Apoptotic Cleavage of Rabaptin-5-like Proteins and a Model for Rabaptin-5 Inactivation in Apoptosis

Intracellular membrane transport from the plasma membrane is one of the processes affected in apoptotic cells. Apoptotic inhibition of endosomal transport occurs due to cleavage of Rabaptin-5, an effector of small GTPase Rab5, which results in inhibition of early endosome fusion. Recently several novel Rabaptin-5-like proteins were identified. We investigated whether Rabaptin-5-like proteins, Rabaptin-5? and Rabaptin-5?, are also cleaved in apoptosis and found that both proteins are cleaved in apoptotic cell extracts by caspase-3-related proteases. This suggests that functional inactivation of these proteins is necessary for apoptotic cell death. We also mapped a novel, N-terminal, putative Rab5 binding site in Rabaptin-5-like proteins, which becomes physically separated from the previously known C-terminal Rab5 binding site after apoptotic cleavage of these proteins. Presence of the second Rab5 binding site provides a new insight into Rabaptin-5 function in early endosome fusion and a mechanistic model for functional inactivation of Rabaptin-5 in apoptosis.     

Caspase Cleavage of Keratin 18 and Reorganization of Intermediate Filaments during Epithelial Cell Apoptosis

Keratins 8 (K8) and 18 (K18) are major components of intermediate filaments (IFs) of simple epithelial cells and tumors derived from such cells. Structural cell changes during apoptosis are mediated by proteases of the caspase family. During apoptosis, K18 IFs reorganize into granular structures enriched for K18 phosphorylated on serine 53. K18, but not K8, generates a proteolytic fragment during drug- and UV light–induced apoptosis; this fragment comigrates with K18 cleaved in vitro by caspase-6, -3, and -7. K18 is cleaved by caspase-6 into NH₂-terminal, 26-kD and COOH-terminal, 22-kD fragments; caspase-3 and -7 additionally cleave the 22-kD fragment into a 19-kD fragment. The cleavage site common for the three caspases was the sequence VEVD/A, located in the conserved L1-2 linker region of K18. The additional site for caspases-3 and -7 that is not cleaved efficiently by caspase-6 is located in the COOH-terminal tail domain of K18. Expression of K18 with alanine instead of serine at position 53 demonstrated that cleavage during apoptosis does not require phosphorylation of serine 53. However, K18 with a glutamate instead of aspartate at position 238 was resistant to proteolysis during apoptosis. Furthermore, this cleavage site mutant appears to cause keratin filament reorganization in stably transfected clones. The identification of the L1-2 caspase cleavage site, and the conservation of the same or very similar sites in multiple other intermediate filament proteins, suggests that the processing of IFs during apoptosis may be initiated by a similar caspase cleavage.     

Iron-Dependent Oxidative Inactivation with Affinity Cleavage of Pyruvate Kinase

Treatment of rabbit muscle pyruvate kinase with iron/ascorbate caused an inactivation with the cleavage of peptide bond. The inactivation or fragmentation of the enzyme was prevented by addition of Mg²⁺, catalase, and mannitol, but ADP and PEP the substrates did not show any effect. Protective effect of catalase and mannitol suggests that hydroxyl radical produced through the ferrous ion-dependent reduction of oxygen is responsible for the inactivation/fragmentation of the enzyme. SDS-PAGE and TOF-MS analysis confirmed five pairs of fragments, which were determined to result from the cleavage of the Lys114-Gly115, Glu117-Ile118, Asp177-Gly178, Gly207-Val208, and Phe243-Ile244 bonds of the enzyme by amino-terminal sequencing analysis. Protection of the enzyme by Mg²⁺ implies the identical binding sites of Fe²⁺ and Mg²⁺, but the cleavage sites were discriminated from the cofactor Mg²⁺-binding sites. Considering amino acid residues interacting with metal ions and tertiary structure, Fe²⁺ ion may bind to Asp177 neighboring to Gly207 and Glu117 neighboring to Lys114 and Phe243, causing the peptide cleavage by hydroxyl radical. Iron-dependent oxidative inactivation/fragmentation of pyruvate kinase can explain the decreased glycolytic flux under aerobic conditions. Intracellular free Mg²⁺ concentrations are responsible for the control of cellular respiration and glycolysis.     

