Phosphoregulation of the Ceramide Transport Protein CERT at Serine 315 in the Interaction with VAMP-associated Protein (VAP) for Inter-organelle Trafficking of Ceramide in Mammalian Cells

The ceramide transport protein CERT mediates the inter-organelle transport of ceramide for the synthesis of sphingomyelin, presumably through endoplasmic reticulum (ER)-Golgi membrane contact sites. CERT has a short peptide motif named FFAT, which associates with the ER-resident membrane protein VAP. We show that the phosphorylation of CERT at serine 315, which is adjacent to the FFAT motif, markedly enhanced the interaction of CERT with VAP. The phosphomimetic CERT S315E mutant exhibited higher activity to support the ER-to-Golgi transport of ceramide than the wild-type control in a semi-intact cell system, and this enhanced activity was abrogated when its FFAT motif was deleted. The level of phosphorylation of CERT at Ser-315 increased in HeLa cells treated with a sphingolipid biosynthesis inhibitor or exogenous sphingomyelinase. Expression of CERT S315E induced intracellular punctate structures, to which CERT and VAP were co-localized, and the occurrence of the structure was dependent on both phosphatidylinositol 4-monophosphate binding and VAP binding activities of CERT. Phosphorylation of another region (named a serine-rich motif) in CERT is known to down-regulate the activity of CERT. Analysis of various CERT mutant constructs showed that the de-phosphorylation of the serine-rich motif and the phosphorylation of Ser-315 likely have the additive contribution to enhance the activity of CERT. These results demonstrate that the phosphorylation of CERT at the FFAT motif-adjacent serine affected its affinity for VAP, which may regulate the inter-organelle trafficking of ceramide in response to the perturbation of cellular sphingomyelin and/or other sphingolipids.     

The Lifetime of UDP-galactose:Ceramide Galactosyltransferase Is Controlled by a Distinct Endoplasmic Reticulum-associated Degradation (ERAD) Regulated by Sigma-1 Receptor Chaperones

The glycosphingolipid biosynthesis is initiated by monoglycosylation of ceramides, the action of which is catalyzed either by UDP-glucose:ceramide glucosyltransferase or by UDP-galactose:ceramide galactosyltransferase (CGalT). CGalT is expressed predominantly at the endoplasmic reticulum (ER) of oligodendrocytes and is responsible for synthesizing galactosylceramides (GalCer) that play an important role in regulation of axon conductance. However, despite the importance of ceramide monoglycosylation enzymes in a spectrum of cellular functions, the mechanism that fine tunes activities of those enzymes is largely unknown. In the present study, we demonstrated that the sigma-1 receptor (Sig-1R) chaperone, the mammalian homologue of a yeast C8-C7 sterol isomerase, controls the protein level and activity of the CGalT enzyme via a distinct ER-associated degradation system involving Insig. The Sig-1R forms a complex with Insig via its transmembrane domain partly in a sterol-dependent manner and associates with CGalT at the ER. The knockdown of Sig-1Rs dramatically prolonged the lifetime of CGalT without affecting the trimming of N-linked oligosaccharides at CGalT. The increased lifetime leads to the up-regulation of CGalT protein as well as elevated enzymatic activity in CHO cells stably expressing CGalT. Knockdown of Sig-1Rs also decreased CGalT degradation endogenously expressed in D6P2T-schwannoma cells. Our data suggest that Sig-1Rs negatively regulate the activity of GalCer synthesis under physiological conditions by enhancing the degradation of CGalT through regulation of the dynamics of Insig in the lipid-activated ER-associated degradation system. The GalCer synthesis may thus be influenced by sterols at the ER.     

Murine diacylglycerol acyltransferase-2 (DGAT2) can catalyze triacylglycerol synthesis and promote lipid droplet formation independent of its localization to the endoplasmic reticulum

