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1.
Dejgaard S Nicolay J Taheri M Thomas DY Bergeron JJ 《Current issues in molecular biology》2004,6(1):29-42
The endoplasmic reticulum (ER) is the major site for folding and sorting of newly synthesized secretory cargo proteins. One central regulator of this process is the quality control machinery, which retains and ultimately disposes of misfolded secretory proteins before they can exit the ER. The ER quality control process is highly effective and mutations in cargo molecules are linked to a variety of diseases. In mammalian cells, a large number of secretory proteins, whether membrane bound or soluble, are asparagine (N)-glycosylated. Recent attention has focused on a sugar transferase, UDP-Glucose: glycoprotein glucosyl transferase (UGGT), which is now recognized as a constituent of the ER quality control machinery. UGGT is capable of sensing the folding state of glycoproteins and attaches a single glucose residue to the Man9GlcNAc2 glycan of incompletely folded or misfolded glycoproteins. This enables misfolded glycoproteins to rebind calnexin and reenter productive folding cycles. Prolonging the time of glucose addition on misfolded glycoproteins ultimately results in either the proper folding of the glycoprotein or its presentation to an ER associated degradation machinery. 相似文献
2.
Bilodeau J Désilets A McDuff FO St-Pierre C Barbar E Leduc R Lavigne P 《FEBS letters》2011,585(12):1910-1914
Proper folding is a crucial step for the trafficking of proteins through the secretory pathway. We hypothesized that the secretory granules of endocrine cells provide optimal folding conditions of prohormone precursors for cleavage. Here, using circular dichroism and in vitro processing on purified prourotensin II (ProUII), we show that the precursor undergoes pH- and Ca(2+)-dependent conformational and stability changes. ProUII has a stable tertiary structure at pH 5.5 in presence of Ca(2+) and is correctly cleaved in these conditions by prohormone convertases. Taken together, our results support the notion that precursors may need to be optimally folded in the lumen of secretory granules for their processing. 相似文献
3.
Membrane glycoprotein folding, oligomerization and intracellular transport: effects of dithiothreitol in living cells. 总被引:27,自引:2,他引:25
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Using influenza hemagglutinin (HA0) and vesicular stomatitis virus G protein as model proteins, we have analyzed the effects of dithiothreitol (DTT) on conformational maturation and transport of glycoproteins in the secretory pathway of living cells. While DTT caused reduction of folding intermediates and misfolded proteins in the endoplasmic reticulum (ER), it did not affect molecules that had already acquired a mature trimeric conformation, whether present in the ER or elsewhere. The conversion to DTT resistance was therefore a pre-Golgi event. Reduction of folding intermediates was dependent on the intactness of the ER and on metabolic energy, suggesting cooperativity between DTT and ER folding factors. DTT did not inhibit most cellular functions, including ATP synthesis and protein transport within the secretory pathway. The results established DTT as an effective tool for analyzing the folding and compartmental distribution of proteins with disulfide bonds. 相似文献
4.
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. 相似文献
5.
Chevet E Cameron PH Pelletier MF Thomas DY Bergeron JJ 《Current opinion in structural biology》2001,11(1):120-124
The endoplasmic reticulum is the entry point into the secretory pathway. To acquire a correct conformation, secretory proteins encounter the endoplasmic reticulum molecular machines of folding, quality control, signaling and disposal, which function as an integrated mechanism. The creation of such a molecular network, spatially regulated, suggests how the endoplasmic reticulum promotes the release of correctly folded secretory proteins. 相似文献
6.
Pathways for protein disulphide bond formation 总被引:16,自引:0,他引:16
The folding of many secretory proteins depends upon the formation of disulphide bonds. Recent advances in genetics and cell biology have outlined a core pathway for disulphide bond formation in the endoplasmic reticulum (ER) of eukaryotic cells. In this pathway, oxidizing equivalents flow from the recently identified ER membrane protein Ero1p to secretory proteins via protein disulphide isomerase (PDI). Contrary to prior expectations, oxidation of glutathione in the ER competes with oxidation of protein thiols. Contributions of PDI homologues to the catalysis of oxidative folding will be discussed, as will similarities between eukaryotic and prokaryotic disulphide-bond-forming systems. 相似文献
7.
Kaufman RJ 《Trends in biochemical sciences》2004,29(3):152-158
The process of protein secretion is intimately linked to the rate and potential of proper folding and assembly of secretory proteins. The efficiency of protein folding is communicated to the cytoplasm via several signal transduction pathways. This regulates the rate of polypeptide chain synthesis and induction of genes encoding functions that reduce protein-folding load on the endoplasmic reticulum (ER). This review summarizes recent insights into the mechanisms that couple protein translation with protein folding in the ER. 相似文献
8.
