Journal: Nature Cell Biology

Article Title: A chaperone-proteasome-based fragmentation machinery is essential for aggrephagy

doi: 10.1038/s41556-025-01747-1

Figure Lengend Snippet: 3D tomogram reconstructions of control, SAR405-treated, PSMC5-depleted and DNAJB6-depleted dualPIM cells. 3D tomograms of an area of interest with a dualPIM aggregate. Supplementary Video 3 corresponds to Fig. 4a, siCtr-treated cells.

Article Snippet: The following primary antibodies (and dilutions) were used: rabbit anti-BAG3 (Abcam, ab47124, 1:1,000); mouse anti-GAPDH (Fitzgerald Industries International, 10R-G109a, 1:1,0000); rabbit anti-RB1CC1/FIP200 (Proteintech, 17250-1-AP, 1:1,000 WB, 1:50 IF); rabbit anti vinculin (Cell Signaling, 13901, 1:1,000); rabbit anti-TAX1BP1 (Millipore, HPA024432, 1:1,000 WB, 1:100 IF); rabbit anti-CALCOCO2/NDP52 (Millipore, HPA023195, 1:1,000 WB, 1:100 IF); rat anti-HSC70 (Enzo Life Sciences, ADI-SPA-815, 1:1,000 WB); mouse anti-HSP70 (Enzo Life Sciences, ADI-SPA-810, 1:1,000 WB, 1:100 IF); mouse anti-APG2 (Santa Cruz Biotechnology, sc-365366, 1:1,000 WB); mouse anti HSPBP1 (Novus Biologicals, NBP 2-01168, 1:1,000 WB); mouse anti-HSPB1 (Enzo Life Sciences, ADI-SPA-800, 1:1,000 WB); mouse anti-HSPB7 (Abnova H00027129-M01, 1:1,000 WB); rabbit anti-DNAJA1 (Abcam, ab126774, 1:1,000 WB); mouse anti DNAJA2 (Sigma-Aldrich, WH0010294M1, 1:1,000 WB); rabbit DNAJB1 (Atlas Antibodies, HPA063247, 1:1,000 WB); rabbit anti-DNAJB2 (Atlas Antibodies, HPA036268, 1:1,000 WB); rabbit anti-DNAJB6 (a kind gift from Ineke Braakman, Utrecht University, 1:1,000 WB, 1:50 IF); rabbit anti-ATG16L (MBL International, PM040, 1:1,000 WB, 1:50 IF); rabbit anti-LC3 (Novus Biologicals, NB600-1384, 1:1,000 WB); mouse anti-tubulin (Sigma-Aldrich, T5168, 1:10,000 WB); mouse anti-actin (Merck, MAB1501, 1:10,000 WB); guinea pig anti-p62/SQSTM1 (Progen, GP62-C, 1:200 IF); mouse anti-p62/SQSTM1 (Abcam, ab56416, 1:2,000 WB); mouse anti-LAMP1 (BP Biosciences, 555798, 1:100 IF); mouse anti-RFP (ChromoTek, 6g6, 1:2,000 WB); mouse anti-V5 (Thermo Fisher Scientific, R960-25, 1:2,000 WB, 1:200 IF); rabbit anti-PSMC1 (Merck, HPA000872, 1:1,000 WB, 1:50 IF); rabbit anti-PSMC2 (Cell Signaling, 14395S, 1:1,000 WB); rabbit anti-PSMC3 (Cell Signaling, 13923S, 1:1,000 WB); rabbit anti-PSMC4 (Proteintech, 11389-1-AP); rabbit anti-PSMC5 (Merck, HPA064293, 1:1,000 WB, 1:50 IF); rabbit anti-PSMC6 (Proteintech, 15839-1-AP, 1:1,000 WB); rabbit anti-PSMB5 (Merck, HPA049518, 1:1,000 WB, 1:50 IF); rabbit anti-PSMB2 (Merck, HPA026324, 1:1,000 WB, 1:50 IF); mouse anti-PSMA7 (Enzo Life Sciences, BML-PW8110, 1:50 IF); mouse anti-PSMC2 (Enzo Life Sciences, BML-PW8825, 1:3,000 WB).

Techniques:

Journal: Nature Cell Biology

Article Title: A chaperone-proteasome-based fragmentation machinery is essential for aggrephagy

doi: 10.1038/s41556-025-01747-1

Figure Lengend Snippet: PSMC5-depleted and DNAJB6-depleted dualPIM cells. 3D tomograms of an area of interest with a dualPIM aggregate. Supplementary Video 4 corresponds to Fig. 4a, SAR405-treated cells.

Article Snippet: The following primary antibodies (and dilutions) were used: rabbit anti-BAG3 (Abcam, ab47124, 1:1,000); mouse anti-GAPDH (Fitzgerald Industries International, 10R-G109a, 1:1,0000); rabbit anti-RB1CC1/FIP200 (Proteintech, 17250-1-AP, 1:1,000 WB, 1:50 IF); rabbit anti vinculin (Cell Signaling, 13901, 1:1,000); rabbit anti-TAX1BP1 (Millipore, HPA024432, 1:1,000 WB, 1:100 IF); rabbit anti-CALCOCO2/NDP52 (Millipore, HPA023195, 1:1,000 WB, 1:100 IF); rat anti-HSC70 (Enzo Life Sciences, ADI-SPA-815, 1:1,000 WB); mouse anti-HSP70 (Enzo Life Sciences, ADI-SPA-810, 1:1,000 WB, 1:100 IF); mouse anti-APG2 (Santa Cruz Biotechnology, sc-365366, 1:1,000 WB); mouse anti HSPBP1 (Novus Biologicals, NBP 2-01168, 1:1,000 WB); mouse anti-HSPB1 (Enzo Life Sciences, ADI-SPA-800, 1:1,000 WB); mouse anti-HSPB7 (Abnova H00027129-M01, 1:1,000 WB); rabbit anti-DNAJA1 (Abcam, ab126774, 1:1,000 WB); mouse anti DNAJA2 (Sigma-Aldrich, WH0010294M1, 1:1,000 WB); rabbit DNAJB1 (Atlas Antibodies, HPA063247, 1:1,000 WB); rabbit anti-DNAJB2 (Atlas Antibodies, HPA036268, 1:1,000 WB); rabbit anti-DNAJB6 (a kind gift from Ineke Braakman, Utrecht University, 1:1,000 WB, 1:50 IF); rabbit anti-ATG16L (MBL International, PM040, 1:1,000 WB, 1:50 IF); rabbit anti-LC3 (Novus Biologicals, NB600-1384, 1:1,000 WB); mouse anti-tubulin (Sigma-Aldrich, T5168, 1:10,000 WB); mouse anti-actin (Merck, MAB1501, 1:10,000 WB); guinea pig anti-p62/SQSTM1 (Progen, GP62-C, 1:200 IF); mouse anti-p62/SQSTM1 (Abcam, ab56416, 1:2,000 WB); mouse anti-LAMP1 (BP Biosciences, 555798, 1:100 IF); mouse anti-RFP (ChromoTek, 6g6, 1:2,000 WB); mouse anti-V5 (Thermo Fisher Scientific, R960-25, 1:2,000 WB, 1:200 IF); rabbit anti-PSMC1 (Merck, HPA000872, 1:1,000 WB, 1:50 IF); rabbit anti-PSMC2 (Cell Signaling, 14395S, 1:1,000 WB); rabbit anti-PSMC3 (Cell Signaling, 13923S, 1:1,000 WB); rabbit anti-PSMC4 (Proteintech, 11389-1-AP); rabbit anti-PSMC5 (Merck, HPA064293, 1:1,000 WB, 1:50 IF); rabbit anti-PSMC6 (Proteintech, 15839-1-AP, 1:1,000 WB); rabbit anti-PSMB5 (Merck, HPA049518, 1:1,000 WB, 1:50 IF); rabbit anti-PSMB2 (Merck, HPA026324, 1:1,000 WB, 1:50 IF); mouse anti-PSMA7 (Enzo Life Sciences, BML-PW8110, 1:50 IF); mouse anti-PSMC2 (Enzo Life Sciences, BML-PW8825, 1:3,000 WB).

Techniques:

Journal: Nature Cell Biology

Article Title: A chaperone-proteasome-based fragmentation machinery is essential for aggrephagy

doi: 10.1038/s41556-025-01747-1

Figure Lengend Snippet: PSMC5-depleted and DNAJB6-depleted dualPIM cells. 3D tomograms of an area of interest with a dualPIM aggregate. Supplementary Video 5 corresponds to Fig. 4a, siDNAJB6-treated cells.

Article Snippet: The following primary antibodies (and dilutions) were used: rabbit anti-BAG3 (Abcam, ab47124, 1:1,000); mouse anti-GAPDH (Fitzgerald Industries International, 10R-G109a, 1:1,0000); rabbit anti-RB1CC1/FIP200 (Proteintech, 17250-1-AP, 1:1,000 WB, 1:50 IF); rabbit anti vinculin (Cell Signaling, 13901, 1:1,000); rabbit anti-TAX1BP1 (Millipore, HPA024432, 1:1,000 WB, 1:100 IF); rabbit anti-CALCOCO2/NDP52 (Millipore, HPA023195, 1:1,000 WB, 1:100 IF); rat anti-HSC70 (Enzo Life Sciences, ADI-SPA-815, 1:1,000 WB); mouse anti-HSP70 (Enzo Life Sciences, ADI-SPA-810, 1:1,000 WB, 1:100 IF); mouse anti-APG2 (Santa Cruz Biotechnology, sc-365366, 1:1,000 WB); mouse anti HSPBP1 (Novus Biologicals, NBP 2-01168, 1:1,000 WB); mouse anti-HSPB1 (Enzo Life Sciences, ADI-SPA-800, 1:1,000 WB); mouse anti-HSPB7 (Abnova H00027129-M01, 1:1,000 WB); rabbit anti-DNAJA1 (Abcam, ab126774, 1:1,000 WB); mouse anti DNAJA2 (Sigma-Aldrich, WH0010294M1, 1:1,000 WB); rabbit DNAJB1 (Atlas Antibodies, HPA063247, 1:1,000 WB); rabbit anti-DNAJB2 (Atlas Antibodies, HPA036268, 1:1,000 WB); rabbit anti-DNAJB6 (a kind gift from Ineke Braakman, Utrecht University, 1:1,000 WB, 1:50 IF); rabbit anti-ATG16L (MBL International, PM040, 1:1,000 WB, 1:50 IF); rabbit anti-LC3 (Novus Biologicals, NB600-1384, 1:1,000 WB); mouse anti-tubulin (Sigma-Aldrich, T5168, 1:10,000 WB); mouse anti-actin (Merck, MAB1501, 1:10,000 WB); guinea pig anti-p62/SQSTM1 (Progen, GP62-C, 1:200 IF); mouse anti-p62/SQSTM1 (Abcam, ab56416, 1:2,000 WB); mouse anti-LAMP1 (BP Biosciences, 555798, 1:100 IF); mouse anti-RFP (ChromoTek, 6g6, 1:2,000 WB); mouse anti-V5 (Thermo Fisher Scientific, R960-25, 1:2,000 WB, 1:200 IF); rabbit anti-PSMC1 (Merck, HPA000872, 1:1,000 WB, 1:50 IF); rabbit anti-PSMC2 (Cell Signaling, 14395S, 1:1,000 WB); rabbit anti-PSMC3 (Cell Signaling, 13923S, 1:1,000 WB); rabbit anti-PSMC4 (Proteintech, 11389-1-AP); rabbit anti-PSMC5 (Merck, HPA064293, 1:1,000 WB, 1:50 IF); rabbit anti-PSMC6 (Proteintech, 15839-1-AP, 1:1,000 WB); rabbit anti-PSMB5 (Merck, HPA049518, 1:1,000 WB, 1:50 IF); rabbit anti-PSMB2 (Merck, HPA026324, 1:1,000 WB, 1:50 IF); mouse anti-PSMA7 (Enzo Life Sciences, BML-PW8110, 1:50 IF); mouse anti-PSMC2 (Enzo Life Sciences, BML-PW8825, 1:3,000 WB).

