Biology subjects

Ivanov, A.

Publications and source records attributed to Ivanov, A..

At least 19 recordsLinked to original sources

bioRxiv2026

Intermolecular interactions of CYP3A enzymes and cytosolic sulfotransferases (SULT1E1, SULT2A1)

Cytochrome P450 enzymes of the CYP3A subfamily (CYP3A4 and CYP3A5) and cytosolic sulfotransferases (SULT1E1 and SULT2A1) play key roles in the sequential oxidative and conjugative metabolism of steroid hormones and xenobiotics. While their coordinated transcriptional regulation and co-expression patterns are well documented, direct physical interactions between these phase I and phase II enzymes have not previously been demonstrated. In this study, we combined systems biology analysis, SPR biosensing, activity assays, and AlphaFold2 modeling to investigate PPIs between CYP3A4/3A5 and SULT1E1/2A1. STRINGdb and transcriptomic data suggested potential associations, but direct evidence was lacking. SPR confirmed high-affinity binding (Kd 0.29-1.8 nM); the SULT2A1-CYP3A5 pair gave 0.29 nM, while SULT2A1-CYP3A4 gave 1.8 nM. Notably, the presence of the sulfo group donor SULT PAPS, had minimal effect on binding affinity. Functional assays demonstrated that SULTs slightly inhibited CYP3A4 activity while moderately enhancing CYP3A5 activity in the presence of SULT1E1, both with and without PAPS, whereas the addition of CYPs leads to selectively increase in SULT2A1 activity in vitro by 38% (CYP3A4) and 108% (CYP3A5), without affecting SULT1E1. AlphaFold2 models revealed stable interfaces (715-1856 square angstroms) with 10-16 H-bonds and salt bridges. The results of our in silico tunnel analysis suggested a plausible alignment of substrate exit channels from CYPs with entry pathways to SULTs, supporting a mechanistic basis for substrate channeling in CYP-SULT complexes. These results indicate that the enzymes may form functional multienzyme complexes, offering new insights into coordinated steroid/xenobiotic metabolism and drug clearance.

molecular biology↗

bioRxiv2026

HAP40 functions as a proteostasis regulator by controlling huntingtin interactions and its release into the extracellular space

Huntingtin-associated protein 40 (HAP40) forms a stable protein complex with huntingtin (HTT). Its cellular function and how HAP40 loss influences mutant HTT (mHTT) abundance and pathobiology are currently unclear. Here, using diverse cellular models and OMICs methods, we demonstrate that HAP40 is an obligate interaction partner of full-length HTT and through its binding controls the abundance of HTT-associated proteins, indicating that it functions as HTT interaction regulatory unit. Also, loss of HAP40 in mHTT-expressing striatal cells impairs autophagosome-lysosome flux, triggers massive transcriptional dysregulation, including the activation of the CLEAR network, demonstrating that it functions as proteostasis regulator that acts on quality control pathways. Finally, mHTT-expressing cells lacking HAP40 showed increased secretion of mHTT through the ER-to-Golgi route, indicating that striatal cells reduce intracellular mHTT-induced proteotoxicity through activation of secretory pathways. Together, these results establish HAP40 as a critical proteostasis regulator that through controlling HTT interactions maintains cellular homeostasis.

cell biology↗

bioRxiv2026

Diverse Ligands for Mycobacterial CYP124 Identified from Plant and Marine Compounds

Tuberculosis persists as a major global health threat, significantly exacerbated by the rise of drug-resistant strains. Cytochrome P450 of 124 family CYP124 from Mycobacterium tuberculosis (CYP124), implicated in host sterol metabolism and bacterial virulence, represents an emerging and promising therapeutic target. While its precise physiological role was previously debated, CYP124s confirmed ability to metabolize immunomodulatory host sterols underscores its pharmacological relevance. Utilizing surface plasmon resonance binding assays and UV-Vis spectral titration screening, we identified nine novel non-azole ligands for CYP124 from a library of 32 plant-derived and marine natural compounds. Among these hits, (25S)-5-cholestane-3{beta},4{beta},6,7,8,15{beta},16{beta},26-octaol (termed 15{beta}-octaol) and henricioside H2 (HD-4) induced characteristic difference spectra and formed long-lived inhibitory complexes with CYP124, exhibiting dissociation half-lives of 181 min and 65 min, respectively. However, their inhibitory potency was moderate, with IC50 values of approximately 86 M for 15{beta}-octaol and exceeding 100 M for HD-4. Complementary in silico molecular docking and analysis identified key conserved hydrophobic residues within the CYP124 active site crucial for ligand binding, suggesting a shared pharmacophore. Furthermore, structural similarity analysis revealed that 37 human endogenous metabolites, including known immunoregulatory sterols, bear resemblance to the identified CYP124 ligands. This finding points towards a potential sterol-mediated interplay at the host-pathogen interface. Collectively, these results provide a foundation for the future development of mechanism-based CYP124 inhibitors as therapeutics against multidrug-resistant tuberculosis.

