Formation Of Oligomers Research Articles

Elevated serum mtDNA in COVID-19 patients is linked to SARS-CoV-2 envelope protein targeting mitochondrial VDAC1, inducing apoptosis and mtDNA release

Mitochondria dysfunction is implicated in cell death, inflammation, and autoimmunity. During viral infections, some viruses employ different strategies to disrupt mitochondria-dependent apoptosis, while others, including SARS-CoV-2, induce host cell apoptosis to facilitate replication and immune system modulation. Given mitochondrial DNAs (mtDNA) role as a pro-inflammatory damage-associated molecular pattern in inflammatory diseases, we examined its levels in the serum of COVID-19 patients and found it to be high relative to levels in healthy donors. Furthermore, comparison of serum protein profiles between healthy individuals and SARS-CoV-2-infected patients revealed unique bands in the COVID-19 patients. Using mass spectroscopy, we identified over 15 proteins, whose levels in the serum of COVID-19 patients were 4- to 780-fold higher. As mtDNA release from the mitochondria is mediated by the oligomeric form of the mitochondrial-gatekeeper—the voltage-dependent anion-selective channel 1 (VDAC1)—we investigated whether SARS-CoV-2 protein alters VDAC1 expression. Among the three selected SARS-CoV-2 proteins, small envelope (E), nucleocapsid (N), and accessory 3b proteins, the E-protein induced VDAC1 overexpression, VDAC1 oligomerization, cell death, and mtDNA release. Additionally, this protein led to mitochondrial dysfunction, as evidenced by increased mitochondrial ROS production and cytosolic Ca2+ levels. These findings suggest that SARS-CoV-2 E-protein induces mitochondrial dysfunction, apoptosis, and mtDNA release via VDAC1 modulation. mtDNA that accumulates in the blood activates the cGAS-STING pathway, triggering inflammatory cytokine and chemokine expression that contribute to the cytokine storm and tissue damage seen in cases of severe COVID-19.

7542 Androgen Receptor Oligomerization State Allows Cells to Exhibit Non-Linear Dose Responses to Androgens Resulting in Distinct Biological Outputs

Abstract Disclosure: R. Safi: None. S.E. Wardell: None. P. Watkinson: None. X. Qin: None. M. Lee: None. S. Park: None. T. Krebs: None. E. Dolan: None. T. Tsuji: None. S. Nayak: None. M. Khater: None. M. Newlin: None. M. Kirkland: None. Y. Xie: None. H. Long: None. E. Fink: None. S.W. Fanning: None. S. Runyon: None. M. Brown: None. S. Xu: None. K. Owzar: None. J.D. Norris: None. D.P. McDonnell: None. An unanswered fundamental question in androgen action is how cells recognize and respond in a non-linear fashion to different levels of androgens to elicit distinct biological outputs such as occurs in prostate cancer (PCa) cells. Indeed, most PCa express the androgen receptor (AR), and tumor growth and progression are facilitated by exceedingly low levels of systemic or intratumorally produced androgens. Paradoxically, high dose androgens have been used to treat patients with late-stage metastatic PCa. We have determined that androgen dose regulates the relative cellular abundance of AR monomers and dimers in cells and that the biological output of each oligomeric form of the receptor is different. Using in vitro systems that model exposure from castrate (low dose; LD) to eugonadal levels and above (high dose; HD) of androgens, we have determined that the global changes in gene expression are substantially different, with the changes induced by LD androgens (monomeric AR) being associated with cell proliferation, and HD androgens (dimeric AR) inducing a differentiated phenotype. A similar distinction in response to androgen levels was observed in vivo. Using a chemical biology strategy, we identified ligands which freeze AR in a monomeric form and demonstrate that these compounds faithfully recapitulate the LD biology exhibited by physiological AR agonists. Mechanistically, we determined that monomeric AR facilitates a non-genomic activation of the mTOR signaling pathway to drive proliferation, while the dimer suppresses c-MYC expression, inhibit proliferation, and drive a transcriptional program associated with a differentiated phenotype. These findings highlight the inherent liabilities in current approaches used to inhibit AR-action in prostate cancer and are instructive as to strategies that can be used to develop new therapeutics for this disease and other androgenopathies. Presentation: 6/3/2024

Structural Insight into Melatonin's Influence on the Conformation of Aβ42 Dimer Studied by Molecular Dynamics Simulation.

