Mutations in the SPTLC2 subunit of serine palmitoyltransferase cause hereditary sensory and autonomic neuropathy type I

Hereditary sensory and autonomic neuropathy type I (HSAN-I) is an axonal peripheral neuropathy associated with progressive distal sensory loss and severe ulcerations. Mutations in the first subunit of the enzyme serine palmitoyltransferase (SPT) have been associated with HSAN-I. The SPT enzyme catalyzes the first and rate-limiting step in the de novo sphingolipid synthesis pathway. However, different studies suggest the implication of other genes in the pathology of HSAN-I. Therefore, we screened the two other known subunits of SPT, SPTLC2 and SPTLC3, in a cohort of 78 HSAN patients. No mutations were found in SPTLC3, but we identified three heterozygous missense mutations in the SPTLC2 subunit of SPT in four families presenting with a typical HSAN-I phenotype. We demonstrate that these mutations result in a partial to complete loss of SPT activity in vitro and in vivo. Moreover, they cause the accumulation of the atypical and neurotoxic sphingoid metabolite 1-deoxy-sphinganine. Our findings extend the genetic heterogeneity in HSAN-I and enlarge the group of HSAN neuropathies associated with SPT defects. We further show that HSAN-I is consistently associated with an increased formation of the neurotoxic 1-deoxysphinganine, suggesting a common pathomechanism for HSAN-I.     

The structure of serine palmitoyltransferase; gateway to sphingolipid biosynthesis

Sphingolipid biosynthesis commences with the condensation of L-serine and palmitoyl-CoA to produce 3-ketodihydrosphingosine (KDS). This reaction is catalysed by the PLP-dependent enzyme serine palmitoyltransferase (SPT; EC 2.3.1.50), which is a membrane-bound heterodimer (SPT1/SPT2) in eukaryotes such as humans and yeast and a cytoplasmic homodimer in the Gram-negative bacterium Sphingomonas paucimobilis. Unusually, the outer membrane of S. paucimobilis contains glycosphingolipid (GSL) instead of lipopolysaccharide (LPS), and SPT catalyses the first step of the GSL biosynthetic pathway in this organism. We report here the crystal structure of the holo-form of S. paucimobilis SPT at 1.3 A resolution. The enzyme is a symmetrical homodimer with two active sites and a monomeric tertiary structure consisting of three domains. The PLP cofactor is bound covalently to a lysine residue (Lys265) as an internal aldimine/Schiff base and the active site is composed of residues from both subunits, located at the bottom of a deep cleft. Models of the human SPT1/SPT2 heterodimer were generated from the bacterial structure by bioinformatics analysis. Mutations in the human SPT1-encoding subunit have been shown to cause a neuropathological disease known as hereditary sensory and autonomic neuropathy type I (HSAN1). Our models provide an understanding of how these mutations may affect the activity of the enzyme.     

Identification and characterization of a novel testis-specific gene CKT2, which encodes a substrate for protein kinase CK2

Protein kinase CK2 is a serine/threonine kinase known to phosphorylate numerous substrates. CK2 is implicated in several physiologic and pathologic processes, particularly in cancer biology. CK2 is comprised of several subunits, including CK2α, CK2α′ and CK2β. Inactivation of CK2α′ leads to chromatin degeneration of germ cells, resulting in male sterility. To identify additional targets of CK2α′ in testes and to determine the role of CK2α′ in germ cell nuclear integrity, GST pull-down and protein–protein interaction assays were conducted. A novel testis-specific gene, CKT2 (CK2 Target protein 2), was found whose product interacts with and is phosphorylated by CK2 in vitro and in vivo. CKT2 is a 30.2 kDa protein with one coiled-coil domain and six putative phosphorylation sites. High expression of CKT2 correlated with chromatin condensation of spermatids in murine testes. Findings reported herein demonstrate that CKT2 is a target protein of native CK2α′ in testes and suggest that CKT2 plays a role in chromatin regulation of male germ cells.     

