Many p53 missense mutations possess dominant-negative activity and oncogenic gain of function. We report that for structurally destabilized p53 mutants, these effects result from mutant-induced coaggregation of wild-type p53 and its paralogs p63 and p73, thereby also inducing a heat-shock response. Aggregation of mutant p53 resulted from self-assembly of a conserved aggregation-nucleating sequence within the hydrophobic core of the DNA-binding domain, which becomes exposed after mutation. Suppressing the aggregation propensity of this sequence by mutagenesis abrogated gain of function and restored activity of wild-type p53 and its paralogs. In the p53 germline mutation database, tumors carrying aggregation-prone p53 mutations have a significantly lower frequency of wild-type allele loss as compared to tumors harboring nonaggregating mutations, suggesting a difference in clonal selection of aggregating mutants. Overall, our study reveals a novel disease mechanism for mutant p53 gain of function and suggests that, at least in some respects, cancer could be considered an aggregation-associated disease.

Alzheimer's disease (AD)-linked mutations in Presenilins (PSEN) and the amyloid precursor protein (APP) lead to production of longer amyloidogenic Aβ peptides. The shift in Aβ length is fundamental to the disease; however, the underlying mechanism remains elusive. Here, we show that substrate shortening progressively destabilizes the consecutive enzyme-substrate (E-S) complexes that characterize the sequential γ-secretase processing of APP. Remarkably, pathogenic PSEN or APP mutations further destabilize labile E-S complexes and thereby promote generation of longer Aβ peptides. Similarly, destabilization of wild-type E-S complexes by temperature, compounds, or detergent promotes release of amyloidogenic Aβ. In contrast, E-Aβ stabilizers increase γ-secretase processivity. Our work presents a unifying model for how PSEN or APP mutations enhance amyloidogenic Aβ production, suggests that environmental factors may increase AD risk, and provides the theoretical basis for the development of γ-secretase/substrate stabilizing compounds for the prevention of AD.

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Molecular chaperones are essential elements of the protein quality control machinery that governs translocation and folding of nascent polypeptides, refolding and degradation of misfolded proteins, and activation of a wide range of client proteins. The prokaryotic heat-shock protein DnaK is the E. coli representative of the ubiquitous Hsp70 family, which specializes in the binding of exposed hydrophobic regions in unfolded polypeptides. Accurate prediction of DnaK binding sites in E. coli proteins is an essential prerequisite to understand the precise function of this chaperone and the properties of its substrate proteins. In order to map DnaK binding sites in protein sequences, we have developed an algorithm that combines sequence information from peptide binding experiments and structural parameters from homology modelling. We show that this combination significantly outperforms either single approach. The final predictor had a Matthews correlation coefficient (MCC) of 0.819 when assessed over the 144 tested peptide sequences to detect true positives and true negatives. To test the robustness of the learning set, we have conducted a simulated cross-validation, where we omit sequences from the learning sets and calculate the rate of repredicting them. This resulted in a surprisingly good MCC of 0.703. The algorithm was also able to perform equally well on a blind test set of binders and non-binders, of which there was no prior knowledge in the learning sets. The algorithm is freely available at http://limbo.vib.be.

The possible link between hIAPP accumulation and β-cell death in diabetic patients has inspired numerous studies focusing on amyloid structures and aggregation pathways of this hormone. Recent studies have reported on the importance of early oligomeric intermediates, the many roles of their interactions with lipid membrane, pH, insulin and zinc on the mechanism of aggregation of hIAPP. weight reduction was observed. In a phase-1 clinical trial in which AM833 was combined with the GLP-1-agonist Semaglutid, 20 weeks of treatment, participants lost an average of 17.1 % body weight. Overweight and obesity are risk factors for T2D and cardiovascular disease and the presented weight loss exceed reductions observed for GLP-1 and metformin. It should be noted that an IAPP derivative, pramlintide (symlin) is used, in addition to insulin, in patients with insulindependent T1D and T2D that have been difficult to regulate with insulin by a single drug. 42 Cross-correlation with other diseasesEpidemiological studies link diabetes with neurodegenerative diseases, including Alzheimer's disease [43][44][45][46] and Parkinson's disease. [47][48][49] The link is unclear, but the three conditions are multifactorial and have protein aggregation in their pathophysiology in common. If protein aggregation constitutes the link between these diseases, however, still needs to be proven. The Rotterdam study 50 , published in 1999, showed that patients with T2D have an almost doubled risk of developing dementia and Alzheimer's disease (AD). Data from the ULSAM study (Uppsala Longitudinal study of adult men) showed that already a moderate disturbance in the first-phase insulin release at the age of 55 increased the risk of developing AD thirty years later. 51 This suggests that diabetes precedes AD. In fact, another type of diabetes has been proposed [52][53][54] , which is type 3 diabetes (T3DM) which is an AD associated insulin resistance, also described as "brain diabetes phenotype". A considerable number of biophysical studies have shown a close link between the amyloid-forming proteins IAPP and Aβ. [55][56][57][58] IAPP immunoreactivity is present in formic acid brain extracts from patients with AD 59 and exhibited pattern on the western blot ranges from dimers to 16 mers. 60 The laddering pattern suggests that IAPP is present in the Aβ amyloid deposit. Proximity ligation assay (PLA) using two primary antibodies can be applied to determine the co-deposition of proteins. The positive PLA signal obtained after the combination of anti-IAPP and anti-Aβ antibodies points to the co-deposition of IAPP and Aβ in vivo. To confirm that the peptides interact in vivo, hIAPP transgenic mice were injected with preformed fibrils of Aβ followed by high-fat feeding for ten months. 60 Mice injected with preformed Aβ fibrils developed amyloid in 15% of the islets, which is comparable to mice injected with preformed fibrils of proIAPP. In mice injected with preformed IAPP fibrils, IAPP amyloid was found in 24 % of the islets. Control mice injected with fib...

Aggregation is a sequence-specific process, nucleated by short aggregation-prone regions (APRs) that can be exploited to induce aggregation of proteins containing the same APR. Here, we find that most APRs are unique within a proteome, but that a small minority of APRs occur in many proteins. When aggregation is nucleated in bacteria by such frequently occurring APRs, it leads to massive and lethal inclusion body formation containing a large number of proteins. Buildup of bacterial resistance against these peptides is slow. In addition, the approach is effective against drug-resistant clinical isolates of Escherichia coli and Acinetobacter baumannii, reducing bacterial load in a murine bladder infection model. Our results indicate that redundant APRs are weak points of bacterial protein homeostasis and that targeting these may be an attractive antibacterial strategy.

Small heat shock proteins are molecular chaperones capable of maintaining denatured proteins in a folding-competent state. We have previously shown that missense mutations in the small heat shock protein HSPB1 (HSP27) cause distal hereditary motor neuropathy and axonal Charcot-Marie-Tooth disease. Here we investigated the biochemical consequences of HSPB1 mutations that are known to cause peripheral neuropathy. In contrast to other chaperonopathies, our results revealed that particular HSPB1 mutations presented higher chaperone activity compared with wild type. Hyperactivation of HSPB1 was accompanied by a change from its wild-type dimeric state to a monomer without dissociation of the 24-meric state. Purification of protein complexes from wild-type and HSPB1 mutants showed that the hyperactive isoforms also presented enhanced binding to client proteins. Furthermore, we show that the wild-type HSPB1 protein undergoes monomerization during heat-shock activation, strongly suggesting that the monomer is the active form of the HSPB1 protein.