真核细胞非经典蛋白分泌途径

在生物体中, 细胞间的信息传递是细胞生长、分化、发育、增殖、凋亡等生命活动的基本保证, 而蛋白分泌是细胞间信息传递的重要方式。大多数分泌蛋白都是通过内质网-高尔基体(ER-Golgi)途径分泌的。然而越来越多的研究表明, 存在着一类无信号肽的分泌蛋白, 这类蛋白不依赖ER-Golgi途径就能分泌到细胞外发挥功能, 被称为非经典分泌蛋白。非经典蛋白的分泌有其特有的机制, 它对ER-Golgi分泌途径是一种必要和有益的补充。非经典分泌与细胞增殖、免疫反应、肿瘤形成、传染病病理学等密切相关。文章旨在对非经典分泌蛋白的特点、分泌机制及生物学意义进行概述。     

信号肽序列及其在蛋白质表达中的应用

信号肽在蛋白分泌的过程中起重要作用,分泌性蛋白质合成后由信号肽引导其穿过合成所在的细胞到其他组织细胞中。可以利用因特网在线工具和信号序列捕获系统来判定基因序列中是否含有信号肽序列。外源蛋白的表达形式多为细胞内不溶性表达(包涵体),少数为细胞外分泌表达。利用信号肽来引导外源蛋白分泌可避免因包涵体复性带来的困难。研究表明,多种外源基因连接上信号肽后在原核表达系统如大肠杆菌、L型细菌、芽孢杆菌和乳酸杆菌中等都得到了分泌表达;信号肽也广泛应用于真核表达系统如毕赤酵母和昆虫杆状病毒表达系统,以提高蛋白的表达量。     

全基因组预测糙皮侧耳经典胞外分泌蛋白

糙皮侧耳(平菇)作为目前木质纤维素降解模式真菌,其产生的胞外分泌蛋白在植物纤维素、半纤维素及木质素降解过程中发挥着重要作用。为了给蛋白质组学鉴定糙皮侧耳胞外分泌蛋白实验提供参考,利用常见的生物信息学工具SignalP、ProtComp、TMHMM、big-PI Fungal Predictor和TargetP对糙皮侧耳全基因组中的12 186条蛋白质序列进行经典分泌蛋白预测,同时,对上述分泌蛋白的氨基酸分布、信号肽长度和切割位点以及其理化性质等进行分析。结果表明,糙皮侧耳全基因组中含有359个经典分泌蛋白,其氨基酸长度主要集中于101~600之间;信号肽长度集中在18~21之间;信号肽切割位点属于A-X-A类型,除此之外还含有5条具有RR-motif的信号肽。对这些分泌蛋白进行功能注释,结果表明225个蛋白质为未知功能蛋白质;114个为已知功能蛋白质,主要功能注释集中于木质纤维素降解酶类,包括纤维素酶、半纤维素酶、木质素降解相关酶等。以上结果表明通过上述生物信息学分析实现了对糙皮侧耳全基因组经典分泌蛋白的有效预测。     

嗜酸氧化亚铁硫杆菌基因组分泌蛋白的初步分析

利用信号肽预测软件SignalP v3.0、跨膜螺旋结构预测软件TMHMM v2.0和非经典分泌蛋白预测软件SecretomeP对嗜酸氧化亚铁硫杆菌全基因组的3 218个氨基酸序列进行预测分析.结果表明在嗜酸氧化亚铁硫杆菌中有507个蛋白为分泌蛋白,其中分泌型信号肽120个(其中有9个为RR-motif亚组型信号肽),脂蛋白信号肽3个,Prepilin-like信号肽4个,非经典分泌蛋白380个.并对分泌型信号肽的长度分布、氨基酸使用频率和酶切位点的氨基酸使用频率作了统计.得分最高的100个非经典分泌蛋白中,有36个具有功能分类,主要是参与细胞壁、能量代谢及转运和结合的蛋白质.嗜酸氧化亚铁硫杆菌的这507个分泌蛋白所参与的生化过程可能发生在膜外的周质空间或是菌体外的场所,为该物种与矿物相互作用,以及对环境做出响应服务.     

