The effect of amyloidogenic peptides on bacterial aging correlates with their intrinsic aggregation propensity

The formation of aggregates by misfolded proteins is thought to be inherently toxic, affecting cell fitness. This observation has led to the suggestion that selection against protein aggregation might be a major constraint on protein evolution. The precise fitness cost associated with protein aggregation has been traditionally difficult to evaluate. Moreover, it is not known if the detrimental effect of aggregates on cell physiology is generic or depends on the specific structural features of the protein deposit. In bacteria, the accumulation of intracellular protein aggregates reduces cell reproductive ability, promoting cellular aging. Here, we exploit the cell division defects promoted by the intracellular aggregation of Alzheimer's-disease-related amyloid β peptide in bacteria to demonstrate that the fitness cost associated with protein misfolding and aggregation is connected to the protein sequence, which controls both the in vivo aggregation rates and the conformational properties of the aggregates. We also show that the deleterious impact of protein aggregation on bacterial division can be buffered by molecular chaperones, likely broadening the sequential space on which natural selection can act. Overall, the results in the present work have potential implications for the evolution of proteins and provide a robust system to experimentally model and quantify the impact of protein aggregation on cell fitness.     

Reconsidering the mechanism of polyglutamine peptide aggregation

There are at least nine neurodegenerative diseases associated with proteins that contain an unusually expanded polyglutamine domain, the best known of which is Huntington's disease. In all of these diseases, the mutant protein aggregates into neuronal inclusions; it is generally, although not universally, believed that protein aggregation is an underlying cause of the observed neuronal degeneration. In an effort to examine the role of polyglutamine in facilitating protein aggregation, investigators have used synthetic polyglutamine peptides as model systems. Analysis of kinetic data led to the conclusions that aggregation follows a simple nucleation-elongation mechanism characterized by a significant lag time, during which the peptide is monomeric, and that the nucleus is a monomer in a thermodynamically unfavorable conformation [Chen, S. M., et al. (2002) Proc. Natl. Acad. Sci. U.S.A. 99, 11884-11889]. We re-examined this hypothesis by measuring the aggregation kinetics of the polyglutamine peptide K2Q23K2, using sedimentation, static and dynamic light scattering, and size exclusion chromatography. Our data show that during the lag time in sedimentation kinetics, there is substantial organization of the peptide into soluble linear aggregates. These aggregates have no regular secondary structure as measured by circular dichroism but have particle dimensions and morphologies similar to those of mature insoluble aggregates. The soluble aggregates constitute approximately 30% of the total peptide mass, form rapidly, and continue to grow over a period of hours to days, eventually precipitating. Once insoluble aggregates form, loss of monomer from the solution phase continues. Our data support an assembly mechanism for polyglutamine peptide more complex than that previously proposed.     

DnaK prevents human insulin amyloid fiber formation on hydrophobic surfaces

We have developed a multiwell-based protein aggregation assay to study the kinetics of insulin adsorption and aggregation on hydrophobic surfaces and to investigate the molecular mechanisms involved. Protein-surface interaction progresses in two phases: (1) a lag phase during which proteins adsorb and prefibrillar aggregates form on the material surface and (2) a growth phase during which amyloid fibers form and then are progressively released into solution. We studied the effect of three bacterial chaperones, DnaK, DnaJ, and ClpB, on insulin aggregation kinetics. In the presence of ATP, the simultaneous presence of DnaK, DnaJ, and ClpB allows good protection of insulin against aggregation. In the absence of ATP, DnaK alone is able to prevent insulin aggregation. Furthermore, DnaK binds to insulin adsorbed on hydrophobic surfaces. This process is slowed in the presence of ATP and can be enhanced by the cochaperone DnaJ. The peptide LVEALYL, derived from the insulin B chain, is known to promote fast aggregation in a concentration- and pH-dependent manner in solution. We show that it also shortens the lag phase for insulin aggregation on hydrophobic surfaces. As this peptide is also a known DnaK substrate, our data indicate that the peptide and the chaperone might compete for a common site during the process of insulin aggregation on hydrophobic surfaces.     

