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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.
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
4.
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
5.
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
6.
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
7.
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
8.
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
9.
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
10.
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
11.
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
13.
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
14.
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
15.
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
16.
J Phys Chem B ; 126(42): 8391-8403, 2022 Oct 27.
Artigo em Inglês | MEDLINE | ID: mdl-36255318

RESUMO

Hepatitis C virus (HCV) is the second viral agent that causes the majority of chronic hepatic infections worldwide, following Hepatitis B virus (HBV) infection. HCV infection comprises several steps, from the attachment to the receptors to the delivery of the viral genetic material and replication inside the cells. Tetraspanin CD81 is a key entry factor for HCV as it accompanies the virus during attachment and internalization through clathrin-mediated endocytosis. HCV-CD81 binding takes place through the viral glycoprotein E2. We performed full-atom molecular dynamics simulations reproducing the pH conditions that occur during the viral attachment to the hepatocytes (pH 7.4) and internalization (pH 6.2-4.6). We observed that changing the pH from 7.4 to 6.2 triggers a large conformational change in the binding orientation between E2core (E2core corresponds to residues 412-645 of the viral glycoprotein E2) and CD81LEL (CD81LEL corresponds to residues 112-204 of CD81) that occurs even more rapidly at low pH 4.6. This pH-induced switching mechanism has never been observed before and could allow the virus particles to sense the right moment during the maturation of the endosome to start fusion.


Assuntos
Hepacivirus , Hepatite C , Humanos , Tetraspanina 28/química , Tetraspanina 28/metabolismo , Hepacivirus/metabolismo , Proteínas do Envelope Viral/química , Clatrina/metabolismo
17.
Cell Mol Biol Lett ; 27(1): 90, 2022 Oct 11.
Artigo em Inglês | MEDLINE | ID: mdl-36221072

RESUMO

BACKGROUND: E2F1 is a transcription factor that regulates cell cycle progression. It is highly expressed in most cancer cells and activates transcription of cell cycle-related kinases. Stathmin1 and transforming acidic coiled-coil-containing protein 3 (TACC3) are factors that enhance the stability of spindle fiber. METHODS: The E2F1-mediated transcription of transforming acidic coiled-coil-containing protein 3 (TACC3) and stathmin1 was examined using the Cancer Genome Atlas (TCGA) analysis, quantitative polymerase chain reaction (qPCR), immunoblotting, chromatin immunoprecipitation (ChIP), and luciferase reporter. Protein-protein interaction was studied using co-IP. The spindle structure was shown by immunofluorescence. Phenotype experiments were performed through MTS assay, flow cytometry, and tumor xenografts. Clinical colorectal cancer (CRC) specimens were analyzed based on immunohistochemistry. RESULTS: The present study showed that E2F1 expression correlates positively with the expression levels of stathmin1 and TACC3 in colorectal cancer (CRC) tissues, and that E2F1 transactivates stathmin1 and TACC3 in CRC cells. Furthermore, protein kinase A (PKA)-mediated phosphorylation of stathmin1 at Ser16 is essential to the phosphorylation of TACC3 at Ser558, facilitating the assembly of TACC3/clathrin/α-tubulin complexes during spindle formation. Overexpression of Ser16-mutated stathmin1, as well as knockdown of stathmin1 or TACC3, lead to ectopic spindle poles including disorganized and multipolar spindles. Overexpression of wild-type but not Ser16-mutated stathmin1 promotes cell proliferation in vitro and tumor growth in vivo. Consistently, a high level of E2F1, stathmin1, or TACC3 not only associates with tumor size, lymph node metastasis, TNM stage, and distant metastasis, but predicts poor survival in CRC patients. CONCLUSIONS: E2F1 drives the cell cycle of CRC by promoting spindle assembly, in which E2F1-induced stathmin1 and TACC3 enhance the stability of spindle fiber.