biochemistry↗

bioRxiv2025

Tracking gene expression of single mitochondria in live neurons using nanotweezers

Neurons are highly polarised cells that depend on mitochondria for energy and signalling homeostasis. Importantly, energy and signalling requirements vary considerably across individual neurons both spatially and temporally. Therefore, to fully understand neuronal mitochondria, methods are needed to analyse mitochondria in live cells over time. The nanotweezer, a minimally invasive single-cell sampling technique, enables precise extraction a individual mitochondria from defined subcellular locations. Here, we combine single-mitochondrial extraction from live neurons with mitochondrial gene expression tracking and mtDNA profiling. By tracking mitochondrial gene expression in the same neurons over time, we reveal a downregulation of mitochondrial genes MT-ND1 and MT-ATP6 following exposure to -synuclein aggregates, independent of the proximity of the aggregates to the sampled mitochondria. Our approach provides precise, dynamic measurements of mitochondrial composition and gene expression in vivo at single-organelle resolution, enabling mechanistic studies of neuronal mitochondrial heterogeneity and its perturbation in models of neurodegeneration.

neuroscience↗

bioRxiv2025

Different data analysis models for detecting miRNAs using nanopores

Nanopore sensors offer exceptional sensitivity for detecting single molecules, making them ideal for early disease diagnostics. In this study, we present a multiplexed nanopore-based assay that combines DNA-barcoded probes with advanced computational analysis to detect microRNAs (miRNAs) with high specificity and quantitative accuracy. Each probe binds selectively to its target biomarker and generates a characteristic delay in the ionic current signal upon translocation through the nanopore, enabling label-free detection. We evaluated three analytical strategies for classifying delayed versus non-delayed events: (1) moving standard deviation (MSD), (2) spectral entropy (SE), and (3) a convolutional neural network (CNN). While MSD and SE rely on manually defined thresholds and exhibit limited sensitivity, the CNN model, trained on image representations of raw current traces, achieved near-perfect classification performance across all metrics. Grad-CAM visualisation confirmed that the CNN focused on biophysically relevant signal regions, enhancing interpretability and generalisability. All methods produced sigmoidal concentration-response curves consistent with expected binding kinetics, and nanopore-derived delay metrics closely matched RT-qPCR validation data. All three methods were capable of distinguishing between signal classes; however, the CNN model demonstrated superior sensitivity and robustness. This work highlights the importance of data interpretation in nanopore sensing and presents a comparative framework for binary event classification. The findings pave the way for the development of machine learning-driven nanopore diagnostics capable of detecting diverse biomarker types at the single-molecule level.

biophysics↗

bioRxiv2025

Degeneracy in Astrocytic Potassium Buffering: A Minimal Model Capturing the Interplay Between Local and Long-Range Mechanisms

Maintaining extracellular potassium (K+) homeostasis is critical for neuronal function, and astrocytes achieve this through a combination of local uptake and long-range spatial buffering. While degeneracy--the ability of different mechanisms to achieve the same function--is a fundamental property of biological systems, its role in astrocytic potassium buffering has remained unexplored. We present a minimal mathematical model that identifies essential buffering mechanisms while ensuring tractability and interpretability. Incorporating Kir channels and gap junction coupling, the model reproduces experimentally observed astrocyte membrane dynamics under various pharmacological conditions Parameter exploration reveals two levels of degeneracy. At the single-cell level, multiple parameter configurations yield similar membrane potential dynamics, indicating flexibility in local and spatial buffering contributions. At the functional level, despite variations in astrocyte morphology and buffering efficiency, homeostasis of extracellular K+ is restored, demonstrating homeostatic degeneracy. These findings highlight the robustness of astrocytic potassium regulation, showing that diverse buffering strategies ensure stability. Our work establishes a theoretical framework for understanding how astrocytic heterogeneity contributes to robust ionic homeostasis and offers perspectives for studying pathological conditions where buffering mechanisms are impaired.

