D．QU:細胞死亡通道(細胞凋亡,自噬,壞死與其他)之全面介紹!
1.膜死亡受體通路之"細胞凋亡通路悖論"（例如CD95):
Chen L, Park SM, Tumanov AV, ET AL:CD95 promotes tumour growth,Nature. 2010 May 27;465(7297):492-6.
2.線粒體通路之"細胞凋亡通路悖論"(例如p53,PUMA等）:
1).PUMA
Labi V, Erlacher M, Krumschnabel G, ET AL Apoptosis of leukocytes triggered by acute DNA damage promotes lymphoma formation. Genes Dev. 2010 Aug 1;24(15):1602-7.
2).p53
Michalak EM, Vandenberg CJ, Delbridge AR, ET AL:Apoptosis-promoted tumorigenesis: gamma-irradiation-induced thymic lymphomagenesis requires Puma-driven leukocyte death,Genes Dev. 2010 Aug 1;24(15):1608-13.
Yongqiang Chen, Meghan B. Azad, Spencer B. Gibson:Methods for detecting autophagy and determining autophagy-induced cell death;Can. J. Physiol. Pharmacol. 88(3): 285–295 (2010)
Abstract:
Autophagy is an intracellular lysosomal degradation process, which in the case of macroautophagy, is characterized by the formation of double-membraned autophagosomes.
Enhanced under stress conditions, autophagy can function to promote cell survival or cell death depending on the type of cellular stress.
Interest in autophagy has increased substantially in the past several years as new research implicates this 「self-eating」 pathway in cell growth, development, and many human diseases.
Various methods have been developed for detecting autophagy; however, the implementation of these methods and the interpretation of the results often vary between studies, and a more standardized approach is required.
In this review, we summarize the current methods available for detecting autophagy and for determining its contribution to cell death.
Furthermore, we discuss the critical points for the successful application of these methods based on experiences from our laboratory and from other research groups.
Fas是一種跨膜蛋白，屬於腫瘤壞死因子受體超家族成員，它與FasL結合可以啟動凋亡信號的轉導引起細胞凋亡。它的活化包括一系列步驟：首先配體誘導受體三聚體化，然後在細胞膜上形成凋亡誘導複合物，這個複合物中包括帶有死亡結構域的Fas相關蛋白FADD。 Fas又稱CD95，是由325個氨基酸組成的受體分子，Fas一旦和配體FasL結合，可通過Fas分子啟動致死性信號轉導，最終引起細胞一系列特徵性變化，使細胞死亡。Fas作為一種普遍表達的受體分子，可出現於多種細胞表面，但FasL的表達卻有其特點，通常只出現於活化的T細胞和NK細胞，因而已被活化的殺傷性免疫細胞，往往能夠最有效地以凋亡途徑置靶細胞於死地。 Fas分子胞內段帶有特殊的死亡結構域（DD, death domain）。三聚化的Fas和FasL結合后，使三個Fas分子的死亡結構域相聚成簇，吸引了胞漿中另一種帶有相同死亡結構域的蛋白FADD。FADD是死亡信號轉錄中的一個連接蛋白，它由兩部分組成：C端（DD結構域）和N端（DED）部分。DD結構域負責和Fas分子胞內段上的DD結構域結合，該蛋白再以DED連接另一個帶有DED的後續成分，由此引起N段DED隨即與無活性的半胱氨酸蛋白酶8（caspase8）酶原發生同嗜性交聯，聚合多個caspase8的分子，caspase8分子逐由單鏈酶原轉成有活性的雙鏈蛋白，進而引起隨後的級聯反應，即Caspases，後者作為酶原而被激活，引起下面的級聯反應。細胞發生凋亡。因而TNF誘導的細胞凋亡途徑與此類似
3）凋亡抑制蛋白（IAPs,inhibitors of Apoptosis protien）為一組具有抑制凋亡作用的蛋白質，首先是從桿狀病毒基因組克隆到，發現能夠抑制由病毒感染引起的宿主細胞死亡應答。其特性是有大約20氨基酸組成的功能區，這對IAPs抑制凋亡是必需要的，它們主要抑制Caspase3,-7，而不結合它的酶原，對Caspase則即可以結合活化的，又可結合酶原，進而抑制細胞凋亡。
一.程序性細胞死亡(PCD)包括細胞凋亡（apoptosis）、自噬性細胞死亡（autophagic cell death）、類凋亡（paraptosis）、有絲分裂災難（mitotic catastrophe）、脹亡（oncosis）、凋亡樣程序性細胞死亡（model of apoptosis-like）和壞死樣程序性細胞死亡（model of necrosis-like）等。
Frank Madeo is in the Institute of Molecular Biosciences, University of Graz, 8010 Graz, Austria.
Abstract
Organismal lifespan can be extended by genetic manipulation of cellular processes such as histone acetylation, the insulin/IGF-1 (insulin-like growth factor 1) pathway or the p53 system. Longevity-promoting regimens, including caloric restriction and inhibition of TOR with rapamycin, resveratrol or the natural polyamine spermidine, have been associated with autophagy (a cytoprotective self-digestive process) and in some cases were reported to require autophagy for their effects. We summarize recent developments that outline these links and hypothesize that clearing cellular damage by autophagy is a common denominator of many lifespan-extending manipulations.
Kraft C, Peter M, Hofmann K：Selective autophagy: ubiquitin-mediated recognition and beyond，Nat Cell Biol. 2010 Sep;12(9):836-41.
文章標題：選擇性自噬：泛素介導的認同及其超越.
Claudine Kraft and Matthias Peter are in the Institute of Biochemistry, ETH Zürich, Schafmattstrasse 18, CH-8093 Zürich, Switzerland. matthias.peter@bc.biol.ethz.ch.
Abstract
Eukaryotic cells use autophagy and the ubiquitin-proteasome system as their major protein degradation pathways. Whereas the ubiquitin-proteasome system is involved in the rapid degradation of proteins, autophagy pathways can selectively remove protein aggregates and damaged or excess organelles. Proteasome-mediated degradation requires previous ubiquitylation of the cargo, which is then recognized by ubiquitin receptors directing it to 26S proteasomes. Although autophagy has long been viewed as a random cytoplasmic degradation system, the involvement of ubiquitin as a specificity factor for selective autophagy is rapidly emerging. Recent evidence also suggests active crosstalk between proteasome-mediated degradation and selective autophagy. Here, we discuss the molecular mechanisms that link autophagy and the proteasome system, as well as the emerging roles of ubiquitin and ubiquitin-binding proteins in selective autophagy. On the basis of the evolutionary history of autophagic ubiquitin receptors, we propose a common origin for metazoan ubiquitin-dependent autophagy and the cytoplasm-to-vacuole targeting pathway of yeast.
Yang Z, Klionsky DJ.：Eaten alive: a history of macroautophagy. Nat Cell Biol. 2010 Sep;12(9):814-22.
the Life Sciences Institute, University of Michigan, 210 Washtenaw Avenue, Ann Arbor, MI 48109-2216, USA, the Department of Molecular, Cellular and Developmental Biology, 830 North University Avenue, Natural Science Building (Kraus) Ann Arbor, MI 48109-1048, USA and the Department of Biological Chemistry 1150 West Medical Center Drive, Ann Arbor, MI 48109-5606, USA.
