Biology subjects

Dufour, A.

Publications and source records attributed to Dufour, A..

At least 19 recordsLinked to original sources

bioRxiv2026

Mitochondrial-targeted therapy with elamipretide preserves cardiac function and prevents late mortality in murine sepsis-induced cardiac dysfunction.

Sepsis-induced cardiac dysfunction (SICD) occurs in nearly half of septic patients, is associated with increased mortality, and lacks targeted therapy. Emerging evidence implicates impaired mitochondrial function and metabolic inflexibility as central contributors to myocardial depression. Here, we characterized SICD in a murine model of polymicrobial sepsis and evaluated the therapeutic potential of the cardiolipin-stabilizing peptide elamipretide (Ela). Sepsis induced marked impairments in cardiac performance, accompanied by reductions in cardiac cardiolipin content, impaired mitochondrial respiratory capacity localized to complex I, and altered substrate utilization. Integration of stable isotope metabolic flux tracing with lipidomic, metabolomic, and proteomic analyses identified a convergent metabolic bottleneck at the level of the electron transport system. This defect was associated with upstream accumulation of acetyl-CoA, Co-A esters, and ketone bodies, consistent with impaired oxidative flux and energetic failure. Administration of a single early dose of Ela restored cardiolipin content, complex I function, normalized metabolic flux, improved cardiac function during both acute sepsis and recovery, and completely prevented late sepsis-related mortality. These findings identify cardiolipin-dependent mitochondrial dysfunction as a central pathogenic mechanism underlying SICD and position mitochondrial-targeted therapy as a promising therapeutic strategy in sepsis. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=130 SRC="FIGDIR/small/736409v1_ufig1.gif" ALT="Figure 1"> View larger version (47K): org.highwire.dtl.DTLVardef@1ca5b47org.highwire.dtl.DTLVardef@2ecfc2org.highwire.dtl.DTLVardef@149ccb9org.highwire.dtl.DTLVardef@1fbcb6_HPS_FORMAT_FIGEXP M_FIG C_FIG Ela improves SICD by stabilizing cardiolipin species and improving mitochondrial complex I function. SICD depicted in red denotes conditions altered compared to healthy control cardiomyocyte, SICD+Ela depicted in green denotes changes relative to SICD. SICD, sepsis-induced cardiac dysfunction; ELA, elamipretide; ADP, adenosine diphosphate; ATP, adenosine triphosphate; ROS, reactive oxygen species.

physiology↗

bioRxiv2026

Multi omics reveals mesodermal fate bias and enables predictive cell state control in human pluripotent stem cell biomanufacturing

Human induced pluripotent stem cells can retain conventional markers of pluripotency while acquiring environmentally dependent molecular states that may influence their developmental competence. Yet, how biomanufacturing conditions reorganize the interconnected networks governing cell state remains poorly characterized. Here, we first examined compensation of hiPSCs to static aggregation and dynamic agitation by proteomics. We then examined hiPSCs during single-passage culture in stirred-suspension bioprocesses under varying oxygen and agitation conditions. Intracellular metabolomic and transcriptomic profiling revealed distinct responses across culture configuration, hydrodynamic exposure, and oxygenation. Transition into dynamic culture broadly remodeled mitochondrial organization, carbon allocation, mechanotransduction, proteostasis, and developmental regulation. Increasing agitation produced a persistent response involving cellular architecture, growth-factor signaling, genome maintenance, and Epiblast- and lineage-aligned programs, whereas oxygenation elicited a smaller and more transient metabolic and transcriptional response. Through comparison with a human gastrulation reference, we show that these adaptations intersect with natural developmental programs without reproducing coherent epiblast state or lineage commitment. These findings demonstrate that bioprocess conditions can preserve core pluripotent identity while remodeling broader molecular states associated with developmental responsiveness, providing perspective on evaluating and optimizing hiPSC quality in biomanufacturing beyond restricted marker panels.

bioengineering↗

bioRxiv2025

Therapeutic modulation of the calpastatin/calpain pathway restores calpain-mediated synaptic proteolysis and preserves motor neurons survival and function in C9orf72 ALS

