RESUMO
BACKGROUND: Radiation-induced fibrosis (RIF) is an important late complication of radiation therapy, and the resulting damaging effects of RIF can significantly impact reconstructive outcomes. There is currently a paucity of effective treatment options available, likely due to the continuing knowledge gap surrounding the cellular mechanisms involved. In this study, detailed analyses of irradiated and non-irradiated human skin samples were performed incorporating histological and single-cell transcriptional analysis to identify novel features guiding development of skin fibrosis following radiation injury. METHODS: Paired irradiated and contralateral non-irradiated skin samples were obtained from six female patients undergoing post-oncologic breast reconstruction. Skin samples underwent histological evaluation, immunohistochemistry, and biomechanical testing. Single-cell RNA sequencing was performed using the 10X single cell platform. Cells were separated into clusters using Seurat in R. The SingleR classifier was applied to ascribe cell type identities to each cluster. Differentially expressed genes characteristic to each cluster were then determined using non-parametric testing. RESULTS: Comparing irradiated and non-irradiated skin, epidermal atrophy, dermal thickening, and evidence of thick, disorganized collagen deposition within the extracellular matrix of irradiated skin were readily appreciated on histology. These histologic features were associated with stiffness that was higher in irradiated skin. Single-cell RNA sequencing revealed six predominant cell types. Focusing on fibroblasts/stromal lineage cells, five distinct transcriptional clusters (Clusters 0-4) were identified. Interestingly, while all clusters were noted to express Cav1, Cluster 2 was the only one to also express Cav2. Immunohistochemistry demonstrated increased expression of Cav2 in irradiated skin, whereas Cav1 was more readily identified in non-irradiated skin, suggesting Cav1 and Cav2 may act antagonistically to modulate fibrotic cellular responses. CONCLUSION: In response to radiation therapy, specific changes to fibroblast subpopulations and enhanced Cav2 expression may contribute to fibrosis. Altogether, this study introduces a novel pathway of caveolin involvement which may contribute to fibrotic development following radiation injury.
Assuntos
Caveolina 1 , Fibroblastos , Análise de Célula Única , Pele , Humanos , Feminino , Fibroblastos/efeitos da radiação , Fibroblastos/metabolismo , Caveolina 1/metabolismo , Caveolina 1/genética , Caveolina 1/biossíntese , Pele/efeitos da radiação , Pele/patologia , Pele/metabolismo , Neoplasias da Mama/radioterapia , Neoplasias da Mama/patologia , Caveolina 2/metabolismo , Caveolina 2/genética , Lesões por Radiação/patologia , Lesões por Radiação/metabolismo , Fibrose , Pessoa de Meia-IdadeRESUMO
In order to identify how differential gene expression in the trabecular meshwork (TM) contributes to racial disparities of caveolar protein expression, TM dysfunction and development of primary open angle glaucoma (POAG), RNA sequencing was performed to compare TM tissue obtained from White and Black POAG surgical (trabeculectomy) specimens. Healthy donor TM tissue from White and Black donors was analyzed by PCR, qPCR, immunohistochemistry staining, and Western blot to evaluate SDPR (serum deprivation protein response; Cavin 2) and CAV1/CAV2 (Caveolin 1/Caveolin 2). Standard transmission electron microscopy (TEM) and immunogold labeled studies were performed. RNA sequencing demonstrated reduced SDPR expression in TM from Black vs White POAG patients' surgical specimens, with no significant expression differences in other caveolae-associated genes, confirmed by qPCR analysis. No racial differences in SDPR gene expression were noted in healthy donor tissue by PCR analysis, but there was greater expression as compared to specimens from patients with glaucoma. Analysis of SDPR protein expression confirmed specific expression in the TM regions, but not in adjacent tissues. TEM studies of TM specimens from healthy donors did not demonstrate any racial differences in caveolar morphology, but a significant reduction of caveolae with normal morphology and immuno-gold staining of SDPR were noted in glaucomatous TM as compared to TM from healthy donors. Linkage of SDPR expression levels in TM, POAG development, and caveolar ultrastructural morphology may provide the basis for a novel pathway of exploration of the pathologic mechanisms of glaucoma. Differential gene expression of SDPR in TM from Black vs White subjects with glaucoma may further our understanding of the important public health implications of the racial disparities of this blinding disease.
