Journal: Communications Biology

Article Title: Ibetazol, a novel inhibitor of importin β1-mediated nuclear import

doi: 10.1038/s42003-024-07237-8

Figure Lengend Snippet: A Rationale for the importin α1 localization screen. Importin α1 acts as adapter protein between importin β1 and its cargos and, in steady state, it resides predominantly in the nucleus. Compounds that interfere with the importin β1-importin α1 interaction will result in importin α1 cytoplasmic accumulation. B Left: Localization of importin α1-mNeonGreen in endogenously tagged HeLa Kyoto cells upon knockdown of KPNB1 or upon treatment with 50 µM CDD-01 for 2 h as compared to vehicle control (DMSO). Right: Untagged importin α1 shows similar relocalization to the cytoplasm upon compound treatment as confirmed by immunofluorescence (IF) staining in wild type cells. mNGreen: mNeonGreen tag; AF488: Alexa Fluor™ 488 label. Bar: 20 µm. C Overview of the applied screening cascade. A primary screen (single dose) based on importin α1-mNeonGreen localization in Hela Kyoto cells is followed by dose-response confirmation and chemical validation (independent resynthesis) of the initial hits in the same cell line. One chemical series was further characterized in three importin β1 dependent import assays (translocation of NLS cMyc dependent mNeongreen reporter, p65 and SREBP1-mNeongreen) and two counter screens (exportin-1 dependent export and transportin-1/2 dependent import). D Chemical structure of compounds CDD-01, CDD-02, ibetazol (CDD-03) and CDD-04. E Dose-response curve for importin α1-mNeonGreen relocalization upon 2 h compound treatment. %HIGH: percentage of cells with predominant nuclear localization of mNeonGreen (ratio of intensity in nucleus over cytoplasm is >1.4). Curves where fit by non-linear regression using GraphPad Prism, n = 5 wells from 2 independent experiments. Inset: treatment with reference compound importazole (IPZ) at 50 µM for 2 h does not induce relocalization of importin α1-mNeonGreen to the cytoplasm.

Article Snippet: Adenoviral mediated knockdown of KPNB1 employing Ad-U6-h- KPNB1 -shRNA (Vector Biolabs), which results in clear cytoplasmic relocalization of the importin α1-mNeonGreen fusion protein, was used as a positive control.

Techniques: Knockdown, Control, Immunofluorescence, Staining, Biomarker Discovery, Translocation Assay

Journal: medRxiv

Article Title: Genetic SNUPN variants cause spinocerebellar atrophy by disrupting global splicing in Purkinje cells

doi: 10.1101/2024.07.11.24310169

Figure Lengend Snippet: (a) T1-weighted sagittal midline images of a control individual (Control), Patient 1, Patient 2, and Patient 3.at age range of 10-15 years-old. Images of all patients show marked dilation of the cerebellar interfolia spaces and a marked decrease in size of the superior and inferior vermis (arrows). (b) The inheritance pattern of affected individuals is consistent with a fully penetrant autosomal recessive disorder. Squares and circles indicate males and females, respectively. Black-filled and open symbols indicate affected individuals a nd unaffected relatives, respectively. * denotes individual enforced whole exome sequencing (WES). (c) The location of identified snurportin-1 variants. Regions related to import/export into/from the nucleus are shown in orange and blue, r espectively. R55W is located at the binding domain with importinβ, and R204Q is located in the CRM1 binding domain. (d) Localization of wt and mutant GFP-snurportin-1 in HeLa cells after treatment with nuclear import and export inhibitors. Scale bar, 50 μm.

Article Snippet: Binding of the snurportin-1 fusion proteins and recombinant human importin beta (Novus Biologicals) was measured by following a series of different importin beta concentrations (7.4, 22.2, 66.7, 200, and 600 nM) to the snurportin-1 fragments (2 μg/ml) fixed on a sensor chip with glutathione S-transferase antibodies.

Techniques: Control, Sequencing, Binding Assay, Mutagenesis

Journal: Science Advances

Article Title: Antiviral activity of a purine synthesis enzyme reveals a key role of deamidation in regulating protein nuclear import

doi: 10.1126/sciadv.aaw7373

Figure Lengend Snippet: ( A ) 293T cells were transfected with plasmids containing FLAG-tagged RTA-WT and the indicated V5-tagged importins. WCLs were incubated with anti-V5 antibody. The precipitated proteins and WCLs were analyzed by immunoblotting with indicated antibodies. ( B ) Glutathione agarose loaded with GST or recombinant GST–importin β1 was incubated with purified RTA. Precipitated proteins and RTA (input) were analyzed by immunoblotting with anti-RTA antibody, while GST and GST–importin β1 were analyzed by Coomassie staining (bottom). ( C ) SLK/iBAC.RTA-WT cells were induced with doxycycline (1 μg/ml) for 24 hours and then transfected with a plasmid containing EGFP-bimax2 for 24 hours. Cells were analyzed by immunofluorescence staining and microscopy. ( D ) Glutathione agarose loaded with GST fusions containing either importin β1 (imp-β1) or β2 (imp-β2) was incubated with WCLs containing RTA-WT (WT) or RTA-DD (DD). Precipitated proteins and WCLs (Input) were analyzed by immunoblotting with anti-RTA antibody (right). GST–importin β1 and GST–importin β2 were analyzed by Coomassie staining. ( E ) 293T cells were transfected with plasmids containing RTA-WT (WT) or RTA-DD (DD) mutant. WCLs were prepared and precipitated with control immunoglobulin G (IgG) or antibody against importin β1 (Imp-β1). Precipitated proteins and WCLs were analyzed by immunoblotting with indicated antibodies. ( F ) iSLK/rKSHV.219 cells were induced with doxycycline (0.5 μg/ml) and sodium butyrate (1 mM) for the indicated times. Immunoprecipitation and immunoblotting were performed as described in (E). ( G ) iSLK/rKSHV.219 cells were transduced with control lentivirus (CTL) or lentivirus encoding shRNA against PFAS, followed by doxycycline and sodium butyrate induction for 72 hours. Cells were harvested for cellular fractionation to obtain cytosolic (C) and nuclear (N) fractions that, along with WCLs, were analyzed by immunoblotting with indicated antibodies. The results shown in (A), (B), and (D) to (G) represent three independent experiments ( n = 3).

