Understanding the rules governing NCX1 palmitoylation

No abstract available

Maleimide scavenging enhances determination of protein S-palmitoylation state in acyl-exchange methods.

S-palmitoylation (S-acylation) is an emerging dynamic post-translational modification of cysteine residues within proteins.Current assays for protein S-palmitoylation involve either in vivo labelling or chemical cleavage of S-palmitoyl groupsto reveal a free cysteine sulfhydryl that can be subsequently labelled with an affinity handle (acyl-exchange). Assays for protein S-palmitoylation using acylexchange chemistry therefore require blocking of non-S-palmitoylated cysteines, typically using Nethylmaleimide, to prevent non-specific detection. This in turn necessitates multiple precipitation based clean-up steps to remove reagents between stages, often leading to variable sample loss, reduced signal or protein aggregation. These combine to reduce the sensitivity, reliability and accuracy of these assays and also requires a substantial amount of time to perform. By substituting these precipitation steps with chemical scavenging of N-ethylmaleimide by 2,3-dimethyl-1,3-butadiene in an aqueous Diels-Alder 4+2 cyclo-addition reaction it is possible to greatly improve sensitivity and accuracy while reducing hands-on and overall time required for assays

Control of protein palmitoylation by regulating substrate recruitment to a zDHHC-protein acyltransferase

Although palmitoylation regulates numerous cellular processes, as yet efforts to manipulate this post-translational modification for therapeutic gain have proved unsuccessful. The Na-pump accessory sub-unit phospholemman (PLM) is palmitoylated by zDHHC5. Here, we show that PLM palmitoylation is facilitated by recruitment of the Na-pump α sub-unit to a specific site on zDHHC5 that contains a juxtamembrane amphipathic helix. Site-specific palmitoylation and GlcNAcylation of this helix increased binding between the Na-pump and zDHHC5, promoting PLM palmitoylation. In contrast, disruption of the zDHHC5-Na-pump interaction with a cell penetrating peptide reduced PLM palmitoylation. Our results suggest that by manipulating the recruitment of specific substrates to particular zDHHC-palmitoyl acyl transferases, the palmitoylation status of individual proteins can be selectively altered, thus opening the door to the development of molecular modulators of protein palmitoylation for the treatment of disease

Insights into the molecular basis of the palmitoylation and depalmitoylation of NCX1

Catalyzed by zDHHC-PAT enzymes and reversed by thioesterases, protein palmitoylation is the only post-translational modification recognized to regulate the sodium/calcium exchanger NCX1. NCX1 palmitoylation occurs at a single site at position 739 in its large regulatory intracellular loop. An amphipathic ɑ-helix between residues 740-756 is a critical for NCX1 palmitoylation. Given the rich background of the structural elements involving in NCX1 palmitoylation, the molecular basis of NCX1 palmitoylation is still relatively poorly understood. Here we found that (1) the identity of palmitoylation machinery of NCX1 controls its spatial organization within the cell, (2) the NCX1 amphipathic ɑ-helix directly interacts with zDHHC-PATs, (3) NCX1 is still palmitoylated when it is arrested in either Golgi or ER, indicating that NCX1 is a substrate for multiple zDHHC-PATs, (4) the thioesterase APT1 but not APT2 as a part of NCX1-depalmitoylation machinery governs subcellular organization of NCX1, (5) APT1 catalyzes NCX1 depalmitoylation in the Golgi but not in the ER. We also report that NCX2 and NCX3 are dually palmitoylated, with important implications for substrate recognition and enzyme catalysis by zDHHC-PATs. Our results could support new molecular or pharmacological strategies targeting the NCX1 palmitoylation and depalmitoylation machinery

Greasing the wheels or a spanner in the works?:Regulation of the cardiac sodium pump by palmitoylation

The ubiquitous sodium/potassium ATPase (Na pump) is the most abundant primary active transporter at the cell surface of multiple cell types, including ventricular myocytes in the heart. The activity of the Na pump establishes transmembrane ion gradients that control numerous events at the cell surface, positioning it as a key regulator of the contractile and metabolic state of the myocardium. Defects in Na pump activity and regulation elevate intracellular Na in cardiac muscle, playing a causal role in the development of cardiac hypertrophy, diastolic dysfunction, arrhythmias and heart failure. Palmitoylation is the reversible conjugation of the fatty acid palmitate to specific protein cysteine residues; all subunits of the cardiac Na pump are palmitoylated. Palmitoylation of the pump’s accessory subunit phospholemman (PLM) by the cell surface palmitoyl acyl transferase DHHC5 leads to pump inhibition, possibly by altering the relationship between the pump catalytic α subunit and specifically bound membrane lipids. In this review, we discuss the functional impact of PLM palmitoylation on the cardiac Na pump and the molecular basis of recognition of PLM by its palmitoylating enzyme DHHC5, as well as effects of palmitoylation on Na pump cell surface abundance in the cardiac muscle. We also highlight the numerous unanswered questions regarding the cellular control of this fundamentally important regulatory process

Greasing the wheels or a spanner in the works?:Regulation of the cardiac sodium pump by palmitoylation

The ubiquitous sodium/potassium ATPase (Na pump) is the most abundant primary active transporter at the cell surface of multiple cell types, including ventricular myocytes in the heart. The activity of the Na pump establishes transmembrane ion gradients that control numerous events at the cell surface, positioning it as a key regulator of the contractile and metabolic state of the myocardium. Defects in Na pump activity and regulation elevate intracellular Na in cardiac muscle, playing a causal role in the development of cardiac hypertrophy, diastolic dysfunction, arrhythmias and heart failure. Palmitoylation is the reversible conjugation of the fatty acid palmitate to specific protein cysteine residues; all subunits of the cardiac Na pump are palmitoylated. Palmitoylation of the pump's accessory subunit phospholemman (PLM) by the cell surface palmitoyl acyl transferase DHHC5 leads to pump inhibition, possibly by altering the relationship between the pump catalytic α subunit and specifically bound membrane lipids. In this review, we discuss the functional impact of PLM palmitoylation on the cardiac Na pump and the molecular basis of recognition of PLM by its palmitoylating enzyme DHHC5, as well as effects of palmitoylation on Na pump cell surface abundance in the cardiac muscle. We also highlight the numerous unanswered questions regarding the cellular control of this fundamentally important regulatory process.</p