Triacylglycerol (TG) is the major form of stored energy in eukaryotic organisms and is synthesized by two distinct acyl-CoA:diacylglycerol acyltransferase (DGAT) enzymes, DGAT1 and DGAT2. Both DGAT enzymes reside in the endoplasmic reticulum (ER), but DGAT2 also co-localizes with mitochondria and lipid droplets. In this report, we demonstrate that murine DGAT2 is part of a multimeric complex consisting of several DGAT2 subunits. We also identified the region of DGAT2 responsible for its localization to the ER. A DGAT2 mutant lacking both its transmembrane domains, although still associated with membranes, was absent from the ER and instead localized to mitochondria. Unexpectedly, this mutant was still active and capable of interacting with lipid droplets to promote TG storage. Additional experiments indicated that the ER targeting signal was present in the first transmembrane domain (TMD1) of DGAT2. When fused to a fluorescent reporter, TMD1, but not TMD2, was sufficient to target mCherry to the ER. Finally, the interaction of DGAT2 with lipid droplets was dependent on the C terminus of DGAT2. DGAT2 mutants, in which regions of the C terminus were either truncated or specific regions were deleted, failed to co-localize with lipid droplets when cells were oleate loaded to stimulate TG synthesis. Our findings demonstrate that DGAT2 is capable of catalyzing TG synthesis and promote its storage in cytosolic lipid droplets independent of its localization in the ER.     

Stearoyl-CoA Desaturase 1 Activity Is Required for Autophagosome Formation

Autophagy is one of the major degradation pathways for cytoplasmic components. The autophagic isolation membrane is a unique membrane whose content of unsaturated fatty acids is very high. However, the molecular mechanisms underlying formation of this membrane, including the roles of unsaturated fatty acids, remain to be elucidated. From a chemical library consisting of structurally diverse compounds, we screened for novel inhibitors of starvation-induced autophagy by measuring LC3 puncta formation in mouse embryonic fibroblasts stably expressing GFP-LC3. One of the inhibitors we identified, 2,5-pyridinedicarboxamide, N2,N5-bis[5-[(dimethylamino)carbonyl]-4-methyl-2-thiazolyl], has a molecular structure similar to that of a known stearoyl-CoA desaturase (SCD) 1 inhibitor. To determine whether SCD1 inhibition influences autophagy, we examined the effects of the SCD1 inhibitor 28c. This compound strongly inhibited starvation-induced autophagy, as determined by LC3 puncta formation, immunoblot analyses of LC3, electron microscopic observations, and p62/SQSTM1 accumulation. Overexpression of SCD1 or supplementation with oleic acid, which is a catalytic product of SCD1 abolished the inhibition of autophagy by 28c. Furthermore, 28c suppressed starvation-induced autophagy without affecting mammalian target of rapamycin activity, and also inhibited rapamycin-induced autophagy. In addition to inhibiting formation of LC3 puncta, 28c also inhibited formation of ULK1, WIPI1, Atg16L, and p62/SQSTM1 puncta. These results suggest that SCD1 activity is required for the earliest step of autophagosome formation.     

Topological Orientation of Acyl-CoA:Diacylglycerol Acyltransferase-1 (DGAT1) and Identification of a Putative Active Site Histidine and the Role of the N Terminus in Dimer/Tetramer Formation

Acyl CoA:diacylglycerol acyltransferase (DGAT) is an integral membrane protein of the endoplasmic reticulum that catalyzes the synthesis of triacylglycerols. Two DGAT enzymes have been identified (DGAT1 and DGAT2) with unique roles in lipid metabolism. DGAT1 is a multifunctional acyltransferase capable of synthesizing diacylglycerol, retinyl, and wax esters in addition to triacylglycerol. Here, we report the membrane topology for murine DGAT1 using protease protections assays and indirect immunofluorescence in conjunction with selective permeabilization of cellular membranes. Topology models based on prediction algorithms suggested that DGAT1 had eight transmembrane domains. In contrast, our data indicate that DGAT1 has three transmembrane domains with the N terminus oriented toward the cytosol. The C-terminal region of DGAT1, which accounts for ∼50% of the protein, is present in the endoplasmic reticulum lumen and contains a highly conserved histidine residue (His-426) that may be part of the active site. Mutagenesis of His-426 to alanine impaired the ability of DGAT1 to synthesize triacylglycerols as well as retinyl and wax esters in an in vitro acyltransferase assay. Finally, we show that the N-terminal domain of DGAT1 is not required for the catalytic activity of DGAT1 but, instead, may be involved in regulating enzyme activity and dimer/tetramer formation.     

