p53/CEP-1 increases or decreases lifespan, depending on level of mitochondrial bioenergetic stress

Mitochondrial pathologies underlie a number of life-shortening diseases in humans. In the nematode Caenorhabditis elegans, severely reduced expression of mitochondrial proteins involved in electron transport chain-mediated energy production also leads to pathological phenotypes, including arrested development and/or shorter life; in sharp contrast, mild suppression of these same proteins extends lifespan. In this study, we show that the C. elegans p53 ortholog cep-1 mediates these opposite effects. We found that cep-1 is required to extend longevity in response to mild suppression of several bioenergetically relevant mitochondrial proteins, including frataxin – the protein defective in patients with Friedreich's Ataxia. Importantly, we show that cep-1 also mediates both the developmental arrest and life shortening induced by severe mitochondrial stress. These findings support an evolutionarily conserved function for p53 in modulating organismal responses to mitochondrial dysfunction and suggest that metabolic checkpoint responses may play a role in longevity control and in human mitochondrial-associated diseases.     

Cis requirements for transposition of Tc1-like transposons in C. elegans

The Caenorhabditis elegans transposons Tc1 and Tc3 are able to transpose in heterologous systems such as human cell lines and zebrafish. Because these transposons might be useful vectors for transgenesis and mutagenesis of diverse species, we determined the minimal cis requirements for transposition. Deletion mapping of the transposon ends shows that fewer than 100 bp are sufficient for transposition of Tc3. Unlike Tc1, Tc3 has a second, internal transposase binding site at each transposon end. We found that these binding sites play no major role in the transposition reaction, since they can be deleted without reduction of the transposition frequency. Site-directed mutagenesis was performed on the conserved terminal base pairs at the Tc3 ends. The four terminal base pairs at the ends of the Tc3 inverted repeats were shown to be required for efficient transposition. Finally, increasing the length of the transposon from 1.9 kb to 12.5 kb reduced the transposition frequency by 20-fold, both in vivo and in vitro. Received: 21 April 1999 / Accepted: 10 June 1999     

线虫中的小分子热休克蛋白HSP12.1具有类分子伴侣活性

很多种类的小分子热休克蛋白(small heat shock protein,sHSP)都能在胁迫条件下抑制蛋白质的聚集,显示出了类分子伴侣活性,这种活性是ATP非依赖型的.从已经进行的实验发现,线虫C.elegans中最小的小分子热休克蛋白家族成员HSP12.1具有类分子伴侣活性,以胰岛素、乙醇脱氢酶和溶菌酶做底物发现HSP12.1能够一定程度地抑制底物的热聚集,虽然这种活性较一些经典的分子伴侣蛋白(线虫中的HSP16.1)要低.与此不同,另外3种和其分子质量相近的sHSP12s(HSP12.2、HSP12.3和HSP12.6)却没有检测出这样的类分子伴侣活性,虽然它们在一级结构上有很高的相似性.另外,在大肠杆菌中表达HSP12.1蛋白能够提高细菌在高温环境下的生存率,45℃处理后的生存率比未表达HSP12.1的菌高4倍左右,不过在线虫中是否发挥同样的功能还不是很清楚.从研究结果来看,C端“尾巴”结构域对sHSP发挥类分子伴侣活性不是必要的,在HSP12.1中没有C端“尾巴”结构域也有类分子伴侣活性就证明了这一点.N端结构域可能在发挥类分子伴侣活性中发挥比较重要的作用,当然α-crystallin结构域也可能参与到发挥这样的功能当中.     

Genome-wide analysis of germ cell proliferation in C.elegans identifies VRK-1 as a key regulator of CEP-1/p53

Proliferating germ cells in Caenorhabditiselegans provide a useful model system for deciphering fundamental mechanisms underlying the balance between proliferation and differentiation. Using gene expression profiling, we identified approximately 200 genes upregulated in the proliferating germ cells of C. elegans. Functional characterization using RNA-mediated interference demonstrated that over forty of these factors are required for normal germline proliferation and development. Detailed analysis of two of these factors defined an important regulatory relationship controlling germ cell proliferation. We established that the kinase VRK-1 is required for normal germ cell proliferation, and that it acts in part to regulate CEP-1(p53) activity. Loss of cep-1 significantly rescued the proliferation defects of vrk-1 mutants. We suggest that VRK-1 prevents CEP-1 from triggering an inappropriate cell cycle arrest, thereby promoting germ cell proliferation. This finding reveals a previously unsuspected mechanism for negative regulation of p53 activity in germ cells to control proliferation.     