neuroscience↗

bioRxiv2025

MetaFX: feature extraction from whole-genome metagenomic sequencing data

MotivationMicrobial communities consist of thousands of microorganisms and viruses and have a tight connection with an environment, such as gut microbiota modulation of host body metabolism. However, the direct relationship between the presence of certain microorganism and host state often remains unknown. Toolkits using reference-based approaches are limited to microbes present in databases. Reference-free methods often require enormous resources for metagenomic assembly or results in many poorly interpretable features based on k-mers. ResultsHere we present MetaFX - an open-source library for feature extraction from whole-genome metagenomic sequencing data and classification of groups of samples. Using a large volume of metagenomic samples deposited in databases, MetaFX compares samples grouped by metadata criteria (e.g. disease, treatment, etc) and constructs genomic features distinct for certain types of communities. Features constructed based on statistical k-mer analysis and de Bruijn graphs partition. Those features are used in machine learning models for classification of novel samples. Extracted features can be visualised on de Bruijn graphs and annotated for providing biological insights. We demonstrate the utility of MetaFX by building classification models for 590 human gut samples with inflammatory bowel disease. Our results outperform the previous research disease prediction accuracy up to 17%, and improves classification results compared to taxonomic analysis by 9 {+/-} 10% on average. AvailabilityMetaFX is a feature extraction toolkit applicable for metagenomic datasets analysis and samples classification. The source code, test data, and relevant information for MetaFX are freely accessible at https://github.com/ctlab/metafx under the MIT License. Contactabivanov@itmo.ru

bioinformatics↗

bioRxiv2025

Upstream open reading frame inactivation augments GATA4 translation and cardiomyocyte hypertrophy in mice

Upstream open reading frames (uORFs) are short peptide-encoding sequences located in the 5 untranslated region (5 UTR) of mRNAs, enabling translational repression of main (m)ORFs. While uORFs are found in [~]50% of mRNAs in humans, our understanding of their biological function remains limited. This study aims to elucidate the role of the uORF in the 5 UTR of the Gata4 (GATA binding protein 4) gene in cardiac biology by inactivating its start codon ({Delta}uORF) in the mouse genome. Our investigation reveals that mice with Gata4 uORF inactivation manifest spontaneous cardiac hypertrophy without apparent fibrosis as they age. Utilizing single-nucleus RNA sequencing (snRNA-seq), we uncovered significant transcriptional variations between wild-type (WT) and {Delta}ORF mice. Notably, mRNAs associated with sarcomeres and contractile functions show heightened expression levels, reflecting the hypertrophic phenotype. Notably, at least nine upregulated genes are GATA4-bound targets in mouse ventricles. Functional assessments of isolated primary adult cardiomyocytes confirmed enhanced hypertrophy and contractility in {Delta}ORF mice. Additionally, we employed single-nucleus transposase-accessible chromatin (snATAC)-seq to investigate changes in chromatin accessibility. Our results indicated increased accessibility within specific transcription-regulatory elements linked to elevated gene transcription. These putative cis-regulatory elements (pCREs) are significantly enriched in MEF2 (myocyte enhancer factor 2) binding motifs. In vitro luciferase reporter assays further supported the regulatory potential of three of these pCREs, highlighting their role in the transcriptional enhancement of three GATA4 target genes bound by MEF2 and GATA4. These findings illuminate the role of uORF in negatively regulating GATA4 protein expression and cardiomyocyte hypertrophy at the organismal level and provide a novel therapeutic target for cardiac pathogenesis.

biochemistry↗

bioRxiv2025

Assessing PARP trapping dynamics in ovarian cancer using a CRISPR-engineered FRET biosensor