Article Snippet: Antibodies against FLAG (M2, Sigma), importin α1 (sc-101292, Santa Cruz Biotechnology), importin β1 (NB100-94993, Novus Biologicals), importin β2 (sc-32314, Santa Cruz Biotechnology), human influenza hemagglutinin (HA) (MMS-101P, BioLegend), goat anti-rabbit/mouse immunoglobulin G (H+L) Alexa Fluor 488 (A-11034 and A32723, Invitrogen), tubulin (DM1A, Cell Signaling), and histone H3 (1B1B2, Cell Signaling) were purchased from the indicated suppliers.

Techniques: Transfection, Incubation, Western Blot, Recombinant, Purification, Staining, Plasmid Preparation, Immunofluorescence, Microscopy, Mutagenesis, Control, Immunoprecipitation, Transduction, shRNA, Cell Fractionation

Journal: Science Advances

Article Title: Antiviral activity of a purine synthesis enzyme reveals a key role of deamidation in regulating protein nuclear import

doi: 10.1126/sciadv.aaw7373

Figure Lengend Snippet: ( A ) Alignment of RTA proteins of KSHV, RRV, EBV, HVS, and MHV68 shows the bipartite NLS and the two deamidation sites corresponding to N37 and N225 of KSHV RTA. ( B ) 293T stable cells carrying control shRNA or PFAS shRNA were transfected with a plasmid containing RRV RTA (rRTA), EBV RTA (eRTA), HVS RTA (hRTA), or MHV68 RTA (mRTA). WCLs were prepared at 30 hours after transfection and analyzed by two-dimensional gel electrophoresis and immunoblotted for RTA (left). WCLs were analyzed by immunoblotting with antibodies against PFAS and RTA (right). ( C ) 293T cells transfected with plasmids containing rRTA, eRTA, hRTA, or mRTA. WCLs were precipitated with a control IgG or antibody against importin β1. Precipitated proteins and WCLs were analyzed by immunoblotting with indicated antibodies. ( D ) Glutathione agarose loaded with GST or GST–importin β1 (GST–imp β1) were incubated with WCLs prepared from 293T cells transfected with a plasmid containing eRTA, hRTA, or mRTA, without or with a plasmid containing PFAS-ED. Precipitated proteins and WCLs were analyzed by immunoblotting with indicated antibodies. ( E ) 293T cells were transfected with wild type (WT) or the deamidated mutant (DD/D) of rRTA, hRTA, or eRTA. Sites of N>D mutations were highlighted in (A). Nuclear (N) and cytosolic (C) fractions were obtained by sequential centrifugation and analyzed by immunoblotting with indicated antibodies. WCLs were analyzed for the expression of RTA wild type and the DD/D mutant (right panels). The results shown in (B) to (E) represent three independent experiments ( n = 3).

Article Snippet: Antibodies against FLAG (M2, Sigma), importin α1 (sc-101292, Santa Cruz Biotechnology), importin β1 (NB100-94993, Novus Biologicals), importin β2 (sc-32314, Santa Cruz Biotechnology), human influenza hemagglutinin (HA) (MMS-101P, BioLegend), goat anti-rabbit/mouse immunoglobulin G (H+L) Alexa Fluor 488 (A-11034 and A32723, Invitrogen), tubulin (DM1A, Cell Signaling), and histone H3 (1B1B2, Cell Signaling) were purchased from the indicated suppliers.

Techniques: Control, shRNA, Transfection, Plasmid Preparation, Two-Dimensional Gel Electrophoresis, Electrophoresis, Western Blot, Incubation, Mutagenesis, Centrifugation, Expressing

Journal: eLife

Article Title: KPNB1 mediates PER/CRY nuclear translocation and circadian clock function

doi: 10.7554/eLife.08647

Figure Lengend Snippet: ( A ) Immunoprecipitation (IP) analysis of interactions of KPNB1 with PER2. U2 OS cells were transfected with Venus-tagged PER2, and then immunoprecipitated with anti-GFP antibody (IP: αGFP). The immunoprecipitates were analyzed by immunoblotting with anti-KPNB1 (IB: αKPNB1) or anti-GFP antibody (IB: αGFP). Representative images from three independent experiments are shown. ( B ) Immunofluorescence (IF) analyses of subcellular colocalization of PER2 with KPNB1. U2 OS cells expressing PER2-V were fixed and immunostained with antibody to endogenous KPNB1. The representative images were captured by fluorescence imaging microscopy using specific filter sets for FITC (green; PER-V), TRITC (Red; αKPNB1), and DAPI (Blue; Nuclei). See . Scale bar: 10 µm. ( C ) Coimmunoprecipitation of PER2 with endogenous KPNB1. Liver extracts were immunoprecipitated (IP) with anti-KPNB1 or control IgG antibodies, and the immunoprecipitated proteins were probed with antibodies to PER2, KPNB1 as well as GAPDH as negative control and IgG light chain as a positive control for the IP. Representative images from three independent experiments are shown. ( D ) Immunoblotting analysis using cytoplasmic and nuclear extracts prepared from mouse liver tissues collected at 4 hr interval as indicated for 24 hr in constant darkness (CT: Circadian time). Anti-PER2, anti-CRY1, and anti-KPNB1 antibodies were used for detecting endogenous PER2, CRY1, and KPNB1 proteins. Anti-Tubulin antibody (αTubulin) for cytoplasmic fraction (Cyto) marker and anti-Lamin (αLamin) for nuclear fraction (Nuc) marker were used for loading controls respectively. Representative images from two independent experiments are shown. ( E ) Circadian expressions of endogenous clock gene mRNAs ( Per1 , Dbp , Nr1d1 , Nr1d2 ) were determined by quantitative RT-PCR analysis of mouse liver tissue samples collected at 4 hr interval as indicated for 24 hr in constant darkness (CT; Circadian time). The data presented are the means ± S.E. of triplicate samples. DOI: http://dx.doi.org/10.7554/eLife.08647.006

