ABSTRACT
Introduction: N-glycosylation is a post-translational modification that is highly important for the development of monoclonal antibodies (mAbs), as it regulates their biological activity, particularly in terms of immune effector functions. While typically added at the Fc level, approximately 15-25% of circulating antibodies exhibit glycosylation in the Fab domains as well. To the best of our knowledge, cetuximab (Erbitux®) is the only therapeutic antibody presenting Fab glycosylation approved world-wide targeting the epidermal growth factor receptor for the treatment of metastatic-colorectal and head and neck cancers. Additionally, it can trigger antibody-dependent cell cytotoxicity (ADCC), a response that typically is influenced by N-glycosylation at Fc level. However, the role of Fab glycosylation in cetuximab remains poorly understood. Hence, this study aims to investigate the structural role of Fab glycosylation on the conformational behavior of cetuximab. Methods: The study was performed in silico via accelerated molecular dynamics simulations. The commercial cetuximab was compared to its form without Fab glycosylation and structural descriptors were evaluated to establish conformational differences. Results: The results clearly show a correlation between the Fab glycosylation and structural descriptors that may modulate the conformational freedom of the antibody, potentially affecting Fc effector functions, and suggesting a negative role of Fab glycosylation on the interaction with FcγRIIIa. Conclusion: Fab glycosylation of cetuximab is the most critical challenge for biosimilar development, but the differences highlighted in this work with respect to its aglycosylated form can improve the knowledge and represent also a great opportunity to develop novel strategies of biotherapeutics.
Subject(s)
Cetuximab , Immunoglobulin Fab Fragments , Molecular Dynamics Simulation , Cetuximab/immunology , Glycosylation , Humans , Immunoglobulin Fab Fragments/immunology , Immunoglobulin Fab Fragments/chemistry , Immunoglobulin Fab Fragments/metabolism , Antibody-Dependent Cell Cytotoxicity/immunology , Computer Simulation , Antineoplastic Agents, Immunological/immunology , Antineoplastic Agents, Immunological/pharmacology , Protein Conformation , ErbB Receptors/immunology , ErbB Receptors/metabolismABSTRACT
Cytochrome P450 (CYP450) enzymes comprise a highly diverse superfamily of heme-thiolate proteins that responsible for catalyzing over 90 % of enzymatic reactions associated with xenobiotic metabolism in humans. Accurately predicting whether chemicals are substrates or inhibitors of different CYP450 isoforms can aid in pre-selecting hit compounds for the drug discovery process, chemical toxicology studies, and patients treatment planning. In this work, we investigated in silico studies on CYP450s specificity over past twenty years, categorizing these studies into structure-based and ligand-based approaches. Subsequently, we utilized 100 of the most frequently prescribed drugs to test eleven machine learning-based prediction models which were published between 2015 and 2024. We analyzed various aspects of the evaluated models, such as their datasets, algorithms, and performance. This will give readers with a comprehensive overview of these prediction models and help them choose the most suitable one to do prediction. We also provide our insights for future research trend in both structure-based and ligand-based approaches in this field.
ABSTRACT
The activity of sirtuin 1 (SIRT1, a member of the NAD+-dependent deacetylases family) decreases during aging as NAD+ levels naturally decline, thus increasing the risk of several age-associated diseases. Several sirtuin-activating compounds (STACs) have been developed to counteract the age-associated reduction in SIRT1 activity, and some of them are currently under development in clinical trials. STACs induce SIRT1 activation, either through allosteric activation of the enzyme in the presence of NAD+, or by increasing NAD+ levels by inhibiting its degradation or by supplying a key precursor in biosynthesis. In this study, we have identified (E)-2'-des-methyl sulindac analogues as a novel class of STACs that act also in the absence of NAD+, a peculiar behavior demonstrated through enzymatic and mass spectrometry experiments, both in vitro and in cell lines. The activation of the SIRT1 pathway was confirmed in vivo through gene expression and metabolomics analysis. Our data suggest that these compounds could serve as candidate leads for a novel therapeutic strategy aimed at addressing a key metabolic deficiency that may contribute to metabolic and age-associated diseases.
