Structural Biology of Calcitonin: From Aqueous Therapeutic Properties to Amyloid Aggregation

Under appropriate conditions, peptides and proteins can assemble from their native state into prefibrillar oligomers and then mature into fibrillar aggregates. This transition forms the molecular basis of several pathologies, often related to the deposition of these amyloid fibrils. Several hormone peptides involved in fundamental biological processes have the tendency to self-assemble into amyloid fibrils, resulting in a loss of their native functions, and more importantly, entailing devastating consequences, such as the formation of amyloid depositions. Calcitonin is a 32 amino-acid hormone peptide that can be considered a molecular paradigm for the central events associated with hormone misfolding. Calcitonin in its native form is involved in various physiological functions, including mediating calcium homeostasis and maintaining bone structure. It is the latter function that has motivated the use of calcitonin as an aqueous therapeutic agent for the treatment of bone-related pathologies such as osteoporosis and Paget's disease. Despite some success as a therapeutic, calcitonin's ability to control these diseases is limited by its aggregation along the canonical amyloid aggregation pathway, compromising its long-term stability as a therapeutic agent. A better understanding of the misfolding process would not only provide the structural basis to improve calcitonin's long-term stability and activity as a therapeutic, but also provide valuable insights into pathological aggregation of other amyloids. In this work, we review the physiological roles of calcitonin, its structure, and aggregation process, and consider the effects of calcitonin's structure on its role as a therapeutic.     

Correctors Rescue CFTR Mutations in Nucleotide‐Binding Domain 1 (NBD1) by Modulating Proteostasis

We evaluated whether small molecule correctors could rescue four nucleotide‐binding domain 1 (NBD1) mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene (A455E, S492F, ΔI507, and R560T). We first transfected Cos‐7 cells (green monkey kidney cells) with A455E, S492F, ΔI507, or R560T and created HEK‐293 (human embryonic kidney cells) cell lines stably expressing these CFTR mutations. The mutants showed lowered protein expression, instability at physiological temperature, and rapid degradation. After treatment with correctors CFFT‐002, CFFT‐003, C3, C4, and/or C18, the combination of C18+C4 showed the most correction and resulted in increased CFTR residing in the plasma membrane. We found a profound decrease in binding of CFTR to histone deacetylases (HDAC) 6 and 7 and heat shock proteins (Hsps) 27 and 40. Silencing Hsp27 or 40 rescued the mutants, but no additional amount of CFTR was rescued when both proteins were knocked down simultaneously. Thus, CFTR mutations in NBD1 can be rescued by a combination of correctors, and the treatment alters the interaction between mutated CFTR and the endoplasmic reticulum machinery.     

Hydration of Simple Model Peptides in Aqueous Osmolyte Solutions

The biology and chemistry of proteins and peptides are inextricably linked with water as the solvent. The reason for the high stability of some proteins or uncontrolled aggregation of others may be hidden in the properties of their hydration water. In this study, we investigated the effect of stabilizing osmolyte–TMAO (trimethylamine N-oxide) and destabilizing osmolyte–urea on hydration shells of two short peptides, NAGMA (N-acetyl-glycine-methylamide) and diglycine, by means of FTIR spectroscopy and molecular dynamics simulations. We isolated the spectroscopic share of water molecules that are simultaneously under the influence of peptide and osmolyte and determined the structural and energetic properties of these water molecules. Our experimental and computational results revealed that the changes in the structure of water around peptides, caused by the presence of stabilizing or destabilizing osmolyte, are significantly different for both NAGMA and diglycine. The main factor determining the influence of osmolytes on peptides is the structural-energetic similarity of their hydration spheres. We showed that the chosen peptides can serve as models for various fragments of the protein surface: NAGMA for the protein backbone and diglycine for the protein surface with polar side chains.     

The Mechanism of Mitochondrial Injury in Alpha-1 Antitrypsin Deficiency Mediated Liver Disease

Alpha-1 antitrypsin deficiency (AATD) is caused by a single mutation in the SERPINA1 gene, which culminates in the accumulation of misfolded alpha-1 antitrypsin (ZAAT) within the endoplasmic reticulum (ER) of hepatocytes. AATD is associated with liver disease resulting from hepatocyte injury due to ZAAT-mediated toxic gain-of-function and ER stress. There is evidence of mitochondrial damage in AATD-mediated liver disease; however, the mechanism by which hepatocyte retention of aggregated ZAAT leads to mitochondrial injury is unknown. Previous studies have shown that ER stress is associated with both high concentrations of fatty acids and mitochondrial dysfunction in hepatocytes. Using a human AAT transgenic mouse model and hepatocyte cell lines, we show abnormal mitochondrial morphology and function, and dysregulated lipid metabolism, which are associated with hepatic expression and accumulation of ZAAT. We also describe a novel mechanism of ZAAT-mediated mitochondrial dysfunction. We provide evidence that misfolded ZAAT translocates to the mitochondria for degradation. Furthermore, inhibition of ZAAT expression restores the mitochondrial function in ZAAT-expressing hepatocytes. Altogether, our results show that ZAAT aggregation in hepatocytes leads to mitochondrial dysfunction. Our findings suggest a plausible model for AATD liver injury and the possibility of mechanism-based therapeutic interventions for AATD liver disease.     

