Abstract
CTP:phosphocholine cytidylyltransferase (CCT) is the key regulatory enzyme in phosphatidylcholine (PC) synthesis and is activated by binding to PC-deficient membranes. Mutations in the gene encoding CCTα (PCYT1A) cause three distinct pathologies in humans: lipodystrophy, spondylometaphyseal dysplasia with cone-rod dystrophy (SMD-CRD), and isolated retinal dystrophy. Previous analyses showed that for some disease-linked PCYT1A variants steady state levels of CCTα and PC synthesis were reduced in patient fibroblasts, but other variants impaired PC synthesis with little effect on CCT levels. To explore the impact on CCT stability and function we expressed WT and mutant CCTs in COS-1 cells, which have very low endogenous CCT. Over-expression of two missense variants in the catalytic domain (V142M and P150A) generated aggregated enzymes that could not be refolded after solubilization by denaturation. Other mutations in the catalytic core that generated CCTs with reduced solubility could be purified. Five variants destabilized the catalytic domain-fold as assessed by lower transition temperatures for unfolding, and three of these manifested defects in substrate Km values. A mutation (R223S) in a signal-transducing linker between the catalytic and membrane-binding domains also impaired enzyme kinetics. E280del, a single amino acid deletion in the autoinhibitory helix increased the constitutive (lipid-independent) enzyme activity ∼4-fold. This helix also participates in membrane binding, and surprisingly E280del enhanced the enzyme's response to anionic lipid vesicles ∼4-fold. These in vitro analyses on purified mutant CCTs will complement future measurements of their impact on PC synthesis in cultured cells and in tissues with a stringent requirement for CCTα.
Highlights
CTP:phosphocholine cytidylyltransferase (CCT) is the key regulatory enzyme in phosphatidylcholine (PC) synthesis and is activated by binding to PC-deficient membranes
The amphipathic helix-membrane interaction is selective for membrane surfaces with packlarge unilamellar vesicles; lipid droplets (LDs), lipid droplet; DMEM, Dulbecco’s modified Eagle’s medium; TBS, Tris-buffered saline; SMD-CRD, spondylometaphyseal dysplasia with cone-rod dystrophy
Several of the CCT mutations were expressed at levels similar to WT CCT in this system, or within a factor of two: V142M, P150A, Y240H, A93T, A99V, A99T, E129K, R223S, and E280del (Fig. 2)
Summary
They include single nucleotide variants (encoding A93T, A99V, A99T, S114T, E129K, V142M, P150A, F191L, R223S, Y240H, and R283STOP); a codon deletion in the autoinhibitory region of the regulatory domain (encoding E280del); three frameshift mutations (S323Rfs*38, S333Lfs*164, and S331Pfs*?); and one splice site mutation (L299*17). The protein levels of mutant CCTs associated with lipodystrophy (V142M, E280del, and S333Lfs*164) are barely detectable in patient fibroblasts [20], the SMD-CRD–linked alleles A99V and E129K result in only modestly reduced CCT levels [24] (Table 1). Each of the catalytic domain mutations could destabilize the packing of the catalytic-fold, leading to impairment of enzyme function. To explore this hypothesis we purified CCT enzymes with mutations associated with all three conditions. Most of the mutants analyzed had defects in one or more of these properties
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