Biology subjects

Marcaida, M. J.

Publications and source records attributed to Marcaida, M. J..

12 recordsLinked to original sources

bioRxiv2026

Redox-regulated cysteine acylation governs β-lactam sensing by the Vibrio histidine kinase VbrK

The ability of pathogens to develop resistance mechanisms makes the continuous search for novel therapeutic targets indispensable. Resistance-activating systems are promising targets for restoring the efficacy of existing antibiotics. VbrK/VbrR is a two-component system from Vibrio parahaemolyticus reported as the first sensing system in gram-negatives that directly detects {beta}-lactam antibiotics. {beta}-Lactam-induced activation of this system results in the expression of the serine {beta}-lactamase CARB. In this study, we provide insights into the mechanism of {beta}-lactam binding to the periplasmic sensor domain of the histidine kinase VbrK. Our results demonstrate that the interaction depends on the redox state of cysteines C86 and C107, highlighting the role of disulfide bond dynamics in modulating ligand recognition. We further show that formation of a non-covalent complex leads to acylation of the sensor domain by {beta}-lactams, a modification that is slowly reversed through de-acylation, yielding the hydrolyzed {beta}-lactam ring and allowing for recovery from induction once the antibiotic has been depleted from the environment. Together, these findings reveal a previously unrecognized redox- and covalent chemistry-dependent mode of {beta}-lactam interaction with histidine kinases, providing a molecular framework to understand how VbrK detects and responds to {beta}-lactam antibiotics, and opening new avenues to prevent manifestation of resistance. Short broader audience statementBacterial pathogens can sense the presence of antibiotics and trigger resistance responses, making infections increasingly difficult to treat. In this study, we uncover a previously unknown mechanism by which {beta}-lactam antibiotics interact with a sensor protein that activates resistance in Vibrio parahaemolyticus. These findings pave the way for designing new compounds that can block this interaction and thus restore the effectiveness of {beta}-lactam antibiotics against gastrointestinal infections caused by resistant bacteria.

microbiology↗

bioRxiv2025

Sub-Nanomolar Detection and Discrimination of Microcystin Congeners Using Aerolysin Nanopores

Climate-driven disruptions in aquatic ecosystems are amplifying cyanotoxin production, threatening drinking and recreational water safety. Monitoring of these toxins is challenged by requirements of the low {micro}g/L detections limits and structural diversity. Here, we employ aerolysin nanopores to distinguish seven of the most prevalent microcystin congeners, both individually and in mixtures, at environmentally relevant concentrations. Importantly, we showed that aerolysin enables the detection of microcystins in different lake water samples at concentrations below the World Health Organizations intervention thresholds, reaching picomolar sensitivity. Moreover, combining experiments and molecular dynamics simulations, we further investigated the microcystin sensing mechanism, suggesting that the ionic current blockage is primarily governed by K238 in aerolysin, while dwell time is regulated by R220 constriction site. Our results open the way to use the nanopore sensing technology for real-time monitoring of microcystins in drinking water sources and surface waters.

bioengineering↗

bioRxiv2025

Molecular Regulation and Physiological Role of 1 GOLPH3-mediated Golgi retention

The Golgi complex serves as the central hub of the biosynthetic pathway, where anterograde and retrograde trafficking converge. How cargo and Golgi-resident proteins traverse this organelle has long been debated. Recent studies have identified a molecular machinery that sorts resident proteins into retrograde-directed COPI vesicles during cisternal maturation. Golgi phosphoprotein 3 (GOLPH3) is a key component of this system; however, its physiological relevance and regulatory mechanisms remain poorly defined. Here, we show that GOLPH3 depletion in mice disrupts both protein and lipid glycosylation, causes partially penetrant embryonic lethality, and severely impairs growth and bone mineralization. At the molecular level, we find that GOLPH3 is regulated by functionally antagonistic S-acylation events that control the topology of its membrane association. To mediate retrograde trafficking of Golgi-resident glycosyltransferases, GOLPH3 must bind their cytosolic tails. This occurs via a negatively charged surface region, which is correctly oriented only in one of the S-acylated GOLPH3 conformations. Together, these findings reveal a lipid-mediated regulatory mechanism for intra-Golgi trafficking and establish the critical role of GOLPH3 in vertebrate development.

cell biology↗

bioRxiv2024

Lumen charge governs ion transport in β-barrel nanopores

{beta}-barrel nanopores are involved in crucial biological processes, from ATP export in mitochondria to bacterial resistance, and represent a promising platform for emerging sequencing technologies. However, in contrast to ion channels, the understanding of the fundamental principles governing ion transport through these nanopores remains in its early stages. In this study, we integrate experimental, numerical, and theoretical approaches to elucidate ion transport mechanisms in these biological nanopores. We identify and characterise two distinct nonlinear phenomena: open-pore rectification and gating. Through extensive mutation analysis of aerolysin nanopores, we demonstrate that open-pore rectification is caused by ionic accumulation driven by the distribution of lumen charges. Additionally, we provide converging evidence suggesting that gating is controlled by electric fields dissociating counterions from lumen charges, promoting local structural deformations. Our findings establish a rigorous framework for the characterisation and understanding of biological ion transport processes, enabling the design of adaptable biosensors. We illustrate this by optimizing an aerolysin mutant for computing applications, paving the way for novel nanofluidic technologies.

