scholarly journals Incorporation of bisphosphonates into adenine nucleotides by amoebae of the cellular slime mould Dictyostelium discoideum

1994 ◽  
Vol 303 (1) ◽  
pp. 303-311 ◽  
Author(s):  
M J Rogers ◽  
X Ji ◽  
R G G Russell ◽  
G M Blackburn ◽  
M P Williamson ◽  
...  
Keyword(s):  
Bone Resorption ◽  
Dictyostelium Discoideum ◽  
Adenine Nucleotides ◽  
Side Chains ◽  
Short Side ◽  
Slime Mould ◽  
Trna Synthetases ◽  
Aminoacyl Trna Synthetases ◽  
Antiresorptive Agents

Bisphosphonates are a class of synthetic pyrophosphate analogues. Some are known to be potent inhibitors of osteoclast-mediated bone resorption in vivo, but their mechanisms of action are unclear. The order of potency of bisphosphonates as inhibitors of bone resorption closely matches the order of potency as inhibitors of growth of amoebae of the slime mould Dictyostelium discoideum, indicating that bisphosphonates may have a mechanism of action that is similar in both osteoclasts and Dictyostelium. Methylenebisphosphonate and several halogenated derivatives, which have low potency as antiresorptive agents and as growth inhibitors of Dictyostelium, are metabolized intracellularly by Dictyostelium amoebae into methylene-containing adenine nucleotides. We have used a combination of n.m.r. and f.p.l.c. analysis to determine whether incorporation into nucleotides is a feature of other bisphosphonates, especially those that are potent antiresorptive agents. Only bisphosphonates with short side chains or of low potency are incorporated into adenine nucleotides, whereas those with long side chains or of high potency are not metabolized. Bisphosphonate metabolism in cell-free extracts of Dictyostelium was accompanied by inhibition of aminoacylation of tRNA by several aminoacyl-tRNA synthetases. These enzymes were barely affected by the bisphosphonates that were not metabolized. The results indicate that some bisphosphonates are not metabolically inert analogues of pyrophosphate and appear to be metabolized by aminoacyl-tRNA synthetases. The cellular effects of some bisphosphonates may be the result of their incorporation into adenine nucleotides or inhibition of aminoacyl-tRNA synthetases, although the potent bisphosphonates appear to act by a different mechanism.

scholarly journals Lysyl-tRNA synthetase from Escherichia coli K12. Chromatographic heterogeneity and the lysU-gene product

1987 ◽  
Vol 248 (1) ◽  
pp. 43-51 ◽  
Author(s):  
J Charlier ◽  
R Sanchez
Keyword(s):  
Escherichia Coli ◽  
Gene Product ◽  
Activity Ratio ◽  
Deae Cellulose ◽  
Trna Synthetase ◽  
Trna Synthetases ◽  
E Coli ◽  
Aminoacyl Trna Synthetases

In contrast with most aminoacyl-tRNA synthetases, the lysyl-tRNA synthetase of Escherichia coli is coded for by two genes, the normal lysS gene and the inducible lysU gene. During its purification from E. coli K12, lysyl-tRNA synthetase was monitored by its aminoacylation and adenosine(5′)tetraphospho(5′)adenosine (Ap4A) synthesis activities. Ap4A synthesis was measured by a new assay using DEAE-cellulose filters. The heterogeneity of lysyl-tRNA synthetase (LysRS) was revealed on hydroxyapatite; we focused on the first peak, LysRS1, because of its higher Ap4A/lysyl-tRNA activity ratio at that stage. Additional differences between LysRS1 and LysRS2 (major peak on hydroxyapatite) were collected. LysRS1 was eluted from phosphocellulose in the presence of the substrates, whereas LysRS2 was not. Phosphocellulose chromatography was used to show the increase of LysRS1 in cells submitted to heat shock. Also, the Mg2+ optimum in the Ap4A-synthesis reaction is much higher for LysRS1. LysRS1 showed a higher thermostability, which was specifically enhanced by Zn2+. These results in vivo and in vitro strongly suggest that LysRS1 is the heat-inducible lysU-gene product.

scholarly journals Effects of liver regeneration on tRNA contents and aminoacyl-tRNA synthetase activities and sedimentation patterns