综述: 秀丽线虫胰岛素类生长因子和雷帕霉素受体信号通路对衰老的调节作用

衰老表现为随着时间推移而带来的功能上的衰退和死亡率的上升.利用模式生物,研究人员已经证明,衰老受高度保守的信号通路所调控,而且遗传与环境因素的改变可以显著延长寿命并延缓功能上的衰退.作为一种模式生物,秀丽线虫由于其遗传操作的简单性以及基因组的高度保守性,已被广泛应用于现代生物学研究中.许多关于衰老的分子机理最初是在秀丽线虫中被阐明的.本文总结了秀丽线虫中高度保守的胰岛素类生长因子(IGF-1)和雷帕霉素受体(TOR)这两条信号通路调控衰老的研究进展,并对未来的研究方向展开了评述.     

Apurinic/apyrimidinic endonuclease 1, p53, and thioredoxin are linked in control of aging in C. elegans

Deletions in mitochondrial DNA (mtDNA) accumulate during aging. Expression of the Caenorhabditis elegans apurinic/apyrimidinic endonuclease 1 (APE1) ortholog exo‐3, involved in DNA repair, is reduced by 45% (P < 0.05) during aging of C. elegans. Suppression of exo‐3 by treatment with RNAi resulted in a threefold increase in mtDNA deletions (P < 0.05), twofold enhanced generation of reactive oxygen species (ROS) (P < 0.01), distortion of the structural integrity of the nervous system, reduction of head motility by 43% (P < 0.01) and whole animal motility by 38% (P < 0.05). Suppression of exo‐3 significantly reduced life span: mean life span decreased from 18.5 ± 0.4 to 15.4 ± 0.1 days (P < 0.001) and maximum life span from 25.9 ± 0.4 to 23.2 ± 0.1 days (P = 0.001). Additional treatment of exo‐3‐suppressed animals with a mitochondrial uncoupler decreased ROS levels, reduced neuronal damage, and increased motility and life span. Additional suppression of the C. elegans p53 ortholog cep‐1 in exo‐3 RNAi‐treated animals similarly decreased ROS levels, preserved neuronal integrity, and increased motility and life span. In wild‐type animals, suppression of cep‐1, involved in downregulation of exo‐3, increased expression of exo‐3 without a significant effect on ROS levels, preserved neuronal integrity, and increased motility and life span. Suppression of the C. elegans thioredoxin orthologs trx‐1 and trx‐2, involved in the redox chaperone activity of exo‐3, overrides the protective effect of cep‐1 RNAi treatment on neuronal integrity, neuronal function, mean and maximum life span. These results show that APE1/EXO‐3, p53/CEP‐1, and thioredoxin affect each other and that these interactions determine aging as well as neuronal structure and function.     

计算机视觉在线虫进食行为自动分析中的应用

秀丽隐杆线虫被广泛地用作研究基因与行为关系的绝佳模式生物.线虫的咽部神经元回路控制着复杂的进食行为.为了研究进食行为的分子机制,有必要对线虫进食行为表型分析鉴定.然而,目前为止,几乎所有的线虫进食行为表型鉴定都是通过人眼来判断.因为其泵入食物的肌肉运动频率高,该行为的分析是很困难而且效率低下的.为解决这个问题,我们设计了基于计算机视觉技术的自动化成像系统来高通量分析线虫进食行为表型.此成像系统对进食表型的检测准确率达到98%以上,并使得连续可靠地分析其表型细微变化成为可能.同时,在保证高准确率的前提下单位时间内分析数据的效率比人工分析提高了3倍.     

