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1.
Plant Signal Behav ; 18(1): 2163337, 2023 Dec 31.
Artigo em Inglês | MEDLINE | ID: mdl-36603596

RESUMO

In eukaryotes, EPSINs are Epsin N-terminal Homology (ENTH) domain-containing proteins that serve as monomeric clathrin adaptors at the plasma membrane (PM) or the trans-Golgi Network (TGN)/early endosomes (EE). The model plant Arabidopsis thaliana encodes for seven ENTH proteins, of which so far, only AtEPSIN1 (AtEPS1) and MODIFIED TRANSPORT TO THE VACUOLE1 (AtMTV1) localize to the TGN/EE and contribute to cargo trafficking to both the cell surface and the vacuole. However, relatively little is known about role(s) of any plant EPSIN in governing physiological responses. We have recently shown that AtEPS1 is a positive modulator of plant immune signaling and pattern-triggered immunity against flagellated Pseudomonas syringae pv. tomato (Pto) DC3000 bacteria. In eps1 mutants, impaired immune responses correlate with reduced accumulation of the receptor FLAGELLIN SENSING2 (AtFLS2) and the convergent immune co-receptor BRASSINOSTEROID INSENTIVE1-ASSOCIATED RECEPTOR KINASE1 (AtBAK1) in the PM. Here, we report that in contrast to AtEPS1, the TGN/EE-localized AtMTV1 did not contribute significantly to immunity against pathogenic Pto DC3000 bacteria. We also compared the amino acid sequences, peptide motif structures and in silico tertiary structures of the ENTH domains of AtEPS1 and AtMTV1 in more detail. We conclude that despite sharing the classical tertiary alpha helical ENTH-domain structure and clathrin-binding motifs, the overall low amino acid identity and differences in peptide motifs may explain their role(s) in trafficking of some of the same as well as distinct cargo components to their site of function, with the latter potentially contributing to differences in physiological responses.


Assuntos
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Pseudomonas syringae , Proteínas Adaptadoras de Transporte Vesicular/química , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Imunidade Vegetal/fisiologia , Clatrina/metabolismo
2.
Proc Natl Acad Sci U S A ; 120(2): e2205199120, 2023 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-36598941

RESUMO

Assembly of protein complexes is facilitated by assembly chaperones. Alpha and gamma adaptin-binding protein (AAGAB) is a chaperone governing the assembly of the heterotetrameric adaptor complexes 1 and 2 (AP1 and AP2) involved in clathrin-mediated membrane trafficking. Here, we found that before AP1/2 binding, AAGAB exists as a homodimer. AAGAB dimerization is mediated by its C-terminal domain (CTD), which is critical for AAGAB stability and is missing in mutant proteins found in patients with the skin disease punctate palmoplantar keratoderma type 1 (PPKP1). We solved the crystal structure of the dimerization-mediating CTD, revealing an antiparallel dimer of bent helices. Interestingly, AAGAB uses the same CTD to recognize and stabilize the γ subunit in the AP1 complex and the α subunit in the AP2 complex, forming binary complexes containing only one copy of AAGAB. These findings demonstrate a dual role of CTD in stabilizing resting AAGAB and binding to substrates, providing a molecular explanation for disease-causing AAGAB mutations. The oligomerization state transition mechanism may also underlie the functions of other assembly chaperones.


Assuntos
Proteínas Adaptadoras de Transporte Vesicular , Ceratodermia Palmar e Plantar , Humanos , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Proteínas de Transporte/genética , Ceratodermia Palmar e Plantar/genética , Ceratodermia Palmar e Plantar/metabolismo , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Clatrina/metabolismo , Complexo 2 de Proteínas Adaptadoras/genética , Complexo 2 de Proteínas Adaptadoras/metabolismo
3.
FASEB J ; 37(2): e22764, 2023 02.
Artigo em Inglês | MEDLINE | ID: mdl-36624697

