Protein Conformational Diseases Research Articles

Trehalose Promotes Clearance of Proteotoxic Aggregation of Neurodegenerative Disease-Associated Aberrant Proteins.

Accumulation of misfolded proteins compromises overall cellular health and fitness. The failure to remove misfolded proteins is a critical reason for their unwanted aggregation in dense cellular protein pools. The accumulation of various inclusions serves as a clinical feature for neurodegenerative diseases. Previous findings suggest that different cellular compartments can store these abnormal inclusions. Studies of transgenic mice and cellular models of neurodegenerative diseases indicate that depleted chaperone capacity contributes to the aggregation of damaged or aberrant proteins, which consequently disturb proteostasis and cell viability. However, improving these abnormal proteins' selective elimination is yet to be well understood. Still, molecular strategies that can promote the effective degradation of abnormal proteins without compromising cellular viability are unclear. Here, we reported that the trehalose treatment elevates endogenous proteasome levels and enhances the activities of the proteasome. Trehalose-mediated proteasomal activation elevates the removal of both bona fide misfolded and various neurodegenerative disease-associated proteins. Our current study suggests that trehalose may retain a proteasome activation potential, which seems helpful in the solubilization of different mutant misfolded proteins, improving cell viability. These results reveal a possible molecular approach to reduce the overload of intracellular misfolded proteins, and such cytoprotective functions may play a critical role against protein conformational diseases.

Application of Proteostasis Regulators in GABAA Receptor Misfolding Diseases

Protein homeostasis (proteostasis) relies on an orchestrated balance between protein synthesis, folding, assembly, trafficking, and degradation to ensure normal physiological function. Protein misfolding diseases often arise as a result of deficient proteostasis. Proteostasis regulators enhance the proteostasis network capacity to correct the folding, thus promoting the trafficking and restoring the function of disease-associated variants. Here, we focus on the application of proteostasis regulators to promote the folding, assembly, and trafficking of pathogenic γ-aminobutyric acid type A (GABAA) receptor variants. Since clinical variants in GABAA receptors often lead to neurological diseases, such as epilepsy, proteostasis regulators have the promise to be developed as novel therapeutics to target misfolding-prone GABAA receptors to treat their protein conformational diseases.

Pharmacological Chaperones and Protein Conformational Diseases: Approaches of Computational Structural Biology.

Whenever a protein fails to fold into its native structure, a profound detrimental effect is likely to occur, and a disease is often developed. Protein conformational disorders arise when proteins adopt abnormal conformations due to a pathological gene variant that turns into gain/loss of function or improper localization/degradation. Pharmacological chaperones are small molecules restoring the correct folding of a protein suitable for treating conformational diseases. Small molecules like these bind poorly folded proteins similarly to physiological chaperones, bridging non-covalent interactions (hydrogen bonds, electrostatic interactions, and van der Waals contacts) loosened or lost due to mutations. Pharmacological chaperone development involves, among other things, structural biology investigation of the target protein and its misfolding and refolding. Such research can take advantage of computational methods at many stages. Here, we present an up-to-date review of the computational structural biology tools and approaches regarding protein stability evaluation, binding pocket discovery and druggability, drug repurposing, and virtual ligand screening. The tools are presented as organized in an ideal workflow oriented at pharmacological chaperones' rational design, also with the treatment of rare diseases in mind.

Protein conformation characterization via a silicon resonator-based optical sensor based on the combination of wavelength interrogation and dual polarization detection.

