RESUMO
Wnt/ß-catenin signaling is a primary pathway for stem cell maintenance during tissue renewal and a frequent target for mutations in cancer. Impaired Wnt receptor endocytosis due to loss of the ubiquitin ligase RNF43 gives rise to Wnt-hypersensitive tumors that are susceptible to anti-Wnt-based therapy. Contrary to this paradigm, we identify a class of RNF43 truncating cancer mutations that induce ß-catenin-mediated transcription, despite exhibiting retained Wnt receptor downregulation. These mutations interfere with a ubiquitin-independent suppressor role of the RNF43 cytosolic tail that involves Casein kinase 1 (CK1) binding and phosphorylation. Mechanistically, truncated RNF43 variants trap CK1 at the plasma membrane, thereby preventing ß-catenin turnover and propelling ligand-independent target gene transcription. Gene editing of human colon stem cells shows that RNF43 truncations cooperate with p53 loss to drive a niche-independent program for self-renewal and proliferation. Moreover, these RNF43 variants confer decreased sensitivity to anti-Wnt-based therapy. Our data demonstrate the relevance of studying patient-derived mutations for understanding disease mechanisms and improved applications of precision medicine.
Assuntos
Caseína Quinase I/metabolismo , Neoplasias/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Via de Sinalização Wnt , Caseína Quinase I/genética , Células HEK293 , Humanos , Neoplasias/genética , Neoplasias/patologia , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Ubiquitina-Proteína Ligases/genética , beta Catenina/genética , beta Catenina/metabolismoRESUMO
Mammalian Wnt proteins are believed to act as short-range signals, yet have not been previously visualized in vivo. Self-renewal, proliferation and differentiation are coordinated along a putative Wnt gradient in the intestinal crypt. Wnt3 is produced specifically by Paneth cells. Here we have generated an epitope-tagged, functional Wnt3 knock-in allele. Wnt3 covers basolateral membranes of neighbouring stem cells. In intestinal organoids, Wnt3-transfer involves direct contact between Paneth cells and stem cells. Plasma membrane localization requires surface expression of Frizzled receptors, which in turn is regulated by the transmembrane E3 ligases Rnf43/Znrf3 and their antagonists Lgr4-5/R-spondin. By manipulating Wnt3 secretion and by arresting stem-cell proliferation, we demonstrate that Wnt3 mainly travels away from its source in a cell-bound manner through cell division, and not through diffusion. We conclude that stem-cell membranes constitute a reservoir for Wnt proteins, while Frizzled receptor turnover and 'plasma membrane dilution' through cell division shape the epithelial Wnt3 gradient.
Assuntos
Membrana Celular/metabolismo , Mucosa Intestinal/citologia , Nicho de Células-Tronco , Células-Tronco/citologia , Células-Tronco/metabolismo , Via de Sinalização Wnt , Proteína Wnt3/metabolismo , Alelos , Animais , Adesão Celular , Divisão Celular , Difusão , Feminino , Receptores Frizzled/metabolismo , Técnicas de Introdução de Genes , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Masculino , Camundongos , Organoides/citologia , Organoides/metabolismo , Celulas de Paneth/citologia , Celulas de Paneth/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteína Wnt3/genéticaRESUMO
Wnt/ß-catenin signaling controls development and adult tissue homeostasis by regulating cell proliferation and cell fate decisions. Wnt binding to its receptors Frizzled (FZD) and low-density lipoprotein-related 6 (LRP6) at the cell surface initiates a signaling cascade that leads to the transcription of Wnt target genes. Upon Wnt binding, the receptors assemble into large complexes called signalosomes that provide a platform for interactions with downstream effector proteins. The molecular basis of signalosome formation and regulation remains elusive, largely due to the lack of tools to analyze its endogenous components. Here, we use internally tagged Wnt3a proteins to isolate and characterize activated, endogenous Wnt receptor complexes by mass spectrometry-based proteomics. We identify the single-span membrane protein TMEM59 as an interactor of FZD and LRP6 and a positive regulator of Wnt signaling. Mechanistically, TMEM59 promotes the formation of multimeric Wnt-FZD assemblies via intramembrane interactions. Subsequently, these Wnt-FZD-TMEM59 clusters merge with LRP6 to form mature Wnt signalosomes. We conclude that the assembly of multiprotein Wnt signalosomes proceeds along well-ordered steps that involve regulated intramembrane interactions.
