Biology subjects

Vadas, O.

Publications and source records attributed to Vadas, O..

14 recordsLinked to original sources

bioRxiv2026

A transporter essential for apical secretory organelles homeostasis in Toxoplasma gondii

Apicomplexan parasites rely on the rhoptries and micronemes to coordinate egress, motility, and host cell invasion. In Toxoplasma gondii, mechanistic insights into the control of these organelles lipid composition remain sparse. Although nearly 60 T. gondii Major Facilitator Superfamily (MFS) are predicted, many remain functionally uncharacterized. The MFS protein HAP12 was previously annotated as an apicoplast protein contributing to invasion and organelle inheritance. Based on localization and phylogenetic analyses, we show that HAP12 (renamed TFP5), is predominantly associated with the Golgi, with additional signal in the ELC and apical compartments, without convincing evidence of a major apicoplast-associated pool. Furthermore, TFP5 clusters with a subgroup of MFS transporter facilitator proteins (TFPs) involved in the morphological integrity of secretory apical organelles. TFP5 depletion results in defective microneme secretion and pronounced alterations in rhoptry morphology and positioning. Structural analyses revealed that TFP5 is strongly similar to MFSD2-like lipid transporters, while lipidomic analyses showed substantial changes in parasite lipid composition following TFP5 depletion. Together, these findings support a model in which TFP5 regulates lipid homeostasis required for the function of apical secretory organelles, thereby critically contributing to parasite virulence.

microbiology↗

bioRxiv2026

RNase E resolves toxic condensates by counteracting phase separation of Type II RhlB helicases

In many Proteobacteria, the RNA helicase RhlB is a component of the RNA degradosome, a multi-protein complex involved in RNA processing and degradation. Within this complex, RhlB interacts with the scaffold endoribonuclease RNase E. In Escherichia coli, allosteric activation of RhlB by RNase E has defined the current paradigm for RhlB regulation. Here we identify a distinct clade of RhlB helicases, exemplified by Pseudomonas aeruginosa RhlB, which we designate Type II. Unlike Type I RhlB, Type II RhlB helicases contain an N-terminal intrinsically disordered region that drives RNA-dependent liquid-liquid phase separation and enhances RhlB activity. Biochemical, structural, and functional analyses show that P. aeruginosa RNase E binds RhlB through an interface distinct from that described in the E. coli model and, rather than stimulating activity, antagonizes RhlB phase separation. Excessive RhlB condensation impairs bacterial growth at low temperature, and RNase E-mediated control of RhlB condensation maintains growth under these conditions. Together, these findings reveal that conserved RNA degradosome components can engage in distinct regulatory interactions across species and identify condensate dissolution as a novel mechanism regulating RNA helicase activity.

microbiology↗

bioRxiv2026

Molecular basis of mitogen-activated protein kinase ERK2 activation by its upstream kinase MEK1

The RAS-RAF-MEK-ERK mitogen-activated protein kinase (MAPK) pathway relays extracellular signals into a cellular response and its dysregulation leads to many pathologies, particularly cancer. Here, we determined cryo-EM structures of the MAP2K MEK1 activating its substrate MAPK ERK2, the final event in the cascade. We define the molecular details of specificity and phosphoryl transfer to the tyrosine of the ERK2 activation loop and examine the mechanism of substrate recognition using solution techniques and molecular dynamics. Binding of the substrate MAPK leads to release of the MAP2K catalytic machinery, a mechanism enhanced in many gain of function disease mutations. Further, we observe that ERK2 release is not required for nucleotide exchange, suggesting how a processive mechanism could proceed. Our data advance the understanding of MAPK signalling and provide a starting point for drug development. One-Sentence SummaryCryo-EM structures of the MEK1-ERK2 complex reveal details of cellular signal transmission.

molecular biology↗

bioRxiv2026

MyoL-Dependent Coupling of Preconoidal Rings to Conoid Is Required for Motility in Toxoplasma gondii

