Alternaria alternata is a common fungus strongly related with severe allergic asthma, with 80% of affected individuals being sensitized solely to its major allergen Alt a 1. Here, we assessed the function of Alt a 1 as an innate defense protein binding to micronutrients, such as iron-quercetin complexes (FeQ2), and its impact on antigen presentation in vitro. Binding of Alt a 1 to FeQ2 was determined in docking calculations. Recombinant Alt a 1 was generated, and binding ability, as well as secondary and quaternary structure, assessed by UV-VIS, CD, and DLS spectroscopy. Proteolytic functions were determined by casein and gelatine zymography. Uptake of empty apo- or ligand-filled holoAlt a 1 were assessed in human monocytic THP1 cells under the presence of dynamin and clathrin-inhibitors, activation of the Arylhydrocarbon receptor (AhR) using the human reporter cellline AZ-AHR. Human PBMCs were stimulated and assessed for phenotypic changes in monocytes by flow cytometry. Alt a 1 bound strongly to FeQ2 as a tetramer with calculated Kd values reaching pico-molar levels and surpassing affinities to quercetin alone by a factor of 5000 for the tetramer. apoAlt a 1 but not holoAlta 1 showed low enzymatic activity against casein as a hexamer and gelatin as a trimer. Uptake of apo- and holo-Alt a 1 occurred partly clathrin-dependent, with apoAlt a 1 decreasing labile iron in THP1 cells and holoAlt a 1 facilitating quercetin-dependent AhR activation. In human PBMCs uptake of holoAlt a 1 but not apoAlt a 1 significantly decreased the surface expression of the costimulatory CD86, but also of HLADR, thereby reducing effective antigen presentation. We show here for the first time that the presence of nutritional iron complexes, such as FeQ2, significantly alters the function of Alt a 1 and dampens the human immune response, thereby supporting the notion that Alt a 1 only becomes immunogenic under nutritional deprivation.
- MeSH
- alergeny * MeSH
- Alternaria metabolismus MeSH
- bronchiální astma * MeSH
- kaseiny MeSH
- klathrin MeSH
- lidé MeSH
- quercetin MeSH
- železo metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
The efficacy of current antimalarial drugs is threatened by reduced susceptibility of Plasmodium falciparum to artemisinin, associated with mutations in pfkelch13 Another gene with variants known to modulate the response to artemisinin encodes the μ subunit of the AP-2 adaptin trafficking complex. To elucidate the cellular role of AP-2μ in P. falciparum, we performed a conditional gene knockout, which severely disrupted schizont organization and maturation, leading to mislocalization of key merozoite proteins. AP-2μ is thus essential for blood-stage replication. We generated transgenic P. falciparum parasites expressing hemagglutinin-tagged AP-2μ and examined cellular localization by fluorescence and electron microscopy. Together with mass spectrometry analysis of coimmunoprecipitating proteins, these studies identified AP-2μ-interacting partners, including other AP-2 subunits, the K10 kelch-domain protein, and PfEHD, an effector of endocytosis and lipid mobilization, but no evidence was found of interaction with clathrin, the expected coat protein for AP-2 vesicles. In reverse immunoprecipitation experiments with a clathrin nanobody, other heterotetrameric AP-complexes were shown to interact with clathrin, but AP-2 complex subunits were absent.IMPORTANCE We examine in detail the AP-2 adaptin complex from the malaria parasite Plasmodium falciparum In most studied organisms, AP-2 is involved in bringing material into the cell from outside, a process called endocytosis. Previous work shows that changes to the μ subunit of AP-2 can contribute to drug resistance. Our experiments show that AP-2 is essential for parasite development in blood but does not have any role in clathrin-mediated endocytosis. This suggests that a specialized function for AP-2 has developed in malaria parasites, and this may be important for understanding its impact on drug resistance.
