RESUMO
Smoothened (SMO) transduces the Hedgehog (Hh) signal across the plasma membrane in response to accessible cholesterol. Cholesterol binds SMO at two sites: one in the extracellular cysteine-rich domain (CRD) and a second in the transmembrane domain (TMD). How these two sterol-binding sites mediate SMO activation in response to the ligand Sonic Hedgehog (SHH) remains unknown. We find that mutations in the CRD (but not the TMD) reduce the fold increase in SMO activity triggered by SHH. SHH also promotes the photocrosslinking of a sterol analog to the CRD in intact cells. In contrast, sterol binding to the TMD site boosts SMO activity regardless of SHH exposure. Mutational and computational analyses show that these sites are in allosteric communication despite being 45 angstroms apart. Hence, sterols function as both SHH-regulated orthosteric ligands at the CRD and allosteric ligands at the TMD to regulate SMO activity and Hh signaling.
Assuntos
Cisteína , Proteínas Hedgehog , Colesterol/metabolismo , Proteínas Hedgehog/química , Ligantes , Esteróis/químicaRESUMO
A long-standing mystery in vertebrate Hedgehog signaling is how Patched 1 (PTCH1), the receptor for Hedgehog ligands, inhibits the activity of Smoothened, the protein that transmits the signal across the membrane. We previously proposed (Kinnebrew et al., 2019) that PTCH1 inhibits Smoothened by depleting accessible cholesterol from the ciliary membrane. Using a new imaging-based assay to directly measure the transport activity of PTCH1, we find that PTCH1 depletes accessible cholesterol from the outer leaflet of the plasma membrane. This transport activity is terminated by binding of Hedgehog ligands to PTCH1 or by dissipation of the transmembrane potassium gradient. These results point to the unexpected model that PTCH1 moves cholesterol from the outer to the inner leaflet of the membrane in exchange for potassium ion export in the opposite direction. Our study provides a plausible solution for how PTCH1 inhibits SMO by changing the organization of cholesterol in membranes and establishes a general framework for studying how proteins change cholesterol accessibility to regulate membrane-dependent processes in cells.
Assuntos
Membrana Celular/metabolismo , Colesterol/metabolismo , Receptor Patched-1/genética , Receptor Smoothened/genética , Animais , Escherichia coli , Humanos , Camundongos , Receptor Patched-1/metabolismo , Receptor Smoothened/metabolismoRESUMO
The pore-forming protein gasdermin D (GSDMD) executes lytic cell death called pyroptosis to eliminate the replicative niche of intracellular pathogens. Evolution favors pathogens that circumvent this host defense mechanism. Here, we show that the Shigella ubiquitin ligase IpaH7.8 functions as an inhibitor of GSDMD. Shigella is an enteroinvasive bacterium that causes hemorrhagic gastroenteritis in primates, but not rodents. IpaH7.8 contributes to species specificity by ubiquitinating human, but not mouse, GSDMD and targeting it for proteasomal degradation. Accordingly, infection of human epithelial cells with IpaH7.8-deficient Shigella flexneri results in increased GSDMD-dependent cell death compared with wild type. Consistent with pyroptosis contributing to murine disease resistance, eliminating GSDMD from NLRC4-deficient mice, which are already sensitized to oral infection with Shigella flexneri, leads to further enhanced bacterial replication and increased disease severity. This work highlights a species-specific pathogen arms race focused on maintenance of host cell viability.
Assuntos
Proteínas de Bactérias/metabolismo , Disenteria Bacilar/metabolismo , Proteínas de Ligação a Fosfato/metabolismo , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Shigella flexneri/enzimologia , Ubiquitina-Proteína Ligases/metabolismo , Animais , Proteínas de Bactérias/genética , Disenteria Bacilar/genética , Disenteria Bacilar/microbiologia , Células Epiteliais/metabolismo , Células Epiteliais/microbiologia , Feminino , Interações Hospedeiro-Patógeno , Humanos , Camundongos , Camundongos Knockout , Proteínas de Ligação a Fosfato/genética , Proteínas Citotóxicas Formadoras de Poros/genética , Proteólise , Shigella flexneri/genética , Shigella flexneri/fisiologia , Ubiquitina-Proteína Ligases/genéticaRESUMO
Previously we proposed that transmission of the hedgehog signal across the plasma membrane by Smoothened is triggered by its interaction with cholesterol (Luchetti et al., 2016). But how is cholesterol, an abundant lipid, regulated tightly enough to control a signaling system that can cause birth defects and cancer? Using toxin-based sensors that distinguish between distinct pools of cholesterol, we find that Smoothened activation and Hedgehog signaling are driven by a biochemically-defined, small fraction of membrane cholesterol, termed accessible cholesterol. Increasing cholesterol accessibility by depletion of sphingomyelin, which sequesters cholesterol in complexes, amplifies Hedgehog signaling. Hedgehog ligands increase cholesterol accessibility in the membrane of the primary cilium by inactivating the transporter-like protein Patched 1. Trapping this accessible cholesterol blocks Hedgehog signal transmission across the membrane. Our work shows that the organization of cholesterol in the ciliary membrane can be modified by extracellular ligands to control the activity of cilia-localized signaling proteins.
