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Nature Communications Jun 2024Viral genetic diversity presents significant challenges in developing antivirals with broad-spectrum activity and high barriers to resistance. Here we report development...
Viral genetic diversity presents significant challenges in developing antivirals with broad-spectrum activity and high barriers to resistance. Here we report development of proteolysis targeting chimeras (PROTACs) targeting the dengue virus envelope (E) protein through coupling of known E fusion inhibitors to ligands of the CRL4 E3 ubiquitin ligase. The resulting small molecules block viral entry through inhibition of E-mediated membrane fusion and interfere with viral particle production by depleting intracellular E in infected Huh 7.5 cells. This activity is retained in the presence of point mutations previously shown to confer partial resistance to the parental inhibitors due to decreased inhibitor-binding. The E PROTACs also exhibit broadened spectrum of activity compared to the parental E inhibitors against a panel of mosquito-borne flaviviruses. These findings encourage further exploration of targeted protein degradation as a differentiated and potentially advantageous modality for development of broad-spectrum direct-acting antivirals.
Topics: Humans; Proteolysis; Animals; Antiviral Agents; Flavivirus; Virus Internalization; Dengue Virus; Culicidae; Ubiquitin-Protein Ligases; Viral Envelope Proteins; Cell Line
PubMed: 38898037
DOI: 10.1038/s41467-024-49161-9 -
The Journal of Biological Chemistry Jun 2024Phospholipase A2 receptor 1 (PLA2R1) is a 180-kDa transmembrane protein that plays a role in inflammation and cancer, and is the major autoantigen in membranous...
Phospholipase A2 receptor 1 (PLA2R1) is a 180-kDa transmembrane protein that plays a role in inflammation and cancer, and is the major autoantigen in membranous nephropathy (MN), a rare but severe autoimmune kidney disease. A soluble form of PLA2R1 has been detected in mouse and human serum. It is likely produced by proteolytic shedding of membrane-bound PLA2R1 but the mechanism is unknown. Here, we show that human PLA2R1 is cleaved by A Disintegrin And Metalloprotease 10 (ADAM10) and ADAM17 in HEK293 cells, mouse embryonic fibroblasts and human podocytes. By combining site-directed mutagenesis and sequencing, we determined the exact cleavage site within the extracellular juxtamembrane stalk of human PLA2R1. Orthologs and paralogs of PLA2R1 are also shed. By using pharmacological inhibitors and genetic approaches with RNA interference and knock-out cellular models, we identified a major role of ADAM10 in the constitutive shedding of PLA2R1, and a dual role of ADAM10 and ADAM17 in the stimulated shedding. We did not observe evidence for cleavage by β- or γ-secretase, suggesting that PLA2R1 may not be a substrate for Regulated Intramembrane Proteolysis. PLA2R1 shedding occurs constitutively and can be triggered by the calcium ionophore ionomycin, the protein kinase C inducer PMA, cytokines and lipopolysaccharides, in vitro and in vivo. Altogether, our results show that PLA2R1 is a novel substrate for ADAM10 and ADAM17, producing a soluble form that is increased in inflammatory conditions and likely exerts various functions in physiological and pathophysiological conditions including inflammation, cancer and MN.
PubMed: 38897568
DOI: 10.1016/j.jbc.2024.107480 -
RSC Advances Jun 2024Monoclonal antibodies (mAbs) are pivotal therapeutic agents for various diseases, and effective treatment hinges on attaining a specific threshold concentration of mAbs...