稻瘟病菌含信号肽小蛋白的计算机分析

结合计算机技术和生物信息学的方法,采用组合的信号肽分析软件SignalPv3.0、TargetPv1.1、Big-PIpredictor、TMHMMv2.0和SecretomeP对已公布的1486个稻瘟菌(magnaporthegrisea)小蛋白基因的N-端氨基酸序列进行信号肽分析,同时系统分析了信号肽的类型及结构。分析结果表明,在1486个稻瘟病菌小蛋白中,119个具有N-端信号肽的典型分泌蛋白。其中116个具有分泌型信号肽,1个具RR-motif型信号肽,2个具信号肽酶II型信号肽。在稻瘟病菌基因组中,分泌型小蛋白的序列是高度趋异的,仅出现少数氨基酸组成完全一致的信号肽,为进一步确认具有相同信号肽的分泌蛋白是否具有同源性,分别用BLAST2SEQUENCES对具有相同信号肽的分泌蛋白进行了序列对比。结果表明,具有相同信号肽的分泌蛋白同源性非常高。同时还采用Sublocv1.0对1486个小蛋白的亚细胞位置进行了预测,结果显示小蛋白的可能功能场所包括细胞质、细胞外、线立体和细胞核,功能场所位于细胞核的小蛋白是最多的。     

哺乳动物非经典分泌信号肽介导重组EGFP蛋白向毕赤酵母细胞壁转运

为探索哺乳动物非经典分泌信号肽在毕赤酵母表达系统中引导重组蛋白分泌的作用,本研究将一段来源于小鼠同源异型框蛋白(En2)的分泌信号序列(SS)融合至EGFP蛋白的N端,在毕赤酵母中表达。实验结果显示SS信号肽能通过一种不同于经典的内质网-高尔基体分泌通路的方式将EGFP蛋白分泌至细胞膜表面,与α交配因子前导肽相比,显著降低了细胞的内质网压力。本研究提示哺乳动物非经典分泌信号肽可作为递送重组蛋白至酵母膜表面的一项工具。     

利用非经典分泌蛋白质实现脂肪酶A的分泌表达

【目的】以枯草芽孢杆菌脂肪酶A(Lipase A)为报告蛋白,尝试利用4种非经典分泌蛋白质及其前50个氨基酸作为分泌信号以实现其分泌表达。【方法】我们扩增了脂肪酶A的编码基因和非经典分泌蛋白质的编码序列,构建了8种针对脂肪酶A的分泌表达载体,并转化至枯草芽孢杆菌WB800菌株,通过测定重组菌株的酶活、利用蛋白质电泳和免疫印迹等技术检测脂肪酶A的分泌情况【结果】以Pdh A的氨基酸序列和Sod A、Eno的前50氨基酸序列作为分泌信号的重组菌株较好的实现了脂肪酶A的分泌表达。【结论】部分非经分泌蛋白质的编码基因或其前50个氨基酸序列能够引导脂肪酶A分泌至细胞外。     