The accumulation of amyloid-beta (Aβ) oligomers is recognized as a potential culprit in Alzheimer's disease (AD). Experimental studies show that melatonin, a hormone that mainly regulates circadian rhythm and sleep, can interact with Aβ peptides and disrupt the formation of oligomers. However, how melatonin inhibits the oligomerization of soluble Aβ is unclear. Here, by computational simulations, we investigate the effect of different levels of melatonin on the conformation of the Aβ42 dimer. We find that the conformation of the Aβ42 dimer is dependent on melatonin levels. When melatonin is absent, the dimer mainly forms a parallel β-sheet in the CHC region. When one melatonin molecule is present, the overall conformation of the dimer does not change much, but the N-terminal of the dimer tends to adopt antiparallel β-sheets. When two melatoinin molecules are present, the Aβ42 dimer exhibits significant structural change, especially in its central region, resulting in a more compact conformation, and forms parallel β-sheets in the C-terminal. This conformational difference induced by different levels of melatoinin can shed light on the protective role of melatonin.

Study on the hydrothermal gradient extraction of hemicellulose by a flow-through reactor

The hydrothermal gradient extraction process based on the hemicellulose constituent units is important for obtaining high quality hemicellulose products. The hydrothermal extraction of sawdust hemicellulose was performed under both non-isothermal and isothermal operations using a flow-through reactor for investigating the extraction patterns. The results show that there were significant differences in the major forms of hemicellulose units at different extraction stages. For glucose, xylose, and galactose units, the selectivity of oligomeric form decreased gradually with increasing temperature, whereas it decreased and then increased under thermostatic operation. The selectivity of the mannose oligomeric form decreased and then increased in both operation modes, reaching a trough at 170 °C (96.81 %) and 60 min (55.21 %), respectively. The molecular weight of extracted hemicelluloses were mainly distributed below 70,000 Da, and gradually decreased with temperature, but increased with time. The results contribute to the quantitative and qualitative understanding of the hemicellulose gradient extraction process.

Isolicoflavonol ameliorates acute liver injury via inhibiting NLRP3 inflammasome activation through boosting Nrf2 signaling in vitro and in vivo

BackgroundNOD like receptor pyrin domain containing 3 (NLRP3) inflammasome is involved in innate immunity, and related to liver injury. However, no inflammasome inhibitors are clinically available until now. Our previous research suggests that isolicoflavonol (ILF), isolated from Macaranga indica, is a potent NLRP3 inflammasome inhibitor, but its mechanism is unclear. MethodsFluorescent imaging and Western blot assay were used to ascertain the effects of ILF on pyroptosis and NLRP3 inflammasome activation in macrophages. Next, Nrf2 signal pathway, its downstream gene transcription and expression were further investigated. ML385, a Nrf2 inhibitor, was used to verify whether ILF targets Nrf2 signaling. A carbon tetrachloride induced liver injury model was introduced to evaluate the liver protection activity of ILF in mice. ResultsThis work revealed that ILF inhibited macrophage LDH release and IL-1β secretion in a dose-dependent manner. ILF had no significant cytotoxic effect on macrophage, it reduced pyroptosis and Gasdermin D N-terminal fragment formation. Moreover, ILF inhibited IL-1β maturation and Caspase-1 cleavage, but did not affect NLRP3, pro-Caspase-1, pro-IL-1β and ASC expression. ILF decreased ASC speck rate and reduced ASC oligomer formation. ILF decreased aggregated JC-1 formation restoring mitochondria membrane potential. In addition, ILF increased Nrf2 expression, extended Nrf2 lifespan and upregulated Nrf2 signaling pathway in macrophages whether the NLRP3 inflammasome was activated or not. Besides, ILF increased Nrf2 nuclear translocation, maintained a high proportion of Nrf2 in the nucleus, and upregulated ARE-related gene transcription and expression. Furthermore, Nrf2 signal inhibition attenuated compound ILF-mediated inhibition of pyroptosis, inflammasome activation and upregulation of Nrf2 signaling. ILF in a liver injury mouse model inhibited NLRP3 inflammasome activation and enhanced Nrf2 signaling. ConclusionOur study verified that ILF ameliorates liver injury via inhibiting NLRP3 inflammasome activation through boosting Nrf2 signaling, and highlighted that ILF is a potent anti-inflammatory drug for inflammasome-related liver diseases.

