Control of protein stability by post-translational modifications

Abstract

Post-translational modifications (PTMs) can occur on specific amino acids localized within regulatory domains of target proteins, which control a protein's stability. These regions, called degrons, are often controlled by PTMs, which act as signals to expedite protein degradation (PTM-activated degrons) or to forestall degradation and stabilize a protein (PTM-inactivated degrons). We summarize current knowledge of the regulation of protein stability by various PTMs. We aim to display the variety and breadth of known mechanisms of regulation as well as highlight common themes in PTM-regulated degrons to enhance potential for identifying novel drug targets where druggable targets are currently lacking.

Fig. 1. Examples of two opposite archetypal regulatory modules: the PTM-activated degron and PTM-inactivated degron.

a PTM-activated degron: Writer enzymes generate PTMs at specific residues within target proteins that are recognized by an E3 ubiquitin ligase complex for proteasome degradation. PTM-activated degron is a natively inactive degron structure, modified, or otherwise activated by the addition of one or more PTMs, ultimately leading to the proteolytic degradation of a protein. b PTM-inactivated degron: PTMs added to specific sites on protein substrates to inhibit their interaction with the protein degradation machinery. PTM-inactivated degron is a destabilization motif that is inactivated by the addition of one or more PTMs.

Fig. 2. Molecular function of lysine and arginine methylation in control of protein stability.

Lysine and arginine methylation regulates the function of protein by altering stability. Of note, lysine methylation converts protein stability negatively by methyl-degron (light green) and arginine methylation mainly increases protein stabilization by methyl-inactivated degron (dark green). Methyltransferase EZH2, G9a, and SETD7 modify lysine site via mono-methylation and further stimulate poly-ubiquitination of substrates by reader protein such as DCAF1 and L3MBTL3.

Fig. 3. Diverse molecular mechanisms of PTM-control of protein degradation.

Various different molecular mechanisms for PTMs changing a protein’s proteolytic stability have been found. DTL (for Denticleless protein homolog, also called CDT2) is the human orthologue of yeast Sic1 and itself a substrate adapter of the DCX E3 ligase complex. DTL is phosphorylated at T464, which promotes interaction with 14-3-3, thus shielding it from degradation by SCF^FBXO11,.

Fig. 4. Protein stability controlled by methylation, acetylation, hydroxylation, and SUMOylation.

a HIF-1α is regulated by acetyl-inactivated degron, hydroxyl-activated degron, and methyl-activated degron. p300 acetyltransferase generates acetyl-inactivated degron of HIF-1α and HIF-1α acetylation triggers stabilization in acetylation-dependent manner. Because hydroxylation stimulates methyl-activated degron of HIF-1α, hydroxylation reduces protein stability of HIF-1α directly or indirectly. b Examples of the regulation of protein stability by SUMOylation. SUMOylation of PML-RAR induces poly-ubiquitination and further degradation of PML-RAR. In contrast, SUMOylation and multiple phosphorylations of tau cooperatively increase protein stability of Tau.

Fig. 5. PTM cross-talk at degrons.

a Examples of the regulation of MYC stability by different PTMs. In many cases, PTM networks are formed as one PTM acts as a priming factor for the next, by establishing protein-protein-interaction interfaces. b Examples of the regulation of p53 stability by PTMs. Note, multiple other PTMs have also been shown to affect p53 turnover in addition to the ones shown.

Fig. 6. Proteomic profiling of effects of PTM modulating enzymes on proteolytic stability.

Schematic for using multiplexed proteome dynamics profiling for detecting the effects of PTM writer and eraser enzymes on protein degradation. Cells are cultured in light SILAC medium or heavy SILAC medium. Following a switch to heavy SILAC medium (replicate 1) or light SILAC medium (replicate 2), and subsequent inhibition of writer or eraser enzymes, samples are collected after a suitable time point, lysed, digested with trypsin, labeled with isobaric mass tag reagents (TMT), and pooled. Next, samples are processed by mass spectrometry. Tandem mass spectra generated from light and heavy SILAC signals will contain TMT reporter ions that can be used for relative quantification of changes in degradation rates between the different conditions (control, inhibition of writer enzyme, inhibition of reader enzyme) from the two biological replicates.