Contrasting secretory processing of simultaneously expressed heterologous proteins in Saccharomyces cerevisiae

In this study, secretory processing of cell-surface displayed Aga2p fusions to bovine pancreatic trypsin inhibitor (BPTI) and the single chain Fv (scFv) antibody fragment D1.3 are examined. BPTI is more efficiently processed than D1.3 both when secreted and surface-displayed, and D1.3 expression imparts a greater amount of secretory stress on the cell as assayed by a reporter of the unfolded protein response (UPR). Surprisingly, simultaneous expression of the two proteins in the same cell somewhat improves BPTI surface display while decreasing D1.3 surface display with minimal effect on UPR activation. Furthermore, co-expression leads to the accumulation of punctate vacuolar aggregates of D1.3 and increased secretion of the D1.3-Aga2p fusion into the supernatant. Overexpression of the folding chaperones protein disulfide isomerase (PDI) and BiP largely mitigates the D1.3 surface expression decrease, suggesting that changes in vacuolar and cell surface targeting may be due, in part, to folding inefficiency. Titration of constitutive UPR expression across a broad range progressively decreases surface display of both proteins as UPR increases. D1.3-Aga2p traffic through the late secretory pathway appears to be strongly affected by overall secretory load as well as folding conditions in the ER.     

Protein targeting and secretion in filamentous fungi

Although the application of filamentous fungi, such asAspergillus niger for the production of extracellular proteins is well established for several decades, hardly any information is available about the molecular mechanisms of the process of protein secretion in these organisms.Two lines of research initiated towards a systematic analysis of the mechanism of protein targeting and secretion are presented in this paper.1 — To study routing and targeting of proteins in filamentous fungi the availability of a versatile reporter/carrier protein will be of considerable importance. Experiments towards the identification of such a protein are presented.2 — In analogy to the situation inSaccharomyces cerevisiae, the availability of defined (conditional) mutations in the secretion pathway will provide very important information about the organisation of the pathway. Therefore, based on results obtained inS. cerevisiae, the cloning of several fungal secretion genes was started. The results of the cloning and characterisation of one of these genes is presented.     

后基因组时代的丝状真菌基因组学与代谢工程

丝状真菌不仅是传统发酵工业中抗生素、酶制剂和有机酸的主要生产者,而且也是代谢工程育种中异源蛋白表达的重要细胞工厂。丝状真菌的遗传修饰和代谢工程研究是现代工业生物技术领域最具活力的研究方向之一。特别是与细菌和酵母相比,丝状真菌在细胞生长、营养需求、环境适应性、翻译后修饰、蛋白分泌能力和生物安全性等方面具有显著的优势。文章综述了丝状真菌作为异源蛋白表达系统在基因组学技术研究和代谢工程研究方面的最新进展。作者在分析丝状真菌基因组结构、特点的基础上,阐述了比较基因组学、蛋白质组学、转录组学和代谢组学等对丝状真菌的代谢途径重构、新型蛋白挖掘和代谢工程育种中的作用和意义。另一方面,作者分析了丝状真菌在表达外源蛋白时遇到的瓶颈问题,总结了丝状真菌代谢工程育种中的常用策略包括异源基因的融合表达、反义核酸技术、蛋白分泌途径改造、密码子优化和蛋白酶缺陷宿主的选育等技术和手段。最后,对该领域的发展趋势进行了展望。     

Early endosomes motility in filamentous fungi: How and why they move

Elongate hyphae of filamentous fungi grow predominantly at their tips, whereas organelles are positioned in the subapical parts of the cell. Organelle positioning and long-distance intracellular communication involves active, energy-dependent transport along microtubules (MTs). This is mediated by specialized molecular motors, named kinesins and dynein, which utilize ATP hydrolysis to “walk” along the tubulin polymers. Work in the basidiomycete Ustilago maydis and the ascomycete Aspergillus nidulans has shown that early endosomes (EEs) are one of the major cargos of MT-dependent motors in fungi. EEs are part of the early endocytic pathway, and their motility behavior and the underlying transport machinery is well understood. However, the physiological role of constant bi-directional EE motility remains elusive. Recent reports, conducted in the corn smut fungus U. maydis, have provided novel insights into the cellular function of EE motility. They show that EE motility is crucial for the distribution of the protein synthesis machinery, and also that EEs transmit signals during plant infection that trigger the production of fungal effector proteins, required for successful invasion into host plants.     

