scholarly journals Hypomyelinating Leukodystrophy 15 (HLD15)-Associated Mutation of EPRS1 Leads to Its Polymeric Aggregation in Rab7-Positive Vesicle Structures, Inhibiting Oligodendroglial Cell Morphological Differentiation

Polymers ◽  
2021 ◽  
Vol 13 (7) ◽  
pp. 1074
Author(s):  
Sui Sawaguchi ◽  
Mizuki Goto ◽  
Yukino Kato ◽  
Marina Tanaka ◽  
Kenji Tago ◽  
...  
Keyword(s):  
Morphological Differentiation ◽  
Proteolipid Protein ◽  
Trna Synthetase ◽  
Structural Protein ◽  
Aminoacyl Trna Synthetase ◽  
Wild Type ◽  
Mutant Proteins ◽  
Trna Species ◽  
Pelizaeus Merzbacher Disease ◽  
Hypomyelinating Leukodystrophy

Pelizaeus–Merzbacher disease (PMD), also known as hypomyelinating leukodystrophy 1 (HLD1), is an X-linked recessive disease affecting in the central nervous system (CNS). The gene responsible for HLD1 encodes proteolipid protein 1 (plp1), which is the major myelin structural protein produced by oligodendroglial cells (oligodendrocytes). HLD15 is an autosomal recessive disease affecting the glutamyl-prolyl-aminoacyl-tRNA synthetase 1 (eprs1) gene, whose product, the EPRS1 protein, is a bifunctional aminoacyl-tRNA synthetase that is localized throughout cell bodies and that catalyzes the aminoacylation of glutamic acid and proline tRNA species. Here, we show that the HLD15-associated nonsense mutation of Arg339-to-Ter (R339X) localizes EPRS1 proteins as polymeric aggregates into Rab7-positive vesicle structures in mouse oligodendroglial FBD-102b cells. Wild-type proteins, in contrast, are distributed throughout the cell bodies. Expression of the R339X mutant proteins, but not the wild-type proteins, in cells induces strong signals regulating Rab7. Whereas cells expressing the wild-type proteins exhibited phenotypes with myelin web-like structures bearing processes following the induction of differentiation, cells expressing the R339X mutant proteins did not. These results indicate that HLD15-associated EPRS1 mutant proteins are localized in Rab7-positive vesicle structures where they modulate Rab7 regulatory signaling, inhibiting cell morphological differentiation. These findings may reveal some of the molecular and cellular pathological mechanisms underlying HLD15.

scholarly journals Bi-allelic Mutations in EPRS, Encoding the Glutamyl-Prolyl-Aminoacyl-tRNA Synthetase, Cause a Hypomyelinating Leukodystrophy

2018 ◽  
Vol 102 (4) ◽  
pp. 676-684 ◽  
Author(s):  
Marisa I. Mendes ◽  
Mariana Gutierrez Salazar ◽  
Kether Guerrero ◽  
Isabelle Thiffault ◽  
Gajja S. Salomons ◽  
...  
Keyword(s):  
Trna Synthetase ◽  
Aminoacyl Trna Synthetase ◽  
Hypomyelinating Leukodystrophy

scholarly journals Hypomyelinating Leukodystrophy 7 (HLD7)-Associated Mutation of POLR3A Is Related to Defective Oligodendroglial Cell Differentiation, Which Is Ameliorated by Ibuprofen

2021 ◽  
Vol 14 (1) ◽  
pp. 11-33
Author(s):  
Sui Sawaguchi ◽  
Kenji Tago ◽  
Hiroaki Oizumi ◽  
Katsuya Ohbuchi ◽  
Masahiro Yamamoto ◽  
...  
Keyword(s):  
Protein Expression ◽  
Rna Polymerase ◽  
Rna Polymerase Iii ◽  
Mtor Signaling ◽  
Marker Protein ◽  
Wild Type ◽  
Mutant Proteins ◽  
Oligodendroglial Cell ◽  
Hypomyelinating Leukodystrophy ◽  
In Cells