RESUMO

Endocytosis is an essential biological process for nutrient absorption and intercellular communication; it can also be used to accelerate the cellular internalization of drug delivery carriers. Clarifying the cellular uptake mechanisms of unidentified endogenous and exogenous molecules and designing new effective drug delivery systems require an accurate, specific endocytosis analysis methodology. Therefore, we developed a method to specifically evaluate cellular internalization via three main endocytic pathways: clathrin- and caveolae-mediated endocytosis, and macropinocytosis. We first revealed that most known endocytosis inhibitors had no specific inhibitory effect or were cytotoxic. Second, we successfully established an alternative method using small interfering RNA to knock down dynamin-2 and caveolin-1, which are necessary for clathrin- and caveolae-mediated endocytosis, in HeLa cells. Third, we established another method to specifically analyze macropinocytosis using rottlerin on A431 cells. Finally, we validated the proposed methods by testing the cellular internalization of a biological molecule (insulin) and carriers (nanoparticles and cell-penetrating peptides). Through this study, we established versatile methods to precisely and specifically evaluate endocytosis of newly developed biopharmaceuticals or drug delivery systems.


Assuntos
Endocitose , Pinocitose , Humanos , Células HeLa , RNA Interferente Pequeno/genética , Clatrina/genética , Cavéolas
4.
Int J Mol Sci ; 24(2)2023 Jan 06.
Artigo em Inglês | MEDLINE | ID: mdl-36674638

RESUMO

In recent years, rare-earth-doped upconverting nanoparticles (UCNPs) have been widely used in different life sciences due to their unique properties. Nanoparticles have become a multifunctional and promising new approach to neurobiological disorders and have shown extraordinary application potential to overcome the problems related to conventional treatment strategies. This study evaluated the internalization mechanisms, bio-distribution, and neurotoxicity of NaYF4:20%Yb3+,2%Er3+ UCNPs in rat organotypic hippocampal slices. TEM results showed that UCNPs were easily internalized by hippocampal cells and co-localized with selected organelles inside neurons and astrocytes. Moreover, the UCNPs were taken into the neurons via clathrin- and caveolae-mediated endocytosis. Propidium iodide staining and TEM analysis did not confirm the adverse effects of UCNPs on hippocampal slice viability and morphology. Therefore, UCNPs may be a potent tool for bio-imaging and testing new therapeutic strategies for brain diseases in the future.


Assuntos
Diagnóstico por Imagem , Nanopartículas , Ratos , Animais , Neurônios , Clatrina
5.
Life Sci Alliance ; 6(4)2023 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-36717249

RESUMO

PKCßII, a conventional PKC family member, plays critical roles in the regulation of a variety of cellular functions. Here, we employed loss-of-function approaches and mutants of PKCßII with altered phosphorylation and protein interaction behaviors to identify the cellular mechanisms underlying the activation of PKCßII. Our results show that 3-phosphoinositide-dependent protein kinase-1 (PDK1)-mediated constitutive phosphorylation of PKCßII at the activation loop (T500) is required for phorbol ester-induced nuclear entry and subsequent Mdm2-mediated ubiquitination of PKCßII, whereas ubiquitination of PKCßII is required for the PDK1-mediated inducible phosphorylation of PKCßII at T500 in the nucleus. After moving out of the nucleus, PKCßII interacts with actin, undergoes inducible mTORC2-mediated phosphorylation at the turn motif (T641), interacts with clathrin, and then translocates to the plasma membrane. This overall cascade of cellular events intertwined with the phosphorylation at critical residues and Mdm2-mediated ubiquitination in the nucleus and along with interactions with actin and clathrin plays roles that encompass the core processes of PKC activation.


