ERM Proteins and Cdk5 in Cellular Senescence

Cellular senescence is a tumor-suppressive process instigated by proliferation in the absence of telomere replication, by cellular stresses such as oncogene activation, or by activation of the retinoblastoma tumor suppressor protein, pRb. This process is characterized by an irreversible cell cycle exit, a unique morphology, and expression of senescence-associated-b-galactosidase (SA-b-gal). Despite the potential biological importance of cellular senescence, little is known of the mechanisms leading to the senescent phenotype. We have recently discovered that expression of active pRb induces expression and altered localization of the ERM family member ezrin, an actin-binding protein involved in membrane-cytoskeletal signaling. pRb expression results in the stimulation of cdk5-mediated phosphorylation of ezrin with subsequent membrane association and induction of cell shape changes, linking pRb activity to cytoskeletal regulation in senescent cells. Cdk5 activity increases in senescing cells and is required for expression of SA-b-gal and for actin polymerization accompanying acquisition of the senescent morphology. These results begin to illuminate the mechanisms underlying induction of senescence and the senescent shape change and describe new pathways that may contribute to the ability of senescent cells to influence tumor growth.     

核因子Y在细胞衰老中的作用

核因子Ｙ(ｎｕｃｌｅａｒｆａｃｔｏｒＹ ,ＮＦ Ｙ)也称作ＣＢＦ(ＣＣＡＡＴｂｏｘｂｉｎｄｉｎｇｆａｃｔｏｒ)、ＣＰ1 ,是结合ＣＣＡＡＴ盒的转录因子。它能识别真核基因启动子区域的 5′ ＣＴＧＡＴＴＧＧＹＹＲＲ 3′或 5′ ＹＹＲＲＣＣＡＡＴＣＡＧ 3′共有序列。有功能的ＮＦ Ｙ是一个异源三聚体蛋白 ,它包含三个不同的亚单位Ａ、Ｂ、Ｃ。细胞衰老是由细胞分裂的次数决定的。控制细胞分裂的机制之一是调控Ｇ1 /Ｓ期的基因表达。当细胞衰老时 ,由于Ｇ1 /Ｓ基因调控的转变 ,细胞不能进入Ｓ期而只停留在Ｇ1期。ＮＦ Ｙ正是通过与Ｇ1 /Ｓ…     

Characterization of Cytoplasmic and Nuclear Mutations Affecting Chlorophyll and Chlorophyll-Binding Proteins during Senescence in Soybean

Soybean plants (Glycine max [L.] Merr. cv Clark) carrying nuclear and cytoplasmic “stay-green” mutations, which affect senescence, were examined. Normally, the levels of chlorophyll (Chl) a and b decline during seedfill and the Chl a/b ratio decreases during late pod development in cv Clark. Plants homozygous for both the d₁ and d₂ recessive alleles, at two different nuclear loci, respectively, retained most (64%) of their Chl a and b and exhibited no change in their Chl a/b ratio. Combination of G (a dominant nuclear allele in a third locus causing only the seed coat to stay green during senescence) with d₁d₂ further inhibited the loss of Chl in the leaf. Whereas the thylakoid proteins seem to be degraded in normal Clark leaves during late pod development, they were not substantially diminished in d₁d₂ and Gd₁d₂ leaves. In plants carrying a cytoplasmic mutation, cytG, Chl declined in parallel with normal cv Clark; however, the cytG leaves had a much higher level of Chl b, and somewhat more Chl a, remaining at abscission, enough to color the leaves green. In cytG, most thylakoid proteins were degraded, but the Chl a/b-binding polypeptides of the light-harvesting complex in photosystem II (LHCII), and their associated Chl a and b molecules, were not. Thus, the combination of d₁ and d₂ causes broad preservation of the thylakoid proteins, whereas cytG appears to selectively preserve LHCII. The cytG mutation may be useful in elucidating the sequence of events involved in the degradation of LHCII proteins and their associated pigments during senescence.     

