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TDP43 - Coggle Diagram

- - - - Resi 1–77
      - forms dimers & oligomers: single dissociation constant of ~70 mM (No cooperativity subunit binding)
        
        reversible TDP-43 polymer formation through NTD
        
        required for splicing activity**
        
        contribute to phase separation via liquid droplet formation
        
        contribute to cytoplasmic SG formation
      - site of 1 of 3 predicted mitochondrial targeting seq (F35 – L41) (in the loop following the a-helix)
      - conserved phosphosite S48
      - potential weak,nt binding
        
        X3 increased binding affinity for TDP-43 to ssDNA (TG)6 in the presence of NTD1-105 compared to only RRMs
        
        HSQC-NMR >> seq-specific(?) binding of folded form
        
        to RNA (UG)6
        
        ssDNA (TT)6 (?)
        
        ssDNA (TG)6
      - 6 b-strands &1 a-helix make ub-like b-grasp fold
        
        Like DIX domain of Axin 1
        
        NTD & DIX similarities
        
        very similar struc: differing by a moderate rotation & shift btw subunits, ~due to diff in experimental conditions
        
        The DIX domain is known to facilitate both homo- & heterooligomerization > dimeric NTD structures revealed a head-to-tail subunits configuration
        
        upon dimerization, a surf-accessible &mobile loop becomes more structured as it participates in the interface
        
        Phosphosite S48 is shifted toward the interface where it is involved in H-bonds
        
        mimicking phosphorylation at this site disrupts self
        association
        of the NTD and affects splicing function
        one of three mitochondrial targeting
        sequences
      - L27A & L28A
        
        monomeric TDP-43
        
        decreased(L27A) and abolished(L28A)
        TDP-43 splicing function
        
        induced a partial or
        complete cytoplasm localization
    - - RRM-nt binding
        
        2 highly conserved short seq motifs for nt binding
        
        RNP-1 (octa seq)
        
        KGFGFVRF in RRM1
        
        RAFAFVTF in RRM2
        
        RNP-2 (hexa seq)
        
        LIVLGL in RRM1
        
        VFVGRC in RRM2
        
        RRM1: Resi 106–177, RRM2: Resi 192–259
        
        RRM1 & 2 fold & binding mode
        
        5-stranded beta sheet stacked against 2 a-helices
        
        Stacking interaction of conserved b-sheet aromatic & hydrophobic residues w/ the bases & sugar rings of ssRNA or DNA
        
        5'>3' direction unlike typical tandem 4-stranded RRMs
        
        both have a suppl b-strand (necessary for struc stability
        
        preferential binding of TDP-
        43 to (TG)/(UG) sequences
        
        RRM1 bind other motifs, consistent w/ A3(GG)4A3,
        via a distinct binding site implicating R151
        
        R151A: No impact on seq recog of RRM1-
        RRM2 tandem construct while E246/D247 substitution decrease RNA seq specificity
        
        E246, especially D247 crucial in the struc of RRM2 & the binding of TDP-43 by hiding R151
        
        Cooperation btw RRM1/ RRM2 >> TDP-43 affinity increases w/ nt length: large affinity increase btw 4 & 6 nt long, corresponding to the 22 Å distance btw both nt binding sites
        
        RRM1: sufficient & essential for proper nt binding (affinity=~ low nM range for the canonical UG-rich seq) unlike RRM2
      - Amyloidogenic cores
        
        ability to misfold & participate in nucleation
        or propagation of TDP-43 aggregation in ALS
        
        Two ROIs identified in ALS patients but not in well-folded non-pathological TDP-43
        
        Resi 166–173 in RRM1
        
        in a reasonably accessible
        loop (50% accessibility)
        
        D169G
        
        stabilized TDP-43 & induced a loss of H-bond
        w/ T115 resulting in a local conformational change
        
        disease linked
        
        no impact on aggregate formation but increased caspase cleavage
        
        Resi 246–255 in
        RRM2
        
        lie between RRM1-RRM2
        & not exposed in the folded state
        
        In vitro: participate in the
        formation fibrillar structures
        
        minimal seq EDLIIKGISV is necessary & deletion of the
        1st + the 2nd resi >> reduced aggregation
        