Article Snippet: Anti-KPNB1 (A300-482A) for immunoblotting analysis was obtained from Bethyl Labratories (Montgomery, TX, United States), and Anti-KPNB1 (ab2811) for immunoprecipiation with mouse liver extracts or immunostaining of U2 OS cells was purchased from Abcam (Cambridge, MA, United States).

Techniques: Immunoprecipitation, Transfection, Western Blot, Immunofluorescence, Expressing, Fluorescence, Imaging, Microscopy, Control, Negative Control, Positive Control, Marker, Quantitative RT-PCR

Journal: eLife

Article Title: KPNB1 mediates PER/CRY nuclear translocation and circadian clock function

doi: 10.7554/eLife.08647

Figure Lengend Snippet: ( A ) IP analysis of interactions of KPNB1 with PER1, PER2, CRY1, and CRY2. U2 OS Cells were transfected with Venus-tagged PER1, PER2, CRY1, and CRY2 (PER1-V, PER2-V, CRY1-V, CRY2-V) and then immunoprecipitated with anti-GFP antibody (IP: αGFP). The immunoprecipitates were analyzed by immunoblotting with anti-KPNB1(IB: αKPNB1) or anti-GFP antibody (IB: αGFP). Representative images from three independent experiments are shown. ( B ) The intensity of the immunoprecipited protein bands in the data shown in ( A ) was quantified by densitometry (ImageJ), and the data are shown as means ± S.E. of three independent experiments. ( C ) IF analyses of subcellular colocalization of PER2 with KPNB1. U2 OS cells expressing PER2-V were fixed and immunostained with antibody to endogenous KPNB1. The representative images were captured by fluorescence imaging microscopy using specific filter sets for FITC (green; PER2-V), TRITC (Red; αKPNB1), and DAPI (Blue; Nuclei). Scale bar: 10 μm. DOI: http://dx.doi.org/10.7554/eLife.08647.007

Article Snippet: Anti-KPNB1 (A300-482A) for immunoblotting analysis was obtained from Bethyl Labratories (Montgomery, TX, United States), and Anti-KPNB1 (ab2811) for immunoprecipiation with mouse liver extracts or immunostaining of U2 OS cells was purchased from Abcam (Cambridge, MA, United States).

Techniques: Transfection, Immunoprecipitation, Western Blot, Expressing, Fluorescence, Imaging, Microscopy

Journal: eLife

Article Title: KPNB1 mediates PER/CRY nuclear translocation and circadian clock function

doi: 10.7554/eLife.08647

Figure Lengend Snippet: ( A ) Immunoblot analysis of KPNB1 knockdown efficiency with anti-KPNB1 (αKPNB1) in control (siCTL) and KPNB1 siRNA (siKPNB1)-treated U2 OS cells. Anti-GAPDH antibody (αGAPDH) was used as a loading control. ( B ) Bioluminescence recordings of dexamethasone (Dex)-synchronized control (siCTL) and increasing dose of KPNB1 siRNA (siKPNB1 0.5×/1×)-treated U2 OS cells expressing Per2 promoter-driven destabilized luciferase (left; p Per2 -dLuc). The Bioluminescence recordings were detrended by a 24-hr moving average subtraction (right). ( C ) Altered rhythmic expression of KPNB1, PER1 , PER 2, and CRY1 transcripts by KPNB1 depletion. mRNA expressions of the target genes were determined by quantitative RT-PCR in control or KPNB1-depleted U2 OS cells over the course of 44 hr after 24 hr upon Dex treatment. The data are shown with the mean value of triplicate samples in each time point. ( D ) KPNB1 knockdown alters rhythmic nuclear accumulation of PER and CRY proteins. Immunoblotting analysis using cytoplasmic and nuclear extracts prepared from control or KPNB1-depleted U2 OS cells collected at 4 hr interval over the course of 44 hr after 24 hr of Dex treatment. Anti-PER2, anti-CRY1, anti CRY2, and anti-KPNB1 antibodies were used for detecting endogenous PER2, CRY1, CRY2 and KPNB1 proteins. Anti-Tubulin antibody (αTubulin) for cytoplasmic fraction (Cyto) marker and anti-TATA binding protein (αTBP) for nuclear fraction (Nuc) marker were used for loading controls respectively. Representative images from two independent experiments are shown. DOI: http://dx.doi.org/10.7554/eLife.08647.011

Article Snippet: Anti-KPNB1 (A300-482A) for immunoblotting analysis was obtained from Bethyl Labratories (Montgomery, TX, United States), and Anti-KPNB1 (ab2811) for immunoprecipiation with mouse liver extracts or immunostaining of U2 OS cells was purchased from Abcam (Cambridge, MA, United States).

