TAR DNA-binding protein 43 (TDP-43, transactive response DNA binding protein 43
kDa) is a
protein that in humans is encoded by the TARDBPgene.[5]
Structure
TDP-43 is 414
amino acid residues long. It consists of 4
domains: an N-terminal domain spanning residues 1-76 (NTD) with a well-defined
fold that has been shown to form a
dimer or
oligomer;[6][7] 2 highly conserved folded
RNA recognition motifs spanning residues 106-176 (RRM1) and 191-259 (RRM2), respectively, required to bind target
RNA and
DNA;[8] an unstructured C-terminal domain encompassing residues 274-414 (CTD), which contains a
glycine-rich region, is involved in protein-protein interactions, and harbors most of the
mutations associated with familial
amyotrophic lateral sclerosis.[9]
The entire protein devoid of large solubilising tags has been purified.[10] The full-length protein is a dimer.[10] The dimer is formed due to a self-interaction between two NTD domains,[6][7] where the dimerisation can be propagated to form higher-order oligomers.[6]
In December 2021 the structure of TDP-43 was resolved with
cryo-EM[11][12] but shortly after it was argued that in the context of
FTLD-TDP the protein involved could be
TMEM106B (which has been also resolved with cryo-EM), rather than of TDP-43.[13][14]
N-Terminal domain (NTD)
The
NTD located between residues 1 and 76 is involved in TDP-43
polymerization.[15] Indeed, dimers are formed by head-to-head interactions between NTDs, and the polymer thus obtained allows for
pre-mRNAsplicing.[16] However, further
oligomerization brings to more toxic accumulates. This process of polymerization into dimers, larger forms or just stabilizing monomers is dependent on TDP-43 conformational equilibrium between monomers, homodimers and oligomers. Hence, in TDP-43 diseased
cells, TDP-43's over-expression leads to the NTD showing high propensity to aggregate. Contrary to this, in normal cells, normal levels of TDP-43 allow for folded NTD, preventing aggregates and polymers formation.
More recently, this domain was found to have a
ubiquitin-like structure. It bears 27,6% of homology with
Ubiquitin-1 and a
β1-β2-
α1-β3-β4-β5-β6 + 2*
SO42- form.[17] Ubiquitin-like domain are usually associated with a greater affinity for
RNA/
DNA. However, in the unique case of TDP-43, the Ubiquitin-like NTD binds directly to
ssDNA. This interaction permits the conformational equilibrium cited higher to shift towards non-aggregated forms.[18]
The domain spanning from [1,80] has a
solenoid-like structure which sterically impedes interactions between aggregation prone
C-term regions.[16]
All of this raises the possibility that NTD and the
RNA Recognition Motifs (later on defined) could cooperatively interact with nucleic acids to accomplish TDP-43's physiological functions.[19]
Mitochondrial localization signal
There are six
mitochondrial localization signals[20] to be accounted on TDP-43's
amino acid sequence, although only M1, M3, and M5 were shown to be essential for mitochondrial localization. Indeed, their ablation leads to a lessened mitochondrial localization.
These localizing sequences are found on the following amino acids:
The
nuclear localization signal (NLS) domain is located between residues 82 and 98 is of critical importance in
ALS, and such is witnessed by the depletion or the mutations (notably A90V) of this domain, which cause loss-of-function from nucleus and promote aggregating, two processes very likely to conduct to TDP-43's toxic gain of function.[16]
It is thereby of the utmost importance to note that TDP-43's nuclear localization is absolutely critical for it to fulfill its physiological functions.[19]
RNA recognition motif
The
RNA recognition motif ranges between residues 105 and 181, much like many
hnRNPs, TDP-43's RRMs encompass highly conserved motifs of primary importance for fulfilling their function. Both RRMs follow this pattern: β1-α1-β2-β3-α2-β4-β5,[16] which allows them to bind to both
RNA and
DNA onto
UG/
TG-repeats of
3'UTR (Untranslated Terminal Regions) end of
mRNA/DNA.[15]
These sequences mainly ensure mRNA processing,
RNA export and RNA stabilizing. It is notably thanks to these sequences that TDP-43 importantly binds to its own mRNA regulates its very own
solubility and
polymerization.