Assuntos
Actinas , Clatrina , Proteína Quinase C beta , Proteínas Proto-Oncogênicas c-mdm2 , Actinas/metabolismo , Clatrina/metabolismo , Fosforilação , Proteína Quinase C beta/metabolismo , Ubiquitinação , Proteínas Proto-Oncogênicas c-mdm2/metabolismo
6.
Nanoscale Horiz ; 8(2): 256-269, 2023 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-36594629

RESUMO

The paramount relevance of clathrin-coated pits (CCPs) to receptor-mediated endocytosis of nanoparticles, extracellular vesicles, and viruses has made them the focus of many studies; however, the role of CCP geometry in the ligand-receptor interactions between multivalent nanoparticles and cells has not been investigated. We hypothesized the general dependence of nanoparticle binding energy on local membrane curvature to be expandable to the specific case of ligand-functionalized nanoparticles binding cell membranes, in the sense that membrane structures whose curvature matches that of the particle (e.g., CCPs) signficantly contribute to binding avidity. We investigated this hypothesis with nanoparticles that bind multivalently to angiotensin II receptor type 1, which is subject to clathrin-mediated endocytosis. When we used cholesterol extraction to prevent the action of CCPs, we found a 67 to 100-fold loss in avidity. We created a theoretical model that predicts this decrease based on the loss of ligand-receptor interactions when CCPs, which perfectly match nanoparticle geometry, are absent. Our findings shed new light on how cells "see" nanoparticles. The presence or absence of CPPs is so influential on how cells interact with nanoparticles that the number of particles required to be visible to cells changes by two orders of magnitude depending on CCP presence.


Assuntos
Clatrina , Nanopartículas , Clatrina/metabolismo , Ligantes , Membrana Celular/metabolismo , Endocitose
7.
Prog Mol Biol Transl Sci ; 194: 1-18, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36631188

RESUMO

Endocytosis is a cellular process which mediates receptor internalization, nutrient uptake, and the regulation of cell signaling. Microorganisms (many bacteria and viruses) and toxins also use the same process and enter the cells. Generally, endocytosis is considered in the three forms such as phagocytosis (cell eating), pinocytosis (cell drinking), and highly selective receptor-mediated endocytosis (clathrin-dependent and independent). Several endocytic routes exist in an analogous, achieving diverse functions. Most studies on endocytosis have used transformed cells in culture. To visualize the receptor internalization, trafficking, and signaling in subcellular organelles, a green fluorescent protein-tagged receptor has been utilized. It also helps to visualize the endocytosis effects in live-cell imaging. Confocal laser microscopy increases our understanding of receptor endocytosis and signaling. Site-directed mutagenesis studies demonstrated that many short-sequence motifs of the cytoplasmic domain of receptors significantly play a vital role in receptor internalization, subcellular trafficking, and signaling. However, other factors also regulate receptor internalization through clathrin-coated vesicles. Receptor endocytosis can occur through clathrin-dependent and clathrin-independent pathways. This chapter briefly discusses the internalization, trafficking, and signaling of various receptors in normal conditions. In addition, it also highlights the malfunction of the receptor in disease conditions.


Assuntos
Endocitose , Transdução de Sinais , Humanos , Endocitose/fisiologia , Clatrina/metabolismo , Transporte Biológico
8.
Prog Mol Biol Transl Sci ; 194: 79-107, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36631202

RESUMO

Insulin signaling controls multiple aspects of animal physiology. At the cell surface, insulin binds and activates the insulin receptor (IR), a receptor tyrosine kinase. Insulin promotes a large conformational change of IR and stabilizes the active conformation. The insulin-activated IR triggers signaling cascades, thus controlling metabolism, growth, and proliferation. The activated IR undergoes internalization by clathrin- or caveolae-mediated endocytosis. The IR endocytosis plays important roles in insulin clearance from blood, and distribution and termination of the insulin signaling. Despite decades of extensive studies, the mechanism and regulation of IR endocytosis and its contribution to pathophysiology remain incompletely understood. Here we discuss recent findings that provide insights into the molecular mechanisms and regulatory pathways that mediate the IR endocytosis.