Membrane association and polar localization of the Legionella pneumophila T4SS DotO ATPase mediated by two nonredundant receptors

The Legionella pneumophila Dot/Icm type IVB secretion system (T4BSS) is a large, multisubunit complex that exports a vast array of substrates into eukaryotic host cells. DotO, a distant homolog of the T4ASS ATPase VirB4, associates with the bacterial inner membrane despite lacking hydrophobic transmembrane domains. Employing a genetic approach, we found DotO's membrane association is mediated by three inner-membrane Dot/Icm components, IcmT, and a combined DotJ-DotI complex (referred to as DotJI). Although deletion of icmT or dotJI individually does not affect DotO's membrane association, the simultaneous inactivation of all three genes results in increased amounts of soluble DotO. Nevertheless, deleting each receptor separately profoundly affects positioning of DotO, disrupting its link with the Dot/Icm complex at the bacterial poles, rendering the receptors nonredundant. Furthermore, a collection of dotO point mutants that we isolated established that DotO's N-terminal domain interacts with the membrane receptors and is involved in dimerization, whereas DotO's C-terminal ATPase domain primarily contributes to the protein's formation of oligomers. Modeling data revealed the complex interaction between DotO and its receptors is responsible for formation of DotO's unique "hexamer of dimers" configuration, which is a defining characteristic of VirB4 family members.

Computational Insights Into the Mechanism of EGCG's Binding and Inhibition of the TDP-43 Aggregation.

Misfolding and aggregation of TAR DNA-binding protein, TDP-43, is linked to devastating proteinopathies such as ALS. Therefore, targeting TDP-43's aggregation is significant for therapeutics. Recently, green tea polyphenol, EGCG, was observed to promote non-toxic TDP-43 oligomer formation disallowing TDP-43 aggregation. Here, we investigated if the anti-aggregation effect of EGCG is mediated via EGCG's binding to TDP-43. In silico molecular docking and molecular dynamics (MD) simulation suggest a strong binding of EGCG with TDP-43's aggregation-prone C-terminal domain (CTD). Three replicas, each having 800 ns MD simulation of the EGCG-TDP-43-CTD complex, yielded a high negative binding free energy (ΔG) inferring a stable complex formation. Simulation snapshots show that EGCG forms close and long-lasting contacts with TDP-43's Phe-313 and Ala-341 residues, which were previously identified for monomer recruitment in CTD's aggregation. Notably, stable physical interactions between TDP-43 and EGCG were also detected invitro using TTC staining and isothermal titration calorimetry which revealed a high-affinity binding site of EGCG on TDP-43 (Kd, 7.8 μM; ΔG, -6.9 kcal/mol). Additionally, TDP-43 co-incubated with EGCG was non-cytotoxic when added to HEK293 cells. In summary, EGCG's binding to TDP-43 and blocking of residues important for aggregation can be a possible mechanism of its anti-aggregation effects on TDP-43.

Simultaneous Regulation of Organic and Inorganic Components in Interphase by Fiber Separator for High-Stable Sodium Metal Batteries.

Uncontrollable side reactions at the metal interface have been identified as the root cause of the formation of a fragile solid electrolyte interphase, leading to irreversible sodium loss in sodium metal batteries. Here, we proposed an interface engineering strategy that employed a carboxyl functionalized cellulose separator to provide strong dipole moments and induce the cleavage of P-F bond to construct a solid electrolyte interphase (SEI) rich in NaF. In addition, we employed nuclear magnetic resonance technology confirmed that the separator with strong dipole moments prevented the reduction of organic solvents by attracting electrons, thereby inhibiting the formation of organic oligomers. SEI with high NaF content and few oligomers is smooth and robust, obviously decreasing the interface impedance of the Na anode. The symmetric Na||Na cells, equipped with the functionalized separator, efficiently operated for 1400 hours with a stable 72 mV overpotential at 0.25 mA cm-2, exhibiting low energy barrier and fast ion transport kinetics. The Na||Na3V2(PO4)3 cell also showed stable cycling performance, with the capacity remaining at 94.83 % of the initial capacity after 1000 cycles at 1C. The proposed separator could control the formation and composition of SEI, paving the way for the development of long-life sodium metal batteries.

