RESUMEN
Mass-spectrometry-enabled ADP-ribosylation workflows are developing rapidly, providing researchers a variety of ADP-ribosylome enrichment strategies and mass spectrometric acquisition options. Despite the growth spurt in upstream technologies, systematic ADP-ribosyl (ADPr) peptide mass spectral annotation methods are lacking. HCD-dependent ADP-ribosylome studies are common, but the resulting MS2 spectra are complex, owing to a mixture of b/y-ions and the m/p-ion peaks representing one or more dissociation events of the ADPr moiety (m-ion) and peptide (p-ion). In particular, p-ions that dissociate further into one or more fragment ions can dominate HCD spectra but are not recognized by standard spectral annotation workflows. As a result, annotation strategies that are solely reliant upon the b/y-ions result in lower spectral scores that in turn reduce the number of reportable ADPr peptides. To improve the confidence of spectral assignments, we implemented an ADPr peptide annotation and scoring strategy. All MS2 spectra are scored for the ADPr m-ions, but once spectra are assigned as an ADPr peptide, they are further annotated and scored for the p-ions. We implemented this novel workflow to ADPr peptides enriched from the liver and spleen isolated from mice post 4 h exposure to systemic IFN-γ. HCD collision energy experiments were first performed on the Orbitrap Fusion Lumos and the Q Exactive, with notable ADPr peptide dissociation properties verified with CID (Lumos). The m-ion and p-ion series score distributions revealed that ADPr peptide dissociation properties vary markedly between instruments and within instrument collision energy settings, with consequences on ADPr peptide reporting and amino acid localization. Consequentially, we increased the number of reportable ADPr peptides by 25% (liver) and 17% (spleen) by validation and the inclusion of lower confidence ADPr peptide spectra. This systematic annotation strategy will streamline future reporting of ADPr peptides that have been sequenced using any HCD/CID-based method.
Asunto(s)
Péptidos , Bazo , Adenosina Difosfato , Animales , Interferón gamma , Iones , Hígado , Ratones , Péptidos/química , Bazo/químicaRESUMEN
Conventional drug screening methods search for a limited number of small molecules that directly interact with the target protein. This process can be slow, cumbersome and has driven the need for developing new drug screening approaches to counter rapidly emerging diseases such as COVID-19. We propose a pipeline for drug repurposing combining in silico drug candidate identification followed by in vitro characterization of these candidates. We first identified a gene target of interest, the entry receptor for the SARS-CoV-2 virus, angiotensin converting enzyme 2 (ACE2). Next, we employed a gene expression profile database, L1000-based Connectivity Map to query gene expression patterns in lung epithelial cells, which act as the primary site of SARS-CoV-2 infection. Using gene expression profiles from 5 different lung epithelial cell lines, we computationally identified 17 small molecules that were predicted to decrease ACE2 expression. We further performed a streamlined validation in the normal human epithelial cell line BEAS-2B to demonstrate that these compounds can indeed decrease ACE2 surface expression and to profile cell health and viability upon drug treatment. This proposed pipeline combining in silico drug compound identification and in vitro expression and viability characterization in relevant cell types can aid in the repurposing of FDA-approved drugs to combat rapidly emerging diseases.
RESUMEN
AIMS: Proteostasis maintains protein homeostasis and participates in regulating critical cardiometabolic disease risk factors including proprotein convertase subtilisin/kexin type 9 (PCSK9). Endoplasmic reticulum (ER) remodeling through release and incorporation of trafficking vesicles mediates protein secretion and degradation. We hypothesized that ER remodeling that drives mitochondrial fission participates in cardiometabolic proteostasis. METHODS AND RESULTS: We used in vitro and in vivo hepatocyte inhibition of a protein involved in mitochondrial fission, dynamin-related protein 1 (DRP1). Here, we show that DRP1 promotes remodeling of select ER microdomains by tethering vesicles at ER. A DRP1 inhibitor, mitochondrial division inhibitor 1 (mdivi-1) reduced ER localization of a DRP1 receptor, mitochondrial fission factor, suppressing ER remodeling-driven mitochondrial fission, autophagy, and increased mitochondrial calcium buffering and PCSK9 proteasomal degradation. DRP1 inhibition by CRISPR/Cas9 deletion or mdivi-1 alone or in combination with statin incubation in human hepatocytes and hepatocyte-specific Drp1-deficiency in mice reduced PCSK9 secretion (-78.5%). In HepG2 cells, mdivi-1 increased low-density lipoprotein receptor via c-Jun transcription and reduced PCSK9 mRNA levels via suppressed sterol regulatory binding protein-1c. Additionally, mdivi-1 reduced macrophage burden, oxidative stress, and advanced calcified atherosclerotic plaque in aortic roots of diabetic Apoe-deficient mice and inflammatory cytokine production in human macrophages. CONCLUSIONS: We propose a novel tethering function of DRP1 beyond its established fission function, with DRP1-mediated ER remodeling likely contributing to ER constriction of mitochondria that drives mitochondrial fission. We report that DRP1-driven remodeling of select ER micro-domains may critically regulate hepatic proteostasis and identify mdivi-1 as a novel small molecule PCSK9 inhibitor.