Hypomyelinating leukodystrophy 7 (HLD7) is an autosomal recessive oligodendroglial cell-related myelin disease, which is associated with some nucleotide mutations of the RNA polymerase 3 subunit a (polr3a) gene. POLR3A is composed of the catalytic core of RNA polymerase III synthesizing non-coding RNAs, such as rRNA and tRNA. Here, we show that an HLD7-associated nonsense mutation of Arg140-to-Ter (R140X) primarily localizes POLR3A proteins as protein aggregates into lysosomes in mouse oligodendroglial FBD−102b cells, whereas the wild type proteins are not localized in lysosomes. Expression of the R140X mutant proteins, but not the wild type proteins, in cells decreased signaling through the mechanistic target of rapamycin (mTOR), controlling signal transduction around lysosomes. While cells harboring the wild type constructs exhibited phenotypes with widespread membranes with myelin marker protein expression following the induction of differentiation, cells harboring the R140X mutant constructs did not exhibit them. Ibuprofen, a non-steroidal anti-inflammatory drug (NSAID), which is also known as an mTOR signaling activator, ameliorated defects in differentiation with myelin marker protein expression and the related signaling in cells harboring the R140X mutant constructs. Collectively, HLD7-associated POLR3A mutant proteins are localized in lysosomes where they decrease mTOR signaling, inhibiting cell morphological differentiation. Importantly, ibuprofen reverses undifferentiated phenotypes. These findings may reveal some of the pathological mechanisms underlying HLD7 and their amelioration at the molecular and cellular levels.

scholarly journals PLP1 Mutations in Patients with Multiple Sclerosis: Identification of a New Mutation and Potential Pathogenicity of the Mutations

2018 ◽  
Vol 7 (10) ◽  
pp. 342 ◽  
Author(s):  
Nancy Cloake ◽  
Jun Yan ◽  
Atefeh Aminian ◽  
Michael Pender ◽  
Judith Greer
Keyword(s):  
Multiple Sclerosis ◽  
Clinical Symptoms ◽  
Point Mutations ◽  
Human Leukocyte ◽  
In Silico Analysis ◽  
Proteolipid Protein ◽  
Wild Type ◽  
Exon 2 ◽  
Mutant Proteins ◽  
The Unfolded Protein Response

PLP1 is located on the X-chromosome and encodes myelin proteolipid protein (PLP), the most abundant protein in central nervous system myelin. Generally, point mutations in PLP1 result in X-linked dysmyelinating disorders, such as Pelizaeus-Merzbacher disease (PMD) or spastic paraplegia type 2 (SPG2). However, several case studies have identified patients with missense point mutations in PLP1 and clinical symptoms and signs compatible with a diagnosis of multiple sclerosis (MS). To investigate if PLP1 mutations occur relatively frequently in MS, we sequenced the coding regions of PLP1 in 22 female MS patients who had developed disease after the age of 40 and in 42 healthy women, and identified a missense mutation in exon 2 of PLP1 resulting in a Leu30Val mutation in the protein in one of the MS patients. mCherry-tagged plasmids containing wild type or mutant PLP1 sequences of PLP, including two known PMD/SPG2-related mutations as positive controls, were constructed and transfected into Cos-7 cells. In comparison with cells transfected with wild type PLP1, all mutations caused significant accumulation of PLP in the endoplasmic reticulum of the cells and induction of the unfolded protein response—a mechanism that leads to apoptosis of cells expressing mutant proteins. Additionally, in silico analysis of the binding of peptides containing the Leu30Val mutation to the human leukocyte antigen (HLA) molecules carried by the patient harboring this mutation suggested that the mutation could produce several novel immunogenic epitopes in this patient. These results support the idea that mutations in myelin-related genes could contribute to the development of MS in a small proportion of patients.

scholarly journals Studies on chemical modification of thionucleosides in the transfer ribonucleic acid of Escherichia coli