Subject(s)
NAD , Sirtuin 1 , Sirtuin 1/metabolism , NAD/metabolism , Animals , Humans , Enzyme Activators/pharmacology , Cell Line , Mice , Male , Mice, Inbred C57BL , Drug DiscoveryABSTRACT
The Organic Cation Transporter Novel 1 (OCTN1), also known as SLC22A4, is widely expressed in various human tissues, and involved in numerous physiological and pathological processes remains. It facilitates the transport of organic cations, zwitterions, with selectivity for positively charged solutes. Ergothioneine, an antioxidant compound, and acetylcholine (Ach) are among its substrates. Given the lack of experimentally solved structures of this protein, this study aimed at generating a reliable 3D model of OCTN1 to shed light on its substrate-binding preferences and the role of sodium in substrate recognition and transport. A chimeric model was built by grafting the large extracellular loop 1 (EL1) from an AlphaFold-generated model onto a homology model. Molecular dynamics simulations revealed domain-specific mobility, with EL1 exhibiting the highest impact on overall stability. Molecular docking simulations identified cytarabine and verapamil as highest affinity ligands, consistent with their known inhibitory effects on OCTN1. Furthermore, MM/GBSA analysis allowed the categorization of substrates into weak, good, and strong binders, with molecular weight strongly correlating with binding affinity to the recognition site. Key recognition residues, including Tyr211, Glu381, and Arg469, were identified through interaction analysis. Ach demonstrated a low interaction energy, supporting the hypothesis of its one-directional transport towards to outside of the membrane. Regarding the role of sodium, our model suggested the involvement of Glu381 in sodium binding. Molecular dynamics simulations of systems at increasing levels of Na+ concentrations revealed increased sodium occupancy around Glu381, supporting experimental data associating Na+ concentration to molecule transport. In conclusion, this study provides valuable insights into the 3D structure of OCTN1, its substrate-binding preferences, and the role of sodium in the recognition. These findings contribute to the understanding of OCTN1 involvement in various physiological and pathological processes and may have implications for drug development and disease management.
Subject(s)
Molecular Docking Simulation , Molecular Dynamics Simulation , Organic Cation Transport Proteins , Humans , Organic Cation Transport Proteins/chemistry , Organic Cation Transport Proteins/metabolism , Organic Cation Transport Proteins/genetics , Symporters/chemistry , Symporters/metabolism , Binding Sites , Protein Binding , Ergothioneine/chemistry , Ergothioneine/metabolism , Sodium/metabolism , Sodium/chemistry , Computer Simulation , Acetylcholine/metabolism , Acetylcholine/chemistry , LigandsABSTRACT
Inflammation is a physiological condition characterized by a complex interplay between different cells handled by metabolites and specific inflammatory-related molecules. In some pathological situations, inflammation persists underlying and worsening the pathological state. Over the years, two membrane transporters namely OCTN1 (SLC22A4) and OCTN2 (SLC22A5) have been shown to play specific roles in inflammation. These transporters form the OCTN subfamily within the larger SLC22 family. The link between these proteins and inflammation has been proposed based on their link to some chronic inflammatory diseases such as asthma, Crohn's disease (CD), and rheumatoid arthritis (RA). Moreover, the two transporters show the ability to mediate the transport of several compounds including carnitine, carnitine derivatives, acetylcholine, ergothioneine, and gut microbiota by-products, which have been specifically associated with inflammation for their anti- or proinflammatory action. Therefore, the absorption and distribution of these molecules rely on the presence of OCTN1 and OCTN2, whose expression is modulated by inflammatory cytokines and transcription factors typically activated by inflammation. In the present review, we wish to provide a state of the art on OCTN1 and OCTN2 transport function and regulation in relationships with inflammation and inflammatory diseases focusing on the metabolic signature collected in different body districts and gene polymorphisms related to inflammatory diseases.
