scholarly journals Availability of Arg, but Not tRNA, Is a Rate-Limiting Factor for Intracellular Arginylation

2021 ◽  
Vol 23 (1) ◽  
pp. 314
Author(s):  
Irem Avcilar-Kucukgoze ◽  
Brittany MacTaggart ◽  
Anna Kashina
Keyword(s):  
Amino Acid ◽  
Posttranslational Modification ◽  
Cultured Cells ◽  
Limiting Factor ◽  
Strong Binding ◽  
Biological Importance ◽  
Rate Limiting ◽  
In Cells

Protein arginylation, mediated by arginyltransferase ATE1, is a posttranslational modification of emerging biological importance that consists of transfer of the amino acid Arg from tRNA to protein and peptide targets. ATE1 can bind tRNA and exhibits specificity toward particular tRNA types, but its dependence on the availability of the major components of the arginylation reaction has never been explored. Here we investigated key intracellular factors that can potentially regulate arginylation in vivo, including several tRNA types that show strong binding to ATE1, as well as availability of free Arg, in an attempt to identify intracellular rate limiting steps for this enzyme. Our results demonstrate that, while modulation of tRNA levels in cells does not lead to any changes in intracellular arginylation efficiency, availability of free Arg is a potentially rate-limiting factor that facilitates arginylation if added to the cultured cells. Our results broadly outline global pathways that may be involved in the regulation of arginylation in vivo.

Reevaluation of amino acid stimulation of protein synthesis in murine- and human-derived skeletal muscle cells assessed by independent techniques

2005 ◽  
Vol 288 (5) ◽  
pp. E1028-E1037 ◽  
Author(s):  
Britt-Marie Iresjö ◽  
Elisabeth Svanberg ◽  
Kent Lundholm
Keyword(s):  
Protein Synthesis ◽  
Amino Acid ◽  
Cultured Cells ◽  
Incorporation Rate ◽  
Synthesis Methods ◽  
Distribution Analysis ◽  
Western Blots ◽  
Initiation Factors ◽  
Initiation Of Translation

Murine L6 and human rhabdomyosarcoma cells were cultured standardized in low (0.28 mM) and normal (9 mM) amino acid (AA) concentrations to reevaluate by independent methods to what extent AA activate initiation of protein synthesis. Methods used were incorporation of radioactive AA into proteins, distribution analysis of RNA in density gradient, and Western blots on initiation factors of translation of proteins in cultured cells as well as in vivo (gastrocnemius, C57Bl mice) during starvation/refeeding. Incorporation rate of AA gave incorrect results in a variety of conditions, where phenylalanine stimulated the incorporation rate of phenylalanine into proteins, but not of tyrosine, and tyrosine stimulated incorporation of tyrosine but not of phenylalanine. Similar problems were observed when [35S]methionine was used for labeling of fractionated cellular proteins. However, the methods entirely independent of labeled AA incorporation indicated that essential AA activate initiation of translation, whereas nonessential AA did not. Branched-chain AA and glutamine, in combination with some other AA, also stimulated initiation of translation. Starvation/refeeding in vitro agreed qualitatively with results in vivo evaluated by initiation factors. Insulin at physiological concentrations (100 μM/ml) did not stimulate global protein synthesis at low or normal AA concentrations but did so at supraphysiological levels (3 mU/ml), confirmed by independent methods. Our results reemphasize that labeled AA should be used with caution for quantification of protein synthesis, since the precursor pool(s) for protein synthesis is not in complete equilibrium with surrounding AA. “Flooding” tracee experiments did not overcome this problem.

scholarly journals Monosaccharide transport in the mammary gland of the intact lactating rat

1984 ◽  
Vol 218 (1) ◽  
pp. 213-219 ◽  
Author(s):  
L C Threadgold ◽  
N J Kuhn
Keyword(s):  
Mammary Gland ◽  
Glucose Transport ◽  
Intracellular Water ◽  
Limiting Factor ◽  
Transport Capacity ◽  
Tissue Water ◽  
Monosaccharide Transport ◽  
Rate Limiting ◽  
Total Tissue