Apicoplast and endoplasmic reticulum cooperate in fatty acid biosynthesis in apicomplexan parasite Toxoplasma gondii

Apicomplexan parasites are responsible for high impact human diseases such as malaria, toxoplasmosis, and cryptosporidiosis. These obligate intracellular pathogens are dependent on both de novo lipid biosynthesis as well as the uptake of host lipids for biogenesis of parasite membranes. Genome annotations and biochemical studies indicate that apicomplexan parasites can synthesize fatty acids via a number of different biosynthetic pathways that are differentially compartmentalized. However, the relative contribution of each of these biosynthetic pathways to total fatty acid composition of intracellular parasite stages remains poorly defined. Here, we use a combination of genetic, biochemical, and metabolomic approaches to delineate the contribution of fatty acid biosynthetic pathways in Toxoplasma gondii. Metabolic labeling studies with [(13)C]glucose showed that intracellular tachyzoites synthesized a range of long and very long chain fatty acids (C14:0-26:1). Genetic disruption of the apicoplast-localized type II fatty-acid synthase resulted in greatly reduced synthesis of saturated fatty acids up to 18 carbons long. Ablation of type II fatty-acid synthase activity resulted in reduced intracellular growth that was partially restored by addition of long chain fatty acids. In contrast, synthesis of very long chain fatty acids was primarily dependent on a fatty acid elongation system comprising three elongases, two reductases, and a dehydratase that were localized to the endoplasmic reticulum. The function of these enzymes was confirmed by heterologous expression in yeast. This elongase pathway appears to have a unique role in generating very long unsaturated fatty acids (C26:1) that cannot be salvaged from the host.     

Importin beta interacts with the endoplasmic reticulum-associated degradation machinery and promotes ubiquitination and degradation of mutant alpha1-antitrypsin

The mechanism by which misfolded proteins in the endoplasmic reticulum (ER) are retrotranslocated to the cytosol for proteasomal degradation is still poorly understood. Here, we show that importin β, a well established nucleocytoplasmic transport protein, interacts with components of the retrotranslocation complex and promotes ER-associated degradation (ERAD). Knockdown of importin β specifically inhibited the degradation of misfolded ERAD substrates but did not affect turnover of non-ERAD proteasome substrates. Genetic studies and in vitro reconstitution assays demonstrate that importin β is critically required for ubiquitination of mutant α1-antitrypsin, a luminal ERAD substrate. Furthermore, we show that importin β cooperates with Ran GTPase to promote ubiquitination and proteasomal degradation of mutant α1-antitrypsin. These results establish an unanticipated role for importin β in ER protein quality control.     

Phosphorylation of Serine Palmitoyltransferase Long Chain-1 (SPTLC1) on Tyrosine 164 Inhibits Its Activity and Promotes Cell Survival

In BCR-ABL-expressing cells, sphingolipid metabolism is altered. Because the first step of sphingolipid biosynthesis occurs in the endoplasmic reticulum (ER), our objective was to identify ABL targets in the ER. A phosphoproteomic analysis of canine pancreatic ER microsomes identified 49 high scoring phosphotyrosine-containing peptides. These were then categorized in silico and validated in vitro. We demonstrated that the ER-resident human protein serine palmitoyltransferase long chain-1 (SPTLC1), which is the first enzyme of sphingolipid biosynthesis, is phosphorylated at Tyr¹⁶⁴ by the tyrosine kinase ABL. Inhibition of BCR-ABL using either imatinib or shRNA-mediated silencing led to the activation of SPTLC1 and to increased apoptosis in both K562 and LAMA-84 cells. Finally, we demonstrated that mutation of Tyr¹⁶⁴ to Phe in SPTLC1 increased serine palmitoyltransferase activity. The Y164F mutation also promoted the remodeling of cellular sphingolipid content, thereby sensitizing K562 cells to apoptosis. Our observations provide a mechanistic explanation for imatinib-mediated cell death and a novel avenue for therapeutic strategies.     

