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Teaser, summary, work performed and final results

Periodic Reporting for period 3 - 20SComplexity (An integrative approach to uncover the multilevel regulation of 20S proteasome degradation)

Teaser

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...

Summary

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.

Work performed

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. The results of this work were published in:
• 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
Moreover, we have determined that the activity of DJ-1 is conserved throughout evolution. This lead us to the identification of a highly conserved motif, located at the N-terminal of the protein. Based on this result and in combination with a structural search we have identified a family of proteins with similar ability to coordinate the 20S proteasome. This novel family is currently been investigated.

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. The results were compiled in the following joint publication:
• 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.

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.
Results from this work have been recently published:
• 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

Final results

During the course of the project we have developed several generic native mass spectrometry (MS) methods, as described below:

Developing a native mass spectrometry approach for unraveling the heterogeneity of endogenous protein complexes. (Ben-Nissan et al. (2017). Anal. Chem. 89(8):4708-15)
In-depth MS investigation of the diversity of protein complexes requires analysis across all levels of protein organization, from intact assembly, through its constituent subunits, to the primary sequence of each protein. Sequence analysis is needed, in order to identify the uniqueness of each proteoform, including the type and positions of various post-translational modifications. Subunit analysis is essential for exposing the multiple proteoforms that exist in each subunit, whereas characterization of the intact protein complex reveals both the stoichiometry of subunits and the ensemble of compositions generated through the assembly of different subunit variants. Combining these layers of complexity also sheds light on the cross-talk between subunit proteoforms within the protein assembly and the possible combinatorial use of PTMs.
Only an instrument capable of performing three steps of mass spectrometry fragmentation (MS3), would enable such multilevel analysis. The MS1 spectrum would depict the multiple, coexisting states of the intact assembly. Trapping the ions and dissociation of the protein complex ions into its composing subunits (MS2) would reveal the heterogeneity that exists within individual subunits. Consequently, selecting a specific type of monomer ion and fragmenting it within a collision cell dedicated to this purpose will enable its identification and PTM mapping (MS3). Despite remarkable technological advances in native MS, as far as we know, the aforementioned fragmentation pathway is not yet available for large protein complexes. Therefore, we sought to expand the boundaries of current Technology by creating such a capability, using the recently introduced Orbitrap Extended Mass Range (EMR) platform, which was modified for efficient transmission and detection of high molecular weight ions, and coupled with the advantages of high mass accuracy and high resolving power.
In collaborations with Dr. Alexander Makarov, Orbitrap inventor and director of research in life sciences MS at Thermo Scientific, and Dr. Mikhail Belov, chief executive manager at Spectroglyph LLC, modifications were introduced into our Orbitrap EMR platform to enable MS3 analysis. We demonstrated this approach on the yeast homotetrameric FBP1 complex, the rate-limiting enzyme in gluconeogenesis. We show that the complex responds differently to changes in growth conditions by tuning phosphorylation dynamics. Our methodology deciphers, on a single instrument and in a single measurement, the stoichiometry, kinetics, and exact position of modifications, contributing to the exposure of the multilevel diversity of protein complexes.

Structural characterization of missense mutations using high resolution mass spectrometry ((Ben-Nissan et al. (2016) J. Am. Soc. Mass Spectrom. 27(6):1062-70)
Missense mutations that lead to the expression of mutant proteins carrying single amino acid substitutions underlie numerous diseases. Unlike gene lesions, insertions, deletions, nonsense mutations or modified RNA splicing, all of which affect the length of a polypeptide, or determine whether a polypeptide is translated at all, missense mutations exert more subtle effects on protein structure, which are often difficult to evaluate. Here, we took advantage of the spectral resolution afforded by our modified EMR Orbitrap platform, to generate a mass spectrometry-based approach relying on simultaneous measurements of the WT protein and its missense variants. This approach not only considerably shortens the analysis time due to concurrent data acquisition but, more importantly, enables direct comparisons between the wild-type protein and the variants, enabling identificatio