Subject(s)
Inflammation , Organic Cation Transport Proteins , Solute Carrier Family 22 Member 5 , Symporters , Humans , Inflammation/metabolism , Solute Carrier Family 22 Member 5/metabolism , Solute Carrier Family 22 Member 5/genetics , Animals , Organic Cation Transport Proteins/metabolism , Organic Cation Transport Proteins/genetics , Ergothioneine/metabolism , Crohn Disease/metabolism , Crohn Disease/genetics , Crohn Disease/pathology , Arthritis, Rheumatoid/metabolism , Arthritis, Rheumatoid/genetics , Gastrointestinal Microbiome , Carnitine/metabolism , Asthma/metabolism , Asthma/genetics , Acetylcholine/metabolismABSTRACT
Angiopoietin-like protein 3 (ANGPTL3) is a plasmatic protein that plays a crucial role in lipoprotein metabolism by inhibiting the lipoprotein lipase (LPL) and the endothelial lipase (EL) responsible for the hydrolysis of phospholipids on high-density lipoprotein (HDL). Interest in developing new pharmacological therapies aimed at inhibiting ANGPTL3 has been growing due to the hypolipidemic and antiatherogenic profile observed in its absence. The goal of this study was the in silico characterization of the interaction between ANGPTL3 and EL. Because of the lack of any structural information on both the trimeric coiled-coil N-terminal domain of ANGPTL3 and the EL homodimer as well as data regarding their interactions, the first step was to obtain the three-dimensional model of these two proteins. The models were then refined via molecular dynamics (MD) simulations and used to investigate the interaction mechanism. The analysis of interactions in different docking poses and their refinement via MD allowed the identification of three specific glutamates of ANGPTL3 that recognize a positively charged patch on the surface of EL. These ANGPTL3 key residues, i.e., Glu154, Glu157, and Glu160, could form a putative molecular recognition site for EL. This study paves the way for future investigations aimed at confirming the recognition site and at designing novel inhibitors of ANGPTL3.
Subject(s)
Angiopoietin-Like Protein 3 , Lipase , Angiopoietin-like Proteins , Lipase/metabolism , Lipoprotein Lipase/metabolism , Lipoproteins, HDL/metabolism , Phospholipids/metabolism , Triglycerides , Angiopoietins/metabolismABSTRACT
Innervation of the hypothalamic median eminence by Gonadotropin-Releasing Hormone (GnRH) neurons is vital to ensure puberty onset and successful reproduction. However, the molecular and cellular mechanisms underlying median eminence development and pubertal timing are incompletely understood. Here we show that Semaphorin-6A is strongly expressed by median eminence-resident oligodendrocytes positioned adjacent to GnRH neuron projections and fenestrated capillaries, and that Semaphorin-6A is required for GnRH neuron innervation and puberty onset. In vitro and in vivo experiments reveal an unexpected function for Semaphorin-6A, via its receptor Plexin-A2, in the control of median eminence vascular permeability to maintain neuroendocrine homeostasis. To support the significance of these findings in humans, we identify patients with delayed puberty carrying a novel pathogenic variant of SEMA6A. In all, our data reveal a role for Semaphorin-6A in regulating GnRH neuron patterning by tuning the median eminence vascular barrier and thereby controlling puberty onset.
Subject(s)
Gonadotropin-Releasing Hormone , Semaphorins , Humans , Gonadotropin-Releasing Hormone/metabolism , Median Eminence/metabolism , Capillary Permeability , Neurons/metabolism , Puberty , Semaphorins/genetics , Semaphorins/metabolismABSTRACT
Since the 1980s, it has been known that the administration of ganglioside GM1 to cultured cells induced or enhanced neuronal differentiation. GM1 mechanism of action relies on its direct interaction and subsequent activation of the membrane tyrosine kinase receptor, TrkA, which naturally serves as NGF receptor. This process is mediated by the sole oligosaccharide portion of GM1, the pentasaccharide ß-Gal-(1-3)-ß-GalNAc-(1-4)-[α-Neu5Ac-(2-3)]-ß-Gal-(1-4)-ß-Glc. Here we detailed the minimum structural requirements of the oligosaccharide portion of GM1 for mediating the TrkA dependent neuritogenic processing. By in vitro and in silico biochemical approaches, we demonstrated that the minimal portion of GM1 required for the TrkA activation is the inner core of the ganglioside's oligosaccharide ß-Gal-(1-3)-ß-GalNAc-(1-4)-[α-Neu5Ac-(2-3)]-ß-Gal. The addition of a sialic acid residue at position 3 of the outer galactose of the GM1 oligosaccharide, which forms the oligosaccharide of GD1a, prevented the interaction with TrkA and the resulting neuritogenesis. On the contrary, the addition of a fucose residue at position 2 of the outer galactose, forming the Fucosyl-GM1 oligosaccharide, did not prevent the TrkA-mediated neuritogenesis.