Assuntos
Colesterol/metabolismo , Cílios/metabolismo , Proteínas Hedgehog/metabolismo , Transdução de Sinais , Animais , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , HumanosRESUMO
The morphogen Sonic Hedgehog (SHH) patterns tissues during development by directing cell fates in a concentration-dependent manner. The SHH signal is transmitted across the membrane of target cells by the heptahelical transmembrane protein Smoothened (SMO), which activates the GLI family of transcription factors through a mechanism that is undefined in vertebrates. Using CRISPR-edited null alleles and small-molecule inhibitors, we systematically analyzed the epistatic interactions between SMO and three proteins implicated in SMO signaling: the heterotrimeric G protein subunit GαS, the G protein-coupled receptor kinase 2 (GRK2), and the GαS-coupled receptor GPR161. Our experiments uncovered a signaling mechanism that modifies the sensitivity of target cells to SHH and consequently changes the shape of the SHH dose-response curve. In both fibroblasts and spinal neural progenitors, the loss of GPR161, previously implicated as an inhibitor of basal SHH signaling, increased the sensitivity of target cells across the entire spectrum of SHH concentrations. Even in cells lacking GPR161, GRK2 was required for SHH signaling, and Gαs, which promotes the activation of protein Kinase A (PKA), antagonized SHH signaling. We propose that the sensitivity of target cells to Hedgehog morphogens, and the consequent effects on gene expression and differentiation outcomes, can be controlled by signals from G protein-coupled receptors that converge on Gαs and PKA.
Assuntos
Proteínas Hedgehog/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais , Receptor Smoothened/metabolismo , Animais , Células Cultivadas , Cromograninas/genética , Cromograninas/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/genética , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Epistasia Genética , Quinase 2 de Receptor Acoplado a Proteína G/genética , Quinase 2 de Receptor Acoplado a Proteína G/metabolismo , Subunidades alfa Gs de Proteínas de Ligação ao GTP/genética , Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Células HEK293 , Proteínas Hedgehog/genética , Humanos , Camundongos , Células NIH 3T3 , Receptores Acoplados a Proteínas G/genética , Receptor Smoothened/genéticaRESUMO
The Hedgehog (Hh) pathway plays a central role in the development of multicellular organisms, guiding cell differentiation, proliferation and survival. While many components of the vertebrate pathway were discovered two decades ago, the mechanism by which the Hh signal is transmitted across the plasma membrane remains mysterious. This fundamental task in signalling is carried out by Smoothened (SMO), a human oncoprotein and validated cancer drug target that is a member of the G-protein coupled receptor protein family. Recent structural and functional studies have advanced our mechanistic understanding of SMO activation, revealing its unique regulation by two separable but allosterically-linked ligand-binding sites. Unexpectedly, these studies have nominated cellular cholesterol as having an instructive role in SMO signalling.
Assuntos
Proteínas de Drosophila/metabolismo , Proteínas Hedgehog/metabolismo , Vertebrados/metabolismo , Animais , Sítios de Ligação , Drosophila melanogaster , Humanos , Ligantes , Transdução de SinaisRESUMO
Cholesterol is necessary for the function of many G-protein coupled receptors (GPCRs). We find that cholesterol is not just necessary but also sufficient to activate signaling by the Hedgehog (Hh) pathway, a prominent cell-cell communication system in development. Cholesterol influences Hh signaling by directly activating Smoothened (SMO), an orphan GPCR that transmits the Hh signal across the membrane in all animals. Unlike many GPCRs, which are regulated by cholesterol through their heptahelical transmembrane domains, SMO is activated by cholesterol through its extracellular cysteine-rich domain (CRD). Residues shown to mediate cholesterol binding to the CRD in a recent structural analysis also dictate SMO activation, both in response to cholesterol and to native Hh ligands. Our results show that cholesterol can initiate signaling from the cell surface by engaging the extracellular domain of a GPCR and suggest that SMO activity may be regulated by local changes in cholesterol abundance or accessibility.