未折叠蛋白反应在胰腺癌发生发展中的作用及意义

胰腺癌是一种致死率相当高的消化系统肿瘤,其起病隐蔽导致早期诊断困难。近期研究发现,内质网应激 (endoplasmic reticulum stress,ERS) 状态下的未折叠蛋白反应 (unfolded protein response,UPR) 通路的调节作用,对于胰腺癌发生发展至关重要。UPR通路伴侣蛋白 GRP78 抑制了胰腺导管腺癌 (pancreatic adenocarcinoma,PDAC)细胞的凋亡,并增强了其化学抗性和耐药性。而 UPR 途径及其调节因子对于血管内皮生长因子 (vascular endothelial growth factor,VEGF) 的调节作用,有助于胰腺癌抵抗缺血缺氧环境。尝试靶向 UPR 途径关键调节因子的药物来控制胰腺癌的研究,可以为胰腺癌的治疗开辟新的途径。本文通过对近年来 UPR 在胰腺癌发生发展中的作用及意义进行综述,希望为通过调控 UPR 通路作为针对治疗胰腺癌的关键过程的一种新型抗癌方法研究提供参考。     

The unfolded protein response in hereditary haemochromatosis

To cope with the accumulation of unfolded or misfolded proteins the endoplasmic reticulum (ER) has evolved specific signalling pathways collectively called the unfolded protein response (UPR). Elucidation of the mechanisms governing ER stress signalling has linked this response to the regulation of diverse physiologic processes as well as to the progression of a number of diseases. Interest in hereditary haemochromatosis (HH) has focused on the study of proteins implicated in iron homeostasis and on the identification of new alleles related with the disease. HFE has been amongst the preferred targets of interest, since the discovery that its C282Y mutation was associated with HH. However, the discrepancies between the disease penetrance and the frequency of this mutation have raised the possibility that its contribution to disease progression might go beyond the mere involvement in regulation of cellular iron uptake. Recent findings revealed that activation of the UPR is a feature of HH and that this stress response may be involved in the genesis of immunological anomalies associated with the disease. This review addresses the connection of the UPR with HH, including its role in MHC-I antigen presentation pathway and possible implications for new clinical approaches to HH.     

The synthesis of phaseolin: a model for the study of the plant secretory pathway

Abstract

Phascolin, the major seed storage protein of common bean (Phaseolus vulgaris), has been for many years one of the main working horses for studying protein synthesis, trafficking and structural maturation in the secretory pathway of higher plants. Recently, phaseolin has been used as a tool to determine molecular interactions between chaperones and newly-synthesised wild-type or structurally-defective secretory proteins in plant cells. Despite the vast amount of information available on the structure and the cell biology of phaseolin, the determinants for its sorting to the vacuole are still unknown.     

ERS和microRNA间的相互作用与肿瘤

内质网是细胞蛋白质翻译后修饰和折叠的重要场所. 多种外界因素诸如热激、病毒感染和低氧等均可导致内质网功能受损,表现为细胞中未折叠或者发生错误折叠的蛋白质在内质网腔内大量聚集,这种聚集将会引发细胞产生应激反应,称为内质网应激. MicroRNAs 是一类内源性非编码 RNAs,通过调控基因的表达来发挥重要的功能. 越来越多的研究表明,内质网应激和microRNAs之间通过相互作用参与诸多重要的生理过程. 本文综述了内质网应激和microRNAs两者的相互作用与肿瘤发生发展的关系,以期对肿瘤发生发展的过程调控机制有更为深入的理解,为发展新的肿瘤治疗方案提供思路.     

丝状真菌次级代谢产物生物合成的表观遗传调控

丝状真菌产生的次级代谢产物是新药的重要来源之一,其生物合成过程受到众多因素的调控。最近的研究表明,表观遗传对多种丝状真菌次级代谢产物的生物合成具有调控作用。DNA和组蛋白的甲基化与乙酰化修饰是目前所知的丝状真菌主要的表观遗传调控形式。通过过表达或缺失相关表观修饰基因和利用小分子表观遗传试剂改变丝状真菌染色体的修饰形式,不仅可以提高多种已知次级代谢产物产量,而且可以通过激活沉默的生物合成基因簇诱导丝状真菌产生新的未知代谢产物。丝状真菌表观遗传学正逐渐成为真菌菌株改良的新策略以及挖掘真菌次级代谢产物合成潜力的强有力手段。     

