Reticulons家族蛋白研究进展

Reticulons（RTNs）蛋白是一类广泛存在于真菌、植物及动物等真核生物的膜蛋白,主要定位于内质网,尤其是管状内质网,原核生物中至今尚未发现其同系物。哺乳动物基因组中有RTN1、RTN2、RTN3及RTN4共四类相互独立的基因,因启动子及选择性剪接方式不同每个基因可产生不同转录本。RTNs家族成员氨基端序列高度可变且大小显著不同,羧基端的约200个氨基酸残基则高度保守,被称为内质网蛋白同源结构域（reticulon-homology domain,RHD）。RTNs高度可变的氨基端赋予其各成员物种特异性及细胞特异性的功能,羧基端的RHD则是其执行基本细胞功能的基础。越来越多的研究结果表明,RTNs可参与蛋白转运、参与膜结构形态发生或稳定及细胞分裂、构成内质网膜通道或转运体、调节细胞凋亡、调节β分泌酶（β-site APP cleaving enzyme1,BACE1）活性及抑制神经再生等。该文就RTNs家族各成员基因和蛋白的结构特性以及功能特点的研究进展进行简要综述。     

RTNs家族研究进展

编码内质网蛋白家族的基因(RTNs)是一类广泛存在于真核生物的基因家族,它有着特殊的拓扑结构。RTNs蛋白的羧基端存在一个大约200个氨基酸的保守区(RHD),此保守区包含2个疏水区段。RTNs蛋白定位内质网膜上,高等脊椎动物中枢神经受损时,RTN4-A／Nogo-A起抑制神经生长的作用,RTNs蛋白家族其余成员的功能还不是很清楚,可能与胞内运输,细胞分裂和细胞凋亡等有关。现就RTNs成员的基本结构、表达分布、亚细胞定位、拓扑结构和RTNs蛋白的功能等进行综述。     

Nogo/RTN4 isoforms and RTN3 expression protect SH-SY5Y cells against multiple death insults

Among the members of the reticulon (RTN) family, Nogo-A/RTN4A, a prominent myelin-associated neurite growth inhibitory protein, and RTN3 are highly expressed in neurons. However, neuronal cell-autonomous functions of Nogo-A, as well as other members of the RTN family, are unclear. We show here that SH-SY5Y neuroblastoma cells stably over-expressing either two of the three major isoforms of Nogo/RTN4 (Nogo-A and Nogo-B) or a major isoform of RTN3 were protected against cell death induced by a battery of apoptosis-inducing agents (including serum deprivation, staurosporine, etoposide, and H₂O₂) compared to vector-transfected control cells. Nogo-A, -B, and RTN3 are particularly effective in terms of protection against H₂O₂-induced increase in intracellular reactive oxygen species levels and ensuing apoptotic and autophagic cell death. Expression of these RTNs upregulated basal levels of Bax, activated Bax, and activated caspase 3, but did not exhibit an enhanced ER stress response. The protective effect of RTNs is also not dependent on classical survival-promoting signaling pathways such as Akt and Erk kinase pathways. Neuron-enriched Nogo-A/Rtn4A and RTN3 may, therefore, exert a protective effect on neuronal cells against death stimuli, and elevation of their levels during injury may have a cell-autonomous survival-promoting function.     

RTN4-C基因在肝癌组织中的表达及其对SMMC7721细胞生长和凋亡的影响

RTN4-C属于编码内质网蛋白的基因家族(RTNs)。为研究RTN4-C在肝癌及其癌旁组织中的表达情况及其对SMMC7721肝癌细胞株的影响,利用RT-PCR检测RTN4-C在肝癌及其癌旁组织中的表达。构建RTN4-C-pcDNA3.1真核表达重组质粒,用脂质体介导转染SMMC7721肝癌细胞,通过G418稳定筛选,建立转染RTN4-C/SMMC7721稳定细胞株。MTT法测定转染前后细胞生长改变、吖啶橙染色检测细胞凋亡改变、免疫细胞化学检测肿瘤相关蛋白p53、Hsp70和c-Fos表达改变。结果表明,RTN4-C/SMMC7721细胞生长减慢,与对照组相比,第2、3、4d细胞生长抑制率分别为33·8%±0·026、56·2%±0·094、34·8%±0·077;凋亡明显增多。突变型p53蛋白从核内转移到细胞质且表达减弱,c-Fos、Hsp70蛋白表达量明显减弱。提示RTN4-C基因在肝癌组织中存在表达差异,促进突变型p53的核转移并减少其表达量、抑制癌基因c-Fos和Hsp70的表达,从而抑制SMMC7721细胞的生长,促进其凋亡。     

