RESUMO
Through a network of progressively maturing vesicles, the endosomal system connects the cell's interior with extracellular space. Intriguingly, this network exhibits a bilateral architecture, comprised of a relatively immobile perinuclear vesicle "cloud" and a highly dynamic peripheral contingent. How this spatiotemporal organization is achieved and what function(s) it curates is unclear. Here, we reveal the endoplasmic reticulum (ER)-located ubiquitin ligase Ring finger protein 26 (RNF26) as the global architect of the entire endosomal system, including the trans-Golgi network (TGN). To specify perinuclear vesicle coordinates, catalytically competent RNF26 recruits and ubiquitinates the scaffold p62/sequestosome 1 (p62/SQSTM1), in turn attracting ubiquitin-binding domains (UBDs) of various vesicle adaptors. Consequently, RNF26 restrains fast transport of diverse vesicles through a common molecular mechanism operating at the ER membrane, until the deubiquitinating enzyme USP15 opposes RNF26 activity to allow vesicle release into the cell's periphery. By drawing the endosomal system's architecture, RNF26 orchestrates endosomal maturation and trafficking of cargoes, including signaling receptors, in space and time.
Assuntos
Retículo Endoplasmático/metabolismo , Endossomos/metabolismo , Membranas Intracelulares/metabolismo , Proteínas de Neoplasias/metabolismo , Linhagem Celular Tumoral , Células Dendríticas/citologia , Células Dendríticas/metabolismo , Humanos , Macrófagos/citologia , Macrófagos/metabolismo , Proteína Sequestossoma-1/metabolismo , Vesículas Transportadoras/metabolismo , Proteases Específicas de Ubiquitina/metabolismoRESUMO
HLA class I (HLA-I) glycoproteins drive immune responses by presenting antigens to cognate CD8+ T cells. This process is often hijacked by tumors and pathogens for immune evasion. Because options for restoring HLA-I antigen presentation are limited, we aimed to identify druggable HLA-I pathway targets. Using iterative genome-wide screens, we uncovered that the cell surface glycosphingolipid (GSL) repertoire determines effective HLA-I antigen presentation. We show that absence of the protease SPPL3 augmented B3GNT5 enzyme activity, resulting in upregulation of surface neolacto-series GSLs. These GSLs sterically impeded antibody and receptor interactions with HLA-I and diminished CD8+ T cell activation. Furthermore, a disturbed SPPL3-B3GNT5 pathway in glioma correlated with decreased patient survival. We show that the immunomodulatory effect could be reversed through GSL synthesis inhibition using clinically approved drugs. Overall, our study identifies a GSL signature that inhibits immune recognition and represents a potential therapeutic target in cancer, infection, and autoimmunity.
Assuntos
Ácido Aspártico Endopeptidases/metabolismo , Linfócitos T CD8-Positivos/imunologia , Glioma/imunologia , Glicoesfingolipídeos/metabolismo , Glicosiltransferases/metabolismo , Antígenos HLA/metabolismo , Antígenos de Histocompatibilidade Classe I/metabolismo , Imunoterapia/métodos , Apresentação de Antígeno , Ácido Aspártico Endopeptidases/genética , Linhagem Celular Tumoral , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Glioma/mortalidade , Glicoesfingolipídeos/imunologia , Antígenos HLA/imunologia , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Ativação Linfocitária , Transdução de Sinais , Análise de Sobrevida , Evasão TumoralRESUMO
MHC class II molecules (MHC-II) present peptides to T helper cells to facilitate immune responses and are strongly linked to autoimmune diseases. To unravel processes controlling MHC-II antigen presentation, we performed a genome-wide flow cytometry-based RNAi screen detecting MHC-II expression and peptide loading followed by additional high-throughput assays. All data sets were integrated to answer two fundamental questions: what regulates tissue-specific MHC-II transcription, and what controls MHC-II transport in dendritic cells? MHC-II transcription was controlled by nine regulators acting in feedback networks with higher-order control by signaling pathways, including TGFß. MHC-II transport was controlled by the GTPase ARL14/ARF7, which recruits the motor myosin 1E via an effector protein ARF7EP. This complex controls movement of MHC-II vesicles along the actin cytoskeleton in human dendritic cells (DCs). These genome-wide systems analyses have thus identified factors and pathways controlling MHC-II transcription and transport, defining targets for manipulation of MHC-II antigen presentation in infection and autoimmunity.
