Biology subjects

Schmitt, A.

Publications and source records attributed to Schmitt, A..

34 records · Page 2Linked to original sources

bioRxiv2023

Structures of two main components of the virophage and Marseilleviridae virions extend the range of unrelated viruses using fiber head as common receptor binding fold

The detailed proteomic analysis of Marseilleviridae icosahedral capsids revealed that the two most abundant protein components of the virions were the Major Capsid Protein (MCP) and the product of an ORFan gene conserved in all Marseilleviridae. The noumeavirus NMV_189 3D structure revealed a common fold with fiber head proteins used by a variety of viruses to recognize their cellular receptor. However, the trimeric structure of NMV_189 uniquely lacking a tail domain, presented a deep concave site suggesting it could be directly anchored to the pseudo-hexagonal capsomers of the virion. This was confirmed by the unambiguous fit of the structure in the melbournevirus 4.4 [A] cryo-EM map. In parallel, our structural genomic study of zamilon vitis virophage capsid proteins revealed that Zav_19 shared the same trimeric fiber head fold, but presented an N-terminal tail with a unique {beta}-prism fold. The fiber head fold thus appears to be conserved in all types of non-enveloped icosahedral virions independently of their genomic contents (dsDNA, ssRNA, dsRNA). This could be a testimony of a common origin or the result of convergent evolution for receptor binding function. IMPORTANCEGiant viruses and their associated virophages exhibit a large proportion ([≥]60%) of orphan genes, i.e. genes without homologs in databases, and thus a vast majority of their proteins are of unknown function. The structural characterization of two ORFans, NMV_189 and Zav_19, both major components of noumeavirus and zamilon virophage capsids, respectively, revealed that despite a total lack of sequence homology, the two proteins share a common trimeric fold typical of viral receptor binding proteins and could be responsible for host receptor recognition. These two structures extend the range of unrelated viruses using fiber head structures as common receptor binding fold.

microbiology↗

bioRxiv2023

Functional synapses between small cell lung cancer and glutamatergic neurons

Small cell lung cancer (SCLC) is a highly aggressive type of lung cancer, characterized by rapid proliferation, early metastatic spread, clinical recurrence and high rate of mortality. Using in vivo insertional mutagenesis screening in conjunction with cross-species genomic and transcriptomic validation, we identified a strong and consistent signal for neuronal, synaptic, and glutamatergic signaling gene sets in murine and human SCLC. We show that SCLC cells have the ability to develop intimate contacts with neuronal glutamatergic terminals in vitro, in autochthonous primary lung tumors and in brain-engrafted tumors. These contacts can develop into bona fide synapses, allowing SCLC cells to receive glutamatergic inputs. Fitting with a potential oncogenic role of neuron-SCLC interactions, we show that SCLC cells derive a robust proliferation advantage when co-cultured with neurons. Moreover, the repression of glutamate release and the stimulation of the inhibitory glutamate receptor GRM8 displayed therapeutic efficacy in an autochthonous mouse model of SCLC. Therefore, following malignant transformation, SCLC cells appear to hijack glutamatergic signaling to sustain tumor growth, thereby exposing a novel entry route for therapeutic intervention.

cancer biology↗

bioRxiv2022

Structural foundation for the role of enterococcal PrgB in conjugation, biofilm formation and virulence

Type 4 Secretion Systems are a main driver for the spread of antibiotic resistance genes and virulence factors in bacteria. In Gram-positives, these secretion systems often rely on surface adhesins to enhance cellular aggregation and mating pair formation. One of the best studied adhesins is PrgB from the conjugative plasmid pCF10 of Enterococcus faecalis, which has been shown to play major roles in conjugation, biofilm formation and importantly also in bacterial virulence. Since prgB orthologs exist on a large number of conjugative plasmids in various different species, this makes PrgB a model protein for this widespread virulence factor. After characterizing the polymer adhesin domain of PrgB previously, we here report the structure for almost the entire remainder of PrgB, which reveals that PrgB contains four immunoglobulin-like domains. Based on this new insight, we re-evaluate previously studied variants and present new in vivo data where specific domains or conserved residues have been removed. For the first time, we can show a decoupling of cellular aggregation from biofilm formation and conjugation in prgB mutant phenotypes. Based on the presented data, we propose a new functional model to explain how PrgB mediates its different functions. We hypothesize that the Ig-like domains act as a rigid stalk that presents the polymer adhesin domain at the right distance from the cell wall.