Assuntos
Caveolina 1 , Glaucoma de Ângulo Aberto , Malha Trabecular , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Negro ou Afro-Americano/genética , Caveolina 1/genética , Caveolina 1/metabolismo , Caveolina 2/genética , Caveolina 2/metabolismo , Glaucoma de Ângulo Aberto/genética , Glaucoma de Ângulo Aberto/metabolismo , Glaucoma de Ângulo Aberto/patologia , Glaucoma de Ângulo Aberto/etnologia , Malha Trabecular/metabolismo , Malha Trabecular/patologia , Brancos , População Branca/genéticaRESUMO
Here, we show that insulin induces palmitoylation turnover of Caveolin-2 (Cav-2) in adipocytes. Acyl protein thioesterases-1 (APT1) catalyzes Cav-2 depalmitoylation, and zinc finger DHHC domain-containing protein palmitoyltransferase 21 (ZDHHC21) repalmitoylation of the depalmitoylated Cav-2 for the turnover, thereby controlling insulin receptor (IR)-Cav-2-insulin receptor substrate-1 (IRS-1)-Akt-driven signaling. Insulin-induced palmitoylation turnover of Cav-2 facilitated glucose uptake and fat storage through induction of lipogenic genes. Cav-2-, APT1-, and ZDHHC21-deficient adipocytes, however, showed increased induction of lipolytic genes and glycerol release. In addition, white adipose tissues from insulin sensitive and resistant obese patients exhibited augmented expression of LYPLA1 (APT1) and ZDHHC20 (ZDHHC20). Our study identifies the specific enzymes regulating Cav-2 palmitoylation turnover, and reveals a new mechanism by which insulin-mediated lipid metabolism is controlled in adipocytes.
Assuntos
Adipócitos , Caveolina 2 , Proteínas Substratos do Receptor de Insulina , Insulina , Metabolismo dos Lipídeos , Lipoilação , Receptor de Insulina , Humanos , Adipócitos/metabolismo , Animais , Proteínas Substratos do Receptor de Insulina/metabolismo , Proteínas Substratos do Receptor de Insulina/genética , Camundongos , Caveolina 2/metabolismo , Caveolina 2/genética , Receptor de Insulina/metabolismo , Receptor de Insulina/genética , Insulina/metabolismo , Obesidade/metabolismo , Obesidade/genética , Tioléster Hidrolases/metabolismo , Tioléster Hidrolases/genética , Aciltransferases/metabolismo , Aciltransferases/genética , Transdução de Sinais , Resistência à Insulina , Células 3T3-L1 , MasculinoRESUMO
Endometriotic cells exhibit a notable degree of invasiveness and some characteristics of tissue remodeling underlying lesion formation. In this regard, do matrix metalloproteinases 14 (MMP14) and other related genes such as SPARC-like protein 1 (SPARCL1), caveolin 2 (CAV2), and clusterin (CLU) exert any significant influence in the processes of endometriosis development and pathophysiology is not apparent. We aim to assess whether these genes could serve as potential diagnostic biomarkers in endometriosis. Microarray-based gene expression analysis was performed on total RNA extracted from endometriotic tissue samples treated with and without gonadotropin-releasing hormone agonist (GnRHa). The GnRHa untreated patients were considered the control group. The validation of genes was performed using quantitative real-time polymerase chain reaction (qRT-PCR). qRT-PCR analysis showed significant downregulation in the expression of MMP14 (p = 0.024), CAV2 (p = 0.017), and upregulation of CLU (p = 0.005) in endometriosis patients treated with GnRHa. SPARCL1 did not show any significant (p = 0.30) change in the expression compared to the control group. These data have the potential to contribute to the comprehension of the molecular pathways implicated in the remodeling of the extracellular matrix, which is a vital step for the physiology of the endometrium. Based on the result, it is concluded that changes in the expression of MMP14, CAV2, and CLU post-treatment imply their role in the pathophysiology of endometriosis and may serve as a potential diagnostic biomarker of endometriosis in response to GnRHa treatment in patients with ovarian endometrioma.
Assuntos
Endometriose , Feminino , Humanos , Endometriose/patologia , Clusterina/genética , Clusterina/metabolismo , Caveolina 2/metabolismo , Metaloproteinase 14 da Matriz/genética , Metaloproteinase 14 da Matriz/metabolismo , Endométrio/patologia , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas da Matriz Extracelular/genéticaRESUMO
Here, we identify that Caveolin-2 (Cav-2), an integral membrane protein, controls adipocyte hypertrophy in association with nuclear lamina. In the hypertrophy stage of adipogenesis, pY19-Cav-2 association with lamin A/C facilitated the disengagement of CCAAT/enhancer-binding protein α (C/EBPα) and peroxisome proliferator-activated receptor γ (PPARγ) from lamin A/C and repressed Cav-2 promoter at the nuclear periphery for epigenetic activation of Cav-2, and thereby promoted C/EBPα and PPARγ-induced adipocyte hypertrophy. Stable expression of Cav-2 was required and retained by phosphorylation, deubiquitination, and association with lamin A/C for the adipocyte hypertrophy. However, obese adipocytes exhibited augmented Cav-2 stability resulting from the up-regulation of lamin A/C over lamin B1, protein tyrosine phosphatase 1B (PTP1B), and nuclear deubiquitinating enzyme (DUB), Uchl5. Our findings show a novel epigenetic regulatory mechanism of adipocyte hypertrophy by Cav-2 at the nuclear periphery.