Techniques:

Journal: Nature Cell Biology

Article Title: A chaperone-proteasome-based fragmentation machinery is essential for aggrephagy

doi: 10.1038/s41556-025-01747-1

Figure Lengend Snippet: PSMC5-depleted and DNAJB6-depleted dualPIM cells. 3D tomograms of an area of interest with a dualPIM aggregate. Supplementary Video 6 corresponds to Fig. 4a, siPSMC5-treated cells.

Article Snippet: The following primary antibodies (and dilutions) were used: rabbit anti-BAG3 (Abcam, ab47124, 1:1,000); mouse anti-GAPDH (Fitzgerald Industries International, 10R-G109a, 1:1,0000); rabbit anti-RB1CC1/FIP200 (Proteintech, 17250-1-AP, 1:1,000 WB, 1:50 IF); rabbit anti vinculin (Cell Signaling, 13901, 1:1,000); rabbit anti-TAX1BP1 (Millipore, HPA024432, 1:1,000 WB, 1:100 IF); rabbit anti-CALCOCO2/NDP52 (Millipore, HPA023195, 1:1,000 WB, 1:100 IF); rat anti-HSC70 (Enzo Life Sciences, ADI-SPA-815, 1:1,000 WB); mouse anti-HSP70 (Enzo Life Sciences, ADI-SPA-810, 1:1,000 WB, 1:100 IF); mouse anti-APG2 (Santa Cruz Biotechnology, sc-365366, 1:1,000 WB); mouse anti HSPBP1 (Novus Biologicals, NBP 2-01168, 1:1,000 WB); mouse anti-HSPB1 (Enzo Life Sciences, ADI-SPA-800, 1:1,000 WB); mouse anti-HSPB7 (Abnova H00027129-M01, 1:1,000 WB); rabbit anti-DNAJA1 (Abcam, ab126774, 1:1,000 WB); mouse anti DNAJA2 (Sigma-Aldrich, WH0010294M1, 1:1,000 WB); rabbit DNAJB1 (Atlas Antibodies, HPA063247, 1:1,000 WB); rabbit anti-DNAJB2 (Atlas Antibodies, HPA036268, 1:1,000 WB); rabbit anti-DNAJB6 (a kind gift from Ineke Braakman, Utrecht University, 1:1,000 WB, 1:50 IF); rabbit anti-ATG16L (MBL International, PM040, 1:1,000 WB, 1:50 IF); rabbit anti-LC3 (Novus Biologicals, NB600-1384, 1:1,000 WB); mouse anti-tubulin (Sigma-Aldrich, T5168, 1:10,000 WB); mouse anti-actin (Merck, MAB1501, 1:10,000 WB); guinea pig anti-p62/SQSTM1 (Progen, GP62-C, 1:200 IF); mouse anti-p62/SQSTM1 (Abcam, ab56416, 1:2,000 WB); mouse anti-LAMP1 (BP Biosciences, 555798, 1:100 IF); mouse anti-RFP (ChromoTek, 6g6, 1:2,000 WB); mouse anti-V5 (Thermo Fisher Scientific, R960-25, 1:2,000 WB, 1:200 IF); rabbit anti-PSMC1 (Merck, HPA000872, 1:1,000 WB, 1:50 IF); rabbit anti-PSMC2 (Cell Signaling, 14395S, 1:1,000 WB); rabbit anti-PSMC3 (Cell Signaling, 13923S, 1:1,000 WB); rabbit anti-PSMC4 (Proteintech, 11389-1-AP); rabbit anti-PSMC5 (Merck, HPA064293, 1:1,000 WB, 1:50 IF); rabbit anti-PSMC6 (Proteintech, 15839-1-AP, 1:1,000 WB); rabbit anti-PSMB5 (Merck, HPA049518, 1:1,000 WB, 1:50 IF); rabbit anti-PSMB2 (Merck, HPA026324, 1:1,000 WB, 1:50 IF); mouse anti-PSMA7 (Enzo Life Sciences, BML-PW8110, 1:50 IF); mouse anti-PSMC2 (Enzo Life Sciences, BML-PW8825, 1:3,000 WB).

Techniques:

Journal: Nature Cell Biology

Article Title: A chaperone-proteasome-based fragmentation machinery is essential for aggrephagy

doi: 10.1038/s41556-025-01747-1

Figure Lengend Snippet: PSMC5-depleted and DNAJB6-depleted dualPIM cells. 3D tomograms of an area of interest with a dualPIM aggregate. Supplementary Video 7 corresponds to Extended Data Fig. 7b, siCtr-treated cells.

Article Snippet: The following primary antibodies (and dilutions) were used: rabbit anti-BAG3 (Abcam, ab47124, 1:1,000); mouse anti-GAPDH (Fitzgerald Industries International, 10R-G109a, 1:1,0000); rabbit anti-RB1CC1/FIP200 (Proteintech, 17250-1-AP, 1:1,000 WB, 1:50 IF); rabbit anti vinculin (Cell Signaling, 13901, 1:1,000); rabbit anti-TAX1BP1 (Millipore, HPA024432, 1:1,000 WB, 1:100 IF); rabbit anti-CALCOCO2/NDP52 (Millipore, HPA023195, 1:1,000 WB, 1:100 IF); rat anti-HSC70 (Enzo Life Sciences, ADI-SPA-815, 1:1,000 WB); mouse anti-HSP70 (Enzo Life Sciences, ADI-SPA-810, 1:1,000 WB, 1:100 IF); mouse anti-APG2 (Santa Cruz Biotechnology, sc-365366, 1:1,000 WB); mouse anti HSPBP1 (Novus Biologicals, NBP 2-01168, 1:1,000 WB); mouse anti-HSPB1 (Enzo Life Sciences, ADI-SPA-800, 1:1,000 WB); mouse anti-HSPB7 (Abnova H00027129-M01, 1:1,000 WB); rabbit anti-DNAJA1 (Abcam, ab126774, 1:1,000 WB); mouse anti DNAJA2 (Sigma-Aldrich, WH0010294M1, 1:1,000 WB); rabbit DNAJB1 (Atlas Antibodies, HPA063247, 1:1,000 WB); rabbit anti-DNAJB2 (Atlas Antibodies, HPA036268, 1:1,000 WB); rabbit anti-DNAJB6 (a kind gift from Ineke Braakman, Utrecht University, 1:1,000 WB, 1:50 IF); rabbit anti-ATG16L (MBL International, PM040, 1:1,000 WB, 1:50 IF); rabbit anti-LC3 (Novus Biologicals, NB600-1384, 1:1,000 WB); mouse anti-tubulin (Sigma-Aldrich, T5168, 1:10,000 WB); mouse anti-actin (Merck, MAB1501, 1:10,000 WB); guinea pig anti-p62/SQSTM1 (Progen, GP62-C, 1:200 IF); mouse anti-p62/SQSTM1 (Abcam, ab56416, 1:2,000 WB); mouse anti-LAMP1 (BP Biosciences, 555798, 1:100 IF); mouse anti-RFP (ChromoTek, 6g6, 1:2,000 WB); mouse anti-V5 (Thermo Fisher Scientific, R960-25, 1:2,000 WB, 1:200 IF); rabbit anti-PSMC1 (Merck, HPA000872, 1:1,000 WB, 1:50 IF); rabbit anti-PSMC2 (Cell Signaling, 14395S, 1:1,000 WB); rabbit anti-PSMC3 (Cell Signaling, 13923S, 1:1,000 WB); rabbit anti-PSMC4 (Proteintech, 11389-1-AP); rabbit anti-PSMC5 (Merck, HPA064293, 1:1,000 WB, 1:50 IF); rabbit anti-PSMC6 (Proteintech, 15839-1-AP, 1:1,000 WB); rabbit anti-PSMB5 (Merck, HPA049518, 1:1,000 WB, 1:50 IF); rabbit anti-PSMB2 (Merck, HPA026324, 1:1,000 WB, 1:50 IF); mouse anti-PSMA7 (Enzo Life Sciences, BML-PW8110, 1:50 IF); mouse anti-PSMC2 (Enzo Life Sciences, BML-PW8825, 1:3,000 WB).

Techniques:

Journal: Nature Cell Biology

Article Title: A chaperone-proteasome-based fragmentation machinery is essential for aggrephagy

doi: 10.1038/s41556-025-01747-1

Figure Lengend Snippet: PSMC5-depleted and DNAJB6-depleted dualPIM cells. 3D tomograms of an area of interest with a dualPIM aggregate. Supplementary Video 8 corresponds to Extended Data Fig. 7b, SAR405-treated cells.

