scholarly journals DHHC7-mediated palmitoylation of the accessory protein barttin critically regulates the functions of ClC-K chloride channels

2020 ◽  
Vol 295 (18) ◽  
pp. 5970-5983 ◽  
Author(s):  
Nataliya Gorinski ◽  
Daniel Wojciechowski ◽  
Daria Guseva ◽  
Dalia Abdel Galil ◽  
Franziska E. Mueller ◽  
...  
Keyword(s):  
Chloride Channels ◽  
Bartter Syndrome ◽  
Post Translational Modification ◽  
Salt Diet ◽  
Type Iv ◽  
Low Salt ◽  
Accessory Subunit ◽  
Palmitoyl Acyltransferase

Barttin is the accessory subunit of the human ClC-K chloride channels, which are expressed in both the kidney and inner ear. Barttin promotes trafficking of the complex it forms with ClC-K to the plasma membrane and is involved in activating this channel. Barttin undergoes post-translational palmitoylation that is essential for its functions, but the enzyme(s) catalyzing this post-translational modification is unknown. Here, we identified zinc finger DHHC-type containing 7 (DHHC7) protein as an important barttin palmitoyl acyltransferase, whose depletion affected barttin palmitoylation and ClC-K-barttin channel activation. We investigated the functional role of barttin palmitoylation in vivo in Zdhhc7−/− mice. Although palmitoylation of barttin in kidneys of Zdhhc7−/− animals was significantly decreased, it did not pathologically alter kidney structure and functions under physiological conditions. However, when Zdhhc7−/− mice were fed a low-salt diet, they developed hyponatremia and mild metabolic alkalosis, symptoms characteristic of human Bartter syndrome (BS) type IV. Of note, we also observed decreased palmitoylation of the disease-causing R8L barttin variant associated with human BS type IV. Our results indicate that dysregulated DHHC7-mediated barttin palmitoylation appears to play an important role in chloride channel dysfunction in certain BS variants, suggesting that targeting DHHC7 activity may offer a potential therapeutic strategy for reducing hypertension.

Generation and analyses of R8L barttin knockin mouse

2011 ◽  
Vol 301 (2) ◽  
pp. F297-F307 ◽  
Author(s):  
Naohiro Nomura ◽  
Masato Tajima ◽  
Noriko Sugawara ◽  
Tetsuji Morimoto ◽  
Yoshiaki Kondo ◽  
...  
Keyword(s):  
Chloride Channels ◽  
Plasma Membranes ◽  
Chloride Transport ◽  
Mdck Cells ◽  
Intracellular Localization ◽  
Bartter Syndrome ◽  
K Channel ◽  
Type Iv ◽  
Knockin Mouse

Barttin, a gene product of BSND, is one of four genes responsible for Bartter syndrome. Coexpression of barttin with ClC-K chloride channels dramatically induces the expression of ClC-K current via insertion of ClC-K-barttin complexes into plasma membranes. We previously showed that stably expressed R8L barttin, a disease-causing missense mutant, is retained in the endoplasmic reticulum (ER) of Madin-Darby canine kidney (MDCK) cells, with the barttin β-subunit remaining bound to ClC-K α-subunits (Hayama A, Rai T, Sasaki S, Uchida S. Histochem Cell Biol 119: 485–493, 2003). However, transient expression of R8L barttin in MDCK cells was reported to impair ClC-K channel function without affecting its subcellular localization. To investigate the pathogenesis in vivo, we generated a knockin mouse model of Bartter syndrome that carries the R8L mutation. These mice display disease-like phenotypes (hypokalemia, metabolic alkalosis, and decreased NaCl reabsorption in distal tubules) under a low-salt diet. Immunofluorescence and immunoelectron microscopy revealed that the plasma membrane localization of both R8L barttin and the ClC-K channel was impaired in these mice, and transepithelial chloride transport in the thin ascending limb of Henle's loop (tAL) as well as thiazide-sensitive chloride clearance were significantly reduced. This reduction in transepithelial chloride transport in tAL, which is totally dependent on ClC-K1/barttin, correlated well with the reduction in the amount of R8L barttin localized to plasma membranes. These results suggest that the major cause of Bartter syndrome type IV caused by R8L barttin mutation is its aberrant intracellular localization.

