As the degradation machinery that is responsible for ~70% of intracellular proteolysis, proteasome complex (26S proteasome) plays a critical roles in maintaining the homeostasis of cellular proteome. Accordingly, misfolded proteins and damaged protein need to be continuously removed to recycle amino acids for new synthesis; in parallel, some key regulatory proteins fulfill their biological functions via selective degradation; furthermore, proteins are digested into peptides for MHC class I antigen presentation. To meet such complicated demands in biological process via spatial and temporal proteolysis, protein substrates have to be recognized, recruited, and eventually hydrolyzed in a well controlled fashion. Thus, 19S regulatory particle pertains a series of important capabilities to address these functional challenges. To recognize protein as designated substrate, 19S complex has subunits that are capable to recognize proteins with a special degradative tag, the ubiquitinylation. It also have subunits that can bind with nucleotides (e.g., ATPs) in order to facilitate the association between 19S and 20S particles, as well as to cause confirmation changes of alpha subunit C-terminals that form the substate entrance of 20S complex. The ATPases subunits assemble into a six-membered ring with a sequence of Rpt1–Rpt5–Rpt4–Rpt3–Rpt6–Rpt2, which interacts with the seven-membered alpha ring of 20S core particle and establishes an asymmetric interface between the 19S RP and the 20S CP. Three C-terminal tails with HbYX motifs of distinct Rpt ATPases insert into pockets between two defined alpha subunits of the CP and regulate the gate opening of the central channels in the CP alpha ring. Evidence showed that ATPase subunit Rpt5, along with other ubuiqintinated 19S proteasome subunits (Rpn13, Rpn10) and the deubiquitinating enzyme Uch37, can be ubiquitinated in situ by proteasome-associating ubiquitination enzymes. Ubiquitination of proteasome subunits can regulates proteasomal activity in response to the alteration of cellular ubiquitination levels.
Background References
1. Zhang JY et al. The Silence of PSMC6 Inhibits Cell Growth and Metastasis in Lung Adenocarcinoma. Biomed Res Int. 2021 Jun
2. Dišlere K et al. 1,4-dihydropyridine derivatives increase mRNA expression of Psma3, Psmb5, and Psmc6 in rats. Arh Hig Rada Toksikol. 2021 Jun
Western blot analysis of PSMC6 on different lysates with Rabbit anti-PSMC6 antibody (HA721270) at 1/1,000 dilution.
Lane 1: Hela cell lysate (10 µg/Lane) Lane 2: 293T cell lysate (10 µg/Lane) Lane 3: SH-SY5Y cell lysate (10 µg/Lane) Lane 4: HepG2 cell lysate (10 µg/Lane) Lane 5: PC-12 cell lysate (10 µg/Lane) Lane 6: NIH/3T3 cell lysate (10 µg/Lane) Lane 7: Mouse kidney tissue lysate (20 µg/Lane) Lane 8: Rat kidney tissue lysate (20 µg/Lane) Lane 9: Rat brain tissue lysate (20 µg/Lane)
Predicted band size: 44 kDa Observed band size: 44 kDa
Exposure time: 2 minutes;
10% SDS-PAGE gel.
Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (HA721270) at 1/1,000 dilution was used in 5% NFDM/TBST at room temperature for 2 hours. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1:300,000 dilution was used for 1 hour at room temperature.
Immunohistochemical analysis of paraffin-embedded human testis tissue with Rabbit anti-PSMC6 antibody (HA721270) at 1/400 dilution.
The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) (high pressure) for 2 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (HA721270) at 1/400 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.
Flow cytometric analysis of SH-SY5Y cells labeling PSMC6.
Cells were fixed and permeabilized. Then stained with the primary antibody (HA721270, 1ug/ml) (red) compared with Rabbit IgG Isotype Control (green). After incubation of the primary antibody at +4℃ for an hour, the cells were stained with a iFluor™ 488 conjugate-Goat anti-Rabbit IgG Secondary antibody (HA1121) at 1/1,000 dilution for 30 minutes at +4℃. Unlabelled sample was used as a control (cells without incubation with primary antibody; black).
Flow cytometric analysis of NIH/3T3 cells labeling PSMC6.
Cells were fixed and permeabilized. Then stained with the primary antibody (HA721270, 1ug/ml) (red) compared with Rabbit IgG Isotype Control (green). After incubation of the primary antibody at +4℃ for an hour, the cells were stained with a iFluor™ 488 conjugate-Goat anti-Rabbit IgG Secondary antibody (HA1121) at 1/1,000 dilution for 30 minutes at +4℃. Unlabelled sample was used as a control (cells without incubation with primary antibody; black).
Flow cytometric analysis of PC-12 cells labeling PSMC6.
Cells were fixed and permeabilized. Then stained with the primary antibody (HA721270, 1ug/ml) (red) compared with Rabbit IgG Isotype Control (green). After incubation of the primary antibody at +4℃ for an hour, the cells were stained with a iFluor™ 488 conjugate-Goat anti-Rabbit IgG Secondary antibody (HA1121) at 1/1,000 dilution for 30 minutes at +4℃. Unlabelled sample was used as a control (cells without incubation with primary antibody; black).
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