内生菌KM-1-2全基因组ORFs信号肽和分泌蛋白预测及功能分析

【目的】内生菌普遍存在于植物中,与宿主在长期的进化中形成了互利共生的关系。目前对内生菌和植物之间的互作机制研究较少,为深入了解银杏叶内生菌KM-1-2与寄主植物作用机制,本研究对其分泌蛋白进行预测,并明确其特征。【方法】组合使用信号肽分析软件Signal P,跨膜螺旋结构分析软件TMHMM 2.0和Phobius,蛋白质细胞定位软件PSORT,亚细胞定位软件Target P和GPI锚定位点分析软件big-PI Predictor,预测KM-1-2基因组范围内所有分泌蛋白,定义为分泌组。【结果】KM-1-2全基因组5299条蛋白序列中发现271个具有典型信号肽的分泌蛋白,占全基因组的2.4%;编码这些蛋白的ORF最短为61 bp,最大为2105 bp,平均为373 bp;引导它们的信号肽长度分布在15–37 aa之间,平均为24 aa。信号肽中出现频率最高的氨基酸依次为丙氨酸、亮氨酸和缬氨酸,信号肽切割类型多属于A-X-A型,即SPI切割类型。共66个蛋白质有功能描述,其中包括26个酶类。这些酶主要包括各种糖苷水解酶、酯酶、蛋白酶、碳氧裂解酶等。【结论】通过上述生物信息学分析方法有效实现了银杏叶内生菌KM-1-2分泌蛋白的预测,这些分泌蛋白功能涉及较多的酶类以及其他未知功能,为进一步研究内生菌和植物的互作提供了基础。     

丝状真菌胞外蛋白高效分泌机制的研究进展

丝状真菌由于其胞外蛋白分泌的高效性,成为生产酶制剂的高效细胞工厂.近年来针对真核生物胞外蛋白分泌途径的研究发现,丝状真菌蛋白的分泌途径相比其他真核生物具有高效分泌的特性.为了研究丝状真菌高效分泌的机制,本文总结了近年来丝状真菌分泌途径的最新研究进展,并且选取了分泌途径中关键环节的数种蛋白进行分析,通过与其他真核生物相关蛋白进行结构与序列比对,推测了丝状真菌胞外蛋白高效分泌的可能机制.     

Caspase-1介导的蛋白质分泌机制新进展

此前多数观点认为内质网一高尔基体运输是经典的分泌型蛋白质分泌出胞的途径。它们要先翻译出一段信号肽，这段信号肽将mRNA连同核糖体转移到内质网上并使合成的蛋白质保存于内质网内，而后通过高尔基体分泌出胞。     

SPRED: A machine learning approach for the identification of classical and non-classical secretory proteins in mammalian genomes

Eukaryotic protein secretion generally occurs via the classical secretory pathway that traverses the ER and Golgi apparatus. Secreted proteins usually contain a signal sequence with all the essential information required to target them for secretion. However, some proteins like fibroblast growth factors (FGF-1, FGF-2), interleukins (IL-1 alpha, IL-1 beta), galectins and thioredoxin are exported by an alternative pathway. This is known as leaderless or non-classical secretion and works without a signal sequence. Most computational methods for the identification of secretory proteins use the signal peptide as indicator and are therefore not able to identify substrates of non-classical secretion. In this work, we report a random forest method, SPRED, to identify secretory proteins from protein sequences irrespective of N-terminal signal peptides, thus allowing also correct classification of non-classical secretory proteins. Training was performed on a dataset containing 600 extracellular proteins and 600 cytoplasmic and/or nuclear proteins. The algorithm was tested on 180 extracellular proteins and 1380 cytoplasmic and/or nuclear proteins. We obtained 85.92% accuracy from training and 82.18% accuracy from testing. Since SPRED does not use N-terminal signals, it can detect non-classical secreted proteins by filtering those secreted proteins with an N-terminal signal by using SignalP. SPRED predicted 15 out of 19 experimentally verified non-classical secretory proteins. By scanning the entire human proteome we identified 566 protein sequences potentially undergoing non-classical secretion. The dataset and standalone version of the SPRED software is available at http://www.inb.uni-luebeck.de/tools-demos/spred/spred.     

The Secretion of an Intrinsically Disordered Protein with Different Secretion Signals in Bacillus subtilis

In this study, a naturally unsecretory intrinsically disordered domain of nucleoskeletal-like protein (Nsp) was attempted to be secreted with different types of secretion signals in Bacillus subtilis. The results showed that Nsp can be secreted efficiently by all selected Sec-type signal peptides. Nsp was successfully exported when fused to Tat-type signal peptides but less efficient than Sec-type. The fusion protein with the non-classical extracellular proteins can be detected in the cell and extracellular milieu. This study further demonstrated that the mature protein plays an important role in protein secretion. Moreover, these results indicated that Nsp could be a useful tool to understand the individual roles of mature proteins and signal peptide in protein secretion, to evaluate the effect of conformation of mature proteins on their export pathway when coupled with Tat-type signal peptide, and to seek the signal of non-classical secretory proteins.     