Assuntos
Receptor de Insulina , Transdução de Sinais , Animais , Receptor de Insulina/metabolismo , Membrana Celular/metabolismo , Insulina , Endocitose/fisiologia , Clatrina/metabolismo
9.
J Immunother Cancer ; 11(1)2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36596591

RESUMO

BACKGROUND: Loss of major histocompatibility complex class I (MHC-I) in tumor cells limits the use of immune checkpoint blockade (ICB) in colorectal cancer. Nevertheless, the regulatory mechanism of MHC-I downregulation in tumor cells has not been fully elucidated. Overexpression of CEMIP in tumor tissues is associated with a poor prognosis in colorectal cancer. Here, in this research, we aim to address the role of CEMIP in mediating MHC-I expression in tumor cells and investigate the underlying regulatory mechanisms. METHOD: Protein levels were analyzed by western blotting. Flow cytometry analysis was used to examine immune cells. Protein-protein interactions were investigated by co-immunoprecipitation and proximity ligation assays. The intracellular trafficking of MHC-I was revealed by an immunofluorescent technique. In addition, the effect of CEMIP on tumor growth and the antitumor efficacy of targeting CEMIP in combination with ICB therapy were evaluated in murine models of colorectal cancer. RESULTS: We reported that CEMIP specifically downregulated the expression of MHC-I on the surface of murine and human colon cancer cells, hindering the cytotoxicity of CD8+ T cells. We also demonstrated that CEMIP restricted CD8+ T-cell antitumor activities both in vitro and in vivo due to impaired MHC-I-mediated antigen presentation. Correspondingly, the combination of CEMIP inhibition and ICB impeded tumor growth and enhanced therapeutic efficacy. Mechanistically, CEMIP acted as an adaptor for the interaction betweenMHC-I and clathrin, which drove MHC-I internalization via clathrin-dependent endocytosis. Furthermore, CEMIP anchored internalized MHC-I to lysosomes for degradation, disrupting the recycling of MHC-I to the cell surface. CONCLUSION: Overall, our study unveils a novel regulatory mechanism of MHC-I on tumor cell surfaces by CEMIP-mediated internalization and degradation. Furthermore, targeting CEMIP provides an effective strategy for colorectal cancer immunotherapy.


Assuntos
Linfócitos T CD8-Positivos , Neoplasias Colorretais , Humanos , Animais , Camundongos , Evasão da Resposta Imune , Antígenos de Histocompatibilidade Classe I , Clatrina/metabolismo
10.
Eur J Med Chem ; 247: 115001, 2023 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-36577213

RESUMO

Wiskostatin (1-(3,6-dibromo-9H-carbazol-9-yl)-3-(dimethylamino)propan-2-ol) (1) is a carbazole-based compound reported as a specific and relatively potent inhibitor of the N-WASP actin remodelling complex (S-isomer EC50 = 4.35 µM; R-isomer EC50 = 3.44 µM). An NMR solution structure showed that wiskostatin interacts with a cleft in the regulatory GTPase binding domain of N-WASP. However, numerous studies have reported wiskostatin's actions on membrane transport and cytokinesis that are independent of the N-WASP-Arp2/3 complex pathway, but offer limited alternative explanation. The large GTPase, dynamin has established functional roles in these pathways. This study reveals that wiskostatin and its analogues, as well as other carbazole-based compounds, are inhibitors of helical dynamin GTPase activity and endocytosis. We characterise the effects of wiskostatin on in vitro dynamin GTPase activity, in-cell endocytosis, and determine the importance of wiskostatin functional groups on these activities through design and synthesis of libraries of wiskostatin analogues. We also examine whether other carbazole-based scaffolds frequently used in research or the clinic also modulate dynamin and endocytosis. Understanding off-targets for compounds used as research tools is important to be able to confidently interpret their action on biological systems, particularly when the target and off-targets affect overlapping mechanisms (e.g. cytokinesis and endocytosis). Herein we demonstrate that wiskostatin is a dynamin inhibitor (IC50 20.7 ± 1.2 µM) and a potent inhibitor of clathrin mediated endocytosis (IC50 = 6.9 ± 0.3 µM). Synthesis of wiskostatin analogues gave rise to 1-(9H-carbazol-9-yl)-3-((4-methylbenzyl)amino)propan-2-ol (35) and 1-(9H-carbazol-9-yl)-3-((4-chlorobenzyl)amino)propan-2-ol (43) as potent dynamin inhibitors (IC50 = 1.0 ± 0.2 µM), and (S)-1-(3,6-dibromo-9H-carbazol-9-yl)-3-(dimethylamino)propan-2-ol (8a) and (R)-1-(3,6-dibromo-9H-carbazol-9-yl)-3-(dimethylamino)propan-2-ol (8b) that are amongst the most potent inhibitors of clathrin mediated endocytosis yet reported (IC50 = 2.3 ± 3.3 and 2.1 ± 1.7 µM, respectively).


