Lupus Sci Med. 2024 Feb 14;11(1):e001078. doi: 10.1136/lupus-2023-001078.

ABSTRACT

BACKGROUND: Juvenile SLE (JSLE) is a complex autoimmune disorder that predominantly affects children and adolescents with several unique challenges, and microRNA-146a (miRNA-146a) might be an interesting anti-inflammatory molecule. Because exosomes in the blood might protect miRNAs, the association between circulating exosomal miRNA-146a and lupus proinflammatory genes, such as IRAK1 and TRAF6, was studied in peripheral blood mononuclear cells from people with JSLE.

METHODS: Blood samples from 12 patients were collected every 3 months until 1 year with the recorded disease activity, and quantitative real-time PCR was used to determine the circulating exosomal miRNA-146a and the gene expression (IRAK1 and TRAF6).

RESULTS: The mean age was 12.60±0.43 years at diagnosis and all patients had a complete response at 12 months. According to the nanoparticle tracking analysis, the abundance of exosomes was significantly lower at 3, 6 and 12 months compared with 0 months, while the level of circulating exosomal miRNA-146a was significantly higher at 12 months than at diagnosis (p<0.001). There was a negative correlation between the level of circulating exosomal miRNA-146a expression and the level of TRAF6 mRNA (r=-0.30, p=0.049). Moreover, there were correlations between circulating exosomal miRNA-146a and disease severity such as SLE Disease Activity Index 2000 score, anti-double-stranded DNA antibody and proteinuria (urine protein-creatinine ratio), respectively. Therefore, increasing the level of circulating exosomal miRNA-146a, which might control TRAF6 mRNA expression, could have an effect on the production of inflammatory cytokines.

CONCLUSION: This suggests that miRNA-146a might serve as a non-invasive biomarker to evaluate the response to treatment in patients with juvenile lupus nephritis.

PMID:38355214 | DOI:10.1136/lupus-2023-001078

Elife. 2024 Feb 14;12:RP87356. doi: 10.7554/eLife.87356.

ABSTRACT

Neurostimulation of the hippocampal formation has shown promising results for modulating memory but the underlying mechanisms remain unclear. In particular, the effects on hippocampal theta-nested gamma oscillations and theta phase reset, which are both crucial for memory processes, are unknown. Moreover, these effects cannot be investigated using current computational models, which consider theta oscillations with a fixed amplitude and phase velocity. Here, we developed a novel computational model that includes the medial septum, represented as a set of abstract Kuramoto oscillators producing a dynamical theta rhythm with phase reset, and the hippocampal formation, composed of biophysically realistic neurons and able to generate theta-nested gamma oscillations under theta drive. We showed that, for theta inputs just below the threshold to induce self-sustained theta-nested gamma oscillations, a single stimulation pulse could switch the network behavior from non-oscillatory to a state producing sustained oscillations. Next, we demonstrated that, for a weaker theta input, pulse train stimulation at the theta frequency could transiently restore seemingly physiological oscillations. Importantly, the presence of phase reset influenced whether these two effects depended on the phase at which stimulation onset was delivered, which has practical implications for designing neurostimulation protocols that are triggered by the phase of ongoing theta oscillations. This novel model opens new avenues for studying the effects of neurostimulation on the hippocampal formation. Furthermore, our hybrid approach that combines different levels of abstraction could be extended in future work to other neural circuits that produce dynamical brain rhythms.

PMID:38354040 | DOI:10.7554/eLife.87356

Neuropsychol Dev Cogn B Aging Neuropsychol Cogn. 2024 Feb 14:1-32. doi: 10.1080/13825585.2024.2315774. Online ahead of print.