Protein conformational abnormality causes cell malfunction. Conformational change of amyloid protein causes neuron malfunction, which renders "protein conformational disease" Alzheimer's disease. Dual polarization interferometry enables to provide one-dimensional structure of a protein biolayer via deconvolution of interference patterns, which in turn is interpreted as the protein molecule conformation. However, it is still challenging to avoid interference patterns becoming faint and obscure sometimes. Resonance wavelength response to the biolayer structure can achieve a very low detection limit due to inherent high Q factor of an optical resonator. Here, we introduce the concept of combining dual polarization detection with wavelength interrogation via a simple and compact resonator-based optical biosensor. Biolayer were probed by the wave of dual polarization and its opto-geometrical parameters were resolved into resonance wavelength shift. Because protein molecule with distinct conformation produced a biolayer with unique thickness and mass density. Amyloid proteins in monomeric and dimeric morphology were respectively characterized. This concept enables protein conformation characterization in an easy and direct paradigm and provides a desirable sensing performance due to sensitive resonance response in the form of the sharp resonance profile occurring in a nonoverlapping spectrum.

Silybins are stereospecific regulators of the 20S proteasome

A reduced proteasome activity tiles excessive amyloid growth during the progress of protein conformational diseases (PCDs). Hence, the development of safe and effective proteasome enhancers represents an attractive target for the therapeutic treatment of these chronic disorders. Here we analyze two natural diastereoisomers belonging to the family of flavonolignans, Sil A and Sil B, by evaluating their capacity to increase proteasome activity. Enzyme assays carried out on yeast 20S (y20S) proteasome and in parallel on a permanently “open gate” mutant (α3ΔN) evidenced that Sil B is a more efficient 20S activator than Sil A. Conversely, in the case of human 20S proteasome (h20S) a higher affinity and more efficient activation is observed for Sil A. Driven by experimental data, computational studies further demonstrated that the taxifolin group of both diastereoisomers plays a crucial role in their anchoring to the α5/α6 groove of the outer α-ring. However, due to the different stereochemistry at C-7“ and C-8” of ring D, only Sil A was able to reproduce the interactions responsible for h20S proteasome activation induced by their cognate regulatory particles. The provided silybins/h20S interaction models allowed us to rationalize their different ability to activate the peptidase activities of h20S and y20S. Our results provide structural details concerning the important role played by stereospecific interactions in driving Sil A and Sil B binding to the 20S proteasome and may support future rational design of proteasome enhancers.

Bacteria-Derived Protein Aggregates Contribute to the Disruption of Host Proteostasis.

Neurodegenerative protein conformational diseases are characterized by the misfolding and aggregation of metastable proteins encoded within the host genome. The host is also home to thousands of proteins encoded within exogenous genomes harbored by bacteria, fungi, and viruses. Yet, their contributions to host protein-folding homeostasis, or proteostasis, remain elusive. Recent studies, including our previous work, suggest that bacterial products contribute to the toxic aggregation of endogenous host proteins. We refer to these products as bacteria-derived protein aggregates (BDPAs). Furthermore, antibiotics were recently associated with an increased risk for neurodegenerative diseases, including Parkinson’s disease and amyotrophic lateral sclerosis—possibly by virtue of altering the composition of the human gut microbiota. Other studies have shown a negative correlation between disease progression and antibiotic administration, supporting their protective effect against neurodegenerative diseases. These contradicting studies emphasize the complexity of the human gut microbiota, the gut–brain axis, and the effect of antibiotics. Here, we further our understanding of bacteria’s effect on host protein folding using the model Caenorhabditis elegans. We employed genetic and chemical methods to demonstrate that the proteotoxic effect of bacteria on host protein folding correlates with the presence of BDPAs. Furthermore, the abundance and proteotoxicity of BDPAs are influenced by gentamicin, an aminoglycoside antibiotic that induces protein misfolding, and by butyrate, a short-chain fatty acid that we previously found to affect host protein aggregation and the associated toxicity. Collectively, these results increase our understanding of host–bacteria interactions in the context of protein conformational diseases.

Exploiting Caenorhabditis elegans to discover human gut microbiota-mediated intervention strategies in protein conformational diseases.

Exploiting Caenorhabditis elegans to discover human gut microbiota-mediated intervention strategies in protein conformational diseases.

Time-off-pick Assay to Measure Caenorhabditis elegans Motility.

