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1.
Cell Mol Life Sci ; 79(1): 67, 2021 Dec 31.
Artigo em Inglês | MEDLINE | ID: mdl-34971429

RESUMO

Mutations in the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) protein lead to persistent lung bacterial infections, mainly due to Pseudomonas aeruginosa, causing loss of respiratory function and finally death of people affected by CF. Unfortunately, even in the era of CFTR modulation therapies, management of pulmonary infections in CF remains highly challenging especially for patients with advanced stages of lung disease. Recently, we identified antimicrobial peptides (AMPs), namely Esc peptides, with potent antipseudomonal activity. In this study, by means of electrophysiological techniques and computational studies we discovered their ability to increase the CFTR-controlled ion currents, by direct interaction with the F508del-CFTR mutant. Remarkably, this property was not explored previously with any AMPs or peptides in general. More interestingly, in contrast with clinically used CFTR modulators, Esc peptides would give particular benefit to CF patients by combining their capability to eradicate lung infections and to act as promoters of airway wound repair with their ability to ameliorate the activity of the channel with conductance defects. Overall, our findings not only highlighted Esc peptides as the first characterized AMPs with a novel property, that is the potentiator activity of CFTR, but also paved the avenue to investigate the functions of AMPs and/or other peptide molecules, for a new up-and-coming pharmacological approach to address CF lung disease.


Assuntos
/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Pneumopatias/tratamento farmacológico , Infecções por Pseudomonas/tratamento farmacológico , Animais , Antibacterianos/metabolismo , Antibacterianos/farmacologia , Bicarbonatos/metabolismo , Cloretos/metabolismo , Fibrose Cística/genética , Fibrose Cística/microbiologia , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Humanos , Transporte de Íons/efeitos dos fármacos , Pneumopatias/microbiologia , Pneumopatias/patologia , Infecções por Pseudomonas/patologia , Pseudomonas aeruginosa/patogenicidade , Ratos , Ratos Endogâmicos F344
2.
Biomolecules ; 11(12)2021 12 20.
Artigo em Inglês | MEDLINE | ID: mdl-34944553

RESUMO

Integrases of different retroviruses assemble as functional complexes with varying multimers of the protein. Retroviral integrases require a divalent metal cation to perform one-step transesterification catalysis. Tetrameric prototype foamy virus (PFV) intasomes assembled from purified integrase and viral DNA oligonucleotides were characterized for their activity in the presence of different cations. While most retroviral integrases are inactive in calcium, PFV intasomes appear to be uniquely capable of catalysis in calcium. The PFV intasomes also contrast with other retroviral integrases by displaying an inverse correlation of activity with increasing manganese beginning at relatively low concentrations. The intasomes were found to be significantly more active in the presence of chloride co-ions compared to acetate. While HIV-1 integrase appears to commit to a target DNA within 20 s, PFV intasomes do not commit to target DNA during their reaction lifetime. Together, these data highlight the unique biochemical activities of PFV integrase compared to other retroviral integrases.


Assuntos
DNA/metabolismo , Integrases/química , Integrases/metabolismo , Spumavirus/enzimologia , Acetatos/metabolismo , Sítios de Ligação , Cloretos/metabolismo , Esterificação , Manganês/metabolismo , Oligonucleotídeos , Spumavirus/química , Proteínas Virais/química , Proteínas Virais/metabolismo
3.
PLoS One ; 16(10): e0257824, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34618824

RESUMO

Infectious gastrointestinal diseases are frequently caused by toxins secreted by pathogens which may impair physiological functions of the intestines, for instance by cholera toxin or by heat-labile enterotoxin. To obtain a functional model of the human intestinal epithelium for studying toxin-induced disease mechanisms, differentiated enterocyte-like Caco-2 cells were co-cultured with goblet cell-like HT29-MTX cells. These co-cultures formed a functional epithelial barrier, as characterized by a high electrical resistance and the presence of physiological intestinal properties such as glucose transport and chloride secretion which could be demonstrated electrophysiologically and by measuring protein expression. When the tissues were exposed to cholera toxin or heat-labile enterotoxin in the Ussing chamber, cholera toxin incubation resulted in an increase in short-circuit currents, indicating an increase in apical chloride secretion. This is in line with typical cholera toxin-induced secretory diarrhea in humans, while heat-labile enterotoxin only showed an increase in short-circuit-current in Caco-2 cells. This study characterizes for the first time the simultaneous measurement of physiological properties on a functional and structural level combined with the epithelial responses to bacterial toxins. In conclusion, using this model, physiological responses of the intestine to bacterial toxins can be investigated and characterized. Therefore, this model can serve as an alternative to the use of laboratory animals for characterizing pathophysiological mechanisms of enterotoxins at the intestinal level.


