Biology subjects

Reuter, N.

Publications and source records attributed to Reuter, N..

11 recordsLinked to original sources

bioRxiv2025

Unveiling the Multifaceted Networks of the Left DLPFC for Precision TMS Targeting

The left dorsolateral prefrontal cortex (lDLPFC) is the standard transcranial magnetic stimulation (TMS) target for treatment-resistant depression (TRD), yet non-response rates remain high. TMS efficacy has been linked to the stimulation sites functional connectivity, particularly its anti-correlation with the subgenual cingulate cortex (SGC). While this pragmatic strategy has demonstrated clinical utility, it offers limited insight into how the lDLPFCs network interactions contribute to site-dependent variability in treatment response. Here, we used connectivity-based parcellation within a region of interest encompassing common TMS targets in the left prefrontal cortex (TMS-PFC) to delineate functionally distinct subregions and characterize their large-scale network connectivity and behavioral associations. Our results revealed a hierarchical organization: a coarse two-pole antagonism between anterior-central and superior-posterior subregions and a finer nine-cluster architecture exposing the heterogeneity along anterior-posterior and ventral-dorsal axes within TMS-PFC. Anterior-central areas were strongly anti-correlated with SGC and default-mode network, positively connected with salience, dorsal attention, and control networks, and associated with cognitive control. In contrast, superior-posterior subregions displayed the inverse pattern, while ventral clusters engaged somatomotor and visual networks, and language-related processes. Central and superior-anterior clusters showed differentiated profiles, including associations with inhibition, social cognition, and perceptual functions. To aid clinical translation, we derived a likelihood map integrating granularities, highlighting the anterior-central subregion as the strongest TMS candidate given its connectivity and behavioral relevance in depression, while indicating that neighboring subregions have distinct functions. These findings underscore the hierarchical and heterogeneous organization of TMS-PFC and provide a network-informed reference for developing individualized, symptom-specific TMS interventions. HighlightsO_LITMS targets within the left PFC encompass nine functionally distinct subregions C_LIO_LISubregions differ in SGC, DMN, salience, attention, and control network connectivity C_LIO_LIAnterior-central and superior-posterior subregions show antagonistic profiles C_LIO_LIA likelihood map identifies the anterior-central subregion as optimal TMS target C_LIO_LIFindings provide a network-informed reference for symptom-specific TMS targeting C_LI

neuroscience↗

bioRxiv2023

A system-wide analysis of lipid transfer proteins delineates lipid mobility in human cells

Lipid transfer proteins (LTPs) maintain the specialised lipid compositions of biological membranes, and many are associated with disease. In eukaryotes, they support organellar functions by transporting lipids between compartmentalised metabolic pathways. However, for the majority of the hundreds of human LTPs, the cargoes remain unknown. We combined biochemical, lipidomic and computational methods to characterize LTP-lipid complexes assembled in cellulo and in an in vitro biochemical assay. We identified bound lipids for about half of the LTPs analysed, and confirmed known cargoes, while discovering new ones for most LTP families. The data represents a systematic resource that captures the general principles of non-vesicular lipid transport in humans. The specificity of LTPs for lipids involves not only the recognition of specific head groups, but also of specific acyl chains. This selectivity defines lipid species within a lipid class with different metabolic or functional fates. The generalised ability of LTPs to form complexes with more than one class of lipids delineates new relationships between lipids and regulatory mechanisms that may contribute to the coordination of metabolism between different organelles. This work represents a resource and a framework for further analyses in different cell types, in pathological states or following various cellular perturbations.

biochemistry↗

bioRxiv2023

A Mechanistic Model for the Release of Ceramide from the CERT START Domain

Ceramide transfer protein CERT is the mediator of non-vesicular transfer of ceramide from ER to Golgi. In CERT, START is the domain responsible for the binding and transport of ceramide. A wealth of structural data has revealed a helix-grip fold surrounding a large hydrophobic holding the ceramide. Yet little is known about the mechanisms by which START releases the ceramide through the polar region and into the packed environment of cellular membranes. As such events do not lend themselves easily to experimental investigations we used multiple unbiased microsecond-long molecular simulations. We propose a membrane-assisted mechanism in which the passage of the ceramide acyl chains is facilitated by the intercalation of a single phosphatidylcholine lipid in the cavity, practically greasing the ceramide way out. We verify using experimental lipidomics data that CERT forms stable complexes with phosphatidylcholine lipids, in addition to ceramide, thus providing a validation for the proposed computational model.

