冠状病毒入侵细胞途径的研究进展

近年来,冠状病毒严重威胁人类和动物的健康。病毒具有专性胞内寄生的特点,其需要利用细胞完成自身的增殖,因此入侵细胞的过程是其感染机制中非常重要的一部分。多项研究表明冠状病毒能通过细胞表面途径和内吞途径入侵细胞。通过对冠状病毒入侵途径研究有助于了解其生命周期,有利于冠状病毒的防治以及新型药物和疫苗的研发。本文对近年来不同冠状病毒的入侵途径及针对入侵途径所开发的潜在药物的相关研究进行总结,以期为全面了解冠状病毒的入侵方式提供参考。     

人偏肺病毒通过活化EGFR促进其入胞的机制研究

研究发现人偏肺病毒入胞过程有脂筏的参与,而EGFR是定位于胞膜脂筏上的一种跨膜糖蛋白,在多种病毒的感染中发挥重要作用,该文主要探索表皮生长因子受体(EGFR)在人偏肺病毒(hMPV)感染中的作用及其相关机制.利用人支气管上皮细胞16HBE,用Western blot检测细胞感染hMPV特定时间后EGFR的活化情况.使用...     

SARS-CoV-2入胞活细胞示踪平台的建立

新型冠状病毒（SARS-CoV-2）由于其高传染性已造成全球大流行。目前关于SARS-CoV-2依赖内吞途径进入细胞的研究偏向于药物抑制实验或固定样品的蛋白共定位实验,而对于其入胞时与细胞内吞结构相互作用的动力学机制研究较少。本研究首先基于慢病毒系统包装出可在生物安全二级实验室（BSL-2）进行操作的SARS-CoV-2假病毒,之后利用膜染料对假病毒的囊膜进行荧光标记,并进行鉴定。通过免疫荧光方法观察到假病毒与网格蛋白包被的内吞结构共定位,进一步在网格蛋白敲低的Caco-2细胞系上进行假病毒感染,检测到荧光素酶活性显著下降,这些结果确定了网格蛋白介导的内吞作用参与假病毒感染。最后基于单病毒示踪技术,通过共聚焦显微镜对假病毒入胞过程进行活细胞实时拍摄,选取两个具有代表性的假病毒粒子依赖网格途径入胞的典型视野,并对假病毒动力学进行分析。本研究成功建立了SARS-CoV-2假病毒入胞活细胞示踪平台,可应用于研究单个假病毒粒子入胞过程动力学机制。该平台对SARS-CoV-2入胞机制的研究具有重大意义。     

细胞内吞的研究进展

细胞外基质的各种分子经细胞膜进入真核细胞是一个复杂的过程。细胞内吞是通过细胞质膜的变形运动将细胞外物质转运入细胞内的过程。不同的细胞内吞途径需要不同的蛋白质分子参与,引起不同的信号转导通路。目前认为细胞内吞和膜转运是细胞对其信号转导过程的一种精密的组织安排,细胞内吞在细胞信号转导,维持机体动态平衡方面起着重要作用。细胞内吞途径通常可以分为网格蛋白依赖的内吞和非网格蛋白依赖的内吞,其中后者包括陷窝蛋白依赖和非陷窝蛋白依赖的内吞,以及巨胞饮介导的内吞。本文将就这几种主要细胞内吞途径及与细胞信号转导通路关系的研究进展予以介绍。     

