Protein degradation is a regulatory process of all living beings that helps to weed out proteins that are faulty in nature. The fault maybe in their sequence or folding. The proteins are degraded via ubiquitin-proteasome system (UPS). The proteasome is made up of an outer activating module and an inner core particle (CP), which in turn is made up of outer α subunits and inner β subunits. The α subunits guard a N-terminal gates pore, which open up when proteasome activators (PA) binds to it. The β subunits have the active sites that can broadly accommodate different faulty proteins for degradation. The CP interacts with 3 types of activators in the eukaryotes: 1) ATPases complex, regulatory particle (RP) and proteasome activating nucleotidase (PAN) 2) PA28/11S family, 3) Blm10/PA200 family.
The objective of the paper under study by Dange et al. focuses on the effects of the yeast proteasome activator Blm10 on proteasomal peptide and protein degradation. The paper shows that mutagenesis of Blm10 C-terminal can result in loss of function phenotype. It also explores that possibility of associating cellular functions of Blm10 in regulating gate opening. Lastly, the scientists have tried to demonstrate that Blm10-CP complexes accelerate protein turnover. For this, the scientists have used tau441 substrate.
The experimental methods include using different strains of yeast. Some of them were cloned with genetically modified C-terminal for mutagenesis study. The proteasomal activity was measured by determining the rate of hydrolysis. For this, Tris-HCl, DTT, glycerol and fluorogenic substrates were used. The peptides for study were dissolved in DMSO before adding into the above reaction mixture. The rate of hydrolysis was determined using spectrophotometric plate reader at 380nm (excited) and read at 460nm (emitted). Phenotype was analyzed by growing the strains on YPD with or without cycloheximide (CHX) and on different carbon sources. In-gel digestion was performed by obtaining 200 μg protein through cryogenic disruption lysis of the cells. Blm10 proteasome complex was separated using IgG affinity gel chromatography. The complex was cleaved from IgG using tobacco etch viral (TEV) protease and run on acrylamide native gels. An in-gel assay was done using fluorogenic substrates. SDS-PAGE was done to confirm presence of complex and purity of Blm10. The eluted and purified proteasome complex was assayed using 3 fluorogenic substrates to measured proteasome activity by measuring Khalf, vmax and R2. In-gel activity assay is performed in 96-well plates using pure CP and Blm10-CP complexes. This was used to determine the efficacy in Tau degradation. This experiment determined which among CP and Blm-CP complex better accelerates the degradation of tau. EDTA, MgCl2, Tris-HCl and recombinant tau were incubated at room temperature. The reaction mixture was supplemented with pure CP or Blm10-CP complex either in presence or in absence of MG132 (a proteasome inhibitor). The incubated mixture is stopped by heating and blotted using immunoblot. The image was analyzed using densitometry.
The results were that the last 7 residues of Blm10 at the C-terminal (ct-Blm10) are crucial for binding to CP and gate opening. These residues caused trypsin-like and caspase like hydrolysis of proteins. Increased hydrolytic rate was seen in wild type strains. Mutant strains with modified penultimate residue (Ala or Val instead of Try) allowed binding to CP, but in loss of function phenotype. In addition, mutation in last 7 residues rendered the strains viable to CHX. The mutant strains have an open gate and ct-Blm10 has no effect on its hydrolysis. When the last seven residues were substituted with the residues found in PA26, Blm10PA26-CP complexes were formed, which shows that there is similar binding affinities to CP. The mutant strain’s own promoter was replaced with an overexpressing promoter GAL1. Upon overexpression of Blm10, it was noticed that cells could tolerate such an activity in low temperatures, but failed to do so under induced oxidative metabolism and high temperatures. The strains, whose proteasome related transcription factors (Rpn4) were removed, died when grown on non-fermentable carbon source. Mutant strains with modified ct-Blm10 are gate-incompetent and were found to be lethal to the cells at higher temperatures. Blm10 capped at both ends of the CP was thought to be inactive. However, this paper proves that such a doubly-capped Blm10-CP complex is still effective in its proteasomal activity. This is shown through X-ray crystallography, which shows partially opened gates at both ends. Tau441 is a natural substrate for Blm10, which degrades tau in absence of ATPase or UPS. Tau441 was seen better degraded by Blm10-CP complex, rather than pure CP.
This study shows that the last 7 (2137-2143) residues are the most important ones for proteasome activity. The mutation at the penultimate residue brings about major changes, but any mutation at the last residue has no effect on the proteasome activity. However, replacing arginine (R2139) with aspartate promotes binding but there is partial loss of function in the cell. According to this paper, the conserved motif of hydrophobic-tyr-X is important for recognition and binding.