Subject(s)
G(M1) Ganglioside , Galactose , G(M1) Ganglioside/chemistry , N-Acetylneuraminic Acid , Oligosaccharides/chemistryABSTRACT
PURPOSE: RPH3A encodes a protein involved in the stabilization of GluN2A subunit of N-methyl-D-aspartate (NMDA)-type glutamate receptors at the cell surface, forming a complex essential for synaptic plasticity and cognition. We investigated the effect of variants in RPH3A in patients with neurodevelopmental disorders. METHODS: By using trio-based exome sequencing, GeneMatcher, and screening of 100,000 Genomes Project data, we identified 6 heterozygous variants in RPH3A. In silico and in vitro models, including rat hippocampal neuronal cultures, have been used to characterize the effect of the variants. RESULTS: Four cases had a neurodevelopmental disorder with untreatable epileptic seizures [p.(Gln73His)dn; p.(Arg209Lys); p.(Thr450Ser)dn; p.(Gln508His)], and 2 cases [p.(Arg235Ser); p.(Asn618Ser)dn] showed high-functioning autism spectrum disorder. Using neuronal cultures, we demonstrated that p.(Thr450Ser) and p.(Asn618Ser) reduce the synaptic localization of GluN2A; p.(Thr450Ser) also increased the surface levels of GluN2A. Electrophysiological recordings showed increased GluN2A-dependent NMDA ionotropic glutamate receptor currents for both variants and alteration of postsynaptic calcium levels. Finally, expression of the Rph3AThr450Ser variant in neurons affected dendritic spine morphology. CONCLUSION: Overall, we provide evidence that missense gain-of-function variants in RPH3A increase GluN2A-containing NMDA ionotropic glutamate receptors at extrasynaptic sites, altering synaptic function and leading to a clinically variable neurodevelopmental presentation ranging from untreatable epilepsy to autism spectrum disorder.
Subject(s)
Autism Spectrum Disorder , Epilepsy , Animals , Humans , Rats , Autism Spectrum Disorder/genetics , Epilepsy/genetics , Mutation, Missense/genetics , N-Methylaspartate/metabolism , Neurons/metabolism , Rabphilin-3AABSTRACT
Ozonolysis is a useful as well as dangerous reaction for performing alkene cleavage. On the other hand, enzymes are considered a more sustainable and safer alternative. Among them, Caulobacter segnis dioxygenase (CsO2) known so far for its ability to catalyze the coenzyme-free oxidation of vinylguaiacol into vanillin, was selected and its substrate scope evaluated towards diverse natural and synthetic stilbenoids. Under optimized conditions, CsO2 catalyzed the oxidative cleavage of the C=C double bonds of various trans-stilbenes, providing that a hydroxyl moiety was necessary in para-position of the phenyl group (e. g., resveratrol and its derivatives) for the reaction to take place, which was confirmed by modelling studies. The reactions occurred rapidly (0.5-3â h) with high conversions (95-99 %) and without formation of by-products. The resveratrol biotransformation was carried out on 50-mL scale thus confirming the feasibility of the biocatalytic system as a preparative method.
