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1.
Adv Exp Med Biol ; 1267: 101-115, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32894479

RESUMO

Pathogenic bacteria colonize or disseminate into cells and tissues by inducing large-scale remodeling of host membranes. The physical phenomena underpinning these massive membrane extension and deformation are poorly understood. Invasive strategies of pathogens have been recently enriched by the description of a spectacular mode of opening of large transendothelial cell macroaperture (TEM) tunnels correlated to the dissemination of EDIN-producing strains of Staphylococcus aureus via a hematogenous route or to the induction of gelatinous edema triggered by the edema toxin from Bacillus anthracis. Remarkably, these highly dynamic tunnels close rapidly after they reach a maximal size. Opening and closure of TEMs in cells lasts for hours without inducing endothelial cell death. Multidisciplinary studies have started to provide a broader perspective of both the molecular determinants controlling cytoskeleton organization at newly curved membranes generated by the opening of TEMs and the physical processes controlling the dynamics of these tunnels. Here we discuss the analogy between the opening of TEM tunnels and the physical principles of dewetting, stemming from a parallel between membrane tension and surface tension. This analogy provides a broad framework to investigate biophysical constraints in cell membrane dynamics and their diversion by certain invasive microbial agents.


Assuntos
Bactérias/patogenicidade , Membrana Celular/microbiologia , Membrana Celular/patologia , Células Endoteliais/microbiologia , Células Endoteliais/patologia , Molhabilidade , Membrana Celular/metabolismo , Edema/metabolismo , Edema/microbiologia , Edema/patologia , Células Endoteliais/metabolismo , Humanos , Tensão Superficial
2.
PLoS Pathog ; 16(9): e1008878, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32946535

RESUMO

As an obligate intracellular pathogen, host cell invasion is paramount to Chlamydia trachomatis proliferation. While the mechanistic underpinnings of this essential process remain ill-defined, it is predicted to involve delivery of prepackaged effector proteins into the host cell that trigger plasma membrane remodeling and cytoskeletal reorganization. The secreted effector proteins TmeA and TarP, have risen to prominence as putative key regulators of cellular invasion and bacterial pathogenesis. Although several studies have begun to unravel molecular details underlying the putative function of TarP, the physiological function of TmeA during host cell invasion is unknown. Here, we show that TmeA employs molecular mimicry to bind to the GTPase binding domain of N-WASP, which results in recruitment of the actin branching ARP2/3 complex to the site of chlamydial entry. Electron microscopy revealed that TmeA mutants are deficient in filopodia capture, suggesting that TmeA/N-WASP interactions ultimately modulate host cell plasma membrane remodeling events necessary for chlamydial entry. Importantly, while both TmeA and TarP are necessary for effective host cell invasion, we show that these effectors target distinct pathways that ultimately converge on activation of the ARP2/3 complex. In line with this observation, we show that a double mutant suffers from a severe entry defect nearly identical to that observed when ARP3 is chemically inhibited or knocked down. Collectively, our study highlights both TmeA and TarP as essential regulators of chlamydial invasion that modulate the ARP2/3 complex through distinct signaling platforms, resulting in plasma membrane remodeling events that are essential for pathogen uptake.


Assuntos
Proteínas de Bactérias , Membrana Celular/metabolismo , Chlamydia trachomatis , Proteína Neuronal da Síndrome de Wiskott-Aldrich/metabolismo , Complexo 2-3 de Proteínas Relacionadas à Actina/genética , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Membrana Celular/genética , Membrana Celular/patologia , Chlamydia trachomatis/genética , Chlamydia trachomatis/metabolismo , Chlamydia trachomatis/patogenicidade , Células HeLa , Humanos , Mutação , Domínios Proteicos , Pseudópodes/genética , Pseudópodes/metabolismo , Proteína Neuronal da Síndrome de Wiskott-Aldrich/genética
3.
Khirurgiia (Mosk) ; (7): 12-17, 2020.
Artigo em Russo | MEDLINE | ID: mdl-32736458

RESUMO

OBJECTIVE: To determine the value of membrane protective effect in intestine and liver cells for the effectiveness of minimally invasive surgery for acute peritonitis. MATERIAL AND METHODS: Patients with acute peritonitis undergoing laparoscopic (n=60) and open (n=50) surgery are analyzed. Functional characteristics of liver and bowel, disorders of homeostasis were evaluated in early postoperative period. RESULTS: Reduced negative impact of surgical aggression on the state of liver and intestine is essential to improve treatment outcomes in patients with acute peritonitis undergoing minimally invasive surgery. Fast recovery of intestine inevitably results reduced release of endotoxins while restoration of liver function is associated with rapid elimination of these toxins. These processes prevent severe intoxication and facilitate accelerated recovery. Functional restoration of liver and bowel is associated with reduced oxidative stress during laparoscopic operations. It is also important because peritonitis causes activation of free-radical processes per se. Therefore, an additional source of oxidative phenomena is extremely undesirable in these cases. CONCLUSION: Laparoscopic surgery for acute peritonitis minimizes surgical aggression and is associated with more favorable recovery of liver and bowel function. Undoubtedly, these findings should be considered to choose surgical approach in this severe category of patients.