A hexanucleotide repeat expansion (GGGGCC) in the C9orf72 gene is the most prevalent genetic cause of ALS, with early neuromuscular junction (NMJ) dysfunction being a key pathological feature. Current therapies provide only limited symptomatic relief, underscoring the need for targeted, mechanism-based interventions. Using a C9orf72 ALS zebrafish model (C9-miR) and patient-derived induced pluripotent stem cell (iPSC) motor neurons, we identified significant downregulation of calpastatin, the endogenous inhibitor of calpains, a calcium-dependent protease family implicated in neurodegeneration. We demonstrate that restoring calpastatin activity with a cell-permeable calpastatin-derived peptide or the small molecule, calpeptin, ameliorates locomotor deficits and NMJ dysfunction in the C9-miR zebrafish model. These interventions enhance synaptic vesicle turnover and quantal release at the NMJ while improving motor neuron excitability and synaptic integrity in iPSC-derived motor neurons. N-terminomic/TAILS mass spectrometry revealed direct calpain-mediated cleavage of synaptic proteins in motor neurons derived from C9orf72 patients. Proteolysis of novel ALS-relevant synaptic and axonal proteins is prevented by calpeptin and calpastatin peptide treatments. Our findings establish the calpastatin as a pivotal regulator of synaptic function in C9orf72-associated ALS and identify it as a promising therapeutic target, offering a novel strategy to restore synaptic transmission and potentially halt disease progression.

neuroscience↗

bioRxiv2025

A platform of robust patient-derived leukemia models covering subgroups for which no cell lines exist

Preclinical cancer research requires robust model systems, especially for poor prognosis entities like acute myeloid leukemia (AML), a highly aggressive blood cancer. Here, primary tumor cells from 137 AML patients of all age groups were transplanted into immune compromised mice to generate patient-derived xenografts (PDX). From these, 23 models enable robust, virtually endless serial re-transplantation and are amenable to lentiviral genetic engineering (*PDX AML models). These models primarily originate from patients with highly aggressive, relapsed disease. Comprehensive genomic, transcriptomic, and epigenomic analyses confirmed that they replicate primary AML biology more faithfully than conventional cell lines. Notably, *PDX AML models include AML subgroups that are underrepresented or absent in existing model systems, such as cytogenetically normal or IDH1/2-mutant AML. They withstand freeze-thaw cycles, making them suitable for broad distribution and reproducibility across research institutions. Luciferase-based in vivo imaging enables real-time monitoring of tumor progression and treatment responses in preclinical trials. Surprisingly, long-term treatment, including repeated cytarabine therapy over a period of one year, showed a gradual reduction in leukemia cell proliferation, which decreased continuously after each treatment block. Collectively, our *PDX models represent a robust, versatile, and relevant platform that holds great promise to accelerate translational research for the benefit of cancer patients. Visual Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=123 SRC="FIGDIR/small/677299v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@1ead31eorg.highwire.dtl.DTLVardef@1ca702corg.highwire.dtl.DTLVardef@18a7137org.highwire.dtl.DTLVardef@edbe6_HPS_FORMAT_FIGEXP M_FIG C_FIG Key PointsO_LIWe present new robust AML PDX models covering subgroups for which no cell lines exist for use in various ex vivo and in vivo applications. C_LIO_LI*PDX models enable serial transplantation, genetic engineering and better representation of primary AML biology than cell lines. C_LIO_LIOne-year in vivo trials mimicking clinical chemotherapy showed surprising gradual decline in leukemia growth after each treatment block. C_LI

cancer biology↗

bioRxiv2025

The role of aging on endothelial cell-cell junctions and pulmonary microvascular permeability

