Hiromi Ando

Understanding the functional significance of ghrelin processing and degradation.

Post-translational modification, cleavage and processing of circulating hormones are common themes in the control of hormone activities. Full-length ghrelin is a 28 amino acid protein that exists in several modified and processed forms, including addition of an acyl moiety at the third serine of the N-terminus. When modified with octanoic acid, the first five N-terminal residues of ghrelin can modulate a signaling pathway via the ghrelin receptor GHSR1a. Although modification via a lipid moiety is essential for binding and activation of GHSR1a by ghrelin, many reports suggest that a desacyl form of ghrelin exists and has synergistic, opposing and distinct properties as compared to the acyl form. Therefore, it is important to clarify the physiological relevance of ghrelin derivatives. Based on lines of evidence from various studies, we propose that a larger proportion of secreted ghrelin is present in the deacylated form and furthermore, that circulating acyl and desacyl forms of ghrelin may be hydrolyzed to form short peptide fragments. Here, we summarize the results of studies aimed at understanding ghrelin processing and its implications for physiological function, as well as our recent findings regarding enzymes in the blood capable of generating processed forms of ghrelin.

Low-density lipoprotein and oxysterols suppress the transcription of CTP:Phosphoethanolamine cytidylyltransferase in vitro

The rate-limiting step in phosphatidylethanolamine (PE) synthesis by the CDP-ethanolamine pathway is the second step, which is catalyzed by CTP:phosphoethanolamine cytidylyltransferase (ET). The rate-limiting step for phosphatidylcholine biosynthesis by the CDP-choline pathway is also the second step, which is catalyzed by CTP:phosphocholine cytidylyltransferase (CT). The transcription of the most active form of CT, CTalpha, in serum-starved cells was stimulated by fetal bovine serum (FBS). Therefore, we were interested in the effects of FBS on the transcription of ET. Unexpectedly, the ET mRNA levels were significantly increased after NIH3T3 cells were cultured in serum-starved medium (0.5% FBS) longer than 8h, and the increase was suppressed by the addition of FBS. Actinomycin-D inhibited the increased ET mRNA levels in serum-starved cells. ET enzyme activities and protein amounts were also increased after serum starvation. These results suggest that FBS contains substances that inhibit the transcription of ET. To identify these substances, cells were incubated with several fractions of FBS separated by molecular sizes. As expected from the results, low-density lipoprotein, 25-hydroxycholesterol (25-OHC), 24-OHC, 27-OHC, 24(S),25-epoxycholesterol and mevalonolactate suppressed the ET mRNA levels in serum-starved cells, similar to 3-hydroxy-3-methylglutaryl-CoA reductase but not CTalpha. These results suggest that oxysterols are important regulating lipids for the suppression of ET transcription and may help maintain the contents of PE and cholesterol at the same ratio in the cellular membrane.

Identification of the N-terminal transmembrane domain of StarD7 and its importance for mitochondrial outer membrane localization and phosphatidylcholine transfer

StarD7 facilitates phosphatidylcholine (PC) transfer to mitochondria, and is essential for mitochondrial homeostasis. However, the molecular mechanism for PC transfer by protein remains poorly understood. Herein, we describe a putative novel transmembrane (TM) domain C-terminal to the mitochondria-targeting signal (MTS) sequence at the N-terminus of StarD7. The mature form of StarD7 is integrated and/or associated onto the outer leaflet of the outer mitochondrial membrane (OMM) in HEPA-1 and HepG2 cells. A truncated form of StarD7 lacking the TM domain is distributed in the inner space of the mitochondria, and cannot reverse mitochondrial abnormalities, such as complex formation and PC content, when re-expressed in StarD7-KO HEPA-1 cells. Re-expression of wild StarD7 can compensate these mitochondrial functions of StarD7-KO HEPA-1 cells. The precursor form of StarD7 is cleaved between Met76 and Ala77, and Ala77 and Ala78 in the TM domain to produce the mature form. These results suggest that StarD7 is anchored onto the OMM through its N-terminal TM domain, and the C-terminal START domain may extend into the cytoplasm and shuttle PC between the ER and OMM at the ER-mitochondria contact sites.