RTN4B interacting protein FAM134C promotes ER membrane curvature and has a functional role in autophagy

The endoplasmic reticulum (ER) is composed of a controlled ratio of sheets and tubules, which are maintained by several proteins with multiple functions. Reticulons (RTNs), especially RTN4, and DP1/Yop1p family members are known to induce ER membrane curvature. RTN4B is the main RTN4 isoform expressed in nonneuronal cells. In this study, we identified FAM134C as a RTN4B interacting protein in mammalian, nonneuronal cells. FAM134C localized specifically to the ER tubules and sheet edges. Ultrastructural analysis revealed that overexpression of FAM134C induced the formation of unbranched, long tubules or dense globular structures composed of heavily branched narrow tubules. In both cases, tubules were nonmotile. ER tubulation was dependent on the reticulon homology domain (RHD) close to the N-terminus. FAM134C plays a role in the autophagy pathway as its level elevated significantly upon amino acid starvation but not during ER stress. Moreover, FAM134C depletion reduced the number and size of autophagic structures and the amount of ER as a cargo within autophagic structures under starvation conditions. Dominant-negative expression of FAM134C forms with mutated RHD or LC3 interacting region also led to a reduced number of autophagic structures. Our results suggest that FAM134C provides a link between regulation of ER architecture and ER turnover by promoting ER tubulation required for subsequent ER fragmentation and engulfment into autophagosomes.     

Analysis of the reticulon gene family demonstrates the absence of the neurite growth inhibitor Nogo-A in fish

Reticulons (RTNs) are a family of evolutionary conserved proteinswith four RTN paralogs (RTN1, RTN2, RTN3, and RTN4) presentin land vertebrates. While the exact functions of RTN1 to RTN3are unknown, mammalian RTN4-A/Nogo-A was shown to inhibit theregeneration of severed axons in the mammalian central nervoussystem (CNS). This inhibitory function is exerted via two distinctregions, one within the Nogo-A–specific N-terminus andthe other in the conserved reticulon homology domain (RHD).In contrast to mammals, fish are capable of CNS axon regeneration.We performed detailed analyses of the fish rtn gene family todetermine whether this regeneration ability correlates withthe absence of the neurite growth inhibitory protein Nogo-A.A total of 7 rtn genes were identified in zebrafish, 6 in pufferfish,and 30 in eight additional fish species. Phylogenetic and syntenicrelationships indicate that the identified fish rtn genes areorthologs of mammalian RTN1, RTN2, RTN3, and RTN4 and that severalparalogous fish genes (e.g., rtn4 and rtn6) resulted from genomeduplication events early in actinopterygian evolution. Accordingly,sequences homologous to the conserved RTN4/Nogo RHD are presentin two fish genes, rtn4 and rtn6. However, sequences comparableto the first 1,000 amino acids of mammalian Nogo-A includinga major neurite growth inhibitory region are absent in zebrafish.This result is in accordance with functional data showing thataxon growth inhibitory molecules are less prominent in fisholigodendrocytes and CNS myelin compared to mammals.     

Mapping of interaction domains mediating binding between BACE1 and RTN/Nogo proteins

BACE1 is a membrane-bound aspartyl protease that specifically cleaves amyloid precursor protein (APP) at the beta-secretase site. Membrane bound reticulon (RTN) family proteins interact with BACE1 and negatively modulate BACE1 activity through preventing access of BACE1 to its cellular APP substrate. Here, we focused our study on RTN3 and further show that a C-terminal QID triplet conserved among mammalian RTN members is required for the binding of RTN to BACE1. Although RTN3 can form homo- or heterodimers in cells, BACE1 mainly binds to the RTN monomer and disruption of the QID triplet does not interfere with the dimerization. Correspondingly, the C-terminal region of BACE1 is required for the binding of BACE1 to RTNs. Furthermore, we show that the negative modulation of BACE1 by RTN3 relies on the binding of RTN3 to BACE1. The knowledge from this study may potentially guide discovery of small molecules that can mimic the effect of RTN3 on the inhibition of BACE1 activity.     

