Epigenetic Modulation of Intestinal Cholesterol Transporter Niemann-Pick C1-like 1 (NPC1L1) Gene Expression by DNA Methylation

Intestinal NPC1L1 transporter is essential for cholesterol absorption and the maintenance of cholesterol homeostasis in the body. NPC1L1 is differentially expressed along the gastrointestinal tract with very low levels in the colon as compared with the small intestine. This study was undertaken to examine whether DNA methylation was responsible for segment-specific expression of NPC1L1. Treatment of mice with 5-azacytidine (i.p.) resulted in a significant dose-dependent increase in NPC1L1 mRNA expression in the colon. The lack of expression of NPC1L1 in the normal colon was associated with high levels of methylation in the area flanking the 3-kb fragment upstream of the initiation site of the mouse NPC1L1 gene in mouse colon as analyzed by EpiTYPER® MassARRAY®. The high level of methylation in the colon was observed in specific CpG dinucleotides and was significantly decreased in response to 5-azacytidine. Similar to mouse NPC1L1, 5-azacytidine treatment also increased the level of human NPC1L1 mRNA expression in the intestinal HuTu-80 cell line in a dose- and time-dependent manner. Silencing the expression of DNA methyltransferase DNMT1, -2, -3A, and -3B alone by siRNA did not affect NPC1L1 expression in HuTu-80 cells. However, the simultaneous attenuation of DNMT1 and -3B expression caused a significant increase in NPC1L1 mRNA expression as compared with control. Also, in vitro methylation of the human NPC1L1 promoter significantly decreased NPC1L1 promoter activity in human intestinal Caco2 cells. In conclusion, our data demonstrated for the first time that DNA methylation in the promoter region of the NPC1L1 gene appears to be a major mechanism underlying differential expression of NPC1L1 along the length of the gastrointestinal tract.     

Human Sodium Phosphate Transporter 4 (hNPT4/SLC17A3) as a Common Renal Secretory Pathway for Drugs and Urate

The evolutionary loss of hepatic urate oxidase (uricase) has resulted in humans with elevated serum uric acid (urate). Uricase loss may have been beneficial to early primate survival. However, an elevated serum urate has predisposed man to hyperuricemia, a metabolic disturbance leading to gout, hypertension, and various cardiovascular diseases. Human serum urate levels are largely determined by urate reabsorption and secretion in the kidney. Renal urate reabsorption is controlled via two proximal tubular urate transporters: apical URAT1 (SLC22A12) and basolateral URATv1/GLUT9 (SLC2A9). In contrast, the molecular mechanism(s) for renal urate secretion remain unknown. In this report, we demonstrate that an orphan transporter hNPT4 (human sodium phosphate transporter 4; SLC17A3) was a multispecific organic anion efflux transporter expressed in the kidneys and liver. hNPT4 was localized at the apical side of renal tubules and functioned as a voltage-driven urate transporter. Furthermore, loop diuretics, such as furosemide and bumetanide, substantially interacted with hNPT4. Thus, this protein is likely to act as a common secretion route for both drugs and may play an important role in diuretics-induced hyperuricemia. The in vivo role of hNPT4 was suggested by two hyperuricemia patients with missense mutations in SLC17A3. These mutated versions of hNPT4 exhibited reduced urate efflux when they were expressed in Xenopus oocytes. Our findings will complete a model of urate secretion in the renal tubular cell, where intracellular urate taken up via OAT1 and/or OAT3 from the blood exits from the cell into the lumen via hNPT4.     

The JmjN Domain of Jhd2 Is Important for Its Protein Stability,and the Plant Homeodomain (PHD) Finger Mediates Its Chromatin Association Independent of H3K4 Methylation

Histone lysine methylation is a dynamic process that plays an important role in regulating chromatin structure and gene expression. Recent studies have identified Jhd2, a JmjC domain-containing protein, as an H3K4-specific demethylase in budding yeast. However, important questions regarding the regulation and functions of Jhd2 remain unanswered. In this study, we show that Jhd2 has intrinsic activity to remove all three states of H3K4 methylation in vivo and can dynamically associate with chromatin to modulate H3K4 methylation levels on both active and repressed genes and at the telomeric regions. We found that the plant homeodomain (PHD) finger of Jhd2 is important for its chromatin association in vivo. However, this association is not dependent on H3K4 methylation and the H3 N-terminal tail, suggesting the presence of an alternative mechanism by which Jhd2 binds nucleosomes. We also provide evidence that the JmjN domain and its interaction with the JmjC catalytic domain are important for Jhd2 function and that Not4 (an E3 ligase) monitors the structural integrity of this interdomain interaction to maintain the overall protein levels of Jhd2. We show that the S451R mutation in human SMCX (a homolog of Jhd2), which has been linked to mental retardation, and the homologous T359R mutation in Jhd2 affect the protein stability of both of these proteins. Therefore, our findings provide a mechanistic explanation for the observed defects in patients harboring this SMCX mutant and suggest the presence of a conserved pathway involving Not4 that modulates the protein stability of both yeast Jhd2 and human SMCX.     

