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1.
Am J Pathol ; 191(3): 475-486, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33345999

RESUMO

Niemann-Pick type C (NPC) disease is a lysosomal storage disorder characterized by cholesterol accumulation caused by loss-of-function mutations in the Npc1 gene. NPC disease primarily affects the brain, causing neuronal damage and affecting motor coordination. In addition, considerable liver malfunction in NPC disease is common. Recently, we found that the depletion of annexin A6 (ANXA6), which is most abundant in the liver and involved in cholesterol transport, ameliorated cholesterol accumulation in Npc1 mutant cells. To evaluate the potential contribution of ANXA6 in the progression of NPC disease, double-knockout mice (Npc1-/-/Anxa6-/-) were generated and examined for lifespan, neurologic and hepatic functions, as well as liver histology and ultrastructure. Interestingly, lack of ANXA6 in NPC1-deficient animals did not prevent the cerebellar degeneration phenotype, but further deteriorated their compromised hepatic functions and reduced their lifespan. Moreover, livers of Npc1-/-/Anxa6-/- mice contained a significantly elevated number of foam cells congesting the sinusoidal space, a feature commonly associated with inflammation. We hypothesize that ANXA6 deficiency in Npc1-/- mice not only does not reverse neurologic and motor dysfunction, but further worsens overall liver function, exacerbating hepatic failure in NPC disease.


Assuntos
Anexina A6/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Hepatopatias/patologia , Longevidade , Animais , Comportamento Animal , Hepatopatias/etiologia , Hepatopatias/metabolismo , Camundongos , Camundongos Knockout
2.
PLoS One ; 15(4): e0231711, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32298357

RESUMO

Actively growing tumors are often histologically associated with Ki67 positivity, while the detection of invasiveness relies on non-quantitative pathologic evaluation of mostly advanced tumors. We recently reported that reduced expression of the Ca2+-dependent membrane-binding annexin A6 (AnxA6) is associated with increased expression of the Ca2+ activated RasGRF2 (GRF2), and that the expression status of these proteins inversely influence the growth and motility of triple negative breast cancer (TNBC) cells. Here, we establish that the reciprocal expression of AnxA6 and GRF2 is at least in part, dependent on inhibition of non-selective Ca2+ channels in AnxA6-low but not AnxA6-high TNBC cells. Immunohistochemical staining of breast cancer tissues revealed that compared to non-TNBC tumors, TNBC tumors express lower levels of AnxA6 and higher Ki67 expression. GRF2 expression levels strongly correlated with high Ki67 in pretreatment biopsies from patients with residual disease and with residual tumor size following chemotherapy. Elevated AnxA6 expression more reliably identified patients who responded to chemotherapy, while low AnxA6 levels were significantly associated with shorter distant relapse-free survival. Finally, the reciprocal expression of AnxA6 and GRF2 can delineate GRF2-low/AnxA6-high invasive from GRF2-high/AnxA6-low rapidly growing TNBCs. These data suggest that AnxA6 may be a reliable biomarker for distant relapse-free survival and response of TNBC patients to chemotherapy, and that the reciprocal expression of AnxA6 and GRF2 can reliably delineate TNBCs into rapidly growing and invasive subsets which may be more relevant for subset-specific therapeutic interventions.


Assuntos
Anexina A6/metabolismo , Canais de Cálcio/metabolismo , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/patologia , Fatores ras de Troca de Nucleotídeo Guanina/metabolismo , Animais , Anexina A6/genética , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Bloqueadores dos Canais de Cálcio/farmacologia , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Feminino , Humanos , Antígeno Ki-67/metabolismo , Camundongos , Metástase Neoplásica/genética , Prognóstico , Transplante Heterólogo , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/mortalidade , Fatores ras de Troca de Nucleotídeo Guanina/genética
3.
Int J Mol Sci ; 21(4)2020 Feb 18.
Artigo em Inglês | MEDLINE | ID: mdl-32085611