1986 ◽  
Vol 236 (1) ◽  
pp. 163-169 ◽  
Author(s):  
U Del Monte ◽  
S Capaccioli ◽  
G Neri Cini ◽  
R Perego ◽  
R Caldini ◽  
...  
Keyword(s):  
Protein Degradation ◽  
Cultured Cells ◽  
Trna Synthetase ◽  
Synthetase Activity ◽  
Regenerating Liver ◽  
Trna Synthetases ◽  
Aminoacyl Trna Synthetases ◽  
Acceptor Capacity ◽  
Increased Activity

The tRNA content and aminoacyl-tRNA synthetases of regenerating liver in the phase of rapid growth were compared with those of livers from both intact and sham-operated rats. At 48 h after hepatectomy, the amount of active tRNA (called ‘total acceptor capacity’) is significantly higher in regenerating liver than in control livers, owing to a general, possibly not uniform, increase in the various tRNA families, which suggests that it may contribute to the increased protein synthesis and to decreased protein degradation as well. The activities of most, but not of all, aminoacyl-tRNA synthetases in cell sap of regenerating liver tend to be greater than normal. Increased activity of histidyl-tRNA synthetase fits in with the possibility that the mechanisms that control the rate of protein degradation through aminoacylation of tRNAHis in cultured cells [Scornik (1983) J. Biol. Chem. 258, 882-886] also operate in the liver and play a role in regeneration. Sedimentation analysis of cell sap in sucrose density gradients shows a shift of prolyl-tRNA synthetase activity toward the high-Mr form in regenerating liver. This change might be related to the positive protein balance and to growth in vivo, since it is also observed in the anaplastic Yoshida ascites hepatoma AH 130.

scholarly journals Drosophila Models for Charcot–Marie–Tooth Neuropathy Related to Aminoacyl-tRNA Synthetases

Genes ◽  
2021 ◽  
Vol 12 (10) ◽  
pp. 1519
Author(s):  
Laura Morant ◽  
Maria-Luise Erfurth ◽  
Albena Jordanova
Keyword(s):  
Motor Neurons ◽  
Clinical Manifestations ◽  
Neuromuscular Disorder ◽  
Charcot Marie Tooth ◽  
Trna Synthetases ◽  
Aminoacyl Trna Synthetases ◽  
Global Protein Synthesis ◽  
Peripheral Sensory ◽  
Drosophila Models

Aminoacyl-tRNA synthetases (aaRS) represent the largest cluster of proteins implicated in Charcot–Marie–Tooth neuropathy (CMT), the most common neuromuscular disorder. Dominant mutations in six aaRS cause different axonal CMT subtypes with common clinical characteristics, including progressive distal muscle weakness and wasting, impaired sensory modalities, gait problems and skeletal deformities. These clinical manifestations are caused by “dying back” axonal degeneration of the longest peripheral sensory and motor neurons. Surprisingly, loss of aminoacylation activity is not a prerequisite for CMT to occur, suggesting a gain-of-function disease mechanism. Here, we present the Drosophila melanogaster disease models that have been developed to understand the molecular pathway(s) underlying GARS1- and YARS1-associated CMT etiology. Expression of dominant CMT mutations in these aaRSs induced comparable neurodegenerative phenotypes, both in larvae and adult animals. Interestingly, recent data suggests that shared molecular pathways, such as dysregulation of global protein synthesis, might play a role in disease pathology. In addition, it has been demonstrated that the important function of nuclear YARS1 in transcriptional regulation and the binding properties of mutant GARS1 are also conserved and can be studied in D. melanogaster in the context of CMT. Taken together, the fly has emerged as a faithful companion model for cellular and molecular studies of aaRS-CMT that also enables in vivo investigation of candidate CMT drugs.

scholarly journals Plasticity and Constraints of tRNA Aminoacylation Define Directed Evolution of Aminoacyl-tRNA Synthetases

2019 ◽  
Vol 20 (9) ◽  
pp. 2294 ◽  
Author(s):  
Ana Crnković ◽  
Oscar Vargas-Rodriguez ◽  
Dieter Söll
Keyword(s):  
Amino Acids ◽  
Directed Evolution ◽  
Active Site ◽  
Nonsense Suppression ◽  
Natural Amino Acid ◽  
Noncanonical Amino Acids ◽  
Trna Synthetases ◽  
Aminoacyl Trna Synthetases ◽  
Genetic Incorporation

