Protein Folding and Misfolding on Surfaces

Protein folding, misfolding and aggregation, as well as the way misfolded and aggregated proteins affects cell viability are emerging as key themes in molecular and structural biology and in molecular medicine. Recent advances in the knowledge of the biophysical basis of protein folding have led to propose the energy landscape theory which provides a consistent framework to better understand how a protein folds rapidly and efficiently to the compact, biologically active structure. The increased knowledge on protein folding has highlighted its strict relation to protein misfolding and aggregation, either process being in close competition with the other, both relying on the same physicochemical basis. The theory has also provided information to better understand the structural and environmental factors affecting protein folding resulting in protein misfolding and aggregation into ordered or disordered polymeric assemblies. Among these, particular importance is given to the effects of surfaces. The latter, in some cases make possible rapid and efficient protein folding but most often recruit proteins/peptides increasing their local concentration thus favouring misfolding and accelerating the rate of nucleation. It is also emerging that surfaces can modify the path of protein misfolding and aggregation generating oligomers and polymers structurally different from those arising in the bulk solution and endowed with different physical properties and cytotoxicities.     

Alchemical Design of Pharmacological Chaperones with Higher Affinity for Phenylalanine Hydroxylase

Phenylketonuria (PKU) is a rare metabolic disease caused by variations in a human gene, PAH, encoding phenylalanine hydroxylase (PAH), and the enzyme converting the essential amino acid phenylalanine into tyrosine. Many PKU-causing variations compromise the conformational stability of the encoded enzyme, decreasing or abolishing its catalytic activity, and leading to an elevated concentration of phenylalanine in the blood, which is neurotoxic. Several therapeutic approaches have been developed to treat the more severe manifestations of the disorder, but they are either not entirely effective or difficult to adhere to throughout life. In a search for novel pharmacological chaperones to treat PKU, a lead compound was discovered (compound IV) that exhibited promising in vitro and in vivo chaperoning activity on PAH. The structure of the PAH-IV complex has been reported. Here, using alchemical free energy calculations (AFEC) on the structure of the PAH-IV complex, we design a new generation of compound IV-analogues with a higher affinity for the enzyme. Seventeen novel analogues were synthesized, and thermal shift and isothermal titration calorimetry (ITC) assays were performed to experimentally evaluate their stabilizing effect and their affinity for the enzyme. Most of the new derivatives bind to PAH tighter than lead compound IV and induce a greater thermostabilization of the enzyme upon binding. Importantly, the correspondence between the calculated alchemical binding free energies and the experimentally determined ΔΔG_b values is excellent, which supports the use of AFEC to design pharmacological chaperones to treat PKU using the X-ray structure of their complexes with the target PAH enzyme.     

A Systematic Investigation of Iminosugar Click Clusters as Pharmacological Chaperones for the Treatment of Gaucher Disease

A series of 18 mono‐ to 14‐valent iminosugars with different ligands, scaffolds, and alkyl spacer lengths have been synthesized and evaluated as inhibitors and pharmacological chaperones of β‐glucocerebrosidase (GCase). Small but significant multivalent effects in GCase inhibition have been observed for two iminosugar clusters. Our study provides strong confirmation that compounds that display the best affinity for GCase are not necessarily the best chaperones. The best chaperoning effect observed for a deprotected iminosugar cluster has been obtained with a tetravalent 1‐deoxynojirimycin (DNJ) analogue (3.3‐fold increase at 10 μM ). In addition, our study provides the first evidence of the high potential of prodrugs for the development of potent pharmacological chaperones. Acetylation of a trivalent DNJ derivative, to give the corresponding acetate prodrug, leads to a pharmacological chaperone that produces higher enzyme activity increases (3.0‐fold instead of 2.4‐fold) at a cellular concentration (1 μM ) reduced by one order of magnitude.     