biophysics↗

bioRxiv2023

Membrane specificity of the human cholesterol transfer protein STARD4

STARD4 regulates cholesterol homeostasis by transferring cholesterol between plasma membrane and endoplasmic reticulum. The STARD4 structure features a helix-grip fold surrounding a large hydrophobic cavity holding the sterol. Its access is controlled by a gate formed by two flexible loops - {Omega}1 and {Omega}4- and the C-terminal -helix. Besides this, little is known about the mechanisms by which STARD4 binds to membranes and extract/releases cholesterol. All available structures of STARD4 are without a bound sterol and display the same closed conformation of the gate. The cholesterol transfer activity of the mouse STARD4 is enhanced in the presence of anionic lipids, and in particular of phosphatidylinositol biphosphates (PIP2) for which two binding sites were proposed on the mouse STARD4 surface. Yet only one of these sites is conserved in human STARD4. We here report the results of a liposome microarray-based assay and microseconds-long molecular dynamics simulations of apo-and holo forms of human STARD4 with complex lipid bilayers mimicking the composition of the donor and acceptor membranes. We show that the binding of apo form of human STARD4 is sensitive to the presence of PIP2 through two specific binding sites, one of which was not identified on mouse STARD4. We report two novel conformations of the gate in holo-STARD4: a yet-unobserved close conformation and an open conformation of {Omega}4 shedding light on the opening/closure mechanism needed for cholesterol uptake/release. Overall, the modulation of human STARD4 membrane-binding by lipid composition, and by the presence of the cargo supports the capacity of human STARD4 to achieve directed transfer between specific organelle membranes.

molecular biology↗

bioRxiv2023

Expression and purification of human neutrophil proteinase 3 from insect cells and characterization of ligand binding

Neutrophil proteinase 3 (PR3) is an important drug target for inflammatory lung diseases such as chronic obstructive pulmonary disease and cystic fibrosis. Drug discovery efforts targeting PR3 require active enzyme for in vitro characterization, such as inhibitor screening, enzymatic assays, and structural studies. Recombinant expression of active PR3 overcomes the need for enzyme supplies from human blood and in addition allows studies on the influence of mutations on enzyme activity and ligand binding. Here, we report the expression of recombinant PR3 (rPR3) using a baculovirus expression system. The purification and activation process described resulted in highly pure and active PR3. The activity of rPR3 in the presence of commercially available inhibitors was compared with human PR3 by using a fluorescence-based enzymatic assay. Purified rPR3 had comparable activity to the native human enzyme, thus being a suitable alternative for enzymatic studies in vitro. Further, we established a surface plasmon resonance-based assay to determine binding affinities and kinetics of PR3 ligands. These methods provide valuable tools for early drug discovery aiming towards treatment of lung inflammation.

biochemistry↗

bioRxiv2022

Dissecting peripheral protein-membrane interfaces

Peripheral membrane proteins (PMPs) include a wide variety of proteins that have in common to bind transiently to the chemically complex interfacial region of membranes through their interfacial binding site (IBS). In contrast to protein-protein or protein-DNA/RNA interfaces, peripheral protein-membrane interfaces are poorly characterized. We collected a dataset of PMP domains representative of the variety of PMP functions: membrane-targeting domains (Annexin, C1, C2, discoidin C2, PH, PX), enzymes (PLA, PLC/D) and lipid-transfer proteins (START). The dataset contains 1328 experimental structures and 1194 AphaFold models. We mapped the amino acid composition and structural patterns of the IBS of each protein in this dataset, and evaluated which were more likely to be found at the IBS compared to the rest of the domains accessible surface. In agreement with earlier work we find that about two thirds of the PMPs in the dataset have protruding hydrophobes (Leu, Ile, Phe, Tyr, Trp and Met) at their IBS. The three aromatic amino acids Trp, Tyr and Phe are a hallmark of PMPs IBS regardless of whether they protrude on loops or not. This is also the case for lysines but not arginines suggesting that, unlike for Arg-rich membrane-active peptides, the less membrane-disruptive lysine is preferred in PMPs. Another striking observation was the over-representation of glycines at the IBS of PMPs compared to the rest of their surface, possibly procuring IBS loops a much-needed flexibility to insert in-between membrane lipids. The analysis of the 9 superfamilies revealed amino acid distribution patterns in agreement with their known functions and membrane-binding mechanisms. Besides revealing novel amino acids patterns at protein-membrane interfaces, our work contributes a new PMP dataset and an analysis pipeline that can be further built upon for future studies of PMPs properties, or for developing PMPs prediction tools using for example, machine learning approaches.

