Vibrational spectroscopy coupled with machine learning sheds light on the cellular effects induced by rationally designed TLR4 agonists.

In this work, we present the potential of Fourier transform infrared (FTIR) microspectroscopy to compare on whole cells, in an unbiased and untargeted way, the capacity of bacterial lipopolysaccharide (LPS) and two rationally designed molecules (FP20 and FP20Rha) to activate molecular circuits of innate immunity. These compounds are important drug hits in the development of vaccine adjuvants and tumor immunotherapeutics. The biological assays indicated that FP20Rha was more potent than FP20 in inducing cytokine production in cells and in stimulating IgG antibody production post-vaccination in mice. Accordingly, the overall significant IR spectral changes induced by the treatment with LPS and FP20Rha were similar, lipids and glycans signals being the most diagnostic, while the effect of the less potent molecule FP20 on cells resulted to be closer to control untreated cells. We propose here the use of FTIR spectroscopy supported by artificial intelligence (AI) to achieve a more holistic understanding of the cell response to new drug candidates while screening them in cells.

Truncation of the constant domain drives amyloid formation by immunoglobulin light chains.

AL amyloidosis is a life-threatening disease caused by deposition of immunoglobulin light chains. While the mechanisms underlying light chains amyloidogenesis in vivo remain unclear, several studies have highlighted the role that tissue environment and structural amyloidogenicity of individual light chains have in the disease pathogenesis. AL natural deposits contain both full-length light chains and fragments encompassing the variable domain (VL) as well as different length segments of the constant region (CL), thus highlighting the relevance that proteolysis may have in the fibrillogenesis pathway. Here, we investigate the role of major truncated species of the disease-associated AL55 light chain that were previously identified in natural deposits. Specifically, we study structure, molecular dynamics, thermal stability, and capacity to form fibrils of a fragment containing both the VL and part of the CL (133-AL55), in comparison with the full-length protein and its variable domain alone, under shear stress and physiological conditions. Whereas the full-length light chain forms exclusively amorphous aggregates, both fragments generate fibrils, although, with different kinetics, aggregate structure, and interplay with the unfragmented protein. More specifically, the VL-CL 133-AL55 fragment entirely converts into amyloid fibrils microscopically and spectroscopically similar to their ex vivo counterpart and increases the amorphous aggregation of full-length AL55. Overall, our data support the idea that light chain structure and proteolysis are both relevant for amyloidogenesis in vivo and provide a novel biocompatible model of light chain fibrillogenesis suitable for future mechanistic studies.

Condensation of the N-terminal domain of human topoisomerase 1 is driven by electrostatic interactions and tuned by its charge distribution.

Liquid-liquid phase separation (LLPS) is pivotal in forming biomolecular condensates, which are crucial in several biological processes. Intrinsically disordered regions (IDRs) are typically responsible for driving LLPS due to their multivalency and high content of charged residues that enable the establishment of electrostatic interactions. In our study, we examined the role of charge distribution in the condensation of the disordered N-terminal domain of human topoisomerase I (hNTD). hNTD is densely charged with oppositely charged residues evenly distributed along the sequence. Its LLPS behavior was compared with that of charge permutants exhibiting varying degrees of charge segregation. At low salt concentrations, hNTD undergoes LLPS. However, LLPS is inhibited by high concentrations of salt and RNA, disrupting electrostatic interactions. Our findings show that, in hNTD, moderate charge segregation promotes the formation of liquid condensates that are sensitive to salt and RNA, whereas marked charge segregation results in the formation of aberrant condensates. Although our study is based on a limited set of protein variants, it supports the applicability of the "stickers-and-spacers" model to biomolecular condensates involving highly charged IDRs. These results may help generate reliable models of the overall LLPS behavior of supercharged polypeptides.

Structural insights and aggregation propensity of a super-stable monellin mutant: A new potential building block for protein-based nanostructured materials.