Assuntos
Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Proteínas de Membrana/metabolismo , Complexos Multiproteicos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Via de Sinalização Wnt/fisiologia , Proteína Wnt3A/metabolismo , Animais , Células HEK293 , Humanos , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Proteínas de Membrana/genética , Camundongos , Complexos Multiproteicos/genética , Proteínas do Tecido Nervoso/genética , Proteína Wnt3A/genéticaRESUMO
LGR5+ stem cells reside at crypt bottoms, intermingled with Paneth cells that provide Wnt, Notch and epidermal growth factor signals. Here we find that the related RNF43 and ZNRF3 transmembrane E3 ubiquitin ligases are uniquely expressed in LGR5+ stem cells. Simultaneous deletion of the two genes encoding these proteins in the intestinal epithelium of mice induces rapidly growing adenomas containing high numbers of Paneth and LGR5+ stem cells. In vitro, growth of organoids derived from these adenomas is arrested when Wnt secretion is inhibited, indicating a dependence of the adenoma stem cells on Wnt produced by adenoma Paneth cells. In the HEK293T human cancer cell line, expression of RNF43 blocks Wnt responses and targets surface-expressed frizzled receptors to lysosomes. In the RNF43-mutant colorectal cancer cell line HCT116, reconstitution of RNF43 expression removes its response to exogenous Wnt. We conclude that RNF43 and ZNRF3 reduce Wnt signals by selectively ubiquitinating frizzled receptors, thereby targeting these Wnt receptors for degradation.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Endocitose , Proteínas Oncogênicas/metabolismo , Receptores Wnt/metabolismo , Células-Tronco/enzimologia , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Via de Sinalização Wnt , Adenoma/metabolismo , Adenoma/patologia , Animais , Proliferação de Células , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Receptores Frizzled/metabolismo , Células HEK293 , Humanos , Lisossomos/metabolismo , Camundongos , Proteínas Oncogênicas/deficiência , Proteínas Oncogênicas/genética , Organoides/citologia , Organoides/metabolismo , Organoides/patologia , Celulas de Paneth/metabolismo , Celulas de Paneth/patologia , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Receptores Wnt/antagonistas & inibidores , Células-Tronco/citologia , Células-Tronco/metabolismo , Proteínas Supressoras de Tumor/deficiência , Proteínas Supressoras de Tumor/genética , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/deficiência , Ubiquitina-Proteína Ligases/genética , Ubiquitinação , Via de Sinalização Wnt/efeitos dos fármacos , beta Catenina/metabolismoRESUMO
Wnt binding to members of the seven-span transmembrane Frizzled (Fz) receptor family controls essential cell fate decisions and tissue polarity during development and in adulthood. The Fz-mediated membrane recruitment of the cytoplasmic effector Dishevelled (Dvl) is a critical step in Wnt/ß-catenin signaling initiation, but how Fz and Dvl act together to drive downstream signaling events remains largely undefined. Here, we use an Fz peptide-based microarray to uncover a mechanistically important role of the bipartite Dvl DEP domain and C terminal region (DEP-C) in binding a three-segmented discontinuous motif in Fz. We show that cooperative use of two conserved motifs in the third intracellular loop and the classic C-terminal motif of Fz is required for DEP-C binding and Wnt-induced ß-catenin activation in cultured cells and Xenopus embryos. Within the complex, the Dvl DEP domain mainly binds the Fz C-terminal tail, whereas a short region at the Dvl C-terminal end is required to bind the Fz third loop and stabilize the Fz-Dvl interaction. We conclude that Dvl DEP-C binding to Fz is a key event in Wnt-mediated signaling relay to ß-catenin. The discontinuous nature of the Fz-Dvl interface may allow for precise regulation of the interaction in the control of Wnt-dependent cellular responses.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Receptores Frizzled/metabolismo , Fosfoproteínas/metabolismo , Transdução de Sinais , Proteínas Wnt/metabolismo , beta Catenina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Sequência de Aminoácidos , Linhagem Celular , Proteínas Desgrenhadas , Polarização de Fluorescência , Receptores Frizzled/química , Humanos , Microscopia Confocal , Dados de Sequência Molecular , Fosfoproteínas/química , Ligação Proteica , Proteínas de XenopusRESUMO
The transmembrane E3 ligases RNF43 and ZNRF3 perform key tumour suppressor roles by inducing endocytosis of members of the Frizzled (FZD) family, the primary receptors for WNT. Loss-of-function mutations in RNF43 and ZNRF3 mediate FZD stabilisation and a WNT-hypersensitive growth state in various cancer types. Strikingly, RNF43 and ZNRF3 mutations are differentially distributed across cancer types, raising questions about their functional redundancy. Here, we compare the efficacy of RNF43 and ZNRF3 of targeting different FZDs for endocytosis. We find that RNF43 preferentially down-regulates FZD1/FZD5/FZD7, whereas ZNRF3 displays a preference towards FZD6. We show that the RNF43 transmembrane domain (TMD) is a key molecular determinant for inducing FZD5 endocytosis. Furthermore, a TMD swap between RNF43 and ZNRF3 re-directs their preference for FZD5 down-regulation. We conclude that RNF43 and ZNRF3 preferentially down-regulate specific FZDs, in part by a TMD-dependent mechanism. In accordance, tissue-specific expression patterns of FZD homologues correlate with the incidence of RNF43 or ZNRF3 cancer mutations in those tissues. Consequently, our data point to druggable vulnerabilities of specific FZD receptors in RNF43- or ZNRF3-mutant human cancers.
