通过核质转运调节细胞凋亡的蛋白质

凋亡,也称Ⅰ型程序性细胞死亡,是细胞在面临严重威胁时发起的保护性主动死亡机制. 凋亡对于个体的生长发育及各种生理功能具有不可或缺的作用. 作为涉及整个细胞的复杂过程,凋亡的顺利进行有赖于众多凋亡相关因子的协调合作与精确调控. 细胞受到凋亡刺激后,核内的某些蛋白质转运出核,将凋亡信号传递到核外,胞质内的多种蛋白质则转运入核,在细胞核这一信息整合的大本营直接发挥作用. 这种双向交流机制在胞核与胞质间建立起密切的联系,同时使得相关蛋白质在特定场所发挥促进或抑制凋亡的作用,确保凋亡信号及时、通畅、有序地传递. 因此,蛋白质的核质转运作为介导胞核与胞质物质交换、信号交流的关键机制,在凋亡过程中就显得尤为重要. 本文主要就核质转运的机制、通过核质转运调节凋亡的蛋白质及其作用机理作一综述.     

Esophageal cancer alters the expression of nuclear pore complex binding protein Hsc70 and eIF5A-1

Nuclear pore complex (NPC) is the only corridor for macromolecules exchange between nucleus and cytoplasm. NPC and its components, nucleoporins, play important role in the diverse physiological processes including macromolecule exchange, chromosome segregation, apoptosis and gene expression. Recent reports also suggest involvement of nucleoporins in carcinogenesis. Applying proteomics, we analyzed expression pattern of the NPC components in a newly established esophageal cancer cell line from Persia (Iran), the high-risk region for esophageal cancer. Our results indicate overexpression of Hsc70 and downregulation of subunit alpha type-3 of proteasome, calpain small subunit 1, and eIF5A-1. Among these proteins, Hsc70 and eIF5A-1 are in direct interaction with NPC and involved in the nucleocytoplasmic exchange. Hsc70 plays a critical role as a chaperone in the formation of a cargo–receptor complex in nucleocytoplasmic transport. On the other hand, it is an NPC-associated protein that binds to nucleoporins and contributes in recycling of the nucleocytoplasmic transport receptors in mammals and affects transport of proteins between nucleus and cytoplasm. The other nuclear pore interacting protein: eIF5A-1 binds to the several nucleoporins and participates in nucleocytoplasmic transport. Altered expression of Hsc70 and eIF5A-1 may cause defects in nucleocytoplasmic transport and play a role in esophageal carcinogenesis.     

Nucleocytoplasmic transport of intrinsically disordered proteins studied by high‐speed super‐resolution microscopy

Both natively folded and intrinsically disordered proteins (IDPs) destined for the nucleus need to transport through the nuclear pore complexes (NPCs) in eukaryotic cells. NPCs allow for passive diffusion of small folded proteins while barricading large ones, unless they are facilitated by nuclear transport receptors. However, whether nucleocytoplasmic transport of IDPs would follow these rules remains unknown. By using a high‐speed super‐resolution fluorescence microscopy, we have measured transport kinetics and 3D spatial locations of transport routes through native NPCs for various IDPs. Our data revealed that the rules executed for folded proteins are not well followed by the IDPs. Instead, both large and small IDPs can passively diffuse through the NPCs. Furthermore, their diffusion efficiencies and routes are differentiated by their content ratio of charged (Ch) and hydrophobic (Hy) amino acids. A Ch/Hy‐ratio mechanism was finally suggested for nucleocytoplasmic transport of IDPs.     

Nucleocytoplasmic transport in apoptosis

The apoptotic demolition of the nucleus is accomplished by diverse proapoptotic factors, most of which are activated in the cytoplasm and gain access to the nucleoplasm during the cell death process. The nucleus is also the main target for genotoxic insult, a potent apoptotic trigger. Signals generated in the nucleus by DNA damage have to propagate to all cellular compartments to ensure the coordinated execution of cell demise. The nucleocytoplasmic shuttling of signalling and execution factors is thus an integral part of the apoptotic programme. Several proteins implicated in apoptotic cell death have been shown to migrate in and out of the nucleus following apoptosis induction. This review summarises the current knowledge on nucleocytoplasmic trafficking of apoptosis-relevant proteins. The effects of apoptosis induction on the nucleocytoplasmic transport machinery are also discussed. Finally, a potential role of nuclear transport as a critical control point of the apoptotic signal cascade is proposed.     