Furanic jet fuels – Water-free aldol condensation of furfural and cyclopentanone

A method for the water-free, large-scale synthesis of bio-jet fuel precursors, distinct from traditional fossil-based sources, is introduced. This approach involves the aldol condensation of furfural with cyclopentanone, producing C10-C15 fuel precursors eligible for further hydrodeoxygenation to high-performance diesel and jet fuel hydrocarbons. In the context of process integration, aldol condensation reactions were conducted under water-free conditions, utilizing excess furfural as the solvent. Evaluation of various commercial catalysts confirmed the feasibility of running in excess furfural. Both basic and acidic catalysts demonstrated significant activity, with CaO and amorphous silica-alumina achieving ≥80 mol% conversion of cyclopentanone and yielding ≥80 mol% selectivity towards the desired fuel components within 5 h of reaction. However, an overlooked aspect is the notable formation of undesired heavy side products. Observations indicated that the high furfural concentration, combined with the use of strong acidic catalysts, were the primary cause of heavy side product formation. The strong base catalyst, CaO, significantly reduced the formation of these oligomers, but did not appear to stop it completely. Interestingly, water content did not appear to play a major role in byproduct selectivity. To further suppress the formation of oligomers, the use of process-owned intermediates as solvents is proposed.

Insight into HTPB pyrolysis mechanism under high-temperature: A reactive molecular dynamics study

Hydroxyl-terminated polybutadiene (HTPB) is widely utilized in solid and hybrid rocket propellants due to its mechanical properties and combustion performance. Insight into its pyrolysis process is key to enhancing combustion efficiency in rocket engines. This study employs ReaxFF molecular dynamics (MD) simulations to explore the pyrolysis mechanism of HTPB under extreme conditions, with temperatures ranging from 1000 K to 2000 K. The results identify the primary degradation products and elucidate their formation mechanisms. The simulation reveals that C-C bond cleavage at polymerization sites is the initial step, followed by the formation of linear oligomers and butadiene. Subsequent reactions, including hydrogenation and dehydrogenation, lead to the generation of smaller molecular species. The kinetic analysis confirms that HTPB pyrolysis follows first-order reaction kinetics, with an activation energy of 8.12 kcal/mol. The findings are compared with existing experimental data, highlighting the influence of thermal environments on pyrolysis mechanisms and product distributions.

The Development of Clioquinol Labeled-Gold Nanoparticles for the Treatment of Alzheimer’s Disease

Clioquinol has emerged as a prominent metal-chelating agent for Alzheimer’s Disease. While clioquinol was once a popular antibiotic for skin infections, it has recently been investigated as a potential therapeutic for Alzheimer’s Disease. Its metal chelating property can restore metal homeostasis and prevent copper (Cu2+) and zinc (Zn2+) from interacting with amyloid-β (Aβ) peptides. Here, we propose labeling gold nanoparticles with clioquinol as a novel therapeutic approach for Alzheimer’s Disease to enable effective delivery of the agent across the blood-brain barrier. Based on previous studies on the individual efficacies of clioquinol and gold nanoparticles, we anticipate that this approach will prevent the formation of Aβ oligomers by disrupting metal-protein interactions. Our proposed clioquinol-labeled gold nanoparticles provide a novel curative approach for Alzheimer’s Disease and insights into subsequent therapeutic developments.

Two major bovine milk whey proteins induce distinct responses in IEC-6 intestinal cells