Lung injury leads to pulmonary microvascular endothelial cell (PMVEC) damage, disruption of cell-cell junctions, and increased permeability. Previously, we demonstrated in a mechanical ventilation-induced model of lung injury that aging exacerbated pulmonary microvascular permeability. Based on this, we hypothesized that aging was associated with increased PMVEC barrier dysfunction due to impaired cell-cell junction integrity. PMVEC were isolated from young and aged mice and cultured to confluence in vitro. Barrier function and cell-cell junction integrity were assessed through electric cell-substrate impedance sensing, XPerT permeability assay, immunofluorescence, and western blot analysis. Further studies were conducted to examine alterations in the proteome, markers of inflammation, and actin cytoskeleton organization. To model injurious conditions, PMVEC were stimulated with inflammatory cytokines; permeability and actin cytoskeletal alterations were subsequently assessed. We observed increased basal permeability in PMVEC from aged mice, which was associated with disrupted cell-surface localization of the adherens junction protein, vascular endothelial (VE)-cadherin. Protein abundance of VE-cadherin was significantly increased with age; however, levels of the adapter protein,{gamma} -catenin, and the tight junction protein, claudin-5, were decreased. Measures of inflammation, including cytokine expression and cell surface abundance of adhesion molecules, did not differ with age. Alterations in actin cytoskeleton organization, characterized by augmented presence of actin stress fibers, were observed in aged PMVEC. Under inflammatory conditions, permeability and actin stress fiber formation were exacerbated with age. It is concluded that aging predisposes PMVEC to elevated injury, due to inherent deficiencies in cell-cell junctions and barrier function, potentially mediated through altered actin cytoskeleton organization. New and NoteworthyCompared with pulmonary microvascular endothelial cells (PMVEC) from young mice, PMVEC isolated from aged mice had higher permeability, which was directly associated with impairments in cell-cell junctions. The higher permeability in aged PMVEC was not associated with augmented inflammatory signaling but was associated with actin cytoskeletal alterations. Following an inflammatory insult, PMVEC from aged mice had further exacerbated permeability. These findings may begin to highlight why older patients exhibit higher mortality during lung injury.

cell biology↗

bioRxiv2025

Fecal proteomics of wild capuchins reveals impacts of season, diet, age, and, sex on gut physiology

Understanding how the physiology of free-ranging mammals is impacted by environmental stressors is a major focus of ecological research. However, the constraints of non-invasive sampling pose serious challenges to the acquisition of physiological data from most species of primates. As a result, little is known about how the gut responds to ecological stimuli at the cellular level in wild populations. Recent research has demonstrated that proteomics could fill this knowledge gap by sequencing and quantifying proteins directly from primate feces. In order to ascertain how the gut of free-ranging white-faced capuchin monkeys (C. imitator) is influenced by environmental heterogeneity, diet, age, and sex, we sequenced 45 fecal proteomes from 24 individuals from the Sector Santa Rosa population in Costa Rica, using liquid chromatography-tandem mass spectrometry with label-free quantification. Fecal proteins assigned to C. imitator were strongly localized to gut tissues and functionally enriched for digestive and immune functions. We identified 41 capuchin candidate proteins linked to seasonality, age, sex, and diet. We also quantified abundances of dietary fruit, dietary insects, helminth gut parasites, and gut microbes. Our results demonstrate the viability of using quantitative fecal proteomics in free-ranging populations of mammals to integrate host physiology, diet, and microbial ecology through non-invasive means.

ecology↗

bioRxiv2025

Autopolyploidy exacerbates dominance masking under negative frequency-dependent selection: evidence from sporophytic self-incompatibility in Arabidopsis arenosa and A. lyrata

Polyploidy is a widespread phenomenon in flowering plants, with up to 30% of extant species being recent polyploids. Whether and how polyploidy modulates the action of natural selection remains debated, and the particular case of balancing selection has been poorly explored. This study investigates the impact of autopolyploidy on sporophytic self-incompatibility in plants, a striking example of a genetic system evolving under a special form of balancing selection (strong negative frequency-dependent selection). Numerical simulations reveal that under strict codominance, the number of S-alleles maintained in tetraploid populations is expected to double as compared to diploid populations. However, under a model with strict hierarchical dominance among alleles, the number of S-alleles increases only slightly, but gene diversity and observed heterozygosity are substantially lower in tetraploids as compared to diploids, due to enhanced dominance masking effects. Empirical data on Arabidopsis arenosa and A. lyrata confirm the latter predictions, showing similar levels of allelic diversity but dramatically lower observed and expected heterozygosity at the self-incompatibility locus in tetraploids compared to diploids. The study highlights the significant impact of autopolyploidy on patterns of diversity at the self-incompatibility locus, emphasizing the increased dominance effect in tetraploids. The results also allow us to reject a scenario of strong founder effects associated with evolution of the polyploid lineages.

evolutionary biology↗

bioRxiv2025

Single-cell omics reveal distinct gene regulatory dynamics underpinning embryonic and extraembryonic lineage functions during pig blastocyst development

