Journal: Disease Markers

Article Title: HSPA5 Inhibitor Meliorate DSS-Induced Colitis through HSPA1A/CHIP

doi: 10.1155/2022/7115181

Figure Lengend Snippet: Primer sequences used for RT-PCR.

Article Snippet: The HSPA5 inhibitor HA15 (batch no. 1609402-14-3) was purchased from the MedChemExpress (New Jersey, USA).

Techniques:

Journal: Disease Markers

Article Title: HSPA5 Inhibitor Meliorate DSS-Induced Colitis through HSPA1A/CHIP

doi: 10.1155/2022/7115181

Figure Lengend Snippet: Expressions of HSPA5 in colonic tissue and the effects of pharmacotherapies on DSS-induced colitis in mice. (a) The colonic protein levels of HSPA5 measured by Western blotting. (b) Representative protein levels of HSPA5/GAPDH. (c) Levels of HSPA5 mRNA in colonic tissue. Data are shown as means ± SD ( n = 3). (d) The disease activity index (DAI) score of each group was monitored daily. (e) Typical colonic appearance of each group. (f) Colon length of each group. (g) Histological analysis (×50, ×200) about pathological changes of colonic tissue under a microscope. (h) Histological score of each group. Data are shown as means ± SD ( n = 8). ∗∗ P < 0.01, ∗∗∗ P < 0.001, vs. DSS group.

Article Snippet: The HSPA5 inhibitor HA15 (batch no. 1609402-14-3) was purchased from the MedChemExpress (New Jersey, USA).

Techniques: Western Blot, Activity Assay, Microscopy

Journal: Autophagy

Article Title: HSPA5 negatively regulates lysosomal activity through ubiquitination of MUL1 in head and neck cancer

doi: 10.1080/15548627.2017.1414126

Figure Lengend Snippet: NTS-induced inhibition of HSPA5 expression and its pivotal role in ER stress or autophagy. (A) FaDu cells were treated with NTS for the indicated times and protein levels were evaluated by western blot assay. (B) Inhibition of NTS-induced ER stress prevents autophagy. GFP-MAP1LC3-II plasmids were transfected into FaDu cells and 24 h later, the cells were pretreated with TUDCA (1 mg/ml) for 1 h. NTS treatment was given for 24 h with or without TUDCA in absence of serum. GFP-MAP1LC3-II puncta were analyzed with a fluorescence microscope (scale bar: 50 μm). (C) HSPA5 was decreased in response to NTS. FaDu cells were treated with NTS for 24 h in the absence of serum, and HSPA5 expression was determined by western blot assay (n = 3). (D) HSPA5 overexpression in HNC tissues. Proteins were isolated from frozen tissues of 6 patients with HNC, and HSPA5 expression level was determined by western blot assay (n = 6; C, cancer tissue; N, normal tissue; P, patient). (E) The immunohistochemistry analysis of HSPA5 in cancer or normal (scale bar: 200 μm). (F and G) HSPA5 overexpression inhibited NTS-induced ER stress, autophagy and cytotoxicity. HSPA5 plasmids were transfected into FaDu cells, and the cells were treated with NTS for 24 h in the absence of serum. Protein levels were evaluated by western blot assay (F) and MTT assay (G; n = 6). (C, D and G) Data are means ± SD. Asterisks indicate statistically significant differences (P < 0.05).

Article Snippet: The following plasmids were obtained from Addgene: pcDNA3.1(+)-HSPA5 (32701, deposited by Richard C. Austin), pRK5-HA-Ubiquitin WT (17608), KO (17603), K33 (17607), K48 (17605), K63 (17606, deposited by Ted Dawson), K6 (22900), K11 (22901), K27 (22902) and K29 (22903, deposited by Sandra Weller). pSELECT-GFP- Hs MAP1LC3-II (psetz-gfplc3) was purchased from InvivoGen.

Techniques: Inhibition, Expressing, Western Blot, Transfection, Fluorescence, Microscopy, Over Expression, Isolation, Immunohistochemistry, MTT Assay

Journal: Autophagy

Article Title: HSPA5 negatively regulates lysosomal activity through ubiquitination of MUL1 in head and neck cancer

doi: 10.1080/15548627.2017.1414126

Figure Lengend Snippet: HSPA5 has a pivotal role in NTS-mediated regulation of lysosomal activity. (A) FaDu cells were seeded in a 48-well plate and treated with NTS for the indicated times. Cells viability was measured by the MTT assay (n = 6). Data are means ± SD. Asterisks indicate statistically significant differences (P < 0.05). (B) FaDu cells were treated with or without NTS for 24 h in the absence of serum and lysosomal activity was measured by FACS analysis (n = 3). Data are means ± SD. Asterisks indicate statistically significant differences (P < 0.05) (*, vs O H). (C) GFP-MAP1LC3-II puncta was accumulated by inhibiting lysosomes. NH4Cl (10 mM), E-64 (10 μM), and NTS were added to GFP-MAP1LC3-II-transfected cells for 24 h in the absence of serum. GFP-MAP1LC3-II puncta were observed by fluorescence microscopy. Scale bar: 20 μm. (D) HSPA5 overexpression inhibits NTS-induced downregulation of lysosome-related proteins or gene expression. FaDu cells were transfected with or without pcDNA3.1-HSPA5 for 24 h, and then cells were treated with NTS for the indicated times. The indicated proteins or gene expression were determined by western blot assay or RT-PCR. (E to G) HSPA5 overexpression inhibits NTS-induced downregulation of lysosomal activity. HSPA5-transfected or nontransfected cells were treated with NTS for 24 h and then lysosomal activity was assessed with FACS analysis (E), TFEB transcription factor activity assay (F) or CTSD (cathepsin D) activity assay (G). (E, F and G) Data are means ± SD. Asterisks indicate statistically significant differences (n = 3, P < 0.05).