Abstract α-Lactalbumin (α-LA) and β-lactoglobulin (β-LG) are major whey proteins in bovine milk. We studied the effects of these molecules on the intestinal cell response by comparing the native form with the denatured form containing oligomers obtained by treatment with 2,2,2-trifluoroethanol (TFE). We previously reported that proteins in native and TFE-treated forms exhibited cell growth stimulation and cytotoxicity, respectively, in undifferentiated rat crypt IEC-6 and human colon Caco-2 cells. However, neither whey protein showed cytotoxicity even in the TFE-treated form in differentiated Caco-2 cells. Only undifferentiated immature intestinal cells can distinguish between these native and denatured proteins. Moreover, α-LA and β-LG exhibited different oligomer formation characteristics during the TFE treatment. In the present study, we compared the effects of native and TFE-treated whey proteins on IEC-6 cells in more detail. The native forms of both whey proteins exhibited cell proliferative effects in a concentration-dependent manner. For the TFE-treated forms, α-LA showed rapid and potent cytotoxicity, whereas β-LG altered cell responses depending on its concentration and exposure time; lower concentration/shorter exposure and higher concentration/longer exposure induced cell growth stimulation and cytotoxicity, respectively. Pre-treatment of the cell membrane with cholesterol suppressed the effects on the cell response only in TFE-treated β-LG (TFE-β-LG). In a preliminary examination using inhibitors of signal transduction, TFE-treated α-LA acted on the intrinsic apoptosis pathway via Bcl-2-associated X and p53, whereas the action of TFE-LG did not require this pathway. Tyrosine phosphorylation is necessary for the cell proliferation effect of both native whey proteins; however, native α-LA, but not native β-LG, also required activation of the pathway with selective epidermal growth factor receptor tyrosine kinase and Janus kinase 2/3. In summary, the two major bovine milk whey proteins induced similar yet discrete responses in undifferentiated intestinal cells. Even when oligomers are formed, β-LG may be much less hazardous to immature intestinal cells than α-LA.

Comprehensive Analysis of Cetuximab Critical Quality Attributes: Impact of Handling on Antigen-Antibody Binding.

Cetuximab, formulated in Erbitux® (5 mg/mL), is a therapeutic monoclonal antibody (mAb) widely used in several cancer treatments. Currently, there is insufficient knowledge about the behavior of cetuximab with regard to the risk associated with its routine handling or unintentional mishandling in hospitals. Forced degradation studies can simulate these conditions and provide insights into the biophysical and biochemical properties of mAbs. In this study, we conducted a deep physicochemical and functional characterization of the critical quality attributes of cetuximab in control samples and under controlled degraded conditions, including freeze-thaw cycles, heat, agitation, and light exposure. To achieve this purpose, we used a set of proper analytical techniques, including CD, IT-FS, DLS, SE/UHPLC-UV, UHPLC-MS/MS, and ELISA, to check functionality based on antigen-antibody binding. The results revealed that light exposure was the stress stimuli with the greatest impact on the drug product, leading to the formation of non-natural oligomers, fragmentation, and oxidation of methionine residues. Additionally, cetuximab (Erbitux®, 5 mg/mL) showed a tendency to aggregate when submitted to 60 °C for 1 h. In terms of functionality, cetuximab (Erbitux®, 5 mg/mL) samples were found to be affected when subjected to freeze-thaw cycles, 60 °C (1 h), and when exposed to light (daylight with room temperature excursion and accelerated light exposure). Thus, we suggest that Erbitux® (5 mg/mL) should be shielded from these environmental conditions, as they compromise both the safety and efficacy of the drug product.

Natural Compounds That Activate the KEAP1/Nrf2 Signaling Pathway as Potential New Drugs in the Treatment of Idiopathic Parkinson's Disease.

Recently, a single-neuron degeneration model has been proposed to understand the development of idiopathic Parkinson's disease based on (i) the extremely slow development of the degenerative process before the onset of motor symptoms and during the progression of the disease and (ii) the fact that it is triggered by an endogenous neurotoxin that does not have an expansive character, limiting its neurotoxic effect to single neuromelanin-containing dopaminergic neurons. It has been proposed that aminochrome is the endogenous neurotoxin that triggers the neurodegenerative process in idiopathic Parkinson's disease by triggering mitochondrial dysfunction, oxidative stress, neuroinflammation, dysfunction of both lysosomal and proteasomal protein degradation, endoplasmic reticulum stress and formation of neurotoxic alpha-synuclein oligomers. Aminochrome is an endogenous neurotoxin that is rapidly reduced by flavoenzymes and/or forms adducts with proteins, which implies that it is impossible for it to have a propagative neurotoxic effect on neighboring neurons. Interestingly, the enzymes DT-diaphorase and glutathione transferase M2-2 prevent the neurotoxic effects of aminochrome. Natural compounds present in fruits, vegetables and other plant products have been shown to activate the KEAP1/Nrf2 signaling pathway by increasing the expression of antioxidant enzymes including DT-diaphorase and glutathione transferase. This review analyzes the possibility of searching for natural compounds that increase the expression of DT-diaphorase and glutathione transferase through activation of the KEAP1/Nrf2 signaling pathway.