Article Snippet: The following primary antibodies (and dilutions) were used: rabbit anti-BAG3 (Abcam, ab47124, 1:1,000); mouse anti-GAPDH (Fitzgerald Industries International, 10R-G109a, 1:1,0000); rabbit anti-RB1CC1/FIP200 (Proteintech, 17250-1-AP, 1:1,000 WB, 1:50 IF); rabbit anti vinculin (Cell Signaling, 13901, 1:1,000); rabbit anti-TAX1BP1 (Millipore, HPA024432, 1:1,000 WB, 1:100 IF); rabbit anti-CALCOCO2/NDP52 (Millipore, HPA023195, 1:1,000 WB, 1:100 IF); rat anti-HSC70 (Enzo Life Sciences, ADI-SPA-815, 1:1,000 WB); mouse anti-HSP70 (Enzo Life Sciences, ADI-SPA-810, 1:1,000 WB, 1:100 IF); mouse anti-APG2 (Santa Cruz Biotechnology, sc-365366, 1:1,000 WB); mouse anti HSPBP1 (Novus Biologicals, NBP 2-01168, 1:1,000 WB); mouse anti-HSPB1 (Enzo Life Sciences, ADI-SPA-800, 1:1,000 WB); mouse anti-HSPB7 (Abnova H00027129-M01, 1:1,000 WB); rabbit anti-DNAJA1 (Abcam, ab126774, 1:1,000 WB); mouse anti DNAJA2 (Sigma-Aldrich, WH0010294M1, 1:1,000 WB); rabbit DNAJB1 (Atlas Antibodies, HPA063247, 1:1,000 WB); rabbit anti-DNAJB2 (Atlas Antibodies, HPA036268, 1:1,000 WB); rabbit anti-DNAJB6 (a kind gift from Ineke Braakman, Utrecht University, 1:1,000 WB, 1:50 IF); rabbit anti-ATG16L (MBL International, PM040, 1:1,000 WB, 1:50 IF); rabbit anti-LC3 (Novus Biologicals, NB600-1384, 1:1,000 WB); mouse anti-tubulin (Sigma-Aldrich, T5168, 1:10,000 WB); mouse anti-actin (Merck, MAB1501, 1:10,000 WB); guinea pig anti-p62/SQSTM1 (Progen, GP62-C, 1:200 IF); mouse anti-p62/SQSTM1 (Abcam, ab56416, 1:2,000 WB); mouse anti-LAMP1 (BP Biosciences, 555798, 1:100 IF); mouse anti-RFP (ChromoTek, 6g6, 1:2,000 WB); mouse anti-V5 (Thermo Fisher Scientific, R960-25, 1:2,000 WB, 1:200 IF); rabbit anti-PSMC1 (Merck, HPA000872, 1:1,000 WB, 1:50 IF); rabbit anti-PSMC2 (Cell Signaling, 14395S, 1:1,000 WB); rabbit anti-PSMC3 (Cell Signaling, 13923S, 1:1,000 WB); rabbit anti-PSMC4 (Proteintech, 11389-1-AP); rabbit anti-PSMC5 (Merck, HPA064293, 1:1,000 WB, 1:50 IF); rabbit anti-PSMC6 (Proteintech, 15839-1-AP, 1:1,000 WB); rabbit anti-PSMB5 (Merck, HPA049518, 1:1,000 WB, 1:50 IF); rabbit anti-PSMB2 (Merck, HPA026324, 1:1,000 WB, 1:50 IF); mouse anti-PSMA7 (Enzo Life Sciences, BML-PW8110, 1:50 IF); mouse anti-PSMC2 (Enzo Life Sciences, BML-PW8825, 1:3,000 WB).

Techniques:

Journal: Nature Cell Biology

Article Title: A chaperone-proteasome-based fragmentation machinery is essential for aggrephagy

doi: 10.1038/s41556-025-01747-1

Figure Lengend Snippet: PSMC5-depleted and DNAJB6-depleted dualPIM cells. 3D tomograms of an area of interest with a dualPIM aggregate. Supplementary Video 9 corresponds to Extended Data Fig. 7b, siDNAJB6-treated cells.

Article Snippet: The following primary antibodies (and dilutions) were used: rabbit anti-BAG3 (Abcam, ab47124, 1:1,000); mouse anti-GAPDH (Fitzgerald Industries International, 10R-G109a, 1:1,0000); rabbit anti-RB1CC1/FIP200 (Proteintech, 17250-1-AP, 1:1,000 WB, 1:50 IF); rabbit anti vinculin (Cell Signaling, 13901, 1:1,000); rabbit anti-TAX1BP1 (Millipore, HPA024432, 1:1,000 WB, 1:100 IF); rabbit anti-CALCOCO2/NDP52 (Millipore, HPA023195, 1:1,000 WB, 1:100 IF); rat anti-HSC70 (Enzo Life Sciences, ADI-SPA-815, 1:1,000 WB); mouse anti-HSP70 (Enzo Life Sciences, ADI-SPA-810, 1:1,000 WB, 1:100 IF); mouse anti-APG2 (Santa Cruz Biotechnology, sc-365366, 1:1,000 WB); mouse anti HSPBP1 (Novus Biologicals, NBP 2-01168, 1:1,000 WB); mouse anti-HSPB1 (Enzo Life Sciences, ADI-SPA-800, 1:1,000 WB); mouse anti-HSPB7 (Abnova H00027129-M01, 1:1,000 WB); rabbit anti-DNAJA1 (Abcam, ab126774, 1:1,000 WB); mouse anti DNAJA2 (Sigma-Aldrich, WH0010294M1, 1:1,000 WB); rabbit DNAJB1 (Atlas Antibodies, HPA063247, 1:1,000 WB); rabbit anti-DNAJB2 (Atlas Antibodies, HPA036268, 1:1,000 WB); rabbit anti-DNAJB6 (a kind gift from Ineke Braakman, Utrecht University, 1:1,000 WB, 1:50 IF); rabbit anti-ATG16L (MBL International, PM040, 1:1,000 WB, 1:50 IF); rabbit anti-LC3 (Novus Biologicals, NB600-1384, 1:1,000 WB); mouse anti-tubulin (Sigma-Aldrich, T5168, 1:10,000 WB); mouse anti-actin (Merck, MAB1501, 1:10,000 WB); guinea pig anti-p62/SQSTM1 (Progen, GP62-C, 1:200 IF); mouse anti-p62/SQSTM1 (Abcam, ab56416, 1:2,000 WB); mouse anti-LAMP1 (BP Biosciences, 555798, 1:100 IF); mouse anti-RFP (ChromoTek, 6g6, 1:2,000 WB); mouse anti-V5 (Thermo Fisher Scientific, R960-25, 1:2,000 WB, 1:200 IF); rabbit anti-PSMC1 (Merck, HPA000872, 1:1,000 WB, 1:50 IF); rabbit anti-PSMC2 (Cell Signaling, 14395S, 1:1,000 WB); rabbit anti-PSMC3 (Cell Signaling, 13923S, 1:1,000 WB); rabbit anti-PSMC4 (Proteintech, 11389-1-AP); rabbit anti-PSMC5 (Merck, HPA064293, 1:1,000 WB, 1:50 IF); rabbit anti-PSMC6 (Proteintech, 15839-1-AP, 1:1,000 WB); rabbit anti-PSMB5 (Merck, HPA049518, 1:1,000 WB, 1:50 IF); rabbit anti-PSMB2 (Merck, HPA026324, 1:1,000 WB, 1:50 IF); mouse anti-PSMA7 (Enzo Life Sciences, BML-PW8110, 1:50 IF); mouse anti-PSMC2 (Enzo Life Sciences, BML-PW8825, 1:3,000 WB).

Techniques:

Journal: Nature Cell Biology

Article Title: A chaperone-proteasome-based fragmentation machinery is essential for aggrephagy

doi: 10.1038/s41556-025-01747-1

Figure Lengend Snippet: PSMC5-depleted and DNAJB6-depleted dualPIM cells. 3D tomograms of an area of interest with a dualPIM aggregate. Supplementary Video 10 corresponds to Extended Data Fig. 7 b, siPSMC5-treated cells.

Article Snippet: The following primary antibodies (and dilutions) were used: rabbit anti-BAG3 (Abcam, ab47124, 1:1,000); mouse anti-GAPDH (Fitzgerald Industries International, 10R-G109a, 1:1,0000); rabbit anti-RB1CC1/FIP200 (Proteintech, 17250-1-AP, 1:1,000 WB, 1:50 IF); rabbit anti vinculin (Cell Signaling, 13901, 1:1,000); rabbit anti-TAX1BP1 (Millipore, HPA024432, 1:1,000 WB, 1:100 IF); rabbit anti-CALCOCO2/NDP52 (Millipore, HPA023195, 1:1,000 WB, 1:100 IF); rat anti-HSC70 (Enzo Life Sciences, ADI-SPA-815, 1:1,000 WB); mouse anti-HSP70 (Enzo Life Sciences, ADI-SPA-810, 1:1,000 WB, 1:100 IF); mouse anti-APG2 (Santa Cruz Biotechnology, sc-365366, 1:1,000 WB); mouse anti HSPBP1 (Novus Biologicals, NBP 2-01168, 1:1,000 WB); mouse anti-HSPB1 (Enzo Life Sciences, ADI-SPA-800, 1:1,000 WB); mouse anti-HSPB7 (Abnova H00027129-M01, 1:1,000 WB); rabbit anti-DNAJA1 (Abcam, ab126774, 1:1,000 WB); mouse anti DNAJA2 (Sigma-Aldrich, WH0010294M1, 1:1,000 WB); rabbit DNAJB1 (Atlas Antibodies, HPA063247, 1:1,000 WB); rabbit anti-DNAJB2 (Atlas Antibodies, HPA036268, 1:1,000 WB); rabbit anti-DNAJB6 (a kind gift from Ineke Braakman, Utrecht University, 1:1,000 WB, 1:50 IF); rabbit anti-ATG16L (MBL International, PM040, 1:1,000 WB, 1:50 IF); rabbit anti-LC3 (Novus Biologicals, NB600-1384, 1:1,000 WB); mouse anti-tubulin (Sigma-Aldrich, T5168, 1:10,000 WB); mouse anti-actin (Merck, MAB1501, 1:10,000 WB); guinea pig anti-p62/SQSTM1 (Progen, GP62-C, 1:200 IF); mouse anti-p62/SQSTM1 (Abcam, ab56416, 1:2,000 WB); mouse anti-LAMP1 (BP Biosciences, 555798, 1:100 IF); mouse anti-RFP (ChromoTek, 6g6, 1:2,000 WB); mouse anti-V5 (Thermo Fisher Scientific, R960-25, 1:2,000 WB, 1:200 IF); rabbit anti-PSMC1 (Merck, HPA000872, 1:1,000 WB, 1:50 IF); rabbit anti-PSMC2 (Cell Signaling, 14395S, 1:1,000 WB); rabbit anti-PSMC3 (Cell Signaling, 13923S, 1:1,000 WB); rabbit anti-PSMC4 (Proteintech, 11389-1-AP); rabbit anti-PSMC5 (Merck, HPA064293, 1:1,000 WB, 1:50 IF); rabbit anti-PSMC6 (Proteintech, 15839-1-AP, 1:1,000 WB); rabbit anti-PSMB5 (Merck, HPA049518, 1:1,000 WB, 1:50 IF); rabbit anti-PSMB2 (Merck, HPA026324, 1:1,000 WB, 1:50 IF); mouse anti-PSMA7 (Enzo Life Sciences, BML-PW8110, 1:50 IF); mouse anti-PSMC2 (Enzo Life Sciences, BML-PW8825, 1:3,000 WB).

Techniques:

Journal: Human Molecular Genetics

Article Title: PSMC5 insufficiency and P320R mutation impair proteasome function

doi: 10.1093/hmg/ddae085

Figure Lengend Snippet: Summary clinical features of individuals with PSMC5 mutations described in this paper.

Article Snippet: Rabbit anti-PSMC5 , Abcam , ab178681.