Asunto(s)
Aterosclerosis/tratamiento farmacológico , Dinaminas/antagonistas & inhibidores , Retículo Endoplásmico/efectos de los fármacos , Hígado/efectos de los fármacos , Mitocondrias Hepáticas/efectos de los fármacos , Inhibidores de PCSK9/farmacología , Proproteína Convertasa 9/metabolismo , Quinazolinonas/farmacología , Animales , Aterosclerosis/enzimología , Aterosclerosis/genética , Aterosclerosis/patología , Modelos Animales de Enfermedad , Dinaminas/genética , Dinaminas/metabolismo , Retículo Endoplásmico/enzimología , Retículo Endoplásmico/genética , Retículo Endoplásmico/patología , Células Hep G2 , Humanos , Hígado/enzimología , Hígado/patología , Ratones Noqueados para ApoE , Mitocondrias Hepáticas/enzimología , Mitocondrias Hepáticas/genética , Mitocondrias Hepáticas/patología , Dinámicas Mitocondriales/efectos de los fármacos , Proproteína Convertasa 9/genética , Complejo de la Endopetidasa Proteasomal , Mapas de Interacción de Proteínas , Proteolisis , Proteostasis , Vías SecretorasRESUMEN
OBJECTIVES: Angiopoietin-like protein 2 (ANGPTL2), a recently identified pro-inflammatory cytokine, is mainly secreted from the adipose tissue. This study aimed to explore the role of ANGPTL2 in adipose tissue inflammation and macrophage activation in a mouse model of diabetes. METHODOLOGY/PRINCIPAL FINDINGS: Adenovirus mediated lacZ (Ad-LacZ) or human ANGPTL2 (Ad-ANGPTL2) was delivered via tail vein in diabetic db/db mice. Ad-ANGPTL2 treatment for 2 weeks impaired both glucose tolerance and insulin sensitivity as compared to Ad-LacZ treatment. Ad-ANGPTL2 treatment significantly induced pro-inflammatory gene expression in white adipose tissue. We also isolated stromal vascular fraction from epididymal fat pad and analyzed adipose tissue macrophage and T lymphocyte populations by flow cytometry. Ad-ANGPTL2 treated mice had more adipose tissue macrophages (F4/80+CD11b+) and a larger M1 macrophage subpopulation (F4/80+CD11b+CD11c+). Moreover, Ad-ANGPTL2 treatment increased a CD8-positive T cell population in adipose tissue, which preceded increased macrophage accumulation. Consistent with our in vivo results, recombinant human ANGPTL2 protein treatment increased mRNA levels of pro-inflammatory gene products and production of TNF-α protein in the human macrophage-like cell line THP-1. Furthermore, Ad-ANGPTL2 treatment induced lipid accumulation and increased fatty acid synthesis, lipid metabolism related gene expression in mouse liver. CONCLUSION: ANGPTL2 treatment promotes macrophage accumulation and activation. These results suggest potential mechanisms for insulin resistance.
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Tejido Adiposo Blanco/metabolismo , Angiopoyetinas/metabolismo , Diabetes Mellitus Experimental/metabolismo , Macrófagos/metabolismo , Obesidad/metabolismo , Linfocitos T/metabolismo , Adenoviridae/genética , Tejido Adiposo Blanco/efectos de los fármacos , Tejido Adiposo Blanco/patología , Proteína 2 Similar a la Angiopoyetina , Proteínas Similares a la Angiopoyetina , Angiopoyetinas/genética , Angiopoyetinas/farmacología , Animales , Antígenos de Diferenciación/genética , Antígenos de Diferenciación/metabolismo , Antígeno CD11b/genética , Antígeno CD11b/metabolismo , Antígeno CD11c/genética , Antígeno CD11c/metabolismo , Movimiento Celular/efectos de los fármacos , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/patología , Vectores Genéticos , Prueba de Tolerancia a la Glucosa , Humanos , Resistencia a la Insulina , Metabolismo de los Lípidos/efectos de los fármacos , Hígado/efectos de los fármacos , Hígado/metabolismo , Hígado/patología , Macrófagos/efectos de los fármacos , Macrófagos/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Obesidad/genética , Obesidad/patología , Transducción de Señal , Linfocitos T/efectos de los fármacos , Linfocitos T/patología , Factor de Necrosis Tumoral alfa/biosíntesis , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
Angiogenesis requires endothelial cell survival and proliferation, which depend upon cytokine stimulation together with integrin-mediated cell adhesion to extracellular matrix; however, the question of which specific integrins are the best targets for suppressing neovascularization is controversial and unresolved. Therefore, we designed experiments to compare contributions of individual integrins from both the alphav and beta1 integrin subfamilies. With immobilized antibodies, we determined that adhesion through integrins alpha1beta1, alpha2beta1, alphavbeta3, and alphavbeta5 each individually supported dermal microvascular endothelial cell survival. Also, substratum coated with collagen I (which binds alpha1beta1 and alpha2beta1) and vitronectin (which binds alphavbeta3 and alphavbeta5) each supported survival. Importantly, substratum coated with combinations of collagen I and vitronectin were most effective at promoting survival, and survival on three-dimensional collagen I gels was strongly enhanced by vitronectin. Vascular endothelial growth factor activation of the p44/p42 mitogen-activated protein kinase pathway, which is required for angiogenesis, was supported by adhesion through either alpha1beta1, alpha2beta1, alphavbeta3, or alphavbeta5, and pharmacologic inhibition of this pathway blocked proliferation and suppressed survival. Therefore, these studies establish that the alpha1beta1, alpha2beta1, alphavbeta3, and alphavbeta5 integrins each support dermal microvascular endothelial cell viability, and that each collaborate with vascular endothelial growth factor to support robust activation of the mitogen-activated protein kinase pathway which mediates both proliferation and survival. Moreover, survival is supported most significantly by extracellular matrices, which engage all of these integrins in combination. Consistent with important complementary and overlapping functions, combined antagonism of these integrins provided superior inhibition of angiogenesis in skin, indicating that multiplicity of integrin involvement should be considered in designing strategies for controlling neovascularization.