Techniques: Western Blot, Knockdown, Control, Expressing, Luciferase, Quantitative RT-PCR, Marker, Binding Assay

Journal: Journal of Cell Science

Article Title: RNA-binding protein HuR regulates nuclear import of protein

doi: 10.1242/jcs.192096

Figure Lengend Snippet: HuR stabilizes mRNA for importin-β1 (Kpnb1) but not for importin-α1. (A,B) Immunoblots of importin-α1 in M2−/− (A) or MCF-7 (B) cells stably expressing shCtrl or shHuR. (C,D) Immunoblots of importin-β1 in M2−/− (C) or MCF-7 (D) cells stably expressing shCtrl or shHuR. (E) Levels of mRNA for importin-β1 (Kpnb1) in M2−/− cells stably expressing shCtrl or shHuR, as measured by Q-PCR and normalized to 18s (mean±s.d.; n=3). *P<0.05, paired Student's t-test. (F) M2−/− cells stably expressing shCtrl or shHuR were treated with actinomycin D (2.5 µg/ml). Levels of Kpnb1 mRNA at various time points following treatment were measured by Q-PCR. Data (mean±s.e.m.; n=3) were normalized to corresponding values at time zero. (G) HuR was immunoprecipitated from M2−/− cells. Kpnb1 mRNA that co-precipitated with HuR was detected by semi-quantitative PCR.

Article Snippet: Q-PCR was performed using a StepOnePlus real-time PCR system with the following TaqMan probes: Elavl1 , Mm00516012_m1; Kpna1 , Mm00434700_m1; Kpnb1 , Mm00434318_m1; Tnpo1 , Mm00839059_g1; Tnpo2 , Mm00520392_m1; Ranbp1 , Mm00650862_m1; Cyp26a1 , Mm00514486_m1; and 18s rRNA (4352930E, Applied Biosystems).

Techniques: Western Blot, Stable Transfection, Expressing, Immunoprecipitation, Real-time Polymerase Chain Reaction

Journal: Cellular and Molecular Life Sciences

Article Title: KPNB1 modulates the Machado–Joseph disease protein ataxin-3 through activation of the mitochondrial protease CLPP

doi: 10.1007/s00018-022-04372-5

Figure Lengend Snippet: Ataxin-3 interacts with karyopherins, but KPNB1 overexpression does not affect the intracellular localization of ataxin-3. a Interaction of wild-type (15Q) and polyQ-expanded (77Q) ataxin-3 with KPNB1 and its partner KPNA3 was validated by GST pull-down assay. GST-tagged 15Q and 77Q ataxin-3 were purified using glutathione beads followed by incubation with wild-type HEK 293T cell lysates. Input as well as pull-down samples were assessed by western blot analysis. Empty GST was applied as negative control. White Bullets (o) indicate nonspecific bands. b Fluorescence microscopy was performed to visualize the subcellular localization of wild-type and polyQ-expanded ataxin-3 upon KPNB1 overexpression. ATXN3 KO HEK 293T cells co-expressing either EGFP-ataxin-3 15Q or EGFP-ataxin-3 148Q and KPNB1 were analyzed 72 h post-transfection. Microscopy indicated that the intracellular localization of neither wild-type (15Q) nor polyQ-expanded (148Q) ataxin-3 was altered upon KPNB1 overexpression. Blue and green channels show DAPI as nuclear counterstain and GFP signals, respectively. 400 × magnification, scale bar = 20 µm. c , d Nucleocytoplasmic fractionation assay demonstrates a reduction of cytoplasmic as well as nuclear wild-type (15Q) and polyQ-expanded (148Q) ataxin-3 upon KPNB1 overexpression. However, no alteration was observed in the localization of ataxin-3. Moreover, accumulation of mainly cytoplasmic ataxin-3 fragments (black arrowheads) in KPNB1 overexpressing cells was observed. ATXN3 KO HEK 293T cells were cotransfected with either 15Q or 148Q ataxin-3 and KPNB1 or empty vectors. 72 h post-transfection, nucleocytoplasmic fractionation was performed followed by western blot analysis. Blots are shown in low and high exposures. White bullet (o) marks nonspecific bands. Ataxin-3 was detected by 1H9 and C-terminal antibodies. The diagrams illustrate the ratio of nuclear and cytoplasmic ataxin-3 as percentage. GAPDH and lamin A/C were applied as cytoplasmic and nuclear loading controls, respectively. c , n = 4, Cyt/Nuc, unpaired t -test, p = 0.6261; d , n = 4, Cyt/Nuc, unpaired t -test, p = 0.5443. CTRL empty vector, Cyt cytoplasmic fraction, Nuc nuclear fraction, fl full-length, frg fragment, C-term C-terminal, Exp exposure. Values are displayed as means ± SEM. ns not significant

Article Snippet: All ataxin-3 constructs contained isoform 3c. pCMV6-XL5 and pCMV6-Entry/myc-DDK vectors (both OriGene Technologies, Rockville, MD, US) were used for overexpressing human KPNB1 and human CLPP, respectively.