Assuntos
Endocitose , Receptores Frizzled , Ubiquitina-Proteína Ligases , Receptores Frizzled/metabolismo , Receptores Frizzled/genética , Humanos , Endocitose/genética , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genética , Células HEK293 , Mutação , Via de Sinalização Wnt/genética , Regulação para Baixo/genéticaRESUMO
The transmembrane receptor LGR5 potentiates Wnt/ß-catenin signaling by binding both secreted R-spondin (RSPOs) and the Wnt tumor suppressors RNF43/ZNRF3, directing clearance of RNF43/ZNRF3 from the cell surface. Besides being widely used as a stem cell marker in various tissues, LGR5 is overexpressed in many types of malignancies, including colorectal cancer. Its expression characterizes a subpopulation of cancer cells that play a crucial role in tumor initiation, progression and cancer relapse, known as cancer stem cells (CSCs). For this reason, ongoing efforts are aimed at eradicating LGR5-positive CSCs. Here, we engineered liposomes decorated with different RSPO proteins to specifically detect and target LGR5-positive cells. Using fluorescence-loaded liposomes, we show that conjugation of full-length RSPO1 to the liposomal surface mediates aspecific, LGR5-independent cellular uptake, largely mediated by heparan sulfate proteoglycan binding. By contrast, liposomes decorated only with the Furin (FuFu) domains of RSPO3 are taken up by cells in a highly specific, LGR5-dependent manner. Moreover, encapsulating doxorubicin in FuFuRSPO3 liposomes allowed us to selectively inhibit the growth of LGR5-high cells. Thus, FuFuRSPO3-coated liposomes allow for the selective detection and ablation of LGR5-high cells, providing a potential drug delivery system for LGR5-targeted anti-cancer strategies.
Assuntos
Lipossomos , Receptores Acoplados a Proteínas G , Receptores Acoplados a Proteínas G/metabolismo , Furina/metabolismo , Via de Sinalização Wnt , Sistemas de Liberação de Medicamentos , Células-Tronco Neoplásicas/metabolismoRESUMO
The mammalian intestine is one of the most rapidly self-renewing tissues, driven by stem cells residing at the crypt bottom. Paneth cells form a major element of the niche microenvironment providing various growth factors to orchestrate intestinal stem cell homeostasis, such as Wnt3. Different Wnt ligands can selectively activate ß-catenin-dependent (canonical) or -independent (noncanonical) signaling. Here, we report that the Dishevelled-associated activator of morphogenesis 1 (Daam1) and its paralogue Daam2 asymmetrically regulate canonical and noncanonical Wnt (Wnt/PCP) signaling. Daam1/2 interacts with the Wnt inhibitor RNF43, and Daam1/2 double knockout stimulates canonical Wnt signaling by preventing RNF43-dependent degradation of the Wnt receptor, Frizzled (Fzd). Single-cell RNA sequencing analysis revealed that Paneth cell differentiation is impaired by Daam1/2 depletion because of defective Wnt/PCP signaling. Together, we identified Daam1/2 as an unexpected hub molecule coordinating both canonical and noncanonical Wnt, which is fundamental for specifying an adequate number of Paneth cells.