Functional analysis of the SUMOylation pathway in Drosophila

2008 ◽  
Vol 36 (5) ◽  
pp. 868-873 ◽  
Author(s):  
Ana Talamillo ◽  
Jonatan Sánchez ◽  
Rosa Barrio
Keyword(s):  
Functional Analysis ◽  
Fine Tuning ◽  
Model Systems ◽  
Post Translational Modification ◽  
Reversible Process ◽  
Cellular Processes ◽  
Tuning Mechanism ◽  
Sumo Conjugation

SUMOylation, a reversible process used as a ‘fine-tuning’ mechanism to regulate the role of multiple proteins, is conserved throughout evolution. This post-translational modification affects several cellular processes by the modulation of subcellular localization, activity or stability of a variety of substrates. A growing number of proteins have been identified as targets for SUMOylation, although, for many of them, the role of SUMO conjugation on their function is unknown. The use of model systems might facilitate the study of SUMOylation implications in vivo. In the present paper, we have compiled what is known about SUMOylation in Drosophila melanogaster, where the use of genetics provides new insights on SUMOylation's biological roles.

scholarly journals SAT-006 A New Concept for the Endocrinology of Pre-Eclampsia: The Role of Spiral Steroids

2020 ◽  
Vol 4 (Supplement_1) ◽  
Author(s):  
Fred I Chasalow ◽  
Ron Bochner
Keyword(s):  
Type A ◽  
Structural Features ◽  
Type B ◽  
Salt Diet ◽  
Breast Cyst ◽  
High K ◽  
Epithelial Na Channels ◽  
Low Salt

Abstract Background: In 1987, Graves observed that during the 3rd trimester, some patients with pre-eclampsia had high levels of unknown materials that could be detected with assays for digoxin (DLM). In 2018, we characterized a new candidate for the DLM, Ionotropin. It is a phosphocholine (PC) ester of a novel steroid with 23 carbon atoms. As Ionotropin shares structural features (a) with spironolactone (both have spiral lactones in the E-ring) and (b) with digoxin (E-ring lactone and 3α-5β configuration), we have proposed that Ionotropin may function as a potassium (K+) sparing diuretic. This suggestion is supported by the observations that [1] patients who cannot make Ionotropin (7-dehydrosterol reductase deficiency) are K+ wasting and [2] breast cyst fluids with high K+ levels also have high Ionotropin levels. Hypothesis: During the 3rd trimester, fetal requirements for K+ reach a maximum, fetal blood pressure increases and aldosterone signaling is blocked. This blockage leads to fetal sodium (Na+) wasting and is essential for formation of amniotic fluid. These events are consistent with a normal role for an unknown endogenous K+ sparing hormone and would be the basis for a modest elevation of maternal DLM during the 3rd trimester. Our hypothesis is that if any of the functions were inadequate, then the fetal-placental unit would synthesize excess PC-spiral steroids; the woman would exhibit symptoms of K+ sparing hormone excess (hypertension and proteinuria) and would be diagnosed with pre-eclampsia. Experimental Results: We have just reported a pilot study associating elevated PC esters of spiral steroids in women with pre-eclampsia. In brief, 12 of 19 women had elevated levels of at least one of the PC steroids (Z-score > 2) when compared to the levels in 20 pregnant women matched for gestational age and fetal sex. There are two basic mechanisms for this dichotomy: (a) there may be episodic secretion with of a DLM with a short half-life or (b) there may be two different underlying biochemical causes. In prior studies, there has been no indication of episodic secretion of DLM similar to that observed with glucocorticoids, Ionotropin or other PC spiral steroids. Discussion: There are two basic types of K+ sparing diuretics. Type A: Spironolactone functions by regulating the NaK-ATPase. Type B: Triamterene functions by blocking synthesis of epithelial Na+ channels. Thus, Type A would have high levels of spiral steroids and Type B would have low levels of spiral steroids. Type A patients would be expected to have higher risk of long-term consequences when compared to the Type B patients. Conclusion: The recognition of the division of pre-eclampsia into two separate diseases might be the key observation for developing Type-specific diagnosis and therapy. For example, a Type A patient might benefit from a low salt diet but that diet would not be expected to benefit a patient with Type B disease.