Assuntos
Lamina Tipo A , PPAR gama , Humanos , Camundongos , Animais , PPAR gama/genética , PPAR gama/metabolismo , Lamina Tipo A/metabolismo , Lâmina Nuclear/metabolismo , Caveolina 2/genética , Caveolina 2/metabolismo , Proteína alfa Estimuladora de Ligação a CCAAT/metabolismo , Adipócitos/metabolismo , Hipertrofia/metabolismo , Diferenciação Celular , Adipogenia/genética , Células 3T3-L1RESUMO
Aging is a major risk factor for common neurodegenerative diseases. Although multiple molecular, cellular, structural, and functional changes occur in the brain during aging, the involvement of caveolin-2 (Cav-2) in brain ageing remains unknown. We investigated Cav-2 expression in brains of aged mice and its effects on endothelial cells. The human umbilical vein endothelial cells (HUVECs) showed decreased THP-1 adhesion and infiltration when treated with Cav-2 siRNA compared to control siRNA. In contrast, Cav-2 overexpression increased THP-1 adhesion and infiltration in HUVECs. Increased expression of Cav-2 and iba-1 was observed in brains of old mice. Moreover, there were fewer iba-1-positive cells in the brains of aged Cav-2 knockout (KO) mice than of wild-type aged mice. The levels of several chemokines were higher in brains of aged wild-type mice than in young wild-type mice; moreover, chemokine levels were significantly lower in brains of young mice as well as aged Cav-2 KO mice than in their wild-type counterparts. Expression of PECAM1 and VE-cadherin proteins increased in brains of old wild-type mice but was barely detected in brains of young wild-type and Cav-2 KO mice. Collectively, our results suggest that Cav-2 expression increases in the endothelial cells of aged brain, and promotes leukocyte infiltration and age-associated neuroinflammation.
Assuntos
Envelhecimento , Caveolina 2 , Doenças Neuroinflamatórias , Animais , Humanos , Camundongos , Encéfalo/metabolismo , Caveolina 2/genética , Caveolina 2/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Camundongos Knockout , Doenças Neuroinflamatórias/genética , RNA Interferente Pequeno/metabolismo , Envelhecimento/patologiaRESUMO
Caveolin-2 is a protein suitable for the study of interactions of caveolins with other proteins and lipids present in caveolar lipid rafts. Caveolin-2 has a lower tendency to associate with high molecular weight oligomers than caveolin-1, facilitating the study of its structural modulation upon association with other proteins or lipids. In this paper, we have successfully expressed and purified recombinant human caveolin-2 using E. coli. The structural changes of caveolin-2 upon interaction with a lipid bilayer of liposomes were characterized using bioinformatic prediction models, circular dichroism, differential scanning calorimetry, and fluorescence techniques. Our data support that caveolin-2 binds and alters cholesterol-rich domains in the membranes through a CARC domain, a type of cholesterol-interacting domain in its sequence. The far UV-CD spectra support that the purified protein keeps its folding properties but undergoes a change in its secondary structure in the presence of lipids that correlates with the acquisition of a more stable conformation, as shown by differential scanning calorimetry experiments. Fluorescence experiments using egg yolk lecithin large unilamellar vesicles loaded with 1,6-diphenylhexatriene confirmed that caveolin-2 adsorbs to the membrane but only penetrates the core of the phospholipid bilayer if vesicles are supplemented with 30% of cholesterol. Our study sheds light on the caveolin-2 interaction with lipids. In addition, we propose that purified recombinant caveolin-2 can provide a new tool to study protein-lipid interactions within caveolae.
Assuntos
Caveolina 1 , Escherichia coli , Humanos , Escherichia coli/metabolismo , Caveolina 1/metabolismo , Caveolina 2/metabolismo , Cavéolas/metabolismo , Colesterol/metabolismo , Microdomínios da Membrana/metabolismo , Bicamadas Lipídicas/metabolismoRESUMO
Here, we show that Caveolin-2 (Cav-2) is an epigenetic regulator for adipogenesis. Upon adipogenic stimulation, inner nuclear membrane (INM)-targeted pY19-Cav-2 interacted with lamin A/C to disengage the repressed Cebpb promoter from lamin A/C, which facilitated the Cebpb promoter association with lamin B1. Consequently, pY19-Cav-2 recruited lysine demethylase 4b (KDM4b) for demethylation of histone H3 lysine 9 trimethylation (H3K9me3) and histone acetyltransferase GCN5 for acetylation of H3K27, and subsequently RNA polymerase II (Pol II) on Cebpb promoter for epigenetic activation of Cebpb, to initiate adipogenesis. Cav-2 knock-down abrogated the Cebpb activation and blocked the Pparg2 and Cebpa activation. Re-expression of Cav-2 restored Cebpb activation and adipogenesis in Cav-2-deficient preadipocytes. Our data identify a new mechanism by which the epigenetic activation of Cebpb is controlled at the nuclear periphery to promote adipogenesis.