Subject(s)
Dioxygenases , Ozone , Stilbenes , Dioxygenases/metabolism , Resveratrol , Stilbenes/chemistryABSTRACT
AIM: The effect of liposomes bi-functionalized with phosphatidic acid and with a synthetic peptide derived from human apolipoprotein E has been evaluated on the aggregation features of different amyloidogenic proteins: human Amyloid ß1-40 (Aß1-40), transthyretin (TTR) variant S52P, human ß2microglobulin (ß2m) variants ΔN6 and D76N, Serum Amyloid A (SAA). METHODS: The formation of fibrillar aggregates of the proteins was investigated by ThioflavinT fluorescence assay and validated by Atomic Force Microscopy. RESULTS: The results show that liposomes are preventing the transition of non-aggregated forms to the fibrillar state, with stronger effects on Aß1-40, ß2m ΔN6 and SAA. Liposomes also induce disaggregation of the amyloid aggregates of all the proteins investigated, with stronger effects on Aß1-40, ß2 D76N and TTR.SPR assays show that liposomes bind Aß1-40 and SAA aggregates with high affinity (KD in the nanomolar range) whereas binding to TTR aggregates showed a lower affinity (KD in the micromolar range). Aggregates of ß2m variants showed both high and low affinity binding sites. Computed Structural analysis of protein fibrillar aggregates and considerations on the multidentate features of liposomes allow to speculate a common mechanism of action, based on binding the ß-stranded peptide regions responsible for the amyloid formation. CONCLUSION: Thus, multifunctional liposomes perform as pharmacological chaperones with anti-amyloidogenic activity, with a promising potential for the treatment of a number of protein-misfolding diseases.Key messageAmyloidosis is a group of diseases, each due to a specific protein misfolding.Anti-amyloidogenic nanoparticles have been gaining the utmost importance as a potential treatment for protein misfolding disorders.Liposomes bi-functionalized with phosphatidic acid and with a synthetic peptide derived from human apolipoprotein E showed anti-amyloidogenic activity.
Subject(s)
Amyloid , Liposomes , Humans , Amyloid/chemistry , Amyloid/metabolism , Protein Aggregates , Molecular Chaperones , Phosphatidic Acids , ApolipoproteinsABSTRACT
N-glycosylation plays a key role in modulating the bioactivity of monoclonal antibodies (mAbs), as well as the light chain (LC) isotype can influence their physicochemical properties. However, investigating the impact of such features on mAbs conformational behavior is a big challenge, due to the very high flexibility of these biomolecules. In this work we investigate, by accelerated molecular dynamics (aMD), the conformational behavior of two commercial immunoglobulins G1 (IgG1), representative of κ and λ LCs antibodies, in both their fucosylated and afucosylated forms. Our results show, through the identification of a stable conformation, how the combination of fucosylation and LC isotype modulates the hinge behavior, the Fc conformation and the position of the glycan chains, all factors potentially affecting the binding to the FcγRs. This work also represents a technological enhancement in the conformational exploration of mAbs, making aMD a suitable approach to clarify experimental results.
Subject(s)
Antibodies, Monoclonal , Immunoglobulin G , Glycosylation , TechnologyABSTRACT
The fulfilment of the European "Farm to Fork" strategy requires a drastic reduction in the use of "at risk" synthetic pesticides; this exposes vulnerable agricultural sectors-among which is the European risiculture-to the lack of efficient means for the management of devastating diseases, thus endangering food security. Therefore, novel scaffolds need to be identified for the synthesis of new and more environmentally friendly fungicides. In the present work, we employed our previously developed 3D model of P. oryzae cytochrome bc1 (cyt bc1) complex to perform a high-throughput virtual screening of two commercially available compound libraries. Three chemotypes were selected, from which a small collection of differently substituted analogues was designed and synthesized. The compounds were tested as inhibitors of the cyt bc1 enzyme function and the mycelium growth of both strobilurin-sensitive (WT) and -resistant (RES) P. oryzae strains. This pipeline has permitted the identification of thirteen compounds active against the RES cyt bc1 and five compounds that inhibited the WT cyt bc1 function while inhibiting the fungal mycelia only minimally. Serendipitously, among the studied compounds we identified a new chemotype that is able to efficiently inhibit the mycelium growth of WT and RES strains by ca. 60%, without inhibiting the cyt bc1 enzymatic function, suggesting a different mechanism of action.
