Neurodegeneration-associated protein fragments as short-lived substrates of the N-end rule pathway

Abstract

Protein aggregates are a common feature of neurodegenerative syndromes. Specific protein fragments were found to be aggregated in disorders including Alzheimer's disease, amyotrophic lateral sclerosis, and Parkinson's disease. Here, we show that the natural C-terminal fragments of Tau, TDP43, and α-synuclein are short-lived substrates of the Arg/N-end rule pathway, a processive proteolytic system that targets proteins bearing "destabilizing" N-terminal residues. Furthermore, a natural TDP43 fragment is shown to be metabolically stabilized in Ate1(-/-) fibroblasts that lack the arginylation branch of the Arg/N-end rule pathway, leading to accumulation and aggregation of this fragment. We also found that a fraction of Aβ42, the Alzheimer's disease-associated fragment of APP, is N-terminally arginylated in the brains of 5xFAD mice and is degraded by the Arg/N-end rule pathway. The discovery that neurodegeneration-associated natural fragments of TDP43, Tau, α-synuclein, and APP can be selectively destroyed by the Arg/N-end rule pathway suggests that this pathway counteracts neurodegeneration.

Figure 1. C-Terminal TDP43 Fragments From Aggregates in the Brains of Patients with Frontotemporal Lobal Degeneration (FTLD-TDP) As Short-Lived N-End Rule Substrates

(A) Domain organization of human TDP43. Arrowheads indicate the cleavage sites. RRM1, RRM2, RNA-binding domains. (B) The cleavage site 1 is indicated by an arrowhead, with the P1’ residue (Arg) in red. X²⁰⁸-TDP43^f fragments, produced from ^fDHFR-Ub^R48-X²⁰⁸-TDP43^f (X=Arg, Gln, Glu, Met), were expressed in reticulocyte extract and labeled with ³⁵S-Met/Cys for 10 min at 30°C, followed by a chase, immunoprecipitation with anti-flag antibody, SDS-PAGE, and autoradiography. Indicated molecular masses of proteins in this and other panels include their ~1 kDa flag epitope. (C) Quantification of B using the 33 kDa reference protein ^fDHFR-Ub^R48. (D) Same as B but with X²¹⁹-TDP43^f fragments (X=Asp, Arg-Asp, Met, Val). (E) Quantification of D. (F) Same as B but with X²⁴⁷-TDP43^f fragments (X=Asp, Arg-Asp, Met, Val). (G) Quantification of F. See also Figure S1.

Figure 2. In Vivo Degradation and Aggregation of X²⁴⁷-TDP43^f fragments

(A) Pulse-chase assays with X²⁴⁷-TDP43^f, produced from ^fDHFR-Ub^R48-X²⁴⁷-TDP43^f (X=Asp, Val, Arg-Asp). The fusion proteins were expressed in transiently transfected human HEK-293T cells, which were labeled with ³⁵S-Met/Cys for 15 min at 37°C, followed by a chase, preparation of extracts, immunoprecipitation with anti-flag, SDS-PAGE, and autoradiography. For designations, see the legend to Figure 1B. (B) Quantification of A. (C) Diagram of the mCherry-Ub^R48-Asp²⁴⁷-TDP43^f fusion. The arrowhead indicates the site of cleavage by deubiquitylases (Figure S1C). (D) Representative images of either Ate1^−/−(upper panels) or wt (lower panels) mouse EF cells transfected with a plasmid expressing mCherry-Ub^R48-Asp²⁴⁷-TDP43^f. The mCherry moiety of mCherry-Ub^R48 was detected by red fluorescence, and the Asp²⁴⁷-TDP43^f fragment was detected by indirect immunofluorescence, using anti-flag antibody and a fluorescein-conjugated secondary antibody. White bars indicate 10 µm. (E) Percentage of mCherry-positive cells containing Asp²⁴⁷-TDP43^f aggregates, with surveys of > 1,000 mCherry-positive cells of the wt and Ate1^−/− genotypes. The error bars indicate SEM (standard error of measurement). Statistical analyses employed the unpaired t-test (p <10⁻¹²). See also Figure S2A.