Assuntos
Caveolina 2 , Lamina Tipo A , Caveolina 2/genética , Caveolina 2/metabolismo , Epigênese Genética , Histonas/genética , Histonas/metabolismo , Lamina Tipo A/genética , Lisina/genética , Lâmina Nuclear/metabolismo , RNA Polimerase II/genéticaRESUMO
OBJECTIVE: CAV1 encodes caveolin-1, a major protein of plasma membrane microdomains called caveolae, involved in several signaling pathways. Caveolin-1 is also located at the adipocyte lipid droplet. Heterozygous pathogenic variants of CAV1 induce rare heterogeneous disorders including pulmonary arterial hypertension and neonatal progeroid syndrome. Only one patient was previously reported with a CAV1 homozygous pathogenic variant, associated with congenital generalized lipodystrophy (CGL3). We aimed to further delineate genetic transmission, clinical, metabolic, and cellular characteristics of CGL3. DESIGN/METHODS: In a large consanguineous kindred referred for CGL, we performed next-generation sequencing, as well as clinical, imagery, and metabolic investigations. We studied skin fibroblasts from the index case and the previously reported patient with CGL3. RESULTS: Four patients, aged 8 months to 18 years, carried a new homozygous p.(His79Glnfs*3) CAV1 variant. They all displayed generalized lipodystrophy since infancy, insulin resistance, low HDL-cholesterol, and/or high triglycerides, but no pulmonary hypertension. Two patients also presented at the age of 15 and 18 years with dysphagia due to achalasia, and one patient had retinitis pigmentosa. Heterozygous parents and relatives (n = 9) were asymptomatic, without any metabolic abnormality. Patients' fibroblasts showed a complete loss of caveolae and no protein expression of caveolin-1 and its caveolin-2 and cavin-1 partners. Patients' fibroblasts also displayed insulin resistance, increased oxidative stress, and premature senescence. CONCLUSIONS: The CAV1 null variant investigated herein leads to an autosomal recessive congenital lipodystrophy syndrome. Loss of caveolin-1 and/or caveolae induces specific manifestations including achalasia which requires specific management. Overlapping phenotypic traits between the different CAV1-related diseases require further studies.
Assuntos
Caveolina 1/genética , Acalasia Esofágica/genética , Lipodistrofia Generalizada Congênita/genética , Adolescente , Cavéolas/patologia , Cavéolas/ultraestrutura , Caveolina 1/metabolismo , Caveolina 2/metabolismo , Senescência Celular , Criança , Pré-Escolar , Consanguinidade , Dislipidemias/metabolismo , Acalasia Esofágica/patologia , Feminino , Fibroblastos/patologia , Fibroblastos/ultraestrutura , Homozigoto , Humanos , Lactente , Lipodistrofia Generalizada Congênita/metabolismo , Lipodistrofia Generalizada Congênita/patologia , Masculino , Microscopia Eletrônica de Transmissão , Estresse Oxidativo , Linhagem , Proteínas de Ligação a RNA/metabolismoRESUMO
BACKGROUND: Pancreatic cancer is the fourth leading cause of cancer deaths in the United States both in females and in males, and is projected to become the second deadliest cancer by 2030. The overall 5-year survival rate remains at around 10%. Cancer metabolism and specifically lipid metabolism plays an important role in pancreatic cancer progression and metastasis. Lipid droplets can not only store and transfer lipids, but also act as molecular messengers, and signaling factors. As lipid droplets are implicated in reprogramming tumor cell metabolism and in invasion and migration of pancreatic cancer cells, we aimed to identify lipid droplet-associated genes as prognostic markers in pancreatic cancer. METHODS: We performed a literature search on review articles related to lipid droplet-associated proteins. To select relevant lipid droplet-associated factors, bioinformatics analysis on the GEPIA platform (data are publicly available) was carried out for selected genes to identify differential expression in pancreatic cancer versus healthy pancreatic tissues. Differentially expressed genes were further analyzed regarding overall survival of pancreatic cancer patients. RESULTS: 65 factors were identified as lipid droplet-associated factors. Bioinformatics analysis of 179 pancreatic cancer samples and 171 normal pancreatic tissue samples on the GEPIA platform identified 39 deferentially expressed genes in pancreatic cancer with 36 up-regulated genes (ACSL3, ACSL4, AGPAT2, BSCL2, CAV1, CAV2, CAVIN1, CES1, CIDEC, DGAT1, DGAT2, FAF2, G0S2, HILPDA, HSD17B11, ICE2, LDAH, LIPE, LPCAT1, LPCAT2, LPIN1, MGLL, NAPA, NCEH1, PCYT1A, PLIN2, PLIN3, RAB5A, RAB7A, RAB8A, RAB18, SNAP23, SQLE, VAPA, VCP, VMP1) and 3 down-regulated genes (FITM1, PLIN4, PLIN5). Among 39 differentially expressed factors, seven up-regulated genes (CAV2, CIDEC, HILPDA, HSD17B11, NCEH1, RAB5A, and SQLE) and two down-regulation genes (BSCL2 and FITM1) were significantly associated with overall survival of pancreatic cancer patients. Multivariate Cox regression analysis identified CAV2 as the only independent prognostic factor. CONCLUSIONS: Through bioinformatics analysis, we identified nine prognostic relevant differentially expressed genes highlighting the role of lipid droplet-associated factors in pancreatic cancer.