Cleavage of the Junin Virus Nucleoprotein Serves a Decoy Function To Inhibit the Induction of Apoptosis during Infection

The regulation of apoptosis during infection is an important factor for host survival and, in some cases, also for the virus life cycle. At the same time, mechanisms to prevent the induction of apoptosis have been observed in numerous viral pathogens, but until now the role of apoptosis during arenavirus infection has not been investigated. Junin virus (JUNV) belongs to the New World arenavirus serogroup of the Arenaviridae and is the causative agent of Argentine hemorrhagic fever. We have demonstrated that infection with JUNV in cell culture does not induce apoptosis but leads to cleavage of the nucleoprotein (NP) into discrete products resembling caspase cleavage events. Similar specific NP degradation patterns were also observed in NP-transfected cell lines, and a closer examination of the sequence of NP showed several putative caspase cleavage motifs. Point mutations that abolished these cleavage motifs were consistent with the loss of certain cleavage products. Consistent with these data, further studies showed that treatment with a caspase inhibitor also reduced NP cleavage, indicating that the observed cleavage events were occurring as a result of caspase activity with NP as a substrate. Finally, we showed that expression of NP suppresses the cleavage of caspase 3 in cells treated with an apoptosis activator. Based on these findings, we propose that NP functions as a decoy substrate for caspase cleavage in order to inhibit the induction of apoptosis in JUNV-infected cells.     

A Potential Association between Helicobacter pylori CagA EPIYA and Multimerization Motifs with Cytokeratin 18 Cleavage Rate during Early Apoptosis

Background and Aims: Helicobacter pylori is a highly diverse pathogen, which encounters epithelial cells as the initial defense barrier during its lifelong infection. The structure of epithelial cells can be disrupted through cleavage of microfilaments. Cytokeratin 18 (CK18) is an intermediate filament, the cleavage of which is considered an early event during apoptosis following activation of effector caspases. Methods: Helicobacter pylori strains were isolated from 76 dyspeptic patients. cagA 3’ variable region and CagA protein status were analyzed by PCR and western blotting, respectively. Eight hours post‐co‐culture of AGS cells with different H. pylori strains, flow cytometric analysis was performed using M30 monoclonal antibody specific to CK18 cleavage‐induced neo‐epitope. Results: Higher rates of CK18 cleavage were detected during co‐culture of AGS cells with H. pylori strains bearing greater numbers of cagA EPIYA‐C and multimerization (CM) motifs. On the other hand, H. pylori strains with greater numbers of EPIYA‐B relative to EPIYA‐C demonstrated a decrease in CK18 cleavage rate. Thus, H. pylori‐mediated cleavage of CK18 appeared proportional to the number of CagA EPIYA‐C and CM motifs, which seemed to be downplayed in the presence of EPIYA‐B motifs. Conclusions: Our observation associating the heterogeneity of cagA variants with the potential of H. pylori strains in the induction of CK18 cleavage as an early indication of apoptosis in gastric epithelial cells supports the fact that apoptosis may be a type‐specific trait. However, additional cagA‐targeted experiments are required to clearly identify the role of EPIYA and CM motifs in apoptosis and/or the responsible effector molecules.     