        E246G or D247G pr induced the formation of fibrils
        
        247-DLIIKGISVHI-257 pep: amyloid polymorphism characterized by diff backbone conformations + 7 distinct steric zipper
        arrangements, a common struc feature of amyloid prs
        
        regions 166–173 & 246–255 target of several pathological modifications
        
        caspase cleavage
        
        ,
        oxidation
        
        ubiquitination
      - Former NES
        
        no effect on TDP-43 localization to cytoplasm: XPO1 inhibition by siRNA/inhibitors (leptomycin B - LMB-, selective inhibitors of nuclear export -SINE) or overexpression of XPO1 had
        
        when fused to GFP/YFP did not induce a specific
        localization to cytoplasm of the constructs
        
        weak affinity of for XPO1:uM range
        
        For XPO7 (Exportin 7) & NXF1 (Nuclear RNA export factor 1): passive diffusion mechanism, slowed down by size & inhibited by newly synthesized RNA binding.
        
        neuroprotective
        
        deltaNES (I239A/L243A,L248A/I249A/I250A)
        
        No TDP-43-related toxicity in Drosophila & cell lines
        
        disruption of TDP-43 splicing function
        
        @ the RNA interface
        
        Comparison of TDP43-WT vs TDP43-deltaNES shows a suppression of the known amyloidogenic core @ resi 244–
        255, exposed in ALS patients
        
        F147/149L mutation
        
        disruption of splicing
        
        alleviate overexpressed TDP-43 toxicity
        
        Not @ the RNA interface but @ But, those the interface btw RRM1 & RRM2 (any modification of those contacts seems to lead to loss)
    - - Resi 260–414
      - 50 disease-linked mutations
      - Contains most of the
        phosphorylation sites
      - disordered>>All studies on fragments
      - required for TDP-43 splicing activity
        
        including
        autoregulation
      - interaction site
        with several pr partners, e.g. UBQLN2, FMRP, hnRNP
      - Organization of the CTD
        
        distinct subdomains
        
        2 Gly-aromatic-Ser-rich (GaroS) regions
        
        resi 273–317 & 368–414
        
        similar to regions in FUS
        
        contributing
        to the formation of hydrogels
        
        interact
        w/in RNA granules
        
        resi 286–331: contains an ALS linked mutation (A315E, Q331K)
        & folds into an R-shaped motif
        
        encodes prion-like struc, potentiating cytoplasmic aggregation
        
        low-complexity Gly-rich region
        
        amyloidogenic core divided into
        
        resi 318–340: hydrophobic region
        
        can adopt a helical structure
        
        Thioflavin T-positive filaments
        consistent w/ cross-b architecture
        
        helix-to-sheet transition of the hydrophobic
        region
        
        resi 341–369: Q/N-rich region
        
        form amyloid & amyloid-like aggregates
        
        6 segments forming classic steric zippers & contribute to
        pathogenic aggregation
        
        resi 320–343:Omega-loop region
        
        neurotoxicity
        
        form Low complexity Aromatic-Rick Kinked Struc (LARKS) sensitive to mutation & phosphorylation
        
        stack into kinked b-sheets, forming reversible (Velcro-like) associations (role in pr interactions & reversible association of the CTD + pathogenic aggregation by bringing adjacent amyloid-forming segments together)
        
        extended b-hairpin struc w/ 2 longer regions not occuring simultanously (steric clashes)
        
        1st segment: resi 286–331
        
        2 more items...
        
        2nd segment: resi 311–360
        
        2 more items...
        
        Liquid-liquid phase separation (LLPS) & aggregation
        
        complicated role
        
        C-ter essential for aggregation
        
        CTD combined w/ RRM2 is required for high aggregate accumulation in cellular models (as RRM domains contain amylogenic
        seq elements contributing to aggregation)
        
        constructs lacking the CTD have been shown to aggregate
        
        reversible self-association,
        
        initiate formation of SGs & to which both the NTD and
        CTD seem to contribute
        
        conditions under which TDP-43 undergoes LLPS:
        highly sensitive to the conditions of the experiment
        
        interaction btw CTD & charged nt, specifically ssDNA, increases CTD’s ability to undergo LLPS, maybe via the many aromatic & pi interactions
        
