RESUMEN
Cyclic peptides that inhibit protein-protein interactions have significant advantages over linear peptides and small molecules for modulating cellular signaling networks in cancer and other diseases. However, the permeability barrier of the plasma membrane remains a formidable obstacle to the development of cyclic peptides into applicable drugs. Here, we test the ability of a family of synthetically evolved spontaneous membrane translocating peptides (SMTPs) to deliver phalloidin, a representative bioactive cyclic peptide, to the cytosol of human cells in culture. Phalloidin does not enter cells spontaneously, but if delivered to the cytosol, it inhibits actin depolymerization. We thus use a wound-healing cell mobility assay to assess the biological activity of phalloidin conjugated to three SMTPs that we previously discovered. All three SMTPs can deliver phalloidin to the cell cytosol, and one does so at concentrations as low as 3 µM. Delivery occurs despite the fact that the SMTPs were originally selected based on membrane translocation with no cargo other than a small fluorescent dye. These results show that SMTPs are viable delivery vehicles for cyclic peptides, although their efficiency is moderate. Further, these results suggest that one additional generation of synthetic molecular evolution could be used to optimize SMTPs for the efficient delivery of any bioactive cyclic peptide into cells.
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We study the efficacy of a glucagon-like peptide-1 (GLP-1) and estrogen dual agonist (GLP1-E2) in pancreatic islet protection. GLP1-E2 provides superior protection from insulin-deficient diabetes induced by multiple low-dose streptozotocin (MLD-STZ-diabetes) and by the Akita mutation in mice than a GLP-1 monoagonist. GLP1-E2 does not protect from MLD-STZ-diabetes in estrogen receptor-α (ERα)-deficient mice and fails to prevent diabetes in Akita mice following GLP-1 receptor (GLP-1R) antagonism, demonstrating the requirement of GLP-1R and ERα for GLP1-E2 antidiabetic actions. In the MIN6 ß cell model, GLP1-E2 activates estrogen action following clathrin-dependent, GLP-1R-mediated internalization and lysosomal acidification. In cultured human islet, proteomic bioinformatic analysis reveals that GLP1-E2 amplifies the antiapoptotic pathways activated by monoagonists. However, in cultured mouse islets, GLP1-E2 provides antiapoptotic protection similar to monoagonists. Thus, GLP1-E2 promotes GLP-1 and E2 antiapoptotic signals in cultured islets, but in vivo, additional GLP1-E2 actions in non-islet cells expressing GLP-1R are instrumental to prevent diabetes.
Asunto(s)
Diabetes Mellitus , Islotes Pancreáticos , Animales , Diabetes Mellitus/metabolismo , Receptor alfa de Estrógeno/metabolismo , Estrógenos/metabolismo , Péptido 1 Similar al Glucagón/farmacología , Insulina/metabolismo , Insulina Regular Humana/metabolismo , Islotes Pancreáticos/metabolismo , Ratones , Proteómica , Estreptozocina/toxicidadRESUMEN
The Pleckstrin Homology-like Domain (PHLD) class of proteins are multifunctional proteins. The class is comprised of two families of proteins, PHLDA and PHLDB, each with 3 members. All members of the families possess a pleckstrin homology (PH) domain. Though identified nearly 30 years ago, this class of proteins remains understudied with PHLDA family members receiving most of the research attention. Recent studies have also begun to reveal the functions of the PHLDB family proteins in regulation of p53 and AKT signaling pathways important for cancer and metabolism. This review will discuss current research and offer some prospects on the possible roles of both families in cancer and metabolism.
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Neoplasias/metabolismo , Dominios Homólogos a Pleckstrina/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal/genética , Proteína p53 Supresora de Tumor/metabolismo , Animales , Humanos , Neoplasias/genética , Unión Proteica , Proteínas Proto-Oncogénicas c-akt/genética , Proteína p53 Supresora de Tumor/genéticaRESUMEN
Post-translational modifications (PTMs) play pivotal roles in controlling the stability and activity of the tumor suppressor p53 in response to distinct stressors. Here we report an unexpected finding of a short chain fatty acid modification of p53 in human cells. Crotonic acid (CA) treatment induces p53 crotonylation, but surprisingly reduces its protein, but not mRNA level, leading to inhibition of p53 activity in a dose dependent fashion. Surprisingly this crotonylation targets serine 46, instead of any predicted lysine residues, of p53, as detected in TCEP-probe labeled crotonylation and anti-crotonylated peptide antibody reaction assays. This is further confirmed by substitution of serine 46 with alanine, which abolishes p53 crotonylation in vitro and in cells. CA increases p53-dependent glycolytic activity, and augments cancer cell proliferation in response to metabolic or DNA damage stress. Since serine 46 is only found in human p53, our studies unveil an unconventional PTM unique for human p53, impairing its activity in response to CA. Because CA is likely produced by the gut microbiome, our results also predict that this type of PTM might play a role in early human colorectal neoplasia development by negating p53 activity without mutation of this tumor suppressor gene.