Techniques: Over Expression, Pull Down Assay, Purification, Incubation, Western Blot, Negative Control, Fluorescence, Microscopy, Expressing, Transfection, Fractionation, Plasmid Preparation

Journal: Cellular and Molecular Life Sciences

Article Title: KPNB1 modulates the Machado–Joseph disease protein ataxin-3 through activation of the mitochondrial protease CLPP

doi: 10.1007/s00018-022-04372-5

Figure Lengend Snippet: KPNB1 overexpression leads to a reduction of ataxin-3 protein levels, and mutation of the FxFG binding site in KPNB1 does not abolish this effect on ataxin-3. a , b KPNB1 overexpression decreases soluble levels of both wild-type (15Q) and polyQ-expanded (148Q) ataxin-3 and enhances fragmentation of ataxin-3 in comparison to control. ATXN3 KO HEK 293T cells were cotransfected with either 15Q or 148Q ataxin-3 and KPNB1 or empty vectors for 72 h followed by western blot analysis. Detection of ataxin-3 using two different antibodies (1H9 and C-terminal) which recognize different epitopes revealed that ataxin-3 fragments are C-terminal breakdown products (black arrowheads). Western blot analysis confirmed the overexpression of KPNB1. Blots are shown in low and high exposures. GAPDH was applied as loading control. a , 15Q Atx3, n = 6, one sample t -test, p = 0.0008; KPNB1, n = 6, one sample t -test, p < 0.0001; b , 148Q Atx3, n = 6, one sample t -test, p = 0.0006; KPNB1, n = 5, one sample t -test, p = 0.0349. c , d KPNB1 mutation (I178D) in the FxFG binding site does not change the effect of KPNB1 overexpression on either wild-type or polyQ-expanded ataxin-3. ATXN3 KO HEK 293T cells were cotransfected with either 15Q or 148Q ataxin-3 and wild-type KPNB1 or KPNB1 I178D constructs for 72 h. Western blot analysis displays that the ataxin-3-lowering effect of KPNB1 I178D is comparable to wild-type KPNB1, and it leads to a significant reduction of both 15Q and 148Q ataxin-3 protein levels. GAPDH was applied as loading control. c , n = 5, one sample t -test, CTRL vs KPNB1, p = 0.0030; CTRL vs KPNB1 I178D, p = 0.0086; unpaired t -test, KPNB1 vs KPNB1 I178D, p = 0.4784; d , n = 5, one sample t -test, CTRL vs KPNB1, p = 0.0052; CTRL vs KPNB1 I178D, p = 0.0765; unpaired t -test, KPNB1 vs KPNB1 I178D, p = 0.5367. e Western blot analysis of wild-type HEK 293T cells transfected with either KPNB1 or empty vectors. The result displays that KPNB1 overexpression is accompanied by a reduction in protein levels of endogenous ataxin-3. Vinculin was applied as loading control. n = 6, unpaired t -test, p = 0.0221. f Western blot analysis demonstrates that protein levels of GFP do not change upon KPNB1 overexpression. Wild-type HEK 293T cells were cotransfected with GFP and KPNB1 constructs for 72 h. β-actin was applied as loading control. n = 6, unpaired t -test, p = 0.0756. CTRL empty vector, fl full-length, frg fragment, C-term C-terminal, Exp exposure, egAtx3 endogenous ataxin-3, Rel. relative. Values are displayed as means ± SEM. ns not significant; * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001

Article Snippet: All ataxin-3 constructs contained isoform 3c. pCMV6-XL5 and pCMV6-Entry/myc-DDK vectors (both OriGene Technologies, Rockville, MD, US) were used for overexpressing human KPNB1 and human CLPP, respectively.

Techniques: Over Expression, Mutagenesis, Binding Assay, Western Blot, Construct, Transfection, Plasmid Preparation

Journal: Cellular and Molecular Life Sciences

Article Title: KPNB1 modulates the Machado–Joseph disease protein ataxin-3 through activation of the mitochondrial protease CLPP

doi: 10.1007/s00018-022-04372-5

Figure Lengend Snippet: Knockdown and pharmacological inhibition of KPNB1 elevate ataxin-3 protein levels. a , b Western blot analysis reveals that knockdown of KPNB1 is accompanied by a significant increase in the soluble levels of wild-type (15Q) and polyQ-expanded (148Q) ataxin-3. ATXN3 KO HEK 293T cells were cotransfected with either 15Q or 148Q ataxin-3 and esiKPNB1 or esiLUC (control). Cells were harvested 72 h post-transfection, and cell lysates were subjected to western blotting. GAPDH was applied as loading control. a , n = 5, one sample t -test, p = 0.0567; b , n = 5, one sample t -test, p = 0.0315. c , d KPNB1 was inhibited using 16 µM importazole (IPZ) in either wild-type (15Q) or polyQ-expanded (148Q) ataxin-3 expressing HEK 293T cells. 48 h after treatment, cells were harvested and subjected to western blot analysis. Quantification of blots demonstrates that the soluble levels of both 15Q and 148Q ataxin-3 increase upon IPZ treatment compared with DMSO-treated cells (control). GAPDH was applied as loading control. c , n = 6, one sample t -test, p = 0.0162; d , n = 4, one sample t -test, p = 0.0077. e Western blot analysis shows a significant increase of endogenous ataxin-3 protein levels upon IPZ treatment compared with control. Wild-type HEK 293T cells were treated with either 16 µM IPZ or DMSO for 48 h prior to harvesting. GAPDH was applied as loading control. n = 3, unpaired t -test, p = 0.0009. f Protein levels of ataxin-3 can be rescued by IPZ treatment in KPNB1 overexpressing cells. ATXN3 KO HEK 293T cells were cotransfected with 15Q ataxin-3 and KPNB1 constructs followed by 16 µM IPZ or DMSO treatment for 48 h prior to harvesting. Cells were harvested 72 h post-transfection, and ataxin-3 levels were analyzed by western blotting. GAPDH was applied as loading control. n = 4, one sample t -test, CTRL + DMSO vs KPNB1 + DMSO, p = 0.0129; CTRL + DMSO vs KPNB1 + IPZ, p = 0.0615; unpaired t -test, KPNB1 + DMSO vs KPNB1 + IPZ, p = 0.0021. CTRL empty vector, IPZ importazole, egAtx3 endogenous ataxin-3, Rel. relative. Values are displayed as means ± SEM. ns not significant; * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001

Article Snippet: All ataxin-3 constructs contained isoform 3c. pCMV6-XL5 and pCMV6-Entry/myc-DDK vectors (both OriGene Technologies, Rockville, MD, US) were used for overexpressing human KPNB1 and human CLPP, respectively.