ABSTRACT

Word-finding difficulty (WFD) is a common cognitive complaint in aging, manifesting both in natural speech and in controlled laboratory tests. Various theories of cognitive aging have addressed WFD, and understanding its underlying mechanisms can help to clarify whether it has diagnostic value for neurodegenerative disease. Two influential "information-universal" theories attribute it to rather broad changes in cognition. The processing speed theory posits a general slowdown of all cognitive processes, while the inhibitory deficit hypothesis (IDH) predicts a specific problem in suppressing irrelevant information. One "information specific" theory of language production, the transmission deficit hypothesis (TDH), posits a breakdown in retrieval of phonological word forms from a corresponding lemma. To adjudicate between these accounts, we administered an online gamified covert naming task featuring picture-word interference (PWI), previously validated to elicit similar semantic interference and phonological facilitation effects as overt naming tasks. 125 healthy adults aged 18 to 85 completed the task, along with a battery of executive function tasks and a naturalistic speech sample to quantify WFD in connected speech. PWI effects provided strong support for the TDH but limited support for IDH, in that semantic interference increased and phonological facilitation decreased across the lifespan. However, neither of these effects on single-word retrieval associated with WFD measured in connected speech. Rather, overall reaction time for word retrieval (controlling for psychomotor slowing) was the best predictor of spontaneous WFD and executive function decline, suggesting processing speed as the key factor, and that verbal reaction time may be an important clinical measure.

PMID:38353604 | DOI:10.1080/13825585.2024.2315774

J Pharm Anal. 2024 Jan;14(1):140-148. doi: 10.1016/j.jpha.2023.10.004. Epub 2023 Oct 18.

ABSTRACT

Acylcarnitines are metabolic intermediates of fatty acids and branched-chain amino acids having vital biofunctions and pathophysiological significances. Here, we developed a high-throughput method for quantifying hundreds of acylcarnitines in one run using ultrahigh performance liquid chromatography and tandem mass spectrometry (UPLC-MS/MS). This enabled simultaneous quantification of 1136 acylcarnitines (C0-C26) within 10-min with good sensitivity (limit of detection < 0.7 fmol), linearity (correlation coefficient > 0.992), accuracy (relative error < 20%), precision (coefficient of variation (CV), CV < 15%), stability (CV < 15%), and inter-technician consistency (CV < 20%, n = 6). We also established a quantitative structure-retention relationship (goodness of fit > 0.998) for predicting retention time (tR) of acylcarnitines with no standards and built a database of their multiple reaction monitoring parameters (tR, ion-pairs, and collision energy). Furthermore, we quantified 514 acylcarnitines in human plasma and urine, mouse kidney, liver, heart, lung, and muscle. This provides a rapid method for quantifying acylcarnitines in multiple biological matrices.

PMID:38352947 | PMC:PMC10859589 | DOI:10.1016/j.jpha.2023.10.004

Front Immunol. 2024 Jan 30;15:1297955. doi: 10.3389/fimmu.2024.1297955. eCollection 2024.

ABSTRACT

Mycobacterium avium subspecies paratuberculosis (MAP) is a global concern in modern livestock production worldwide. The available vaccines against paratuberculosis do not offer optimal protection and interfere with the diagnosis of bovine tuberculosis. The aim of this study was to identify immunogenic MAP-specific peptides that do not interfere with the diagnosis of bovine tuberculosis. Initially, 119 peptides were selected by either (1) identifying unique MAP peptides that were predicted to bind to bovine major histocompatibility complex class II (MHC-predicted peptides) or (2) selecting hydrophobic peptides unique to MAP within proteins previously shown to be immunogenic (hydrophobic peptides). Subsequent testing of peptide-specific CD4+ T-cell lines from MAP-infected, adult goats vaccinated with peptides in cationic liposome adjuvant pointed to 23 peptides as being most immunogenic. These peptides were included in a second vaccine trial where three groups of eight healthy goat kids were vaccinated with 14 MHC-predicted peptides, nine hydrophobic peptides, or no peptides in o/w emulsion adjuvant. The majority of the MHC-predicted (93%) and hydrophobic peptides (67%) induced interferon-gamma (IFN-γ) responses in at least one animal. Similarly, 86% of the MHC-predicted and 89% of the hydrophobic peptides induced antibody responses in at least one goat. The immunization of eight healthy heifers with all 119 peptides formulated in emulsion adjuvant identified more peptides as immunogenic, as peptide specific IFN-γ and antibody responses in at least one heifer was found toward 84% and 24% of the peptides, respectively. No peptide-induced reactivity was found with commercial ELISAs for detecting antibodies against Mycobacterium bovis or MAP or when performing tuberculin skin testing for bovine tuberculosis. The vaccinated animals experienced adverse reactions at the injection site; thus, it is recommend that future studies make improvements to the vaccine formulation. In conclusion, immunogenic MAP-specific peptides that appeared promising for use in a vaccine against paratuberculosis without interfering with surveillance and trade tests for bovine tuberculosis were identified by in silico analysis and ex vivo generation of CD4+ T-cell lines and validated by the immunization of goats and cattle. Future studies should test different peptide combinations in challenge trials to determine their protective effect and identify the most MHC-promiscuous vaccine candidates.

