Blanc SVSE 2 - Sciences de la vie, de la santé et des écosystèmes : Biologie cellulaire, développement

Structure/function studies of the nuclear export factor CRM1: cooperativity, regulation and inhibition – NucExp

Getting out of the nucleus

This project studies the structure and function of a human protein responsible for transporting cellular and viral proteins out of the nucleus of the cell. It also studies a class of molecules that inhibit the activity of this protein.

Project Objectives

The protein CRM1 plays a key role in the cell, as it is responsible for transporting hundreds of proteins from the nucleus to the cytoplasm. This activity, called «nuclear export«, is fundamental for every eukaryotic cell, including human cells. However, precisely how CRM1 exports proteins (called «cargos«) from the nucleus is poorly understood. For example, CRM1 needs to associate with a partner protein called Ran before binding to and exporting its cargo, but how Ran permits CRM1 de recognize its cargo is unclear. The activity of CRM1 is also modified by a second partner, RanBP3, which changes the selectivity of CRM1 for its cargo, but the molecular basis of this effect is unknown. Furthermore, certain natural compounds called «leptomycins«, interact with CRM1 and inhibit its nuclear export activity. These compounds are of biomedical interest because they may be developed into new drugs against certain cancers and other pathologies. However, precisely how leptomycins interact with CRM1 is not yet known. The aims of the NucExp project are: to better understand the structure and function of CRM1; to characterize its interactions with Ran and RanBP3; and to elucidate in detail how leptomycins interact with and inhibit CRM1.

The strategy used to study CRM1 and its partners involves a combination of biochemistry, structural biology, molecular biology, and synthetic organic chemistry. We use biochemical methods to isolate and purify the three proteins of interest, CRM1, Ran and RanBP3. We subsequently use X-ray crystallography to study the three-dimensional structure of CRM1 alone and in complex with its two partners, Ran and RanBP3. These studies allow us to formulate specific hypotheses about the molecular mechanism by which these proteins function. We can verify these hypotheses by introducing mutations into the proteins and comparing the activity of the original protein to the mutated one in biochemical and biophysical assays. Using organic chemistry methods we synthesize different leptomycins and their analogs in order to study how these molecules interact with CRM1 and inhibit its activity.

We have determined the crystal structure of a domain of RanBP3 which allowed us to better understand how this protein regulates CRM1 activity by modulating its selectivity for different cargos. This work has been published in a peer-reviewed journal. We have also determined the structure of a large fragment of CRM1 that suggests the mechanism by which Ran increases the affinity of CRM1 for its cargos. We could verify this mechanism by constructing various mutants of CRM1 and testing their ability to associate with different cargos and export them from the nucleus. We have also developed a new methodology for synthesizing fragments of a member of the leptomycin family called Callystatin A. We have produced these fragments on a scale sufficient to test their ability to bind CRM1 and inhibit its activity.

This basic research project will allow us to better understand a fundamental activity of every human cell: the transport of proteins from the nucleus to the cytoplasm. CRM1 is responsible for exporting hundreds of cellular and viral proteins, including proteins involved in cancer and infectious disease. Understanding the precise molecular mechanisms underlying nuclear export is essential for better understanding the pathologies associated with the proteins exported by CRM1. Moreover this project aims to determine, at the atomic level, how leptomycins interact with CRM1 and block its activity. These studies should facilitate the development of a new class of drugs for combatting certain diseases.

1. Langer K, Dian C, Rybin V, Müller CW, Petosa C. (2011) Insights into the function of the CRM1 co-factor RanBP3 from the structure of its Ran-binding domain. PLoS ONE 6(2):e17011.
The results described in this paper help to clarify how a protein partner of CRM1 called RanBP3 facilitates the nuclear export of certain proteins.

2. Petosa C. Ran. In Encyclopedia of Signaling Molecules. Choi S. ed. Springer. New York. Sous presse.
This paper summarizes the different roles played by Ran in the cell, including its role in nuclear export and its interactions with CRM1.

CRM1/Exportin1 mediates the nuclear export of proteins bearing a leucine-rich nuclear export signal (NES). In the nucleus CRM1 associates cooperatively with the NES-bearing cargo and with the small GTPase Ran, forming a ternary complex that translocates to the cytosol and subsequently dissociates when GTP is hydrolyzed. Three aspects of CRM1-mediated export are intriguing:

First, the cooperative nature of nuclear export complex formation.
The presence of either Ran or cargo enhances the affinity of CRM1 for the other binding partner. Recently, two crystal structures of CRM1 bound to a cargo protein in the presence and absence of Ran revealed how CRM1 recognizes the NES motif and interacts with Ran. However, the mechanism underlying cooperativity remains unclear.

Second, the regulation of CRM1 by RanBp3.
RanBp3 is a cofactor of CRM1 that strongly enhances the export of certain NES-bearing cargos and diminishes that of others. The mechanism by which RanBP3 modulates cargo-binding selectivity of CRM1 is unknown.

Third, the inhibition of CRM1 by leptomycin B.
Leptomycin and its analogs are potent antibiotics that are exquisitely specific for CRM1. These compounds form a covalent adduct with a cysteine residue in the NES-binding site of CRM1 and sterically inhibit cargo binding. These compounds are of increasing biomedical interest because of their potential use for the treatment of diverse cancers and inflammatory diseases. However, precisely how these compounds interact with CRM1 is currently unknown, hampering progress in the design of improved compounds for therapeutic use.

Here we describe a new collaboration involving three research groups in Grenoble, Marseilles and Amsterdam that will investigate
1. the structural basis of the cooperativity that characterizes CRM1/Ran/NES complex formation;
2. the mechanism by which RanBP3 regulates CRM1-mediated export;
3. the detailed interactions between leptomycins and CRM1.

The work envisaged is a comprehensive effort that combines expertise in structural biology, biochemistry, cell biology, and synthetic organic chemistry. The project will considerably advance our understanding of CRM1-mediated export and should expedite the development of novel LMB-based therapeutics.

Project coordinator

Monsieur Carlo PETOSA (CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE - DELEGATION REGIONALE RHONE-ALPES SECTEUR ALPES) – carlo.petosa@ibs.fr

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.

Partner

CNRS IBS CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE - DELEGATION REGIONALE RHONE-ALPES SECTEUR ALPES
Université Aix Marseille 3 - ISM2 UNIVERSITE AIX-MARSEILLE III

Help of the ANR 429,764 euros
Beginning and duration of the scientific project: - 36 Months

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