Poly(ADP-ribose) polymerase inhibitors (PARPi) have revolutionised the treatment of ovarian high grade serous carcinoma (HGSC), especially those with defective homologous recombination. However, the emergence of resistance poses a critical challenge, as over 50% of patients relapse within three years. The mechanisms underlying changes in PARP trapping, a central aspect of PARPi efficacy, are not well understood due to limitations in current experimental methodologies. Existing techniques lack resolution and throughput, impeding efforts to study PARP trapping dynamics over time with single-cell resolution. Effective tools to study PARP trapping in live cells are urgently needed to elucidate resistance mechanisms and inform therapeutic strategies. To address this, we used CRISPR-Cas9 gene editing to dual-label endogenous PARP1 with EGFP and mCherryFP in OVCAR4 cells to develop a novel intramolecular FRET-based biosensor that enables real-time, single-cell visualization of PARP trapping dynamics in live cells. High-content fluorescence lifetime imaging microscopy (FLIM) revealed dose dependent PARP trapping upon exposure to PARP inhibitor and distinguished between the trapping efficiencies of four different PARPi (veliparib, olaparib, rucaparib, talazoparib). Moreover, we found reduced PARP trapping in PARPi-resistant models, both in vitro and in vivo, providing critical evidence for altered PARP trapping as a resistance mechanism and illustrating the potential of this FRET biosensor to interrogate resistance mechanisms quantitatively. This PARP trapping biosensor represents a transformative advance, enabling dynamic, high-resolution analysis of mechanisms underlying cancer drug resistance. It provides critical insights into the heterogeneity of PARPi resistance, with implications for developing more effective therapies and improving personalised treatment strategies for ovarian cancer patients. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=58 SRC="FIGDIR/small/642798v1_ufig1.gif" ALT="Figure 1"> View larger version (11K): org.highwire.dtl.DTLVardef@1c5ee67org.highwire.dtl.DTLVardef@1a6d101org.highwire.dtl.DTLVardef@e3a2bborg.highwire.dtl.DTLVardef@d0ba03_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2025

Formation of amyloid-like HTTex1 aggregates in neurons, downregulation of synaptic proteins and early mortality of Huntington's disease flies are causally linked

Amyloidogenic mutant huntingtin exon-1 (mHTTex1) protein aggregates with pathogenic polyglutamine (polyQ) tracts are the potential root cause of Huntingtons disease (HD). Here, we assessed the gain-of-function toxicity of mHTTex1 aggregation in neurons of HD transgenic flies. We show that the rate of mHTTex1 aggregation in neurons and early mortality of HD transgenic flies are correlated. We observed sequestration of key synaptic proteins into amyloid-like mHTTex1 aggregates and a concomitant decrease of their transcript levels, suggesting that progressive mHTTex1 aggregate stress in neurons leads to an impairment of synaptic function. Machine learning-based data analysis revealed that the abundance of synaptic proteins such as the vesicular monoamine transporter Vmat in the brain is predictive of fly survival. RNAi knockdown of Vmat-encoding transcripts in neurons with pathogenic amyloid-like HTTex1Q97 aggregates further shortened the lifespan of HD flies, supporting the hypothesis that mHTTex1 aggregation drives impairment of synaptic processes and pathogenesis of HD.

molecular biology↗

bioRxiv2025

Divergent endothelial mechanisms drive arteriovenous malformations in Alk1 and Smad4 loss-of-function