Molecular Basis of Calpain Cleavage and Inactivation of the Sodium-Calcium Exchanger 1 in Heart Failure

Cardiac sodium (Na⁺)-calcium (Ca²⁺) exchanger 1 (NCX1) is central to the maintenance of normal Ca²⁺ homeostasis and contraction. Studies indicate that the Ca²⁺-activated protease calpain cleaves NCX1. We hypothesized that calpain is an important regulator of NCX1 in response to pressure overload and aimed to identify molecular mechanisms and functional consequences of calpain binding and cleavage of NCX1 in the heart. NCX1 full-length protein and a 75-kDa NCX1 fragment along with calpain were up-regulated in aortic stenosis patients and rats with heart failure. Patients with coronary artery disease and sham-operated rats were used as controls. Calpain co-localized, co-fractionated, and co-immunoprecipitated with NCX1 in rat cardiomyocytes and left ventricle lysate. Immunoprecipitations, pull-down experiments, and extensive use of peptide arrays indicated that calpain domain III anchored to the first Ca²⁺ binding domain in NCX1, whereas the calpain catalytic region bound to the catenin-like domain in NCX1. The use of bioinformatics, mutational analyses, a substrate competitor peptide, and a specific NCX1-Met³⁶⁹ antibody identified a novel calpain cleavage site at Met³⁶⁹. Engineering NCX1-Met³⁶⁹ into a tobacco etch virus protease cleavage site revealed that specific cleavage at Met³⁶⁹ inhibited NCX1 activity (both forward and reverse mode). Finally, a short peptide fragment containing the NCX1-Met³⁶⁹ cleavage site was modeled into the narrow active cleft of human calpain. Inhibition of NCX1 activity, such as we have observed here following calpain-induced NCX1 cleavage, might be beneficial in pathophysiological conditions where increased NCX1 activity contributes to cardiac dysfunction.     

Estrogen-Dependent Proteolytic Cleavage of Semaphorin 4D and Plexin-B1 Enhances Semaphorin 4D-Induced Apoptosis during Postnatal Vaginal Remodeling in Pubescent Mice

Around the fifth week after birth, the vaginal cavity in female mouse pups opens to the overlaying skin. This postnatal tissue remodeling of the genital tract occurs during puberty, and it largely depends upon hormonally induced apoptosis that mainly occurs in the epithelium at the lower part of the mouse vaginal cavity. Previously, we showed that most BALB/c mice lacking the class IV Semaphorin (Sema4D) develop imperforate vagina and hydrometrocolpos; therefore, we reasoned that the absence of Sema4D-induced apoptosis in vaginal epithelial cells may cause the imperforate vagina. Sema4D signals via the Plexin-B1 receptor; nevertheless detailed mechanisms mediating this hormonally triggered apoptosis are not fully documented. To investigate the estrogen-dependent control of Sema4D signaling during the apoptosis responsible for mouse vaginal opening, we examined structural and functional modulation of Sema4D, Plexin-B1, and signaling molecules by analyzing both wild-type and Sema4D−/− mice with or without ovariectomy. Both the release of soluble Sema4D and the conversion of Plexin-B1 by proteolytic processing in vaginal tissue peaked 5 weeks after birth of wild-type BALB/c mice at the time of vaginal opening. Estrogen supplementation of ovariectomized wild-type mice revealed that both the release of soluble Sema4D and the conversion of Plexin-B1 into an active form were estrogen-dependent and concordant with apoptosis. Estrogen supplementation of ovariectomized Sema4D−/− mice did not induce massive vaginal apoptosis in 5-week-old mice; therefore, Sema4D may be an essential apoptosis-inducing ligand that acts downstream of estrogen action in vaginal epithelium during this postnatal tissue remodeling. Analysis of ovariectomized mice also indicated that Sema4D contributed to estrogen-dependent dephosphorylation of Akt and ERK at the time of vaginal opening. Based on our results, we propose that apoptosis in vaginal epithelium during postnatal vaginal opening is induced by enhanced Sema4D signaling that is caused by estrogen-dependent structural changes of Sema4D and Plexin-B1.     

