Post-translational modification (PTM) of proteins includes the covalent addition of various lipids (e.g., fatty acids, isoprenoids, and cholesterol), which increases protein hydrophobicity to influence their localization and function. While this process is important to normal cell signaling, many proteins have gained attention as targets for lipid modifications due to misregulations observed in disease states (Figure 1). For example, Sonic hedgehog protein (Shh) is modified by cholesterylation before undergoing N-palmitoylation, which regulates signaling that is important for embryonic patterning and stem cell biology. The signaling protein Wnt can be modified by S-palmitoylation, which controls the transport of this ligand to its receptor during critical events in embryonic development, cell proliferation, and insulin sensitivity. The Ras superfamily of GTPases undergoes farnesylation, geranylgeranylation, and S-palmitoleoylation, which plays a central role in cancer development. Therapies targeting these modifications have the potential to impact a range of pathologies from cancer to viral infection. Therefore, developing specific tools to study them is essential. Cayman offers a variety of chemical tools to identify and quantify protein lipidation. The most widely applicable are alkyne or azide-tagged lipid analogs that can be used in cell culture and in vitro studies as a chemical method for labeling and isolating lipidated proteins. Below we cover the basic biology of two of the most prominent lipidation modifications—acylation and prenylation—and provide an overview of the types of products Cayman offers to study them.

Figure 1.

PROTEIN ACYLATION

Protein acylation is catalyzed by acyltransferases. Palmitoylation, protein modification with palmitic acid, is the most common form of protein acylation, though other lipids, such as stearic acid or arachidonic acid, have been observed. Palmitoylation facilitates the association of proteins with cell membranes, mediates protein trafficking, and can regulate protein stability. S-Palmitoylation on cysteine residues is entirely reversible through the action of deacylases (depalmitoylases). The addition and removal of palmitate regulates protein distribution and function. In contrast to S-palmitoylation, N-palmitoylation is a stable modification of cysteine residues occurring at the protein’s N-terminus.

Wnt proteins are palmitoleoylated on a highly-conserved serine residue by porcupine (PORCN), a membrane-bound O-acyltransferase (MBOAT) that resides in the ER. Palmitoleoylation of Wnt proteins initiates their secretion and binding to the Frizzled receptor (Figure 2). In certain cancers with Wnt sensitivity, the inhibition of PORCN activity has become a therapeutic target for limiting Wnt secretion. The MBOAT, Hedgehog acyltransferase, N-palmitoylates Shh proteins prior to secretion from the endoplasmic reticulum, which is critical for their signaling range and efficacy. Cayman offers a broad collection of PORCN inhibitors, depalmitoylase inhibitors, acyltransferase antibodies, and alkyne-linked palmitic acid probes to help characterize the function of key palmitoylated proteins (see product table below).

Figure 2. Image adapted from Lories, R.J., Corr, M., and Lane, N.E. Nat. Rev. Rheumatol. 9, 328-339 (2013).

PROTEIN PRENYLATION

Prenylation is the enzymatic addition of either a farnesyl or a geranylgeranyl group to a C-terminal cysteine within the recognition sequence known as CaaX box. Proteins that undergo prenylation include those involved in cell cycle progression, oncogenesis, and parasitic infection. The process is catalyzed by three different enzymes: farnesyltransferase (FTase), which adds a 15-carbon farnesyl group to proteins with the CaaX box, and two different geranylgeranyltransferases (GGTase I and RabGGTase) (Figure 3). GGTase adds 20-carbon geranylgeranyl groups to proteins with a CaaX sequence when X is leucine, and RabGGTase acts on Rab proteins, which do not have a CaaX box consensus sequence, with the assistance of a Rab escort protein. The isoprenoid addition drives proteins to associate with the endoplasmic reticular membrane where the converting enzyme RCE1 will cleave three terminal amino acids in order for isoprenylcysteine carboxyl methyltransferase (Icmt) to methylate the C-terminal prenylated cysteine. This sequence of events results in increased membrane affinity of the target proteins and is key for proper membrane association and protein-protein binding. Cayman offers a collection of potent and specific small molecule inhibitors of FTase, GGTase, and Icmt, as well as precursors, substrates, and an azide-linked farnesyl alcohol probe to study the processes of farnesylation and geranylgeranylation (see product table below).

Figure 3. Image adapted from Triola, G.J. Glycom. Lipidom.S2:001 (2011).

CLICK CHEMISTRY PROBES

PORCN INHIBITORS

ACYLTRANSFERASE ANTIBODIES

DEPALMITOYLASE INHIBITORS

FTASE AND GGTASE INHIBITORS

ICMT INHIBITORS

PRECURSORS, DONOR, AND SUBSTRATES TO FARNESYLATION AND GERANYLGERANYLATION