Subject(s)
Ascomycota , Fungicides, Industrial , Cytochromes b/metabolism , Ascomycota/metabolism , Fungicides, Industrial/pharmacology , Strobilurins/pharmacology , Electron Transport Complex IIIABSTRACT
Chaperone-assisted selective autophagy (CASA) is a highly selective pathway for the disposal of misfolding and aggregating proteins. In muscle, CASA assures muscle integrity by favoring the turnover of structural components damaged by mechanical strain. In neurons, CASA promotes the removal of aggregating substrates. A crucial player of CASA is HSPB8 (heat shock protein family B (small) member 8), which acts in a complex with HSPA, their cochaperone BAG3, and the E3 ubiquitin ligase STUB1. Recently, four novel HSPB8 frameshift (fs) gene mutations have been linked to neuromyopathies, and encode carboxy-terminally mutated HSPB8, sharing a common C-terminal extension. Here, we analyzed the biochemical and functional alterations associated with the HSPB8_fs mutant proteins. We demonstrated that HSPB8_fs mutants are highly insoluble and tend to form proteinaceous aggregates in the cytoplasm. Notably, all HSPB8 frameshift mutants retain their ability to interact with CASA members but sequester them into the HSPB8-positive aggregates together with two autophagy receptors SQSTM1/p62 and TAX1BP1. This copartitioning process negatively affects the CASA capability to remove its clients and causes a general failure in proteostasis response. Further analyses revealed that the aggregation of the HSPB8_fs mutants occurs independently of the other CASA members or from the autophagy receptors interaction, but it is an intrinsic feature of the mutated amino acid sequence. HSPB8_fs mutants aggregation alters the differentiation capacity of muscle cells and impairs sarcomere organization. Collectively, these results shed light on a potential pathogenic mechanism shared by the HSPB8_fs mutants described in neuromuscular diseases.Abbreviations : ACD: α-crystallin domain; ACTN: actinin alpha; BAG3: BAG cochaperone 3; C: carboxy; CASA: chaperone-assisted selective autophagy; CE: carboxy-terminal extension; CLEM: correlative light and electron microscopy; CMT2L: Charcot-Marie-Tooth type 2L; CTR: carboxy-terminal region; dHMNII: distal hereditary motor neuropathy type II; EV: empty vector; FRA: filter retardation assay; fs: frameshift; HSPA/HSP70: heat shock protein family A (Hsp70); HSPB1/Hsp27: heat shock protein family B (small) member 1; HSPB8/Hsp22: heat shock protein family B (small) member 8; HTT: huntingtin; KO: knockout; MAP1LC3B/LC3: microtubule associated protein 1 light chain 3 beta; MD: molecular dynamics; MTOC: microtubule organizing center; MYH: myosin heavy chain; MYOG: myogenin; NBR1: NBR1 autophagy cargo receptor; CALCOCO2/NDP52: calcium binding and coiled-coil domain 2; NSC34: Neuroblastoma X Spinal Cord 34; OPTN: optineurin; polyQ: polyglutamine; SQSTM1/p62: sequestosome 1; STUB1/CHIP: STIP1 homology and U-box containing protein 1; TARDBP/TDP-43: TAR DNA binding protein; TAX1BP1: Tax1 binding protein 1; TUBA: tubulin alpha; WT: wild-type.
Subject(s)
Charcot-Marie-Tooth Disease , Neuromuscular Diseases , Humans , Sequestosome-1 Protein/genetics , Sequestosome-1 Protein/metabolism , Autophagy/genetics , Heat-Shock Proteins/metabolism , Charcot-Marie-Tooth Disease/genetics , Ubiquitin-Protein Ligases/metabolism , Adaptor Proteins, Signal Transducing/metabolism , Apoptosis Regulatory Proteins/metabolism , Molecular Chaperones/genetics , Molecular Chaperones/metabolismABSTRACT
Riboflavin is an essential water-soluble vitamin that needs to be provided through the diet because of the conversion into flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN), important cofactors in hundreds of flavoenzymes. The adsorption and distribution of riboflavin is mediated by transmembrane transporters of the SLC52 family, namely RFVT1-3, whose mutations are mainly associated with two diseases, MADD and the Brown-Vialetto-Van Laere syndrome. Interest in RFVTs as pharmacological targets has increased in the last few years due to their overexpression in several cancer cells, which can be exploited both by blocking the uptake of riboflavin into the cancerous cells, and by performing cancer targeted delivery of drugs with a high affinity for RFVTs. In this work, we propose three-dimensional structural models for all three human riboflavin transporters obtained by state-of-the-art artificial intelligence-based methods, which were then further refined with molecular dynamics simulations. Furthermore, two of the most notable mutations concerning RFVT2 and RFVT3 (W31S and N21S, respectively) were investigated studying the interactions between the wild-type and mutated transporters with riboflavin.