StarD7 Protein Deficiency Adversely Affects the Phosphatidylcholine Composition, Respiratory Activity, and Cristae Structure of Mitochondria.

Phosphatidylcholine (PC) is a major phospholipid of mitochondria, comprising 40–50% of both the outer and the inner membranes. However, PC must be imported from its production organelles because mitochondria lack the enzymes essential for PC biosynthesis. In a previous study, we found that StarD7 mediates the intracellular transfer of PC to mitochondria. Therefore, in this study, we analyzed the contribution of StarD7 to the maintenance of mitochondrial phospholipid content and function using siRNA-mediated knockdown and knock-out (KO) of the StarD7 gene in HEPA-1 cells. Real time analysis of respiratory activity demonstrated that the oxygen consumption rate and activity of mitochondrial complexes were impaired in StarD7-KD cells. To confirm these results, we established StarD7-KO HEPA-1 cells by double nicking using CRISPR/Cas9n. As expected, StarD7-KD and -KO cells showed a significant reduction in mitochondrial PC content. The ATP level and growth rate of KO cells were notably lower compared with wild-type cells when cultured in glucose-free galactose-containing medium to force cells to rely on mitochondrial ATP production. In KO cells, the level of the MTCO1 protein, a primary subunit of complex IV, was reduced without a concomitant decrease in its mRNA, but the level was restored when StarD7-I was overexpressed. StarD7-KO cells showed impaired formation of the mitochondrial supercomplexes and exhibited a disorganized cristae structure, with no changes in optic atrophy 1 protein. These findings indicate that StarD7 plays important roles in maintaining the proper composition of mitochondrial phospholipids as well as mitochondrial function and morphogenesis.

Enzymatic and transcriptional regulation of the cytoplasmic acetyl-CoA hydrolase ACOT12

Acyl-CoA thioesterase 12 (ACOT12) is the major enzyme known to hydrolyze the thioester bond of acetyl-CoA in the cytosol in the liver. ACOT12 contains a catalytic thioesterase domain at the N terminus and a steroidogenic acute regulatory protein-related lipid transfer (START) domain at the C terminus. We investigated the effects of lipids (phospholipids, sphingolipids, fatty acids, and sterols) on ACOT12 thioesterase activity and found that the activity was inhibited by phosphatidic acid (PA) in a noncompetitive manner. In contrast, the enzymatic activity of a mutant form of ACOT12 lacking the START domain was not inhibited by the lipids. These results suggest that the START domain is important for regulation of ACOT12 activity by PA. We also found that PA could bind to thioesterase domain, but not to the START domain, and had no effect on ACOT12 dissociation. ACOT12 is detectable in the liver but not in hepatic cell lines such as HepG2, Hepa-1, and Fa2N-4. ACOT12 mRNA and protein levels in rat primary hepatocytes decreased following treatment with insulin. These results suggest that cytosolic acetyl-CoA levels in the liver are controlled by lipid metabolites and hormones, which result in allosteric enzymatic and transcriptional regulation of ACOT12.

Matrix-assisted laser desorption/ionization imaging mass spectrometry reveals changes of phospholipid distribution in induced pluripotent stem cell colony differentiation

AbstractInduced pluripotent stem cells (iPSCs) are opening up new possibilities for medicine. Understanding the regulation of iPSC biology is important when attempting to apply these cells to disease models or therapy. Changes of lipid metabolism in iPSCs were investigated by matrix-assisted laser desorption/ionization time-of-flight imaging mass spectrometry (MALDI-TOF-IMS). Analysis revealed changes of the intensity and distribution of peaks at m/z 782.5 and 798.5 in iPSC colonies during spontaneous differentiation. Two phosphatidylcholines (PCs) were identified: C44H81NO8P, PC(36:4)[M+H]+ at m/z 782.5 and C42H82NO8P, PC(34:1)[M+K]+ at m/z 798.5. The intensity of PC(36:4) showed an inverse relation between undifferentiated and differentiated iPSC colonies. PC(34:1) displayed a diffuse distribution in undifferentiated iPSC colonies, while it showed a concentric distribution in differentiated iPSC colonies, and was localized at the border of the differentiated and undifferentiated areas or the border between undifferentiated iPSC and feeder cells. These findings suggested that the distribution of lipids changes during the growth and differentiation of iPSCs and that MALDI-TOF-IMS was useful for analyzing these changes. PC(36:4) might play a role in maintaining pluripotency, while PC(34:1) might play a role in the differentiation and spread of iPSCs. Graphical AbstractMALDI Imaging for phosphatidylcholine distribution changes during sponteneous differentiaton of induced pluiripotent stem cells colonies