Assuntos
Colesterol/metabolismo , Ouriços/metabolismo , Transdução de Sinais , Receptor Smoothened/agonistas , Animais , Linhagem Celular , Células Epiteliais/fisiologia , Fibroblastos/fisiologia , Humanos , CamundongosRESUMO
Developmental signals of the Hedgehog (Hh) and Wnt families are transduced across the membrane by Frizzledclass G-protein-coupled receptors (GPCRs) composed of both a heptahelical transmembrane domain (TMD) and an extracellular cysteine-rich domain (CRD). How the large extracellular domains of GPCRs regulate signalling by the TMD is unknown. We present crystal structures of the Hh signal transducer and oncoprotein Smoothened, a GPCR that contains two distinct ligand-binding sites: one in its TMD and one in the CRD. The CRD is stacked a top the TMD, separated by an intervening wedge-like linker domain. Structure-guided mutations show that the interface between the CRD, linker domain and TMD stabilizes the inactive state of Smoothened. Unexpectedly, we find a cholesterol molecule bound to Smoothened in the CRD binding site. Mutations predicted to prevent cholesterol binding impair the ability of Smoothened to transmit native Hh signals. Binding of a clinically used antagonist, vismodegib, to the TMD induces a conformational change that is propagated to the CRD, resulting in loss of cholesterol from the CRD-linker domain-TMD interface. Our results clarify the structural mechanism by which the activity of a GPCR is controlled by ligand-regulated interactions between its extracellular and transmembrane domains.
Assuntos
Espaço Extracelular/metabolismo , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/metabolismo , Anilidas/química , Anilidas/metabolismo , Anilidas/farmacologia , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Sítios de Ligação/genética , Colesterol/metabolismo , Colesterol/farmacologia , Cristalografia por Raios X , Cisteína/química , Cisteína/genética , Cisteína/metabolismo , Proteínas Hedgehog/metabolismo , Humanos , Ligantes , Modelos Moleculares , Ligação Proteica/genética , Estabilidade Proteica/efeitos dos fármacos , Estrutura Terciária de Proteína/efeitos dos fármacos , Estrutura Terciária de Proteína/genética , Piridinas/química , Piridinas/metabolismo , Piridinas/farmacologia , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Receptores Acoplados a Proteínas G/genética , Transdução de Sinais/efeitos dos fármacos , Receptor SmoothenedRESUMO
Oxysterols, oxidized metabolites of cholesterol, are endogenous small molecules that regulate lipid metabolism, immune function, and developmental signaling. Although the cell biology of cholesterol has been intensively studied, fundamental questions about oxysterols, such as their subcellular distribution and trafficking pathways, remain unanswered. We have therefore developed a useful method to image intracellular 20(S)-hydroxycholesterol with both high sensitivity and spatial resolution using click chemistry and fluorescence microscopy. The metabolic labeling of cells with an alkynyl derivative of 20(S)-hydroxycholesterol has allowed us to directly visualize this oxysterol by attaching an azide fluorophore through cyclo-addition. Unexpectedly, we found that this oxysterol selectively accumulates in the Golgi membrane using a pathway that is sensitive to ATP levels, temperature, and lysosome function. Although previous models have proposed nonvesicular pathways for the rapid equilibration of oxysterols between membranes, direct imaging of oxysterols suggests that a vesicular pathway is responsible for differential accumulation of oxysterols in organelle membranes. More broadly, clickable alkynyl sterols may represent useful tools for sterol cell biology, both to investigate the functions of these important lipids and to decipher the pathways that determine their cellular itineraries.
Assuntos
Química Click , Corantes Fluorescentes , Complexo de Golgi/metabolismo , Hidroxicolesteróis , Membranas Intracelulares/metabolismo , Animais , Transporte Biológico Ativo/fisiologia , Células CHO , Cricetinae , Cricetulus , Corantes Fluorescentes/química , Corantes Fluorescentes/metabolismo , Hidroxicolesteróis/síntese química , Hidroxicolesteróis/química , Hidroxicolesteróis/metabolismo , Camundongos , Microscopia de Fluorescência , Células NIH 3T3RESUMO
Rab GTPases are master regulators of membrane trafficking events and template the directionality of protein transport through the secretory and endocytic pathways. Certain Rabs recruit the guanine nucleotide exchange factor (GEF) that activates a subsequent acting Rab protein in a given pathway; this process has been termed a Rab cascade. We show here that the medial Golgi-localized Rab33B GTPase has the potential to link functionally to the late Golgi, Rab6 GTPase, by its capacity for association with Ric1 and Rgp1 proteins. In yeast, Ric1p and Rgp1p form a complex that catalyzes guanine nucleotide exchange by Ypt6p, the Rab6 homolog. Human Ric1 and Rgp1 both bind Rab6A with preference for the GDP-bound conformation, characteristic of a GEF. Nevertheless, both Ric1 and Rgp1 proteins are needed to catalyze nucleotide exchange on Rab6A protein. Ric1 and Rgp1 form a complex, but unlike their yeast counterparts, most of the subunits are not associated, and most of the proteins are cytosolic. Loss of Ric1 or Rgp1 leads to destabilization of Rab6, concomitant with a block in Rab6-dependent retrograde transport of mannose 6-phosphate receptors to the Golgi. The C terminus of Ric1 protein contains a distinct binding site for Rab33B-GTP, supporting the existence of a Rab cascade between the medial and trans Golgi. This study thus identifies a GEF for Rab6A in human cells.