        The Role of Charged resi
        
        Arg residues: integral role in changing material properties & dynamics of the droplets formed by TDP-43
        
        electrostatic repulsion modulates the formation of TDP-
        43 aggregation.
        
        link between LLPS and aggregation is unclear,
        
        Time is a key factor
        
        LLPS is important for formation of membraneless organelles
        
        SGs
        
        2 more items...
        
        mutations in CTD alter propensity for LLPS
    - - Resi 78-100
      - Recognized by Importin-a >> active transport of TDP-43 into the nucleus
      - NLS disruption causing TDP43 accumulation
        in the cytoplasm
        
        caspase cleavage at D89
        
        initiation at the alternative start site, M85
        
        Mild:**SNP A90V** >>low-level mislocalization
      - flexible linker >> connection btw NTD & RNA-binding domains is dynamic, allowing
        
        the arrangement of these domains to change with
        
        NTD-self association
        
        post-translational modifications
        
        interactions with other prs.
        
        RNA binding
  - - - cysteninopathia in ALS
        
        dramatic shift in TDP-43 solubility>> direct modification of TDP-43 via Cys oxidation
        
        C173,C175, C198, C244
        
        major redox-regulated
        cys resi
        
        intramolecular and intermolecular
        s-s bond
        
        C173 & C175 is 5.1 Å BUT 173 the least accessible and least likely to be oxidized in full length pr
        
        C198 & C244 is 15.5 Å
        
        sequential oxidation of RRM1 w C173's preferential oxidation & a conformational change allowing C175 modification & a subsequent formation of crosslinked dimers
        
        tandem RRM1-RRM2 struc analysis: C173 & C175 make contacts w/ resi in the RRM1.
        
        Loss of contacts by oxidation exposes amyloidogenic resi 166–173, by loss of control of correct & aberrant folding of TDP-43 in ALS depending on the freedom of their 173 & 175 thiol
        
        proximity of oxidation sites & TDP-43 disease-exposed regions in ALS patients (166–173 and 246–255) & cleavage sites>>the early
        misfolding of TDP-43 by oxidation, upstream of ubiquitination,
        phosphorylation, & fragmentation
        
        disease-linked mutations G348C & S379C, introducing new Cys
        resi in TDP-43, then undergo oxidation>>
        cross-linked TDP-43 species, >> the role of early
        oxidation in TDP-43 misfunction
        
        C39 & C50
        
        22–24 Å apart
        
        Much lesser extent redoc-regulated
        
        intramolecular s-s bonding btw those
        2 resi is very challenging
        
        intramolecular ss-bond formation: unlikely on a native TDP-43 struc
      - ROS>> TDP43 pathology
    - - associated w/ various pathways including RNA processing, cytoskeleton association, & cellular signaling
      - associated with aggregating proteins such as Tau, Huntingtin, SOD1
      - 2 main sites: K145 and K192
        
        acetylation mimics (K145Q and/or K192Q) >>
        formation of nuclear speckles, & cytoplasmic aggregates when
        the TDP-43-nuclear localization signal was impaired
        
        K145
        
        K145Q decreased nt binding translated into decreased splicing function
        
        part of RRM1 RNP-1 motif
        
        K145Q moderately accessible in
        the free protein
        
        moderately accessible in
        the free protein
        
        K192
        
        part of RRM2 RNP-2
        
        acetylation-null mutant (TDP43-2KR) diffuse,
        even in TDP-43 impaired NLS
        
        a recent study didn't find them acetylated
      - a PTM known to modulate pr-nt binding by neutralizing the electrostatic interface.
      - Lys82
        
        found in TDP-43 NLS
        
        impair proper shuttling
        to the nucleus
        
        act as a pathological event
    - - TDP-43 affinity in uM range
      - decrease TDP-43 thermostability & formed Thioflavin-T-positive aggregates, reminiscent of amyloid nuclei
      - Zinc treated
        
        TDP-43 proteinopathy: reduced expression, formation of small nuclear inclusions, & diffuse cytosolic localization.
        