Reticulon 3 is involved in membrane trafficking between the endoplasmic reticulum and Golgi

Reticulons (RTNs) constitute a family of endoplasmic reticulum (ER)-associated proteins with a reticular distribution. Despite the implication of their neuronal isoforms in axonal regeneration, the function of their widely expressed isoforms is largely unknown. In this study, we examined the role of the ubiquitously expressed RTN3 in membrane trafficking. Ectopically expressed RTN3 exhibited heterogeneous patterns; filamentous, reticular, and granular distributions. The ER morphology changed accordingly. In cells where RTN3 displayed a filamentous/reticular distribution, protein transport between the ER and Golgi was blocked, and Golgi proteins were dispersed. In contrast, ERGIC-53, a marker for the ER-Golgi intermediate compartment, accumulated at the perinuclear region, and remained there even after cells were treated with agents that induce redistribution of Golgi proteins to the ER, indicating an inhibition of Golgi-to-ER transport of ERGIC-53. These results suggest that RTN3 plays a role in membrane trafficking in the early secretory pathway.     

Five Arabidopsis Reticulon Isoforms Share Endoplasmic Reticulum Location,Topology, and Membrane-Shaping Properties

The cortical endoplasmic reticulum (ER) in tobacco (Nicotiana tabacum) epidermal cells is a network of tubules and cisternae undergoing dramatic rearrangements. Reticulons are integral membrane proteins involved in shaping ER tubules. Here, we characterized the localization, topology, effect, and interactions of five Arabidopsis thaliana reticulons (RTNs), isoforms 1-4 and 13, in the cortical ER. Our results indicate that RTNLB13 and RTNLB1-4 colocate to and constrict the tubular ER membrane. All five RTNs preferentially accumulate on ER tubules and are excluded from ER cisternae. All isoforms share the same transmembrane topology, with N and C termini facing the cytosol and four transmembrane domains. We show by Förster resonance energy transfer and fluorescence lifetime imaging microscopy that several RTNs have the capacity to interact with themselves and each other, and we suggest that oligomerization is responsible for their residence in the ER membrane. We also show that a complete reticulon homology domain is required for both RTN residence in high-curvature ER membranes and ER tubule constriction, yet it is not necessary for homotypic interactions.     

A metal-binding site in the RTN1-C protein: new perspectives on the physiological role of a neuronal protein

Reticulon 1-C (RTN1-C) is an ER-associated neuronal protein characterized by horse-shoe-like topology with two transmembrane helices and the N- and C-terminal regions which are supposed in the cytosolic side of ER. The physiological role of this protein is not completely clarified, but several studies have suggested its involvement in the neuronal differentiation, membrane vesicle trafficking and induction of apoptosis. The C-terminal region of RTN1-C is characterized by the presence of a H4 histone consensus sequence that makes it able to interact with nucleic acids and HDAC enzymes both in vitro and in vivo. In the present study a potential metal ion binding motif (HxE/D) at the C-terminal of the RTN1-C has been identified and its capability to bind metals investigated by UV-vis, CD, multidimensional NMR spectroscopy and biological assays. The results suggest a possible implication of the metal ions in the mechanisms of formation of the recently observed RTNs multiprotein complexes contributing to understand the structure and function of this neuronal membrane protein, suggesting a possible effect of the metal binding property on its biological function.     

