Tein levels (Fig 5H and Supplementary Fig S8A and B), accompanied by normalization of the intracellular Zn level (Supplementary Fig S8C) as the MG132 remedy does (Supplementary Fig S9). These observations suggested that 26S proteasome inhibitors could restore the impaired intracellular Zn homeostasis by the ZIP13 mutants; thus, the manipulation of 26S proteasome activity by inhibitory compounds may possibly be a therapeutic approach for SCD-EDS brought on by pathogenic mutant ZIP13 proteins. VCP is involved within the degradation on the mutant ZIP13 proteins To additional elucidate the molecular mechanisms involved in standard and pathogenic ZIP13 homeostasis, we isolated ZIP13-associatedmolecules by immunoprecipitation. Of these, we identified VCP/ Cdc48/p97 by mass spectrometric evaluation (Fig 6A). VCP belongs towards the AAA superfamily, which mediates many functions, like the ubiquitination-dependent proteasome technique (Ye et al, 2001, 2004; Richly et al, 2005). Along with ZIP13WT, VCP bound to and co-localized with all the mutant ZIP13G64D protein (Fig 6A ). Intriguingly, more VCP was related with ZIP13G64D than with ZIP13WT (Fig 6B, decrease), indicating that the VCP protein could preferentially interact with all the pathogenic ZIP13G64D protein. To understand VCP’s role within the degradation from the mutant ZIP13 protein, we knocked down VCP by siRNAs or suppressed its function by expressing a dominant-negative type of VCP. VCP siRNAs reduced the protein amount of the endogenous VCP (Fig 6D, middle) and restored the protein level of ZIP13G64D (Fig 6D, upper). In addition, the ectopic expression of dominant-negative VCP, F-VCPE305Q/E578Q, restored the protein amount of ZIP13G64D (Fig 6E). Also, a VCP inhibitor DBeQ (Chou et al, 2011) could suppressAIP: FLAG F-G64D Mock F-WTBIP: V5 Hexokinase drug G64D-V5 WT-VCDG64D-V5 VCP V5 Merge Scrambled siRNAEG64D-V5 F-VCPE305Q/E578QkDaMockVCP siRNA#88VCPInput G64D-VIgHIB : GAPDH VCP/ZIP13 Ratio12 8 4IB : V5 IB : VCP IB : GAPDHIB : V5 IB : FLAG IB : GAPDHABIgLRelative expression Adiponectin Receptor Agonist manufacturer level1.2 1.0 0.8 0.6 0.FWT-V5 CHX CHX four 0G64D-V5 CHX MG132 four two four CHX DBeQ 2WT-V5: CHX G64D-V5: CHX G64D-V5: CHX + MG132 G64D-V5: CHX + DBeQIncubation (hr)Silver stain 119IB : VCPIB: V5 IB: TUBULIN0.2 02 4 CHX therapy (hr)Figure 6. The mutant ZIP13 protein is degraded via a VCP-dependent mechanism. A Identification of VCP/Cdc48/p97 as a ZIP13-associating protein. Whole-cell lysates from 293T cells transfected with FLAG-tagged ZIP13 had been immunoprecipitated with an anti-FLAG antibody, followed by SDS AGE and silver staining. Exceptional bands were cut out and analyzed by TOF/MASS to identify the proteins. A protein band close to 88 kDa was determined to be VCP/Cdc48/p97. VCP was also detected by Western blot applying an anti-VCP antibody (decrease). IgH: heavy chain of IgG; IgL: light chain of IgG; A: SP-uncleaved immature ZIP13 protein; B: SP-cleaved mature ZIP13 protein. B VCP binds to ZIP13. Whole-cell lysates from 293T cells transfected with expression plasmids for V5-tagged ZIP13 proteins had been immunoprecipitated with an anti-V5 antibody, followed by SDS AGE. VCP and ZIP13 proteins have been detected by Western blot utilizing anti-VCP and anti-V5 antibodies, respectively. The VCP/ZIP13 ratio was analyzed using ImageJ software (http://rsbweb.nih.gov/ij/download.html) (bottom). C Confocal pictures of VCP in HeLa cells stably expressing G64D-V5. VCP (green) and G64D-V5 (red) were stained with anti-V5 and anti-VCP antibodies, respectively. D Effect of VCP siRNA on the protein.
