Research

研究内容

1. Identification of sphingolipid-related genes

Almost all reactions in living organisms are catalyzed by enzymes. Identifying the genes encoding the enzymes responsible for individual reactions provides a foundation for subsequent studies in biochemistry, molecular biology, genetics, physiology, and pathophysiology. For example, generating gene knockout (KO) models enables the elucidation of the physiological functions of the gene (or its product, such as a protein or enzyme), as well as the biological roles of the enzyme products (in our case, lipids), and also facilitates understanding of the mechanisms underlying related diseases. Furthermore, once a gene is identified, it can be cloned and overexpressed in appropriate cells or organisms (often with affinity tags), enabling large-scale production and purification of the enzyme, as well as in vitro biochemical analyses.

The Laboratory of Biochemistry (including the former Laboratory of Biomembrane and Biofunctional Chemistry) has identified numerous genes involved in ceramide and sphingolipid metabolism. These include 16 mammalian genes (ELOVL1, HACD1/2, KDSR, CERS3/6, FADS3, CYP4F22, FATP4, PNPLA1, ABHD5, SGPP2, ALDH3A2/B2, TECR, and HACL2) and 6 yeast genes (CSH1, RSB1, HFD1, FAA1/4, MPO1) that function in ceramide and sphingolipid synthesis and modification, the degradation of long-chain bases (see Section 2: Metabolic pathways of long-chain bases and fatty acid α-oxidation, for details), the elongation of very long-chain fatty acids (see Section 3: Production, functions, and pathology of very-long-chain fatty acids, for details), and acylceramide biosynthesis (see Section 4: Ceramide-mediated skin barrier formation, for details). Figure 4 shows the ceramide metabolic pathway along with the genes identified in our laboratory (red, mammalian genes; blue, yeast genes).

Genes identified in our laboratory

Ceramide synthesis and modification

  1. 3-Ketodihydrosphingosine reductase KDSR (mammals): We identified KDSR (also known as FVT-1) as the gene encoding the reductase responsible for the second step of sphingolipid synthesis, the conversion of 3-ketodihydrosphingosine to dihydrosphingosine1). We further demonstrated that mutations in KDSR cause harlequin ichthyosis2).
  2. Ceramide desaturase FADS3 (mammals): We identified FADS3 as the gene encoding a desaturase that introduces the characteristic cis double bond at position 14 of sphingadiene (4,14-sphingadiene)3). Subsequent studies revealed detailed enzymatic properties of FADS3 and the metabolic pathways in which it functions, and showed that ceramide, rather than long-chain bases, serves as its substrate4).
  3. Ceramide synthase CERS3 (mammals): We first demonstrated that CERS3 (also known as LASS3) encodes a ceramide synthase5), and showed that its expression is upregulated during keratinocyte differentiation, suggesting an important role in ceramide production in the skin6). We also performed ceramide profiling of the stratum corneum in ichthyosis patients carrying CERS3 mutations and demonstrated that reduced levels of acylceramides are a major cause of ichthyosis7).
  4. Ceramide synthase CERS6 (mammals): We demonstrated that CERS6 (also known as LASS6) encodes a ceramide synthase and showed that it exhibits high substrate specificity toward C16:0 acyl-CoA8).

Long-chain base degradation pathway

  1. Aldehyde dehydrogenases ALDH3A2 and ALDH3B2 (mammals) and HFD1 (yeast): We identified ALDH3A2, ALDH3B2, and HFD1 as genes encoding aldehyde dehydrogenases that convert long-chain aldehydes, which are produced by sphingosine-1-phosphate (S1P) lyase-mediated cleavage of long-chain base 1-phosphates, into long-chain fatty acids9, 10). During the identification process, we showed that among yeast deletion mutants lacking each of the aldehyde dehydrogenase genes, only the HFD1-deficient strain (hfd1Δ) exhibited impaired degradation of long-chain bases9). We further demonstrated that long-chain base degradation is impaired in cultured mammalian cells carrying an ALDH3A2 mutation9). Although ALDH3B2 is a pseudogene in humans, we demonstrated that Aldh3b2 functions together with Aldh3a2 in the long-chain base degradation pathway in mice, based on analyses of Aldh3a2 KO and Aldh3a2/Aldh3b2 double KO mice11, 12). ALDH3A2 is a causative gene of the neurocutaneous disorder Sjögren-Larsson syndrome (SLS), which is characterized by ichthyosis and neurological abnormalities13).
    2. Acyl-CoA synthetases of the ACSL family (ACSL1, ACSL3, ACSL4, ACSL5, and ACSL6) in mammals, and FAA1 and FAA4 in yeast: We identified ACSL family members and FAA1 and FAA4 as genes encoding acyl-CoA synthetases that convert long-chain fatty acids into acyl-CoAs9, 14). During this identification process, we used yeast deletion mutants lacking individual or combinations of acyl-CoA synthetase genes and showed that the faa1Δ faa4Δ double mutant exhibits a block in the long-chain base degradation pathway9). We further demonstrated that treatment of cultured mammalian cells with an ACSL inhibitor results in a similar impairment of long-chain base degradation14).
  2. Trans-2-enoyl-CoA reductase TECR (mammals): We identified TECR (also known as TER) as a gene encoding a reductase that saturates hexadecenoyl-CoA (trans-2-hexadecenoyl-CoA), which is generated in the degradation pathway of sphingosine/S1P, to produce palmitoyl-CoA15).
  3. 2-Hydroxy fatty acid dioxygenase MPO1 (yeast): We identified MPO1 as a gene involved in the conversion of 2-hydroxy fatty acids, generated during phytosphingosine metabolism, into fatty acids that are shorter by one carbon (fatty acid α-oxidation) 16). This identification was achieved through screening of yeast deletion mutants lacking genes encoding uncharacterized endoplasmic reticulum proteins. The gene was named MPO1, for Metabolism of Phytosphingosine to Odd-numbered fatty acids. Subsequent analyses revealed that Mpo1 is an iron ion-dependent dioxygenase17). We registered this enzyme as EC 1.14.18.12 with the IUBMB.
  4. 2-Hydroxyacyl-CoA lyase HACL2 (mammals): We identified HACL2 as a gene encoding a 2-hydroxyacyl-CoA lyase that functions in the α-oxidation of 2-hydroxy fatty acids generated during phytosphingosine metabolism and fatty acid 2-hydroxylation18). We further demonstrated that, in contrast to its homolog HACL1, which primarily functions in the α-oxidation of branched-chain fatty acids, HACL2 predominantly functions in the α-oxidation of 2-hydroxy fatty acids19).