The cellular prion protein does not affect tau seeding and spreading of sarkosyl-insoluble fractions from Alzheimer’s disease

The cellular prion protein (PrPC) plays many roles in the developing and adult brain. In addition, PrPC binds to several amyloids in oligomeric and prefibrillar forms and may act as a putative receptor of abnormal misfolded protein species. The role of PrPC in tau seeding and spreading is not known. In the present study, we have inoculated well-characterized sarkosyl-insoluble fractions of sporadic Alzheimer’s disease (sAD) into the brain of adult wild-type mice (Prnp+/+), Prnp0/0 (ZH3 strain) mice, and mice over-expressing the secreted form of PrPC lacking their GPI anchor (Tg44 strain). Phospho-tau (ptau) seeding and spreading involving neurons and oligodendrocytes were observed three and six months after inoculation. 3Rtau and 4Rtau deposits from the host tau, as revealed by inoculating Mapt0/0 mice and by using specific anti-mouse and anti-human tau antibodies suggest modulation of exon 10 splicing of the host mouse Mapt gene elicited by exogenous sAD-tau. However, no tau seeding and spreading differences were observed among Prnp genotypes. Our results show that PrPC does not affect tau seeding and spreading in vivo.

The greasy finger region of DTMUV NS1 plays an essential role in NS1 secretion and viral proliferation

Duck Tembusu virus (DTMUV) of the Orthoflavivirus genus poses a significant threat to waterfowl aquaculture. Nonstructural protein 1 (NS1), a multifunctional glycoprotein, exists in various oligomeric forms and performs diverse functions. The Greasy Finger (GF) region within NS1 of other flaviviruses has been shown to be a crucial component of the hydrophobic protrusion aiding in anchoring NS1 to the endoplasmic reticulum (ER). However, detailed studies on the role of the GF region in viral proliferation in vitro and the biological properties of NS1 remain scarce. A series of recombinant DTMUV (rDTMUV) with mutations in the GF region, including NS1-F158A, G159A, F160A, G161A, V162A, L163A, F160R, multipoint mutations (GF-4M), or regional deletions (ΔGF), were rescued using a DNA-based reverse genetics system. Only five rDTMUV variants (G159A, F160A, G161A, V162A, and L163A) could be rescued successfully, and these mutations were found to impair replication, reduce virulence, and decrease plaque size, as shown by growth kinetics, duck embryo virulence, and plaque assays, respectively. Upon examining NS1 expression by western blot, we discovered that secreted NS1 (sNS1) presented in large quantities in the supernatant of cells infected with rDTMUV-NS1-G159A, whereas intracellular NS1 was less abundant. These mutations also impacted the primary forms and secretion rates of NS1 in cases of overexpression by western blot and indirect ELISA. Exception for F160A and G161A, which showed decreased secretion rates, all other mutations increased sNS1 expression, with the most pronounced increase observed in F158A and ΔGF, and rDTMUV with these mutations can't be rescued. Co-localization studies of NS1 with the ER demonstrated that the ΔGF mutation attenuated NS1 anchoring to the ER, thereby inhibiting its intracellular residence and promoting secretion. Although these effects vary between flaviviruses, our data reveal that the GF region of NS1 is crucial for viral proliferation and NS1 secretion.

A method for deriving oligomers from sulfamide monomer and their application as electrolytes

This paper describes the development of a method of catalytic polymerization that was then applied to synthesize sulfate-based oligomers from sulfamide. Two molecules, copper triflate and copper acetate, catalyzed the formation of oligomers with diol monomers. Two oligomers were identified as products of the reactions. Oligomers with sulfate incorporated in the backbone proved to have an ability for ion diffusion. When the oligomers were doped with lithium salt, anion transportation was predominant, as shown by solid-state NMR. The lithium ion was found to be strongly bonded to the backbone, while the anion was highly mobile. To corroborate this finding, we conducted molecular dynamics (MD) simulations, which revealed the structural characteristics and static properties of two electrolytes containing LiTFSI and two different polyethylene oxide oligomers. Interactions between the anion and cation were analyzed through computation of the radial distribution function (RDF) and the spatial distribution function (SDF). Our findings indicate that while the anion presents weak interactions with the polymer chain, the cation interacts strongly with the oligomer backbone.

