Protein folding in the secretory pathway of animal cells

The exit of newly-synthesized proteins from the lumen of the endoplasmic reticulum (ER) is the rate-determining step in protein secretion. Only correctly-folded and fully-assembled proteins exit the ER and progress along the secretory pathway. Folding and assembly in the ER are mediated by a variety of factors including folding catalysts and molecular chaperones. The properties of these factors, and the nature of their interactions with folding substrates, are beginning to be clarified. Little work has been done to characterize these processes and these factors in cell lines employed for large-scale cell culture. Manipulation of these process may permit improvement in yield or productivity of recombinant proteins by cultured animal cells.     

Mechanisms of Intracellular Protein Transport and Targeting in Plant Cells

The specificity of protein targeting processes is the basis of maintaining structural and functional integrity of the cell, enabling the various subcellular compartments to carry out their unique metabolic roles. Studies in plants have progressed markedly in the last 5 years, and many of the specific signals involved in the transport and targeting of proteins to the nucleus, chloroplast, mitochondrion and microbody, and to organelles along the secretory pathway (endoplasmic reticulum [ER], Golgi complex, and vacuole) have been characterized. Exciting prospects include the identification of receptors involved in the recognition of protein targeting signals, mechanisms of vesicle targeting, and the role of mRNA targeting. Although important exceptions exist, a striking feature of the mechanisms and cellular machinery of protein targeting is their universality — among plants, animals, and eukaryotic microorganisms — and even between prokaryotes and eukaryotes. More information is required about the structural features of proteins that allow for their stable accumulation in a particular subcellular compartment, of particular interest to the plant genetic engineer. Our understanding of the rules that govern protein folding and oligomer assembly and how these processes relate to a protein's ultimate stability in the cell is limited.     

Synaptophysin-containing microvesicles transport heat-shock protein hsp60 in insulin-secreting beta cells

58–62 kDa heat-shock proteins (hsp60) are molecular chaperonins involved in the process of protein folding, transmembrane translocation and assembly of oligomeric protein complexes. In eukaryotic cells hsp60 proteins have been found in mitochondria and chloroplasts. However, we have recently documented that, in addition to mitochondria, a hsp60-like protein is present in secretory granules of insulin-secreting beta cells. The pathway by which hsp60 is targeted to secretory granules was unknown. Here we report the existence of microvesicles involved in the transport of hsp60 protein. Immunoelectron microscopy of serial thin-sections of beta cells directly visualized stages associated with hsp60 delivery: attachment of microvesicles to a secretory granule, fusion with the secretory granule membrane and release of hsp60 molecules. Further biochemical and immunological analysis of microvesicles revealed the presence in their membrane of synaptophysin, a major component of synaptic-like microvesicles (SLMV) of neuroendocrine cells. Double immunogold labelling with antibodies to synaptophysin and hsp60 demonstrated co-localization of both proteins in the same microvesicles. Moreover, fusion of synaptophysin-positive microvesicles leaves synaptophysin incorporated, at least transiently, to secretory granule membranes. These findings suggest that, in beta cells, synaptic-like vesicles are involved in the transport and delivery of hsp60 and represent a novel pathway for protein transport and secretion.     

The endoplasmic reticulum and the unfolded protein response

The endoplasmic reticulum (ER) is the site where proteins enter the secretory pathway. Proteins are translocated into the ER lumen in an unfolded state and require protein chaperones and catalysts of protein folding to attain their final appropriate conformation. A sensitive surveillance mechanism exists to prevent misfolded proteins from transiting the secretory pathway and ensures that persistently misfolded proteins are directed towards a degradative pathway. In addition, those processes that prevent accumulation of unfolded proteins in the ER lumen are highly regulated by an intracellular signaling pathway known as the unfolded protein response (UPR). The UPR provides a mechanism by which cells can rapidly adapt to alterations in client protein-folding load in the ER lumen by expanding the capacity for protein folding. In addition, a variety of insults that disrupt protein folding in the ER lumen also activate the UPR. These include changes in intralumenal calcium, altered glycosylation, nutrient deprivation, pathogen infection, expression of folding-defective proteins, and changes in redox status. Persistent protein misfolding initiates apoptotic cascades that are now known to play fundamental roles in the pathogenesis of multiple human diseases including diabetes, atherosclerosis and neurodegenerative diseases.     