Assuntos
Fatores de Troca do Nucleotídeo Guanina/metabolismo , Proteínas de Membrana/metabolismo , Complexos Multiproteicos/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Estabilidade Enzimática/fisiologia , Fatores de Troca do Nucleotídeo Guanina/genética , Células HeLa , Humanos , Proteínas de Membrana/genética , Complexos Multiproteicos/genética , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas rab de Ligação ao GTP/genética , Rede trans-Golgi/genética , Rede trans-Golgi/metabolismoRESUMO
The Amaryllidaceae alkaloid bulbispermine was derivatized to produce a small group of synthetic analogues. These, together with bulbispermine's natural crinine-type congeners, were evaluated in vitro against a panel of cancer cell lines with various levels of resistance to pro-apoptotic stimuli. Bulbispermine, haemanthamine, and haemanthidine showed the most potent antiproliferative activities as determined by the MTT colorimetric assay. Among the synthetic bulbispermine analogues, only the C1,C2-dicarbamate derivative exhibited notable growth inhibitory properties. All active compounds were found not to discriminate between the cancer cell lines based on the apoptosis sensitivity criterion; they displayed similar potencies in both cell types, indicating that the induction of apoptosis is not the primary mechanism responsible for antiproliferative activity in this series of compounds. It was also found that bulbispermine inhibits the proliferation of glioblastoma cells through cytostatic effects, possibly arising from rigidification of the actin cytoskeleton. These findings lead us to argue that crinine-type alkaloids are potentially useful drug leads for the treatment of apoptosis-resistant cancers and glioblastoma in particular.
Assuntos
Alcaloides de Amaryllidaceae/química , Antineoplásicos Fitogênicos/química , Alcaloides de Amaryllidaceae/farmacologia , Antineoplásicos Fitogênicos/farmacologia , Simulação por Computador , Resistencia a Medicamentos Antineoplásicos , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Concentração Inibidora 50 , Estrutura Molecular , Raízes de Plantas/químicaRESUMO
Diversely substituted 2-pyrrolines have been prepared by a novel multicomponent process involving a reaction of various N-(aryl- and alkylsulfonamido)-acetophenones with aldehydes and malononitrile. While the reaction is highly regioselective, it is not stereoselective, generating a mixture of cis and trans 2-pyrrolines. A number of analogs from both cis and trans 2-pyrroline libraries were found to have antiproliferative activity in human cancer cell lines.
Assuntos
Antineoplásicos/química , Antineoplásicos/farmacologia , Pirróis/síntese química , Pirróis/farmacologia , Acetofenonas/química , Aldeídos/química , Antineoplásicos/síntese química , Apoptose/efeitos dos fármacos , Processos de Crescimento Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Cristalografia por Raios X , Ensaios de Seleção de Medicamentos Antitumorais , Células HeLa , Humanos , Isomerismo , Células Jurkat , Nitrilas/química , Pirróis/química , Relação Estrutura-AtividadeRESUMO
Methods for the enantioselective conversion of D-xylose to differentially protected myo-inositol and L-chiro-inositol have been developed. The key transformation is a highly diastereoselective intramolecular SmI(2)-promoted pinacol coupling. The stereoselectivity was extremely dependent on the conditions, suggesting a change in mechanism. Preliminary mechanistic experiments and possible explanations for this behavior are discussed.
RESUMO
Short stereoselective syntheses of various cyclitols, including the derivatives of conduritol B, conduritol F, myo-inositol and chiro-inositol, have been accomplished. The key steps in the syntheses are a ring-closing metathesis process and a diastereodivergent organometallic addition to a D-xylose-derived alde-hyde.