UNC-10在致密核心囊泡分泌过程中的作用

Rim是囊泡分泌活性区中的重要组成蛋白,它与细胞分泌和突触可塑性相关．在秀丽隐感线虫中只存在一种编码Rim的基因即unc-10．我们的研究发现,在线虫中Rim的基因突变unc-10(md1117)会导致致密核心囊泡的分泌缺陷．在活体中,unc-10突变虫系的神经多肽分泌显著下降．此外,在主要分泌致密核心囊泡的ALA神经元内,钙光解释放促发的快相分泌也比野生型减少．运用全内反射荧光显微成像技术,我们观察在unc-10缺失的情况下ALA 神经元中致密核心囊泡的锚定过程,结果显示在细胞膜附近停留的囊泡数目减少,表明囊泡锚定受到阻碍．上述试验结果表明,UNC-10能够影响致密核心囊泡的分泌过程,其机制可能是影响了囊泡的锚定过程．     

An expression screen for RhoGEF genes involved in C. elegans gonadogenesis

The gonad in Caenorhabditis elegans is an important model system for understanding complex morphogenetic processes including cellular movement, cell fusion, cell invasion and cell polarity during development. One class of signaling proteins known to be critical for the cellular events underlying morphogenesis is the Rho family GTPases, particularly RhoA, Rac and Cdc42. In C. elegans orthologues of these genes have been shown to be important for gonad development. In our current study we have extended those findings by examining the patterns of 5′ cis-regulatory element (5′CRE) activity associated with nineteen putative guanine nucleotide exchange factors (GEFs) encoded by the C. elegans genome predicted to activate Rho family GTPases. Here we identify 13 RhoGEF genes that are expressed during gonadogenesis and characterize the cells in which their 5′CREs are active. These data provide the basis for designing experiments to examine Rho GTPase activation during morphogenetic processes central to normal gonad development.     

Expression of a Homeostatic Regulator, Wip1 (Wild-type p53-induced Phosphatase), Is Temporally Induced by c-Jun and p53 in Response to UV Irradiation

Wild-type p53-induced phosphatase (Wip1) is induced by p53 in response to stress, which results in the dephosphorylation of proteins (i.e. p38 MAPK, p53, and uracil DNA glycosylase) involved in DNA repair and cell cycle checkpoint pathways. p38 MAPK-p53 signaling is a unique way to induce Wip1 in response to stress. Here, we show that c-Jun directly binds to and activates the Wip1 promoter in response to UV irradiation. The binding of p53 to the promoter occurs earlier than that of c-Jun. In experiments, mutation of the p53 response element (p53RE) or c-Jun consensus sites reduced promoter activity in both non-stressed and stressed A549 cells. Overexpression of p53 significantly decreased Wip1 expression in HCT116 p53^+/+ cells but increased it in HCT116 p53^−/− cells. Adenovirus-mediated p53 overexpression greatly decreased JNK activity. Up-regulation of Wip1 via the p38 MAPK-p53 and JNK-c-Jun pathways is specific, as demonstrated by our findings that p38 MAPK and JNK inhibitors affected the expression of the Wip1 protein, whereas an ERK inhibitor did not. c-Jun activation occurred much more quickly, and to a greater extent, in A549-E6 cells than in A549 cells, with delayed but fully induced Wip1 expression. These data indicate that Wip1 is activated via both the JNK-c-Jun and p38 MAPK-p53 signaling pathways and that temporal induction of Wip1 depends largely on the balance between c-Jun and p53, which compete for JNK binding. Moreover, our results suggest that JNK-c-Jun-mediated Wip1 induction could serve as a major signaling pathway in human tumors in response to frequent p53 mutation.     