1974 ◽  
Vol 143 (2) ◽  
pp. 285-294 ◽  
Author(s):  
Yarlagadda S. Prasada Rao ◽  
Joseph D. Cherayil
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Chemical Modification ◽  
Gel Filtration ◽  
Trna Synthetase ◽  
Aminoacyl Trna Synthetase ◽  
E Coli ◽  
Chemical Reagents ◽  
Mild Conditions ◽  
Trna Species

35S-labelled tRNA from Escherichia coli was treated with chemical reagents such as CNBr, H2O2, NH2OH, I2, HNO2, KMnO4 and NaIO4, under mild conditions where the four major bases were not affected. Gel filtration of the treated tRNA showed desulphurization to various extents, depending on the nature of the reagent. The treated samples after conversion into nucleosides were chromatographed on a phosphocellulose column. NH2OH, I2 and NaIO4 reacted with all the four thionucleosides of E. coli tRNA, 4-thiouridine (s4U), 5-methylaminomethyl-2-thiouridine (mnm5s2U), 2-thiocytidine (s2C) and 2-methylthio-N6-isopentenyladenosine (ms2i6A), to various extents. CNBr, HNO2 and NaHSO3 reacted with s4U, mnm5s2U and s2C, but not with ms2i6A. KMnO4 and H2O2 were also found to react extensively with thionucleosides in tRNA. Iodine oxidation of 35S-labelled tRNA showed that only 6% of the sulphur was involved in disulphide formation. Desulphurization of E. coli tRNA with CNBr resulted in marked loss of acceptor activities for glutamic acid, glutamine and lysine. Acceptor activities for alanine, arginine, glycine, isoleucine, methionine, phenylalanine, serine, tyrosine and valine were also affected, but to a lesser extent. Five other amino acids tested were almost unaffected. These results indicate the fate of thionucleosides in tRNA when subjected to various chemical reactions and the involvement of sulphur in aminoacyl-tRNA synthetase recognition of some tRNA species of E. coli.

scholarly journals A Temporal Order in 5′- and 3′- Processing of Eukaryotic tRNAHis

2019 ◽  
Vol 20 (6) ◽  
pp. 1384 ◽  
Author(s):  
Marie-Theres Pöhler ◽  
Tracy Roach ◽  
Heike Betat ◽  
Jane Jackman ◽  
Mario Mörl
Keyword(s):  
Temporal Order ◽  
Unique Feature ◽  
Identity Element ◽  
Random Order ◽  
Trna Synthetase ◽  
Aminoacyl Trna Synthetase ◽  
Substrate Preferences ◽  
Positive Elements ◽  
Trna Species ◽  
Important Processing

For flawless translation of mRNA sequence into protein, tRNAs must undergo a series of essential maturation steps to be properly recognized and aminoacylated by aminoacyl-tRNA synthetase, and subsequently utilized by the ribosome. While all tRNAs carry a 3′-terminal CCA sequence that includes the site of aminoacylation, the additional 5′-G-1 position is a unique feature of most histidine tRNA species, serving as an identity element for the corresponding synthetase. In eukaryotes including yeast, both 3′-CCA and 5′-G-1 are added post-transcriptionally by tRNA nucleotidyltransferase and tRNAHis guanylyltransferase, respectively. Hence, it is possible that these two cytosolic enzymes compete for the same tRNA. Here, we investigate substrate preferences associated with CCA and G-1-addition to yeast cytosolic tRNAHis, which might result in a temporal order to these important processing events. We show that tRNA nucleotidyltransferase accepts tRNAHis transcripts independent of the presence of G-1; however, tRNAHis guanylyltransferase clearly prefers a substrate carrying a CCA terminus. Although many tRNA maturation steps can occur in a rather random order, our data demonstrate a likely pathway where CCA-addition precedes G-1 incorporation in S. cerevisiae. Evidently, the 3′-CCA triplet and a discriminator position A73 act as positive elements for G-1 incorporation, ensuring the fidelity of G-1 addition.

scholarly journals The Infantile Leukoencephalopathy-Associated Mutation of C11ORF73/HIKESHI Proteins Generates De Novo Interactive Activity with Filamin A, Inhibiting Oligodendroglial Cell Morphological Differentiation