Techniques: Variant Assay, Reflux

Journal: Human Molecular Genetics

Article Title: PSMC5 insufficiency and P320R mutation impair proteasome function

doi: 10.1093/hmg/ddae085

Figure Lengend Snippet: PSMC5 knockdown results in accumulation of K48-ubiquitinated proteins and apoptotic cell death in HeLa cells. (A) Western blotting of PSMC5 levels after PSMC5 siRNA knockdown using SMARTpool or four deconvoluted oligos. GAPDH served as protein loading control. (B) LDH cytotoxicity assay to measure cell death caused by PSMC5 knockdown. Plots represent mean ± SD (n = 4 independent experiments). P values were calculated using two-tailed, paired Student’s t-test. (C) Western blotting and quantification of PSMC5, K48-ubiquitinated proteins, and cleaved caspase-3 levels in PSMC5-knockdown cells. GAPDH served as protein loading control. Plots represent mean ± SD (n = 4 independent experiments). P values were calculated using two-tailed, paired Student’s t-test. (D) LDH cytotoxicity assay to measure the inhibition effect of ferroptosis inhibitors (ferrostatin-1, liproxstatin-1), necroptosis inhibitors (necrostatin-1, necrosulfonamide), pyroptosis inhibitor (Z-YVAD), and apoptosis inhibitor (Z-VAD) on cell death caused by PSMC5 knockdown. Cell death of drug treatment groups was normalized to DMSO treated wild-type, PSMC5 knockdown with oligo1 or oligo2, respectively. Plots represent mean ± SD (n = 4 independent experiments). P values were calculated using one-way ANOVA with post-hoc Dunnett’s multiple comparison test. * indicates P < 0.05; * * indicates P < 0.01; * * * indicates P < 0.001; NS, not significant.

Article Snippet: Rabbit anti-PSMC5 , Abcam , ab178681.

Techniques: Knockdown, Western Blot, Control, LDH Cytotoxicity Assay, Two Tailed Test, Inhibition, Comparison

Journal: Human Molecular Genetics

Article Title: PSMC5 insufficiency and P320R mutation impair proteasome function

doi: 10.1093/hmg/ddae085

Figure Lengend Snippet: Knockdowns of the 19S RP subunits of the 26S proteasome impairs proteasome function and leads to apoptosis in HeLa cells. (A) LDH cytotoxicity assay to measure cell death caused by PSMC5, PSMC3, PSMD2, or PSMD12 siRNA knockdown. Plots represent mean ± SD (n = 4 independent experiments). P values were calculated using two-tailed, paired Student’s t-test. (B) Western blotting of PSMC5, PSMC3, PSMD2, PSMD12, K48-ubiquitinated proteins, and cleaved caspase-3 levels, and quantification of K48-ubiquitinated proteins and cleaved caspase-3 in PSMC5, PSMC3, PSMD2, or PSMD12-knockdown cells. GAPDH served as protein loading control. Plots represent mean ± SD (n = 4 independent experiments). P values were calculated using two-tailed, paired Student’s t-test. (C) In-gel proteasome chymotrypsin-like (CT-L) activity of the 30S, 26S, and 20S proteasome complexes and quantification thereof in PSMC5, PSMC3, PSMD2, or PSMD12-knockdown cells. The CT-L activity of proteasome complexes is visualized by their ability to cleave the Suc-LLVY-AMC fluorogenic peptide. The CT-L activity of the 30S, 26S, and 20S proteasome complexes of different knockdowns was normalized to the corresponding controls in the quantification plots. Plots represent mean ± SD (n = 3 independent experiments). P values were calculated using two-tailed, paired Student’s t-test. (D) Immunoblotting of the native gel in (C) and probing of the membrane with anti-α1-7 antibody and quantification the amount of 30S, 26S, and 20S proteasome complexes indicated by α1-7. The protein level of α1-7 in the 30S, 26S, and 20S proteasome complexes of different knockdowns was normalized to the corresponding controls in the quantification plots. Plots represent mean ± SD (n = 3 independent experiments). P values were calculated using two-tailed, paired Student’s t-test. * indicates P < 0.05; * * indicates P < 0.01; * * * indicates P < 0.001; NS, not significant.

Article Snippet: Rabbit anti-PSMC5 , Abcam , ab178681.

Techniques: LDH Cytotoxicity Assay, Knockdown, Two Tailed Test, Western Blot, Control, Activity Assay, Membrane

Journal: Human Molecular Genetics

Article Title: PSMC5 insufficiency and P320R mutation impair proteasome function

doi: 10.1093/hmg/ddae085

Figure Lengend Snippet: Transient expression of PSMC5 P320R in PSMC5-knockdown HeLa cells leads to the dissociation of the 19S RP from the 20S CP. (A) LDH cytotoxicity assay to compare cell death in PSMC5-knockdown HeLa cells transiently expressing Flag-PSMC5 WT or PSMC5 P320R . Cell death of control and PSMC5 knockdown groups was normalized to the corresponding vector groups, respectively. Plots represent mean ± SD (n = 5 independent experiments). P values were calculated using two-tailed, paired Student’s t-test. (B) Western blotting of PSMC5, K48-ubiquitinated proteins, and cleaved caspase-3 levels and quantification of K48-ubiquitinated proteins, and cleaved caspase-3 in PSMC5-knockdown HeLa cells transiently expressing Flag-PSMC5 WT or PSMC5 P320R . GAPDH served as protein loading control. The protein levels of K48-ubiquitinated proteins and cleaved caspase-3 were normalized to the corresponding vector groups in the quantification plots. Plots represent mean ± SD (n = 4 independent experiments). P values were calculated using two-tailed, paired Student’s t-test. (C) Domain organization of PSMC5. (D) the structure of 26S proteasome and PSMC5 are shown as ribbon diagrams with PSMC5 highlighted in green and PSMC5 P320 highlighted in red. The ribbon diagrams were generated using PyMOL software based on the solved structure of the human 26S proteasome (PDB 6MSH) . (E) Immunoprecipitation of exogenously expressed Flag-PSMC5 WT or PSMC5 P320R in PSMC5-knockdown HeLa cells and quantification of the 19S RP and 20S CP subunits co-immunoprecipitated with Flag-PSMC5 WT or PSMC5 P320R . PSMC5 (LE) indicates a longer exposure of the PSMC5 blot. * in the blots indicates non-specific bands. The amount of all the preys (PSMC3, PSMD2, PSMD12, α1-7, and β5) co-immunoprecipitated with Flag-PSMC5 was normalized to the corresponding bait Flag-PSMC5 and then normalized to the corresponding Flag-PSMC5 WT groups in the quantification plots. Plots represent mean ± SD (n = 3 independent experiments). P values were calculated using two-tailed, paired Student’s t-test. * indicates P < 0.05; * * indicates P < 0.01; NS, not significant. Endogenous PSMC5 is knocked down using PSMC5 siRNA oligo2 and exogenously expressed PSMC5 WT or PSMC5 P320R are PSMC5 siRNA oligo2-resistant.

Article Snippet: Rabbit anti-PSMC5 , Abcam , ab178681.

Techniques: Expressing, Knockdown, LDH Cytotoxicity Assay, Control, Plasmid Preparation, Two Tailed Test, Western Blot, Generated, Software, Immunoprecipitation

Journal: Human Molecular Genetics

Article Title: PSMC5 insufficiency and P320R mutation impair proteasome function

doi: 10.1093/hmg/ddae085

Figure Lengend Snippet: Engineered BE(2)-M17 cells carrying homozygous or heterozygous PSMC5 P320R show accumulation of K48-ubiquitinated proteins, apoptosis, and the dissociation of the 19S RP from the 20S CP. (A) CellTox green cytotoxicity assay to measure cell death of BE(2)-M17 cells carrying homozygous or heterozygous PSMC5 P320R . Plots represent mean ± SD (n = 6 independent experiments). P values were calculated using two-tailed, paired Student’s t-test. (B) Western blotting and quantification of PSMC5, K48-ubiquitinated proteins, and cleaved caspase-3 levels in BE(2)-M17 cells carrying homozygous or heterozygous PSMC5 P320R . GAPDH served as protein loading control. The average of two repeats shown in one western blot image was taken as a biological replicate. Plots represent mean ± SD (n = 4 independent experiments). P values were calculated using two-tailed, paired Student’s t-test. (C) BE(2)-M17 cells carrying homozygous or heterozygous PSMC5 P320R and wild-type control were transfected with Ub-G76V-GFP-IRES-DsRed, followed by FACS. Plots represent mean ± SD (n = 3 independent experiments). P values were calculated using two-tailed, paired Student’s t-test. (D) Immunoprecipitation of PSMC3 in BE(2)-M17 cells carrying homozygous or heterozygous PSMC5 P320R and quantification of the 19S RP and 20S CP subunits co-immunoprecipitated with PSMC3. * in the blots indicates non-specific bands. The amount of all the preys (PSMC5, PSMD2, PSMD12, α1-7, and β5) co-immunoprecipitated with PSMC3 was normalized to the corresponding PSMC3 and then normalized to the corresponding +/+ groups in the quantification plots. Plots represent mean ± SD (n = 3 independent experiments). P values were calculated using two-tailed, paired Student’s t-test. * indicates P < 0.05; * * indicates P < 0.01; * * * indicates P < 0.001; NS, not significant. +/+: Wild type; P320R/+: Heterozygous PSMC5 P320R ; P320R/P320R: Homozygous PSMC5 P320R . C13, clone 13; C26, clone 26; C28, clone 28; C102, clone 102.

Article Snippet: Rabbit anti-PSMC5 , Abcam , ab178681.

Techniques: CellTox Assay, Cytotoxicity Assay, Two Tailed Test, Western Blot, Control, Transfection, Immunoprecipitation

Journal: Human Molecular Genetics

Article Title: PSMC5 insufficiency and P320R mutation impair proteasome function

doi: 10.1093/hmg/ddae085

Figure Lengend Snippet: Homozygous PSMC5 P320R shows the decrease of 26S and 30S proteasome and the increase of 20S proteasome, and heterozygous PSMC5 P320R shows the decrease of 30S proteasome and the increase of 20S proteasome. (A) Proteasome CT-L activity in BE(2)-M17 cells carrying homozygous or heterozygous PSMC5 P320R was assessed in gels using the Suc-LLVY-AMC fluorogenic peptide and quantification thereof. The CT-L activity of the 30S, 26S, and 20S proteasome complexes in different cell lines was normalized to the corresponding +/+ groups in the quantification plots. Plots represent mean ± SD (n = 6 independent experiments). P values were calculated using two-tailed, paired Student’s t-test. (B) Immunoblotting of the native gel in (A) and probing of the membrane with anti-α1-7 antibody and quantification the amount of 30S, 26S, and 20S proteasome complexes or the ratio of 30S/20S and 26S/20S indicated by α1-7. The protein level of α1-7 in the 30S, 26S, and 20S proteasome complexes or in the ratio of 30S/20S and 26S/20S in different cell lines was normalized to the corresponding +/+ groups in the quantification plots. Plots represent mean ± SD (n = 7 independent experiments). P values were calculated using two-tailed, paired Student’s t-test. (C) Immunoblotting of the native gel in (A) and probing of the membrane with anti-PSMC3 antibody and quantification the amount of 30S and 26S proteasome complexes indicated by PSMC3. The protein level of PSMC3 in the 30S, 26S, and 20S proteasome complexes in different cell lines was normalized to the corresponding +/+ groups in the quantification plots. Plots represent mean ± SD (n = 7 independent experiments). P values were calculated using two-tailed, paired Student’s t-test. * indicates P < 0.05; * * indicates P < 0.01; * * * indicates P < 0.001; NS, not significant. +/+: Wild type; P320R/+: Heterozygous PSMC5 P320R ; P320R/P320R: Homozygous PSMC5 P320R . C13, clone 13; C26, clone 26; C28, clone 28; C102, clone 102.