Figure 3. Neurodegeneration-Associated C-Terminal Fragments of Human α-Synuclein and Tau As Short-Lived N-End Rule Substrates

(A) Domain organization of human α-Synuclein. Arrowhead indicates the metalloprotease cleavage site. (B) The cleavage site is indicated by an arrowhead, with the P1’ Gln (Q) residue in red. X⁷⁹-αSyn^f, produced from ^fDHFR-Ub^R48-X-αSyn^f (X= Gln, Val) in reticulocyte extract, were assayed as described in the legend to Figure 1B. (C) Quantification of B using the reference ^fDHFR-Ub^R48. (D) Domain organization of human Tau-2N. Arrowheads indicate the calpain cleavage sites. (E) Same as B but with X³-Tau-2N^f, produced from ^fDHFR-Ub^R48-X³-Tau-2N^f (X=Glu, Val). (F) Quantification of E. See also Figure S3.

Figure 4. N-Terminal Arginylation of Aβ42, and the Degradation of C-terminally Tagged Aβ42 by the Arg/N-End Rule Pathway

(A) Human APP near its transmembrane domain, indicated by yellow rectangle. Arrowheads indicate the cleavage sites by secretases that yield Aβ42, termed Asp-Aβ42 and indicated by a red square parenthesis. (B) C-terminally tagged Asp-Aβ42^13myc (produced from ^fDHFR-Ub^R48-Asp-Aβ42^13myc) was assayed in reticulocyte extract as described in the legend to Figure 1B, with either no added dipeptides (lanes 1–3), or 5 mM Arg-Ala (lanes 4–6), or Ala-Arg (lanes 7–9). (C) Quantification of B. (D) Nt-arginylation of Asp-Aβ42 by isoforms of mouse Ate1 R-transferase. 0.1 or 1 µg of Ate1-1 (lanes 3, 4), Ate1-2 (lanes 5, 6), Ate1-3 (lanes 7, 8), and Ate1-4 (lanes 9, 10) were incubated for 30 min at 37 °C with 20 µg of chemically synthesized Asp-Aβ42, [¹⁴C]-L-arginine, and other components of the Nt-arginylation assay, followed by SDS/PAGE and autoradiography. Lane 1, complete reaction (including equal amounts of all four Ate1 isoforms, 1 µg total) but no added Asp-Aβ42. Lane 2, same as lane 3 but without Ate1. Upper panel: immunoblotting with antibody to mouse Ate1. Middle panel: autoradiography to detect ¹⁴C-labeled Arg-Asp-Aβ42. Lower panel: immunoblotting with anti-4G8 antibody to detect Asp-Aβ42 or Arg-Asp-Aβ42. (E) Steady-state levels of X-Aβ42^13myc, produced from ^fDHFR-Ub^R48-X-Aβ42^13myc (X=Asp, Val, Arg-Asp, Met), in wt, ate1Δ, and ubr1Δ strains of S. cerevisiae. Upper panels: immunoblotting with anti-myc antibody. Lower panels: immunoblotting with anti-flag antibody. See also Figure S4.

Figure 5. Untagged, N-terminally Arginylated Human Aβ42 in Mouse Brains

(A) Antibody (anti^R-Aβ) specific for RDAEFRHDSGYC was examined by dot assay with Nt-arginylated Aβ and BRCA1 peptides (see the main text). (B) Immunoblotting of extracts from the indicated ate1Δ or ubr1Δ yeast strains expressing X-Aβ42^13myc, produced in vivo from ^fDHFR-Ub^R48-X-Aβ42^13myc (X=Asp, Arg-Asp). Upper panel: immunoblotting with anti^R-Aβ antibody, characterized in A. Lower panel: immunoblotting with anti-myc antibody. (C) X-Aβ42^13myc was produced from ^fDHFR-Ub^R48-X-Aβ42^13myc (X=Arg-Asp, Met, Asp) in the wt, Ate1^−/−, or Ubr1^−/− Ubr2^−/− mouse EF cell lines, followed by SDS-PAGE of cell extracts and immunoblotting with anti-myc antibody. (D) Immunoblotting with anti-4G8 (recognizing total Aβ) of the indicated brain extracts from either 5xFAD or non-Tg (nontransgenic) mice. Also shown are immunoblots with antibody to GAPDH (loading controls). (E) Untagged, Nt-arginylated human Aβ42 in 70% formic acid (FA) extracts from mouse and human brains, detected by immunoblotting with anti^R-Aβ. Lanes 1 and 2, FA extracts from brains of non-Tg and 5xFAD mice, respectively, that were treated with inhibitors of proteasome and neprilysin 12 hrs before harvesting brains. Lane 3 and 4, FA extracts from the cortices of a human AD patient and a non-AD control, respectively. Lane 5, same as lane 2 but from 5xFAD mice that had not been treated with protease inhibitors. Lane 6, in vitro Nt-arginylated Arg-Asp Aβ42 (see Figure 4D), a positive control. See also Figure S4.