Assuntos
Caveolina 2/genética , Regulação Neoplásica da Expressão Gênica , Gotículas Lipídicas/metabolismo , Proteínas de Neoplasias/genética , Neoplasias Pancreáticas/diagnóstico , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Caveolina 2/metabolismo , Biologia Computacional/métodos , Feminino , Perfilação da Expressão Gênica , Humanos , Gotículas Lipídicas/química , Metabolismo dos Lipídeos/genética , Masculino , Invasividade Neoplásica , Proteínas de Neoplasias/classificação , Proteínas de Neoplasias/metabolismo , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/mortalidade , Neoplasias Pancreáticas/patologia , Prognóstico , Transdução de Sinais , Análise de Sobrevida , Neoplasias PancreáticasRESUMO
AIM: This study aimed to identify the functional genes and genetic variants associated with the prognosis of pancreatic ductal adenocarcinoma (PDAC) and reveal the mechanism underlying their prognostic roles. METHODS: First, we implement a two-stage exome-wide association study in a total of 1070 patients to identify the genetic variant correlated with PDAC prognosis. Then we performed fine mapping through bioinformatics analysis and dual-luciferase reporter assays to reveal the causal functional variant and prognostic gene. Next, we established the gene knockdown, knockout, and overexpression cell lines with small interfering RNA, CRISPR/Cas9, and lentivirus, respectively, and investigated the gene function on cell proliferation and migration in vivo and in vitro. Finally, we performed the RNA-seq to elucidate downstream genes and mechanisms altering PDAC prognosis. RESULTS: We identified the CAV1-CAV2 locus tagged by rs8940 was significantly associated with PDAC prognosis, and rs10249656 in the 3'untranslated region of CAV2 was the real functional variant, which upregulated CAV2 expression through abolishing miR-548s binding. We observed upregulated CAV2 in PDAC and the higher expression correlated with worse prognosis. Transient knockdown of CAV2 inhibited PDAC migration without affecting proliferation rate. Knockout of CAV2 suppressed PDAC progression and metastasis, whereas stable overexpression of CAV2 promoted. Overexpressed CAV2 promoted the PDAC progression and metastasis via perturbing genes in the focal adhesion (CCND1, IGTA1, and ZYX) and extracellular matrix organisation (PLOD2, CAST, and ITGA1) pathways mechanically. CONCLUSION: These findings shed light on an important role of CAV2 on PDAC progression and the prognostic impact of its genetic variation.
Assuntos
Carcinoma Ductal Pancreático/metabolismo , Caveolina 2/metabolismo , Movimento Celular , Neoplasias Pulmonares/metabolismo , Neoplasias Pancreáticas/metabolismo , Polimorfismo de Nucleotídeo Único , Animais , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/secundário , Caveolina 2/genética , Linhagem Celular Tumoral , Progressão da Doença , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Matriz Extracelular/patologia , Adesões Focais/genética , Adesões Focais/metabolismo , Adesões Focais/patologia , Regulação Neoplásica da Expressão Gênica , Estudos de Associação Genética , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/secundário , Camundongos Endogâmicos BALB C , Camundongos Nus , MicroRNAs/genética , MicroRNAs/metabolismo , Invasividade Neoplásica , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , RNA-Seq , Transdução de Sinais , Sequenciamento do ExomaRESUMO
N-myristoylation is a ubiquitous protein lipidation in eukaryotes, but regulatory roles for myristoylation on proteins still remain to be explored. Here, we show that N-myristoylation of Caveolin-2 (Cav-2) controls insulin signaling. Alternative translation initiation (ATI)-yielded truncated form of non-N-myristoylable Cav-2ß and various conditional Cav-2 mutants were compared to full-length form of N-myristoylable Cav-2α. Insulin induced insulin receptor (IR) tyrosine kinase-catalyzed Tyr-19 phosphorylation of N-myristoylable M14A Cav-2 and triggered activation of IR signaling cascade. In contrast, insulin induced ubiquitination of non-N-myristoylable M1A and G2A Cav-2 to facilitate protein-tyrosine phosphatase 1B interaction with IR which desensitized IR signaling through internalization. Metabolic labeling and click chemistry showed palmitoylation of M14A but not M1A and G2A Cav-2. Insulin did not induce phosphorylation of M1A and G2A Cav-2 and Cav-2ß. Like Cav-2α, G2A Cav-2 and Cav-2ß formed large homo-oligomers localized in lipid rafts. These findings show Cav-2 N-myristoylation plays a crucial role to coordinate its phosphorylation, palmitoylation, and ubiquitination to control insulin signaling.