        did not cause formation of CTD fragments,
        ubiquitination or phosphorylation of TDP-43
      - known to bind and
        promote in vitro aggregation of Tau, alphasynuclein
        (aSyn) & Amyloid-b Peptide(Ab)
      - Altered zinc homeostasis
        
        a risk factor for several neurodegenerative disorders
        such as ALS or Alzheimer’s disease
        
        relatively poor affinity of zinc for those proteins (in the micromolar range),
        
        direct contribution of zinc to TDP-43 aggregation could lead
        to complexes actively producing ROS similar to Ab and aSyn
        
        amplifying toxicity
    - - a pathological event in ALS
      - no confirmation on where the
        modification occurs on TDP-43
      - increases aggregation of those proteins
      - Prs SUMoylated
        
        Fused in Sarcoma (FUS)
        
        in superoxide dismustase
        1 (SOD1),
        
        TDP-43
      - Prediction by SUMOplot:
        
        K136
        
        30% in the free pr
        
        27% when TDP-43 is in complex with RNA
        
        poor surf accessibility K136 SUMO motif>> unlikely
        
        K84 or K140
        
        more likely to occur on a folded
        TDP-43
        
        SUMO-K84: disrupt TDP-43 NLS & active transport from cytoplasm to nucleus
        
        SUMO-K136: most detrimental by affecting interactions btw RRM1 & RRM2 domains & RNA binding
    - - hallmark feature of cytoplasmic aggregates in ALS/FTLD
      - S403/404 &/or 409/410: consistent ref marker of disease
      - in CTD mostly a later event, & to accumulate on the pr as it is trapped in the cytoplasm over time, depicting attempt from the cellular machinery to trigger degradation
      - At T153& Y155 by MAPK/ERK Kinase: not associated w/ pr
        accumulation & TDP-43 remained soluble NBUT capacity to
        bind nt reduced, consistent w/ a 2nd RNA binding site on RRM1
      - S375G
        
        early onset disease
        
        remove a phospho site >> increased nuclear localization compared to WT
        
        phosphomimic S375E>>cytotoxicity & increased cytoplasmic accumulation
        
        A reversible phosphorylation important for the regulation of TDP-43 cellular redistribution
      - pathogenesis based on phosphorylation alone is possible but is
        probably correlated with the site of phosphorylation and/or the
        phosphorylation machinery:~0.5 of known disease-linked mutation:.
        
        create potential phosphorylated
        sites (new Ser or Thr residues),
        
        remove phoshorylation
        sites (elimination of Ser or Thr residues)
        
        introduce a phosphomimic residue (Asp and Glu)
    - - one of the first pathological modifications of
        TDP-43 that was discovered
      - results in covalent attachment of (8.6 kDa) -ubiquitin-, similarly to SUMOylation, &role into protein degradation.
      - several ubiquitinated sites
        
        proteasome and autophagosome targeting function of TDP-
        43 ubiquitination
        
        K84 and K95 modification
        
        K84 to affect nuclear import
        
        K95 seemed to impact
        CTD phosphorylation
        
        no effect on TDP-43 solubility
        
        K160, K181, and K263 in the RRM domains
        
        K263
        
        modification could result in direct RNA binding reduction
        
        is the most
        accessible to modification
        
        K263E:
        
        K263E exhibits higher propensity to form
        aggregates
        
        enhanced ubiquitination that was
        attributed to ubiquitination to redundant sites
        
        K181 modification
        
        detrimental to TDP-43 struc possibly by
        
        modifying interactions btw RRM1 & RRM2 especially via loss of contact w/ the critical resi D247
        
        Affecting RRM-NTD interactions since it is close to the linker
        
        K160 modification
        
        less detrimental, even though it has potential contacts
        w/ F127 & T157
        
        MIGHT only occur
        post TDP-43 cleavage
    - - result of caspase cleavage at several sites
      - Cleavage at D89-A90
        
        lacks the NTD and disrupts the NLS but is correctly folded
        
        a 35 kDa fragment (TDP-35)
      - at D169-G170 and D174-C175
        
        lacks the
        NTD, NLS and most of RRM1
        
        both associated with the 25 kDa fragment (TDP-25)
        
        located at or in amyloidogenic cores >> cleavage might help in
        exposing those seq
      - at M218-D219 and E246-D247
        
