Graduation Year


Document Type




Degree Name

Doctor of Philosophy (Ph.D.)

Degree Granting Department

Medical Sciences

Major Professor

Robert J. Deschenes, Ph.D.

Committee Member

Yu Chen, Ph.D.

Committee Member

Gloria Ferreira, Ph.D.

Committee Member

Keiran Smalley, Ph.D.

Committee Member

Luc Berthiaume, Ph.D.


Acylation, Erf2, High-throughput Screen (HTS), Enzyme Inhibition, zDHHC


Palmitoylation refers to the covalent attachment of fatty acids, such as palmitate, onto the cysteine residues of proteins. This process may subsequently alter their localization and function. Nearly all of the enzymes that catalyze palmitoylation, zDHHC protein acyl transferases (PATs), are implicated in neurological disorders, infectious diseases, and cancer in humans. Of particular interest to those who study palmitoylation are Ras family GTPas and zDHHC9-GCP16, the zDHHC PAT that palmitoylates Ras proteins. Erf2-Erf4 is the zDHHC PAT that palmitoylates Ras proteins in Saccharomyces cerevisiae. Currently, there are no methods to therapeutically target palmitoylation for the treatment of disease. One of the barriers to identifying a modulator of palmitoylation is the lack of a reliable high-throughput screening system. To date, few assay systems have been developed to examine the kinetics and mechanism of that palmitoylation reaction. This lab has developed a fluorescence-based coupled assay to gain insight into the enzymology, biochemical mechanism, and kinetics of the palmitoylation reaction. This assay may be used to identify specific inhibitors of autopalmitoylation. In the first step of this reaction, the palmitoyl-moiety from palmitoyl-CoA is transferred to the zDHHC9 PAT cysteine side chain to form a palmitoyl:enzyme intermediate. The second step of palmitoylation is the subsequent transfer of the palmitoyl-moiety from the palmitoyl:enzyme intermediate to the cysteine residue of the substrate protein. This fluorescence-based coupled assay was utilized to screen a natural products library and a unique synthetic compound library for inhibitors of Erf2 autopalmitoylation. These screens led to the identification of fungal metabolite extracts and ten bis-cyclic piperazine compounds that inhibit Erf2 autopalmitoylation in the low micromolar range. This effect is similar to known inhibitors of palmitoylation that lack specificity for the palmitoylation reaction itself.