Assuntos
Celulas de Paneth , Via de Sinalização Wnt , Animais , Intestinos , Diferenciação Celular , Células-Tronco/metabolismo , MamíferosRESUMO
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
R-spondins (RSPOs) amplify WNT signaling during development and regenerative responses. We previously demonstrated that RSPOs 2 and 3 potentiate WNT/ß-catenin signaling in cells lacking leucine-rich repeat-containing G-protein coupled receptors (LGRs) 4, 5 and 6 (Lebensohn and Rohatgi, 2018). We now show that heparan sulfate proteoglycans (HSPGs) act as alternative co-receptors for RSPO3 using a combination of ligand mutagenesis and ligand engineering. Mutations in RSPO3 residues predicted to contact HSPGs impair its signaling capacity. Conversely, the HSPG-binding domains of RSPO3 can be entirely replaced with an antibody that recognizes heparan sulfate (HS) chains attached to multiple HSPGs without diminishing WNT-potentiating activity in cultured cells and intestinal organoids. A genome-wide screen for mediators of RSPO3 signaling in cells lacking LGRs 4, 5 and 6 failed to reveal other receptors. We conclude that HSPGs are RSPO co-receptors that potentiate WNT signaling in the presence and absence of LGRs.
Assuntos
Proteoglicanas de Heparan Sulfato/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Via de Sinalização Wnt , Células Cultivadas , Biologia do Desenvolvimento , Proteoglicanas de Heparan Sulfato/genética , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Ligantes , Organoides , Receptores Acoplados a Proteínas G/genética , TrombospondinasRESUMO
The establishment of neuronal polarity is driven by cytoskeletal remodeling that stabilizes and promotes the growth of a single axon from one of the multiple neurites. The importance of the local microtubule stabilization in this process has been revealed however, the external signals initiating the cytoskeletal rearrangements are not completely understood. In this study, we show that local activation of the canonical Wnt pathway regulates neuronal polarity and axonal outgrowth. We found that in the early stages of neuronal polarization, Wnt3a accumulates in one of the neurites of unpolarized cells and thereby could determine axon positioning. Subsequently, Wnt3a localizes to the growing axon, where it activates the canonical Wnt pathway and controls axon positioning and axonal length. We propose a model in which Wnt3a regulates the formation and growth of the axon by activating local intracellular signaling events leading to microtubule remodeling.
RESUMO
Wnt-induced ß-catenin-mediated transcription is a driving force for stem cell self-renewal during adult tissue homeostasis. Enhanced Wnt receptor expression due to mutational inactivation of the ubiquitin ligases RNF43/ZNRF3 recently emerged as a leading cause for cancer development. Consequently, targeting canonical Wnt receptors such as LRP5/6 holds great promise for treatment of such cancer subsets. Here, we employ CIS display technology to identify single-domain antibody fragments (VHH) that bind the LRP6 P3E3P4E4 region with nanomolar affinity and strongly inhibit Wnt3/3a-induced ß-catenin-mediated transcription in cells, while leaving Wnt1 responses unaffected. Structural analysis reveal that individual VHHs variably employ divergent antigen-binding regions to bind a similar surface in the third ß-propeller of LRP5/6, sterically interfering with Wnt3/3a binding. Importantly, anti-LRP5/6 VHHs block the growth of Wnt-hypersensitive Rnf43/Znrf3-mutant intestinal organoids through stem cell exhaustion and collective terminal differentiation. Thus, VHH-mediated targeting of LRP5/6 provides a promising differentiation-inducing strategy for treatment of Wnt-hypersensitive tumors.
