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Project ID: 657725
Funded under: H2020-EU.1.3.2.

Periodic Reporting for period 1 - APCINTERACTIONS (Molecular basis for securin and cyclin ubiquitylation by the anaphase-promoting complex (APC/C))

Reporting period: 2016-03-01 to 2018-02-28

Summary of the context and overall objectives of the project

Equal distribution of the genetic information (each sister chromatid is distributed into the two emerging daughter cells) is the primary goal during cell cycle progression. High accuracy of this process is crucial to maintain the integrity of the genetic information and is achieved by a number of highly specialized proteins and protein complexes. At the very centre of this process is a protein complex termed the anaphase-promoting complex (APC/C). This complex marks a number of regulatory proteins (e. g. securin) for timely degradation via a specialized pathway. Destruction of securin, in turn, leads to the activation of an evolutionary conserved protease named separase. Proteases are molecular scissors that can cleave and therefore destruct protein targets. Separases promote sister chromatid separation in mitosis through cleavage of a protein complex called cohesin, which forms a ring like structure that physically entraps sister chromatids, and thus cleavage of this ring allows for timely sister chromatid separation (when equal distribution of the genetic information is desired).
In summary, all projects carried out during the time of the fellowship aimed at understanding the molecular details of how cells duplicate, a process that is impaired in cancer and hence these studies might lead to the development of new anti-cancer drugs.

Securin is an inhibitor of separase. Previous studies suggested - albeit lacking structural evidence and therefore the atomic details - that securin is likely to bind as a pseudo-substrate to the cleavage site of the separase, thereby inhibiting its activity (Nagao et al., 2006).

Separase has previously been proposed as a promising and clinically applicable proliferation marker (Gurvits et al., BJC 2017). Elevated levels of separase, for instance, has been shown to be linked to aneuploidy (the unequal distribution of sister chromatids) and mammary tumorigenesis (Zhang et al. PNAS 2008). Consequently, separase expression levels could be utilized as a biomarker for treatment decisions in e.g. breast carcinoma treatments.

In this work, we aimed at understanding the molecular mechanisms of securin-mediated separase inhibition in atomic detail. We employed a mixture of biochemical and structural approaches to further characterize the formation of the separase-securin complex, with a main focus on cryo-electron microscopy (cryo-EM) studies.

A second project aims at understanding the molecular details of the interaction of the human separase-securin complex with the APC/C. Such a structure would not only provide critical insights into substrate recognition of the APC/C but would also represent the first structure of the APC/C bound to a natural, full-length substrate determined.

Lastly, we have also directed our efforts towards obtaining a higher resolution structure of the human APC/C complex. The APC/C might represent an interesting drug target in cancer treatment and as such high-resolution structure information will be of invaluable interest.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

Cryo-electron microscopy is a method to determine the molecular structure of proteins and protein complexes that has recently been awarded with the nobel prize in chemistry (2017). It was used in our study to determine the atomic structure of a separase-securin complex from the roundworm C.elegans. These results are potentially interesting for future drug design in human cancers, as structures and molecular mechanisms are often highly conserved in evolution. The results have been published in a prestigious scientific journal.

To obtain such structural information the protein complex of interest needs to be produced in cells, separated from all other contaminants present in the cell and finally imaged in an electron microscope.
In brief, during the time of the fellowship I first established the protein purification as well as the freezing- and imaging conditions of a separase-securin complex from C. elegans. We were then able to determine the first atomic structure of a metazoan complex at 3.7 Å resolution.

We also established the purification of the human separase-securin complex in milligram quantities. This has not been reported in the literature before and provides a major breakthrough in the feasibility of performing activity assays (cleavage assays). In vitro expression of proteins eases the possibility of purifying and testing specifically designed mutants. Testing the wild-type protein complex or specific mutants is vital to fully understand the function of the protein complex of interest and consequently might be important in designing specific inhibitors.

As mentioned above, a second project aims at the determination of the human separase-securin complex bound to the human APC/C. To this end, the purifications of both, the human separase-securin complex as well as a specific version of the APC/C have been optimised and complex formation can be observed. These are very important and challenging initial steps that have already been mastered.

We have also directed our efforts towards obtaining a higher resolution structure of the human APC/C complex. We have modified our data collection and data processing strategies. and initial results are looking very promising and we are hoping to finalise this project in the next few months. To obtain a high-resolution structure would likely provide more biological insights and could potentially aid drug design, as designing specific anti-cancer drugs does indeed require accurate models.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

Future work on this project will include a high-resolution structure determination of the human separase-securin complex. The human separase-securin complex might be a potential drug target in certain breast cancer types and consequently it is of high interest and impact, obtaining a high-resolution structure.

Here, we have already achieved a medium resolution envelope of the structure at around ∼7 Ångstrom, however this resolution does not allow building an atomic model and therefore obtaining higher resolution information is key. During this fellowship we realised that high-resolution information cannot be obtained yet, due to interactions of the protein molecules with the air-water when prepared for cryo-EM studies. This phenomenon is a well-known hurdle in cryo-EM and limits the number of available views that can be imaged of a particular protein complex. As a consequence, structural information is limited to lower resolution or (in extreme cases) impossible to obtain.

Another option to obtain high-resolution structure information on the human separase-securin complex, would be by obtaining a structure of the human APC/C bound to separase-securin. First step of this approach, have already been successful and are part of our current research in the lab.
Future milestones in this project will include the stabilization of this complex using different chemical compounds. To discover the right chemical compound is often a laborious and lengthy process.

A last line of work that is currently very successfully undertaken in our lab is the determination of a high-resolution structure of the APC/C. Here, we expect to get new data within the next few months.

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