Article Snippet: Rabbit anti-PSMC5 , Abcam , ab178681.

Techniques: Activity Assay, Two Tailed Test, Western Blot, Membrane

Journal: Human Molecular Genetics

Article Title: PSMC5 insufficiency and P320R mutation impair proteasome function

doi: 10.1093/hmg/ddae085

Figure Lengend Snippet: Antibodies used in this paper.

Article Snippet: Rabbit anti-PSMC5 , Abcam , ab178681.

Techniques:

Journal: medRxiv

Article Title: Unveiling the crucial neuronal role of the proteasomal ATPase subunit gene PSMC5 in neurodevelopmental proteasomopathies

doi: 10.1101/2024.01.13.24301174

Figure Lengend Snippet: Clinical phenotypes associated with PSMC5 variants. A. To visually represent the phenotypic data from tables S2b-2c, we organized the HPO terms into categories and ranked the top categories based on their prevalence within the cohort. Within each category, circles illustrate the number of phenotypes assessed and documented for each individual. The size of a circle corresponds to the number of phenotypes evaluated for a particular subject. If no phenotypes were assessed, there is no circle present. The color of each circle indicates the proportion of these confirmed phenotypes in a subject, with a spectrum ranging from dark blue (indicating the lowest fraction) to red (indicating the highest fraction). The top categories are arranged in descending order according to the highest average fraction observed among subjects. Notably, the most commonly observed categories among these are nervous system and head and neck anomalies. Intriguingly, there was no apparent correlation between genotype and phenotype, as significant variability in phenotypic expression was observed even among individuals with the same genetic variant. B . Dysmorphic facial features included notably abnormal ear morphology as seen in subjects S3/9/10/13/19/30/32-34 (this feature is present in 19/31 (61%) subjects in the whole cohort), abnormal palpebral fissures (13/35; 37% in the whole cohort) including downslanted ones as in subjects S3/10/13/30, thin upper lip vermilion as in subjects S3/34 (7/35; 20%), abnormal palate as in subject S10 (7/35; 20%), tall or broad forehead as in S3/33/34 (9/35; 26%), epicanthus as in S4/38 (5/35; 14%), and orofacial clefts as in subject S10 (2/35; 6%). C. Facial image analysis using GestaltMatcher. The pairwise rank matrix and hierarchical clustering of 13 PSMC5 subjects. Each column is the result of testing one subject in the column and ranking of the remaining 12 photos in each row. For example, by testing the similarities between S10 and the 7,459 images of affected individuals from GMDB, S31 was ranked 1st and S19 was ranked 7th as being most similar to S10. The red box is the cluster of subjects with at least one match below the rank 50, which indicates this cluster shares a similar facial phenotype.

Article Snippet: Antibodies used in this study were directed against HA (BioLegend, clone HA.11), PSMC5/Rpt6 (Enzo Life Sciences, clone p45-110), α-tubulin (Abcam, clone DM1A), α6 (Enzo Life Sciences, clone MCP20), ubiquitin K48-linked ubiquitin chains (Cell Signaling, clone D9D5), GAPDH (Cell Signaling, clone 14C10), GRP94 (Invitrogen, PA5-27860), ATF6 (Cell Signaling, clone D4Z8V), BNIP3L (Cell Signaling, 12396).

Techniques: Expressing, Variant Assay

Journal: medRxiv

Article Title: Unveiling the crucial neuronal role of the proteasomal ATPase subunit gene PSMC5 in neurodevelopmental proteasomopathies

doi: 10.1101/2024.01.13.24301174

Figure Lengend Snippet:

Article Snippet: Antibodies used in this study were directed against HA (BioLegend, clone HA.11), PSMC5/Rpt6 (Enzo Life Sciences, clone p45-110), α-tubulin (Abcam, clone DM1A), α6 (Enzo Life Sciences, clone MCP20), ubiquitin K48-linked ubiquitin chains (Cell Signaling, clone D9D5), GAPDH (Cell Signaling, clone 14C10), GRP94 (Invitrogen, PA5-27860), ATF6 (Cell Signaling, clone D4Z8V), BNIP3L (Cell Signaling, 12396).

Techniques:

Journal: medRxiv

Article Title: Unveiling the crucial neuronal role of the proteasomal ATPase subunit gene PSMC5 in neurodevelopmental proteasomopathies

doi: 10.1101/2024.01.13.24301174

Figure Lengend Snippet: Structural analysis of PSMC5 variants reveals most missense variants are located in the AAA+ ATPase domain of PSMC5/Rpt6, and affect proteasome dynamics or subunit interactions. A . Schematic representation of the PSMC5 (i.e. RPT6) protein on which the 23 alterations identified in patients with NDD are designated. Approximately half of these mutations fall into three distinct hotspot regions within the AAA+ ATPase domain of PSMC5/RPT6, as indicated. Numbers refer to the sums of unrelated NDD subjects in which the highlighted variant has been identified. Missense variants are colored in blue, frameshift variants in red and splice site variants in purple. B . Basing on the structures 6MSK from RCSB Protein Data Bank, we localized the missense variants identified in the AAA+ ATPase subunit PSMC5/Rpt6 within the 26S proteasomal complex. The variants of interest (spheres) are primarily located in the lower ATPase domain. Part of the 26S proteasome was hidden for better visibility; Rpt6 is shown in cartoon representation.

Article Snippet: Antibodies used in this study were directed against HA (BioLegend, clone HA.11), PSMC5/Rpt6 (Enzo Life Sciences, clone p45-110), α-tubulin (Abcam, clone DM1A), α6 (Enzo Life Sciences, clone MCP20), ubiquitin K48-linked ubiquitin chains (Cell Signaling, clone D9D5), GAPDH (Cell Signaling, clone 14C10), GRP94 (Invitrogen, PA5-27860), ATF6 (Cell Signaling, clone D4Z8V), BNIP3L (Cell Signaling, 12396).

Techniques: Variant Assay

Journal: medRxiv

Article Title: Unveiling the crucial neuronal role of the proteasomal ATPase subunit gene PSMC5 in neurodevelopmental proteasomopathies

doi: 10.1101/2024.01.13.24301174

Figure Lengend Snippet: Suppression of Psmc5 leads to changes in reversal learning in flies and disrupts the balance between excitatory and inhibitory signals in rat hippocampal neurons. A. Pan-neuronal knockdown of Rpt6 in Drosophila did not significantly affect normal olfactory learning (p=0.3059, n=4). B. When faced with the more difficult task of reversal olfactory learning, knocking down Rpt6 pan-neuronally in Drosophila resulted in a significant decrease in performance (p<0.0001, n=4). (C-J) Primary rat hippocampal neurons infected DIV1-DIV14 with scrambled shRNA or PMSC5 knockdown (KD) shRNA delivering lenti-particles that co-express GFP. C. Assessment of PMSC5 fluorescent signal intensity in neurons at DIV14 shows a significant reduction in cells infected (GFP positive) with PMSC5 knockdown (KD) compared to cells infected with scrambled shRNA (n=10, Mann Whitney test, p = 0.0002). D. Exemplary images of neurons infected with scrambled shRNA (left) or PMSC5 shRNA (right) lenti-particles. PMSC5 labeling (arrow) is significantly reduced, confirming the KD of PMSC5. E-F. Infected neurons stained for MAP2 to visualize neuronal morphology. E. No significant difference in dendritic branching measured by the mean number of primary, secondary, and tertiary dendrites per neuron at DIV14 was found comparing scrambled Ctrl and PMSC5 KD conditions (n=6). F. Exemplary images of neurons infected with scrambled shRNA (left) or PMSC5 shRNA (right) lenti-particles. MAP2 labeling is shown in red. G-J. Infected neurons stained for vGlut to visualize excitatory presynaptic compartments or vGat to visualize inhibitory presynapses. G. The number of vGlut positive puncta along the primary dendrites was measured at DIV14. In Psmc5 KD conditions, a significant reduction was observed compared to scrambled Ctrls (n=5, Mann Whitney test, p = 0.0045). H. The number of vGat positive puncta along the primary dendrites was not significantly altered (n=5, Mann Whitney test, p = 0.2988). I-J. Exemplary images of primary dendrites of neurons infected with scrambled shRNA (left) or Psmc5 shRNA (right) lenti- particles. VGlut (full arrow) positive signals per dendrite length are significantly reduced, while VGat (open arrow) positive signals per dendrite length are not altered after Psmc5 KD.

Article Snippet: Antibodies used in this study were directed against HA (BioLegend, clone HA.11), PSMC5/Rpt6 (Enzo Life Sciences, clone p45-110), α-tubulin (Abcam, clone DM1A), α6 (Enzo Life Sciences, clone MCP20), ubiquitin K48-linked ubiquitin chains (Cell Signaling, clone D9D5), GAPDH (Cell Signaling, clone 14C10), GRP94 (Invitrogen, PA5-27860), ATF6 (Cell Signaling, clone D4Z8V), BNIP3L (Cell Signaling, 12396).

Techniques: Infection, shRNA, MANN-WHITNEY, Labeling, Staining

Journal: medRxiv

Article Title: Unveiling the crucial neuronal role of the proteasomal ATPase subunit gene PSMC5 in neurodevelopmental proteasomopathies

doi: 10.1101/2024.01.13.24301174

Figure Lengend Snippet: Overexpression of PSMC5 affects neuronal morphology. A. Representative images of primary hippocampal neurons transfected with the empty vector control (EV), PSMC5 -WT or the different PSMC5 variants. Red indicates the tdTomato blue indicat MAP2. B. Analysis of the total neurite length in the different conditions reveal: (i) a significant increase induced by overexpression of PSMC5-WT compared to empty vector control (One-way ANOVA, F=3.296, p= 0.0014; empty vector versus PSMC5-WT, p=0.0046, Dunnet’s multiple comparison test); (ii) a similar effect as PSMC5-WT induced by overexpression of variants p.(Arg201Trp), p.(Pro320His) and p.(Arg325Trp) (PSMC5-WT versus p.(Arg201Trp), p=0.8; PSMC5-WT versus p.(Pro320His), p=0.8; PSMC5-WT versus p.(Arg325Trp), p=0.5, Dunnet’s multiple comparison test); (iii) a decrease induced by overexpression of variants p.(Ala202Val), p.(Thr207Met), p.(Pro320Arg) and PSMC5-Δex10, compared to PSMC5-WT (PSMC5-WT versus p.(Ala202Val), p=0.0031; PSMC5-WT versus p.(Thr207Met), p=0.02; PSMC5-WT versus p.(Pro320Arg), p=0.006; PSMC5-WT versus PSMC5-Δex10, p=0.03, Dunnet’s multiple comparison test). The effects of these variants were indistinguishable from the empty vector control (empty vector versus p.(Ala202Val), p=0.4; empty vector versus p.(Thr207Met), p=0.9; empty vector versus p.(Pro320Arg), p=0.9; empty vector versus PSMC5-Δex10, p=0.9, Dunnet’s multiple comparison test). C. Analysis of the arborization showed no differences between conditions compared to the empty vector control (One-way ANOVA, F=2.179, p= 0.03; empty vector versus PSMC5-WT, p=0.9, PSMC5-WT versus p.(Arg201Trp), p=0.9; PSMC5-WT versus p.(Ala202Val), p=0.9; PSMC5- WT versus p.(Thr207Met), p=0.6; PSMC5-WT versus p.(Pro320His), p=0.3; PSMC5-WT versus p.(Pro320Arg), p=0.9; PSMC5-WT versus p.(Arg 325Trp), p=0.9; PSMC5-WT versus PSMC5-Δex10, p=0.9, Dunnet’s multiple comparison test). Error bars indicate SEM, n (number of neurons traced): EV=40, PSMC5-WT=30, p.(Arg201Trp)=18 p.(Ala202Val)=20, p.(Thr207Met)=27, p.(Pro320Arg)=29, p.(Pro320Arg)=19, p.(Arg325Trp)=20 and PSMC5-Δex10=20. *p<0.05; **p<0.01. Scale bar: 100μm.