Assuntos
Procedimentos Cirúrgicos Minimamente Invasivos/efeitos adversos , Procedimentos Cirúrgicos Minimamente Invasivos/métodos , Peritonite/cirurgia , Doença Aguda , Membrana Celular/metabolismo , Membrana Celular/patologia , Membrana Celular/fisiologia , Humanos , Mucosa Intestinal/metabolismo , Intestinos/patologia , Intestinos/fisiopatologia , Laparoscopia/efeitos adversos , Laparotomia/efeitos adversos , Fígado/metabolismo , Fígado/patologia , Fígado/fisiopatologia , Estresse Oxidativo/fisiologia , Peritonite/metabolismo , Peritonite/fisiopatologia , Complicações Pós-Operatórias/etiologia , Complicações Pós-Operatórias/prevenção & controle , Recuperação de Função Fisiológica , Toxinas Biológicas/biossíntese , Toxinas Biológicas/metabolismo
4.
Biophys Chem ; 266: 106452, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-32818817

RESUMO

The Envelope (E) protein in SARS Coronavirus (CoV) is a small structural protein, incorporated as part of the envelope. A major fraction of the protein has been known to be associated with the host membranes, particularly organelles related to intracellular trafficking, prompting CoV packaging and propagation. Studies have elucidated the central hydrophobic transmembrane domain of the E protein being responsible for much of the viroporin activity in favor of the virus. However, newer insights into the organizational principles at the membranous compartments within the host cells suggest further complexity of the system. The lesser hydrophobic Carboxylic-terminal of the protein harbors interesting amino acid sequences- suggesting at the prevalence of membrane-directed amyloidogenic properties that remains mostly elusive. These highly conserved segments indicate at several potential membrane-associated functional roles that can redefine our comprehensive understanding of the protein. This should prompt further studies in designing and characterizing of effective targeted therapeutic measures.


Assuntos
Betacoronavirus/fisiologia , Membrana Celular/metabolismo , Infecções por Coronavirus/metabolismo , Pneumonia Viral/metabolismo , Proteínas do Envelope Viral/metabolismo , Sequência de Aminoácidos , Animais , Betacoronavirus/química , Membrana Celular/patologia , Membrana Celular/virologia , Infecções por Coronavirus/patologia , Infecções por Coronavirus/virologia , Interações Hospedeiro-Patógeno , Humanos , Modelos Moleculares , Pandemias , Pneumonia Viral/patologia , Pneumonia Viral/virologia , Domínios Proteicos , Alinhamento de Sequência , Proteínas do Envelope Viral/química
5.
Am J Physiol Heart Circ Physiol ; 319(2): H410-H421, 2020 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-32648820

RESUMO

Cardiac t tubules undergo significant remodeling in various pathological and experimental conditions, which can be associated with mechanical or osmotic stress. In particular, it has been shown that removal of hyposmotic stress can lead to sealing of t tubules. However, the mechanisms underlying the sealing process remain essentially unknown. In this study we used dextran trapping assay to demonstrate that in adult mouse cardiomyocytes, t-tubular sealing can also be induced by hyperosmotic challenge and that both hypo- and hyperosmotic sealing display a clear threshold behavior requiring ≈100 mosmol/L minimal stress. Importantly, during both hypo- and hyperosmotic challenges, the sealing of t tubules occurs only during the shrinking phase. Analysis of the time course of t-tubular remodeling following removal of hyposmotic stress shows that t tubules become sealed essentially instantly, well before any significant reduction in cell size can be observed. Overall, the data support the hypothesis that the critical event in the process of t-tubular sealing during osmotic challenges is detachment (peeling) of the membrane from the underlying cytoskeleton due to suprathreshold stress.NEW & NOTEWORTHY This study provides new insights into how t-tubular membranes respond to osmotic forces. In particular, the data show that osmotically induced sealing of cardiac t tubules is a threshold phenomenon initiated by detachment of t-tubular membrane from the underlying cytoskeleton. The findings are consistent with the hypothesis that final sealing of t tubules is driven by negative hydrostatic intracellular pressure coincident with cell shrinking.