非变性离子淌度质谱在蛋白质结构及构象疾病研究方面的应用

离子淌度质谱技术可以分离空间尺寸或构象不同的离子,能够在近似生理条件下表征蛋白质及其复合物的构象,可提供蛋白质及其复合物的构象稳定性及异质性、化学计量比等多重信息,已成为蛋白质构象及蛋白质-配体相互作用研究的重要手段。非变性离子淌度质谱还具有灵敏捕获蛋白质构象动态转变的特点,适用于低浓度、高异质性的蛋白混合物分析。本文综述了离子淌度质谱的基本原理、获取数据及信息形式、以及在蛋白质构象及蛋白质-配体相互作用研究领域的应用进展,重点关注其在蛋白质错误折叠、聚集动力学及与配体相互作用的应用研究。     

Folding funnels and binding mechanisms

The long-held views on lock-and-key versus induced fit in bindingarose from the notion that a protein exists in a single, moststable conformation, dictated by its sequence. However, in solutionproteins exist in a range of conformations, which may be describedby statistical mechanical laws and their populations followstatistical distributions. Upon binding, the equilibrium willshift in favor of the bound conformation from the ensemble ofconformations around the bottom of the folding funnel. Hencehere we extend the implications and the usefulness of the foldingfunnel concept to explain fundamental binding mechanisms.     

Mimicking Molecular Chaperones to Regulate Protein Folding

Folding and unfolding are essential ways for a protein to regulate its biological activity. The misfolding of proteins usually reduces or completely compromises their biological functions, which eventually causes a wide range of diseases including neurodegeneration diseases, type II diabetes, and cancers. Therefore, materials that can regulate protein folding and maintain proteostasis are of significant biological and medical importance. In living organisms, molecular chaperones are a family of proteins that maintain proteostasis by interacting with, stabilizing, and repairing various non-native proteins. In the past few decades, efforts have been made to create artificial systems to mimic the structure and biological functions of nature chaperonins. Herein, recent progress in the design and construction of materials that mimic different kinds of natural molecular chaperones is summarized. The fabrication methods, construction rules, and working mechanisms of these artificial chaperone systems are described. The application of these materials in enhancing the thermal stability of proteins, assisting de novo folding of proteins, and preventing formation of toxic protein aggregates is also highlighted and explored. Finally, the challenges and potential in the field of chaperone-mimetic materials are discussed.     

Transthyretin Stabilization: An Emerging Strategy for the Treatment of Alzheimer’s Disease?

Transthyretin (TTR), previously named prealbumin is a plasma protein secreted mainly by the liver and choroid plexus (CP) that is a carrier for thyroid hormones (THs) and retinol (vitamin A). The structure of TTR, with four monomers rich in β-chains in a globular tetrameric protein, accounts for the predisposition of the protein to aggregate in fibrils, leading to a rare and severe disease, namely transthyretin amyloidosis (ATTR). Much effort has been made and still is required to find new therapeutic compounds that can stabilize TTR (“kinetic stabilization”) and prevent the amyloid genetic process. Moreover, TTR is an interesting therapeutic target for neurodegenerative diseases due to its recognized neuroprotective properties in the cognitive impairment context and interestingly in Alzheimer’s disease (AD). Much evidence has been collected regarding the neuroprotective effects in AD, including through in vitro and in vivo studies as well as a wide range of clinical series. Despite this supported hypothesis of neuroprotection for TTR, the mechanisms are still not completely clear. The aim of this review is to highlight the most relevant findings on the neuroprotective role of TTR, and to summarize the recent progress on the development of TTR tetramer stabilizers.     

Conformational Analysis of Misfolded Protein Aggregation by FRET and Live-Cell Imaging Techniques

Cellular homeostasis is maintained by several types of protein machinery, including molecular chaperones and proteolysis systems. Dysregulation of the proteome disrupts homeostasis in cells, tissues, and the organism as a whole, and has been hypothesized to cause neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS) and Huntington’s disease (HD). A hallmark of neurodegenerative disorders is formation of ubiquitin-positive inclusion bodies in neurons, suggesting that the aggregation process of misfolded proteins changes during disease progression. Hence, high-throughput determination of soluble oligomers during the aggregation process, as well as the conformation of sequestered proteins in inclusion bodies, is essential for elucidation of physiological regulation mechanism and drug discovery in this field. To elucidate the interaction, accumulation, and conformation of aggregation-prone proteins, in situ spectroscopic imaging techniques, such as Förster/fluorescence resonance energy transfer (FRET), fluorescence correlation spectroscopy (FCS), and bimolecular fluorescence complementation (BiFC) have been employed. Here, we summarize recent reports in which these techniques were applied to the analysis of aggregation-prone proteins (in particular their dimerization, interactions, and conformational changes), and describe several fluorescent indicators used for real-time observation of physiological states related to proteostasis.