Periodic Reporting for period 4 - 20SComplexity (An integrative approach to uncover the multilevel regulation of 20S proteasome degradation)
Berichtszeitraum: 2019-10-01 bis 2020-03-31
Both cellular homeostasis and regulation of cellular functions depend on the finely orchestrated degradation of regulatory proteins. The ubiquitin-dependent 26S proteasomal degradation pathway most likely represents the primary cellular means of protein turnover; however, it is becoming increasingly clear that proteins can also be degraded via an alternative route, mediated solely by the 20S proteasome. Degradation by the 20S proteasome is a passive process that does not require ubiquitin tagging or the presence of the 19S regulatory particle; rather, it relies on the presence of unstructured regions within the protein substrate. Moreover, it has been shown that under oxidative stress, a condition prevalent in almost all cancers, the 20S proteolytic pathway becomes the major degradation route. Hence, cancer cells are predicted to be more sensitive to 20S inhibition than normal cells. Our overall aim is to reveal the multiple regulatory levels that coordinate the 20S proteasome degradation route. Our multidisciplinary strategy involves the application of biochemical approaches coupled to native mass spectrometry and fluorescence microscopy measurements, complemented by in vivo cell biology analyses.
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
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
During the course of the project we have developed several generic native mass spectrometry (MS) methods, as described in the following publications:
1. 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.
2. Sokolovski M, Cveticanin J, Hayoun D, Korobko I, Sharon M and A Horovitz. (2017). Measuring inter-protein pairwise interaction energies from a single native mass spectrum by double-mutant cycle analysis. Nat. Commun. 9;8(1):212.
3. Gan J, Ben-Nissan G, Otonin G, Tarnavsky M, Trudeau D, Noda-Garcia L, Tawfik D S and M Sharon. (2017). Native mass spectrometry of recombinant proteins from crude cell lysates. Anal. Chem. 89(8):4398-404.
4. Ben-Nissan G, Belov M E, Morgenstern D, Levin Y, Dym O, Arkind G, Lipson C, Makarov A A and M. Sharon. (2017). Triple-stage mass spectrometry unravels the heterogeneity of an Endogenous Protein Complex. Anal. Chem. 89(8):4708-15.
5. Cveticanin J, Netzer R, Arkind G, Fleishman SJ, Horovitz A and M Sharon. (2018). Estimating Interprotein pairwise interaction energies in cell lysates from a single native mass spectrum. Anal. Chem. 90(17):10090-94.
6. Ben-Nissan G, Vimer S, Warszawskia S, Katz A, Yona M, Unger T, Peleg Y, Diskin R, Fleishman SJ and M Sharon. (2018). Rapid characterization of secreted recombinant proteins by native mass spectrometry. Commun Biol. 1:213.
7. Vimer S, Ben-Nissan G and M Sharon. (2020). Direct Characterization of Overproduced Proteins by Native Mass Spectrometry. Nat Protoc, 15(2):236-265
8. Vimer S, Ben-Nissan G, Morgenstern D, Kumar-Deshmukh F, Polkinghorn C, Quintyn RS, Vasil’ev YV, Beckman JS, Elad N, Wysocki VH and M Sharon. (2020). Comparative structural analysisof 20S proteasome ortholog protein complexes bynative mass spectrometry. In Press ACS Cent Sci.
1. 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.
2. Sokolovski M, Cveticanin J, Hayoun D, Korobko I, Sharon M and A Horovitz. (2017). Measuring inter-protein pairwise interaction energies from a single native mass spectrum by double-mutant cycle analysis. Nat. Commun. 9;8(1):212.
3. Gan J, Ben-Nissan G, Otonin G, Tarnavsky M, Trudeau D, Noda-Garcia L, Tawfik D S and M Sharon. (2017). Native mass spectrometry of recombinant proteins from crude cell lysates. Anal. Chem. 89(8):4398-404.
4. Ben-Nissan G, Belov M E, Morgenstern D, Levin Y, Dym O, Arkind G, Lipson C, Makarov A A and M. Sharon. (2017). Triple-stage mass spectrometry unravels the heterogeneity of an Endogenous Protein Complex. Anal. Chem. 89(8):4708-15.
5. Cveticanin J, Netzer R, Arkind G, Fleishman SJ, Horovitz A and M Sharon. (2018). Estimating Interprotein pairwise interaction energies in cell lysates from a single native mass spectrum. Anal. Chem. 90(17):10090-94.
6. Ben-Nissan G, Vimer S, Warszawskia S, Katz A, Yona M, Unger T, Peleg Y, Diskin R, Fleishman SJ and M Sharon. (2018). Rapid characterization of secreted recombinant proteins by native mass spectrometry. Commun Biol. 1:213.
7. Vimer S, Ben-Nissan G and M Sharon. (2020). Direct Characterization of Overproduced Proteins by Native Mass Spectrometry. Nat Protoc, 15(2):236-265
8. Vimer S, Ben-Nissan G, Morgenstern D, Kumar-Deshmukh F, Polkinghorn C, Quintyn RS, Vasil’ev YV, Beckman JS, Elad N, Wysocki VH and M Sharon. (2020). Comparative structural analysisof 20S proteasome ortholog protein complexes bynative mass spectrometry. In Press ACS Cent Sci.