植物病原丝状真菌G蛋白偶联受体的研究进展

通过对丝状真菌G蛋白偶联受体(GPCR)的结构、分类以及功能方面进行综述,以期明确丝状真菌与其他模式生物GPCR之间的关系。基于已报道的模式生物及丝状真菌等不同生物中的GPCR,通过SMART保守结构域分析,以及利用Clustal X、MEGA等软件对上述GPCR进行遗传关系分析。明确丝状真菌典型GPCR具有七跨膜结构域,新型GPCR则含有PIPK、RGS等保守结构域,明确不同学者对于GPCR的分类情况,以及新型GPCR所具有的特殊功能,明确模式生物GPCR、丝状真菌GPCR分别各自聚类。丝状真菌中GPCR的数量较模式生物少,不同分类单元中真菌之间GPCR的数量也不尽相同,同时,丝状真菌GPCR除具有典型的七跨膜结构域外,还含有一些其他保守的结构域,上述研究为进一步开展其功能研究提供重要的理论基础。     

Endoplasmic reticulum in health and disease: the 12th International Calreticulin Workshop,Delphi, Greece

Starting from 1994, every 2 years, an international workshop is organized focused on calreticulin and other endoplasmic reticulum chaperones. In 2017, the workshop took place at Delphi Greece. Participants from North and South America, Europe, Asia and Australia presented their recent data and discussed them extensively with their colleagues. Presentations dealt with structural aspects of calreticulin and calnexin, the role of Ca²⁺ in cellular signalling and in autophagy, the endoplasmic reticulum stress and the unfolded protein response, the role of calreticulin in immune responses. Several presentations focused on the role of calreticulin and other ER chaperones in a variety of disease states, including haemophilia, obesity, diabetes, Sjogren's syndrome, Chagas diseases, multiple sclerosis, amyotrophic lateral sclerosis, neurological malignancies (especially glioblastoma), haematological malignancies (especially essential thrombocythemia and myelofibrosis), lung adenocarcinoma, renal pathology with emphasis in fibrosis and drug toxicity. In addition, the role of calreticulin and calnexin in growth and wound healing was discussed, as well as the possible use of extracellular calreticulin as a marker for certain diseases. It was agreed that the 13th International Calreticulin Workshop will be organized in 2019 in Montreal, Quebec, Canada.     

Protein kinase RNA-like ER kinase/eukaryotic translation initiation factor 2α pathway attenuates tumor necrosis factor alpha-induced apoptosis in nucleus pulposus cells by activating autophagy

Cellular loss induced by tumor necrosis factor alpha (TNF-α) contributes to the pathogenesis of intervertebral disc (IVD) degeneration. Cellular stress induced by TNF-α activates several processes to restore cell homeostasis. These processes include autophagy, endoplasmic reticulum stress, and related unfolded protein response (UPR). However, the effect and mechanism of UPR and autophagy regulated by TNF-α in IVD degeneration (IDD) remain unclear. The effect of autophagy on biological changes in nucleus pulposus cells (NPCs) also remains elusive. In this study, rat NPCs were cultured with TNF-α in the presence or absence of the UPR or autophagy pathway small-interfering RNAs. The associated genes and proteins were evaluated through immunofluorescence staining, quantitative real-time polymerase chain reaction (qRT-PCR) and western blot analyses to monitor UPR and autophagy signaling and identify the regulatory mechanism of autophagy by the UPR pathway. Trypan blue exclusion assay, cell flow cytometry, terminal deoxynucleotidyl transferase dUTP nick end labeling staining, qRT-PCR, and western blot analyses were performed to examine the apoptosis of NPCs. The results showed that the acute exposure of TNF-α induced the apoptosis of rat NPCs and activated the protein kinase RNA-like ER kinase/eukaryotic translation initiation factor 2α (PERK/eIF2α) pathway of UPR and initiated autophagy. Silencing the PERK/eIF2α pathway or inhibiting autophagy enhanced the apoptosis of NPCs. Interference of the PERK/eIF2α pathway suppressed the autophagy of rat NPCs under TNF-α stimulation. Taken together, the PERK/eIF2α pathway reinforces the survival of NPCs under TNF-α stimulation by activating autophagy. Therefore, PERK/eIF2α-dependent autophagy could be a novel biological therapeutic target for IDD.     