细胞穿膜肽介导生物大分子入胞机制研究进展

生物大分子药物与传统治疗方式相比作用靶点具有高度的专一性,成为21世纪药物研发中最具发展前景的领域之一,但由于细胞膜的天然屏障作用致使许多潜在的胞内药物靶标无法应用于新药研究。细胞穿膜肽(cell-penetrating peptides,CPP)是一类具有穿膜功能的小分子短肽,可高效携带核酸、蛋白质等生物大分子穿过细胞膜进入胞质发挥功能,在介导生物大分子药物入胞上有着高效、低毒等诸多优势,但仍存在效率低、靶向性差等问题。CPP携带货物分子入胞的方式可以根据是否依赖能量分为直接入胞和内吞。直接入胞依据孔隙形成的方式不同分为四种模型:桶板模型、超环面模型、地毯模型和反向胶团模型。内吞则根据受体的不同又分为巨胞饮、网格蛋白介导的内吞、小窝蛋白介导的内吞、硫酸乙酰肝素蛋白聚糖介导的内吞以及神经毡蛋白-1介导的内吞。CPP自身的类型、浓度、效应分子的物理化学性质以及分子大小都会影响CPP的入胞过程,进而决定CPP携带生物大分子入胞的途径。对CPP介导生物大分子的入胞机制进行综述,为研究更加高效、靶向性强的CPP提供依据,从而推动其在生物、医学领域的应用。     

巨胞饮的机制及功能的研究进展

巨胞饮（macropinocytosis）是内吞的一种形式,指在某些因素刺激下,细胞膜皱褶形成大且不规则的原始内吞小泡,它们被称为巨胞饮体。巨胞饮体的直径一般为0．5～2μm,有时可达5μm。与其它内吞形式的小泡相比,巨胞饮体直径较大,为非选择性地内吞细胞外营养物质和液相大分子提供了一条有效途径。最近的研究表明,巨胞饮具有清除凋亡细胞、参与免疫反应、介导某些病原菌侵袭细胞、更新细胞膜等功能。     

融合蛋白与病毒入膜机制研究进展

包膜病毒感染细胞的第一步即病毒与靶细胞膜的融合,它由病毒包膜上的融合蛋白诱发,融合蛋白与受体分子相互作用后暴露出融合肽,它伸向靶膜使两膜紧密接近后,多肽周围的脂质分子进一步重排,通过中间态最后发生融合,本文将介绍近年来病毒融合蛋白及入膜机制研究进展。     

腺病毒感染及胞内运输途径的研究进展

目前关于腺病毒感染及胞内运输的分子机制研究主要来源于C亚群腺病毒在肿瘤细胞系中的研究结果。腺病毒对靶细胞的感染及胞内运输大致分为几步:病毒与细胞表面受体的特异结合,胞吞介导的病毒内化,病毒逃脱胞内体进入细胞质,病毒沿着微管运输至核孔,病毒基因组入核。病毒胞内运输效率极高,感染后1 h,80%以上的病毒基因组被送至核内。但是腺病毒胞内的运输方式会因以下几个因素变化而产生差异:靶细胞类型,细胞生理状态,病毒血清型。文中对腺病毒感染靶细胞及胞内运输的已有分子机制进行综述,为临床基因治疗用途的病毒载体研发提供思路。     

单病毒示踪SADS-CoV通过网格蛋白介导的内吞入胞

猪急性腹泻综合征冠状病毒（Swine acute diarrhoea syndrome coronavirus, SADS-CoV）是一种能够引起仔猪急性腹泻的新发冠状病毒,因其在体外广泛的物种嗜性,具有潜在的跨种传播风险。本研究首先利用单病毒示踪技术平台实现了对SADS-CoV入胞动态过程的可视化;接着,结合网格蛋白介导的内吞途径（Clathrin-mediated endocytosis, CME）抑制剂（氯丙嗪）和表皮生长因子通路底物15突变体质粒确定了SADS-CoV依赖CME的入胞路径。通过对SADS-CoV依赖CME的入胞动态事件进行可视化分析,发现SADS-CoV入胞后形成的网格蛋白包被的囊泡（Clathrin-coated vesicle, CCV）出现了去包被的现象,并进一步发现SADS-CoV依赖CME内化后可快速进入早期内体,同时CME介导SADS-CoV入胞后的CCV去包被现象是发生在早期内体中。综上,本研究基于单病毒示踪技术,解析了SADS-CoV依赖CME内化后快速进入早期内体中发生CCV去包被的动态过程,进一步加深了对SADS-CoV感染过程的理解,也为抗病毒药物的研发提供理论依据。     