RRM2
RRM2 spans between residues 181 and 261. In pathological conditions, it notably binds to
p65/NF-kB, an
apoptosis implicated factor, and is thus a potential therapeutic target. Moreover it can be burdened with a mutation, D169G, altering a key cleaving site for regulating formation of toxic inclusions.[21]
Nuclear export signal (NES)
The nuclear export signal is located between residues 239 and 251 sequence probably bears a role in TDP-43's shuttling function, and was recently found using a prediction algorithm.[22]
Disordered glycin rich C-terminal domain (CTD)
The Disordered
Glycin Rich C-terminal domain is located between residues 277 and 414. Much like 70 other
RNA binding proteins, TDP-43 bears a
Q/
N rich domain [344, 366] which resembles
yeastprion sequence. This sequence is called a Prion-Like Domain (PLD).[23]
PLDs are low complexity sequences that have been reported to mediate gene regulation via Liquid-Liquid Phase Transition (LLP) thus driving RNP granule assembly.[16] Forming these microscopically visible
RNP granules is thought to induce more effective gene regulatory process.[24]
It is here noted that LLP are reversible phenomenons of de-mixing a solution into two distinct liquid phases, hereby forming granules.
Mutations within the TDP-43 proteins Glycine Rich Region (GRR) have recently been identified as associates that can contribute to various neurodegenerative diseases, with the most notable and common NDD being ALS, about 10% of the mutations causing familial ALS are accredited with the TDP-43 protein [25]
This CTD is often reported to play important role in pathogenic behavior of TDP-43:
RNPs granules could have a role in stress response, and thus, aging, or persistence stress could lead the LLPs to turn into irreversible Liquid Solid Phase separation, pathological aggregates notably found in
ALS neurons.[26]
CTD's disorganized structure can turn into a full fledged
Amyloid-like
Beta-sheet rich structure causing it to adopt
prion-like properties.[16]
Moreover, CTFs are a common maker in diseased
neurons and are argued to be of high toxicity.
However, notice is to be taken that some points are not always consensual. Indeed, due to its
hydrophobic structure, TDP-43 can be hard to analyze, and parts of it remain somewhat vague. Precise sites of
phosphorylation,
methylation, or even binding are still a bit elusive.[16]
Function
TDP-43 is a transcriptional
repressor that binds to chromosomally integrated TAR DNA and represses
HIV-1 transcription. In addition, this protein regulates alternate splicing of the
CFTR gene. In particular, TDP-43 is a splicing factor binding to the intron8/exon9 junction of the CFTR gene and to the intron2/exon3 region of the apoA-II gene.[27][28] A similar pseudogene is present on chromosome 20.[29]
TDP-43 has been shown to bind both DNA and RNA and have multiple functions in transcriptional repression, pre-mRNA splicing and translational regulation. Recent work has characterized the transcriptome-wide binding sites revealing that thousands of RNAs are bound by TDP-43 in neurons.[30]
TDP-43 was originally identified as a transcriptional repressor that binds to chromosomally integrated
trans-activation response element (TAR) DNA and represses
HIV-1 transcription.[5] It was also reported to regulate alternate splicing of the
CFTR gene and the
apoA-II gene.[31][32]
In spinal motor neurons TDP-43 has also been shown in humans to be a low molecular weight neurofilament (hNFL) mRNA-binding protein.[22] It has also shown to be a
neuronal activity response factor in the dendrites of hippocampal neurons suggesting possible roles in regulating mRNA stability, transport and local translation in neurons.[33]
It has been demonstrated that zinc ions are able to induce aggregation of endogenous TDP-43 in cells.[34] Moreover, zinc could bind to RNA binding domain of TDP-43 and induce the formation of amyloid-like aggregates in vitro.[35]
DNA repair
TDP-43 protein is a key element of the
non-homologous end joining (NHEJ) enzymatic pathway that
repairs DNAdouble-strand breaks (DSBs) in pluripotent
stem cell-derived
motor neurons.[36] TDP-43 is rapidly recruited to DSBs where it acts as a scaffold for the further recruitment of the
XRCC4-
DNA ligase protein complex that then acts to seal the DNA breaks. In TDP-43 depleted human neural stem cell-derived motor neurons, as well as in sporadic
ALS patients' spinal cord specimens there is significant DSB accumulation and reduced levels of NHEJ.[36]
Clinical significance
A hyper-
phosphorylated,
ubiquitinated and cleaved form of TDP-43—known as pathologic TDP43—is the major disease protein in
ubiquitin-positive, tau-, and
alpha-synuclein-negative
frontotemporal dementia (FTLD-TDP, previously referred to as FTLD-U[37]) and in
amyotrophic lateral sclerosis (ALS).[38][39] Elevated levels of the TDP-43 protein have also been identified in individuals diagnosed with
chronic traumatic encephalopathy, and has also been associated with ALS leading to the inference that athletes who have experienced multiple
concussions and other types of
head injury are at an increased risk for both encephalopathy and motor neuron disease (ALS).[40] Abnormalities of TDP-43 also occur in an important subset of
Alzheimer's disease patients, correlating with clinical and neuropathologic features indexes.[41] Misfolded TDP-43 is found in the brains of
older adults over age 85 with
limbic-predominant age-related TDP-43 encephalopathy, (LATE), a form of dementia. New monoclonal antibodies, 2G11 and 2H1, have been developed to specify different TDP-43 inclusion types that occur across neurodegenerative diseases, without relying on hyper-phosphorylated epitopes.[42] These antibodies were raised against an epitope within the RRM2 domain (amino acid residues 198–216).[42]
HIV-1, the causative agent of
acquired immunodeficiency syndrome (AIDS), contains an
RNAgenome that produces a chromosomally integrated
DNA during the replicative cycle. Activation of HIV-1 gene expression by the transactivator "Tat" is dependent on an RNA regulatory element (TAR) located "downstream" (i.e. to-be transcribed at a later point in time) of the transcription initiation site.