- MeSH
- adaptorový proteinový komplex 2 genetika metabolismus MeSH
- antimalarika farmakologie MeSH
- artemisininy metabolismus MeSH
- endocytóza fyziologie MeSH
- geneticky modifikované organismy MeSH
- genový knockout MeSH
- klathrin metabolismus MeSH
- léková rezistence MeSH
- membránové proteiny metabolismus MeSH
- Plasmodium falciparum účinky léků genetika metabolismus MeSH
- protozoální proteiny genetika metabolismus MeSH
- schizonti účinky léků genetika metabolismus MeSH
- transport proteinů MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
Clathrin-mediated endocytosis (CME) is key to maintaining the transmembrane protein composition of cells' limiting membranes. During mammalian CME, a reversible phosphorylation event occurs on Thr156 of the μ2 subunit of the main endocytic clathrin adaptor, AP2. We show that this phosphorylation event starts during clathrin-coated pit (CCP) initiation and increases throughout CCP lifetime. μ2Thr156 phosphorylation favors a new, cargo-bound conformation of AP2 and simultaneously creates a binding platform for the endocytic NECAP proteins but without significantly altering AP2's cargo affinity in vitro. We describe the structural bases of both. NECAP arrival at CCPs parallels that of clathrin and increases with μ2Thr156 phosphorylation. In turn, NECAP recruits drivers of late stages of CCP formation, including SNX9, via a site distinct from where NECAP binds AP2. Disruption of the different modules of this phosphorylation-based temporal regulatory system results in CCP maturation being delayed and/or stalled, hence impairing global rates of CME.
- MeSH
- adaptorový proteinový komplex - alfa-podjednotky genetika MeSH
- adaptorový proteinový komplex 2 genetika metabolismus MeSH
- endocytóza genetika MeSH
- fosforylace genetika MeSH
- klathrin genetika metabolismus MeSH
- klathrinové vezikuly genetika metabolismus MeSH
- lidé MeSH
- potažené jamky v buněčné membráně genetika metabolismus MeSH
- třídící nexiny genetika MeSH
- vazba proteinů genetika MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The developmental and epileptic encephalopathies (DEEs) are heterogeneous disorders with a strong genetic contribution, but the underlying genetic etiology remains unknown in a significant proportion of individuals. To explore whether statistical support for genetic etiologies can be generated on the basis of phenotypic features, we analyzed whole-exome sequencing data and phenotypic similarities by using Human Phenotype Ontology (HPO) in 314 individuals with DEEs. We identified a de novo c.508C>T (p.Arg170Trp) variant in AP2M1 in two individuals with a phenotypic similarity that was higher than expected by chance (p = 0.003) and a phenotype related to epilepsy with myoclonic-atonic seizures. We subsequently found the same de novo variant in two individuals with neurodevelopmental disorders and generalized epilepsy in a cohort of 2,310 individuals who underwent diagnostic whole-exome sequencing. AP2M1 encodes the μ-subunit of the adaptor protein complex 2 (AP-2), which is involved in clathrin-mediated endocytosis (CME) and synaptic vesicle recycling. Modeling of protein dynamics indicated that the p.Arg170Trp variant impairs the conformational activation and thermodynamic entropy of the AP-2 complex. Functional complementation of both the μ-subunit carrying the p.Arg170Trp variant in human cells and astrocytes derived from AP-2μ conditional knockout mice revealed a significant impairment of CME of transferrin. In contrast, stability, expression levels, membrane recruitment, and localization were not impaired, suggesting a functional alteration of the AP-2 complex as the underlying disease mechanism. We establish a recurrent pathogenic variant in AP2M1 as a cause of DEEs with distinct phenotypic features, and we implicate dysfunction of the early steps of endocytosis as a disease mechanism in epilepsy.
- MeSH
- adaptorový proteinový komplex - mu-podjednotky genetika MeSH
- adaptorový proteinový komplex 2 genetika MeSH
- dítě MeSH
- endocytóza * MeSH
- epilepsie etiologie patologie MeSH
- klathrin genetika metabolismus MeSH
- kojenec MeSH
- lidé MeSH
- missense mutace * MeSH
- mladiství MeSH
- myši knockoutované MeSH
- myši MeSH
- nemoci mozku etiologie patologie MeSH
- neurovývojové poruchy etiologie patologie MeSH
- předškolní dítě MeSH
- sekvenování exomu MeSH
- zvířata MeSH
- Check Tag
- dítě MeSH
- kojenec MeSH
- lidé MeSH
- mladiství MeSH
- myši MeSH
- předškolní dítě MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
Ritonavir (RIT) is a widely used antiviral drug that acts as an HIV protease inhibitor with emerging potential in anticancer therapies. RIT causes inhibition of P-glycoprotein, which plays an important role in multidrug resistance (MDR) in cancer cells when overexpressed. Moreover, RIT causes mitochondrial dysfunction, leading to decreased ATP production and reduction of caveolin I expression, which can affect cell migration and tumor progression. To increase its direct antitumor activity, decrease severe side effects induced by the use of free RIT and improve its pharmacokinetics, ritonavir 5-methyl-4-oxohexanoate (RTV) was synthesized and conjugated to a tumor-targeted polymer carrier based on a N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer. Here we demonstrated that polymer-bound RTV enhanced the internalization of polymer-RTV conjugates, differing in RTV content from 4 to 15 wt%, in HeLa cancer cells compared with polymer without RTV. The most efficient influx and internalization properties were determined for the polymer conjugate bearing 11 wt% of RTV. This conjugate was internalized by cells using both caveolin- and clathrin-dependent endocytic pathways in contrast to the RTV-free polymer, which was preferentially internalized only by clathrin-mediated endocytosis. Moreover, we found the co-localization of the RTV-conjugate with mitochondria and a significant decrease of ATP production in treated cells. Thus, the impact on mitochondrial mechanism can influence the function of ATP-dependent P-glycoprotein and also the cell viability of MDR cancer cells. Overall, this study demonstrated that the polymer-RTV conjugate is a promising polymer-based nanotherapeutic, suitable for antitumor combination therapy with other anticancer drugs and a potential mitochondrial drug delivery system.