Compromised calnexin function in calreticulin-deficient cells

Calnexin and calreticulin are molecular chaperones, which are involved in the protein folding, assembly, and retention/retrieval. We know that calreticulin-deficiency is lethal in utero, but do not understand the contribution of chaperone function to this phenotype. Here we studied protein folding and chaperone function of calnexin in the absence of calreticulin. We show that protein folding is accelerated and quality control is compromised in calreticulin-deficient cells. Calnexin-substrate association is severely reduced, leading to accumulation of unfolded proteins and a triggering of the unfolded protein response (UPR). PERK and Ire1alpha and eIF2alpha are also activated in calreticulin-deficient cells. We show that the absence of calreticulin can have devastating effects on the function of the others, compromising overall quality control of the secretory pathway and activating UPR-dependent pathways.     

Intracellular interactome of secreted antibody Fab fragment in <Emphasis Type="Italic">Pichia pastoris</Emphasis> reveals its routes of secretion and degradation

Protein translation, translocation, folding, processing, and secretion in eukaryotic cells are complex and not always straightforward processes, e.g., different routes of secretion and degradation exist. Formation of malfolded proteins in the endoplasmic reticulum (ER) can be one of the major bottlenecks for recombinant protein production. In this regard, an in-depth analysis of the interactions of a secreted protein during its pathway through the cell may be beneficial, as realized in this study for the methylotrophic yeast Pichia pastoris. The antibody fragment Fab3H6 used here is the anti-idiotype to the HIV neutralizing antibody 2F5 and is known to be intracellularly degraded in significant amounts when expressed in P. pastoris. The interactome of Fab3H6 was analyzed by using a pull-down mass spectrometry approach, and 23 proteins were found to bind specifically to the antibody fragment. Those allowed concluding that Fab3H6 is post-translationally translocated into the ER and degraded via the proteasome as well as the vacuole. In line with this, the expression of Fab3H6 increased the proteasomal activities by over 20%. Partial inhibition of the proteasome resulted in a significant increase of extracellular Fab3H6. Thus, it seems that ER quality control overshoots its requirements for the recombinant protein expressed and that more than just terminally malfolded protein is degraded by ER-associated degradation. This work will further facilitate our understanding how recombinant proteins behave in the secretory pathway.     

Endoplasmic reticulum stress and fungal pathogenesis

The gateway to the secretory pathway is the endoplasmic reticulum (ER), an organelle that is responsible for the accurate folding, post-translational modification and final assembly of up to a third of the cellular proteome. When secretion levels are high, errors in protein biogenesis can lead to the accumulation of abnormally folded proteins, which threaten ER homeostasis. The unfolded protein response (UPR) is an adaptive signaling pathway that counters a buildup in misfolded and unfolded proteins by increasing the expression of genes that support ER protein folding capacity. Fungi, like other eukaryotic cells that are specialized for secretion, rely upon the UPR to buffer ER stress caused by fluctuations in secretory demand. However, emerging evidence is also implicating the UPR as a central regulator of fungal pathogenesis. In this review, we discuss how diverse fungal pathogens have adapted ER stress response pathways to support the expression of virulence-related traits that are necessary in the host environment.     