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Crotonatos/metabolismo , Procesamiento Proteico-Postraduccional , Serina/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Línea Celular Tumoral , Proliferación Celular , Crotonatos/química , Glucosa/deficiencia , Glucólisis , Humanos , Lisina/metabolismo , Mitocondrias/metabolismo , Proteína p53 Supresora de Tumor/químicaRESUMEN
Conjugated estrogens (CE) delay the onset of type 2 diabetes (T2D) in postmenopausal women, but the mechanism is unclear. In T2D, the endoplasmic reticulum (ER) fails to promote proinsulin folding and, in failing to do so, promotes ER stress and ß cell dysfunction. We show that CE prevent insulin-deficient diabetes in male and in female Akita mice using a model of misfolded proinsulin. CE stabilize the ER-associated protein degradation (ERAD) system and promote misfolded proinsulin proteasomal degradation. This involves activation of nuclear and membrane estrogen receptor-α (ERα), promoting transcriptional repression and proteasomal degradation of the ubiquitin-conjugating enzyme and ERAD degrader, UBC6e. The selective ERα modulator bazedoxifene mimics CE protection of ß cells in females but not in males.
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Diabetes Mellitus/metabolismo , Estrógenos/farmacología , Proinsulina/biosíntesis , Pliegue de Proteína , Proteolisis , Animales , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Diabetes Mellitus/prevención & control , Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/metabolismo , Retículo Endoplásmico/ultraestructura , Estrés del Retículo Endoplásmico/efectos de los fármacos , Degradación Asociada con el Retículo Endoplásmico/efectos de los fármacos , Receptor alfa de Estrógeno/metabolismo , Femenino , Humanos , Indoles/farmacología , Células Secretoras de Insulina/efectos de los fármacos , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/ultraestructura , Masculino , Ratones Endogámicos C57BL , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Mitocondrias/ultraestructura , Pliegue de Proteína/efectos de los fármacos , Estabilidad Proteica/efectos de los fármacos , Proteolisis/efectos de los fármacos , Elementos de Respuesta/genética , Enzimas Ubiquitina-Conjugadoras/metabolismoRESUMEN
OBJECTIVE: Transforming growth factor ß1 (TGFß1) is considered a key factor in fibrogenesis, and blocking TGFß1 signaling pathways diminishes fibrogenesis in animal models. The objective of this study was to determine whether nelfinavir mesylate (NFV), a drug approved by the Food and Drug Administration (FDA) for treating HIV infection, could be repurposed to treat pulmonary fibrosis in patients with systemic sclerosis (SSc). METHODS: Normal human lung, ventricular, and skin fibroblasts as well as lung fibroblasts from SSc patients were used to determine the effects of NFV on fibroblast-to-myofibroblast differentiation mediated by TGFß1. The efficacy of NFV was also evaluated in an animal model of SSc (bleomycin-induced pulmonary fibrosis). In addition, in silico analysis was performed to determine novel off-target effects of NFV. RESULTS: NFV inhibited TGFß1-mediated fibroblast-to-myofibroblast differentiation in lung fibroblasts through inhibition of the TGFß1 canonical pathway. NFV also inhibited differentiation of skin and ventricular fibroblasts and adipocyte precursors into myofibroblasts. Activation of the TGFß1/mechanistic target of rapamycin pathway inhibited autophagy in lung fibroblasts, favoring collagen deposition, and NFV counteracted this effect in a dose-dependent manner. Moreover, NFV significantly reduced lung injury and collagen deposition in an animal model of SSc. In silico analysis of NFV binding proteins revealed new putative beneficial mechanisms of action, consistent with known common pathways in fibrogenesis. CONCLUSION: NFV abrogates TGFß1-mediated fibroblast-to-myofibroblast differentiation and pulmonary fibrosis through off-target protein binding, a finding that supports consideration of this FDA-approved medication as an antifibrotic agent.
