Application of mass spectrometry approaches to the study of the function assembly associated factors and architecture of the proteasome

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One of the amazing properties of the cell is its ability to regenerate and renew itself. Old and damaged proteins are constantly being degraded and new proteins are continuously prepared. The major pathway for degradation is the proteasome pathway. The proteasome is a very large complex of at least 46 proteins that compose the cellular degradation machinery. It is a crucial machine that breaks down proteins in a specific manner. To prevent the proteasome from destroying the cell or itself, it destroys only proteins that have been tagged for destruction. One tag commonly used is a small protein, ubiquitin. The regulatory particle (19S) of the proteasome recognizes the labelled proteins and acts as a 'gatekeeper' only allowing targeted proteins into the catalytic particle (20S), which rapidly breaks them down. A great level of information has been accumulated over the years about this important pathway, resulting in the 2004 Nobel prize for chemistry. However, much remains to be learned, as studying such a complex system is not a trivial task. Massive heterogeneous and dynamic assemblies pose difficulties for current structural biology methods. In our laboratory, we use unique mass spectrometry approaches to investigate very large protein assemblies. Although proteins within the complex are bound by very delicate interactions, we are able to maintain these complexes in the gas phase and examine extremely large assemblies such as the proteasome with molecular masses well over two million. Recently we have recorded spectra and measured the masses of the proteasome catalytic particle bound to target proteins. To interpret the data is very challenging not only because the mass difference between free and protein bound forms of the complex are relatively small, but also in understanding the information available from the spectra. We were surprised to find that we could define not only the precise number of occupied proteins but also their location within the proteasome inner cavities (figure 2). Another challenging aspect of our research was to gain structural information regarding the regulatory particle. Using our mass spectrometry approach we were able to determine the spatial organization of proteins in the lid particle, one of the two subcomplexes that form the regulatory particle These findings prompt much of our future research. We aim to follow how the proteasome building-blocks come together to form this large assembly by recording mass spectra in real-time, monitoring the formation of larger complexes from the individual subunits. One of the unresolved mysteries is the specific biological role of a complex thought to be closely related to the 19S lid, known as the signalosome. We aim to investigate how the proteins are organized within the structure of the signalosome assembly. Once we have this information we would like to compare the overall shape of the siganalosome and lid complexes, as recent findings suggest that they can substitute for one another. To address this question we are developing methods that can measure the cross section of ions directly in the mass spectrometer. We also plan to extend our initial study and understand the full subunit arrangement of the 19S regulatory particle (a total of 18 subunits). Revealing the structural organization of the constituent proteins will also shed light on their biological functions. One of the major attractions of this research is that we can analyze proteasome complexes that have been taken directly out of the cell. We will use specific proteins as 'baits' to isolate proteasome complexes and transiently associated proteins. We will then use mass spectrometry to identify the composition and number of copies of these associated proteins. This information will greatly facilitate the understanding of how the degradation machinery is regulated.