Assuntos
Apresentação de Antígeno , Estudo de Associação Genômica Ampla , Antígenos de Histocompatibilidade Classe II/imunologia , Actinas/metabolismo , Autoimunidade , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Humanos , Miosinas/metabolismo , Interferência de RNARESUMO
The efficacy of anthracycline-based chemotherapeutics, which include doxorubicin and its structural relatives daunorubicin and idarubicin, remains almost unmatched in oncology, despite a side effect profile including cumulative dose-dependent cardiotoxicity, therapy-related malignancies and infertility. Detoxifying anthracyclines while preserving their anti-neoplastic effects is arguably a major unmet need in modern oncology, as cardiovascular complications that limit anti-cancer treatment are a leading cause of morbidity and mortality among the 17 million cancer survivors in the U.S. In this study, we examined different clinically relevant anthracycline drugs for a series of features including mode of action (chromatin and DNA damage), bio-distribution, anti-tumor efficacy and cardiotoxicity in pre-clinical models and patients. The different anthracycline drugs have surprisingly individual efficacy and toxicity profiles. In particular, aclarubicin stands out in pre-clinical models and clinical studies, as it potently kills cancer cells, lacks cardiotoxicity, and can be safely administered even after the maximum cumulative dose of either doxorubicin or idarubicin has been reached. Retrospective analysis of aclarubicin used as second-line treatment for relapsed/refractory AML patients showed survival effects similar to its use in first line, leading to a notable 23% increase in 5-year overall survival compared to other intensive chemotherapies. Considering individual anthracyclines as distinct entities unveils new treatment options, such as the identification of aclarubicin, which significantly improves the survival outcomes of AML patients while mitigating the treatment-limiting side-effects. Building upon these findings, an international multicenter Phase III prospective study is prepared, to integrate aclarubicin into the treatment of relapsed/refractory AML patients.
Assuntos
Aclarubicina , Antraciclinas , Leucemia Mieloide Aguda , Animais , Feminino , Humanos , Masculino , Aclarubicina/farmacologia , Aclarubicina/uso terapêutico , Antraciclinas/uso terapêutico , Antineoplásicos/uso terapêutico , Antineoplásicos/farmacologia , Antineoplásicos/efeitos adversos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/mortalidade , Resultado do TratamentoRESUMO
The endolysosomal system fulfils a myriad of cellular functions predicated on regulated membrane identity progressions, collectively termed maturation. Mature or "late" endosomes are designated by small membrane-bound GTPases Rab7 and Arl8b, which can either operate independently or collaborate to form a joint compartment. Whether, and how, Rab7 and Arl8b resolve this hybrid identity compartment to regain functional autonomy is unknown. Here, we report that Arl8b employs its effector SKIP to instigate inactivation and removal of Rab7 from select membranes. We find that SKIP interacts with Rab7 and functions as its negative effector, delivering the cognate GAP, TBC1D15. Recruitment of TBC1D15 to SKIP occurs via the HOPS complex, whose assembly is facilitated by contacts between Rab7 and the KMI motif of SKIP. Consequently, SKIP mediates reinstatement of single identity Arl8b sub-compartment through an ordered Rab7-to-Arl8b handover, and, together with Rab7's positive effector RILP, enforces spatial, temporal and morphological compartmentalization of endolysosomal organelles.