The late-stage development of the blastocyst before implantation is a unique feature of ungulates. During this period, the epiblast proliferates and remains pluripotent for several days before gastrulation begins. Simultaneously, extra-embryonic tissues undergo significant growth, elongating to several tens of centimeters. However, the mechanisms that regulate and synchronize these processes remain poorly understood. In this study, we analyzed transcriptomic and epigenomic data at the single-cell level from early and late porcine blastocyst cells, spanning from the hatched blastocyst stage (E7) to early (E9) and late ovoid blastocyst (E11). We characterized 15,370 blastocyst cells, clustering them into distinct embryonic and extra-embryonic populations with specific chromatin accessibility landscapes. For each population, we inferred gene regulatory networks based on enhancer-driven gene regulation modules (eRegulons) and validated them through motif occupancy visualization. Our findings reveal strong dynamics in gene regulatory module activity within extra-embryonic tissues at the onset of blastocyst elongation, while gene regulatory activity in the epiblast remains relatively stable.

developmental biology↗

bioRxiv2024

Biofabrication of spatially organized temporo-mandibular fibrocartilage assembloids

The combination of mesenchymal stem cell (MSC) spheroids and polymeric scaffolds has been actively explored for engineering organized hyaline cartilage; however, its application to other types of cartilage remains under-explored. The temporo-mandibular joint (TMJ) fibrocartilage is a highly stratified tissue whose recapitulation remains challenging. In this study, the shape and growth orientation of assembloids were controlled by seeding early mature human adipose-derived MSC spheroids into polymeric scaffolds with a dual architecture of micron-scale fibers. This results in flattened asymmetric tissues with a single-sided articular surface. Structurally, the engineered fibrocartilage mimicked the histotypical organization observed in the native human condylar fibrocartilage, notably featuring a thick fibrous zone with flattened cells. Native-like distribution of general extracellular matrix (ECM) components, including glycosaminoglycans and total collagens, and ECM-specific components, such as type I and II collagens, aggrecan core protein, and fibronectin, were observed. Collagen organization, as demonstrated by polarized light microscopy and scanning electron microscopy at the fibril level, was also found to be similar to that of native human tissue. Zonal-dependent micromechanical properties were identified in both the engineered and native tissues, although lower mechanical properties were observed in the fibrous zone of the engineered tissue. This work provides further evidence that the combination of MSC spheroids and micron-sized fiber polymeric scaffolds is a versatile approach for engineering stratified cartilage and a promising strategy for engineering biomimetic fibrocartilage grafts for TMJ reconstruction.

bioengineering↗

bioRxiv2024

Characteristics of early career health researchers and experiences of burnout during the COVID-19 pandemic in Canada

IntroductionThe COVID-19 pandemic disrupted research globally. How it impacted Canadian early-career health researchers (ECHRs) remains unclear. We administered a survey to understand the composition of ECHRs in Canada, their job experiences, and experiences of burnout during the COVID-19 pandemic. MethodsA cross-sectional survey was conducted in May 2023 of Canadian ECHRs defined as within 7 years of their first independent research position. Quantitative analyses included a description of respondents by research pillar, socio-demographic and workplace characteristics, and the prevalence of burnout, disengagement or exhaustion. Sample characteristics were compared to national data on ECHRs from a Canadian funding agency. Thematic analysis of free-text responses was also conducted. ResultsA total of 225 respondents met the eligibility criteria. Most respondents were assistant professors and characteristics of our sample were like the national data. The COVID-19 pandemic posed many challenges to student recruitment, and emotional support of students, with over half of the respondents reporting a moderate to significant decline in mental health compared to pre-pandemic. A significant proportion of respondents were experiencing high burnout (62%, 95%CI:56-67%), exhaustion (64%, 95%CI: 57-70%) or disengagement (91%, 95%CI: 87-95%). Thematic analysis identified three themes: ongoing benefits/problems preceding the pandemic, unintended outcomes of strategies to manage/prevent/contain COVID-19, and reasons to stay in their current position. ConclusionsOur survey revealed that Canadian ECHRs reported many diverse challenges during the COVID-19 pandemic and high burnout, putting the sustainability of this workforce at risk. Improved systems are needed to understand the long-term impacts and support the future of the Canadian health research ecosystem.

scientific communication and education↗

bioRxiv2024

Identification of a sub-population of synovial mesenchymal stem cells with enhanced treatment efficacy in a rat model of Osteoarthritis