Engineering of chaperone systems and of the unfolded protein response

Production of recombinant proteins in mammalian cells is a successful technology that delivers protein pharmaceuticals for therapies and for diagnosis of human disorders. Cost effective production of protein biopharmaceuticals requires extensive optimization through cell and fermentation process engineering at the upstream and chemical engineering of purification processes at the downstream side of the production process. The majority of protein pharmaceuticals are secreted proteins. Accumulating evidence suggests that the folding and processing of these proteins in the endoplasmic reticulum (ER) is a general rate- and yield limiting step for their production. We will summarize our knowledge of protein folding in the ER and of signal transduction pathways activated by accumulation of unfolded proteins in the ER, collectively called the unfolded protein response (UPR). On the basis of this knowledge we will evaluate engineering approaches to increase cell specific productivities through engineering of the ER-resident protein folding machinery and of the UPR.     

Versatility of the endoplasmic reticulum protein folding factory

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.     

植物表达分泌蛋白的运输及定位

分泌途径主要由内膜系统构成,内质网和高尔基体对于分泌蛋白的运输及定位具有重要作用。分泌蛋白的运输包括顺行途径和逆行途径。蛋白质通过质流和受体介导的途径运输到小泡中。在植物中,分泌蛋白的运输主要通过小泡和相连的小管来完成。分子伴侣和质量控制不仅能优化新合成蛋白的折叠和组装,而且去除了有折叠缺陷的蛋白。分泌蛋白的定位需要特定的信号肽,而高尔基体固有蛋白以依赖跨膜长度的方式,沿着分泌途径的细胞器分布。本文对植物表达分泌蛋白的分泌途径及定位、相关的分子伴侣和质量控制进行了综述。     

Quality control in the secretory assembly line

As a rule, only proteins that have reached a native, folded and assembled structure are transported to their target organelles and compartments within the cell. In the secretory pathway of eukaryotic cells, this type of sorting is particularly important. A variety of molecular mechanisms are involved that distinguish between folded and unfolded proteins, modulate their intracellular transport, and induce degradation if they fail to fold. This phenomenon, called quality control, occurs at several levels and involves different types of folding sensors. The quality control system provides a stringent and versatile molecular sorting system that guaranties fidelity of protein expression in the secretory pathway.     

Mapping N-linked glycosylation sites in the secretome and whole cells of Aspergillus niger using hydrazide chemistry and mass spectrometry

Protein glycosylation (e.g., N-linked glycosylation) is known to play an essential role in both cellular functions and secretory pathways; however, our knowledge of in vivo N-glycosylated sites is very limited for the majority of fungal organisms including Aspergillus niger. Herein, we present the first extensive mapping of N-glycosylated sites in A. niger by applying an optimized solid phase glycopeptide enrichment protocol using hydrazide-modified magnetic beads. The enrichment protocol was initially optimized using both mouse blood plasma and A. niger secretome samples, and it was demonstrated that the protein-level enrichment protocol offered superior performance over the peptide-level protocol. The optimized protocol was then applied to profile N-glycosylated sites from both the secretome and whole cell lysates of A. niger. A total of 847 N-glycosylated sites from 330 N-glycoproteins (156 proteins from the secretome and 279 proteins from whole cells) were confidently identified by LC-MS/MS. The identified N-glycoproteins in the whole cell lysate were primarily localized in the plasma membrane, endoplasmic reticulum, Golgi apparatus, lysosome, and storage vacuoles, supporting the important role of N-glycosylation in the secretory pathways. In addition, these glycoproteins are involved in many biological processes including gene regulation, signal transduction, protein folding and assembly, protein modification, and carbohydrate metabolism. The extensive coverage of N-glycosylated sites and the observation of partial glycan occupancy on specific sites in a number of enzymes provide important initial information for functional studies of N-linked glycosylation and their biotechnological applications in A. niger.     

Secretory pathway quality control operating in Golgi,plasmalemmal, and endosomal systems

Exportable proteins that have significant defects in nascent polypeptide folding or subunit assembly are frequently retained in the endoplasmic reticulum and subject to endoplasmic reticulum-associated degradation by the ubiquitin-proteasome system. In addition to this, however, there is growing evidence for post-endoplasmic reticulum quality control mechanisms in which mutant or non-native exportable proteins may undergo anterograde transport to the Golgi complex and post-Golgi compartments before intracellular disposal. In some instances, these proteins may undergo retrograde transport back to the endoplasmic reticulum with re-targeting to the endoplasmic reticulum-associated degradation pathway; in other typical cases, they are targeted into the endosomal system for degradation by vacuolar/lysosomal proteases. Such quality control targeting is likely to involve recognition of features more commonly expressed in mutant proteins, but may also be expressed by wild-type proteins, especially in cells with perturbation of local environments that are essential for normal protein trafficking and stability in the secretory pathway and at the cell surface .     