Monoclonal antibodies (mAbs) are pivotal therapeutic agents for various diseases, and effective treatment hinges on attaining a specific threshold concentration of mAbs in patients. With the rising adoption of combination therapy involving multiple mAbs, there arises a clinical demand for multiplexing assays capable of measuring the concentrations of these mAbs. However, minimizing the complexity of serum samples while achieving rapid and accurate quantification is difficult. In this work, we introduced a novel method termed nano-surface and molecular orientation limited (nSMOL) proteolysis for the fragment of antigen binding (Fab) region-selective proteolysis of co-administered trastuzumab and pertuzumab based on the pore size difference between the protease nanoparticles (∼200 nm) and the resin-captured antibody (∼100 nm). The hydrolyzed peptide fragments were then quantified using liquid chromatography-tandem mass spectrometry (LC-MS/MS). In this process, the digestion time is shortened, and the produced digestive peptides are greatly reduced, thereby minimizing sample complexity and increasing detection accuracy. Assay linearity was confirmed within the ranges of 0.200-200 μg mL for trastuzumab and 0.300-200 μg mL for pertuzumab. The intra- and inter-day precision was within 9.52% and 8.32%, except for 12.5% and 10.8% for the lower limit of quantitation, and the accuracy (bias%) was within 6.3%. Additionally, other validation parameters were evaluated, and all the results met the acceptance criteria of the guiding principles. Our method demonstrated accuracy and selectivity for the simultaneous determination of trastuzumab and pertuzumab in clinical samples, addressing the limitation of ligand binding assays incapable of simultaneously quantifying mAbs targeting the same receptor. This proposed assay provides a promising technical approach for realizing clinical individualized precise treatment, especially for co-administered mAbs.
PubMed: 38895524
DOI: 10.1039/d4ra03060e -
BioRxiv : the Preprint Server For... Jun 2024The etiology of fetal growth restriction (FGR) is multifactorial, although many cases often involve placental insufficiency. Placental insufficiency is associated with...
The etiology of fetal growth restriction (FGR) is multifactorial, although many cases often involve placental insufficiency. Placental insufficiency is associated with inadequate trophoblast invasion resulting in high resistance to blood flow, decreased availability of nutrients, and increased hypoxia. We have developed a non-viral, polymer-based nanoparticle that facilitates delivery and transient gene expression of ( ) in placental trophoblast for the treatment of placenta insufficiency and FGR. Using the established guinea pig maternal nutrient restriction (MNR) model of placental insufficiency and FGR, the aim of the study was to identify novel pathways in the sub-placenta/decidua that provide insight into the underlying mechanism driving placental insufficiency, and may be corrected with nanoparticle treatment. Pregnant guinea pigs underwent ultrasound-guided sham or nanoparticle treatment at mid-pregnancy, and sub-placenta/decidua tissue was collected 5 days later. Transcriptome analysis was performed using RNA Sequencing on the Illumina platform. The MNR sub-placenta/decidua demonstrated fewer maternal spiral arteries lined by trophoblast, shallower trophoblast invasion and downregulation of genelists involved in the regulation of cell migration. nanoparticle treatment resulted in marked changes to transporter activity in the MNR + sub-placenta/decidua when compared to sham MNR. Under normal growth conditions however, nanoparticle treatment decreased genelists enriched for kinase signaling pathways and increased genelists enriched for proteolysis indicative of homeostasis. Overall, this study identified changes to the sub-placenta/decidua transcriptome that likely result in inadequate trophoblast invasion and increases our understanding of pathways that nanoparticle treatment acts on in order to restore or maintain appropriate placenta function.
PubMed: 38895421
DOI: 10.1101/2024.06.05.597595 -
BioRxiv : the Preprint Server For... Jun 2024Parkinson's disease (PD) and other α-synucleinopathies are characterized by the accumulation of α-synuclein (αS) pathology that can spread via the cell-to-cell...
Parkinson's disease (PD) and other α-synucleinopathies are characterized by the accumulation of α-synuclein (αS) pathology that can spread via the cell-to-cell transmission of αS aggregates. To better understand how various brain cells contribute to the spreading of αS pathology, we examined the metabolism of αS aggreges or pre-formed fibrils (PFFs) in neuronal and glial cells (microglia, astrocytes, and oligodendrocytes). In neurons, while the full-length αS rapidly disappeared following αS PFF uptake, truncated αS accumulated with a half-life of days rather than hours. Epitope mapping and fractionation studies indicate that αS PFF was truncated at the C-terminal region following uptake and remained insoluble/aggregated. In contrast, microglia and astrocytes rapidly metabolized αS PFF as the half-lives of αS PFF in these glial cells were <6 hours. Differential processing of αS by neurons was recapitulated in cell lines as differentiated CLU neuronal cell lines stably accumulate truncated αS while undifferentiated cells rapidly metabolize αS. Immunolocalization and subcellular fractionation studies show that internalized αS PFF is initially localized to endosomes followed by lysosomes. The lysosome is largely responsible for the degradation of internalized αS PFF as the inhibition of lysosomal function leads to the stabilization of αS in all cell types. Significantly, αS PFF causes lysosomal dysfunction in neurons. In summary, we show that neurons are inefficient in metabolizing internalized αS aggregates, partially because αS aggregates cause lysosomal dysfunction, potentially generating aggregation-prone truncated αS. In contrast, glial cells may protect neurons from αS aggregates by rapidly clearing αS aggregates.