biochemistry↗

bioRxiv2022

A recurrent de novo splice site variant involving DNM1 alternative exon 10a causes developmental and epileptic encephalopathy through a dominant-negative mechanism

Heterozygous pathogenic variants in DNM1 cause developmental and epileptic encephalopathy (DEE) due to a dominant-negative mechanism impeding vesicular fission. Thus far, pathogenic variants in DNM1 have been studied using a canonical transcript that includes the alternatively spliced exon 10b. However, after performing RNA sequencing in thirty-nine pediatric brain samples, we find the primary transcript expressed in the brain includes the downstream exon 10a instead. Using this information, we evaluated genotype-phenotype correlations of variants affecting exon 10a and identified a cohort of eleven previously unreported individuals. Eight individuals harbor a recurrent de novo splice site variant, NG_029726.1(NM_001288739.1):c.1197-8G>A, which affects exon 10a and leads to DEE consistent with the classical DNM1 phenotype. We find this splice site variant leads to disease through an unexpected dominant-negative mechanism. Functional testing reveals an in-frame upstream splice acceptor causing insertion of two amino acids predicted to impair oligomerization-dependent activity. This is supported by neuropathological samples showing accumulation of synaptic vesicles adherent to the plasma membrane consistent with impaired vesicular fission. Two additional individuals with missense variants affecting exon 10a, p.(Arg399Trp) and p.(Gly401Asp), had a similar DEE phenotype. In contrast, a single individual with a missense variant affecting exon 10b, p.(Pro405Leu), which is less expressed in the brain, had a correspondingly less severe presentation. Thus, we implicate variants affecting exon 10a as causing the severe DEE typically associated with DNM1-related disorders. We highlight the importance of considering relevant isoforms for disease-causing variants, as well as the possibility of splice site variants acting through a dominant-negative mechanism.

genetics↗

bioRxiv2022

The giant Mimivirus 1.2 Mb genome is elegantly organized into a 30 nm helical protein shield

Mimivirus is the prototype of the Mimiviridae family of giant dsDNA viruses. Little is known about the organization of the 1.2 Mb genome inside the membrane-limited nucleoid filling the [~]0.5 {micro}m icosahedral capsids. Cryo-electron microscopy, cryo-electron tomography and proteomics revealed that it is encased into a [~]30 nm diameter helical protein shell surprisingly composed of two GMC-type oxidoreductases, which also form the glycosylated fibrils decorating the capsid. The genome is arranged in 5- or 6-start left-handed super-helices, with each DNA-strand lining the central channel. This luminal channel of the nucleoprotein fiber is wide enough to accommodate oxidative stress proteins and RNA polymerase subunits identified by proteomics. Such elegant supramolecular organization would represent a remarkable evolutionary strategy for packaging and protecting the genome, in a state ready for immediate transcription upon unwinding in the host cytoplasm. The parsimonious use of the same protein in two unrelated substructures of the virion is unexpected for a giant virus with thousand genes at its disposal. One-Sentence SummaryMimivirus genome organization in the icosahedral virion.

microbiology↗

bioRxiv2021

LRRFIP2 modulates the response to hypoxia during embryonic cardiogenesis

Oxygen is crucial for appropriate embryonic and fetal development, including cardiogenesis. The heart is the first organ formed in the embryo and is required to provide oxygen and nutrients to all cells in the body. Embryonic cardiogenesis is a complex process finely regulated and prone to congenital malformations. It takes place in a hypoxic environment that activates the HIF-1 signaling pathway which mediates cellular and systemic adaptations to low oxygen levels. Since inhibition or overactivation of the HIF-1 signaling pathway in the myocardium lead to severe cardiac malformations and embryonic lethality, it is important that the cellular response to hypoxia be precisely regulated. While many gene regulatory networks involved in embryonic cardiogenesis have been characterized in detail, the modulation of the response of cardiomyocytes (CM) to hypoxia has remained less studied. We identified LRRFIP2 as a new negative cofactor of HIF-1. Indeed, we have shown that the absence of Lrrfip2 expression in a mouse KI model led to an enhance of many HIF-1 target genes including Igfbp3, Bnip3 and Ndufa4l2 in embryonic CM during development. As results, the absence of Lrrfip2 led to the inhibition of the PI3K/Akt survival pathway, growth defects, mitochondrial dysfunction and to a precocious maturation of the embryonic CMs. Altogether, these defects led to the formation of a smaller heart unable to provide sufficient oxygen to the embryo and finally to a severe hypoxia and a precocious lethality. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=199 HEIGHT=200 SRC="FIGDIR/small/472540v1_ufig1.gif" ALT="Figure 1"> View larger version (42K): org.highwire.dtl.DTLVardef@1cb5836org.highwire.dtl.DTLVardef@10c7b97org.highwire.dtl.DTLVardef@86263aorg.highwire.dtl.DTLVardef@137da51_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LILRRFIP2 regulates negatively the HIF-1 activity C_LIO_LILrrfip2 deletion leads to an embryonic lethality between E11.5 and E13.5 C_LIO_LILRRFIP2 controls ROS production and CM maturation C_LI