Assuntos
Fatores de Ribosilação do ADP/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Fatores de Ribosilação do ADP/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Compartimento Celular , Endossomos/metabolismo , Proteínas Ativadoras de GTPase/genética , Células HEK293 , Humanos , Lisossomos/metabolismo , Ligação Proteica , Transporte Proteico , Proteínas rab de Ligação ao GTP/genética , proteínas de unión al GTP Rab7RESUMO
The invariant chain (Ii, also known as CD74) is a multifunctional regulator of adaptive immune responses and is responsible for sorting major histocompatibility complex class I and class II (MHCI and MHCII, respectively) molecules, as well as other Ii-associated molecules, to a specific endosomal pathway. When Ii is expressed, endosomal maturation and proteolytic degradation of proteins are delayed and, in non-antigen presenting cells, the endosomal size increases, but the molecular mechanisms underlying this are not known. We identified that a SNARE, Vti1b, is essential for regulating these Ii-induced effects. Vti1b binds to Ii and is localized at the contact sites of fusing Ii-positive endosomes. Furthermore, truncated Ii lacking the cytoplasmic tail, which is not internalized from the plasma membrane, relocates Vti1b to the plasma membrane. Knockout of Ii in an antigen-presenting cell line was found to speed up endosomal maturation, whereas silencing of Vti1b inhibits the Ii-induced maturation delay. Our results suggest that Ii, by interacting with the SNARE Vti1b in antigen-presenting cells, directs specific Ii-associated SNARE-mediated fusion in the early part of the endosomal pathway that leads to a slower endosomal maturation for efficient antigen processing and MHC antigen loading.
Assuntos
Antígenos de Diferenciação de Linfócitos B , Proteínas SNARE , Animais , Antígenos de Diferenciação de Linfócitos B/genética , Endossomos , Antígenos de Histocompatibilidade Classe II/genética , Humanos , Ratos , Proteínas SNARE/genéticaRESUMO
Upon nuclear envelope (NE) fragmentation in the prometaphase, the nuclear and cytosolic proteomes mix and must be redefined to reinstate homeostasis. Here, by using a molecular GFP ladder, we show that in early mitosis, condensed chromatin excludes cytosolic proteins. When the NE reforms tightly around condensed chromatin in late mitosis, large GFP multimers are automatically excluded from the nucleus. This can be circumvented by limiting DNA condensation with Q15, a condensin II inhibitor. Soluble small and other nuclear localization sequence (NLS)-targeted proteins then swiftly enter the expanding nuclear space. We then examined proteasomes, which are located in the cytoplasm and nucleus. A significant fraction of 20S proteasomes is imported by the importin IPO5 within 20â min of reformation of the nucleus, after which import comes to an abrupt halt. This suggests that maintaining the nuclear-cytosol distribution after mitosis requires chromatin condensation to exclude cytosolic material from the nuclear space, and specialized machineries for nuclear import of large protein complexes, such as the proteasome.
Assuntos
Membrana Nuclear/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Transporte Ativo do Núcleo Celular , Linhagem Celular Tumoral , Cromossomos Humanos , Células HeLa , Homeostase , Humanos , Mitose/fisiologiaRESUMO
MHC class I molecules present a variable but limited repertoire of antigenic peptides for T-cell recognition. Understanding how peptide selection is achieved requires mechanistic insights into the interactions between the MHC I and candidate peptides. We find that, at first encounter, MHC I H-2K(b) considers a wide range of peptides, including those with expanded N termini and unfitting anchor residues. Discrimination occurs in the second step, when noncanonical peptides dissociate with faster exchange rates. This second step exhibits remarkable temperature sensitivity, as illustrated by numerous noncanonical peptides presented by H-2K(b) in cells cultured at 26 °C relative to 37 °C. Crystallographic analyses of H-2K(b)-peptide complexes suggest that a conformational adaptation of H-2K(b) drives the decisive step in peptide selection. We propose that MHC class I molecules consider initially a large peptide pool, subsequently refined by a temperature-sensitive induced-fit mechanism to retain the canonical peptide repertoire.