Assuntos
Caveolina 2/metabolismo , Insulina/fisiologia , Transdução de Sinais , Animais , Caveolina 2/química , Linhagem Celular , Humanos , Lipoilação , Microdomínios da Membrana/metabolismo , Ácido Mirístico/metabolismo , Fosforilação , Ratos , Receptor de Insulina/metabolismo , Tirosina/metabolismo , UbiquitinaçãoRESUMO
Voltage gated calcium channels (VGCCs) regulate neuronal excitability and translate activity into calcium dependent signaling. The α1 subunit of high voltage activated (HVA) VGCCs associates with α2δ accessory subunits, which may affect calcium channel biophysical properties, cell surface expression, localization and transport and are thus important players in calcium-dependent signaling. In vertebrates, the functions of the different combinations of the four α2δ and the seven HVA α1 subunits are incompletely understood, in particular with respect to partially redundant or separate functions in neurons. This study capitalizes on the relatively simpler situation in the Drosophila genetic model containing two neuronal putative α2δ subunits, straightjacket and CG4587, and one Cav1 and Cav2 homolog each, both with well-described functions in different compartments of identified motoneurons. Straightjacket is required for normal Cav1 and Cav2 current amplitudes and correct Cav2 channel function in all neuronal compartments. By contrast, CG4587 does not affect Cav1 or Cav2 current amplitudes or presynaptic function, but is required for correct Cav2 channel allocation to the axonal versus the dendritic domain. We suggest that the two different putative α2δ subunits are required in the same neurons to regulate different functions of VGCCs.
Assuntos
Canais de Cálcio/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila/metabolismo , Neurônios Motores/metabolismo , Animais , Axônios/metabolismo , Caveolina 1/metabolismo , Caveolina 2/metabolismo , Dendritos/metabolismo , Feminino , MasculinoRESUMO
The primary aim of this study was to determine the anti-neuropathic activity of (±)-18-methoxycoronaridine [(±)-18-MC] and (+)-catharanthine in mice by using the oxaliplatin-induced neuropathic pain paradigm and cold plate test. The results showed that both coronaridine congeners induce anti-neuropathic pain activity at a dose of 72 mg/kg (per os), whereas a lower dose (36 mg/kg) of (+)-catharanthine decreased the progress of oxaliplatin-induced neuropathic pain. To determine the underlying molecular mechanism, electrophysiological recordings were performed on α9α10, α3ß4, and α4ß2 nAChRs as well as voltage-gated calcium (CaV2.2) channels modulated by G protein-coupled γ-aminobutyric acid type B receptors (GABABRs). The results showed that (±)-18-MC and (+)-catharanthine competitively inhibit α9α10 nAChRs with potencies higher than that at α3ß4 and α4ß2 nAChRs and directly block CaV2.2 channels without activating GABABRs. Considering the potency of the coronaridine congeners at Cav2.2 channels and α9α10 nAChRs, and the calculated brain concentration of (+)-catharanthine, it is plausible that the observed anti-neuropathic pain effects are mediated by peripheral and central mechanisms involving the inhibition of α9α10 nAChRs and/or CaV2.2 channels.
Assuntos
Analgésicos/administração & dosagem , Caveolina 2/metabolismo , Ibogaína/análogos & derivados , Neuralgia/metabolismo , Receptores Nicotínicos/metabolismo , Alcaloides de Vinca/administração & dosagem , Animais , Células HEK293 , Humanos , Ibogaína/administração & dosagem , Masculino , Potenciais da Membrana/efeitos dos fármacos , Camundongos , Xenopus laevisRESUMO
Ad libitum-fed diets high in fat and carbohydrate (especially fructose) induce weight gain, obesity, and nonalcoholic fatty liver disease (NAFLD) in humans and animal models. However, interpretation is complicated since ad libitum feeding of such diets induces hyperphagia and upregulates expression of liver fatty acid binding protein (L-FABP)-a protein intimately involved in fatty acid and glucose regulation of lipid metabolism. Wild-type (WT) and L-fabp gene ablated (LKO) mice were pair-fed either high-fat diet (HFD) or high-fat/high-glucose diet (HFGD) wherein total carbohydrate was maintained constant but the proportion of glucose was increased at the expense of fructose. In LKO mice, the pair-fed HFD increased body weight and lean tissue mass (LTM) but had no effect on fat tissue mass (FTM) or hepatic fatty vacuolation as compared to pair-fed WT counterparts. These LKO mice exhibited upregulation of hepatic proteins in fatty acid uptake and cytosolic transport (caveolin and sterol carrier protein-2), but lower hepatic fatty acid oxidation (decreased serum ß-hydroxybutyrate). LKO mice pair-fed HFGD also exhibited increased body weight; however, these mice had increased FTM, not LTM, and increased hepatic fatty vacuolation as compared to pair-fed WT counterparts. These LKO mice also exhibited upregulation of hepatic proteins in fatty acid uptake and cytosolic transport (caveolin and acyl-CoA binding protein, but not sterol carrier protein-2), but there was no change in hepatic fatty acid oxidation (serum ß-hydroxybutyrate) as compared to pair-fed WT counterparts.