Genetic incorporation of noncanonical amino acids (ncAAs) has become a powerful tool to enhance existing functions or introduce new ones into proteins through expanded chemistry. This technology relies on the process of nonsense suppression, which is made possible by directing aminoacyl-tRNA synthetases (aaRSs) to attach an ncAA onto a cognate suppressor tRNA. However, different mechanisms govern aaRS specificity toward its natural amino acid (AA) substrate and hinder the engineering of aaRSs for applications beyond the incorporation of a single l-α-AA. Directed evolution of aaRSs therefore faces two interlinked challenges: the removal of the affinity for cognate AA and improvement of ncAA acylation. Here we review aspects of AA recognition that directly influence the feasibility and success of aaRS engineering toward d- and β-AAs incorporation into proteins in vivo. Emerging directed evolution methods are described and evaluated on the basis of aaRS active site plasticity and its inherent constraints.

scholarly journals A developmentally controlled change in the post-translational modifications on the lysosomal α-mannosidase of the cellular slime mould Dictyostelium discoideum

1987 ◽  
Vol 243 (3) ◽  
pp. 739-746 ◽  
Author(s):  
B R Moore ◽  
G Vladutiu ◽  
S J Free
Keyword(s):  
Dictyostelium Discoideum ◽  
Human Fibroblasts ◽  
Amino Acid Sequences ◽  
Double Labelling ◽  
Early Form ◽  
Cellular Slime Mould ◽  
Slime Mould ◽  
Cellular Slime ◽  
Late Form

During the development of the cellular slime mould Dictyostelium discoideum, a second form of a number of lysosomal enzymes begins to accumulate. The second (‘late’) form of these enzymes differs from the pre-existing (‘early’) form in post-translational modification. Pulse-chase experiments using [35S]methionine show that the late form of alpha-mannosidase-1 is made by synthesis de novo starting 8 h after the onset of development. These experiments show there is no interconversion between early and late forms in vivo. A one-dimensional peptide map indicated that the early and late forms of alpha-mannosidase have similar amino acid sequences. The two forms have a similar half-life in vivo when measured during the same period of development. Double-labelling studies were performed with 35SO4 and [3H]leucine or 32PO4 and [3H]leucine. and these studies indicated that the oligosaccharides present on the early form of alpha-mannosidase contained more sulphate and phosphate than did those on the late form. The early enzyme had a 10-fold higher 35S/3H ratio and a 4-fold higher 32P/3H ratio. Endocytosis experiments using early and late alpha-mannoside showed that the early form was efficiently taken up by human fibroblasts, whereas the late form was poorly endocytosed. This suggests that the late form lacks the mannose 6-phosphate residue required for efficient uptake.

scholarly journals Protein synthesis and loss of viability in rye embryos. The lability of transferase enzymes during senescence

1973 ◽  
Vol 135 (3) ◽  
pp. 405-410 ◽  
Author(s):  
B. E. Roberts ◽  
D. J. Osborne
Keyword(s):  
Protein Synthesis ◽  
Free Protein ◽  
Trna Synthetases ◽  
Aminoacyl Trna Synthetases ◽  
Activity Of Enzymes ◽  
Eventual Loss ◽  
Soluble Components

Poor germination and eventual loss of viability of rye grains on storage reflects a decreasing ability of embryos to synthesize protein in vivo. Cell-free protein-synthesizing systems from low viability embryo stocks exhibit lesions in the soluble components of the post-ribosomal supernatant fractions. tRNA and aminoacyl-tRNA synthetases are not impaired. The activity of enzymes concerned with transfer of phenylalanyl-tRNA from the aminoacyl to the peptidyl site on the ribosome is slightly decreased. The transfer enzymes (transferase I) involved in the binding of phenylalanyl-tRNA to the aminoacyl site show a loss of activity that closely reflects the loss of viability and the decline of protein synthesis of the embryo in vivo. The inactivation of labile transferase I components may be a major factor leading to senescence and loss of viability in rye grains.

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