Aggregation kinetics of interrupted polyglutamine peptides

Abnormally expanded polyglutamine domains are associated with at least nine neurodegenerative diseases, including Huntington's disease. Expansion of the glutamine region facilitates aggregation of the impacted protein, and aggregation has been linked to neurotoxicity. Studies of synthetic peptides have contributed substantially to our understanding of the mechanism of aggregation because the underlying biophysics of polyglutamine-mediated association can be probed independent of their context within a larger protein. In this report, interrupting residues were inserted into polyglutamine peptides (Q20), and the impact on conformational and aggregation properties was examined. A peptide with two alanine residues formed laterally aligned fibrillar aggregates that were similar to the uninterrupted Q20 peptide. Insertion of two proline residues resulted in soluble, nonfibrillar aggregates, which did not mature into insoluble aggregates. In contrast, insertion of a β-turn template _DPG rapidly accelerated aggregation and resulted in a fibrillar aggregate morphology with little lateral alignment between fibrils. These results are interpreted to indicate that (a) long-range nonspecific interactions lead to the formation of soluble oligomers, while maturation of oligomers into fibrils requires conformational conversion and (b) that soluble oligomers dynamically interact with each other, while insoluble aggregates are relatively inert. Kinetic analysis revealed that the increase in aggregation caused by the _DPG insert is inconsistent with the nucleation-elongation mechanism of aggregation featuring a monomeric β-sheet nucleus. Rather, the data support a mechanism of polyglutamine aggregation by which monomers associate into soluble oligomers, which then undergo slow structural rearrangement to form sedimentable aggregates.     

Aggregation of misfolded proteins can be a selective process dependent upon peptide composition

Intracellular aggregation of misfolded proteins is observed in a number of human diseases, in particular, neurologic disorders in which expanded tracts of polyglutamine residues play a central role. A variety of other proteins are prone to aggregation when mutated, indicating that this process is a common pathologic mechanism for inherited disorders. However, little is known about the relationship between the sequence of aggregating peptides and the specificity of intracellular accumulation. Here we demonstrate that substitution of two residues eliminates aggregation of a 111-amino acid peptide derived from the C-terminal portion of the cystic fibrosis transmembrane conductance regulator (CFTR). We also show that fusion to a reporter protein considerably alters the subcellular distribution of aggregating peptide. When fused to green fluorescent protein, the peptide containing amino acids 1370-1480 of CFTR accumulates in large perinuclear or nuclear aggregates. The same CFTR fragment devoid of green fluorescent protein localizes predominantly to discrete accumulations associated with mitochondria. Importantly, both types of accumulation are dependent on the presence of the same two amino acids within the CFTR sequence. Co-expression studies show that both CFTR-derived proteins can co-localize in large cytoplasmic/nuclear aggregates. However, neither CFTR construct accumulates in intracellular inclusions formed by N-terminal fragment of huntingtin. In addition to unique accumulation patterns, each aggregating peptide shows differences in association with chaperone proteins. Thus, our results indicate that the process of intracellular aggregation can be a selective process determined by the composition of the aggregating peptides.     

Inhibition of insulin fibrillogenesis with targeted peptides

Under conditions of acidic pH and elevated temperature, insulin partially unfolds and aggregates into highly structured amyloid fibrils. Aggregation of insulin leads to loss of activity and can trigger an unwanted immune response. Compounds that prevent protein aggregation have been used to stabilize insulin; these compounds generally suppress aggregation only at relatively high inhibitor concentrations. For example, effective inhibition of aggregation of 0.5 mM insulin required arginine concentrations of > or =100 mM. Here, we investigate a targeted approach toward inhibiting insulin aggregation. VEALYL, corresponding to residues B12-17 of full-length insulin, was identified as a short peptide that interacts with full-length insulin. A hybrid peptide was synthesized that contained this binding domain and hexameric arginine; this peptide significantly reduced the rate of insulin aggregation at near-equimolar concentrations. An effective binding domain and N-terminal placement of the arginine hexamer were necessary for inhibitory activity. The data were analyzed using a simple two-step model of aggregation kinetics. These results are useful not only in identifying an insulin aggregation inhibitor but also in extending a targeted protein strategy for modifying aggregation of amyloidogenic proteins.     

