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

Abstract

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.

Intrinsic α helix propensities compact hydrodynamic radii in intrinsically disordered proteins

“…N ranged from 40 to 260 and net charge from 1 to 43, where low net charge was from mixing positive and negative groups (e.g., SNAP25 and securin) or from relatively few charged residues (e.g., ShB‐C). The dataset includes IDPs known to aggregate under certain conditions (e.g., CFTR‐R and α‐synuclein) or form dimers (e.g., sml1), as well as IDPs that have high monomeric solubility (e.g., p53 TAD and prothymosin‐α). Sequence content among dataset IDPs was generally diverse (see Fig.…”

Transmembrane Helices 7 and 8 Confer Aggregation Sensitivity to the Cystic Fibrosis Transmembrane Conductance Regulator

“…Instead, two other structural elements of CFTR have been pinpointed in NBD2 and TMD1. A distinct C-terminal region of 110 amino acids in NBD2 confers aggregating propensity when fused to GFP [55][56][57]. Its significance for stability in the context of full-length CFTR has not been shown, however, and these 110 residues may well require the rest of NBD2 to acquire a native, more stable fold.…”

The composition of the polyglutamine‐containing proteins influences their co‐aggregation properties

“…Moreover, fusions with unrelated reporter proteins are frequently used to study protein self‐ and co‐aggregation in vivo . Since many studies have shown that modifying the peptide's sequence by fusing it with the reporter protein may influence its ability to self‐aggregate (Stenoien et al, 1999; Thomas and Maule, 2000; Milewski et al, 2002; Link et al, 2006; Bulone et al, 2006; Bąk et al, 2007, Rich and Varadaraj, 2007), it is conceivable that such fusion may also affect the ability of fused proteins to specifically co‐aggregate with other proteins, although no previous reports have investigated this issue.…”

“…Among the models of non‐poly‐Q‐mediated protein aggregation, the model based on the intracellular aggregation of the C‐terminal fragment of the CFTR (cystic fibrosis transmembrane conductance regulator) shows remarkable dependence on the presence of a short amino acid sequence (Milewski et al, 2002; Bąk et al, 2007). When fused to GFP (green fluorescence protein), this CFTR‐derived fragment leads to the formation of typical insoluble electron‐dense protein aggregates, while the non‐fused peptide exhibits a very unique accumulation pattern with numerous small aggregates dispersed through the cytoplasm and associated with mitochondria (Milewski et al, 2002).…”