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

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 p58alpha 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 alpha-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 alpha-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.

[1]  Jörg Gsponer,et al.  Molecular dynamics simulations of protein folding from the transition state , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[2]  Christopher M. Dobson,et al.  Protein-misfolding diseases: Getting out of shape , 2002, Nature.

[3]  E. Mandelkow,et al.  Assembly of tau protein into Alzheimer paired helical filaments depends on a local sequence motif ((306)VQIVYK(311)) forming beta structure. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[4]  S. Betts,et al.  Mutational effects on inclusion body formation. , 1997, Advances in protein chemistry.

[5]  J. Richardson,et al.  Natural β-sheet proteins use negative design to avoid edge-to-edge aggregation , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[6]  D. Otzen,et al.  Designed protein tetramer zipped together with a hydrophobic Alzheimer homology: a structural clue to amyloid assembly. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[7]  M. Mulkerrin,et al.  pH dependence of the reversible and irreversible thermal denaturation of gamma interferons. , 1989, Biochemistry.

[8]  P. S. Kim,et al.  Measurement of the β-sheet-forming propensities of amino acids , 1994, Nature.

[9]  Fabrizio Chiti,et al.  Studies of the aggregation of mutant proteins in vitro provide insights into the genetics of amyloid diseases , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[10]  J. Bernhagen,et al.  Identification of a penta- and hexapeptide of islet amyloid polypeptide (IAPP) with amyloidogenic and cytotoxic properties. , 2000, Journal of molecular biology.

[11]  R. Ellis,et al.  Medicine: Danger — misfolding proteins , 2002, Nature.

[12]  L. Regan,et al.  A systematic exploration of the influence of the protein stability on amyloid fibril formation in vitro. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[13]  Luis Serrano,et al.  Elucidating the folding problem of helical peptides using empirical parameters , 1994, Nature Structural Biology.

[14]  I D Campbell,et al.  Amyloid fibril formation by an SH3 domain. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[15]  Carl W. Cotman,et al.  Common Structure of Soluble Amyloid Oligomers Implies Common Mechanism of Pathogenesis , 2003, Science.

[16]  M. Hecht,et al.  Nature disfavors sequences of alternating polar and non-polar amino acids: implications for amyloidogenesis. , 2000, Journal of molecular biology.

[17]  Peer Bork,et al.  SMART: identification and annotation of domains from signalling and extracellular protein sequences , 1999, Nucleic Acids Res..

[18]  L Serrano,et al.  Development of the multiple sequence approximation within the AGADIR model of alpha-helix formation: comparison with Zimm-Bragg and Lifson-Roig formalisms. , 1997, Biopolymers.

[19]  M. Hurle,et al.  Prolines and amyloidogenicity in fragments of the Alzheimer's peptide beta/A4. , 1995, Biochemistry.

[20]  Andreas Hoenger,et al.  De novo designed peptide-based amyloid fibrils , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[21]  L. Tjernberg,et al.  Medin: an integral fragment of aortic smooth muscle cell-produced lactadherin forms the most common human amyloid. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[22]  M. Hecht,et al.  De novo amyloid proteins from designed combinatorial libraries. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[23]  Kresten Lindorff-Larsen,et al.  Calculation of mutational free energy changes in transition states for protein folding. , 2003, Biophysical journal.

[24]  D Baker,et al.  Long-range order in the src SH3 folding transition state. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[25]  C. Dobson Protein Folding and Disease: a view from the first Horizon Symposium , 2003, Nature Reviews Drug Discovery.

[26]  J. Kelly,et al.  The alternative conformations of amyloidogenic proteins and their multi-step assembly pathways. , 1998, Current opinion in structural biology.

[27]  Elena Orlova,et al.  Cryo‐electron microscopy structure of an SH3 amyloid fibril and model of the molecular packing , 1999, The EMBO journal.

[28]  W. Kabsch,et al.  Dictionary of protein secondary structure: Pattern recognition of hydrogen‐bonded and geometrical features , 1983, Biopolymers.

[29]  L Serrano,et al.  Protein engineering as a strategy to avoid formation of amyloid fibrils , 2000, Protein science : a publication of the Protein Society.