Functional analysis of mutations in the PIS gene, which encodes Saccharomyces cerevisiae phosphatidylinositol synthase

Abstract The PIS gene for an enzyme phosphatidylinositol synthase having an increased K _m for myo-inositol, was isolated from Saccharomyces cerevisiae . The mutant PIS gene contained a CAA codon at position 114 instead of the CAC codon observed in the wild-type gene, resulting in alteration of the amino acid from His to Gln. Oligonucleotide mediated site-directed mutagenesis of PIS at codon 114 revealed that mutant genes with codons for Ala, Thr and Leu could support yeast cell growth in vivo, but those for Asp, Lys and Tyr could not. All mutant enzymes when expressed in Escherichia coli showed greatly reduced in vitro activity.     

Purification and characterization of hevain,a serine protease from Hevea brasiliensis

A serine protease of MW 69000 has been isolated, in homogeneous form, from the latex of Hevea brasiliensis. The enzyme, named hevain, has only limited esterolytic and proteolytic abilities, a maximum activity in the pH range 6.5–7.5, and a pI of 4.3. Hevain has a notably high content of acidic amino acids, while the aromatic residues are present in relatively minor amounts.     

Cardiomyocyte specific deficiency of serine palmitoyltransferase subunit 2 reduces ceramide but leads to cardiac dysfunction

The role of serine palmitoyltransferase (SPT) and de novo ceramide biosynthesis in cardiac ceramide and sphingomyelin metabolism is unclear. To determine whether the de novo synthetic pathways, rather than ceramide uptake from circulating lipoproteins, is important for heart ceramide levels, we created cardiomyocyte-specific deficiency of Sptlc2, a subunit of SPT. Heart-specific Sptlc2-deficient (hSptlc2 KO) mice had a >35% reduction in ceramide, which was limited to C18:0 and very long chain ceramides. Sphingomyelinase expression, and levels of sphingomyelin and diacylglycerol were unchanged. But surprisingly phospholipids and acyl CoAs contained increased saturated long chain fatty acids. hSptlc2 KO mice had decreased fractional shortening and thinning of the cardiac wall. While the genes regulating glucose and fatty acid metabolism were not changed, expression of cardiac failure markers and the genes involved in the formation of extracellular matrices were up-regulated in hSptlc2 KO hearts. In addition, ER-stress markers were up-regulated leading to increased apoptosis. These results suggest that Sptlc2-mediated de novo ceramide synthesis is an essential source of C18:0 and very long chain, but not of shorter chain, ceramides in the heart. Changes in heart lipids other than ceramide levels lead to cardiac toxicity.     

Ceramide 1-phosphate inhibits serine palmitoyltransferase and blocks apoptosis in alveolar macrophages

We previously reported that incubation of bone-marrow derived macrophages in the absence of macrophage-colony stimulating factor (M-CSF), a cytokine that is essential for their growth and survival, resulted in stimulation of acid sphingomyelinase, accumulation of ceramides, and induction of apoptosis [A. Gomez-Munoz et al. 2004. Ceramide 1-phosphate blocks apoptosis through inhibition of acid sphingomyelinase in macrophages. J Lipid Res 45: 99–105]. Here, we show that alveolar NR8383 macrophages, which are not dependent on M-CSF for viability, undergo apoptosis when they are incubated in the absence of serum. NR8383 cells showed increased levels of ceramides under apoptotic conditions, but in contrast to bone marrow macrophage acid and neutral sphingomyelinases were only slightly activated. We found that the major mechanism for ceramide generation in NR8383 macrophages was stimulation of their synthesis de novo. This action involved activation of serine palmitoyltransferase (SPT), the key regulatory enzyme of this pathway. A relevant finding was that ceramide 1-phosphate (C1P) inhibited SPT activity and ceramide accumulation leading to inhibition of apoptosis. Furthermore, C1P enhanced the activity of antiapoptotic protein kinase B and its downstream effector nuclear factor kappa B. These observations add a new dimension to the understanding of the pro-survival actions of C1P in mammalian cells.     