Assuntos
Membrana Celular/patologia , Tamanho Celular , Citoesqueleto/patologia , Miócitos Cardíacos/patologia , Pressão Osmótica , Vacúolos/patologia , Animais , Membrana Celular/metabolismo , Citoesqueleto/metabolismo , Feminino , Masculino , Camundongos Endogâmicos C57BL , Miócitos Cardíacos/metabolismo , Fatores de Tempo , Vacúolos/metabolismo
6.
Artigo em Inglês | MEDLINE | ID: mdl-32348180

RESUMO

The ubiquitous calpains, calpain-1 and -2, play important roles in Ca2+-dependent membrane repair. Mechanically active tissues like skeletal muscle are particularly reliant on mechanisms to repair and remodel membrane injury, such as those caused by eccentric damage. We demonstrate that calpain-1 and -2 are master effectors of Ca2+-dependent repair of mechanical plasma membrane scrape injuries, although they are dispensable for repair/removal of small wounds caused by pore-forming agents. Using CRISPR gene-edited human embryonic kidney 293 (HEK293) cell lines, we established that loss of both calpains-1 and -2 (CAPNS1-/-) virtually ablates Ca2+-dependent repair of mechanical scrape injuries but does not affect injury or recovery from perforation by streptolysin-O or saponin. In contrast, cells with targeted knockout of either calpain-1 (CAPN1-/-) or -2 (CAPN2-/-) show near-normal repair of mechanical injuries, inferring that both calpain-1 and calpain-2 are equally capable of conducting the cascade of proteolytic cleavage events to reseal a membrane injury, including that of the known membrane repair agent dysferlin. A severe muscular dystrophy in a murine model with skeletal muscle knockout of Capns1 highlights vital roles for calpain-1 and/or -2 for health and viability of skeletal muscles not compensated for by calpain-3 (CAPN3). We propose that the dystrophic phenotype relates to loss of maintenance of plasma membrane/cytoskeletal networks by calpains-1 and -2 in response to directed and dysfunctional Ca2+-signaling, pathways hyperstimulated in the context of membrane injury. With CAPN1 variants associated with spastic paraplegia, a severe dystrophy observed with muscle-specific loss of calpain-1 and -2 activity identifies CAPN2 and CAPNS1 as plausible candidate neuromuscular disease genes.


Assuntos
Calpaína/deficiência , Membrana Celular/enzimologia , Músculo Esquelético/enzimologia , Distrofia Muscular do Cíngulo dos Membros/enzimologia , Distrofia Muscular Animal/enzimologia , Animais , Proteínas de Bactérias/farmacologia , Sinalização do Cálcio , Calpaína/genética , Membrana Celular/efeitos dos fármacos , Membrana Celular/patologia , Modelos Animais de Doenças , Disferlina/deficiência , Disferlina/genética , Feminino , Células HEK293 , Humanos , Masculino , Camundongos Knockout , Músculo Esquelético/patologia , Distrofia Muscular do Cíngulo dos Membros/genética , Distrofia Muscular do Cíngulo dos Membros/patologia , Distrofia Muscular Animal/genética , Distrofia Muscular Animal/patologia , Saponinas/farmacologia , Índice de Gravidade de Doença , Estreptolisinas/farmacologia
7.
Mol Pharmacol ; 97(6): 365-376, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32234808

RESUMO

Proteinase-activated receptors (PARs) are a four-member family of G-protein-coupled receptors that are activated via proteolysis. PAR4 is a member of this family that is cleaved and activated by serine proteinases such as thrombin, trypsin, and cathepsin-G. PAR4 is expressed in a variety of tissues and cell types, including platelets, vascular smooth muscle cells, and neuronal cells. In studying PAR4 signaling and trafficking, we observed dynamic changes in the cell membrane, with spherical membrane protrusions that resemble plasma membrane blebbing. Since nonapoptotic membrane blebbing is now recognized as an important regulator of cell migration, cancer cell invasion, and vesicular content release, we sought to elucidate the signaling pathway downstream of PAR4 activation that leads to such events. Using a combination of pharmacological inhibition and CRISPR/CRISPR-associated protein 9 (Cas9)-mediated gene editing approaches, we establish that PAR4-dependent membrane blebbing occurs independently of the Gα q/11- and Gα i-signaling pathways and is dependent on signaling via the ß-arrestin-1/2 and Ras homolog family member A (RhoA) signaling pathways. Together these studies provide further mechanistic insight into PAR4 regulation of cellular function. SIGNIFICANCE STATEMENT: We find that the thrombin receptor PAR4 triggers cell membrane blebbing in a RhoA-and ß-arrestin-dependent manner. In addition to identifying novel cellular responses mediated by PAR4, these data provide further evidence for biased signaling in PAR4 since membrane blebbing was dependent on some, but not all, signaling pathways activated by PAR4.