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Antirretrovirales/farmacología , Diferenciación Celular/efectos de los fármacos , Nelfinavir/farmacología , Fibrosis Pulmonar/tratamiento farmacológico , Esclerodermia Sistémica/tratamiento farmacológico , Animales , Técnicas de Cultivo de Célula , Simulación por Computador , Modelos Animales de Enfermedad , Fibroblastos/efectos de los fármacos , Fibroblastos/patología , Humanos , Pulmón/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Fibrosis Pulmonar/complicaciones , Esclerodermia Sistémica/complicaciones , Transducción de Señal/efectos de los fármacos , Piel/patología , Factor de Crecimiento Transformador beta1/farmacologíaRESUMEN
We previously used an orthogonal high-throughput screen to select peptides that spontaneously cross synthetic lipid bilayers without bilayer disruption. Many of the 12-residue spontaneous membrane translocating peptides (SMTPs) selected from the library contained a 5-residue consensus motif, LRLLR in positions 5-9. We hypothesized that the conserved motif could be a necessary and sufficient minimal motif for translocation. To test this and to explore the mechanism of spontaneous membrane translocation, we synthesized seven arginine placement variants of LRLLRWC and compared their membrane partitioning, translocation, and perturbation to one of the parent SMTPs, called "TP2". Several motif variant peptides translocate into synthetic vesicles with rates that are similar to TP2. However, the peptide containing the selected motif, LRLLRWC, was not the fastest; sequence context is also important for translocation efficiency. Although none of these peptides permeabilize bilayers, the motif peptides translocate faster at higher peptide to lipid ratios, suggesting that bilayer perturbation and/or cooperative interactions are important for their translocation. On the other hand, TP2 translocates slower as its concentration is increased, suggesting that TP2 translocates as a monomer and is inhibited by lateral interactions in the membrane. TP2 and the LRLLR motif peptide induce lipid translocation, suggesting that lipids chaperone them across the bilayer. The other motif peptides do not induce lipid flip-flop, suggesting an alternate mechanism. Concatenated motifs translocate slower than the motifs alone. Variants of TP2 with shorter and longer arginine side-chain analogs translocate slower than TP2. In summary, these results suggest that multiple patterns of leucine and arginine can support spontaneous membrane translocation, and that sequence context is important for the contribution of the motifs. Because motifs do not make simple, additive contributions to spontaneous translocation, rational engineering of novel SMTPs will remain difficult, providing even more reason to pursue SMTP discovery with synthetic molecular evolution.
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Arginina , Membrana Celular/metabolismo , Péptidos de Penetración Celular/química , Péptidos de Penetración Celular/metabolismo , Secuencia de Consenso , Leucina , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Potenciales de la Membrana , Transporte de ProteínasRESUMEN
The Ebola virus (EBOV) genome encodes a partly conserved 40-residue nonstructural polypeptide, called the delta peptide, that is produced in abundance during Ebola virus disease (EVD). The function of the delta peptide is unknown, but sequence analysis has suggested that delta peptide could be a viroporin, belonging to a diverse family of membrane-permeabilizing small polypeptides involved in replication and pathogenesis of numerous viruses. Full-length and conserved C-terminal delta peptide fragments permeabilize the plasma membranes of nucleated cells of rodent, dog, monkey, and human origin; increase ion permeability across confluent cell monolayers; and permeabilize synthetic lipid bilayers. Permeabilization activity is completely dependent on the disulfide bond between the two conserved cysteines. The conserved C-terminal portion of the peptide is biochemically stable in human serum, and most serum-stable fragments have full activity. Taken together, the evidence strongly suggests that Ebola virus delta peptide is a viroporin and that it may be a novel, targetable aspect of Ebola virus disease pathology.IMPORTANCE During the unparalleled West African outbreak of Ebola virus disease (EVD) that began in late 2013, the lack of effective countermeasures resulted in chains of serial infection and a high mortality rate among infected patients. A better understanding of disease pathology is desperately needed to develop better countermeasures. We show here that the Ebola virus delta peptide, a conserved nonstructural protein produced in large quantities by infected cells, has the characteristics of a viroporin. This information suggests a critical role for the delta peptide in Ebola virus disease pathology and as a possible target for novel countermeasures.
RESUMEN
To better understand the sequence-structure-function relationships that control the activity and selectivity of membrane-permeabilizing peptides, we screened a peptide library, based on the archetypal pore-former melittin, for loss-of-function variants. This was accomplished by assaying library members for failure to cause leakage of entrapped contents from synthetic lipid vesicles at a peptide-to-lipid ratio of 1:20, 10-fold higher than the concentration at which melittin efficiently permeabilizes the same vesicles. Surprisingly, about one-third of the library members are inactive under these conditions. In the negative peptides, two changes of hydrophobic residues to glycine were especially abundant. We show that loss-of-function activity can be completely recapitulated by a single-residue change of the leucine at position 16 to glycine. Unlike the potently cytolytic melittin, the loss-of-function peptides, including the single-site variant, are essentially inactive against phosphatidylcholine vesicles and multiple types of eukaryotic cells. Loss of function is shown to result from a shift in the binding-folding equilibrium away from the active, bound, α-helical state toward the inactive, unbound, random-coil state. Accordingly, the addition of anionic lipids to synthetic lipid vesicles restored binding, α-helical secondary structure, and potent activity of the "negative" peptides. While nontoxic to mammalian cells, the single-site variant has potent bactericidal activity, consistent with the anionic nature of bacterial membranes. The results show that conformational fine-tuning of helical pore-forming peptides is a powerful way to modulate their activity and selectivity.