RESUMO

Annexin A6 (AnxA6) is the largest member of the annexin family of proteins present in matrix vesicles (MVs). MVs are a special class of extracellular vesicles that serve as a nucleation site during cartilage, bone, and mantle dentin mineralization. In this study, we assessed the localization of AnxA6 in the MV membrane bilayer using native MVs and MV biomimetics. Biochemical analyses revealed that AnxA6 in MVs can be divided into three distinct groups. The first group corresponds to Ca2+-bound AnxA6 interacting with the inner leaflet of the MV membrane. The second group corresponds to AnxA6 localized on the surface of the outer leaflet. The third group corresponds to AnxA6 inserted in the membrane's hydrophobic bilayer and co-localized with cholesterol (Chol). Using monolayers and proteoliposomes composed of either dipalmitoylphosphatidylcholine (DPPC) to mimic the outer leaflet of the MV membrane bilayer or a 9:1 DPPC:dipalmitoylphosphatidylserine (DPPS) mixture to mimic the inner leaflet, with and without Ca2+, we confirmed that, in agreement with the biochemical data, AnxA6 interacted differently with the MV membrane. Thermodynamic analyses based on the measurement of surface pressure exclusion (πexc), enthalpy (ΔH), and phase transition cooperativity (Δt1/2) showed that AnxA6 interacted with DPPC and 9:1 DPPC:DPPS systems and that this interaction increased in the presence of Chol. The selective recruitment of AnxA6 by Chol was observed in MVs as probed by the addition of methyl-ß-cyclodextrin (MßCD). AnxA6-lipid interaction was also Ca2+-dependent, as evidenced by the increase in πexc in negatively charged 9:1 DPPC:DPPS monolayers and the decrease in ΔH in 9:1 DPPC:DPPS proteoliposomes caused by the addition of AnxA6 in the presence of Ca2+ compared to DPPC zwitterionic bilayers. The interaction of AnxA6 with DPPC and 9:1 DPPC:DPPS systems was distinct even in the absence of Ca2+ as observed by the larger change in Δt1/2 in 9:1 DPPC:DPPS vesicles as compared to DPPC vesicles. Protrusions on the surface of DPPC proteoliposomes observed by atomic force microscopy suggested that oligomeric AnxA6 interacted with the vesicle membrane. Further work is needed to delineate possible functions of AnxA6 at its different localizations and ways of interaction with lipids.


Assuntos
Anexina A6/metabolismo , Calcificação Fisiológica , Matriz Extracelular/metabolismo , Vesículas Extracelulares/metabolismo , 1,2-Dipalmitoilfosfatidilcolina/química , Varredura Diferencial de Calorimetria , Colesterol/metabolismo , Humanos , Bicamadas Lipídicas/metabolismo , Microdomínios da Membrana/metabolismo , Microscopia de Força Atômica , Proteolipídeos/metabolismo
4.
Cell Mol Life Sci ; 77(14): 2839-2857, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31664461

RESUMO

Cholesterol accumulation in late endosomes is a prevailing phenotype of Niemann-Pick type C1 (NPC1) mutant cells. Likewise, annexin A6 (AnxA6) overexpression induces a phenotype reminiscent of NPC1 mutant cells. Here, we demonstrate that this cellular cholesterol imbalance is due to AnxA6 promoting Rab7 inactivation via TBC1D15, a Rab7-GAP. In NPC1 mutant cells, AnxA6 depletion and eventual Rab7 activation was associated with peripheral distribution and increased mobility of late endosomes. This was accompanied by an enhanced lipid accumulation in lipid droplets in an acyl-CoA:cholesterol acyltransferase (ACAT)-dependent manner. Moreover, in AnxA6-deficient NPC1 mutant cells, Rab7-mediated rescue of late endosome-cholesterol export required the StAR-related lipid transfer domain-3 (StARD3) protein. Electron microscopy revealed a significant increase of membrane contact sites (MCS) between late endosomes and ER in NPC1 mutant cells lacking AnxA6, suggesting late endosome-cholesterol transfer to the ER via Rab7 and StARD3-dependent MCS formation. This study identifies AnxA6 as a novel gatekeeper that controls cellular distribution of late endosome-cholesterol via regulation of a Rab7-GAP and MCS formation.


Assuntos
Anexina A6/genética , Colesterol/genética , Proteínas Ativadoras de GTPase/genética , Doença de Niemann-Pick Tipo C/genética , Proteínas rab de Ligação ao GTP/genética , Animais , Células CHO , Proteínas de Transporte/genética , Membrana Celular/genética , Membrana Celular/metabolismo , Colesterol/metabolismo , Cricetulus , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Endossomos/genética , Endossomos/metabolismo , Humanos , Proteínas de Membrana/genética , Doença de Niemann-Pick Tipo C/metabolismo , Doença de Niemann-Pick Tipo C/patologia , Domínios Proteicos/genética , Transporte Proteico/genética , RNA Interferente Pequeno/genética
5.
J Clin Invest ; 129(11): 4657-4670, 2019 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-31545299

RESUMO

Membrane repair is essential to cell survival. In skeletal muscle, injury often associates with plasma membrane disruption. Additionally, muscular dystrophy is linked to mutations in genes that produce fragile membranes or reduce membrane repair. Methods to enhance repair and reduce susceptibility to injury could benefit muscle in both acute and chronic injury settings. Annexins are a family of membrane-associated Ca2+-binding proteins implicated in repair, and annexin A6 was previously identified as a genetic modifier of muscle injury and disease. Annexin A6 forms the repair cap over the site of membrane disruption. To elucidate how annexins facilitate repair, we visualized annexin cap formation during injury. We found that annexin cap size positively correlated with increasing Ca2+ concentrations. We also found that annexin overexpression promoted external blebs enriched in Ca2+ and correlated with a reduction of intracellular Ca2+ at the injury site. Annexin A6 overexpression reduced membrane injury, consistent with enhanced repair. Treatment with recombinant annexin A6 protected against acute muscle injury in vitro and in vivo. Moreover, administration of recombinant annexin A6 in a model of muscular dystrophy reduced serum creatinine kinase, a biomarker of disease. These data identify annexins as mediators of membrane-associated Ca2+ release during membrane repair and annexin A6 as a therapeutic target to enhance membrane repair capacity.


