Mannose metabolism inhibition sensitizes acute myeloid leukaemia cells to therapy by driving ferroptotic cell death.

Resistance to standard and novel therapies remains the main obstacle to cure in acute myeloid leukaemia (AML) and is often driven by metabolic adaptations which are therapeutically actionable. Here we identify inhibition of mannose-6-phosphate isomerase (MPI), the first enzyme in the mannose metabolism pathway, as a sensitizer to both cytarabine and FLT3 inhibitors across multiple AML models. Mechanistically, we identify a connection between mannose metabolism and fatty acid metabolism, that is mediated via preferential activation of the ATF6 arm of the unfolded protein response (UPR). This in turn leads to cellular accumulation of polyunsaturated fatty acids, lipid peroxidation and ferroptotic cell death in AML cells. Our findings provide further support to the role of rewired metabolism in AML therapy resistance, unveil a connection between two apparently independent metabolic pathways and support further efforts to achieve eradication of therapy-resistant AML cells by sensitizing them to ferroptotic cell death.

Regulation and function of the palmitoyl-acyltransferase ZDHHC5.

Protein palmitoylation (S-acylation) has emerged as an important player in a range of cellular processes, and as a result, the palmitoyl-acyltransferase (PAT) enzymes which mediate this modification have entered into the spotlight. Palmitoyltransferase ZDHHC5 (ZDHHC5) is among the more unique members of the PAT family as it is mainly localised to the plasma membrane and contains an extended cytoplasmic domain with several regulatory features. ZDHHC5 plays a vital role in a wide range of processes in different cell types. In this review, we offer a summary of the functions of ZDHHC5 in synaptic plasticity, cardiac function, cell adhesion and fatty acid uptake, among other processes. We also explore recent work has revealed several mechanisms to control the activity, localisation and function of ZDHHC5.

S-acylated Golga7b stabilises DHHC5 at the plasma membrane to regulate cell adhesion.

S-acylation (palmitoylation) is the only fully reversible lipid modification of proteins; however, little is known about how protein S-acyltransferases (PATs) that mediate it are regulated. DHHC5 is a PAT that is mainly localised at the plasma membrane with roles in synaptic plasticity, massive endocytosis and cancer cell growth/invasion. Here, we demonstrate that DHHC5 binds to and palmitoylates a novel accessory protein Golga7b. Palmitoylation of Golga7b prevents clathrin-mediated endocytosis of DHHC5 and stabilises it at the plasma membrane. Proteomic analysis of the composition of DHHC5/Golga7b-associated protein complexes reveals a striking enrichment in adhesion proteins, particularly components of desmosomes. We show that desmoglein-2 and plakophilin-3 are substrates of DHHC5 and that DHHC5 and Golga7b are required for localisation of desmoglein-2 to the plasma membrane and for desmosomal patterning. Loss of DHHC5/Golga7b causes functional impairments in cell adhesion, suggesting these proteins have a wider role in cell adhesion beyond desmosome assembly. This work uncovers a novel mechanism of DHHC5 regulation by Golga7b and demonstrates a role for the DHHC5/Golga7b complex in the regulation of cell adhesion.

Quantitative Analysis of Protein S-Acylation Site Dynamics Using Site-Specific Acyl-Biotin Exchange (ssABE).

Protein S-acylation (palmitoylation) is a reversible lipid modification that is increasingly recognized as an important regulator of protein function, including membrane association, trafficking, and subcellular localization. Most proteomic methods to study palmitoylation allow characterization of putative palmitoylated proteins but do not permit identification of individual sites of palmitoylation. We have recently adapted the Acyl-Biotin Exchange (ABE) method that is routinely used for palmitoyl-proteome characterization, to permit global S-acylation site analysis. This site-specific ABE (ssABE) protocol, when combined with SILAC-based quantification, allows both the large-scale identification of palmitoylation sites and quantitative profiling of palmitoylation site changes. This approach enables palmitoylation to be studied at a systems level comparable to other more intensively studied post-translational modifications.

Global, site-specific analysis of neuronal protein S-acylation.

Protein S-acylation (palmitoylation) is a reversible lipid modification that is an important regulator of dynamic membrane-protein interactions. Proteomic approaches have uncovered many putative palmitoylated proteins however, methods for comprehensive palmitoylation site characterization are lacking. We demonstrate a quantitative site-specific-Acyl-Biotin-Exchange (ssABE) method that allowed the identification of 906 putative palmitoylation sites on 641 proteins from mouse forebrain. 62% of sites map to known palmitoylated proteins and 102 individual palmitoylation sites are known from the literature. 54% of palmitoylation sites map to synaptic proteins including many GPCRs, receptors/ion channels and peripheral membrane proteins. Phosphorylation sites were also identified on a subset of peptides that were palmitoylated, demonstrating for the first time co-identification of these modifications by mass spectrometry. Palmitoylation sites were identified on over half of the family of palmitoyl-acyltransferases (PATs) that mediate protein palmitoylation, including active site thioester-linked palmitoyl intermediates. Distinct palmitoylation motifs and site topology were identified for integral membrane and soluble proteins, indicating potential differences in associated PAT specificity and palmitoylation function. ssABE allows the global identification of palmitoylation sites as well as measurement of the active site modification state of PATs, enabling palmitoylation to be studied at a systems level.