How are the Non-classically Secreted Bacterial Proteins Released into the Extracellular Milieu?

Most bacterial proteins that are destined to leave the cytoplasm are exported across the cell membrane to their sites of function. These proteins are generally exported via the classical secretion pathway, in which the signal peptide plays a central role. However, some bacterial proteins have been found in the extracellular milieu without any apparent signal peptide. As none of the classical secretion systems is involved in their secretion, this occurrence is termed non-classical protein secretion. The mechanism or mechanisms responsible for non-classical secretion are contentious. This review compiles evidence from the debate over whether the release of the non-classically secreted proteins is the result of cell lysis and discusses how these proteins are exported to the exterior of the cell.     

Genetic screen for signal peptides in Hydra reveals novel secreted proteins and evidence for non-classical protein secretion

We have screened a Hydra cDNA library for sequences encoding N-terminal signal peptides using the yeast invertase secretion vector pSUC [Jacobs et al., 1997. A genetic selection for isolating cDNAs encoding secreted proteins. Gene 198, 289-296]. We isolated and sequenced 907 positive clones; 88% encoded signal peptides; 12% lacked signal peptides. By searching the Hydra EST database we identified full-length sequences for the selected clones. These encoded 37 known proteins with signal peptides and 40 novel Hydra-specific proteins with signal peptides. Localization of two signal peptide-containing sequences, VEGF and ferritin, to the secretory pathway was confirmed with GFP fusion proteins. In addition, we isolated 105 clones which lacked signal peptides but which supported invertase secretion from yeast. Isolation of plasmids from these clones and retransformation in invertase-negative yeast cells confirmed the phenotype. A GFP fusion protein of one such clone encoding the foot morphogen pedibin was localized to the cytoplasm in transfected Hydra cells and did not enter the ER/Golgi secretory pathway. Secretion of pedibin and other proteins lacking signal peptides appears to occur by a non-classical protein secretion route.     

Extracellular sunflower proteins: evidence on non-classical secretion of a jacalin-related lectin

Extracellular proteins from sunflower seedlings were analyzed by electrophoresis followed by peptide mass fingerprinting. Tentative identification revealed novel proteins for this crop. A significant number of those proteins were not expected to be extracellular because they lacked the typical signal peptide responsible for secretion. In silico analysis showed that some members of this group presented the characteristic disordered structures of certain non-classical and leaderless mammalian secretory proteins. Among these proteins, a putative jacalin-related lectin (Helja) with a mannose binding domain was further isolated from extracellular fluids by mannose-affinity chromatography, thus validating its identification. Besides, immunolocalization assays confirmed its extracellular location. These results showed that a lectin, not predicted to be secreted in strict requirement of the N-terminal signal peptide, occurs in a sunflower extracellular compartment. The implications of this finding are discussed.     

Protein secretion in bacteria.

Most secretory proteins are synthesized as precursors with an amino-terminal signal peptide. Genetic identification of proteins essential for signal peptide dependent translocation to the Escherichia coli periplasm has led to the biochemical dissection of the secretion pathway. Additional mechanisms exist in Gram-negative bacteria for protein secretion to the extracellular environment.     

