Aggregation of Misfolded Proteins Can Be a Selective Process Dependent upon Peptide Composition

Milewski, Michał; Mickle, John E.; Forrest, John K.; Stanton, Bruce A.; Cutting, Garry R.

doi:10.1074/jbc.m205420200

Cited by 21 publications

(28 citation statements)

References 56 publications

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“…All the constructs were expressed at very similar levels, as could be judged from the similar transfection rates and comparable results of Western blot analysis. In agreement with previously published data [22], the removal of the ag region (Δag) significantly reduced the aggregation rate of the CFTR-derived peptides (Fig. 1C).…”

Section: Resultssupporting

confidence: 82%

“…Although the aggregation of CFTR seems not to be causally related to the pathogenesis of cystic fibrosis, it was the first protein for which the formation of aggresomes was described [17]. It was also demonstrated that intracellular aggregation of the CFTR-derived C-terminal peptides depends on the presence of a nine-amino acid sequence, termed the ag region [22,23]. The existence of such a relatively simple peptide model provides a unique opportunity to study…”

Section: Introductionmentioning

confidence: 99%

“…This exposes the need for new protein models that would facilitate the exploration of the sequence-specific mechanisms governing the aggregation process. Cystic fibrosis transmembrane conductance regulator (CFTR), a protein that is defective in cystic fibrosis, is commonly used to study protein aggregation [17][18][19][20][21][22]. Although the aggregation of CFTR seems not to be causally related to the pathogenesis of cystic fibrosis, it was the first protein for which the formation of aggresomes was described [17].…”

Section: Introductionmentioning

confidence: 99%

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The CFTR-derived peptides as a model of sequence-specific protein aggregation

Bąk¹,

Cutting

Milewski³

2007

Cellular and Molecular Biology Letters

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Protein aggregation is a hallmark of a growing group of pathologies known as conformational diseases. Although many native or mutated proteins are able to form aggregates, the exact amino acid sequences involved in the process of aggregation are known only in a few cases. Hence, there is a need for different model systems to expand our knowledge in this area. The so-called ag region was previously found to cause the aggregation of the C-terminal fragment of the cystic fibrosis transmembrane conductance regulator (CFTR). To investigate whether this specific amino acid sequence is able to induce protein aggregation irrespective of the amino acid context, we altered its position within the CFTR-derived C-terminal peptide and analyzed the localization of such modified peptides in transfected mammalian cells. Insertion of the ag region into a different amino acid background affected not only the overall level of intracellular protein aggregation, but also the morphology and subcellular localization of aggregates, suggesting that sequences other than the ag region can substantially influence the peptide’s behavior. Also, the introduction of a short dipeptide (His-Arg) motif, a crucial component of the ag region, into different locations within the C-terminus of CFTR lead to changes in the aggregation pattern that were less striking, although still statistically significant. Thus, our results indicate that even subtle alterations within the aggregating peptide can affect many different aspects of the aggregation process.

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Section: Resultssupporting

confidence: 82%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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The CFTR-derived peptides as a model of sequence-specific protein aggregation

Bąk¹,

Cutting

Milewski³

2007

Cellular and Molecular Biology Letters

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“…Moreover, the process of aggregate formation can be examined in detail in vitro and correlated with in vivo events. This simple system using an E. coli expression system serves as a paradigm for similar analyses of aggregation in eukaryotic cells, including mammalian cells, and should complement active work on aggregation in these systems (7,35). Thus, it may provide an experimental platform for studies of amyloidogenesis and other pathological protein aggregation events and enable tests of therapeutic approaches to counteract these processes.…”

Section: Discussionmentioning

confidence: 99%

Monitoring protein stability and aggregationin vivoby real-time fluorescent labeling

Ignatova

Gierasch

2003

Proc. Natl. Acad. Sci. U.S.A.