Assuntos
Antígenos de Histocompatibilidade Classe I/química , Peptídeos/química , Sequência de Aminoácidos , Cristalografia por Raios X , Entropia , Cinética , Peptídeos/imunologiaRESUMO
Peptide splicing, in which two distant parts of a protein are excised and then ligated to form a novel peptide, can generate unique MHC class I-restricted responses. Because these peptides are not genetically encoded and the rules behind proteasomal splicing are unknown, it is difficult to predict these spliced Ags. In the current study, small libraries of short peptides were used to identify amino acid sequences that affect the efficiency of this transpeptidation process. We observed that splicing does not occur at random, neither in terms of the amino acid sequences nor through random splicing of peptides from different sources. In contrast, splicing followed distinct rules that we deduced and validated both in vitro and in cells. Peptide ligation was quantified using a model peptide and demonstrated to occur with up to 30% ligation efficiency in vitro, provided that optimal structural requirements for ligation were met by both ligating partners. In addition, many splicing products could be formed from a single protein. Our splicing rules will facilitate prediction and detection of new spliced Ags to expand the peptidome presented by MHC class I Ags.
Assuntos
Apresentação de Antígeno , Antígenos de Histocompatibilidade Classe I/imunologia , Peptídeos/imunologia , Complexo de Endopeptidases do Proteassoma/química , Processamento de Proteína , Sequência de Aminoácidos , Humanos , Dados de Sequência Molecular , Complexo de Endopeptidases do Proteassoma/imunologiaRESUMO
Trafficking of cargo through the endosomal system depends on endosomal fusion events mediated by SNARE proteins, Rab-GTPases, and multisubunit tethering complexes. The CORVET and HOPS tethering complexes, respectively, regulate early and late endosomal tethering and have been characterized in detail in yeast where their sequential membrane targeting and assembly is well understood. Mammalian CORVET and HOPS subunits significantly differ from their yeast homologues, and novel proteins with high homology to CORVET/HOPS subunits have evolved. However, an analysis of the molecular interactions between these subunits in mammals is lacking. Here, we provide a detailed analysis of interactions within the mammalian CORVET and HOPS as well as an additional endosomal-targeting complex (VIPAS39-VPS33B) that does not exist in yeast. We show that core interactions within CORVET and HOPS are largely conserved but that the membrane-targeting module in HOPS has significantly changed to accommodate binding to mammalian-specific RAB7 interacting lysosomal protein (RILP). Arthrogryposis-renal dysfunction-cholestasis (ARC) syndrome-associated mutations in VPS33B selectively disrupt recruitment to late endosomes by RILP or binding to its partner VIPAS39. Within the shared core of CORVET/HOPS, we find that VPS11 acts as a molecular switch that binds either CORVET-specific TGFBRAP1 or HOPS-specific VPS39/RILP thereby allowing selective targeting of these tethering complexes to early or late endosomes to time fusion events in the endo/lysosomal pathway.