BackgroundHereditary hemorrhagic telangiectasia is a genetic disorder caused by loss-of-function mutations in components of the bone morphogenetic protein signaling pathway, leading to arteriovenous malformations. Most prior work has treated BMP-component mutations as mechanistically interchangeable, yet whether distinct genes converge on a shared mechanism remains unclear. We aimed to understand the molecular relationship between BMP signaling and endothelial flow response that leads to AVM formation. MethodsWe expose human endothelial monolayers treated with siRNA against SMAD4 or ALK1 to laminar flow and analyze flow-responsive transcriptomics, flow-responsive BMP signaling activation dynamics, cell polarity and morphology. We analyze the cell-autonomous and non-cell-autonomous migration dynamics of ECs treated with siSMAD4 or siALK1. Using the postnatal mouse retina model, we study EC distribution changes over time in mosaic settings, and assess the remodelling capabilities of Smad4iECKO or Alk1iECKO, relative to littermate controls. ResultsThis study shows that mutations in SMAD4 or ALK1 lead to fundamentally distinct mechanisms of malformation formation. SMAD4 deficiency enhances endothelial responses to blood flow, including transcriptional activation and migration against flow, causing excessive capillary pruning and the development of single large shunts. In contrast, ALK1 deficiency disrupts flow sensing, impairs cell polarization and migration, and promotes a persistent angiogenic state, resulting in dense, hypervascularized networks. RNA sequencing reveals that these transcriptional changes precede flow onset, suggesting early defects in endothelial fate specification. Mosaic in vitro models show that mutant cells co-opt neighboring wild-type cells, while in vivo tracking confirms mutation-specific migration behavior. ConclusionsThese findings reveal divergent cellular programs driving arteriovenous malformations and underscore the need for gene-specific diagnostic and therapeutic strategies. Clinical PerspectiveO_ST_ABSWhat is New?C_ST_ABSO_LIThis study identifies gene-specific mechanisms of AVM formation in endothelial cells with SMAD4 or ALK1 loss-of-function, two major mutations underlying Hereditary Hemorrhagic Telangiectasia. C_LIO_LIUsing multiple side-by-side analyses, the study reveals that SMAD4 deficiency enhances flow-mediated remodeling, while ALK1 deficiency impairs flow sensing and promotes angiogenic persistence. C_LIO_LIRNA sequencing showed that several transcriptional differences precede the onset of flow, indicating early divergence in endothelial cell fate specification and mechanosensory behavior. C_LIO_LIMosaic culture and retinal models demonstrate that mutant cells exert distinct non-cell-autonomous effects on wild-type endothelial cells. C_LI What Are the Clinical Implications?O_LIThis work challenges the prevailing assumption of a unified pathogenic pathway in HHT and supports a model of mutation-specific AVM pathogenesis, with distinct cellular and molecular drivers. C_LIO_LIThe findings provide a mechanistic basis for the variable clinical presentation seen in patients with different HHT mutations. C_LIO_LIIdentification of early transcriptional signatures, such as persistent ANGPT2 or DPP4 upregulation, may offer new biomarkers or therapeutic targets for early intervention before AVMs become clinically symptomatic. C_LIO_LIPersonalized therapeutic strategies could reduce reliance on invasive treatments and shift toward mechanism-based, precision medicine approaches in HHT and related vascular disorders. C_LI

developmental biology↗

bioRxiv2024

Intestinal stem cells enhance local mucosal immunity through apoptotic body phagocytosis

Modulation of immune tone at mucosal surfaces is critical to maintain homeostasis while facilitating the handling of emerging threats. One dynamic component of immune modulation is the phagocytosis and clearance of apoptotic bodies known as efferocytosis that inhibits inflammation by promoting its resolution. Here, we evaluated the effects of apoptotic body phagocytosis by intestinal epithelial stem and progenitor cells (ISCs). Unexpectedly, instead of immunomodulation through efferocytosis, this process elevated local immune system activity. To achieve this result, ISCs actively engaged apoptotic bodies in a unique fashion, leading to their engulfment and ultimate delivery to lysosomes for processing. We found that ISCs were capable of actively recruiting inert material such as apoptotic bodies by using actin-based intrinsic biomechanical processes. Uptake of apoptotic bodies was facilitated by complement factor C3 produced by apoptotic bodies themselves. ISCs in turn generated signals heightening T cell activity that was driven in part by ISC-generated TNF. Taken together, uptake of apoptotic bodies by ISCs produced a local inflammatory alert to specific immune cells. This altered paradigm for the response to phagocytosed apoptotic bodies fits the needs of active mucosal surfaces and demonstrates that efferocytosis as currently defined is not a universal response of all cell types.

cell biology↗

bioRxiv2024

Functional contribution of astrocytic Kir4.1 channels to spasticity after spinal cord injury

Spasticity, a prevalent motor issue characterized by network hyperexcitability, causes pain and discomfort, with existing treatments offering limited relief. While past research has focused on neuronal factors, the role of astrocytes in spasticity has been overlooked. This study explores the potential of restoring astrocytic potassium (K+) uptake to reduce spasticity following SCI. Astrocytes buffer extracellular K+ via Kir4.1 channels, preventing neuronal hyperexcitability. Following spinal cord injury (SCI), Kir4.1 levels decrease at the injury site, though the consequences and mechanisms of this reduction within the motor output area have not been investigated. Utilizing advanced techniques, we demonstrate that lumbar astrocytes in a juvenile thoracic SCI mouse model switch to reactive phenotype, displaying morpho-functional and pro-inflammatory changes. These astrocytes also experience NBCe1-mediated intracellular acidosis, leading to Kir4.1 dysfunction and impaired K+ uptake. Enhancing Kir4.1 function reduces spasticity in SCI mice, revealing new therapeutic targets for neurological diseases associated with neuronal hyperexcitability. HighlightsO_LILumbar astrocytes adopt a reactive phenotype following a thoracic SCI C_LIO_LINBCe1-mediated acidosis in astrocytes disrupts Kir4.1 function post-SCI. C_LIO_LIImpaired K+ uptake leads to motoneuron hyperexcitability post-SCI. C_LIO_LIEnhanced astroglial Kir4.1 function reduces spastic-like symptoms in SCI mice. C_LI

