Characterization of the intracellular proteolytic cleavage of myocilin and identification of calpain II as a myocilin-processing protease

MYOC, a gene involved in different types of glaucoma, encodes myocilin, a secreted glycoprotein of unknown function, consisting of an N-terminal leucine-zipper-like domain, a central linker region, and a C-terminal olfactomedin-like domain. Recently, we have shown that myocilin undergoes an intracellular endoproteolytic processing. We show herein that the proteolytic cleavage in the linker region splits the two terminal domains. The C-terminal domain is secreted to the culture medium, whereas the N-terminal domain mainly remains intracellularly retained. In transiently transfected 293T cells, the cleavage was prevented by calpain inhibitors, such as calpeptin, calpain inhibitor IV, and calpastatin. Since calpains are calcium-activated proteases, we analyzed how changes in either intra- or extracellular calcium affected the cleavage of myocilin. Intracellular ionomycin-induced calcium uptake enhanced myocilin cleavage, whereas chelation of extracellular calcium by EGTA inhibited the proteolytic processing. Calpains I and II cleaved myocilin in vitro. However, in cells in culture, only RNA interference knockdown of calpain II reduced myocilin processing. Subcellular fractionation and digestion of the obtained fractions with proteinase K showed that full-length myocilin resides in the lumen of the endoplasmic reticulum together with a subpopulation of calpain II. These data revealed that calpain II is responsible for the intracellular processing of myocilin in the lumen of the endoplasmic reticulum. We propose that this cleavage might regulate extracellular interactions of myocilin, contributing to the control of intraocular pressure.     

Distribution of insulin receptors between plasmalemma and intracellular compartments: effect of amino acids

Insulin receptor regulation was studied in the rat erythroblastic leukemic (EBL) cell in primary culture. After 1-2-hr incubations in medium containing 12 essential amino acids, glutamine, and serine, EBL cell protein synthesis and insulin receptor concentrations were increased compared to cells incubated without serine. Deficiency of medium isoleucine in the presence of serine rapidly decreased protein synthesis and insulin binding to intact cells. Supplementation of deficient media with serine or isoleucine had no effect on total insulin receptor numbers measured in solubilized cell preparations. Increased insulin binding following serine exposure was seen with binding assays at both 4 and 37 degrees C. Dissociation experiments to quantitate intracellular ligand after 37 degrees C binding assays showed increased in both surface binding and intracellular [125I]insulin accumulation. These data combined with previous observations suggest that amino acids essential for this cell are required for the rapid synthesis of a labile regulatory protein which facilitates the redistribution and/or recycling of insulin receptors.     

Relationship between intracellular amino acids and protein synthesis in the extensor digitorum longus muscle of rats

1. The incorporation into protein, and the accumulation into the free amino acid pools, of radioactive l-leucine and glycine was studied in rat extensor digitorum longus muscle. 2. The tissue was incubated first with (14)C-labelled and then with (3)H-labelled amino acid. 3. The experimental results were consistent with a model based on the premise that the amino acids in protein were incorporated directly from the extracellular pool.     

Effect of exogenous amino acids on the intracellular content of proline and other amino acids in Daucus carota cells

The effect of tryptophan on the biosynthesis of proline has been investigated. Cells of Daucus carota grown in B5 medium supplemented with 5×10^–4M tryptophan acquired the ability to grow in the presence of inhibitory concentrations of azetidine-2-carboxylic acid, an analog of proline. When trp was added to carrot cell cultures at sub-growth inhibiting concentrations, overproduction of intracellular free proline was observed. An increase was also observed for lys, his, ala, leu and phe. Likewise, the addition of asparagine, glutamic acid and phenylalanine to the medium stimulated the intracellular increase of free proline and other amino acids.Abbreviations A2CA azetidine-2-carboxylic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - 5MT 5-methyltryptophan - P5C pyrroline-5-carboxylic acid - f.wt. fresh weight - d.wt. dry weight     

Free amino acids in the early cleavage stages of the rabbit egg

In the first half of gestation, mouse yolk sac contains levels of N-acetyl-β-hexosaminidase activity and a ratio of N-acetyl-β-hexosaminidase: β-glucuronidase much higher than those of other embryonic tissues or the decidua. The properties of the yolk sac enzyme are very similar to those in the other tissues, suggesting that the observed difference in activity is quantitative rather than qualitative. In blastocyst cultures, the vesicular growths derived from the inner cell mass possess patterns of N-acetyl-β-hexosaminidase activity similar to yolk sac in vivo, but different from trophoblast cells developing in the same cultures. It is therefore proposed that the vesicular growths contain at least some differentiated yolk sac cells.     