Assuntos
Proteína-5 Relacionada a Receptor de Lipoproteína de Baixa Densidade/química , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/química , Organoides/efeitos dos fármacos , Anticorpos de Domínio Único/química , Células-Tronco/efeitos dos fármacos , Proteína Wnt3A/genética , Animais , Sítios de Ligação , Diferenciação Celular , Linhagem Celular Tumoral , Proliferação de Células , Cristalografia por Raios X , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Regulação da Expressão Gênica , Células HEK293 , Humanos , Intestino Delgado/citologia , Intestino Delgado/efeitos dos fármacos , Intestino Delgado/metabolismo , Proteína-5 Relacionada a Receptor de Lipoproteína de Baixa Densidade/antagonistas & inibidores , Proteína-5 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Proteína-5 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/antagonistas & inibidores , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/genética , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade/metabolismo , Camundongos , Modelos Moleculares , Organoides/citologia , Organoides/metabolismo , Ligação Proteica , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Anticorpos de Domínio Único/genética , Anticorpos de Domínio Único/metabolismo , Células-Tronco/citologia , Células-Tronco/metabolismo , Transcrição Gênica , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Proteína Wnt3A/metabolismo , beta Catenina/genética , beta Catenina/metabolismoRESUMO
Blepharocheilodontic syndrome (BCDS) consists of lagophthalmia, ectropion of the lower eyelids, distichiasis, euryblepharon, cleft lip/palate and dental anomalies and has autosomal dominant inheritance with variable expression. We identified heterozygous variants in two genes of the cadherin-catenin complex, CDH1, encoding E-cadherin, and CTNND1, encoding p120 catenin delta1 in 15 of 17 BCDS index patients, as was recently described in a different publication. CDH1 plays an essential role in epithelial cell adherence; CTNND1 binds to CDH1 and controls the stability of the complex. Functional experiments in zebrafish and human cells showed that the CDH1 variants impair the cell adhesion function of the cadherin-catenin complex in a dominant-negative manner. Variants in CDH1 have been linked to familial hereditary diffuse gastric cancer and invasive lobular breast cancer; however, no cases of gastric or breast cancer have been reported in our BCDS cases. Functional experiments reported here indicated the BCDS variants comprise a distinct class of CDH1 variants. Altogether, we identified the genetic cause of BCDS enabling DNA diagnostics and counseling, in addition we describe a novel class of dominant negative CDH1 variants.
Assuntos
Antígenos CD/genética , Caderinas/genética , Cateninas/genética , Fenda Labial/genética , Fissura Palatina/genética , Ectrópio/genética , Mutação , Anormalidades Dentárias/genética , Adolescente , Adulto , Animais , Antígenos CD/metabolismo , Caderinas/metabolismo , Cateninas/metabolismo , Adesão Celular , Criança , Pré-Escolar , Fenda Labial/patologia , Fissura Palatina/patologia , Ectrópio/patologia , Feminino , Humanos , Células MCF-7 , Masculino , Ligação Proteica , Anormalidades Dentárias/patologia , Peixe-Zebra , delta CateninaRESUMO
Salmonella typhimurium survives and replicates intracellular in a membrane-bound compartment, the Salmonella-containing vacuole (SCV). In HeLa cells, the SCV matures through interactions with the endocytic pathway, but Salmonella avoids fusion with mature lysosomes. The exact mechanism of the inhibition of phagolysosomal fusion is not understood. Rab GTPases control several proteins involved in membrane fusion and vesicular transport. The small GTPase Rab7 regulates the transport of and fusion between late endosomes and lysosomes and associates with the SCV. We show that the Rab7 GTPase cycle is not affected on the SCV. We then manipulated a pathway downstream of the small GTPase Rab7 in HeLa cells infected with Salmonella. Expression of the Rab7 effector RILP induces recruitment of the dynein/dynactin motor complex to the SCV. Subsequently, SCV fuse with lysosomes. As a result, the intracellular replication of Salmonella is inhibited. Activation of dynein-mediated vesicle transport can thus control intracellular survival of Salmonella.
Assuntos
Vesículas Citoplasmáticas/metabolismo , Dineínas/metabolismo , Salmonella typhimurium/crescimento & desenvolvimento , Vacúolos/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Transporte Biológico , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Vesículas Citoplasmáticas/microbiologia , Complexo Dinactina , Células HeLa , Humanos , Lisossomos/metabolismo , Lisossomos/microbiologia , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Motores Moleculares/metabolismo , Transfecção , Vacúolos/microbiologia , Proteínas rab de Ligação ao GTP/metabolismo , proteínas de unión al GTP Rab7RESUMO
Tumor vaccine development aimed at stimulating the cellular immune response focuses mainly on MHC class I molecules. This is not surprising since most tumors do not express MHC class II or CD1 molecules. Nevertheless, the most successful targets for cancer immunotherapy, leukemia and melanoma, often do express MHC class II molecules, which leaves no obvious reason to ignore MHC class II molecules as a mediator in anticancer immune therapy. We review the current state of knowledge on the process of MHC class II-restricted antigen presentation and subsequently discuss the consequences of MHC class II expression on tumor surveillance and the induction of an efficient MHC class II mediated antitumor response in vivo and after vaccination.
