Project description:We compared the expression changes in Ewing sarcoma cell lines following treatment with 2 known 20S proteasome inhibitors versus 2 novel compounds
Project description:The 20S proteasome is responsible for the catalytic activity of all proteasome complexes. Structural constraints mean that only unfolded, extended polypeptide chains may enter the catalytic core of the 20S proteasome. Here we conducted a comprehensive analysis of the 20S CP substrates in-vitro. We revealed that the 20S CP substrates are highly structually disordered. The 20S proteasome substrate group, termed 20S-IDPome are characterized by having significantly more protein binding partners, more post-translational modification sites and are highly enriched for RNA binding proteins. Remarkably, we found that low complexity proteins with prion-like domain which interact with GR or PR di-peptide repeats are the most preferential 20S proteasome substrates. Our finding suggests roles of the 20S proteasome in gene transcription and formation of phase-separated granules.
Project description:For many years, the ubiquitin-26S proteasome degradation pathway was considered the principal route for proteasomal degradation. However, it is now becoming clear that proteins can also be targeted for degradation by an ubiquitin-independent mechanism mediated by the core 20S proteasome itself. The fact that half of cellular proteasomes are free 20S complexes suggests that degradation by this complex is not limited to rare cases. Identifying 20S proteasome substrates is challenging, as different pools of the same protein can be sent to degradation via either 20S or 26S proteasomes. Hence, current knowledge regarding the repertoire of 20S proteasome substrates mainly originates from individual case studies. Here, in an effort to unravel the global repertoire of substrates degraded by the 20S proteasome, we used an advanced mass spectrometry approach coupled with biochemical and cellular analysis. Our analysis enabled the identification of hundreds of 20S proteasome substrates. In addition to proteins that are degraded to completion, we also identified proteins that undergo specific cleavage by the 20S proteasome, at their N- or C- termini, to possibly tune their function. We also found that 20S substrates are significantly enriched with RNA- and DNA-binding proteins that contain intrinsically disordered regions. The vast majority of them are localized in the nucleus and stress granules. Further, we demonstrate that oxidized proteasomes have reduced proteolytic activity compared to naïve proteasomes, which we propose is an adaptive advantage under conditions of cellular stress. Whereas oxidized protein substrates, rather than being folded proteins that lost their native structure due to the stress, actually display a higher degree of structural-disorder than naïve proteins. In summary, here we shed light on the nature of the 20S substrates, providing critical insight into the biological role of the 20S proteasome.
Project description:Proteasomes undergo dynamic changes in their types and activities during mammalian spermatogenesis. While the spermatogenesis-specific 20S proteasome (s20S), characterized by PSMA8 substitution of PSMA7, is known to be essential for meiosis I completion, the functions of the constitutive PSMA7-containing 20S proteasomes (c20S) in spermatogenesis remain poorly understood. Here, we show that c20S proteasomes are required for the maintenance and differentiation of spermatogonia. PSMA7 is ubiquitously expressed in male germ cells beginning at the early spermatogonial stage, preceding PSMA8 expression. Conditional ablation of Psma7 using Stra8-Cre impairs proteasomal degradation in differentiating spermatogonia, leading to male infertility. Single-cell RNA sequencing analysis reveals that PSMA7-deleted germ cells are arrested at the differentiating spermatogonia stage and fail to enter meiosis. Notably, sufficient overexpression of PSMA8 restores normal spermatogenesis in Psma7-null germ cells, suggesting a potential complementarity of s20S to c20S. Therefore, our results add critical insights into the complex regulation of proteasomal degradation during spermatogenesis.
Project description:The yeast 20S proteasome was analyzed using native mass spectrometry combined with UVPD, HCD, and vT-ESI. Nano-LC-MS-EThcD was used to characterize the primary sequence features of the 20S subunits.
Project description:Careful removal of unwanted proteins is necessary for cell survival. The primary constitutive intracellular protease is the 26S proteasome complex, very often found in equilibrium with its catalytic core particle – the 20S subcomplex. Protein degradation by the former is tightly regulated due to prior selection of substrates by ubiquitination, whereas the latter is most likely confined to substrates with an inherent unstructured stretch. Understanding the interplay between 26S and 20S proteasomes is challenging due to their common catalytic sites. By using MS analyses, we define the 20S and 26S substrate preferences, and ascribe a unique property to 20S in degrading ubiquitin. High-end mass-spectroscopy of intracellular peptidome defines signature activities of 20S and finds their contribution to proteostasis under severe hypoxia and in human failing heart.
Project description:KRAS is a well known tumor associated antigen. It’s a GTPase that functions as molecular switch in regulatory pathways responsible for proliferation and survival. It’s frequently mutated in a variety of cancers. One of the possible driver mutations is G12V substitution, which impairs KRAS GTPase activity and renders the mutants persistently in the GTP-bound active form, thereby promoting tumorigenesis and tumor malignancy. Its high frequency in cancers makes KRAS an attractive target for immunotherapy. We performed in vitro digestions of synthetic polypeptide corresponding to KRAS2-35 carrying the mutation with purified 20S proteasome. Samples were measured by LC-MS/MS.