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Palmitoyl acyltransferase Aph2 in cardiac function and the development of cardiomyopathy.

ABSTRACT: Protein palmitoylation regulates many aspects of cell function and is carried out by acyl transferases that contain zf-DHHC motifs. The in vivo physiological function of protein palmitoylation is largely unknown. Here we generated mice deficient in the acyl transferase Aph2 (Ablphilin 2 or zf-DHHC16) and demonstrated an essential role for Aph2 in embryonic/postnatal survival, eye development, and heart development. Aph2(-/-) embryos and pups showed cardiomyopathy and cardiac defects including bradycardia. We identified phospholamban, a protein often associated with human cardiomyopathy, as an interacting partner and a substrate of Aph2. Aph2-mediated palmitoylation of phospholamban on cysteine 36 differentially alters its interaction with PKA and protein phosphatase 1 α, augmenting serine 16 phosphorylation, and regulates phospholamban pentamer formation. Aph2 deficiency results in phospholamban hypophosphorylation, a hyperinhibitory form. Ablation of phospholamban in Aph2(-/-) mice histologically and functionally alleviated the heart defects. These findings establish Aph2 as a critical in vivo regulator of cardiac function and reveal roles for protein palmitoylation in the development of other organs including eyes.

SUBMITTER: Zhou T

PROVIDER: S-EPMC4697436 | biostudies-other | 2015 Dec

REPOSITORIES: biostudies-other

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Project description:BackgroundHypertrophic (HCM) and dilated (DCM) cardiomyopathies result from sarcomeric protein mutations, including cardiac troponin T (cTnT, TNNT2). We determined whether TNNT2 mutations cause cardiomyopathies by altering cTnT function or quantity; whether the severity of DCM is related to the ratio of mutant to wildtype cTnT; whether Ca(2+) desensitization occurs in DCM; and whether absence of cTnT impairs early embryonic cardiogenesis.Methods and findingsWe ablated Tnnt2 to produce heterozygous Tnnt2(+/-) mice, and crossbreeding produced homozygous null Tnnt2(-/-) embryos. We also generated transgenic mice overexpressing wildtype (TG(WT)) or DCM mutant (TG(K210Delta)) Tnnt2. Crossbreeding produced mice lacking one allele of Tnnt2, but carrying wildtype (Tnnt2(+/-)/TG(WT)) or mutant (Tnnt2(+/-)/TG(K210Delta)) transgenes. Tnnt2(+/-) mice relative to wildtype had significantly reduced transcript (0.82+/-0.06[SD] vs. 1.00+/-0.12 arbitrary units; p = 0.025), but not protein (1.01+/-0.20 vs. 1.00+/-0.13 arbitrary units; p = 0.44). Tnnt2(+/-) mice had normal hearts (histology, mass, left ventricular end diastolic diameter [LVEDD], fractional shortening [FS]). Moreover, whereas Tnnt2(+/-)/TG(K210Delta) mice had severe DCM, TG(K210Delta) mice had only mild DCM (FS 18+/-4 vs. 29+/-7%; p<0.01). The difference in severity of DCM may be attributable to a greater ratio of mutant to wildtype Tnnt2 transcript in Tnnt2(+/-)/TG(K210Delta) relative to TG(K210Delta) mice (2.42+/-0.08, p = 0.03). Tnnt2(+/-)/TG(K210Delta) muscle showed Ca(2+) desensitization (pCa(50) = 5.34+/-0.08 vs. 5.58+/-0.03 at sarcomere length 1.9 microm, p<0.01), but no difference in maximum force generation. Day 9.5 Tnnt2(-/-) embryos had normally looped hearts, but thin ventricular walls, large pericardial effusions, noncontractile hearts, and severely disorganized sarcomeres.ConclusionsAbsence of one Tnnt2 allele leads to a mild deficit in transcript but not protein, leading to a normal cardiac phenotype. DCM results from abnormal function of a mutant protein, which is associated with myocyte Ca(2+) desensitization. The severity of DCM depends on the ratio of mutant to wildtype Tnnt2 transcript. cTnT is essential for sarcomere formation, but normal embryonic heart looping occurs without contractile activity.

Project description:Polyglutamine expansions of huntingtin protein are responsible for the Huntington neurological disorder. HIP14 protein has been shown to interact with huntingtin. HIP14 and a HIP14-like protein, HIP14L, with a 69% similarity reside in the Golgi and possess palmitoyl acyltransferase activity through innate cysteine-rich domains, DHHC. Here, we used microarray analysis to show that reduced extracellular magnesium concentration increases HIP14L mRNA suggesting a role in cellular magnesium metabolism. Because HIP14 was not on the microarray platform, we used real-time reverse transcriptase-PCR to show that HIP14 and HIP14L transcripts were up-regulated 3-fold with low magnesium. Western analysis with a specific HIP14 antibody also showed that endogenous HIP14 protein increased with diminished magnesium. Furthermore, we demonstrate that when expressed in Xenopus oocytes, HIP14 and HIP14L mediate Mg2+ uptake that is electrogenic, voltage-dependent, and saturable with Michaelis constants of 0.87 +/- 0.02 and 0.74 +/- 0.07 mm, respectively. Diminished magnesium leads to an apparent increase in HIP14-green fluorescent protein and HIP14L-green fluorescent fusion proteins in the Golgi complex and subplasma membrane post-Golgi vesicles of transfected epithelial cells. We also show that inhibition of palmitoylation with 2-bromopalmitate, or deletion of the DHHC motif HIP14DeltaDHHC, diminishes HIP14-mediated Mg2+ transport by about 50%. Coexpression of an independent protein acyltransferase, GODZ, with the deleted HIP14DeltaDHHC mutant restored Mg2+ transport to values observed with wild-type HIP14. Although we did not directly measure palmitoylation of HIP14 in these studies, the data are consistent with a regulatory role of autopalmitoylation in HIP14-mediated Mg2+ transport. We conclude that the huntingtin interacting protein genes, HIP14 and HIP14L, encode Mg2+ transport proteins that are regulated by their innate palmitoyl acyltransferases thus fulfilling the characteristics of "chanzymes."

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Palmitoyl acyltransferase Aph2 in cardiac function and the development of cardiomyopathy.

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