as well as increased activity of PRMT5 toward PDCD4. During nutrient deprivation, levels of methylated PDCD4 promote cell viability, which is dependent on an enhanced interaction with eIF4A. Upon recovery from nutrient deprivation, levels of methylated PDCD4 are regulated by phosphorylation, which controls both the localization and stability of methylated PDCD4. This study reveals that, in response to particular environmental cues, the role of PDCD4 is up-regulated and is advantageous for cell viability. These findings suggest that the methylated form of PDCD4 promotes tumor viability during nutrient deprivation, ultimately allowing the tumor to grow more aggressively.     

The Autoregulatory Feedback Loop of MicroRNA-21/Programmed Cell Death Protein 4/Activation Protein-1 (MiR-21/PDCD4/AP-1) as a Driving Force for Hepatic Fibrosis Development

Sustained activation of hepatic stellate cells (HSCs) leads to hepatic fibrosis, which is characterized by excessive collagen production, and for which there is no available drug clinically. Despite tremendous progress, the cellular activities underlying HSC activation, especially the driving force in the perpetuation stage, are only partially understood. Recently, microRNA-21 (miR-21) has been found to be prevalently up-regulated during fibrogenesis in different tissues, although its detailed role needs to be further elucidated. In the present study, miR-21 expression was examined in human cirrhotic liver samples and in murine fibrotic livers induced by thioacetamide or carbon tetrachloride. A dramatic miR-21 increase was noted in activated HSCs. We further found that miR-21 maintained itself at constant high levels by using a microRNA-21/programmed cell death protein 4/activation protein-1 (miR-21/PDCD4/AP-1) feedback loop. Disrupting this loop with miR-21 antagomir or AP-1 inhibitors significantly suppressed fibrogenic activities in HSCs and ameliorated liver fibrosis. In contrast, reinforcing this loop with small interfering RNA (siRNA) against PDCD4 promoted fibrogenesis in HSCs. Further analysis indicated that the up-regulated miR-21 promoted the central transforming growth factor-β (TGF-β) signaling pathway underlying HSC activation. In summary, we suggest that the miR-21/PDCD4/AP-1 autoregulatory loop is one of the main driving forces for hepatic fibrosis progression. Targeting this aberrantly activated feedback loop may provide a new therapeutic strategy and facilitate drug discovery against hepatic fibrosis.     

Overexpression of the OsPDCD5 Gene Induces Programmed Cell Death in Rice

The primary aim of the present study was to investigate the overexpression of the rice （Oryza sativa L.） programmed cell death 5 （OsPDCD5） gene in rice plant. Constitutive expression of OsPDCD5 from the cauliflower mosaic virus （CaMV） 35S promoter induced programmed cell death （PCD） in transgenic rice. Programmed cell death was accompanied by typical features, including inhibition of developmental growth, a reduction of fresh weight, degradation of total protein content, and fragmentation of genomic DNA. These results suggest that OsPDCD5 plays an essential role in the regulation of PCD in rice plants.     

毛竹茎秆纤维细胞发育过程中的细胞程序性死亡

利用TUNEL检测、细胞学及细胞化学方法,对毛竹茎秆纤维细胞发育过程中的细胞程序性死亡进行了研究。在次生壁形成的早期,纤维细胞出现染色质凝聚、细胞器膨胀、液泡膜解体和细胞质泡状化等典型的细胞程序性死亡形态学特征;TUNEL检测反应呈阳性,显示此时的纤维细胞核DNA发生了片段化。此时,在纤维细胞裂解的液泡膜、降解的细胞质和凝聚的染色质上具有ATPase活性。纤维细胞质的Ca^2＋水平会随着次生壁的形成而逐渐升高,随后Ca^2＋聚集成块状。在初生壁形成后期,纤维细胞染色质上的酸性磷酸酶（APase）活性增强。随着纤维次生壁的持续增厚,ATPase、酸性磷酸酶和Ca^2＋将在裂解的细胞质和凝聚的染色质上持续存在多年。结果表明,毛竹茎秆纤维细胞的次生壁形成过程是一个主动自溶的细胞程序性死亡过程。初生壁形成后期染色质上酸性磷酸酶活性增强及次生壁形成期胞质Ca^2＋的聚集,与纤维细胞的程序性死亡密切相关。ATPase,Ca^2＋和APase参与了纤维细胞程序性死亡过程中原生质体的降解。     

Analysis of Expression of Programmed Cell Death 1 Ligand 1 (PD-L1) in Malignant Pleural Mesothelioma (MPM)

Background

The increasing incidence and poor outcome associated with MPM requires finding effective treatment for this disease. PD1/PD-L1 pathway plays a central role in tumor immune evasion and appears to be predictive and prognostic marker. PD-L1 is expressed in many different human cancers but its role in MPM has yet to be established. The aim of this study is to evaluate the expression of PD-L1 in MPM.

Methods

119 MPM patients (p) from two institutions between November 2002 and February 2014 were reviewed. Formalin-fixed, paraffin-embedded tissue was stained with anti-PD-L1 (clone E1L3N). Cases showing more than 1% of tumor cells expression of PD-L1 were considered positive.

Results

PD-L1 was analyzed in 77 p with tumor tissue available and was positive in 20.7% p (14 samples in membrane, 16 in cytoplasm and 4 in immune infiltrate). PD-L1 intensity was weak in 56.2%, moderate in 25% and strong in 18.7% p. There was a significant relationship between PD-L1 expression and histology (PD-L1 expression 37.5% in no-epithelioid tumor and 13.2% in epithelioid; p=0.033). The median survival in p PD-L1 positive was 4.79 vs 16.3 months in p PD-L1 negative (p=0.012).

Conclusions

We have shown PD-L1 is expressed in 20% of patients, associated with no epithelioid histology and poor prognostic in MPM. Our results suggest PD-L1 warrants further exploration in selecting p for immunotherapy.