bioinformatics↗

bioRxiv2021

Specificity of Loxosceles α clade phospholipase D enzymes for choline-containing lipids: role of a conserved aromatic cage

Spider venom GDPD-like phospholipases D (SicTox) have been identified to be one of the major toxins in recluse spider venom. They are divided into two major clades: the clade and the {beta} clade. Most clade toxins present high activity against lipids with choline head groups such as sphingomyelin, while activities in {beta} clade toxins vary and include preference for substrates containing ethanolamine headgroups (Sicarius terrosus, St_{beta}IB1). A structural comparison of available PLDs structures reveals a conserved aromatic cage in the clade. To test the potential influence of the aromatic cage on membrane-lipid specificity we performed molecular-dynamics (MD) simulations of the binding of several PLDs onto lipid bilayers containing choline headgroups; two SicTox from the clade, Loxosceles intermedia IA1 (Li_IA) and Loxosceles laeta III1 (Ll_III1), and one from the {beta} clade, St_{beta}IB1. The simulation results reveal that the aromatic cage captures a choline-headgroup and suggest that the cage plays a major role in lipid specificity. We also simulated an engineered St_{beta}IB1, where we introduced the aromatic cage, and this led to binding with choline-containing lipids. Moreover, a multiple sequence alignment revealed the conservation of the aromatic cage among the clade PLDs. Here, we confirmed the membrane binding site of and {beta} clade PLDs on choline and ethanolamine-containing bilayers, respectively. Furthermore, our results suggest a major role in choline lipid recognition of the aromatic cage of the clade PLDs. The MD simulation results are supported by in vitro liposome binding assay experiments.

bioinformatics↗

bioRxiv2020

Classification and phylogeny for the annotation of novel eukaryotic GNAT acetyltransferases

The enzymes of the GCN5-related N-acetyltransferase (GNAT) superfamily count more than 870 000 members through all kingdoms of life and share the same structural fold. GNAT enzymes transfer an acyl moiety from acyl coenzyme A to a wide range of substrates including aminoglycosides, serotonin, glucosamine-6-phosphate, protein N-termini and lysine residues of histones and other proteins. The GNAT subtype of protein N-terminal acetyltransferases (NATs) alone targets a majority of all eukaryotic proteins stressing the omnipresence of the GNAT enzymes. Despite the highly conserved GNAT fold, sequence similarity is quite low between members of this superfamily even when substrates are similar. Furthermore, this superfamily is phylogenetically not well characterized. Thus functional annotation based on homology is unreliable and strongly hampered for thousands of GNAT members that remain biochemically uncharacterized. Here we used sequence similarity networks to map the sequence space and propose a new classification for eukaryotic GNAT acetyltransferases. Using the new classification, we built a phylogenetic tree, representing the entire GNAT acetyltransferase superfamily. Our results show that protein NATs have evolved more than once on the GNAT acetylation scaffold. We use our classification to predict the function of uncharacterized sequences and verify by in vitro protein assays that two fungi genes encode NAT enzymes targeting specific protein N-terminal sequences, showing that even slight changes on the GNAT fold can lead to change in substrate specificity. In addition to providing a new map of the relationship between eukaryotic acetyltransferases the classification proposed constitutes a tool to improve functional annotation of GNAT acetyltransferases. Author SummaryEnzymes of the GCN5-related N-acetyltransferase (GNAT) superfamily transfer an acetyl group from one molecule to another. This reaction is called acetylation and is one of the most common reactions inside the cell. The GNAT superfamily counts more than 870 000 members through all kingdoms of life. Despite sharing the same fold the GNAT superfamily is very diverse in terms of amino acid sequence and substrates. The eight N-terminal acetyltransferases (NatA, NatB, etc.. to NatH) are a GNAT subtype which acetylates the free amine group of polypeptide chains. This modification is called N-terminal acetylation and is one of the most abundant protein modifications in eukaryotic cells. This subtype is also characterized by a high sequence diversity even though they share the same substrate. In addition the phylogeny of the superfamily is not characterized. This hampers functional annotation based on homology, and discovery of novel NATs. In this work we set out to solve the problem of the classification of eukaryotic GCN5-related acetyltransferases and report the first classification framework of the superfamily. This framework can be used as a tool for annotation of all GCN5-related acetyltransferases. As an example of what can be achieved we report in this paper the computational prediction and in vitro verification of the function of two previously uncharacterized N-terminal acetyltransferases. We also report the first acetyltransferase phylogenetic tree of the GCN5 superfamily. It indicates that N-terminal acetyltransferases do not constitute one homogeneous protein family, but that the ability to bind and acetylate protein N-termini had evolved more than once on the same acetylation scaffold. We also show that even small changes in key positions can lead to altered enzyme specificity.

