Oligomeric Protein Research Articles

Neuroprotective effects and mechanisms of action of artemisinin in retinal ganglion cells in a mouse model of traumatic optic neuropathy

IntroductionTraumatic optic neuropathy is known to be a critical condition that can cause blindness; however, the specific mechanism underlying optic nerve injury is unclear. Recent studies have reported that artemisinin, considered vital in malaria treatment, can also be used to treat neurodegenerative diseases; however, its precise role and mechanism of action remain unknown. Therefore, in this study, we aimed to investigate the impact and probable mechanism of action of artemisinin in retinal ganglion cells (RGCs) in a mouse model of traumatic optic neuropathy induced by optic nerve crush (ONC). MethodsONC was induced in the left eye of mice by short-term clamping of the optic nerve; oral artemisinin was administered daily. The neuroprotective effect of the drug was assessed using Tuj-1 staining in RGCs. In addition, the inflammatory response and the expression levels of phosphorylated tau protein and tau oligomers were observed using RT-qPCR, TUNEL assay, and fluorescence staining to investigate the underlying mechanisms. ResultsArtemisinin increased the survival rate of RGCs 14 days after ONC. Artemisinin significantly reduced the levels of inflammatory factors such as CXCL10, CXCR3, and IL-1β in the retina and decreased the apoptosis of RGCs. Moreover, downregulation of the phosphorylation of tau proteins and the expression of tau oligomers were observed after artemisinin treatment. ConclusionOur results suggest that artemisinin can increase the survival rate of RGCs after ONC and reduce their apoptosis. This effect may be achieved by inhibiting the inflammatory response it triggers and downregulating tau protein phosphorylation and tau oligomer expression. These findings suggest the potential application of artemisinin as a therapeutic agent for neuropathy.

Nano-optogenetics for Disease Therapies.

Optogenetic, known as the method of 21 centuries, combines optic and genetic engineering to precisely control photosensitive proteins for manipulation of a broad range of cellular functions, such as flux of ions, protein oligomerization and dissociation, cellular intercommunication, and so on. In this technique, light is conventionally delivered to targeted cells through optical fibers or micro light-emitting diodes, always suffering from high invasiveness, wide-field illumination facula, strong absorption, and scattering by nontargeted endogenous substance. Light-transducing nanomaterials with advantages of high spatiotemporal resolution, abundant wireless-excitation manners, and easy functionalization for recognition of specific cells, recently have been widely explored in the field of optogenetics; however, there remain a few challenges to restrain its clinical applications. This review summarized recent progress on light-responsive genetically encoded proteins and the myriad of activation strategies by use of light-transducing nanomaterials and their disease-treatment applications, which is expected for sparking helpful thought to push forward its preclinical and translational uses.

GluN2A or GluN2B subunits of the NMDA receptor contribute to changes in neuronal excitability and impairments in LTP in the hippocampus of aging mice but do not mediate detrimental effects of oligomeric Aβ (1-42).