Protein fibrillation is commonly associated with pathologic amyloidosis. However, under appropriate conditions several proteins form fibrillar structures in vitro that can be used for biotechnological applications. MNEI and its variants, firstly designed as single chain derivatives of the sweet protein monellin, are also useful models for protein fibrillary aggregation studies. In this work, we have drawn attention to a protein dubbed Mut9, already characterized as a "super stable" MNEI variant. Comparative analysis of the respective X-ray structures revealed how the substitutions present in Mut9 eliminate several unfavorable interactions and stabilize the global structure. Molecular dynamic predictions confirmed the presence of a hydrogen-bonds network in Mut9 which increases its stability, especially at neutral pH. Thioflavin-T (ThT) binding assays and Fourier transform infrared (FTIR) spectroscopy indicated that the aggregation process occurs both at acidic and neutral pH, with and without addition of NaCl, even if with a different kinetics. Accordingly, Transmission Electron Microscopy (TEM) showed a fibrillar organization of the aggregates in all the tested conditions, albeit with some differences in the quantity and in the morphology of the fibrils. Our data underline the great potential of Mut9, which combines great stability in solution with the versatile conversion into nanostructured biomaterials.

HspB8 interacts with BAG3 in a "native-like" conformation forming a complex that displays chaperone-like activity.

The HspB8-BAG3 complex plays an important role in the protein quality control acting alone or within multi-components complexes. To clarify the mechanism underlying its activity, in this work we used biochemical and biophysical approaches to study the tendency of both proteins to auto-assemble and to form the complex. Solubility and Thioflavin T assays, Fourier transform infrared spectroscopy and atomic force microscopy analyses clearly showed the tendency of HspB8 to self-assemble at high concentration and to form oligomers in a "native-like" conformation; otherwise, BAG3 aggregates poorly. Noteworthy, also HspB8 and BAG3 associate in a "native-like" conformation, forming a stable complex. Furthermore, the high difference between dissociation constant values of HspB8-HspB8 interaction with respect to the binding to BAG3 obtained by surface plasmon resonance confirms that HspB8 is an obligated partner of BAG3 in vivo. Lastly, both proteins alone or in the complex are able to bind and affect the aggregation of the Josephin domain, the structured domain that triggers the ataxin-3 fibrillation. In particular, the complex displayed higher activity than HspB8 alone. All this considered, we can assert that the two proteins form a stable assembly with chaperone-like activity that could contribute to the physiological role of the complex in vivo.

Addressing Critical Issues Related to Storage and Stability of the Vault Nanoparticle Expressed and Purified from Komagataella phaffi.

The vault nanoparticle is a eukaryotic assembly consisting of 78 copies of the 99-kDa major vault protein. They generate two cup-shaped symmetrical halves, which in vivo enclose protein and RNA molecules. Overall, this assembly is mainly involved in pro-survival and cytoprotective functions. It also holds a remarkable biotechnological potential for drug/gene delivery, thanks to its huge internal cavity and the absence of toxicity/immunogenicity. The available purification protocols are complex, partly because they use higher eukaryotes as expression systems. Here, we report a simplified procedure that combines human vault expression in the yeast Komagataella phaffii, as described in a recent report, and a purification process we have developed. This consists of RNase pretreatment followed by size-exclusion chromatography, which is far simpler than any other reported to date. Protein identity and purity was confirmed by SDS-PAGE, Western blot and transmission electron microscopy. We also found that the protein displayed a significant propensity to aggregate. We thus investigated this phenomenon and the related structural changes by Fourier-transform spectroscopy and dynamic light scattering, which led us to determine the most suitable storage conditions. In particular, the addition of either trehalose or Tween-20 ensured the best preservation of the protein in native, soluble form.

Cheese-whey permeate improves the fitness of Escherichia coli cells during recombinant protein production.

BACKGROUND: Escherichia coli cells are the most frequently used hosts in recombinant protein production processes and mainly require molecules such as IPTG or pure lactose as inducers of heterologous expression. A possible way to reduce the production costs is to replace traditional inducers with waste materials such as cheese whey permeate (CWP). CWP is a secondary by-product generated from the production of the valuable whey proteins, which are obtained from ultrafiltration of cheese whey, a main by-product of the dairy industry, which is rich in lactose. RESULTS: The effects of CWP collected from an Italian plant were compared with those of traditional inducers on the production of two model proteins (i.e., green fluorescent protein and the toxic Q55 variant of ataxin-3), in E. coli BL21 (DE3) cells. It was found that the high lactose content of CWP (165 g/L) and the antioxidant properties of its micronutrients (vitamins, cofactors and osmolytes) sustain production yields similar to those obtained with traditional inducers, accompanied by the improvement of cell fitness. CONCLUSIONS: CWP has proven to be an effective and low-cost alternative inducer to produce recombinant proteins. Its use thus combines the advantage of exploiting a waste product with that of reducing the production costs of recombinant proteins.

