Effect of lysine to alanine mutations on the phosphate activation and BPTES inhibition of glutaminase.

The GLS1 gene encodes a mitochondrial glutaminase that is highly expressed in brain, kidney, small intestine and many transformed cells. Recent studies have identified multiple lysine residues in glutaminase that are sites of N-acetylation. Interestingly, these sites are located within either a loop segment that regulates access of glutamine to the active site or the dimer:dimer interface that participates in the phosphate-dependent oligomerization and activation of the enzyme.

pH-responsive, gluconeogenic renal epithelial LLC-PK1-FBPase+cells: a versatile in vitro model to study renal proximal tubule metabolism and function.

Ammoniagenesis and gluconeogenesis are prominent metabolic features of the renal proximal convoluted tubule that contribute to maintenance of systemic acid-base homeostasis. Molecular analysis of the mechanisms that mediate the coordinate regulation of the two pathways required development of a cell line that recapitulates these features in vitro. By adapting porcine renal epithelial LLC-PK1 cells to essentially glucose-free medium, a gluconeogenic subline, termed LLC-PK1-FBPase(+) cells, was isolated.

Proximal tubule function and response to acidosis.

The human kidneys produce approximately 160-170 L of ultrafiltrate per day. The proximal tubule contributes to fluid, electrolyte, and nutrient homeostasis by reabsorbing approximately 60%-70% of the water and NaCl, a greater proportion of the NaHCO3, and nearly all of the nutrients in the ultrafiltrate. The proximal tubule is also the site of active solute secretion, hormone production, and many of the metabolic functions of the kidney.

Proteomic profiling and pathway analysis of the response of rat renal proximal convoluted tubules to metabolic acidosis.

Metabolic acidosis is a relatively common pathological condition that is defined as a decrease in blood pH and bicarbonate concentration. The renal proximal convoluted tubule responds to this condition by increasing the extraction of plasma glutamine and activating ammoniagenesis and gluconeogenesis. The combined processes increase the excretion of acid and produce bicarbonate ions that are added to the blood to partially restore acid-base homeostasis.

Proteomic profiling of the mitochondrial inner membrane of rat renal proximal convoluted tubules.

The proximal convoluted tubule is the primary site of renal fluid, electrolyte, and nutrient reabsorption, processes that consume large amounts of adenosine-5'-triphosphate. Previous proteomic studies have profiled the adaptions that occur in this segment of the nephron in response to the onset of metabolic acidosis. To extend this analysis, a proteomic workflow was developed to characterize the proteome of the mitochondrial inner membrane of the rat renal proximal convoluted tubule.

Response of the mitochondrial proteome of rat renal proximal convoluted tubules to chronic metabolic acidosis.

Metabolic acidosis is a common clinical condition that is caused by a decrease in blood pH and bicarbonate concentration. Increased extraction and mitochondrial catabolism of plasma glutamine within the renal proximal convoluted tubule generates ammonium and bicarbonate ions that facilitate the excretion of acid and partially restore acid-base balance. Previous studies identified only a few mitochondrial proteins, including two key enzymes of glutamine metabolism, which are increased during chronic acidosis.

Concurrent binding and modifications of AUF1 and HuR mediate the pH-responsive stabilization of phosphoenolpyruvate carboxykinase mRNA in kidney cells.

Onset of metabolic acidosis leads to a pronounced increase in renal expression of phosphoenolpyruvate carboxykinase (PEPCK). This response, which is mediated in part by stabilization of PEPCK mRNA, is effectively modeled by treating LLC-PK(1)-F(+)-9C cells with an acidic medium. siRNA knockdown of HuR prevented the pH-responsive increase in PEPCK mRNA half-life suggesting that HuR is necessary for this response.

Proteomic profiling of the effect of metabolic acidosis on the apical membrane of the proximal convoluted tubule.

The physiological response to the onset of metabolic acidosis requires pronounced changes in renal gene expression. Adaptations within the proximal convoluted tubule support the increased extraction of plasma glutamine and the increased synthesis and transport of glucose and of NH(4)(+) and HCO(3)(-) ions. Many of these adaptations involve proteins associated with the apical membrane.

BPTES inhibition of hGA(124-551), a truncated form of human kidney-type glutaminase.

The initial transcript of the GLS1 gene undergoes alternative splicing to produce two glutaminase variants (KGA and GAC) that contain unique C-terminal sequences. A truncated form of human glutaminase (hGA(124-551)) that lacks either C-terminal sequence was expressed in E.Coli and purified.

