We have made significant progress in all 3 aims of the original proposal:
Aim 1: Elucidate the molecular switch that activates the 20S proteasome.
To unravel the activation mechanism of the 20S proteasome, we searched for regulators that can regulate this complex. This lead us to the discovery of a novel and specific regulator, DJ-1, a protein associated with Parkinson’s disease. The protein physically binds the 20S, though not the 26S, proteasome and prevents its proteolytic activity. Consequently, DJ-1 stabilizes cellular levels of 20S proteasome substrates, as we showed for α-synuclein and p53.
• Moscovitz O, Ben-Nissan G, Polack D, Zaroock O and M Sharon. (2015). The Parkinson's-Associated Protein DJ-1 Regulates the 20S Proteasome. Nat. Commun. 6:6609
Aim 2: Uncover the mechanisms of 20S proteasome regulation
We have taken three different experimental approaches in order to reveal the molecular details that underlie the activity of the 20S proteasome regulators: i) peptide mapping, ii) electron microscopy analysis iii) hydrogen exchange mass spectrometry (in collaboration with David Schriemer, Calgary Canada). Our findings suggest that the 20S regulator impact the gate opening capacity of the 20S proteasome. In addition, we have studied the structural properties of the 20S regulator, DJ-1, and two missense mutants of this protein, DJ-1A104T and DJ-1D149A, which lead to early-onset familial Parkinson’s disease. We discovered that DJ-1D149A is more capable of inhibiting the 20S proteasome, in comparison to both DJ-1A104T and DJ-1WT, these observations may suggest that following its association with the 20S proteasome, DJ-1 undergoes structural rearrangements that enable its inhibitory function. The more relaxed structure of DJ-1D149A may more readily promote such a structural transition.
• Ben-Nissan G, Spectror A, Taranavsky M and M Sharon. (2016). Structural Characterization of Missense Mutations Using High Resolution Mass Spectrometry: a Case Study of the Parkinson's-Related Protein, DJ-1. J. Am. Soc. Mass Spectrom. 27(6):1062-70.
Remarkably, we have also discovered a new family of 20S proteasome regulators and provided the first demonstration that degradation by the 20S proteasome is not a simple and random process, but rather a highly regulated and coordinated mechanism. This family which we have named Catalytic Core Regulators (CCRs) consists of 17 small proteins of 20-30 kDa, many of which are enzymes (such as CBR3, NQO2, PGDH and RBBP9) or key signaling proteins (as NRas, KRas, HRas and RhoA. Beyond their primary functions, we showed that these proteins have an additional moonlighting activity – they specifically bind to the 20S, affect protein degradation and influence the cellular levels of 20S proteasome substrates. Interestingly, the vast majority of CCRs can be found also in the extracellular milieu, raising the possibility that they regulate extracellular proteasomes.
• Olshina MA, Arkind G, Deshmukh FK, Fainer I, Taranavsky M, Hayat D, Ben-Dor S, Ben-Nissan G and Sharon M. (2020). Regulation of the 20S Proteasome by a Novel Family of Inhibitory Proteins. Antioxid. Redox. Signal. 32(9):636-55.
Aim 3: Investigate the reactivity of the 20S proteasome to various cellular cues.
We have focused our efforts on the investigating the activity of the 20S proteasome under oxidative stress. Under such conditions, the 20S proteasome is known to be the major degradation machinery, likely due to its higher resistance to oxidation, and the sensitivity of the ubiquitinylation machinery to redox conditions. We discovered that the 20S proteasome specifically cleaves p53 to generate the Δ40p53 isoform lacking the first 40 amino acids. Under oxidative stress, Δ40p53 levels are increased in a 20S proteasome-dependent manner, leading to reduction in p53 transcriptional activity.
• Solomon H, Bräuning B, Rabani S, Goldfinger N, Moscovitz O, Shakked Z, Rotter V and M Sharon. (2017). Post-translational Regulation of p53 Function through 20S Proteasome Mediated Cleavage. Cell Death Diff. 24(12):2187-98