Article Snippet: The following plasmids were obtained from Addgene: pcDNA3.1(+)-HSPA5 (32701, deposited by Richard C. Austin), pRK5-HA-Ubiquitin WT (17608), KO (17603), K33 (17607), K48 (17605), K63 (17606, deposited by Ted Dawson), K6 (22900), K11 (22901), K27 (22902) and K29 (22903, deposited by Sandra Weller). pSELECT-GFP- Hs MAP1LC3-II (psetz-gfplc3) was purchased from InvivoGen.

Techniques: Activity Assay, MTT Assay, Transfection, Fluorescence, Microscopy, Over Expression, Expressing, Western Blot, Reverse Transcription Polymerase Chain Reaction

Journal: Autophagy

Article Title: HSPA5 negatively regulates lysosomal activity through ubiquitination of MUL1 in head and neck cancer

doi: 10.1080/15548627.2017.1414126

Figure Lengend Snippet: HSPA5 inhibits NTS-induced apoptosis through intracellular ROS control. (A) NTS-induced cytotoxicity was prevented by the caspase protease inhibitor, Z-VAD-FMK. FaDu cells were pretreated with Z-VAD-FMK (10 μM) for 1 h and then the NTS treatment was given for a further 24 h. Cell viability was assessed by MTT assay. Data are means ± SD. Asterisks indicate statistically significant differences (n = 6, P < 0.05). (B and C) HSPA5-transfected cells exhibited inhibition of NTS-induced mitochondrial apoptosis signaling and the alleviation of MMP alteration. FaDu cells were transfected with or without pcDNA3.1-HSPA5 for 24 h and then NTS treatment was given for 24 h. The indicated protein levels were determined by western blot (B) and MMP alteration was evaluated by JC-1 assay (C, healthy, red; apoptotic, green). JC-1-stained live cells were observed by fluorescence microscopy in 5 fields captured randomly and red/green fluorescent density ratio was quantified (n = 3; scale bar: 20 μm). Data are means ± SD. Asterisks indicate statistically significant differences (P < 0.05). (D) The ROS increment induced by NTS was inhibited by HSPA5 overexpression. NTS treatment was given to pcDNA3.1-HSPA5-transfected or nontransfected cells for 24 h and then intracellular ROS levels were determined by FACS analysis (n = 3). Data are means ± SD. Asterisks indicate statistically significant differences (P < 0.05). (E and F) NTS induced translocation of HSPA5 to mitochondria. (E) FaDu cells were seeded on cover-slips and then NTS treatment was given for 8 h in the absence of serum. After cell fixing, mitochondria (green) or endogenous HSPA5 (red) were stained with anti-TOMM20 or anti-HSPA5 antibodies. Green and red signal intensity was measured by confocal microscopy (allow). (F) NTS treatment was given to FaDu cells for the indicated times. After cellular fractionation, HSPA5 levels were assessed by western blot (Whole: whole cells lysate; cyto, cytosolic fraction; mito, mitochondria fraction).

Article Snippet: The following plasmids were obtained from Addgene: pcDNA3.1(+)-HSPA5 (32701, deposited by Richard C. Austin), pRK5-HA-Ubiquitin WT (17608), KO (17603), K33 (17607), K48 (17605), K63 (17606, deposited by Ted Dawson), K6 (22900), K11 (22901), K27 (22902) and K29 (22903, deposited by Sandra Weller). pSELECT-GFP- Hs MAP1LC3-II (psetz-gfplc3) was purchased from InvivoGen.

Techniques: Protease Inhibitor, MTT Assay, Transfection, Inhibition, Western Blot, Staining, Fluorescence, Microscopy, Over Expression, Translocation Assay, Confocal Microscopy, Cell Fractionation