Abstract
Macroautophagy (hereafter autophagy), or 'self-eating', is a conserved cellular pathway that controls protein and organelle degradation, and has essential roles in survival, development and homeostasis. Autophagy is also integral to human health and is involved in physiology, development, lifespan and a wide range of diseases, including cancer, neurodegeneration and microbial infection. Although research on this topic began in the late 1950s, substantial progress in the molecular study of autophagy has taken place during only the past 15 years. This review traces the key findings that led to our current molecular understanding of this complex process.
Mizushima N, Yoshimori T, Levine B.：Methods in mammalian autophagy research. Cell. 2010 Feb 5;140(3):313-26.
Department of Physiology and Cell Biology, Tokyo Medical and Dental University, Tokyo 113-8519, Japan. nmizu.phy2@***.ac.jp
Abstract
Autophagy has been implicated in many physiological and pathological processes. Accordingly, there is a growing scientific need to accurately identify, quantify, and manipulate the process of autophagy. However, as autophagy involves dynamic and complicated processes, it is often analyzed incorrectly. In this Primer, we discuss methods to monitor autophagy and to modulate autophagic activity, with a primary focus on mammalian macroautophagy.
Klionsky DJ, Emr SD.：Autophagy as a regulated pathway of cellular degradation.
，Science. 2000 Dec 1;290(5497):1717-21.
Department of Biology, University of Michigan, 830 North University, Ann Arbor, MI 48109-1048, USA. klionsky@umich.edu
Abstract
Macroautophagy is a dynamic process involving the rearrangement of subcellular membranes to sequester cytoplasm and organelles for delivery to the lysosome or vacuole where the sequestered cargo is degraded and recycled. This process takes place in all eukaryotic cells.
It is highly regulated through the action of various kinases, phosphatases, and guanosine triphosphatases (GTPases). The core protein machinery that is necessary to drive formation and consumption of intermediates in the macroautophagy pathway includes a ubiquitin-like protein conjugation system and a protein complex that directs membrane docking and fusion at the lysosome or vacuole. Macroautophagy plays an important role in developmental processes, human disease, and cellular response to nutrient deprivation.
Abstract
Dying cells release and expose at their surface molecules that signal to the immune system. We speculate that combinations of these molecules determine the route by which dying cells are engulfed and the nature of the immune response that their death elicits.
Abstract
Dying cells release and expose at their surface molecules that signal to the immune system. We speculate that combinations of these molecules determine the route by which dying cells are engulfed and the nature of the immune response that their death elicits.
Tooze SA, Yoshimori ：The origin of the autophagosomal membrane. Nat Cell Biol. 2010 Sep;12(9):831-5.
Sharon A. Tooze is in the Secretory Pathways Laboratory, London Research Institute Cancer Research UK, 44 Lincoln's Inn Fields, London, WC2A 3PX, U.K. sharon.tooze@cancer.org.uk.
Abstract
Macroautophagy is initiated by the formation of the phagophore (also called the isolation membrane). This membrane can both selectively and non-selectively engulf cytosolic components, grow and close around the sequestered components and then deliver them to a degradative organelle, the lysosome. Where this membrane comes from and how it grows is not well understood. Since the discovery of autophagy in the 1950s the source of the membrane has been investigated, debated and re-investigated, with the consensus view oscillating between a de novo assembly mechanism or formation from the membranes of the endoplasmic reticulum (ER) or the Golgi. In recent months, new information has emerged that both the ER and mitochondria may provide a membrane source, enlightening some older findings and revealing how complex the initiation of autophagy may be in mammalian cells.
Mizushima N, Levine B.:Autophagy in mammalian development and differentiation;Nat Cell Biol. 2010 Sep;12(9):823-30.
Noboru Mizushima is in the Department of Physiology and Cell Biology, Tokyo Medical and Dental University, Tokyo 113-8519, Japan.
Abstract
It has been known for many decades that autophagy, a conserved lysosomal degradation pathway, is highly active during differentiation and development. However, until the discovery of the autophagy-related (ATG) genes in the 1990s, the functional significance of this activity was unknown. Initially, genetic knockout studies of ATG genes in lower eukaryotes revealed an essential role for the autophagy pathway in differentiation and development. In recent years, the analyses of systemic and tissue-specific knockout models of ATG genes in mice has led to an explosion of knowledge about the functions of autophagy in mammalian development and differentiation. Here we review the main advances in our understanding of these functions.
Klionsky DJ, Abeliovich H, Agostinis P,ET AL:Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes.Autophagy. 2008 Feb 16;4(2):151-75. Epub 2007 Nov 21.
Life Sciences Institute, University of Michigan, Ann Arbor, Michigan 48109-2216, USA. klionsky@umich.edu
Comment in:
Autophagy. 2008 Feb 16;4(2):139-40.
Abstract
Research in autophagy continues to accelerate,(1) and as a result many new scientists are entering the field. Accordingly, it is important to establish a standard set of criteria for monitoring macroautophagy in different organisms. Recent reviews have described the range of assays that have been used for this purpose.(2,3) There are many useful and convenient methods that can be used to monitor macroautophagy in yeast, but relatively few in other model systems, and there is much confusion regarding acceptable methods to measure macroautophagy in higher eukaryotes. A key point that needs to be emphasized is that there is a difference between measurements that monitor the numbers of autophagosomes versus those that measure flux through the autophagy pathway; thus, a block in macroautophagy that results in autophagosome accumulation needs to be differentiated from fully functional autophagy that includes delivery to, and degradation within, lysosomes (in most higher eukaryotes) or the vacuole (in plants and fungi). Here, we present a set of guidelines for the selection and interpretation of the methods that can be used by investigators who are attempting to examine macroautophagy and related processes, as well as by reviewers who need to provide realistic and reasonable critiques of papers that investigate these processes. This set of guidelines is not meant to be a formulaic set of rules, because the appropriate assays depend in part on the question being asked and the system being used. In addition, we emphasize that no individual assay is guaranteed to be the most appropriate one in every situation, and we strongly recommend the use of multiple assays to verify an autophagic response.
Galluzzi L, Morselli E, Kepp O, ET AL:Mitochondrial gateways to cancer
,Mol Aspects Med. 2010 Feb;31(1):1-20. Epub 2009 Aug 19.
INSERM, U848, Institut Gustave Roussy, PR1, 39 Rue Camille Desmoulins, F-94805 Villejuif, France.
Abstract
Mitochondria are required for cellular survival, yet can also orchestrate cell death. The peculiar biochemical properties of these organelles, which are intimately linked to their compartmentalized ultrastructure, provide an optimal microenvironment for multiple biosynthetic and bioenergetic pathways. Most intracellular ATP is generated by mitochondrial respiration, which also represents the most relevant source of intracellular reactive oxygen species. Mitochondria participate in a plethora of anabolic pathways, including cholesterol, cardiolipin, heme and nucleotide biosynthesis. Moreover, mitochondria integrate numerous pro-survival and pro-death signals, thereby exerting a decisive control over several biochemical cascades leading to cell death, in particular the intrinsic pathway of apoptosis. Therefore, it is not surprising that cancer cells often manifest the deregulation of one or several mitochondrial functions. The six classical hallmarks of cancer (i.e., limitless replication, self-provision of proliferative stimuli, insensitivity to antiproliferative signals, disabled apoptosis, sustained angiogenesis, invasiveness/metastatic potential), as well as other common features of tumors (i.e., avoidance of the immune response, enhanced anabolic metabolism, disabled autophagy) may directly or indirectly implicate deregulated mitochondria. In this review, we discuss several mechanisms by which mitochondria can contribute to malignant transformation and tumor progression.