New steps toward the nucleocytoplasmic traffic of macromolecules

The nucleocytoplasmic transport of functional molecules is mediated bidirectionally through the nuclear pore complex (NPC), which spans the double membranes of the nuclear envelope. It has recently been shown that signaling between the nucleus and the cytoplasm plays a key role in coordinating the cellular processes such as the cell cycle and cell differentiation (Yoneda, 2000). As the result of recent extensive analysis, significant progress has been made in our understanding of the fundamental mechanism of nuclear transport of proteins and RNAs and numerous transport factors have now been identified. In this special issue of review articles, we focus on our rapid growing knowledge of nucleocytoplasmic transport, especially the translocation of proteins through the NPC and mRNA export, and review this exciting field from various points of view including cell biology, structural biology and yeast genetics.     

Global enhancement of nuclear localization-dependent nuclear transport in transformed cells

Fundamental to eukaryotic cell function, nucleocytoplasmic transport can be regulated at many levels, including through modulation of the importin/exportin (Imp/Exp) nuclear transport machinery itself. Although Imps/Exps are overexpressed in a number of transformed cell lines and patient tumor tissues, the efficiency of nucleocytoplasmic transport in transformed cell types compared with nontransformed cells has not been investigated. Here we use quantitative live cell imaging of 3 isogenic nontransformed/transformed cell pairs to show that nuclear accumulation of nuclear localization signal (NLS)-containing proteins, but not their NLS-mutated derivatives, is increased up to 7-fold in MCF10CA1h human epithelial breast carcinoma cells and in simian virus 40 (SV40)-transformed fibroblasts of human and monkey origin, compared with their nontransformed counterparts. The basis for this appears to be a significantly faster rate of nuclear import in transformed cell types, as revealed by analysis using fluorescence recovery after photobleaching for the human MCF10A/MCF10CA1h cell pair. Nuclear accumulation of NLS/nuclear export signal-containing (shuttling) proteins was also enhanced in transformed cell types, experiments using the nuclear export inhibitor leptomycin B demonstrating that efficient Exp-1-mediated nuclear export was not impaired in transformed compared with nontransformed cells. Enhanced nuclear import and export efficiencies were found to correlate with 2- to 4-fold higher expression of specific Imps/Exps in transformed cells, as indicated by quantitative Western blot analysis, with ectopic expression of Imps able to enhance NLS nuclear accumulation levels up to 5-fold in nontransformed MCF10A cells. The findings indicate that transformed cells possess altered nuclear transport properties, most likely due to the overexpression of Imps/Exps. The findings have important implications for the development of tumor-specific drug nanocarriers in anticancer therapy.     

Identification of Cargo Proteins Specific for Importin-β with Importin-α Applying a Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC)-based in Vitro Transport System

The human importin (Imp)-β family consists of 21 nucleocytoplasmic transport carrier proteins, which transport thousands of proteins (cargoes) across the nuclear envelope through nuclear pores in specific directions. To understand the nucleocytoplasmic transport in a physiological context, the specificity of cargoes for their cognate carriers should be determined; however, only a limited number of nuclear proteins have been linked to specific carriers. To address this biological question, we recently developed a novel method to identify carrier-specific cargoes. This method includes the following three steps: (i) the cells are labeled by stable isotope labeling by amino acids in cell culture (SILAC); (ii) the labeled cells are permeabilized, and proteins in the unlabeled cell extracts are transported into the nuclei of the permeabilized cells by a particular carrier; and (iii) the proteins in the nuclei are quantitatively identified by LC-MS/MS. The effectiveness of this method was demonstrated by the identification of transportin (Trn)-specific cargoes. Here, we applied this method to identify cargo proteins specific for Imp-β, which is a predominant carrier that exclusively utilizes Imp-α as an adapter for cargo binding. We identified candidate cargoes, which included previously reported and potentially novel Imp-β cargoes. In in vitro binding assays, most of the candidate cargoes bound to Imp-β in one of three binding modes: directly, via Imp-α, or via other cargoes. Thus, our method is effective for identifying a variety of Imp-β cargoes. The identified Imp-β and Trn cargoes were compared, ensuring the carrier specificity of the method and illustrating the complexity of these transport pathways.     

