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N-terminal acetylation and methylation differentially affect the function of MYL9.

ABSTRACT: Deciphering the histone code has illustrated that acetylation or methylation on the same residue can have analogous or opposing roles. However, little is known about the interplay between these post-translational modifications (PTMs) on the same nonhistone residues. We have recently discovered that N-terminal acetyltransferases (NATs) and N-terminal methyltransferases (NRMTs) can have overlapping substrates and identified myosin regulatory light chain 9 (MYL9) as the first confirmed protein to occur in either ?-amino-methylated (N?-methyl) or ?-amino-acetylated (N?-acetyl) states in vivo Here we aim to determine if these PTMs function similarly or create different MYL9 proteoforms with distinct roles. We use enzymatic assays to directly verify MYL9 is a substrate of both NRMT1 and NatA and generate mutants of MYL9 that are exclusive for N?-acetylation or N?-methylation. We then employ eukaryotic cell models to probe the regulatory functions of these N?-PTMs on MYL9. Our results show that, contrary to prevailing dogma, neither of these modifications regulate the stability of MYL9. Rather, exclusive N?-acetylation promotes cytoplasmic roles of MYL9, while exclusive N?-methylation promotes the nuclear role of MYL9 as a transcription factor. The increased cytoplasmic activity of N?-acetylated MYL9 corresponds with increased phosphorylation at serine 19, a key MYL9 activating PTM. Increased nuclear activity of N?-methylated MYL9 corresponds with increased DNA binding. N?-methylation also results in a decrease of interactions between the N-terminus of MYL9 and a host of cytoskeletal proteins. These results confirm that N?-acetylation and N?-methylation differentially affect MYL9 function by creating distinct proteoforms with different internal PTM patterns and binding properties.

SUBMITTER: Nevitt C

PROVIDER: S-EPMC6442934 | biostudies-literature | 2018 Oct

REPOSITORIES: biostudies-literature

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N-terminal acetylation and methylation differentially affect the function of MYL9.

Nevitt Chris C Tooley John G JG Schaner Tooley Christine E CE

The Biochemical journal 20181022 20

Deciphering the histone code has illustrated that acetylation or methylation on the same residue can have analogous or opposing roles. However, little is known about the interplay between these post-translational modifications (PTMs) on the same nonhistone residues. We have recently discovered that N-terminal acetyltransferases (NATs) and N-terminal methyltransferases (NRMTs) can have overlapping substrates and identified myosin regulatory light chain 9 (MYL9) as the first confirmed protein to o ...[more]

PMID: 30242065

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Project description:T-type calcium channels (Cav3.1 to Cav3.3) regulate low-threshold calcium spikes, burst firing and rhythmic oscillations of neurons and are involved in sensory processing, sleep, and hormone and neurotransmitter release. Here, we examined four heterozygous missense variants in CACNA1I, encoding the Cav3.3 channel, in patients with variable neurodevelopmental phenotypes. The p.(Ile860Met) variant, affecting a residue in the putative channel gate at the cytoplasmic end of the IIS6 segment, was identified in three family members with variable cognitive impairment. The de novo p.(Ile860Asn) variant, changing the same amino acid residue, was detected in a patient with severe developmental delay and seizures. In two additional individuals with global developmental delay, hypotonia, and epilepsy, the variants p.(Ile1306Thr) and p.(Met1425Ile), substituting residues at the cytoplasmic ends of IIIS5 and IIIS6, respectively, were found. Because structure modelling indicated that the amino acid substitutions differentially affect the mobility of the channel gate, we analysed possible effects on Cav3.3 channel function using patch-clamp analysis in HEK293T cells. The mutations resulted in slowed kinetics of current activation, inactivation, and deactivation, and in hyperpolarizing shifts of the voltage-dependence of activation and inactivation, with Cav3.3-I860N showing the strongest and Cav3.3-I860M the weakest effect. Structure modelling suggests that by introducing stabilizing hydrogen bonds the mutations slow the kinetics of the channel gate and cause the gain-of-function effect in Cav3.3 channels. The gating defects left-shifted and increased the window currents, resulting in increased calcium influx during repetitive action potentials and even at resting membrane potentials. Thus, calcium toxicity in neurons expressing the Cav3.3 variants is one likely cause of the neurodevelopmental phenotype. Computer modelling of thalamic reticular nuclei neurons indicated that the altered gating properties of the Cav3.3 disease variants lower the threshold and increase the duration and frequency of action potential firing. Expressing the Cav3.3-I860N/M mutants in mouse chromaffin cells shifted the mode of firing from low-threshold spikes and rebound burst firing with wild-type Cav3.3 to slow oscillations with Cav3.3-I860N and an intermediate firing mode with Cav3.3-I860M, respectively. Such neuronal hyper-excitability could explain seizures in the patient with the p.(Ile860Asn) mutation. Thus, our study implicates CACNA1I gain-of-function mutations in neurodevelopmental disorders, with a phenotypic spectrum ranging from borderline intellectual functioning to a severe neurodevelopmental disorder with epilepsy.

Dataset Information

N-terminal acetylation and methylation differentially affect the function of MYL9.

Publications

N-terminal acetylation and methylation differentially affect the function of MYL9.

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