Maiuri MC, Galluzzi L, Morselli E,ET AL:Autophagy regulation by p53
,Curr Opin Cell Biol. 2010 Apr;22(2):181-5. Epub 2010 Jan 13.
INSERM, U848, F-94805 Villejuif, France.
Abstract
Autophagy is an evolutionarily conserved catabolic pathway that is involved in numerous physiological processes and in multiple pathological conditions including cancer. Autophagy is regulated by an intricate network of signaling cascades that have not yet been entirely disentangled. Accumulating evidence indicates that p53, the best-characterized human tumor suppressor protein, can modulate autophagy in a dual fashion, depending on its subcellular localization. On the one hand, p53 functions as a nuclear transcription factor and transactivates proapoptotic, cell cycle-arresting and proautophagic genes. On the other hand, cytoplasmic p53 can operate at mitochondria to promote cell death and can repress autophagy via poorly characterized mechanisms. This review focuses on the recently discovered function of p53 as a master regulator of autophagy.
He C, Levine B.:The Beclin 1 interactome.Curr Opin Cell Biol. 2010 Apr;22(2):140-9. Epub 2010 Jan 22.
Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, TX 75390, United States.
Abstract
The mammalian ortholog of yeast Atg6/Vps30, Beclin 1, is an essential autophagy protein that has been linked to diverse biological processes, including immunity, development, tumor suppression, lifespan extension, and protection against certain cardiac and neurodegenerative diseases. In recent years, major advances have been made in identifying components of functionally distinct Beclin 1/class III phosphatidylinositol 3-kinase complexes, in characterizing the molecular regulation of interactions between Beclin 1 and the autophagy inhibitors, Bcl-2/BcL-X(L), and in uncovering a role for viral antagonists of Beclin 1 in viral pathogenesis. The rapidly growing list of components of the 'Beclin 1 interactome' supports a model in which autophagy, and potentially other membrane trafficking functions of Beclin 1, are governed by differential interactions with different binding partners in different physiological or pathophysiological contexts.
曲 度譯：Apg1p：在釀酒酵母自噬過程中所需的一種新的蛋白激酶
Matsuura A, Tsukada M, Wada Y,ET AL:Apg1p, a novel protein kinase required for the autophagic process in Saccharomyces cerevisiae.Gene. 1997 Jun 19;192(2):245-50
Department of Biology, Graduate School of Arts and Sciences, University of Tokyo, Komaba, Meguro-ku, Japan.
Abstract
Autophagic protein degradation includes bulk protein turnover with dynamic membrane reorganization, in which formation of novel organelles autophagosomes play key roles.
We have shown that Saccharomyces cerevisiae performs the autophagy in the vacuole, a lytic compartment of yeast, in response to various kinds of nutrient starvation.
Here we show that the APG1 gene, involved in the autophagic process in yeast, encodes a novel type of Ser/Thr protein kinase.
Our results provide direct evidence for involvement of protein phosphorylation in regulation of the autophagic process.
We found overall homology of Apglp with C. elegans Unc-51 protein, suggesting that homologous molecular mechanisms, conserved from unicellular to multicellular organisms, are involved in dynamic membrane flow.
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摘要：
細胞自噬蛋白質的降解作用涉及散在的蛋白質轉變成動態膜的重組作用；
在該作用中，新型細胞器自噬體的形成起著一種關鍵作用。
我們已經顯示，釀酒酵母形成液泡中的自嗜現象，與一種酵母裂解成分對各種營養缺乏狀態之反應相關。
在本文中，我們顯示APG1基因，在酵母自噬過程中，參與了一種新的類型的絲氨酸/蘇氨酸蛋白激酶（Ser/Thr protein kinase）的編碼。
Liang XH, Kleeman LK, Jiang HH,ET ALrotection against fatal Sindbis virus encephalitis by beclin, a novel Bcl-2-interacting protein.J Virol. 1998 Nov;72(11):8586-96.
Departments of Medicine, Columbia University College of Physicians and Surgeons, New York, New York 10032, USA.
Abstract
bcl-2, the prototypic cellular antiapoptotic gene, decreases Sindbis virus replication and Sindbis virus-induced apoptosis in mouse brains, resulting in protection against lethal encephalitis.
To investigate potential mechanisms by which Bcl-2 protects against central nervous system Sindbis virus infection, we performed a yeast two-hybrid screen to identify Bcl-2-interacting gene products in an adult mouse brain library.
We identified a novel 60-kDa coiled-coil protein, Beclin, which we confirmed interacts with Bcl-2 in mammalian cells, using fluorescence resonance energy transfer microscopy.
To examine the role of Beclin in Sindbis virus pathogenesis, we constructed recombinant Sindbis virus chimeras that express full-length human Beclin (SIN/beclin), Beclin lacking the putative Bcl-2-binding domain (SIN/beclinDeltaBcl-2BD), or Beclin containing a premature stop codon near the 5' terminus (SIN/beclinstop).
The survival of mice infected with SIN/beclin was significantly higher (71%) than the survival of mice infected with SIN/beclinDeltaBcl-2BD (9%) or SIN/beclinstop (7%) (P < 0.001).
The brains of mice infected with SIN/beclin had fewer Sindbis virus RNA-positive cells, fewer apoptotic cells, and lower viral titers than the brains of mice infected with SIN/beclinDeltaBcl-2BD or SIN/beclinstop.
These findings demonstrate that Beclin is a novel Bcl-2-interacting cellular protein that may play a role in antiviral host defense.
Levine B, Yuan J.：Autophagy in cell death: an innocent convict? J Clin Invest. 2005 Oct;115(10):2679-88.
曲 度譯:細胞死亡中的自噬：一個無辜的犯人?
Division of Infectious Diseases, Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9113, USA. beth.levin@utsouthwestern.edu
The visualization of autophagosomes in dying cells has led to the belief that autophagy is a nonapoptotic form of programmed cell death.
This concept has now been evaluated using cells and organisms deficient in autophagy genes.
Most evidence indicates that, at least in cells with intact apoptotic machinery, autophagy is primarily a pro-survival rather than a pro-death mechanism.
This review summarizes the evidence linking autophagy to cell survival and cell death, the complex interplay between autophagy and apoptosis pathways, and the role of autophagy-dependent survival and death pathways in clinical diseases.
Levine B, Yuan J.：Autophagy in cell death: an innocent convict? J Clin Invest. 2005 Oct;115(10):2679-88.
曲 度譯:細胞死亡中的自噬：一個無辜的犯人?
Division of Infectious Diseases, Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9113, USA. beth.levin@utsouthwestern.edu
The visualization of autophagosomes in dying cells has led to the belief that autophagy is a nonapoptotic form of programmed cell death.
This concept has now been evaluated using cells and organisms deficient in autophagy genes.
Most evidence indicates that, at least in cells with intact apoptotic machinery, autophagy is primarily a pro-survival rather than a pro-death mechanism.
This review summarizes the evidence linking autophagy to cell survival and cell death, the complex interplay between autophagy and apoptosis pathways, and the role of autophagy-dependent survival and death pathways in clinical diseases.
In 2000, it was suggested to me that "Autophagy will be the wave of the future; it will become the new apoptosis."
Few people would have agreed at the time, but this statement turned out to be prophetic, and this process of 'self-eating' rapidly exploded as a research field, as scientists discovered connections to cancer, neurodegeneration and even lifespan extension.
Amazingly, the molecular breakthroughs in autophagy have taken place during only the past decade.
Abstract
Autophagy, or cellular self-digestion, is a cellular pathway involved in protein and organelle degradation, with an astonishing number of connections to human disease and physiology.