Caenorhabditis elegans is a simple metazoan that is often used as a model organism to study various human ailments with impaired motility phenotypes, including protein conformational diseases. Numerous motility assays that measure neuro-muscular function have been employed using C. elegans . Here, we describe "time-off-pick" (TOP), a novel assay for assessing motility in C. elegans . TOP is conducted by sliding an eyebrow hair under the mid-section of the worm and counting the number of seconds it takes for the worm to crawl completely off. The time it takes for the worm to crawl off the eyebrow hair is proportional to the severity of its motility defect. Other readouts of motility include crawling or swimming phenotypes, and although widely established, have some limitations. For example, worms that are roller mutants are less suitable for crawling or swimming assays. We demonstrated that our novel TOP assay is sensitive to age-dependent changes in motility, thus, providing another more inclusive method to assess motor function in C. elegans . Graphical abstract: Conceptual overview of the "time-off-pick" (TOP) assay. Various C. elegans models exhibit age-dependent defects in motility. The time it takes for a worm to crawl off of an eyebrow pick that is slid under its mid-section is measured in TOP seconds. A greater TOP is indicative of a greater motility defect. Eventually, worms with phenotypes that lead to paralysis will not be able to leave the pick.

Detecting the insoluble protein aggregates in live cells using an AIE derivative of fluorescent protein chromophore

Many incurable or unmanageable human protein conformational diseases are associated with the misfolding or aggregation of the aberrantly processed or mutant proteins. In this work, we report an aggregation-induced emission (AIE) derivative of fluorescent protein chromophore used to detect the insoluble protein aggregates in live cells. Based on the 4-hydroxybenzylidene-imidazolinone (HBI), we designed and synthesized a series of AIEgens that span a wide range of viscosity coefficients (χ), thus mimicking the viscous microenvironment of a wide variety of amorphous protein aggregates. The mechanism of these AIEgens were systematically investigated using a combination of photophysical studies, computational analyses and structure-function studies. With the aid of the AggTag method, the optimized probe was used to realize the imaging of the aggregated proteome under the control of the proteostasis network. Besides, we also described the formation and decomposition of protein aggregates under the control of small molecule proteostasis regulators. Briefly, we developed a series of AIEgens that explore varying viscosity sensitivities to visualize protein aggregation in live cells as well as study other biological processes that associated with local viscosity changes.

Automating Aggregate Quantification in <em>Caenorhabditis elegans</em>

A rise in the prevalence of neurodegenerative protein conformational diseases (PCDs) has fostered a great interest in this subject over the years. This increased attention has called for the diversification and improvement of animal models capable of reproducing disease phenotypes observed in humans with PCDs. Though murine models have proven invaluable, they are expensive and are associated with laborious, low-throughput methods. Use of the Caenorhabditis elegans nematode model to study PCDs has been justified by its relative ease of maintenance, low cost, and rapid generation time, which allow for high-throughput applications. Additionally, high conservation between the C. elegans and human genomes makes this model an invaluable discovery tool. Nematodes that express fluorescently tagged tissue-specific polyglutamine (polyQ) tracts exhibit age- and polyQ length-dependent aggregation characterized by fluorescent foci. Such reporters are often employed as proxies to monitor changes in proteostasis across tissues. Manual aggregate quantification is time-consuming, limiting experimental throughput. Furthermore, manual foci quantification can introduce bias, as aggregate identification can be highly subjective. Herein, a protocol consisting of worm culturing, image acquisition, and data processing was standardized to support high-throughput aggregate quantification using C. elegans that express intestine-specific polyQ. By implementing a C. elegans-based image processing pipeline using CellProfiler, an image analysis software, this method has been optimized to separate and identify individual worms and enumerate their respective aggregates. Though the concept of automation is not entirely unique, the need to standardize such procedures for reproducibility, elimination of bias from manual counting, and increase throughput is high. It is anticipated that these methods can drastically simplify the screening process of large bacterial, genomic, or drug libraries using the C. elegans model.

Automating Aggregate Quantification in Caenorhabditis elegans.