Assuntos
Membrana Celular/metabolismo , Membrana Celular/patologia , Receptores de Trombina/metabolismo , beta-Arrestinas/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Animais , Sistemas CRISPR-Cas , Forma Celular , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Músculo Liso Vascular/metabolismo , Ratos , Ratos Endogâmicos WKY , Receptores Acoplados a Proteínas-G/metabolismo , Receptores de Trombina/agonistas , Transdução de Sinais
8.
Nature ; 580(7801): 106-112, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32238932

RESUMO

Radial glial progenitor cells (RGPs) are the major neural progenitor cells that generate neurons and glia in the developing mammalian cerebral cortex1-4. In RGPs, the centrosome is positioned away from the nucleus at the apical surface of the ventricular zone of the cerebral cortex5-8. However, the molecular basis and precise function of this distinctive subcellular organization of the centrosome are largely unknown. Here we show in mice that anchoring of the centrosome to the apical membrane controls the mechanical properties of cortical RGPs, and consequently their mitotic behaviour and the size and formation of the cortex. The mother centriole in RGPs develops distal appendages that anchor it to the apical membrane. Selective removal of centrosomal protein 83 (CEP83) eliminates these distal appendages and disrupts the anchorage of the centrosome to the apical membrane, resulting in the disorganization of microtubules and stretching and stiffening of the apical membrane. The elimination of CEP83 also activates the mechanically sensitive yes-associated protein (YAP) and promotes the excessive proliferation of RGPs, together with a subsequent overproduction of intermediate progenitor cells, which leads to the formation of an enlarged cortex with abnormal folding. Simultaneous elimination of YAP suppresses the cortical enlargement and folding that is induced by the removal of CEP83. Together, these results indicate a previously unknown role of the centrosome in regulating the mechanical features of neural progenitor cells and the size and configuration of the mammalian cerebral cortex.


Assuntos
Centrossomo/metabolismo , Córtex Cerebral/citologia , Córtex Cerebral/embriologia , Células Ependimogliais/citologia , Células-Tronco Neurais/citologia , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Proteínas de Ciclo Celular/metabolismo , Membrana Celular/metabolismo , Membrana Celular/patologia , Proliferação de Células , Centríolos/metabolismo , Córtex Cerebral/patologia , Feminino , Masculino , Camundongos , Proteínas Associadas aos Microtúbulos/deficiência , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Microtúbulos/patologia , Neurogênese
9.
J Pathol ; 250(5): 656-666, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32086805

RESUMO

The cells of the mononuclear phagocyte system (MPS) constitute a dispersed organ, which is distributed throughout the body. Macrophages in different tissues display distinctive mosaic phenotypes as resident and recruited cells of embryonic and bone marrow origin, respectively. They help to maintain homeostasis during development and throughout adult life, yet contribute to the pathogenesis of many disease processes, including inflammation, innate and adaptive immunity, metabolic disorders, and cancer. Heterogeneous tissue macrophage populations display a wide variety of surface molecules to recognise and respond to host, microbial, and exogenous ligands in their environment; their receptors mediate the uptake and destruction of effete and dying host cells and pathogens, as well as contribute trophic and secretory functions within every organ in the body. Apart from local cellular interactions, macrophage surface molecules and products serve to mobilise and coordinate systemic humoral and cellular responses. Their use as antigen markers in pathogenesis and as potential drug targets has lagged in clinical pathology and human immunotherapy. In this review, we summarise the properties of selected surface molecules expressed on macrophages in different tissues and disease processes, to provide a functional basis for diagnosis, further research, and treatment. © 2020 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.


Assuntos
Imunidade Adaptativa/imunologia , Membrana Celular/patologia , Inflamação/patologia , Macrófagos/patologia , Receptores de Superfície Celular/metabolismo , Animais , Comunicação Celular/imunologia , Membrana Celular/imunologia , Humanos , Inflamação/imunologia , Macrófagos/imunologia , Receptores de Superfície Celular/imunologia
10.
Sci Rep ; 10(1): 1463, 2020 01 29.
Artigo em Inglês | MEDLINE | ID: mdl-31996706

RESUMO

Microbes develop several strategies to survive in the adverse condition such as biofilm formation, attaining non-dividing state, altering drug target or drug, thereby increases the burden of drug dosage. To combat these issues, nanoparticles have shown an alternative approach for new treatment strategy but synthesis via chemical synthetic route limits their application in biomedical field. Here, green method for the synthesis of gold nanoparticles using sophorolipid (SL) is discussed that is characterized by various techniques. Initially, the antimicrobial activity was checked against metabolically active state of microbes; Gram-positive Staphylococcus aureus and Gram-negative Vibrio cholerae using XTT assay and growth kinetics assay. Results suggested higher efficacy of nanoparticles for Gram-negative, therefore further analyzed against Escherichia coli that confirmed its potency for the same. AuNPs-SL also signifies its efficiency at least metabolically active state; non dividing cells and biofilm of these microbes. Induced morphological changes were studied by SEM that revealed AuNPs-SL led to disruption of cell membrane and leakage of intracellular fluid to the surroundings. Inhibition of respiratory enzymes activity also plays a crucial role in bactericidal action as indicated by LDH assay. Synergy of AuNPs-SL with different antibiotics was also analyzed using checkerboard assay. These results suggested the possible use of AuNPs-SL as an antimicrobial therapy in the field of nanomedicine.