Assuntos
Endossomos/metabolismo , Complexos Multiproteicos/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Substituição de Aminoácidos , Artrogripose/genética , Artrogripose/metabolismo , Artrogripose/patologia , Proteínas Relacionadas à Autofagia , Linhagem Celular , Colestase/genética , Colestase/metabolismo , Colestase/patologia , Endossomos/genética , Endossomos/patologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Complexos Multiproteicos/genética , Mutação de Sentido Incorreto , Insuficiência Renal/genética , Insuficiência Renal/metabolismo , Insuficiência Renal/patologia , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismoRESUMO
C-type lectins are key players in immune regulation by driving distinct functions of antigen-presenting cells. The C-type lectin CLEC16A gene is located at 16p13, a susceptibility locus for several autoimmune diseases, including multiple sclerosis. However, the function of this gene and its potential contribution to these diseases in humans are poorly understood. In this study, we found a strong upregulation of CLEC16A expression in the white matter of multiple sclerosis patients (n = 14) compared to non-demented controls (n = 11), mainly in perivascular leukocyte infiltrates. Moreover, CLEC16A levels were significantly enhanced in peripheral blood mononuclear cells of multiple sclerosis patients (n = 69) versus healthy controls (n = 46). In peripheral blood mononuclear cells, CLEC16A was most abundant in monocyte-derived dendritic cells, in which it strongly co-localized with human leukocyte antigen class II. Treatment of these professional antigen-presenting cells with vitamin D, a key protective environmental factor in multiple sclerosis, downmodulated CLEC16A in parallel with human leukocyte antigen class II. Knockdown of CLEC16A in distinct types of model and primary antigen-presenting cells resulted in severely impaired cytoplasmic distribution and formation of human leucocyte antigen class II-positive late endosomes, as determined by immunofluorescence and electron microscopy. Mechanistically, CLEC16A participated in the molecular machinery of human leukocyte antigen class II-positive late endosome formation and trafficking to perinuclear regions, involving the dynein motor complex. By performing co-immunoprecipitations, we found that CLEC16A directly binds to two critical members of this complex, RILP and the HOPS complex. CLEC16A silencing in antigen-presenting cells disturbed RILP-mediated recruitment of human leukocyte antigen class II-positive late endosomes to perinuclear regions. Together, we identify CLEC16A as a pivotal gene in multiple sclerosis that serves as a direct regulator of the human leukocyte antigen class II pathway in antigen-presenting cells. These findings are a first step in coupling multiple sclerosis-associated genes to the regulation of the strongest genetic factor in multiple sclerosis, human leukocyte antigen class II.
Assuntos
Endossomos/metabolismo , Predisposição Genética para Doença/genética , Antígenos de Histocompatibilidade Classe II/biossíntese , Lectinas Tipo C/fisiologia , Proteínas de Transporte de Monossacarídeos/fisiologia , Esclerose Múltipla/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Adolescente , Adulto , Idoso , Células Apresentadoras de Antígenos/efeitos dos fármacos , Células Apresentadoras de Antígenos/metabolismo , Estudos de Casos e Controles , Células Cultivadas , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/metabolismo , Feminino , Técnicas de Silenciamento de Genes , Humanos , Lectinas Tipo C/genética , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/ultraestrutura , Masculino , Pessoa de Meia-Idade , Proteínas de Transporte de Monossacarídeos/genética , Transporte Proteico/genética , RNA Interferente Pequeno/farmacologia , Regulação para Cima/efeitos dos fármacos , Vitamina D/farmacologia , Substância Branca/metabolismo , Adulto JovemRESUMO
Late endosomes and lysosomes are dynamic organelles that constantly move and fuse to acquire cargo from early endosomes, phagosomes and autophagosome. Defects in lysosomal dynamics cause severe neurodegenerative and developmental diseases, such as Niemann-Pick type C disease and ARC syndrome, yet little is known about the regulation of late endosomal fusion in a mammalian system. Mammalian endosomes destined for fusion need to be transported over very long distances before they tether to initiate contact. Here, we describe that lysosomal tethering and transport are combined processes co-regulated by one multi-protein complex: RAB7-RILP-ORP1L. We show that RILP directly and concomitantly binds the tethering HOPS complex and the p150(Glued) subunit of the dynein motor. ORP1L then functions as a cholesterol-sensing switch controlling RILP-HOPS-p150(Glued) interactions. We show that RILP and ORP1L control Ebola virus infection, a process dependent on late endosomal fusion. By combining recruitment and regulation of both the dynein motor and HOPS complex into a single multiprotein complex, the RAB7-RILP-ORP1L complex efficiently couples and regulates the timing of microtubule minus-end transport and fusion, two major events in endosomal biology.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Colesterol/metabolismo , Endossomos/metabolismo , Receptores de Esteroides/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Linhagem Celular Tumoral , Dineínas/genética , Dineínas/metabolismo , Humanos , Melanoma/genética , Melanoma/metabolismo , Receptores de Esteroides/genética , Vesículas Transportadoras/metabolismo , Proteínas rab de Ligação ao GTP/genéticaRESUMO
