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Characterization of the upstream and intron promoters of the gene encoding TAR DNA-binding protein.

ABSTRACT: TAR DNA-binding protein (TDP-43, encoded by TARDBP) is a multifunctional protein that regulates transcription and RNA metabolism by binding DNA or RNA. TDP-43 has been implicated in the pathogenesis of amyotrophic lateral sclerosis (ALS) because abnormal accumulation of cleaved and phosphorylated C-terminal fragments of TDP-43 in motor neurons is a pathological hallmark of ALS. Here, we cloned and analyzed the promoter region of the TARDBP gene. TARDBP upstream sequences and/or intron/luciferase constructs were generated, and their promoter activity was experimentally assessed. The upstream region predictably exhibited promoter activity and identified putative cis-acting elements, including the i-motif, was relevant for the regulation of TDP-43 expression. The cellular abundance of TDP-43 is strictly controlled, and its constancy is critically important for motor neuron survival. A machinery serving to maintain a constant level of TDP-43 is autoregulation via control of mRNA stability, a negative feedback system involving binding to the 3' untranslated region of its own pre-mRNA. However, whether transcriptional mechanisms contribute to TDP-43 autoregulation is unclear. We further showed that TDP-43 negatively regulates the TARDBP promoter and, surprisingly, that disease-causing TDP-43 mutants lacked this regulatory activity. These results allowed the elucidation of a novel transcriptional autoregulatory mechanism of TDP-43.

SUBMITTER: Hasegawa-Ogawa M

PROVIDER: S-EPMC8062691 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Biophysical characterization of full-length TAR DNA-binding protein (TDP-43) phase separation.

Project description:Amyotrophic lateral sclerosis and frontotemporal lobar degeneration with ubiquitin-positive inclusions are associated with deposition of cytosolic inclusion bodies of TAR DNA-binding protein 43 (TDP-43) in brain and motor neurons. We induced phase separation of purified full-length TDP-43 devoid of large tags using a solution-jump method, and monitored it with an array of biophysical techniques. The tetramethylrhodamine-5-maleimide- or Alexa488-labeled protein formed rapidly (<1 min) apparently round, homogeneous and 0.5-1.0 μm wide assemblies, when imaged using confocal fluorescence, bright-field, and stimulated emission depletion microscopy. The assemblies, however, had limited internal diffusion, as assessed with fluorescence recovery after photobleaching, and did not coalesce, but rather clustered into irregular bunches, unlike those formed by the C-terminal domain. They were enriched with α-helical structure, with minor contributions of β-sheet/random structure, had a red-shifted tryptophan fluorescence and did not bind thioflavin T. By monitoring with turbidimetry both the formation of the spherical species and their further clustering under different experimental conditions, we carried out a multiparametric analysis of the two phenomena. In particular, both processes were found to be promoted by high protein concentrations, salts, crowding agents, weakly by reducing agents, as the pH approached a value of 6.0 from either side (corresponding to the TDP-43 isoionic point), and as the temperature approached a value of 31°C from either side. Important differences were found with respect to the TDP-43 C-terminal domain. Our multiparametric results also provide explanations to some of the solubility data obtained on full-length TDP-43 that were difficult to explain following the multiparametric analysis acquired on the C-terminal domain.

| S-EPMC9703588 | biostudies-literature

Characterization of alternative isoforms and inclusion body of the TAR DNA-binding protein-43.

Project description:TAR DNA-binding protein-43 (TDP-43) has been recently identified as a major component of the ubiquitinated inclusions found in frontotemporal lobar degeneration with ubiquitin-positive inclusions and in amyotrophic lateral sclerosis, diseases that are collectively termed TDP-43 proteinopathies. Several amyotrophic lateral sclerosis-linked mutations of the TDP-43 gene have also been identified; however, the precise molecular mechanisms underlying the neurodegeneration remain unclear. To investigate the biochemical characteristics of TDP-43, we examined truncation, isoforms, and cytoplasmic inclusion (foci) formation using TDP-43-expressing cells. Under apoptosis, caspase-3 generates two 35-kDa (p35f) and 25-kDa (p25f) fragments. However, in caspase-3(-/-) cells, novel caspase-3-independent isoforms of these two variants (p35iso and p25iso) were also detected under normal conditions. With a deletion mutant series, the critical domains for generating both isoforms were determined and applied to in vitro transcription/translation, revealing alternate in-frame translation start sites downstream of the natural initiation codon. Subcellular localization analysis indicated that p35 (p35f and p35iso) expression leads to the formation of stress granules, cellular structures that package mRNA and RNA-binding proteins during cell stress. After applying proteasome inhibitors, aggresomes, which are aggregates of misfolded proteins, were formed in the cytoplasm of cells expressing p35. Collectively, this study demonstrates that the 35-kDa isoforms of TDP-43 assemble in stress granules, suggesting that TDP-43 plays an important role in translation, stability, and metabolism of mRNA. Our findings provide new biological and pathological insight into the development of TDP-43 proteinopathies.

| S-EPMC2804209 | biostudies-literature

Identification and characterization of ubiquitinylation sites in TAR DNA-binding protein of 43 kDa (TDP-43).