scholarly journals Update on Peripheral Arterial Vasodilation, Ascites and Hepatorenal Syndrome in Cirrhosis

2000 ◽  
Vol 14 (suppl d) ◽  
pp. 112D-121D ◽  
Author(s):  
Mladen Knotek ◽  
Boris Rogachev ◽  
Robert W Schrier
Keyword(s):  
Hepatorenal Syndrome ◽  
Decompensated Cirrhosis ◽  
Arterial Tree ◽  
Salt Diet ◽  
Bacterial Peritonitis ◽  
Low Salt ◽  
Intrahepatic Portosystemic Shunt ◽  
Peripheral Arterial ◽  
Orally Active

In cirrhosis of the liver, according to the peripheral arterial vasodilation hypothesis, relative underfilling of the arterial tree triggers a neurohumoral response (activation of renin-angiotensinaldosterone system, sympathetic nervous system, nonosmotic release of vasopressin) aimed at restoring circulatory integrity by promoting renal sodium and water retention. Evidence has accumulated for a major role of increased vascular production of nitric oxide as the primary cause of arterial vasodilation in cirrhosis. Ascites is a common complication in cirrhosis. Treatment of ascites consists of a low salt diet with diuretics, and paracentesis together with plasma volume expanders in diuretic-resistant patients. Progression of cirrhosis may result in hepatorenal syndrome, a state of functional renal failure that carries an ominous prognosis. Orthotopic liver transplantation has remained the only curative treatment for patients with advanced liver disease; other modalities such as transjugular intrahepatic portosystemic shunt or vasopressin analogues may serve as a bridge to transplantation. Another complication of decompensated cirrhosis is spontaneous bacterial peritonitis, the incidence of which can be reduced by primary or secondary antibiotic prophylaxis by using orally active antibiotics.

scholarly journals In vivo vizualisation of mono-ADP-ribosylation by dPARP16 upon amino-acid starvation

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Angelica Aguilera-Gomez ◽  
Marinke M van Oorschot ◽  
Tineke Veenendaal ◽  
Catherine Rabouille
Keyword(s):  
Amino Acid ◽  
Metabolic Stress ◽  
Amino Acid Starvation ◽  
Critical Event ◽  
Post Translational Modification ◽  
Adp Ribosylation ◽  
Body Component ◽  
Necessary And Sufficient

PARP catalysed ADP-ribosylation is a post-translational modification involved in several physiological and pathological processes, including cellular stress. In order to visualise both Poly-, and Mono-, ADP-ribosylation in vivo, we engineered specific fluorescent probes. Using them, we show that amino-acid starvation triggers an unprecedented display of mono-ADP-ribosylation that governs the formation of Sec body, a recently identified stress assembly that forms in Drosophila cells. We show that dPARP16 catalytic activity is necessary and sufficient for both amino-acid starvation induced mono-ADP-ribosylation and subsequent Sec body formation and cell survival. Importantly, dPARP16 catalyses the modification of Sec16, a key Sec body component, and we show that it is a critical event for the formation of this stress assembly. Taken together our findings establish a novel example for the role of mono-ADP-ribosylation in the formation of stress assemblies, and link this modification to a metabolic stress.

Abstract 294: Evaluation of the Role of the NAD(P)H Oxidase Subunit p67phox in the Regulation of Glomerular Filtration Rate in Dahl Salt-sensitive Rats

Hypertension ◽  
2013 ◽  
Vol 62 (suppl_1) ◽  
Author(s):  
Louise C Evans ◽  
Robert P Ryan ◽  
Terry Kurth ◽  
Meredith Skelton ◽  
Allen W Cowley
Keyword(s):  
Nadph Oxidase ◽  
Pressor Response ◽  
High Salt ◽  
Null Mutation ◽  
High Salt Diet ◽  
Salt Diet ◽  
Low Salt ◽  
Species Production ◽  
Stable Control