Role of AUF1 and HuR in the pH-responsive stabilization of phosphoenolpyruvate carboxykinase mRNA in LLC-PK₁-F⁺ cells.

Onset of metabolic acidosis leads to a rapid and pronounced increase in expression of phosphoenolpyruvate carboxykinase (PEPCK) in rat renal proximal convoluted tubules. This adaptive response is modeled by treating a clonal line of porcine LLC-PK(1)-F(+) cells with an acidic medium (pH 6.9, 9 mM HCO(3)(-)).

Importance of manual validation for the identification of phosphopeptides using a linear ion trap mass spectrometer.

Accurate determination of protein phosphorylation is challenging, particularly for researchers who lack access to a high-accuracy mass spectrometer. In this study, multiple protocols were used to enrich phosphopeptides, and a rigorous filtering workflow was used to analyze the resulting samples. Phosphopeptides were enriched from cultured rat renal proximal tubule cells using three commonly used protocols and a dual method that combines separate immobilized metal affinity chromatography (IMAC) and titanium dioxide (TiO(2)) chromatography, termed dual IMAC (DIMAC).

Proteomic analysis of brush-border membrane vesicles isolated from purified proximal convoluted tubules.

The renal proximal convoluted tubule is the primary site of water, electrolyte and nutrient reabsorption and of active secretion of selected molecules. Proteins in the apical brush-border membrane facilitate these functions and initiate some of the cellular responses to altered renal physiology. The current study uses two-dimensional liquid chromatography/mass spectrometry to compare brush border membrane vesicles isolated from rat renal cortex (BBMV(CTX)) and from purified proximal convoluted tubules (BBMV(PCT)).

Zeta-crystallin: a tale of two cells.

Szutkowska et al. demonstrate that zeta-crystallin plays an essential role in the stabilization of the Na(+)/K(+)/2Cl(-) cotransporter mRNA in the medullary thick ascending limb. However, differential effects of experiments using small interfering RNA to knock down zeta-crystallin in proximal tubule and thick ascending limb cells suggest that additional proteins must contribute to the rapid turnover and selective stabilization of the various mRNAs during metabolic acidosis.

TGF-beta signaling and its effect on glutaminase expression in LLC-PK1-FBPase+ cells.

During systemic acidosis, renal proximal tubular cells exhibit enhanced rates of bicarbonate and ammonium ion synthesis and undergo extensive hypertrophy. The former adaptations are accomplished, in part, by increased expression of glutaminase (GA). LLC-PK(1)-FBPase+ cells, a gluconeogenic line of porcine kidney cells, exhibit a rapid activation of the ERK1/2 and p38 MAPK pathways and a two- to threefold increase in GA mRNA when transferred to acidic medium (pH 6.

Novel mechanism of inhibition of rat kidney-type glutaminase by bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl)ethyl sulfide (BPTES).

The release of GA (mitochondrial glutaminase) from neurons following acute ischaemia or during chronic neurodegenerative diseases may contribute to the propagation of glutamate excitotoxicity. Thus an inhibitor that selectively inactivates the released GA may limit the accumulation of excess glutamate and minimize the loss of neurological function that accompanies brain injury. The present study examines the mechanism of inactivation of rat KGA (kidney GA isoform) by the small-molecule inhibitor BPTES [bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl)ethyl sulfide].

Glutamatergic or GABAergic neuron-specific, long-term expression in neocortical neurons from helper virus-free HSV-1 vectors containing the phosphate-activated glutaminase, vesicular glutamate transporter-1, or glutamic acid decarboxylase promoter.

Many potential uses of direct gene transfer into neurons require restricting expression to one of the two major types of forebrain neurons, glutamatergic or GABAergic neurons. Thus, it is desirable to develop virus vectors that contain either a glutamatergic or GABAergic neuron-specific promoter. The brain/kidney phosphate-activated glutaminase (PAG), the product of the GLS1 gene, produces the majority of the glutamate for release as neurotransmitter, and is a marker for glutamatergic neurons.

Proteomic analysis of the adaptive response of rat renal proximal tubules to metabolic acidosis.

Proximal tubules were isolated from control and acidotic rats by collagenase digestion and Percoll density gradient centrifugation. Western blot analysis indicated that the tubules were approximately 95% pure. The samples were analyzed by two-dimensional difference gel electrophoresis (DIGE) and DeCyder software was used to quantify the temporal changes in proteins that exhibit enhanced or reduced expression.