For example, autophagic dysfunction is associated with cancer, neurodegeneration, microbial infection and ageing.
Paradoxically, although autophagy is primarily a protective process for the cell, it can also play a role in cell death. Understanding autophagy may ultimately allow scientists and clinicians to harness this process for the purpose of improving human health.
Abstract
Autophagy, or cellular self-digestion, is a cellular pathway involved in protein and organelle degradation, with an astonishing number of connections to human disease and physiology.
For example, autophagic dysfunction is associated with cancer, neurodegeneration, microbial infection and ageing.
Paradoxically, although autophagy is primarily a protective process for the cell, it can also play a role in cell death. Understanding autophagy may ultimately allow scientists and clinicians to harness this process for the purpose of improving human health.
He C, Klionsky DJ.：Regulation mechanisms and signaling pathways of autophagy. Annu Rev Genet. 2009;43:67-93.
曲 度譯:細胞自噬的調控機制和信號通路
Life Sciences Institute and Departments of Molecular, Cellular and Developmental Biology, and Biological Chemistry, University of Michigan, Ann Arbor, Michigan 48109, USA.
Autophagy is a process of self-degradation of cellular components in which double-membrane autophagosomes sequester organelles or portions of cytosol and fuse with lysosomes or vacuoles for breakdown by resident hydrolases.
Autophagy is upregulated in response to extra- or intracellular stress and signals such as starvation, growth factor deprivation, ER stress, and pathogen infection. Defective autophagy plays a significant role in human pathologies, including cancer, neurodegeneration, and infectious diseases.
We present our current knowledge on the key genes composing the autophagy machinery in eukaryotes from yeast to mammalian cells and the signaling pathways that sense the status of different types of stress and induce autophagy for cell survival and homeostasis.
We also review the recent advances on the molecular mechanisms that regulate the autophagy machinery at various levels, from transcriptional activation to post-translational protein modification.
Maiuri MC, Zalckvar E, Kimchi A,ET AL：Self-eating and self-killing: crosstalk between autophagy and apoptosis. Nat Rev Mol Cell Biol. 2007 Sep;8(9):741-52.
INSERM, U848, F-94805 Villejuif, France.
Abstract
The functional relationship between apoptosis ('self-killing') and autophagy ('self-eating') is complex in the sense that, under certain circumstances, autophagy constitutes a stress adaptation that avoids cell death (and suppresses apoptosis), whereas in other cellular settings, it constitutes an alternative cell-death pathway.
Autophagy and apoptosis may be triggered by common upstream signals, and sometimes this results in combined autophagy and apoptosis; in other instances, the cell switches between the two responses in a mutually exclusive manner.
On a molecular level, this means that the apoptotic and autophagic response machineries share common pathways that either link or polarize the cellular responses.
The term apoptosis is proposed for a hitherto little recognized mechanism of controlled cell deletion, which appears to play a complementary but opposite role to mitosis in the regulation of animal cell populations.
Its morphological features suggest that it is an active, inherently programmed phenomenon, and it has been shown that it can be initiated or inhibited by a variety of environmental stimuli, both physiological and pathological.
The structural changes take place in two discrete stages. The first comprises nuclear and cytoplasmic condensation and breaking up of the cell into a number of membrane-bound, ultrastructurally well-preserved fragments.
In the second stage these apoptotic bodies are shed from epithelial-lined surfaces or are taken up by other cells, where they undergo a series of changes resembling in vitro autolysis within phagosomes, and are rapidly degraded by lysosomal enzymes derived from the ingesting cells.
Apoptosis seems to be involved in cell turnover in many healthy adult tissues and is responsible for focal elimination of cells during normal embryonic development.
It occurs spontaneously in untreated malignant neoplasms, and participates in at least some types of therapeutically induced tumour regression.
It is implicated in both physiological involution and atrophy of various tissues and organs. It can also be triggered by noxious agents, both in the embryo and adult animal.
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42.Activity of the Novel Dual Phosphatidylinositol 3-Kinase/Mammalian Target of Rapamycin Inhibitor NVP-BEZ235 against T-Cell Acute Lymphoblastic Leukemia.
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In its simplest form, a DRACO is a chimeric protein with one domain that binds to viral dsRNA and a second domain (e.g., a procaspase-binding domain or a procaspase) that induces apoptosis when two or more DRACOs crosslink on the same dsRNA.
If viral dsRNA is present inside a cell, DRACOs will bind to the dsRNA and induce apoptosis of that cell. If viral dsRNA is not present inside the cell, DRACOs will not crosslink and apoptosis will not occur.
A serious threat is posed by viral pathogens, including clinical viruses (HIV, hepatitis viruses, etc.), natural emerging viruses (avian and swine influenza strains, SARS, etc.), and viruses relevant to potential bioterrorism (Ebola, smallpox, etc.). Unfortunately, there are relatively few prophylactics or therapeutics for these viruses, and most which do exist can be divided into three broad categories [1]–[3]:
(1) Specific inhibitors of a virus associated target (e.g., HIV protease inhibitors, RNAi) generally must be developed for each virus or viral strain, are prone to resistance if a virus mutates the drug target, are not immediately available for emerging or engineered viral threats, and can have unforeseen adverse effects.
(2) Vaccines also require a new vaccine to be developed for each virus or viral strain, must be administered before or in some cases soon after exposure to be effective, are not immediately available for emerging or engineered viral threats, can have unforeseen adverse effects, and are difficult to produce for certain pathogens (e.g., HIV).
(3) Interferons and other pro- or anti-inflammatories are less virus specific, but still are only useful against certain viruses, and they can have serious adverse effects through their interactions with the immune and endocrine systems.
To overcome these shortcomings of existing approaches, we have developed and demonstrated a novel antiviral approach that is effective against a very broad spectrum of viruses, nontoxic in vitro and in vivo, and potentially suitable for either prophylactic or therapeutic administration. Our approach, which we call a Double-stranded RNA (dsRNA) Activated Caspase Oligomerizer (DRACO), is designed to selectively and rapidly kill virus-infected cells while not harming uninfected cells.
Our DRACO approach combines two natural cellular processes. The first process involves dsRNA detection in the interferon pathway. Most viruses have double- or single-stranded RNA (ssRNA) genomes and produce long dsRNA helices during transcription and replication; the remainder of viruses have DNA genomes and typically produce long dsRNA via symmetrical transcription [4]–[5]. In contrast, uninfected mammalian cells generally do not produce long dsRNA (greater than ~21–23 base pairs) [4]–[5]. Natural cellular defenses exploit this difference in order to detect and to attempt to counter viral infections [6]–[7]. For example, protein kinase R (PKR) contains an N-terminal domain with two dsRNA binding motifs (dsRBM 1 and 2) and a C-terminal kinase domain [8]–[9]. Binding of multiple PKR proteins to dsRNA with a length of at least 30–50 base pairs [5] activates the PKRs via trans-autophosphorylation; activated PKR then phosphorylates eIF-2α, thereby inhibiting translation of viral (and cellular) proteins. Other examples of proteins that detect viral dsRNA include 2′,5′-oligoadenylate (2–5A) synthetases [10], RNase L (activated via dimerization by 2–5A produced by 2–5A synthetases in response to dsRNA [11]), TLR 3 [12], interferon-inducible ADAR1 [13], and RIG-I and Mda-5 [6]–[7].