PMID:38352876 | PMC:PMC10861761 | DOI:10.3389/fimmu.2024.1297955

Heliyon. 2024 Feb 2;10(3):e25487. doi: 10.1016/j.heliyon.2024.e25487. eCollection 2024 Feb 15.

ABSTRACT

Given emerging food supply challenges for the world population, Genetic Modified Organisms (GMOs) are referred to as a solution to the upcoming food security crisis. Besides technological advancement, other significant components such as public Awareness play an important role in national and international scientific regulations. Towards this, this study evaluated Tehranian consumers' Awareness (a sample including 946 respondents) about GMOs' risks and benefits, trust in governmental regulation, and the ways to obtain information about GMOs. Specific questionnaires were designed and distributed among participants in four districts in Tehran, and the collected data were used to conduct descriptive and inferential statistics by applying the ANOVA test. The Findings showed that 39 % with a p-value <0.01 of the public is unaware of GMOs in Tehran despite 20 years of commercialization, consumption, and controversial debate about GMOs in media and social networks. Therefore, the goals of public Awareness of science concerning biotechnology have not been met yet. Based on these findings, it can be concluded that public Awareness is not a crucial component in biotechnology advancement, and the other factors, including policymakers' desire, may have more weight.

PMID:38352779 | PMC:PMC10862675 | DOI:10.1016/j.heliyon.2024.e25487

Front Genet. 2024 Jan 30;15:1371473. doi: 10.3389/fgene.2024.1371473. eCollection 2024.

NO ABSTRACT

PMID:38352165 | PMC:PMC10861723 | DOI:10.3389/fgene.2024.1371473

EMBO Rep. 2024 Feb 13. doi: 10.1038/s44319-024-00076-y. Online ahead of print.

ABSTRACT

Membrane adenylyl cyclase AC8 is regulated by G proteins and calmodulin (CaM), mediating the crosstalk between the cAMP pathway and Ca2+ signalling. Despite the importance of AC8 in physiology, the structural basis of its regulation by G proteins and CaM is not well defined. Here, we report the 3.5 Å resolution cryo-EM structure of the bovine AC8 bound to the stimulatory Gαs protein in the presence of Ca2+/CaM. The structure reveals the architecture of the ordered AC8 domains bound to Gαs and the small molecule activator forskolin. The extracellular surface of AC8 features a negatively charged pocket, a potential site for unknown interactors. Despite the well-resolved forskolin density, the captured state of AC8 does not favour tight nucleotide binding. The structural proteomics approaches, limited proteolysis and crosslinking mass spectrometry (LiP-MS and XL-MS), allowed us to identify the contact sites between AC8 and its regulators, CaM, Gαs, and Gβγ, as well as to infer the conformational changes induced by these interactions. Our results provide a framework for understanding the role of flexible regions in the mechanism of AC regulation.

PMID:38351373 | DOI:10.1038/s44319-024-00076-y

Biotechnol Biofuels Bioprod. 2024 Feb 13;17(1):23. doi: 10.1186/s13068-024-02469-6.

ABSTRACT

BACKGROUND: Marine cyanobacteria offer many sustainability advantages, such as the ability to fix atmospheric CO2, very fast growth and no dependence on freshwater for culture. Cyanobacterial biomass is a rich source of sugars and proteins, two essential nutrients for culturing any heterotroph. However, no previous study has evaluated their application as a feedstock for fungal bioprocesses.