Journal: Autophagy

Article Title: HSPA5 negatively regulates lysosomal activity through ubiquitination of MUL1 in head and neck cancer

doi: 10.1080/15548627.2017.1414126

Figure Lengend Snippet: MUL1 negatively regulates HSPA5. (A) NTS induced ubiquitination of HSPA5. NTS treatment was given to FaDu cells for 24 h, in the absence of serum and MG132 (10 μM) was included for 8 h before cell harvest. Endogenous HSPA5 was immunoprecipitated by an anti-HSPA5 antibody and the determination of ubiquitin-conjugated HSPA5 was performed with an anti-ubiquitin antibody. (B) NTS induced UPS of HSPA5. FaDu cells were treated with NTS for each indicated time. A pretreatment of MG132 (10 μM) was given for 1 h before the NTS treatment. Endogenous HSPA5 or MUL1 was evaluated by western blot assay. (C and D) MUL1 knockdown inhibited NTS-mediated HNC cellular events or HSPA5 ubiquitination. FaDu cells were transfected with scrambled RNAs or MUL1 siRNA and 24 h later, NTS treatment was given for 24 h in the absence of serum. The cells were subjected to western blot assay with the indicated antibodies (C) or Ni-NTA His affinity-isolation ubiquitination assay (D). (E) MUL1 knockdown prevented NTS-induced lysosome inhibition. NTS treatment was given for 24 h to scrambled RNA- or MUL1 siRNA-transfected cells. Lysosomal activity was assessed with FACS analysis (n = 3). Data are means ± SD. Asterisks indicate statistically significant differences (P < 0.05). (F) MUL1 was suppressed in HNC regions. MUL1 expression in HNC patients was analyzed by immunohistochemistry using anti-MUL1 antibody. (G) HSPA5 or MUL1 expression levels were different between tissues from cancer patients or healthy controls. Proteins were isolated from tissues of 14 patients with HNC, and HSPA5 or MUL1 expression levels were determined by western blot (C, cancer tissue; N, normal tissue; P, patient). Data are means ± SD. Asterisks indicate statistically significant differences (P < 0.05).

Article Snippet: The following plasmids were obtained from Addgene: pcDNA3.1(+)-HSPA5 (32701, deposited by Richard C. Austin), pRK5-HA-Ubiquitin WT (17608), KO (17603), K33 (17607), K48 (17605), K63 (17606, deposited by Ted Dawson), K6 (22900), K11 (22901), K27 (22902) and K29 (22903, deposited by Sandra Weller). pSELECT-GFP- Hs MAP1LC3-II (psetz-gfplc3) was purchased from InvivoGen.

Techniques: Immunoprecipitation, Western Blot, Transfection, Isolation, Ubiquitin Assay, Inhibition, Activity Assay, Expressing, Immunohistochemistry

Journal: Autophagy

Article Title: HSPA5 negatively regulates lysosomal activity through ubiquitination of MUL1 in head and neck cancer

doi: 10.1080/15548627.2017.1414126

Figure Lengend Snippet: Lysine 446 of HSPA5 is a site of MUL1-mediated K48-linked ubiquitination. (A) HSPA5 interacted with MUL1. At 24 h after cotransfection with the indicated plasmids, FaDu cells were treated with MG132 (10 μM) for 8 h before cell harvest and then subjected to immunoprecipitation with an anti-GFP antibody, followed by western blot with the indicated antibodies. (B) MUL1 induced K48-linked ubiquitination of HSPA5. FaDu cells were cotransfected with plasmids as indicated. At 24 h after cotransfection, cells were treated with MG132 (10 μM) for 8 h and further subjected to ubiquitination assay. (C) K446 is a putative ubiquitination site for MUL1. Each indicated plasmid was transfected into FaDu cells together with or without EGFP-MUL1 and 24 h later, cells were treated with MG132 (10 μM) for 8 h and further subjected to Ni-NTA His affinity-isolation ubiquitination assay. (D) The effect of MUL1 on HSPA5 protein stability was inhibited in the HSPA5K446R mutant, compared to wild-type HSPA5. K446R mutant was inhibited MUL1-dependent protein stability compared with wild type of HSPA5. WT HSPA5-MYC-His or HSPA5K446R-MYC-His plasmids were transfected into FaDu cells together with or without EGFP-MUL1 dose dependently and 24 h later, the level of each indicated protein was determined by western blot. (E) The half-life of the K446R mutant protein was compared with the wild type of HSPA5. WT HSPA5-MYC-His or HSPA5K446R-MYC-His plasmids were transfected into BEAS-2B cells and 24 h later, treatment with CHX (20 μg/ml) was included for each indicated time. Endogenous MUL1 or exogenous HSPA5 were detected using anti-MUL1 or anti-MYC antibodies. (F) K446R did not induce ubiquitination from the NTS. Cells transfected with each indicated plasmid, were treated with NTS for 24 h and the ubiquitinated form of HSPA5 was detected by Ni-NTA His affinity isolation assay.

Article Snippet: The following plasmids were obtained from Addgene: pcDNA3.1(+)-HSPA5 (32701, deposited by Richard C. Austin), pRK5-HA-Ubiquitin WT (17608), KO (17603), K33 (17607), K48 (17605), K63 (17606, deposited by Ted Dawson), K6 (22900), K11 (22901), K27 (22902) and K29 (22903, deposited by Sandra Weller). pSELECT-GFP- Hs MAP1LC3-II (psetz-gfplc3) was purchased from InvivoGen.

