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My research has been
supported by a Marie-Curie International Incoming Fellowship (2008-2010;FP7-MC-IIF-219452).
Below is a summarised version of a recent report detailing the outcomes of
the project, written for the general public: Introduction
Cells
are the fundamental building blocks of all living organisms. Although there are
many different mature cell types within a particular organism (e.g., blood,
kidney and nerve cells), they nearly all contain the same ensemble of DNA
sequences. Genes encoded within these DNA sequences are activated or
deactivated in different cell types, depending on the functional requirement
of the cell. The DNA is located within the cells nucleus, and this region can
be visualised under a microscope by using fluorescent stains that
specifically interact with DNA (Figure 1). Other stains (antibody-based) can
be used to reveal internal nuclear regions such as nucleoli and Cajal bodies
that contain specific protein and DNA molecules, demonstrating that the
nucleus is highly organised.
Figure 1: The cell nucleus can be
visualised under the microscope using specific stains for DNA (left). The
same nucleus can be co-stained to reveal subnuclear structures such as
nucleoli (No) and Cajal bodies (CB). Cells
respond to changes in their environment, such as stress, in order to either
adapt and therefore survive the change, or die to prevent build-up of unhealthy
cells. Many cellular stress responses involve changes within the cell nucleus
and ultimately result in changes to which genes are activated. These changes
are mediated by protein molecules that have received signals to change either
their structure, location within the cell or interactions with other molecules.
For example, it has been shown that some proteins become attached (or
post-translationally modified) to a particular class of protein molecules
collectively referred to as SUMOs (small ubiquitin-like modifiers) in either
normal or stress conditions. These stress response pathways are
fundamental processes that operate in most cell types, and thus it is
important to understand these at a highly detailed, molecular level. Aim and approach
-
To understand changes that occur
within the cell nucleus and subnuclear regions such as nucleoli in normal and
stress conditions -
Conduct a screen to identify
proteins in nucleoli that can be modified by SUMO using the latest proteomic
technology -
Confirm the results of the screen
and obtain molecular insight about the role of SUMOylation for some of the
identified target proteins. Results and conclusions
Our screen identified the proteins Nop58, dyskerin, Nhp2 and Nopp140 as major candidates for modification by SUMO in the nucleolus. These proteins are involved in one of the major functions of the nucleolus, namely to generate ribosomes. Ribosomes are machines that synthesise new protein molecules. Although protein synthesis takes place outside the nucleus, ribosomes are largely put together in nucleoli. We
confirmed Nop58 and Nhp2 are indeed modified by SUMO, in both cell-free
reactions and analyses within living cells. Nop58 and Nhp2 are made up of 529
and 153 amino acids, respectively, and we next identified precisely which
amino acids were attached to SUMO, namely two (K467 and K497) in Nop58 and
one (K5) in Nhp2. Related proteins known as Nop56 and nhpx that are composed
of very similar amino acid sequences to Nop58 and Nhp2, respectively, were
shown not to be SUMOylated. This demonstrated that we had uncovered a very
specific mechanism to regulate the function of proteins such as Nop58 and
Nhp2. Finally, we discovered that although SUMO-Nop58 appeared to localise
correctly to nucleoli and Cajal bodies, its interactions with one of its
major binding partners (small nucleolar RNA) appeared to be stronger than
that of Nop58 alone. This suggested that SUMOylation of proteins such as
Nop58 could indirectly play a role in ribosome biogenesis, which provides a
major new insight into this field. The successful conclusion of the project
was underlined by these new results being accepted for publication in a
leading international journal, Molecular Cell. Impact, Target Groups and
Socio-Economic Impact
We have discovered the existence of molecular pathways that link proteins involved in ribosome biogenesis, SUMOylation and the nucleus/subnuclear bodies. Since ribosome biogenesis occurs in nearly every cell type, these findings advance our current understanding of fundamental cellular pathways. In the short term, this project will be of greatest interest for researchers interested in understanding basic molecular processes, such as ribosomal assembly and posttranslational modification of nuclear proteins. However, in the long term, our findings could be targeted toward pharmaceutical companies or academic researchers interested in developing therapeutic agents, especially if we can establish that different levels of SUMOylated Nop58 are related to particular disease states. Back to Group
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