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News

05Jul 2019

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Solving a Molecular Scissors Mystery

A Netherlands Cancer Institute team, co-led by Thijn Brummelkamp and Anastassis (Tassos) Perrakis, reported independently, but almost simultaneously with three more groups from all over the world, on the crystal structure and mechanism of a peculiar molecular end-tail of the microtubules that constitute the cell skeleton.

A cell skeleton is made of cables called microtubules. These allow a cell to maintain its shape, move to different places and transport molecules through its interior. Microtubules also play a key role in cell division.

The frequently used cancer therapeutic paclitaxel, aimed at cells that are dividing, specifically acts on microtubules and thereby affects their detyrosination. In addition, detyrosynation of tubulin has been implicated in cardiac dysfunction, correct segregation of chromosomes during mitosis, and mental retardation.

Microtubules are continuously modified to serve different purposes within the cell. For this, their tyrosine tails are cut and put back by different enzymes. After researchers from the Netherlands Cancer Institute and Oncode Institute, in 2017, found the identity of the scissors that remove the tail, an apparent race was launched to solve the next piece of the puzzle: to determine the 3D structure of these molecular scissors.

This month the Netherlands Cancer Institute team, co-led by Thijn Brummelkamp and Anastassis (Tassos) Perrakis, independently but almost simultaneously with three more groups from all over the world, are reporting on the crystal structure and mechanism of these peculiar molecular end-tail scissors. Tassos Perrakis: 'This means that a beautiful consensus is emerging, supported by complementary experiments which together have been constructing an exciting story.'  

Athanasios Adamopoulos et al., 'Crystal structure of the VASH1-SVBP complex, a 2 tubulin tyrosine carboxypeptidase', Nature Structural & Molecular Biology, 1 July 2019.

Kevin C. Slep. 'Cytoskeletal cryptography: structure and mechanism of an eraser', Nature Structural & Molecular Biology3 July 2019 (News & Views). 

 

Juli2019 molecuul Tassos


Caption:In orange the Vash1 molecule, and in yellow the "arrow" of SVBP penetrating it through the middle. The magenta is the tubulin tail, with the tyrosine pointing downwards, just before it gets cleaved. (courtesy of Tassos Perrakis)

More information:

The cell skeleton

Our cells all have a cell skeleton, made of an extensive network of tubulin cables, also called microtubules. This skeleton allows a cell to maintain its shape, move to different places and transport molecules through its interior. Microtubules also play a key role in cell division, by allowing the cell to meticulously align its chromosomes before dividing them amongst daughter cells.

Cutting off the last tyrosine

The cell modifies these cables continuously, to give them specific  roles. The first modification of microtubules, described 40 years ago, is the removal of the last tyrosine amino acid of the alpha tubulin subunit, in a process called detyrosination.

Such biochemical reactions are typically carried out by single enzymes called proteases. Last year, the Brummelkamp lab in the Netherlands Cancer Institute identified how, unexpectedly, a complex of two proteins working together  - the VASH1/SVBP complex -  was needed to carry out this reaction of cutting off the final tyrosine amino acid. However, what this complex would look like and how it would work remained an unsolved riddle.

The mechanism of action

'The next exciting step for us became to unravel the exact mechanism of action of the VASH1/SVBP complex', says X-ray crystallography expert Tassos Perrakis, who co-led the new study. 'What we wanted to know was: Which specific protein residues are responsible for making the complex between the two proteins, which residues recognize the terminal tyrosine and the glutamate-rich tail of the tubulin peptide, and how does the enzyme complex help the chemical reaction to cleave the tyrosine to take place?'

X-ray crystallography and genetic experiments

In the Perrakis lab, Nassos Adamopoulos, together with Tatjana Heidebrecht, grew crystals of the VASH1/SVBP enzyme and determined the structure of the complex to atomic resolution, by X-ray crystallography. Nassos Adamopoulos: 'Finally, we identified a new curious structural class of cellular proteases that involve a helical "chaperone" that drills through the middle of the protease subunit and allows it to function.' 

Function of the protein

But that was not all. To find the exact residues needed for function, the Perrakis group used computational docking techniques and site-directed mutagenesis, to suggest experiments carried out by Lisa Landskron from the Brummelkamp lab, monitoring the activity of the complex within cells. A couple of rounds of hypotheses testing, iterating between computational and cell biology experiments, were enough to identify the exact residues that are responsible for the function of this protein. 

Overall mission: Finding regulators of key cellular processes

In a longstanding NKI-collaboration, the Brummelkamp and Perrakis research teams seek new regulators of key cellular processes. Once they have identified these regulators, using a genetics approach, they study their function and action in molecular detail. A major example is the modifications and regulation of the tubulin modifications discussed here. Their current quest has not yet ended. Thijn Brummelkamp: 'There must be at least one more protein in the cell, in addition to this unusual complex of VASH1/SVBP, that cuts the last amino acid of tubulin. Currently we are focusing on identifying this.'

Read also:

 The three other papers on the same mechanism:

 

 

 

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