Assuntos
Dinamina I , Dinaminas , Dinamina I/química , Dinamina I/metabolismo , Dinaminas/farmacologia , Carbazóis/farmacologia , GTP Fosfo-Hidrolases , Actinas , Clatrina/metabolismo , Clatrina/farmacologia , Endocitose
11.
J Cell Biol ; 222(2)2023 Feb 06.
Artigo em Inglês | MEDLINE | ID: mdl-36469001

RESUMO

Volume electron microscopy is an important imaging modality in contemporary cell biology. Identification of intracellular structures is a laborious process limiting the effective use of this potentially powerful tool. We resolved this bottleneck with automated segmentation of intracellular substructures in electron microscopy (ASEM), a new pipeline to train a convolutional neural network to detect structures of a wide range in size and complexity. We obtained dedicated models for each structure based on a small number of sparsely annotated ground truth images from only one or two cells. Model generalization was improved with a rapid, computationally effective strategy to refine a trained model by including a few additional annotations. We identified mitochondria, Golgi apparatus, endoplasmic reticulum, nuclear pore complexes, caveolae, clathrin-coated pits, and vesicles imaged by focused ion beam scanning electron microscopy. We uncovered a wide range of membrane-nuclear pore diameters within a single cell and derived morphological metrics from clathrin-coated pits and vesicles, consistent with the classical constant-growth assembly model.


Assuntos
Processamento de Imagem Assistida por Computador , Microscopia Eletrônica , Redes Neurais de Computação , Clatrina , Retículo Endoplasmático/ultraestrutura , Complexo de Golgi/ultraestrutura , Microscopia Eletrônica/métodos , Mitocôndrias/ultraestrutura , Poro Nuclear/ultraestrutura , Cavéolas/ultraestrutura , Biologia Celular
12.
Methods Mol Biol ; 2557: 3-15, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36512205

RESUMO

Fluorescence imaging of live cells allows for the observation of dynamic processes inside cells in real time. Here we describe a strategy to image clathrin-coated vesicle dynamics in a single focal plane at the trans-Golgi network of the yeast Saccharomyces cerevisiae. This method can be readily adapted for live cell imaging of a diverse set of dynamic processes within cells.


Assuntos
Saccharomyces cerevisiae , Rede trans-Golgi , Vesículas Revestidas por Clatrina , Complexo de Golgi , Clatrina
13.
Methods Mol Biol ; 2557: 619-633, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36512241

RESUMO

The function and integrity of epithelial cells depends on the polarized localization of transmembrane proteins at either apical or basolateral plasma membrane domains. To facilitate sorting to the basolateral domain, columnar epithelial cells express the tissue-specific AP-1B complex in addition to the ubiquitously expressed AP-1A. Both AP-1A and AP-1B are heterotetrameric clathrin adaptor protein complexes that are closely related. Here we describe a biochemical method to separate AP-1B from AP-1A clathrin-coated vesicles by immunoprecipitation from clathrin-coated vesicle pellets that were obtained by ultracentrifugation and analyzed by SDS-PAGE and western blot using fluorescently labeled secondary antibodies.