- MeSH
- adenosintrifosfát biosyntéza MeSH
- chemorezistence účinky léků MeSH
- endocytóza účinky léků MeSH
- HeLa buňky MeSH
- kaveolin 1 biosyntéza genetika MeSH
- klathrin farmakologie MeSH
- koncentrace vodíkových iontů MeSH
- lidé MeSH
- methakryláty chemie MeSH
- nanostruktury chemie MeSH
- P-glykoprotein účinky léků metabolismus MeSH
- polymery MeSH
- protinádorové látky aplikace a dávkování chemie MeSH
- ritonavir aplikace a dávkování analogy a deriváty chemie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Clathrin-mediated endocytosis (CME) is a cellular trafficking process in which cargoes and lipids are internalized from the plasma membrane into vesicles coated with clathrin and adaptor proteins. CME is essential for many developmental and physiological processes in plants, but its underlying mechanism is not well characterized compared with that in yeast and animal systems. Here, we searched for new factors involved in CME in Arabidopsis thaliana by performing tandem affinity purification of proteins that interact with clathrin light chain, a principal component of the clathrin coat. Among the confirmed interactors, we found two putative homologs of the clathrin-coat uncoating factor auxilin previously described in non-plant systems. Overexpression of AUXILIN-LIKE1 and AUXILIN-LIKE2 in Arabidopsis caused an arrest of seedling growth and development. This was concomitant with inhibited endocytosis due to blocking of clathrin recruitment after the initial step of adaptor protein binding to the plasma membrane. By contrast, auxilin-like1/2 loss-of-function lines did not present endocytosis-related developmental or cellular phenotypes under normal growth conditions. This work contributes to the ongoing characterization of the endocytotic machinery in plants and provides a robust tool for conditionally and specifically interfering with CME in Arabidopsis.
- MeSH
- Arabidopsis genetika metabolismus MeSH
- endocytóza genetika fyziologie MeSH
- klathrin genetika metabolismus MeSH
- proteiny huseníčku genetika metabolismus MeSH
- semenáček genetika metabolismus MeSH
- transport proteinů MeSH
- vazba proteinů MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The fibroblast growth factor receptors (FGFRs) are important oncogenes promoting tumor progression in many types of cancer, such as breast, bladder, and lung cancer as well as multiple myeloma and rhabdomyosarcoma. However, little is known about how these receptors are internalized and down-regulated in cells. We have here applied proximity biotin labeling to identify proteins involved in FGFR4 signaling and trafficking. For this purpose we fused a mutated biotin ligase, BirA*, to the C-terminal tail of FGFR4 (FGFR4-BirA*) and the fusion protein was stably expressed in U2OS cells. Upon addition of biotin to these cells, proteins in proximity to the FGFR4-BirA* fusion protein became biotinylated and could be isolated and identified by quantitative mass spectrometry. We identified in total 291 proteins, including 80 proteins that were enriched in samples where the receptor was activated by the ligand (FGF1), among them several proteins previously found to be involved in FGFR signaling (e.g., FRS2, PLCγ, RSK2 and NCK2). Interestingly, many of the identified proteins were implicated in endosomal transport, and by precise annotation we were able to trace the intracellular pathways of activated FGFR4. Validating the data by confocal and three-dimensional structured illumination microscopy analysis, we concluded that FGFR4 uses clathrin-mediated endocytosis for internalization and is further sorted from early endosomes to the recycling compartment and the trans-Golgi network. Depletion of cells for clathrin heavy chain led to accumulation of FGFR4 at the cell surface and increased levels of active FGFR4 and PLCγ, while AKT and ERK signaling was diminished, demonstrating that functional clathrin-mediated endocytosis is required for proper FGFR4 signaling. Thus, this study reveals proteins and pathways involved in FGFR4 transport and signaling that provide possible targets and opportunities for therapeutic intervention in FGFR4 aberrant cancer.
