Biochemistry students learn that proteins are composed of the 20 biological amino acids linked together by peptide bonds. In reality, many of these 20 amino acids can also be chemically modified inside of cells, creating a profound diversity of possible structures. In addition to the many known enzyme-catalyzed post-translational modifications (phosphorylation, ubiquitylation, acetylation, etc.), some amino acid sidechains can react nonenzymatically with cellular carbonyl-containing compounds. Some of these compounds become abundant during conditions of metabolic or oxidative stress, such as lipid aldehydes. Carbonylation reactions between proteins and lipid aldehydes occur via standard organic chemistry mechanisms, not enzymatic catalysis. The carbonylated proteins are usually not functional and have to be removed by cellular proteolysis mechanisms.
Identification of modified proteins following bacterial expression. The graph shows mass spectrometry data of an unmodified protein (black) and a modified protein which has a mass increase of 258 daltons. The increased mass indicates the covalent addition of phosphogluconate, a bacterial metabolite. From Alnaas et al, J. Biol. Chem. 2021, 296, 100159.
The lipid-binding lysine cluster on Slp-4 reacts with carbonyl compounds. A ribbon diagram of the structure of the Slp-4 C2A domain is shown in green. Membrane-binding lysine and arginine residues are shown as blue sticks, and the membrane-inserting phenylalanine is shown as gray sticks. The two identified sites of phosphgluconoylation were both within the PIP2-binding lysine cluster. From Knight et al, ASBMB Today 2021.
In our recent JBC paper, we reported that the lysine cluster of the Slp-4 C2A domain is especially susceptible to reacting with carbonyl compounds in the bacteria that we were using to express the protein. Similar patterns have been reported for other C2 domains. Although C2 domains did not evolve to function in bacteria, the observation of these reaction products raises several questions: How general is the carbonyl reactivity of C2 domain lysine clusters? What eukaryotic compounds do these proteins react with? Is this reactivity important in eukaryotic cells under oxidative stress, for example in inflamed pancreatic islets during the onset of type 1 diabetes? How does lysine modification alter protein function? We are exploring these questions in our ongoing research in collaboration with Dr. Colin Shearn, an expert in protein carbonylation. We are also collaborating with diabetes experts thanks to a Pilot/Feasibility Grant from the CU Diabetes Research Center, and with proteomics experts on the CU Anschutz Medical Campus.