PubMed: 38895363
DOI: 10.1101/2024.06.05.597615 -
Zoological Research Jul 2024The organ-specific toxicity resulting from microplastic (MP) exposure has been extensively explored, particularly concerning the gut, liver, testis, and lung. However,...
The organ-specific toxicity resulting from microplastic (MP) exposure has been extensively explored, particularly concerning the gut, liver, testis, and lung. However, under natural conditions, these effects are not restricted to specific organs or tissues. Investigating whether MP exposure presents a systemic threat to an entire organism, impacting factors such as lifespan, sleep, and fecundity, is essential. In this study, we investigated the effects of dietary exposure to two different doses of MPs (1-5 μm) using the terrestrial model organism . Results indicated that the particles caused gut damage and remained within the digestive system. Continuous MP exposure significantly shortened the lifespan of adult flies. Even short-term exposure disrupted sleep patterns, increasing the length of daytime sleep episodes. Additionally, one week of MP exposure reduced ovary size, with a trend towards decreased egg-laying in mated females. Although MPs did not penetrate the brain or ovaries, transcriptome analysis revealed altered gene expression in these tissues. In the ovary, Gene Ontology (GO) analysis indicated genotoxic effects impacting inflammation, circadian regulation, and metabolic processes, with significant impacts on extracellular structure-related pathways. In the brain, GO analysis identified changes in pathways associated with proteolysis and carbohydrate metabolism. Overall, this study provides compelling evidence of the systemic negative effects of MP exposure, highlighting the urgent need to address and mitigate environmental MP pollution.
Topics: Animals; Drosophila melanogaster; Female; Ovary; Longevity; Sleep; Microplastics; Male; Organ Size
PubMed: 38894523
DOI: 10.24272/j.issn.2095-8137.2024.038 -
Zoological Research Jul 2024As ectotherms, fish are highly sensitive to temperature fluctuations, which can profoundly impact their reproductive cycles. In this study, we investigated the fertility...
As ectotherms, fish are highly sensitive to temperature fluctuations, which can profoundly impact their reproductive cycles. In this study, we investigated the fertility and histological characteristics of zebrafish ( ) ovaries exposed to a temperature gradient ranging from the thermopreferendum temperature of the species, 27°C, to lower temperatures of 22°C, 20°C, and 13°C over a period of two weeks. Comparative metabolomic (six biological replicates for each temperature) and transcriptomic (four biological replicates for each temperature) analyses were conducted under the four temperature conditions. Results indicated that lower temperatures inhibited oocyte development and differential metabolites were involved in steroid hormone production, antioxidant function, and lipid and protein catabolism. Disrupted reproductive hormones, increased proteolysis, and lipid degradation significantly impeded oocyte development and egg maturation. Notably, a significant increase in bile acid content was noted in the ovaries of the cold-treated fish, indicating that bile acids play a critical role in ovarian failure. Overall, these findings provide valuable insights into the mechanisms governing the reproductive response of fish to cold stress.
Topics: Animals; Zebrafish; Female; Bile Acids and Salts; Ovary; Cold Temperature; Metabolomics
PubMed: 38894522
DOI: 10.24272/j.issn.2095-8137.2023.369 -
Molecules (Basel, Switzerland) Jun 2024The Omicron BA.5 variant of SARS-CoV-2 is known for its high transmissibility and its capacity to evade immunity provided by vaccine protection against the (original)...
Non-Glycosylated SARS-CoV-2 Omicron BA.5 Receptor Binding Domain (RBD) with a Native-like Conformation Induces a Robust Immune Response with Potent Neutralization in a Mouse Model.