Late endosomal multivesicular bodies (MVBs) are complicated organelles with various subdomains located at the limiting membrane and the internal vesicles (ILVs). ILVs accumulate tetraspanins such as CD63 and CD82 that might form protein assemblies, including major histocompatibility complex class II (MHC-II) and its chaperone human leukocyte antigen (HLA)-DM. Here, we studied the effect of four late endosomal tetraspanin proteins on MHC-II expression. Silencing CD9, CD63 and CD81 enhanced MHC-II expression whereas silencing CD82 did not. No effect on peptide loading was observed. Using confocal FRET technology, we measured the dynamics of CD63 and CD82 interaction with MHC-II and its chaperone HLA-DM. CD63-CD82 interactions remained unaltered in the two MVB subdomains whereas the interactions between CD63 or CD82 homologous pairs changed. CD63 stably associated with MHC-II, and CD82 with HLA-DM, on both MVB subdomains whereas the CD82-MHC-II and CD63-HLA-DM interactions changed. These data visualize for the first time the protein dynamics of tetraspanin assemblies in MVB subdomains. CD63, unlike CD82, stably interacts with MHC-II at both MVB subdomains and controls MHC-II expression.
Assuntos
Antígenos HLA-D/metabolismo , Proteína Kangai-1/metabolismo , Corpos Multivesiculares/metabolismo , Tetraspanina 30/metabolismo , Linhagem Celular , Membrana Celular/metabolismo , Cloroquina/farmacologia , Difusão , Transferência Ressonante de Energia de Fluorescência , Células HEK293 , Antígenos HLA-DR/metabolismo , Humanos , Corpos Multivesiculares/efeitos dos fármacos , Corpos Multivesiculares/ultraestrutura , Tetraspaninas/metabolismoRESUMO
Cholesterol is an essential lipid in eukaryotic cells and is present in membranes of all intracellular compartments. A major source for cellular cholesterol is internalized lipoprotein particles that are transported toward acidic late endosomes (LE) and lysosomes. Here the lipoprotein particles are hydrolyzed, and free cholesterol is redistributed to other organelles. The LE can contain over half of the cellular cholesterol and, as a major sorting station, can contain many cholesterol-binding proteins from the ABCA, STARD, and ORP families. Here, we show that metastatic lymph node 64 (MLN64, STARD3) and oxysterol-binding protein-related protein 1L (ORP1L) define two subpopulations of LE. MLN64 is present on a LE containing the cholesterol transporter ABCA3, whereas ORP1L localizes to another population of LE containing Niemann Pick type C1 (NPC1), a cholesterol exporter. Endocytosed cargo passes through MLN64/ABCA3-positive compartments before it reaches ORP1L/NPC1-positive LE. The MLN64/ABCA3 compartments cycle between LE and plasma membrane and frequently contact "later" ORP1L/NPC1-containing LE. We propose two stages of cholesterol handling in late endosomal compartments: first, cholesterol enters MLN64/ABCA3-positive compartments from where it can be recycled to the plasma membrane, and later, cholesterol enters ORP1L/NPC1 endosomes that mediate cholesterol export to the endoplasmic reticulum.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Proteínas de Transporte/metabolismo , Colesterol/metabolismo , Endossomos/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de Membrana/metabolismo , Receptores de Esteroides/metabolismo , Membrana Celular/metabolismo , Células Cultivadas , Retículo Endoplasmático/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Proteína C1 de Niemann-PickRESUMO
With the emergence of multidrug resistant (MDR) bacteria, it is imperative to develop new intervention strategies. Current antibiotics typically target pathogen rather than host-specific biochemical pathways. Here we have developed kinase inhibitors that prevent intracellular growth of unrelated pathogens such as Salmonella typhimurium and Mycobacterium tuberculosis. An RNA interference screen of the human kinome using automated microscopy revealed several host kinases capable of inhibiting intracellular growth of S. typhimurium. The kinases identified clustered in one network around AKT1 (also known as PKB). Inhibitors of AKT1 prevent intracellular growth of various bacteria including MDR-M. tuberculosis. AKT1 is activated by the S. typhimurium effector SopB, which promotes intracellular survival by controlling actin dynamics through PAK4, and phagosome-lysosome fusion through the AS160 (also known as TBC1D4)-RAB14 pathway. AKT1 inhibitors counteract the bacterial manipulation of host signalling processes, thus controlling intracellular growth of bacteria. By using a reciprocal chemical genetics approach, we identified kinase inhibitors with antibiotic properties and their host targets, and we determined host signalling networks that are activated by intracellular bacteria for survival.