Assuntos
Anti-Infecciosos/farmacologia , Biofilmes/crescimento & desenvolvimento , Membrana Celular/patologia , Cólera/tratamento farmacológico , Química Verde/métodos , Ácidos Oleicos/farmacologia , Vibrio cholerae/fisiologia , Processos de Crescimento Celular , Ouro , Nanopartículas Metálicas
11.
Toxicology ; 432: 152364, 2020 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-31927068

RESUMO

Environmental exposure to nanoparticles (NPs) has significantly increased in the last decades, mostly due to increased environmental pollution and frequent use of NP containing consumer products. Such NPs may enter our body and cause various health-related problems. The brain is a particularly problematic accumulation site due to its physiological and anatomical restrictions. Several mechanisms of NP neurotoxicity have already been identified, however not enough is known especially regarding toxicity of engineered/industrial NPs. The focus of this in vitro study was on analysis of neurotoxicity of different engineered NPs, with which we come into contact in our daily lives; SiO2 NPs, food grade (FG) TiO2 NPs, TiO2 P25 and silver NPs as examples of industrial NPs, and polyacrylic acid (PAA) coated cobalt ferrite NPs as an example of biomedical NPs. All short term exposure experiments (24-72 h) were performed on SH-SY5Y human neuroblastoma cell line in vitro using higher (25-50 µg/ml) as well as lower (2-10 µg/ml), concentrations that are more relevant for in vivo NPs exposure. We show that NPs can cause neurotoxicity through different mechanisms, such as membrane damage, cell cycle interference, ROS formation and accumulation of autophagosomes, depending on their physico-chemical properties and stability in physiological media. Low, in vivo achievable concentrations of NPs induced only minor or no changes in vitro, however prolonged exposure and accumulation in vivo could negatively affect the cells. This was also shown in case of autophagy dysfunction for TiO2 P25 NPs and decrease of cell viability for TiO2 FG NPs, which were only evident after 72 h of incubation.


Assuntos
Nanopartículas/toxicidade , Células-Tronco Neurais/efeitos dos fármacos , Resinas Acrílicas , Autofagossomos/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Membrana Celular/efeitos dos fármacos , Membrana Celular/patologia , Sobrevivência Celular , Cobalto , Compostos Férricos , Alimentos , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Nanopartículas Metálicas/toxicidade , Síndromes Neurotóxicas/patologia , Espécies Reativas de Oxigênio/metabolismo , Titânio/toxicidade
12.
FASEB J ; 34(1): 1665-1678, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31914676

RESUMO

Bacterial infectious diseases can lead to death or to serious illnesses. These outcomes are partly the consequence of pore-forming toxins, which are secreted by the pathogenic bacteria (eg, pneumolysin of Streptococcus pneumoniae). Pneumolysin binds to cholesterol within the plasma membrane of host cells and assembles to form trans-membrane pores, which can lead to Ca2+ influx and cell death. Membrane repair mechanisms exist that limit the extent of damage. Immune cells which are essential to fight bacterial infections critically rely on survival mechanisms after detrimental pneumolysin attacks. This study investigated the susceptibility of different immune cell types to pneumolysin. As a model system, we used the lymphoid T-cell line Jurkat, and myeloid cell lines U937 and THP-1. We show that Jurkat T cells are highly susceptible to pneumolysin attack. In contrast, myeloid THP-1 and U937 cells are less susceptible to pneumolysin. In line with these findings, human primary T cells are shown to be more susceptible to pneumolysin attack than monocytes. Differences in susceptibility to pneumolysin are due to (I) preferential binding of pneumolysin to Jurkat T cells and (II) cell type specific plasma membrane repair capacity. Myeloid cell survival is mostly dependent on Ca2+ induced expelling of damaged plasma membrane areas as microvesicles. Thus, in myeloid cells, first-line defense cells in bacterial infections, a potent cellular repair machinery ensures cell survival after pneumolysin attack. In lymphoid cells, which are important at later stages of infections, less efficient repair mechanisms and enhanced toxin binding renders the cells more sensitive to pneumolysin.


