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In malignant cancer, excessive amounts of mutant p53 often lead to its aggregation, a feature that was recently identified as druggable. Here, we describe that the induction of a heat-shock response by Foldlin, a small-molecule tool compound, reduced the protein levels of misfolded/aggregated mutant p53 while contact mutants or wild-type p53 remained largely unaffected. Foldlin also prevented the formation of stress-induced p53 nuclear inclusion bodies. In spite of our inability to identify a specific molecular target, Foldlin also reduced protein levels of aggregating SOD1 variants. Finally, by screening a library of 778 FDA-approved compounds for their ability to reduce misfolded mutant p53, we identified the proteasome inhibitor Bortezomib with similar cellular effects as Foldlin. Overall, the induction of a cellular heat shock response seems an effective strategy to deal with pathological protein aggregation. It remains to be seen however, how this strategy can be translated to a clinical setting.

Summary The amyloid-like aggregation propensity present in most globular proteins is generally considered to be a secondary side effect resulting from the requirements of protein stability. Here, we demonstrate, however, that mutations in the globular and amyloid state are thermodynamically correlated rather than simply associated. In addition, we show that the standard genetic code couples this structural correlation into a tight evolutionary relationship. We illustrate the extent of this evolutionary entanglement of amyloid propensity and globular protein stability. Suppressing a 600-Ma-conserved amyloidogenic segment in the p53 core domain fold is structurally feasible but requires 7-bp substitutions to concomitantly introduce two aggregation-suppressing and three stabilizing amino acid mutations. We speculate that, rather than being a corollary of protein evolution, it is equally plausible that positive selection for amyloid structure could have been a driver for the emergence of globular protein structure., Graphical Abstract, Highlights • Mutations in the globular and amyloid state are thermodynamically correlated • The genetic code tightens this relationship between the amyloid and native state • Strongly amyloidogenic sequences in globular proteins are deeply conserved • Positive selection of amyloid propensity will favor globular protein stability, Langenberg et al. show that amyloid propensity favors protein stability. This results from the energetic correlation of mutation in the native and amyloid state. The genetic code tightens this relationship so that stable amyloidogenic sequences are deeply conserved. Positive selection of amyloidogenic sequences could therefore have favored the evolution of globular structure.

Caspase-3 and -7 are considered functionally redundant proteases with similar proteolytic specificities. We performed a proteome-wide screen on a mouse macrophage lysate using the N-terminal combined fractional diagonal chromatography technology and identified 46 shared, three caspase-3-specific, and six caspase-7-specific cleavage sites. Further analysis of these cleavage sites and substitution mutation experiments revealed that for certain cleavage sites a lysine at the P5 position contributes to the discrimination between caspase-7 and -3 specificity. One of the caspase-7-specific substrates, the 40 S ribosomal protein S18, was studied in detail. The RPS18-derived P6–P5′ undecapeptide retained complete specificity for caspase-7. The corresponding P6–P1 hexapeptide still displayed caspase-7 preference but lost strict specificity, suggesting that P′ residues are additionally required for caspase-7-specific cleavage. Analysis of truncated peptide mutants revealed that in the case of RPS18 the P4–P1 residues constitute the core cleavage site but that P6, P5, P2′, and P3′ residues critically contribute to caspase-7 specificity. Interestingly, specific cleavage by caspase-7 relies on excluding recognition by caspase-3 and not on increasing binding for caspase-7.

Previous vaccination trials with calves have shown that intramuscular immunization with natively purified activation-associated secreted proteins (ASPs) of Ostertagia ostertagi induces protection against a homologous challenge infection with a 74% reduction in cumulative faecal egg counts and a significant reduction in worm length. Recently, O. ostertagi ASP1 was recombinantly expressed using a baculovirus system and tested in a vaccination trial. However, immunized calves failed to recognize native ASPs and no protection was observed. These results suggest an important structural difference between the baculo r-ASP1 and its native counterpart. Therefore, we investigated whether glycans and/or structural epitopes are key features in the induction of a protective immune response. The results show that ASPs carry two hybrid N-glycosylations with a complex alpha-1,3-arm, an unprocessed alpha-1,6-arm and an alpha-1,6-fucose core. While removal of these glycans had little effect on antibody recognition by vaccinated animals, denaturing and reducing the proteins dramatically reduced recognition, suggesting the importance of conformational protein backbone epitopes.

In G protein-coupled receptors (GPCRs), the interaction between the cytosolic ends of transmembrane helix 3 (TM3) and TM6 was shown to play an important role in the transition from inactive to active states. According to the currently prevailing model, constructed for rhodopsin and structurally related receptors, the arginine of the conserved "DRY" motif located at the cytosolic end of TM3 (R3.50) would interact with acidic residues in TM3 (D/E3.49) and TM6 (D/E6.30) at the resting state and shift out of this polar pocket upon agonist stimulation. However, 30% of GPCRs, including all chemokine receptors, contain a positively charged residue at position 6.30 which does not support an interaction with R3.50. We have investigated the role of R6.30 in this receptor family by using CCR5 as a model. R6.30D and R6.30E substitutions, which allow an ionic interaction with R3.50, resulted in an almost silent receptor devoid of constitutive activity and strongly impaired in its ability to bind chemokines but still able to internalize. R6.30A and R6.30Q substitutions, allowing weaker interactions with R3.50, preserved chemokine binding but reduced the constitutive activity and the functional response to chemokines. These results indicate that the constitutive and ligand-promoted activity of CCR5 can be modified by modulating the interaction between the DRY motif in TM3 and residues in TM6 suggesting that the overall structure and activation mechanism are well conserved in GPCRs. However, the molecular interactions locking the inactive state must be different in receptors devoid of D/E6.30.