[Show abstract][Hide abstract] ABSTRACT: Different neuropeptide hormones, which are either too small to adopt a stable conformation or are predicted to be intrinsically disordered, are synthesized as larger precursors containing a prodomain in addition to an N-terminal signal peptide. We analyzed the biogenesis of three unstructured neuropeptide hormones and observed that translocation of these precursors into the lumen of the endoplasmic reticulum (ER) is critically dependent on the presence of the prodomain. The hormone domains could be deleted from the precursors without interfering with ER import and secretion, whereas constructs lacking the prodomain remained in the cytosol. Domain-swapping experiments revealed that the activity of the prodomains to promote productive ER import resides in their ability to adopt an α-helical structure. Removal of the prodomain from the precursor did not interfere with co-translational targeting of the nascent chain to the Sec61 translocon but with its subsequent productive translocation into the ER lumen. Our study reveals a novel function of prodomains to enable import of small or intrinsically disordered secretory proteins into the ER based on their ability to adopt an α-helical conformation.
Journal of Biological Chemistry 03/2013; · 4.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Protein targeting to specified cellular compartments is essential to maintain cell function and homeostasis. In eukaryotic cells, two major pathways rely on N-terminal signal peptides to target proteins to either the endoplasmic reticulum (ER) or mitochondria. In this study, we show that the ER signal peptides of the prion protein-like protein shadoo, the neuropeptide hormone somatostatin and the amyloid precursor protein have the property to mediate alternative targeting to mitochondria. Remarkably, the targeting direction of these signal peptides is determined by structural elements within the nascent chain. Each of the identified signal peptides promotes efficient ER import of nascent chains containing α-helical domains, but targets unstructured polypeptides to mitochondria. Moreover, we observed that mitochondrial targeting by the ER signal peptides correlates inversely with ER import efficiency. When ER import is compromised, targeting to mitochondria is enhanced, whereas improving ER import efficiency decreases mitochondrial targeting. In conclusion, our study reveals a novel mechanism of dual targeting to either the ER or mitochondria that is mediated by structural features within the nascent chain.
[Show abstract][Hide abstract] ABSTRACT: Prion diseases are characterized by the conformational transition of the cellular prion protein (PrP(C)) into an aberrant protein conformer, designated scrapie-prion protein (PrP(Sc)). A causal link between protein misfolding and neurodegeneration has been established for a variety of neurodegenerative disease, such as Alzheimer's disease, Parkinson's disease and polyglutamine diseases, but there is an ongoing debate about the nature of the neurotoxic species and how non-native conformers can damage neuronal populations. PrP is normally imported into the endoplasmic reticulum (ER) and targeted to the outer leaflet of the plasma membrane via a glycosylphosphatidylinositol (GPI) anchor. However, several conditions, such as ER stress or some pathogenic mutations in the PrP gene, can induce the mislocalization of PrP in the cytosol, where it has a neurotoxic potential as demonstrated in cell culture and transgenic mouse models. In this review we focus on intrinsic factors and cellular pathways implicated in the import of PrP into the ER and its mistargeting to the cytosol. The findings summarized here not only reveal a complex regulation of the biogenesis of PrP, but also provide interesting new insight into toxic activities of pathogenic protein conformers and quality control pathways of ER-targeted proteins.
Current issues in molecular biology 09/2009; 12(2):109-18. · 3.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Co-translational import into the endoplasmic reticulum (ER) is primarily controlled by N-terminal signal sequences that mediate targeting of the ribosome-nascent chain complex to the Sec61/translocon and initiate the translocation process. Here we show that after targeting to the translocon the secondary structure of the nascent polypeptide chain can significantly modulate translocation efficiency. ER-targeted polypeptides dominated by unstructured domains failed to efficiently translocate into the ER lumen and were subjected to proteasomal degradation via a co-translocational/preemptive pathway. Productive ER import could be reinstated by increasing the amount of alpha-helical domains, whereas more effective ER signal sequences had only a minor effect on ER import efficiency of unstructured polypeptides. ER stress and overexpression of p58(IPK) promoted the co-translocational degradation pathway. Moreover polypeptides with unstructured domains at their N terminus were specifically targeted to proteasomal degradation under these conditions. Our study indicates that extended unstructured domains are signals to dispose ER-targeted proteins via a co-translocational, preemptive quality control pathway.