Assuntos
Vesículas Revestidas por Clatrina , Clatrina , Vesículas Revestidas por Clatrina/metabolismo , Clatrina/metabolismo , Fator de Transcrição AP-1/metabolismo , Polaridade Celular/fisiologia , Proteínas Adaptadoras de Transporte Vesicular , Western Blotting , Imunoprecipitação
14.
Cells ; 11(24)2022 12 10.
Artigo em Inglês | MEDLINE | ID: mdl-36552756

RESUMO

Genome-wide association studies (GWAS) have identified the PICALM (Phosphatidylinositol binding clathrin-assembly protein) gene as the most significant genetic susceptibility locus after APOE and BIN1. PICALM is a clathrin-adaptor protein that plays a critical role in clathrin-mediated endocytosis and autophagy. Since the effects of genetic variants of PICALM as AD-susceptibility loci have been confirmed by independent genetic studies in several distinct cohorts, there has been a number of in vitro and in vivo studies attempting to elucidate the underlying mechanism by which PICALM modulates AD risk. While differential modulation of APP processing and Aß transcytosis by PICALM has been reported, significant effects of PICALM modulation of tau pathology progression have also been evidenced in Alzheimer's disease models. In this review, we summarize the current knowledge about PICALM, its physiological functions, genetic variants, post-translational modifications and relevance to AD pathogenesis.


Assuntos
Doença de Alzheimer , Proteínas Monoméricas de Montagem de Clatrina , Humanos , Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Clatrina/metabolismo , Loci Gênicos , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Proteínas Monoméricas de Montagem de Clatrina/genética , Proteínas Monoméricas de Montagem de Clatrina/metabolismo
15.
Int J Mol Sci ; 23(23)2022 Nov 24.
Artigo em Inglês | MEDLINE | ID: mdl-36499010

RESUMO

Vertebrate and fly rhodopsins are prototypical GPCRs that have served for a long time as model systems for understanding GPCR signaling. Although all rhodopsins seem to become phosphorylated at their C-terminal region following activation by light, the role of this phosphorylation is not uniform. Two major functions of rhodopsin phosphorylation have been described: (1) inactivation of the activated rhodopsin either directly or by facilitating binding of arrestins in order to shut down the visual signaling cascade and thus eventually enabling a high-temporal resolution of the visual system. (2) Facilitating endocytosis of activated receptors via arrestin binding that in turn recruits clathrin to the membrane for clathrin-mediated endocytosis. In vertebrate rhodopsins the shutdown of the signaling cascade may be the main function of rhodopsin phosphorylation, as phosphorylation alone already quenches transducin activation and, in addition, strongly enhances arrestin binding. In the Drosophila visual system rhodopsin phosphorylation is not needed for receptor inactivation. Its role here may rather lie in the recruitment of arrestin 1 and subsequent endocytosis of the activated receptor. In this review, we summarize investigations of fly rhodopsin phosphorylation spanning four decades and contextualize them with regard to the most recent insights from vertebrate phosphorylation barcode theory.


Assuntos
Drosophila , Rodopsina , Animais , Rodopsina/metabolismo , Drosophila/metabolismo , Arrestina/metabolismo , Arrestinas/metabolismo , Fosforilação , Clatrina/metabolismo
17.
Toxins (Basel) ; 14(11)2022 Nov 08.
Artigo em Inglês | MEDLINE | ID: mdl-36356021

RESUMO

Curcin and Curcin C, both of the ribosome-inactivating proteins of Jatropha curcas, have apparent inhibitory effects on the proliferation of osteosarcoma cell line U20S. However, the inhibitory effect of the latter is 13-fold higher than that of Curcin. The mechanism responsible for the difference has not been studied. This work aimed to understand and verify whether there are differences in entry efficiency and pathway between them using specific endocytosis inhibitors, gene silencing, and labeling techniques such as fluorescein isothiocyanate (FITC) labeling. The study found that the internalization efficiency of Curcin C was twice that of Curcin for U2OS cells. More than one entering pathway was adopted by both of them. Curcin C can enter U2OS cells through clathrin-dependent endocytosis and macropinocytosis, but clathrin-dependent endocytosis was not an option for Curcin. The low-density lipoprotein receptor-related protein 1 (LRP1) was found to mediate clathrin-dependent endocytosis of Curcin C. After LRP1 silencing, there was no significant difference in the 50% inhibitory concentration (IC50) and endocytosis efficiency between Curcin and Curcin C on U2OS cells. These results indicate that LRP1-mediated endocytosis is specific to Curcin C, thus leading to higher U2OS endocytosis efficiency and cytotoxicity than Curcin.