Assuntos
Toxinas Bacterianas/metabolismo , Toxina da Cólera/metabolismo , Doenças Transmissíveis/microbiologia , Gastroenteropatias/microbiologia , Toxinas Bacterianas/química , Células CACO-2 , Cloretos/metabolismo , Toxina da Cólera/química , Técnicas de Cocultura , Doenças Transmissíveis/patologia , Enterotoxinas/química , Enterotoxinas/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Gastroenteropatias/patologia , Glucose/metabolismo , Células HT29 , Humanos , Mucosa Intestinal/efeitos dos fármacos
4.
Nat Commun ; 12(1): 6035, 2021 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-34654800

RESUMO

Between 6-20% of the cellular proteome is under circadian control and tunes mammalian cell function with daily environmental cycles. For cell viability, and to maintain volume within narrow limits, the daily variation in osmotic potential exerted by changes in the soluble proteome must be counterbalanced. The mechanisms and consequences of this osmotic compensation have not been investigated before. In cultured cells and in tissue we find that compensation involves electroneutral active transport of Na+, K+, and Cl- through differential activity of SLC12A family cotransporters. In cardiomyocytes ex vivo and in vivo, compensatory ion fluxes confer daily variation in electrical activity. Perturbation of soluble protein abundance has commensurate effects on ion composition and cellular function across the circadian cycle. Thus, circadian regulation of the proteome impacts ion homeostasis with substantial consequences for the physiology of electrically active cells such as cardiomyocytes.


Assuntos
Fenômenos Fisiológicos Celulares , Ritmo Circadiano/fisiologia , Transporte de Íons/fisiologia , Osmose , Animais , Sistema Cardiovascular/patologia , Células Cultivadas , Cloretos/metabolismo , Fibroblastos , Homeostase , Pulmão , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Potássio/metabolismo , Proteoma , Sódio/metabolismo , Membro 2 da Família 12 de Carreador de Soluto/genética
5.
Exp Cell Res ; 408(1): 112837, 2021 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-34547255

RESUMO

Myelodysplastic syndrome (MDS) is a heterogeneous group of clonal hematopoietic neoplasms. The progression of malignancy is closely associated with immune regulation. Macrophages are indispensable tissue components and have been proposed to play a role in the pathophysiology of hematopoietic malignancies. However, the specific role of macrophages in the development of MDS remains unclear. Here, we investigated the characteristics and phenotypic evolution of macrophages from patients with MDS. Macrophages from patients with MDS expressed CD68, CD86 and CD163. Furthermore, MDS macrophages exhibited more M2-related characteristics. Moreover, a number of phenotype-associated genes in MDS macrophages exhibited diverse responses to iron overload or iron chelation upon stimulation by ferric chloride or deferoxamine (DFO, an iron chelator). Ferric chloride polarized MDS macrophages to exhibit more M1-related characteristics, a phenomenon that could be partially reversed by DFO. Therefore, this study reveals the characteristics and phenotypic evolution of MDS macrophages and broadens the knowledge of macrophage plasticity in hematopoietic malignancies.


Assuntos
Sobrecarga de Ferro/patologia , Ferro/metabolismo , Macrófagos/patologia , Síndromes Mielodisplásicas/patologia , Adulto , Idoso , Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/metabolismo , Microambiente Celular/fisiologia , Cloretos/metabolismo , Feminino , Compostos Férricos/metabolismo , Humanos , Sobrecarga de Ferro/metabolismo , Masculino , Pessoa de Meia-Idade , Receptores de Superfície Celular/metabolismo
6.
Biomed Khim ; 67(4): 338-346, 2021 Jul.
Artigo em Russo | MEDLINE | ID: mdl-34414892