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Integrina alfaV/fisiología , Integrina beta1/fisiología , Neovascularización Fisiológica/fisiología , Piel/irrigación sanguínea , Animales , Línea Celular , Supervivencia Celular/fisiología , Colágeno Tipo I/fisiología , Combinación de Medicamentos , Factores de Crecimiento Endotelial/fisiología , Endotelio Vascular/fisiología , Activación Enzimática , Humanos , Integrina alfa1beta1/fisiología , Integrina alfa2beta1/fisiología , Integrina alfaVbeta3/fisiología , Integrinas/fisiología , Péptidos y Proteínas de Señalización Intercelular/fisiología , Linfocinas/fisiología , Microcirculación , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Receptores de Vitronectina/fisiología , Spodoptera , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial Vascular , Vitronectina/fisiologíaRESUMEN
Collagen I provokes endothelial cells to assume a spindle-shaped morphology and to align into solid cord-like assemblies. These cords closely imitate the solid pre-capillary cords of embryonic angiogenesis, raising interesting questions about underlying mechanisms. Studies described here identify a critical mechanism beginning with collagen I ligation of integrins alpha(1)beta(1) and alpha(2)beta(1), followed by suppression of cyclic AMP and cyclic AMP (cAMP)-dependent protein kinase A, and marked induction of actin polymerization to form prominent stress fibers. In contrast to collagen I, laminin-1 neither suppressed cAMP nor protein kinase A activity nor induced actin polymerization or changes in cell shape. Moreover, fibroblasts did not respond to collagen I with changes in cAMP, actin polymerization, or cell shape, thus indicating that collagen signaling, as observed in endothelial cells, does not extend to all cell types. Pharmacological elevation of cAMP blocked collagen-induced actin polymerization and formation of cords by endothelial cells; conversely, pharmacological suppression of either cAMP or protein kinase A induced actin polymerization. Collectively, these studies identify a previously unrecognized and critical mechanism, involving suppression of cAMP-dependent protein kinase A and induction of actin polymerization, through which collagen I drives endothelial cell organization into multicellular pre-capillary cords.
Asunto(s)
Actinas/metabolismo , Colágeno Tipo I/farmacología , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Endotelio Vascular/efectos de los fármacos , Morfogénesis/fisiología , Animales , Anticuerpos Monoclonales/metabolismo , Camptotecina/farmacología , Tamaño de la Célula , Células Cultivadas , Factores de Crecimiento Endotelial/metabolismo , Endotelio Vascular/citología , Endotelio Vascular/fisiología , Inhibidores Enzimáticos/farmacología , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Humanos , Integrina alfa1beta1/metabolismo , Integrina alfa2beta1/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Linfocinas/metabolismo , Factor A de Crecimiento Endotelial Vascular , Factores de Crecimiento Endotelial VascularRESUMEN
The mechanisms that control organization of endothelial cells (ECs) into new blood vessels are poorly understood. We hypothesized that the GTPase Rho, which regulates cytoskeletal architecture, is important for EC organization during neovascularization. To test this hypothesis, we designed a highly versatile mouse skin model that used vascular endothelial growth factor-expressing cells together with packaging cells producing retroviruses encoding RhoA GTPase mutants. In this animal model, dominant negative N19RhoA selectively impaired assembly of ECs into new blood vessels; and, in contrast, active V14RhoA stimulated ECs to form blood vessels with functional lumens. In vitro, dominant negative N19RhoA reduced EC actin stress fibers and prevented ECs from contracting and reorganizing into precapillary cords within collagen gels. In contrast, active V14RhoA promoted EC stress fiber formation, contractility, and organization into cords. Neither N19RhoA nor V14RhoA significantly affected EC proliferation or migration in vitro; and, similarly, neither mutant significantly affected EC density during angiogenesis in vivo. Thus, these studies identify a critical and selective role for Rho activity in regulating EC assembly into new blood vessels, and they identify both negative and positive manipulation of Rho activity, respectively, as strategies for suppressing or promoting the organizational stages of neovascularization.