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Schey, Garrett

Garrett Schey

University of Minnesota

Talk Session: SESSION 1: POST-TRANSLATIONAL MODIFICATIONS
Date: Sunday, June 12, 2022
Talk Time: 08:55 am – 09:10 am
Talk Title: Expanding the Scope of Extended Farnesyltransferase Substrates with a MALDI/MS Screening Approach

Protein prenylation is a post-translational modification where a 15 carbon farnesyl or 20 carbon geranylgeranyl isoprenoid is appended to the C terminal end of a protein by either farnesyltransferase, FTase, or geranylgeranyl transferase type 1, respectively. In the canonical understanding of FTase, the isoprenoids are attached to the Cysteine residue of a four amino acid CaaX box sequence. However, recent work has shown that five amino acid sequences can be recognized, such as the pentapeptide CMIIM. This new discovery greatly increases the number of potential FTase substrates, as the enzyme is already known to tolerate a wide variety of amino acids in the canonical CaaX box.

With the goal of developing a more rapid and methodical method to evaluate potential substrates, we envisioned using MALDI to assay libraries of 10 peptides at a time, varying one amino acid in the CaaaX box to all 20 canonical amino acids over two libraries, utilizing both yeast and rat FTase. Through this method we observed over 30 hits in the mass spectrum and chose eleven for further evaluation. Nine of these sequencesare novel substrates for FTase, with several meeting or surpassing the in vitro efficiency of the benchmark sequence CMIIM. Additionally, in vivo experiments in yeast demonstrate that proteins bearing these sequences can be efficiently prenylated in a biological context. Searching the human genome for pentapeptide CaaaX sequences found several hits that prenylated with similar efficiency to a native CaaX sequence, raising the possibility of relevance of these sequences in humans.

Garrett Schey, talk image 2

Garrett Schey
Garrett Schey, talk image 1
Garrett Schey, talk image 3
Garrett Schey, talk image 4