Project description:TAR DNA-binding protein of 43 kDa (TDP-43) forms pathological aggregates in neurodegenerative diseases, particularly in certain forms of frontotemporal dementia and amyotrophic lateral sclerosis. Pathological modifications of TDP-43 include proteolytic fragmentation, phosphorylation, and ubiquitinylation. A pathognomonic TDP-43 C-terminal fragment (CTF) spanning amino acids 193-414 contains only four lysine residues that could be potentially ubiquitinylated. Here, serial mutagenesis of these four lysines to arginine revealed that not a single residue is responsible for the ubiquitinylation of mCherry-tagged CTF. Removal of all four lysines was necessary to suppress ubiquitinylation. Interestingly, Lys-408 substitution enhanced the pathological phosphorylation of the immediately adjacent serine residues 409/410 in the context of mCherry-CTF. Thus, Lys-408 ubiquitinylation appears to hinder Ser-409/410 phosphorylation in TDP-43 CTF. However, we did not observe the same effect for full-length TDP-43. We extended the mutagenesis study to full-length TDP-43 and performed MS. Ubiquitinylated lysine residues were identified in the nuclear localization sequence (NLS; Lys-84 and Lys-95) and RNA-binding region (mostly Lys-160, Lys-181, and Lys-263). Mutagenesis of Lys-84 confirmed its importance as the major determinant for nuclear import, whereas Lys-95 mutagenesis did not significantly affect TDP-43's nucleo-cytoplasmic distribution, solubility, aggregation, and RNA-processing activities. Nevertheless, the K95A mutant had significantly reduced Ser-409/410 phosphorylation, emphasizing the suspected interplay between TDP-43 ubiquitinylation and phosphorylation. Collectively, our analysis of TDP-43 ubiquitinylation sites indicates that the NLS residues Lys-84 and Lys-95 have more prominent roles in TDP-43 function than the more C-terminal lysines and suggests a link between specific ubiquitinylation events and pathological TDP-43 phosphorylation.

| S-EPMC6187624 | biostudies-literature

Progranulin mediates caspase-dependent cleavage of TAR DNA binding protein-43.

Project description:TAR DNA binding protein-43 (TDP-43) is the pathologic substrate of neuronal and glial inclusions in frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTDL-U) and in amyotrophic lateral sclerosis (ALS). Mutations in the progranulin gene (PGRN) have been shown to cause familial FTLD-U. The relationship between progranulin and TDP-43 and their respective roles in neurodegeneration is unknown. We report that progranulin mediates proteolytic cleavage of TDP-43 to generate approximately 35 and approximately 25 kDa species. Suppression of PGRN expression with small interfering RNA leads to caspase-dependent accumulation of TDP-43 fragments that can be inhibited with caspase inhibitor treatment. Cells treated with staurosporine also induced caspase-dependent cleavage and redistribution of TDP-43 from its nuclear localization to cytoplasm. Altered cleavage and redistribution of TDP-43 in cell culture models are similar to findings in FTLD-U and ALS. The results suggest that abnormal metabolism of TDP-43 mediated by progranulin may play a pivotal role in neurodegeneration.

| S-EPMC6673167 | biostudies-literature

Amyotrophic lateral sclerosis-plus syndrome with TAR DNA-binding protein-43 pathology.

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| S-EPMC2702763 | biostudies-literature

Identification and characterization of pbpC, the gene encoding Bacillus subtilis penicillin-binding protein 3.

Project description:Penicillin-binding proteins (PBPs) are enzymes involved in the synthesis of peptidoglycan structures in Bacillus subtilis such as the vegetative cell wall and the spore cortex. The B. subtilis sequencing project has identified a gene (orf16, EMBL accession number D38161) which exhibits significant sequence similarity to genes encoding class B high-molecular-weight PBPs. We have found that orf16 encodes PBP3 and have renamed this locus pbpC. Transcriptional fusions to lacZ were used to demonstrate that pbpC is transcribed primarily during log-phase growth, with lower amounts expressed during sporulation. During spore germination and outgrowth, pbpC expression resumes coincident with an increase in the optical density of the culture. The major promoter for pbpC is located just upstream of the gene; a low level of expression during sporulation appears to originate from much further upstream. Loss of PBP3 does not produce any detectable change in phenotype with respect to cell morphology, growth, sporulation, spore heat resistance, or spore germination and outgrowth. This was also true when the pbpC mutation was combined with mutations affecting other PBP-encoding genes to produce double mutants. These findings are consistent with previous evidence that many PBPs of B. subtilis have redundant functions within the cell.

| S-EPMC178458 | biostudies-literature

Characterization of devH, a gene encoding a putative DNA binding protein required for heterocyst function in Anabaena sp. strain PCC 7120.

