Fig 2: Suppression of the aggregate formation of neurodegenerative disease-associated proteins by PJA1. (A) Schematic presentation of the experiments for adenovirus-transduced protein aggregate formation. The 1464R-derived neuronal cells were infected with adenoviruses expressing DsRed- and FLAG-tagged genes of interest (GOI), i.e. human wild-type (WT) and P525L FUS (B), WT and G93A SOD1 (C), WT and A53T a-synuclein (D), ataxin-3 Q28 and Q84 (E), and huntingtin exon 1 Q23 and Q74 (F), and EGFP-tagged PJA1 or PJA1?R, followed by incubation with 0.5 µm MG-132. (B1–4) Suppression of FUS aggregate formation by PJA1. (B1) Western blot analysis of 1464R-derived neuronal cells infected with adenoviruses expressing DsRed- and FLAG-tagged human WT and P525L FUS (AxDsRhFUSWTFL, AxDsRhFUSP525LFL) and EGFP-tagged PJA1 (AxhPJA1EGFP) or PJA1?R (AxhPJA1?REGFP) in the presence f MG-132. (B2) Densitometric analysis of FLAG Western blot data of RIPA-insoluble WT and P525L FUS versus PJA1 and PJA1?R (PJA1D) (arrowheads in B1; n = 3) calibrated by GAPDH signals. Data are expressed as relative density compared with AxEGFP-treated control samples in the presence of MG132. Results are presented as mean ± SD (*P < 0.05). (B3) The co-immunoprecipitation (Co-IP) assay showing that PJA1 and PJA1?R bind to WT and P525L FUS that are ubiquitinated (arrowheads). (B4) Fluorescence microscopy of TuJ1-immunoreactive neurons infected with adenoviruses expressing DsRed-tagged human P525L FUS and EGFP-tagged human PJA1 in the presence of MG-132. The nucleus was counterstained with Hoechst 33342. (C1–4) Suppression of SOD1 aggregate formation by PJA1. (C1) Western blot analysis of 1464R-derived neuronal cells infected with adenoviruses expressing DsRed- and FLAG-tagged human WT and G93A SOD1 (AxDsRhSOD1WTFL, AxDsRhSOD1G93AFL) and EGFP-tagged PJA1 (AxhPJA1EGFP) or PJA1?R (AxhPJA1?REGFP) in the presence of MG-132. (C2) Densitometric analysis of the FLAG Western blot data of RIPA-insoluble WT and G93A SOD1 versus PJA1 and PJA1?R (PJA1D) (arrowheads in C1; n = 3) calibrated by GAPDH signals. Data are expressed as relative density compared with AxEGFP-treated control samples in the presence of MG132. Results are presented as mean ± SD (*P < 0.05). (C3) The Co-IP assay showing that PJA1 and PJA1?R bind to WT and G93A SOD1 that are ubiquitinated (arrowheads). (C4) Fluorescence microscopy of TuJ1-immunoreactive neurons infected with adenoviruses expressing DsRed-tagged human G93A SOD1 and EGFP-tagged human PJA1 in the presence of MG-132. The nucleus was counterstained with Hoechst 33342. (D1–4) Suppression of a-synuclein aggregate formation by PJA1. (D1) Western blot analysis of 1464R-derived neuronal cells infected with adenoviruses expressing DsRed- and FLAG-tagged human WT and A53T a-synuclein (AxDsRhSNCAWTFL, AxDsRhSNCAA53TFL) and EGFP-tagged PJA1 (AxhPJA1EGFP) or PJA1?R (AxhPJA1?REGFP) in the presence of MG-132. (D2) Densitometric analysis of the FLAG and phosphorylated a-synuclein Western blot data of RIPA-insoluble WT and A53T a-synuclein versus PJA1 and PJA1?R (PJA1D) (arrowheads in D1; n = 3) calibrated by GAPDH signals. Data are expressed as relative density compared with AxEGFP-treated control samples in the presence of MG132. Results are presented as mean ± SD (*P < 0.05). (D3) The Co-IP assay showing that PJA1 and PJA1?R bind to WT and A53T a-synuclein that are ubiquitinated (arrowheads). (D4) Fluorescence microscopy of TuJ1-immunoreactive neurons infected with adenoviruses expressing DsRed-tagged human A53T a-synuclein and