Impaired nutrient signaling and body weight control in a Na+ neutral amino acid cotransporter (Slc6a19)-deficient mouse

Amino acid uptake in the intestine and kidney is mediated by a variety of amino acid transporters. To understand the role of epithelial neutral amino acid uptake in whole body homeostasis, we analyzed mice lacking the apical broad-spectrum neutral (0) amino acid transporter B(0)AT1 (Slc6a19). A general neutral aminoaciduria was observed similar to human Hartnup disorder which is caused by mutations in SLC6A19. Na(+)-dependent uptake of neutral amino acids into the intestine and renal brush-border membrane vesicles was abolished. No compensatory increase of peptide transport or other neutral amino acid transporters was detected. Mice lacking B(0)AT1 showed a reduced body weight. When adapted to a standard 20% protein diet, B(0)AT1-deficient mice lost body weight rapidly on diets containing 6 or 40% protein. Secretion of insulin in response to food ingestion after fasting was blunted. In the intestine, amino acid signaling to the mammalian target of rapamycin (mTOR) pathway was reduced, whereas the GCN2/ATF4 stress response pathway was activated, indicating amino acid deprivation in epithelial cells. The results demonstrate that epithelial amino acid uptake is essential for optimal growth and body weight regulation.     

SLC6A14 (ATB0,+) protein, a highly concentrative and broad specific amino acid transporter, is a novel and effective drug target for treatment of estrogen receptor-positive breast cancer

SLC6A14, also known as ATB(0,+), is an amino acid transporter with unique characteristics. It transports 18 of the 20 proteinogenic amino acids. However, this transporter is expressed only at low levels in normal tissues. Here, we show that the transporter is up-regulated specifically in estrogen receptor (ER)-positive breast cancer, demonstrable with primary human breast cancer tissues and human breast cancer cell lines. SLC6A14 is an estrogen/ER target. The transport features of SLC6A14 include concentrative transport of leucine (an activator of mTOR), glutamine (an essential amino acid for nucleotide biosynthesis and substrate for glutaminolysis), and arginine (an essential amino acid for tumor cells), suggesting that ER-positive breast cancer cells up-regulate SLC6A14 to meet their increased demand for these amino acids. Consequently, treatment of ER-positive breast cancer cells in vitro with α-methyl-DL-tryptophan (α-MT), a selective blocker of SLC6A14, induces amino acid deprivation, inhibits mTOR, and activates autophagy. Prolongation of the treatment with α-MT causes apoptosis. Addition of an autophagy inhibitor (3-methyladenine) during α-MT treatment also induces apoptosis. These effects of α-MT are specific to ER-positive breast cancer cells, which express the transporter. The ability of α-MT to cause amino acid deprivation is significantly attenuated in MCF-7 cells, an ER-positive breast cancer cell line, when SLC6A14 is silenced with shRNA. In mouse xenograft studies, α-MT by itself is able to reduce the growth of the ER-positive ZR-75-1 breast cancer cells. These studies identify SLC6A14 as a novel and effective drug target for the treatment of ER-positive breast cancer.     

A novel non-SET domain multi-subunit methyltransferase required for sequential nucleosomal histone H3 methylation by the mixed lineage leukemia protein-1 (MLL1) core complex