Apicomplexan parasites are unified by their apical complex, a structure of cytoskeletal elements and secretory organelles. At its core lies the conoid, made of spiraling tubulin fibers essential for parasite motility and invasion. In Toxoplasma gondii, conoid extrusion and retraction through the apical polar ring is powered by Myosin H, ultimately regulating gliding motility. The conoid is capped by three preconoidal rings (PCRs), critical for extrusion and motility as they anchor formin 1, which produces filamentous actin needed for both processes. We demonstrate here that PCR composition and stability differ between daughter and mature cells and that Pcr2 and Pcr3 adopt a half-ring localization. The non-functional head containing Myosin-like protein MyoL, a previously unrecognized PCR component, is essential for gliding motility and invasion. Its depletion partially detaches PCRs from the conoid, highlighting its role as a mechanical tether. Targeted mutagenesis identifies the neck and tail regions of MyoL as critical for localization and function. Together, these findings illuminate the architecture and functional organization of the PCR in T. gondii, a structure conserved across several Apicomplexa.

microbiology↗

bioRxiv2025

Structural and dynamic basis of indirect apoptosis inhibition by Bcl-xL: a case study with Bid

Intrinsic apoptosis is a form of cell death which is activated, executed and inhibited by the Bcl-2 protein family. The structural basis of the inhibition mechanisms remains elusive. Here, we characterize the ensemble structural model of the inhibitory Bcl-xL/tBid complex at the mitochondrial membrane by probing inter-residue distances and dynamic solvent accessibilities complemented by integrative modelling and molecular dynamics simulations. We show that Bcl-xL and tBid form a heterodimer anchored to the membrane by the C-terminal helix of Bcl-xL. The BH3 domain of tBid is wedged between the exposed hydrophobic groove of Bcl-xL and the membrane headgroups, while tBids C-terminal helices remain dynamically engaged with the bilayer. This dynamic architecture sheds light on the mechanism of indirect inhibition of apoptosis. Significance StatementProgrammed cell death, or apoptosis, is a fundamental process that eliminates damaged cells. However, cancer cells often evade this process by overexpressing anti-apoptotic Bcl-2 family proteins, which neutralise their pro-apoptotic counterparts. The structural details of the molecular mechanisms governing this inhibition have remained elusive and up to now, only partial structures could be obtained with high-resolution methods. In this study, we provide the structural model of the inhibitory complex formed by the anti-apoptotic protein Bcl-xL and the pro-apoptotic protein Bid at the mitochondrial membrane using experimental constraints and molecular dynamics simulations. Our work provides the structural basis of one of the inhibitory mechanisms of cell death, offering a new framework for developing strategies to overcome therapeutic resistance in cancer.

biophysics↗

bioRxiv2024

RNG2 tethers the conoid to the apical polar ring in Toxoplasma gondii: a key mechanism in parasite motility and invasion

In Toxoplasma gondii, the conoid is a dynamic organelle composed of spiraling tubulin fibers that extrudes during egress, gliding motility, and invasion. This organelle traverses the apical polar ring (APR) in response to calcium waves and plays a critical role in controlling parasite motility. While the actomyosin-dependent extrusion of the conoid is beginning to be understood, the mechanism by which it is anchored apically to the APR remains unclear. RNG2, a protein localized at both the conoid and the APR, has emerged as a key candidate for this function. By combining iterative ultrastructure expansion microscopy and immunoelectron microscopy we discovered that RNG2 forms 22 tethers between the APR and the conoid. The unique biochemical properties of RNG2, including several proteolytic processing events and its ability to form concatenations, enable it to function as a dynamic bridge between these structures. Conditional depletion of RNG2 resulted in the conoid organelle detaching from the APR without compromising the integrity of its structure, thereby confirming RNG2 essential tethering role. Although microneme secretion remains normal, parasites lacking RNG2 were unable to move and impaired in rhoptry discharge, highlighting the conoids crucial role in parasite motility and invasion. RNG2 is a pivotal protein that ensures conoid functionality in Coccidia.

microbiology↗

bioRxiv2024

Soluble αβ-tubulins reversibly sequester TTC5 to regulate tubulin mRNA decay

Microtubules, built from heterodimers of - and {beta}-tubulins, control cell shape, mediate intracellular transport and power cell division. The concentration of {beta}-tubulins is tightly controlled through a post-transcriptional mechanism involving selective and regulated degradation of tubulin-encoding mRNAs. Degradation is initiated by TTC5, which recognizes tubulin-synthesizing ribosomes and recruits downstream effectors to trigger mRNA deadenylation. Here, we have investigated how cells regulate TTC5 activity. Biochemical and structural proteomic approaches reveal that under normal conditions, soluble {beta}-tubulins bind to and sequester TTC5, preventing it from engaging nascent tubulins at translating ribosomes. We identify the flexible C-terminal tail of TTC5 as a molecular switch, toggling between soluble {beta}-tubulin-bound and nascent tubulin-bound states. Loss of sequestration by soluble {beta}-tubulins constitutively activates TTC5, leading to diminished tubulin mRNA levels and compromised microtubule-dependent chromosome segregation during cell division. Our findings provide a paradigm for how cells regulate the activity of a specificity factor to adapt posttranscriptional regulation of gene expression to cellular needs.