Assuntos
Toxinas Bacterianas/metabolismo , Estruturas da Membrana Celular/metabolismo , Estruturas da Membrana Celular/patologia , Membrana Celular/metabolismo , Membrana Celular/patologia , Micropartículas Derivadas de Células/metabolismo , Micropartículas Derivadas de Células/patologia , Cálcio/metabolismo , Morte Celular/fisiologia , Linhagem Celular Tumoral , Sobrevivência Celular/fisiologia , Humanos , Células Jurkat , Monócitos/metabolismo , Monócitos/patologia , Células Mieloides/metabolismo , Células Mieloides/patologia , Streptococcus pneumoniae/patogenicidade , Células THP-1 , Células U937
13.
FASEB J ; 34(1): 1620-1636, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31914692

RESUMO

Acute kidney injury (AKI) is the leading cause of renal failure, and quite a few patients will advance to chronic kidney disease (CKD) in the long term. Here, we explore the roles and mechanisms of tubular epithelial cells (TECs) during repeated cisplatin (CP) induced AKI to CKD transition (AKI-CKD). Previously, we reported that murine double minute 2 (MDM2), an E3-ubiquitin ligase, is involved in tubulointerstitial fibrosis. However, whether tubular MDM2 is implicated in AKI-CKD is undefined. Currently, we confirmed that during AKI-CKD, MDM2 shifts from nucleus to cell membrane in TECs both in vivo and in vitro. Whereas regulating MDM2 distribution chemically or genetically has a prominent impact on tubular disorders. And then we investigated the mechanisms of the above findings. First, in the nucleus, repeated CP administration leads to MDM2 reduction with escalated p53 and cell cycle G2/M arrest. On the other hand, multiple CP treatment increases the level of membranous MDM2 with ensuing integrin ß8 degradation and TGF-ß1 activation. More interestingly, anchoring MDM2 on cell membranes can mimic the reduction of integrin ß8 arousing by repeated CP exposure. Collectively, our findings provided the evidence that tubular MDM2 subcellular shuttling is involved in AKI-CKD through p53-G2/M arrest and integrin ß8 mediated TGF-ß1 activation.


Assuntos
Lesão Renal Aguda/tratamento farmacológico , Lesão Renal Aguda/metabolismo , Cisplatino/farmacologia , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Insuficiência Renal Crônica/tratamento farmacológico , Insuficiência Renal Crônica/metabolismo , Lesão Renal Aguda/patologia , Animais , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Membrana Celular/patologia , Movimento Celular/efeitos dos fármacos , Movimento Celular/fisiologia , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Núcleo Celular/patologia , Células Cultivadas , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Humanos , Cadeias beta de Integrinas/metabolismo , Rim/efeitos dos fármacos , Rim/metabolismo , Rim/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Insuficiência Renal Crônica/patologia , Fator de Crescimento Transformador beta1/metabolismo , Proteína Supressora de Tumor p53/metabolismo
14.
Sci Rep ; 10(1): 1358, 2020 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-31992816

RESUMO

We previously demonstrated that cyclic guanosine monophosphate (cGMP) stimulates amyloid precursor protein (APP) and beta-secretase (BACE1) approximation in neuronal endo-lysosomal compartments, thus boosting the production of amyloid-ß (Aß) peptides and enhancing synaptic plasticity and memory. Here, we further investigated the mechanism by which cGMP regulates the subcellular localization of APP and BACE1, finding that the cyclic nucleotide inhibits the activity of Rab5, a small GTPase associated with the plasma membrane and early endosomes. Accordingly, we also found that expression of a dominant-negative Rab5 mutant increases both APP-BACE1 approximation and Aß extracellular levels, therefore mimicking the effects induced by cGMP. These results reveal a functional correlation between the cGMP/Aß pathway and the activity of Rab5 that may contribute to the understanding of Alzheimer's disease pathophysiology.


Assuntos
Doença de Alzheimer/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Ácido Aspártico Endopeptidases/metabolismo , Membrana Celular/metabolismo , Sistemas do Segundo Mensageiro , Proteínas rab5 de Ligação ao GTP/metabolismo , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Secretases da Proteína Precursora do Amiloide/genética , Precursor de Proteína beta-Amiloide/genética , Animais , Ácido Aspártico Endopeptidases/genética , Linhagem Celular , Membrana Celular/genética , Membrana Celular/patologia , GMP Cíclico , Endossomos/genética , Endossomos/metabolismo , Endossomos/patologia , Camundongos , Proteínas rab5 de Ligação ao GTP/genética
15.
J Photochem Photobiol B ; 203: 111732, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31864089

RESUMO

Investigating the dynamics of different biomolecules in the cellular milieu through microscopic imaging has gained paramount importance in the last decade. Continuous developments in the field of microscopy are paralleled by the design and synthesis of fluorophores that target specific compartments within a cell. In this study, we have synthesized four fluorescent styrene derivatives, a neutral styrylpridine, three cationic styrylpyridinium probes with and without cholesterol tether, and investigated their absorption, emission, and cellular imaging properties. The fluorophores show solvatochromic emission attributed to intramolecular charge transfer from donor to acceptor with an emission range of 500-600 nm. The fluorescent cholesterol conjugate labels plasma membrane effectively while the fluorophores devoid of the cholesterol tether label mitochondria. Cholesterol conjugate also shows strong interaction with liposome membrane. Furthermore, the fluorophores alsotrack the mitochondria in live cells with high specificity. Cell viability assay showed overall non-toxic nature of the probes even at higher fluorophore concentrations. Through sidearm modifications, keeping the fluorescent core intact, we successfully targeted specific subcellular compartments of neuronal (N2a) and non-neuronal (HeLa) mammalian cell lines. This strategy of using a single molecular scaffold with subtle substitutions could be ideal in generating a variety of fluorophores targeting other subcellular compartments.