Phosphorylation-regulated cleavage of the reticulon protein Nogo-B by caspase-7 at a noncanonical recognition site

Reticulons (RTNs) are a large family of transmembrane proteins present throughout the eukaryotic domain in virtually every cell type. Despite their wide distribution, their function is still mostly unknown. RTN4, also termed Nogo, comes in three isoforms, Nogo-A, -B, and -C. While Nogo-A has been described as potent inhibitor of nerve growth, Nogo-B has been implicated in vascular remodeling and regulation of apoptosis. We show here that Nogo-B gets cleaved by caspase-7, but not caspase-3, during apoptosis at a caspase nonconsensus site. By a combination of MS and site-directed mutagenesis we demonstrate that proteolytic processing of Nogo-B is regulated by phosphorylation of Ser(16) within the cleavage site. We present cyclin-dependent kinase (Cdk)1 and Cdk2 as kinases that phosphorylate Nogo-B at Ser(16) in vitro. In vivo, cleavage of Nogo-B is markedly increased in Schwann cells in a lesion model of the rat sciatic nerve. Taken together, we identified an RTN protein as one out of a selected number of caspase targets during apoptosis and as a novel substrate for Cdk1 and 2. Furthermore, our data support a functionality of caspase-7 that is distinct from closely related caspase-3.     

Reticulon 2 promotes gastric cancer metastasis via activating endoplasmic reticulum Ca2+ efflux-mediated ERK signalling

Gastric cancer ranks fourth for mortality globally among various malignant tumours, and invasion and metastasis are the major reason leading to its poor prognosis. Recently, accumulating studies revealed the role of reticulon proteins in cell growth and transmigration. However, the expression and biological function of reticulon proteins in human gastric cancer remain largely unclear. Herein, we explored the potential role of reticulon 2 (RTN2) in the progression of gastric cancer. Tissue microarray was used to determine the expression levels of RTN2 in 267 gastric cancer patients by immunohistochemistry. Gastric cancer cell lines were utilised to examine the influences of RTN2 on cellular migration and invasion abilities, epithelial-to-mesenchymal transition (EMT) and signalling pathway. In vivo studies were also performed to detect the effect of RTN2 on tumour metastasis. We found that RTN2 expression was notably upregulated in tumour tissues compared to pericarcinomatous tissues. High RTN2 expression was positively correlated with patients’ age, vessel invasion, tumour invasion depth, lymph node metastasis and TNM stage. Besides, high RTN2 staining intensity was associated with adverse survival which was further identified as an independent prognostic factor for gastric cancer patients by multivariate analysis. And the predictive accuracy was also improved when incorporated RTN2 into the TNM-staging system. RTN2 could promote the proliferation, migration and invasion of gastric cancer cells in vitro and lung metastasis in vivo. Mechanistically, RTN2 interacted with IP3R, and activated ERK signalling pathway via facilitating Ca²⁺ release from the endoplasmic reticulum, and subsequently drove EMT in gastric cancer cells. These results proposed RTN2 as a novel promotor and potential molecular target for gastric cancer therapies.Subject terms: Gastric cancer, Calcium signalling     

双转基因小鼠模型中microRNA-153对RTN4的表达调控

目的探讨mir-153在阿尔茨海默病发病机制中的作用。方法通过microRNA芯片及Real-timePCR检测APPswe/PSΔE9双转基因小鼠脑内mir-153的表达;构建高表达mir-153的稳转细胞系,通过western-blot检测稳转细胞系内RTN4的蛋白表达;构建野生型及突变型RTN4的3’UTR荧光素酶报告载体,分别将其与mir-153表达载体或microRNA阴性对照载体共转染入293T细胞内,检测海肾荧光素酶的相对活性以验证mir-153对RTN4 mRNA的作用靶点。结果 microRNA芯片及Real-time PCR检测均证实3月龄APPswe/PSΔE9双转基因小鼠脑内mir-153的表达较同龄野生对照显著降低;在高表达mir-153的稳转细胞系内RTN4的蛋白水平明显降低;共转染野生型RTN4的3’UTR/mir-153表达载体可使海肾荧光素酶相对活性较共转染野生型RTN4的3’UTR/miRNA阴性对照质粒组显著降低（P〈0.05）,而共转染突变型RTN4的3’UTR/mir-153表达载体组则较之无明显差异。结论在3月龄APPswe/PSΔE9双转基因小鼠脑内存在mir-153的异常表达;mir-153可调控RTN4的蛋白表达;mir-153对RTN4的表达调控是通过结合RTN4 3’UTR 839-845碱基处的作用位点而实现的。     