Assuntos
Apresentação de Antígeno/imunologia , Transformação Celular Neoplásica/imunologia , Antígenos de Histocompatibilidade Classe II/imunologia , Chaperonas Moleculares/imunologia , Vacinas Anticâncer , Vigilância Imunológica , Imunoterapia/métodosRESUMO
Signaling cascades depend on scaffold proteins that regulate the assembly of multiprotein complexes. Missense mutations in scaffold proteins are frequent in human cancer, but their relevance and mode of action are poorly understood. Here we show that cancer point mutations in the scaffold protein Axin derail Wnt signaling and promote tumor growth in vivo through a gain-of-function mechanism. The effect is conserved for both the human and Drosophila proteins. Mutated Axin forms nonamyloid nanometer-scale aggregates decorated with disordered tentacles, which 'rewire' the Axin interactome. Importantly, the tumor-suppressor activity of both the human and Drosophila Axin cancer mutants is rescued by preventing aggregation of a single nonconserved segment. Our findings establish a new paradigm for misregulation of signaling in cancer and show that targeting aggregation-prone stretches in mutated scaffolds holds attractive potential for cancer treatment.
Assuntos
Proteína Axina/genética , Proteína Axina/metabolismo , Neoplasias/genética , Mutação Puntual , Agregados Proteicos , Via de Sinalização Wnt , Sequência de Aminoácidos , Animais , Proteína Axina/análise , Proteína Axina/ultraestrutura , Linhagem Celular , Drosophila/química , Drosophila/genética , Drosophila/metabolismo , Drosophila/ultraestrutura , Proteínas de Drosophila/análise , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Células HEK293 , Humanos , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Mutação de Sentido Incorreto , Neoplasias/metabolismo , Neoplasias/patologia , Conformação Proteica , Mapas de Interação de Proteínas , Espalhamento a Baixo Ângulo , Alinhamento de Sequência , Difração de Raios XRESUMO
Insulin stimulates glucose transport by recruiting the GLUT4 glucose transporter to the plasma membrane. Here we use total internal reflection fluorescence microscopy to show that two trafficking motifs of GLUT4, a FQQI motif and a TELE-based motif, target GLUT4 to specialized vesicles that accumulate adjacent to the plasma membrane of unstimulated adipocytes. Mutations of these motifs redistributed GLUT4 to transferrin-containing recycling vesicles adjacent to the plasma membrane, and the degree of redistribution correlated with the increases of the GLUT4 mutants in the plasma membrane of basal adipocytes. These results establish that GLUT4 defaults to recycling endosomes when trafficking to specialized vesicles is disrupted, supporting the hypothesis that the specialized vesicles are derived from an endosomal compartment. Insulin stimulates both the accumulation of GLUT4 in the evanescent field and the fraction of this GLUT4 that is inserted into the plasma membrane. Unexpectedly, these two steps are differentially affected by the development of insulin resistance. We ascribe this selective insulin resistance to inherent differences in the sensitivities of GLUT4 vesicle accumulation and insertion into the plasma membrane to insulin. Differences in insulin sensitivities of various processes may be a general mechanism for the development of the physiologically important phenomenon of selective insulin resistance.
Assuntos
Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Transportador de Glucose Tipo 4/metabolismo , Resistência à Insulina , Insulina/farmacologia , Vesículas Transportadoras/efeitos dos fármacos , Vesículas Transportadoras/metabolismo , Células 3T3-L1 , Adipócitos/efeitos dos fármacos , Adipócitos/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Endocitose/efeitos dos fármacos , Transportador de Glucose Tipo 4/química , Humanos , Camundongos , Microscopia de Fluorescência , Dados de Sequência Molecular , Proteínas Mutantes/metabolismo , Mutação/genética , Transporte Proteico/efeitos dos fármacos , Transferrina/metabolismoRESUMO
Insulin stimulates glucose uptake by regulating translocation of the GLUT4 glucose transporter from intracellular compartments to the plasma membrane. In the absence of insulin GLUT4 is actively sequestered away from the general endosomes into GLUT4-specialized compartments, thereby controlling the amount of GLUT4 at the plasma membrane. Here, we investigated the role of the aminopeptidase IRAP in GLUT4 trafficking. In unstimulated IRAP knockdown adipocytes, plasma membrane GLUT4 levels are elevated because of increased exocytosis, demonstrating an essential role of IRAP in GLUT4 retention. Current evidence supports the model that AS160 RabGAP, which is required for basal GLUT4 retention, is recruited to GLUT4 compartments via an interaction with IRAP. However, here we show that AS160 recruitment to GLUT4 compartments and AS160 regulation of GLUT4 trafficking were unaffected by IRAP knockdown. These results demonstrate that AS160 is recruited to membranes by an IRAP-independent mechanism. Consistent with a role independent of AS160, we showed that IRAP functions in GLUT4 sorting from endosomes to GLUT4-specialized compartments. This is revealed by the relocalization of GLUT4 to endosomes in IRAP knockdown cells. Although IRAP knockdown has profound effects on GLUT4 traffic, GLUT4 knockdown does not affect IRAP trafficking, demonstrating that IRAP traffics independent of GLUT4. In sum, we show that IRAP is both cargo and a key regulator of the insulin-regulated pathway.