Subject(s)
Artificial Intelligence , Hearing Loss, Sensorineural , Humans , Riboflavin/metabolism , Membrane Transport Proteins/genetics , Membrane Transport Proteins/metabolism , Hearing Loss, Sensorineural/genetics , Structure-Activity Relationship , Flavin Mononucleotide , Flavin-Adenine Dinucleotide/metabolismABSTRACT
Toxic aggregates of α-synuclein (αsyn) are considered key drivers of Parkinson's disease (PD) pathology. In early PD, αsyn induces synaptic dysfunction also modulating the glutamatergic neurotransmission. However, a more detailed understanding of the molecular mechanisms underlying αsyn-triggered synaptic failure is required to design novel therapeutic interventions. Here, we described the role of Rabphilin-3A (Rph3A) as novel target to counteract αsyn-induced synaptic loss in PD. Rph3A is a synaptic protein interacting with αsyn and involved in stabilizing dendritic spines and in promoting the synaptic retention of NMDA-type glutamate receptors. We found that in vivo intrastriatal injection of αsyn-preformed fibrils in mice induces the early loss of striatal synapses associated with decreased synaptic levels of Rph3A and impaired Rph3A/NMDA receptors interaction. Modulating Rph3A striatal expression or interfering with the Rph3A/αsyn complex with a small molecule prevented dendritic spine loss and rescued associated early motor defects in αsyn-injected mice. Notably, the same experimental approaches prevented αsyn-induced synaptic loss in vitro in primary hippocampal neurons. Overall, these findings indicate that approaches aimed at restoring Rph3A synaptic functions can slow down the early synaptic detrimental effects of αsyn aggregates in PD.
Subject(s)
Parkinson Disease , alpha-Synuclein , Adaptor Proteins, Signal Transducing/metabolism , Animals , Mice , Nerve Tissue Proteins , Parkinson Disease/drug therapy , Parkinson Disease/metabolism , Receptors, N-Methyl-D-Aspartate/metabolism , Vesicular Transport Proteins/metabolism , alpha-Synuclein/metabolism , Rabphilin-3AABSTRACT
NONO and SFPQ are involved in multiple nuclear processes (e.g., pre-mRNA splicing, DNA repair, and transcriptional regulation). These proteins, along with NEAT1, enable paraspeckle formation, thus promoting multiple myeloma cell survival. In this paper, we investigate NONO and SFPQ dimer stability, highlighting the hetero- and homodimer structural differences, and model their interactions with RNA, simulating their binding to a polyG probe mimicking NEAT1guanine-rich regions. We demonstrated in silico that NONO::SFPQ heterodimerization is a more favorable process than homodimer formation. We also show that NONO and SFPQ RRM2 subunits are primarily required for protein-protein interactions with the other DBHS protomer. Simulation of RNA binding to NONO and SFPQ, beside validating RRM1 RNP signature importance, highlighted the role of ß2 and ß4 strand residues for RNA specific recognition. Moreover, we demonstrated the role of the NOPS region and other protomer's RRM2 ß2/ß3 loop in strengthening the interaction with RNA. Our results, having deepened RNA and DBHS dimer interactions, could contribute to the design of small molecules to modulate the activity of these proteins. RNA-mimetics, able to selectively bind to NONO and/or SFPQ RNA-recognition site, could impair paraspeckle formation, thus representing a first step towards the discovery of drugs for multiple myeloma treatment.