An in situ and in vitro investigation of cytoplasmic TDP-43 inclusions reveals the absence of a clear amyloid signature.

Introduction: Several neurodegenerative conditions are associated with a common histopathology within neurons of the central nervous system, consisting of the deposition of cytoplasmic inclusions of TAR DNA-binding protein 43 (TDP-43). Such inclusions have variably been described as morphologically and molecularly ordered aggregates having amyloid properties, as filaments without the cross-ß-structure and dye binding specific for amyloid, or as amorphous aggregates with no defined structure and fibrillar morphology.Aims and Methods: Here we have expressed human full-length TDP-43 in neuroblastoma x spinal cord 34 (NSC-34) cells to investigate the morphological, structural, and tinctorial properties of TDP-43 inclusions in situ. We have used last-generation amyloid diagnostic probes able to cross the cell membrane and detect amyloid in the cytoplasm and have adopted Raman and Fourier transform infrared microspectroscopies to study in situ the secondary structure of the TDP-43 protein in the inclusions. We have then used transmission electron microscopy to study the morphology of the TDP-43 inclusions.Results: The results show the absence of amyloid dye binding, the lack of an enrichment of cross-ß structure in the inclusions, and of a fibrillar texture in the round inclusions. The aggregates formed in vitro from the purified protein under conditions in which it is initially native also lack all these characteristics, ruling out a clear amyloid-like signature.Conclusions: These findings indicate a low propensity of TDP-43 to form amyloid fibrils and even non-amyloid filaments, under conditions in which the protein is initially native and undergoes its typical nucleus-to-cell mislocalization. It cannot be excluded that filaments emerge on the long time scale from such inclusions, but the high propensity of the protein to form initially other types of inclusions appear to be an essential characteristic of TDP-43 proteinopathies.KEY MESSAGESCytoplasmic inclusions of TDP-43 formed in NSC-34 cells do not stain with amyloid-diagnostic dyes, are not enriched with cross-ß structure, and do not show a fibrillar morphology.TDP-43 assemblies formed in vitro from pure TDP-43 do not have any hallmarks of amyloid.

A Global Picture of Molecular Changes Associated to LPS Treatment in THP-1 Derived Human Macrophages by Fourier Transform Infrared Microspectroscopy.

Macrophages are among the first immune cells involved in the initiation of the inflammatory response to protect the host from pathogens. THP-1 derived macrophages (TDM) are used as a model to study the pro-inflammatory effects of lipopolysaccharide (LPS) exposure. Intact TDM cells were analysed by Fourier transform infrared (FTIR) microspectroscopy, supported by multivariate analysis, to obtain a snapshot of the molecular events sparked by LPS stimulation in macrophage-like cells. This spectroscopic analysis enabled the untargeted identification of the most significant spectral components affected by the treatment, ascribable mainly to lipid, protein, and sulfated sugar bands, thus stressing the fundamental role of these classes of molecules in inflammation and in immune response. Our study, therefore, shows that FTIR microspectroscopy enabled the identification of spectroscopic markers of LPS stimulation and has the potential to become a tool to assess those global biochemical changes related to inflammatory and anti-inflammatory stimuli of synthetic and natural immunomodulators different from LPS.

Contribution of Infrared Spectroscopy to the Understanding of Amyloid Protein Aggregation in Complex Systems.

Infrared (IR) spectroscopy is a label-free and non-invasive technique that probes the vibrational modes of molecules, thus providing a structure-specific spectrum. The development of infrared spectroscopic approaches that enable the collection of the IR spectrum from a selected sample area, from micro- to nano-scale lateral resolutions, allowed to extend their application to more complex biological systems, such as intact cells and tissues, thus exerting an enormous attraction in biology and medicine. Here, we will present recent works that illustrate in particular the applications of IR spectroscopy to the in situ characterization of the conformational properties of protein aggregates and to the investigation of the other biomolecules surrounding the amyloids. Moreover, we will discuss the potential of IR spectroscopy to the monitoring of cell perturbations induced by protein aggregates. The essential support of multivariate analyses to objectively pull out the significant and non-redundant information from the spectra of highly complex systems will be also outlined.