The Michaelis-Menten equation for the utilization of competing substrates was applied to the uptake of 2-deoxy[3H]glucose into the mammary gland of anaesthetized lactating rats. Intracellular water was calculated from total tissue water and sucrose space. Fed rats had a mean transport capacity of 2.2 mumol/min per g of tissue, giving an actual glucose transport in vivo of 1.1 mumol/min per g. Transport decreased by 90% on overnight starvation and returned to normal by 2 h of re-feeding. Similar changes were observed in the 1 min or 5 min transport of circulating 3-O-methylglucose. Transport of 3-O-methylglucose in starved rats was restored towards normal by insulin. In fed rats it increased between parturition and day 12 of lactation. The findings support the proposal that transport is a rate-limiting factor in the mammary utilization of carbohydrate.

scholarly journals The herbicidally active compound N-2-(5-chloro-pyridyl) aminomethylene bisphosphonic acid acts by inhibiting both glutamine and aromatic amino acid biosynthesis

2000 ◽  
Vol 27 (7) ◽  
pp. 677 ◽  
Author(s):  
Giuseppe Forlani ◽  
Barbara Lejczak ◽  
Pawel Kafarski
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Active Compound ◽  
Cultured Cells ◽  
Shikimate Pathway ◽  
Glutamate Synthase ◽  
Phenylalanine Level ◽  
Bisphosphonic Acid

The effect of the herbicidally active compound N-2-(5-chloro-pyridyl)aminomethylene bisphosphonic acid (Cl-pyr-AMBPA), previously found in vitro to inhibit the activity of the first enzyme in the shikimate pathway 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase, was investigated in vivo on suspension cultured cells of Nicotiana plumbaginifolia Viviani. Amino acid pool measurement showed an actual reduction of tyrosine, tryptophan and phenylalanine level following the addition of the compound to the growth medium. However, an even stronger effect was noticed for other amino acids, mainly glutamine. When the activity of the enzymes involved in the glutamate cycle was measured in the presence of Cl-pyr-AMBPA, glutamate synthase was unaffected, while glutamine synthetase was significantly inhibited. Contrary to the herbicide phosphinothricin, the inhibitor bound reversibly to the enzyme. Kinetic analysis accounted for an inhibition of uncompetitive type with respect to ammonium, glutamate and ATP, withKivalues of 113, 97 and 39 M, respectively. Only the exogenous supply of a mixture of glutamine and aromatic amino acids relieved cell growth inhibition, suggesting that the phytotoxic properties of Cl-pyr-AMBPA are due to inhibition of key enzymes in both the corresponding pathways.

scholarly journals Cloning and Characterization of a Novel Subunit of Protein Serine/Threonine Phosphatase 4 from Mesangial Cells

2001 ◽  
Vol 12 (12) ◽  
pp. 2601-2608 ◽  
Author(s):  
Takehiko Wada ◽  
Toshio Miyata ◽  
Reiko Inagi ◽  
Masaomi Nangaku ◽  
Masako Wagatsuma ◽  
...  
Keyword(s):  
Amino Acid ◽  
Large Scale ◽  
Cultured Cells ◽  
Mesangial Cells ◽  
Spindle Pole ◽  
Regulatory Subunit ◽  
Glomerular Diseases ◽  
Regulatory Subunits ◽  
Novel Approach

ABSTRACT. Mesangial cells play an important role in maintaining glomeruli structure and function and in the pathogenesis of glomerular diseases. With a novel approach using a rapid large-scale DNA sequencing strategy and computerized data processing, a new human gene, PP4Rmegwas cloned. The full-length cDNA clone of human PP4Rmegcoded for a novel 950-amino acid protein, which was similar to a subunit of protein serine/threonine phosphatase 4 (PP4). Recombinant PP4Rmegproduced in COS-7 cells bound to the catalytic subunit of PP4. PP4Rmegis therefore structurally and functionally related to the recently reported regulatory subunit of PP4, PP4R1. Amino acid sequence analysis of rat PP4Rmeghomologue revealed that the sequences were well conserved between human and rat (86.3% identity). Northern blot analyses of human tissues and cultured cells demonstrated that the regulatory subunits were expressed abundantly in human cultured mesangial cells, although their expression was relatively ubiquitous.In situhybridization studies in normal human renal tissues confirmed their expression in glomeruliin vivo. The expression was upregulated in glomeruli of anti-Thy1 glomerulonephritis rats before mesangial proliferation. These data demonstrate that PP4Rmegis a novel regulatory subunit of PP4, which is expressed ubiquitously but abundantly in mesangial cells. Its pathophysiologic role in mesangial cells and glomerulus remains unknown. As PP4 is an essential protein for nucleation, growth, and stabilization of microtubules at centrosomes/spindle pole bodies during cell division, PP4Rmegmay play a role in regulation of mitosis in mesangial cells.