Tissue-Specific Distribution of DNA-Binding Nuclear Proteins

Abstract. The DNA-binding capacity of nuclear proteins of mouse cells was examined by the protein-blotting method. Under conditions in which the lac repressor specifically binds to the lac operator, the DNA-binding nuclear proteins from different tissues showed a tissue-specific distribution, suggesting that the species and amounts of nuclear proteins with DNA binding activity differ in different tissues.
When cloned eukaryotic genes were used for binding, eukaryotic DNA showed stronger binding than prokaryotic DNA. Competition experiments suggested that many nuclear proteins have different DNA binding properties from that of the prokaryotic repressor.     

The N-Terminal Methionine of Cellular Proteins as a Degradation Signal

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Degradation of Specific Nuclear Proteins Occurs in the Cytoplasm in Saccharomyces cerevisiae

The ubiquitin/proteasome system has been characterized extensively, although the site of nuclear substrate turnover has not been established definitively. We report here that two well-characterized nuclear proteins are stabilized in nuclear export mutants in Saccharomyces cerevisiae. The requirement for nuclear export defines a new regulatory step in intracellular proteolysis.     

Calcium- and Calmodulin-Promoted Phosphorylation of Membrane Proteins during Senescence in Apples

Sodium dodecylsulfate-polyacrylamide gel electrophoresis ofmicrosomal membrane proteins from post-climacteric apples atan early and an advanced stage of senescence showed only slightqualitative changes in the protein pattern. Though there wasa 30% reduction in the total microsomal protein content in applesat an advanced stage of senescence, a polypeptide with 18,000molecular weight increased in quantity during senescence. Invitro phosphorylation of several proteins was promoted by calciumin membranes from apples at an early stage of senescence. Phosphorylationof proteins with molecular weights of 95,000, 91,000, 53,000and 50,000 was promoted by calcium and calmodulin. Phosphorylationof these proteins increased with increasing calcium concentration.Proteins with molecular weights of 53,000 and 50,000 showedmarked promotion of phosphorylation over the calcium-promotedlevel when the amount of calmodulin in the assay mixture wasincreased. Calcium- and calmodulin-promoted phosphorylationof membrane proteins showed considerable decrease when the appleswere at an advanced stage of senescence. Moreover, increasingthe concentrations of calcium and calmodulin in the assay mixturedid not have any promoting effect on the phosphorylation ofthese proteins. Phosphoprotein phosphatase activity as measuredby the loss of label from phosphorylated proteins followingchase with cold ATP, did not differ to a great extent in membranepreparations from normal and senesced apples. Hydrolysis ofATP by senesced apple membrane preparation, however, was foundto be relatively higher. The significance of these observationsin relation to senescence is discussed. ¹ Scientific Paper No. 7084, College of Agriculture and HomeEconomics, Washington State University, Pullman, Project 0321.² Supported in part by grants from the Washington State TreeFruit Research Commission, and National Science Foundation GrantPCM-8208408.     

Thermosensitivity of Nuclear RNA Polymerase during the in vitro Senescence of Mouse Fibroblasts

Embryonic mouse fibroblasts divide approximately twelve times in vitro prior to cessation of mitotic activity. During this period of cellular senescence the thermosensitivity of the RNA polymerase activity of isolated nuclei has been examined as a means of detecting the possible accumulation of defective enzyme molecules, as has been found by other workers for several cytoplasmic enzymes during the ageing of human fibroblasts in vitro.
The total RNA polymerase activity of nuclei isolated from old (10th generation) cells is more thermoresistant than that of young (2nd generation) cells. However, the net RNA polymerase activity of nuclei from non-dividing (confluent) cells is more thermoresistant than that of exponentially growing cells of the same age. When allowance is made for the state of growth of the cultures, little difference is seens in the thermosensitivity of the activities of nuclei from old and young cells. Neither is there any difference between the thermosensitivity of the net activity of an established line of murine fibroblasts (L-cells) and cells in primary culture.
Preheating nuclei increases the inhibition of their total RNA polymerase activity by or-α-amanitin, indicating that RNA polymerase II is the most heat resistance species present. There appears to be no difference between the thermosensitivity of the α-amanitin sensitive and resistance species of the enzyme in the nuclei of old and young cells.
It is concluded that old cells resemble non-dividing young cells in containing a higher proportion of RNA polymerase II in their nuclei, resulting in greater thermoresistance of the total RNA polymerase activity over that of exponentially growing cells. However, there appears to be no increase in thermosensitivity of the enzymes with age.     