Protein activity in bacterial inclusion bodies correlates with predicted aggregation rates

Recent data show that protein aggregation as bacterial inclusion bodies does not necessarily imply loss of biological activity. Here, we investigate the effect of a large set of single-point mutants of an aggregation-prone protein on its specific activity once deposited in inclusion bodies. The activity of such aggregates significantly correlates with the predicted aggregation rates for each mutant, suggesting that rationally tuning the kinetic competition between folding and aggregation might result in highly active, inclusion bodies. The exploration of this technology during recombinant protein production would have a significant biotechnological value.     

Role of DNA in Bacterial Aggregation

The role of DNA in bacterial aggregation was determined using various types of DNA and Escherichia coli, a good model for investigating the correlation between added polymer and bacterial aggregation and adsorption of polymer to bacterial surfaces. The results of the aggregation assay suggest that extracellular DNA indeed increased the aggregation percentage of E. coli, but this effect was dependent on DNA concentration and length. Moreover, DNA promoted bacterial aggregation in a type-nonspecific way. The combined results of the aggregation assay and the adsorption assay show further that the promotion of E. coli aggregation by DNA occurred along with adsorption of DNA to E. coli. Consequently, the possible mechanisms for DNA-promoted bacterial aggregation are discussed. Using fluorescent-labeled DNA, we mapped DNA within the E. coli aggregates. Subsequently, introduction of DNase I broke up the DNA-involved E. coli aggregates. These results suggest that DNA functions as a molecular bridge to promote E. coli aggregation.     

Inclusion bodies: specificity in their aggregation process and amyloid-like structure

The accumulation of aggregated protein in the cell is associated with the pathology of many diseases and constitutes a major concern in protein production. Intracellular aggregates have been traditionally regarded as nonspecific associations of misfolded polypeptides. This view is challenged by studies demonstrating that, in vitro, aggregation often involves specific interactions. However, little is known about the specificity of in vivo protein deposition. Here, we investigate the degree of in vivo co-aggregation between two self-aggregating proteins, Abeta42 amyloid peptide and foot-and-mouth disease virus VP1 capsid protein, in prokaryotic cells. In addition, the ultrastructure of intracellular aggregates is explored to decipher whether amyloid fibrils and intracellular protein inclusions share structural properties. The data indicate that in vivo protein aggregation exhibits a remarkable specificity that depends on the establishment of selective interactions and results in the formation of oligomeric and fibrillar structures displaying amyloid-like properties. These features allow prokaryotic Abeta42 intracellular aggregates to act as effective seeds in the formation of Abeta42 amyloid fibrils. Overall, our results suggest that conserved mechanisms underlie protein aggregation in different organisms. They also have important implications for biotechnological and biomedical applications of recombinant polypeptides.     

New trends in aggregating tags for therapeutic protein purification

The rapid growth of the therapeutic protein market calls for more efficient purification methods. Various aggregating tags have recently emerged as simple, fast, cost-effective and column-free technologies for protein (and peptide) purification. In general, these column-free protein purification technologies involve the use of aggregating tags that induce the target protein into insoluble aggregates. These aggregates can be easily separated from soluble impurities and the target protein or peptide is then liberated by a cleavage process. This review summarizes the current state-of-the-art in using aggregating tags for protein purification. The methods are here categorized as follows: (1) tags that allow soluble expression of target protein in vivo and induce aggregation in vitro; (2) tags that induce soluble expression and self-assembling of target protein on insoluble biological polyester beads in vivo; (3) tags that induce formation of inactive aggregates in vivo; (4) tags that induce formation of active aggregates in vivo.     

Dual roles of the transmembrane protein p23/TMP21 in the modulation of amyloid precursor protein metabolism

Alzheimer's disease (AD) is characterized by the presence of two histopathological hallmarks; the senile plaques, or extracellular deposits mainly composed of amyloid-β peptide (Aβ), and the neurofibrillary tangles, or intraneuronal inclusions composed of hyperphosphorylated tau protein. Since Aβ aggregates are found in the pathological cases, several strategies are under way to develop drugs that interact with Aβ to reduce its assembly. One of them is 3-amino-1-propane sulfonic acid (Tramiprosate, 3-APS, Alzhemed?), that was developed as a sulfated glycosaminoglycan mimetic, that could interact with Aβ peptide, preventing its aggregation. However, little is known about the action of 3-APS on tau protein aggregation. In this work, we have tested the action of 3-APS on cell viability, microtubule network, actin organization and tau aggregation. Our results indicate that 3-APS favours tau aggregation, in tau transfected non-neuronal cells, and in neuronal cells. We also found that 3-APS does not affect the binding of tau to microtubules but may prevent the formation of tau-actin aggregates. We like to emphasize the importance of testing on both types of pathology (amyloid and tau) the potential drugs to be used for AD treatment.     