Mig6 Is a Sensor of EGF Receptor Inactivation that Directly Activates c-Abl to Induce Apoptosis during Epithelial Homeostasis

A fundamental aspect of epithelial homeostasis is the dependence on specific growth factors for cell survival, yet the underlying mechanisms remain obscure. We found an "inverse" mode of receptor tyrosine kinase signaling that directly links ErbB receptor inactivation to the induction of apoptosis. Upon ligand deprivation Mig6 dissociates from the ErbB receptor and binds to and activates the tyrosine kinase c-Abl to trigger p73-dependent apoptosis in mammary epithelial cells. Deletion of Errfi1 (encoding Mig6) and inhibition or RNAi silencing of c-Abl causes impaired apoptosis and luminal filling of mammary ducts. Mig6 activates c-Abl by binding to the kinase domain, which is prevented in the presence of epidermal growth factor (EGF) by Src family kinase-mediated phosphorylation on c-Abl-Tyr488. These results reveal a receptor-proximal switch mechanism by which Mig6 actively senses EGF deprivation to directly activate proapoptotic c-Abl. Our findings challenge the common belief that deprivation of growth factors induces apoptosis passively by lack of mitogenic signaling.     

毛细血管扩张性共济失调突变蛋白在细胞凋亡过程中被Caspase┐3降解

新近的研究揭示：ｃａｓｐａｓｅ蛋白酶在细胞凋亡中起着死亡执行者的重要功能．一些蛋白相继被证明在细胞凋亡中可被ｃａｓｐａｓｅ特异切割,其中参与ＤＮＡ损伤修复过程的聚ＡＤＰ核糖聚合酶（ＰＡＲＰ）以及ＤＮＡ依赖的蛋白激酶（ＤＮＡ－ＰＫ）,在细胞凋亡过程中被ｃａｓｐａｓｅ选择性切割具有特殊的功能意义．为探索与ＤＮＡ－ＰＫ催化亚基有较高同源性,含有ｃａｓｐａｓｅ切割位点,且功能上目前也被认为是感受ＤＮＡ损伤和参与信号传导途径的ＡＴＭ（Ａｔａｘｉａｔｅｌａｎｇ－ｉｅｃｔａｓｉａｍｕｔａｔｅｄ）蛋白,是否在凋亡过程中也可被切割而降解？应用体外转录与翻译系统获得ＡＴＭ蛋白的ＰＩ３Ｋ结构域,同时通过建立无细胞反应体系获得含ｃａｓｐａｓｅ活性的细胞抽提液,将两者在体外共同保温．结果发现：ＡＴＭ蛋白与ｃａｓｐａｓｅ－３能免疫共沉淀,ＡＴＭ蛋白的ＰＩ３Ｋ结构域可被ｃａｓｐａｓｅ－３特异切割,并观察到辐射诱发细胞调亡中ＡＴＭ蛋白的降解．从而进一步证实了ＤＮＡ损伤修复的抑制,促进细胞凋亡的发生．     

胎盘发生过程中的细胞凋亡

细胞凋亡是一种正常的生理现象,在胚泡着床过程中,伴随有大量细胞凋亡。研究表明,胎盘发生过程中的细胞凋亡,对调控子宫内膜基质细胞的蜕膜化和滋养层细胞的浸润以形成胎盘具有重要意义;另外,由Fas/FasL系统介导的细胞凋亡可能与母体对胎儿的免疫耐受性有关。本文主要评述细胞凋亡的一般通路以及胎盘发生过程中细胞凋亡的调控。     

Cleavage of Cdc6 by caspase-3 promotes ATM/ATR kinase-mediated apoptosis of HeLa cells

We show that caspase-3 cleaves Cdc6 at D(290)/S and D(442)/G sites, producing p32-tCdc6 (truncated Cdc6) and p49-tCdc6, respectively, during etoposide- or tumor necrosis factor (TNF)-alpha-induced apoptosis. The expression of these tCdc6 proteins, p32- and p49-tCdc6, promotes etoposide-induced apoptosis. The expression of tCdc6 perturbs the loading of Mcm2 but not Orc2 onto chromatin and activates ataxia telangiectasia mutated (ATM) and ATM and Rad-3 related (ATR) kinase activities with kinetics similar to that of the phosphorylation of Chk1/2. The activation kinetics are consistent with elevated cellular levels of p53 and mitochondrial levels of Bax. The tCdc6-induced effects are all suppressed to control levels by expressing a Cdc6 mutant that cannot be cleaved by caspase-3 (Cdc6-UM). Cdc6-UM expression attenuates the TNF-alpha-induced activation of ATM and caspase-3 activities. When ATM or ATR is down-expressed by using the small interfering RNA technique, the TNF-alpha- or tCdc6-induced activation of caspase-3 activities is suppressed in the cells. These results suggest that tCdc6 proteins act as dominant-negative inhibitors of replication initiation and that they disrupt chromatin structure and/or induce DNA damage, leading to the activation of ATM/ATR kinase activation and p53-Bax-mediated apoptosis.     