Oxidative Protein Folding in the Secretory Pathway and Redox Signaling Across Compartments and Cells
The endoplasmic reticulum (ER) is central for many essential cellular activities, such as folding, assembly and quality control of secretory and membrane proteins, disulfide bond formation, glycosylation, lipid biosynthesis, Ca2+ storage and signaling. In addition, this multifunctional organelle integrates many adaptive and/or maladaptive signaling cues reporting on metabolism, proteostasis, Ca2+ and redox homeostasis. We are beginning to understand how these functions and pathways are integrated with one another to regulate homeostasis at cell, tissue and organism levels. The mechanisms underlying the introduction of the proper set of disulfide bonds into secretory proteins (oxidative folding) are strictly related to redox homeostasis, ER stress sensing and signaling and provide a good example of the integration systems operative in the early secretory compartment. 相似文献
9.
未折叠蛋白反应的信号转导 总被引:6,自引:0,他引:6
在内质网中,分泌性蛋白、跨膜蛋白和内质网驻留蛋白折叠成天然构象,经过修饰后,形成有活性的功能性蛋白质。如果蛋白质在内质网内的折叠受到抑制,造成未折叠蛋白聚集,将引起内质网应激。激活未折叠蛋白反应(unfolded protein response,UPR),使蛋白质的生物合成减少,内质网的降解功能增强,从而降低内质网负担,维持细胞内的稳态。如果内质网应激持续存在,则可能诱发细胞凋亡。研究表明,未折叠蛋白反应能在多种肿瘤细胞中发生,并能促进肿瘤细胞的生长。本文对未折叠蛋白反应与肿瘤研究的最新进展进行综述。 相似文献
10.
Inability to meet protein folding demands within the endoplasmic reticulum (ER) activates the unfolded protein response (UPR), a signaling pathway with both adaptive and apoptotic outputs. While some secretory cell types have a remarkable ability to increase protein folding capacity, their upper limits can be reached when pathological conditions overwhelm the fidelity and/or output of the secretory pathway. Irremediable 'ER stress' induces apoptosis and contributes to cell loss in several common human diseases, including type 2 diabetes and neurodegeneration. Researchers have begun to elucidate the molecular switches that determine when ER stress is too great to repair and the signals that are then sent from the UPR to execute the cell. 相似文献
11.
The folding, transport and modification of recombinant proteins in the constitutive secretory pathway of eukaryotic cell expression systems are reported to be a bottleneck in their production. We have utilised a proteomic approach to investigate the processes catalysed by proteins constituting the secretory pathway to further our understanding of those processes involved in high-level antibody secretion. We used GS-NS0 cell populations differing in qmAb to prepare enriched microsome fractions from each cell population at mid-exponential growth phase. These were analysed by 2-D PAGE to characterise the microsome protein component and test the hypothesis that bottlenecks in recombinant protein synthesis exist in these compartments, which are alleviated in high producers by the up-regulation of key secretory pathway proteins. Proteins whose abundance changed in a statistically significant manner with increasing qmAb were involved in a range of cellular functions: energy metabolism, mAb folding/assembly, cytoskeletal organisation and protein turnover. Amongst these were BiP and PDI, chaperones resident in the ER that interact with nascent immunoglobulins during their folding/assembly. However, our results suggest that there are diverse mechanisms by which these cells achieve qmAb. The results imply that cell-engineering strategies for improving qmAb should target proteins associated with altered functional phenotype identified in this study. 相似文献
12.
The EDEM and Yos9p families of lectin-like ERAD factors 总被引:2,自引:0,他引:2
Protein quality control pathways monitor the folding of newly synthesized proteins throughout the cell. Irreversibly misfolded proteins are sorted and degraded to neutralize their potential toxicity. In the secretory pathway, multiple strategies have evolved to test the wide diversity of molecules that traffic through the endoplasmic reticulum. The organelle has adapted the use of N-linked glycans to signal protein folding states. The signals are read by the EDEM and Yos9 protein families that take substrates out of folding cycles for degradation. 相似文献
13.
The endoplasmic reticulum (ER) is dedicated to import, folding and assembly of all proteins that travel along or reside in the secretory pathway of eukaryotic cells. Folding in the ER is special. For instance, newly synthesized proteins are N-glycosylated and by default form disulfide bonds in the ER, but not elsewhere in the cell. In this review, we discuss which features distinguish the ER as an efficient folding factory, how the ER monitors its output and how it disposes of folding failures. 相似文献
14.
Efficient protein folding and trafficking are essential for high-level production of secretory proteins. Slow folding or misfolding of proteins can lead to secretory bottlenecks that reduce productivity. We previously examined the expression of a hyperthermophilic tetramer Pyrococcus furiosus beta-glucosidase in the yeast Saccharomyces cerevisiae. A secretory bottleneck was found in the endoplasmic reticulum, presumably due to beta-glucosidase misfolding. By increasing expression temperature from 30 degrees C up to 40 degrees C, secretion yields increased by as much as 440% per cell to greater than 100 mg/L at 37 degrees C. We examined the effect of temperature on beta-glucosidase folding and secretion and determined that increased expression temperature decreased intracellularly retained, insoluble beta-glucosidase. Likewise, stress on the cell caused by beta-glucosidase expression was found to be greatly reduced at 37 degrees C compared to 30 degrees C. Levels of the abundant endoplasmic reticulum chaperone, BiP, were relatively unchanged at these temperatures during heterologous expression. Using cycloheximide to inhibit new protein synthesis, we determined that the increase in secretion is likely due to the effect of temperature on the beta-glucosidase itself rather than the cell's response to elevated temperatures. We believe that this is the first evidence of in vivo effects of temperature on the secretion of hyperthermophilic proteins. 相似文献
15.