Glucosylceramide Mimics: Highly Potent GCase Inhibitors and Selective Pharmacological Chaperones for Mutations Associated with Types 1 and 2 Gaucher Disease

A series of iminoxylitol derivatives carrying a C‐linked di‐O‐acyl or di‐O‐alkyl glyceryl substituent were prepared and characterized. All of these compounds, which were designed as glucosylceramide (GlcCer) mimics, were nanomolar inhibitors of lysosomal β‐glucosidase (glucocerebrosidase, GCase). Two of these pseudoglycolipids were further evaluated for their ability to enhance the activity of mutant GCase in human Gaucher cells. Although the di‐O‐hexyl ether was surprisingly devoid of chaperoning activity on both N370S and L444P GCases, the di‐O‐decanoyl ester was a potent chaperone of the L444P hydrolase, capable of increasing the residual activity of the enzyme by a factor of two at a very low concentration (50 nM ); such a significant effect on the L444P mutation in human fibroblasts has not yet been observed. In heat‐stress studies, the diether was found to be much more effective in stabilizing the wild‐type enzyme than the diester. Four representative pseudoglycolipids were also assayed as inhibitors of GlcCer synthase, because such compounds could find use in the substrate reduction therapy approach to treat lysosomal storage diseases, but these compounds revealed only moderate activity. As efficient pharmacological chaperones, new structures such as the di‐C₁₀‐ester constitute leads for the development of therapeutic agents for types 2 and 3 Gaucher disease, the most severe neuronopathic forms of this lysosomal disease.     

Protein Misfolding and Aggregation in the Brain: Common Pathogenetic Pathways in Neurodegenerative and Mental Disorders

Mental disorders represent common brain diseases characterized by substantial impairments of social and cognitive functions. The neurobiological causes and mechanisms of psychopathologies still have not been definitively determined. Various forms of brain proteinopathies, which include a disruption of protein conformations and the formation of protein aggregates in brain tissues, may be a possible cause behind the development of psychiatric disorders. Proteinopathies are known to be the main cause of neurodegeneration, but much less attention is given to the role of protein impairments in psychiatric disorders’ pathogenesis, such as depression and schizophrenia. For this reason, the aim of this review was to discuss the potential contribution of protein illnesses in the development of psychopathologies. The first part of the review describes the possible mechanisms of disruption to protein folding and aggregation in the cell: endoplasmic reticulum stress, dysfunction of chaperone proteins, altered mitochondrial function, and impaired autophagy processes. The second part of the review addresses the known proteins whose aggregation in brain tissue has been observed in psychiatric disorders (amyloid, tau protein, α-synuclein, DISC-1, disbindin-1, CRMP1, SNAP25, TRIOBP, NPAS3, GluA1, FABP, and ankyrin-G).     

Potential for Protein Kinase Pharmacological Regulation in Flaviviridae Infections

Protein kinases (PKs) are enzymes that catalyze the transfer of the terminal phosphate group from ATP to a protein acceptor, mainly to serine, threonine, and tyrosine residues. PK catalyzed phosphorylation is critical to the regulation of cellular signaling pathways that affect crucial cell processes, such as growth, differentiation, and metabolism. PKs represent attractive targets for drugs against a wide spectrum of diseases, including viral infections. Two different approaches are being applied in the search for antivirals: compounds directed against viral targets (direct-acting antivirals, DAAs), or against cellular components essential for the viral life cycle (host-directed antivirals, HDAs). One of the main drawbacks of DAAs is the rapid emergence of drug-resistant viruses. In contrast, HDAs present a higher barrier to resistance development. This work reviews the use of chemicals that target cellular PKs as HDAs against virus of the Flaviviridae family (Flavivirus and Hepacivirus), thus being potentially valuable therapeutic targets in the control of these pathogens.     

Protein Misfolding and Aggregation: The Relatedness between Parkinson’s Disease and Hepatic Endoplasmic Reticulum Storage Disorders