Neuronal extracellular vesicles influence the expression, degradation and oligomeric state of fructose 1,6-bisphosphatase 2 in astrocytes affecting their glycolytic capacity

Fructose 1,6-bisphosphatase 2 (Fbp2) is a regulatory enzyme of gluco- and glyconeogenesis which, in the course of evolution, acquired non-catalytic functions. Fbp2 promotes cell survival during calcium stress, regulates glycolysis via inhibition of Hif-1α activity, and is indispensable for the formation of long-term potentiation in hippocampus. In hippocampal astrocytes, the amount of Fbp2 protein is reduced by signals delivered in neuronal extracellular vesicles (NEVs) through an unknown mechanism. The physiological role of Fbp2 (determined by its subcellular localization/interactions) depends on its oligomeric state and thus, we asked whether the cargo of NEVs is sufficient to change also the ratio of Fbp2 dimer/tetramer and, consequently, influence astrocyte basal metabolism. We found that the NEVs cargo reduced the Fbp2 mRNA level, stimulated the enzyme degradation and affected the cellular titers of different oligomeric forms of Fbp2. This was accompanied with increased glucose uptake and lactate release by astrocytes. Our results revealed that neuronal signals delivered to astrocytes in NEVs provide the necessary balance between enzymatic and non-enzymatic functions of Fbp2, influencing not only its amount but also subcellular localization. This may allow for the metabolic adjustments and ensure protection of mitochondrial membrane potential during the neuronal activity-related increase in astrocytic [Ca2+].

Single-Molecule Characterization of Heterogeneous Oligomer Formation during Co-Aggregation of 40- and 42-Residue Amyloid-β.

The two most abundant isoforms of amyloid-β (Aβ) are the 40- (Aβ40) and 42-residue (Aβ42) peptides. Since they coexist and there is a correlation between toxicity and the ratio of the two isoforms, quantitative characterization of their interactions is crucial for understanding the Aβ aggregation mechanism. In this work, we follow the aggregation of individual isoforms in a mixture using single-molecule FRET spectroscopy by labeling Aβ42 and Aβ40 with the donor and acceptor fluorophores, respectively. We found that there are two phases of aggregation. The first phase consists of coaggregation of Aβ42 with a small amount of Aβ40, while the second phase results mostly from aggregation of Aβ40. We also found that the aggregation of Aβ42 is slowed by Aβ40 while the aggregation of Aβ40 is accelerated by Aβ42 in a concentration-dependent manner. The formation of oligomers was monitored by incubating mixtures in a plate reader and performing a single-molecule free-diffusion experiment at several different stages of aggregation. The detailed properties of the oligomers were obtained by maximum likelihood analysis of fluorescence bursts. The FRET efficiency distribution is much broader than that of the Aβ42 oligomers, indicating the diversity in isoform composition of the oligomers. Pulsed interleaved excitation experiments estimate that the fraction of Aβ40 in the co-oligomers in a 1:1 mixture of Aβ42 and Aβ40 varies between 0 and 20%. The detected oligomers were mostly co-oligomers especially at the physiological ratio of Aβ42 and Aβ40 (1:10), suggesting the critical role of Aβ40 in oligomer formation and aggregation.

Probing the structural stability of R-phycocyanin under pressure.

The red macroalgae Porphyra, commonly known as Nori, is widely used as food around the world due to its high nutrient content, including the significant abundance of colored phycobiliproteins (PBPs). Among these, R-phycocyanin (R-PC) stands out for its vibrant purple color and numerous bioactive properties, making it a valuable protein for the food industry. However, R-PC's limited thermal stability necessitates alternative processing methods to preserve its color and bioactive properties. Our study aimed to investigate the in-situ stability of oligomeric R-PC under high pressure (HP) conditions (up to 4000 bar) using a combination of absorption, fluorescence, and small-angle X-ray scattering (SAXS) techniques. The unfolding of R-PC is a multiphase process. Initially, low pressure induces conformational changes in the R-PC oligomeric form (trimers). As pressure increases above 1600 bar, these trimers dissociate into monomers, and at pressures above 3000 bar, the subunits begin to unfold. When returned to atmospheric pressure, R-PC partially refolds, retaining 50% of its original color absorbance. In contrast, heat treatment causes irreversible and detrimental effects on R-PC color, highlighting the advantages of HP treatment in preserving both the color and bioactive properties of R-PC compared to heat treatment.