RESUMO

Hyperhomocysteinemia is a risk factor for many diseases, including reproductive disorders in men. L-carnitine is used in medical practice to correct impaired bioenergetic conditions; in patients with idiopathic forms of infertility its effects are associated with improvement of the sperm parameters. However, the effect of exogenous L-carnitine on the level of homocysteine in the gonadal tissues, as a risk factor for impaired fertility, has not been investigated yet. The aim of this study was to investigate activity of bioenergetic enzymes in the epididymal mitochondrial fraction, the dynamics of changes in the cytoplasmic and mitochondrial lactate levels and LDH activity, the total carnitine content, as well as the oxidative status of these cells under conditions of oxidative stress caused by hyperhomocysteinemia, and to assess the effect of carnitine chloride on these parameters under conditions of methionine administration to male Wistar rats. Methionine administration to animals for three weeks at a dose of 3 g/kg, resulted in development of the severe forms of hyperhomocysteinemia with serum homocysteine concentrations exceeding 100 µmol/L. This was accompanied by a decrease in the activity of enzymes involved in the bioenergetic processes of the cell: tissue respiration (succinate dehydrogenase) and oxidative phosphorylation (H+-ATPase) in the epididymal head and tail. The change in lactate metabolism included an increase in its level in both the mitochondrial and cytoplasmic fractions of the epididymal head and mitochondria of the epididymal tail, and also simultaneous statistically significant decrease in LDH activity in the mitochondria and cytoplasm of the epididymal head. In male rats with severe hyperhomocysteinemia, an increase in the activity of mitochondrial SOD accompanied by an increase in the carbonylation of mitochondrial proteins in the head and tail of the epididymis was noted. Modeling of hyperhomocysteinemia under conditions of carnitine chloride of administration led to different reactions of the cells of the studied tissues assayed in the epididymal head and tail homogenate. In the epididymal head, carnitine chloride promoted an increase in the mitochondrial lactate concentration and a decrease in the cytoplasmic lactate concentration, as well as an increase in the LDH activity associated with the mitochondrial fraction. These changes were accompanied by an increase in the activity of H+-ATPase in the epididymal, thus suggesting that carnitine chloride stimulated lactate transport of into the mitochondria and its use as an energy substrate under conditions of oxidative stress caused by hyperhomocysteinemia. In the tail tissues, the changes were protective in nature and were associated with a decrease in the formation of oxidatively modified proteins.


Assuntos
Epididimo , Hiper-Homocisteinemia , Animais , Carnitina/metabolismo , Cloretos/metabolismo , Epididimo/metabolismo , Humanos , Hiper-Homocisteinemia/tratamento farmacológico , Hiper-Homocisteinemia/metabolismo , Ácido Láctico/metabolismo , Masculino , Mitocôndrias , Ratos , Ratos Wistar
7.
Int J Mol Sci ; 22(16)2021 Aug 09.
Artigo em Inglês | MEDLINE | ID: mdl-34445284

RESUMO

TMEM16F is involved in several physiological processes, such as blood coagulation, bone development and virus infections. This protein acts both as a Ca2+-dependent phospholipid scramblase and a Ca2+-activated ion channel but several studies have reported conflicting results about the ion selectivity of the TMEM16F-mediated current. Here, we have performed a detailed side-by-side comparison of the ion selectivity of TMEM16F using the whole-cell and inside-out excised patch configurations to directly compare the results. In inside-out configuration, Ca2+-dependent activation was fast and the TMEM16F-mediated current was activated in a few milliseconds, while in whole-cell recordings full activation required several minutes. We determined the relative permeability between Na+ and Cl¯ (PNa/PCl) using the dilution method in both configurations. The TMEM16F-mediated current was highly nonselective, but there were differences depending on the configuration of the recordings. In whole-cell recordings, PNa/PCl was approximately 0.5, indicating a slight preference for Cl¯ permeation. In contrast, in inside-out experiments the TMEM16F channel showed a higher permeability for Na+ with PNa/PCl reaching 3.7. Our results demonstrate that the time dependence of Ca2+ activation and the ion selectivity of TMEM16F depend on the recording configuration.


Assuntos
Anoctaminas/metabolismo , Proteínas de Transferência de Fosfolipídeos/metabolismo , Animais , Ânions/metabolismo , Anoctaminas/genética , Cátions/metabolismo , Cloretos/metabolismo , Células HEK293 , Humanos , Transporte de Íons , Camundongos , Permeabilidade , Proteínas de Transferência de Fosfolipídeos/genética , Sódio/metabolismo
8.
Nat Commun ; 12(1): 4546, 2021 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-34315884

RESUMO

The NLRP3 inflammasome mediates the production of proinflammatory cytokines and initiates inflammatory cell death. Although NLRP3 is essential for innate immunity, aberrant NLRP3 inflammasome activation contributes to a wide variety of inflammatory diseases. Understanding the pathways that control NLRP3 activation will help develop strategies to treat these diseases. Here we identify WNK1 as a negative regulator of the NLRP3 inflammasome. Macrophages deficient in WNK1 protein or kinase activity have increased NLRP3 activation and pyroptosis compared with control macrophages. Mice with conditional knockout of WNK1 in macrophages have increased IL-1ß production in response to NLRP3 stimulation compared with control mice. Mechanistically, WNK1 tempers NLRP3 activation by balancing intracellular Cl- and K+ concentrations during NLRP3 activation. Collectively, this work shows that the WNK1 pathway has a critical function in suppressing NLRP3 activation and suggests that pharmacological inhibition of this pathway to treat hypertension might have negative clinical implications.