Osteoarthritis (OA) is a painful and debilitating disease which has no cure and there are no treatments which can predictably stop/reverse its progression. Treating this disease is particularly difficult since the articular cartilage lacks intrinsic repair capacity even though mesenchymal stem cells (MSCs) are present in the joint environment and have robust chondrogenic potential. We have previously shown that there is heterogeneity of MSC sub-types within the human synovium, yet it remains unclear if any of these MSC types can regenerate cartilage and/or impact OA disease progression. Therefore, we have undertaken this study focusing on clonally derived MSC populations derived from the synovium of normal and OA patients to characterize if any MSC populations can positively impact OA disease trajectory in a rat model of OA. MSCs were clonally isolated by indexed flow cytometry, expanded in culture and then characterized for differentiation capacity and by quantitative proteomics. MSC clones were then transplanted into a xenograft rat OA model and treatment effect was determined by histology and immunofluorescence outcomes. We identified heterogeneity in putative MSCs derived from within and between patient groups (normal vs. OA) and the ability of these cells to effect repair in a rat OA model. However, these different sub-types of MSCs could not be distinguished by traditional cell surface markers showing the need for a better understanding of these populations at the single cell level. Using an unbiased proteomics approach, CD47 was identified a novel marker of human MSCs. Using the same rat model of OA, CD47Hi expressing cells were found to have robust treatment efficacy and directly contributed to the formation of new articular cartilage tissue. Characterizing MSCs is essential to understand which sub-types are appropriate for further clinical investigation. If OA patients still have functional MSCs in their synovium, then it is possible these cells can be exploited for cartilage regeneration / OA treatment strategies.

cell biology↗

bioRxiv2024

Detection of titanium nanoparticles in human, animal and infant formula milk

The sustainability of mammals on Earth relies on milk. During lactation, maternal exposure to pollutants like metal nanoparticles (NPs) can affect offspring development and survival. Despite being banned from food applications in Europe due suspected toxicity, titanium dioxide (TiO2) NPs are still massively manufactured for countless other uses. While contamination of ecosystems is well documented, contamination of mammals remains underexplored. Here, we used synchrotron X-ray fluorescence and single particle inductively coupled plasma mass spectrometry to analyse human, animal, and infant formula milk. Titanium containing micro- and nano-particles were detected in all samples, regardless of the species, location, and processing. We identified varying concentrations, sizes, and combinations of rutile and anatase TiO2, ilmenite FeTiO3 and possibly titanite CaTiSiO5 or pseudobrookite Fe2TiO5. These findings suggest that milk serves as a carrier for titanium-containing nanomaterials to expose newborns on a daily basis until weaning.

physiology↗

bioRxiv2024

Redox Signaling Mediates Differentiation of Adipose Progenitors in Response to Inflammatory Cytokines in the Adipose Tissue Secretome

Adipogenesis, the terminal differentiation of adipose progenitor cells (APCs), is critical in maintaining the functional integrity of adipose depots under obesogenic conditions. It is thought that a signal arising from the adipose microenvironment triggers APC differentiation; yet the identity and source of the signal remains unknown. This study sought to uncover the signal responsible for activating adipogenesis. Redox signaling was shown to influence adipogenesis in primary murine APCs treated with pharmacologic agents to manipulate the levels of reactive oxygen species (ROS). Increased generation of superoxide (O2-) and hydrogen peroxide (H2O2) via redox cyclers amplified both early and late APC differentiation, while ROS scavengers and antioxidants blunted differentiation. The impact of specifically targeting H2O2 with the antioxidant, catalase, or a catalase inhibitor, was restricted to lipid accumulation in late adipogenesis. Protein was concentrated from conditioned media of adipose tissue explants cultured ex vivo to capture signals within the adipose secretome. Differentiation was enhanced in APCs cultured in the presence of the adipose secretome, an effect that was diminished with scavenging of ROS and amplified when the secretome was collected from mice fed a high fat diet for 8 weeks. Proteomic analysis revealed that the adipose secretome from animals on a high fat diet was enriched in pathways related to immune cell-mediated inflammation, with interleukin 6 (IL-6) as a central regulator of differentially expressed proteins. A multiplex assay to measure cytokines confirmed higher IL-6 in the adipose secretome of high fat-fed animals. Exposure of APCs to IL-6 increased adipogenesis, while treatment of APCs with an IL-6 blocking antibodies diminished the adipogenic effect of the adipose secretome. Together, these findings substantiate a role for redox signaling in the regulation of adipogenesis and identify IL-6 as a novel activator of adipogenesis that may mediate APC differentiation via generation of ROS under obesogenic conditions.

systems biology↗

bioRxiv2024

EMMPRIN confers metabolic advantage for monocytes and macrophages to promote disease in a model of multiple sclerosis