Assuntos
Anexina A6/farmacologia , Cálcio/metabolismo , Membrana Celular/metabolismo , Músculo Esquelético/lesões , Distrofia Muscular Animal/prevenção & controle , Animais , Anexina A6/genética , Membrana Celular/patologia , Feminino , Masculino , Camundongos , Camundongos Knockout , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Distrofia Muscular Animal/genética , Distrofia Muscular Animal/metabolismo , Distrofia Muscular Animal/patologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologia
6.
J Cell Biochem ; 120(12): 19310-19317, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31502705

RESUMO

PURPOSE: The aim of this study was to determine the biological function of pyridine nucleotide-disulfide oxidoreductase domain 1 (PYROXD1), a recently discovered protein, in colon cancer cell line HCT116. METHODS: The small interfering RNA (siRNA) was designed rationally on the basis of the target sequence against PYROXD1. Relative PYROXD1 mRNA levels were measured by a quantitative real-time polymerase chain reaction. Flow cytometry was performed to monitor tumor cells proliferation and apoptosis after siRNA transfection. RESULTS: Knockdown of PYROXD1 arrested the cell cycle, and induced late apoptosis in colon cancer cell line HCT116 DISCUSSION: Taken together, these results revealed the critical roles of PYROXD1 in regulating cell cycle and apoptosis and possibly will signify its therapeutic potential for targeting colorectal cancer models.


Assuntos
Neoplasias do Colo/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/metabolismo , RNA Interferente Pequeno/metabolismo , Anexina A6/genética , Anexina A6/metabolismo , Apoptose/genética , Apoptose/fisiologia , Ciclo Celular/genética , Ciclo Celular/fisiologia , Pontos de Checagem do Ciclo Celular/genética , Pontos de Checagem do Ciclo Celular/fisiologia , Neoplasias do Colo/genética , Citometria de Fluxo , Células HCT116 , Humanos , Oxirredutases atuantes sobre Doadores de Grupo Enxofre/genética , RNA Interferente Pequeno/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa
7.
Med Sci Sports Exerc ; 51(11): 2201-2209, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31107349

RESUMO

INTRODUCTION: The purpose of this study was to determine the effect of growth restriction on the biological regulation of physical activity. METHODS: Using a cross-fostering, protein-restricted nutritive model, mice were growth-restricted during either gestation (GUN; N = 3 litters) or postnatal life (PUN; N = 3 litters). At 21 d of age, all mice pups were weaned and fed a nonrestrictive healthy diet for the remainder of the study. At 45 d of age, mice were individually housed in cages with free moving running wheels to assess physical activity engagement. At day 70, mice were euthanized, and the nucleus accumbens was analyzed for dopamine receptor 1 expression. Skeletal muscle fiber type and cross-sectional area of the soleus, extensor digitorom longus, and diaphragm were analyzed by immunohistochemistry. The soleus from the other hindleg was evaluated for calsequestrin 1 and annexin A6 expression. RESULTS: The PUN female mice (15,365 ± 8844 revolutions per day) had a reduction (P = 0.0221) in wheel revolutions per day as compared with the GUN (38,667 ± 8648 revolutions per day) and CON females (36,421.0 ± 6700 revolutions per day). The PUN female mice also expressed significantly higher dopamine receptor 1 compared (P = 0.0247) to the other groups. The PUN female soleus had a higher expression of calsequestrin 1, along with more type IIb fibers (P = 0.0398). CONCLUSIONS: Growth restriction during lactation reduced physical activity in female mice by reducing the central drive to be active and displayed a more fatigable skeletal muscle phenotype.


Assuntos
Transtornos do Crescimento/fisiopatologia , Condicionamento Físico Animal/fisiologia , Animais , Anexina A6/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Modelos Animais de Doenças , Feminino , Transtornos do Crescimento/etiologia , Transtornos do Crescimento/metabolismo , Masculino , Desnutrição/complicações , Camundongos , Fibras Musculares Esqueléticas/citologia , Fibras Musculares Esqueléticas/metabolismo , Receptores Dopaminérgicos/metabolismo , Corrida/fisiologia
8.
Nat Cell Biol ; 21(2): 190-202, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30598531

RESUMO

Cytotoxic chemotherapy is an effective treatment for invasive breast cancer. However, experimental studies in mice also suggest that chemotherapy has pro-metastatic effects. Primary tumours release extracellular vesicles (EVs), including exosomes, that can facilitate the seeding and growth of metastatic cancer cells in distant organs, but the effects of chemotherapy on tumour-derived EVs remain unclear. Here we show that two classes of cytotoxic drugs broadly employed in pre-operative (neoadjuvant) breast cancer therapy, taxanes and anthracyclines, elicit tumour-derived EVs with enhanced pro-metastatic capacity. Chemotherapy-elicited EVs are enriched in annexin A6 (ANXA6), a Ca2+-dependent protein that promotes NF-κB-dependent endothelial cell activation, Ccl2 induction and Ly6C+CCR2+ monocyte expansion in the pulmonary pre-metastatic niche to facilitate the establishment of lung metastasis. Genetic inactivation of Anxa6 in cancer cells or Ccr2 in host cells blunts the pro-metastatic effects of chemotherapy-elicited EVs. ANXA6 is detected, and potentially enriched, in the circulating EVs of breast cancer patients undergoing neoadjuvant chemotherapy.