Extra- and intracellular amino acids in the hippocampus during development of hepatic encephalopathy

Fulminant hepatic failure was induced in rabbits by intravenous administration of galactosamine hydrochloride. The animals were sacrified after 45 h and the hippocampus analyzed for free amino acids. In addition, free amino acids were measured in plasma and in the extracellular fluid of the hippocampus 20, 30 and 45 h after galactosamine injection. The extracellular fluid compartment was analyzed by slow perfusion of a thin dialysis tube which was implanted in the hippocampus one day prior to galactosamine administration. The amino acid concentration in the extracellular fluid agreed fairly well with that of the cerebrospinal fluid in the control situation. During development of hepatic failure, the plasma concentrations of all amino acids increased. The changes in extracellular amino acids were smaller, except for phosphoethanolamine and glutamate. The concentration ratio intra/extracellular amino acids decreased in the hippocampus for amino acids with a normally high concentration gradient.     

Stimulation of glycogen synthesis in hepatocytes by added amino acids is related to the total intracellular content of amino acids

Katz et al. [Katz, J., Golden, S. & Wals, P.A. (1976) Proc. Natl Acad. Sci. USA 73, 3433-3437] were the first to report that in hepatocytes isolated from fasted rats and incubated with either dihydroxyacetone, glucose or other sugars, glycogen synthesis was greatly accelerated by addition of amino acids. We have looked for possible mediators responsible for this effect and have tested the effect of alanine, proline, asparagine, glutamine or a combination of ammonia with either pyruvate or lactate in activating glycogen synthesis from dihydroxyacetone. The following observations were made. 1. Stimulation of glycogen synthesis by alanine, proline or asparagine does not require production of glutamine since the effect also occurs in periportal hepatocytes which lack glutamine synthetase. 2. Under various conditions, stimulation of glycogen synthesis by added amino acids directly correlated with increases in the intracellular content of amino acids, expressed in osmotic equivalents. 3. 3-Mercaptopicolinic acid, the inhibitor of phosphoenolpyruvate carboxykinase, further enhances stimulation of glycogen synthesis by amino acids because it increases the intracellular accumulation of aspartate and glutamate. 4. The previously reported enhancement by leucine of the stimulation of glycogen synthesis by glutamine [Chen. K. S. & Lardy, H. A. (1985) J. Biol. Chem. 260, 14683-14688] can be ascribed to inhibition of urea synthesis by leucine which results in accumulation of glutamate and of ammonia, the essential activator of glutaminase. It is concluded that activation of glycogen synthesis by added amino acids is due to an increase in intracellular osmolarity following their uptake and the accumulation of intracellular catabolites. This results in an increase in hepatic volume which stimulates glycogen synthesis [Baquet, A., Hue, L., Meijer, A. J., van Woerkom, G. M. & Plomp, P. J. A. M. (1990) J. Biol. Chem. 265, 955-959].     

Effects of the metabolites of the branched-chain amino acids and cysteamine on the glycine cleavage system

The effects of ten metabolites of the branched-chain amino acids, CoA and cysteamine (beta-mercaptoethylamine), on the glycine cleavage system were investigated with the liver extracts. It was found that CoA derivatives including tiglyl CoA, isobutyryl CoA, succinyl CoA, methylmalonyl CoA, isovaleryl CoA, propionyl CoA and CoA itself and cysteamine significantly inhibited the glycine cleavage system of the liver extracts. Further studies on the glycine-14CO2 exchange catalyzed by p-protein and H-protein purified from chicken liver indicated that tiglyl CoA inhibited the activity of P-protein in an apparently competitive manner with respect to H-protein, and that cysteamine inhibited the activity of P-protein in two ways, by increasing the Km value for glycine and changing Vmax.     

Pinocytosis and intracellular degradation of exogenous protein: modulation by amino acids

Intracellular degradation of exogenous (serum) proteins provides a source of amino acids for cellular protein synthesis. Pinocytosis serves as the mechanism for delivering exogenous protein to the lysosomes, the major site of intracellular degradation of exogenous protein. To determine whether the availability of extracellular free amino acids altered pinocytic function, we incubated monolayers of pulmonary alveolar macrophages with the fluid-phase marker, [14C]sucrose, and we dissected the pinocytic process by kinetic analysis. Additionally, intracellular degradation of endogenous and exogenous protein was monitored by measuring phenylalanine released from the cell monolayers in the presence of cycloheximide. Results revealed that in response to a subphysiological level of essential amino acids or to amino acid deprivation, (a) the rate of fluid-phase pinocytosis increased in such a manner as to preferentially increase both delivery to and size of an intracellular compartment believed to be the lysosomes, (b) the degradation of exogenously supplied albumin increased, and (c) the fraction of phenylalanine derived from degradation of exogenous albumin and reutilized for de novo protein synthesis increased. Thus, modulation of the pinosome-lysosome pathway may represent a homeostatic mechanism sensitive to the availability of extracellular free amino acids.     

Disease-causing mutations in proteins: structural analysis of the CYP1B1 mutations causing primary congenital glaucoma in humans

In this communication, we report an in-depth structure-based analysis of the human CYP1b1 protein carrying disease-causing mutations that are discovered in patients suffering from primary congenital glaucoma (PCG). The "wild-type" and the PCG mutant structures of the human CYP1b1 protein obtained from comparative modeling were subjected to long molecular dynamics simulations with an intention of studying the possible impact of these mutations on the protein structure and hence its function. Analysis of time evolution as well as time averaged values of various structural properties--especially of those of the functionally important regions: the heme binding region, substrate binding region, and substrate access channel--gave some insights into the possible structural characteristics of the disease mutant and the wild-type forms of the protein. In a nutshell, compared to the wild-type the core regions in the mutant structures are associated with subtle but significant changes, and the functionally important regions seem to adopt such structures that are not conducive for the wild-type-like functionality.     