Assuntos
Alcaloides , Jatropha , Osteossarcoma , Toxinas Biológicas , Humanos , Proteínas Inativadoras de Ribossomos Tipo 1/farmacologia , Jatropha/genética , Jatropha/metabolismo , Proteínas Inativadoras de Ribossomos/metabolismo , Toxinas Biológicas/metabolismo , Alcaloides/metabolismo , Clatrina/metabolismo , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Proteína-1 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo
18.
Virol J ; 19(1): 163, 2022 10 17.
Artigo em Inglês | MEDLINE | ID: mdl-36253859

RESUMO

BACKGROUND: Hepatitis delta virus (HDV), a satellite virus of hepatitis B virus (HBV), is a small, defective RNA virus strongly associated with the most severe form of hepatitis and progressive chronic liver disease and cirrhosis. Chronic hepatitis D, resulting from HBV/HDV coinfection, is considered to be the most severe form of viral hepatitis and affects 12-20 million people worldwide. Involved in the endocytosis and exocytosis of cellular and viral proteins, clathrin contributes to the pathogenesis and morphogenesis of HDV. Previously, we demonstrated that HDV-I and -II large hepatitis delta antigens (HDAg-L) possess a putative clathrin box that interacts with clathrin heavy chain (CHC) and supports HDV assembly. METHODS: Virus assembly and vesicular trafficking of HDV virus-like particles (VLPs) were evaluated in Huh7 cells expressing HDV-I, -II and -III HDAg-L and hepatitis B surface antigen (HBsAg). To elucidate the interaction motif between HDAg-L and CHC, site-directed mutagenesis was performed to introduce mutations into HDAg-L and CHC and analyzed using coimmunoprecipitation or pull-down assays. RESULTS: Comparable to HDV-I virus-like particles (VLPs), HDV-III VLPs were produced at a similar level and secreted into the medium via clathrin-mediated post-Golgi vesicular trafficking. Mutation at F27 or E33 of CHC abolished the binding of CHC to the C-terminus of HDV-III HDAg-L. Mutation at W207 of HDV-III HDAg-L inhibited its association with CHC and interfered with HDV-III VLP formation. We elucidated mechanism of the binding of HDV-III HDAg-L to CHC and confirmed the pivotal role of clathrin binding in the assembly of genotype III HDV. CONCLUSIONS: A novel W box which was identified at the C terminus of HDV-III HDAg-L is known to differ from the conventional clathrin box but also interacts with CHC. The novel W box of HDAg-L constitutes a new molecular target for anti-HDV-III therapeutics.


Assuntos
Antígenos de Superfície da Hepatite B , Vírus Delta da Hepatite , Clatrina/metabolismo , Cadeias Pesadas de Clatrina/genética , Cadeias Pesadas de Clatrina/metabolismo , Genótipo , Antígenos de Superfície da Hepatite B/metabolismo , Vírus da Hepatite B/genética , Vírus Delta da Hepatite/genética , Antígenos da Hepatite delta/química , Antígenos da Hepatite delta/genética , Antígenos da Hepatite delta/metabolismo , Humanos , RNA Viral/metabolismo , Proteínas Virais/genética , Replicação Viral
19.
BMC Med ; 20(1): 359, 2022 10 21.
Artigo em Inglês | MEDLINE | ID: mdl-36266654