EGFP-tagged human PJA1 in the presence of MG-132. The nucleus was counterstained with Hoechst 33342. (E1–4) Suppression of ataxin-3 aggregate formation by PJA1. (E1) Western blot analysis of 1464R-derived neuronal cells infected with adenoviruses expressing DsRed- and FLAG-tagged human ataxin-3 Q28 and Q84 (AxDsRhATXN3Q28FL, AxDsRATXN3Q84FL) and EGFP-tagged PJA1 (AxhPJA1EGFP) or PJA1?R (AxhPJA1?REGFP) in the presence of MG-132. (E2) Densitometric analysis of the FLAG Western blot data of RIPA-insoluble ataxin-3 Q28 and Q84 versus PJA1 and PJA1?R (PJA1D) (arrowheads in E1; n = 3) calibrated by GAPDH signals. Data are expressed as relative density compared with AxEGFP-treated control samples in the presence of MG132. Results are presented as mean ± SD (*P < 0.05). (E3) The Co-IP assay showing that PJA1 and PJA1?R bind to ataxin-3 Q28 and Q84 that are ubiquitinated (arrowheads). (E4) Fluorescence microscopy of 1464R-derived TuJ1-immunoreactive neurons infected with adenoviruses expressing DsRed-tagged human ataxin-3 Q84 and EGFP-tagged human PJA1 in the presence of MG-132. The nucleus was counterstained with Hoechst 33342. (F1–4) Suppression of ataxin-3 aggregate formation by PJA1. (F1) Western blot analysis of 1464R-derived neuronal cells infected with adenoviruses expressing DsRed- and FLAG-tagged huntingtin exon 1/Q23 and Q74 (AxDsRhHttEx1Q23FL, AxDsRhHttEx1Q74FL) and EGFP-tagged PJA1 (AxhPJA1EGFP) or PJA1?R (AxhPJA1?REGFP) in the absence or presence of MG-132. (F2) Densitometric analysis of the FLAG Western blot data of RIPA-insoluble huntingtin exon 1 Q23 and Q74 versus PJA1 and PJA1?R (PJA1D) (arrowheads in F1; n = 3) calibrated by GAPDH signals. Data are expressed as relative density compared with AxEGFP-treated control samples in the presence of MG132. Results are presented as mean ± SD (*P < 0.05). (E3) The Co-IP assay showing that PJA1 and PJA1?R bind to huntingtin exon 1/Q23 and Q74 that are ubiquitinated (arrowheads). (E4) Fluorescence microscopy of 1464R-derived TuJ1-immunoreactive neurons infected with adenoviruses expressing DsRed-tagged human huntingtin exon 1 Q74 and EGFP-tagged human PJA1 in the presence of MG-132. The nucleus was counterstained with Hoechst 33342.

Fig 3: Immunohistochemistry of PJA1 in human brains. (A–D) PJA1-immunoreactive cerebellar Purkinje cells (A), temporal cortical neurons (B), dopaminergic neurons in substantia nigra pars compacta (C), and hippocampal CA2 neurons (D) in a non-diseased 74-year-old male control case. (E) PJA1-immunoreactive anterior horn motor neurons containing PJA1-negative hyaline inclusions (arrows) in the cervical spinal cord in a SOD1-amyotrophic lateral sclerosis (ALS) case. (F, G) PJA1-immunoreactive, neuromelanin-bearing dopaminergic neurons containing PJA1-negative Lewy bodies (arrows) and PJA1-positive Marinesco bodies (arrowheads) in pars compacta of substantia nigra of a Parkinson's disease (PD) case. (H) A PJA1-immunoreactive neuron containing PJA1-negative cortical Lewy body in the amygdala of a PD case (arrow). (I, J) PJA1-immunoreactive neurons containing PJA1-negative (I), polyglutamine (1C2)-positive nuclear inclusions (J; arrows) in the amygdala of a Huntington's disease case. (K, L) A PJA1-immunoreactive pontine neuron containing PJA1- (K) and 1C2- (L) positive nuclear inclusions (arrows) in the pons of Machado–Joseph disease (MJD) case. (M–O) Double immunofluorescence of a pontine neuron containing PJA1- (M) and 1C2- (N) positive nuclear inclusions (arrows) in an MJD case. Scale bars: 50 µm (A–D); 20 µm (E-O).