Differential regulation of endoplasmic reticulum stress by protein tyrosine phosphatase 1B and T cell protein tyrosine phosphatase

Protein-tyrosine phosphatase 1B (PTP1B) and T cell protein-tyrosine phosphatase (TCPTP) are closely related intracellular phosphatases implicated in the control of glucose homeostasis. PTP1B and TCPTP can function coordinately to regulate protein tyrosine kinase signaling, and PTP1B has been implicated previously in the regulation of endoplasmic reticulum (ER) stress. In this study, we assessed the roles of PTP1B and TCPTP in regulating ER stress in the endocrine pancreas. PTP1B and TCPTP expression was determined in pancreases from chow and high fat fed mice and the impact of PTP1B and TCPTP over- or underexpression on palmitate- or tunicamycin-induced ER stress signaling assessed in MIN6 insulinoma β cells. PTP1B expression was increased, and TCPTP expression decreased in pancreases of mice fed a high fat diet, as well as in MIN6 cells treated with palmitate. PTP1B overexpression or TCPTP knockdown in MIN6 cells mitigated palmitate- or tunicamycin-induced PERK/eIF2α ER stress signaling, whereas PTP1B deficiency enhanced ER stress. Moreover, PTP1B deficiency increased ER stress-induced cell death, whereas TCPTP deficiency protected MIN6 cells from ER stress-induced death. ER stress coincided with the inhibition of Src family kinases (SFKs), which was exacerbated by PTP1B overexpression and largely prevented by TCPTP knockdown. Pharmacological inhibition of SFKs ameliorated the protective effect of TCPTP deficiency on ER stress-induced cell death. These results demonstrate that PTP1B and TCPTP play nonredundant roles in modulating ER stress in pancreatic β cells and suggest that changes in PTP1B and TCPTP expression may serve as an adaptive response for the mitigation of chronic ER stress.     

Slc35c2 Promotes Notch1 Fucosylation and Is Required for Optimal Notch Signaling in Mammalian Cells

Mammalian Notch receptors require modification by fucose on epidermal growth factor-like (EGF) repeats of their extracellular domain to respond optimally to signal induction by canonical Notch ligands. Inactivation of the Golgi GDP-fucose transporter Slc35c1 in mouse or human does not cause marked defects in Notch signaling during development, and shows milder fucosylation defects than those observed in mice unable to synthesize GDP-fucose, indicating the existence of another mechanism for GDP-fucose transport into the secretory pathway. We show here that fibroblasts from mice or humans lacking Slc35c1 exhibit robust Notch signaling in co-culture signaling assays. A potential candidate for a second GDP-fucose transporter is the related gene Slc35c2. Overexpression of Slc35c2 reduces expression of the fucosylated epitopes Lewis X and sialylated Lewis X in CHO cells, indicating competition with Slc35c1. The fucosylation of a Notch1 EGF repeat fragment that occurs in the endoplasmic reticulum was increased in CHO transfectants overexpressing Slc35c2. In CHO cells with low levels of Slc35c2, both Delta1- and Jagged1-induced Notch signaling were reduced, and the fucosylation of a Notch1 fragment was also decreased. Immunofluorescence microscopy of rat intestinal epithelial cells and HeLa cells, and analysis of rat liver membrane fractions showed that Slc35c2 is primarily colocalized with markers of the cis-Golgi network and endoplasmic reticulum-Golgi intermediate compartment (ERGIC). The combined results suggest that Slc35c2 is either a GDP-fucose transporter that competes with Slc35c1 for GDP-fucose, or a factor that otherwise enhances the fucosylation of Notch and is required for optimal Notch signaling in mammalian cells.     

Dual role of ancient ubiquitous protein 1 (AUP1) in lipid droplet accumulation and endoplasmic reticulum (ER) protein quality control

Quality control of endoplasmic reticulum proteins involves the identification and engagement of misfolded proteins, dislocation of the misfolded protein across the endoplasmic reticulum (ER) membrane, and ubiquitin-mediated targeting to the proteasome for degradation. Ancient ubiquitous protein 1 (AUP1) physically associates with the mammalian HRD1-SEL1L complex, and AUP1 depletion impairs degradation of misfolded ER proteins. One of the functions of AUP1 in ER quality control is to recruit the soluble E2 ubiquitin-conjugating enzyme UBE2G2. We further show that the CUE domain of AUP1 regulates polyubiquitylation and facilitates the interaction of AUP1 with the HRD1 complex and with dislocation substrates. AUP1 localizes both to the ER and to lipid droplets. The AUP1 expression level affects the abundance of cellular lipid droplets and as such represents the first protein with lipid droplet regulatory activity to be linked to ER quality control. These findings indicate a possible connection between ER protein quality control and lipid droplets.     