A rise in the prevalence of neurodegenerative protein conformational diseases (PCDs) has fostered a great interest in this subject over the years. This increased attention has called for the diversification and improvement of animal models capable of reproducing disease phenotypes observed in humans with PCDs. Though murine models have proven invaluable, they are expensive and are associated with laborious, low-throughput methods. Use of the Caenorhabditis elegans nematode model to study PCDs has been justified by its relative ease of maintenance, low cost, and rapid generation time, which allow for high-throughput applications. Additionally, high conservation between the C. elegans and human genomes makes this model an invaluable discovery tool. Nematodes that express fluorescently tagged tissue-specific polyglutamine (polyQ) tracts exhibit age- and polyQ length-dependent aggregation characterized by fluorescent foci. Such reporters are often employed as proxies to monitor changes in proteostasis across tissues. Manual aggregate quantification is time-consuming, limiting experimental throughput. Furthermore, manual foci quantification can introduce bias, as aggregate identification can be highly subjective. Herein, a protocol consisting of worm culturing, image acquisition, and data processing was standardized to support high-throughput aggregate quantification using C. elegans that express intestine-specific polyQ. By implementing a C. elegans-based image processing pipeline using CellProfiler, an image analysis software, this method has been optimized to separate and identify individual worms and enumerate their respective aggregates. Though the concept of automation is not entirely unique, the need to standardize such procedures for reproducibility, elimination of bias from manual counting, and increase throughput is high. It is anticipated that these methods can drastically simplify the screening process of large bacterial, genomic, or drug libraries using the C. elegans model.

What can we learn from β-cell failure biomarker application in diabetes in childhood? A systematic review.

BACKGROUNDThe prevalence of diabetes as a catastrophic disease in childhood is growing in the world. The search for novel biomarkers of β-cell failure has been an elusive task because it requires several clinical and biochemical measurements in order to integrate the risk of metabolic syndrome.AIMTo determine which biomarkers are currently used to identify β-cell failure among children and adolescents with high risk factors for diabetes mellitus.METHODSThis systematic review was carried out using a modified version of the PICO protocol (Participants/Intervention/Comparison/Outcome). Once our research question was established, terms were individually researched on three different databases (PubMed, BIREME and Web of Science). The total articles obtained underwent a selection process from which the 78 most relevant articles were retrieved to undergo further analysis. They were assessed individually according to quality criteria.RESULTSFirst, we made the classification of the β-cell-failure biomarkers by the target tissue and the evolution of the disease, separating the biomarkers in relation to the types of diabetes. Second, we demonstrated that most biomarkers currently used as early signs of β-cell failure are those that concern local or systemic inflammation processes and oxidative stress as well as those related to endothelial dysfunction processes. Third, we explored the novelties of diabetes as a protein conformational disease and the novel biomarker called real human islet amyloid polypeptide amyloid oligomers. Finally, we ended with a discussion about the best practice of validation and individual control of using different types of biomarkers in type 1 and type 2 diabetes in order to assess the role they play in the progress of diabetes in childhood.CONCLUSIONThis review makes widely evident that most biomarkers currently used as early signs of β-cell failure are those that concern local or systemic inflammation processes and oxidative stress as well as those related to endothelial dysfunction processes. Landing in the clinical practice we propose that real human islet amyloid polypeptide amyloid oligomers is good for identifying patients with β-cell damage and potentially could substitute many biomarkers.

Recent Insights into the Interplay of Alpha-Synuclein and Sphingolipid Signaling in Parkinson's Disease.