Studies in rodent models have revealed that oligomeric beta-amyloid protein [Aβ (1-42)] plays an important role in the pathogenesis of Alzheimer's disease. Early elevations in hippocampal neuronal excitability caused by Aβ (1-42) have been proposed to be mediated via enhanced activation of GluN2B-containing N-methyl-D-aspartate receptors (NMDAR). To what extent GluN2A or GluN2B-containing NMDAR contribute to Aβ (1-42)-mediated impairments of hippocampal function in advanced rodent age is unclear. Here, we assessed hippocampal long-term potentiation (LTP) and neuronal responses 4-5 weeks after bilateral intracerebral inoculation of 8-15 month old GluN2A+/- or GluN2B+/- transgenic mice with oligomeric Aβ (1-42), or control peptide. Whole-cell patch-clamp recordings in CA1 pyramidal neurons revealed a more positive resting membrane potential and increased total spike time in GluN2A+/-, but not GluN2B+/--hippocampi following treatment with Aβ (1-42) compared to controls. Action potential 20%-width was increased, and the descending slope was reduced, in Aβ-treated GluN2A+/-, but not GluN2B+/- hippocampi. Sag ratio was increased in Aβ-treated GluN2B+/--mice. Firing frequency was unchanged in wt, GluN2A+/-, and GluN2B+/-hippocampi after Aβ-treatment. Effects were not significantly different from responses detected under the same conditions in wt littermates, however. LTP that lasted for over 2 h in wt hippocampal slices was significantly reduced in GluN2A+/- and was impaired for 15 min in GluN2B+/--hippocampi compared to wt littermates. Furthermore, LTP (>2 h) was significantly impaired in Aβ-treated hippocampi of wt littermates compared to wt treated with control peptide. LTP induced in Aβ-treated GluN2A+/- and GluN2B+/--hippocampi was equivalent to LTP in control peptide-treated transgenic and Aβ-treated wt animals. Taken together, our data indicate that knockdown of GluN2A subunits subtly alters membrane properties of hippocampal neurons and reduces the magnitude of LTP. GluN2B knockdown reduces the early phase of LTP but leaves later phases intact. Aβ (1-42)-treatment slightly exacerbates changes in action potential properties in GluN2A+/--mice. However, the vulnerability of the aging hippocampus to Aβ-mediated impairments of LTP is not mediated by GluN2A or GluN2B-containing NMDAR.

Structure of HIV-1 RRE stem-loop II identifies two conformational states of the high-affinity Rev binding site

During HIV infection, specific RNA-protein interaction between the Rev response element (RRE) and viral Rev protein is required for nuclear export of intron-containing viral mRNA transcripts. Rev initially binds the high-affinity site in stem-loop II, which promotes oligomerization of additional Rev proteins on RRE. Here, we present the crystal structure of RRE stem-loop II in distinct closed and open conformations. The high-affinity Rev-binding site is located within the three-way junction rather than the predicted stem IIB. The closed and open conformers differ in their non-canonical interactions within the three-way junction, and only the open conformation has the widened major groove conducive to initial Rev interaction. Rev binding assays show that RRE stem-loop II has high- and low-affinity binding sites, each of which binds a Rev dimer. We propose a binding model, wherein Rev-binding sites on RRE are sequentially created through structural rearrangements induced by Rev-RRE interactions.

Equine-assisted service’s effect on cartilage and skeletal biomarkers for adults and older adults with arthritis: A pilot study

ObjectiveThe object of this study was to determine the effect of EAS (Equine-Assisted Services) on arthritis conditions, as measured by the sTnT (Skeletal troponin) and COMP (cartilage oligomeric matrix proteins) biomarkers, compared to an exercise attention control intervention. DesignThis was a secondary analysis of a randomized clinical trial comparing equine-assisted therapy to exercise education attention-control on cartilage and skeletal biomarkers in adults with arthritis. Twenty-one adults (Mage = 64 years) with arthritis who attended rheumatology clinics in the midwestern United States participated. ResultsNo changes were found in sTnT from baseline to week six within either intervention nor were there differences in changes between the two groups (p = 0.91). COMP increased from baseline to week six for both conditions, suggesting increased deterioration of cartilage and joints. Although the attention-control condition demonstrated larger increases in cartilage oligomeric matrix proteins level, compared to the EAS condition, these differences were not statistically (p = 0.58) or clinically significant (i.e., trivial effect, d = −0.16). When 3 outliers were removed, the differences in changes between EAT and attention-control group could be arguably of clinical significance (d = - 0.33), suggesting that the attention-control group demonstrated larger increases in levels of COMP than those in the EAS condition, though this difference was not statistically significant (p = 0.28). ConclusionAlthough equine-assisted therapy may reduce pain and improve quality of life for adults with arthritis, findings here are not fully corroborated with biomarkers.