In vivo Nitrate Reductase Activity in Leaves of Pearl Millet, Pennisetum americanum (L.) Leeke

1987 ◽  
Vol 14 (2) ◽  
pp. 125 ◽  
Author(s):  
SV Chanda ◽  
AK Joshi ◽  
PN Krishnan ◽  
YD Singh
Keyword(s):  
Nitrate Reductase ◽  
Reductase Activity ◽  
Potassium Phosphate ◽  
Limiting Factor ◽  
Growth Stages ◽  
In Vivo Assay ◽  
Rate Limiting ◽  
Nr Activity

In the in vivo assay of nitrate reductase (NR) in P. americanum leaves, addition of 1% (v/v) Triton X-100, potassium phosphate buffer (80 mM, pH 7.4) and 1.13 mM NADH to the assay medium resulted in maximum activity. With increasing concentration of NADH, saturation-type kinetics were observed. Based on this data metabolic pool concentration for NADH and apparent Km for nitrate reductase were determined. In field studies with cultivars BJ-104, J-104 and 5141-A of P. americanum, the relative limitation of NO3-, NADH and nitrate reductase in NO3- assimilation was determined. NR activity was measured by four modifications of the in vivo assay technique (with NO3-, with NADH, without NO3- and NADH and with both NO3- and NADH additions to the reaction mixture) and with one in vitro technique. For all the cultivars, NADH was the major rate-limiting factor for in vivo assay during early growth stages, while at later stages, NO3- was limiting. At no stage was NR rate-limiting. It is concluded that NR activity alone may not serve as biochemical marker for improved efficiency of utilisation of nitrogen in P. americanum.

scholarly journals Phosphorylation of UT-A1 on serine 486 correlates with membrane accumulation and urea transport activity in both rat IMCDs and cultured cells

2010 ◽  
Vol 298 (4) ◽  
pp. F935-F940 ◽  
Author(s):  
Janet D. Klein ◽  
Mitsi A. Blount ◽  
Otto Fröhlich ◽  
Chad E. Denson ◽  
Xiaoxiao Tan ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Amino Acid ◽  
Cell Lines ◽  
Specific Antibody ◽  
Cultured Cells ◽  
Apical Plasma Membrane ◽  
Wild Type ◽  
Amino Acid Peptide ◽  
In The Wild

Vasopressin is the primary hormone regulating urine-concentrating ability. Vasopressin phosphorylates the UT-A1 urea transporter in rat inner medullary collecting ducts (IMCDs). To assess the effect of UT-A1 phosphorylation at S486, we developed a phospho-specific antibody to S486-UT-A1 using an 11 amino acid peptide antigen starting from amino acid 482 that bracketed S486 in roughly the center of the sequence. We also developed two stably transfected mIMCD3 cell lines: one expressing wild-type UT-A1 and one expressing a mutated form of UT-A1, S486A/S499A, that is unresponsive to protein kinase A. Forskolin stimulates urea flux in the wild-type UT-A1-mIMCD3 cells but not in the S486A/S499A-UT-A1-mIMCD3 cells. The phospho-S486-UT-A1 antibody identified UT-A1 protein in the wild-type UT-A1-mIMCD3 cells but not in the S486A/S499A-UT-A1-mIMCD3 cells. In rat IMCDs, forskolin increased the abundance of phospho-S486-UT-A1 (measured using the phospho-S486 antibody) and of total UT-A1 phosphorylation (measured by 32P incorporation). Forskolin also increased the plasma membrane accumulation of phospho-S486-UT-A1 in rat IMCD suspensions, as measured by biotinylation. In rats treated with vasopressin in vivo, the majority of the phospho-S486-UT-A1 appears in the apical plasma membrane. In summary, we developed stably transfected mIMCD3 cell lines expressing UT-A1 and an S486-UT-A1 phospho-specific antibody. We confirmed that vasopressin increases UT-A1 accumulation in the apical plasma membrane and showed that vasopressin phosphorylates UT-A1 at S486 in rat IMCDs and that the S486-phospho-UT-A1 form is primarily detected in the apical plasma membrane.