Short-chain alcohols inactivate an immobilized industrial lipase through two different mechanisms.

Broadly used in biocatalysis as acyl acceptors or (co)-solvents, short-chain alcohols often cause irreversible loss of enzyme activity. Understanding the mechanisms of inactivation is a necessary step toward the optimization of biocatalytic reactions and the design of enzyme-based sustainable processes. The functional and structural responses of an immobilized enzyme, Novozym 435 (N-435), exposed to methanol, ethanol, and tert-butanol, are explored in this work. N-435 consists of Candida antarctica lipase B (CALB) adsorbed on polymethacrylate beads and finds application in a variety of processes involving the presence of short-chain alcohols. The nature of the N-435 material required the development of an ad hoc method of structural analysis, based on Fourier transform infrared microspectroscopy, which was complemented by catalytic activity assays and by morphological observation by transmission electron microscopy. The inactivation of N-435 was found to be highly dependent on alcohol concentration and occurs through two different mechanisms. Short-chain alcohols induce conformational changes leading to CALB aggregation, which is only partially prevented by immobilization. Moreover, alcohol modifies the texture of the solid support promoting the enzyme release. Overall, knowledge of the molecular mechanisms underlying N-435 inactivation induced by short-chain alcohols promises to overcome the limitations that usually occur during industrial processes.

Characterization of the Conformational Properties of Soluble and Insoluble Proteins by Fourier Transform Infrared Spectroscopy.

The FTIR (micro-)spectroscopy method applied to the study of the structural properties of different soluble and insoluble proteins will be illustrated. In particular, we will discuss the procedure to analyze proteins in form of hydrated films and in solution by means of attenuated total reflection (ATR) measurements. Moreover, we will describe the procedure to characterize bacterial inclusion bodies (IBs) and amyloid deposits within human tissues by means of FTIR microspectroscopy.

Tear-Based Vibrational Spectroscopy Applied to Amyotrophic Lateral Sclerosis.

Biofluid analysis by optical spectroscopy techniques is attracting considerable interest due to its potential to revolutionize diagnostics and precision medicine, particularly for neurodegenerative diseases. However, the lack of effective biomarkers combined with the unaccomplished identification of convenient biofluids has drastically hampered optical advancements in clinical diagnosis and monitoring of neurodegenerative disorders. Here, we show that vibrational spectroscopy applied to human tears opens a new route, offering a non-invasive, label-free identification of a devastating disease such as amyotrophic lateral sclerosis (ALS). Our proposed approach has been validated using two widespread techniques, namely, Fourier transform infrared (FTIR) and Raman microspectroscopies. In conjunction with multivariate analysis, this vibrational approach made it possible to discriminate between tears from ALS patients and healthy controls (HCs) with high specificity (∼97% and ∼100% for FTIR and Raman spectroscopy, respectively) and sensitivity (∼88% and ∼100% for FTIR and Raman spectroscopy, respectively). Additionally, the investigation of tears allowed us to disclose ALS spectroscopic markers related to protein and lipid alterations, as well as to a reduction of the phenylalanine level, in comparison with HCs. Our findings show that vibrational spectroscopy is a new potential ALS diagnostic approach and indicate that tears are a reliable and non-invasive source of ALS biomarkers.

Single-Tear Proteomics: A Feasible Approach to Precision Medicine.

Lacrimal fluid is an attractive source of noninvasive biomarkers, the main limitation being the small sample amounts typically collected. Advanced analytical methods to allow for proteomics profiling from a few microliters are needed to develop innovative biomarkers, with attractive perspectives of applications to precision medicine. This work describes an effective, analytical pipeline for single-tear analysis by ultrahigh-resolution, shotgun proteomics from 23 healthy human volunteers, leading to high-confidence identification of a total of 890 proteins. Highly reproducible quantification was achieved by either peak intensity, peak area, or spectral counting. Hierarchical clustering revealed a stratification of females vs. males that did not emerge from previous studies on pooled samples. Two subjects were monitored weekly over 3 weeks. The samples clustered by withdrawal time of day (morning vs. afternoon) but not by follow-up week, with elevated levels of components of the immune system in the morning samples. This study demonstrates feasibility of single-tear quantitative proteomics, envisaging contributions of this unconventional body fluid to individualized approaches in biomedicine.

Transcriptomic analysis of Rhodococcus opacus R7 grown on polyethylene by RNA-seq.