neuroscience↗

bioRxiv2023

Microglia undergo transcriptional, translational and functional adaptations to dark and light phases in laboratory mice

Microglia cells are increasingly recognized to contribute to brain health and disease. Preclinical studies using laboratory rodents are essential to advance our understanding of the physiological and pathophysiological functions of these cells in the central nervous system. Rodents are nocturnal animals, and they are mostly maintained in a defined light-dark cycle within animal facilities, with many laboratories investigating microglial molecular and functional profiles during the animals light (sleep) phase. However, only a few studies have considered possible differences in microglial functions between the active and sleep phases. Based on initial evidence suggesting that microglial intrinsic clock genes can affect their phenotype, we sought to investigate differences in transcriptional, proteotype and functional profiles of microglia between light (sleep) and dark (active) phases, and how these changes are affected in pathological models. We found marked transcriptional and proteotype differences between microglia harvested during the light or dark phase. Amongst others, these differences related to genes and proteins associated with immune responses, motility, and phagocytosis, which were reflected by functional alterations in microglial synaptic pruning and response to bacterial stimuli. Possibly accounting for such circadian changes, we found RNA and protein regulation in SWI/SNF and NuRD chromatin remodeling complexes between light and dark phases. Importantly, we show that microglial circadian transcriptional changes are impaired in a model of immune-mediated neurodevelopmental disorders. Our findings emphasize the importance of considering circadian factors in studying microglial cells and indicate that implementing a circadian perspective is pivotal for advancing our understanding of their physiological and pathophysiological roles in brain health and disease. This may also open novel avenues towards therapeutic strategies for modulating microglial functions during specific windows of the active or sleep phase.

neuroscience↗

bioRxiv2023

Increased beta2-adrenergic signaling is a targetable stimulus essential for bone healing by promoting callus neovascularization

Traumatic brain injury (TBI) is associated with a hyperadrenergic state and paradoxically causes systemic bone loss while accelerating fracture healing. Here, we identify the beta2-adrenergic receptor (Adrb2) as a central mediator of these skeletal manifestations. While the negative effects of TBI on the unfractured skeleton can be explained by the established impact of Adrb2 signaling on bone formation, Adrb2 promotes neovascularization of the fracture callus under conditions of high sympathetic tone, including TBI and advanced age. Mechanistically, norepinephrine stimulates the expression of Vegfa and Cgrp primarily in periosteal cells via Adrb2, both of which synergistically promote the formation of osteogenic type-H vessels in the fracture callus. Accordingly, the beneficial effect of TBI on bone repair is abolished in mice lacking Adrb2 or Cgrp, and aged Adrb2-deficient mice without TBI develop fracture nonunions despite high bone formation in uninjured bone. Pharmacologically, the Adrb2 antagonist propranolol impairs, and the agonist formoterol promotes fracture healing in aged mice by regulating callus neovascularization. Clinically, intravenous beta-adrenergic sympathomimetics are associated with improved callus formation in trauma patients with long bone fractures. Thus, Adrb2 is a novel target for promoting bone healing, and widely used beta-blockers may cause fracture nonunion under conditions of increased sympathetic tone. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=95 SRC="FIGDIR/small/548550v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@cea57corg.highwire.dtl.DTLVardef@d29642org.highwire.dtl.DTLVardef@186ea5aorg.highwire.dtl.DTLVardef@83eaae_HPS_FORMAT_FIGEXP M_FIG Artwork was created in BioRender. C_FIG

cell biology↗

bioRxiv2023

A proteomics analysis of 5xFAD mouse brain regions reveals the lysosome-associated protein Arl8b as a candidate biomarker for Alzheimer's disease