developmental biology↗

bioRxiv2021

Epigenetic therapy suppresses endocrine-resistant breast tumour growth by re-wiring ER-mediated 3D chromatin interactions

Three-dimensional (3D) epigenome remodelling is an important mechanism of gene deregulation in cancer. However, its potential as a target to overcome therapy resistance remains largely unaddressed. Here we show that FDA-approved epigenetic therapy Decitabine (5-Aza-mC) suppresses tumour growth in preclinical metastatic ER+ breast tumour xenograft models. Decitabine-induced genome-wide DNA hypomethylation results in large-scale 3D epigenome deregulation, including de-compaction of higher order chromatin structure and loss of topologically associated domain boundary insulation. Significant DNA hypomethylation at ER-enhancer elements was associated with gain in ER binding, creation of ectopic 3D enhancer-promoter interactions and concordant activation of ER-mediated transcription pathways. Importantly long-term withdrawal of epigenetic therapy partially restores methylation at ER-enhancer elements, resulting in loss of ectopic 3D enhancer-promoter interactions and associated gene repression. Our study illustrates how epigenetic therapy has potential to target ER+ endocrine-resistant breast cancer by DNA methylation-dependent rewiring of 3D chromatin interactions associated with suppression of tumour growth.

molecular biology↗

bioRxiv2021

A fast Myh super enhancer dictates adult muscle fiber phenotype through competitive interactions with the fast Myh genes

The contractile properties of adult myofibers are shaped by their Myosin heavy chain (MYH) isoform content. We identify by snATAC-seq a 42kb super-enhancer (SE) at the locus regrouping the fast Myh (fMyh) genes. By 4C-seq we show that active fMyh promoters interact with the SE by DNA looping, leading to the activation of a single promoter per nucleus. A rainbow mouse transgenic model of the locus including the SE recapitulates the endogenous spatio-temporal expression of adult fMyh genes. In situ deletion of the SE by CRISPR/Cas9 editing demonstrates its major role in the control of associated fMyh genes, and deletion of two fMyh genes at the locus reveals an active competition of the promoters for the shared SE. Last, by disrupting the organization of fMyh, we uncover positional heterogeneity within limb skeletal muscles that may underlie selective muscle susceptibility to damage in certain myopathies.

cell biology↗

bioRxiv2021

Genotype and age influence cortical-FKBP5 at multiple regulatory and single-cell-type levels in severe psychiatric disorders

Identification and characterisation of novel targets for treatment is a priority in the field of psychiatry. FKBP5 is a gene with decades of evidence suggesting its pathogenic role in a subset of psychiatric patients, with potential to be leveraged as a therapeutic target for these individuals. While it is widely reported that FKBP5/FKBP51 mRNA/protein (FKBP5/1) expression is impacted by psychiatric disease state, risk genotype and age, it is not known in which cell-types and sub-anatomical areas of the human brain this occurs. This knowledge is critical to propel FKBP5/1-targeted treatment development. Here, we performed an extensive, large-scale postmortem study (n=1024) of FKBP5/1 examining prefrontal cortex (BA9, BA11, BA24) derived from subjects that lived with schizophrenia, major depression or bipolar disorder. With an extensive battery of RNA (bulk RNA sequencing, single-nucleus RNA sequencing, microarray, qPCR, RNAscope) and protein (immunoblot, immunohistochemistry) analysis approaches, we thoroughly investigated the effects of disease-state, aging and genotype on cortical FKBP5/1 expression including in a cell-type specific manner. We identified consistently heightened FKBP5/1 levels in psychopathology and with age, but not genotype, with these effects strongest in schizophrenia. Using single-nucleus RNA sequencing (snRNAseq) and targeted histology, we established that these disease- and aging-effects on FKBP5/1 expression were most pronounced in excitatory supragranular neurons. We then found that this increase in FKBP5 levels likely impacts on synaptic plasticity, as FKBP5 gex levels strongly and inversely correlated with dendritic mushroom spine density and brain-derived neurotrophic factor (BDNF) levels in supragranular neurons. These findings pinpoint a novel cellular and molecular mechanism that has significant potential to open a new avenue of FKBP51 drug development to treat cognitive symptoms in psychiatric disorders.