Subject(s)
DNA-Binding Proteins , Multiple Myeloma , PTB-Associated Splicing Factor , RNA , DNA-Binding Proteins/metabolism , Dimerization , Humans , Multiple Myeloma/genetics , Multiple Myeloma/metabolism , PTB-Associated Splicing Factor/metabolism , Protein Subunits/metabolism , RNA/metabolism , RNA Splicing Factors/metabolism , RNA-Binding Proteins/metabolismABSTRACT
Interferon beta (IFNß) is a well-known cytokine, belonging to the type I family, that exerts antiviral, immunomodulatory, and antiproliferative activity. It has been reported that the artificially deamidated form of recombinant IFNß-1a at Asn25 position shows an increased biological activity. As a deepening of the previous study, the molecular mechanism underlying this biological effect was investigated in this work by combining experimental and computational techniques. Specifically, the binding to IFNAR1 and IFNAR2 receptors and the canonical pathway of artificially deamidated IFNß-1a molecule were analyzed in comparison to the native form. As a result, a change in receptor affinity of deamidated IFNß-1a with respect to the native form was observed, and to better explore this molecular interaction, molecular dynamics simulations were carried out. Results confirmed, as previously hypothesized, that the N25D mutation can locally change the interaction network of the mutated residue but also that this effect can be propagated throughout the molecule. In fact, many residues not involved in the interaction with IFNAR1 in the native form participate to the recognition in the deamidated molecule, enhancing the binding to IFNAR1 receptor and consequently an increase of signaling cascade activation. In particular, a higher STAT1 phosphorylation and interferon-stimulated gene expression was observed under deamidated IFNß-1a cell treatment. In conclusion, this study increases the scientific knowledge of deamidated IFNß-1a, deciphering its molecular mechanism, and opens new perspectives to novel therapeutic strategies.
Subject(s)
Antiviral Agents , Interferon-beta , Antiviral Agents/metabolism , Immunologic Factors , Interferon beta-1a , Interferon-beta/metabolism , Interferons , Signal TransductionABSTRACT
On October 21-22, 2020 the HESI (Health and Environmental Sciences Institute) Protein Allergens, Toxins, and Bioinformatics Committee, and the Society of Toxicology Food Safety Specialty Section co-hosted a virtual workshop titled "From Protein Toxins to Applied Toxicological Testing". The workshop focused on the safety assessment of novel proteins contained in foods and feeds, was globally represented by over 200 stakeholder attendees, and featured contributions from experts in academia, government and non-government organizations, and agricultural biotechnology developers from the private sector. A range of topics relevant to novel protein safety were discussed, including: the state of protein toxin biology, modes and mechanisms of action, structures and activity, use of bioinformatic analyses to assess the safety of a protein, and ways to leverage computational biology with in silico approaches for protein toxin identification/characterization. Key outcomes of the workshop included the appreciation of the complexity of developing a definition for a protein toxin when viewed from the perspective of food and feed safety, confirming the need for a case-by-case hypothesis-driven interpretation of bioinformatic results that leverages additional metadata rather than an alignment threshold-driven interpretation, and agreement that a "toxin protein database" is not necessary, as the bioinformatic needs for toxin detection may be accomplished by existing databases such as Pfam and UniProtKB/Swiss-Prot. In this paper, a path forward is proposed.
Subject(s)
Computational Biology , Food Safety , Allergens/chemistry , Allergens/toxicity , Biotechnology/methods , Databases, ProteinABSTRACT
Serum progesterone sulfates were evaluated in the etiology of gestational diabetes mellitus (GDM). Serum progesterone sulfates were measured using ultra-performance liquid chromatography-tandem mass spectrometry in four patient cohorts: 1) the Hyperglycemia and Adverse Pregnancy Outcomes study; 2) London-based women of mixed ancestry and 3) U.K.-based women of European ancestry with or without GDM; and 4) 11-13 weeks pregnant women with BMI ≤25 or BMI ≥35 kg/m2 with subsequent uncomplicated pregnancies or GDM. Glucose-stimulated insulin secretion (GSIS) was evaluated in response to progesterone sulfates in mouse islets and human islets. Calcium fluorescence was measured in HEK293 cells expressing transient receptor potential cation channel subfamily M member 3 (TRPM3). Computer modeling using Molecular Operating Environment generated three-dimensional structures of TRPM3. Epiallopregnanolone sulfate (PM5S) concentrations were reduced in GDM (P < 0.05), in women with higher fasting plasma glucose (P < 0.010), and in early pregnancy samples from women who subsequently developed GDM with BMI ≥35 kg/m2 (P < 0.05). In islets, 50 µmol/L PM5S increased GSIS by at least twofold (P < 0.001); isosakuranetin (TRPM3 inhibitor) abolished this effect. PM5S increased calcium influx in TRPM3-expressing HEK293 cells. Computer modeling and docking showed identical positioning of PM5S to the natural ligand in TRPM3. PM5S increases GSIS and is reduced in GDM serum. The activation of GSIS by PM5S is mediated by TRPM3 in both mouse and human islets.