Techniques: Cotransfection, Immunoprecipitation, Western Blot, Ubiquitin Assay, Plasmid Preparation, Transfection, Isolation, Mutagenesis

Journal: Autophagy

Article Title: HSPA5 negatively regulates lysosomal activity through ubiquitination of MUL1 in head and neck cancer

doi: 10.1080/15548627.2017.1414126

Figure Lengend Snippet: MUL1 KO HNC cells show resistance to NTS treatment in a xenograft in vivo model. Human FaDu (5 × 106 cells) HNC cells were injected subcutaneously into BALB/c nu/nu mice and the mice were treated with NTS every day for 14 d (PBS, n = 5; NTS, n = 8). Tumor volume and images are shown in Figure S12A. (A) Immunohistochemical analysis of HSPA5, p-AKT, MAP1LC3-II, TFEB, LAMP1, and cleaved CASP3 (scale bar: 100 μm). Protein levels were analyzed and quantified by western blot assay in Figures S12B. (B) NTS induced increases in level of the interaction between MUL1 and HSPA5 in NTS-treated mice tumors. The interaction of MUL1 and HSPA5 induced by NTS was assessed by PLA in mice tissues. Arrows indicate PLA-positive (+) cells (scale bar: 60 μm). Numbers of PLA (+) cells were counted at x200 magnification in 5 randomly chosen fields. Data are means ± SD. Asterisks indicate statistically significant differences (P < 0.05). The distribution of PLA (+) cells in whole tumor (Figure S13A) or immunostaining between HSPA5 and MUL1 (Figure S13B) is represented in Figure S13. (C) TEM of NTS-treated tumor. Arrows indicate autophagic vesicles (scale bar: 2,000 nm). (D) MUL1 knockout (KO) HNC cells showed resistance to NTS treatment. MUL1 KO cells were generated by the CRISPR/Cas9 system (Figure S14A to S14E). FaDu cells with wild-type MUL1 (WT) or MUL1 KO (each 5 × 106 cells) were injected subcutaneously into BALB/c nu/nu mice and the mice were treated with NTS every day for 15 d. Data are means ± SD. Asterisks indicate statistically significant differences (P < 0.05). (E) Immunohistochemical analysis of MUL1 or HSPA5 in tumors (scale bar: 100 μm). MUL1- or HSPA5-positive stain intensity was measured and quantified by ImageJ program. Data are means ± SD. Asterisks indicate statistically significant differences (P < 0.05).

Article Snippet: The following plasmids were obtained from Addgene: pcDNA3.1(+)-HSPA5 (32701, deposited by Richard C. Austin), pRK5-HA-Ubiquitin WT (17608), KO (17603), K33 (17607), K48 (17605), K63 (17606, deposited by Ted Dawson), K6 (22900), K11 (22901), K27 (22902) and K29 (22903, deposited by Sandra Weller). pSELECT-GFP- Hs MAP1LC3-II (psetz-gfplc3) was purchased from InvivoGen.

Techniques: In Vivo, Injection, Immunohistochemical staining, Western Blot, Immunostaining, Knock-Out, Generated, CRISPR, Staining

Journal: Autophagy

Article Title: HSPA5 negatively regulates lysosomal activity through ubiquitination of MUL1 in head and neck cancer

doi: 10.1080/15548627.2017.1414126

Figure Lengend Snippet: Schema of the MUL1-HSPA5 axis in HNC. HSPA5 shows a protective mechanism through control of lysosomal activity; however, NTS-mediated MUL1 induces HSPA5 ubiquitination at the K446 residue and leads to alteration in the signal from survival to apoptosis, through lysosome inhibition. NAC, N-acetylcysteine.

Article Snippet: The following plasmids were obtained from Addgene: pcDNA3.1(+)-HSPA5 (32701, deposited by Richard C. Austin), pRK5-HA-Ubiquitin WT (17608), KO (17603), K33 (17607), K48 (17605), K63 (17606, deposited by Ted Dawson), K6 (22900), K11 (22901), K27 (22902) and K29 (22903, deposited by Sandra Weller). pSELECT-GFP- Hs MAP1LC3-II (psetz-gfplc3) was purchased from InvivoGen.

Techniques: Activity Assay, Inhibition

Journal: Autophagy

Article Title: Crosstalk between HSPA5 arginylation and sequential ubiquitination leads to AKT degradation through autophagy flux