bioinformatics↗

bioRxiv2018

Dynamics-function relationship of N-terminal acetyltransferases: the β6β7 loop modulates substrate accessibility to the catalytic site

N-terminal acetyltransferases (NATs) are enzymes catalysing the transfer of the acetyl from Ac-CoA to the N-terminus of proteins, one of the most common protein modifications. Unlike NATs, lysine acetyltransferases (KATs) transfer an acetyl onto the amine group of internal lysines. To date, not much is known on the exclusive substrate specificity of NATs towards protein N-termini. All the NATs and some KATs share a common fold called GNAT. The main difference between NATs and KATs is an extra hairpin loop found only in NATs called {beta}6{beta}7 loop. It covers the active site as a lid. The hypothesized role of the loop is that of a barrier restricting the access to the catalytic site and preventing acetylation of internal lysines. We investigated the dynamics-function relationships of all available structures of NATs covering the three domains of life. Using elastic network models and normal mode analysis, we found a common dynamics pattern conserved through the GNAT fold; a rigid V-shaped groove, formed by the {beta}4 and {beta}5 strands and three relatively more dynamic loops 12, {beta}3{beta}4 and {beta}6{beta}7. We identified two independent dynamical domains in the GNAT fold, which is split at the {beta}5 strand. We characterized the {beta}6{beta}7 hairpin loop slow dynamics and show that its movements are able to significantly widen the mouth of the ligand binding site thereby influencing its size and shape. Taken together our results show that NATs may have access to a broader ligand specificity range than anticipated.\n\nAuthor summaryN-terminal acetylation concerns 80% of eukaryotic proteins and is achieved by enzymes called the N-terminal acetyltransferases (NATs). They belong to the large family of acetyltransferases and adopt the GNAT fold. Interestingly most lysine acetyltransferases (KATs), which acetylate specifically internal lysines, share the same fold. Rationale for the ligand recognition by the GNAT enzymes remains unclear. Proteins are dynamic entities that utilize their structural flexibility to carry out functions in living cells. By studying the dynamics throughout the entire NATs family, we found that the slow dynamics of the fold is strongly conserved. We also revealed the mobility of the active site lid, namely the {beta}-hairpin loop {beta}6{beta}7, which is one of the main structural differences between the NATs and the KATs. The size and shape of the ligand binding site depend on movements of that {beta}-hairpin loop. We suggest that in attempts of mapping NATs specificity or ligand design the fold flexibility should be taken into consideration.

bioinformatics↗

bioRxiv2018

ZIKA VIRUS INFECTION AT MID-GESTATION RESULTS IN FETAL CEREBRAL CORTICAL INJURY AND FETAL DEATH IN THE OLIVE BABOON.