Assuntos
Dieta Hiperlipídica/efeitos adversos , Proteínas de Ligação a Ácido Graxo/genética , Glucose/administração & dosagem , Ácido 3-Hidroxibutírico/sangue , Animais , Peso Corporal/efeitos dos fármacos , Proteínas de Transporte/metabolismo , Caveolina 2/metabolismo , Ácidos Graxos/análise , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Técnicas de Inativação de Genes , Masculino , CamundongosRESUMO
Malignant gliomas are the most common tumor in central nervous system with poor prognosis. Due to the limitation of histological classification in earlier diagnosis and individualized medicine, it is necessary to combine the molecular signatures and the pathological characteristics of gliomas. Lots of microRNAs presented abnormal expression in gliomas and modulated gliomas development. Exploration the miRNAs profile is helpful for the diagnosis, therapy and prognosis of gliomas. It has been demonstrated that miR-144 plays important roles in solid tumors. However, the detail mechanisms remained unrevealed. In this study, we have demonstrated the level of miR-144 decreased in glioma tissues from patients, especially in gliomas with higher grades. MiR-144 was also validated have lower expression in glioma cell lines compared with cortical neuron cell by using qRT-PCR. The in vitro functional experiment indicated miR-144 improved gliomas progression through repressing proliferation, sensitizing to chemotherapeutics and inhibiting metastasis. We further identified fibroblast growth factor 7 (FGF7) and Caveolin 2 (CAV2) were target genes of miR-144 by luciferase reporter assay and western blotting. The mechanisms study suggested forced FGF7 expression elevated Akt activation and decreased reactive oxygen species (ROS) generation. The MTT and cell cycle assay indicated miR-144 suppressed glioma cells proliferation through modulating FGF mediated Akt signaling pathway. Meanwhile, miR-144 promoted Temozolomide (TMZ) induced apoptosis in glioma cells via increasing ROS production by using FACS. On the other hand, CAV2, as another target of miR-144, accelerated glioma cells migration and invasion via promoting glioma cells EMT progress. Retrieved expression of FGF7 or CAV2 rescued the proliferation and migration function mediated by miR-144. Furthermore, the in vivo experiments in PDX models displayed the anti-tumor function of miR-144, which could be retrieved by overexpression of FGF7 and CAV2. Taken together, these findings indicated miR-144 acted as a potential target against gliomas progression and uncovered a novel regulatory mechanism, which may provide a new therapeutic strategy and prognostic indicator for gliomas.
Assuntos
Caveolina 2/metabolismo , Fator 7 de Crescimento de Fibroblastos/metabolismo , Glioma/metabolismo , Glioma/patologia , MicroRNAs/metabolismo , Animais , Apoptose/genética , Apoptose/fisiologia , Western Blotting , Caveolina 2/genética , Ciclo Celular/genética , Ciclo Celular/fisiologia , Linhagem Celular , Linhagem Celular Tumoral , Movimento Celular/genética , Movimento Celular/fisiologia , Proliferação de Células/genética , Proliferação de Células/fisiologia , Fator 7 de Crescimento de Fibroblastos/genética , Humanos , Técnicas In Vitro , Masculino , Camundongos , Camundongos Endogâmicos BALB C , MicroRNAs/genética , Espécies Reativas de Oxigênio/metabolismoRESUMO
Caveolae consist in lipid raft domains composed of caveolin proteins, cholesterol, glycosphingolipids, and GPI-anchored proteins. Caveolin proteins present three different types, caveolin 1 (CAV-1), caveolin 2 (CAV-2) and caveolin 3 (CAV-3), with a very similar structure and amino acid composition. The native caveolin proteins oxidation mechanism was investigated for the first time, at a glassy carbon electrode, using cyclic, square wave and differential pulse voltammetry. The three native caveolin proteins oxidation mechanism presented only one tyrosine and tryptophan amino acid residues oxidation peak. Denatured caveolin proteins presented also the tyrosine, tryptophan and cysteine amino acid residues oxidation peaks. The reverse cholesterol transport is related to caveolae and caveolin proteins, and CAV-1 is directly connected to cholesterol transport. The influence of cholesterol on the three caveolin proteins electrochemical behaviour was evaluated. In the absence and in the presence of cholesterol, significant differences in the CAV-1 oxidation peak current were observed.