Localization,topology, and function of the LCB1 subunit of serine palmitoyltransferase in mammalian cells

Serine palmitoyltransferase (SPT), the enzyme catalyzing the initial step in the biosynthesis of sphingolipids, comprises two different subunits, LCB1 and LCB2. LCB1 has a single highly hydrophobic domain near the N terminus. Chinese hamster ovary cell mutant LY-B cells are defective in SPT activity because of the lack of expression of an endogenous LCB1 subunit. Stable expression of LCB1 having an epitope tag at either the N or C terminus restored SPT activity of LY-B cells, suggesting that the epitope tag did not affect the localization or topology of LCB1. Indirect immunostaining showed that the N- and C-terminal epitopes are oriented toward the lumenal and cytosol side, respectively, at the endoplasmic reticulum. Interestingly, there was far less LCB2 in LY-B cells than in wild-type cells, and the amount of LCB2 in LY-B cells was restored to the wild-type level by transfection with LCB1 cDNA. In addition, overproduction of the LCB2 subunit required co-overproduction of the LCB1 subunit. These results indicated that the LCB1 subunit is most likely an integral protein having a single transmembrane domain with a lumenal orientation of its N terminus in the endoplasmic reticulum and that the LCB1 subunit is indispensable for the maintenance of the LCB2 subunit in mammalian cells.     

Functional characterization of the MKC1 gene of Candida albicans, which encodes a mitogen-activated protein kinase homolog related to cell integrity.

Mitogen-activated protein (MAP) kinases represent a group of serine/threonine protein kinases playing a central role in signal transduction processes in eukaryotic cells. Using a strategy based on the complementation of the thermosensitive autolytic phenotype of slt2 null mutants, we have isolated a Candida albicans homolog of Saccharomyces cerevisiae MAP kinase gene SLT2 (MPK1), which is involved in the recently outlined PKC1-controlled signalling pathway. The isolated gene, named MKC1 (MAP kinase from C. albicans), coded for a putative protein, Mkc1p, of 58,320 Da that displayed all the characteristic domains of MAP kinases and was 55% identical to S. cerevisiae Slt2p (Mpk1p). The MKC1 gene was deleted in a diploid Candida strain, and heterozygous and homozygous strains, in both Ura+ and Ura- backgrounds, were obtained to facilitate the analysis of the function of the gene. Deletion of the two alleles of the MKC1 gene gave rise to viable cells that grew at 28 and 37 degrees C but, nevertheless, displayed a variety of phenotypic traits under more stringent conditions. These included a low growth yield and a loss of viability in cultures grown at 42 degrees C, a high sensitivity to thermal shocks at 55 degrees C, an enhanced susceptibility to caffeine that was osmotically remediable, and the formation of a weak cell wall with a very low resistance to complex lytic enzyme preparations. The analysis of the functions downstream of the MKC1 gene should contribute to understanding of the connection of growth and morphogenesis in pathogenic fungi.     

Functional analysis of TcCTLP-5C2, a chymotrypsin-like serine protease needed for molting in Tribolium castaneum

In a previous study, we have characterized a gene family encoding chymotrypsin-like proteases from the red flour beetle, Tribolium castaneum (TcCTLPs). We identified 14 TcCTLP genes that were predominantly expressed in the midgut, where they presumably function in digestion. Two genes (TcCTLP-6C and TcCTLP-5C₂), however, additionally showed considerable expression in the carcass, and RNAi studies demonstrated that they are required for molting (Broehan et al., 2010; Insect Biochem. Mol. Biol 40, 274-83). Thus, the enzyme has distinct functions in different physiological environments. To study molecular adaptations that facilitate enzyme function in different environments, we performed an in-depth analysis of the molecular and enzymatic properties of TcCTLP-5C_2. We expressed different mutated versions of TcCTLP-5C₂ in form of factor Xa activatable pro-enzymes in insect cells using a baculoviral expression system, and purified the recombinant proteins by affinity chromatography. By measuring and comparing the enzyme activities, we obtained information about the significance of single amino acid residues in motifs that determine substrate specificity and pH tolerance. Further, we showed that TcCTLP-5C₂ is modified by N-glycosylation at amino acid position N137, which lies opposite to the catalytic cleft. Comparison of the enzymatic properties of non-glycosylated and glycosylated TcCTLP-5C₂ versions showed that N-glycosylation decreases V_max (maximum velocity) and k_cat (turnover) while leaving the K_m (specificity) unchanged. Thus, we provide evidence that N-glycosylation alters catalytic behavior by allosteric effects presumably due to altered structural dynamics as observed for chemically glycosylated enzymes.     