The second natural process used by our approach is one of the last steps in the apoptosis pathway [14], in which complexes containing intracellular apoptosis signaling molecules, such as apoptotic protease activating factor 1 (Apaf-1) [15]–[16] or FLICE-activated death domain (FADD) [17]–[18], simultaneously bind multiple procaspases. The procaspases transactivate via cleavage, activate additional caspases in the cascade, and cleave a variety of cellular proteins [14], thereby killing the cell.
Many viruses attempt to counter these defenses. A wide variety of viruses target dsRNA-induced signaling proteins, including IPS-1, interferon response factors (IRFs), interferons and interferon receptors, JAK/STAT proteins, and eIF-2α [19]–[20]. Some viral products attempt to sequester dsRNA (e.g., poxvirus E3L [21]) or to directly interfere with cellular dsRNA binding domains (e.g., HIV TAR RNA [19]–[20]). Virtually all viruses that inhibit apoptosis do so by targeting early steps in the pathway, for example by inhibiting p53, mimicking anti-apoptotic Bcl-2, or interfering with death receptor signaling [22]–[23]. Among the few viral proteins that directly inhibit one or more caspases are African swine fever virus A224L (which inhibits caspase 3) [24], poxvirus CrmA (which inhibits caspases 1, 8, and 10 but not others) [25], and baculovirus p35 (which inhibits several caspases but is relatively ineffective against caspase 9) [25].
Because PKR activation and caspase activation function in similar ways and involve proteins that have separate domains with well-defined functions, these two processes can be combined to circumvent most viral blockades [26]–[27]. In its simplest form, a DRACO is a chimeric protein with one domain that binds to viral dsRNA and a second domain (e.g., a procaspase-binding domain or a procaspase) that induces apoptosis when two or more DRACOs crosslink on the same dsRNA. If viral dsRNA is present inside a cell, DRACOs will bind to the dsRNA and induce apoptosis of that cell. If viral dsRNA is not present inside the cell, DRACOs will not crosslink and apoptosis will not occur.
For delivery into cells in vitro or in vivo, DRACOs can be fused with proven protein transduction tags, including a sequence from the HIV TAT protein [28], the related protein transduction domain 4 (PTD) [29], and polyarginine (ARG) [30]. These tags have been shown to carry large cargo molecules into both the cytoplasm and the nucleus of all cell types in vitro and in vivo, even across the blood-brain barrier.
Results and Discussion
We produced DRACOs with different dsRNA detection domains, apoptosis induction domains, and transduction tags (Figure 1). The dsRNA detection domains included PKR1–181, PKR1–181 with dsRBM 1 (NTE3L), dsRBM 2 (CTE3L), or dsRBM 1 and 2 (2×E3L) replaced by the dsRNA binding motif from poxvirus E3L, and RNaseL1–335 (which binds to 2–5A produced by endogenous cellular 2–5A synthetases in response to viral dsRNA). The apoptosis induction domains included FADD1–90 Death Effector Domain (DED, which binds to procaspase 8), Apaf-11–97 caspase recruitment domain (CARD, which binds to procaspase 9), and murine Apaf-11–97 (mApaf) CARD. Except for mApaf, all domains refer to the human sequence. Isolated dsRNA detection domains and apoptosis induction domains were produced as negative controls. Mutant DRACOs with deleterious K64E [9] and homologous K154E mutations in the PKR domain were also produced as negative controls. Proteins were produced with TAT, PTD, or ARG tags on the N terminus, C terminus, or both termini. Proteins were expressed in BL21(DE3)pLysS Rosetta E. coli. An empty expression vector was transformed into the E. coli and the same purification protocol was followed, resulting in control extract without DRACOs. 作者: huabin 時間: 2015-4-20 06:33
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Figure 2.DRACOs penetrated cells and persisted for days.
(A) DRACOs with PTD or TAT tags entered H1-HeLa cells more readily than DRACO without a transduction tag. 400 nM PKR-Apaf
DRACO was added to medium for 1 hour, and then cells were trypsinized and washed to remove any DRACO on the cell surface.
Cells were lysed and analyzed for DRACO by westerns using anti-His6 antibodies. Lysate from approximately 105 cells was loaded
in each lane. A known amount of purified PKR-Apaf DRACO was used as a standard as indicated. (B) DRACOs entered HeLa cells
within 10 minutes and reached a maximum after 1.5 hours. 400 nM TAT-PKR-Apaf DRACO was added to medium for the specified
time, and then cells were analyzed as in (A). (C) DRACOs persisted within HeLa cells for at least 8 days. 500 nM PTD-PKR-Apaf
DRACO was added to cell medium for 1 hour, and then cells were put into DRACO-free medium. After the specified number of days,
cells were analyzed as in (A).
doi:10.1371/journal.pone.0022572.g002
Figure 3.DRACOs mediated apoptosis in cells containing dsRNA.
L929 cells transfected with both DRACO and poly(I):poly(C) dsRNA exhibited apoptosis within 24 hours, whereas cells that received
only DRACO did not. Caspase inhibitors eliminated DRACO-mediated apoptosis in the presence of dsRNA.
doi:10.1371/journal.pone.0022572.g003
We measured the viability of normal human lung fibroblast (NHLF) cells that had been treated with PKR-Apaf DRACOs or negative
controls and then challenged with 130 plaque forming units (pfu) per well rhinovirus 1B (Figures 4, S2, S3).
Untreated cell populations succumbed to virus within days, indicating that any innate cellular responses were ineffective against the
virus or blocked by the virus. DRACOs with PTD, TAT, and ARG tags prevented significant cytopathic effects (CPE) in virus-challenged
cell populations by rapidly killing any initially infected cells, thereby terminating the infection in its earliest stages. DRACOs had no
apparent toxicity in unchallenged cells. Isolated PKR1–181 and Apaf-11–97 domains were nontoxic but not antiviral, even when added
simultaneously (but not covalently linked). DRACO with deleterious amino acid changes also had little efficacy. Likewise, an amount
of purified bacterial extract (without DRACOs) approximately 10-fold greater than the average volume of DRACOs added to cells was
nontoxic and not efficacious, demonstrating that any remaining bacterial contaminants such as lipopolysaccharide did not affect
the cells or produce antiviral activity. Thus the antiviral efficacy
appears to require intact functional DRACOs. Tests using DRACOs with protein transduction tags on the N terminus, C terminus,
or both termini indicated that N-terminal tags generally worked the best (data not shown). DRACOs with transduction tags penetrated cells and were antiviral when
administered by themselves (Figures 2, S2A), but efficacy was enhanced by co-administration with Roche FuGene 6 to maximize uptake (Figure S2B), so FuGene was used in experiments unless otherwise noted. Cell viability measured 7 days post infection (dpi) showed little difference if DRACO-containing medium was removed 3 dpi after untreated cells had widespread CPE; there was no relapse of viral CPE in treated cells
after DRACOs were withdrawn (Figure 4B).
Figure 4.DRACOs were effective against rhinovirus 1B in NHLF cells.
(A) 100 nM DRACO was effective against 130 pfu/well rhinovirus, whereas 100 nM negative controls were not (12 dpi). (B) Cell
viability measured 7 dpi showed little difference if 100 nM DRACO-containing medium was removed 3 dpi when untreated cells
had widespread CPE from 130 pfu/well rhinovirus 1B; there was no relapse of viral CPE in treated cells after DRACOs were
withdrawn. (C) 1 dose of 25 nM PTD-PKR-Apaf DRACO was effective against rhinovirus 1B in NHLF cells when it was added from
6 days before infection to 3 days after infection. (Complete viral CPE in untreated cell populations required 3–4 days in our
experiments, and for these experiments a significant fraction of cells were still uninfected 3 dpi.) Cell viability was measured 14 dpi.
doi:10.1371/journal.pone.0022572.g004
DRACOs were added approximately 24 hours before virus unless otherwise noted, but other dosing times were tested (Figure 4C).