neuroscience↗

bioRxiv2020

Enterococcal PrgA extends far outside the cell and provides surface exclusion to protect against unwanted conjugation

Horizontal gene transfer between Gram-positive bacteria leads to a rapid spread of virulence factors and antibiotic resistance. This transfer is often facilitated via Type 4 Secretion Systems (T4SS), which frequently are encoded on conjugative plasmids. However, donor cells that already contain a particular conjugative plasmid resist acquisition of a 2nd copy of said plasmid. They utilize different mechanisms, including surface exclusion for this purpose. Enterococcus faecalis PrgA, encoded by the conjugative plasmid pCF10, is a surface protein that has been implicated to play a role in both virulence and surface exclusion, but the mechanism by which this is achieved has not been fully explained. Here, we report the structure of full-length PrgA, which shows that PrgA protrudes far out from the cell wall (approximately 40 nm), where it presents a protease domain. In vivo experiments show that PrgA provides a physical barrier to cellular adhesion, thereby reducing cellular aggregation. This function of PrgA contributes to surface exclusion, reducing the uptake of its cognate plasmid by approximately one order of magnitude. Using variants of PrgA with mutations in the catalytic site we show that the surface exclusion effect is dependent on the activity of the protease domain of PrgA. In silico analysis suggest that PrgA can interact with another enterococcal adhesin, PrgB, and that these two proteins have co-evolved. PrgB is a strong virulence factor, and PrgA is involved in post-translational processing of PrgB. Finally, competition mating experiments show that PrgA provides a significant fitness advantage to plasmid-carrying cells.

biochemistry↗

bioRxiv2019

Ferroptotic pores induce Ca2+ fluxes and ESCRT-III activation to modulate cell death kinetics

Ferroptosis is an iron-dependent form of regulated necrosis associated with lipid peroxidation. Despite its key role in the inflammatory outcome of ferroptosis, little is known about the molecular events leading to the disruption of the plasma membrane during this type of cell death. Here we show that a sustained increase in cytosolic Ca2+ is a hallmark of ferroptosis that precedes complete bursting of the cell. We report that plasma membrane damage leading to ferroptosis is associated with membrane nanopores of few nanometers in radius and that ferroptosis, but not lipid peroxidation, can be delayed by osmoprotectants. Importantly, Ca2+ fluxes during ferroptosis correlate with the activation of ESCRT-III-mediated membrane repair, which counterbalances the kinetics of cell death and modulates the inflammatory signature of ferroptosis. Our findings with ferroptosis provide a unifying concept that sustained high levels of cytosolic Ca2+ prior to plasma membrane disruption are a common feature of regulated necrosis and position ESCRT-III as a general protective mechanism in these inflammatory cell death pathways.

cell biology↗

bioRxiv2019

Efficient chromosome-scale haplotype-resolved assembly of human genomes

Haplotype-resolved or phased sequence assembly provides a complete picture of genomes and complex genetic variations. However, current phased assembly algorithms either fail to generate chromosome-scale phasing or require pedigree information, which limits their application. We present a method that leverages long accurate reads and long-range conformation data for single individuals to generate chromosome-scale phased assembly within a day. Applied to three public human genomes, PGP1, HG002 and NA12878, our method produced haplotype-resolved assemblies with contig NG50 up to 25 Mb and phased [~]99.5% of heterozygous sites to 98-99% accuracy, outperforming other approaches in terms of both contiguity and phasing completeness. We demonstrate the importance of chromosome-scale phased assemblies to discover structural variants (SVs), including thousands of new transposon insertions, and of highly polymorphic and medically important regions such as HLA and KIR. Our improved method will enable high-quality precision medicine and facilitate new studies of individual haplotype variation and population diversity.

bioinformatics↗

bioRxiv2019

Air-interfaced colonization model suggests a commensal-like interaction of Neisseria meningitidis with the epithelium, which benefit from colonization by Streptococcus mitis.

