Saakshi Bangera, DY Patil School of Biotechnology and Bioinformatics
Microtubules are the constituents of the cytoskeleton that help maintain the shape and structure of a cell as well as mediate intracellular transportation. They essentially are polymers of tubulin units and require post-translational modifications (PTMs) to function correctly. They undergo several modifications like methylation, acetylation and phosphorylation of specific residues. PTMs along with the tubulin codes of isoforms of α- and β-tubulin help microtubules perform various cellular functions. In this study, controlled mutation of SETD2 was done to analyse and understand its role in the working of microtubules.
A well-known hypothesis called the ‘tubulin code’ states that certain tubulin isotypes along with a variety of post-translational modifications define the properties and function of microtubules. This concept is analogous to the histone code in chromatin. A crucial contributor of tubulin codes is SETD2 (SET domain containing 2), a lysine methyltransferase dependent on S-adenosyl-methionine. This methyltransferase is responsible for histone-3 lysine 36 trimethylation (H3K36me3) which happens to be associated with gene transcription in chromatin. This ability of SETD2 makes the enzyme unique because, in human cells, there is no such enzyme for lysine 36.
In α-tubulin, SETD2 can methylate the Lysine 40 residue (αTubK40me3). αTubK40me3, in dividing cells, localizes at the negative ends of microtubules that regulate the spindle fibers. Inactivity of SETD2 is problematic because it results in mitotic defects (multipolar spindles, micronuclei), eventually causing reduction of H3K36me3 and αTubK40me3 methylation.
In this report, a truncated version of SETD2 (tSETD2) successfully restored the post-translational modifications (histone and lysine methylation) in cells with inactive SETD2.
Following results were obtained through biochemical analysis
SETD2 methylates tubulin in vitro
A FLAG-tagged version of tSETD2 (tSETD2-FLAG) was introduced to undo the inactivation of SETD2 in mammalian cells. tSETD2-FLAG localized in the nucleus during interphase and scattered throughout the daughter cells.
tSTED2 shows higher activity towards tubulin dimers than microtubule polymers
The relative ability of tSTED2 to methylate microtubules and tubulin dimers was measured. For this, microtubules were maintained in a polymerized state using taxol and the dimers were maintained in an unpolymerized state using podophyllotoxin.
Measurement of enzyme activity of tSETD2-FLAG
The production of the S-adenosyl homocysteine – a product of SETD2 was assayed to measure the enzymatic activity of tSETD2-FLAG. Histone peptide and tubulin peptide both demonstrated increased fluorescence over time, which proves that the reconstituted protein shows activity towards tubulin substrate.
SETD2 only methylates α-tubulin lysine 40
The known methylation site – lysine 40 was mutated to alanine and the αβ-tubulin dimers were purified. Their ability to polymerize into microtubules was observed and it was confirmed that only K40 is methylated by SETD2.
Mutation in SRI domain of tSETD2 alters the ability of tubulin binding and methylation
By methyltransferase assay and purified tSETD2 proteins, it was confirmed that mutation of R2510H in the SRI domain eliminates the ability of tSETD2-FLAG to bind to and methylate tubulin.
SRI domain is critical to recognize substrates for tubulin
To test this, the SRI residues were identified and targeted to generate tSTED2-FLAG variants with alanine mutations along with a deleted variant as a control. The control and the mutant variant, both were purified from mammalian cells.
SETD2 recognizes tubulin via α-tubulin’s C-terminal tail
It was reported that this recognition involves similar charge-charge interactions between positively-charged residues of the SRI domain and negatively-charged C-terminal tails of tubulin.
Significance of the study in cancer treatment
SETD2 can be successfully labelled as an essential component of the microtubular machinery and eventually mitosis. Our knowledge about its role opens new doors to improve the treatment of cancer patients since cancers of the lung, kidney and bladder exhibit loss of activity of STED2. A thorough understanding of tubulin methylation by SETD2 can help propel the anti-cancer drug development to a further point.
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Source:
- Kearns S. et al, Molecular determinants for α-tubulin methylation by SETD2, Journal of Biological chemistry (2021) DOI: https://doi.org/10.1016/j.jbc.2021.100898
- Schuhmacher, M.K., Beldar, S., Khella, M.S. et al. Sequence specificity analysis of the SETD2 protein lysine methyltransferase and discovery of a SETD2 super-substrate. Commun Biol3, 511 (2020). DOI: https://doi.org/10.1038/s42003-020-01223-6
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