Techniques: Inhibition, Western Blot, Transfection, Expressing, Construct, Plasmid Preparation

Journal: Cellular and Molecular Life Sciences

Article Title: KPNB1 modulates the Machado–Joseph disease protein ataxin-3 through activation of the mitochondrial protease CLPP

doi: 10.1007/s00018-022-04372-5

Figure Lengend Snippet: KPNB1 overexpression decreases the stability of ataxin-3, and observed ataxin-3 fragments are not mediated by the activation of known MJD-associated proteases or proteolytic pathways. a , b Modulation of ataxin-3 stability was analyzed upon KPNB1 overexpression using the Tet-off system. Western blot analysis revealed a significant reduction in the stability of both wild-type (15Q) and polyQ-expanded (77Q) ataxin-3 in KPNB1 overexpressing cells 48 h after termination of ataxin-3 expression compared with control. ATXN3 KO HEK 293T cells were cotransfected with either pTRE-15Q or pTRE-77Q ataxin-3 and KPNB1 or empty vectors. 24 h post-transfection, ataxin-3 expression was shut off using doxycycline (Dox) at different time intervals (0 h, 6 h, 24 h, and 48 h). GAPDH was applied as loading control. a , n = 3, paired t -test, CTRL vs KPNB1 (6 h), p = 0.6029; CTRL vs KPNB1 (24 h), p = 0.6730; CTRL vs KPNB1 (48 h), p = 0.0132; b , n = 4, paired t -test, CTRL vs KPNB1 (6 h), p = 0.3859; CTRL vs KPNB1 (24 h), p = 0.0202; CTRL vs KPNB1 (48 h), p = 0.0427. c Activation of calpains and caspases upon KPNB1 overexpression was evaluated by detecting cleavage of their common substrate α-spectrin protein in wild-type HEK 293T cells. Western blot analysis demonstrates no alteration in the levels of either full-length (white arrowhead) or breakdown products of α-spectrin (black arrowhead). Moreover, ratio between cleaved and full-length protein remained comparable to control in KPNB1 overexpressing cells. GAPDH was applied as loading control. n = 6, unpaired t -test, p = 0.6296. d Breakdown products of α-spectrin (black arrowhead) are induced in ionomycin (IM) treated cells confirming the activation of calpains compared with control. ATXN3 KO HEK 293T cells were cotransfected with either 15Q or 77Q ataxin-3 followed by KPNB1 overexpression or 1 µM IM treatment for 1 h. β-actin was applied as loading control. e IM administration results in the activation of endogenous calpains and cleavage of ataxin-3. Western blot analysis displays that ataxin-3 fragments induced by KPNB1 overexpression (blue boxes and arrowheads) are not comparable to fragments mediated by calpain cleavage (red boxes and arrowheads). Red and blue channels indicate ataxin-3 detected by 1H9 and C-terminal antibodies, respectively. f Western blot analysis demonstrates that inhibition of calpains and caspases or blocking of autophagy and proteasomal degradation do not prevent ataxin-3 fragmentation in KPNB1 overexpressing cells. ATXN3 KO HEK 293T cells cotransfected with 15Q ataxin-3 and KPNB1 or empty vectors were incubated with 10 µM calpain inhibitor III (CI-III), 10 µM caspase inhibitor (Q-VD-OPh), 50 nM autophagy inhibitor bafilomycin A1 (BafA1), or 10 µM proteasomal inhibitor lactacystin (Lac) for 16 h prior to harvesting. Cells incubated with DMSO were considered as control. The diagram illustrates quantification of ataxin-3 fragments in KPNB1 overexpressing cells. GAPDH was applied as loading control. n = 5, one sample t -test, DMSO vs CI-III, p = 0.3708; DMSO vs Q-VD, p = 0.3136; DMSO vs BafA1, p = 0.4063; DMSO vs Lac, p = 0.2682. CTRL empty vector, fl full-length, frg fragment, Dox Doxycycline, C-term C-terminal, f-Nt fragment-N-terminal, IM ionomycin, CI-III calpain inhibitor III, BafA1 bafilomycin A1, Lac lactacystin, Rel. relative. Values are displayed as means ± SEM. ns not significant; * p ≤ 0.05

Article Snippet: All ataxin-3 constructs contained isoform 3c. pCMV6-XL5 and pCMV6-Entry/myc-DDK vectors (both OriGene Technologies, Rockville, MD, US) were used for overexpressing human KPNB1 and human CLPP, respectively.

