44 research outputs found
Localization of the regulatory particle subunit Semi in the 26S proteasome
No full textThe ubiquitin-proteasome system is responsible for regulated protein degradation in the cell with the 26S proteasome acting as its executive arm. The molecular architecture of this 2.5 MDa complex has been established recently, with the notable exception of the small acidic subunit Semi. Here, we localize the C-terminal helix of Semi binding to the PCI domain of the subunit Rpn7 using cryo-electron microscopy single particle reconstruction of proteasomes purified from yeast cells with semi deletion. The approximate position of the N-terminal region of Semi bridging the cleft between Rpn7 and Rpn3 was inferred based on site-specific cross-linking data of the 26S proteasome. Our structural studies indicate that Semi can assume different conformations in different contexts, which supports the idea that Semi functions as a molecular glue stabilizing the Rpn3/Rpn7 heterodimer. (C) 2013 Elsevier Inc. All rights reserved
Glucocorticoids enhance intestinal glucose uptake via the dimerized glucocorticoid receptor in enterocytes
No full textGlucocorticoid (GC) treatment of inflammatory disorders, such as inflammatory bowel disease, causes deranged metabolism, in part by enhanced intestinal resorption of glucose. However, the underlying molecular mechanism is poorly understood. Hence, we investigated transcriptional control of genes reported to be involved in glucose uptake in the small intestine after GC treatment and determined effects of GC on electrogenic glucose transport from transepithelial currents. GR(villinCre) mice lacking the GC receptor (GR) in enterocytes served to identify the target cell of GC treatment and the requirement of the GR itself; GR(dim) mice impaired in dimerization and DNA binding of the GR were used to determine the underlying molecular mechanism. Our findings revealed that oral administration of dexamethasone to wild-type mice for 3 d increased mRNA expression of serum- and GC-inducible kinase 1, sodium-coupled glucose transporter 1, and Na(+)/H(+) exchanger 3, as well as electrogenic glucose transport in the small intestine. In contrast, GR(villinCre) mice did not respond to GC treatment, neither with regard to gene activation nor to glucose transport. GR(dim) mice were also refractory to GC, because dexamethasone treatment failed to increase both, gene expression and electrogenic glucose transport. In addition, the rise in blood glucose levels normally observed after GC administration was attenuated in both mutant mouse strains. We conclude that enhanced glucose transport in vivo primarily depends on gene regulation by the dimerized GR in enterocytes, and that this mechanism contributes to GC-induced hyperglycemia
Deferasirox (Exjade®) significantly improves cardiac T2* in heavily iron-overloaded patients with β-thalassemia major
No full textNoninvasive measurement of tissue iron levels can be assessed using T2* magnetic resonance imaging (MRI) to identify and monitor patients with iron overload. This study monitored cardiac siderosis using T2* MRI in a cohort of 19 heavily iron-overloaded patients with β-thalassemia major receiving iron chelation therapy with deferasirox over an 18-month period. Overall, deferasirox therapy significantly improved mean ± standard deviation cardiac T2* from a baseline of 17.2±10.8 to 21.5±12.8 ms (+25.0percent; P=0.02). A concomitant reduction in median serum ferritin from a baseline of 5,497 to 4,235 ng-mL (-23.0percent; P=0.001), and mean liver iron concentration from 24.2±9.0 to 17.6± 12.9 mg Fe-g dry weight (-27.1percent; P=0.01) was also seen. Improvements were seen in patients with various degrees of cardiac siderosis, including those patients with a baseline cardiac T2* of 10 ms, indicative of high cardiac iron burden. These findings therefore support previous observations that deferasirox is effective in the removal of myocardial iron with concomitant reduction in total body iron. © The Author(s) 2009.Anderson LJ, 2006, ACTA HAEMATOL-BASEL, V115, P106, DOI 10.1159-000089475; Anderson LJ, 2001, EUR HEART J, V22, P2171, DOI 10.1053-euhj.2001.2822; Borgna-Pignatti C, 2004, HAEMATOLOGICA, V89, P1187; BRITTENHAM GM, 1994, NEW ENGL J MED, V331, P567, DOI 10.1056-NEJM199409013310902; Cappellini MD, 2006, BLOOD, V107, P3455, DOI 10.1182-blood-2005-08-3430; Daar S, 2009, HAEMATOL-HEMATOL J, V94, P140, DOI 10.3324-haematol.13845; ELEFTHERIOU P, 2006, HAEMATOLOGICA S1, V91, P999; Garbowski M, 2008, BLOOD, V112, P116; Kolnagou A, 2006, HEMOGLOBIN, V30, P219, DOI 