丙型肝炎病毒入胞抑制剂的研究进展

丙肝感染是全球性的公共卫生问题之一,目前尚无预防丙肝病毒感染的疫苗,联合应用靶向丙肝不同复制阶段的药物,即所谓的"鸡尾酒"疗法可能会有更好的疗效。丙肝病毒进入宿主细胞的过程是药物干预的重要环节,这个过程是由丙肝病毒的包膜蛋白与宿主因子作用介导的,其中病毒的包膜蛋白包括E1、E2,宿主因子包括硫酸乙酰肝素(Heparan sulfate,HS)、膜蛋白CD81、B族I型清道夫受体(Scavenger receptor class B type I,SR-BI)、两种紧密连接蛋白Occludin(OCLD)和Claudin(CLDN)、低密度脂蛋白受体(Low densitity lipoprotein receptor,LDLR)、树突细胞特异性细胞间黏附分子-3-结合非整合素分子(Dendritic cell-specific ICAM-3-grab-bing nonintegrin,DCSIGN)、肝/淋巴细胞特异性细胞间粘附分子-3-结合整合素分子(Liver/lymph node specific ICAM-3-grabbing integrin,L-SIGN)和转铁蛋白1受体(Transferrin receptor 1,TfR1)等。病毒的包膜蛋白和这些宿主因子都可以作为靶向丙肝入胞抑制剂的作用靶点,许多研究表明丙肝入胞抑制剂作为一种新颖和有发展前景的化合物与作用于复制阶段药物联合应用能够更有效的治疗丙肝。本文综述了自20世纪90年代以来发现的靶点和靶向丙肝病毒进入宿主细胞的化合物。     

Targeting of nanoparticles to the clathrin-mediated endocytic pathway

Nanoparticles (NPs) are considered attractive carriers for gene therapy and drug delivery owing to their minor toxic effect and their ability to associate and internalize into mammalian cells. In this study, we compared the endocytosis into HeLa cells of NPs exposing either a negative or positive charge on their surface. The exposed charge significantly affected their ability to internalize as well as the cellular endocytosis mechanism utilized. Negatively charged NPs show an inferior rate of endocytosis and do not utilize the clathrin-mediated endocytosis pathway. On the other hand, positively charged NPs internalize rapidly via the clathrin-mediated pathway. When this pathway is blocked, NPs activate a compensatory endocytosis pathway that results in even higher accumulation of NPs. Overall, the addition of a positive charge to NPs may improve their potential as nanoparticulate carriers for drug delivery.     

Agonist-dependent internalization of the angiotensin II type one receptor (AT1): role of C-terminus phosphorylation in recruitment of beta-arrestins

β-Arrestins play a role in AT₁ endocytosis by binding the cytoplasmic, C-terminus region T332–S338, the major site of angiotensin II (Ang II)-induced phosphorylation. However, the processes responsible for recruiting β-arrestin to the activated receptor are poorly defined. In this study, we used CHO-K1 and HEK 293 cells expressing wild-type or mutant AT₁ to investigate two possibilities: activated AT₁ induces global relocation of β-arrestins to the plasma membrane or the phosphorylated C-terminus acts as bait to attract β-arrestins. Results obtained using high osmolarity and dominant-negative β-arrestin confirmed that internalization of AT₁ in both CHO-K1 and HEK 293 cells is predominately via clathrin-mediated endocytosis involving β-arrestin, and substitution of T332, S335, T336 and S338 with alanine to preclude phosphorylation markedly attenuated AT₁ internalization. Confocal microscopy revealed that wild-type AT₁ induced a time-dependent translocation of GFP-tagged β-arrestins 1 and 2 to the cell surface. In contrast, the TSTS/A mutant did not traffic β-arrestin 1 at all, and only trafficked β-arrestin 2 weakly. Results of rescue-type experiments were consistent with the idea that both β-arrestins are able to interact with the non-phosphorylated receptor, albeit with much lower affinity and β-arrestin 1 less so than β-arrestin 2. In conclusion, this study shows that the high affinity binding of β-arrestins to the phosphorylated C-terminus is the predominant mechanism of agonist-induced β-arrestin recruitment to the cell surface and AT₁ receptor.     