小鼠早期胚胎发育过程中细胞凋亡及凋亡基因表达的检测

小鼠早期胚胎发育过程中凋亡现象大量存在,细胞凋亡与凋亡基因表达有关。应用彗星电泳法检测小鼠早期胚胎凋亡情况;应用巢式RT-PCR、免疫组化的方法检测了Bcl-2家族成员(Bax、Bcl-2、Bak、Bcl-xl)的表达变化情况。结果显示:随着胚胎细胞数目的增加,凋亡比率逐渐增大;Bax表达量在整个过程中基本不变,Bcl-2表达量逐渐上调,Bak、Bcl-xl的表达量逐渐降低。对小鼠早期胚胎发育过程中的基因表达研究对于揭示早期胚胎发育的机制有重大的意义。     

Electrical Characteristics of Triturus Egg Cells during Cleavage

The membrane potential in the blastomeres of dividing Triturus egg cells increases progressively from the first cleavage to the late morula stages. Both the animal and vegetal poles show the same increasing trend in potential; there is no significant potential difference between them. Upon first cell cleavage, the total resistance of the egg cell surface in contact with the exterior decreases to about one-tenth of its value before cleavage, and then remains rather constant up to the late morula stage. The specific resistance of this membrane surface drops rather abruptly upon first cleavage, and rises progressively during the morula stage. The resistance of the junctional membrane surface of the blastomeres, that is, the membrane formed at the former planes of cleavage, is small in relation to that of the cell surface in contact with the exterior. As a result, the blastomeres are electrically coupled throughout all stages of embryonic development examined.     

Dismantling Cell–Cell Contacts during Apoptosis Is Coupled to a Caspase-dependent Proteolytic Cleavage of β-Catenin

Cell death by apoptosis is a tightly regulated process that requires coordinated modification in cellular architecture. The caspase protease family has been shown to play a key role in apoptosis. Here we report that specific and ordered changes in the actin cytoskeleton take place during apoptosis.

In this context, we have dissected one of the first hallmarks in cell death, represented by the severing of contacts among neighboring cells. More specifically, we provide demonstration for the mechanism that could contribute to the disassembly of cytoskeletal organization at cell–cell adhesion. In fact, β-catenin, a known regulator of cell–cell adhesion, is proteolytically processed in different cell types after induction of apoptosis. Caspase-3 (cpp32/apopain/yama) cleaves in vitro translated β-catenin into a form which is similar in size to that observed in cells undergoing apoptosis. β-Catenin cleavage, during apoptosis in vivo and after caspase-3 treatment in vitro, removes the amino- and carboxy-terminal regions of the protein. The resulting β-catenin product is unable to bind α-catenin that is responsible for actin filament binding and organization. This evidence indicates that connection with actin filaments organized at cell–cell contacts could be dismantled during apoptosis. Our observations suggest that caspases orchestrate the specific and sequential changes in the actin cytoskeleton occurring during cell death via cleavage of different regulators of the microfilament system.