biophysics↗

bioRxiv2024

Multiplexed lifetime imaging of single molecules with a gated single-photon camera

Fluorescence lifetime imaging microscopy (FLIM) is a powerful tool to discriminate fluorescent molecules or probe their nanoscale environment. Traditionally, FLIM uses time-correlated single-photon counting (TCSPC), which is precise but intrinsically low-throughput due to its dependence on point detectors. Although time-gated cameras have demonstrated the potential for high-throughput FLIM in bright samples with dense labeling, their use in single-molecule microscopy has not been explored extensively. Here, we report fast and accurate single-molecule FLIM with a commercial time-gated single-photon camera. Our optimized acquisition scheme achieves single-molecule lifetime measurements with a precision only about three times less than TCSPC, while imaging with a large number of pixels (512x512) allowing for the spatial multiplexing of over 3000 molecules. With this approach, we demonstrate parallelized lifetime measurements of large numbers of labeled pore-forming proteins on supported lipid bilayers, and temporal single-molecule Forster resonance energy transfer measurements at 5-25 Hz. This method holds considerable promise for the advancement of multi-target single-molecule localization microscopy and biopolymer sequencing.

biophysics↗

bioRxiv2024

Aerolysin nanopore structure revealed at high resolution in lipid environment

Aerolysin is a {beta}-pore-forming toxin produced by most Aeromonas bacteria which has attracted large attention in the field of nanopore sensing due to its narrow and charged pore lumen. Structurally similar proteins, belonging to the aerolysin-like family, are present throughout all kingdoms of life, but very few of them have been structurally characterized in a lipid environment. Here we present the first high-resolution atomic cryo-EM structures of aerolysin pre-pore and pore in a membrane-like environment. These structures allow the identification of key interactions, which are relevant for the pore formation and for positioning the pore barrel into the membrane with the anchoring {beta}-turn motif now finally observed. Moreover, we elucidate at high resolution the architecture of key mutations and precisely identify four constriction rings in the pore lumen that are highly relevant for nanopore sensing experiments.

biochemistry↗

bioRxiv2024

Exploring new nanopore sensors from the aerolysin family

Aerolysin-like proteins are a family of {beta}-pore-forming toxins which are widely present in all kingdoms of life. Recently, this family of proteins is gaining attention because of their biotechnological application as nanopore sensors for sensing and sequencing of biomolecules. Here, we explore the possibilities of using the knowledge of the sequence and structure of proteins to screen and explore new potential nanopore candidates. However, in spite of the conserved structural fold, the sequence identity in this family is very low. This complicates their sequence alignment, hindering the understanding of their pore structure and properties, therefore limiting further biotechnological applications. In an attempt to further understand the properties of aerolysin-like pores, we analyzed the pore structure of three family members, Clostridium perfringens epsilon toxin (ETX), Laetiporus sulphureus lectin (LSL) and Bacillus thuringiensis parasporin-2, comparing it to aerolysin. Their structure and sensing capabilities for ssDNA were first assessed by in silico methods. Moreover, ETX was characterized experimentally in planar lipid membranes for the detection of biomolecules. We found that ETX can form three distinct pore conformations, each presenting a specific open pore current, and only one of them being able to translocate ssDNA. When the ssDNA translocate through ETX, the depth of current blockage is higher compared to aerolysin which indicates a higher sensitivity for molecular sensing. Our findings open a new venue for improving and diversifying nanopore capabilities for molecular sensing.

biophysics↗

bioRxiv2023

Missense mutations in CMS22 patients reveal that PREPL has both enzymatic and non-enzymatic functions

Congenital myasthenic syndrome-22 (CMS22) is a rare genetic disorder caused by mutations in the prolyl endopeptidase-like (PREPL) gene. Since previous reports only described patients with deletions and nonsense mutations in PREPL, nothing is known about the effect of missense mutations in the pathology of CMS22. In this study, we have characterized missense mutations in PREPL in three CMS22 patients, all with hallmark phenotypes. Biochemical evaluation revealed that these missense mutations do not impair hydrolase activity, thereby challenging the conventional diagnostic criteria. Structural analysis shows that the mutations affect regions most likely involved in intra-protein or protein-protein interactions. Indeed, binding to a selected group of known interactors was differentially reduced for the three mutants. The importance of non-hydrolytic functions of PREPL was investigated in catalytically inactive PREPL p.Ser559Ala cell lines which showed that hydrolytic activity of PREPL is needed for normal mitochondrial function but not for regulating AP1-mediated transport in the trans-Golgi network. In conclusion, these studies show that CMS22 can be caused not only by deletion and truncation of PREPL but also by missense mutations that do not necessarily result in a loss of hydrolytic activity of PREPL. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=111 SRC="FIGDIR/small/572145v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@13dc0bborg.highwire.dtl.DTLVardef@13865d6org.highwire.dtl.DTLVardef@1a5ed3borg.highwire.dtl.DTLVardef@79fdf5_HPS_FORMAT_FIGEXP M_FIG C_FIG

genetics↗

bioRxiv2021

Viral tolerance in Aedes aegypti relies on the negative cooperativity between Toll5A and the cytokine ligand Spz1C