- MeSH
- barvení a značení MeSH
- biotinylace MeSH
- endocytóza MeSH
- endozomy metabolismus MeSH
- klathrin metabolismus MeSH
- lidé MeSH
- mikroskopie metody MeSH
- nádorové buněčné linie MeSH
- receptor fibroblastových růstových faktorů, typ 4 metabolismus MeSH
- signální transdukce MeSH
- trans-Golgiho síť metabolismus MeSH
- transport proteinů MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Plant cell morphogenesis involves concerted rearrangements of microtubules and actin microfilaments. We previously reported that FH1, the main Arabidopsis thaliana housekeeping Class I membrane-anchored formin, contributes to actin dynamics and microtubule stability in rhizodermis cells. Here we examine the effects of mutations affecting FH1 (At3g25500) on cell morphogenesis and above-ground organ development in seedlings, as well as on cytoskeletal organization and dynamics, using a combination of confocal and variable angle epifluorescence microscopy with a pharmacological approach. Homozygous fh1 mutants exhibited cotyledon epinasty and had larger cotyledon pavement cells with more pronounced lobes than the wild type. The pavement cell shape alterations were enhanced by expression of the fluorescent microtubule marker GFP-microtubule-associated protein 4 (MAP4). Mutant cotyledon pavement cells exhibited reduced density and increased stability of microfilament bundles, as well as enhanced dynamics of microtubules. Analogous results were also obtained upon treatments with the formin inhibitor SMIFH2 (small molecule inhibitor of formin homology 2 domains). Pavement cell shape in wild-type (wt) and fh1 plants in some situations exhibited a differential response towards anti-cytoskeletal drugs, especially the microtubule disruptor oryzalin. Our observations indicate that FH1 participates in the control of microtubule dynamics, possibly via its effects on actin, subsequently influencing cell morphogenesis and macroscopic organ development.
- MeSH
- aktiny metabolismus MeSH
- Arabidopsis cytologie účinky léků metabolismus MeSH
- biologické markery metabolismus MeSH
- biologické modely MeSH
- cytoskelet účinky léků metabolismus MeSH
- fluorescence MeSH
- klathrin metabolismus MeSH
- kotyledon účinky léků metabolismus MeSH
- membránové proteiny metabolismus MeSH
- mikrofilamenta účinky léků metabolismus MeSH
- mikrotubuly účinky léků metabolismus MeSH
- mutace genetika MeSH
- proteiny huseníčku metabolismus MeSH
- semenáček účinky léků růst a vývoj metabolismus MeSH
- thioketony farmakologie MeSH
- tvar buňky * účinky léků MeSH
- uracil analogy a deriváty farmakologie MeSH
- zelené fluorescenční proteiny metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The major brassinosteroid (BR) receptor of Arabidopsis BRASSINOSTEROID INSENSITIVE1 (BRI1) plays fundamental roles in BR signaling, but the molecular mechanisms underlying the effects of BR on BRI1 internalization and assembly state remain unclear. Here, we applied variable angle total internal reflection fluorescence microscopy and fluorescence cross-correlation spectroscopy to analyze the dynamics of GFP-tagged BRI1. We found that, in response to BR, the degree of co-localization of BRI1-GFP with AtFlot1-mCherry increased, and especially BR stimulated the membrane microdomain-associated pathway of BRI1 internalization. We also verified these observations in endocytosis-defective chc2-1 mutants and the AtFlot1 amiRNA 15-5 lines. Furthermore, examination of the phosphorylation status of bri1-EMS-suppressor 1 and measurement of BR-responsive gene expression revealed that membrane microdomains affect BR signaling. These results suggest that BR promotes the partitioning of BRI1 into functional membrane microdomains to activate BR signaling.
- MeSH
- Arabidopsis cytologie metabolismus MeSH
- brassinosteroidy farmakologie MeSH
- časoprostorová analýza * MeSH
- difuze MeSH
- endocytóza účinky léků MeSH
- klathrin metabolismus MeSH
- membránové mikrodomény účinky léků metabolismus MeSH
- multimerizace proteinu účinky léků MeSH
- pohyb těles MeSH
- proteinkinasy metabolismus MeSH
- proteiny huseníčku metabolismus MeSH
- rostlinné buňky účinky léků metabolismus MeSH
- signální transdukce účinky léků MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