Assuntos
Doxorrubicina/uso terapêutico , Vesículas Extracelulares/efeitos dos fármacos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Mamárias Experimentais/tratamento farmacológico , Paclitaxel/uso terapêutico , Animais , Anexina A6/metabolismo , Linhagem Celular Tumoral , Quimiocina CCL2/metabolismo , Vesículas Extracelulares/metabolismo , Feminino , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/secundário , Neoplasias Mamárias Experimentais/metabolismo , Neoplasias Mamárias Experimentais/patologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Nus , Camundongos Transgênicos
9.
Reprod Biomed Online ; 38(3): 442-454, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30612956

RESUMO

RESEARCH QUESTION: Are there proteomic differences between endometrial stromal cells of repeated implantation failure (RIF), recurrent pregnancy loss (RPL) and normal fertile women, and is there differential protein expression upon decidualization? DESIGN: This exploratory study investigated the proteome of in-vitro cultured endometrial stromal cells of women with RIF (n = 4), women with RPL (n = 3) and normal fertile women (n = 4), comparing day 0 with 5 days of decidualization. Total proteins extracted from cell lysates were analysed by high-definition mass spectrometry. Data analysis was performed using significance analysis of microarray in R (P < 0.05; false discovery rate [FDR] 10%). RESULTS: In the RIF group, ANXA6, PSMC5 and FSCN1 were up-regulated (1.9-fold, 2.5-fold and 1.9-fold, respectively), whereas PBXIP1 was down-regulated (7.7-fold) upon decidualization. In the RPL group, RPS25 and ACADVL were down-regulated (1.9-fold and 2.4-fold, respectively; FDR 10%) between the non-decidualized and the decidualized samples. In the normal fertile group VIM and RPL23A were down-regulated (1.9-fold and 2.4-fold, respectively). Comparing ratios of expression of decidualized over non-decidualized samples in the different groups revealed six differentially expressed proteins: DUX4L2, CNPY4, PDE7A, CTSK, PCBP2 and PSMD4. Comparison of RPL versus normal fertile in the decidualized condition revealed serotransferrin to be differentially expressed. The changes in expression levels for serotransferrin, ANX6, ACDVL and VIM were confirmed by western blot. CONCLUSIONS: Results show a varying response of endometrial stromal cells in distinct clinical groups (RIF, RPL and normal fertile) upon in-vitro decidualization. Serotransferrin could serve as a marker for the aberrant decidualization process in RPL.


Assuntos
Aborto Habitual/metabolismo , Implantação do Embrião/fisiologia , Endométrio/metabolismo , Infertilidade Feminina/metabolismo , Células Estromais/metabolismo , ATPases Associadas a Diversas Atividades Celulares/metabolismo , Acil-CoA Desidrogenase de Cadeia Longa/metabolismo , Adulto , Anexina A6/metabolismo , Proteínas de Transporte/metabolismo , Feminino , Fertilidade/fisiologia , Humanos , Proteínas dos Microfilamentos/metabolismo , Gravidez , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteoma , Proteômica , Proteínas Ribossômicas/metabolismo , Transferrina/metabolismo , Vimentina/metabolismo
10.
Carcinogenesis ; 40(8): 998-1009, 2019 08 22.
Artigo em Inglês | MEDLINE | ID: mdl-30590459

RESUMO

The epidermal growth factor receptor (EGFR) is a major oncogene in triple-negative breast cancer (TNBC), but the use of EGFR-targeted tyrosine kinase inhibitors (TKI) and therapeutic monoclonal antibodies is associated with poor response and acquired resistance. Understanding the basis for the acquired resistance to these drugs and identifying biomarkers to monitor the ensuing resistance remain a major challenge. We previously showed that reduced expression of annexin A6 (AnxA6), a calcium-dependent membrane-binding tumor suppressor, not only promoted the internalization and degradation of activated EGFR but also sensitized TNBC cells to EGFR-TKIs. Here, we demonstrate that prolong (>3 days) treatment of AnxA6-low TNBC cells with lapatinib led to AnxA6 upregulation and accumulation of cholesterol in late endosomes. Basal extracellular signal-regulated kinase 1 and 2 (ERK1/2) activation was EGFR independent and significantly higher in lapatinib-resistant MDA-MB-468 (LAP-R) cells. These cells were more sensitive to cholesterol depletion than untreated control cells. Inhibition of lapatinib-induced upregulation of AnxA6 by RNA interference (A6sh) or withdrawal lapatinib from LAP-R cells not only reversed the accumulation of cholesterol in late endosomes but also led to enrichment of plasma membranes with cholesterol, restored EGFR-dependent activation of ERK1/2 and sensitized the cells to lapatinib. These data suggest that lapatinib-induced AnxA6 expression and accumulation of cholesterol in late endosomes constitute an adaptive mechanism for EGFR-expressing TNBC cells to overcome prolong treatment with EGFR-targeted TKIs and can be exploited as an option to inhibit and/or monitor the frequently observed acquired resistance to these drugs.