Assuntos
Cloretos/metabolismo , Inflamassomos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Piroptose , Proteína Quinase 1 Deficiente de Lisina WNK/metabolismo , Animais , Caspase 1/metabolismo , Feminino , Imidazóis/farmacologia , Imunidade Inata/efeitos dos fármacos , Interleucina-1beta/metabolismo , L-Lactato Desidrogenase/metabolismo , Lipopolissacarídeos/farmacologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Modelos Biológicos , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Potássio/metabolismo , Piroptose/efeitos dos fármacos , Pirrolidinas/farmacologia , Tamoxifeno/farmacologia , Proteína Quinase 1 Deficiente de Lisina WNK/antagonistas & inibidores
9.
Biochemistry ; 60(32): 2463-2470, 2021 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-34319067

RESUMO

The role of glutamate in excitatory neurotransmission depends on its transport into synaptic vesicles by the vesicular glutamate transporters (VGLUTs). The three VGLUT isoforms exhibit a complementary distribution in the nervous system, and the knockout of each produces severe, pleiotropic neurological effects. However, the available pharmacology lacks sensitivity and specificity, limiting the analysis of both transport mechanism and physiological role. To develop new molecular probes for the VGLUTs, we raised six mouse monoclonal antibodies to VGLUT2. All six bind to a structured region of VGLUT2, five to the luminal face, and one to the cytosolic. Two are specific to VGLUT2, whereas the other four bind to both VGLUT1 and 2; none detect VGLUT3. Antibody 8E11 recognizes an epitope spanning the three extracellular loops in the C-domain that explains the recognition of both VGLUT1 and 2 but not VGLUT3. 8E11 also inhibits both glutamate transport and the VGLUT-associated chloride conductance. Since the antibody binds outside the substrate recognition site, it acts allosterically to inhibit function, presumably by restricting conformational changes. The isoform specificity also shows that allosteric inhibition provides a mechanism to distinguish between closely related transporters.


Assuntos
Anticorpos Monoclonais/imunologia , Proteínas Vesiculares de Transporte de Glutamato/imunologia , Regulação Alostérica/imunologia , Animais , Cloretos/metabolismo , Epitopos/química , Epitopos/imunologia , Ácido Glutâmico/metabolismo , Células HEK293 , Humanos , Isoformas de Proteínas/imunologia , Proteína Vesicular 1 de Transporte de Glutamato/química , Proteína Vesicular 1 de Transporte de Glutamato/imunologia , Proteína Vesicular 2 de Transporte de Glutamato/química , Proteína Vesicular 2 de Transporte de Glutamato/imunologia , Proteínas Vesiculares de Transporte de Glutamato/química , Xenopus laevis
10.
Int J Mol Sci ; 22(11)2021 Jun 02.
Artigo em Inglês | MEDLINE | ID: mdl-34199520

RESUMO

Autosomal dominant polycystic kidney disease (ADPKD) is caused by loss of function of PKD1 (polycystin 1) or PKD2 (polycystin 2). The Ca2+-activated Cl- channel TMEM16A has a central role in ADPKD. Expression and function of TMEM16A is upregulated in ADPKD which causes enhanced intracellular Ca2+ signaling, cell proliferation, and ion secretion. We analyzed kidneys from Pkd1 knockout mice and found a more pronounced phenotype in males compared to females, despite similar levels of expression for renal tubular TMEM16A. Cell proliferation, which is known to be enhanced with loss of Pkd1-/-, was larger in male when compared to female Pkd1-/- cells. This was paralleled by higher basal intracellular Ca2+ concentrations in primary renal epithelial cells isolated from Pkd1-/- males. The results suggest enhanced intracellular Ca2+ levels contributing to augmented cell proliferation and cyst development in male kidneys. Enhanced resting Ca2+ also caused larger basal chloride currents in male primary cells, as detected in patch clamp recordings. Incubation of mouse primary cells, mCCDcl1 collecting duct cells or M1 collecting duct cells with dihydrotestosterone (DHT) enhanced basal Ca2+ levels and increased basal and ATP-stimulated TMEM16A chloride currents. Taken together, the more severe cystic phenotype in males is likely to be caused by enhanced cell proliferation, possibly due to enhanced basal and ATP-induced intracellular Ca2+ levels, leading to enhanced TMEM16A currents. Augmented Ca2+ signaling is possibly due to enhanced expression of Ca2+ transporting/regulating proteins.