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Péptidos/química , Secuencia de Aminoácidos , Antibacterianos/farmacología , Ensayos de Selección de Medicamentos Antitumorales , Eritrocitos/efectos de los fármacos , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Datos de Secuencia Molecular , Péptidos/farmacología , Conformación ProteicaRESUMEN
The permeability barrier imposed by cellular membranes limits the access of exogenous compounds to the interior of cells. Researchers and patients alike would benefit from efficient methods for intracellular delivery of a wide range of membrane-impermeant molecules, including biochemically active small molecules, imaging agents, peptides, peptide nucleic acids, proteins, RNA, DNA, and nanoparticles. There has been a sustained effort to exploit cell penetrating peptides (CPPs) for the delivery of such useful cargoes in vitro and in vivo because of their biocompatibility, ease of synthesis, and controllable physical chemistry. Here, we discuss the many mechanisms by which CPPs can function, and describe a taxonomy of mechanisms that could be help organize future efforts in the field.
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Membrana Celular/metabolismo , Péptidos de Penetración Celular/clasificación , Péptidos de Penetración Celular/metabolismo , Animales , Membrana Celular/química , Péptidos de Penetración Celular/química , HumanosRESUMEN
Peptides that self-assemble, at low concentration, into bilayer-spanning pores which allow the passage of macromolecules would be beneficial in multiple areas of biotechnology. However, there are few, if any, natural or designed peptides that have this property. Here we show that the 26-residue peptide "MelP5", a synthetically evolved gain-of-function variant of the bee venom lytic peptide melittin identified in a high-throughput screen for small molecule leakage, enables the passage of macromolecules across bilayers under conditions where melittin and other pore-forming peptides do not. In surface-supported bilayers, MelP5 forms unusually high conductance, equilibrium pores at peptide:lipid ratios as low as 1:25000. The increase in bilayer conductance due to MelP5 is dramatically higher, per peptide, than the increase due to the parent sequence of melittin or other peptide pore formers. Here we also develop two novel assays for macromolecule leakage from vesicles, and we use them to characterize MelP5 pores in bilayers. We show that MelP5 allows the passage of macromolecules across vesicle membranes at peptide:lipid ratios as low as 1:500, and under conditions where neither osmotic lysis nor gross vesicle destabilization occur. The macromolecule-sized, equilibrium pores formed by MelP5 are unique as neither melittin nor other pore-forming peptides release macromolecules significantly under the same conditions. MelP5 thus appears to belong to a novel functional class of peptide that could form the foundation of multiple potential biotechnological applications.
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Membrana Dobles de Lípidos/metabolismo , Meliteno/química , Meliteno/metabolismo , Secuencia de Aminoácidos , Meliteno/síntesis química , Datos de Secuencia Molecular , Porosidad , Estructura Secundaria de ProteínaRESUMEN
Root hairs are fast-growing tubular protrusions on root epidermal cells that play important roles in water and nutrient uptake in plants. The tip-focused polarized growth of root hairs is accomplished by the secretion of newly synthesized materials to the tip via the polarized membrane trafficking mechanism. Here, we report the function of two different types of plasma membrane (PM) Qa-SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors), SYP123 and SYP132, in the growth of root hair in Arabidopsis. We found that SYP123, but not SYP132, localizes in the tip region of root hairs by recycling between the brefeldin A (BFA)-sensitive endosomes and the PM of the expanding tip in an F-actin-dependent manner. The vesicle-associated membrane proteins VAMP721/722/724 also exhibited tip-focused localization in root hairs and formed ternary SNARE complexes with both SYP123 and SYP132. These results demonstrate that SYP123 and SYP132 act in a coordinated fashion to mediate tip-focused membrane trafficking for root hair tip growth.