Influenza entry pathways in polarized MDCK cells

In non-polarized cell culture models, influenza virus has been shown to enter host cells via multiple endocytic pathways, including classical clathrin-mediated endocytic routes (CME), clathrin- and caveolae-independent routes and macropinocytosis. However, little is known about the entry route of influenza virus in differentiated epithelia, in vivo site of infection for influenza virus. Here, we show that in polarized Madin–Darby canine kidney type II (MDCK II) cells, influenza virus has a specific utilization of the clathrin-mediated endocytic pathway and requires Eps15 for host cell entry.     

Effects of dynamin inactivation on pathways of anthrax toxin uptake

Internalization and traffic to acidic endosomes of anthrax lethal factor (LF) and protective antigen (PA), bound to the anthrax toxin receptor (ATR), is required for LF translocation into the cytosol, where it can elicit its toxic effects. Dynamin is required for clathrin-mediated endocytosis, and long-term disruption of dynamin function blocks internalization of PA. We have used LFn-DTA, a surrogate of LF consisting of the N-terminal domain of LF fused to the catalytic subunit of diphtheria toxin, to differentiate the effects of acute and long-term block of dynamin function on LFn-DTA toxicity. Both forms of interference reduce LFn-DTA toxicity only partially, consistent with alternative routes for LFn-DTA endocytosis. In contrast, a long-term block of dynamin activity results in a further interference with LFn-DTA toxicity that is consistent with an altered endosomal environment, probably an increase in endosomal pH.     

Structure determination of clathrin coats to subnanometer resolution by single particle cryo-electron microscopy

Clathrin triskelions can assemble into lattices of different shapes, sizes and symmetries. For many years, the structures of clathrin lattices have been studied by single particle cryo-electron microscopy, which probed the architecture of the D6 hexagonal barrel clathrin coat at the molecular level. By introducing additional image processing steps we have recently produced a density map for the D6 barrel clathrin coat at subnanometer resolution, enabling us to generate an atomic model for this lattice [Fotin, A., Cheng, Y., Sliz, P., Grigorieff, N., Harrison, S.C., Kirchhausen, T., Walz, T., 2004. Molecular model for a complete clathrin lattice from electron cryomicroscopy. Nature 432, 573-579]. We describe in detail here the image processing steps that we have added to produce a density map at this high resolution. These procedures should be generally applicable and may thus help determine the structures of other large protein assemblies to higher resolution by single particle cryo-electron microscopy.     

Utilization of fluorescently-labeled tetracysteine-tagged proteins to study virus entry by live cell microscopy

Viruses exploit cellular machinery to gain entry and initiate their replication cycle within host cells. The development of methods to visualize virus entry in live cells has provided new insights to the cellular processes involved in virus entry and the intracellular locations where viral payloads are deposited. The use of fluorescently labeled virus and high-resolution microscopy is currently the method of choice to study virus entry in live cells. While fluorescent protein fusions (e.g. viral proteins fused to GFP) have been used, the labeling of viral proteins that contain a small tetracysteine (tc) tag with biarsenical fluorescent compounds (e.g. FlAsH, ReAsH, Lumio-x) offers several advantages over conventional xFP-fusion constructs. This article describes methods for generating fluorescently labeled viruses encoding tc-tagged proteins that are suitable for the study of virus entry in live cells by fluorescence microscopy. Critical parameters required to quantify fluorescence signals from the labeled, tc-tagged proteins in individual virus particles during the entry process and the subsequent fate of the labeled viral proteins after virus uncoating are also described.     