doi: 10.1080/15548627.2020.1740529

Figure Lengend Snippet: R-HSPA5 functions as a delivery determinant for AKT and SQSTM1 to autophagosomes. (a) Cycloheximide degradation assay of R-HSPA5, p-AKT, and AKT in atg5 KO mouse embryonic fibroblasts (MEFs) in the absence or presence of PI (bortezomib, 10 nM). Cells were treated with 50 μg/ml cycloheximide for different periods of time. Protein expression was assessed by western blot analysis. (b) R-HSPA5 is associated with the lysosomal degradation of AKT. JJN3 cells were treated with PI (bortezomib, 10 nM) for times indicated. Cells were pretreated with chloroquine (50 μM) for 1 h before PI treatment. Protein levels were evaluated by western blot analysis. (c) AKT1 interacts with R-HSPA5. JJN3 cells were transfected with AKT1-MYC-His. After 24 h post-transfection, cells treated with bafilomycin (100 nM) for 1 h and then, PI (bortezomib, 10 nM), and thapsigargin (200 nM) incubation for an additional 12 h. AKT1 and R-HSPA5 binding was evaluated by Ni-NTA His pull-down assay. (d) Scrambled or ATE1-knockdown JJN3 cells were treated with PI (bortezomib, 10 nM), followed by western blot. (e) FaDu cells were transfected with scrambled or ATE1-specific siRNA, followed by treatment for 12 h with PI (bortezomib, 40 nM). Colocalization analysis between R-HSPA5 (green) and AKT (red). Yellow = merge/colocalization. Larger white boxes in the images are enlargements of the area indicated by the smaller white boxes; scale bars: 10 μm. (f) Quantification of the colocalization of AKT and R-HSPA5 puncta. Merged images from Figure 3(e) were analyzed for AKT:R-HSPA5 colocalization using NIS Elements software and Pearson’s correlation coefficient. Data represent the mean ± SD of 10 randomly selected cells per condition; *P < 0.05, n.s. (not significant). (g) JJN3 cells were treated with PI (bortezomib, 10 nM) or co-treated with NAC (10 mM), followed by western blot. (h) Colocalization of cytoplasmic R-HSPA5 puncta with SQSTM1 in PI (bortezomib, 40 nM)-treated FaDu cells. (i) Colocalization of R-HSPA5 puncta with AKT in FaDu cells as determined by immunofluorescence, using anti-HSPA5 (green) and anti-AKT (red) antibodies. Yellow = merge/colocalization. Larger white boxes in the images are enlargements of the area indicated by the smaller white boxes; scale bars: 10 μm

Article Snippet: The primary antibodies used included those against p-AKT (Cell Signaling Technology, S473, 9271), p-AKT (Cell Signaling Technology, T308, 13,038), AKT (Cell Signaling Technology, 9272), PARP1 (Cell signaling Technology, 9532), cleaved CAPS3 (Cell Signaling Technology, 9664), GAPDH (Cell Signaling Technology, 5174), Myc (Cell Signaling Technology, 2276 and 2272), MAP1LC3A (Cell Signaling Technology, 4599), MAP1LC3B (Cell Signaling Technology, 3868), Ub (Cell Signaling Technology, 3936), HA (Cell Signaling Technology, 3724), His (Cell Signaling Technology, 12,698), HSPA5 (Cell Signaling Technology, 3177), ATG5 (Cell Signaling Technology, 12,994), ATG12 (Cell Signaling Technology, 2010), ATG16L1 (Cell Signaling Technology, 8089), BECN1 (Cell Signaling Technology, 3495), p-GSK3B (Cell Signaling Technology, S9, 9323), GSK3B (Cell Signaling Technology, 12,456), USP7 (Cell Signaling Technology, 4833), BAX (Cell Signaling Technology, 2772), Total-BID (Cell Signaling Technology, 2002), p-MTOR (Cell Signaling Technology, S2448, 5536), MTOR (Cell Signaling Technology, 2983), p-RPS6KB1 (Cell Signaling Technology, T389, 9234), RPS6KB1 (Cell Signaling Technology, 2708), p-RPS6 (Cell Signaling Technology, S240/244, 5364), p-RPS6 (Cell Signaling Technology, S235/236, 4858), LAMP1 (Cell Signaling Technology, 9091), TFEB (Cell Signaling Technology, 37,785), Flag (Sigma-Aldrich, M2, F3165), FK2 (Millipore, Burlington, MA, USA, 04–263), TUBA (Millipore, CP06), MUL1 (abcam, ab209263 and ab84067), CTSL/V/K/H (abcam, ab133641), GFP (Santa Cruz Biotechnology, B-2, sc-9996), NEK2 (Santa Cruz Biotechnology, D-8, sc-55,601), ATE1 (Santa Cruz Biotechnology, E-6, sc-271,220), and R-HSPA5 (Abfrontier, AR05-PA0001).

Techniques: Degradation Assay, Expressing, Western Blot, Transfection, Incubation, Binding Assay, Pull Down Assay, Knockdown, Software, Immunofluorescence

Journal: Autophagy

Article Title: Crosstalk between HSPA5 arginylation and sequential ubiquitination leads to AKT degradation through autophagy flux