Protein folding in the endoplasmic reticulum: lessons from the human chorionic gonadotropin beta subunit.
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R. W. Ruddon S. A. Sherman E. Bedows 《Protein science : a publication of the Protein Society》1996,5(8):1443-1452
There have been few studies of protein folding in the endoplasmic reticulum of intact mammalian cells. In the one case where the in vivo and in vitro folding pathways of a mammalian secretory protein have been compared, the folding of the human chorionic gonadotropin beta subunit (hCG-beta), the order of formation of the detected folding intermediates is the same. The rate and efficiency with which multidomain proteins such as hCG-beta fold to native structure in intact cells is higher than in vitro, although intracellular rates of folding of the beta subunit can be approached in vitro in the presence of an optimal redox potential and protein disulfide isomerase. Understanding how proteins fold in vivo may provide a new way to diagnose and treat human illnesses that occur due to folding defects. 相似文献
16.
Lori A. Rutkevich Myrna F. Cohen-Doyle Ulf Brockmeier David B. Williams 《Molecular biology of the cell》2010,21(18):3093-3105
To examine the relationship between protein disulfide isomerase family members within the mammalian endoplasmic reticulum, PDI, ERp57, ERp72, and P5 were depleted with high efficiency in human hepatoma cells, either singly or in combination. The impact was assessed on the oxidative folding of several well-characterized secretory proteins. We show that PDI plays a predominant role in oxidative folding because its depletion delayed disulfide formation in all secretory proteins tested. However, the phenotype was surprisingly modest suggesting that other family members are able to compensate for PDI depletion, albeit with reduced efficacy. ERp57 also exhibited broad specificity, overlapping with that of PDI, but with preference for glycosylated substrates. Depletion of both PDI and ERp57 revealed that some substrates require both enzymes for optimal folding and, furthermore, led to generalized protein misfolding, impaired export from the ER, and degradation. In contrast, depletion of ERp72 or P5, either alone or in combination with PDI or ERp57 had minimal impact, revealing a narrow substrate specificity for ERp72 and no detectable role for P5 in oxidative protein folding. 相似文献
17.
Chevet E Jakob CA Thomas DY Bergeron JJ 《Seminars in cell & developmental biology》1999,10(5):473-480
The endoplasmic reticulum (ER) is an intracellular compartment devoted to the synthesis, segregation and folding of soluble and membrane secretory proteins. Some mutations in these proteins lead to their incorrect or incomplete folding in the ER. The ER has a quality control system which detects misfolded proteins and then specifies their fate. Some mutated proteins are retained in the ER wherein they accumulate (Russell bodies for misfolded immunoglobulin heavy chains, the PiZZ for alpha 1-antitrypsin), others are retrotranslocated from the ER and degraded by the cytosolic proteasomal system, and yet other proteins are eventually secreted (in AZC-treated cells). In this review we summarize the role of ER resident proteins in quality control of mutated secretory proteins. 相似文献
18.
19.
Background
The Sec-dependent protein export apparatus of Escherichia coli is very efficient at correctly identifying proteins to be exported from the cytoplasm. Even bacterial strains that carry prl mutations, which allow export of signal sequence-defective precursors, accurately differentiate between cytoplasmic and mutant secretory proteins. It was proposed previously that the basis for this precise discrimination is the slow folding rate of secretory proteins, resulting in binding by the secretory chaperone, SecB, and subsequent targeting to translocase. Based on this proposal, we hypothesized that a cytoplasmic protein containing a mutation that slows its rate of folding would be recognized by SecB and therefore targeted to the Sec pathway. In a Prl suppressor strain the mutant protein would be exported to the periplasm due to loss of ability to reject non-secretory proteins from the pathway. 相似文献20.
The folding stability of insulin is positively correlated with the expression yield of the precursor expressed in yeast. Insulin assembles into dimers and hexamers in a concentration-dependent manner and amino acid substitutions that impair the ability of insulin to associate into dimers concomitantly decrease the expression yield (excluding substitutions that enhance folding stability). In contrast, introduction of an amino substitution that enhances the self-association of insulin improved the yeast expression yield. In the monomeric state the majority of the non-polar residues of insulin are exposed to the solvent and assembly into dimers and hexamers shields these from contact with the solvent. It is proposed that self-association enhances the flux of insulin through the secretory pathway by increasing the hydrophilicity, decreasing the surface area as well as decreasing the molar concentration in the secretory pathway. 相似文献