RESULTS: In this work, we cultured the marine cyanobacterium Synechococcus sp. PCC 7002 in a 3-L externally illuminated bioreactor with working volume of 2 L with a biomass productivity of ~ 0.8 g L-1 day-1. Hydrolysis of the biomass with acids released proteins and hydrolyzed glycogen while hydrolysis of the biomass with base released only proteins but did not hydrolyze glycogen. Among the different acids tested, treatment with HNO3 led to the highest release of proteins and glucose. Cyanobacterial biomass hydrolysate (CBH) prepared in HNO3 was used as a medium to produce cellulase enzyme by the Penicillium funiculosum OAO3 strain while CBH prepared in HCl and treated with charcoal was used as a medium for citric acid by Aspergillus tubingensis. Approximately 50% higher titers of both products were obtained compared to traditional media.

CONCLUSIONS: These results show that the hydrolysate of marine cyanobacteria is an effective source of nutrients/proteins for fungal bioprocesses.

PMID:38350992 | PMC:PMC10863111 | DOI:10.1186/s13068-024-02469-6

Comput Biol Chem. 2024 Feb 7;109:108022. doi: 10.1016/j.compbiolchem.2024.108022. Online ahead of print.

ABSTRACT

Studying gene regulatory networks associated with cancer provides valuable insights for therapeutic purposes, given that cancer is fundamentally a genetic disease. However, as the number of genes in the system increases, the complexity arising from the interconnections between network components grows exponentially. In this study, using Boolean logic to adjust the existing relationships between network components has facilitated simplifying the modeling process, enabling the generation of attractors that represent cell phenotypes based on breast cancer RNA-seq data. A key therapeutic objective is to guide cells, through targeted interventions, to transition from the current cancer attractor to a physiologically distinct attractor unrelated to cancer. To achieve this, we developed a computational method that identifies network nodes whose inhibition can facilitate the desired transition from one tumor attractor to another associated with apoptosis, leveraging transcriptomic data from cell lines. To validate the model, we utilized previously published in vitro experiments where the downregulation of specific proteins resulted in cell growth arrest and death of a breast cancer cell line. The method proposed in this manuscript combines diverse data sources, conducts structural network analysis, and incorporates relevant biological knowledge on apoptosis in cancer cells. This comprehensive approach aims to identify potential targets of significance for personalized medicine.

PMID:38350182 | DOI:10.1016/j.compbiolchem.2024.108022

Anal Chem. 2024 Feb 13. doi: 10.1021/acs.analchem.3c04654. Online ahead of print.

ABSTRACT

The accurate prediction of tandem mass spectra from molecular structures has the potential to unlock new metabolomic discoveries by augmenting the community's libraries of experimental reference standards. Cheminformatic spectrum prediction strategies use a "bond-breaking" framework to iteratively simulate mass spectrum fragmentations, but these methods are (a) slow due to the need to exhaustively and combinatorially break molecules and (b) inaccurate as they often rely upon heuristics to predict the intensity of each resulting fragment; neural network alternatives mitigate computational cost but are black-box and not inherently more accurate. We introduce a physically grounded neural approach that learns to predict each breakage event and score the most relevant subset of molecular fragments quickly and accurately. We evaluate our model by predicting spectra from both public and private standard libraries, demonstrating that our hybrid approach offers state-of-the-art prediction accuracy, improved metabolite identification from a database of candidates, and higher interpretability when compared to previous breakage methods and black-box neural networks. The grounding of our approach in physical fragmentation events shows especially great promise for elucidating natural product molecules with more complex scaffolds.

PMID:38349970 | DOI:10.1021/acs.analchem.3c04654

Cell Rep. 2024 Feb 12;43(2):113774. doi: 10.1016/j.celrep.2024.113774. Online ahead of print.

ABSTRACT

Long interspersed nuclear element-1 (L1 or LINE-1) is a highly abundant mobile genetic element in both humans and mice, comprising almost 20% of each genome. L1s are silenced by several mechanisms, as their uncontrolled expression has the potential to induce genomic instability. However, L1s are paradoxically expressed at high levels in differentiating neural progenitor cells. Using in vitro and in vivo techniques to modulate L1 expression, we report that L1s play a critical role in both human and mouse brain development by regulating the rate of neural differentiation in a reverse-transcription-independent manner.