Assuntos
Membrana Celular/patologia , Mitocôndrias/patologia , Piridinas/química , Animais , Células COS , Sobrevivência Celular/efeitos dos fármacos , Chlorocebus aethiops , Células HeLa , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Piridinas/farmacologia
16.
Chemosphere ; 239: 124523, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31499308

RESUMO

Arsenic (As) is a toxic metalloid that severely hampers plant growth and also poses health risks for humans through the food chain. Although nitric oxide (NO) is known to improve plant resistance to multiple stresses including metal toxicity, little is known about its role in the As tolerance of hyperaccumulator plants. This study investigates the role of the exogenously applied NO donor, sodium nitroprusside (SNP), in improving the As tolerance of Isatis cappadocica, which has been reported to hyperaccumulate As. Exposure to toxic As concentrations significantly increases NO production and damages the cell membrane, as indicated by increased hydrogen peroxide (H2O2) and malondialdehyde (MDA) concentrations, thereby reducing plant growth. However, the addition of SNP improves growth and alleviates As-induced oxidative stress by enhancing the activity of superoxide dismutase (SOD), ascorbate peroxidase (APX), glutathione reductase (GR), glutathione S-transferase (GST), glutathione (GSH), as well as proline and thiol concentrations, thereby confirming the beneficial role played by NO in increasing As stress tolerance. Furthermore, the As-induced decrease in growth and the increase in oxidative stress were more marked in the presence of bovine hemoglobin (Hb; a NO scavenger) and N(G)-nitro-l-arginine methyl ester (l-NAME; a NO synthase inhibitor), thus demonstrating the protective role of NO against As toxicity. The reduction in NO concentrations by l-NAME suggests that NOS-like activity is involved in the generation of NO in response to As in I. cappadocica.


Assuntos
Antioxidantes/metabolismo , Arsênico/toxicidade , Isatis/efeitos dos fármacos , Óxido Nítrico/metabolismo , Nitroprussiato/farmacologia , Animais , Ascorbato Peroxidases/metabolismo , Bovinos , Membrana Celular/patologia , Glutationa/metabolismo , Glutationa Redutase/metabolismo , Peróxido de Hidrogênio/metabolismo , Isatis/metabolismo , Malondialdeído/metabolismo , Doadores de Óxido Nítrico , Estresse Oxidativo/efeitos dos fármacos , Superóxido Dismutase/metabolismo
17.
Adv Exp Med Biol ; 1131: 93-129, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31646508

RESUMO

Plasma membrane Ca2+ transport ATPases (PMCA1-4, ATP2B1-4) are responsible for removing excess Ca2+ from the cell in order to keep the cytosolic Ca2+ ion concentration at the low level essential for normal cell function. While these pumps take care of cellular Ca2+ homeostasis they also change the duration and amplitude of the Ca2+ signal and can create Ca2+ gradients across the cell. This is accomplished by generating more than twenty PMCA variants each having the character - fast or slow response, long or short memory, distinct interaction partners and localization signals - that meets the specific needs of the particular cell-type in which they are expressed. It has become apparent that these pumps are essential to normal tissue development and their malfunctioning can be linked to different pathological conditions such as certain types of neurodegenerative and heart diseases, hearing loss and cancer. In this chapter we summarize the complexity of PMCA regulation and function under normal and pathological conditions with particular attention to recent developments of the field.


Assuntos
Membrana Celular , ATPases Transportadoras de Cálcio da Membrana Plasmática , Animais , Membrana Celular/enzimologia , Membrana Celular/patologia , Citosol/metabolismo , Homeostase/fisiologia , Humanos , ATPases Transportadoras de Cálcio da Membrana Plasmática/genética , ATPases Transportadoras de Cálcio da Membrana Plasmática/metabolismo
18.
Adv Exp Med Biol ; 1131: 699-718, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31646531