One dose of PTD-PKR-Apaf DRACO was efficacious against rhinovirus 1B in NHLF cells when added up to 6 days before infection,
supporting the western data (Figure 2C) that DRACO persisted inside cells for at least 8 days. Up to 3 days after infection, one
DRACO dose could still rescue a significant percentage of the cell population. After 3 days, virtually all of the cells had already been
killed or at least infected by the virus.
Additional DRACO designs exhibited efficacy against rhinovirus (Figure 5A). Other effective dsRNA detection domains included
NTE3L, CTE3L, 2×E3L, and RNaseL1–335. Other effective apoptotic domains included FADD1–90, mApaf11–97, and procaspases
[26]–[27]. Although the initial performance of these alternate DRACOs was generally inferior to that of PKR-Apaf human DRACO
in these experiments, better performance might be achieved with further optimization. These results demonstrate that the alternate
DRACO designs are nontoxic and efficacious against virus, and they support the DRACO mechanism of action.
作者: huabin 時間: 2015-4-20 06:36
(接上面)
Figure 5.DRACOs were effective against rhinovirus 1B and other viruses.
(A) Multiple 100 nM DRACOs were effective against 130 pfu/well rhinovirus (4 dpi). Even better performance of these alternate DRACOs might be achieved with further optimization. (B) PKR-Apaf DRACOs reduced the viral titer in supernatant from NHLF cells challenged with 300 pfu/well rhinovirus 1B to undetectable levels. PKR and Apaf-1 domains not covalently linked increased viral titers somewhat, possibly by interfering with the antiviral activity of endogenous wild-type PKR and Apaf-1. Cells were treated with 100 nM DRACO or controls. Supernatants were collected 4 dpi and their viral titers determined by serial dilution onto fresh 96-well NHLF plates. (C) The EC50 for PTD-PKR-Apaf DRACO was 2–3 nM against 130 pfu/well rhinovirus 1B in NHLF cells (measured 3 dpi), and 50 pfu/well murine encephalomyelitis (3 dpi) and 50 pfu/well murine adenovirus (11 dpi) in L929 cells.
doi:10.1371/journal.pone.0022572.g005
In addition to improving survival of the cell population, DRACOs reduced viral titers from virus-challenged cells (Figures 5B, S4). One dose of PKR-Apaf DRACO administered to NHLF cells 24 hours before 300 pfu/well rhinovirus 1B eliminated any measurable viral titer in cell supernatant samples collected 4 dpi.
The median effective concentration for DRACOs with PTD, TAT, and ARG tags against a variety of viruses was 2–3 nM, as illustrated for PTD-PKR-Apaf DRACO against rhinovirus 1B, murine encephalomyelitis, and murine adenovirus (Figures 5C).
DRACOs were effective against a broad spectrum of other viruses in a variety of cell types (Tables 1–2). DRACOs were effective against rhinoviruses 2 and 30 in NHLF cells (data not shown) and rhinovirus 14 in HeLa cells (Figure S4). DRACOs were effective against murine adenovirus in L929 cells if added before or up to at least 72 hours after virus (Figures 6, S5), demonstrating efficacy against a DNA virus (Figures 6A, S5), in murine cells (using human apoptotic DRACO domains to recruit endogenous murine procaspases), when treatment is delayed until significantly after infection (Figure 6B), and with a variety of DRACO designs (Figure 6C). DRACOs were effective against murine encephalomyelitis in L929 cells regardless of whether the DRACO-containing medium was removed 3 dpi (Figure 7A), whether DRACOs were added before or after infection (Figure 7B), and which DRACOs were used (Figures 7C, S6). DRACOs were effective in Vero E6 cells against Amapari and Tacaribe, arenaviruses that are closely related to lymphocytic choriomeningitis virus (LCMV), Lassa, and Junin viruses (Figures 8A, S7, S8). Likewise, DRACOs were effective against Guama strain Be An 277 (Figures 8B, S9); comparable results were obtained for Guama strain Be Ar 12590 (data not shown). Guama virus is a significant human pathogen and is closely related to other bunyaviruses such as Rift Valley fever, hantavirus, and Crimean-Congo virus. DRACOs were similarly effective against dengue type 2 (New Guinea C) hemorrhagic fever virus, a major human pathogen that is very closely related to other flaviviruses such as West Nile virus, Yellow fever virus, and Omsk virus (Figures 8C, S10, S11). DRACOs were also effective against H1N1 influenza A/PR/8/34 in normal human hepatocytes (Figure S12 left), reovirus 3 in BALB/3T3 murine cells (Figure S12 center), and adenovirus 5 in AD293 cells (Figure S12 right).
Figure 6.DRACOs were effective against murine adenovirus in L929 cells.
(A) 100 nM DRACOs were effective against 50 pfu/well murine adenovirus, whereas all negative controls were not (16 dpi). (B) 100 nM PTD-PKR-Apaf DRACO was effective if added before or up to at least 72 hours after adenovirus (16 dpi). (C) Multiple 100 nM DRACOs were effective against 50 pfu/well murine adenovirus (11 dpi). Even better performance of these alternate DRACOs might be achieved with further optimization.
doi:10.1371/journal.pone.0022572.g006
Figure 7.DRACOs were effective against murine encephalomyelitis in L929 cells.
(A) 100 nM DRACOs were effective against 50 pfu/well encephalomyelitis. Cell viability measured 6 dpi showed little difference if DRACO-containing medium was removed 3 dpi when untreated cells had widespread CPE; there was no relapse of viral CPE in treated cells after DRACOs were withdrawn. (B) 100 nM PTD-PKR-Apaf DRACO was effective if added before, simultaneously with, or up to at least 6 hours after encephalomyelitis. (C) Multiple 100 nM DRACOs were effective against 50 pfu/well murine encephalomyelitis (4 dpi). Even better performance of these alternate DRACOs might be achieved with further optimization.
doi:10.1371/journal.pone.0022572.g007
Figure 9.DRACOs appeared promising when administered via intraperitoneal (i.p.) injection in proof-of-concept trials with adult BALB/c mice.
(A) 2.5 mg PTD-PKR-Apaf DRACO administered i.p. penetrated the liver, kidney, and lungs and persisted for at least 48 hours. Averages of 3 mice per data point are plotted, and error bars show s.e.m. (B) PTD-PKR-Apaf and TAT-PKR-Apaf DRACOs administered i.p. from day -1 through day 3 greatly reduced the morbidity and day-2 lung viral titers in mice challenged intranasally (i.n.) with 1.3 LD50 influenza H1N1 A/PR/8/34. (C) PTD-RNaseL-Apaf, TAT-RNaseL-Apaf, and ARG-RNaseL-Apaf DRACOs administered i.p. from day -1 through day 3 greatly reduced the morbidity and day-2 lung viral titers in mice challenged i.n. with 0.3 LD50 influenza H1N1 A/PR/8/34.
doi:10.1371/journal.pone.0022572.g009
Figure 10.DRACOs appeared promising when administered via intranasal (i.n.) injection in proof-of-concept trials with adult BALB/c mice.