Assuntos
Anexina A6/genética , Lapatinib/farmacologia , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/genética , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Lapatinib/efeitos adversos , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Transdução de Sinais/efeitos dos fármacos , Ativação Transcricional/efeitos dos fármacos , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/patologia
11.
PLoS One ; 13(8): e0201310, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30110341

RESUMO

Annexin A6 (AnxA6) controls cholesterol and membrane transport in endo- and exocytosis, and modulates triglyceride accumulation and storage. In addition, AnxA6 acts as a scaffolding protein for negative regulators of growth factor receptors and their effector pathways in many different cell types. Here we investigated the role of AnxA6 in the regulation of whole body lipid metabolism and insulin-regulated glucose homeostasis. Therefore, wildtype (WT) and AnxA6-knockout (KO) mice were fed a high-fat diet (HFD) for 17 weeks. During the course of HFD feeding, AnxA6-KO mice gained less weight compared to controls, which correlated with reduced adiposity. Systemic triglyceride and cholesterol levels of HFD-fed control and AnxA6-KO mice were comparable, with slightly elevated high density lipoprotein (HDL) and reduced triglyceride-rich lipoprotein (TRL) levels in AnxA6-KO mice. AnxA6-KO mice displayed a trend towards improved insulin sensitivity in oral glucose and insulin tolerance tests (OGTT, ITT), which correlated with increased insulin-inducible phosphorylation of protein kinase B (Akt) and ribosomal protein S6 kinase (S6) in liver extracts. However, HFD-fed AnxA6-KO mice failed to downregulate hepatic gluconeogenesis, despite similar insulin levels and insulin signaling activity, as well as expression profiles of insulin-sensitive transcription factors to controls. In addition, increased glycogen storage in livers of HFD- and chow-fed AnxA6-KO animals was observed. Together with an inability to reduce glucose production upon insulin exposure in AnxA6-depleted HuH7 hepatocytes, this implicates AnxA6 contributing to the fine-tuning of hepatic glucose metabolism with potential consequences for the systemic control of glucose in health and disease.


Assuntos
Anexina A6/deficiência , Gorduras na Dieta/farmacologia , Gluconeogênese/efeitos dos fármacos , Glucose/metabolismo , Glicogênio/metabolismo , Fígado/metabolismo , Adiposidade/efeitos dos fármacos , Animais , Anexina A6/metabolismo , Gorduras na Dieta/efeitos adversos , Gluconeogênese/genética , Glucose/genética , Glicogênio/genética , Resistência à Insulina , Lipídeos/sangue , Fígado/patologia , Masculino , Camundongos , Camundongos Knockout , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Quinases S6 Ribossômicas/genética , Proteínas Quinases S6 Ribossômicas/metabolismo
12.
mBio ; 9(4)2018 07 24.
Artigo em Inglês | MEDLINE | ID: mdl-30042202

RESUMO

To transfer the viral genome into the host cell cytoplasm, internalized influenza A virus (IAV) particles depend on the fusion of the IAV envelope with host endosomal membranes. The antiviral host interferon (IFN) response includes the upregulation of interferon-induced transmembrane protein 3 (IFITM3), which inhibits the release of the viral content into the cytosol. Although IFITM3 induction occurs concomitantly with late endosomal/lysosomal (LE/L) cholesterol accumulation, the functional significance of this process is not well understood. Here we report that LE/L cholesterol accumulation itself plays a pivotal role in the early antiviral defense. We demonstrate that inducing LE/L cholesterol accumulation is antiviral in non-IFN-primed cells, restricting incoming IAV particles and impairing mixing of IAV/endosomal membrane lipids. Our results establish a protective function of LE/L cholesterol accumulation and suggest endosomal cholesterol balance as a possible antiviral target.IMPORTANCE With annual epidemics occurring in all parts of the world and the risk of global outbreaks, influenza A virus (IAV) infections remain a major threat to public health. Infected host cells detect viral components and mount an interferon (IFN)-mediated response to restrict virus propagation and spread of infection. Identification of cellular factors and underlying mechanisms that establish such an antiviral state can provide novel strategies for the development of antiviral drugs. The contribution of LE/L cholesterol levels, especially in the context of the IFN-induced antiviral response, has remained controversial so far. Here, we report that accumulation of cholesterol in the LE/L compartment contributes to the IFN-induced host cell defense against incoming IAV. Our results establish cholesterol accumulation in LE/L per se as a novel antiviral barrier and suggest the endosomal cholesterol balance as a putative druggable host cell factor in IAV infection.