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Proteínas de Arabidopsis/metabolismo , Arabidopsis/crecimiento & desarrollo , Arabidopsis/metabolismo , Raíces de Plantas/crecimiento & desarrollo , Proteínas Qa-SNARE/metabolismo , Actinas/metabolismo , Arabidopsis/efectos de los fármacos , Brefeldino A/farmacología , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Recuperación de Fluorescencia tras Fotoblanqueo , Proteínas Fluorescentes Verdes/metabolismo , Modelos Biológicos , Complejos Multiproteicos/metabolismo , Mutación/genética , Fenotipo , Raíces de Plantas/citología , Raíces de Plantas/efectos de los fármacos , Unión Proteica/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , Proteínas Recombinantes de Fusión/metabolismo , Proteínas SNARE/metabolismo , Tiazolidinas/farmacologíaRESUMEN
Direct cellular entry of potentially useful polar compounds into cells is prevented by the hydrophobic barrier of the membrane. Toward circumventing this barrier, we used high throughput screening to identify a family of peptides that carry membrane-impermeant cargos across synthetic membranes. Here we characterize the plasma membrane translocation of these peptides with polar cargos under a variety of conditions. The spontaneous membrane-translocating peptides (SMTPs) delivered the zwitterionic, membrane-impermeant dye tetramethylrhodamine (TAMRA) into cells even when the conditions were not permissive for endocytosis. They also delivered the larger, anionic membrane-impermeant dye Alexa Fluor 546 but did not deliver a quantum dot nanoparticle. Under all conditions, the SMTP-cargo filled the cytoplasm with a diffuse, non-punctate fluorescence that was partially excluded from the nucleus. D-amino acid peptides behaved identically in vitro, ruling out proteolysis as an important factor in the diffuse cellular distribution. Thus, cytosolic delivery of SMTP-cargo conjugates is dominated by direct membrane translocation. This is in sharp contrast to Arg9-TAMRA, a representative highly cationic, cell-penetrating peptide, which entered cells only when endocytosis was permitted. Arg9-TAMRA triggered large scale endocytosis and did not appreciably escape the endosomal compartments in the 1-h timescales we studied. When injected into mice, SMTP-TAMRA conjugates were found in many tissues even after 2 h. Unconjugated TAMRA was rapidly cleared and did not become systemically distributed. SMTPs are a platform that could improve delivery of many polar compounds to cells, in the laboratory or in the clinic, including those that would otherwise be rejected as drugs because they are membrane-impermeant.
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Membrana Celular/metabolismo , Citosol/metabolismo , Péptidos/metabolismo , Rodaminas/metabolismo , Secuencia de Aminoácidos , Animales , Transporte Biológico , Células CHO , Supervivencia Celular/efectos de los fármacos , Cricetinae , Cricetulus , Sistemas de Liberación de Medicamentos/métodos , Endocitosis , Femenino , Colorantes Fluorescentes/administración & dosificación , Colorantes Fluorescentes/química , Colorantes Fluorescentes/metabolismo , Ratones , Ratones Endogámicos BALB C , Microscopía Confocal , Péptidos/administración & dosificación , Péptidos/química , Compuestos de Quinolinio/administración & dosificación , Compuestos de Quinolinio/química , Compuestos de Quinolinio/metabolismo , Reproducibilidad de los Resultados , Rodaminas/administración & dosificación , Rodaminas/químicaRESUMEN
Membrane associated proteins SNAREs (soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptors) provide the minimal fusion machinery necessary for cellular vesicles to fuse to target organelle membranes in eukaryotic cells. Despite the conserved nature of the fusion machinery in all eukaryotes, it still remains challenging to identify functional SNARE pairs in higher plants. We developed a method based on a split-luciferase complementation assay for detecting changes in SNARE-SNARE interaction by luminescence within Arabidopsis protoplasts that express recombinant proteins at physiological levels in 96-well plates. The reliability of the assay was confirmed by three experiments. First, reduction of the SNARE-SNARE interaction caused by a single amino acid substitution adjacent to the SNARE motif in endosome-localized AtVAM3/SYP22 (syntaxin of plant 22) was detected by a reduction of luminescence. Second, reduction of the interaction between plasma-membrane localized SYP121 and VAMP722 in response to sodium azide was detected in real-time. Third, the results of 21 SNARE pairs investigated by this method largely agreed with the results from previously reported co-immunoprecipitation assays. Using the method, we newly identified the interaction between SYP121 and VAMP722 was significantly increased when the protoplasts were incubated in the light. Microscopic observation of transgenic Arabidopsis expressing GFP-SYP121 (green fluorescent protein tagged SYP121) from its own promoter suggested that the plasma-membrane localization of GFP-SYP121 is maintained by light. These suggested that the vesicle trafficking pathway mediated by SYP121 might be regulated by light in Arabidopsis. In general, this article demonstrated the method that can generate new biological insight of the SNARE protein interactions in plant cells.