Caveolin-1 frame-shift mutation induces multifunctional alterations in human fibroblasts that are exosome-mediated

Caveolins, Cav1-3, are oligomeric proteins critical for the formation of membrane caveolae that regulate cell physiology. A de novo frameshift mutation (Cav1F160X) in Cav1 (Cav1M) was described in a patient with a progeria-like phenotype, lipodystrophy, and pulmonary hypertension. We hypothesize that Cav1M presents as a dominant negative protein that perturbs cellular physiology, particularly nuclear morphology, mitochondrial function, plasma membrane structure, and exocytic behavior. Transmission electron microscopy (TEM) of patient-derived dermal fibroblasts (Cav1M) showed abnormal nuclear shape, increased rough endoplasmic reticulum, and an increased number of autophagosomes. Cav1M cells present an abnormal mitochondrial size and shape, accompanied by a reduced mitochondrial capacity for energy production detected by Seahorse real-time oximetry analysis. In addition, we observed in the patient’s fibroblasts an altered pattern of Cav1 expression and distribution, with less Cav1 in caveolae membrane fractions, together with a reduced membrane cholesterol concentration and number of caveolae. Electron paramagnetic spectroscopy shows an altered localization of proteins commonly found in caveolae fractions and increased membrane fluidity. Interestingly, Cav1M fibroblasts showed 4-fold increase in exosome secretion. Nanoparticle tracking and TEM analysis revealed a significant reduction in nanoparticle size in Cav1M-derived exosomes. Normal human dermal fibroblasts (HDF) treated to express Cav1M (Cav1M1-159) or exogenously exposed to exosomes isolated from Cav1M fibroblasts showed altered nuclear morphology, exosome secretion profile, proteomics, and miRNA analysis. Our results indicate that mutations in the C-terminus of Cav1 lead to significant cellular alterations at different levels that can lead to morpho-functional features, energetic imbalances, membrane ultrastructure abnormalities, and exosome secretion and signaling alterations. These findings suggest a novel role of Cav1 that links several aspects of cell physiology, from plasma and nuclear membrane dynamics to exocytic behavior, that have potential implications in cellular communication impacting physiology and pathophysiology. 5R01HL091071. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Asymptotic analysis of particle cluster formation in the presence of anchoring sites

The aggregation or clustering of proteins and other macromolecules plays an important role in the formation of large-scale molecular assemblies within cell membranes. Examples of such assemblies include lipid rafts, and postsynaptic domains (PSDs) at excitatory and inhibitory synapses in neurons. PSDs are rich in scaffolding proteins that can transiently trap transmembrane neurotransmitter receptors, thus localizing them at specific spatial positions. Hence, PSDs play a key role in determining the strength of synaptic connections and their regulation during learning and memory. Recently, a two-dimensional (2D) diffusion-mediated aggregation model of PSD formation has been developed in which the spatial locations of the clusters are determined by a set of fixed anchoring sites. The system is kept out of equilibrium by the recycling of particles between the cell membrane and interior. This results in a stationary distribution consisting of multiple clusters, whose average size can be determined using an effective mean-field description of the particle concentration around each anchored cluster. In this paper, we derive corrections to the mean-field approximation by applying the theory of diffusion in singularly perturbed domains. The latter is a powerful analytical method for solving two-dimensional (2D) and three-dimensional (3D) diffusion problems in domains where small holes or perforations have been removed from the interior. Applications range from modeling intracellular diffusion, where interior holes could represent subcellular structures such as organelles or biological condensates, to tracking the spread of chemical pollutants or heat from localized sources. In this paper, we take the bounded domain to be the cell membrane and the holes to represent anchored clusters. The analysis proceeds by partitioning the membrane into a set of inner regions around each cluster, and an outer region where mean-field interactions occur. Asymptotically matching the inner and outer stationary solutions generates an asymptotic expansion of the particle concentration, which includes higher-order corrections to mean-field theory that depend on the positions of the clusters and the boundary of the domain. Motivated by a recent study of light-activated protein oligomerization in cells, we also develop the analogous theory for cluster formation in a three-dimensional (3D) domain. The details of the asymptotic analysis differ from the 2D case due to the contrasting singularity structure of 2D and 3D Green’s functions.Graphical abstract2D model of diffusion-based protein cluster formation in the presence of anchoring cites and particle recycling. a A set of N anchoring sites at positions xj\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$${\ extbf{x}}_j$$\\end{document}, j=1,…,N\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$j=1,\\ldots ,N$$\\end{document}, in a bounded domain Ω\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$$\\Omega $$\\end{document}. b Diffusing particles accumulate at the anchoring sites resulting in the formation of particle aggregates or clusters Uj\\documentclass[12pt]{minimal} \\usepackage{amsmath} \\usepackage{wasysym} \\usepackage{amsfonts} \\usepackage{amssymb} \\usepackage{amsbsy} \\usepackage{mathrsfs} \\usepackage{upgreek} \\setlength{\\oddsidemargin}{-69pt} \\begin{document}$${{\\mathcal {U}}}_j$$\\end{document}. c The clusters are dynamically maintained by a combination of lateral diffusion outside the clusters and particle recycling