scholarly journals The Gene for Aromatase, a Rate-Limiting Enzyme for Local Estrogen Biosynthesis, Is a Downstream Target Gene of Runx2 in Skeletal Tissues

2010 ◽  
Vol 30 (10) ◽  
pp. 2365-2375 ◽  
Author(s):  
Jae-Hwan Jeong ◽  
Youn-Kwan Jung ◽  
Hyo-Jin Kim ◽  
Jung-Sook Jin ◽  
Hyun-Nam Kim ◽  
...  
Keyword(s):  
Cultured Cells ◽  
Steroid Hormone ◽  
Biological Significance ◽  
Bone Homeostasis ◽  
Mineral Density ◽  
Aromatase Gene ◽  
Estrogen Biosynthesis ◽  
Rate Limiting

ABSTRACT The essential osteoblast-related transcription factor Runx2 and the female steroid hormone estrogen are known to play pivotal roles in bone homeostasis; however, the functional interaction between Runx2- and estrogen-mediated signaling in skeletal tissues is minimally understood. Here we provide evidence that aromatase (CYP19), a rate-limiting enzyme responsible for estrogen biosynthesis in mammals, is transcriptionally regulated by Runx2. Consistent with the presence of multiple Runx2 binding sites, the binding of Runx2 to the aromatase promoter was demonstrated in vitro and confirmed in vivo by chromatin immunoprecipitation assays. The bone-specific aromatase promoter is activated by Runx2, and endogenous aromatase gene expression is upregulated by Runx2 overexpression, establishing the aromatase gene as a target of Runx2. The biological significance of the Runx2 transcriptional control of the aromatase gene is reflected by the enhanced estrogen biosynthesis in response to Runx2 in cultured cells. Reduced in vivo expression of skeletal aromatase gene and low bone mineral density are evident in Runx2 mutant mice. Collectively, these findings uncover a novel link between Runx2-mediated osteoblastogenic processes and the osteoblast-mediated biosynthesis of estrogen as an osteoprotective steroid hormone.

scholarly journals Role of fructose 2,6-bisphosphate in mammary gland of fed, starved and re-fed lactating rats

1985 ◽  
Vol 232 (3) ◽  
pp. 931-934 ◽  
Author(s):  
S Ward ◽  
N J Kuhn
Keyword(s):  
Mammary Gland ◽  
Intracellular Concentration ◽  
Limiting Factor ◽  
Maximal Rate ◽  
Physiological Conditions ◽  
Highly Sensitive ◽  
Lactating Mammary Gland ◽  
Rate Limiting

The fructose 2,6-bisphosphate (Fru-2,6-P2) content and intracellular concentration of lactating mammary gland was measured in fed, starved and re-fed rats. There was little or no change on starvation, and about 1.5-fold rise on re-feeding, contrasting with estimated glycolytic changes of about 10-fold. The 6-phosphofructokinase (PFK-1) activity of mammary extracts was highly sensitive to added Fru-2,6-P2 under all conditions examined, and appeared to approach saturation at physiological concentrations of this effector. The activity of mammary PFK-1 measured under optimal and ‘physiological’ conditions suggested that this enzyme operates in vivo at about 24% of maximal rate, and is likely to be an important rate-limiting factor in mammary glycolysis.

scholarly journals Posttranslational modification of the RHO of plants protein RACB by phosphorylation and cross-kingdom conserved ubiquitination

2020 ◽  
Author(s):  
Lukas Weiß ◽  
Tina Reiner ◽  
Julia Mergner ◽  
Bernhard Kuster ◽  
Attila Fehér ◽  
...  
Keyword(s):  
Amino Acid ◽  
Posttranslational Modification ◽  
Individual Case ◽  
Bound State ◽  
Amino Acid Residues ◽  
Nucleotide Exchange ◽  
Protein Activity ◽  
Developmental Defects