RESUMO

BACKGROUND: The severe fever with thrombocytopenia syndrome disease (SFTS), caused by the novel tick-borne SFTS virus (SFTSV), was listed among the top 10 priority infectious disease by World Health Organization due to the high fatality rate of 5-30% and the lack of effective antiviral drugs and vaccines and therefore raised the urgent need to develop effective anti-SFTSV drugs to improve disease treatment. METHODS: The antiviral drugs to inhibit SFTSV infection were identified by screening the library containing 1340 FDA-approved drugs using the SFTSV infection assays in vitro. The inhibitory effect on virus entry and the process of clathrin-mediated endocytosis under different drug doses was evaluated based on infection assays by qRT-PCR to determine intracellular viral copies, by Western blot to characterize viral protein expression in cells, and by immunofluorescence assays (IFAs) to determine virus infection efficiencies. The therapeutic effect was investigated in type I interferon receptor defective A129 mice in vivo with SFTSV infection, from which lesions and infection in tissues caused by SFTSV infection were assessed by H&E staining and immunohistochemical analysis. RESULTS: Six drugs were identified as exerting inhibitory effects against SFTSV infection, of which anidulafungin, an antifungal drug of the echinocandin family, has a strong inhibitory effect on SFTSV entry. It suppresses SFTSV internalization by impairing the late endosome maturation and decreasing virus fusion with the membrane. SFTSV-infected A129 mice had relieving symptoms, reduced tissue lesions, and improved disease outcomes following anidulafungin treatment. Moreover, anidulafungin exerts an antiviral effect in inhibiting the entry of other viruses including SARS-CoV-2, SFTSV-related Guertu virus and Heartland virus, Crimean-Congo hemorrhagic fever virus, Zika virus, and Herpes simplex virus 1. CONCLUSIONS: The results demonstrated that the antifungal drug, anidulafungin, could effectively inhibit virus infection by interfering with virus entry, suggesting it may be utilized for the clinical treatment of infectious viral diseases, in addition to its FDA-approved use as an antifungal. The findings also suggested to further evaluate the anti-viral effects of echinocandins and their clinical importance for patients with infection of viruses, which may promote therapeutic strategies as well as treatments and improve outcomes pertaining to various viral and fungal diseases.


Assuntos
Anidulafungina , Infecções por Bunyaviridae , Viroses , Animais , Camundongos , Anidulafungina/farmacologia , Anidulafungina/uso terapêutico , Antifúngicos/farmacologia , Antifúngicos/uso terapêutico , Antivirais/farmacologia , Antivirais/uso terapêutico , Infecções por Bunyaviridae/tratamento farmacológico , Clatrina , Receptor de Interferon alfa e beta , SARS-CoV-2 , Proteínas Virais , Viroses/tratamento farmacológico
20.
Nature ; 610(7933): 761-767, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-36261523

RESUMO

Stimulator of interferon genes (STING) functions downstream of cyclic GMP-AMP synthase in DNA sensing or as a direct receptor for bacterial cyclic dinucleotides and small molecules to activate immunity during infection, cancer and immunotherapy1-10. Precise regulation of STING is essential to ensure balanced immune responses and prevent detrimental autoinflammation11-16. After activation, STING, a transmembrane protein, traffics from the endoplasmic reticulum to the Golgi, where its phosphorylation by the protein kinase TBK1 enables signal transduction17-20. The mechanism that ends STING signalling at the Golgi remains unknown. Here we show that adaptor protein complex 1 (AP-1) controls the termination of STING-dependent immune activation. We find that AP-1 sorts phosphorylated STING into clathrin-coated transport vesicles for delivery to the endolysosomal system, where STING is degraded21. We identify a highly conserved dileucine motif in the cytosolic C-terminal tail (CTT) of STING that, together with TBK1-dependent CTT phosphorylation, dictates the AP-1 engagement of STING. A cryo-electron microscopy structure of AP-1 in complex with phosphorylated STING explains the enhanced recognition of TBK1-activated STING. We show that suppression of AP-1 exacerbates STING-induced immune responses. Our results reveal a structural mechanism of negative regulation of STING and establish that the initiation of signalling is inextricably associated with its termination to enable transient activation of immunity.


Assuntos
Complexo 1 de Proteínas Adaptadoras , Clatrina , Complexo 1 de Proteínas Adaptadoras/química , Complexo 1 de Proteínas Adaptadoras/metabolismo , Complexo 1 de Proteínas Adaptadoras/ultraestrutura , Clatrina/metabolismo , Microscopia Crioeletrônica , DNA/metabolismo , Imunidade Inata , Proteínas Serina-Treonina Quinases , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Proteínas de Membrana/ultraestrutura , Motivos de Aminoácidos , Endossomos/metabolismo , Lisossomos/metabolismo , Fosforilação
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