Temporal Profiling Establishes a Dynamic S-Palmitoylation Cycle

Abstract

S-palmitoylation is required for membrane anchoring, proper trafficking, and the normal function of hundreds of integral and peripheral membrane proteins. Previous bioorthogonal pulse-chase proteomics analyses identified Ras family GTPases, polarity proteins, and G proteins as rapidly cycling S-palmitoylated proteins sensitive to depalmitoylase inhibition, yet the breadth of enzyme regulated dynamic S-palmitoylation largely remains a mystery. Here, we present a pulsed bioorthogonal S-palmitoylation assay for temporal analysis of S-palmitoylation dynamics. Low concentration hexadecylfluorophosphonate (HDFP) inactivates the APT and ABHD17 families of depalmitoylases, which dramatically increases alkynyl-fatty acid labeling and stratifies S-palmitoylated proteins into kinetically distinct subgroups. Most surprisingly, HDFP treatment does not affect steady-state S-palmitoylation levels, despite inhibiting all validated depalmitoylating enzymes. S-palmitoylation profiling of APT1/APT2 mouse brains similarly show no change in S-palmitoylation levels. In comparison with hydroxylamine-switch methods, bioorthogonal alkynyl fatty acids are only incorporated into a small fraction of dynamic S-palmitoylated proteins, raising the possibility that S-palmitoylation is more stable than generally characterized. Overall, disrupting depalmitoylase activity enhances alkynyl fatty acid incorporation, but does not greatly affect steady state S-palmitoylation across the proteome.

Protein palmitoylation and cancer

“…However, it appears that in cells APT2 is a cytosolic enzyme while APT1 is localized to the mitochondria, which may explain the selective effect of APT2 inhibition on the palmitoylation of cytosolic proteins . APT1 and APT2 are not the only thioesterases in the cell and in fact do not impact the palmitoylation of key cancer‐related proteins in the cell ; ABHD17A, ABHD17B, and ABHD17C are novel depalmitoylation enzymes that regulate the palmitoylation status of NRAS, for example . Finally, palmitoyl protein thioesterases 1 and 2 (PPT1/2) localize to and deacylate proteins in the lysosome.…”

“…By contrast, protein S‐palmitoylation (hereafter simply palmitoylation), which is the thioesterification of a sixteen‐carbon saturated fatty acid (palmitate) to an internal cysteine residue, is a reversible modification. Indeed, proteins can cycle between palmitoylated and de‐palmitoylated forms on timescales that range from seconds to hours . Dynamic palmitoylation can impact protein localization, accumulation, secretion, stability, and function by altering membrane affinity .…”

Dynamic palmitoylation events following T-cell receptor signaling

“…The understanding of palmitoylated SNARE proteins has generally been restricted to those lacking transmembrane domains (TMDs), such as SNAP-25 and the vesicular SNARE Ykt6, where the addition of hydrophobic palmitate assists in membrane recruitment 20 . However, the vesicular synaptobrevin-family SNARE protein VAMP7, despite featuring a single TMD at its C-terminus, has been reported to be palmitoylated in a large number of different mammalian global palmitome studies 21 – 30 . The observation that many of the proteins found to be palmitoylated are in fact integral membrane proteins (up to ~50–60% of proteins enriched by ABE) suggests the mechanism of regulated palmitoylation is subtler than the simpler model of membrane recruitment 31 .…”

ABHD17 enzymes regulate dynamic plasma membrane palmitoylation and N-Ras-dependent cancer growth

“…HDFP is a broad-spectrum serine hydrolase inhibitor, targeting multiple depalmitoylases including LYPLA1/2, PPT1, ABHD10 and ABHD17A/B/C 17 . This compound 13,17,23 , along with other long-chain FPs 24,25 , have been shown to preserve the palmitoylation state of several dynamically palmitoylated proteins, including Ras proteins 17, 20 , in mammalian cells.…”