cell biology↗

bioRxiv2023

Structural and regulatory insights into the glideosome-associated connector from Toxoplasma gondii

The phylum of Apicomplexa groups intracellular parasites that employ substratedependent gliding motility to invade host cells, egress from the infected cells and cross biological barriers. The glideosome associated connector (GAC) is a conserved protein essential to this process. GAC facilitates the association of actin filaments with surface transmembrane adhesins and the efficient transmission of the force generated by myosin translocation of actin to the cell surface substrate. Here, we present the crystal structure of Toxoplasma gondii GAC and reveal a unique, supercoiled armadillo repeat region that adopts a closed ring conformation. Characterisation of the membrane binding interface within the C-terminal PH domain as well as an N-terminal fragment necessary for association with F-actin suggest that GAC adopts multiple conformations. A multi-conformational model for assembly of GAC within the glideosome is proposed.

biochemistry↗

bioRxiv2022

Inactivation of Influenza A virus by pH conditions encountered in expiratory aerosol particles results from localized conformational changes within Haemagglutinin and Matrix 1 proteins.

Multiple respiratory viruses including Influenza A virus (IAV) can be transmitted via expiratory aerosol particles, and aerosol pH was recently identified as a major factor influencing airborne virus infectivity. For indoor air, small exhaled aerosols undergo rapid acidification to pH [~]4. IAV is known to be sensitive to mildly acidic conditions encountered within host endosomes, however, it is unknown whether the same mechanisms could mediate viral inactivation within the more acidic aerosol micro-environment. Here, we identified that transient exposure to pH 4 caused IAV inactivation by a two-stage process, with an initial sharp decline in infectious titers that was mainly attributed to premature attainment of the post-fusion conformation of viral protein haemagglutinin (HA). Changes to HA were observed by hydrogen-deuterium exchange coupled to mass spectrometry (HDX-MS) as early as 10 seconds post-exposure to acidic conditions. In addition, virion integrity was partially but irreversibly affected by acidic conditions. This was attributed to a progressive unfolding of the internal matrix protein 1 (M1), and aligned with a more gradual decline in viral infectivity with time. In contrast, no acid-mediated changes to the genome or lipid envelope were detected. Our HDX-MS data are in agreement with other more labor-intensive structural analysis techniques such as X-ray crystallography, highlighting the usefulness of whole-virus HDX-MS for multiplexed protein analyses, even within enveloped viruses such as IAV. Improved understanding of respiratory virus fate within exhaled aerosols constitutes a global public health priority, and information gained here could aid development of novel strategies to control the airborne persistence of seasonal and/or pandemic influenza in the future.

microbiology↗

bioRxiv2022

Conoid extrusion serves as gatekeeper for entry of glideosome components into the pellicular space to control motility and invasion in Apicomplexa

Members of the Apicomplexa are defined by apical cytoskeletal structures and secretory or-ganelles, tailored for motility and invasion. Gliding is powered by actomyosin-dependent rearward translocation of apically secreted transmembrane adhesins. In Toxoplasma gondii, the conoid, composed of a cone of spiraling tubulin fibers and apposed preconoidal rings (PCRs), is an enigmatic, dynamic organelle of undefined function. Here we mapped five new components of the PCRs and deduce that the structure serves as a pivotal hub for actin polymerization and glideosome assembly. F-actin produced by Formin1 on the PCRs is used by Myosin H to generate the force for conoid extrusion. A set of B-box-type zinc finger domain containing proteins conserved in Apicomplexa is indispensable for PCRs formation, conoid extrusion and motility in Toxoplasma and Plasmodium. Conoid dynamics directs the flux of F-actin to the pellicular space, acting as dynamic gatekeeper to tightly control parasite motility during invasion and egress.

microbiology↗

bioRxiv2021

Pantothenate biosynthesis is critical for chronic infection by the neurotropic parasite Toxoplasma gondii