Effects of constitutively active and dominant negative MAPK kinase (MKK) 3 and MKK6 on the pH-responsive increase in phosphoenolpyruvate carboxykinase mRNA.

Metabolic acidosis is partially compensated by a pronounced increase in renal catabolism of glutamine. This adaptive response is sustained, in part, through increased expression of phosphoenolpyruvate carboxykinase (PEPCK). Previous inhibitor studies suggested that the pH-responsive increase in PEPCK mRNA in LLC-PK1-FBPase+ cells is mediated by a p38 mitogen-activated protein kinase (MAPK).

Role of deadenylation and AUF1 binding in the pH-responsive stabilization of glutaminase mRNA.

During chronic metabolic acidosis, increased expression of renal glutaminase (GA) results from selective stabilization of the GA mRNA. This response is mediated by a direct repeat of an 8-base adenylate-uridylate (AU) sequence that binds zeta-crystallin and functions as a pH response element (pH-RE). A tetracycline-responsive promoter system was developed in LLC-PK(1)-F(+) cells to perform pulse-chase analysis of the turnover of a chimeric beta-globin (betaG) mRNA that contains 960 bp of the 3'-UTR of GA mRNA including the pH-RE.

cAMP-dependent stabilization of phosphoenolpyruvate carboxykinase mRNA in LLC-PK1-F+ kidney cells.

Phosphoenolpyruvate carboxykinase (PEPCK) catalyzes a rate-limiting step in hepatic and renal gluconeogenesis. In the kidney, PEPCK expression is enhanced during metabolic acidosis and in response to ANG II and parathyroid hormone. The effect of the latter hormone is mediated, in part, by cAMP.

3'-Untranslated region of phosphoenolpyruvate carboxykinase mRNA contains multiple instability elements that bind AUF1.

Phosphoenolpyruvate carboxykinase (PEPCK) is regulated solely by alterations in gene expression that involve changes in rates of PEPCK mRNA transcription and degradation. A tetracycline-responsive promoter system was used to quantify the half-life of various chimeric beta-globin-PEPCK (betaG-PCK) mRNAs in LLC-PK -F(+) cells. The control betaG mRNA was extremely stable (t(1/2) = 5 days).

Glutamine metabolism: Role in acid-base balance*.

The intent of this review is to provide a broad overview of the interorgan metabolism of glutamine and to discuss in more detail its role in acid-base balance. Muscle, adipose tissue, and the lungs are the primary sites of glutamine synthesis and release. During normal acid-base balance, the small intestine and the liver are the major sites of glutamine utilization.

Mitochondrial glutaminase enhances extracellular glutamate production in HIV-1-infected macrophages: linkage to HIV-1 associated dementia.

Dysfunction in mononuclear phagocyte (MP, macrophages and microglia) immunity is thought to play a significant role in the pathogenesis of HIV-1 associated dementia (HAD). In particular, elevated extracellular concentrations of the excitatory neurotransmitter glutamate, produced by MP as a consequence of viral infection and immune activation, can induce neuronal injury. To determine the mechanism by which MP-mediated neuronal injury occurs, the concentration and rates of production of extracellular glutamate were measured in human monocyte-derived macrophage (MDM) supernatants by reverse phase high-performance liquid chromatography (RP-HPLC).

Bacterial expression, purification, and characterization of rat kidney-type mitochondrial glutaminase.

The human gene that encodes the kidney-type glutaminase (KGA) spans 84-kb, contains 19 exons, and encodes two alternatively spliced mRNAs. Various segments of the rat KGA cDNA were PCR amplified and cloned into a bacterial expression vector to determine whether the N- and C- terminal ends of the glutaminase protein were essential for activity. A recombinant glutaminase, lacking the coding sequence contained in exon 1, was found to be fully active.

pH-responsive stabilization of glutamate dehydrogenase mRNA in LLC-PK1-F+ cells.

During chronic metabolic acidosis, the adaptive increase in rat renal ammoniagenesis is sustained, in part, by increased expression of mitochondrial glutaminase (GA) and glutamate dehydrogenase (GDH) enzymes. The increase in GA activity results from the pH-responsive stabilization of GA mRNA. The 3'-untranslated region (3'-UTR) of GA mRNA contains a direct repeat of an eight-base AU-rich element (ARE) that binds zeta-crystallin/NADPH:quinone reductase (zeta-crystallin) with high affinity and functions as a pH-response element.