Techniques: Over Expression, Activation Assay, Western Blot, Expressing, Transfection, Inhibition, Blocking Assay, Incubation, Plasmid Preparation

Journal: Cellular and Molecular Life Sciences

Article Title: KPNB1 modulates the Machado–Joseph disease protein ataxin-3 through activation of the mitochondrial protease CLPP

doi: 10.1007/s00018-022-04372-5

Figure Lengend Snippet: KPNB1 modulation affects ataxin-3 aggregation levels. a , b Fluorescence microscopy was conducted to visualize the alteration of polyQ-expanded ataxin-3 aggregation in KPNB1 overexpressing cells. ATXN3 KO HEK 293T cells co-expressing EGFP ataxin-3 148Q and KPNB1 were fixed 72 h post-transfection, and the number of GFP-positive (EGFP + ) cells with and without aggregates was counted manually in 20 fields of vision. The analysis showed a decrease of cells with ataxin-3 aggregates upon KPNB1 overexpression. Blue, green, and red channels show DAPI as nuclear counterstain, GFP, and KPNB1 signals, respectively. White arrowheads mark ataxin-3 aggregates. 400 × magnification, scale bar = 20 µm. The diagram shows the percentage of aggregates in EGFP + cells. n = 4, unpaired t -test, p = 0.0123. c Filter retardation assay of ATXN3 KO HEK 293T cells co-expressing polyQ-expanded (148Q) ataxin-3 and KPNB1 for 72 h demonstrated a reduction of polyQ-expanded ataxin-3 aggregates compared with control. n = 6, one sample t -test, p = 0.0004. d PrestoBlue assay of ATXN3 KO HEK 293T cells cotransfected with either 15Q or 148Q ataxin-3 and KPNB1 or empty vectors. The viability of cells expressing 148Q ataxin-3 was rescued upon KPNB1 overexpression. Viability was normalized to 15Q ataxin-3 expressing cells. n = 4, one sample t -test, 15Q + CTRL vs 148Q + CTRL, p = 0.0144; 15Q + CTRL vs 148Q + KPNB1, p = 0.0882; paired t -test, 148Q + CTRL vs 148Q + KPNB1, p = 0.0138. e Filter retardation assay of ATXN3 KO HEK 293T cells co-expressing 148Q ataxin-3 and either wild-type or KPNB1 I178D indicates a decrease in the aggregation of 148Q ataxin-3. n = 6, one sample t -test, CTRL vs KPNB1, p = 0.0030; CTRL vs KPNB1 I178D, p = 0.0247; unpaired t -test, KPNB1 vs KPNB1 I178D, p = 0.1883. f Knockdown of KPNB1 using esiRNA shows an increased load of aggregated 148Q ataxin-3 using filter retardation assay. ATXN3 KO HEK 293T cells were cotransfected for 72 h with 148Q ataxin-3 and esiKPNB1 or esiLUC as control. n = 5, one sample t -test, p = 0.0054. g Filter retardation assay shows increased ataxin-3 aggregation in ATXN3 KO HEK 293T cells expressing 148Q ataxin-3 for 72 h and treated with 16 µM IPZ for 48 h. Values were normalized to DMSO-treated cells. n = 5, one sample t -test, p = 0.0279. CTRL empty vector, IPZ importazole, Rel. relative. Values are displayed as means ± SEM. ns not significant; * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001

Article Snippet: All ataxin-3 constructs contained isoform 3c. pCMV6-XL5 and pCMV6-Entry/myc-DDK vectors (both OriGene Technologies, Rockville, MD, US) were used for overexpressing human KPNB1 and human CLPP, respectively.

Techniques: Fluorescence, Microscopy, Expressing, Transfection, Over Expression, Prestoblue Assay, esiRNA, Plasmid Preparation

Journal: Cellular and Molecular Life Sciences

Article Title: KPNB1 modulates the Machado–Joseph disease protein ataxin-3 through activation of the mitochondrial protease CLPP

doi: 10.1007/s00018-022-04372-5

Figure Lengend Snippet: KPNB1 lowers ataxin-3 levels via activation of the mitochondrial protease CLPP. a – c Label-free quantitative proteomics of cells expressing ataxin-3 reveals activation of mitochondrial protease CLPP upon KPNB1 overexpression. Volcano plots indicating the upregulated and downregulated proteins in ATXN3 KO HEK 293T cells cotransfected with either wild-type (15Q) or polyQ-expanded (148Q) ataxin-3 and KPNB1 or empty vectors. Volcano plots illustrate a direct comparison between the p- value and fold-change of the proteins. Purple circles show proteins with significantly altered levels upon KPNB1 overexpression. The thick dashed line represents the cut-off value at p = 0.05. n = 3, two-sample t -test, p ≤ 0.05. d Venn diagram displays the number of common and unique proteins which have been significantly upregulated or downregulated upon KPNB1 overexpression. e , f Western blot analysis demonstrates that both 15Q and 148Q ataxin-3 protein levels can be rescued partly by knockdown of CLPP in KPNB1 overexpressing cells. ATXN3 KO HEK 293T cells were cotransfected with either 15Q or 148Q ataxin-3, KPNB1, and esiCLPP or esiLUC as control. Cells were harvested 72 h post-transfection and protein levels assessed by western blotting. GAPDH was applied as loading control. e , 15Q Atx3, n = 6, one sample t -test, CTRL + esiLUC vs KPNB1 + esiLUC, p < 0.0001; CTRL + esiLUC vs KPNB1 + esiCLPP, p = 0.0056; unpaired t -test, KPNB1 + esiLUC vs KPNB1 + esiCLPP, p = 0.0264; CLPP, n = 6, one sample t -test, CTRL + esiLUC vs KPNB1 + esiLUC, p = 0.1619; CTRL + esiLUC vs KPNB1 + esiCLPP, p < 0.0001; unpaired t -test, KPNB1 + esiLUC vs KPNB1 + esiCLPP, p < 0.0001; f n = 6, one sample t -test, CTRL + esiLUC vs KPNB1 + esiLUC, p < 0.0001; CTRL + esiLUC vs KPNB1 + esiCLPP, p = 0.0020; unpaired t -test, KPNB1 + esiLUC vs KPNB1 + esiCLPP, p = 0.0219. g Knockdown of CLPP in ATXN3 KO HEK 293T cells cotransfected with 148Q ataxin-3 and KPNB1 for 72 h counteracts the KPNB1-induced lowering of polyQ-expanded ataxin-3 aggregates compared with control. n = 6, one sample t -test, CTRL + esiLUC vs KPNB1 + esiLUC, p = 0.0005; CTRL + esiLUC vs KPNB1 + esiCLPP, p = 0.0107; paired t -test, KPNB1 + esiLUC vs KPNB1 + esiCLPP, p = 0.0187. CTRL empty vector, FC fold-change, Rel. relative. Values are displayed as means ± SEM. ns not significant; * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001

Article Snippet: All ataxin-3 constructs contained isoform 3c. pCMV6-XL5 and pCMV6-Entry/myc-DDK vectors (both OriGene Technologies, Rockville, MD, US) were used for overexpressing human KPNB1 and human CLPP, respectively.