Assuntos
Anoctamina-1/genética , Doenças Renais Policísticas/genética , Canais de Cátion TRPP/genética , Animais , Cálcio/metabolismo , Sinalização do Cálcio/genética , Proliferação de Células/genética , Cloretos/metabolismo , Di-Hidrotestosterona/farmacologia , Modelos Animais de Doenças , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Rim/metabolismo , Rim/patologia , Masculino , Camundongos , Camundongos Knockout , Fenótipo , Doenças Renais Policísticas/metabolismo , Doenças Renais Policísticas/patologia , Caracteres Sexuais
11.
PLoS Biol ; 19(7): e3001355, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34319985

RESUMO

Sensing and response to environmental cues, such as pH and chloride (Cl-), is critical in enabling Mycobacterium tuberculosis (Mtb) colonization of its host. Utilizing a fluorescent reporter Mtb strain in a chemical screen, we have identified compounds that dysregulate Mtb response to high Cl- levels, with a subset of the hits also inhibiting Mtb growth in host macrophages. Structure-activity relationship studies on the hit compound "C6," or 2-(4-((2-(ethylthio)pyrimidin-5-yl)methyl)piperazin-1-yl)benzo[d]oxazole, demonstrated a correlation between compound perturbation of Mtb Cl- response and inhibition of bacterial growth in macrophages. C6 accumulated in both bacterial and host cells, and inhibited Mtb growth in cholesterol media, but not in rich media. Subsequent examination of the Cl- response of Mtb revealed an intriguing link with bacterial growth in cholesterol, with increased transcription of several Cl--responsive genes in the simultaneous presence of cholesterol and high external Cl- concentration, versus transcript levels observed during exposure to high external Cl- concentration alone. Strikingly, oral administration of C6 was able to inhibit Mtb growth in vivo in a C3HeB/FeJ murine infection model. Our work illustrates how Mtb response to environmental cues can intersect with its metabolism and be exploited in antitubercular drug discovery.


Assuntos
Antituberculosos/farmacologia , Desenvolvimento de Medicamentos , Mycobacterium tuberculosis/efeitos dos fármacos , Animais , Antituberculosos/química , Cloretos/metabolismo , Colesterol/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Macrófagos/microbiologia , Camundongos , Testes de Sensibilidade Microbiana , Mycobacterium tuberculosis/crescimento & desenvolvimento , Relação Estrutura-Atividade
12.
Biomed Pharmacother ; 139: 111615, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34243598

RESUMO

BACKGROUND: Severe acidosis deteriorates cardiac injury. Rat coronary arteries (RCAs) are unusually hypercontractive to extracellular (o) acidosis (EA). TMEM16A-encoded anoctamin 1 (ANO1), a Ca2+-activated chloride channel (CaCC), plays an important role in regulating coronary arterial tension. PURPOSE: We tested the possibility that the activation of CaCCs in the arterial smooth muscle cell (ASMC) contributes to EA-induced RCA constriction. METHODS: ANO1 expression was detected with immunofluorescence staining and Western blot. TMEM16A mRNA was assessed with quantitative Real-Time PCR. Cl- currents and membrane potentials were quantified with a patch clamp. The vascular tension was recorded with a myograph. Intracellular (i) level of Cl- and Ca2+ was measured with fluorescent molecular probes. RESULTS: ANO1 was expressed in all tested arterial myocytes, but was much more abundant in RCA ASMCs as compared with ASMCs isolated from rat cerebral basilar, intrarenal and mesenteric arteries. EA reduced [Cl-]i levels, augmented CaCC currents exclusively in RCA ASMCs and depolarized RCA ASMCs to a greater extent. Cl- deprivation, which depleted [Cl-]i by incubating the arteries or their ASMCs in Cl--free bath solution, decreased EA-induced [Cl-]i reduction, diminished EA-induced CaCC augmentation and time-dependently depressed EA-induced RCA constriction. Inhibitor studies showed that these EA-induced effects including RCA constriction, CaCC current augmentation, [Cl-]i reduction and/or [Ca2+]i elevation were depressed by various Cl- channel blockers, [Ca2+]i release inhibitors and L-type voltage-gated Ca2+ channel inhibitor nifedipine. ANO1 antibody attenuated all observed changes induced by EA in RCA ASMCs. CONCLUSION: The greater activity of RCA ASMC CaCCs complicated with an enhanced Ca2+ mobilization from both [Ca2+]i release and [Ca2+]o influx plays a pivotal role in the distinctive hypercontractility of RCAs to acidosis. Translation of these findings to human beings may lead to a new conception in our understanding and treating cardiac complications in severe acidosis.


Assuntos
Acidose/metabolismo , Anoctamina-1/metabolismo , Vasos Coronários/metabolismo , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Vasoconstrição/fisiologia , Acidose/tratamento farmacológico , Animais , Cálcio/metabolismo , Canais de Cloreto/metabolismo , Cloretos/metabolismo , Vasos Coronários/efeitos dos fármacos , Masculino , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Artérias Mesentéricas/efeitos dos fármacos , Artérias Mesentéricas/metabolismo , Células Musculares/efeitos dos fármacos , Células Musculares/metabolismo , Músculo Liso Vascular/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , Nifedipino/farmacologia , Técnicas de Patch-Clamp/métodos , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Vasoconstrição/efeitos dos fármacos
13.
J Phys Chem B ; 125(29): 7975-7984, 2021 07 29.
Artigo em Inglês | MEDLINE | ID: mdl-34260231