Article Snippet: Antibodies used in this study were directed against HA (BioLegend, clone HA.11), PSMC5/Rpt6 (Enzo Life Sciences, clone p45-110), α-tubulin (Abcam, clone DM1A), α6 (Enzo Life Sciences, clone MCP20), ubiquitin K48-linked ubiquitin chains (Cell Signaling, clone D9D5), GAPDH (Cell Signaling, clone 14C10), GRP94 (Invitrogen, PA5-27860), ATF6 (Cell Signaling, clone D4Z8V), BNIP3L (Cell Signaling, 12396).

Techniques: Over Expression, Transfection, Plasmid Preparation, Comparison

Journal: medRxiv

Article Title: Unveiling the crucial neuronal role of the proteasomal ATPase subunit gene PSMC5 in neurodevelopmental proteasomopathies

doi: 10.1101/2024.01.13.24301174

Figure Lengend Snippet: T cells from NDD subjects carrying PSMC5 heterozygous variants exhibit abnormalities in their proteasome expression and/or activity profiles. A . T cells expanded from PBMC isolated from NDD subjects S6/11/12/21/32 as well as related controls (father and/or mother) and heathy donors (noted ‘un. control’ for unrelated controls) were lysed in RIPA buffer prior to SDS-PAGE/western-blotting analysis using antibodies specific PSMC5/RPT6 and GAPDH (loading control). Arrow indicates full-length endogenous PSMC5/RPT6 at the expected size of approximately 45 kDa detected in all samples. PSMC5/Rpt6 staining of T cell lysates from subjects 6/11/21/32 uncovered a further shorter PSMC5/Rpt6 immunoreactive band migrating at ∼30kDa (denoted as a low-molecular mass (LMM) species by the square brackets) following prolonged exposure times, whereas T cells from subject S12 exhibited an extra PSMC5/Rpt6 signal running a little bit lighter than PSMC5/Rpt6 expected size just below 40 kDa (indicated by an asterisk). Lower panel: the RPT6 and GAPDH immunoreactive bands were quantified by densitometry. Data are presented as RPT6 full-length and low molecular weight (LMW) species on GAPDH ratio mean values ± SD for the control (n=9) and patients (PSMC5 lesions, n=5) groups, as indicated. Statistical analysis was performed using the Mann-Whitney U test where *** indicates p <0.001. B . Resting T cells from NDD subjects S6/11/12/21/32/33 as well as related (subject’s father and/or mother, as indicated) and unrelated (healthy donor) control T cells were analyzed for proteasome function and abundance. They were subjected to non- denaturing protein extraction using TSDG buffer prior to native-PAGE/western-blotting analysis using monoclonal antibodies specific for the α6 and PSMC5/RPT6 subunits, as indicated. Proteasome complexes (20S and 26S) were also visualized by in-gel activity assay using the Suc-LLVY-AMC fluorogenic peptide reflecting chymotrypsin-like activity. Lower panel: the LLVY-AMC fluorescent signals as well as the α6 and RPT6 immunoreactive bands in 30S and/or 26S proteasome complexes were quantified by densitometry and presented as fold change values in patients S6, S11, S12, S21, S32 and S33 versus the control group for each gel whose densitometry measurements were set to 1 (grid line). Columns indicate foldchange mean values ± SD of the patient group (n=6) for LLVY-AMC, α6 and RPT6 in 30S and/or 26S complexes, as indicated. Statistical analysis was performed using the Mann-Whitney U test where * indicates p<0.05.

Article Snippet: Antibodies used in this study were directed against HA (BioLegend, clone HA.11), PSMC5/Rpt6 (Enzo Life Sciences, clone p45-110), α-tubulin (Abcam, clone DM1A), α6 (Enzo Life Sciences, clone MCP20), ubiquitin K48-linked ubiquitin chains (Cell Signaling, clone D9D5), GAPDH (Cell Signaling, clone 14C10), GRP94 (Invitrogen, PA5-27860), ATF6 (Cell Signaling, clone D4Z8V), BNIP3L (Cell Signaling, 12396).

Techniques: Expressing, Activity Assay, Isolation, SDS Page, Western Blot, Staining, Molecular Weight, MANN-WHITNEY, Protein Extraction, Clear Native PAGE

Journal: medRxiv

Article Title: Unveiling the crucial neuronal role of the proteasomal ATPase subunit gene PSMC5 in neurodevelopmental proteasomopathies

doi: 10.1101/2024.01.13.24301174

Figure Lengend Snippet: T cells from NDD subjects with PSMC5 heterozygous variants are characterized by disrupted protein homeostasis, increased mitophagy rates and a specific lipid signature. A . T cells expanded from PBMC isolated from NDD subjects S6/11/12/21/32 were stained with 1 µM of the PROTEOSTAT® dye prior to flow cytometry analysis using the B3 (PerCP-Vio 700) channel. PROTEOSTAT®, a dye that specifically intercalates into the cross-beta spine of quaternary protein structures typically found in misfolded and aggregated proteins. All T cells with PSMC5 variants exhibited increased aggresome formation, as evidenced by increased PROTEOSTAT® fluorescence intensity detected using flow cytometry. Shown are one representative histogram overlay plot of the five T cell NDD subject samples over a control one (left panel), the individual mean fluorescence intensity (MFI) values of the patient ( PSMC5 lesions, n=5) and control (n=5) groups (middle panel) (* p <0.05, Mann-Whitney U test) and the foldchange of the. aggresome propensity factor (APF) for each NDD subject sample compared to the mean value of the five controls (right panel). B . T cells from NDD subjects S1/6/11/12/21/32, relatives (subject’s father, mother and/or brother) and unrelated controls (healthy donors) were subjected to RIPA-mediated protein extraction and subsequent SDS-PAGE/western-blotting using antibodies directed against ubiquitin, β-actin (loading control) and GAPDH (loading control), as indicated. Right panel: the ubiquitin, β-actin immunoreactive bands were quantified by densitometry and data are presented as ubiquitin/loading control mean values ±SD for both control (n=6) and patient (PSMC5 lesion, n=6) groups, as indicated. Statistical analysis was performed using the Mann-Whitney test with ** indicating p<0.01. C. In an attempt to deepen our understanding of the involvement of proteasomes in the regulation of lipid metabolism, we undertook an untargeted lipidomic analysis of T cells from seven unrelated PSMC5 Subjects (S1, S6, S11, S12, S19, S21 and S33) together with their six relative controls (probands’ brother, father and/or mother). Principal component analysis (PCA) plots were first generated to visualize lipid distribution and identify specific patterns across control and patient samples with positive (left panel) and negative (right panel) ion modes. The control and patient groups are presented in red and blue, respectively. D . Fold change analysis showing the lipid classes undergoing significant changes in T cells derived from NDD patients when compared to those from healthy donors. E . T cells expanded from PBMC isolated from the six NDD subjects S1/6/11/12/21/33 as well as from related and unrelated healthy donors (n=7) were incubated overnight with 100 nM of the Mtphagy dye prior to flow cytometry analysis using the B4 (PE-Vio 770) channel. Shown are the percentage values of the patient (PSMC5 lesions) and control groups (** p <0.01, Mann-Whitney U test).

Article Snippet: Antibodies used in this study were directed against HA (BioLegend, clone HA.11), PSMC5/Rpt6 (Enzo Life Sciences, clone p45-110), α-tubulin (Abcam, clone DM1A), α6 (Enzo Life Sciences, clone MCP20), ubiquitin K48-linked ubiquitin chains (Cell Signaling, clone D9D5), GAPDH (Cell Signaling, clone 14C10), GRP94 (Invitrogen, PA5-27860), ATF6 (Cell Signaling, clone D4Z8V), BNIP3L (Cell Signaling, 12396).

Techniques: Isolation, Staining, Flow Cytometry, Fluorescence, MANN-WHITNEY, Protein Extraction, SDS Page, Western Blot, Generated, Derivative Assay, Incubation

Journal: medRxiv

Article Title: Unveiling the crucial neuronal role of the proteasomal ATPase subunit gene PSMC5 in neurodevelopmental proteasomopathies

doi: 10.1101/2024.01.13.24301174

Figure Lengend Snippet: Gene expression analysis reveals a specific gene signature and a spontaneous type I IFN response in T cells from NDD subjects with PSMC5 heterozygous variants. A. The heat map indicates the fold-change of expression of immune-related genes between T cell samples from five subjects with NDD (S1/11/12/21/32) and five heathy individuals (probands’ fathers and unrelated donors). Our profiling was based on a panel of 700 immune system genes, using the previously described NanoString nCounter® technology . Differentially expressed genes are hierarchically clustered. Upregulated and downregulated genes are represented in red and green, respectively. Forty genes were differentially expressed, almost all upregulated. The vast majority of them belonged to the large family of type I interferon (IFN)-stimulated genes (ISGs), including in particular the signal transducers STAT1 and STAT2 and members of the ISG15 conjugation machinery (e.g. UBE2L6 and HERC5). D . A comparative expression analysis of seven ISG ( IFIT1 , IFI27 , IFI44 , IFI44L , ISG15 , MX1 and RSAD2 ) was done by quantitative real-time PCR (see Materials & Methods), as previously described between T cells from PSMC5 subjects S1/11/12/21/32/33, unrelated healthy donors (HD) and subjects’ parents. Type I IFN score was determined for each sample by calculating the median of the normalized fold-change values of the seven ISG relative to one control calibrator. Shown are the IFN scores of each group of donor and parental controls (n=8; left panel) and affected individuals (n=6; right panel). (*p<0.05, Mann-Whitney U test). E . We evaluated the potential contribution of PKR to the initiation of type I IFN responses in NDD patients with PSMC5 variants. To this end, T cell from subjects S1/11/12/21/32/33 were exposed to PKR specific inhibitor C16 for 12 h prior to quantification of ISG transcripts by qPCR. T cells expanded from PBMC isolated from NDD subjects were subjected were subjected to an 8-hr treatment with DMSO (vehicle) or the PKR specific inhibitor C16 (3 µM), 4µ8C (100 µM), H-151 (2 µM), baricitinib (1 µM) or A92 (10 µM). Then RNAs were extracted and submitted to RT-qPCR for ISG gene expression analysis. Shown are the median values of the IFN scores calculated for each T cell patient sample (n=6) under different treatment conditions (* p <0.05, Mann-Whitney U test).