Techniques: Activation Assay, Expressing, Over Expression, Western Blot, Transfection, Plasmid Preparation

Journal: Cellular and Molecular Life Sciences

Article Title: KPNB1 modulates the Machado–Joseph disease protein ataxin-3 through activation of the mitochondrial protease CLPP

doi: 10.1007/s00018-022-04372-5

Figure Lengend Snippet: CLPP modulation affects ataxin-3 protein levels. a , b Western blot analysis reveals that knockdown of CLPP is accompanied by an increase in protein levels of both wild-type (15Q) and polyQ-expanded (148Q) ataxin-3. ATXN3 KO HEK 293T cells were cotransfected with either 15Q or 148Q ataxin-3 and esiCLPP or esiLUC (control) for 72 h. GAPDH was used as loading control. a , n = 6, one sample t -test, p < 0.0001; b , n = 6, one sample t -test, p = 0.0009. c , d CLPP overexpression reduces both 15Q and 148Q ataxin-3 protein levels and its effect is enhanced by KPNB1 overexpression. ATXN3 KO HEK 293T cells were cotransfected with either 15Q or 148Q ataxin-3 and CLPP or both CLPP and KPNB1 constructs for 72 h. GAPDH was applied as loading control. c , n = 4, one-way ANOVA with Tukey’s post-test, CTRL vs CLPP, p = 0.0089; CTRL vs CLPP + KPNB1, p < 0.0001; CLPP vs CLPP + KPNB1, p = 0.0009; d, n = 4, one-way ANOVA with Tukey’s post-test, CTRL vs CLPP, p = 0.0004; CTRL vs CLPP + KPNB1, p < 0.0001; CLPP vs CLPP + KPNB1, p < 0.0001. CTRL empty vector, C-term C-terminal, Rel. relative. Values are displayed as means ± SEM. ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001

Article Snippet: All ataxin-3 constructs contained isoform 3c. pCMV6-XL5 and pCMV6-Entry/myc-DDK vectors (both OriGene Technologies, Rockville, MD, US) were used for overexpressing human KPNB1 and human CLPP, respectively.

Techniques: Western Blot, Over Expression, Construct, Plasmid Preparation

Journal: Cellular and Molecular Life Sciences

Article Title: KPNB1 modulates the Machado–Joseph disease protein ataxin-3 through activation of the mitochondrial protease CLPP

doi: 10.1007/s00018-022-04372-5

Figure Lengend Snippet: MJD transgenic mouse models and iPSCs of MJD patients exhibit reduced KPNB1 protein levels. a Western blot analysis of whole brain protein extracts of 15-month-old YAC transgenic mice indicates a significant reduction of KPNB1 protein levels compared with controls. β-actin was used as loading control. n = 4–5, unpaired t -test, p = 0.0257. b Western blot analysis of cortical lysates of 5-month-old CaMKII/MJD77 transgenic mice confirms decreased protein levels of KPNB1 in comparison to controls. β-actin was applied as loading control. n = 4–6, unpaired t -test, p = 0.0175. c Western blot analysis of induced pluripotent stem cells (iPSCs) from three MJD patients and three healthy controls demonstrates a reduction of both KPNB1 and CLPP protein levels. β-actin was applied as loading control. n = 3 replicates of 3 patients and 3 controls, unpaired t -test, KPNB1, p = 0.0410; CLPP, p = 0.0325. WT wild-type, iPSCs induced pluripotent stem cells, CTRL healthy control, Rel. relative. Values are displayed as means ± SEM. * p ≤ 0.05

Article Snippet: All ataxin-3 constructs contained isoform 3c. pCMV6-XL5 and pCMV6-Entry/myc-DDK vectors (both OriGene Technologies, Rockville, MD, US) were used for overexpressing human KPNB1 and human CLPP, respectively.

Techniques: Transgenic Assay, Western Blot

Journal: Cellular and Molecular Life Sciences

Article Title: KPNB1 modulates the Machado–Joseph disease protein ataxin-3 through activation of the mitochondrial protease CLPP

doi: 10.1007/s00018-022-04372-5

Figure Lengend Snippet: Schematic overview of the proposed ataxin-3 degrading pathway mediated by KPNB1 and CLPP. a KPNB1 overexpression activates mitochondrial protease CLPP, leading to the degradation and reduction of ataxin-3 protein levels. Likewise, overexpression of CLPP has a comparable effect on ataxin-3 protein levels. Cleavage of peptides by the protease component of the Clp complex is an ATP-dependent process . b KPNB1 knockdown and pharmacological inhibition as well as CLPP knockdown prevent ataxin-3 degradation via the proposed pathway. OE overexpression, KD knockdown, Inh. inhibition

Article Snippet: All ataxin-3 constructs contained isoform 3c. pCMV6-XL5 and pCMV6-Entry/myc-DDK vectors (both OriGene Technologies, Rockville, MD, US) were used for overexpressing human KPNB1 and human CLPP, respectively.

Techniques: Over Expression, Inhibition