        E246-D247located at or in amyloidogenic cores >> cleavage might help in
        exposing those seq
        
        disruption or elimination of NLS
      - disruption or elimination of TDP-43 NLS,
        trapping the protein in the cytoplasm
      - enhancing protein aggregation
      - early unfolding initiated by other PTMs, for example oxidation of C173 or C244, might result in exposure of those regions, increasing cleavage activity
      - disease-linked mutations, A90V & D169G link those cleavage sites to pathological forms of TDP-43.
    - - PARP (generate long
        and highly negatively charged linear or branched polymers)
        
        1 of the PAR-binding modules is the PAR-binding motif ((PBM: [HKR]1-X2-X3-[AIQVY]4-[KR]5-[KR]6-[AILV]7-[FILPV]8) where K, D & E can be modifie)
        
        two regions of interest with 80 and 63%
        of fitting to the canonical PBM which overlap
        with the bipartite NLS of TDP-43
        
        Multiple PBMs in the same pr
        
        can increase interaction of target pr w/
        PAR conjugation system
      - linked to DNA damage repair, unfolded protein response
        and cellular stress response
      - mammalian SG might contain PAR
      - tankyrase (a PAR catalase) reduced degeneration of the Drosophila eye linked to expression of human TDP-43
      - TDP-43-delta PBM
        
        induce a cytoplasmic localization of the pr
        
        excluded the pr from SG assembly
      - PARylation, close to TDP-43 NLS
        
        coherent with the need to avoid active
        shuttling to nucleus when the protein is needed in stress granules
        
        steric hindrance
        to mask TDP-43 NLS
      - form LLPS in the presence of PAR while TDP-43-deltaPBM produced an aberrant phase transition
      - TDP-35 & TDP-25: lacking partially or totally the PAR binding
        
        not able to co-localize to SG nor undergo correct
        liquid phase demixing
    - - mitochondrial targeting sequences
        in TDP-43, named M1, M3, and M5
        
        Deletion of each of signal
        
        did not abolish it
        
        greatly reduced TDP-43 mitochondrial
        localization
        
        the need for
        multiple sequences in the import process
        
        mitochondrial import of TDP-43 occurred through
        TOM70/TIM22 complexes
        
        to mediate translocation of
        pr that do not carry a classical matrix-targeting signal &
        bind multiple internal mitochondrial motifs
        
        except M5(located in the unstructured
        CTD), none of the mitochondrial signal are surface
        accessible in the well-folded pr
        
        M3 at thent binding site (RNP-2 of RRM1) & accessibility of
        the mitochondrial signals are greatly affected by RNA binding
      - pathological translocation of TDP-43 in mitochondria
        
        TDP-43 abnormally interacts with the mitochondrial chaperone Hsp60 in ALS (like FUS)
        
        Hsp70 in mitochondrial import bind TDP43 & dissolve aggregates & rescue toxicity
        
        Hsp70 is reduced and mislocalized to aggregates in
        SOD1 mutants mice
        
        defects in cytosolic Hsp70 led to enhanced
        entry of misfolded proteins into mitochondria and elevated
        mitochondrial stress in yeast
        
        abnormal Hsp70 could lead
        to exacerbated mislocalization of TDP-43 in mitochondria
      - RNA is neuroprotective
        
        loss of RNA binding could promote TDP-43 to localize in mitochondria
      - Mutations in TDP-43 could contribute to mislocalization to mitochondria
        
        G298S, occurring in M5
        
        increase mitochondrial import in patient fibroblasts and cell lines
        
        This mutation might thus make the mitochondrial
        signal more accessible.
        
        misfolding or unfolding, either local or total, of TDP-43 could help in exposing the mitochondrial targeting seq
        
        subsequent interaction
        with mitochondrial import machinery
        
        phospho-mimic G298D induced increased
        mitochondrial TDP-43 and phospho-null G298A reduced it.
        
        C39 occurs
        in M1
        
        oxidized
        
        which might help in
        TDP-43 unfolding.
        
        acetylation
        at K145
        
        at the N-terminus of the M3 signal and is
        the first residues of RNP-1 in RRM1