Isolation and characterization of the fission yeast gene rpa42 +, which encodes a subunit shared by RNA polymerases I and III

Eukaryotic RNA polymerases I and III share two distinct α-related subunits that show limited homology to the α subunit of Escherichia coli RNA polymerase, which forms a homodimer to nucleate the assembly of prokaryotic RNA polymerase. To gain insight into the functions of α-related subunits in eukaryotes, we have previously identified the α-related small subunit RPA17 of RNA polymerase I (and III) in Schizosaccharomyces pombe, and have shown that it is a functional homolog of Saccharomyces cerevisiae AC19. In an extension of that study, we have now isolated and characterized rpa42 ⁺, which encodes the α-related large subunit RPA42 of S. pombe RNA polymerase I, by virtue of the fact that its product interacts with RPA17 in the yeast two-hybrid system. We have found that rpa42 ⁺ encodes a polypeptide with an apparent molecular mass of 42?kDa, which shows 58% identity to the AC40 subunit shared by RNA polymerases I and III in S. cerevisiae. Furthermore, we have shown that rpa42 ⁺ complements a temperature-sensitive mutation in RPC40 the gene that encodes AC40 in S. cerevisiae and which is essential for cell growth. Finally, we have shown that neither RPA42 nor RPA17 can self-associate. These results provide evidence that the two distinct α-related subunits, RPA42 and RPA17, of RNA polymerases I and III are functionally conserved between S. pombe and S. cerevisiae, and suggest that heterodimer formation between them is essential for the assembly of RNA polymerases I and III in eukaryotes.     

Isolation and characterization of the fission yeast gene rpa42+, which encodes a subunit shared by RNA polymerases I and III

Eukaryotic RNA polymerases I and III share two distinct α-related subunits that show limited homology to the α subunit of Escherichia coli RNA polymerase, which forms a homodimer to nucleate the assembly of prokaryotic RNA polymerase. To gain insight into the functions of α-related subunits in eukaryotes, we have previously identified the α-related small subunit RPA17 of RNA polymerase I (and III) in Schizosaccharomyces pombe, and have shown that it is a functional homolog of Saccharomyces cerevisiae AC19. In an extension of that study, we have now isolated and characterized rpa42 ⁺, which encodes the α-related large subunit RPA42 of S. pombe RNA polymerase I, by virtue of the fact that its product interacts with RPA17 in the yeast two-hybrid system. We have found that rpa42 ⁺ encodes a polypeptide with an apparent molecular mass of 42 kDa, which shows 58% identity to the AC40 subunit shared by RNA polymerases I and III in S. cerevisiae. Furthermore, we have shown that rpa42 ⁺ complements a temperature-sensitive mutation in RPC40 the gene that encodes AC40 in S. cerevisiae and which is essential for cell growth. Finally, we have shown that neither RPA42 nor RPA17 can self-associate. These results provide evidence that the two distinct α-related subunits, RPA42 and RPA17, of RNA polymerases I and III are functionally conserved between S. pombe and S. cerevisiae, and suggest that heterodimer formation between them is essential for the assembly of RNA polymerases I and III in eukaryotes. Received: 20 April 1999 / Accepted: 26 July 1999