Effect of arginine deficiency on arginine-dependent post-translational protein modifications in mice

Transgenic mice that overexpress arginase-I in their small-intestinal enterocytes suffer from a pronounced, but selective decrease in circulating arginine levels during the suckling period, resulting in impaired growth and development of hair, muscle and immune system. In the present study, we tested the hypothesis that the arginine-deficiency phenotype is caused by arginine-specific post-translational modifications, namely, an increase in the degree of mono-ADP-ribosylation of proteins because of reduced competition by free arginine residues and/or an increase in protein-tyrosine nitration because of an increased O2- production by NO synthases in the presence of limiting amounts of arginine. Arginine ADP-ribosylation and tyrosine nitration of proteins in the affected organs were assayed by Western blot analysis, using specific anti-ADP-ribosylarginine and protein-nitrotyrosine antisera. The composition of the group of proteins that were preferentially arginine ADP-ribosylated or tyrosine-nitrated in the respective organs was strikingly similar. Arginine-deficient mice differed from their controls in a reduced ADP-ribosylation of a 130 kDa and a 65 kDa protein in skin and an increased protein nitration of an 83 kDa protein in bone marrow and a 250 kDa protein in spleen. Since only 20 % of the visualised proteins were differentially modified in a subset of the affected organs, our findings appear to rule out these prominent arginine-dependent post-translational protein modifications as mediators of the characteristic phenotype of severely arginine-deficient mice.<br/

The 3'-UTR of the glutamine-synthetase gene interacts specifically with upstream regulatory elements, contains mRNA-instability elements and is involved in glutamine sensing

Glutamine synthetase (GS) is expressed at various levels in a wide range of tissues, suggesting that a complex network of modules regulates its expression. We explored the interactions between the upstream enhancer, regulatory regions in the first intron, and the 3'-untranslated region and immediate downstream genomic sequences of the GS gene (the GS "tail"), and compared the results with those obtained previously in conjunction with the bovine growth hormone (bGH) tail. The statistical analysis of these interactions revealed that the GS tail was required for full enhancer activity of the combination of the upstream enhancer and either the middle or the 3'-intron element. The GS tail also prevented a productive interaction between the upstream enhancer and the 5'-intron element, whereas the bGH tail did not, suggesting that the 5'-intron element is a regulatory element that needs to be silenced for full GS expression. Using the CMV promoter/enhancer and transfection experiments, we established that the 2.8 kb GS mRNA polyadenylation signal is approximately 10-fold more efficient than the 1.4 kb mRNA signal. Because the steady-state levels of both mRNAs are similar, the intervening conserved elements destabilize the long mRNA. Indeed, one but not all constructs containing these elements had a shorter half life in FTO-2B cells. A construct containing only 300 bases before and 100 bases after the 2.8 kb mRNA polyadenylation site sufficed for maximal expression. A stretch of 21 adenines inside this fragment conferred, in conjunction with the upstream enhancer and the 3'-part of the first intron, sensitivity of GS expression to ambient glutamin

Expression of thyroid hormone receptors A and B in developing rat tissues; evidence for extensive posttranscriptional regulation

The perinatal changes in the pattern of expression of the thyroid hormone receptor (TR) isoforms TRalpha (1) TRalpha (2), TRbeta (1), and TRbeta (2) were investigated using in situ hybridization and immunohistochemistry, and RT-PCR and western blotting as visualization and quantification techniques respectively. In liver, lung, and kidney, TRalpha mRNA was expressed in the stromal and TRbeta mRNA in the parenchymal component of the tissues. When compared with liver, TRalpha mRNA concentrations were tenfold higher in lung, kidney, and intestine, and 100-fold higher in brain, with TRalpha (2) mRNA concentrations exceeding those of TRalpha (1) 5-to 10-fold. Tissue TRbeta (1) mRNA concentrations were similar in liver, lung, and brain, and 3- to 5-fold higher in kidney and intestine. None of the TRbeta (2) mRNA could be detected outside the pituitary. Tissue TRalpha (2) and TRbeta (1) protein levels reached adult levels at 5 days before birth, whereas TRalpha (1) protein peaked after birth. Because of the distinct time-course of thyroid hormone-binding receptors TRalpha (1) and TRbeta (1), we speculate that an initiating, TRbeta (1)-mediated signaling from the parenchyma is followed by a TRalpha (1)-mediated response in the stroma. When compared with organs with a complementary parenchymal-stromal expression pattern, organs with extensive cellular co-expression of TRalpha and TRbeta (brain and intestinal epithelium) were characterized by a very low TRalpha protein: mRNA ratio, implying a low translational efficiency of TR mRNA or a high turnover of TR protein. The data indicate that the TR-dependent regulatory cascades are controlled differently in organs with a complementary tissue expression pattern and in those with cellular co-expression of the TRalpha and TRbeta genes. AD - Department of Pediatric Surgery, Erasmus MC-Sophia, Rotterdam, The Netherlands

Cellular concentrations of glutamine synthetase in murine organs

Glutamine synthetase (GS) is the only enzyme that can synthesize glutamine, but it also functions to detoxify glutamate and ammonia. Organs with high cellular concentrations of GS appear to function primarily to remove glutamate or ammonia, whereas those with a low cellular concentration appear to primarily produce glutamine. To validate this apparent dichotomy and to clarify its regulation, we determined the GS concentrations in 18 organs of the mouse. There was a &gt;100-fold difference in GS mRNA, protein, and enzyme-activity levels among organs, whereas there was only a 20-fold difference in the GS protein:mRNA ratio, suggesting extensive transcriptional and posttranscriptional regulation. In contrast, only small differences in the GS enzyme activity : protein ratio were found, indicating that posttrans lational regulation is of minor importance. The cellular concentration of GS was determined by relating the relative differences in cellular GS concentration, detected using image analysis of immunohistochemically stained tissue sections, to the biochemical data. There was a &gt;1000-fold difference in cellular concentrations of GS between GS-positive cells in different organs, and cellular concentrations were up to 20x higher in subpopulations of cells within organs than in whole organs. GS activity was highest in pericentral hepatocytes (~485 micromol.g&ndash;1.min&ndash;1), followed in descending order by epithelial cells in the epididymal head, Leydig cells in the testicular interstitium, epithelial cells of the uterine tube, acid-producing parietal cells in the stomach, epithelial cells of the S3 segment of the proximal convoluted tubule of the kidney, astrocytes of the central nervous tissue, and adipose tissue. GS activity in muscle amounted to only 0.4 micromol.g&ndash;1.min&ndash;1. Our findings confirmed the postulated dichotomy between cellular concentration and GS functio