(A) 0.5 mg PKR-Apaf DRACO administered i.n. to adult BALB/c mice penetrated the lungs and persisted over 24 hours. Averages of 3 mice per data point are plotted, and error bars show s.e.m. (B) PTD-PKR-Apaf, TAT-PKR-Apaf, and ARG-PKR-Apaf DRACOs administered i.n. on day 0 reduced the morbidity in mice challenged i.n. with 1 LD50 influenza H1N1 A/PR/8/34.
doi:10.1371/journal.pone.0022572.g010
Based on these encouraging initial animal trials, future work should be done to test and optimize antiviral efficacy, pharmacokinetics, and absence of toxicity in vitro and in vivo. Future experiments can further characterize and optimize dsRNA binding, apoptosis induction, cellular transduction, and other DRACO properties. More extensive trials are also needed to determine how long after infection DRACOs can be used successfully, or if DRACOs are useful against chronic viral infections without producing unacceptable levels of cell death in vivo.
DRACOs should be effective against numerous clinical and NIAID priority viruses, due to the broad-spectrum sensitivity of the dsRNA detection domain, the potent activity of the apoptosis induction domain, and the novel direct linkage between the two which viruses have never encountered. We have demonstrated that DRACOs are effective against viruses with DNA, dsRNA, positive-sense ssRNA, and negative-sense ssRNA genomes; enveloped and non-enveloped viruses; viruses that replicate in the cytoplasm and viruses that replicate in the nucleus; human, bat, and rodent viruses; and viruses that use a variety of cellular receptors (Table 1).
作者: huabin 時間: 2015-4-20 06:37
Materials and Methods
Ethics statement for mouse trials
This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Committee on Animal Care of MIT (Assurance Number: A-3125-01). Guidelines to minimize suffering were followed, and avertin anesthetic was used for intranasal procedures.
Cloning
E.F. Meurs provided PKR cDNA and Y. Shi donated human Apaf-1 cDNA. RNaseL1–335 sequence was cloned from HeLa cells. PKR-E3L, FADD1–90 (with L75A and L76A to prevent spontaneous self-association [18]) and murine Apaf-11–97 sequences came from BioBasic. Genes for DRACOs and controls were constructed using PCR and restriction cloning. TAT (YGRKKRRQRRR), PTD-4 (YARAAARQARA), and ARG (R9) tags were incorporated at N- and/or C-termini. Genes were inserted into pET100/D-TOPO (Invitrogen).
Protein production
Each vector was transformed into Rosetta BL21(DE3)pLysS E. coli (EMD Biosciences), bacteria were plated on Luria broth (LB) agar with 100 ?g ml?1 ampicillin and 34 ?g ml?1 chloramphenicol, and plates were incubated overnight at 37°C. One colony was inoculated into ampicillin-chloramphenicol LB and grown overnight (37°C, 225 r.p.m.), then diluted 1:30 into ampicillin-chloramphenicol LB and incubated (30°C, 225 r.p.m.) until OD600 reached 1.0. 0.5 mM isopropyl β-D-1-thiogalactopyranoside was added and flasks were incubated overnight. E. coli were recovered by centrifugation (5,000 r.p.m., 30 min., 4°C) and lysed by sonication, and His6-tagged proteins were purified using Ni-NTA agarose (Invitrogen) following the manufacturer's protocols. Proteins were eluted into 1.5× PBS with 300 mM imidazole and 10% (vol/vol) glycerol, concentrated with Amicon-15 (10 kDa cutoff, 3,000 g) to >5 mg/ml, and filter-sterilized. Protein concentrations were measured relative to BSA standards by Bradford assay (BioRad) and Gel Doc densitometry.
Cells
L929 (CCL-1), NIH/3T3 (CRL-1658), BALB/3T3 (CCL-163), H1-HeLa (CRL-1958), MDCK (CCL-34), and Vero E6 (CRL-1586) (ATCC) and AD293 (Stratagene) were cultured in complete DMEM (Gibco). Normal human lung fibroblasts, small airway epithelial cells, osteoblasts, hepatocytes, and aortic smooth muscle cells (Lonza) were cultured in cell-specific media (Lonza).
Viruses
Dengue type 2 (New Guinea C, VR-1584), Amapari (VR-477), Tacaribe (VR-1272), Guama (Be An 277, VR-407; Be Ar 12590, VR-420), murine adenovirus (VR-550), Theiler's murine encephalomyelitis (VR-57), reovirus 3 (VR-824), influenza H1N1 A/PR/8/34 (ATCC VR-1469), influenza H1N1 A/WS/33 (ATCC VR-1520), rhinovirus 1B (VR-481), rhinovirus 2 (VR-482), rhinovirus 14 (VR-284), and rhinovirus 30 (VR-505) were obtained from ATCC. Adenovirus 5 was obtained from Stratagene. Influenza A/PR/8/34 for animal trials was donated by P. Palese.
Cell assays
Contact-inhibited cells were grown to 50–80% confluence and non-contact inhibited cells to 20–50% confluence in 96-well plates with 100 ?l/well medium. DRACOs or controls were added to columns of wells, 8 wells/column. Except in Figs. 2 and S2, 0.4–1% (vol/vol) Roche FuGene 6 was co-administered with DRACOs and controls to optimize cellular uptake. Wells received virus approximately 24 hours after DRACO unless otherwise noted. On selected days, cell viability in each plate was measured using CellTiter 96 (Promega). Assay schedules, viral doses, and other parameters were optimized for different cell/virus systems. Micrographs were taken in 24-well plates under similar conditions.
DRACO cell penetration assays
Cells in 24-well plates were incubated with DRACOs for varying lengths of time, then trypsinized, washed thoroughly in PBS, and lysed. Lysate from approximately 105 cells was loaded in each lane. DRACOs were detected via westerns using mouse anti-His6 (Invitrogen) and goat anti-mouse IgG HRP (Jackson).
Apoptosis assays
70% confluent 96-well L929 plates were treated with 10 ?M Z-VAD-FMK pan-caspase inhibitor or 20 ?M Z-LEHD-FMK caspase-9 inhibitor (R&D Systems), then 75 ?M camptothecin (Calbiochem) or 100 nM DRACOs with or without 25 ng/well poly(I):poly(C) dsRNA (Sigma) transfected using FuGene (Roche) following manufacturers' protocols. After 24 hours, apoptosis was determined using Caspase-Glo 3/7 (Promega).
Viral titers
Titers were determined by serial dilutions onto 96-well NHLF (for rhinovirus 1B) or H1-HeLa (for rhinovirus 14) plates, with 8 wells per 10-fold dilution and with the number of wells exhibiting CPE measured 5 dpi. Reed-Muench titers were calculated from the results (1 TCID50≈0.7 pfu). Error bars indicate s.e.m. from 3 trials.
Statistical analysis
CellTiter 96 cell viabilities were normalized to 100% for untreated uninfected and 0% for untreated virus-killed cells. Graphs indicate averages (n = 8) with s.e.m. Experiments were repeated at least 3 times with similar results.
Mouse trials
7-week-old female BALB/c mice (Charles River) received DRACO i.n. (~0.5 mg in 50 ?l) or i.p. (0.8–2.5 mg in 200 ?l). Mice were challenged i.n. with 0.3–1.3 LD50 influenza H1N1 A/PR/8/34. Mice received DRACO i.p. once daily on days -1 and 1–3 and twice on day 0, or just one i.n. DRACO dose simultaneously with virus. Lungs were harvested on day 2 and viral titers determined by serial dilutions onto 96-well MDCK plates. For pharmacokinetics, organs were harvested at designated times, then sonicated into 1 ml PBS with 1% Triton X-100. 1 mg organ solution was mixed with 2× Laemmeli buffer, boiled 5 min., and run on a 10–20% SDS PAGE gel with a standard curve of purified DRACO, followed by western blots with anti-Apaf (Millipore) and HEP-labeled anti-rabbit IgG (Jackson Immunoresearch). Blots were developed with Pierce luminescent reagent and exposed to film. DRACO bands were quantitated by Gel Doc densitometry vs. the standards.