doi: 10.1080/15548627.2020.1740529

Figure Lengend Snippet: USP7 prevents bortezomib-induced AKT ubiquitination and degradation. (a) OPM2 cells were co-transfected with AKT1-MYC-His, HA-Ub, or Flag-USP7. A pretreatment of chloroquine (50 μM) was given for 1 h before the bortezomib (10 nM) treatment for 12 h. Cells were lysed, and AKT1 protein was immunoprecipitated by His pull-down assay, and ubiquitination levels were analyzed using an anti-HA antibody by western blot. (b) OPM2 cells were transfected with mock or Flag-USP7. Then, 24 h later, cells were treated with bortezomib (10 nM) for 12 h, lysed, and the indicated proteins were determined by western blot. (c) USP7 interacts with p-AKT. OPM2 cells were lysed, and p-AKT was immunoprecipitated using an anti-p-AKT antibody. western blots were probed with anti-p-AKT and USP7 antibodies. (d) USP7 inhibits the colocalization of AKT with LAMP1. RPMI8226 cells were transfected with mock or Flag-USP7. After 24 h, cells were pretreated for 1 h with chloroquine (50 μM), prior to the addition of bortezomib (10 nM) for 8 h. Cells were fixed and labeled with anti-p-AKT and LAMP1 antibodies. Yellow = merge/colocalization. Larger white boxes in the images are enlargements of the area indicated by the smaller white boxes; scale bars: 10 μm. (e) Quantification of the colocalization of p-AKT and LAMP1. Merged images from Figure 5(d) were analyzed for p-AKT:LAMP1 colocalization using NIS Elements software and Pearson’s correlation coefficient. Data represent the mean ± SD of 10 randomly selected cells per condition; *P < 0.05, n.s. (not significant). (f) The autophagic targeting of ubiquitinated AKT and R-HSPA5 is impaired in USP7-overexpressed cells. OPM2 cells were transfected with mock or Flag-USP7 in a dose-dependent manner. After 24 h, cells treated with the presence of chloroquine (50 μM) for 1 h and were additionally treated with bortezomib (10 nM) or thapsigargin (200 nM) for a further 12 h. Proteins were subjected to immunoprecipitation with an anti-p-AKT antibody, followed by western blot with the indicated antibodies. (g) MUL1 overexpression induces G2/M cell cycle arrest in bortezomib-resistance MM cells. RPMI8226/BTZ cells were transfected with mock or pCMV6-MUL1 for 48 h, and cell cycle analysis was processed after 70% ethanol fixation and PI staining. (h) MUL1 overexpression suppresses cell growth in bortezomib-resistance MM cells. RPMI8226/BTZ were transfected with mock or pCMV6-MUL1. At 48 h after transfection, the cells were harvested for western blot. Inhibition of cell growth was measured by the MTS assay on each indicated day (D1, 24 h after transfection; D2, 48 h after transfection). Data represent the mean ± SD. Asterisks indicate statistically significant differences (n = 6, *P < 0.05)

Article Snippet: The primary antibodies used included those against p-AKT (Cell Signaling Technology, S473, 9271), p-AKT (Cell Signaling Technology, T308, 13,038), AKT (Cell Signaling Technology, 9272), PARP1 (Cell signaling Technology, 9532), cleaved CAPS3 (Cell Signaling Technology, 9664), GAPDH (Cell Signaling Technology, 5174), Myc (Cell Signaling Technology, 2276 and 2272), MAP1LC3A (Cell Signaling Technology, 4599), MAP1LC3B (Cell Signaling Technology, 3868), Ub (Cell Signaling Technology, 3936), HA (Cell Signaling Technology, 3724), His (Cell Signaling Technology, 12,698), HSPA5 (Cell Signaling Technology, 3177), ATG5 (Cell Signaling Technology, 12,994), ATG12 (Cell Signaling Technology, 2010), ATG16L1 (Cell Signaling Technology, 8089), BECN1 (Cell Signaling Technology, 3495), p-GSK3B (Cell Signaling Technology, S9, 9323), GSK3B (Cell Signaling Technology, 12,456), USP7 (Cell Signaling Technology, 4833), BAX (Cell Signaling Technology, 2772), Total-BID (Cell Signaling Technology, 2002), p-MTOR (Cell Signaling Technology, S2448, 5536), MTOR (Cell Signaling Technology, 2983), p-RPS6KB1 (Cell Signaling Technology, T389, 9234), RPS6KB1 (Cell Signaling Technology, 2708), p-RPS6 (Cell Signaling Technology, S240/244, 5364), p-RPS6 (Cell Signaling Technology, S235/236, 4858), LAMP1 (Cell Signaling Technology, 9091), TFEB (Cell Signaling Technology, 37,785), Flag (Sigma-Aldrich, M2, F3165), FK2 (Millipore, Burlington, MA, USA, 04–263), TUBA (Millipore, CP06), MUL1 (abcam, ab209263 and ab84067), CTSL/V/K/H (abcam, ab133641), GFP (Santa Cruz Biotechnology, B-2, sc-9996), NEK2 (Santa Cruz Biotechnology, D-8, sc-55,601), ATE1 (Santa Cruz Biotechnology, E-6, sc-271,220), and R-HSPA5 (Abfrontier, AR05-PA0001).

Techniques: Ubiquitin Proteomics, Transfection, Immunoprecipitation, Pull Down Assay, Western Blot, Labeling, Software, Over Expression, Cell Cycle Assay, Staining, Inhibition, MTS Assay