Dysfunction of cellular homeostasis can lead to misfolding of proteins thus acquiring conformations prone to polymerization into pathological aggregates. This process is associated with several disorders, including neurodegenerative diseases, such as Parkinson’s disease (PD), and endoplasmic reticulum storage disorders (ERSDs), like alpha-1-antitrypsin deficiency (AATD) and hereditary hypofibrinogenemia with hepatic storage (HHHS). Given the shared pathophysiological mechanisms involved in such conditions, it is necessary to deepen our understanding of the basic principles of misfolding and aggregation akin to these diseases which, although heterogeneous in symptomatology, present similarities that could lead to potential mutual treatments. Here, we review: (i) the pathological bases leading to misfolding and aggregation of proteins involved in PD, AATD, and HHHS: alpha-synuclein, alpha-1-antitrypsin, and fibrinogen, respectively, (ii) the evidence linking each protein aggregation to the stress mechanisms occurring in the endoplasmic reticulum (ER) of each pathology, (iii) a comparison of the mechanisms related to dysfunction of proteostasis and regulation of homeostasis between the diseases (such as the unfolded protein response and/or autophagy), (iv) and clinical perspectives regarding possible common treatments focused on improving the defensive responses to protein aggregation for diseases as different as PD, and ERSDs.     

Strategies for the Inhibition of Protein Aggregation in Human Diseases

Protein misfolding and aggregation has been related to several human disorders, generally termed protein aggregation diseases. These diseases include neurodegenerative disorders such as Alzheimer's, Parkinson's, and Huntington's diseases and peripheral disorders such as systemic amyloidosis and type 2 diabetes. The complexity of the aggregation processes and the intertwined events account for the fact that no effective disease‐modifying treatments for these disorders are currently available. Nevertheless, in‐depth research into the aggregation processes has recently yielded major insights into some key mechanisms of aggregation‐mediated cell toxicity, offering new targets for drug development. In addition, recent findings in the field have identified similar features, revealing the possibility of shared mechanisms and hence potential common approaches for intervention. This review aims to give an overview of potential strategies for tackling protein aggregation and its associated toxicity, focusing on protein aggregation in human disease.     

Regulation by Different Types of Chaperones of Amyloid Transformation of Proteins Involved in the Development of Neurodegenerative Diseases

The review highlights various aspects of the influence of chaperones on amyloid proteins associated with the development of neurodegenerative diseases and includes studies conducted in our laboratory. Different sections of the article are devoted to the role of chaperones in the pathological transformation of alpha-synuclein and the prion protein. Information about the interaction of the chaperonins GroE and TRiC as well as polymer-based artificial chaperones with amyloidogenic proteins is summarized. Particular attention is paid to the effect of blocking chaperones by misfolded and amyloidogenic proteins. It was noted that the accumulation of functionally inactive chaperones blocked by misfolded proteins might cause the formation of amyloid aggregates and prevent the disassembly of fibrillar structures. Moreover, the blocking of chaperones by various forms of amyloid proteins might lead to pathological changes in the vital activity of cells due to the impaired folding of newly synthesized proteins and their subsequent processing. The final section of the article discusses both the little data on the role of gut microbiota in the propagation of synucleinopathies and prion diseases and the possible involvement of the bacterial chaperone GroE in these processes.     

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.     

Alpha Synuclein only Forms Fibrils In Vitro when Larger than its Critical Size of 70 Monomers

The aggregation of α-synuclein into small soluble aggregates and then fibrils is important in the development and spreading of aggregates through the brain in Parkinson's disease. Fibrillar aggregates can grow by monomer addition and then break into fragments that could spread into neighboring cells. The rate constants for fibril elongation and fragmentation have been measured but it is not known how large an aggregate needs to be before fibril formation is thermodynamically favorable. This critical size is an important parameter controlling at what stage in an aggregation reaction fibrils can form and replicate. We determined this value to be approximately 70 monomers using super-resolution and atomic force microscopy imaging of individual α-synuclein aggregates formed in solution over long time periods. This represents the minimum size for a stable α-synuclein fibril and we hypothesis the formation of aggregates of this size in a cell represents a tipping point at which rapid replication occurs.     