Monocytes and monocyte-derived macrophages have important roles in the initiation and progression of multiple sclerosis (MS). These cells undergo metabolic reprogramming to generate immunophenotypes that promote leukocyte infiltration, axonal degeneration and demyelination, worsening MS pathology. The mechanisms that dictate metabolic programs in monocytes and macrophages in MS remain unclear. We previously reported that extracellular matrix metalloproteinase inducer (EMMPRIN, CD147), a glycoprotein that acts as a chaperone of monocarboxylate transporter 4 (MCT4), assisted with glycolysis-driven pro-inflammatory phenotype in macrophages in experimental autoimmune encephalomyelitis (EAE), an animal model of MS. Using newly-generated CCR2CreERT2:EMMPRINfl/fl (CCR2:EMMP) mice, we report that presymptomatic deletion of EMMPRIN in CCR2+ monocytes prevented or reduced clinical disability of EAE. This was correspondent with decreased infiltration of leukocytes into the CNS. Single cell RNA-seq of blood monocytes from EAE and proteomics analysis of macrophages from CCR2:EMMP-/- mice revealed significant alterations in metabolic programs, particularly reduced glycolysis and elevated mitochondrial electron transport and fatty acid oxidation, which were linked to their reduced pro-inflammatory traits. Our findings implicate EMMPRIN as a key regulator of metabolic pathways that exacerbate pro-inflammatory functions of monocytes in MS.

immunology↗

bioRxiv2024

Extracellular Matrix-Induced Genes May Reduce Response to Rapamycin in LAM

RationaleLymphangioleiomyomatosis (LAM) is a rare cystic lung disease driven by nodules containing TSC2-/- LAM cells and recruited LAM associated fibroblasts (LAFs). Although rapamycin reduces lung function loss, some patients continue to decline meaning additional therapies are needed. ObjectivesTo investigate how the LAM nodule environment affects LAM cell proliferation and the response to rapamycin. MethodsChanges in advanced LAM were identified using shotgun proteomics and immunohistochemistry in tissue from carefully phenotyped patients. Genes potentially associated with rapamycin insensitivity of cells grown on LAF-derived extracellular matrix were identified by RNA sequencing and validated using repurposed pharmacologic inhibitors. Main ResultsMore advanced disease was associated with increasing nodules adjacent to lung cysts and greater decline in forced expiratory volume in 1 sec (FEV1) when treated with rapamycin (p=0.005). In late-stage LAM, proteomics identified upregulation of pathways associated with accumulation of activated fibroblasts, including extracellular matrix deposition, glucose metabolism and the actin cytoskeleton. Picrosirius red staining and immunohistochemistry confirmed deposition of extracellular matrix within LAM nodules. The growth of TSC2-/- model LAM cells was increased on LAF-derived extracellular matrix (LAF ECM), and incompletely supressed by rapamycin (p<0.0001). RNA sequencing of cells grown on LAF ECM identified upregulation of pathways driving cell cycle control, transcription and metabolism in cells. Tractable, pro-proliferative, rapamycin insensitive genes included CDK7, GAS6 and PLAU. Repurposed inhibitors of these pathways inhibited LAM cell proliferation and enhanced the anti-proliferative effect of rapamycin. ConclusionsExtracellular matrix deposited by LAM associated fibroblasts upregulates expression of genes which potentially blunt the response to rapamycin, but offer additional therapeutic opportunities for patients with established LAM.

