Basal expression of copper transporter 1 in intestinal epithelial cells is regulated by hypoxia-inducible factor 2α

Hypoxia, via stabilization of HIF2α, regulates the expression of the intestinal iron transporters DMT1 and ferroportin. Here we investigated whether the intestinal copper importer Ctr1 was also regulated by hypoxia. Copper uptake and Ctr1 mRNA expression were significantly increased in Caco-2 cells exposed to hypoxia. To determine whether HIF2α was involved in regulation of Ctr1 expression, we employed three models of HIF2α knockdown (chemical suppression of HIF2α translation in Caco-2 cells; HIF2α-siRNA-treated HuTu80 cells; HIF2α-intestinal knockout mice); Ctr1 mRNA expression was decreased in all three models under normoxic conditions. HIF2α translational inhibitor did not alter Ctr1 expression under hypoxic conditions. We conclude that basal expression of Ctr1 is regulated by HIF2α; however, the induction by hypoxia is a HIF2α-independent event.     

Dynamic relocation of the TORC1–Gtr1/2–Ego1/2/3 complex is regulated by Gtr1 and Gtr2

TORC1 regulates cellular growth, metabolism, and autophagy by integrating various signals, including nutrient availability, through the small GTPases RagA/B/C/D in mammals and Gtr1/2 in budding yeast. Rag/Gtr is anchored to the lysosomal/vacuolar membrane by the scaffold protein complex Ragulator/Ego. Here we show that Ego consists of Ego1 and Ego3, and novel subunit Ego2. The ∆ego2 mutant exhibited only partial defects both in Gtr1-dependent TORC1 activation and Gtr1 localization on the vacuole. Ego1/2/3, Gtr1/2, and Tor1/Tco89 were colocalized on the vacuole and associated puncta. When Gtr1 was in its GTP-bound form and TORC1 was active, these proteins were preferentially localized on the vacuolar membrane, whereas when Gtr1 was in its GDP-bound form, they were mostly localized on the puncta. The localization of TORC1 to puncta was further facilitated by direct binding to Gtr2, which is involved in suppression of TORC1 activity. Thus regulation of TORC1 activity through Gtr1/Gtr2 is tightly coupled to the dynamic relocation of these proteins.     

Activation of decapping involves binding of the mRNA and facilitation of the post-binding steps by the Lsm1-7–Pat1 complex

Decapping is a critical step in the conserved 5′-to-3′ mRNA decay pathway of eukaryotes. The hetero-octameric Lsm1-7–Pat1 complex is required for normal rates of decapping in this pathway. This complex also protects the mRNA 3′-ends from trimming in vivo. To elucidate the mechanism of decapping, we analyzed multiple lsm1 mutants, lsm1-6, lsm1-8, lsm1-9, and lsm1-14, all of which are defective in decapping and 3′-end protection but unaffected in Lsm1-7–Pat1 complex integrity. The RNA binding ability of the mutant complex was found to be almost completely lost in the lsm1-8 mutant but only partially impaired in the other mutants. Importantly, overproduction of the Lsm1-9p- or Lsm1-14p-containing (but not Lsm1-8p-containing) mutant complexes in wild-type cells led to a dominant inhibition of mRNA decay. Further, the mRNA 3′-end protection defect of lsm1-9 and lsm1-14 cells, but not the lsm1-8 cells, could be partly suppressed by overproduction of the corresponding mutant complexes in those cells. These results suggest the following: (1) Decapping requires both binding of the Lsm1-7–Pat1 complex to the mRNA and facilitation of the post-binding events, while binding per se is sufficient for 3′-end protection. (2) A major block exists at the post-binding steps in the lsm1-9 and lsm1-14 mutants and at the binding step in the lsm1-8 mutant. Consistent with these ideas, the lsm1-9, 14 allele generated by combining the mutations of lsm1-9 and lsm1-14 alleles had almost fully lost the RNA binding activity of the complex and behaved like the lsm1-8 mutant.     

Studies on endocytic mechanisms of the Menkes copper-translocating P-type ATPase (ATP7A; MNK) – Endocytosis of the Menkes protein

The human X-linked recessive copper deficiency disorder, Menkes disease, is caused by mutations in the ATP7A (MNK) gene, which encodes a transmembrane copper-transporting P-type ATPase (MNK). The MNK protein is localised to the Golgi apparatus and relocalises to the plasma membrane when copper levels are elevated. Previous studies have identified a C-terminal di-leucine endocytic motif (L1487L1488) in MNK, thought to direct it into the clathrin-mediated endocytic pathway. To determine whether MNK is internalised via clathrin-dependent endocytosis, this pathway was blocked in MNK-overexpressing HeLa cells by the transient expression of dominant negative dynamin and Eps15 mutants. MNK internalisation was not inhibited in such cells. MNK internalisation was inhibited in cells treated with hypertonic sucrose that not only blocked clathrin-mediated endocytosis but also fluid-phase endocytosis. These studies, together with earlier studies on the requirement for L1487L1488, suggest that MNK can utilise both clathrin-dependent and clathrin-independent endocytosis in HeLa cells.     