RESUMO

Canonical descriptions of multistep biomolecular transformations generally follow a single-pathway viewpoint, with a series of transitions through intermediates converting reactants to products or repeating a conformational cycle. However, mounting evidence suggests that more complexity and pathway heterogeneity are mechanistically relevant due to the statistical distribution of multiple interconnected rate processes. Making sense of such pathway complexity remains a significant challenge. To better understand the role and relevance of pathway heterogeneity, we herein probe the chemical reaction network of a Cl-/H+ antiporter, ClC-ec1, and analyze reaction pathways using multiscale kinetic modeling (MKM). This approach allows us to describe the nature of the competing pathways and how they change as a function of pH. We reveal that although pH-dependent Cl-/H+ transport rates are largely regulated by the charge state of amino acid E148, the charge state of E203 determines relative contributions from coexisting pathways and can shift the flux pH-dependence. The selection of pathways via E203 explains how ionizable mutations (D/H/K/R) would impact the ClC-ec1 bioactivity from a kinetic perspective and lends further support to the indispensability of an internal glutamate in ClC antiporters. Our results demonstrate how quantifying the kinetic selection of competing pathways under varying conditions leads to a deeper understanding of the Cl-/H+ exchange mechanism and can suggest new approaches for mechanistic control.


Assuntos
Antiporters , Ácido Glutâmico , Antiporters/metabolismo , Canais de Cloreto/metabolismo , Cloretos/metabolismo , Concentração de Íons de Hidrogênio , Cinética
14.
Molecules ; 26(13)2021 Jul 05.
Artigo em Inglês | MEDLINE | ID: mdl-34279441

RESUMO

This paper is focused on the in vivo release and biocompatibility evaluation in rats of some novel systems entrapping zinc chloride in lipid vesicles. The particles were prepared by zinc chloride immobilization inside lipid vesicles made using phosphatidylcholine, stabilized with 0.5% chitosan solution, and dialyzed for 10 h to achieve a neutral pH. The submicrometric systems were physico-chemically characterized. White Wistar rats, assigned into four groups of six animals each, were treated orally with a single dose, as follows: Group I (control): deionized water 0.3 mL/100 g body weight; Group II (Zn): 2 mg/kg body weight (kbw) zinc chloride; Group III (LV-Zn): 2 mg/kbw zinc chloride in vesicles; Group IV (LVC-Zn): 2 mg/kbw zinc chloride in vesicles stabilized with chitosan. Haematological, biochemical, and immune parameters were assessed after 24 h and 7 days, and then liver fragments were collected for histopathological examination. The use of zinc submicrometric particles-especially those stabilized with chitosan-showed a delayed zinc release in rats. No substantial changes to blood parameters, plasma biochemical tests, serum complement activity, or peripheral neutrophils phagocytic capacity were noted; moreover, the tested substances did not induce liver architectural disturbances. The obtained systems provided a delayed release of zinc, and showed good biocompatibility in rats.


Assuntos
Quitosana/química , Cloretos/análise , Cloretos/metabolismo , Lipídeos/química , Lipossomos/química , Compostos de Zinco/análise , Compostos de Zinco/metabolismo , Animais , Feminino , Masculino , Teste de Materiais , Ratos , Ratos Wistar
15.
PLoS One ; 16(7): e0246224, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34228751

RESUMO

Chloride intracellular channels (CLICs) are a unique family of evolutionarily conserved metamorphic proteins, switching between stable conformations based on redox conditions. CLICs have been implicated in a wide variety biological processes including ion channel activity, apoptosis, membrane trafficking, and enzymatic oxidoreductase activity. Understanding the molecular mechanisms by which CLICs engage in these activities is an area of active research. Here, the sole Drosophila melanogaster ortholog, Clic, was targeted for RNAi knockdown to identify genes and biological processes associated with Clic expression. Clic knockdown had a substantial impact on global transcription, altering expression of over 7% of transcribed Drosophila genes. Overrepresentation analysis of differentially expressed genes identified enrichment of Gene Ontology terms including Cytoplasmic Translation, Oxidation-Reduction Process, Heme Binding, Membrane, Cell Junction, and Nucleolus. The top term, Cytoplasmic Translation, was enriched almost exclusively with downregulated genes. Drosophila Clic and vertebrate ortholog Clic4 have previously been tied to ethanol sensitivity and ethanol-regulated expression. Clic knockdown-responsive genes from the present study were found to overlap significantly with gene sets from 4 independently published studies related to ethanol exposure and sensitivity in Drosophila. Bioinformatic analysis of genes shared between these studies revealed an enrichment of genes related to amino acid metabolism, protein processing, oxidation-reduction processes, and lipid particles among others. To determine whether the modulation of ethanol sensitivity by Clic may be related to co-regulated oxidation-reduction processes, we evaluated the effect of hyperoxia on ethanol sedation in Clic knockdown flies. Consistent with previous findings, Clic knockdown reduced acute ethanol sedation sensitivity in flies housed under normoxia. However, this effect was reversed by exposure to hyperoxia, suggesting a common set of molecular-genetic mechanism may modulate each of these processes. This study suggests that Drosophila Clic has a major influence on regulation of oxidative stress signaling and that this function overlaps with the molecular mechanisms of acute ethanol sensitivity in the fly.