Disulfiram mediated anti-tumour effect in pituitary neuroendocrine tumours by inducing cuproptosis

ContextMedical treatment plays a critical role in pituitary neuroendocrine tumour (PitNET) treatment. Dopamine agonists and somatostatin receptor agonists are the only known drugs for effectively treating PitNET. Thus, the identification of potential therapeutic targets and drugs is urgently needed. ObjectiveTo discover potential drugs that can suppress PitNET growth and to further investigate the underlying mechanism involved. MethodsHigh-throughput drug screening of primary cultures of 17 patient-derived PitNETs was performed to identify potential therapeutic compounds. Cell viability assays, Western blot analysis and flow cytometry were used to investigate pituitary neuroendocrine tumour cell lines and patient-derived PitNET cultures in vitro. In vivo drug efficacy was examined in a mouse xenograft model. ResultsSeventeen primary PitNET samples were collected for high-throughput drug screening, and a class of copper ionophores that can effectively inhibit cell growth, such as zinc pyrithione, elesclomol, and disulfiram (DSF), was identified. Subsequent experiments initially validated the dose-dependent cell growth-suppressing effect of these copper ionophores on AtT20, GH3, and MMQ cells and several primary PitNET cell lines. Moreover, we confirmed that the cytotoxic effect of DSF depends on the presence of copper. Additionally, we determined that cell death occurs via cuproptosis, with events such as Fe-S cluster protein loss, dihydrolipoyl transacetylase oligomerization and heat shock protein 70 upregulation. Finally, we verified the cytotoxic effects of DSF in vivo. ConclusionThe present study revealed copper ionophores as a potential class of drugs for PitNET treatment. DSF induced PitNET cell death via cuproptosis and might be a promising option for PitNET therapy.

Synthesizing Amino Acids Modified with Reactive Carbonyls in Silico to Assess Structural Effects Using Molecular Dynamics Simulations.

Protein carbonylation by reactive aldehydes derived from lipid peroxidation leads to cross-linking, oligomerization, and aggregation of proteins, causing intracellular damage, impaired cell functions, and, ultimately, cell death. It has been described in aging and several age-related chronic conditions. However, the basis of structural changes related to the loss of function in protein targets is still not well understood. Hence, a route to the in silico construction of new parameters for amino acids carbonylated with reactive carbonyl species derived from fatty acid oxidation is described. The Michael adducts for Cys, His, and Lys with 4-hydroxy-2-nonenal (HNE), 4-hydroxy-2-hexenal (HHE), and a furan ring form for 4-Oxo-2-nonenal (ONE), were built, while malondialdehyde (MDA) was directly attached to each residue. The protocol describes details for the construction, geometry optimization, assignment of charges, missing bonds, angles, dihedral angles parameters, and its validation for each modified residue structure. As a result, structural effects induced by the carbonylation with these lipid derivatives have been measured by molecular dynamics simulations on different protein systems such as the thioredoxin enzyme, bovine serum albumin and the membrane Zu-5-ankyrin domain employing root-mean-square deviation (RMSD), root mean square fluctuation (RMSF), structural secondary prediction (DSSP) and the solvent-accessible surface area analysis (SASA), among others.