Studies were performed in male Dahl salt-sensitive (SS) rats with a null mutation in the p67 phox gene (SSp67 phox -/-) and wild type littermates (SS-WT) aged 10-12 weeks (n=5/6 /group). In previous studies we have shown that this important subunit of NADPH oxidase is upregulated in the SS rat. A new method for sequential measurement of GFR in conscious rats was applied during the consumption of a high salt diet. Utilizing a miniaturized device, disappearance curves of fluorescein isothiocynate (FITC)-sinistrin were determined by transcutaneous excitation and real time detection of the emitted light through the skin. The rats were surgically prepared with femoral venous catheters for the administration of (FITC)-sinistrin and carotid catheters for MAP measurement by telemetry. Baseline measurement of GFR and MAP were made daily to obtain 2 stable control days on low salt (LS, 0.4% NaCl). High salt (HS, 4.0% NaCl) measurements were made on day 2,5,7,14 and 21. In SS-WT rats, HS resulted in a progressive and significant increase in MAP (from 126±2 mmHg during LS to 158±11 mmHg by d21 HS). GFR decreased significantly from 1.50±0.03 ml/min/100g bwgt during LS to 1.26±0.02 by d21 HS. Notably, a significant increase in blood pressure was observed at d7 HS in the SS-WT preceeding the reduction in GFR which did not occur until d14 HS. In rats with the null mutation of the p67 phox gene, the pressor response to HS was blunted, MAP averaged 131±5 mmHg by d21, and there was no significant change in GFR (LS values 1.54±0.07 ml/min/100g bwgt comparable to d21 HS values of 1.41±0.04). In summary, when p67 phox is not functional in the SS rat, the hypertensive response to HS is blunted and GFR is maintained. We conclude that reactive oxygen species production via NADPH oxidase plays a major role in the eventual reduction of GFR in SS rats following the development of hypertension.

scholarly journals Role of the gonads in hypertension-prone rats.

1975 ◽  
Vol 142 (3) ◽  
pp. 748-759 ◽  
Author(s):  
L K Dahl ◽  
K D Knudsen ◽  
E V Ohanian ◽  
M Muirhead ◽  
R Tuthill
Keyword(s):  
Blood Pressure ◽  
Growth Hormone ◽  
Opposite Effect ◽  
Ovarian Function ◽  
Pressure Effect ◽  
High Salt Diet ◽  
Salt Diet ◽  
Pituitary Growth Hormone ◽  
Low Salt

In a genetically hypertension-prone (S) strain of rats it was observed previously that males generally developed hypertension more rapidly on a high salt diet than did females although final pressure ultimately were similar in both sexes. A genetic study had shown that there was no sex-linkage involved in setting blood pressure levels, so it was thought that the gonads might be involved. In the present work, castration of males had no effect on blood pressure but in the females it caused a rise in pressure that could not be distinguished from that in males, both on a high and low salt diet. Castration resulted in greater growth in females than in controls, whereas it had the opposite effect in males. It was speculated that these changes were due to influences on pituitary growth hormone with castration increasing the net output of growth hormone (or enhancing receptor sensitivity to it) in the female and the opposite in the male. From the work of others, there are some data compatible with such an interpretation. Experimentally, growth hormone will induce hypertension in rats. Therefore, it is conceivable that growth hormone is involved in the increment in hypertension observed in these castrate females. Because the effect on blood pressure was observed in castrate females on both high and low NaCl diets, it was considered unlikely that the blood pressure effect was simply due to increased NaCl intake in the food associated with greater growth. It was suggested that this rise in blood pressure with cessation of ovarian function might bear on the unsettled question of "menopausal" hypertension in women: in the genetically susceptible individual an increase in growth hormone associated with declining ovarian funtion in the menopause could provide the stimulus for the appearance of hypertension some years earlier than would otherwise have been the case.

scholarly journals Pseudomonas aeruginosa as a cause of infectious diarrhoea

1998 ◽  
Vol 121 (1) ◽  
pp. 237-241 ◽  
Author(s):  
P. A. ADLARD ◽  
S. M. KIROV ◽  
K. SANDERSON ◽  
G. E. COX
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Signs And Symptoms ◽  
Free Cell ◽  
Persistent Infections ◽  
Infectious Diarrhoea ◽  
Type Iv ◽  
Underlying Illness