Journal: Autophagy

Article Title: Crosstalk between HSPA5 arginylation and sequential ubiquitination leads to AKT degradation through autophagy flux

doi: 10.1080/15548627.2020.1740529

Figure Lengend Snippet: Sequential ubiquitination of residues K284 to K214 plays a vital role in AKT1 degradation. (a) AKT1 lysine (K) residues, K284 and K214, are putative protease inhibitor ubiquitination sites. Eleven lysine residues in the kinase domain of AKT1 were replaced with alanine (R) and each indicated plasmids were transfected into JJN3 cells. At 24 h after transfection, cells were pretreated with NH4Cl (25 mM) for 1 h, prior to the addition of PI (bortezomib, 10 nM) for 8 h. Ubiquitinated form of AKT1 was performed by Ni-NTA His pull-down ubiquitination assay. (b) RPMI8226 cells were transfected HA-Ub K48 together with AKT1 WT-MYC-His or AKT1 mutants (AKT1K214 R-MYC-His or AKT1K284 R-MYC-His). After 24 h, cells were treated with NH4Cl (25 mM) for 1 h, and then, PI (bortezomib, 10 nM) was treated for an additional 8 h. White box in merge pictures showed the colocalization of K48 ubiquitin-positive inclusions with AKT1 WT-MYC-His (but not AKT1K214 R-MYC-His or AKT1K284 R-MYC-His). Yellow = merge/colocalization. Larger white boxes in the images are enlargements of the area indicated by the smaller white boxes; scale bars: 10 μm. (c) K284/K214 mutant is sensitively degraded compared with AKT1 WT under proteasome inhibition. JJN3 cells were transfected with AKT1 WT-MYC-His, AKT1 K214-MYC-His, AKT1 K284-MYC-His, or AKT1 K284/K214-MYC-His. At 24 h after transfection, cells were treated with PI (bortezomib, 10 nM) for the indicated times, and p-AKT1 (S473), p-AKT1 (T308), p-GSK3B, GSK3B or GAPDH levels were determined by western blot. (* or ** means exo- or endogenous p-AKT1). (d) Under proteasome inhibitory status, MUL1 is finally attachment of K48 ubiquitin linkages to the K214 of AKT1. JJN3 cells were co-transfected with the EGFP-MUL1 together with the AKT1 K214-MYC-His, AKT1 K284-MYC-His or AKT1 K284/K214-MYC-His. After 30 h, cells were treated with chloroquine (50 μM) for 1 h, prior to the addition of PI (bortezomib, 10 nM) for the indicated times. Ubiquitinated AKT1 was identified by Ni-NTA His pull-down ubiquitination assay. (e) Fully ubiquitinated AKT1 (in which both K284 and K214 residues are ubiquitinated) can bind to R-HSPA5. JJN3 cells were transfected with AKT1 K214-MYC-His, AKT1 K284-MYC-His, or AKT1 K284/K214-MYC-His plasmids. Then, 24 h later, cells were pretreated with 25 mM NH4Cl 25 mM for 1 h prior to incubating the cells for 12 h with 200 nM thapsigargin, with or without 10 nM bortezomib (PI). AKT1-R-HSPA5 binding was evaluated by Ni-NTA His pull-down assay. (f) Silencing of MUL1 avoids PI-mediated K284/K214 AKT1 degradation. After transfection with scrambled RNAs or MUL1 siRNA, AKT1 K214-MYC-His, AKT1 K284-MYC-His, or AKT1 K284/K214-MYC-His, JJN3 cells were treated with PI (bortezomib, 10 nM) for 12 h. The indicated protein levels were determined by western blot. (g) MUL1 WT or mul1 KO cells were transfected with AKT1 K214-MYC-His, AKT1 K284-MYC-His, or AKT1 K284/K214-MYC-His. After 24 h, cells were treated with PI (bortezomib, 10 nM) for 24 h. The cells were subjected to western blot. (h) The cell growth of AKT1K284 R,K214 R-expressing cells is less affected by PI. JJN3 cells were transfected with mock, AKT1 WT-MYC-His, or AKT1K284 R,K214 R-MYC-His. At 24 h after transfection, cells were treated with PI (bortezomib, 10 nM) for 24 h. Cell viability was assessed by the MTS assay. Data represent the mean ± SD. Asterisks indicate statistically significant differences (n = 6, P < 0.05)

Article Snippet: The primary antibodies used included those against p-AKT (Cell Signaling Technology, S473, 9271), p-AKT (Cell Signaling Technology, T308, 13,038), AKT (Cell Signaling Technology, 9272), PARP1 (Cell signaling Technology, 9532), cleaved CAPS3 (Cell Signaling Technology, 9664), GAPDH (Cell Signaling Technology, 5174), Myc (Cell Signaling Technology, 2276 and 2272), MAP1LC3A (Cell Signaling Technology, 4599), MAP1LC3B (Cell Signaling Technology, 3868), Ub (Cell Signaling Technology, 3936), HA (Cell Signaling Technology, 3724), His (Cell Signaling Technology, 12,698), HSPA5 (Cell Signaling Technology, 3177), ATG5 (Cell Signaling Technology, 12,994), ATG12 (Cell Signaling Technology, 2010), ATG16L1 (Cell Signaling Technology, 8089), BECN1 (Cell Signaling Technology, 3495), p-GSK3B (Cell Signaling Technology, S9, 9323), GSK3B (Cell Signaling Technology, 12,456), USP7 (Cell Signaling Technology, 4833), BAX (Cell Signaling Technology, 2772), Total-BID (Cell Signaling Technology, 2002), p-MTOR (Cell Signaling Technology, S2448, 5536), MTOR (Cell Signaling Technology, 2983), p-RPS6KB1 (Cell Signaling Technology, T389, 9234), RPS6KB1 (Cell Signaling Technology, 2708), p-RPS6 (Cell Signaling Technology, S240/244, 5364), p-RPS6 (Cell Signaling Technology, S235/236, 4858), LAMP1 (Cell Signaling Technology, 9091), TFEB (Cell Signaling Technology, 37,785), Flag (Sigma-Aldrich, M2, F3165), FK2 (Millipore, Burlington, MA, USA, 04–263), TUBA (Millipore, CP06), MUL1 (abcam, ab209263 and ab84067), CTSL/V/K/H (abcam, ab133641), GFP (Santa Cruz Biotechnology, B-2, sc-9996), NEK2 (Santa Cruz Biotechnology, D-8, sc-55,601), ATE1 (Santa Cruz Biotechnology, E-6, sc-271,220), and R-HSPA5 (Abfrontier, AR05-PA0001).

