Fig. 3

The comprehensive kinetic model illustrates dual lipidation interplay within the TME signaling pathways. A detailed representation of dual lipidation dynamics in T-cell receptor (TCR) signal transduction, mitochondrial antiviral signaling and Ras-dependent proliferative signaling within the TME. Initially, the proto-oncogene tyrosine-protein kinase Lck undergoes N-myristoylation. Subsequently, Lck undergoes reversible S-palmitoylation at cysteine residues, primarily catalyzed by ZDHHC within the Golgi apparatus. Palmitoylation significantly enhances Lck membrane affinity, promoting localization into lipid raft microdomains. Upon antigen presentation by antigen-presenting cells (APCs), dual lipidation anchors Lck at the plasma membrane, where it phosphorylates ITAM motifs on CD3 chains, initiating the T-cell activation cascade. Downstream signaling involves adaptor proteins (LAT, SLP- 76) whose S-palmitoylation stabilizes signaling assemblies within lipid rafts, enhancing the activity of PLCγ1, triggering secondary messenger production and signal amplification. A parallel dual lipidation mechanism governs small GTPases, notably HRAS and NRAS, involving initial irreversible S-prenylation catalyzed by FTase. This prenylation event primes Ras proteins for subsequent reversible S-palmitoylation via ZDHHC enzymes at the Golgi apparatus, enhancing their membrane microdomains: affinity and facilitating efficient trafficking to the lipid raft of plasma membrane (PM). At the PM, palmitoylated Ras isoforms upload GTP and tranlocated to the disordered (non-raft) regions of the PM then activate downstream effectors (RAF/MEK/ERK pathway), which promote oncogenic signaling cascades critical for proliferation and survival in the TME. S-palmitoylation dynamically modulates Ras localization between distinct cellular compartments. Thioesterase enzymes (APT1/2) reversibly remove the palmitoyl groups, enabling Ras isoforms to cycle between the PM and Golgi. This continuous palmitoylation–depalmitoylation cycling tightly regulates Ras signaling activity and membrane occupancy. The small GTPase Rac1 similarly undergoes dual lipidation cycles. Following viral infection, Rac1 translocates into cholesterol-enriched microdomains within mitochondria-associated membranes (MAMs), where it inhibits the interaction between MAVS and the E3 ligase Trim31. This prevents Trim31-mediated ubiquitination of MAVS, thereby blocking MAVS aggregation and downstream antiviral activation. ABHD: α/β-Hydrolase domain-containing protein; APC: Antigen-presenting cell; APT: Acyl-protein thioesterase; ERK: Extracellular signal-regulated kinase; Fyn: Proto-oncogene tyrosine-protein kinase Fyn; LAT: Linker for activation of T cells; Lck: Lymphocyte-specific protein tyrosine kinase; MAVS: Mitochondrial antiviral-signaling protein; MEK: Mitogen-activated protein kinase kinase; MHC: Major histocompatibility complex; PLCγ1: Phospholipase C gamma 1; Rac1: Ras-related C3 botulinum toxin substrate 1; RAF: Rapidly accelerated fibrosarcoma kinase; RAS: Rat sarcoma viral oncogene homolog; SLP- 76: SH2 domain-containing leukocyte protein of 76 kDa; TCR: T-cell receptor; Trim31: Tripartite motif-containing protein 31