Assuntos
Colesterol/metabolismo , Endossomos/metabolismo , Interações Hospedeiro-Patógeno , Evasão da Resposta Imune , Vírus da Influenza A/fisiologia , Células A549 , Anexina A6/genética , Linhagem Celular , Endossomos/virologia , Humanos , Interferons/imunologia , Lisossomos/metabolismo , Lisossomos/virologia , Proteínas de Membrana/genética , Proteínas de Ligação a RNA/genética , Regulação para Cima , Replicação Viral
13.
Sci Signal ; 11(535)2018 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-29921656

RESUMO

Mechanically activated, slowly adapting currents in sensory neurons have been linked to noxious mechanosensation. The conotoxin NMB-1 (noxious mechanosensation blocker-1) blocks such currents and inhibits mechanical pain. Using a biotinylated form of NMB-1 in mass spectrometry analysis, we identified 67 binding proteins in sensory neurons and a sensory neuron-derived cell line, of which the top candidate was annexin A6, a membrane-associated calcium-binding protein. Annexin A6-deficient mice showed increased sensitivity to mechanical stimuli. Sensory neurons from these mice showed increased activity of the cation channel Piezo2, which mediates a rapidly adapting mechano-gated current linked to proprioception and touch, and a decrease in mechanically activated, slowly adapting currents. Conversely, overexpression of annexin A6 in sensory neurons inhibited rapidly adapting currents that were partially mediated by Piezo2. Furthermore, overexpression of annexin A6 in sensory neurons attenuated mechanical pain in a mouse model of osteoarthritis, a disease in which mechanically evoked pain is particularly problematic. These data suggest that annexin A6 can be exploited to inhibit chronic mechanical pain.


Assuntos
Anexina A6/fisiologia , Conotoxinas/metabolismo , Mecanotransdução Celular , Dor/prevenção & controle , Fragmentos de Peptídeos/metabolismo , Células Receptoras Sensoriais/fisiologia , Animais , Artrite Experimental/etiologia , Artrite Experimental/fisiopatologia , Biotinilação , Células Cultivadas , Canais Iônicos/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Osteoartrite/etiologia , Osteoartrite/fisiopatologia , Dor/metabolismo , Dor/patologia
14.
PLoS One ; 13(5): e0197690, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29771996

RESUMO

Annexin A6 (AnxA6) is expressed in articular chondrocytes at levels higher than in other mesenchymal cell types. However, the role of AnxA6 in articular chondrocytes is not known. Here we show that complete lack of AnxA6 functions resulted in increased ß-catenin activation in Wnt3a-treated murine articular chondrocytes, whereas AnxA6 expressing articular chondrocytes showed decreased ß-catenin activation. High expression of AnxA6 in human articular chondrocytes showed the highest inhibition of Wnt/ß-catenin signaling. Inhibition of Wnt/ß-catenin signaling activity by AnxA6 together with cytosolic Ca2+ was achieved by interfering with the plasma membrane association of the Wnt signaling complex. AnxA6 also affected the cross-talk between Wnt/ß-catenin signaling and NF-κB signaling by decreasing ß-catenin activity and increasing NF-κB activity in Wnt3a-, interleukin-1beta (IL-1ß)-, and combined Wnt3a/IL-1ß-treated cells. Wnt3a treatment increased the mRNA levels of catabolic markers (cyclooxygenase-2, interleukin-6, inducible nitric oxide synthase) to a much lesser degree than IL-1ß treatment in human articular chondrocytes, and decreased the mRNA levels of matrix metalloproteinase-13 (MMP-13) and articular cartilage markers (aggrecan, type II collagen). Furthermore, Wnt3a decreased the mRNA levels of catabolic markers and MMP-13 in IL-1ß-treated human articular chondrocytes. High expression of AnxA6 resulted in decreased mRNA levels of catabolic markers, and increased MMP-13 and articular cartilage marker mRNA levels in Wnt3a-treated human articular chondrocytes, whereas leading to increased mRNA levels of catabolic markers and MMP-13 in human articular chondrocytes treated with IL-1ß, or combined Wnt3a and IL-1ß. Our findings define a novel role for AnxA6 in articular chondrocytes via its modulation of Wnt/ß-catenin and NF-κB signaling activities and the cross-talk between these two signaling pathways.


Assuntos
Anexina A6/genética , Anexina A6/metabolismo , Cartilagem Articular/metabolismo , NF-kappa B/metabolismo , Via de Sinalização Wnt , Animais , Biomarcadores/metabolismo , Cálcio/metabolismo , Cartilagem Articular/citologia , Membrana Celular/metabolismo , Células Cultivadas , Citosol/metabolismo , Regulação da Expressão Gênica , Camundongos
15.
J Biol Chem ; 293(21): 8065-8076, 2018 05 25.
Artigo em Inglês | MEDLINE | ID: mdl-29636414

RESUMO

During neuronal development, the microtubule-associated protein tau becomes enriched in the axon, where it remains concentrated in the healthy brain. In tauopathies such as Alzheimer's disease, tau redistributes from the axon to the somatodendritic compartment. However, the cellular mechanism that regulates tau's localization remains unclear. We report here that tau interacts with the Ca2+-regulated plasma membrane-binding protein annexin A2 (AnxA2) via tau's extreme N terminus encoded by the first exon (E1). Bioinformatics analysis identified two conserved eight-amino-acids-long motifs within E1 in mammals. Using a heterologous yeast system, we found that disease-related mutations and pseudophosphorylation of Tyr-18, located within E1 but outside of the two conserved regions, do not influence tau's interaction with AnxA2. We further observed that tau interacts with the core domain of AnxA2 in a Ca2+-induced open conformation and interacts also with AnxA6. Moreover, lack of E1 moderately increased tau's association rate to microtubules, consistent with the supposition that the presence of the tau-annexin interaction reduces the availability of tau to interact with microtubules. Of note, intracellular competition through overexpression of E1-containing constructs reduced tau's axonal enrichment in primary neurons. Our results suggest that the E1-mediated tau-annexin interaction contributes to the enrichment of tau in the axon and is involved in its redistribution in pathological conditions.