Missing-in-metastasis protein promotes internalization of magnetic nanoparticles via association with clathrin light chain and Rab7

Background

Magnetic nanoparticles (MNPs) have been widely used in biomedical applications. Proper control of the duration of MNPs in circulation promises to improve further their applications, in particularly drug delivery. It is known that the uptake of tissue-associated MNPs is mainly carried out by macrophages. Yet, the molecular mechanism to control MNPs internalization in macrophages remains to be elusive. Missing-in-metastasis (MIM) is a scaffolding protein that is highly expressed in macrophages and regulates receptor-mediated endocytosis. We hypothesize that uptake of MNPs may also involve the function of MIM.

Towards understanding the molecular mechanism of the endocytosis-like process in the bacterium Gemmata obscuriglobus

An endocytosis-like process of protein uptake in the planctomycete Gemmata obscuriglobus is a recently discovered process unprecedented in the bacterial world. The molecular mechanisms underlying this process are not yet characterized. A homolog of the MC (membrane-coating) proteins of eukaryotes has been proposed to be involved in the mechanism of this process, but its relationship to eukaryote proteins is controversial. However, a number of other proteins of G. obscuriglobus with domains homologous to those involved in endocytosis in eukaryotes can also be identified. Here we critically evaluate current bioinformatic knowledge, and suggest practical experimental steps to overcome the limits of bioinformatics in elucidating the molecular mechanism of endocytosis in bacteria. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey.     

Alcohol consumption impairs hepatic protein trafficking: mechanisms and consequences

Alcoholic liver disease is a major biomedical health concern in the United States. Despite considerable research efforts aimed at understanding the progression of the disease, the specific mechanisms leading to alcohol-induced damage remain elusive. Numerous proteins are known to have alcohol-induced alterations in their dynamics. Defining these defects in protein trafficking is an active area of research. In general, two trafficking pathways are affected: transport of newly synthesized secretory or membrane glycoproteins from the Golgi to the basolateral membrane and clathrin-mediated endocytosis from the sinusoidal surface. Both impaired secretion and internalization require ethanol metabolism and are likely mediated by acetaldehyde. Although the mechanisms by which ethanol exposure impairs protein trafficking are not fully understood, recent work implicates alcohol-induced modifications on tubulin or components of the clathrin machinery as potential mediators. Furthermore, the physiological ramifications of impaired protein trafficking are not fully understood. In this review, we will list and discuss the proteins whose trafficking patterns are known to be impaired by ethanol exposure. We will then describe what is known about the possible mechanisms leading to impaired protein trafficking and how disrupted protein trafficking alters liver function and may explain clinical features of the alcoholic patient.     

Inhibition of dynamin prevents CCL2-mediated endocytosis of CCR2 and activation of ERK1/2

The magnitude and duration of G protein-coupled receptor (GPCR) signals are regulated through desensitization mechanisms. In leukocytes, ligand binding to chemokine receptors leads to Ca²⁺ mobilization and ERK activation through pertussis toxin-sensitive G proteins, as well as to phosphorylation of the GPCR. After interaction with the endocytic machinery (clathrin, adaptin), the adaptor β-arrestin recognizes the phosphorylated GPCR tail and quenches signaling to receptors. The molecular mechanisms that lead to receptor endocytosis are not universal amongst the GPCR, however, and the precise spatial and temporal events in the internalization of the CCR2 chemokine receptor remain unknown. Here we show that after ligand binding, CCR2 internalizes rapidly and reaches early endosomes, and later, lysosomes. Knockdown of clathrin by RNA interference impairs CCR2 internalization, as does treatment with the dynamin inhibitor, dynasore. Our results show that CCR2 internalization uses a combination of clathrin-dependent and -independent pathways, as observed for other chemokine receptors. Moreover, the use of dynasore allowed us to confirm the existence of a dynamin-sensitive element that regulates ERK1/2 activation. Our results indicate additional complexity in the link between receptor internalization and cell signaling.