Aedes aegypti has evolved to become an efficient vector for arboviruses but the mechanisms of host-pathogen tolerance are unknown. Immunoreceptor Toll and its ligand Spaetzle have undergone duplication which may allow neofunctionalization and adaptation. Here we present cryo-EM structures and biophysical characterisation of low affinity Toll5A complexes that display transient but specific interactions with Spaetzle1C, forming asymmetric complexes, with only one ligand clearly resolved. Loop structures of Spaetzle1C and Toll5A intercalate, temporarily bridging the receptor C-termini to promote signalling. By contrast unbound receptors form head-to-head homodimers that keep the juxtamembrane regions far apart in an inactive conformation. Interestingly the transcriptional signature of Spaetzle1C differs from other Spaetzle cytokines and controls genes involved in innate immunity, metabolism and tissue regeneration. Taken together our results explain how upregulation of Spaetzle1C in the midgut and Toll5A in the salivary gland shape the concomitant immune response.

immunology↗

bioRxiv2020

Molecular mode of action of an Acyl Protein thioesterase

Many biochemical reactions occur at the membrane interfaces. The proper control of these reactions requires spatially and temporally controlled recruitment of protein complexes. These assemblies are largely regulated by post-translational modifications and a frequent one is S-acylation, which consists of the addition of medium length acyl chains. Reversibility of this modification is ensured by acyl protein thioesterases (APTs), which are poorly understood enzymes. Using a combination of computational, structural, biochemical, and cellular approaches, we dissect the mode of action of a major cellular thioesterase, APT2 (LYPLA2). We show that for APT2 to encounter its targets, it must interact with membranes by two consecutive steps, the insertion of a hydrophobic loop and subsequent S-acylation by the ZDHHC3 or ZDHHC7 palmitoyltransferases. Once bound, APT2 deforms the lipid bilayer to extract the acyl chain bound to its substrate, capturing it in a hydrophobic pocket and allowing hydrolysis. Deacylation releases APT2, allowing it to bind to other membranes, but also renders it vulnerable to ubiquitination and proteasomal degradation. This molecular understanding of APT2 paves the way to understand the dynamics of APT2-mediated depalmitoylation throughout the endomembrane system.

cell biology↗

bioRxiv2019

Btk SH2-kinase interface is critical for allosteric kinase activation and its targeting inhibits B-cell neoplasms

Brutons tyrosine kinase (Btk) is a key component for B-cell maturation and activation. Btk loss-of-function mutations cause human X-linked agammaglobulinemia (XLA). In contrast, constitutive Btk signaling drives several B-cell neoplasms, which may be treated with tyrosine kinase inhibitors (TKIs). Here, we uncovered the molecular mechanism by which a subset of XLA mutations in the SH2 domain strongly perturbs Btk activation. Using a combination of molecular dynamics (MD) simulations and small-angle X-ray scattering (SAXS), we discovered an allosteric interface between the SH2 and kinase domain to which multiple XLA mutations map and which is required for Btk activation. As allosteric interactions provide unique targeting opportunities, we developed an engineered repebody protein binding to the Btk SH2 domain and able to disrupt the SH2-kinase interaction. The repebody prevented activation of wild-type and TKI-resistant Btk, inhibited Btk-dependent signaling and proliferation of malignant B-cells. Therefore, the SH2-kinase interface is critical for Btk activation and a targetable site for allosteric inhibition.

cancer biology↗

bioRxiv2019

The polyQ expansion modulates the configuration and phosphorylation of huntingtin

The polyQ-expansion at the N-terminus of huntingtin (HTT) is the prime cause of Huntingtons disease. The recent cryo-EM structure of HTT with HAP40 provides information on the proteins prominent HEAT-repeats. Here, we present analyses of the impact of polyQ-length on the conformation of HTT by cryo-EM, the domain-interactions by cross-linking mass spectrometry and the phosphorylation of HTT. The cryo-EM analysis of normal (Q23-) and disease (Q78-) type HTTs in their apo forms shows that the structures of apo HTTs significantly differ from the structure of HTT-HAP40, and that the polyQ expansion induces global structural changes consisting of significant domain movements of the C-HEAT domain relative to the N-HEAT domain. In addition, we show that the polyQ-expansion alters the phosphorylation pattern across the full-length HTT and that the specific phosphorylation (Ser2116p) in turn affects the global structure of HTT, which influences the activity of polyQ-expanded HTT. These results provide a molecular basis for the effect of the N-terminal polyQ segment on HTT structure and activity, that may be important for the cell-selective toxicity of mutant HTT.

neuroscience↗