Gene expression within the context of eukaryotic chromatin is regulated by enzymes that catalyze histone lysine methylation. Histone lysine methyltransferases that have been identified to date possess the evolutionarily conserved SET or Dot1-like domains. We previously reported the identification of a new multi-subunit histone H3 lysine 4 methyltransferase lacking homology to the SET or Dot1 family of histone lysine methyltransferases. This enzymatic activity requires a complex that includes WRAD (WDR5, RbBP5, Ash2L, and DPY-30), a complex that is part of the MLL1 (mixed lineage leukemia protein-1) core complex but that also exists independently of MLL1 in the cell. Here, we report that the minimal complex required for WRAD enzymatic activity includes WDR5, RbBP5, and Ash2L and that DPY-30, although not required for enzymatic activity, increases the histone substrate specificity of the WRAD complex. We also show that WRAD requires zinc for catalytic activity, displays Michaelis-Menten kinetics, and is inhibited by S-adenosyl-homocysteine. In addition, we demonstrate that WRAD preferentially methylates lysine 4 of histone H3 within the context of the H3/H4 tetramer but does not methylate nucleosomal histone H3 on its own. In contrast, we find that MLL1 and WRAD are required for nucleosomal histone H3 methylation, and we provide evidence suggesting that each plays distinct structural and catalytic roles in the recognition and methylation of a nucleosome substrate. Our results indicate that WRAD is a new H3K4 methyltransferase with functions that include regulating the substrate and product specificities of the MLL1 core complex.     

Activation of the Protein Deacetylase SIRT6 by Long-chain Fatty Acids and Widespread Deacylation by Mammalian Sirtuins

Mammalian sirtuins (SIRT1 through SIRT7) are members of a highly conserved family of NAD⁺-dependent protein deacetylases that function in metabolism, genome maintenance, and stress responses. Emerging evidence suggests that some sirtuins display substrate specificity toward other acyl groups attached to the lysine ϵ-amine. SIRT6 was recently reported to preferentially hydrolyze long-chain fatty acyl groups over acetyl groups. Here we investigated the catalytic ability of all sirtuins to hydrolyze 13 different acyl groups from histone H3 peptides, ranging in carbon length, saturation, and chemical diversity. We find that long-chain deacylation is a general feature of mammalian sirtuins, that SIRT1 and SIRT2 act as efficient decrotonylases, and that SIRT1, SIRT2, SIRT3, and SIRT4 can remove lipoic acid. These results provide new insight into sirtuin function and a means for cellular removal of an expanding list of endogenous lysine modifications. Given that SIRT6 is a poor deacetylase in vitro, but binds and prefers to hydrolyze long-chain acylated peptides, we hypothesize that binding of certain free fatty acids (FFAs) could stimulate deacetylation activity. Indeed, we demonstrate that several biologically relevant FFAs (including myristic, oleic, and linoleic acids) at physiological concentrations induce up to a 35-fold increase in catalytic efficiency of SIRT6 but not SIRT1. The activation mechanism is consistent with fatty acid inducing a conformation that binds acetylated H3 with greater affinity. Binding of long-chain FFA and myristoylated H3 peptide is mutually exclusive. We discuss the implications of discovering endogenous, small-molecule activators of SIRT6.     

Role of Transmembrane Domain 8 in Substrate Selectivity and Translocation of SteT,a Member of the l-Amino Acid Transporter (LAT) Family

System l-amino acid transporters (LAT) belong to the amino acid, polyamine, and organic cation superfamily of transporters and include the light subunits of heteromeric amino acid transporters and prokaryotic homologues. Cysteine reactivity of SteT (serine/threonine antiporter) has been used here to study the substrate-binding site of LAT transporters. Residue Cys-291, in transmembrane domain 8 (TM8), is inactivated by thiol reagents in a substrate protectable manner. Surprisingly, DTT activated the transporter by reducing residue Cys-291. Cysteine-scanning mutagenesis of TM8 showed DTT activation in the single-cysteine mutants S287C, G294C, and S298C, lining the same α-helical face. S-Thiolation in Escherichia coli cells resulted in complete inactivation of the single-cysteine mutant G294C. l-Serine blocked DTT activation with an EC₅₀ similar to the apparent K_M of this mutant. Thus, S-thiolation abolished substrate translocation but not substrate binding. Mutation of Lys-295, to Cys (K295C) broadened the profile of inhibitors and the spectrum of substrates with the exception of imino acids. A structural model of SteT based on the structural homologue AdiC (arginine/agmatine antiporter) positions residues Cys-291 and Lys-295 in the putative substrate binding pocket. All this suggests that Lys-295 is a main determinant in the recognition of the side chain of SteT substrates. In contrast, Gly-294 is not facing the surface, suggesting conformational changes involving TM8 during the transport cycle. Our results suggest that TM8 sculpts the substrate-binding site and undergoes conformational changes during the transport cycle of SteT.