Plastic waste management has become a global issue. Polyethylene (PE) is the most abundant synthetic plastic worldwide, and one of the most resistant to biodegradation. Indeed, few bacteria can degrade polyethylene. In this paper, the transcriptomic analysis unveiled for the first time Rhodococcus opacus R7 complex genetic system based on diverse oxidoreductases for polyethylene biodegradation. The RNA-seq allowed uncovering genes putatively involved in the first step of oxidation. In-depth investigations through preliminary bioinformatic analyses and enzymatic assays on the supernatant of R7 grown in the presence of PE confirmed the activation of genes encoding laccase-like enzymes. Moreover, the transcriptomic data allowed identifying candidate genes for the further steps of short aliphatic chain oxidation including alkB gene encoding an alkane monooxygenase, cyp450 gene encoding cytochrome P450 hydroxylase, and genes encoding membrane transporters. The PE biodegradative system was also validated by FTIR analysis on R7 cells grown on polyethylene.

Pathological ATX3 Expression Induces Cell Perturbations in E. coli as Revealed by Biochemical and Biophysical Investigations.

Amyloid aggregation of human ataxin-3 (ATX3) is responsible for spinocerebellar ataxia type 3, which belongs to the class of polyglutamine neurodegenerative disorders. It is widely accepted that the formation of toxic oligomeric species is primarily involved in the onset of the disease. For this reason, to understand the mechanisms underlying toxicity, we expressed both a physiological (ATX3-Q24) and a pathological ATX3 variant (ATX3-Q55) in a simplified cellular model, Escherichia coli. It has been observed that ATX3-Q55 expression induces a higher reduction of the cell growth compared to ATX3-Q24, due to the bacteriostatic effect of the toxic oligomeric species. Furthermore, the Fourier transform infrared microspectroscopy investigation, supported by multivariate analysis, made it possible to monitor protein aggregation and the induced cell perturbations in intact cells. In particular, it has been found that the toxic oligomeric species associated with the expression of ATX3-Q55 are responsible for the main spectral changes, ascribable mainly to the cell envelope modifications. A structural alteration of the membrane detected through electron microscopy analysis in the strain expressing the pathological form supports the spectroscopic results.

Conversion of sugar beet residues into lipids by Lipomyces starkeyi for biodiesel production.

BACKGROUND: Lipids from oleaginous yeasts emerged as a sustainable alternative to vegetable oils and animal fat to produce biodiesel, the biodegradable and environmentally friendly counterpart of petro-diesel fuel. To develop economically viable microbial processes, the use of residual feedstocks as growth and production substrates is required. RESULTS: In this work we investigated sugar beet pulp (SBP) and molasses, the main residues of sugar beet processing, as sustainable substrates for the growth and lipid accumulation by the oleaginous yeast Lipomyces starkeyi. We observed that in hydrolysed SBP the yeast cultures reached a limited biomass, cellular lipid content, lipid production and yield (2.5 g/L, 19.2%, 0.5 g/L and 0.08 g/g, respectively). To increase the initial sugar availability, cells were grown in SBP blended with molasses. Under batch cultivation, the cellular lipid content was more than doubled (47.2%) in the presence of 6% molasses. Under pulsed-feeding cultivation, final biomass, cellular lipid content, lipid production and lipid yield were further improved, reaching respectively 20.5 g/L, 49.2%, 9.7 g/L and 0.178 g/g. Finally, we observed that SBP can be used instead of ammonium sulphate to fulfil yeasts nitrogen requirement in molasses-based media for microbial oil production. CONCLUSIONS: This study demonstrates for the first time that SBP and molasses can be blended to create a feedstock for the sustainable production of lipids by L. starkeyi. The data obtained pave the way to further improve lipid production by designing a fed-batch process in bioreactor.

Exploring the use of leucine zippers for the generation of a new class of inclusion bodies for pharma and biotechnological applications.