Molecular studies have provided increasing evidence that Parkinson’s disease (PD) is a protein conformational disease, where the spread of alpha-synuclein (ASN) pathology along the neuraxis correlates with clinical disease outcome. Pathogenic forms of ASN evoke oxidative stress (OS), neuroinflammation, and protein alterations in neighboring cells, thereby intensifying ASN toxicity, neurodegeneration, and neuronal death. A number of evidence suggest that homeostasis between bioactive sphingolipids with opposing function—e.g., sphingosine-1-phosphate (S1P) and ceramide—is essential in pro-survival signaling and cell defense against OS. In contrast, imbalance of the “sphingolipid biostat” favoring pro-oxidative/pro-apoptotic ceramide-mediated changes have been indicated in PD and other neurodegenerative disorders. Therefore, we focused on the role of sphingolipid alterations in ASN burden, as well as in a vast range of its neurotoxic effects. Sphingolipid homeostasis is principally directed by sphingosine kinases (SphKs), which synthesize S1P—a potent lipid mediator regulating cell fate and inflammatory response—making SphK/S1P signaling an essential pharmacological target. A growing number of studies have shown that S1P receptor modulators, and agonists are promising protectants in several neurological diseases. This review demonstrates the relationship between ASN toxicity and alteration of SphK-dependent S1P signaling in OS, neuroinflammation, and neuronal death. Moreover, we discuss the S1P receptor-mediated pathways as a novel promising therapeutic approach in PD.

Colonization of the Caenorhabditis elegans gut with human enteric bacterial pathogens leads to proteostasis disruption that is rescued by butyrate.

Protein conformational diseases are characterized by misfolding and toxic aggregation of metastable proteins, often culminating in neurodegeneration. Enteric bacteria influence the pathogenesis of neurodegenerative diseases; however, the complexity of the human microbiome hinders our understanding of how individual microbes influence these diseases. Disruption of host protein homeostasis, or proteostasis, affects the onset and progression of these diseases. To investigate the effect of bacteria on host proteostasis, we used Caenorhabditis elegans expressing tissue-specific polyglutamine reporters that detect changes in the protein folding environment. We found that colonization of the C. elegans gut with enteric bacterial pathogens disrupted proteostasis in the intestine, muscle, neurons, and the gonad, while the presence of bacteria that conditionally synthesize butyrate, a molecule previously shown to be beneficial in neurodegenerative disease models, suppressed aggregation and the associated proteotoxicity. Co-colonization with this butyrogenic strain suppressed bacteria-induced protein aggregation, emphasizing the importance of microbial interaction and its impact on host proteostasis. Further experiments demonstrated that the beneficial effect of butyrate depended on the bacteria that colonized the gut and that this protective effect required SKN-1/Nrf2 and DAF-16/FOXO transcription factors. We also found that bacteria-derived protein aggregates contribute to the observed disruption of host proteostasis. Together, these results reveal the significance of enteric infection and gut dysbiosis on the pathogenesis of protein conformational diseases and demonstrate the potential of using butyrate-producing microbes as a preventative and treatment strategy for neurodegenerative disease.

Identification of Natural Products as Potential Pharmacological Chaperones for Protein Misfolding Diseases.

Defective protein folding and accumulation of misfolded proteins is associated with neurodegenerative, cardiovascular, secretory, and metabolic disorders. Efforts are being made to identify small-molecule modulators or structural-correctors for conformationally destabilized proteins implicated in various protein aggregation diseases. Using a metastable-reporter-based primary screen, we evaluated pharmacological chaperone activity of a diverse class of natural products. We found that a flavonoid glycoside (C-10, chrysoeriol-7-O-β-D-glucopyranoside) stabilizes metastable proteins, prevents its aggregation, and remodels the oligomers into protease-sensitive species. Data was corroborated with additional secondary screen with disease-specific pathogenic protein. In vitro and cell-based experiments showed that C-10 inhibits α-synuclein aggregation which is implicated in synucleinopathies-related neurodegeneration. C-10 interferes in its structural transition into β-sheeted fibrils and mitigates α-synuclein aggregation-associated cytotoxic effects. Computational modeling suggests that C-10 binds to unique sites in α-synuclein which may interfere in its aggregation amplification. These findings open an avenue for comprehensive SAR development for flavonoid glycosides as pharmacological chaperones for metastable and aggregation-prone proteins implicated in protein conformational diseases.

The Autophagy Pathway: A Critical Route in the Disposal of Alpha 1-Antitrypsin Aggregates That Holds Many Mysteries.