A functional antibody lacking N-linked glycans is efficiently folded, assembled and secreted by tobacco mesophyll protoplasts

A potential drawback in the use of plants as an expression platform for pharmaceutical proteins such as antibodies is that plant-specific N-glycosylation can result in proteins with altered function and potential antigenicity. In many cases, the N-glycans are essential for the correct folding, assembly and transport of the recombinant proteins. We tested whether progressive removal of glycosylation sites had a detrimental effect on the synthesis, assembly and secretion of a plant-made immunoglobulin G, Guy's 13. Our results indicate that the plant secretory pathway can cope well with aglycosylated antibody chains. The immunoglobulin without N-linked glycans is correctly assembled and secreted by tobacco protoplasts. Capture enzyme-linked immunosorbent assay also shows that antigen-binding properties are unaffected. Our results therefore suggest one possible alternative to the engineering of a humanized glycosylation machinery in plants.     

An atypical protein disulfide isomerase from the protozoan parasite Leishmania containing a single thioredoxin-like domain

In higher eukaryotes, secretory proteins are under the quality control of the endoplasmic reticulum for their proper folding and release into the secretory pathway. One of the proteins involved in the quality control is protein disulfide isomerase, which catalyzes the formation of protein disulfide bonds. As a first step toward understanding the endoplasmic reticulum quality control of secretory proteins in lower eukaryotes, we have isolated a protein disulfide isomerase gene from the protozoan parasite Leishmania donovani. The parasite enzyme shows high sequence homology with homologs from other organisms. However, unlike the four thioredoxin-like domains found in most protein disulfide isomerases, of which two contain an active site, the leishmanial enzyme possesses only one active site present in a single thioredoxin-like domain. When expressed in Escherichia coli, the recombinant parasite enzyme shows both oxidase and isomerase activities. Replacement of the two cysteins with alanines in its active site results in loss of both enzymatic activities. Further, overexpression of the mutated/inactive form of the parasite enzyme in L. donovani significantly reduced their release of secretory acid phosphatases, suggesting that this single thioredoxin-like domain protein disulfide isomerase could play a critical role in the Leishmania secretory pathway.     

Redox signaling loops in the unfolded protein response

The endoplasmic reticulum (ER) is the first compartment of secretory pathway. It plays a major role in ER chaperone-assisted folding and quality control, including post-translational modification such as disulfide bond formation of newly synthesized secretory proteins. Protein folding and assembly takes place in the ER, where redox conditions are distinctively different from the other organelles and are favorable for disulfide formation. These reactions generate the production of reactive oxygen species (ROS) as a byproduct of thiol/disulfide exchange reaction among ER oxidoreductin 1 (Ero1), protein disulfide isomerase (PDI) and ER client proteins, during the formation of disulfide bonds in nascent or incorrectly folded proteins. When uncontrolled, this phenomenon perturbs ER homeostasis, thus aggravating the accumulation of improperly folded or unfolded proteins in this compartment (ER stress). This results in the activation of an adaptive mechanism named the unfolded protein response (UPR). In mammalian cells, the UPR is mediated by three ER-resident membrane proteins (PERK, IRE1 and ATF6) and regulates the expression of the UPR target genes, which themselves encode ER chaperones, folding enzymes, pro-apoptotic proteins and antioxidants, with the objective of restoring ER homeostatic balance. In this review, we will describe redox dependent activation (ER) and amplification (cytosol) loops that control the UPR and the consequences these regulatory loops have on cell fate and physiology.