Supporting Information
Figure S1.
DRACOs entered normal human lung fibroblasts. NHLF cells were incubated overnight with 500 nM PTD-PKR-Apaf DRACO labeled with Lumio (Invitrogen), washed with Hank's balanced salt solution, and photographed with a fluorescent microscope to compare (A) untreated and (B) DRACO-treated cells. DRACOs appeared to be distributed throughout each cell in both the cytoplasm and the nucleus.
(TIF)
Figure S2.
FuGene co-administration with DRACOs improved cellular uptake and antiviral efficacy. (A) 100 nM DRACOs with PTD, TAT, and ARG protein transduction tags were effective against rhinovirus 1B in NHLF cells without FuGene co-administration. Cell viability was measured 3 days after infection with 130 pfu/well. (B) Co-administration of FuGene with DRACOs lowered the EC50 of DRACOs, as shown here for PTD-PKR Apaf DRACO against 130 pfu/well rhinovirus 1B in NHLFs.
(TIF)
Figure S3.
200 nM PTD-PKR-Apaf DRACO was effective against rhinovirus 1B in NHLF cells. Representative photographs were taken 20 days after challenge with 300 pfu/well. Scale bar = 50 ?m.
(TIF)
Figure S4.
DRACOs decreased the viral titer of rhinovirus 14 in H1-HeLa cells. One 120 nM dose of PTD-PKR-Apaf DRACO administered to cells 24 hours before or simultaneously with 10 pfu/well rhinovirus 14 eliminated any measurable titer 3 dpi. One DRACO dose administered 24 or 30 hours after infection halved the 3-dpi viral titer.
(TIF)
Figure S5.
200 nM PTD-PKR-Apaf DRACO was effective against murine adenovirus in L929 cells. Representative photographs were taken 15 days after challenge with 30 pfu/well. Scale bar = 25 ?m.
(TIF)
Figure S6.
200 nM PTD-PKR-Apaf DRACO was effective against murine encephalomyelitis in L929 cells. Representative photographs were taken 21 days after challenge with 50 pfu/well. Scale bar = 25 ?m.
(TIF)
Figure S7.
100 nM PTD-PKR-Apaf DRACO was effective against Amapari arenavirus in Vero E6 cells. Representative photographs were taken 11 days after challenge with 300 pfu/well. Scale bar = 100 ?m.
(TIF)
Figure S8.
100 nM PTD-PKR-Apaf DRACO was effective against Tacaribe arenavirus in Vero E6 cells. Photographs were taken 8 days after challenge with 140 pfu/well. Scale bar = 100 ?m.
(TIF)
Figure S9.
200 nM PTD-PKR-Apaf DRACO was effective against Guama Be An 277 bunyavirus in Vero E6 cells. Photographs were taken 4 days after challenge with 30 pfu/well. Scale bar = 100 ?m.
(TIF)
Figure S10.
200 nM PKR-Apaf DRACO was effective against dengue flavivirus in Vero E6 cells. Cell viability was measured 18 days after challenge with 16 pfu/well.
(TIF)
Figure S11.
100 nM PTD-PKR-Apaf DRACO was effective against dengue flavivirus in Vero E6 cells. Photographs were taken 7 days after challenge with 160 pfu/well. Scale bar = 100 ?m.
(TIF)
Figure S12.
DRACOs were effective against a broad spectrum of other viruses in a variety of cell types. Left four photos: 100 nM PKR-Apaf DRACO was effective against H1N1 influenza A/PR/8/34 in normal human hepatocytes. Untreated cells challenged with 105 pfu/well died within 3 days, whereas treated challenged cells were cultured for 72 days with no sign of viral CPE. Center four photos: 100 nM PTD-PKR-Apaf DRACO was effective against reovirus 3 in BALB/3T3 murine cells. Photographs were taken 11 days after challenge with 30 pfu/well reovirus 3. Right four photos: 200 nM PTD-2×E3L-Apaf DRACO was effective against adenovirus 5 in human embryonic kidney AD293 cells. Fluorescent microscope photographs were taken 4 days after challenge with 25 pfu/well adenovirus 5 expressing enhanced green fluorescent protein (EGFP). Scale bars = 50 ?m.
(TIF) 作者: huabin 時間: 2015-4-20 06:38
Acknowledgments
We thank
E.F. Meurs (Institut Pasteur) for PKR cDNA,
Y. Shi (Princeton University) for human Apaf-1 cDNA,
P. Palese (Mount Sinai School of Medicine) for A/PR/8/34 influenza,
and MIT Division of Comparative Medicine for mouse facilities and advice.
We are grateful to
G. Johnson for assistance in the lab,
B. Lemus for producing the EGFP adenovirus,
and E. Schwoebel, C. Cabrera, G. Beltz, and S. Chiang for helpful discussions.
Author Contributions
Conceived and designed the experiments:
THR. Performed the experiments: THR. Wrote the paper: THR. Designed DRACOs: THR. Conducted cell assays: CZ TB. Lead for animal trials: TB. Lead for protein production and purification: SW. Assisted with experiments: JP BZ.
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作者: huabin 時間: 2015-4-20 06:39
Jerry E. Chipuk, Gavin P. McStay, Archana Bharti,ET AL:Sphingolipid Metabolism Cooperates with BAK and BAX to Promote the Mitochondrial Pathway of Apoptosis,Cell, Volume 148, Issue 5, 988-1000, 2 March 2012
•Highlights
•Mitochondria devoid of heterotypic membranes are resistant to cytochrome c release
•Mitochondrial sphingolipids promote BAK/BAX activation and cytochrome c release
•Sphingosine-1-PO4 and hexadecenal coordinate BAK and BAX activation, respectively
•Inhibition of sphingolipid metabolism blocks cytochrome c release and apoptosis
Summary
Mitochondria are functionally and physically associated with heterotypic membranes, yet little is known about how these interactions impact mitochondrial outer-membrane permeabilization (MOMP) and apoptosis. We observed that dissociation of heterotypic membranes from mitochondria inhibited BAK/BAX-dependent cytochrome c (cyto c) release. Biochemical purification of neutral sphingomyelinases that correlated with MOMP sensitization suggested that sphingolipid metabolism coordinates BAK/BAX activation. Using purified lipids and enzymes, sensitivity to MOMP was achieved by in vitro reconstitution of the sphingolipid metabolic pathway. Sphingolipid metabolism inhibitors blocked MOMP from heavy membrane preparations but failed to influence MOMP in the presence of sphingolipid-reconstituted, purified mitochondria. Furthermore, the sphingolipid products, sphingosine-1-PO4 and hexadecenal, cooperated specifically with BAK and BAX, respectively. Sphingolipid metabolism was also required for cellular responses to apoptosis. Our studies suggest that BAK/BAX activation and apoptosis are coordinated through BH3-only proteins and a specific lipid milieu that is maintained by heterotypic membrane-mitochondrial interactions. 作者: huabin 時間: 2015-4-20 06:48 標題: 曲度: 哈默羅夫的靈魂出竅理論