Assuntos
Cistinil Aminopeptidase/metabolismo , Endossomos/efeitos dos fármacos , Endossomos/metabolismo , Transportador de Glucose Tipo 4/metabolismo , Insulina/farmacologia , Células 3T3-L1 , Adipócitos/efeitos dos fármacos , Adipócitos/metabolismo , Animais , Cistinil Aminopeptidase/química , Endocitose/efeitos dos fármacos , Exocitose/efeitos dos fármacos , Proteínas Ativadoras de GTPase/metabolismo , Técnicas de Silenciamento de Genes , Espaço Intracelular/efeitos dos fármacos , Espaço Intracelular/metabolismo , Camundongos , Estrutura Terciária de Proteína , Transporte Proteico/efeitos dos fármacosRESUMO
The small GTPase Rab7 controls fusion and transport of late endocytic compartments. A critical mediator is the Rab7 effector RILP that recruits the minus-end dynein-dynactin motor complex to these compartments. We identified a natural occurring splice variant of RILP (RILPsv) lacking only 27 amino acids encoded by exon VII. Both variants bind Rab7, prolong its GTP-bound state, and induce clustering of late endocytic compartments. However, RILPsv does not recruit the dynein-dynactin complex, implicating exon VII in motor recruitment. Clustering might still occur via dimerization, since both RILP and RILPsv are able to form hetero- and homo-dimers. Moreover, both effectors compete for Rab7 binding but with different outcome for dynein-dynactin recruitment and transport. Hence, RILPsv provides an extra dimension to the control of vesicle fusion and transport by the small GTPase Rab7.
Assuntos
Proteínas de Transporte/metabolismo , Dineínas/metabolismo , Endocitose , Lisossomos/metabolismo , Lisossomos/ultraestrutura , Melanoma/metabolismo , Proteínas Motores Moleculares/metabolismo , Proteínas Adaptadoras de Transdução de Sinal , Sítios de Ligação , Linhagem Celular Tumoral , Variação Genética/genética , Humanos , Melanoma/ultraestrutura , Proteínas Motores Moleculares/ultraestrutura , Ligação Proteica , Processamento de Proteína/genética , Relação Estrutura-Atividade , Proteínas rab de Ligação ao GTP/metabolismo , proteínas de unión al GTP Rab7RESUMO
Melanosomes are lysosome-related organelles that synthesize, store and transport melanin. In epidermal melanocytes, melanosomes mature and are transferred to surrounding keratinocytes, which is essential for skin and coat colour. Mouse coat colour mutants reveal a critical role for the small GTPase Rab27a, which recruits myosin Va through its effector protein melanophilin/Slac2a. Here we have studied how two different Rab GTPases control two motor proteins during subsequent phases in transport of melanosomes. We show that the small GTPase Rab7 mainly associates with early and intermediate stage melanosomes and Rab27a to intermediate and mature melanosomes. Rab27a is found in an active state on mature melanosomes in the tips of the dendrites. The Rab7-Rab7-interacting lysosomal protein-dynein pathway only controls early and intermediate stage melanosomes because the mature melanosomes lack Rab7 and associate with the actin network through Rab27a recruited MyoVa. Thus two Rab proteins regulate two different motor proteins, thereby controlling complementary phases in melanosome biogenesis: Rab7 controls microtubule-mediated transport of early and Rab27a the subsequent actin-dependent transport of mature melanosomes.