Zika virus (ZIKV) infection during pregnancy in humans is associated with an increased incidence of congenital anomalies including microcephaly as well as fetal death and miscarriage and collectively has been referred to a Congenital Zika Syndrome (CZS). Animal models for ZIKV infection in pregnancy have been developed including mice and macaques. While microcephaly has been achieved in mice via direct injection of ZIKV into the fetal brain or via interference with interferon signaling, in macaques the primary fetal CZS outcome are ocular defects. In the present study we develope the olive baboon (Papio anubis), as a model for the vertical transfer of ZIKV during pregnancy. We infected four mid-gestation, timed-pregnant baboons with the French Polynesian ZIKV isolate (104 ffu) and examined the acute phase of vertical transfer by stopping the study of one dam at 7 days post infection (dpi), two at 14 dpi and one at 21 dpi. All dams exhibited mild to moderate rash and conjunctivitis; three of four dams exhibited viremia at 7 dpi. Of the three dams studied to 14 to 21 days, only one still exhibited viremia on day 14. Vertical transfer of ZIKV to the fetus was found in two pregnancies; in one, vertical transfer was associated with fetal death at [~]14 dpi. In the other, vertical transfer was observed at 21 dpi. Both fetuses had ZIKV RNA in the fetal cerebral cortex as well as other tissues. The 21 dpi fetal cerebral cortex exhibited notable defects in radial glia, radial glial fibers, loss and or damage of immature oligodendrocytes and a loss in neuroprogenitor cells (NPCs). In addition, indices of pronounced neuroinflammation were observed including astrogliosis, increased microglia and IL-6 expression. The dams studied to 14 dpi (n=2) and 21 dpi (n=1) exhibited a anti-ZIKV IgM response and IgG response (21 dpi) that included transfer of the IgG to the fetal compartment (cord blood). The severity of systemic inflammatory response (cytokines and chemokines) reflected the vertical transfer of ZIKV in the two pregnancies. As such, these events likely represent the early mechanisms that lead to microcephaly and/or other CNS pathologies in a primate infected with ZIKV and are the first to be described in a non-human primate during the acute phase of ZIKV infection with a contemporaneous ZIKV strain. The baboon thus represents a major NHP for advancing as a model for ZIKV induced brain pathologies to contrast and compare to humans as well as other NHPs such as macaques.\n\nAUTHOR SUMMARYZika virus is endemic in the Americas, primarily spread through mosquitos and sexual contact. Zika virus infection during pregnancy in women is associated with a variety of fetal pathologies now referred to as Congenital Zika Syndrome (CZS), with the most severe pathology being fetal microcephaly. Developing model organisms that faithfully recreate Zika infection in humans is critical for future development of treatments and preventions. In our present study, we infected Olive baboons at mid-gestation with Zika virus and studied the acute period of viremia and transfer of Zika virus to the fetus during the first three weeks after infection to better understand the timing and mechanisms leading to CZS. We observed Zika virus transfer to fetuses resulting in fetal death in one pregnancy and in a second pregnancy, significant damage to the frontal cortex of the fetal brain consistent with development of microcephaly, closely resembling infection in pregnant women. Our baboon model differs from macaque non-human primate models where the primary fetal outcome during pregnancy following infection with contemporary strains of Zika virus is ocular pathology. Thus, the baboon provides a promising new non-human primate model to further compare and contrast the consequences of Zika virus infection in pregnancy to humans and macaques to better understand the disease.

neuroscience↗

bioRxiv2017

Task-related effective connectivity reveals that the cortical rich club gates cortex-wide communication

Higher cognition may require the globally coordinated integration of specialized brain regions into functional networks. A collection of structural cortical hubs - referred to as the rich club - has been hypothesized to support task-specific functional integration. In the present paper, we use a whole-cortex model to estimate directed interactions between 68 cortical regions from fMRI activity for four different tasks (reflecting different cognitive domains) and resting state. We analyze the state-dependent input and output effective connectivity of the structural rich club and relate these to whole-cortex dynamics and network reconfigurations. We find that the cortical rich club exhibits an increase in outgoing effective connectivity during task performance as compared to rest while incoming connectivity remains constant. Increased outgoing connectivity targets a sparse set of peripheral regions with specific regions strongly overlapping between tasks. At the same time, community detection analyses reveal massive reorganizations of interactions among peripheral regions, including those serving as target of increased rich club output. This suggests that while peripheral regions may play a role in several tasks, their concrete interplay might nonetheless be task-specific. Furthermore, we observe that whole-cortex dynamics are faster during task as compared to rest. The decoupling effects usually accompanying faster dynamics appear to be counteracted by the increased rich club outgoing effective connectivity. Together our findings speak to a gating mechanism of the rich club that supports fast-paced information exchange among relevant peripheral regions in a task-specific and goal-directed fashion, while constantly listening to the whole network.

neuroscience↗