227

239

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In vivo fluorescent labeling of an expressed protein has enabled the observation of its stability and aggregation directly in bacterial cells. Mammalian cellular retinoic acid-binding protein I (CRABP I) was mutated to incorporate in a surface-exposed omega loop the sequence Cys-Cys-Gly-Pro-Cys-Cys, which binds specifically to a biarsenical fluorescein dye (FlAsH). Unfolding of labeled tetra-Cys CRABP I is accompanied by enhancement of FlAsH fluorescence, which made it possible to determine the free energy of unfolding of this protein by urea titration in cells and to follow in real time the formation of inclusion bodies by a slow-folding, aggregationprone mutant (FlAsH-labeled P39A tetra-Cys CRABP I). Aggregation in vivo displayed a concentration-dependent apparent lag time similar to observations of protein aggregation in purified in vitro model systems.protein aggregation ͉ protein folding ͉ fluorescence P rotein folding is an exquisitely optimized process that normally succeeds in producing functional molecules in vivo, despite physicochemical conditions that are very challenging. The heteropolymeric nature of the polypeptide chain encodes a great diversity of complex architectures of native proteins but also presents many side chain groups that are marginally soluble in aqueous solution. Not surprisingly, aggregation of newly synthesized proteins emerges as a process that competes with folding in vivo. In bacterial cells, aggregation of partially folded intermediates manifests itself in the production of insoluble inclusion bodies and often occurs when an exogenous protein is overexpressed (1). Several human diseases, including Alzheimer's, Huntington's, and spongiform encephalopathies, are characterized by the formation of insoluble protein aggregates (2). These distinctive aggregated states seem to be formed from partially folded, partially unfolded, or other nonnative intermediates and not from the native state. Thus, considerable research has been dedicated to the goal of achieving a fundamental understanding of protein aggregation, and much progress has been achieved in several systems in vitro (3). By contrast, relatively little is known about how this process occurs in vivo. Complicating research on protein folding and aggregation in vivo is the need to study these processes in the complex and concentrated environment of the cell. It is therefore highly desirable to develop methods to observe the fates of proteins directly in cells, including their thermodynamic stabilities, their kinetics of folding, the nature of folding intermediates and the energy landscape of folding, and the effects of mutations. Here, we have taken advantage of new approaches to labeling protein molecules in the cell with fluorescent dyes (4) to monitor the synthesis, folding, and aggregation of a protein whose in vitro folding has been well characterized.Cellular retinoic acid-binding protein I (CRABP I) is a 136-residue member of the large family of intracellular lipidbinding proteins, which fold into ␤-barrel structures (Fi...

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“…Milewski et al (39) have suggested that the intracellular accumulation of misfolded proteins is a selective process determined by the sequences of the aggregating polypeptides, and demonstrated that substitution of two amino acids could eliminate the aggregation of a cystic fibrosis transmembrane conductance regulator-derived polypeptide. A theoretical study based on the acylphosphatase protein proposed that variations that specifically perturb the rate of aggregation are located in certain regions of the protein sequence, each of which has a relatively high hydrophobicity and propensity to form ␤-sheets (40).…”

Section: Turnover Of Wild Type and Variant Scad Enzymes In Scad-deficmentioning

confidence: 99%

Misfolding, Degradation, and Aggregation of Variant Proteins

Pedersen

Bross

Winter

et al. 2003

Journal of Biological Chemistry

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Short chain acyl-CoA dehydrogenase (SCAD) deficiency is an inborn error of the mitochondrial fatty acid metabolism caused by rare variations as well as common susceptibility variations in the SCAD gene. Earlier studies have shown that a common variant SCAD protein (R147W) was impaired in folding, and preliminary experiments suggested that the variant protein displayed prolonged association with chaperonins and delayed formation of active enzyme. Accordingly, the molecular pathogenesis of SCAD deficiency may rely on intramitochondrial protein quality control mechanisms, including degradation and aggregation of variant SCAD proteins. In this study we investigated the processing of a set of disease-causing variant SCAD proteins (R22W, G68C, W153R, R359C, and Q341H) and two common variant proteins (R147W and G185S) that lead to reduced SCAD activity. All SCAD proteins, including the wild type, associate with mitochondrial hsp60 chaperonins; however, the variant SCAD proteins remained associated with hsp60 for prolonged periods of time. Biogenesis experiments at two temperatures revealed that some of the variant proteins (R22W, G68C, W153R, and R359C) caused severe misfolding, whereas others (R147W, G185S, and Q341H) exhibited a less severe temperaturesensitive folding defect. Based on the magnitude of in vitro defects, these SCAD proteins are characterized as folding-defective variants and mild folding variants, respectively. Pulse-chase experiments demonstrated that the variant SCAD proteins either triggered proteolytic degradation by mitochondrial proteases or, especially at elevated temperature, aggregation of non-native conformers. The latter finding may indicate that accumulation of aggregated SCAD proteins may play a role in the pathogenesis of SCAD deficiency.

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Aggregation of Misfolded Proteins Can Be a Selective Process Dependent upon Peptide Composition

Cited by 21 publications

References 56 publications

The CFTR-derived peptides as a model of sequence-specific protein aggregation

The CFTR-derived peptides as a model of sequence-specific protein aggregation

Monitoring protein stability and aggregationin vivoby real-time fluorescent labeling

Misfolding, Degradation, and Aggregation of Variant Proteins

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