Fig 4: PJA1 facilitates the binding of the SMC5/6 complex to viral and episomal DNAs. (A and B) 293T cells were plated in 6-cm dishes and cotransfected with 3 µg CMV-Luc and then with 2 µg pFlag-NSE1 (A) or 2 µg pcDNA3.1-PJA1B (B). NSE4 binding of the episomal reporter or GAPDH gene was monitored by ChIP assays using anti-NSE4 antibody. The level of immunoprecipitated DNA was determined by RT-qPCR. (C) 293T cells were plated in 6-well plates and cotransfected with 0.5 µg pCAGGS-HA-NSE4 and 0.5 µg pFlag-SMC5, pFlag-NSE1, or pFlag-NSE3 with or without 0.5 µg pcDNA3.1-PJA1?R. (D) 293T cells were plated in 6-well plates and cotransfected with 0.5 µg pCAGGS-HA-NSE4 and 0.5 µg pFlag-NSE1 and then with pcDNA3.1-PJA1B?R at different concentrations (0, 0.5, and 1.5 µg). Cells were lysed in RIPA lysis buffer. The immunoprecipitates and whole-cell lysates were analyzed by Western blotting with anti-HA or anti-Flag antibody. (E and F) HepG2 cells stably expressing PJA1B were plated in 6-cm dishes and infected with HSV-1 at an MOI of 10 for 8 h. The binding of NSE4 to the VP16 gene promoter (E) or UL36 gene (F) of HSV-1 was monitored by a ChIP assay using beads only, rabbit IgG, or anti-NSE4 antibody. Data are shown as means ± SD and correspond to results of a representative experiment out of three performed. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Fig 5: DNA topoisomerases are involved in PJA1-mediated restriction of viral and episomal DNAs. (A) 293T cells were plated in 12-well plates and transfected with 1 µg sh-NC, sh-SMC6, or sh-PJA1 for 48 h. SMC6, PJA1, NSE4, NSE1, and GAPDH proteins expressed in the transfected cells and in 293T control or 293T(PJA1-KO) cells were detected by Western blot analyses using the corresponding antibodies. WT, wild type. (B) 293T cells were plated in 24-well plates, cotransfected with 0.2 µg pCMV-Luc and 0.2 µg sh-SMC6 or sh-NC for 48 h, and then transfected with 0.2 µg pCAGGS or pCAGGS-HA-PJA1B for 48 h. Luciferase activities were measured. (C) Two stable cell lines were generated, in which sh-NC and sh-SMC6 were stably expressed in HepG2 cells. The stable cells were cotransfected with 0.1 µg pCMV-Luc and pCAGGS-HA-PJA1B at different concentrations (0, 0.02, 0.05, and 0.1 µg) for 48 h. Luciferase activities were measured. (D to G) 293T cells were plated in 24-well plates and cotransfected with 0.3 µg pCAGGS-PJA1B and 0.2 µg pHBV-Enh1-Luc for 24 h. The transfected cells were treated with different inhibitors (D); with topotecan or idarubicin at 5 µM for 10 h (E); or with topotecan (F) or idarubicin (G) at concentrations of 1, 5, and 10 µM for 10 h. Luciferase activity was measured, and results are expressed as primary data (left) and fold induction relative to the control (right). (H) 293T cells were plated in 24-well plates and transfected with 150 µM siR-NC, siR-Top1, siR-Top2a, or siR-Top2b for 48 h. The Top1 (left), Top2a (middle), Top2b (right), and GAPDH mRNA levels were determined by RT-qPCR, and results are expressed as fold induction relative to the control. (I) 293T cells were plated in 24-well plates; cotransfected with 0.1 µg pEnh1-Luc and 150 µM siR-NC, siR-Top1, siR-Top2a, or siR-Top2b for 24 h; and then transfected with 0.2 µg pCAGGS or pCAGGS-HA-PJA1B for 24 h. Luciferase activity was measured, and results are expressed as primary data (left) and fold induction relative to the control (right). Data are shown as means ± SD and correspond to results of a representative experiment out of three performed. ns, nonsignificant; *, P < 0.05; **, P < 0.01; ***, P < 0.001. (J and K) Diagrams of proposed models for PJA1 function. (J) Under normal conditions, the NSE1/NSE3/NSE4 subcomplex ensures that the SMC5/6 complex maintains the host chromosome. (K) In response to viral and episomal DNAs, PJA1 replaces NSE1 to form the PJA1/NSE3/NSE4 subcomplex, which converts the function of the SMC5/6 complex to the restriction of viral (V), extrachromosomal (E), and plasmid (P) DNA molecules.