An Inducible System for Rapid Degradation of Specific Cellular Proteins Using Proteasome Adaptors

A common way to study protein function is to deplete the protein of interest from cells and observe the response. Traditional methods involve disrupting gene expression but these techniques are only effective against newly synthesized proteins and leave previously existing and stable proteins untouched. Here, we introduce a technique that induces the rapid degradation of specific proteins in mammalian cells by shuttling the proteins to the proteasome for degradation in a ubiquitin-independent manner. We present two implementations of the system in human culture cells that can be used individually to control protein concentration. Our study presents a simple, robust, and flexible technology platform for manipulating intracellular protein levels.     

Delay of Membrane Lipid Degradation by Calcium Treatment during Cabbage Leaf Senescence

Cabbage leaf discs (Brassica oleracea L., Capitata group) were floated adaxial side up in 0, 0.05, or 0.25 m CaCl₂ solutions at 15°C for 14 d in the dark. To assess whether the delay of senescence by calcium treatment involved protection of membrane lipids, chlorophyll and protein content and the lipid composition of the membranes were determined during incubation. Chlorophyll and protein content decreased with time, in correlation with a reduction in the amount of phospholipids. The degree of unsaturation of phospholipids and free fatty acids decreased, whereas the ratio of sterol to phospholipid increased. The proportions of phospholipid classes did not change during senescence. The catabolism of phospholipids was delayed by 0.05 m calcium, but accelerated by 0.25 m, as compared to the untreated control. Based on the levels of the lipid intermediates, phospholipase D, phosphatidic acid phosphatase, lipolytic acyl hydrolase, and lipoxygenase appeared to be involved in the breakdown of phospholipids during senescence. Phospholipase D and phosphatidic acid phosphatase may be directly influenced by calcium. The calcium treatment apparently did not affect the activity of acyl hydrolase. Lipoxygenase, responsible for the peroxidation of the polyunsaturated fatty acids, was probably indirectly influenced by calcium. We conclude that the delay of senescence of cabbage leaf discs by calcium treatment involved protection of membrane lipids from degradation.     

Cellular Distribution of Calmodulin and Calmodulin-Binding Proteins in Vicia faba L.

The distribution of calmodulin (CaM) and CaM-binding proteins within Vicia faba was investigated. Both CaM and CaM-binding proteins were found to be differentially distributed among organs, tissues, and protoplast types. CaM levels, on a per protein basis, were found to be the highest in leaf epidermis, containing 3-fold higher levels of CaM than in total leaf. Similarly, guard cell and epidermal cell protoplasts were also found to have higher levels of CaM than mesophyll cell protoplasts. ¹²⁵I-CaM blot overlay assays were performed to qualitatively examine CaM-binding proteins in these protoplast types as well as in whole tissues and organs. CaM-binding proteins with M_r 52,000, 78,000, and 115,000 were common in all metabolically active plant parts. Unique CaM-binding protein bands were detected in guard cell protoplasts (M_r 39,000, 88,000), stems (M_r 45,000, 60,000, 64,000), and roots (M_r 62,000), suggesting the presence of specialized CaM-dependent processes in these cells and organs.     