Kinetic properties of p53 phosphorylation by the human vaccinia-related kinase 1

The vaccinia-related kinase 1 (VRK1) protein is a nuclear Ser-Thr kinase that phosphorylates p53 in Thr18. We have determined the enzyme properties regarding its different substrates. VRK1 has a high affinity for ATP (K(m) 50 microM) and is thus saturated by the intracellular concentration of ATP in vivo. VRK1 uses preferentially magnesium, but is also functional with manganese and zinc. The VRK1 protein is autophosphorylated in multiple residues without effect on its activity. One autophosphorylated residue, T355, is within the VRK1 regulatory carboxy terminus. The kinase phosphorylates p53 with a K(m) of 1 microM and is well suited to respond to the variations of intracellular p53 concentration, which fluctuates as a response to different types of cellular stress.     

Hepatic stellate cell promoted hepatoma cell invasion via the HGF/c-Met signaling pathway regulated by p53

The biological behaviors of hepatocellular carcinoma (HCC) are complex mainly due to heterogeneity of progressive genetic and epigenetic mutations as well as tumor environment. Hepatocyte growth factor (HGF)/c-Met signaling pathway is regarded to be a prototypical example for stromal-epithelial interactions during developmental morphogenesis, wound healing, organ regeneration and cancer progression. And p53 plays as an important regulator of Met-dependent cell motility and invasion. Present study showed that 2 HCC cell lines, Hep3B and HepG2, displayed different invasive capacity when treated with HGF which was secreted by hepatic stellate cells (HSCs). We found that HGF promoted Hep3B cells invasion and migration as well as epithelial-mesenchymal transition (EMT) occurrence because Hep3B was p53 deficient, which leaded to the c-Met over-expression. Then we found that HGF/c-Met promoted Hep3B cells invasion and migration by upregulating Snail expression. In conclusion, HGF/c-Met signaling is enhanced by loss of p53 expression, resulting in increased ability of invasion and migration by upregulating the expression of Snail.     

Retrofitting ampicillin resistant vectors by recombination for use in generating C. elegans transgenic animals by bombardment

Caenorhabditis elegans is an important model organism for modern biologic research. An essential aspect of C. elegans research is the production of transgenic animals for study. These are often generated via microinjection, but biolistic bombardment has become increasingly popular. However, many of the plasmids previously generated for use in microinjection are not readily used for bombardment due to the lack of a convenient marker. The unc-119 gene is often used as a marker since unc-119 rescue can be observed at low magnification, allowing rescued animals to be easily distinguished from the larger number of non-rescued animals. Here we report the use of homologous recombination in Escherichia coli as a method to insert a cassette containing the unc-119 gene into commonly used plasmids at the site of the ampicillin resistance gene which is simpler than other methods like subcloning. These cassettes are flanked by regions homologous to the 5′ and 3′ ends of the ampicillin resistance gene and contain either the unc-119 gene and the kanamycin resistance gene or a unc-119:mCherry fusion gene and the kanamycin resistance gene. The resulting plasmids may be used for biolistic bombardment to yield animals that display unc-119 rescue, and also express the recipient plasmid transgene.     

A novel in vivo post-translational modification of p53 by PARP-1 in MPTP-induced parkinsonism

Sporadic Parkinson's disease (PD) affects primarily dopaminergic neurons of the substantia nigra pars compacta. There is evidence of necrotic and apoptotic neuronal death in PD, but the mechanisms behind selected dopaminergic neuronal death remain unknown. The tumor suppressor protein p53 functions to selectively destroy stressed or abnormal cells during life and development by means of necrosis and apoptosis. Activation of p53 leads to death in a variety of cells including neurons. p53 is a target of the nuclear enzyme Poly(ADP-ribose)polymerase (PARP), and PARP is activated following DNA damage that occurs following 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced neurotoxicity. MPTP is the favored in vivo model of PD, and reproduces the pathophysiology, anatomy and biochemistry of PD. p53 protein normally exhibits a fleeting half-life, and regulation of p53 stability and activation is achieved mainly by post-translational modification. We find that p53 is heavily poly(ADP-ribosyl)ated by PARP-1 following MPTP intoxication. This post-translational modification serves to stabilize p53 and alters its transactivation of downstream genes. These influences of PARP-1 on p53 may underlie the mechanisms of MPTP-induced parkinsonism and other models of neuronal death.