The journal Nature Structural and Molecular Biology published a discovery of researchers from IBioBA and the biotech company Genentech in the United States. They developed a technique for identifying neddylated proteins that elucidates a problem that had not been solved for years

Knowing the molecular basis of both the functions of the cells and communication between their internal compartments allows us to understand the possible alterations occurring in disease conditions. This knowledge is one of the first steps for the development of therapeutic strategies for different pathologies.

IBioBA and Genentech researchers developed a method to detect which are the target proteins of Nedd8, a molecule that is necessary for the normal development of the synaptic connections between neurons and the processes of memory and learning. This innovative method was tested in cofilin, a protein that has a relevant role in early neuronal development since it participates in the intracellular process that takes place during the formation of neurons.

 

The unknown is revealed

 

 The normal functioning of the cells requires a complex protein machinery where each protein develops a specific action. These protein functions are finely regulated by subtle modifications (called post-translational) that consist of the binding to these proteins of small regulatory molecules. Among them is Nedd8, present in all cells of the organism and whose mechanism of action has been under investigation for some years.

Damián Refojo, Sebastián Giusti and Raquel Becerra.

This molecule participates in a biochemical event called neddylation by which it binds to proteins to regulate its functions. However, although this modification is abundant in the cell, both the proteins to which it binds and its function on each one have not been clarified because of the lack of a technique with the sensitive to identify its targets.

Cover. Volume 27 Issue 2, February 2020. Image: Wolfgang Zwanzger / Alamy Stock Photo. Cover Design: Erin Dewalt.

Molecular biology specialists have known for some time that neddylation occurs in a type of enzyme called cullines responsible for controlling cell duplication. In 2015, the Molecular Neurobiology group of IBioBA, headed by Dr. Damián Refojo detected that Nedd8 is also present in neurons, a cell type that does not duplicate, and discovered that neddylation is necessary for the normal development and maintenance of synaptic connections between neurons and memory and learning processes. But due to the lack of a sensitive technology, they had not been able to identify all involved proteins modified by neddylation that participate in those processes. “For this reason there has been, for years, a great controversy regarding neddylation. Most researchers consider that Nedd8 only regulates the function of cullinas and others consider that Nedd8 binds and controls many other proteins,” says Damián Refojo, co-director of this study and head of the Molecular Neurobiology Laboratory (IBioBA). This study validates this second hypothesis.

For several years, researchers from Dr. Refojo’s group and their collaborators dedicated their efforts to develop an adequate variant of a technique called mass spectrometry that would allow neddylated proteins to be identified. Through this technique they managed to identify hundreds of proteins modified by Nedd8 and developed the first catalog of neddylated proteins.

Damián Refojo states “the role of Nedd8 on each target protein can now be studied and, as a consequence, gradually determine its physiological relevance in each type of cell.” And adds: “Today we know that neddylation plays an important role in cancer, liver disease and cognitive functions. We are confident that this work will allow us to better understand how Nedd8 exerts its molecular functions and thus contribute to the development of new strategies that can improve these pathologies.”

Test of the method with cofilin

With the technique already created it was necessary to move to the next step: demonstrate the function of Nedd8 on some relevant protein. “Among the 341 molecules identified we chose one of the most actively modified, cofilin. This protein is directly involved on the formation of actin networks, a kind of intracellular skeleton whose growth and plasticity is essential for neurons to acquire their branched morphology and can interconnect with each other”, says Raquel Becerra, co-author of this study. The researchers noted that when loss of neddylation occurs, cofilin undergoes functional changes that affect the morphology of the neuron that could lead to problems associated with neurodevelopment diseases or memory and learning functions.

(Left) Image of a normal developing neuron that begins to emit prologations (in green) that will then allow it to form synaptic connections. These extensions arise from growth cones formed by actin networks (in red) controlled, in turn, by the neddilated Cofilin protein. (Right) When cofilin protein cannot be neddylated, the neuron loses the organization of its actin networks and develops in an abnormal way.

It remains to discover which physiological consequences are produced by neddylation in the other proteins of the catalog created thanks to the joint effort between argentine scientists of IBioBA and Genentech. This will allow continuing the task of understanding intracellular processes that could derive in possible therapeutic targets in disease conditions.

International cooperation

The study was co-directed by Dr. Damián Refojo from the Molecular Neurobiology Laboratory of the IBioBA, and two scientists from the biotechnology company Genentech (United States), Dr. Donald S. Kirkpatrick from Microchemistry Laboratory and Dr. Morgan Sheng from Neurosciences Laboratory.

The experiments were carried out by Raquel Becerra (IBioBA) and Annette Vogl (Genentech) with the collaboration of Sebastian Giusti and Patricio Yankilevich (IBioBA) and Lilian Phu from Genentech. In addition, they received support from the CIBION bionanoscience laboratory head by Dr. Fernando Stefani.

Annette M. Vogl, Donald S. Kirkpatrick and Morgan Sheng.
AUTHORS
  • Annette M. Vogl (Genentech)
  • Lilian Phu (Genentech)
  • Raquel Becerra (IBioBA)
  • Sebastian A. Giusti (IBioBA)
  • Erik Verschueren (Genentech)
  • Trent B. Hinkle (Genentech)
  • Martín D. Bordenave (CIBION)
  • Max Adrian (Genentech)
  • Amy Heidersbach (Genentech)
  • Patricio Yankilevich (IBioBA)
  • Fernando D. Stefani (CIBION)
  • Wolfgang Wurst (Helmholtz Zentrum München)
  • Casper C. Hoogenraad (Genentech)
  • Donald S. Kirkpatrick (Genentech)
  • Damian Refojo (IBioBA)
  • Morgan Sheng (Genentech)