BackgroundAlzheimers disease (AD) is characterized by the accumulation of amyloid-{beta} (A{beta}) peptides in intra- and extracellular deposits. How A{beta} aggregates perturb the proteome in brains of patients and AD transgenic mouse models, however, remains largely unclear. State-of-the-art mass spectrometry (MS) methods can comprehensively detect proteomic alterations in neurodegenerative disorders, providing relevant insights unobtainable with transcriptomics investigations. Analyses of the relationship between progressive A{beta} aggregation and protein abundance changes in brains of 5xFAD transgenic mice have not been reported previously. MethodsWe quantified progressive A{beta} aggregation in hippocampus and cortex of 5xFAD mice and controls with immunohistochemistry and biochemical membrane filter assays. Protein changes in different mouse tissues were analysed by MS-based proteomics using label-free quantification (LFQ); resulting MS data were processed using an established pipeline. Results were contrasted with existing proteomic data sets from postmortem AD patient brains. Finally, abundance changes in the candidate marker Arl8b were validated in CSF from AD patients and controls using ELISAs. ResultsExperiments revealed a more rapid accumulation of A{beta}42 peptides in hippocampus than in cortex of 5xFAD mice, accompanied by many more protein abundance changes in hippocampus than in cortex, indicating that A{beta}42 aggregate deposition is associated with brain region-specific proteome perturbations. Generating time-resolved data sets, we defined A{beta} aggregate-correlated and anticorrelated proteome changes, a fraction of which was conserved in postmortem AD patient brain tissue, suggesting that proteome changes in 5xFAD mice mimic disease relevant changes in human AD. We detected a positive correlation between A{beta}42 aggregate deposition in the hippocampus of 5xFAD mice and the abundance of the lysosome-associated small GTPase Arl8b, which accumulated together with axonal lysosomal membranes in close proximity of extracellular A{beta} plaques in 5xFAD brains. Abnormal aggregation of Arl8b was observed in AD brain tissue. Arl8b protein levels were significantly increased in cerebrospinal fluid (CSF) of AD patients, a clinically accessible body fluid. ConclusionsWe report a comprehensive biochemical and proteomic investigation of hippocampal and cortical brain tissue derived from 5xFAD transgenic mice, providing a valuable resource to the neuroscientific community. We identified Arl8b, with significant abundance changes in 5xFAD and AD patient brains. Arl8b might enable the measurement of progressive lysosome accumulation in AD patients and have clinical utility as a candidate biomarker. Data are available via ProteomeXchange with identifier PXD030348.

molecular biology↗

bioRxiv2022

Highly multiplexed detection of microRNAs, proteins and small molecules using barcoded molecular probes and nanopore sequencing

Currently, most blood tests in a clinical setting only investigate a handful of markers. A low-cost, rapid, and highly multiplexed platform for the quantitative detection of blood biomarkers has the potential to advance clinical diagnostics beyond the single biomarker paradigm. In this study, we perform nanopore sequencing of barcoded molecular probes that have been engineered to recognise a panel of biological targets (miRNAs, proteins, and small molecules such as neurotransmitters), allowing for highly multiplexed simultaneous detection. Our workflow is rapid, from sample preparation to results in 1 hour. We also demonstrate that the strategy can be used to detect biomarkers directly from human serum without extraction or amplification. The established method is easily adaptable, as the number and type of targets detected can be greatly expanded depending on the application required.

biophysics↗

bioRxiv2022

Structural insights into 3Fe-4S ferredoxins diversity in M.tuberculosis highlighted by a first redox complex with P450.

Ferredoxins are small iron-sulfur proteins and key players in essential metabolic pathways. Among all types, 3Fe-4S ferredoxins are less studied mostly due to anaerobic requirements. Their complexes with cytochrome P450 redox partners have not been structurally characterized. In the present work, we solved the structures of both 3Fe-4S ferredoxins from M. tuberculosis - Fdx alone and the fusion FdxE-CYP143. Our SPR analysis demonstrated a high affinity binding of FdxE to CYP143. According to SAXS data, the same complex is present in solution. The structure reveals extended multipoint interactions and the shape/charge complementarity of redox partners. Furthermore, FdxE binding induced conformational changes in CYP143 as evident from the solved CYP143 structure alone. The comparison of FdxE-CYP143 and modeled Fdx-CYP51 complexes further revealed the specificity of ferredoxins. Our results illuminate the diversity of electron transfer complexes for the production of different secondary metabolites.

biochemistry↗