AbsoluRATE: An in-silico method to predict the aggregation kinetics of native proteins

Protein aggregation has two aspects, namely, mechanistic and kinetics. Understanding protein aggregation kinetics is critical for prediction of progression of diseases caused by amyloidosis, accumulation of aggregates in biotherapeutics during storage and engineering commercial nano-biomaterials. In this work, we have collected experimentally determined absolute protein aggregation rates and developed an SVM based regression model to predict absolute rates of protein and peptide aggregation near-physiological conditions. The regression model achieved a correlation coefficient of 0.72 with MAE of 0.91 (natural log of k_app, where k_app is in hour^?1) using leave-one-out cross-validation on a dataset of 82 non-redundant proteins/peptides. The model accounts for the experimental conditions (such as temperature, pH, ionic and protein concentration) and sequence-based properties. The amino acid sequence features revealed by this model as being important for aggregation kinetics, are also associated with the aggregation mechanism. In particular, inherent aggregation propensity of the protein/peptide sequence and number of aggregation prone regions (APRs) unpunctuated by the gatekeeping residues, were found to play important roles in the prediction of the absolute aggregation rates. This analysis shows that mechanism and kinetics of protein aggregation are coupled via common sequence attributes. The aggregation kinetic prediction method developed in this work is available at https://web.iitm.ac.in/bioinfo2/absolurate-pred/index.html.     

Amyloid-linked cellular toxicity triggered by bacterial inclusion bodies

The aggregation of proteins in the form of amyloid fibrils and plaques is the characteristic feature of some pathological conditions ranging from neurodegenerative disorders to systemic amyloidoses. The mechanisms by which the aggregation processes result in cell damage are under intense investigation but recent data indicate that prefibrillar aggregates are the most proximate mediators of toxicity rather than mature fibrils. Since it has been shown that prefibrillar forms of the nondisease-related misfolded proteins are highly toxic to cultured mammalian cells we have studied the cytoxicity associated to bacterial inclusion bodies that have been recently described as protein deposits presenting amyloid-like structures. We have proved that bacterial inclusion bodies composed by a misfolding-prone beta-galactosidase fusion protein are clearly toxic for mammalian cells but the beta-galactosidase wild type enzyme forming more structured thermal aggregates does not impair cell viability, despite it also binds and enter into the cells. These results are in the line that the most cytotoxic aggregates are early prefibrilar assemblies but discard the hypothesis that the membrane destabilization is the key event to subsequent disruption of cellular processes, such as ion balance, oxidative state and the eventually cell death.     

The CFTR-derived peptides as a model of sequence-specific protein aggregation

Protein aggregation is a hallmark of a growing group of pathologies known as conformational diseases. Although many native or mutated proteins are able to form aggregates, the exact amino acid sequences involved in the process of aggregation are known only in a few cases. Hence, there is a need for different model systems to expand our knowledge in this area. The so-called ag region was previously found to cause the aggregation of the C-terminal fragment of the cystic fibrosis transmembrane conductance regulator (CFTR). To investigate whether this specific amino acid sequence is able to induce protein aggregation irrespective of the amino acid context, we altered its position within the CFTR-derived C-terminal peptide and analyzed the localization of such modified peptides in transfected mammalian cells. Insertion of the ag region into a different amino acid background affected not only the overall level of intracellular protein aggregation, but also the morphology and subcellular localization of aggregates, suggesting that sequences other than the ag region can substantially influence the peptide’s behavior. Also, the introduction of a short dipeptide (His-Arg) motif, a crucial component of the ag region, into different locations within the C-terminus of CFTR lead to changes in the aggregation pattern that were less striking, although still statistically significant. Thus, our results indicate that even subtle alterations within the aggregating peptide can affect many different aspects of the aggregation process.     