The maintenance of proteome homeostasis, or proteostasis, is crucial for preserving cellular functions and for cellular adaptation to environmental challenges and changes in physiological conditions. The capacity of cells to maintain proteostasis requires precise control and coordination of protein synthesis, folding, conformational maintenance, and clearance. Thus, protein degradation by the ubiquitin–proteasome system (UPS) or the autophagy–lysosomal system plays an essential role in cellular functions. However, failure of the UPS or the autophagic process can lead to the development of various diseases (aging-associated diseases, cancer), thus both these pathways have become attractive targets in the treatment of protein conformational diseases, such as alpha 1-antitrypsin deficiency (AATD). The Z alpha 1-antitrypsin (Z-AAT) misfolded variant of the serine protease alpha 1-antitrypsin (AAT) is caused by a structural change that predisposes it to protein aggregation and dramatic accumulation in the form of inclusion bodies within liver hepatocytes. This can lead to clinically significant liver disease requiring liver transplantation in childhood or adulthood. Treatment of mice with autophagy enhancers was found to reduce hepatic Z-AAT aggregate levels and protect them from AATD hepatotoxicity. To date, liver transplantation is the only curative therapeutic option for patients with AATD-mediated liver disease. Therefore, the development and discovery of new therapeutic approaches to delay or overcome disease progression is a top priority. Herein, we review AATD-mediated liver disease and the overall process of autophagy. We highlight the role of this system in the regulation of Z-variant degradation and its implication in AATD-medicated liver disease, including some open questions that remain challenges in the field and require further elucidation. Finally, we discuss how manipulation of autophagy could provide multiple routes of therapeutic benefit in AATD-mediated liver disease.

Isomeric Effect of Nano-Inhibitors on Aβ40 Fibrillation at The Nano-Bio Interface.

Chemical and physical properties of nanobio interface substantially affect the conformational transitions of adjacent biomolecules. Previous studies have reported the chiral effect and charge effect of nanobio interface on the misfolding, aggregation, and fibrillation of amyloid protein. However, the isomeric effect of nanobio interface on protein/peptides amyloidosis is still unclear. Here, three isomeric nanobio interfaces were designed and fabricated based on the same sized gold nanoclusters (AuNCs) modified with 4-mercaptobenzoic acid (p-MBA), 3-mercaptobenzoic acid (m-MBA), and 2-mercaptobenzoic acid (o-MBA). Then three isomeric AuNCs were employed as models to explore the isomeric effect on the misfolding, aggregation, and fibrillation of Aβ40 at nanobio interfaces. Site-specific replacement experiments on the basis of theoretical analysis revealed the possible mechanism of Aβ40 interacting with isomeric ligands of AuNCs at the nanobio interfaces. The distance and orientation of -COOH group from the surface of AuNCs can affect the electrostatic interaction between isomeric ligands and the positively charged residues (R5, K16, and K28) of Aβ40, which may affect the inhibition efficiency of isomeric AuNCs on protein amyloidosis. Actually, the amyloid fibrillation kinetics results together with atomic force microscope (AFM) images, dynamic light scattering (DLS) results and circular dichroism (CD) spectra indeed proved that all the three isomeric AuNCs could inhibit the misfolding, aggregation and fibrillation of Aβ40 in a dose-dependent manner, and the inhibition efficiency was definitely different from each other. The inhibition efficiency of o-MBA-AuNCs was higher than that of m-MBA-AuNCs and p-MBA-AuNCs at the same dosage. These results provide an insight for isomeric effect at nanobio interfaces, and open an avenue for structure-based nanodrug design target Alzheimer's disease (AD) and even other protein conformational diseases.

Mechanistic Insights into the Role of Molecular Chaperones in Protein Misfolding Diseases: From Molecular Recognition to Amyloid Disassembly.