PMID:38349791 | DOI:10.1016/j.celrep.2024.113774

STAR Protoc. 2024 Feb 12;5(1):102880. doi: 10.1016/j.xpro.2024.102880. Online ahead of print.

ABSTRACT

Type 2 diabetes (T2D) is a multifactorial disease that slowly and inconspicuously progresses over years. Here, we present a protocol for analyzing slow progression dynamics of T2D with obesity. We describe steps for using software to exploit the differences between the timescales of the metabolic variables and using numerical continuation and bifurcation analysis. We detail procedures to analyze bi-stable system dynamics and identify the thresholds that separate healthy and diabetic states. For complete details on the use and execution of this protocol, please refer to Yildirim et al. (2023).1.

PMID:38349789 | DOI:10.1016/j.xpro.2024.102880

STAR Protoc. 2024 Feb 12;5(1):102876. doi: 10.1016/j.xpro.2024.102876. Online ahead of print.

ABSTRACT

Here, we present a protocol for estimating nuclear transport parameters in single cells. We describe steps for performing four consecutive fluorescence recovery after photobleaching experiments, fitting the obtained data to an ordinary differential equations model, and statistical analysis of the fittings using a specialized R package. This protocol permits the estimation of import and export rates, nuclear or cytosolic fixed fractions, and total number of molecules. For complete details on the use and execution of this protocol, please refer to Durrieu et al.1.

PMID:38349788 | DOI:10.1016/j.xpro.2024.102876

Cytotherapy. 2024 Feb 13:S1465-3249(24)00046-X. doi: 10.1016/j.jcyt.2024.01.014. Online ahead of print.

ABSTRACT

BACKGROUND AIMS: Age-related macular degeneration (AMD) is the most common cause of blindness in elderly patients within developed countries, affecting more than 190 million worldwide. In AMD, the retinal pigment epithelial (RPE) cell layer progressively degenerates, resulting in subsequent loss of photoreceptors and ultimately vision. There is currently no cure for AMD, but therapeutic strategies targeting the complement system are being developed to slow the progression of the disease.

METHODS: Replacement therapy with pluripotent stem cell-derived (hPSC) RPEs is an alternative treatment strategy. A cell therapy product must be produced in accordance with Good Manufacturing Practices at a sufficient scale to facilitate extensive pre-clinical and clinical testing. Cryopreservation of the final cell product is therefore highly beneficial, as the manufacturing, pre-clinical and clinical testing can be separated in time and location.

RESULTS: We found that mature hPSC-RPE cells do not survive conventional cryopreservation techniques. However, replating the cells 2-5 days before cryopreservation facilitates freezing. The replated and cryopreserved hPSC-RPE cells maintained their identity, purity and functionality as characteristic RPEs, shown by cobblestone morphology, pigmentation, transcriptional profile, RPE markers, transepithelial resistance and pigment epithelium-derived factor secretion. Finally, we showed that the optimal replating time window can be tracked noninvasively by following the change in cobblestone morphology.

CONCLUSIONS: The possibility of cryopreserving the hPSC-RPE product has been instrumental in our efforts in manufacturing and performing pre-clinical testing with the aim for clinical translation.

PMID:38349309 | DOI:10.1016/j.jcyt.2024.01.014

mBio. 2024 Feb 13:e0340923. doi: 10.1128/mbio.03409-23. Online ahead of print.