Arabidopsis STAY-GREEN2 Is a Negative Regulator of Chlorophyll Degradation during Leaf Senescence

Chlorophyll （Chl） degradation causes leaf yellowing during senescence or under stress conditions. For Chl breakdown, STAY-GREEN1 （SGR1） interacts with Chl catabolic enzymes （CCEs） and light-harvesting complex II （LHCII） at the thylakoid membrane, possibly to allow metabolic channeling of potentially phototoxic Chl breakdown intermediates. Among these Chl catabolic components, SGR1 acts as a key regulator of leaf yellowing. In addition to SGR1 （At4g22920）, the Arabidopsis thaliana genome contains an additional homolog, SGR2 （At4g11910）, whose biological function remains elusive. Under senescence-inducing conditions, SGR2 expression is highly up-regulated, similarly to SGR1 expression. Here we show that SGR2 function counteracts SGR1 activity in leaf Chl degradation; SGR2-overexpressing plants stayed green and the sgr2-1 knockout mutant exhibited early leaf yellowing under age-, dark-, and stress-induced senescence conditions. Like SGR1, SGR2 interacted with LHCII but, in contrast to SGR1, SGR2 interactions with CCEs were very limited. Furthermore, SGR1 and SGR2 formed homo- or heterodimers, strongly suggesting a role for SGR2 in negatively regulat- ing Chl degradation by possibly interfering with the proposed CCE-recruiting function of SGR1. Our data indicate an antagonistic evolution of the functions of SGR1 and SGR2 in Arabidopsis to balance Chl catabolism in chloroplasts with the dismantling and remobilizing of other cellular components in senescing leaf cells.     

Translatable mRNAs for Chloroplast-Targeted Proteins in Detached Radish Cotyledons during Senescence in Darkness

A protein-import system prepared with isolated chloroplastswas used to monitor changes in levels of mRNAs for chloroplast-targetedproteins during dark-induced leaf senescence. Biologically activechloroplasts were isolated from young (9-day-old) and aged (14-day-old)radish cotyledons. Poly(A)⁺-RNA was prepared from radish cotyledonsthat had been detached from seedlings and placed in darknessto accelerate senescence. The RNA was translated in a wheatgerm system, and the products were added to an import systemprepared with chloroplasts from young cotyledons. Electrophoreticanalysis of the imported proteins suggested that most chloroplast-targeted proteins decreased in abundance during dark treatmentof cotyledons. However, the relative abundance of 38 stromaland three thylakoid proteins increased transiently or continuouslyamong the products of translation of RNA isolated during thecourse of senescence. The efficiency of the uptake of precursorproteins by chloroplasts isolated from aged cotyledons was lowerthan that by chloroplasts from young tissue. The chloroplastsfrom aged cotyledons more efficiently imported at least onestromal protein and one thylakoid protein than chloroplastsfrom the young tissue. The relative abundance of these two proteinsincreased among the products of translation of RNA from senescingcotyledons when tested in the uptake system with chloroplastsfrom young cotyledons. These results suggest that some nucleargenes for chloroplast-targeted proteins are expressed in senescingcotyledons more efficiently than in young tissue, and that themachinery for import of proteins into chloroplasts changes duringaging of the tissue to allow more efficient import of certainproteins that may be responsible for the senescence of the chloroplasts. ¹Present address: Kihara Institute for Biological Research,Yokohama City University, Mutsukawa 3-122-20, Minami-ku, Yokohama,232 Japan     

Co-Crystallization with Conformation-Specific Designed Ankyrin Repeat Proteins Explains the Conformational Flexibility of BCL-W

BCL-W is a member of the BCL-2 family of anti-apoptotic proteins. A key event in the regulation of apoptosis is the heterodimerization between anti-apoptotic and pro-apoptotic family members, which involves a conserved surface-exposed groove on the anti-apoptotic proteins. Crystal structures of the ligand binding-competent conformation exist for all anti-apoptotic family members, with the exception of BCL-W, due to the flexibility of the BCL-W groove region. Existing structures had suggested major deviations of the BCL-W groove region from the otherwise structurally highly related remaining anti-apoptotic family members. To capture its ligand binding-competent conformation by counteracting the conformational flexibility of the BCL-W groove, we had selected high-affinity groove-binding designed ankyrin repeat proteins (DARPins) using ribosome display. We now determined two high-resolution crystal structures of human BCL-W in complex with different DARPins at resolutions 1.5 and 1.85 Å, in which the structure of BCL-W is virtually identical, and BCL-W adopts a conformation extremely similar to the ligand-free conformation of its closest relative BCL-XL in both structures. However, distinct differences to all previous BCL-W structures are evident, notably in the ligand-binding region. We provide the first structural explanation for the conformational flexibility of the BCL-W groove region in comparison to other BCL-2 family members. Due to the importance of the anti-apoptotic BCL-2 family as drug targets, the presented crystal structure of ligand binding-competent BCL-W may serve as a valuable basis for structure-based drug design in the future and provides a missing piece for the structural characterization of this protein family.     