Age-dependent alterations in the proteostasis network are crucial in the progress of prevalent neurodegenerative diseases, such as Alzheimer’s, Parkinson’s, or amyotrophic lateral sclerosis, which are characterized by the presence of insoluble protein deposits in degenerating neurons. Because molecular chaperones deter misfolded protein aggregation, regulate functional phase separation, and even dissolve noxious aggregates, they are considered major sentinels impeding the molecular processes that lead to cell damage in the course of these diseases. Indeed, members of the chaperome, such as molecular chaperones and co-chaperones, are increasingly recognized as therapeutic targets for the development of treatments against degenerative proteinopathies. Chaperones must recognize diverse toxic clients of different orders (soluble proteins, biomolecular condensates, organized protein aggregates). It is therefore critical to understand the basis of the selective chaperone recognition to discern the mechanisms of action of chaperones in protein conformational diseases. This review aimed to define the selective interplay between chaperones and toxic client proteins and the basis for the protective role of these interactions. The presence and availability of chaperone recognition motifs in soluble proteins and in insoluble aggregates, both functional and pathogenic, are discussed. Finally, the formation of aberrant (pro-toxic) chaperone complexes will also be disclosed.

New insight into enzymatic hydrolysis of peptides with site-specific amino acid d-isomerization

The isomerization of l-amino acids in peptides and proteins into d-configuration under physiological conditions would affect the physiological dysfunction and caused protein conformational diseases. The presence of d-amino acids might change the higher-order structure of proteins and triggered abnormal aggregation. In order to better understand this phenomenon and promote degradation, we systematically studied the enzymatic hydrolysis of a series of peptides obtained by replacing l-amino acids in different positions of template peptide KYNETWRSED with d-amino acids under the action of Protease K. The results showed that, compared with normal peptide, isomerization of different amino acids had different effects on the anti-enzymatic hydrolysis of the peptides, especially d-tryptophan at position 6, which significantly inhibited enzymatic hydrolysis. The analysis of the peptide cleavage site revealed that the efficiency of enzymatic hydrolysis mainly depended on the isomerization of the amino acids at a specific site of the peptide cleavage. Further studies showed that the enzymatic hydrolysis of substrates could be facilitated by optimized reaction conditions such as temperature, pH, addition of metal ions, and change of buffer. In this way the accumulation of disease-associated d-amino acid containing polypeptides/proteins could be prevented.

Protein-conformational diseases in childhood: Naturally-occurring hIAPP amyloid-oligomers and early β-cell damage in obesity and diabetes.

Background and aimsThis is the first time that obesity and diabetes mellitus (DM) as protein conformational diseases (PCD) are reported in children and they are typically diagnosed too late, when β-cell damage is evident. Here we wanted to investigate the level of naturally-ocurring or real (not synthetic) oligomeric aggregates of the human islet amyloid polypeptide (hIAPP) that we called RIAO in sera of pediatric patients with obesity and diabetes. We aimed to reduce the gap between basic biomedical research, clinical practice-health decision making and to explore whether RIAO work as a potential biomarker of early β-cell damage.Materials and methodsWe performed a multicentric collaborative, cross-sectional, analytical, ambispective and blinded study; the RIAO from pretreated samples (PTS) of sera of 146 pediatric patients with obesity or DM and 16 healthy children, were isolated, measured by sound indirect ELISA with novel anti-hIAPP cytotoxic oligomers polyclonal antibody (MEX1). We carried out morphological and functional studied and cluster-clinical data driven analysis.ResultsWe demonstrated by western blot, Transmission Electron Microscopy and cell viability experiments that RIAO circulate in the blood and can be measured by ELISA; are elevated in serum of childhood obesity and diabetes; are neurotoxics and works as biomarkers of early β-cell failure. We explored the range of evidence-based medicine clusters that included the RIAO level, which allowed us to classify and stratify the obesity patients with high cardiometabolic risk.ConclusionsRIAO level increases as the number of complications rises; RIAOs > 3.35 μg/ml is a predictor of changes in the current indicators of β-cell damage. We proposed a novel physio-pathological pathway and shows that PCD affect not only elderly patients but also children. Here we reduced the gap between basic biomedical research, clinical practice and health decision making.