Coenzyme A (CoA) is an essential molecule acting in metabolism, post-translational modification, and regulation of gene expression. While all organisms synthesize CoA, many, including humans, are unable to produce its precursor, pantothenate. Intriguingly, like most plants, fungi and bacteria, parasites of the coccidian subgroup of Apicomplexa, including the human and animal pathogen Toxoplasma gondii, possess all the enzymes required for de novo synthesis of pantothenate. Here, the importance of CoA and pantothenate biosynthesis for the acute and chronic stages of T. gondii infection was dissected through genetic, biochemical and metabolomic approaches, revealing that CoA synthesis is essential for T. gondii tachyzoites, due to the parasites inability to salvage CoA or intermediates of the pathway. In contrast, de novo pantothenate synthesis was only partially active in T. gondii tachyzoites, making the parasite reliant on Pan uptake. However, Pan synthesis proved to be crucial for the establishment of chronic infection, offering a promising target for intervention against the persistent stage of T. gondii.

microbiology↗

bioRxiv2021

Structure of the phosphoinositide 3-kinase p110γ-p101 complex reveals molecular mechanism of GPCR activation

The class IB phosphoinositide 3-kinase (PI3K), PI3K{gamma}, is a master regulator of immune cell function, and a promising drug target for both cancer and inflammatory diseases. Critical to PI3K{gamma} function is the association of the p110{gamma} catalytic subunit to either a p101 or p84 regulatory subunit, which mediates activation by G-protein coupled receptors (GPCRs). Here, we report the cryo-EM structure of a heterodimeric PI3K{gamma} complex, p110{gamma}-p101. This structure reveals a unique assembly of catalytic and regulatory subunits that is distinct from other class I PI3K complexes. p101 mediates activation through its G{beta}{gamma} binding domain, recruiting the heterodimer to the membrane and allowing for engagement of a secondary G{beta}{gamma} binding site in p110{gamma}. Multiple oncogenic mutations mapped to these novel interfaces and enhanced G{beta}{gamma} activation. A nanobody that specifically binds to the p101-G{beta}{gamma} interface blocks activation providing a novel tool to study and target p110{gamma}-p101-specific signaling events in vivo.

biochemistry↗

bioRxiv2020

SUV39 SET domains mediate crosstalk of heterochromatic histone marks

The SUV39 class of methyltransferase enzymes deposits histone H3 lysine 9 di- and trimethylation (H3K9me2/3), the hallmark of constitutive heterochromatin. How these enzymes are regulated to mark specific genomic regions as heterochromatic is poorly understood. Clr4 is the sole H3K9me2/3 methyltransferase in the fission yeast S. pombe and recent evidence suggests that ubiquitination of lysine 14 on histone H3 (H3K14ub) plays a key role in H3K9 methylation. However, the molecular mechanism of this regulation and its role in heterochromatin formation remains to be determined. Our structure-function approach shows that the H3K14ub substrate binds specifically and tightly to the catalytic domain of Clr4, and thereby stimulates the enzyme by over 250-fold. Mutations that disrupt this mechanism lead to a loss of H3K9me2/3 and abolish heterochromatin silencing similar to clr4 deletion. Comparison with mammalian SET domain proteins suggests that the Clr4 SET domain harbours a conserved sensor for H3K14ub, which mediates licensing of heterochromatin formation.

molecular biology↗

bioRxiv2020

Naturally acquired blocking human monoclonal antibodies to Plasmodium vivax reticulocyte binding protein 2b

Plasmodium vivax preferentially invades reticulocytes and recognition of these cells is mediated by P. vivax Reticulocyte Binding Protein 2b (PvRBP2b) binding to human Transferrin receptor 1 (TfR1) and Transferrin (Tf). Longitudinal cohort studies in Papua New Guinea, Thailand and Brazil show that PvRBP2b antibodies are correlated with protection against P. vivax infection and disease. Here, we isolated and characterized anti-PvRBP2b human monoclonal antibodies from two individuals in Cambodia with natural P. vivax infection. These antibodies bind with high affinities and map to different regions of PvRBP2b. Several human antibodies blocked PvRBP2b binding to reticulocytes and inhibited complex formation with human TfR1-Tf. We describe different structural mechanisms for functional inhibition, including either steric hindrance with TfR1-Tf or the reticulocyte membrane. These results show that naturally acquired human antibodies against PvRBP2b can inhibit its function which is important for P. vivax invasion.

microbiology↗