Figure 1.
Identification of HYPK-interacting partners using pull-down coupled with MALDI-mass spectrometry.
Flow chart for pull-down experiment using purified 6HN-HYPK as bait (A); separation of pulled-down proteins by 1D SDS-PAGE using HeLa SCL as prey (B); 2D SDS-PAGE using STHdhQ111/HdhQ111 SCL as prey (C), 2D SDS-PAGE with STHdhQ7/HdhQ7 SCL as prey (D); probability based MOWSE score and parent MS and subsequent MS/MS spectra for HSP90AB1 (E). From the MOWSE score distribution, it is evident that the peptide mass fingerprint search identifying HSP90AB1 (MOWSE score 139) was significant (p≤0.05). MALDI spectra for 5 other proteins and details of MALDI-mass analyses for the identification of HYPK-interacting proteins are provided as Figures S1, S2, S3, S4, and S5 and Table S3 respectively.
Table 1.
HYPK-interacting proteins identified in the present study and obtained earlier.
Figure 2.
Validation of interaction by co-immunoprecipitation (co-IP) and confocal microscopy.
Validation of interaction between HYPK and EEF1A1 by co-IP using anti-HYPK antibody (endogenous EEF1A1 was detected) along with subcellular co-localization of HYPK-GFP with EEF1A1-DsRed (A). The interaction of HYPK with HSPA8, LMNB2, CALM1, HSPA1A, VIM and MLF2 are shown (B, C, D, E, F and G respectively). The co-IP of Neuro2A SCL with anti-HYPK antibody precipitated endogenous HSPA1A (immunoblot probed with anti-HSPA1A; E). Anti-HYPK antibody was used to immunoprecipitate the HYPK-complex from Neuro2A SCL overexpressing HSPA8-DsRed (B), LMNB2-DsRed (C), CALM1-DsRed (D) or VIM-DsRed (F) and the immunoblots were probed with anti-DsRed antibody. Interaction between HYPK and MLF2 was confirmed by probing the HYPK-immunoprecipitated complex with anti-GFP antibody (G). The co-IP blots shown in A, E and F were re-probed with anti-HYPK antibody confirming HYPK in the immunoprecipitate. The dots showing co-localization of HYPK with EEF1A1 (A), LMNB2 (C) and MLF2 (G) are indicated by arrows. The immunoblot showing interaction between endogenous HYPK and endogenous HSPA1A was stripped and re-probed with anti-HIP1 antibody. The absence of HIP1 in the immunoprecipitate confirmed that HYPK did not interact with HIP1 (E). Scale bars (5 µm) for the confocal images are indicated in each panel.
Table 2.
Squared values of the Pearson Correlation coefficient (Rp) for determination of co-localization of HYPK with its interacting partners.
Figure 3.
Formation of high molecular weight HYPK-complex.
High molecular weight HYPK-complex formation by endogenous HYPK with EEF1A1 (AI) and HSPA1A/HSP70 (AII) in Neuro2A cells using native-PAGE western analysis; results obtained by similar experiments in STHdhQ7/HdhQ7 and STHdhQ111/HdhQ111 with LMNB2 (BI) and HTT (BII); result obtained by re-probing the blot shown in B with anti-HYPK antibody is shown in BIII, showing the difference in migration of purified HYPK and complex-bound HYPK; similar co-IP experiments in STHdhQ7/HdhQ7 (C) along with Ponceau staining of the nitrocellulose membrane (CI). The blot is probed with anti-TP53 (CII) and anti-RELA (CIII) antibodies. In all the cases, anti-HYPK antibody was used to pull-down and the immunoprecipitate elute was analyzed to detect the interacting proteins. The loading wells in A and C are marked by arrows.
Figure 4.
Preparation of HYPK knock-down neuronal cell lines using the antisense construct (HYPK-U61).
Validation of HYPK knock-down in mRNA and protein levels in Neuro2A (A) and STHdhQ7/HdhQ7 (B) cell lines. Expression of both mRNA and protein levels were measured by RT-PCR or SDS-PAGE western analysis in these HYPK knocked-down stable mouse cell lines. Upon exogenous expression of 83Q-DsRed in a dose dependent manner (100 ng and 200 ng), the percentage of cells containing mutant HTT aggregates in presence and absence of endogenous HYPK in STHdhQ7/HdhQ7 cell lines are compared (C). The ‘n’ and ‘p’ values for Student’s two-tailed t test are indicated in the bar diagrams along with the mean and standard deviation.
Figure 5.
Cell growth and cell survival in presence and absence of HYPK.
Comparison of the growth curves of control and HYPK downregulated Neuro2A (A) and STHdhQ7/HdhQ7 (B) cell lines and effect of exogenous expression of HYPK-DsRed and HSPA8-DsRed in these HYPK knocked-down neuronal cell lines. Comparison of BrdU incorporation in STHdhQ7/HdhQ7 cells overexpressing DsRed and HYPK-DsRed in a dose dependent manner (transfection with 300 ng and 500 ng of plasmid) (C). The difference in BrdU incorporation in STHdhQ7/HdhQ7 cells in absence of HYPK and in presence of HYPK as well as HSPA8 (500 ng) are shown (D). The effect of mutant Htt (83Q-DsRed) on cell survival of control and HYPK downregulated STHdhQ7/HdhQ7 cells and its effect in presence of exogenous addition of HYPK-DsRed in these cells are shown (E). Flow cytometry analysis showing distribution of STHdhQ7/HdhQ7 cells in different phases of cell cycle (upon 7-AAD staining) in presence and absence of HYPK (F). The cell cycle analysis was performed using CellQuest Pro software as described in Materials and Methods section.The ‘n’ and ‘p’ values for Student’s two-tailed t test are indicated in the bar diagrams along with the mean and standard deviation. The distributions of cell population in different phases are provided in Table 3.
Table 3.
Distribution of cell populations in different cell cycle phases in presence and absence of HYPK in STHdhQ7/HdhQ7 cell lines.
Figure 6.
Effect of HYPK on recovery of heat denatured luciferase activity.
The in vivo chaperone activities of Neuro2A-U61 and Neuro2A HYPK-U61 cells in presence and absence of HYPK (before administration of heat shock) are compared (A). As mentioned in the Materials and Methods section, the cells were subjected to heat shock at 43°C for 1 h (HS) and re-incubated at 37°C CO2 incubator for the next 6h (HS+R). In every case, heat shock was administered 24h post-transfection of Tet+Luc+ve cells with experimental constructs. Comparison of luciferase activities in Neuro2A-U61 cells in presence and absence of HYPK-DsRed in No HS, immediately after heat shock (HS) and HS+R conditions (B). These luciferase activities were also compared upon overexpression of HSPA8-DsRed and 3 other HYPK partners in Neuro2A-U61 cells (B). Such drop and recovery of the luciferase activities upon heat treatment of HYPK downregulated Neuro2A (Neuro2A-HYPK U61) cells and effect of HYPK and its interacting partners are shown (C). The ‘n’ and ‘p’ values for Student’s two-tailed t test are indicated in the bar diagrams along with the mean and standard deviation.