Journal of Biological Chemistry 07/2009; 284(36):24384-93. · 4.65 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A hallmark in prion diseases is the conformational transition of the cellular prion protein (PrP(C)) into a pathogenic conformation, designated scrapie prion protein (PrP(Sc)), which is the essential constituent of infectious prions. Here, we show that epigallocatechin gallate (EGCG) and gallocatechin gallate, the main polyphenols in green tea, induce the transition of mature PrP(C) into a detergent-insoluble conformation distinct from PrP(Sc). The PrP conformer induced by EGCG was rapidly internalized from the plasma membrane and degraded in lysosomal compartments. Isothermal titration calorimetry studies revealed that EGCG directly interacts with PrP leading to the destabilizing of the native conformation and the formation of random coil structures. This activity was dependent on the gallate side chain and the three hydroxyl groups of the trihydroxyphenyl side chain. In scrapie-infected cells EGCG treatment was beneficial; formation of PrP(Sc) ceased. However, in uninfected cells EGCG interfered with the stress-protective activity of PrP(C). As a consequence, EGCG-treated cells showed enhanced vulnerability to stress conditions. Our study emphasizes the important role of PrP(C) to protect cells from stress and indicate efficient intracellular pathways to degrade non-native conformations of PrP(C).
Journal of Neurochemistry 09/2008; 107(1):218-29. · 3.97 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The infectious agent in prion diseases is an aberrant-folded isoform of the cellular prion protein (PrPC). This scrapie-related prion protein (PrPSc) has an increased beta-sheet content, is detergent insoluble and proteinase K resistant, and accumulates in prion-infected organisms and cells. In vitro, PrPSc self-aggregates into amyloid fibrils. However, there is no direct experimental proof for the occurrence of PrPSc-containing fibrils in vivo or in cell cultures. Applying atomic force microscopy (AFM) to scrapie-infected mouse neuroblastoma (ScN2a) cells, we discovered growing patch-like assemblies of amyloid-like fibrillar structures on the cell surfaces. Immunofluorescence and AFM images showed heterogeneous accumulation and aggregation of PrPSc in ScN2a cell cultures. The percentage of cells having characteristic fibrils on their surface increased with time after scrapie infection. These endogeneous fibrils had lengths from 0.5 to 3 microm and protruded from the cell surface by 108 +/- 30 nm, and thus resembled the heterogeneous shapes and networks of in vitro prepared amyloid fibrils.
Pflügers Archiv - European Journal of Physiology 05/2008; 456(1):83-93. · 4.87 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Conversion of cellular prion protein (PrP(C)) into the pathological conformer (PrP(Sc)) has been studied extensively by using recombinantly expressed PrP (rPrP). However, due to inherent difficulties of expressing and purifying posttranslationally modified rPrP variants, only a limited amount of data is available for membrane-associated PrP and its behavior in vitro and in vivo. Here, we present an alternative route to access lipidated mouse rPrP (rPrP(Palm)) via two semisynthetic strategies. These rPrP variants studied by a variety of in vitro methods exhibited a high affinity for liposomes and a lower tendency for aggregation than rPrP. In vivo studies demonstrated that double-lipidated rPrP is efficiently taken up into the membranes of mouse neuronal and human epithelial kidney cells. These latter results enable experiments on the cellular level to elucidate the mechanism and site of PrP-PrP(Sc) conversion.
[Show abstract][Hide abstract] ABSTRACT: Protein misfolding is linked to different neurodegenerative disorders like Alzheimer's disease, polyglutamine, and prion diseases. We investigated the cytotoxic effects of aberrant conformers of the prion protein (PrP) and show that toxicity is specifically linked to misfolding of PrP in the cytosolic compartment and involves binding of PrP to the anti-apoptotic protein Bcl-2. PrP targeted to different cellular compartments, including the cytosol, nucleus, and mitochondria, adopted a misfolded and partially proteinase K-resistant conformation. However, only in the cytosol did the accumulation of misfolded PrP induce apoptosis. Apoptotic cell death was also induced by two pathogenic mutants of PrP, which are partially localized in the cytosol. A mechanistic analysis revealed that the toxic potential is linked to an internal domain of PrP (amino acids 115-156) and involves coaggregation of cytosolic PrP with Bcl-2. Increased expression of the chaperones Hsp70 and Hsp40 prevented the formation of PrP/Bcl-2 coaggregates and interfered with PrP-induced apoptosis. Our study reveals a compartment-specific toxicity of PrP misfolding that involves coaggregation of Bcl-2 and indicates a protective role of molecular chaperones.
Molecular Biology of the Cell 09/2006; 17(8):3356-68. · 4.60 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A hallmark of prion diseases in mammals is a conformational transition of the cellular prion protein (PrP(C)) into a pathogenic isoform termed PrP(Sc). PrP(C) is highly conserved in mammals, moreover, genes of PrP-related proteins have been recently identified in fish. While there is only little sequence homology to mammalian PrP, PrP-related fish proteins were predicted to be modified with N-linked glycans and a C-terminal glycosylphosphatidylinositol (GPI) anchor. We biochemically characterized two PrP-related proteins from zebrafish in cultured cells and show that both zePrP1 and zeSho2 are imported into the endoplasmic reticulum and are post-translationally modified with complex glycans and a C-terminal GPI anchor.
Biochemical and Biophysical Research Communications 04/2006; 341(1):218-24. · 2.41 Impact Factor