RESUMO

Calcium exchanges and homeostasis are finely regulated between cellular organelles and in response to physiological signals. Besides ionophores, including voltage-gated Ca2+ channels, ionotropic neurotransmitter receptors, or Store-operated Ca2+ entry, activity of regulatory intracellular proteins finely tune Calcium homeostasis. One of the most intriguing, by its unique nature but also most promising by the therapeutic opportunities it bears, is the sigma-1 receptor (Sig-1R). The Sig-1R is a chaperone protein residing at mitochondria-associated endoplasmic reticulum (ER) membranes (MAMs), where it interacts with several partners involved in ER stress response, or in Ca2+ exchange between the ER and mitochondria. Small molecules have been identified that specifically and selectively activate Sig-1R (Sig-1R agonists or positive modulators) at the cellular level and that also allow effective pharmacological actions in several pre-clinical models of pathologies. The present review will summarize the recent data on the mechanism of action of Sig-1R in regulating Ca2+ exchanges and protein interactions at MAMs and the ER. As MAMs alterations and ER stress now appear as a common track in most neurodegenerative diseases, the intracellular action of Sig-1R will be discussed in the context of the recently reported efficacy of Sig-1R drugs in pathologies like Alzheimer's disease, Parkinson's disease, Huntington's disease, or amyotrophic lateral sclerosis.


Assuntos
Membrana Celular , Estresse do Retículo Endoplasmático , Doenças Neurodegenerativas , Receptores sigma , Membrana Celular/metabolismo , Membrana Celular/patologia , Humanos , Mitocôndrias/metabolismo , Doenças Neurodegenerativas/fisiopatologia , Receptores sigma/metabolismo
19.
J Pharm Biomed Anal ; 177: 112888, 2020 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-31563758

RESUMO

Metabolomics measurements of seminal plasma are widely used in diagnosis and finding of molecular mechanisms of male infertility. However, so far the limitation of metabolome coverage of analytical methods hinders comprehensive metabolite biomarker finding. Moreover, the widely used case-control comparison is not enough to unveil the detailed correlations of the metabolic changes with different sperm abnormalities. In this work, we aimed to have comprehensive metabolic profiling of seminal plasma to find the metabolomics difference between healthy controls and infertility case samples with different semen abnormities by liquid chromatography-mass spectrometry (LC-MS) detection with previously established new sample preparation procedure. Among 624 detected metabolite features, 63 potential biomarkers in various metabolite classes were found for infertility in seminal plasma by multivariate analysis. Interestingly, different infertility forms have different potential biomarkers with few in common, and most of potential biomarkers were found in oligo-astheno-teratospermia samples. To further find the association of the metabolomic changes with specific sperm abnormality, sperm parameters including sperm concentration, sperm deformity rate and sperm motility were also collected, and multivariate linear regression was used to find correlations between sperm parameters and potential biomarkers. Finally, levels of 17 metabolites were found to be significantly correlated with sperm parameters. Most of correlations agreed with previously reported mechanisms of infertility, such as correlation of acylcarnitines with sperm concentration and sperm deformity, and correlation of some antioxidants with sperm deformity rate and sperm motility. Some correlations were reported for the first time, such as negative correlations of isopentenyl pyrophosphate, 2-phosphoglyceric acid and γ-glutamyl-Se-methylselenocysteine with sperm deformity rate, and negative correlation of creatine riboside with sperm concentration. All the potential biomarkers were involved in 14 metabolic pathways playing important role in energy production, antioxidation, hormone regulation and sperm membrane. These results proved previously reported molecular mechanism (such as oxidative stress and energy production) and also gave new possible clues to the pathology of male infertility, which will benefit future etiology, diagnosis and treatment of male infertility.


Assuntos
Infertilidade Masculina/diagnóstico , Sêmen/metabolismo , Espermatozoides/patologia , Adulto , Biomarcadores/metabolismo , Estudos de Casos e Controles , Membrana Celular/patologia , Metabolismo Energético , Voluntários Saudáveis , Humanos , Infertilidade Masculina/patologia , Masculino , Metaboloma , Metabolômica/métodos , Estresse Oxidativo , Contagem de Espermatozoides , Motilidade Espermática , Espermatozoides/citologia , Espermatozoides/metabolismo , Adulto Jovem
20.
Oxid Med Cell Longev ; 2019: 8010614, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31781351

RESUMO

Acute kidney injury (AKI) is a heterogeneous group of critical disease conditions with high incidence and mortality. Vasoconstriction, oxidative stress, apoptosis, and inflammation are generally thought to be the main pathogenic mechanisms of AKI. Ferroptosis is a type of iron-dependent nonapoptotic cell death characterized by membrane lipid peroxide accumulation and polyunsaturated fatty acid consumption, and it plays essential roles in many diseases, including cancers and neurologic diseases. Recent studies have revealed an emerging role of ferroptosis in the pathophysiological processes of AKI. Here, in the present review, we summarized the most recent discoveries on the role of ferroptosis in the pathogenesis of AKI as well as its therapeutic potential in AKI.


Assuntos
Lesão Renal Aguda/metabolismo , Membrana Celular/metabolismo , Ácidos Graxos Insaturados/metabolismo , Ferroptose , Peroxidação de Lipídeos , Lesão Renal Aguda/patologia , Lesão Renal Aguda/terapia , Animais , Membrana Celular/patologia , Humanos
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