BACKGROUND: Inclusion bodies (IBs) are biologically active protein aggregates forming natural nanoparticles with a high stability and a slow-release behavior. Because of their nature, IBs have been explored to be used as biocatalysts, in tissue engineering, and also for human and animal therapies. To improve the production and biological efficiency of this nanomaterial, a wide range of aggregation tags have been evaluated. However, so far, the presence in the IBs of bacterial impurities such as lipids and other proteins coexisting with the recombinant product has been poorly studied. These impurities could strongly limit the potential of IB applications, being necessary to control the composition of these bacterial nanoparticles. Thus, we have explored the use of leucine zippers as alternative tags to promote not only aggregation but also the generation of a new type of IB-like protein nanoparticles with improved physicochemical properties. RESULTS: Three different protein constructs, named GFP, J-GFP-F and J/F-GFP were engineered. J-GFP-F corresponded to a GFP flanked by two leucine zippers (Jun and Fos); J/F-GFP was formed coexpressing a GFP fused to Jun leucine zipper (J-GFP) and a GFP fused to a Fos leucine zipper (F-GFP); and, finally, GFP was used as a control without any tag. All of them were expressed in Escherichia coli and formed IBs, where the aggregation tendency was especially high for J/F-GFP. Moreover, those IBs formed by J-GFP-F and J/F-GFP constructs were smaller, rougher, and more amorphous than GFP ones, increasing surface/mass ratio and, therefore, surface for protein release. Although the lipid and carbohydrate content were not reduced with the addition of leucine zippers, interesting differences were observed in the protein specific activity and conformation with the addition of Jun and Fos. Moreover, J-GFP-F and J/F-GFP nanoparticles were purer than GFP IBs in terms of protein content. CONCLUSIONS: This study proved that the use of leucine zippers strategy allows the formation of IBs with an increased aggregation ratio and protein purity, as we observed with the J/F-GFP approach, and the formation of IBs with a higher specific activity, in the case of J-GFP-F IBs. Thus, overall, the use of leucine zippers seems to be a good system for the production of IBs with more promising characteristics useful for pharma or biotech applications.

Understanding the self-assembly pathways of a single chain variant of monellin: A first step towards the design of sweet nanomaterials.

Peptides and proteins possess an inherent tendency to self-assemble, prompting the formation of amyloid aggregates from their soluble and functional states. Amyloids are linked to many devastating diseases, but self-assembling proteins can also represent formidable tools to produce new and sustainable biomaterials for biomedical and biotechnological applications. The mechanism of fibrillar aggregation, which influences the morphology and the properties of the protein aggregates, depend on factors such as pH, ionic strength, temperature, agitation, and protein concentration. We have here used intensive mechanical agitation, with or without beads, to prompt the aggregation of the single-chain derivative of the plant protein monellin, named MNEI, which is a well characterized sweet protein. Transmission electron microscopy confirmed the formation of fibrils several micrometers long, morphologically different from the previously characterized fibers of MNEI. Changes in the protein secondary structures during the aggregation process were monitored by Fourier transform infrared spectroscopy, which detected differences in the conformation of the final aggregates obtained under mechanical agitation. Moreover, soluble oligomers could be detected in the early phases of aggregation by polyacrylamide gel-electrophoresis. These findings emphasize the existence of multiple pathways of fibrillar aggregation for MNEI, which could be exploited for the design of innovative protein-based biomaterials.

Probing structural changes during amyloid aggregation of the sweet protein MNEI.

Protein self-assembly is a ubiquitous phenomenon, traditionally studied for its links to amyloid pathologies, which has also gained attention as its physiological roles and possible biotechnological applications emerged over time. It is also known that varying the conditions to which proteins are exposed can lead to aggregate polymorphism. To understand the factors that trigger aggregation and/or direct it toward specific outcomes, we performed a multifaceted structural characterization of the thermally induced self-assembly process of MNEI, a model protein able to form amyloid aggregates under nondenaturing conditions. MNEI is also known for its extreme sweetness which, combined with a considerable thermal stability, makes the protein a promising alternative sweetener. Fourier-transformed infrared spectroscopy and electron microscopy data showed that the presence of NaCl accelerates the kinetics of fibrillar aggregation, while disfavoring the population of off-pathway states that are instead detected by native gel electrophoresis at low ionic strength. NMR studies revealed how NaCl modulates the self-assembling mechanism of MNEI, switching the process from soluble oligomeric forms to fibrils. Comparative analysis demonstrated that the presence of NaCl induces local differences in the protein dynamics and surface accessibility, without altering the native fold. We identified the regions most affected by the presence of NaCl, which control the aggregation process, and represent hot spots on the protein surface for the rational design of new mutants with controlled aggregation propensity.