AbstractSmall RHO-type G-proteins act as signaling hubs and master regulators of polarity in eukaryotic cells. Their activity is tightly controlled, as defective RHO signaling leads to aberrant growth and developmental defects. Two major pathways regulate G-protein activity: canonical switching of the nucleotide bound state and posttranslational modification (PTM). PTMs can support or suppress RHO signaling, depending on each individual case. In plants, regulation of Rho of plants (ROPs) has been shown to act through nucleotide exchange and hydrolysis, as well as through lipid modification, but there is little data available on phosphorylation or ubiquitination of ROPs. Hence, we applied proteomic analyses to identify PTMs of the barley ROP RACB. We observed in vitro phosphorylation by barley ROP Binding Kinase 1 and in vivo ubiquitination of RACB. Comparative analyses of the newly identified RACB phosphosites and human RHO protein phosphosites revealed conservation of modified amino acid residues, but no overlap of actual phosphorylation patterns. However, the identified RACB ubiquitination site is present in all ROPs from Hordeum vulgare, Arabidopsis thaliana and Oryza sativa. Since this highly conserved amino acid residue is likewise ubiquitinated in mammalian Rac1 and Rac3, we suggest that RHO family proteins from different kingdoms could be generally regulated by ubiquitination of this site.

scholarly journals Novel Small Molecule Targeting the Hemagglutinin Stalk of Influenza Viruses

2019 ◽  
Vol 93 (17) ◽  
Author(s):  
Jin Il Kim ◽  
Sangmoo Lee ◽  
Gong Yeal Lee ◽  
Sehee Park ◽  
Joon-Yong Bae ◽  
...  
Keyword(s):  
Influenza Virus ◽  
Amino Acid ◽  
Membrane Fusion ◽  
Small Molecule ◽  
Screening Method ◽  
Influenza Viruses ◽  
Influenza B ◽  
Challenge Study ◽  
In Cells

ABSTRACTCombating influenza is one of the perennial global public health issues to be managed. Antiviral drugs are useful for the treatment of influenza in the absence of an appropriate vaccine. However, the appearance of resistant strains necessitates a constant search for new drugs. In this study, we investigated novel anti-influenza drug candidates usingin vitroandin vivoassays. We identified anti-influenza hit compounds using a high-throughput screening method with a green fluorescent protein-tagged recombinant influenza virus. Through subsequent analyses of their cytotoxicity and pharmacokinetic properties, one candidate (IY7640) was selected for further evaluation. In a replication kinetics analysis, IY7640 showed greater inhibitory effects during the early phase of viral infection than the viral neuraminidase inhibitor oseltamivir. In addition, we observed that hemagglutinin (HA)-mediated membrane fusion was inhibited by IY7640 treatment, indicating that the HA stalk region, which is highly conserved across various (sub)types of influenza viruses, may be the molecular target of IY7640. In an escape mutant analysis in cells, amino acid mutations were identified at the HA stalk region of the 2009 pandemic H1N1 (pH1N1) virus. Even though thein vivoefficacy of IY7640 did not reach complete protection in a lethal challenge study in mice, these results suggest that IY7640 has potential to be developed as a new type of anti-influenza drug.IMPORTANCEAnti-influenza drugs with broad-spectrum efficacy against antigenically diverse influenza viruses can be highly useful when no vaccines are available. To develop new anti-influenza drugs, we screened a number of small molecules and identified a strong candidate, IY7640. When added at the time of or after influenza virus infection, IY7640 was observed to successfully inhibit or reduce viral replication in cells. We subsequently discovered that IY7640 targets the stalk region of the influenza HA protein, which exhibits a relatively high degree of amino acid sequence conservation across various (sub)types of influenza viruses. Furthermore, IY7640 was observed to block HA-mediated membrane fusion of H1N1, H3N2, and influenza B viruses in cells. Although it appears less effective against strains other than H1N1 subtype viruses in a challenge study in mice, we suggest that the small molecule IY7640 has potential to be optimized as a new anti-influenza drug.

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