Transcriptional suppression of CTP:phosphoethanolamine cytidylyltransferase by 25-hydroxycholesterol is mediated by nuclear factor-Y and Yin Yang 1

Pcyt2 (CTP:phosphoethanolamine cytidylyltransferase) is the rate-limiting enzyme in mammalian PE (phosphatidylethanolamine) biosynthesis. Previously, we reported that Pcyt2 mRNA levels increased in several types of cells after serum starvation, an effect that could be suppressed by supplementation with low-density lipoprotein or 25-HC (25-hydroxycholesterol). Transcription of Hmgcr, which encodes 3-hydroxy-3-methylglutaryl-CoA reductase, is also suppressed by 25-HC in the same dose-dependent manner. Nevertheless, a sterol-regulatory element was not detected in the Pcyt2 promoter region. The important element for transcriptional control of Pcyt2 by 25-HC (1.25 μM) was determined to reside between -56 and -36 on the basis of analysis with several Pcyt2 promoter deletion-luciferase reporters in NIH 3T3 cells. Using the yeast one-hybrid system, we found that NF-Y (nuclear factor-Y) binds at C(-37)CAAT(-41) and YY1 (Yin Yang1) binds at C(-42)AT(-40) in the Pcyt2 promoter. Endogenous NF-Y and YY1 bind clearly and competitively to these sites and are important for basal Pcyt2 transcription. Moreover, NF-Y binds to the Hmgcr promoter at C(-14)CA(-12) in gel-shift analysis, and suppression of the basal luciferase activity of the Hmgcr promoter-reporter construct (-30/+61) by 25-HC was abolished when C(-14)CA(-12) was mutated. Furthermore, transcriptional suppression of Pcyt2 by 25-HC was reduced following knockdown targeting of NF-YA or YY1. ChIP analysis revealed that 25-HC inhibited the interaction between NF-Y and RNA polymerase II on the Pcyt2 and Hmgcr promoters. On the basis of these results, we conclude that NF-Y and YY1 are important for the basal transcription of Pcyt2 and that NF-Y is involved in the inhibitory effects of 25-HC on Pcyt2 transcription.

Identification of nuclear localization and nuclear export signals in Ets2, and the transcriptional regulation of Ets2 and CTP:phosphocholine cytidylyltransferase α in tetradecanoyl-13-acetate or macrophage-colony stimulating factor stimulated RAW264 cells

PC is made via the CDP-choline pathway, in which CTP:phosphocholine cytidylyltransferase alpha (CTalpha), encoded by Pcyt1a, is the rate-limiting enzyme whose mRNA expression is strictly regulated. Previously, we reported that Ets1 enhanced and Net repressed CTalpha transcription by binding at the Ets binding site (-49/-47) in the Pcyt1a promoter. In this study, we asked if an Ets1 analogue, Ets2, also regulates CTalpha transcription and investigated the importance of its nuclear localization signal (NLS) and nuclear export signal (NES). Ets2 is primarily detected in the nucleus. Various mutated Ets2 proteins fused with enhanced green fluorescent protein were constructed to identify the NLS and NES in Ets2. Mutation of Ets2 at amino acids 404-410 results in a protein that is evenly distributed in the cell. Interestingly, an Ets2 protein deleted at the C-terminus (amino acids 1-392 present) was localized to the cytoplasm and site-specific mutation in the region 364-372 of this construct resulted in cytoplasmic and nuclear distribution. These results suggest that the NLS in Ets2 is between amino acids 404 and 410, and that the NES is between amino acids 364 and 372. Ets2 enhanced, but the mutant forms of Ets2 had little effects on the transcription of a CTalpha-reporter construct. When RAW264 cells, murine macrophage cell-line, were stimulated with 12-O-tetradecanoylphorbol-13-acetate (TPA) or macrophage-colony stimulating factor, the transcription of CTalpha was enhanced accompanied by increased mRNA of Ets2. These results suggest that the induction of Ets2 is important for CTalpha transcription by TPA and macrophage-colony stimulating factor.