Project description:The devH gene was identified in a screen for Anabaena sp. strain PCC 7120 sequences whose transcripts increase in abundance during a heterocyst development time course. The product of devH contains a helix-turn-helix motif similar to the DNA binding domain of members of the cyclic AMP receptor protein family, and the protein is most closely related to the cyanobacterial transcriptional activator NtcA. devH transcripts are barely detectable in vegetative cells and are induced approximately fivefold after nitrogen starvation. This induction is absent in the two developmental mutants hetR and ntcA. The gene is expressed as monocistronic transcripts with multiple 5' termini, and the approximately 500-bp region 5' to devH was shown to have promoter activity in vivo. The devH gene was insertionally inactivated by the integration of plasmid sequences within the open reading frame. Nitrogen starvation of the devH mutant induces heterocysts of wild-type morphology, but the mutant is inviable in the absence of fixed nitrogen and unable to reduce acetylene aerobically.

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Project description:The TAR DNA Binding Protein (TARDBP) gene has become relevant after the discovery of its several pathogenic mutations. The lack of evolutionary history is in contrast to the amount of studies found in the literature. This study investigated the evolutionary dynamics associated with the retrotransposition of the TARDBP gene in primates. We identified novel retropseudogenes that likely originated in the ancestors of anthropoids, catarrhines, and lemuriformes, i.e. the strepsirrhine clade that inhabit Madagascar. We also found species-specific retropseudogenes in the Philippine tarsier, Bolivian squirrel monkey, capuchin monkey and vervet. The identification of a retropseudocopy of the TARDBP gene overlapping a lncRNA that is potentially expressed opens a new avenue to investigate TARDBP gene regulation, especially in the context of TARDBP associated pathologies.

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Virus-mediated delivery of antibody targeting TAR DNA-binding protein-43 mitigates associated neuropathology.

Project description:The cytoplasmic aggregation of TAR DNA-binding protein-43 (TDP-43) is a hallmark of degenerating neurons in amyotrophic lateral sclerosis (ALS) and subsets of frontotemporal dementia (FTD). In order to reduce TDP-43 pathology, we generated single-chain (scFv) antibodies against the RNA recognition motif 1 (RRM1) of TDP-43, which is involved in abnormal protein self-aggregation and interaction with p65 NF-κB. Virus-mediated delivery into the nervous system of a scFv antibody, named VH7Vk9, reduced microgliosis in a mouse model of acute neuroinflammation and mitigated cognitive impairment, motor defects, TDP-43 proteinopathy, and neuroinflammation in transgenic mice expressing ALS-linked TDP-43 mutations. These results suggest that antibodies targeting the RRM1 domain of TDP-43 might provide new therapeutic avenues for the treatment of ALS and FTD.

| S-EPMC6436898 | biostudies-literature

TAR DNA-Binding Protein 43 is Cleaved by the Protease 3C of Enterovirus A71.

Project description:Enterovirus A71 (EV-A71) is one of the etiological pathogens leading to hand, foot, and mouth disease (HFMD), which can cause severe neurological complications. The neuropathogenesis of EV-A71 infection is not well understood. The mislocalization and aggregation of TAR DNA-binding protein 43 (TDP-43) is the pathological hallmark of amyotrophic lateral sclerosis (ALS). However, whether TDP-43 was impacted by EV-A71 infection is unknown. This study demonstrated that TDP-43 was cleaved during EV-A71 infection. The cleavage of TDP-43 requires EV-A71 replication rather than the activated caspases due to viral infection. TDP-43 is cleaved by viral protease 3C between the residues 331Q and 332S, while mutated TDP-43 (Q331A) was not cleaved. In addition, mutated 3C which lacks the protease activity failed to induce TDP-43 cleavage. We also found that TDP-43 was translocated from the nucleus to the cytoplasm, and the mislocalization of TDP-43 was induced by viral protease 2A rather than 3C. Taken together, we demonstrated that TDP-43 was cleaved by viral protease and translocated to the cytoplasm during EV-A71 infection, implicating the possible involvement of TDP-43 in the pathogenesis of EV-A71infection.

| S-EPMC7973337 | biostudies-literature

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