Enhancement of Procollagen Biosynthesis by p180 through Augmented Ribosome Association on the Endoplasmic Reticulum in Response to Stimulated Secretion

A coiled-coil microtubule-bundling protein, p180, was originally reported as a ribosome-binding protein on the rough endoplasmic reticulum (ER) and is highly expressed in secretory tissues. Recently, we reported a novel role for p180 in the trans-Golgi network (TGN) expansion following stimulated collagen secretion. Here, we show that p180 plays a key role in procollagen biosynthesis and secretion in diploid fibroblasts. Depletion of p180 caused marked reductions of secreted collagens without significant loss of the ER membrane or mRNA. Metabolic labeling experiments revealed that the procollagen biosynthetic activity was markedly affected following p180 depletion. Moreover, loss of p180 perturbs ascorbate-stimulated de novo biosynthesis mainly in the membrane fraction with a preferential secretion defect of large proteins. At the EM level, one of the most prominent morphological features of p180-depleted cells was insufficient ribosome association on the ER membranes. In contrast, the ER of control cells was studded with numerous ribosomes, which were further enhanced by ascorbate. Similarly biochemical analysis confirmed that levels of membrane-bound ribosomes were altered in a p180-dependent manner. Taken together, our data suggest that p180 plays crucial roles in enhancing collagen biosynthesis at the entry site of the secretory compartments by a novel mechanism that mainly involves facilitating ribosome association on the ER.     

An Assembly of Proteins and Lipid Domains Regulates Transport of Phosphatidylserine to Phosphatidylserine Decarboxylase 2 in Saccharomyces cerevisiae

Saccharomyces cerevisiae uses multiple biosynthetic pathways for the synthesis of phosphatidylethanolamine. One route involves the synthesis of phosphatidylserine (PtdSer) in the endoplasmic reticulum (ER), the transport of this lipid to endosomes, and decarboxylation by PtdSer decarboxylase 2 (Psd2p) to produce phosphatidylethanolamine. Several proteins and protein motifs are known to be required for PtdSer transport to occur, namely the Sec14p homolog PstB2p/Pdr17p; a PtdIns 4-kinase, Stt4p; and a C2 domain of Psd2p. The focus of this work is on defining the protein-protein and protein-lipid interactions of these components. PstB2p interacts with a protein encoded by the uncharacterized gene YPL272C, which we name Pbi1p (PstB2p-interacting 1). PstB2p, Psd2, and Pbi1p were shown to be lipid-binding proteins specific for phosphatidic acid. Pbi1p also interacts with the ER-localized Scs2p, a binding determinant for several peripheral ER proteins. A complex between Psd2p and PstB2p was also detected, and this interaction was facilitated by a cryptic C2 domain at the extreme N terminus of Psd2p (C2-1) as well the previously characterized C2 domain of Psd2p (C2-2). The predicted N-terminal helical region of PstB2p was necessary and sufficient for promoting the interaction with both Psd2p and Pbi1p. Taken together, these results support a model for PtdSer transport involving the docking of a PtdSer donor membrane with an acceptor via specific protein-protein and protein-lipid interactions. Specifically, our model predicts that this process involves an acceptor membrane complex containing the C2 domains of Psd2p, PstB2p, and Pbi1p that ligate to Scs2p and phosphatidic acid present in the donor membrane, forming a zone of apposition that facilitates PtdSer transfer.     

Inhibition of Golgi Apparatus Glycosylation Causes Endoplasmic Reticulum Stress and Decreased Protein Synthesis

Nucleotide sugar transporters of the Golgi apparatus play an essential role in the glycosylation of proteins, lipids, and proteoglycans. Down-regulation of expression of the transporters for CMP-sialic acid, GDP-fucose, or both unexpectedly resulted in accumulation of glycoconjugates in the Golgi apparatus rather than in the plasma membrane. Pulse-chase experiments with radiolabeled sugars and amino acids showed decreased synthesis and secretion of both nonglycoproteins and glycoproteins. Further studies revealed that the above silencing induced endoplasmic reticulum stress and inhibited protein translation initiation. Together these results suggest that global inhibition of Golgi apparatus glycosylation may lead to important secondary metabolic changes, unrelated to glycosylation.     