细胞核质转运及其受体在植物抗病防御反应中的调控作用

植物病害严重威胁全球粮食生产,研究植物对病原菌防御机制和病原菌对寄主作物的侵染过程和分子机制,有助于改良植物种源使其获得持久抗性。近年来, 日渐增多的研究表明, 一些抗病蛋白需要转移到细胞核内才能启动免疫反应,进而发挥抗病防御作用,而细胞核质转运受体是实现这些抗病蛋白核质转运必不可少的“载体”。因此,细胞核质转运及转运...     

Regulated nucleocytoplasmic trafficking of viral gene products: a therapeutic target?

The study of viral proteins and host cell factors that interact with them has represented an invaluable contribution to understanding of the physiology as well as associated pathology of key eukaryotic cell processes such as cell cycle regulation, signal transduction and transformation. Similarly, knowledge of nucleocytoplasmic transport is based largely on pioneering studies performed on viral proteins that enabled the first sequences responsible for the facilitated transport through the nuclear pore to be identified. The study of viral proteins has also enabled the discovery of several nucleocytoplasmic regulatory mechanisms, the best characterized being through phosphorylation. Recent delineation of the mechanisms whereby phosphorylation regulates nuclear import and export of key viral gene products encoded by important human pathogens such as human cytomegalovirus dengue virus and respiratory syncytial virus has implications for the development of antiviral therapeutics. In particular, the development of specific and effective kinase inhibitors makes the idea of blocking viral infection by inhibiting the phosphorylation-dependent regulation of viral gene product nuclear transport a real possibility. Additionally, examination of a chicken anemia virus (CAV) protein able to target selectively into the nucleus of tumor but not normal cells, as specifically regulated by phosphorylation, opens the exciting possibility of cancer cell-specific nuclear targeting. The study of nucleoplasmic transport may thus enable the development not only of new antiviral approaches, but also contribute to anti-cancer strategies.     

The Ran GTPase system in fission yeast affects microtubules and cytokinesis in cells that are competent for nucleocytoplasmic protein transport

Misregulation of the evolutionarily conserved GTPase Ran in fission yeast results in defects in several cellular processes in cells that are competent for nucleocytoplasmic protein transport. These results suggest that transport is neither the only nor the primary Ran-dependent process in living cells. The ability of Ran to independently regulate multiple cellular processes in vivo is demonstrated by showing that (i) eight different transport-competent RanGEF (guanine nucleotide exchange factor) mutants have defects in mitotic spindle formation; (ii) the RanGEF temperature-sensitive mutant pim1-d1 has abnormal actin ring structures at the septum. Overexpression of Imp2p, which specifically destabilizes these structures, restores viability. (iii) Ran-dependent processes differ in their requirements for active Ran in vivo. Microtubule function, cytokinesis, and nuclear envelope structure are the Ran-dependent processes most sensitive to the amount of Ran protein in the cell, whereas nucleocytoplasmic protein transport is the most robust. Therefore, the ability of Ran from Schizosaccharomyces pombe to independently regulate multiple cellular processes may reflect differences in its interactions with the binding proteins that mediate these functions and explain the complex phenotypic consequences of its misregulation in vivo.     

植物核质运输与其先天免疫研究进展

植物为了抵御病原菌的侵染而进化出一套独特的先天免疫系统,它主要通过定位在细胞膜或细胞质上的受体介导并激活下游抗病基因表达而实现,但在这些信号传递过程中,细胞质的信号向核传递需要核质运输相关元件的参与。虽然目前只有个别核质运输的信号元件被证实参与了植物的先天免疫信号传递过程,但越来越多的研究表明核质运输是连接抗病基因表达和信号识别受体的一个主要方式。研究发现,病原菌的效应因子也可以利用植物核质运输机制侵入到宿主细胞核内,调控敏感基因的表达,干扰植物的免疫反应。该文对近年来国内外有关植物的核质运输机制、各层次免疫反应需要核质运输作用、核质运输相关蛋白在免疫反应中的作用等方面对核质运输参与植物先天免疫反应研究的研究进展进行综述,并指出该领域未来研究的主要内容和方向。     

Nucleocytoplasmic trafficking of proteins: With or without Ran?