Assuntos
Anexina A2/metabolismo , Anexina A6/metabolismo , Axônios/metabolismo , Microtúbulos/metabolismo , Proteínas tau/metabolismo , Animais , Anexina A2/genética , Anexina A6/genética , Membrana Celular/metabolismo , Células Cultivadas , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Células PC12 , Fosforilação , Ligação Proteica , Ratos , Proteínas tau/genética
16.
Postepy Biochem ; 64(3): 190-195, 2018 Oct 25.
Artigo em Inglês | MEDLINE | ID: mdl-30656904

RESUMO

Annexins, calcium- and membrane-binding proteins, have been extensively studied at the Nencki Institute since early 1990s, in terms of their structure, potential ligands and functions in the organism, with emphasis on mineralization processes in norm and pathology. The results of recently performed studies have revealed that annexins are playing essential roles in membrane organization. In this review we characterize the largest member of the annexin family of proteins, annexin A6 (AnxA6), in respect to its cholesterol and nucleotide binding properties, as well as intracellular pH sensing and ability to change membrane permeability to ions. Furthermore, we discuss biological functions of AnxA6 such as participation in membrane lateral organization, cell membrane repair and regulation of vesicular transport.


Assuntos
Anexina A6/metabolismo , Membrana Celular/metabolismo , Colesterol/metabolismo , Endocitose , Exocitose , Nucleotídeos/metabolismo , Animais , Transporte Biológico , Humanos
17.
Sci Rep ; 7(1): 15129, 2017 11 09.
Artigo em Inglês | MEDLINE | ID: mdl-29123177

RESUMO

HAMLET (human α-lactalbumin made lethal to tumour cells) is a complex of α-lactalbumin (aLA) and oleic acid (OA) which kills transformed cells, while leaving fully differentiated cells largely unaffected. Other protein-lipid complexes show similar anti-cancer potential. We call such complexes liprotides. The cellular impact of liprotides, while intensely investigated, remains unresolved. To address this, we report on the cell-killing mechanisms of liprotides prepared by incubating aLA with OA for 1 h at 20 or 80 °C (lip20 and lip80, respectively). The liprotides showed similar cytotoxicity against MCF7 cells, though lip80 acts more slowly, possibly due to intermolecular disulphide bonds formed during preparation. Liprotides are known to increase the fluidity of a membrane and transfer OA to vesicles, prompting us to focus on the effect of liprotides on the cell membrane. Extracellular Ca2+ influx is important for activation of the plasma membrane repair system, and we found that removal of Ca2+ from the medium enhanced the liprotides' killing effect. Liprotide cytotoxicity was also increased by knockdown of Annexin A6 (ANXA6), a protein involved in plasma membrane repair. We conclude that MCF7 cells counteract liprotide-induced membrane permeabilization by activating their plasma membrane repair system, which is triggered by extracellular Ca2+ and involves ANXA6.


Assuntos
Antineoplásicos/farmacologia , Membrana Celular/efeitos dos fármacos , Proteolipídeos/farmacologia , Anexina A6/metabolismo , Cálcio/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Meios de Cultura/química , Humanos , Lactalbumina/farmacologia , Células MCF-7 , Ácido Oleico/farmacologia , Temperatura
18.
Nat Commun ; 8(1): 1623, 2017 11 20.
Artigo em Inglês | MEDLINE | ID: mdl-29158488

RESUMO

Efficient cell membrane repair mechanisms are essential for maintaining membrane integrity and thus for cell life. Here we show that the Ca2+- and phospholipid-binding proteins annexin A4 and A6 are involved in plasma membrane repair and needed for rapid closure of micron-size holes. We demonstrate that annexin A4 binds to artificial membranes and generates curvature force initiated from free edges, whereas annexin A6 induces constriction force. In cells, plasma membrane injury and Ca2+ influx recruit annexin A4 to the vicinity of membrane wound edges where its homo-trimerization leads to membrane curvature near the edges. We propose that curvature force is utilized together with annexin A6-mediated constriction force to pull the wound edges together for eventual fusion. We show that annexin A4 can counteract various plasma membrane disruptions including holes of several micrometers indicating that induction of curvature force around wound edges is an early key event in cell membrane repair.