Quantitative and spatio‐temporal features of protein aggregation in Escherichia coli and consequences on protein quality control and cellular ageing

The aggregation of proteins as a result of intrinsic or environmental stress may be cytoprotective, but is also linked to pathophysiological states and cellular ageing. We analysed the principles of aggregate formation and the cellular strategies to cope with aggregates in Escherichia coli using fluorescence microscopy of thermolabile reporters, EM tomography and mathematical modelling. Misfolded proteins deposited at the cell poles lead to selective re‐localization of the DnaK/DnaJ/ClpB disaggregating chaperones, but not of GroEL and Lon to these sites. Polar aggregation of cytosolic proteins is mainly driven by nucleoid occlusion and not by an active targeting mechanism. Accordingly, cytosolic aggregation can be efficiently re‐targeted to alternative sites such as the inner membrane in the presence of site‐specific aggregation seeds. Polar positioning of aggregates allows for asymmetric inheritance of damaged proteins, resulting in higher growth rates of damage‐free daughter cells. In contrast, symmetric damage inheritance of randomly distributed aggregates at the inner membrane abrogates this rejuvenation process, indicating that asymmetric deposition of protein aggregates is important for increasing the fitness of bacterial cell populations.     

Concentration dependent Cu2+ induced aggregation and dityrosine formation of the Alzheimer's disease amyloid-beta peptide

The Amyloid beta peptide (Abeta) of Alzheimer's diseases (AD) is closely linked to the progressive cognitive decline associated with the disease. Cu2+ ions can induce the de novo aggregation of the Abeta peptide into non-amyloidogenic aggregates and the production of a toxic species. The mechanism by which Cu2+ mediates the change from amyloid material toward Cu2+ induced aggregates is poorly defined. Here we demonstrate that the aggregation state of Abeta1-42 at neutral pH is governed by the Cu2+:peptide molar ratio. By probing amyloid content and total aggregation, we observed a distinct Cu2+ switching effect centered at equimolar Cu2+:peptide ratios. At sub-equimolar Cu2+:peptide molar ratios, Abeta1-42 forms thioflavin-T reactive amyloid; conversely, at supra-equimolar Cu2+:peptide molar ratios, Abeta1-42 forms both small spherical oligomers approximately 10-20 nm in size and large amorphous aggregates. We demonstrate that these insoluble aggregates form spontaneously via a soluble species without the presence of an observable lag phase. In seeding experiments, the Cu2+ induced aggregates were unable to influence fibril formation or convert into fibrillar material. Aged Cu2+ induced aggregates are toxic when compared to Abeta1-42 aged in the absence of Cu2+. Importantly, the formation of dityrosine crosslinked Abeta, by the oxidative modification of the peptide, only occurs at equimolar molar ratios and above. The formation of dityrosine adducts occurs following the initiation of aggregation and hence does not drive the formation of the Cu2+ induced aggregates. These results define the role Cu2+ plays in modulating the aggregation state and toxicity of Abeta1-42.     

Exploring New Biological Functions of Amyloids: Bacteria Cell Agglutination Mediated by Host Protein Aggregation

Antimicrobial proteins and peptides (AMPs) are important effectors of the innate immune system that play a vital role in the prevention of infections. Recent advances have highlighted the similarity between AMPs and amyloid proteins. Using the Eosinophil Cationic Protein as a model, we have rationalized the structure-activity relationships between amyloid aggregation and antimicrobial activity. Our results show how protein aggregation can induce bacteria agglutination and cell death. Using confocal and total internal reflection fluorescence microscopy we have tracked the formation in situ of protein amyloid-like aggregates at the bacteria surface and on membrane models. In both cases, fibrillar aggregates able to bind to amyloid diagnostic dyes were detected. Additionally, a single point mutation (Ile13 to Ala) can suppress the protein amyloid behavior, abolishing the agglutinating activity and impairing the antimicrobial action. The mutant is also defective in triggering both leakage and lipid vesicle aggregation. We conclude that ECP aggregation at the bacterial surface is essential for its cytotoxicity. Hence, we propose here a new prospective biological function for amyloid-like aggregates with potential biological relevance.     