10.1080-03630260600642542; Noetzli LJ, 2008, BLOOD, V112, P2973, DOI 10.1182-blood-2008-04-148767; PENNELL D, 2008, BLOOD, V112, P3874; Pennell DJ, 2008, BLOOD, V112, P3873; Porter J, 2008, EUR J HAEMATOL, V80, P168, DOI 10.1111-j.1600-0609.2007.00985.x; St Pierre TG, 2005, BLOOD, V105, P855, DOI 10.1182-blood-2004-01-0177; Taher A, 2009, EUR J HAEMATOL, V82, P458, DOI 10.1111-j.1600-0609.2009.01228.x; Vichinsky E, 2007, BRIT J HAEMATOL, V136, P501, DOI 10.1111-j.1365-2141.2006.06455.x; Westwood M, 2003, J MAGN RESON IMAGING, V18, P33, DOI 10.1002-jmri.10332; Wood JC, 2004, BLOOD, V104, p111A; Wood JC, 2008, BLOOD, V112, P3882; Wood JC, 2006, TRANSL RES, V148, P272, DOI 10.1016-j.trsl.2006.05.005; ZURLO MG, 1989, LANCET, V2, P2719222
Unraveling new mechanisms of intestinal and renal ion transport : lessons from mouse models
No full textStructural and function analysis of the 19S regulatory particle from Drosophila melanogaster 26S proteasome
No full textStrukturelle und funktionelle Untersuchungen der 19S Untereinheiten regulatorischen Partikel aus dem 26S-Proteasome
No full textThe 26S proteasome executes targeted protein degradation, a process essential for
eukaryotic cells. It consists of proteolytic and regulatory subcomplexes, named CP and
RP, respectively. The latter provides poly-ubiquitin removal and substrate unfolding
activities. In the present thesis crystal structures of the Rpn6 subunit and the
Rpn8-Rpn11 heterodimer complex and their localization in the holocomplex are reported,
which provided crucial insights into the structure and function of the RP.Das 26S Proteasom dient dem gezielten Abbau zellulärer Proteine. Dieser Prozess ist
essentiell in eukaryotische Zellen. Es besteht aus proteolytischen und regulatorischen
Proteinkomplexen (CP und RP). Letztere spalten Polyubiquitinketten ab und entfalten die
Substrate. In der vorgelegten Arbeit werden Kristallstrukturen der Rpn6 Untereinheit und
des Rpn8-Rpn11-Heterodimers sowie deren Lage im Holokomplex beschrieben, welche
entscheidende Erkenntnisse zur Struktur und Funktion des RP lieferten
The solute carrier SLC16A12 is critical for creatine and guanidinoacetate handling in the kidney
No full textA heterozygous mutation (c.643C.A; p.Q215X) in the creatine transporter SLC16A12 was proposed to cause a syndrome with juvenile cataracts, microcornea and glucosuria in humans. To further explore the role of SLC16A12 in renal physiology and decipher the mechanism underlying the phenotype of humans with the SLC16A12 mutation, we studied Slc16a12 knock-out (KO) rats. Slc16a12 KO rats had lower plasma levels and increased absolute and fractional urinary excretion of creatine and its precursor guanidinoacetate (GAA). Slc16a12 KO rats displayed lower plasma and urinary creatinine levels, but GFR was normal. The phenotype of heterozygous rats was indistinguishable from wild-type (WT) rats. Renal artery to vein (RAV) concentration differences in WT rats were negative for GAA and positive for creatinine. However, RAV differences for GAA were similar in Slc16a12 KO rats, indicating incomplete compensation of urinary GAA losses by renal GAA synthesis. Together, our results reveal that Slc16a12 in the basolateral membrane of the proximal tubule is critical for reabsorption of creatine and GAA. Our data suggest a dominant-negative mechanism underlying the phenotype of humans affected by the heterozygous SLC16A12 mutation. Furthermore, in the absence of Slc16a12, urinary losses of GAA are not adequately compensated by increased tubular synthesis, caused by feedback inhibition of the rate limiting enzyme L-arginine:glycine amidinotransferase by creatine in proximal tubular cells
Elevated FGF23 Levels in Mice Lacking the Thiazide-Sensitive NaCl cotransporter (NCC)
No full textAbstract Fibroblast growth factor 23 (FGF23) participates in the orchestration of mineral metabolism by inducing phosphaturia and decreasing the production of 1,25(OH)2D3. It is known that FGF23 release is stimulated by aldosterone and extracellular volume depletion. To characterize this effect further in a model of mild hypovolemia, we studied mice lacking the thiazide sensitive NaCl cotransporter (NCC). Our data indicate that NCC knockout mice (KO) have significantly higher FGF23, PTH and aldosterone concentrations than corresponding wild type (WT) mice. However, 1,25(OH)2D3, fractional phosphate excretion and renal brush border expression of the sodium/phosphate co-transporter 2a were not different between the two genotypes. In addition, renal expression of FGF23 receptor FGFR1 and the co-receptor Klotho were unaltered in NCC KO mice. FGF23 transcript was increased in the bone of NCC KO mice compared to WT mice, but treatment of primary murine osteoblasts with the NCC inhibitor hydrochlorothiazide did not elicit an increase of FGF23 transcription. In contrast, the mineralocorticoid receptor blocker eplerenone reversed excess FGF23 levels in KO mice but not in WT mice, indicating that FGF23 upregulation in NCC KO mice is primarily aldosterone-mediated. Together, our data reveal that lack of renal NCC causes an aldosterone-mediated upregulation of circulating FGF23