The heterotrimeric G protein subunits Gαqand Gβ1have lysophospholipase D activity

In a previous study we purified a novel lysoPLD (lysophospholipase D) which converts LPC (lysophosphatidylcholine) into a bioactive phospholipid, LPA (lysophosphatidic acid), from the rat brain. In the present study, we identified the purified 42 and 35 kDa proteins as the heterotrimeric G protein subunits Gαq and Gβ1 respectively. When FLAG-tagged Gαq or Gβ1 was expressed in cells and purified, significant lysoPLD activity was observed in the microsomal fractions. Levels of the hydrolysed product choline increased over time, and the Mg2+ dependency and substrate specificity of Gαq were similar to those of lysoPLD purified from the rat brain. Mutation of Gαq at amino acids Lys52, Thr186 or Asp205, residues that are predicted to interact with nucleotide phosphates or catalytic Mg2+, dramatically reduced lysoPLD activity. GTP does not compete with LPC for the lysoPLD activity, indicating that these substrate-binding sites are not identical. Whereas the enzyme activity of highly purified FLAG-tagged Gαq overexpressed in COS-7 cells was ~4 nmol/min per mg, the activity from Neuro2A cells was 137.4 nmol/min per mg. The calculated Km and Vmax values for lysoPAF (1-O-hexadecyl-sn-glycero-3-phosphocholine) obtained from Neuro2A cells were 21 μM and 0.16 μmol/min per mg respectively, similar to the enzyme purified from the rat brain. These results reveal a new function for Gαq and Gβ1 as an enzyme with lysoPLD activity. Tag-purified Gα11 also exhibited a high lysoPLD activity, but Gαi and Gαs did not. The lysoPLD activity of the Gα subunit is strictly dependent on its subfamily and might be important for cellular responses. However, treatment of Hepa-1 cells with Gαq and Gα11 siRNAs (small interfering RNAs) did not change lysoPLD activity in the microsomal fraction. Clarification of the physiological relevance of lysoPLD activity of these proteins will need further studies.

The heterotrimeric G protein subunits Gα(q) and Gβ(1) have lysophospholipase D activity.

In a previous study we purified a novel lysoPLD (lysophospholipase D) which converts LPC (lysophosphatidylcholine) into a bioactive phospholipid, LPA (lysophosphatidic acid), from the rat brain. In the present study, we identified the purified 42 and 35 kDa proteins as the heterotrimeric G protein subunits Gαq and Gβ1 respectively. When FLAG-tagged Gαq or Gβ1 was expressed in cells and purified, significant lysoPLD activity was observed in the microsomal fractions. Levels of the hydrolysed product choline increased over time, and the Mg2+ dependency and substrate specificity of Gαq were similar to those of lysoPLD purified from the rat brain. Mutation of Gαq at amino acids Lys52, Thr186 or Asp205, residues that are predicted to interact with nucleotide phosphates or catalytic Mg2+, dramatically reduced lysoPLD activity. GTP does not compete with LPC for the lysoPLD activity, indicating that these substrate-binding sites are not identical. Whereas the enzyme activity of highly purified FLAG-tagged Gαq overexpressed in COS-7 cells was ~4 nmol/min per mg, the activity from Neuro2A cells was 137.4 nmol/min per mg. The calculated Km and Vmax values for lysoPAF (1-O-hexadecyl-sn-glycero-3-phosphocholine) obtained from Neuro2A cells were 21 μM and 0.16 μmol/min per mg respectively, similar to the enzyme purified from the rat brain. These results reveal a new function for Gαq and Gβ1 as an enzyme with lysoPLD activity. Tag-purified Gα11 also exhibited a high lysoPLD activity, but Gαi and Gαs did not. The lysoPLD activity of the Gα subunit is strictly dependent on its subfamily and might be important for cellular responses. However, treatment of Hepa-1 cells with Gαq and Gα11 siRNAs (small interfering RNAs) did not change lysoPLD activity in the microsomal fraction. Clarification of the physiological relevance of lysoPLD activity of these proteins will need further studies.