Distinct Roles for Key Karyogamy Proteins during Yeast Nuclear Fusion

During yeast mating, cell fusion is followed by the congression and fusion of the two nuclei. Proteins required for nuclear fusion are found at the surface (Prm3p) and within the lumen (Kar2p, Kar5p, and Kar8p) of the nuclear envelope (NE). Electron tomography (ET) of zygotes revealed that mutations in these proteins block nuclear fusion with different morphologies, suggesting that they act in different steps of fusion. Specifically, prm3 zygotes were blocked before formation of membrane bridges, whereas kar2, kar5, and kar8 zygotes frequently contained them. Membrane bridges were significantly larger and occurred more frequently in kar2 and kar8, than in kar5 mutant zygotes. The kinetics of NE fusion in prm3, kar5, and kar8 mutants, measured by live-cell fluorescence microscopy, were well correlated with the size and frequency of bridges observed by ET. However the kar2 mutant was defective for transfer of NE lumenal GFP, but not diffusion within the lumen, suggesting that transfer was blocked at the NE fusion junction. These observations suggest that Prm3p acts before initiation of outer NE fusion, Kar5p may help dilation of the initial fusion pore, and Kar2p and Kar8p act after outer NE fusion, during inner NE fusion.     

辐射诱导细胞衰老的机制研究进展

辐射诱导细胞衰老属于胁迫诱导的早熟性衰老。研究表明在外界应激条件下,衰老的细胞会出现肿胀、增殖减弱、周期受阻、β-半乳糖苷酶活性增强、p53-p21和p16-RB信号通路以及调控衰老的miRNAs被激活等特点。本文综述了辐射诱导细胞衰老的过程,并着重介绍了细胞衰老的两条信号通路p53-p21和p16-RB以及miRNAs对细胞的调控,为进一步的基础研究提供理论参考。     

Resistance to Degradation and Cellular Distribution are Important Features for the Antitumor Activity of Gomesin

Many reports have shown that antimicrobial peptides exhibit anticancer abilities. Gomesin (Gm) exhibits potent cytotoxic activity against cancer cells by a membrane pore formation induced after well-orchestrated intracellular mechanisms. In this report, the replacements of the Cys by Ser or Thr, and the use D-amino acids in the Gm structure were done to investigate the importance of the resistance to degradation of the molecule with its cytotoxicity. [Thr^2,6,11,15]-Gm, and [Ser^2,6,11,15]-Gm exhibits low cytotoxicity, and low resistance to degradation, and after 24 h are present in localized area near to the membrane. Conversely, the use of D-amino acids in the analogue [D-Thr^2,6,11,15]-D-Gm confers resistance to degradation, increases its potency, and maintained this peptide spread in the cytosol similarly to what happens with Gm. Replacements of Cys by Thr and Gln by L- or D-Pro ([D-Thr^2,6,11,15, Pro⁹]-D-Gm, and [Thr^2,6,11,15, D-Pro⁹]-Gm), which induced a similar β-hairpin conformation, also increase their resistance to degradation, and cytotoxicity, but after 24 h they are not present spread in the cytosol, exhibiting lower cytotoxicity in comparison to Gm. Additionally, chloroquine, a lysosomal enzyme inhibitor potentiated the effect of the peptides. Furthermore, the binding and internalization of peptides was determined, but a direct correlation among these factors was not observed. However, cholesterol ablation, which increase fluidity of cellular membrane, also increase cytotoxicity and internalization of peptides. β-hairpin spatial conformation, and intracellular localization/target, and the capability of entry are important properties of gomesin cytotoxicity.