Mutations in the TARDBP gene are associated with neurodegenerative disorders including
frontotemporal lobar degeneration and
amyotrophic lateral sclerosis (ALS).[43] In particular, the TDP-43 mutants M337V and Q331K are being studied for their roles in ALS.[44][45][46] While the aberrant mislocalization and cytoplasmic aggregation of TDP-43 characterizes FTLD with TDP-43 pathology (FTLD-TDP), recent work suggests the amyloid fibrils found in human FTLD-TDP brains are composed of transmembrane lysosomal protein
TMEM106b rather than TDP-43.[47] Cytoplasmic TDP-43 pathology is the dominant histopathological feature of
multisystem proteinopathy.[48] The N-terminal domain, which contributes importantly to the aggregation of the C-terminal region, has a novel structure with two negatively charged loops.[49] A recent study has demonstrated that cellular stress can trigger the abnormal cytoplasmic mislocalisation of TDP-43 in spinal motor neurons in vivo, providing insight into how TDP-43 pathology may develop in sporadic ALS patients.[50]
^Alcalde AI, Barcina Y, Larralde J, Ilundain A (March 1986). "Role of calcium in the phloretin effects on sugar transport in rat small intestine". Revista Espanola de Fisiologia. 42 (1): 23–28.
doi:
10.1038/nsmb.2698.
PMID2424061.
S2CID13783277.
^Fan AC, Leung AK (2016). "RNA Granules and Diseases: A Case Study of Stress Granules in ALS and FTLD". In Yeo GW (ed.). RNA Processing. Advances in Experimental Medicine and Biology. Vol. 907. Cham: Springer International Publishing. pp. 263–296.
doi:
10.1007/978-3-319-29073-7_11.
ISBN978-3-319-29071-3.
PMC5247449.
PMID27256390.
^Kwong LK, Neumann M, Sampathu DM, Lee VM, Trojanowski JQ (July 2007). "TDP-43 proteinopathy: the neuropathology underlying major forms of sporadic and familial frontotemporal lobar degeneration and motor neuron disease". Acta Neuropathologica. 114 (1): 63–70.
doi:
10.1007/s00401-007-0226-5.
PMID17492294.
S2CID20773388.
Kwong LK, Neumann M, Sampathu DM, Lee VM, Trojanowski JQ (July 2007). "TDP-43 proteinopathy: the neuropathology underlying major forms of sporadic and familial frontotemporal lobar degeneration and motor neuron disease". Acta Neuropathologica. 114 (1): 63–70.
doi:
10.1007/s00401-007-0226-5.
PMID17492294.
S2CID20773388.
Maruyama K, Sugano S (January 1994). "Oligo-capping: a simple method to replace the cap structure of eukaryotic mRNAs with oligoribonucleotides". Gene. 138 (1–2): 171–174.
doi:
10.1016/0378-1119(94)90802-8.
PMID8125298.
Suzuki Y, Yoshitomo-Nakagawa K, Maruyama K, Suyama A, Sugano S (October 1997). "Construction and characterization of a full length-enriched and a 5'-end-enriched cDNA library". Gene. 200 (1–2): 149–156.
doi:
10.1016/S0378-1119(97)00411-3.
PMID9373149.