     

Cleavage of β-Catenin and Plakoglobin and Shedding of VE-Cadherin during Endothelial Apoptosis: Evidence for a Role for Caspases and Metalloproteinases

Growth factor deprivation of endothelial cells induces apoptosis, which is characterized by membrane blebbing, cell rounding, and subsequent loss of cell–matrix and cell–cell contacts. In this study, we show that initiation of endothelial apoptosis correlates with cleavage and disassembly of intracellular and extracellular components of adherens junctions. β-Catenin and plakoglobin, which form intracellular links between vascular endothelial cadherin (VE-cadherin) and actin-binding α-catenin in adherens junctions, are cleaved in apoptotic cells. In vitro incubations of cell lysates and immunoprecipitates with recombinant caspases indicate that CPP32 and Mch2 are involved, possibly by initiating proteolytic processing. Cleaved β-catenin from lysates of apoptotic cells does not bind to endogenous α-catenin, whereas plakoglobin retains its binding capacity. The extracellular portion of the adherens junctions is also altered during apoptosis because VE-cadherin, which mediates endothelial cell–cell interactions, dramatically decreases on the surface of cells. An extracellular fragment of VE-cadherin can be detected in the conditioned medium, and this “shedding” of VE-cadherin can be blocked by an inhibitor of metalloproteinases. Thus, cleavage of β-catenin and plakoglobin and shedding of VE-cadherin may act in concert to disrupt structural and signaling properties of adherens junctions and may actively interrupt extracellular signals required for endothelial cell survival.     

静脉注射免疫球蛋白制备中的病毒灭活

在静脉注射免疫球蛋白（ＩＶＩＧ）的制备中,采用有机溶剂结合表面活性剂（Ｓ／Ｄ）处理或６０℃１０小时液态加热（巴氏灭活法）的方法对组分Ⅱ（ＩｇＧ）进行病毒灭活处理,灭活效果用指示病毒进行了评价,结果表明：Ｓ／Ｄ法可有效灭活脂包膜病毒ＶＳＶ和Ｓｉｎｄｂｉｓ,巴氏灭活法则对上述病毒以及Ｖａｃｃｉｎｉａ和Ｅｃｈｏ病毒均有较好的灭活作用。经病毒灭活处理的免疫球蛋白,在理化及生物学特性上基本未受到不利影响,用两种方法分别处理组分Ⅱ后制备的ＩＶＩＧ,其主要特性指标均符合该制品的有关质量规定。     

Inactivation of bacteriophage PM2 during storage

The kinetics of bacteriophage inactivation in the medium that is optimal for its storage has been studied at temperatures from 4 to 55 degrees C. The plot of Arrhenius dependence of the constant of inactivation rate consists of the two linear parts with the energies of activation Ea = 25 kcal/mol for 4-37 degrees C and Ea = 91 kcal/mol for 37-55 degrees C. The DNA of inactivated bacteriophage remained mostly in superspiralized form and completely preserved its biological activity as tested by transfection in spheroplasts. The analysis of inactivation kinetics suggests ageing of virions cultivated at 4 degrees C. The addition of watersoluble antioxidant amoxipin did not change the inactivation kinetics. The addition of antioxidant ionol with twin-80 increased the inactivation that was paralleled by the bacteriophage DNA degradation.     

Inactivation of alpha-ketoglutarate dehydrogenase during enzyme-catalyzed reaction

Alpha-Ketoglutarate dehydrogenase is inactivated during the enzymatic reaction. This inactivation is revealed both in a model system with an artificial electron acceptor and in the overall reaction catalyzed by the alpha-ketoglutarate dehydrogenase complex. Neither the substrate depletion and the product accumulation nor the changing of the alpha-ketoglutarate dehydrogenase oligomeric structure induces the inactivation. There are two independent mechanisms of alpha-ketoglutarate dehydrogenase inactivation in the course of the enzymatic reaction which are consistent with the two stages of the process. The first one corresponds to an essential decrease in activity during several minutes from the beginning of the reaction. This process can be reversed, occurs only during catalysis and manifests itself in the same degree both in the model system and during the functioning of the alpha-ketoglutarate dehydrogenase complex. The second stage is slow, irreversible and more apparent in the model system; it coincides with the inactivation due to the alpha-ketoglutarate dehydrogenase preincubation with hexacyanoferrate alone. The data obtained provide evidence that irreversible inactivation of alpha-ketoglutarate dehydrogenase during the enzymatic reaction is caused by the oxidant.