Proteins and RNAs move between the nucleus and cytoplasm by translocation through nuclear pore complexes in the nuclear envelope. To do this, they require specific targeting signals, energy, and a cellular apparatus that catalyzes their transport. Several of the factors involved in nucleocytoplasmic trafficking of proteins have been identified and characterized in some detail. The emerging picture for nuclear transport proposes a central role for the small GTPase Ran and proteins with which it interacts. In particular, asymmetric distribution of these proteins between nucleus and cytoplasm appears to be responsible for the vectorial nature of nucleocytoplasmic transport. Here, we summarize the role of Ran and Ran-binding proteins in nuclear trafficking of proteins with classical nuclear localisation signals. We also discuss examples of the growing number of alternative pathways that are involved in transport of proteins across the nuclear envelope. BioEssays 21:579–589, 1999. © 1999 John Wiley & Sons, Inc.     

The importin β binding domain as a master regulator of nucleocytoplasmic transport

Specific and efficient recognition of import cargoes is essential to ensure nucleocytoplasmic transport. To this end, the prototypical karyopherin importin β associates with import cargoes directly or, more commonly, through import adaptors, such as importin α and snurportin. Adaptor proteins bind the nuclear localization sequence (NLS) of import cargoes while recruiting importin β via an N-terminal importin β binding (IBB) domain. The use of adaptors greatly expands and amplifies the repertoire of cellular cargoes that importin β can efficiently import into the cell nucleus and allows for fine regulation of nuclear import. Accordingly, the IBB domain is a dedicated NLS, unique to adaptor proteins that functions as a molecular liaison between importin β and import cargoes. This review provides an overview of the molecular role played by the IBB domain in orchestrating nucleocytoplasmic transport. Recent work has determined that the IBB domain has specialized functions at every step of the import and export pathway. Unexpectedly, this stretch of ~ 40 amino acids plays an essential role in regulating processes such as formation of the import complex, docking and translocation through the nuclear pore complex (NPC), release of import cargoes into the cell nucleus and finally recycling of import adaptors and importin β into the cytoplasm. Thus, the IBB domain is a master regulator of nucleocytoplasmic transport, whose complex molecular function is only recently beginning to emerge. This article is part of a Special Issue entitled: Regulation of Signaling and Cellular Fate through Modulation of Nuclear Protein Import.     

Caspase-mediated cleavage of importin-alpha increases its affinity for MCM and downregulates DNA synthesis by interrupting the binding of MCM to chromatin

Importin-alpha is essential for classical nucleocytoplasmic transport of nuclear proteins. Here, we report that importin-alpha is cleaved by caspases during apoptosis, generating importin-alpha lacking an IBB domain. This truncated importin-alpha binds tightly to the MCM replication licensing factor and, thus, prevents its binding to chromatin and downregulates DNA synthesis. Together, our data reveal for the first time that a dying cell effectively salvages limited supplies of cellular energy to ensure an orderly process of its own demise by simultaneously downregulating nucleocytoplasmic protein transport and DNA synthesis. Strikingly, cells can achieve this multi-task process by simply cleaving-off a key nuclear import protein.     

Yeast Ran-binding protein Yrb1p is required for efficient proteolysis of cell cycle regulatory proteins Pds1p and Sic1p

Ubiquitin-dependent proteolysis of specific target proteins is required for several important steps during the cell cycle. Degradation of such proteins is strictly cell cycle-regulated and triggered by two large ubiquitin ligases, termed anaphase-promoting complex (APC) and Skp1/Cullin/F-box complex (SCF). Here we show that yeast Ran-binding protein 1 (Yrb1p), a predominantly cytoplasmic protein implicated in nucleocytoplasmic transport, is required for cell cycle regulated protein degradation. Depletion of Yrb1p results in the accumulation of unbudded G(1) cells and of cells arrested in mitosis implying a function of Yrb1p in the G(1)/S transition and in the progression through mitosis. Temperature-sensitive yrb1-51 mutants are defective in APC-mediated degradation of the anaphase inhibitor protein Pds1p and in degradation of the cyclin-dependent kinase inhibitor Sic1p, a target of SCF. Thus, Yrb1p is crucial for efficient APC- and SCF-mediated proteolysis of important cell cycle regulatory proteins. We have identified the UBS1 gene as a multicopy suppressor of yrb1-51 mutants. Ubs1p is a nuclear protein, and its deletion is synthetic lethal with a yrb1-51 mutation. Interestingly, UBS1 was previously identified as a multicopy suppressor of cdc34-2 mutants, which are defective in SCF activity. We suggest that Ubs1p may represent a link between nucleocytoplasmic transport and ubiquitin ligase activity.     