肝细胞生成素——一种非经典分泌蛋白

肝细胞生成素(hepatopoietin ,HPO)是一种分泌蛋白.为了研究肝细胞生成素的分泌途径,利用SignalP软件分析了HPO的氨基酸序列,但HPO序列中没有经典分泌蛋白的信号肽.Western印迹实验证明,HPO能以双体形式从细胞中分泌出来.特异性体外阻断实验表明,布雷菲尔德菌素A(brefeldinA)和莫能菌素(monensin)都不能阻断HPO的分泌,说明HPO并不通过经典的内质网 高尔基体(ER -Golgi)途径分泌;优降糖(glyburide)对HPO的分泌没有抑制作用,说明HPO的分泌并不是由ABC1(ATP bindingcassette)转运子介导的;DNP和NH4Cl也不能刺激HPO的分泌,说明内体 溶酶体系统不参与HPO的分泌.上述结果表明,HPO是一种非经典分泌蛋白(non classicalsecretoryprotein) ,能以双体形式从细胞中分泌出来.但和已知的非经典分泌蛋白IL -1β不同,HPO的分泌并不是通过ABC1转运子介导的,内体 溶酶体系统也不参与其分泌.     

Genetic screen for signal peptides in Hydra reveals novel secreted proteins and evidence for non-classical protein secretion

We have screened a Hydra cDNA library for sequences encoding N-terminal signal peptides using the yeast invertase secretion vector pSUC [Jacobs et al., 1997. A genetic selection for isolating cDNAs encoding secreted proteins. Gene 198, 289–296]. We isolated and sequenced 907 positive clones; 88% encoded signal peptides; 12% lacked signal peptides. By searching the Hydra EST database we identified full-length sequences for the selected clones. These encoded 37 known proteins with signal peptides and 40 novel Hydra-specific proteins with signal peptides. Localization of two signal peptide-containing sequences, VEGF and ferritin, to the secretory pathway was confirmed with GFP fusion proteins. In addition, we isolated 105 clones which lacked signal peptides but which supported invertase secretion from yeast. Isolation of plasmids from these clones and retransformation in invertase-negative yeast cells confirmed the phenotype. A GFP fusion protein of one such clone encoding the foot morphogen pedibin was localized to the cytoplasm in transfected Hydra cells and did not enter the ER/Golgi secretory pathway. Secretion of pedibin and other proteins lacking signal peptides appears to occur by a non-classical protein secretion route.     

Comparison of signal peptides for efficient protein secretion in the baculovirus-silkworm system

The baculovirus-silkworm expression system is widely used as a mass production system for recombinant secretory proteins. However, the final yields of some recombinant proteins are not sufficient for industrial use. In this study, we focused on the signal peptide as a key factor for improving the efficiency of protein production. Endoplasmic reticulum (ER) translocation of newly synthesized proteins is the first stage of the secretion pathway; therefore, the selection of an efficient signal peptide would lead to the efficient secretion of recombinant proteins. The Drosophila Bip and honeybee melittin signal peptides have often been used in this system, but to the best of our knowledge, there has been no study comparing secretion efficiency between exogenous and endogenous signal peptides. In this study we employed signal peptides from 30K Da and SP2 proteins as endogenous signals, and compared secretion efficiency with those of exogenous or synthetic origins. We have found that the endogenous secretory signal from the 30K Da protein is the most efficient for recombinant secretory protein production in the baculovirus-silkworm expression system.     

Human secretory signal peptide description by hidden Markov model and generation of a strong artificial signal peptide for secreted protein expression

A hidden Markov model (HMM) has been used to describe, predict, identify, and generate secretory signal peptide sequences. The relative strengths of artificial secretory signals emitted from the human signal peptide HMM (SP-HMM) correlate with their HMM bit scores as determined by their effectiveness to direct alkaline phosphatase secretion. The nature of the signal strength is in effect the closeness to the consensus. The HMM bit score of 8 is experimentally determined to be the threshold for discriminating signal sequences from non-secretory ones. An artificial SP-HMM generated signal sequence of the maximum model bit score (HMM + 38) was selected as an ideal human signal sequence. This signal peptide (secrecon) directs strong protein secretion and expression. We further ranked the signal strengths of the signal peptides of the known human secretory proteins by SP-HMM bit scores. The applications of high-bit scoring HMM signals in recombinant protein production and protein engineering are discussed.