Pseudomonas aeruginosa is not generally considered a cause of infectious diarrhoea. However, it was the predominant organism isolated from the faeces of 23 unrelated, hospital outpatients investigated in the course of a year for persistent (>1 week duration) diarrhoea. To investigate the possible aetiological role of P. aeruginosa, these patient histories were reviewed and a selection of their faecal isolates were investigated in vitro (n[ges ]10) and in vivo (n=2) for virulence. The patients had a mean age of 60 years, were receiving antibiotics and/or had an underlying illness. Extensive microbiological investigations identified no other potential or recognized enteropathogen in the faeces of 20 of these patients. More than 40% of the isolates tested were able to adhere to HEp-2 cells and exhibited twitching motility (type IV pili), properties indicative of their ability to colonize the human intestine. Cytotoxic activity was demonstrated in bacterium-free cell supernatants of over 80% of isolates; supernatants of four isolates tested in infant mice were weakly enterotoxigenic. Two isolates intragastrically inoculated into clindamycin pre-treated rats established persistent infections and induced signs and symptoms of enteritis. Overall these findings suggest that P. aeruginosa can cause diarrhoea particularly in immunodeficient individuals.

scholarly journals The Bacterial Second Messenger Cyclic di-GMP Regulates Brucella Pathogenesis and Leads to Altered Host Immune Response

2016 ◽  
Vol 84 (12) ◽  
pp. 3458-3470 ◽  
Author(s):  
Mike Khan ◽  
Jerome S. Harms ◽  
Fernanda M. Marim ◽  
Leah Armon ◽  
Cherisse L. Hall ◽  
...  
Keyword(s):  
Immune Response ◽  
Second Messenger ◽  
Host Immune Response ◽  
Vital Role ◽  
Host Cells ◽  
Content Type ◽  
Type Iv

Brucella species are facultative intracellular bacteria that cause brucellosis, a chronic debilitating disease significantly impacting global health and prosperity. Much remains to be learned about how Brucella spp. succeed in sabotaging immune host cells and how Brucella spp. respond to environmental challenges. Multiple types of bacteria employ the prokaryotic second messenger cyclic di-GMP (c-di-GMP) to coordinate responses to shifting environments. To determine the role of c-di-GMP in Brucella physiology and in shaping host- Brucella interactions, we utilized c-di-GMP regulatory enzyme deletion mutants. Our results show that a Δ bpdA phosphodiesterase mutant producing excess c-di-GMP displays marked attenuation in vitro and in vivo during later infections. Although c-di-GMP is known to stimulate the innate sensor STING, surprisingly, the Δ bpdA mutant induced a weaker host immune response than did wild-type Brucella or the low-c-di-GMP guanylate cyclase Δ cgsB mutant. Proteomics analysis revealed that c-di-GMP regulates several processes critical for virulence, including cell wall and biofilm formation, nutrient acquisition, and the type IV secretion system. Finally, Δ bpdA mutants exhibited altered morphology and were hypersensitive to nutrient-limiting conditions. In summary, our results indicate a vital role for c-di-GMP in allowing Brucella to successfully navigate stressful and shifting environments to establish intracellular infection.

scholarly journals Understanding the Role of Protein Glycation in the Amyloid Aggregation Process

2021 ◽  
Vol 22 (12) ◽  
pp. 6609
Author(s):  
Ivana Sirangelo ◽  
Clara Iannuzzi
Keyword(s):  
Protein Structure ◽  
Protein Function ◽  
The Body ◽  
Protein Glycation ◽  
Amyloid Formation ◽  
Aggregation Process ◽  
Amyloid Aggregation ◽  
Post Translational Modification

Protein function and flexibility is directly related to the native distribution of its structural elements and any alteration in protein architecture leads to several abnormalities and accumulation of misfolded proteins. This phenomenon is associated with a range of increasingly common human disorders, including Alzheimer and Parkinson diseases, type II diabetes, and a number of systemic amyloidosis characterized by the accumulation of amyloid aggregates both in the extracellular space of tissues and as intracellular deposits. Post-translational modifications are known to have an active role in the in vivo amyloid aggregation as able to affect protein structure and dynamics. Among them, a key role seems to be played by non-enzymatic glycation, the most unwanted irreversible modification of the protein structure, which strongly affects long-living proteins throughout the body. This study provided an overview of the molecular effects induced by glycation on the amyloid aggregation process of several protein models associated with misfolding diseases. In particular, we analyzed the role of glycation on protein folding, kinetics of amyloid formation, and amyloid cytotoxicity in order to shed light on the role of this post-translational modification in the in vivo amyloid aggregation process.

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