Rhinovirus 3C Protease Facilitates Specific Nucleoporin Cleavage and Mislocalisation of Nuclear Proteins in Infected Host Cells

Human Rhinovirus (HRV) infection results in shut down of essential cellular processes, in part through disruption of nucleocytoplasmic transport by cleavage of the nucleoporin proteins (Nups) that make up the host cell nuclear pore. Although the HRV genome encodes two proteases (2A and 3C) able to cleave host proteins such as Nup62, little is known regarding the specific contribution of each. Here we use transfected as well as HRV-infected cells to establish for the first time that 3C protease is most likely the mediator of cleavage of Nup153 during HRV infection, while Nup62 and Nup98 are likely to be targets of HRV2A protease. HRV16 3C protease was also able to elicit changes in the appearance and distribution of the nuclear speckle protein SC35 in transfected cells, implicating it as a key mediator of the mislocalisation of SC35 in HRV16-infected cells. In addition, 3C protease activity led to the redistribution of the nucleolin protein out of the nucleolus, but did not affect nuclear localisation of hnRNP proteins, implying that complete disruption of nucleocytoplasmic transport leading to relocalisation of hnRNP proteins from the nucleus to the cytoplasm in HRV-infected cells almost certainly requires 2A in addition to 3C protease. Thus, a specific role for HRV 3C protease in cleavage and mislocalisation of host cell nuclear proteins, in concert with 2A, is implicated for the first time in HRV pathogenesis.     

Rab-dependent cellular trafficking and amyotrophic lateral sclerosis

Rab GTPases are becoming increasingly implicated in neurodegenerative disorders, although their role in amyotrophic lateral sclerosis (ALS) has been somewhat overlooked. However, dysfunction of intracellular transport is gaining increasing attention as a pathogenic mechanism in ALS. Many previous studies have focused axonal trafficking, and the extreme length of axons in motor neurons may contribute to their unique susceptibility in this disorder. In contrast, the role of transport defects within the cell body has been relatively neglected. Similarly, whilst Rab GTPases control all intracellular membrane trafficking events, their role in ALS is poorly understood. Emerging evidence now highlights this family of proteins in ALS, particularly the discovery that C9orf72 functions in intra transport in conjunction with several Rab GTPases. Here, we summarize recent updates on cellular transport defects in ALS, with a focus on Rab GTPases and how their dysfunction may specifically target neurons and contribute to pathophysiology. We discuss the molecular mechanisms associated with dysfunction of Rab proteins in ALS. Finally, we also discuss dysfunction in other modes of transport recently implicated in ALS, including nucleocytoplasmic transport and the ER-mitochondrial contact regions (MAM compartment), and speculate whether these may also involve Rab GTPases.     

Heat shock protein 10 and signal transduction: a "capsula eburnea" of carcinogenesis?

To date, little is known either about the physical interactions of heat shock protein 10 (Hsp10) with other proteins within the cell or its involvement in signal transduction pathways. Hsp10 has been considered mainly as a partner of Hsp60 in the Hsp60/10 protein folding machine. Only recently, Hsp10 was reported to interact with proteins involved in deoxyribonucleic acid checkpoint inactivation, termination of M-phase, messenger ribonucleic acid export, import of nuclear proteins, nucleocytoplasmic transport, and pheromone signaling pathways. At the same time, Hsp10 expression can be up-regulated in cancer cells, because it accumulates as the cell transformation progresses. Recent data suggest that Hsp10 may be not only a component of the folding machine but also an active player of the cell signaling network, influencing cell cycle, nucleocytoplasmic transport, and metabolism, with putative roles in the lack of cell differentiation and in the inhibition of apoptosis. In this review, we revise the involvement of Hsp10 in signal transduction pathways and its possible role in cancer etiology.