Assuntos
Anexina A4/metabolismo , Anexina A6/metabolismo , Membrana Celular/química , Membrana Celular/metabolismo , Anexina A4/genética , Anexina A6/genética , Cálcio/metabolismo , Membrana Celular/genética , Células HeLa , Humanos , Membranas Artificiais , Fosfolipídeos/metabolismo , Cicatrização
19.
PLoS Genet ; 13(10): e1007070, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-29065150

RESUMO

Genetic disruption of the dystrophin complex produces muscular dystrophy characterized by a fragile muscle plasma membrane leading to excessive muscle degeneration. Two genetic modifiers of Duchenne Muscular Dystrophy implicate the transforming growth factor ß (TGFß) pathway, osteopontin encoded by the SPP1 gene and latent TGFß binding protein 4 (LTBP4). We now evaluated the functional effect of these modifiers in the context of muscle injury and repair to elucidate their mechanisms of action. We found that excess osteopontin exacerbated sarcolemmal injury, and correspondingly, that loss of osteopontin reduced injury extent both in isolated myofibers and in muscle in vivo. We found that ablation of osteopontin was associated with reduced expression of TGFß and TGFß-associated pathways. We identified that increased TGFß resulted in reduced expression of Anxa1 and Anxa6, genes encoding key components of the muscle sarcolemma resealing process. Genetic manipulation of Ltbp4 in dystrophic muscle also directly modulated sarcolemmal resealing, and Ltbp4 alleles acted in concert with Anxa6, a distinct modifier of muscular dystrophy. These data provide a model in which a feed forward loop of TGFß and osteopontin directly impacts the capacity of muscle to recover from injury, and identifies an intersection of genetic modifiers on muscular dystrophy.


Assuntos
Genes Modificadores , Proteínas de Ligação a TGF-beta Latente/fisiologia , Músculo Esquelético/fisiologia , Distrofia Muscular Animal/genética , Osteopontina/metabolismo , Animais , Anexina A1/genética , Anexina A1/metabolismo , Anexina A6/genética , Anexina A6/metabolismo , Feminino , Regulação da Expressão Gênica , Masculino , Camundongos , Camundongos Endogâmicos DBA , Camundongos Knockout , Músculo Esquelético/lesões , Distrofia Muscular Animal/metabolismo , Distrofia Muscular Animal/patologia , Osteopontina/genética , Receptores de Fatores de Crescimento Transformadores beta/genética , Receptores de Fatores de Crescimento Transformadores beta/metabolismo , Recuperação de Função Fisiológica , Sarcolema/fisiologia
20.
Br J Cancer ; 117(9): 1326-1335, 2017 Oct 24.
Artigo em Inglês | MEDLINE | ID: mdl-28881357

RESUMO

BACKGROUND: Discovery and validation of new antibody tractable targets is critical for the development of new antibody therapeutics to address unmet needs in oncology. METHODS: A highly invasive clonal variant of the MDA-MB-435S cell line was used to generate monoclonal antibodies (MAbs), which were screened for anti-invasive activity against aggressive cancer cells in vitro. The molecular target of selected inhibitory MAb 9E1 was identified using immunoprecipitation/liquid chromatography-tandem mass spectrometry. The potential anti-tumour effects of MAb 9E1 were investigated in vitro together with immunohistochemical analysis of the 9E1 target antigen in normal and cancer tissues. RESULTS: MAb 9E1 significantly decreases invasion in pancreatic, lung squamous and breast cancer cells and silencing of its target antigen, which was revealed as AnxA6, leads to markedly reduced invasive capacity of pancreatic and lung squamous cancer in vitro. IHC using MAb 9E1 revealed that AnxA6 exhibits a high prevalence of membrane immunoreactivity across aggressive tumour types with restricted expression observed in the majority of normal tissues. In pancreatic ductal adenocarcinoma, high AnxA6 IHC score correlated with the presence of tumour budding at the invasive front of tumours (P=0.082), the presence of perineural invasion (P= <0.0001) and showed a weak correlation with reduced survival (P=0.2242). CONCLUSIONS: This study highlights the use of phenotypic hybridoma screening as an effective strategy to select a novel function-blocking MAb, 9E1 with anti-cancer activity in vitro. Moreover, through characterisation of the 9E1 target antigen, AnxA6, our findings support further investigation of AnxA6 as a potential candidate target for antibody-mediated inhibition of pancreatic cancer.


Assuntos
Anexina A6/metabolismo , Anticorpos Monoclonais/imunologia , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/metabolismo , Carcinoma Ductal Pancreático/metabolismo , Carcinoma de Células Escamosas/metabolismo , Neoplasias Pulmonares/metabolismo , Neoplasias Pancreáticas/metabolismo , Animais , Anexina A6/antagonistas & inibidores , Anexina A6/imunologia , Neoplasias da Mama/imunologia , Neoplasias da Mama/patologia , Carcinoma Ductal Pancreático/imunologia , Carcinoma Ductal Pancreático/patologia , Carcinoma de Células Escamosas/imunologia , Carcinoma de Células Escamosas/patologia , Feminino , Humanos , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/patologia , Camundongos , Estadiamento de Neoplasias , Neoplasias Pancreáticas/imunologia , Neoplasias Pancreáticas/patologia , Prognóstico , Taxa de Sobrevida
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