Selenoprotein K is a novel target of m-calpain, and cleavage is regulated by Toll-like receptor-induced calpastatin in macrophages

Calpains are proteolytic enzymes that modulate cellular function through cleavage of targets, thereby modifying their actions. An important role is emerging for calpains in regulating inflammation and immune responses, although specific mechanisms by which this occurs have not been clearly defined. In this study, we identify a novel target of calpain, selenoprotein K (SelK), which is an endoplasmic reticulum transmembrane protein important for Ca(2+) flux in immune cells. Calpain-mediated cleavage of SelK was detected in myeloid cells (macrophages, neutrophils, and dendritic cells) but not in lymphoid cells (B and T cells). Both m- and μ-calpain were capable of cleaving immunoprecipitated SelK, but m-calpain was the predominant isoform expressed in mouse immune cells. Consistent with these results, specific inhibitors were used to show that only m-calpain cleaved SelK in macrophages. The cleavage site in SelK was identified between Arg(81) and Gly(82) and the resulting truncated SelK was shown to lack selenocysteine, the amino acid that defines selenoproteins. Resting macrophages predominantly expressed cleaved SelK and, when activated through different Toll-like receptors (TLRs), SelK cleavage was inhibited. We found that decreased calpain cleavage was due to TLR-induced up-regulation of the endogenous inhibitor, calpastatin. TLR-induced calpastatin expression not only inhibited SelK cleavage, but cleavage of another calpain target, talin. Moreover, the expression of the calpain isoforms and calpastatin in macrophages were different from T and B cells. Overall, our findings identify SelK as a novel calpain target and reveal dynamic changes in the calpain/calpastatin system during TLR-induced activation of macrophages.     

Premature ligand-receptor interaction during biosynthesis limits the production of growth factor midkine and its receptor LDL receptor-related protein 1

Protein production within the secretory pathway is accomplished by complex but organized processes. Here, we demonstrate that the growth factor midkine interacts with LDL receptor-related protein 1 (LRP1) at high affinity (K(d) value, 2.7 nm) not only at the cell surface but also within the secretory pathway during biosynthesis. The latter premature ligand-receptor interaction resulted in aggregate formation and consequently suppressed midkine secretion and LRP1 maturation. We utilized an endoplasmic reticulum (ER) retrieval signal and an LRP1 fragment, which strongly bound to midkine and the LRP1-specialized chaperone receptor-associated protein (RAP), to construct an ER trapper. The ER trapper efficiently trapped midkine and RAP and mimicked the premature ligand-receptor interaction, i.e. suppressed maturation of the ligand and receptor. The ER trapper also diminished the inhibitory function of LRP1 on platelet-derived growth factor-mediated cell migration. Complementary to these results, an increased expression of RAP was closely associated with midkine expression in human colorectal carcinomas (33 of 39 cases examined). Our results suggest that the premature ligand-receptor interaction plays a role in protein production within the secretory pathway.     

Opposite Cross-Talk by Oleate and Palmitate on Insulin Signaling in Hepatocytes through Macrophage Activation

Chronic low grade inflammation in adipose tissue during obesity is associated with an impairment of the insulin signaling cascade. In this study, we have evaluated the impact of palmitate or oleate overload of macrophage/Kupffer cells in triggering stress-mediated signaling pathways, in lipoapoptosis, and in the cross-talk with insulin signaling in hepatocytes. RAW 264.7 macrophages or Kupffer cells were stimulated with oleate or palmitate, and levels of M1/M2 polarization markers and the lipidomic profile of eicosanoids were analyzed. Whereas proinflammatory cytokines and total eicosanoids were elevated in macrophages/Kupffer cells stimulated with palmitate, enhanced arginase 1 and lower leukotriene B₄ (LTB₄) levels were detected in macrophages stimulated with oleate. When hepatocytes were pretreated with conditioned medium (CM) from RAW 264.7 or Kupffer cells loaded with palmitate (CM-P), phosphorylation of stress kinases and endoplasmic reticulum stress signaling was increased, insulin signaling was impaired, and lipoapoptosis was detected. Conversely, enhanced insulin receptor-mediated signaling and reduced levels of the phosphatases protein tyrosine phosphatase 1B (PTP1B) and phosphatase and tensin homolog (PTEN) were found in hepatocytes treated with CM from macrophages stimulated with oleate (CM-O). Supplementation of CM-O with LTB₄ suppressed insulin sensitization and increased PTP1B and PTEN. Furthermore, LTB₄ decreased insulin receptor tyrosine phosphorylation in hepatocytes, activated the NFκB pathway, and up-regulated PTP1B and PTEN, these effects being mediated by LTB₄ receptor BTL1. In conclusion, oleate and palmitate elicit an opposite cross-talk between macrophages/Kupffer cells and hepatocytes. Whereas CM-P interferes at the early steps of insulin signaling, CM-O increases insulin sensitization, possibly by reducing LTB₄.     