丝状真菌G蛋白信号途径的研究进展

丝状真菌在工业、农业、医药等领域具有重要经济价值,一些亦可导致人类及动、植物疾病,造成经济损失.G蛋白信号途径是真核生物中普遍存在的细胞跨膜信号转导途径.近年来,丝状真菌中G蛋白信号途径的研究发展很快,相关报道表明该信号途径参与感应并传递多种胞外信号刺激,对丝状真菌的生长、分化、繁殖、致病性及真菌毒素等次生代谢产物合成有重要的调控作用.本文就丝状真菌中G蛋白信号途径的基本组成及其生理功能的研究现状进行简要综述.     

Plant endoplasmin supports the protein secretory pathway and has a role in proliferating tissues

Endoplasmin is a molecular chaperone of the heat-shock protein 90 class located in the endoplasmic reticulum and its activity is poorly characterized in plants. We assessed the ability of endoplasmin to alleviate stress via its transient overexpression in tobacco protoplasts treated with tunicamycin, an inhibitor of glycosylation and inducer of the unfolded protein response (UPR). Endoplasmin supported the secretion of a model secretory protein but was less effective than BiP, the endoplasmic reticulum member of the heat-shock protein 70 family. Consistently, immunoprecipitation experiments with in vivo radioactively labelled proteins using an antiserum prepared against Arabidopsis endoplasmin showed that a much smaller number of newly synthesized polypeptides associated with endoplasmin than with BiP. Synthesis of endoplasmin was enhanced by UPR inducers in tobacco seedlings but not protoplasts. As BiP synthesis was induced in both systems, we conclude that the UPR acts differently, at least in part, on the expression of the two chaperones. Endoplasmin was not detectable in extracts of leaves and stems of the Arabidopsis endoplasmin T-DNA insertion mutant shepherd . However, the chaperone is present, albeit at low levels, in shepherd mutant callus, mature roots and tunicamycin-treated seedlings, demonstrating that the mutation is leaky. Reduced endoplasmin in the shepherd mutant has no effect on BiP protein levels in callus or mature roots, leaves and stems, but is compensated by increased BiP in seedlings. This increase occurs in proliferating rather than expanding leaf cells, indicating an important role for endoplasmin in proliferating plant tissues.     

LaeA在丝状真菌次级代谢、生长发育和其他重要生物过程中的作用

LaeA是在构巢曲霉中首次鉴定的一种全局调控因子,其同源蛋白在丝状真菌中广泛存在,具有高度的保守性。LaeA及其同源蛋白序列存在S-腺苷甲硫氨酸结合基序,是一种甲基转移酶,可能影响组蛋白修饰,导致染色体结构的改变,进而调控一系列基因的表达。大量研究表明,LaeA及其同源蛋白参与调控丝状真菌多种次级代谢产物的生物合成,影响丝状真菌的生长发育和形态分化,甚至在丝状真菌生产有机酸和一些工业酶的过程中也发挥着重要作用。本文综述了LaeA及其同源蛋白的作用机制,以及该蛋白在丝状真菌次级代谢、生长发育和其他重要生物过程中的作用,并对存在的问题及应用前景进行讨论与展望。     

The architecture of functional modules in the Hsp90 co-chaperone Sti1/Hop

Sti1/Hop is a modular protein required for the transfer of client proteins from the Hsp70 to the Hsp90 chaperone system in eukaryotes. It binds Hsp70 and Hsp90 simultaneously via TPR (tetratricopeptide repeat) domains. Sti1/Hop contains three TPR domains (TPR1, TPR2A and TPR2B) and two domains of unknown structure (DP1 and DP2). We show that TPR2A is the high affinity Hsp90-binding site and TPR1 and TPR2B bind Hsp70 with moderate affinity. The DP domains exhibit highly homologous α-helical folds as determined by NMR. These, and especially DP2, are important for client activation in vivo. The core module of Sti1 for Hsp90 inhibition is the TPR2A-TPR2B segment. In the crystal structure, the two TPR domains are connected via a rigid linker orienting their peptide-binding sites in opposite directions and allowing the simultaneous binding of TPR2A to the Hsp90 C-terminal domain and of TPR2B to Hsp70. Both domains also interact with the Hsp90 middle domain. The accessory TPR1-DP1 module may serve as an Hsp70-client delivery system for the TPR2A-TPR2B-DP2 segment, which is required for client activation in vivo.