ABSTRACT

Candida albicans can cause mucosal infections in humans. This includes oropharyngeal candidiasis, which is commonly observed in human immunodeficiency virus infected patients, and vulvovaginal candidiasis (VVC), which is the most frequent manifestation of candidiasis. Epithelial cell invasion by C. albicans hyphae is accompanied by the secretion of candidalysin, a peptide toxin that causes epithelial cell cytotoxicity. During vaginal infections, candidalysin-driven tissue damage triggers epithelial signaling pathways, leading to hyperinflammatory responses and immunopathology, a hallmark of VVC. Therefore, we proposed blocking candidalysin activity using nanobodies to reduce epithelial damage and inflammation as a therapeutic strategy for VVC. Anti-candidalysin nanobodies were confirmed to localize around epithelial-invading C. albicans hyphae, even within the invasion pocket where candidalysin is secreted. The nanobodies reduced candidalysin-induced damage to epithelial cells and downstream proinflammatory responses. Accordingly, the nanobodies also decreased neutrophil activation and recruitment. In silico mathematical modeling enabled the quantification of epithelial damage caused by candidalysin under various nanobody dosing strategies. Thus, nanobody-mediated neutralization of candidalysin offers a novel therapeutic approach to block immunopathogenic events during VVC and alleviate symptoms.IMPORTANCEWorldwide, vaginal infections caused by Candida albicans (VVC) annually affect millions of women, with symptoms significantly impacting quality of life. Current treatments are based on anti-fungals and probiotics that target the fungus. However, in some cases, infections are recurrent, called recurrent VVC, which often fails to respond to treatment. Vaginal mucosal tissue damage caused by the C. albicans peptide toxin candidalysin is a key driver in the induction of hyperinflammatory responses that fail to clear the infection and contribute to immunopathology and disease severity. In this pre-clinical evaluation, we show that nanobody-mediated candidalysin neutralization reduces tissue damage and thereby limits inflammation. Implementation of candidalysin-neutralizing nanobodies may prove an attractive strategy to alleviate symptoms in complicated VVC cases.

PMID:38349176 | DOI:10.1128/mbio.03409-23

mSystems. 2024 Feb 13:e0125723. doi: 10.1128/msystems.01257-23. Online ahead of print.

ABSTRACT

Limosilactobacillus reuteri, a probiotic microbe instrumental to human health and sustainable food production, adapts to diverse environmental shifts via dynamic gene expression. We applied the independent component analysis (ICA) to 117 RNA-seq data sets to decode its transcriptional regulatory network (TRN), identifying 35 distinct signals that modulate specific gene sets. Our findings indicate that the ICA provides a qualitative advancement and captures nuanced relationships within gene clusters that other methods may miss. This study uncovers the fundamental properties of L. reuteri's TRN and deepens our understanding of its arginine metabolism and the co-regulation of riboflavin metabolism and fatty acid conversion. It also sheds light on conditions that regulate genes within a specific biosynthetic gene cluster and allows for the speculation of the potential role of isoprenoid biosynthesis in L. reuteri's adaptive response to environmental changes. By integrating transcriptomics and machine learning, we provide a system-level understanding of L. reuteri's response mechanism to environmental fluctuations, thus setting the stage for modeling the probiotic transcriptome for applications in microbial food production.IMPORTANCEWe have studied Limosilactobacillus reuteri, a beneficial probiotic microbe that plays a significant role in our health and production of sustainable foods, a type of foods that are nutritionally dense and healthier and have low-carbon emissions compared to traditional foods. Similar to how humans adapt their lifestyles to different environments, this microbe adjusts its behavior by modulating the expression of genes. We applied machine learning to analyze large-scale data sets on how these genes behave across diverse conditions. From this, we identified 35 unique patterns demonstrating how L. reuteri adjusts its genes based on 50 unique environmental conditions (such as various sugars, salts, microbial cocultures, human milk, and fruit juice). This research helps us understand better how L. reuteri functions, especially in processes like breaking down certain nutrients and adapting to stressful changes. More importantly, with our findings, we become closer to using this knowledge to improve how we produce more sustainable and healthier foods with the help of microbes.

PMID:38349131 | DOI:10.1128/msystems.01257-23

Front Genet. 2024 Jan 29;15:1270387. doi: 10.3389/fgene.2024.1270387. eCollection 2024.