Assuntos
Espaço Intracelular/microbiologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Salmonella typhimurium/crescimento & desenvolvimento , Animais , Antibacterianos/química , Antibacterianos/farmacologia , Linhagem Celular Tumoral , Humanos , Espaço Intracelular/efeitos dos fármacos , Isoquinolinas/química , Isoquinolinas/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/microbiologia , Redes e Vias Metabólicas , Camundongos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/antagonistas & inibidores , Interferência de RNA , Salmonella typhimurium/efeitos dos fármacos , Sulfonamidas/química , Sulfonamidas/farmacologiaRESUMO
CD70 is a TNF-related transmembrane molecule expressed by mature dendritic cells (DCs), which present antigens to T cells via major histocompatibility complex (MHC) molecules. In DCs, CD70 localizes with MHC class II molecules in late endosomal vesicles, known as MHC class II compartments (MIICs). MIICs are transported to the immune synapse when a DC contacts an antigen-specific CD4(+) T cell. Consequently, MHC class II and CD70 are simultaneously exposed to the T cell. Thereby, T-cell activation via the antigen receptor and CD70-mediated co-stimulation are synchronized, apparently to optimize the proliferative response. We report here that the invariant chain (Ii), a chaperone known to transport MHC class II to MIICs, performs a similar function for CD70. CD70 was found to travel by default to the plasma membrane, whereas Ii coexpression directed it to late endosomes and/or lysosomes. In cells containing the MHC class II presentation pathway, CD70 localized to MIICs. This localization relied on Ii, since transport of CD70 from the Golgi to MIICs was impeded in Ii-deficient DCs. Biophysical and biochemical studies revealed that CD70 and Ii participate in an MHC-class-II-independent complex. Thus, Ii supports transport of both MHC class II and CD70 to MIICs and thereby coordinates their delivery to CD4(+) T cells.
Assuntos
Antígenos de Diferenciação de Linfócitos B/metabolismo , Ligante CD27/metabolismo , Células Dendríticas/metabolismo , Endossomos/metabolismo , Antígenos de Histocompatibilidade Classe II/metabolismo , Animais , Apresentação de Antígeno , Antígenos de Diferenciação de Linfócitos B/genética , Antígenos de Diferenciação de Linfócitos B/imunologia , Ligante CD27/imunologia , Linfócitos T CD4-Positivos/imunologia , Células Dendríticas/patologia , Complexo de Golgi/metabolismo , Células HeLa , Antígenos de Histocompatibilidade Classe II/genética , Antígenos de Histocompatibilidade Classe II/imunologia , Humanos , Melanoma Experimental , Camundongos , Camundongos Knockout , Transporte Proteico/genéticaRESUMO
The small GTPase Rab7 controls late endocytic transport by the minus end-directed motor protein complex dynein-dynactin, but how it does this is unclear. Rab7-interacting lysosomal protein (RILP) and oxysterol-binding protein-related protein 1L (ORP1L) are two effectors of Rab7. We show that GTP-bound Rab7 simultaneously binds RILP and ORP1L to form a RILP-Rab7-ORP1L complex. RILP interacts directly with the C-terminal 25-kD region of the dynactin projecting arm p150(Glued), which is required for dynein motor recruitment to late endocytic compartments (LEs). Still, p150(Glued) recruitment by Rab7-RILP does not suffice to induce dynein-driven minus-end transport of LEs. ORP1L, as well as betaIII spectrin, which is the general receptor for dynactin on vesicles, are essential for dynein motor activity. Our results illustrate that the assembly of microtubule motors on endosomes involves a cascade of linked events. First, Rab7 recruits two effectors, RILP and ORP1L, to form a tripartite complex. Next, RILP directly binds to the p150(Glued) dynactin subunit to recruit the dynein motor. Finally, the specific dynein motor receptor Rab7-RILP is transferred by ORP1L to betaIII spectrin. Dynein will initiate translocation of late endosomes to microtubule minus ends only after interacting with betaIII spectrin, which requires the activities of Rab7-RILP and ORP1L.