Assuntos
Neoplasias Colorretais , Fuso Acromático , Ciclo Celular , Clatrina/metabolismo , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Fator de Transcrição E2F1/genética , Fator de Transcrição E2F1/metabolismo , Humanos , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Fuso Acromático/genética , Fuso Acromático/metabolismo , Fatores de Transcrição/metabolismo , Tubulina (Proteína)/metabolismo
18.
Plant Signal Behav ; 17(1): 2129296, 2022 12 31.
Artigo em Inglês | MEDLINE | ID: mdl-36200597

RESUMO

Arabidopsis DYNAMIN-RELATED PROTEIN1A (AtDRP1A) and AtDRP2B are large GTPases that function together in endocytosis for effective cytokinesis, cell enlargement and development. A recent study shows that these DRPs contribute to ligand-induced endocytosis of the immune receptor FLAGELLIN SENSING2 (AtFLS2) to modulate flg22-immune signaling, and they are required for immunity against Pseudomonas syringae pv. tomato bacteria. Here, we demonstrate that atdrp1a and atdrp2b single mutants showed increased susceptibility to Botrytis cinerea indicating that AtDRP1A and AtDRP2B are necessary for effective resistance against this necrotrophic fungus. Thus, we expanded our limited understanding of clathrin endocytic accessory proteins in immunity against plant pathogens.


Assuntos
Proteínas de Arabidopsis , Arabidopsis , Micoses , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Botrytis , Clatrina/metabolismo , Clatrina/farmacologia , Dinaminas/genética , Dinaminas/metabolismo , Flagelina/farmacologia , Regulação da Expressão Gênica de Plantas , Ligantes , Doenças das Plantas/microbiologia , Pseudomonas syringae
19.
Viruses ; 14(10)2022 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-36298756

RESUMO

Caveolin-1 (Cav-1) is the basic component of caveolae, a specialized form of lipid raft that plays an essential role in endocytic viral entry. However, the evidence of direct involvement of caveolae and Cav-1 in avian reovirus (ARV) entry remains insufficient. In this study, the membrane lipid rafts were isolated as detergent-resistant microdomains (DRMs) by sucrose gradient centrifugation, and the capsid protein σB of ARV was found to associate with Cav-1 in DRMs fractions. Additionally, the interaction between ARV σB protein and Cav-1 was demonstrated by immunofluorescence co-localization and co-immunoprecipitation assays. Furthermore, we found that the internalization of ARV is sensitive to caveolae and dynamin inhibitors, while it is insensitive to clathrin inhibitors. In conclusion, these results indicate that the ARV σB protein interacts with Cav-1 during dynamin-dependent caveolae-mediated endocytosis for the entry of ARV.


Assuntos
Caveolina 1 , Orthoreovirus Aviário , Caveolina 1/metabolismo , Cavéolas/metabolismo , Detergentes , Proteínas do Capsídeo/metabolismo , Microdomínios da Membrana/metabolismo , Endocitose , Clatrina/metabolismo , Dinaminas/metabolismo , Sacarose/metabolismo , Lipídeos de Membrana/metabolismo
20.
Cells ; 11(19)2022 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-36231005

RESUMO

The transport of low-density lipoprotein (LDL) through the endothelium is a key step in the development of atherosclerosis, but it is notorious that phenotypic differences exist between endothelial cells originating from different vascular beds. Endothelial cells forming the blood-brain barrier restrict paracellular and transcellular passage of plasma proteins. Here, we systematically compared brain versus aortic endothelial cells towards their interaction with LDL and the role of proteins known to regulate the uptake of LDL by endothelial cells. Both brain endothelial cells and aortic endothelial cells bind and internalize LDL. However, whereas aortic endothelial cells degrade very small amounts of LDL and transcytose the majority, brain endothelial cells degrade but do not transport LDL. Using RNA interference (siRNA), we found that the LDLR-clathrin pathway leads to LDL degradation in either endothelial cell type. Both loss- and gain-of-function experiments showed that ALK1, which promotes transcellular LDL transport in aortic endothelial cells, also limits LDL degradation in brain endothelial cells. SR-BI and caveolin-1, which promote LDL uptake and transport into aortic endothelial cells, limit neither binding nor association of LDL to brain endothelial cells. Together, these results indicate distinct LDL trafficking by brain microvascular endothelial cells and aortic endothelial cells.


Assuntos
Células Endoteliais , Lipoproteínas LDL , Encéfalo/metabolismo , Caveolina 1/metabolismo , Clatrina/metabolismo , Células Endoteliais/metabolismo , Endotélio/metabolismo , Lipoproteínas LDL/metabolismo , RNA Interferente Pequeno/metabolismo
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