Assuntos
Canais de Cloreto/deficiência , Canais de Cloreto/genética , Cloretos/metabolismo , Drosophila melanogaster/citologia , Etanol/farmacologia , Perfilação da Expressão Gênica , Espaço Intracelular/metabolismo , Animais , Drosophila melanogaster/metabolismo , Técnicas de Silenciamento de Genes , Espaço Intracelular/efeitos dos fármacos , Oxirredução/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos
16.
Biochim Biophys Acta Biomembr ; 1863(10): 183667, 2021 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-34111414

RESUMO

An amphiphilic calix[6]arene, alone or complexed with an axle to form a pseudo-rotaxane, has been embedded into liposomes prepared from 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and the permeability of the membrane-doped liposomes towards Cl- ions has been evaluated by using lucigenin as the fluorescent probe. The pseudo-rotaxane promotes transmembrane transport of Cl- ions more than calix[6]arene does. Surprisingly, the quenching of lucigenin was very fast for liposomes doped with the positively charged axle alone. Molecular dynamics (MD) simulations and quantum-chemical calculations were also carried out for providing a semi-quantitative support to the experimental results.


Assuntos
Calixarenos/metabolismo , Cloretos/metabolismo , Ionóforos/metabolismo , Bicamadas Lipídicas , Lipossomos , Biologia Computacional/métodos , Simulação de Dinâmica Molecular , Relação Estrutura-Atividade
17.
Sci Rep ; 11(1): 13127, 2021 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-34162897

RESUMO

The widely expressed two-pore homodimeric inward rectifier CLC-2 chloride channel regulates transepithelial chloride transport, extracellular chloride homeostasis, and neuronal excitability. Each pore is independently gated at hyperpolarized voltages by a conserved pore glutamate. Presumably, exiting chloride ions push glutamate outwardly while external protonation stabilizes it. To understand the mechanism of mouse CLC-2 opening we used homology modelling-guided structure-function analysis. Structural modelling suggests that glutamate E213 interacts with tyrosine Y561 to close a pore. Accordingly, Y561A and E213D mutants are activated at less hyperpolarized voltages, re-opened at depolarized voltages, and fast and common gating components are reduced. The double mutant cycle analysis showed that E213 and Y561 are energetically coupled to alter CLC-2 gating. In agreement, the anomalous mole fraction behaviour of the voltage dependence, measured by the voltage to induce half-open probability, was strongly altered in these mutants. Finally, cytosolic acidification or high extracellular chloride concentration, conditions that have little or no effect on WT CLC-2, induced reopening of Y561 mutants at positive voltages presumably by the inward opening of E213. We concluded that the CLC-2 gate is formed by Y561-E213 and that outward permeant anions open the gate by electrostatic and steric interactions.


Assuntos
Canais de Cloreto/química , Ativação do Canal Iônico , Sequência de Aminoácidos , Animais , Bovinos , Canais de Cloreto/genética , Canais de Cloreto/metabolismo , Cloretos/metabolismo , Humanos , Camundongos , Mutação , Estrutura Terciária de Proteína , Alinhamento de Sequência , Relação Estrutura-Atividade
18.
Elife ; 102021 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-34061021

RESUMO

Genetic mutation of the human BEST1 gene, which encodes a Ca2+-activated Cl- channel (BEST1) predominantly expressed in retinal pigment epithelium (RPE), causes a spectrum of retinal degenerative disorders commonly known as bestrophinopathies. Previously, we showed that BEST1 plays an indispensable role in generating Ca2+-dependent Cl- currents in human RPE cells, and the deficiency of BEST1 function in patient-derived RPE is rescuable by gene augmentation (Li et al., 2017). Here, we report that BEST1 patient-derived loss-of-function and gain-of-function mutations require different mutant to wild-type (WT) molecule ratios for phenotypic manifestation, underlying their distinct epigenetic requirements in bestrophinopathy development, and suggesting that some of the previously classified autosomal dominant mutations actually behave in a dominant-negative manner. Importantly, the strong dominant effect of BEST1 gain-of-function mutations prohibits the restoration of BEST1-dependent Cl- currents in RPE cells by gene augmentation, in contrast to the efficient rescue of loss-of-function mutations via the same approach. Moreover, we demonstrate that gain-of-function mutations are rescuable by a combination of gene augmentation with CRISPR/Cas9-mediated knockdown of endogenous BEST1 expression, providing a universal treatment strategy for all bestrophinopathy patients regardless of their mutation types.