Assuntos
Dineínas/metabolismo , Endocitose/fisiologia , Endossomos/metabolismo , Microtúbulos/metabolismo , Proteínas Motores Moleculares/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Transporte/metabolismo , Complexo Dinactina , Endossomos/ultraestrutura , Células HeLa , Humanos , Substâncias Macromoleculares , Proteínas Associadas aos Microtúbulos/metabolismo , Modelos Moleculares , Transporte Proteico/fisiologia , Receptores de Esteroides , Transdução de Sinais/fisiologia , Espectrina/metabolismo , proteínas de unión al GTP Rab7RESUMO
Using a novel approach that detects changes in the conformation of ERalpha, we studied the efficacy of anti-estrogens to inactivate ERalpha under different experimental conditions. We show that phosphorylation of serine-305 in the hinge region of ERalpha by protein kinase A (PKA) induced resistance to tamoxifen. Tamoxifen bound but then failed to induce the inactive conformation, invoking ERalpha-dependent transactivation instead. PKA activity thus induces a switch from antagonistic to agonistic effects of tamoxifen on ERalpha. In clinical samples, we found that downregulation of a negative regulator of PKA, PKA-RIalpha, was associated with tamoxifen resistance prior to treatment. Activation of PKA by downregulation of PKA-RIalpha converts tamoxifen from an ERalpha inhibitor into a growth stimulator, without any effect on ICI 182780 (Fulvestrant).
Assuntos
Neoplasias da Mama/patologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Resistencia a Medicamentos Antineoplásicos , Estradiol/análogos & derivados , Receptores de Estrogênio/química , Tamoxifeno/farmacologia , Antineoplásicos Hormonais/farmacologia , Sítios de Ligação , Western Blotting , Neoplasias da Mama/enzimologia , Divisão Celular , Linhagem Celular Tumoral , Regulação para Baixo , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Estradiol/farmacologia , Moduladores de Receptor Estrogênico/farmacologia , Receptor alfa de Estrogênio , Feminino , Transferência Ressonante de Energia de Fluorescência , Fulvestranto , Humanos , Luciferases/metabolismo , Microscopia Confocal , Modelos Biológicos , Hibridização de Ácido Nucleico , Análise de Sequência com Séries de Oligonucleotídeos , Fosforilação , Conformação Proteica , Estrutura Terciária de Proteína , Interferência de RNA , Receptores de Estrogênio/metabolismo , Serina/química , Fatores de Tempo , Ativação Transcricional , TransfecçãoRESUMO
MHC class I molecules present peptides from endogenous proteins. Ags can also be presented when derived from extracellular sources in the form of apoptotic bodies. Cross-presentation of such Ags by dendritic cells is required for proper CTL responses. The fate of Ags in cells initiated for apoptosis is unclear as is the mechanism of apoptosis-derived Ag transfer into dendritic cells. Here we show that novel Ags can be generated by caspases and be presented by MHC class I molecules of apoptotic cells. Since gap junctions function until apoptotic cells remodel to form apoptotic bodies, transfer and cross-presentation of apoptotic peptides by neighboring and dendritic cells occurs. We thus define a novel phase in classical Ag presentation and cross-presentation by MHC class I molecules: presentation of Ags created by caspase activities in cells in apoptosis.