Anti-apoptotic activity of Bcl-2 is enhanced by its interaction with RTN3

Bcl-2 is known as a critical inhibitor of apoptosis triggered by a broad range of stimuli, mainly acting on the mitochondria. It can interact with many members of the Bcl-2 family, influence mitochondrial membrane permeability and modulate cell apoptosis. RTN3, a member of the reticulon (RTN) family, was predominantly localized on the endoplasmic reticulum (ER). Its N- and C-termini, both facing the cytoplasm, can recruit some proteins to the ER to modulate some physiological functions. We found that RTN3, which does not belong to the Bcl-2 family, can interact with Bcl-2 on the ER. In normal HeLa cells, ectopic overexpressed Bcl-2 could reduce the cell apoptosis induced by overexpressed RTN3. When the HeLa cells stably expressing Bcl-2 were treated with tunicamycin, endogenous RTN3 increased in the cell microsomal fraction. This change increased the Bcl-2 in microsomal fractions and also in the mitochondrial fractions where the anti-apoptotic activity of Bcl-2 mainly acts. These results suggest that RTN3 could bind with Bcl-2 and mediate its accumulation in mitochondria, which modulate the anti-apoptotic activity of Bcl-2.     

RTN3与细胞凋亡

RTN3是RTN家族的成员之一,因其主要定位于内质网,所以用reticulon来命名.RTN3与RTN4B是RTN家族中目前已知的、唯一一对具有凋亡诱发功能的基因.过表达的RTN3介导了真核细胞的三大凋亡信号转导通路:死亡受体途径、线粒体途径、内质网途径,并使之交联形成凋亡调控网络;RTN3可与RTN4B相互作用,形成同源或异源二聚来调控细胞凋亡.另外,过表达RTN3还参与了细菌的类凋亡作用.RTN3广泛表达于多种组织,其过表达诱发凋亡的机制的总结将让人们更好的了解RTN3及其家族,完善细胞凋亡的信号转导研究.     

Multifunctional nanocomplexes for gene transfer and gene therapy

DNA formulated into aggregates with polycationic reagents are referred to by a variety of terms including non-viral vectors, synthetic vectors, lipoplexes, polyplexes and more recently nanoparticles. The capacity for delivery of multiple genes, genomic-sized constructs and siRNA delivery, with a diversity of possible formulations, as well as the possibilities of improved efficiency of in vivo gene deliveries, means that nanoparticles, or nanocomplexes to reflect self-assembling systems, will be investigated with increasing vigour in the coming years. This review briefly outlines the applications and challenges for nanoparticle technologies in the field of gene therapy then focuses on the development of a specific kind of formulation, receptor-targeted nanocomplex (RTN), that we have found to be particularly useful in our gene therapy research. An overriding guiding concept that has emerged in the development of synthetic nanodelivery systems is the idea to develop formulations and structures that mimic viruses, whilst retaining the safety elements of synthetic, non-viral systems. RTNs have been optimised and developed for airway epithelial transfection, leading towards gene therapy for cystic fibrosis and for vascular transfection in vein grafts used in bypass surgery. The modular design of the RTN platform further allows for the testing of specific hypotheses relating to the structure and functional role of components in the formation of stable particles and in the transfection pathway, leading to their ultimate disassembly in the nucleus.     

RTN4对于结肠癌细胞增殖的调控作用

2018年全球癌症统计调查显示,结直肠癌约占患癌新病例的12.1%。因此,寻找新的结肠癌发生有关的基因,发现新的治疗靶点显得尤为迫切。通过数据库分析发现,RTN4基因的表达水平与结肠癌患者生存率的相关性具有统计学意义。针对RTN4基因构建其干扰质粒,将慢病毒作为载体转染结肠癌HCT116细胞中构建敲低RTN4的结肠癌细胞系,最后检测了低表达后RTN4基因的细胞增殖。结果发现,敲低RTN4基因后显著促进了结肠癌细胞HCT116的增殖,研究通过Western blot观察敲低RTN4后HCT116细胞自噬通路相关蛋白p62和LC3的表达情况,发现与对照组相比较,敲低RTN4组LC3转化量（LC3-II/LC3-I）增多,而p62蛋白减少。研究分析了RTN4的潜在抑癌作用,发现敲低RTN4基因会显著增强结肠癌细胞的增殖能力,并且诱导自噬,说明RTN4可能与激活LC3/p62自噬途径有关。