Genome-Wide Identification and Expression Analysis of Heat Shock Protein 70 (HSP70) Gene Family in Pumpkin (Cucurbita moschata) Rootstock under Drought Stress Suggested the Potential Role of these Chaperones in Stress Tolerance

Heat shock protein 70s (HSP70s) are highly conserved proteins that are involved in stress responses. These chaperones play pivotal roles in protein folding, removing the extra amounts of oxidized proteins, preventing protein denaturation, and improving the antioxidant system activities. This conserved family has been characterized in several crops under drought stress conditions. However, there is no study on HSP70s in pumpkin (Cucurbita moschata). Therefore, we performed a comprehensive analysis of this gene family, including phylogenetic relationship, motif and gene structure analysis, gene duplication, collinearity, and promoter analysis. In this research, we found 21 HSP70s that were classified into five groups (from A to E). These genes were mostly localized in the cytoplasm, chloroplast, mitochondria, nucleus, and endoplasmic reticulum (ER). We could observe more similarity in closely linked subfamilies in terms of motifs, the number of introns/exons, and the corresponding cellular compartments. According to the collinearity analysis, gene duplication had occurred as a result of purifying selection. The results showed that the occurrence of gene duplication for all nine gene pairs was due to segmental duplication (SD). Synteny analysis revealed a closer relationship between pumpkin and cucumber than pumpkin and Arabidopsis. Promoter analysis showed the presence of various cis-regulatory elements in the up-stream region of the HSP70 genes, such as hormones and stress-responsive elements, indicating a potential role of this gene family in stress tolerance. We furtherly performed the gene expression analysis of the HSP70s in pumpkin under progressive drought stress. Pumpkin is widely used as a rootstock to improve stress tolerance, as well as fruit quality of cucumber scion. Since stress-responsive mobile molecules translocate through vascular tissue from roots to the whole plant body, we used the xylem of grafted materials to study the expression patterns of the HSP70 (potentially mobile) gene family. The results indicated that all CmoHSP70s had very low expression levels at 4 days after stress (DAS). However, the genes showed different expression patterns by progressing he drought period. For example, the expression of CmoHSP70-4 (in subgroup E) and CmoHSP70-14 (in subgroup C) sharply increased at 6 and 11 DAS, respectively. However, the expression of all genes belonging to subgroup A did not change significantly in response to drought stress. These findings indicated the diverse roles of this gene family under drought stress and provided valuable information for further investigation on the function of this gene family, especially under stressful conditions.     

Identification of mNeonGreen as a pH-Dependent,Turn-On Fluorescent Protein Sensor for Chloride

Chloride-sensitive fluorescent proteins generated from laboratory evolution have a characteristic tyrosine residue that interacts with a chloride ion and π-stacks with the chromophore. However, the engineered yellow-green fluorescent protein mNeonGreen lacks this interaction but still binds chloride, as seen in a recently reported crystal structure. Based on its unique coordination sphere, we were curious if chloride could influence the optical properties of mNeonGreen. Here, we present the structure-guided identification and spectroscopic characterization of mNeonGreen as a turn-on fluorescent protein sensor for chloride. Our results show that chloride binding lowers the chromophore pK_a and shifts the equilibrium away from the weakly fluorescent phenol form to the highly fluorescent phenolate form, resulting in a pH-dependent, turn-on fluorescence response. Moreover, through mutagenesis, we link this sensing mechanism to a non-coordinating residue in the chloride binding pocket. This discovery sets the stage to further engineer mNeonGreen as a new fluorescent protein-based tool for imaging cellular chloride.     

Impact of Environmental Risk Factors on Mitochondrial Dysfunction,Neuroinflammation, Protein Misfolding,and Oxidative Stress in the Etiopathogenesis of Parkinson’s Disease

As a prevalent progressive neurodegenerative disorder, Parkinson’s disease (PD) is characterized by the neuropathological hallmark of the loss of nigrostriatal dopaminergic (DAergic) innervation and the appearance of Lewy bodies with aggregated α-synuclein. Although several familial forms of PD have been reported to be associated with several gene variants, most cases in nature are sporadic, triggered by a complex interplay of genetic and environmental risk factors. Numerous epidemiological studies during the past two decades have shown positive associations between PD and several environmental factors, including exposure to neurotoxic pesticides/herbicides and heavy metals as well as traumatic brain injury. Other environmental factors that have been implicated as potential risk factors for PD include industrial chemicals, wood pulp mills, farming, well-water consumption, and rural residence. In this review, we summarize the environmental toxicology of PD with the focus on the elaboration of chemical toxicity and the underlying pathogenic mechanisms associated with exposure to several neurotoxic chemicals, specifically 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), rotenone, paraquat (PQ), dichloro-diphenyl-trichloroethane (DDT), dieldrin, manganese (Mn), and vanadium (V). Our overview of the current findings from cellular, animal, and human studies of PD provides information for possible intervention strategies aimed at halting the initiation and exacerbation of environmentally linked PD.     