Effect of VBIT-4 on the functional activity of isolated mitochondria and cell viability

VBIT-4 is a new inhibitor of the oligomerization of VDAC proteins of the outer mitochondrial membrane preventing the development of oxidative stress, mitochondrial dysfunction, and cell death in various pathologies. However, as a VDAC inhibitor, VBIT-4 may itself cause mitochondrial dysfunction in healthy cells. The article examines the effect of VBIT-4 on the functional activity of rat liver mitochondria and cell cultures. We have demonstrated that high concentrations of VBIT-4 (15–30 μM) suppressed mitochondrial respiration in state 3 and 3UDNP driven by substrates of complex I and II. VBIT-4 induced depolarization of organelles fueled by substrates of complex I but not complex II of the respiratory chain. VBIT-4 has been found to inhibit the activity of complexes I, III, and IV of the respiratory chain. Molecular docking demonstrated that VBIT-4 interacts with the rotenone-binding site in complex I with similar affinity. 15–30 μM VBIT-4 caused an increase in H2O2 production in mitochondria, decreased the Ca2+ retention capacity, but increased the time of Ca2+-dependent mitochondrial swelling. We have found that the incubation of breast adenocarcinoma (MCF-7) with 30 μM VBIT-4 for 48 h led to the decrease of the mitochondrial membrane potential, an increase in ROS production and death of MCF-7 cells. The mechanism of action of VBIT-4 on mitochondria and cells is discussed.

Implementation of FRET Spectrometry Using Temporally Resolved Fluorescence: A Feasibility Study.

Förster resonance energy transfer (FRET) spectrometry is a method for determining the quaternary structure of protein oligomers from distributions of FRET efficiencies that are drawn from pixels of fluorescence images of cells expressing the proteins of interest. FRET spectrometry protocols currently rely on obtaining spectrally resolved fluorescence data from intensity-based experiments. Another imaging method, fluorescence lifetime imaging microscopy (FLIM), is a widely used alternative to compute FRET efficiencies for each pixel in an image from the reduction of the fluorescence lifetime of the donors caused by FRET. In FLIM studies of oligomers with different proportions of donors and acceptors, the donor lifetimes may be obtained by fitting the temporally resolved fluorescence decay data with a predetermined number of exponential decay curves. However, this requires knowledge of the number and the relative arrangement of the fluorescent proteins in the sample, which is precisely the goal of FRET spectrometry, thus creating a conundrum that has prevented users of FLIM instruments from performing FRET spectrometry. Here, we describe an attempt to implement FRET spectrometry on temporally resolved fluorescence microscopes by using an integration-based method of computing the FRET efficiency from fluorescence decay curves. This method, which we dubbed time-integrated FRET (or tiFRET), was tested on oligomeric fluorescent protein constructs expressed in the cytoplasm of living cells. The present results show that tiFRET is a promising way of implementing FRET spectrometry and suggest potential instrument adjustments for increasing accuracy and resolution in this kind of study.

Thermodynamics of oligomerization and Helix-to-sheet structural transition of amyloid β-protein on anionic phospholipid vesicles

Understanding oligomerization and aggregation of the amyloid-β protein is important to elucidate the pathological mechanisms of Alzheimer's disease, and lipid membranes play critical roles in this process. In addition to studies reported by other groups, our group has also reported that the negatively-charged lipid bilayers with a high positive curvature induced α-helix-to-β-sheet conformational transitions of amyloid-β-(1–40) upon increase in protein density on the membrane surface and promoted amyloid fibril formation of the protein. Herein, we investigated detailed mechanisms of the conformational transition and oligomer formation of the amyloid-β protein on the membrane surface. Changes in the fractions of the three protein conformers (free monomer, membrane-bound α-helix-rich conformation, and β-sheet-rich conformation) were determined from the fluorescent spectral changes of the tryptophan probe in the protein. The helix-to-sheet structural transition on the surface was described by a thermodynamic model of octamer formation driven by entropic forces including hydrophobic interactions. These findings provide useful information for understanding the self-assembly of amyloidogenic proteins on lipid membrane surfaces.

Endothelial nucleotide-binding oligomerization domain-like receptor protein 3 inflammasome regulation in atherosclerosis.

