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...

Nna1 is a recently described gene product that has sequence similarity with metallocarboxypeptidases. In the present study, five additional Nna1-like genes were identified in the mouse genome and named cytosolic carboxypeptidase (CCP) 2 through 6. Modeling suggests that the carboxypeptidase domain folds into a structure that resembles metallocarboxypeptidases of the M14 family, with all necessary residues for catalytic activity and broad substrate specificity. All CCPs are abundant in testis and also expressed in brain, pituitary, eye, and other mouse tissues. In brain, Nna1/CCP1, CCP5, and CCP6 are broadly distributed, whereas CCP2 and 3 exhibit restricted patterns of expression. Nna1/CCP1, CCP2, CCP5, and CCP6 were found to exhibit a cytosolic distribution, with a slight accumulation of CCP5 in the nucleus. Based on the above results, we hypothesized that Nna1/CCP1 and CCP2-6 function in the processing of cytosolic proteins such as alpha-tubulin, which is known to be modified by the removal of a C-terminal tyrosine. Analysis of the forms of alpha tubulin in the olfactory bulb of mice lacking Nna1/CCP1 showed the absence of the detyrosinylated form in the mitral cells. Taken together, these results are consistent with a role for Nna1/CCP1 and the related CCPs in the processing of tubulin.

The process by which small proteins fold to their native conformations has been intensively studied over the last few decades. In this field, the particular chemistry of disulfide bond formation has facilitated the characterization of the oxidative folding of numerous small, disulfide-rich proteins with results that illustrate a high diversity of folding mechanisms, differing in the heterogeneity and disulfide pairing nativeness of their intermediates. In this review, we combine information on the folding of different protein models together with the recent structural determinations of major intermediates to provide new molecular clues in oxidative folding. Also, we turn to analyze the role of disulfide bonds in misfolding and protein aggregation and their implications in amyloidosis and conformational diseases.2