Medicines ◽  
2021 ◽  
Vol 8 (2) ◽  
pp. 9
Author(s):  
Kohei Hattori ◽  
Kenji Tago ◽  
Shiori Memezawa ◽  
Arisa Ochiai ◽  
Sui Sawaguchi ◽  
...  
Keyword(s):  
De Novo ◽  
Transmembrane Proteins ◽  
Morphological Differentiation ◽  
Filamin A ◽  
Wild Type ◽  
Mutant Proteins ◽  
Cytoskeletal Network ◽  
Interactive Activity ◽  
Shed Light ◽  
Immunoblotting Technique

Background: Genetic hypomyelinating diseases are a heterogeneous group of disorders involving the white matter. One infantile hypomyelinating leukoencephalopathy is associated with the homozygous variant (Cys4-to-Ser (C4S)) of the c11orf73 gene. Methods: We observed that in mouse oligodendroglial FBD-102b cells, the C4S mutant proteins but not the wild type ones of C11orf73 are microscopically localized in the lysosome. And, they downregulate lysosome-related signaling in an immunoblotting technique. Results: The C4S mutant proteins specifically interact with Filamin A, which is known to anchor transmembrane proteins to the actin cytoskeleton; the C4S mutant proteins and Filamin A are also observed in the lysosome fraction. While parental FBD-102b cells and cells harboring the wild type constructs exhibit morphological differentiation, cells harboring C4S mutant constructs do not. It may be that morphological differentiation is inhibited because expression of these C4S mutant proteins leads to defects in the actin cytoskeletal network involving Filamin A. Conclusions: The findings that leukoencephalopathy-associated C11ORF73 mutant proteins specifically interact with Filamin A, are localized in the lysosome, and inhibit morphological differentiation shed light on the molecular and cellular pathological mechanisms that underlie infantile hypomyelinating leukoencephalopathy.

scholarly journals Two essential regions for tRNA recognition in Bacillus subtilis tryptophanyl-tRNA synthetase

2002 ◽  
Vol 365 (3) ◽  
pp. 749-756 ◽  
Author(s):  
Jie JIA ◽  
Feng XU ◽  
Xianglong CHEN ◽  
Li CHEN ◽  
Youxin JIN ◽  
...  
Keyword(s):  
Amino Acids ◽  
Bacillus Subtilis ◽  
Kinetic Parameters ◽  
Computer Modelling ◽  
Trna Synthetase ◽  
Deletion Mutants ◽  
High Homology ◽  
Wild Type ◽  
Mutant Proteins ◽  
Trna Recognition

Bacillus subtilis tryptophanyl-tRNA synthetase (TrpRS) is a homodimeric enzyme. A model for its ability to recognize tRNATrp in B. subtilis was proposed by using computer modelling. This was based on the the fact that there is high homology among bacterial TrpRSs [Chen, Jiang, Jin and Wang (2001) Acta Biochim. Biophys. Sinica 33, 687–690], in which the enzyme dimer binds to two tRNATrp molecules and each tRNATrp is bound to two different domains across the surface of the dimer. In this work, three deletion mutants of TrpRS were constructed and their products were purified. After determining the kinetic parameters of the mutants in the two-step reaction, it was found that the relative activities of wild-type and mutant enzymes had changed little in the ATP-pyrophosphate exchange reaction. In contrast, the activities of three mutant proteins were much decreased in the tRNATrp aminoacylation assay. Deletion of residues 108–122 and residues 234–238 caused 44% and 80% reductions in the activity, respectively. When both regions were deleted, the aminoacylation activity of the TrpRS mutant was too low to be determined using tRNATrp at the limiting concentration. Gel-retardation assays showed that the acceptor minihelix and the anticodon microhelix were recognized by the domains of TrpRS spanning residues 108–122 and residues 234–238 respectively. In addition, the deletion of amino acids 234–238 affected the normal induced expression of TrpRS at 37°C. In conclusion, residues 108–122 and 234–238 were found essential for tRNATrp recognition.

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