The activation of nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) inflammasome in endothelial cells (ECs) contributes to vascular inflammation in atherosclerosis. Considering the high glycolytic rate of ECs, we delineated whether and how glycolysis determines endothelial NLRP3 inflammasome activation in atherosclerosis. Our results demonstrated a significant up-regulation of 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3), a key regulator of glycolysis, in human and mouse atherosclerotic endothelium, which positively correlated with NLRP3 levels. Atherosclerotic stimuli up-regulated endothelial PFKFB3 expression via sterol regulatory element-binding protein 2 (SREBP2) transactivation. EC-selective haplodeficiency of Pfkfb3 in Apoe-/- mice resulted in reduced endothelial NLRP3 inflammasome activation and attenuation of atherogenesis. Mechanistic investigations revealed that PFKFB3-driven glycolysis increased the NADH content and induced oligomerization of C-terminal binding protein 1 (CtBP1), an NADH-sensitive transcriptional co-repressor. The monomer form, but not the oligomer form, of CtBP1 was found to associate with the transcriptional repressor Forkhead box P1 (FOXP1) and acted as a transrepressor of inflammasome components, including NLRP3, caspase-1, and interleukin-1β (IL-1β). Interfering with NADH-induced CtBP1 oligomerization restored its binding to FOXP1 and inhibited the glycolysis-dependent up-regulation of NLRP3, Caspase-1, and IL-1β. Additionally, EC-specific overexpression of NADH-insensitive CtBP1 alleviates atherosclerosis. Our findings highlight the existence of a glycolysis-dependent NADH/CtBP/FOXP1-transrepression pathway that regulates endothelial NLRP3 inflammasome activation in atherogenesis. This pathway represents a potential target for selective PFKFB3 inhibitors or strategies aimed at disrupting CtBP1 oligomerization to modulate atherosclerosis.

Photobleaching FRET-FLIM-ICS for quaternary structure quantification on cells. Theory and simulations

The oligomerization of proteins is an important biological control mechanism and has several functions in activity and stability of enzymes, structural proteins, ion channels and transcription factors. The determination of the relevant oligomeric states in terms of geometry (spatial extent), oligomer size (monomer or dimer or oligomer) and affinity (amounts of monomer, dimer and oligomer) is a challenging biophysical problem. Förster resonance energy transfer and fluorescence fluctuation spectroscopy are powerful tools that are sensitive to proximity and oligomerization respectively. Here it is proposed to combine image-based lifetime-detected Forster resonance energy transfer with image correlation spectroscopy and photobleaching to determine distances, oligomer sizes and oligomer distributions. Simulations for simple oligomeric forms illustrate the potential to improve the discrimination between different quaternary states in the cellular milieu.

Selective Hypoxia-Sensitive Oxomer Formation by FIH Prevents Binding of the NF-κB Inhibitor IκBβ to NF-κB Subunits.

Pharmacologic inhibitors of cellular hydroxylase oxygen sensors are protective in multiple preclinical invivo models of inflammation. However, the molecular mechanisms underlying this regulation are only partly understood, preventing clinical translation. We previously proposed a new mechanism for cellular oxygen sensing: oxygen-dependent, (likely) covalent protein oligomer (oxomer) formation. Here, we report that the oxygen sensor factor inhibiting HIF (FIH) forms an oxomer with the NF-κB inhibitor β (IκBβ). The formation of this protein complex required FIH enzymatic activity and was prevented by pharmacologic inhibitors. Oxomer formation was highly hypoxia-sensitive and very stable. No other member of the IκB protein family formed an oxomer with FIH, demonstrating that FIH-IκBβ oxomer formation was highly selective. In contrast to the known FIH-dependent oxomer formation with the deubiquitinase OTUB1, FIH-IκBβ oxomer formation did not occur via an IκBβ asparagine residue, but depended on the amino acid sequence VAERR contained within a loop between IκBβ ankyrin repeat domains 2 and 3. Oxomer formation prevented IκBβ from binding to its primary interaction partners p65 and c-Rel, subunits of NF-κB, the master regulator of the cellular transcriptional response to pro-inflammatory stimuli. We therefore propose that FIH-mediated oxomer formation with IκBβ contributes to the hypoxia-dependent regulation of inflammation.

