Short amino acid stretches can mediate amyloid formation in globular proteins: The Src homology 3 (SH3) case

Abstract

Protein misfolding and deposition underlie an increasing number of debilitating human disorders. We have shown that model proteins unrelated to disease, such as the Src homology 3 (SH3) domain of the p58␣ subunit of bovine phosphatidyl-inositol-3-kinase (PI3-SH3), can be converted in vitro into assemblies with structural and cytotoxic properties similar to those of pathological aggregates. By contrast, homologous proteins, such as ␣-spectrin-SH3, lack the capability of forming amyloid fibrils at a measurable rate under any of the conditions we have so far examined. However, transplanting a small sequence stretch (6 aa) from PI3-SH3 to ␣-spectrin-SH3, comprising residues of the diverging turn and adjacent RT loop, creates an amyloidogenic protein closely similar in its behavior to the original PI3-SH3. Analysis of specific PI3-SH3 mutants further confirms the involvement of this region in conferring amyloidogenic properties to this domain. Moreover, the inclusion in this stretch of two consensus residues favored in SH3 sequences substantially inhibits aggregation. These findings show that short specific amino acid stretches can act as mediators or facilitators in the incorporation of globular proteins into amyloid structures, and they support the suggestion that natural protein sequences have evolved in part to code for structural characteristics other than those included in the native fold, such as avoidance of aggregation.protein misfolding ͉ protein aggregation ͉ protein evolution T he spontaneous conversion of soluble proteins or protein fragments into aggregates and amyloid fibrils is a challenging problem in biological and medical sciences. An increasing body of evidence supports the anomalous misassembly of proteins into insoluble deposits as the fundamental cause behind a growing number of debilitating human disorders, such as Alzheimer's disease and Parkinson's disease, type II diabetes, and the transmissible spongiform encephalopathies (1-4). Important clues to understand the molecular basis of amyloid diseases and, more generally, the biological significance of protein aggregation have emerged recently from observations made in proteins unrelated to human disease, which have been found to convert in vitro into aggregates with structural and cytotoxic properties indistinguishable from those exhibited by amyloid assemblies associated with pathological conditions (5-14).The Src homology 3 (SH3) domain of the p58␣ subunit of phosphatidyl-inositol-3Ј-kinase (PI3-SH3) is one of the best characterized examples of a small globular protein unrelated to any known pathological condition that can form amyloid fibrils in vitro (5,15,16). Aggregated species obtained from this protein have been found to be cytotoxic when added to cell cultures (13). This observation suggests that the protein aggregates underlying different human disorders could show similar mechanisms of cytotoxicity and more generally that during evolution nature had to develop strategies to avoid protein misassembly to preserve the viability of liv...

Experimental characterization of disordered and ordered aggregates populated during the process of amyloid fibril formation

“…Work on a homologous protein of the PI3-SH3 domain, the ␣-spectrin SH3 domain, and several PI3-SH3 mutants has suggested that residues located in part of the RT loop and adjacent divergent turn (DT) in the native state, act as ''mediators'' or ''facilitators'' in the conversion of the soluble PI3-SH3 into amyloid fibrils (22). Other studies using molecular dynamics simulations to probe aggregation of the Src-SH3 domain concluded that the RT loop sequence constitutes the initial core structure for fibril formation (23).…”

Amyloidogenic sequences in native protein structures

“…45 Investigations into the effect of mutations altering the charge state of a protein without altering its hydrophobicity or secondary structural propensity showed that aggregation was favoured by those mutations which brought about a reduction in the net charge. 11,46,47 Similarly, in vivo experiments have shown that mutations decreasing the positive charge on a molecule increase the aggregation propensity and mutations increasing the net charge result in decreased aggregation. 48 Amyloidogenic proteins which are predominantly a-helical, must undergo an a-helix to b-sheet conversion during the formation of amyloid fibrils.…”

“…1,2,[8][9][10] In some cases, the aggregates formed from these nondisease-associated proteins have been shown to have cytotoxic properties similar to those of pathological aggregates. 11 A diverse group of proteins has been observed to form amyloid-like fibrils having specific physiological functions in nonpathological conditions in a wide range of organisms, such as proteins of the eggshell chorion in the silk moth, spidroin in the spider, Pmel17, which plays a central amyloid has been dubbed functional amyloid to distinguish it from the amyloid associated with disease conditions, but from a structural point of view both types of amyloid are the same. 16,17 The definitive characteristic of amyloid fibrils formed in all the aforementioned cases is the socalled ''cross-b'' structure as revealed by X-ray diffraction, in which the polypeptide chain is organised into b-sheets arranged parallel to the longitudinal fibril axis, with constituent b-strands perpendicular to the fibril axis.…”

Sequence dependent aggregation of peptides and fibril formation

“…The sequence determinant of amyloid formation has been studied with various experi-mental [16][17][18][19][20][21][22] and theoretical [23][24][25][26] approaches. It has been shown that the overall hydropho-bicity 18 and net charge 20 of a peptide, to some extent, may impact the aggregation rate.…”

“…It has been shown that the overall hydropho-bicity 18 and net charge 20 of a peptide, to some extent, may impact the aggregation rate. There are increasing evidence that the capability of a protein to form amyloids strongly depends on certain short amino acid stretches in the sequence [17][18][19] . To support a proteome-wide search for aggregation-prone peptide segments, a number of predictors have been made available [27][28][29] .…”