ABSTRACT

Preserving data privacy is an important concern in the research use of patient data. The DataSHIELD suite enables privacy-aware advanced statistical analysis in a federated setting. Despite its many applications, it has a few open practical issues: the complexity of hosting a federated infrastructure, the performance penalty imposed by the privacy-preserving constraints, and the ease of use by non-technical users. In this work, we describe a case study in which we review different breast cancer classifiers and report our findings about the limits and advantages of such non-disclosive suite of tools in a realistic setting. Five independent gene expression datasets of breast cancer survival were downloaded from Gene Expression Omnibus (GEO) and pooled together through the federated infrastructure. Three previously published and two newly proposed 5-year cancer-free survival risk score classifiers were trained in a federated environment, and an additional reference classifier was trained with unconstrained data access. The performance of these six classifiers was systematically evaluated, and the results show that i) the published classifiers do not generalize well when applied to patient cohorts that differ from those used to develop them; ii) among the methods we tried, the classification using logistic regression worked better on average, closely followed by random forest; iii) the unconstrained version of the logistic regression classifier outperformed the federated version by 4% on average. Reproducibility of our experiments is ensured through the use of VisualSHIELD, an open-source tool that augments DataSHIELD with new functions, a standardized deployment procedure, and a simple graphical user interface.

PMID:38348453 | PMC:PMC10859452 | DOI:10.3389/fgene.2024.1270387

J Mol Model. 2024 Feb 13;30(3):68. doi: 10.1007/s00894-024-05875-7.

ABSTRACT

CONTEXT: Adipose triglyceride lipase (ATGL), a key enzyme responsible for lipolysis, catalyzes the first step of lipolysis and converts triglycerides to diacylglycerols and free fatty acids (FFA). Our previous work suggested that phillyrin treatment improves insulin resistance in HFD-fed mice, which was associated with ATGL inhibition. In this study, using docking simulation, we explored the binding pose of phillyrin and atglistatin (a mouse ATGL inhibitor) to ATGL in mouse. From the docking results, the interactions with Ser47 and Asp166 were speculated to have caused phillyrin to inhibit ATGL in mice. Further, molecular dynamics simulation of 100 ns and MM-GBSA were conducted for the protein-ligand complex, which indicated that the system was stable and that phillyrin displayed a better affinity to ATGL than did atglistatin throughout the simulation period. Moreover, the results of pharmacological validation were consistent with those of the in silico simulations. In summary, our study illustrates the potential of molecular docking to accurately predict the binding protein produced by AlphaFold and suggests that phillyrin is a potential small molecule that targets and inhibits ATGL enzymatic activity.

METHODS: The ATGL-predicted protein structure, verified by PROCHECK, was determined using AlphaFold. Molecular docking, molecular dynamics simulation, and prime molecular mechanic-generalized born surface area were performed using LigPrep, Desmond, and prime MM-GBSA modules of Schrödinger software release 2021-2, respectively. For pharmacological validation, immunoblotting was performed to assess ATGL protein expression. The fluorescence intensity and glycerol concentration were quantified to evaluate the efficiency of phillyrin in inhibiting ATGL.

PMID:38347278 | DOI:10.1007/s00894-024-05875-7

Nat Cell Biol. 2024 Feb 12. doi: 10.1038/s41556-024-01354-6. Online ahead of print.

ABSTRACT

Clathrin-mediated endocytosis is an essential cellular internalization pathway involving the dynamic assembly of clathrin and accessory proteins to form membrane-bound vesicles. The evolutionarily ancient TSET-TPLATE complex (TPC) plays an essential, but ill-defined role in endocytosis in plants. Here we show that two highly disordered TPC subunits, AtEH1 and AtEH2, function as scaffolds to drive biomolecular condensation of the complex. These condensates specifically nucleate on the plasma membrane through interactions with anionic phospholipids, and facilitate the dynamic recruitment and assembly of clathrin, as well as early- and late-stage endocytic accessory proteins. Importantly, condensation promotes ordered clathrin assemblies. TPC-driven biomolecular condensation thereby facilitates dynamic protein assemblies throughout clathrin-mediated endocytosis. Furthermore, we show that a disordered region of AtEH1 controls the material properties of endocytic condensates in vivo. Alteration of these material properties disturbs the recruitment of accessory proteins, influences endocytosis dynamics and impairs plant responsiveness. Our findings reveal how collective interactions shape endocytosis.

PMID:38347182 | DOI:10.1038/s41556-024-01354-6