Nuclear envelope phosphatase 1-regulatory subunit 1 (formerly TMEM188) is the metazoan Spo7p ortholog and functions in the lipin activation pathway

Lipin-1 catalyzes the formation of diacylglycerol from phosphatidic acid. Lipin-1 mutations cause lipodystrophy in mice and acute myopathy in humans. It is heavily phosphorylated, and the yeast ortholog Pah1p becomes membrane-associated and active upon dephosphorylation by the Nem1p-Spo7p membrane complex. A mammalian ortholog of Nem1p is the C-terminal domain nuclear envelope phosphatase 1 (CTDNEP1, formerly "dullard"), but its Spo7p-like partner is unknown, and the need for its existence is debated. Here, we identify the metazoan ortholog of Spo7p, TMEM188, renamed nuclear envelope phosphatase 1-regulatory subunit 1 (NEP1-R1). CTDNEP1 and NEP1-R1 together complement a nem1Δspo7Δ strain to block endoplasmic reticulum proliferation and restore triacylglycerol levels and lipid droplet number. The two human orthologs are in a complex in cells, and the amount of CTDNEP1 is increased in the presence of NEP1-R1. In the Caenorhabditis elegans embryo, expression of nematode CTDNEP1 and NEP1-R1, as well as lipin-1, is required for normal nuclear membrane breakdown after zygote formation. The expression pattern of NEP1-R1 and CTDNEP1 in human and mouse tissues closely mirrors that of lipin-1. CTDNEP1 can dephosphorylate lipins-1a, -1b, and -2 in human cells only in the presence of NEP1-R1. The nuclear fraction of lipin-1b is increased when CTDNEP1 and NEP1-R1 are co-expressed. Therefore, NEP1-R1 is functionally conserved from yeast to humans and functions in the lipin activation pathway.     

Specific enzyme complex of beta-1,4-galactosyltransferase-II and glucuronyltransferase-P facilitates biosynthesis of N-linked human natural killer-1 (HNK-1) carbohydrate

Human natural killer-1 (HNK-1) carbohydrate is highly expressed in the nervous system and is involved in synaptic plasticity and dendritic spine maturation. This unique carbohydrate, consisting of a sulfated trisaccharide (HSO(3)-3GlcAβ1-3Galβ1-4GlcNAc-), is biosynthesized by the successive actions of β-1,4-galactosyltransferase (β4GalT), glucuronyltransferase (GlcAT-P and GlcAT-S), and sulfotransferase (HNK-1ST). A previous study showed that mice lacking β4GalT-II, one of seven β4GalTs, exhibited a dramatic loss of HNK-1 expression in the brain, although β4GalT-I-deficient mice did not. Here, we investigated the underlying molecular mechanism of the regulation of HNK-1 expression. First, focusing on a major HNK-1 carrier, neural cell adhesion molecule, we found that reduced expression of an N-linked HNK-1 carbohydrate caused by a deficiency of β4GalT-II is not likely due to a general loss of the β1,4-galactose residue as an acceptor for GlcAT-P. Instead, we demonstrated by co-immunoprecipitation and endoplasmic reticulum-retention analyses using Neuro2a (N2a) cells that β4GalT-II physically and specifically associates with GlcAT-P. In addition, we revealed by pulldown assay that Golgi luminal domains of β4GalT-II and GlcAT-P are sufficient for the complex to form. With an in vitro assay system, we produced the evidence that the kinetic efficiency k(cat)/K(m) of GlcAT-P in the presence of β4GalT-II was increased about 2.5-fold compared with that in the absence of β4GalT-II. Finally, we showed that co-expression of β4GalT-II and GlcAT-P increased HNK-1 expression on various glycoproteins in N2a cells, including neural cell adhesion molecule. These results indicate that the specific enzyme complex of β4GalT-II with GlcAT-P plays an important role in the biosynthesis of HNK-1 carbohydrate.