Techniques: Ubiquitin Proteomics, Protease Inhibitor, Transfection, Mutagenesis, Inhibition, Western Blot, Binding Assay, Pull Down Assay, Expressing, MTS Assay

Journal: Autophagy

Article Title: Crosstalk between HSPA5 arginylation and sequential ubiquitination leads to AKT degradation through autophagy flux

doi: 10.1080/15548627.2020.1740529

Figure Lengend Snippet: Novel proteolysis mechanisms of AKT through crosstalk between R-HSPA5 and a sequentially ubiquitinated form of AKT. Unfolded and/or misfolded proteins are targeted by ubiquitin for degradation by the proteasome or lysosome pathways. The proteasome inhibitor leads to the accumulation of excessive misfolded/ubiquitinated protein aggregates. Proteasome inhibition facilitates the formation of ubiquitinated proteins, induced R-HSPA5 [21] or MUL1 (steps 1 and 2). In various oxidative conditions, MUL1 induces AKT degradation by UPS via K48-linked ubiquitination at K284 residue [15] (step 3). However, AKT could not induce degradation because proteasome is inhibited by PI, therefore, ubiquitinated AKT is able to lysosomal degradation, for ubiquitin chains linked to another lysine (K214) of AKT by MUL1, generating unique coding signatures of the K48 linkages. (step 4). Sequential ubiquitination of AKT from K284 to K214 acts as a substrate-specific mark for lysosomal degradation through autophagosome. The resulting fully ubiquitinated form of AKT at both K284 and K214 could bind with R-HAPS5 (step 5). This enables selective recruitment of cargo along with R-HSPA5, SQSTAM1, and MAP1LC3 on the autophagic membranes [21,37] (step 6). Following vesicle closure, the autophagosome fuses (step 7) with the lysosome whereby the engulfed material is degraded (step 8). In this model, the connection between MUL1-mediated sequential ubiquitination of AKT and arginylation of HSPA5 is essential for the lysosomal degradation of AKT under proteasome inhibitory cellular stress

Article Snippet: The primary antibodies used included those against p-AKT (Cell Signaling Technology, S473, 9271), p-AKT (Cell Signaling Technology, T308, 13,038), AKT (Cell Signaling Technology, 9272), PARP1 (Cell signaling Technology, 9532), cleaved CAPS3 (Cell Signaling Technology, 9664), GAPDH (Cell Signaling Technology, 5174), Myc (Cell Signaling Technology, 2276 and 2272), MAP1LC3A (Cell Signaling Technology, 4599), MAP1LC3B (Cell Signaling Technology, 3868), Ub (Cell Signaling Technology, 3936), HA (Cell Signaling Technology, 3724), His (Cell Signaling Technology, 12,698), HSPA5 (Cell Signaling Technology, 3177), ATG5 (Cell Signaling Technology, 12,994), ATG12 (Cell Signaling Technology, 2010), ATG16L1 (Cell Signaling Technology, 8089), BECN1 (Cell Signaling Technology, 3495), p-GSK3B (Cell Signaling Technology, S9, 9323), GSK3B (Cell Signaling Technology, 12,456), USP7 (Cell Signaling Technology, 4833), BAX (Cell Signaling Technology, 2772), Total-BID (Cell Signaling Technology, 2002), p-MTOR (Cell Signaling Technology, S2448, 5536), MTOR (Cell Signaling Technology, 2983), p-RPS6KB1 (Cell Signaling Technology, T389, 9234), RPS6KB1 (Cell Signaling Technology, 2708), p-RPS6 (Cell Signaling Technology, S240/244, 5364), p-RPS6 (Cell Signaling Technology, S235/236, 4858), LAMP1 (Cell Signaling Technology, 9091), TFEB (Cell Signaling Technology, 37,785), Flag (Sigma-Aldrich, M2, F3165), FK2 (Millipore, Burlington, MA, USA, 04–263), TUBA (Millipore, CP06), MUL1 (abcam, ab209263 and ab84067), CTSL/V/K/H (abcam, ab133641), GFP (Santa Cruz Biotechnology, B-2, sc-9996), NEK2 (Santa Cruz Biotechnology, D-8, sc-55,601), ATE1 (Santa Cruz Biotechnology, E-6, sc-271,220), and R-HSPA5 (Abfrontier, AR05-PA0001).

Techniques: Ubiquitin Proteomics, Inhibition, Residue