Disease-linked mutations in the phosphatidylcholine regulatory enzyme CCTα impair enzymatic activity and fold stability

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.

Loss of function variants inPCYT1Acausing spondylometaphyseal dysplasia with cone/rod dystrophy have broad consequences on lipid metabolism, chondrocyte differentiation, and lipid droplet formation

“…The protein levels in this cell line were only slightly higher than those of p.Ser323Argfs*38 homozygotes, indicating that the p.Ser114Thr allele has similarly adverse consequences on protein production and/or stability, at least when in combination with the p.Ser323Argfs*38 allele. This is consistent with findings from Cornell et al (2019), showing that cDNA transfection of p.Ser114Thr into COS cells generated no signal above background, unlike transfection with WT PCYT1A or other alleles, perhaps due to misfolding and subsequent degradation [25]. Finally, fibroblasts homozygous for p.Glu129Lys had ~30% of wild-type steady-state CCTα levels and similar levels of PC incorporation (22% of wild-type).…”

“…Nonetheless, because the p.Ala99Val allele decreased PC incorporation more severely than steady-state expression, we can conclude that this allele reduces the catalytic activity of CCTα independently of its effects on protein levels. This result is supported by recent kinetic studies of purified CCTα, which demonstrate impaired catalytic activity of p.Ala99Val and other SMD-CRD alleles in the catalytic domain [25]. In the crystal structure of the orthologous rat CCTα protein, this alanine inserts into a hydrophobic pocket that cannot easily accommodate a bulkier residue such as valine and leads to lower thermal stability [25,35], so the consequences of this allele are also consistent with our expectations based on the structure of the enzyme.…”

“…Finally, fibroblasts homozygous for p.Glu129Lys had ~30% of wild-type steady-state CCTα levels and similar levels of PC incorporation (22% of wild-type). This glutamic acid residue is pivotal in hydrogen-bonding interactions between two α-helices of the catalytic domain Rossman fold, and the p.Glu129Lys mutation destabilizes that domain, lowering the T m for unfolding by 6°C [25,35].…”

“…The catalytic domain mutant enzymes have defects in folding stability based on thermal denaturation of purified enzymes, and several have impaired catalytic function in vitro . For example, p.Ala99Val, p.Ala99Thr, p.Glu129Lys, and p.Arg223Ser have higher K m values for both substrates, CTP and phosphocholine, and lower V max values, suggesting that in cells expressing these enzymes the rate of CDP-choline production would be slower [25].…”

Remodeling of the interdomain allosteric linker upon membrane binding of CCTα pulls its active site close to the membrane surface

“…Possibly, one or more of these arginines contribute to charge facilitation in the active site at some step in the catalytic cycle. In support of this, Arg-223 is mutated to a serine in a CCT␣ allele linked to the rare human skeletal and retinal disease, SMD-CRD (32), with a consequent 16-fold drop in k cat /K m (33).…”