Neisseria meningitidis is an inhabitant of the nasopharynx, from which it is transmitted from person to person or disseminates in the blood and becomes a harmful pathogen. In this work, we addressed the colonization of the nasopharyngeal niche by focusing on the interplay between meningococci and the mucus that lines the mucosa of the host. Using Calu-3 cells grown in air-interfaced culture, we studied the meningococcal colonization of the mucus and the host response. Our results suggested that N. meningitidis behaved like commensal bacteria in mucus, without interacting with human cells or actively transmigrating through the cell layer. As such, meningococci did not trigger a strong innate immune response from the Calu-3 cells. Finally, we have shown that this model is suitable for studying interaction of N. meningitidis with other bacteria living in the nasopharynx, and that Streptococcus mitis but not Moraxella catarrhalis can promote meningococcal growth in this model.

microbiology↗

bioRxiv2019

Common DNA sequence variation influences 3-dimensional conformation of the human genome

The 3-dimensional (3D) conformation of chromatin inside the nucleus is integral to a variety of nuclear processes including transcriptional regulation, DNA replication, and DNA damage repair. Aberrations in 3D chromatin conformation have been implicated in developmental abnormalities and cancer. Despite the importance of 3D chromatin conformation to cellular function and human health, little is known about how 3D chromatin conformation varies in the human population, or whether DNA sequence variation between individuals influences 3D chromatin conformation. To address these questions, we performed Hi-C on Lymphoblastoid Cell Lines (LCLs) from 20 individuals. We identified thousands of regions across the genome where 3D chromatin conformation varies between individuals and found that this conformational variation is often accompanied by variation in gene expression, histone modifications, and transcription factor (TF) binding. Moreover, we found that DNA sequence variation influences several features of 3D chromatin conformation including loop strength, contact insulation, contact directionality and density of local cis contacts. We mapped hundreds of Quantitative Trait Loci (QTLs) associated with 3D chromatin features and found evidence that some of these same variants are associated at modest levels with other molecular phenotypes as well as complex disease risk. Our results demonstrate that common DNA sequence variants can influence 3D chromatin conformation, pointing to a more pervasive role for 3D chromatin conformation in human phenotypic variation than previously recognized.

genomics↗

bioRxiv2018

Integration of phased Hi-C and molecular phenotype data to study genetic and epigenetic effects on chromatin looping

While genetic variation at chromatin loops is relevant for human disease, the relationships between loop strength, genetics, gene expression, and epigenetics are unclear. Here, we quantitatively interrogate this relationship using Hi-C and molecular phenotype data across cell types and haplotypes. We find that chromatin loops consistently form across multiple cell types and quantitatively vary in strength, instead of exclusively forming within only one cell type. We show that large haplotype loop imbalance is primarily associated with imprinting and copy number variation, rather than genetically driven traits such as allele-specific expression. Finally, across cell types and haplotypes, we show that subtle changes in chromatin loop strength are associated with large differences in other molecular phenotypes, with a 2-fold change in looping corresponding to a 100-fold change in gene expression. Our study suggests that regulatory genetic variation could mediate its effects on gene expression through subtle modification of chromatin loop strength.

genomics↗

bioRxiv2018

Integrating Hi-C links with assembly graphs for chromosome-scale assembly

Long-read sequencing and novel long-range assays have revolutionized de novo genome assembly by automating the reconstruction of reference-quality genomes. In particular, Hi-C sequencing is becoming an economical method for generating chromosome-scale scaffolds. Despite its increasing popularity, there are limited open-source tools available. Errors, particularly inversions and fusions across chromosomes, remain higher than alternate scaffolding technologies. We present a novel open-source Hi-C scaffolder that does not require an a priori estimate of chromosome number and minimizes errors by scaffolding with the assistance of an assembly graph. We demonstrate higher accuracy than the state-of-the-art methods across a variety of Hi-C library preparations and input assembly sizes. The Python and C++ code for our method is openly available at https://github.com/machinegun/SALSA\n\nAuthor summaryHi-C technology was originally proposed to study the 3D organization of a genome. Recently, it has also been applied to assemble large eukaryotic genomes into chromosome-scale scaffolds. Despite this, there are few open source methods to generate these assemblies. Existing methods are also prone to small inversion errors due to noise in the Hi-C data. In this work, we address these challenges and develop a method, named SALSA2. SALSA2 uses sequence overlap information from an assembly graph to correct inversion errors and provide accurate chromosome-scale assemblies.

bioinformatics↗