Oligomerization of the IHF Protein in the Presence of Metal Cations

Oligomerization of the IHF Protein in the Presence of Metal Cations

Anti-inflammatory effect of Trichospira verticillata via suppression of the NLRP3 inflammasome in neutrophilic asthma.

Trichospira verticillata is an annual herb that belongs to the family Asteraceae. Trichospira verticillata extract (TVE) elicits anti-plasmodial activity; however, there has been no detailed report about its anti-inflammatory effects and molecular mechanisms. In addition, herbal plants exhibit anti-inflammatory effects by suppressing the NLRP3 inflammasome. Therefore, the primary goal of this study was to examine the effects of TVE on NLRP3 inflammasome activation by measuring interleukin-1β (IL-1β) secretion. We treated lipopolysaccharides (LPS)-primed J774A.1 and THP-1 cells with TVE, which attenuated NLRP3 inflammasome activation. Notably, TVE did not affect nuclear factor-kappa B (NF-κB) signalling or intracellular reactive oxygen species (ROS) production and potassium efflux, suggesting that it inactivates the NLRP3 inflammasome via other mechanisms. Moreover, TVE suppressed the formation of apoptosis-associated speck-like protein (ASC) speck and oligomerization. Immunoprecipitation data revealed that TVE reduced the binding of NLRP3 to NIMA-related kinase 7 (NEK7), resulting in reduced ASC oligomerization and speck formation. Moreover, TVE alleviated neutrophilic asthma (NA) symptoms in mice. This study demonstrates that TVE modulates the binding of NLPR3 to NEK7, thereby reporting novel insights into the mechanism by which TVE inhibits NLRP3 inflammasome. These findings suggest TVE as a potential therapeutic of NLRP3 inflammasome-mediated diseases, particularly NA.

Gold Nanoparticle-Decorated Catalytic Micromotor-Based Aptassay for Rapid Electrochemical Label-Free Amyloid-β42 Oligomer Determination in Clinical Samples from Alzheimer's Patients.

Micromotor (MM) technology offers a valuable and smart on-the-move biosensing microscale approach in clinical settings where sample availability is scarce in the case of Alzheimer's disease (AD). Soluble amyloid-β protein oligomers (AβO) (mainly AβO42) that circulate in biological fluids have been recognized as a molecular biomarker and therapeutic target of AD due to their high toxicity, and they are correlated much more strongly with AD compared to the insoluble Aβ monomers. A graphene oxide (GO)-gold nanoparticles (AuNPs)/nickel (Ni)/platinum nanoparticles (PtNPs) micromotors (MMGO-AuNPs)-based electrochemical label-free aptassay is proposed for sensitive, accurate, and rapid determination of AβO42 in complex clinical samples such as brain tissue, cerebrospinal fluid (CSF), and plasma from AD patients. An approach that implies the in situ formation of AuNPs on the GO external layer of tubular MM in only one step during MM electrosynthesis was performed (MMGO-AuNPs). The AβO42 specific thiolated-aptamer (AptAβO42) was immobilized in the MMGO-AuNPs via Au-S interaction, allowing for the selective recognition of the AβO42 (MMGO-AuNPs-AptAβO42-AβO42). AuNPs were smartly used not only to covalently bind a specific thiolated-aptamer for the design of a label-free electrochemical aptassay but also to improve the final MM propulsion performance due to their catalytic activity (approximately 2.0× speed). This on-the-move bioplatform provided a fast (5 min), selective, precise (RSD < 8%), and accurate quantification of AβO42 (recoveries 94-102%) with excellent sensitivity (LOD = 0.10 pg mL-1) and wide linear range (0.5-500 pg mL-1) in ultralow volumes of the clinical sample of AD patients (5 μL), without any dilution. Remarkably, our MM-based bioplatform demonstrated the competitiveness for the determination of AβO42 in the target samples against the dot blot analysis, which requires more than 14 h to provide qualitative results only. It is also important to highlight its applicability to the potential analysis of liquid biopsies as plasma and CSF samples, improving the reliability of the diagnosis given the heterogeneity and temporal complexity of neurodegenerative diseases. The excellent results obtained demonstrate the analytical potency of our approach as a future tool for clinical/POCT (Point-of-care testing) routine scenarios.