Orthogonal cross-seeding: an approach to explore protein aggregates in living cells

Protein aggregation is associated with the pathology of many diseases, especially neurodegenerative diseases. A variety of structurally polymorphic aggregates or preaggregates including amyloid fibrils is accessible to any aggregating protein. Preaggregates are now believed to be the toxic culprits in pathologies rather than mature aggregates. Although clearly valuable, understanding the mechanism of formation and the structural characteristics of these prefibrillar species is currently lacking. We report here a simple new approach to map the nature of the aggregate core of transient aggregated species directly in the cell. The method is conceptually based on the highly discriminating ability of aggregates to recruit new monomeric species with equivalent molecular structure. Different soluble segments comprising parts of an amyloidogenic protein were transiently pulse-expressed in a tightly controlled, time-dependent manner along with the parent aggregating full-length protein, and their recruitment into the insoluble aggregate was monitored immunochemically. We used this approach to determine the nature of the aggregate core of the metastable aggregate species formed during the course of aggregation of a chimera containing a long polyglutamine repeat tract in a bacterial host. Strikingly, we found that different segments of the full-length protein dominated the aggregate core at different times during the course of aggregation. In its simplicity, the approach is also potentially amenable to screen also for compounds that can reshape the aggregate core and induce the formation of alternative nonamyloidogenic species.     

Targeted control of kinetics of beta-amyloid self-association by surface tension-modifying peptides

Brain tissue from Alzheimer's patients contains extracellular senile plaques composed primarily of deposits of fibrillar aggregates of beta-amyloid peptide. beta-Amyloid aggregation is postulated to be a major factor in the onset of this neurodegenerative disease. Recently proposed is the hypothesis that oligomeric intermediates, rather than fully formed insoluble fibrils, are cytotoxic. Previously, we reported the discovery of peptides that accelerate beta-amyloid aggregation yet inhibit toxicity in vitro, in support of this hypothesis. These peptides contain two domains: a recognition element designed to bind to beta-amyloid and a disrupting element that alters beta-amyloid aggregation kinetics. Here we show that the aggregation rate-enhancing activity of the disrupting element correlates strongly with its ability to increase surface tension of aqueous solutions. Using the Hofmeister series as a guide, we designed a novel peptide with terminal side-chain trimethylammonium groups in the disrupting domain. The derivatized peptide greatly increased solvent surface tension and accelerated beta-amyloid aggregation kinetics by severalfold. Equivalent increases in surface tension in the absence of a recognition domain had no effect on beta-amyloid aggregation. These results suggest a novel strategy for targeting localized changes in interfacial energy to specific proteins, as a way to selectively alter protein folding, stability, and aggregation.     

Strong inhibition of peptide amyloid formation by a fatty acid

The aggregation of peptides into amyloid fibrils is associated with several diseases, including Alzheimer’s and Parkinson’s disease. Because hydrophobic interactions often play an important role in amyloid formation, the presence of various hydrophobic or amphiphilic molecules, such as lipids, may influence the aggregation process. We have studied the effect of a fatty acid, linoleic acid, on the fibrillation process of the amyloid-forming model peptide NACore (GAVVTGVTAVA). NACore is a peptide fragment spanning residue 68–78 of the protein α-synuclein involved in Parkinson’s disease. Based primarily on circular dichroism measurements, we found that even a very small amount of linoleic acid can substantially inhibit the fibrillation of NACore. This inhibitory effect manifests itself through a prolongation of the lag phase of the peptide fibrillation. The effect is greatest when the fatty acid is present from the beginning of the process together with the monomeric peptide. Cryogenic transmission electron microscopy revealed the presence of nonfibrillar clusters among NACore fibrils formed in the presence of linoleic acid. We argue that the observed inhibitory effect on fibrillation is due to co-association of peptide oligomers and fatty acid aggregates at the early stage of the process. An important aspect of this mechanism is that it is nonmonomeric peptide structures that associate with the fatty acid aggregates. Similar mechanisms of action could be relevant in amyloid formation occurring in vivo, where the aggregation takes place in a lipid-rich environment.