Incomplete dRTA in kidney stone formers: diagnostic performance of furosemide/fludrocortisone testing and non-provocative clinical parameters
No full textBackground and objectives: Incomplete distal renal tubular acidosis is a
well-known cause of calcareous nephrolithiasis but the prevalence is
unknown, mostly due to lack of accepted diagnostic tests and criteria. The
ammonium chloride test is considered as gold standard for the diagnosis of
incomplete distal renal tubular acidosis, but the furosemide/fludrocortisone
test was recently proposed as an alternative. Due to the lack of rigorous
comparative studies, the validity of the furosemide/fludrocortisone test in
stone formers remains unknown. In addition, the performance of
conventional, non-provocative parameters in predicting incomplete distal
renal tubular acidosis has not been studied.
Design, setting, participants, and measurements: We conducted a
prospective study in an unselected cohort of 170 stone formers that
underwent sequential ammonium chloride and furosemide/fludrocortisone
testing.
Results: Using the ammonium chloride test as gold standard, the
prevalence of incomplete distal renal tubular acidosis was 7.78 %.
Sensitivity and specificity of the furosemide/fludrocortisone test FF
test were 77 % and 85 %, respectively, yielding a positive predictive
value of 30 % and a negative predictive value of 98 %. Testing of several
non-provocative clinical parameters in the prediction of incomplete distal
renal tubular acidosis revealed fasting morning urinary pH and plasma
potassium as the most discriminative parameters. The combination of a
fasting morning urinary threshold pH <5.3 with a plasma potassium
threshold >3.8 mmolmEq/l yielded a negative predictive value of 98 %
with a sensitivity of 85 % and a specificity of 77 % for the diagnosis of
incomplete distal renal tubular acidosis.
Conclusions: The furosemide/fludrocortisone test can be used for
incomplete distal renal tubular acidosis screening in stone formers, but an
abnormal furosemide/fludrocortisone test result needs confirmation by
ammonium chloride testing. Our data furthermore indicate that incomplete
distal renal tubular acidosis can reliably be excluded in stone formers by
use of non-provocative clinical parameters
Changes in V-ATPase subunits of human urinary exosomes reflect the renal response to acute acid/alkali loading and the defects in distal renal tubular acidosis
No full textIn the kidney, final urinary acidification is achieved by V-ATPases expressed in type A intercalated cells. The B1 subunit of the V-ATPase is required for maximal urinary acidification, while the role of the homologous B2 subunit is less clear. Here we examined the effect of acute acid/alkali loading in humans on B1 and B2 subunit abundance in urinary exosomes in normal individuals and of acid loading in patients with distal renal tubular acidosis (dRTA). Specificities of B1 and B2 subunit antibodies were verified by yeast heterologously expressing human B1 and B2 subunits, and murine wild-type and B1-deleted kidney lysates. Acute ammonium chloride loading elicited systemic acidemia, a drop in urinary pH, and increased urinary ammonium excretion. Nadir urinary pH was achieved at four to five hours, and exosomal B1 abundance was significantly increased at two through six hours after ammonium chloride loading. After acute equimolar sodium bicarbonate loading, blood and urinary pH rose rapidly, with a concomitant reduction of exosomal B1 abundance within two hours, which remained lower throughout the test. In contrast, no change in exosomal B2 abundance was found following acid or alkali loading. In patients with inherited or acquired distal RTA, the urinary B1 subunit was extremely low or undetectable and did not respond to acid loading in urine, whereas no change in B2 subunit was found. Thus, both B1 and B2 subunits of the V-ATPase are detectable in human urinary exosomes, and acid and alkali loading or distal RTA cause changes in the B1 but not B2 subunit abundance in urinary exosomes