A myristoyl switch at the plasma membrane triggers cleavage and oligomerization of Mason-Pfizer monkey virus matrix protein.

For most retroviruses, including HIV, association with the plasma membrane (PM) promotes the assembly of immature particles, which occurs simultaneously with budding and maturation. In these viruses, maturation is initiated by oligomerization of polyprotein precursors. In contrast, several retroviruses, such as Mason-Pfizer monkey virus (M-PMV), assemble in the cytoplasm into immature particles that are transported across the PM. Therefore, protease activation and specific cleavage must not occur until the pre-assembled particle interacts with the PM. This interaction is triggered by a bipartite signal consisting of a cluster of basic residues in the matrix (MA) domain of Gag polyprotein and a myristoyl moiety N-terminally attached to MA. Here, we provide evidence that myristoyl exposure from the MA core and its insertion into the PM occurs in M-PMV. By a combination of experimental methods, we show that this results in a structural change at the C-terminus of MA allowing efficient cleavage of MA from the downstream region of Gag. This suggests that, in addition to the known effect of the myristoyl switch of HIV-1 MA on the multimerization state of Gag and particle assembly, the myristoyl switch may have a regulatory role in initiating sequential cleavage of M-PMV Gag in immature particles.

Abstract 4376: The pseudokinase PTK7 is a negative regulator of EPHA2 in colorectal cancer

Abstract Colorectal cancer (CRC) remains a major public health issue. Identification of biomarkers predicting high risk of recurrence and discovery of novel targets for therapy are urgently needed1. The cell surface receptor PTK7 is a poorly described evolutionary conserved member of the receptor tyrosine kinase superfamily that was first identified in human colon carcinoma and melanoma2-3. Accumulated data highlighted the implication of PTK7 in diverse cancer-related signaling pathways4-6 and, despite the lack of kinase activity, anti-PTK7 inhibitors are currently in development at preclinical and clinical stages7-9. The dual aim of this project is to increase the basic knowledge about PTK7 and to evaluate its role in CRC in order to propose in the future novel therapeutics directed against PTK7. With the aim to discover novel PTK7 associated pathways, we used a proximity biotinylation strategy coupled to mass spectrometry in CRC cells. Among the partners identified, we found that the pseudokinase PTK7 interacts with the active receptor tyrosine kinase EphrinA2 (EPHA2). Similarly to PTK7, EPHA2 expression correlates with poor prognosis in CRC10. In this work, we also demonstrate that PTK7 acts as a negative regulator of EPHA2 activation through the promotion of distinct oligomeric protein complexes and the modulation of EPHA2 phosphorylation. We also shed in light on the role of PTK7 in EPHA2 K63-linked ubiquitination and its subsequent endosomal sorting and lysosomal degradation. References 1 Punt CJ et al, Nat. Rev. Clin. Oncol., 2016; 2 Lhoumeau AC et al, Cell Cycle, 2011; 3 Lhoumeau AC et al, PLoS One, 2015; 4 Puppo F et al, EMBO reports, 2011; 5 Martinez S et al, J. Biol. Chem., 2015; 6 Daulat AM and Borg JP, Trends in Cancer, 2017; 7 Damelin M et al, Sci. Transl. Med., 2017; 8 Jie Y et al, Front. Immunol., 2021; 9 Maitland ML et al, Clin. Cancer. Res., 2021; 10 Dunne P et al, Clin. Cancer. Res., 2016; 11 Cioce M and Fazio VM, Cancers, 2021. Citation Format: Jean-Paul Borg, Charlotte Dessaux, Luc Camoin, Stéphane Audebert, Emilie Baudelet, Avais Daulat. The pseudokinase PTK7 is a negative regulator of EPHA2 in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4376.