Apolipoprotein M expression increases the size of nascent pre�� HDL formed by ATP binding cassette transporter A1

Apolipoprotein M (apoM) is a novel apolipoprotein that is reportedly necessary for preβ HDL formation; however, its detailed function remains unknown. We investigated the biogenesis and properties of apoM and its effects on the initial steps of nascent preβ HDL assembly by ABCA1 in HEK293 cells. Transiently transfected apoM was localized primarily in the endomembrane compartment. Pulse-chase analyses demonstrated that apoM is inefficiently secreted, relative to human serum albumin, and that ∼50% remains membrane-associated after extraction with sodium carbonate, pH 11.5. To investigate the role of apoM in nascent preβ HDL formation, ABCA1-expressing or control cells, transfected with empty vector, apoM, or C-terminal epitope-tagged apoM (apoM-C-FLAG), were incubated with ¹²⁵I-apoA-I for 24 h. Conditioned media were harvested and fractionated by fast-protein liquid chromatography (FPLC) to monitor HDL particle size. Preβ HDL particles were formed effectively in the absence of apoM expression; however, increased apoM expression stimulated the formation of larger-sized nascent preβ HDLs. Immunoprecipitation with anti-apoA-I antibody followed by apoM Western blot analysis revealed that little secreted apoM was physically associated with preβ HDL. Our results suggest that apoM is an atypical secretory protein that is not necessary for ABCA1-dependent preβ HDL formation but does stimulate the formation of larger-sized preβ HDL. We propose that apoM may function catalytically at an intracellular site to transfer lipid onto preβ HDL during or after their formation by ABCA1.     

A structural and functional perspective on the evolution of the heme–copper oxidases

The heme–copper oxidases (HCOs) catalyze the reduction of O₂ to water, and couple the free energy to proton pumping across the membrane. HCOs are divided into three sub-classes, A, B and C, whose order of emergence in evolution has been controversial. Here we have analyzed recent structural and functional data on HCOs and their homologues, the nitric oxide reductases (NORs). We suggest that the C-type oxidases are ancient enzymes that emerged from the NORs. In contrast, the A-type oxidases are the most advanced from both structural and functional viewpoints, which we interpret as evidence for having evolved later.     

Morphine promotes apoptosis via TLR2, and this is negatively regulated by β-arrestin 2

We have previously reported that morphine induces apoptosis. However, the underlying molecular mechanisms remain to be elucidated. Toll-like receptor 2 (TLR2), a key immune receptor in the TLR family, modulates cell survival and cell death in various systems. Evidence indicates that β-arrestin 2 acts as a negative regulator of innate immune activation by TLRs. Here, we investigated the roles of TLR2, the downstreaming mediator MyD88, and β-arrestin 2 in morphine-induced apoptosis. We showed that overexpression of TLR2 in HEK293 cells caused a significant increase in apoptosis after morphine treatment. Inhibition of MyD88 by transfecting dominant negative MyD88 or overexpression of β-arrestin 2 by transfecting β-arrestin 2 full length plasmid in TLR2 overexpressing HEK293 cells attenuated morphine-induced apoptosis. Our study thus demonstrates that TLR2 signaling mediates the morphine-induced apoptosis, and β-arrestin 2 is a negative regulator in morphine-induced, TLR2-mediated apoptosis.     

Survivin is a guardian of the intestinal stem cell niche and its expression is regulated by TGF-β

As an inhibitor of apoptosis (IAP) family member, Survivin is known for its role during regulation of apoptosis. More recently its function as a cell cycle regulator has become evident. Survivin was shown to play a pivotal role during embryonic development and is highly expressed in regenerative tissue as well as in many cancer types. We examined the function of Survivin during mouse intestinal organogenesis and in gut pathophysiology. We found high expression of Survivin in experimentally induced colon cancer in mice but also in colon tumors of humans. Moreover, Survivin was regulated by TGF-β and was found to be highly expressed during mucosal healing following intestinal inflammation. We identified that expression of Survivin is essential early on in life, as specific deletion of Survivin in Villin expressing cells led to embryonic death around day 12 post coitum. Together with our recent study on the role of Survivin in the gut of adult mice our data demonstrate that Survivin is an essential guardian of embryonic gut development and adult gut homeostasis protecting the epithelium from cell death promoting the proliferation of intestinal stem and progenitor cells.     

Neuronal cell type–specific alternative splicing is regulated by the KH domain protein SLM1

The unique functional properties and molecular identity of neuronal cell populations rely on cell type–specific gene expression programs. Alternative splicing represents a powerful mechanism for expanding the capacity of genomes to generate molecular diversity. Neuronal cells exhibit particularly extensive alternative splicing regulation. We report a highly selective expression of the KH domain–containing splicing regulators SLM1 and SLM2 in the mouse brain. Conditional ablation of SLM1 resulted in a severe defect in the neuronal isoform content of the polymorphic synaptic receptors neurexin-1, -2, and -3. Thus, cell type–specific expression of SLM1 provides a mechanism for shaping the molecular repertoires of synaptic adhesion molecules in neuronal populations in vivo.