Proteasome Assembly Assisted by Deubiquitinating Enzyme Ubp6

The 26S proteasome is an energy-dependent protease responsible for the degradation of poly-ubiquitinated proteins in eukaryotic cells. Energy-dependent proteolysis is essential in eukaryotic organisms, and malfunction leads to a variety of disease states, including various cancers and other degenerative diseases such as Alzheimer’s.¹ 26S complexes can be divided into a 20S core particle and a 19S regulatory particle. 20S core particles contain the protease domain sequestered inside barrel-shaped structures formed by the arrangement of 28 subunits into a stack of four heptameric rings. The outer rings, made up of alpha-type subunits, interact with 19S regulatory particles, while the inner rings, made up of beta-type subunits, contain the proteolytic activity. 19S regulatory particles are responsible for the binding, unfolding, deubiquitination, and translocation of substrates to the interior chamber of core particles for degradation.

19S regulatory particles consist of 19 different subunits arranged into two substructures: the base and the lid. The base interacts with the outer rings of the 20S core particle and has 9 different subunits, 6 ATPases that form a ring (Rpt1-6) and 3 non-ATPase subunits (Rpn1, Rpn2, and Rpn13) that interact with different subunits of the ring and bind ubiquitinated substrates of the proteasome. The lid portion of the RP has 10 different subunits (Rpn3, Rpn4-9, 11, 13, and Sem1). Rpn10 is found at the interface between the lid and the base and may help stabilize the complex.²

While much is known about the overall composition of the regulatory particle and the subunit interactions, there is no crystal structure. Recently, a cyro-electron microscopy structure of the 26S complex was solved, but this stucture lacks the atomic resolution of a crystal.³ Biochemical studies have identified several other proteins that interact with the regulatory particle. Some chaperones aid in the assembly of the complex.

A recent report reveals that a deubiquitinating enzyme Ubp6 plays a role in the assembly of the 19S regulatory particle through the activity of removing ubiquitin from substrate proteins.⁴ Nanospray mass spectrometry on an LTQ Orbitrap (Thermo Scientific) of intact 26S proteasome and assembly intermediates revealed the association of Ubp6 with the base substructure of the regulatory particle in complex with other chaperones such as Nas6 and Hsm3. These associations are transient since neither protein was present in mature assembled 26S proteasomes. Knocking out both Hsm3 and Ubp6 severly impaired the growth of yeast at high temperature. Interestingly, the activity of Ubp6 (the removal of ubiquitin chains from substrates) was required for the proper assembly of the base. This is likely because the base of the regulatory particle, and in particular Rpn1, binds polyubiquitin chains, even when not in complex with the other subunits. The presence of these substrates likely interfere with the proper assembly of the regulatory particle and need to be removed prior to the incorporation of the remaining subunits and interaction with the 20S core particle.⁴

Taken together, a new assembly intermediate of the regulatory particle was identified through mass spectrometry. Also, a role for the deubiquitinating activity of Upb6 in the assembly of the base ATPase ring was hypothesized and a phenotype for the double knockout of hsm3 and ubp6 was established. These results provide a more complete understanding of assembly of the 19S regulatory particle and provide insight into possible roles other proteins play during assembly.

References

1. Schwartz, A.L. and A. Ciechanover (2009) ‘Targeting proteins for destruction by the ubiquitin system: Implications for human pathobiology‘, Annual Reviews in Pharmacology and Toxicology, 2009 (49), (pp. 73-96)

2. Seeger, M., et al. (2003) ‘Interaction of the anaphase-promoting complex / cyclosome and proteasome protein complexes with multiubiquitin chain-binding proteins‘, Journal of Biological Chemistry, 278 (19), (pp. 16791-16796)

3. Bohn, S., et al. (2010) ‘Structure of the 26S proteasome from Schizosaccharomyces pombe at subnanometer resolution‘, Proceedings of the National Academy of Sciences USA, 107 (49), (pp. 20992-20997)

4. Sakata, E., et al. (2011) ‘The catalytic activity of Ubp6 enhances maturation of the proteasomal regulatory particle‘, Molecular Cell, 42 (5), (pp. 637-649)