Fatty acid elongation cycle

  1. Fatty acid elongase ELOVL1 (mammals): We identified ELOVL1 as a gene encoding a fatty acid elongase that preferentially elongates C20–C22 acyl-CoAs to C24–C26 acyl-CoAs in the first step of the fatty acid elongation cycle20). We further showed that ELOVL1 is involved in skin barrier formation through the production of ultra-long-chain ceramides and acylceramides in the skin21), in the prevention of dry eye through the production of very long-chain meibum lipids in the tear film22), and in myelin function in the nervous system through the production of very long-chain sphingolipids, particularly galactosylceramides and sulfatides23). We also found that mutations in ELOVL1 cause the neurocutaneous disorder IKSHD syndrome (syndromic ichthyosis)24), and demonstrated by ceramide profiling of the stratum corneum of patients with IKSHD syndrome that reduced levels of acylceramides are a major cause of ichthyosis25).
  2. 3-Hydroxyacyl-CoA dehydratases HACD1 and HACD2 (mammals): We identified HACD1 and HACD2 as genes encoding enzymes that catalyze the dehydration of 3-hydroxyacyl-CoAs to trans-2-enoyl-CoAs, the third step of the fatty acid elongation cycle26), and named them HACD (3-Hydroxy Acyl-CoA Dehydratase). We also first reported that mutations in HACD2 cause myopathy in humans27).

Acylceramide biosynthesis pathway

  1. Fatty acid ω-hydroxylases CYF4F22 (human) and Cyp4f39 (mouse): We identified cytochrome P450 members, CYP4F22 and Cyp4f39, as genes encoding fatty acid ω-hydroxylases that catalyze the ω-hydroxylation of ultra-long-chain fatty acids (C30–C36) and are involved in the production of ω-hydroxylated lipids, such as acylceramides (in the skin)28) and O-acyl-ω-hydroxy fatty acids (OAHFAs; in the tear film)29). CYP4F22 is a causative gene of autosomal recessive congenital ichthyosis. We analyzed several missense variants found in patients and showed that most exhibit reduced ω-hydroxylase activity30). We also showed that acylceramide levels are markedly reduced in the stratum corneum of patients carrying mutations in CYP4F2228, 31).
  2. Transacylase PNPLA1 (mammals): We identified PNPLA1 as a gene encoding a transacylase that transfers linoleic acid from triacylglycerols to ω-hydroxyceramides in the acylceramide biosynthesis pathway32), and registered this enzyme as EC 1.14.18.12 with the IUBMB. PNPLA1 is a causative gene of autosomal recessive congenital ichthyosis. We analyzed several missense mutants found in patients and showed that most exhibit reduced transacylase activity33). We also showed that acylceramide levels are markedly reduced in the stratum corneum of the patient carrying a PNPLA1 mutation34).
  3. PNPLA1 regulator ABHD5 (mammals): We identified ABHD5 as a gene encoding a protein that promotes the recruitment of PNPLA1 to lipid droplets, thereby facilitating the utilization of triacylglycerols as substrates and enhancing PNPLA1-mediated acylceramide production35). ABHD5 is a causative gene of Chanarin–Dorfman syndrome, a syndromic ichthyosis. We analyzed several missense mutants found in patients and showed that most fail to promote PNPLA1-mediated acylceramide production35).
  4. Acyl-CoA synthetase FATP4 (mammals): We identified FATP4 (also known as ACSVL4 and SLC27A4) as a gene encoding an acyl-CoA synthetase that converts ω-hydroxy ultra-long-chain fatty acids, produced by CYP4F22/Cyp4f39, into acyl-CoAs in the acylceramide biosynthesis pathway36).

Others

  1. Long-chain base translocase RSB1 (yeast): We identified RSB1 as a gene encoding a translocase/transporter that exports long-chain bases from cells, through genetic screening in yeast for multicopy suppressors conferring resistance to exogenously added long-chain bases37). We named RSB1 for Resistance to Sphingoid Base . Subsequent analyses revealed that RSB1 expression is induced by changes in plasma membrane lipid asymmetry, and that this signaling involves the Rim101 pathway, in which Rim21 functions as a sensor of lipid asymmetry38–40).
  2. Sphingosine-1-phosphate phosphatase SGPP2 (mammals): We identified SGPP2 (also known as SPP2) as a gene encoding a phosphatase that dephosphorylates S1P to generate sphingosine 41).
  3. IPC mannosyltransferase CSH1 (yeast): We identified CSH1 as a gene encoding a glycosyltransferase that transfers mannose to inositol phosphorylceramide (IPC) to produce mannosylinositol phosphorylceramide (MIPC) 42).42) We named CSH1 for CSG1 Homology.

References

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