[30]  James C. Sacchettini,et al.  Therapeutic strategies for human amyloid diseases , 2002, Nature Reviews Drug Discovery.

[31]  J. King,et al.  Frequencies of amino acid strings in globular protein sequences indicate suppression of blocks of consecutive hydrophobic residues , 2001, Protein science : a publication of the Protein Society.

[32]  S. Aota,et al.  Formation of amyloid-like fibrils by self-association of a partially unfolded fibronectin type III module. , 1998, Journal of molecular biology.

[33]  Vijay S Pande,et al.  Sequence optimization for native state stability determines the evolution and folding kinetics of a small protein. , 2003, Journal of molecular biology.

[34]  Christopher M. Dobson,et al.  Amyloid fibrils from muscle myoglobin , 2001, Nature.

[35]  L. Serrano,et al.  Insights into the origin of the tendency of the PI3-SH3 domain to form amyloid fibrils. , 2002, Journal of molecular biology.

[36]  Y. Kallberg,et al.  Prediction of Amyloid Fibril-forming Proteins* , 2001, The Journal of Biological Chemistry.

[37]  J. Zurdo,et al.  Competing intrachain interactions regulate the formation of β‐sheet fibrils in bovine PrP peptides , 2003, Protein science : a publication of the Protein Society.

[38]  P. Lansbury,et al.  Amyloid fibrillogenesis: themes and variations. , 2000, Current opinion in structural biology.

[39]  M. Picken,et al.  The changing concepts of amyloid. , 2009, Archives of pathology & laboratory medicine.

[40]  Christopher M. Dobson,et al.  Kinetic partitioning of protein folding and aggregation , 2002, Nature Structural Biology.

[41]  Christopher M Dobson,et al.  Exploring amyloid formation by a de novo design. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[42]  J. Pettegrew,et al.  Quantitative evaluation of congo red binding to amyloid-like proteins with a beta-pleated sheet conformation. , 1989, The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society.

[43]  C. Dobson,et al.  Dependence on solution conditions of aggregation and amyloid formation by an SH3 domain. , 2001, Journal of molecular biology.

[44]  J Schultz,et al.  SMART, a simple modular architecture research tool: identification of signaling domains. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[45]  C M Dobson,et al.  Designing conditions for in vitro formation of amyloid protofilaments and fibrils. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[46]  C. Dobson Protein misfolding, evolution and disease. , 1999, Trends in biochemical sciences.

[47]  C. Dobson,et al.  Rationalization of the effects of mutations on peptide andprotein aggregation rates , 2003, Nature.

[48]  L. Serrano,et al.  Non-native local interactions in protein folding and stability: introducing a helical tendency in the all beta-sheet alpha-spectrin SH3 domain. , 1997, Journal of molecular biology.

[49]  C. Dobson,et al.  Inherent toxicity of aggregates implies a common mechanism for protein misfolding diseases , 2002, Nature.

[50]  I. Campbell,et al.  Solution structure and ligand-binding site of the SH3 domain of the p85α subunit of phosphatidylinositol 3-kinase , 1993, Cell.

[51]  R. Wetzel Mutations and off-pathway aggregation of proteins. , 1994, Trends in biotechnology.

[52]  C. Dobson,et al.  Altered aggregation properties of mutant γ‐crystallins cause inherited cataract , 2002 .

[53]  Stefan M. Larson,et al.  The identification of conserved interactions within the SH3 domain by alignment of sequences and structures , 2000, Protein science : a publication of the Protein Society.

[54]  M. Hecht,et al.  Rationally designed mutations convert de novo amyloid-like fibrils into monomeric β-sheet proteins , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[55]  Christine Wurth,et al.  Mutations that reduce aggregation of the Alzheimer's Abeta42 peptide: an unbiased search for the sequence determinants of Abeta amyloidogenesis. , 2002, Journal of molecular biology.

[56]  C. Dobson,et al.  Preparation and characterization of purified amyloid fibrils. , 2001, Journal of the American Chemical Society.

[57]  L Serrano,et al.  Elucidating the folding problem of alpha-helices: local motifs, long-range electrostatics, ionic-strength dependence and prediction of NMR parameters. , 1998, Journal of molecular biology.