Assuntos
Bestrofinas/genética , Mutação com Ganho de Função , Mutação com Perda de Função , Degeneração Retiniana/genética , Bestrofinas/metabolismo , Sistemas CRISPR-Cas , Cloretos/metabolismo , Predisposição Genética para Doença , Terapia Genética , Células HEK293 , Humanos , Potenciais da Membrana , Fenótipo , Degeneração Retiniana/diagnóstico , Degeneração Retiniana/metabolismo , Degeneração Retiniana/terapia , Epitélio Pigmentado da Retina/metabolismo
19.
Int J Mol Sci ; 22(11)2021 May 29.
Artigo em Inglês | MEDLINE | ID: mdl-34072552

RESUMO

Indirect evidence has determined the possibility that microplastics (MP) induce constipation, although direct scientific proof for constipation induction in animals remains unclear. To investigate whether oral administration of polystyrene (PS)-MP causes constipation, an alteration in the constipation parameters and mechanisms was analyzed in ICR mice, treated with 0.5 µm PS-MP for 2 weeks. Significant alterations in water consumption, stool weight, stool water contents, and stool morphology were detected in MP treated ICR mice, as compared to Vehicle treated group. Also, the gastrointestinal (GI) motility and intestinal length were decreased, while the histopathological structure and cytological structure of the mid colon were remarkably altered in treated mice. Mice exposed to MP also showed a significant decrease in the GI hormone concentration, muscarinic acetylcholine receptors (mAChRs) expression, and their downstream signaling pathway. Subsequent to MP treatment, concentrations of chloride ion and expressions of its channel (CFTR and CIC-2) were decreased, whereas expressions of aquaporin (AQP)3 and 8 for water transportation were downregulated by activation of the mitogen-activated protein kinase (MAPK)/nuclear factor (NF)-κB signaling pathway. These results are the first to suggest that oral administration of PS-MP induces chronic constipation through the dysregulation of GI motility, mucin secretion, and chloride ion and water transportation in the mid colon.


Assuntos
Constipação Intestinal/diagnóstico , Constipação Intestinal/etiologia , Microplásticos/efeitos adversos , Fenótipo , Poliestirenos/efeitos adversos , Animais , Comportamento Animal , Biomarcadores , Fenômenos Químicos , Cloretos/metabolismo , Colo/patologia , Colo/ultraestrutura , Modelos Animais de Doenças , Suscetibilidade a Doenças , Hormônios Gastrointestinais/metabolismo , Motilidade Gastrointestinal , Bombas de Íon/metabolismo , Camundongos , Camundongos Endogâmicos ICR , Microplásticos/química , Mucinas/metabolismo , Poliestirenos/química , Transdução de Sinais , Água/metabolismo
20.
Swiss Med Wkly ; 151: w20496, 2021 04 12.
Artigo em Inglês | MEDLINE | ID: mdl-33934316

RESUMO

Cystic fibrosis (CF) is a genetic disease caused by a bi-allelic mutation of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. When the diagnosis cannot be confirmed by a positive sweat test or/and the identification of two CF-causing variants, international guidelines recommend the use of CFTR functional assays. These tests assess whether CFTR activity is normal or diminished/absent through measurement of CFTR-mediated chloride secretion/absorption. CFTR functional assays are not only useful for diagnostic purposes but can also serve as a surrogate outcome for clinical trials of CFTR modulators, which are emerging therapeutic agents designed to correct the malfunctioning protein. In the near future they could also be used as precision-medicine techniques, to help guidance and optimisation of treatment. Until now, sweat testing has been the only CFTR functional assay available in Switzerland. Since 2020, the Centre Hospitalier Universitaire Vaudois (CHUV) at Lausanne and the Lucerne Children’s Hospital perform nasal potential difference measurement. Moreover, The Ecole Polytechnique Fédérale de Lausanne (EPFL) established a reliable procedure to generate adult intestinal organoids, i.e., stem cell-derived in-vitro grown mini tissues, extracted from rectal biopsies, which can be used to assess CFTR function in vitro. This narrative review describes the most popular CFTR functional assays, as well as their indications, limitations and availability in Switzerland.


Assuntos
Fibrose Cística , Cloretos/análise , Cloretos/metabolismo , Fibrose Cística/diagnóstico , Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Humanos , Mutação , Suor/química , Suor/metabolismo , Suíça
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