In Silico Identification and Evaluation of Natural Products as Potential Tumor Necrosis Factor Function Inhibitors Using Advanced Enalos Asclepios KNIME Nodes

Tumor necrosis factor (TNF) is a regulator of several chronic inflammatory diseases, such as rheumatoid arthritis. Although anti-TNF biologics have been used in clinic, they render several drawbacks, such as patients’ progressive immunodeficiency and loss of response, high cost, and intravenous administration. In order to find new potential anti-TNF small molecule inhibitors, we employed an in silico approach, aiming to find natural products, analogs of Ampelopsin H, a compound that blocks the formation of TNF active trimer. Two out of nine commercially available compounds tested, Nepalensinol B and Miyabenol A, efficiently reduced TNF-induced cytotoxicity in L929 cells and production of chemokines in mice joints’ synovial fibroblasts, while Nepalensinol B also abolished TNF-TNFR1 binding in non-toxic concentrations. The binding mode of the compounds was further investigated by molecular dynamics and free energy calculation studies, using and advancing the Enalos Asclepios pipeline. Conclusively, we propose that Nepalensinol B, characterized by the lowest free energy of binding and by a higher number of hydrogen bonds with TNF, qualifies as a potential lead compound for TNF inhibitors’ drug development. Finally, the upgraded Enalos Asclepios pipeline can be used for improved identification of new therapeutics against TNF-mediated chronic inflammatory diseases, providing state-of-the-art insight on their binding mode.     

Identification of Ziyuglycoside II from a Natural Products Library as a STING Agonist

Given the emerging pivotal roles of stimulator of interferon genes (STING) in host pathogen defense and immune-oncology, STING is regarded as a promising target for drug development. Cyclic dinucleotides (CDNs) are the first-generation STING agonists. However, their poor metabolic stability and membrane permeability limits their therapeutic application. In contrast, small-molecule STING agonists show superior properties such as molecular weight, polar character, and delivery diversity. The quest for a potent small-molecular agonist of human STING remains ongoing. In our study, through an IRF/IFN pathway-targeted cell-based screen of a natural products library, we identified a small-molecular STING agonist, Ziyuglycoside II, termed ST12, with potent stimulation of the IRF/IFN and NF-κB pathways. Furthermore, its binding to the C-terminal domain of human STING, detected by bio-layer interferometry, indicates that ST12 is a human STING agonist. Further Tanimoto similarity analysis with existing small-molecule STING agonists indicates that ST12 is a lead compound with a novel core structure for the further optimization.     

Silver Nanoparticles as a Tool for the Study of Spontaneous Aggregation of Immunoglobulin Monoclonal Free Light Chains

Some misfolded proteins, e.g., immunoglobulin monoclonal free light chains (FLC), tend to form fibrils. Protein deposits in tissue may lead to amyloidosis and dysfunction of different organs. There is currently no technique allowing for the identification of FLC that are prone to aggregate. The development of such a method would enable the early selection of patients at high risk of developing amyloidosis. The aim of this study was to investigate whether silver nanoparticles (AgNPs) could be a useful tool to study the process of aggregation of FLC and their susceptibility to form the protein deposits. Mixtures of AgNPs and urine samples from patients with multiple myeloma were prepared. To evaluate the aggregation process of nanoparticles coated with proteins, UV-visible spectroscopy, transmission electron microscopy, and the original laser light scattering method were used. It has been shown that some clones of FLC spontaneously triggered aggregation of the nanoparticles, while in the presence of others, the nanoparticle solution became hyperstable. This is probably due to the structure of the chains themselves, unique protein-AgNPs interactions and perhaps correlates with the tendency of some FLC clones to form deposits. Nanoparticle technology has proven to be helpful in identifying clones of immunoglobulin FLC that tend to aggregate.