DS0401 - Etude des systèmes biologiques, de leur dynamique, des interactions et inter-conversions au niveau moléculaire

Functional dynamics of a nanomachine involved in proteome quality: a combined NMR/SAXS/SANS study of the PAN unfoldase – PROTstretch

PROTstretch

Functional dynamics of a nanomachine involved in proteome quality: a combined NMR/SAXS/SANS study of the PAN unfoldase

Understanding the function and degradation of active protein degradation in cells

The specific degradation of proteins in cells is one of the major mechanisms, along with transcription and expression control, that regulates an ensures a healthy proteome (i.e. the ensemble of proteins) in living cells. A key step in this process is the specific recognition of deleterious proteins and their transfer to the proteolytic machinery. In the present project, we study the conformational dynamics of the specific recognition and unfolding of proteins by an archaeal unfoldase complex, the PAN system. The aim is to understand the specific recognition of the substrate as well as the conformational motions responsible for the unfolding process.

The present project proposes an innovative combination of NMR, SAXS/SANS and fluorescence to study conformational changes of both the unfoldase enzyme PAN and the GFP substrate during the unfolding process. The major techniques are complemented by several biophysical methods such as AUC, SEC-MALLS, SPR etc. to characterize the monodispersity and purity of the samples.

After 18 months, we have a first set of SAXS/SANS data that describes the conformational changes of both the PAN unfoldase and the GFP substrate during the active unfolding process as a function of time. The project has also allowed to stimulate the development of a unique online fluorescence device that allows to record spectroscopic data in parallel to neutrons at the Insitut Laue-Langevin at Grenoble.

We believe that the methodological approach (combination of time-resolved NMR and SAXS/SANS studies) developed in the present ANR project will be applicable to a wide range of complexes that remodel the proteome.

Time-resolved neutron scattering provides new insight into protein substrate processing by a AAA+ unfoldase. (2017) Ibrahim Z, Martel A, Moulin M, Kim HS, Härtlein M, Franzetti B, Gabel F. Sci Rep. 7:40948.

The aim of the present French-German PRCI ANR project is to gain insight into the molecular mode of action of an important class of biomacromolecular machines, the so-called AAA ATPase unfolding machines.

A healthy proteome, i.e. the ensemble of proteins present at a given point in time in a cell, is essential for the correct functioning of any organism. Numerous regulatory mechanisms exist at the gene expression and post-translational levels that control the amount, specificity and activity of proteins in response to variable internal and external environments. In living cells one of these mechanisms consists in trapping and destroying disabled proteins. This function is critical since abnormally folded proteins have a tendency to aggregate and can provoke irreversible damages to the cell. The central challenge for specific protein degradation is to complete the unfolding of proteins that display an abnormal conformational state or that are no longer needed. This task is accomplished by different classes of AAA ATPases unfolding machines (unfoldases) that prepare the proteins for destruction via the proteasomes.

Because of their pivotal position in the protein degradation pathway, unfoldases are now discussed as potential lead compounds for therapeutic intervention. In humans, an altered function in defective protein elimination can cause a number of destructive diseases such as Alzheimer’s or Huntington’s and is also at the basis of infective diseases such as prions. Neurodegenerative diseases, in particular, are more likely with advancing age, which constitutes a present and future health issue of an aging world population. In this context understanding in depth the mode of action of unfoldase machineries represent a major challenge in biomedicine.

“Classical” structural biology techniques, such as crystallography, are limiting when applied to such complex dynamic and oligomeric systems and cannot provide a full understanding of their mode of action. Here we address these questions using a powerful combination of structural biology techniques in solution: small-angle X-ray (SAXS) and neutron (SANS) scattering as well as nuclear magnetic resonance (NMR). We propose to apply this combination of techniques to the PAN system using GFP as a substrate. PAN is the archaeal analog to the eukaryotic 19S system associated with the 26S proteasome. It is a 250 kDa ATP-fueled hexameric unfoldase displaying an intriguing structure in the form of a hollow half-sphere with six appendices. So far, the entire complex has resisted to crystallography and high-resolution structures or homology models are available only for parts of the machinery.

The consortium proposed is in a unique position in France and Germany to contribute significant structural insights into the unfolding function of PAN and to develop new methods of combining complementary techniques for structural molecular biology. Both coordinators are internationally renowned specialists of small-angle scattering and NMR, respectively, and have collaborated in the past on major projects involving large, challenging complexes. Both groups are highly complementary: the Carlomagno group is specialized in structural NMR of large biomolecular systems and has access to high-performance spectrometers, while the Gabel group is expert both in the biochemistry of the PAN system and in the SAXS/SANS techniques. In addition, the Gabel group has the possibility to carry out experiments in a timely manner at the nearby European Synchrotron Radiation Facility (ESRF) and the Institut Laue-Langevin (ILL) neutron source.

Project coordination

Frank GABEL (Institut de Biologie Structurale)

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

Leibniz University Hanover
IBS Institut de Biologie Structurale

Help of the ANR 234,204 euros
Beginning and duration of the scientific project: April 2016 - 36 Months

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