Article Snippet: Antibodies used in this study were directed against HA (BioLegend, clone HA.11), PSMC5/Rpt6 (Enzo Life Sciences, clone p45-110), α-tubulin (Abcam, clone DM1A), α6 (Enzo Life Sciences, clone MCP20), ubiquitin K48-linked ubiquitin chains (Cell Signaling, clone D9D5), GAPDH (Cell Signaling, clone 14C10), GRP94 (Invitrogen, PA5-27860), ATF6 (Cell Signaling, clone D4Z8V), BNIP3L (Cell Signaling, 12396).

Techniques: Expressing, Conjugation Assay, Real-time Polymerase Chain Reaction, MANN-WHITNEY, Isolation, Quantitative RT-PCR

Journal: medRxiv

Article Title: Unveiling the crucial neuronal role of the proteasomal ATPase subunit gene PSMC5 in neurodevelopmental proteasomopathies

doi: 10.1101/2024.01.13.24301174

Figure Lengend Snippet: Clinical phenotypes associated with PSMC5 variants. A. To visually represent the phenotypic data from tables S2b-2c, we organized the HPO terms into categories and ranked the top categories based on their prevalence within the cohort. Within each category, circles illustrate the number of phenotypes assessed and documented for each individual. The size of a circle corresponds to the number of phenotypes evaluated for a particular subject. If no phenotypes were assessed, there is no circle present. The color of each circle indicates the proportion of these confirmed phenotypes in a subject, with a spectrum ranging from dark blue (indicating the lowest fraction) to red (indicating the highest fraction). The top categories are arranged in descending order according to the highest average fraction observed among subjects. Notably, the most commonly observed categories among these are nervous system and head and neck anomalies. Intriguingly, there was no apparent correlation between genotype and phenotype, as significant variability in phenotypic expression was observed even among individuals with the same genetic variant. B . Dysmorphic facial features included notably abnormal ear morphology as seen in subjects S3/9/10/13/19/30/32-34 (this feature is present in 19/31 (61%) subjects in the whole cohort), abnormal palpebral fissures (13/35; 37% in the whole cohort) including downslanted ones as in subjects S3/10/13/30, thin upper lip vermilion as in subjects S3/34 (7/35; 20%), abnormal palate as in subject S10 (7/35; 20%), tall or broad forehead as in S3/33/34 (9/35; 26%), epicanthus as in S4/38 (5/35; 14%), and orofacial clefts as in subject S10 (2/35; 6%). C. Facial image analysis using GestaltMatcher. The pairwise rank matrix and hierarchical clustering of 13 PSMC5 subjects. Each column is the result of testing one subject in the column and ranking of the remaining 12 photos in each row. For example, by testing the similarities between S10 and the 7,459 images of affected individuals from GMDB, S31 was ranked 1st and S19 was ranked 7th as being most similar to S10. The red box is the cluster of subjects with at least one match below the rank 50, which indicates this cluster shares a similar facial phenotype.

Article Snippet: After that, the primary antibody (MAP2 (1:1000, Synaptic Systems #18006); vGlut (1:1000, Synaptic Systems #135304); vGAT (1:200, Synaptic System #131003)); PSMC5 (1:400, Sigma-Aldrich)) in BS was applied, and cells were incubated at 4°C overnight.

Techniques: Expressing, Variant Assay

Journal: medRxiv

Article Title: Unveiling the crucial neuronal role of the proteasomal ATPase subunit gene PSMC5 in neurodevelopmental proteasomopathies

doi: 10.1101/2024.01.13.24301174

Figure Lengend Snippet:

Article Snippet: After that, the primary antibody (MAP2 (1:1000, Synaptic Systems #18006); vGlut (1:1000, Synaptic Systems #135304); vGAT (1:200, Synaptic System #131003)); PSMC5 (1:400, Sigma-Aldrich)) in BS was applied, and cells were incubated at 4°C overnight.

Techniques:

Journal: medRxiv

Article Title: Unveiling the crucial neuronal role of the proteasomal ATPase subunit gene PSMC5 in neurodevelopmental proteasomopathies

doi: 10.1101/2024.01.13.24301174

Figure Lengend Snippet: Structural analysis of PSMC5 variants reveals most missense variants are located in the AAA+ ATPase domain of PSMC5/Rpt6, and affect proteasome dynamics or subunit interactions. A . Schematic representation of the PSMC5 (i.e. RPT6) protein on which the 23 alterations identified in patients with NDD are designated. Approximately half of these mutations fall into three distinct hotspot regions within the AAA+ ATPase domain of PSMC5/RPT6, as indicated. Numbers refer to the sums of unrelated NDD subjects in which the highlighted variant has been identified. Missense variants are colored in blue, frameshift variants in red and splice site variants in purple. B . Basing on the structures 6MSK from RCSB Protein Data Bank, we localized the missense variants identified in the AAA+ ATPase subunit PSMC5/Rpt6 within the 26S proteasomal complex. The variants of interest (spheres) are primarily located in the lower ATPase domain. Part of the 26S proteasome was hidden for better visibility; Rpt6 is shown in cartoon representation.

Article Snippet: After that, the primary antibody (MAP2 (1:1000, Synaptic Systems #18006); vGlut (1:1000, Synaptic Systems #135304); vGAT (1:200, Synaptic System #131003)); PSMC5 (1:400, Sigma-Aldrich)) in BS was applied, and cells were incubated at 4°C overnight.

Techniques: Variant Assay

Journal: medRxiv

Article Title: Unveiling the crucial neuronal role of the proteasomal ATPase subunit gene PSMC5 in neurodevelopmental proteasomopathies

doi: 10.1101/2024.01.13.24301174

Figure Lengend Snippet: Suppression of Psmc5 leads to changes in reversal learning in flies and disrupts the balance between excitatory and inhibitory signals in rat hippocampal neurons. A. Pan-neuronal knockdown of Rpt6 in Drosophila did not significantly affect normal olfactory learning (p=0.3059, n=4). B. When faced with the more difficult task of reversal olfactory learning, knocking down Rpt6 pan-neuronally in Drosophila resulted in a significant decrease in performance (p<0.0001, n=4). (C-J) Primary rat hippocampal neurons infected DIV1-DIV14 with scrambled shRNA or PMSC5 knockdown (KD) shRNA delivering lenti-particles that co-express GFP. C. Assessment of PMSC5 fluorescent signal intensity in neurons at DIV14 shows a significant reduction in cells infected (GFP positive) with PMSC5 knockdown (KD) compared to cells infected with scrambled shRNA (n=10, Mann Whitney test, p = 0.0002). D. Exemplary images of neurons infected with scrambled shRNA (left) or PMSC5 shRNA (right) lenti-particles. PMSC5 labeling (arrow) is significantly reduced, confirming the KD of PMSC5. E-F. Infected neurons stained for MAP2 to visualize neuronal morphology. E. No significant difference in dendritic branching measured by the mean number of primary, secondary, and tertiary dendrites per neuron at DIV14 was found comparing scrambled Ctrl and PMSC5 KD conditions (n=6). F. Exemplary images of neurons infected with scrambled shRNA (left) or PMSC5 shRNA (right) lenti-particles. MAP2 labeling is shown in red. G-J. Infected neurons stained for vGlut to visualize excitatory presynaptic compartments or vGat to visualize inhibitory presynapses. G. The number of vGlut positive puncta along the primary dendrites was measured at DIV14. In Psmc5 KD conditions, a significant reduction was observed compared to scrambled Ctrls (n=5, Mann Whitney test, p = 0.0045). H. The number of vGat positive puncta along the primary dendrites was not significantly altered (n=5, Mann Whitney test, p = 0.2988). I-J. Exemplary images of primary dendrites of neurons infected with scrambled shRNA (left) or Psmc5 shRNA (right) lenti- particles. VGlut (full arrow) positive signals per dendrite length are significantly reduced, while VGat (open arrow) positive signals per dendrite length are not altered after Psmc5 KD.

Article Snippet: After that, the primary antibody (MAP2 (1:1000, Synaptic Systems #18006); vGlut (1:1000, Synaptic Systems #135304); vGAT (1:200, Synaptic System #131003)); PSMC5 (1:400, Sigma-Aldrich)) in BS was applied, and cells were incubated at 4°C overnight.

Techniques: Infection, shRNA, MANN-WHITNEY, Labeling, Staining

Journal: medRxiv

Article Title: Unveiling the crucial neuronal role of the proteasomal ATPase subunit gene PSMC5 in neurodevelopmental proteasomopathies

doi: 10.1101/2024.01.13.24301174

Figure Lengend Snippet: Overexpression of PSMC5 affects neuronal morphology. A. Representative images of primary hippocampal neurons transfected with the empty vector control (EV), PSMC5 -WT or the different PSMC5 variants. Red indicates the tdTomato blue indicat MAP2. B. Analysis of the total neurite length in the different conditions reveal: (i) a significant increase induced by overexpression of PSMC5-WT compared to empty vector control (One-way ANOVA, F=3.296, p= 0.0014; empty vector versus PSMC5-WT, p=0.0046, Dunnet’s multiple comparison test); (ii) a similar effect as PSMC5-WT induced by overexpression of variants p.(Arg201Trp), p.(Pro320His) and p.(Arg325Trp) (PSMC5-WT versus p.(Arg201Trp), p=0.8; PSMC5-WT versus p.(Pro320His), p=0.8; PSMC5-WT versus p.(Arg325Trp), p=0.5, Dunnet’s multiple comparison test); (iii) a decrease induced by overexpression of variants p.(Ala202Val), p.(Thr207Met), p.(Pro320Arg) and PSMC5-Δex10, compared to PSMC5-WT (PSMC5-WT versus p.(Ala202Val), p=0.0031; PSMC5-WT versus p.(Thr207Met), p=0.02; PSMC5-WT versus p.(Pro320Arg), p=0.006; PSMC5-WT versus PSMC5-Δex10, p=0.03, Dunnet’s multiple comparison test). The effects of these variants were indistinguishable from the empty vector control (empty vector versus p.(Ala202Val), p=0.4; empty vector versus p.(Thr207Met), p=0.9; empty vector versus p.(Pro320Arg), p=0.9; empty vector versus PSMC5-Δex10, p=0.9, Dunnet’s multiple comparison test). C. Analysis of the arborization showed no differences between conditions compared to the empty vector control (One-way ANOVA, F=2.179, p= 0.03; empty vector versus PSMC5-WT, p=0.9, PSMC5-WT versus p.(Arg201Trp), p=0.9; PSMC5-WT versus p.(Ala202Val), p=0.9; PSMC5- WT versus p.(Thr207Met), p=0.6; PSMC5-WT versus p.(Pro320His), p=0.3; PSMC5-WT versus p.(Pro320Arg), p=0.9; PSMC5-WT versus p.(Arg 325Trp), p=0.9; PSMC5-WT versus PSMC5-Δex10, p=0.9, Dunnet’s multiple comparison test). Error bars indicate SEM, n (number of neurons traced): EV=40, PSMC5-WT=30, p.(Arg201Trp)=18 p.(Ala202Val)=20, p.(Thr207Met)=27, p.(Pro320Arg)=29, p.(Pro320Arg)=19, p.(Arg325Trp)=20 and PSMC5-Δex10=20. *p<0.05; **p<0.01. Scale bar: 100μm.