Assuntos
Caveolina 1/metabolismo , Caveolina 2/metabolismo , Caveolina 3/metabolismo , Colesterol/metabolismo , Cavéolas/metabolismo , Caveolina 1/química , Caveolina 2/química , Caveolina 3/química , Técnicas Eletroquímicas , Humanos , Oxirredução , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMO
Listeria monocytogenes is a human bacterial pathogen that disseminates through host tissues using a process called cell-to-cell spread. This critical yet understudied virulence strategy resembles a vesicular form of intercellular trafficking that allows L. monocytogenes to move between host cells without escaping the cell. Interestingly, eukaryotic cells can also directly exchange cellular components via intercellular communication pathways (e.g., trans-endocytosis) using cell-cell adhesion, membrane trafficking, and membrane remodeling proteins. Therefore, we hypothesized that L. monocytogenes would hijack these types of host proteins during spread. Using a focused RNA interference screen, we identified 22 host genes that are important for L. monocytogenes spread. We then found that caveolins (CAV1 and CAV2) and the membrane sculpting F-BAR protein PACSIN2 promote L. monocytogenes protrusion engulfment during spread, and that PACSIN2 specifically localizes to protrusions. Overall, our study demonstrates that host intercellular communication pathways may be coopted during bacterial spread and that specific trafficking and membrane remodeling proteins promote bacterial protrusion resolution.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Caveolinas/metabolismo , Listeria monocytogenes/patogenicidade , Listeriose/genética , Listeriose/microbiologia , Células A549 , Actinas/metabolismo , Proteínas de Bactérias/metabolismo , Caveolina 1/metabolismo , Caveolina 2/metabolismo , Endocitose/fisiologia , Células Eucarióticas/metabolismo , Interações Hospedeiro-Patógeno/fisiologia , Humanos , Listeria monocytogenes/metabolismo , Listeriose/metabolismo , Proteínas de Membrana/metabolismo , Transporte Proteico , Interferência de RNA , VirulênciaRESUMO
This study describes the interaction between Cav2 calcium channels and Rabconnectin-3, a di-subunit protein that is associated with synaptic vesicles. Immunostaining reveals that both Rabconnectin-3α (RB-3α) and Rabconnectin-3ß (RB-3ß) are colocalized in mouse hippocampal neurons. Co-immunoprecipitations from brain tissue is consistent with the formation of a protein complex between RB-3α and RB-3ß and both Cav2.2 and the related Cav2.1 calcium channel. The coexpression of either RB-3α or RB-3ß with Cav2.2 calcium channels in tsA-201 cells led to a reduction in Cav2.2 current density without any effects on the voltage-dependence of activation or inactivation. Coexpression of both Rabconnectin-3 subunits did not cause an additive effect on current densities. Finally, the presence of Rabconnectin-3 did not interfere with µ-opioid receptor mediated Gßγ modulation of Cav2.2 channels. Altogether, our findings show that Rabconnectin-3 has the propensity to regulate calcium entry mediated by Cav2.2 channels.
Assuntos
Caveolina 2/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Animais , Proteínas de Ligação ao GTP/metabolismo , Hipocampo/citologia , Humanos , Camundongos , Neurônios/metabolismo , Ligação Proteica , RatosRESUMO
The accumulation of intracellular ß-amyloid peptide (Aß) is important pathological characteristic of Alzheimer's disease (AD). However, the exact underlying molecular mechanism remains to be elucidated. Here, we reported that Nuclear Paraspeckle Assembly Transcript 1 (NEAT1), a long n on-coding RNA, exhibits repressed expression in the early stage of AD and its down-regulation declines neuroglial cell mediating Aß clearance via inhibiting expression of endocytosis-related genes. We find that NEAT1 is associated with P300/CBP complex and its inhibition affects H3K27 acetylation (H3K27Ac) and H3K27 crotonylation (H3K27Cro) located nearby to the transcription start site of many genes, including endocytosis-related genes. Interestingly, NEAT1 inhibition down-regulates H3K27Ac but up-regulates H3K27Cro through repression of acetyl-CoA generation. NEAT1 also mediates the binding between STAT3 and H3K27Ac but not H3K27Cro. Therefore, the decrease of H3K27Ac and/or the increase of H3K27Cro declines expression of multiple related genes. Collectively, this study first reveals the different roles of H3K27Ac and H3K27Cro in regulation of gene expression and provides the insight of the epigenetic regulatory mechanism of NEAT1 in gene expression and AD pathology.