The Omicron BA.5 variant of SARS-CoV-2 is known for its high transmissibility and its capacity to evade immunity provided by vaccine protection against the (original) Wuhan strain. In our prior research, we successfully produced the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein in an expression system. Extensive biophysical characterization indicated that, even without glycosylation, the RBD maintained native-like conformational and biophysical properties. The current study explores the immunogenicity and neutralization capacity of the -expressed Omicron BA.5 RBD using a mouse model. Administration of three doses of the RBD without any adjuvant elicited high titer antisera of up to 7.3 × 10 and up to 1.6 × 10 after a booster shot. Immunization with RBD notably enhanced the population of CD44CD62L T cells, indicating the generation of T cell memory. The in vitro assays demonstrated the antisera's protective efficacy through significant inhibition of the interaction between SARS-CoV-2 and its human receptor, ACE2, and through potent neutralization of a pseudovirus. These findings underscore the potential of our -expressed RBD as a viable vaccine candidate against the Omicron variant of SARS-CoV-2.
Topics: Animals; SARS-CoV-2; Spike Glycoprotein, Coronavirus; Mice; Antibodies, Neutralizing; COVID-19; Angiotensin-Converting Enzyme 2; Humans; COVID-19 Vaccines; Antibodies, Viral; Disease Models, Animal; Protein Domains; Glycosylation; Protein Binding; Female; Escherichia coli; T-Lymphocytes
PubMed: 38893549
DOI: 10.3390/molecules29112676 -
International Journal of Molecular... Jun 2024Multiple myeloma (MM) is a hematologic malignancy caused by the clonal expansion of immunoglobulin-producing plasma cells in the bone marrow and/or extramedullary sites.... (Review)
Review
Multiple myeloma (MM) is a hematologic malignancy caused by the clonal expansion of immunoglobulin-producing plasma cells in the bone marrow and/or extramedullary sites. Common manifestations of MM include anemia, renal dysfunction, infection, bone pain, hypercalcemia, and fatigue. Despite numerous recent advancements in the MM treatment paradigm, current therapies demonstrate limited long-term effectiveness and eventual disease relapse remains exceedingly common. Myeloma cells often develop drug resistance through clonal evolution and alterations of cellular signaling pathways. Therefore, continued research of new targets in MM is crucial to circumvent cumulative drug resistance, overcome treatment-limiting toxicities, and improve outcomes in this incurable disease. This article provides a comprehensive overview of the landscape of novel treatments and emerging therapies for MM grouped by molecular target. Molecular targets outlined include BCMA, GPRC5D, FcRH5, CD38, SLAMF7, BCL-2, kinesin spindle protein, protein disulfide isomerase 1, peptidylprolyl isomerase A, Sec61 translocon, and cyclin-dependent kinase 6. Immunomodulatory drugs, NK cell therapy, and proteolysis-targeting chimera are described as well.
Topics: Humans; Multiple Myeloma; Molecular Targeted Therapy; Antineoplastic Agents; Animals
PubMed: 38892379
DOI: 10.3390/ijms25116192 -
International Journal of Molecular... May 2024This research focuses on the target deconvolution of the natural compound myrianthic acid, a triterpenoid characterized by an ursane skeleton isolated from the roots of...
This research focuses on the target deconvolution of the natural compound myrianthic acid, a triterpenoid characterized by an ursane skeleton isolated from the roots of and from Nutt. (Onagraceae), using MS-based chemical proteomic techniques. Application of drug affinity responsive target stability (DARTS) and targeted-limited proteolysis coupled to mass spectrometry (t-LiP-MS) led to the identification of the enzyme fatty acid synthase (FAS) as an interesting macromolecular counterpart of myrianthic acid. This result, confirmed by comparison with the natural ursolic acid, was thoroughly investigated and validated in silico by molecular docking, which gave a precise picture of the interactions in the MA/FAS complex. Moreover, biological assays showcased the inhibitory activity of myrianthic acid against the FAS enzyme, most likely related to its antiproliferative activity towards tumor cells. Given the significance of FAS in specific pathologies, especially cancer, the myrianthic acid structural moieties could serve as a promising reference point to start the potential development of innovative approaches in therapy.
Topics: Humans; Molecular Docking Simulation; Proteomics; Fatty Acid Synthases; Triterpenes; Antineoplastic Agents; Mass Spectrometry; Cell Line, Tumor; Cell Proliferation; Terpenes
PubMed: 38892106
DOI: 10.3390/ijms25115918