Article Snippet: After that, the primary antibody (MAP2 (1:1000, Synaptic Systems #18006); vGlut (1:1000, Synaptic Systems #135304); vGAT (1:200, Synaptic System #131003)); PSMC5 (1:400, Sigma-Aldrich)) in BS was applied, and cells were incubated at 4°C overnight.

Techniques: Over Expression, Transfection, Plasmid Preparation, Comparison

Journal: medRxiv

Article Title: Unveiling the crucial neuronal role of the proteasomal ATPase subunit gene PSMC5 in neurodevelopmental proteasomopathies

doi: 10.1101/2024.01.13.24301174

Figure Lengend Snippet: T cells from NDD subjects carrying PSMC5 heterozygous variants exhibit abnormalities in their proteasome expression and/or activity profiles. A . T cells expanded from PBMC isolated from NDD subjects S6/11/12/21/32 as well as related controls (father and/or mother) and heathy donors (noted ‘un. control’ for unrelated controls) were lysed in RIPA buffer prior to SDS-PAGE/western-blotting analysis using antibodies specific PSMC5/RPT6 and GAPDH (loading control). Arrow indicates full-length endogenous PSMC5/RPT6 at the expected size of approximately 45 kDa detected in all samples. PSMC5/Rpt6 staining of T cell lysates from subjects 6/11/21/32 uncovered a further shorter PSMC5/Rpt6 immunoreactive band migrating at ∼30kDa (denoted as a low-molecular mass (LMM) species by the square brackets) following prolonged exposure times, whereas T cells from subject S12 exhibited an extra PSMC5/Rpt6 signal running a little bit lighter than PSMC5/Rpt6 expected size just below 40 kDa (indicated by an asterisk). Lower panel: the RPT6 and GAPDH immunoreactive bands were quantified by densitometry. Data are presented as RPT6 full-length and low molecular weight (LMW) species on GAPDH ratio mean values ± SD for the control (n=9) and patients (PSMC5 lesions, n=5) groups, as indicated. Statistical analysis was performed using the Mann-Whitney U test where *** indicates p <0.001. B . Resting T cells from NDD subjects S6/11/12/21/32/33 as well as related (subject’s father and/or mother, as indicated) and unrelated (healthy donor) control T cells were analyzed for proteasome function and abundance. They were subjected to non- denaturing protein extraction using TSDG buffer prior to native-PAGE/western-blotting analysis using monoclonal antibodies specific for the α6 and PSMC5/RPT6 subunits, as indicated. Proteasome complexes (20S and 26S) were also visualized by in-gel activity assay using the Suc-LLVY-AMC fluorogenic peptide reflecting chymotrypsin-like activity. Lower panel: the LLVY-AMC fluorescent signals as well as the α6 and RPT6 immunoreactive bands in 30S and/or 26S proteasome complexes were quantified by densitometry and presented as fold change values in patients S6, S11, S12, S21, S32 and S33 versus the control group for each gel whose densitometry measurements were set to 1 (grid line). Columns indicate foldchange mean values ± SD of the patient group (n=6) for LLVY-AMC, α6 and RPT6 in 30S and/or 26S complexes, as indicated. Statistical analysis was performed using the Mann-Whitney U test where * indicates p<0.05.

Article Snippet: After that, the primary antibody (MAP2 (1:1000, Synaptic Systems #18006); vGlut (1:1000, Synaptic Systems #135304); vGAT (1:200, Synaptic System #131003)); PSMC5 (1:400, Sigma-Aldrich)) in BS was applied, and cells were incubated at 4°C overnight.

Techniques: Expressing, Activity Assay, Isolation, SDS Page, Western Blot, Staining, Molecular Weight, MANN-WHITNEY, Protein Extraction, Clear Native PAGE

Journal: medRxiv

Article Title: Unveiling the crucial neuronal role of the proteasomal ATPase subunit gene PSMC5 in neurodevelopmental proteasomopathies

doi: 10.1101/2024.01.13.24301174

Figure Lengend Snippet: T cells from NDD subjects with PSMC5 heterozygous variants are characterized by disrupted protein homeostasis, increased mitophagy rates and a specific lipid signature. A . T cells expanded from PBMC isolated from NDD subjects S6/11/12/21/32 were stained with 1 µM of the PROTEOSTAT® dye prior to flow cytometry analysis using the B3 (PerCP-Vio 700) channel. PROTEOSTAT®, a dye that specifically intercalates into the cross-beta spine of quaternary protein structures typically found in misfolded and aggregated proteins. All T cells with PSMC5 variants exhibited increased aggresome formation, as evidenced by increased PROTEOSTAT® fluorescence intensity detected using flow cytometry. Shown are one representative histogram overlay plot of the five T cell NDD subject samples over a control one (left panel), the individual mean fluorescence intensity (MFI) values of the patient ( PSMC5 lesions, n=5) and control (n=5) groups (middle panel) (* p <0.05, Mann-Whitney U test) and the foldchange of the. aggresome propensity factor (APF) for each NDD subject sample compared to the mean value of the five controls (right panel). B . T cells from NDD subjects S1/6/11/12/21/32, relatives (subject’s father, mother and/or brother) and unrelated controls (healthy donors) were subjected to RIPA-mediated protein extraction and subsequent SDS-PAGE/western-blotting using antibodies directed against ubiquitin, β-actin (loading control) and GAPDH (loading control), as indicated. Right panel: the ubiquitin, β-actin immunoreactive bands were quantified by densitometry and data are presented as ubiquitin/loading control mean values ±SD for both control (n=6) and patient (PSMC5 lesion, n=6) groups, as indicated. Statistical analysis was performed using the Mann-Whitney test with ** indicating p<0.01. C. In an attempt to deepen our understanding of the involvement of proteasomes in the regulation of lipid metabolism, we undertook an untargeted lipidomic analysis of T cells from seven unrelated PSMC5 Subjects (S1, S6, S11, S12, S19, S21 and S33) together with their six relative controls (probands’ brother, father and/or mother). Principal component analysis (PCA) plots were first generated to visualize lipid distribution and identify specific patterns across control and patient samples with positive (left panel) and negative (right panel) ion modes. The control and patient groups are presented in red and blue, respectively. D . Fold change analysis showing the lipid classes undergoing significant changes in T cells derived from NDD patients when compared to those from healthy donors. E . T cells expanded from PBMC isolated from the six NDD subjects S1/6/11/12/21/33 as well as from related and unrelated healthy donors (n=7) were incubated overnight with 100 nM of the Mtphagy dye prior to flow cytometry analysis using the B4 (PE-Vio 770) channel. Shown are the percentage values of the patient (PSMC5 lesions) and control groups (** p <0.01, Mann-Whitney U test).

Article Snippet: After that, the primary antibody (MAP2 (1:1000, Synaptic Systems #18006); vGlut (1:1000, Synaptic Systems #135304); vGAT (1:200, Synaptic System #131003)); PSMC5 (1:400, Sigma-Aldrich)) in BS was applied, and cells were incubated at 4°C overnight.

Techniques: Isolation, Staining, Flow Cytometry, Fluorescence, MANN-WHITNEY, Protein Extraction, SDS Page, Western Blot, Generated, Derivative Assay, Incubation

Journal: medRxiv

Article Title: Unveiling the crucial neuronal role of the proteasomal ATPase subunit gene PSMC5 in neurodevelopmental proteasomopathies

doi: 10.1101/2024.01.13.24301174

Figure Lengend Snippet: Gene expression analysis reveals a specific gene signature and a spontaneous type I IFN response in T cells from NDD subjects with PSMC5 heterozygous variants. A. The heat map indicates the fold-change of expression of immune-related genes between T cell samples from five subjects with NDD (S1/11/12/21/32) and five heathy individuals (probands’ fathers and unrelated donors). Our profiling was based on a panel of 700 immune system genes, using the previously described NanoString nCounter® technology . Differentially expressed genes are hierarchically clustered. Upregulated and downregulated genes are represented in red and green, respectively. Forty genes were differentially expressed, almost all upregulated. The vast majority of them belonged to the large family of type I interferon (IFN)-stimulated genes (ISGs), including in particular the signal transducers STAT1 and STAT2 and members of the ISG15 conjugation machinery (e.g. UBE2L6 and HERC5). D . A comparative expression analysis of seven ISG ( IFIT1 , IFI27 , IFI44 , IFI44L , ISG15 , MX1 and RSAD2 ) was done by quantitative real-time PCR (see Materials & Methods), as previously described between T cells from PSMC5 subjects S1/11/12/21/32/33, unrelated healthy donors (HD) and subjects’ parents. Type I IFN score was determined for each sample by calculating the median of the normalized fold-change values of the seven ISG relative to one control calibrator. Shown are the IFN scores of each group of donor and parental controls (n=8; left panel) and affected individuals (n=6; right panel). (*p<0.05, Mann-Whitney U test). E . We evaluated the potential contribution of PKR to the initiation of type I IFN responses in NDD patients with PSMC5 variants. To this end, T cell from subjects S1/11/12/21/32/33 were exposed to PKR specific inhibitor C16 for 12 h prior to quantification of ISG transcripts by qPCR. T cells expanded from PBMC isolated from NDD subjects were subjected were subjected to an 8-hr treatment with DMSO (vehicle) or the PKR specific inhibitor C16 (3 µM), 4µ8C (100 µM), H-151 (2 µM), baricitinib (1 µM) or A92 (10 µM). Then RNAs were extracted and submitted to RT-qPCR for ISG gene expression analysis. Shown are the median values of the IFN scores calculated for each T cell patient sample (n=6) under different treatment conditions (* p <0.05, Mann-Whitney U test).

Article Snippet: After that, the primary antibody (MAP2 (1:1000, Synaptic Systems #18006); vGlut (1:1000, Synaptic Systems #135304); vGAT (1:200, Synaptic System #131003)); PSMC5 (1:400, Sigma-Aldrich)) in BS was applied, and cells were incubated at 4°C overnight.

Techniques: Expressing, Conjugation Assay, Real-time Polymerase Chain Reaction, MANN-WHITNEY, Isolation, Quantitative RT-PCR