Impact of separating amino acids between plasma, extracellular and intracellular compartments on estimating protein synthesis in rodents

Summary. Three models representing different separations of amino acid sources were used to simulate experimental specific radioactivity data and to predict protein fractional synthesis rate (FSR). Data were from a pulse dose of ¹⁴C-U Leu given to a non-growing 20 g mouse and a flooding dose of ³H Phe given to a non-growing 200 g rat. Protein synthesis rates estimated using the combined extracellular and intracellular (Ec + Ic) source pool and extracellular and plasma (Ec + Pls) source pool mouse models were 78 and 120% d⁻¹ in liver, 14 and 16% d⁻¹ in brain and 15 and 14% d⁻¹ in muscle. Predicted protein synthesis rates using the Ec + Ic, Ec + Ic + Tr (combined extracellular, intracellular and aminoacyl tRNA source pool) and Ec + Pls rat models were 57, 3.4 and 57% d⁻¹ in gastrocnemius, 58, 71 and 62% d⁻¹ in gut, 8.3, 8.4 and 7.9% d⁻¹ in heart, 32, 23 and 25% d⁻¹ in kidney, 160, 90 and 80% d⁻¹ in liver, 57, 5.5 and 57% d⁻¹ in soleus and 56, 3.4 and 57% d⁻¹ in tibialis. The Ec + Ic + Tr model underestimated protein synthesis rates in mouse tissues (5.0, 27 and 2.5% d⁻¹ for brain, liver and muscle) and rat muscles (3.4, 5.5 and 3.4% d⁻¹ for gastrocnemius, soleus and tibialis). The Ec + Pls model predicted the mouse pulse dose data best and the Ec + Ic model predicted the rat flooding dose data best. Model predictions of FSR imply that identification and separation of the source specific radioactivity is critical to accurately estimate FSR. Received June 11, 2000 Accepted September 26, 2000     

Photometric assay for measuring the intracellular concentration of branched-chain amino acids in bacteria

The changes in intracellular pool of branched-chain amino acids (BCAA) regulate different physiological processes in bacteria. Up to date, the only available photometric test for measuring BCAA concentration was adapted for blood and plasma samples in diagnostic purposes. We have modified this method for use on bacterial cells, and tested its applicability on several model organisms: Lactococcus lactis, Bacillus subtilis and Escherichia coli.     

Arginyl-tRNA synthetase from yeast. Discrimination between 20 amino acids in aminoacylation of tRNA(Arg)-C-C-A and tRNA(Arg)-C-C-A(3'NH2)

For discrimination between arginine and 19 other amino acids in aminoacylation of tRNA(Arg)-C-C-A by arginyl-tRNA synthetase from baker's yeast, discrimination factors (D) have been determined from kcat and Km values. The lowest values were found for Trp, Cys, Lys (D = 800-8500), showing that arginine is 800-8500 times more often incorporated into tRNA(Arg)-C-C-A than noncognate acids at the same amino acid concentrations. The other noncognate amino acids exhibit D values between 10,000 and 60,000. In aminoacylation of tRNA(Arg)-C-C-A(3'NH2) discrimination factors D1 are in the range 10-600. From these values and AMP formation stoichiometry, pretransfer proof-reading factors II1 were determined; from D values and AMP stoichiometry in aminoacylation of tRNA(Arg)-C-C-A, posttransfer proof-reading factors II2 could be calculated, II1 values between 2 and 120 show that pretransfer proof-reading is the main correction step, posttransfer proof-reading (II2 approximately 1-10) plays a marginal role. Initial discrimination factors due to different Gibbs free energies of binding between arginine and the noncognate amino acids were calculated from discrimination and proof-reading factors. According to a two-step binding process, two factors (I1 and I2) were determined. They can be related to hydrophobic interaction forces and hydrogen bonds that are especially formed by the arginine side chain. A hypothetical 'stopper' model of the amino acid recognition site is discussed.     

Arg292----Val] or [Arg292----Leu] mutation enhances the reactivity of Escherichia coli aspartate aminotransferase with aromatic amino acids

Arg292 of E. coli aspartate aminotransferase was substituted with valine or leucine by site-directed mutagenesis. In comparison with the wild-type enzyme, either of the mutant enzymes showed a decrease by over 5 orders of magnitude of kcat/km values for aspartate and glutamate. This supports the contention that Arg292 is important for determining the specificity of this enzyme for dicarboxylic substrates. In contrast, mutant enzymes displayed a 5- to 10-fold increase in kcat/Km values for aromatic amino acids as substrates. Thus, introduction of an uncharged, hydrophobic side chain into position 292 leads to a striking alteration in substrate specificity of this enzyme, thereby improving catalytic efficiency toward aromatic amino acids.