cell biology↗

bioRxiv2024

Kinetic properties of optogenetic DNA editing by LiCre-loxP

Advances in optogenetics now allow to specifically modify the DNA of live cells with light. However, using these technologies successfully requires to know their properties in terms of sensitivity, efficiency, kinetics and mechanism. We previously developed an optogenetic tool made of a single chimeric protein called LiCre that enables the induction of specific changes in the genome with blue light via DNA recombination between loxP sites [1]. Here, we used in vitro and in vivo experiments combined with kinetic modeling to provide a deeper characterization of the photo-activated LiCre-loxP recombination reaction. We find that LiCre binds DNA with high affinity in absence of light stimulus and that this binding is cooperative although not as much as for the Cre recombinase from which LiCre was derived. In yeast, addition of riboflavin to the culture medium had no effect on LiCres efficiency, even when cells over-expressed riboflavin kinase, suggesting that abundance of the flavin mono-nucleotide co-factor is not limiting for the reaction. However, LiCres efficiency in yeast gradually increased when raising temperature from 20{degrees}C to 37{degrees}C. The recombination kinetics observed in live cells are best explained by a model where photo-activation of two or more DNA-bound LiCre units (happening in seconds) can produce (in several minutes) a functional recombination synapse. This model was able to capture the effect of a point mutation altering LiCres light cycle. This deeper understanding of the LiCre-loxP system provides additional knowledge for designing experiments where specific genetic changes are induced in live cells with light. AUTHOR SUMMARYUsing a technology called optogenetics, scientists are now able to change the DNA sequence of live cells by illuminating them with light. In theory, they can trigger in genetically-engineered organisms a mutation of interest in specific cells at a specific time. This practice, however, is not common because optogenetics relies on light-controlled enzymes that are recent and not well characterized. We previously developed one of these enzymes, called LiCre, which recognizes a specific piece of DNA. Following illumination with blue light, LiCre can switch on or off whatever gene is close by. Here, we combined experiments and computational modeling to better understand how, and how fast, LiCre works. We find that, although it is inactive in the dark, LiCre does not need photo-activation to bind to its DNA partner. We estimated the speed at which LiCre gets deactivated in the dark and the speed at which active LiCre molecules modify DNA. We also showed the effect of temperature and illumination dynamics on LiCres efficiency. These results will help design strategies where LiCre can be used to conduct genetic studies at high spatio-temporal resolution, or implement it in industrial and biomedical applications.

bioengineering↗

bioRxiv2023

Cysteine String Protein alpha in Extracellular Vesicle Subtypes: a Proteomic Analysis

Cysteine string protein (CSP /DnaJC5) is a presynaptic J-domain protein (JDP) that prevents neurodegeneration. CSP/DnaJC5 is reported to facilitate export of distinct, highly oligomeric, disease-causing proteins in addition to wild-type TDP-43, tau and -synuclein. Yet, detailed mechanistic knowledge of the full CSP/DnaJC5 secreted proteome is lacking. Understanding the CSP/DnaJC5 export pathway has implications for a growing number of neurodegenerative diseases. In humans, Leu115Arg or Leu116deletion mutations cause adult-onset neuronal ceroid lipofusinosis (ANCL), a rare neurodegenerative disorder. In the present study, we examined extracelular vesicles (EVs) released from CSP/DnaJC5 expressing cells. Cells are known to secrete many types of EVs of different sizes and origins into the extracellular space. EV subpopulations were separated by their sedimentation speed and subjected to proteomic analysis. We find that CSP/DnaJC5 and the CSP/DnaJC5 mutants, Leu115Arg or Leu116del are enriched in multiple EV subpopulations. The exported protein profile is determined by proteomics. We report that several other J-domain proteins (JDPs), such as DnaJC7, DnaJA1 and DnaJA2 are exported and speculate that export of JDPs may facilitate the secretion of diverse client proteins. Our work provides a platform for further inquiry into the role of secreted CSP/DnaJC5 and other JDPs in proteostasis.

cell biology↗

bioRxiv2023

FAIMS-enabled N-terminomics analysis reveals novel legumain substrates in murine spleen

Aberrant levels of the asparaginyl endopeptidase legumain have been linked to inflammation, neurodegeneration and cancer, yet our understanding of this protease is incomplete. Systematic attempts to identify legumain substrates have previously been confined to in vitro studies, which fail to mirror physiological conditions and obscure biologically relevant cleavage events. Using high-field asymmetric waveform ion mobility spectrometry (FAIMS), we developed a sensitive and streamlined approach for proteome and N-terminome analyses in a single analytical method without the need for N-termini enrichment. Compared to unfractionated proteomic analysis, we demonstrate FAIMS fractionation improves neo-N- termini identification by >2.5 fold, resulting in identification of >2,882 unique neo-N-termini from limited sample amounts. Within murine spleens, this approach identifies 6,366 proteins and 2,528 unique neo-N-termini, with 235 cleavage events enriched in wild-type compared to legumain-deficient spleens. Among these, 119 neo-N-termini arose from asparaginyl endopeptidase activities, representing novel putative physiological legumain substrates. The direct cleavage of selected substrates by legumain was confirmed using in vitro assays, providing support for the existence of physiologically relevant extra-lysosomal legumain activity. Combined, these data shed critical light on the functions of legumain and demonstrates the utility of FAIMS as an accessible method to improve depth and quality of N- terminomics studies.

biochemistry↗