N-myristoyltransferase 1 is essential in early mouse development

N-Myristoyltransferase (NMT) transfers myristate to an amino-terminal glycine of many eukaryotic proteins. In yeast, worms, and flies, this enzyme is essential for viability of the organism. Humans and mice possess two distinct but structurally similar enzymes, NMT1 and NMT2. These two enzymes have similar peptide specificities, but no one has examined the functional importance of the enzymes in vivo. To address this issue, we performed both genetic and biochemical studies. Northern blots with RNA from adult mice and in situ hybridization studies of day 13.5 embryos revealed widespread expression of both Nmt1 and Nmt2. To determine whether the two enzymes are functionally redundant, we generated Nmt1-deficient mice carrying a beta-galactosidase marker gene. beta-Galactosidase staining of tissues from heterozygous Nmt1-deficient (Nmt1+/-) mice and embryos confirmed widespread expression of Nmt1. Intercrosses of Nmt1+/- mice yielded no viable homozygotes (Nmt1-/-), and heterozygotes were born at a less than predicted frequency. Nmt1-/- embryos died between embryonic days 3.5 and 7.5. Northern blots revealed lower levels of Nmt2 expression in early development than at later time points, a potential explanation for the demise of Nmt1-/- embryos. To explore this concept, we generated Nmt1-/- embryonic stem (ES) cells. The Nmt2 mRNA could be detected in Nmt1-/- ES cells, but the total NMT activity levels were reduced by approximately 95%, suggesting that Nmt2 contributes little to total enzyme activity levels in these early embryo cells. The Nmt1-/- ES cells were functionally abnormal; they yielded small embryoid bodies in in vitro differentiation experiments and did not contribute normally to organogenesis in chimeric mice. We conclude that Nmt1 is not essential for the viability of mammalian cells but is required for development, likely because it is the principal N-myristoyltransferase in early embryogenesis.     

N-myristoyltransferase in the leukocytic development processes

The lipidic modification of proteins has recently been shown to be of immense importance, although many of the roles of these modifications remain as yet unidentified. One of such key modifications occurring on several proteins is the covalent addition of a 14-carbon long saturated fatty acid, a process termed myristoylation. Myristoylation can occur during both co-translational protein synthesis and posttranslationally, confers lipophilicity to protein molecules, and controls protein functions. The protein myristoylation process is catalyzed by the enzyme N-myristoyltransferase (NMT), which exists as two isoforms: NMT1 and NMT2. NMT1 is essential for growth and development, during which rapid cellular proliferation is required, in a variety of organisms. NMT1 is also reported to be elevated in many cancerous states, which also involve rapid cellular growth, albeit in an unwanted and uncontrolled manner. The delineation of myristoylation-dependent cellular functions is still in a state of infancy, and many of the roles of the myristoylated proteins remain to be established. The development of cells of the leukocytic lineage represents a phase of rapid growth and development, and we have observed that NMT1 plays a role in this process. The current review outlines the roles of NMT1 in the growth and differentiation of the cells of leukocytic origin. The described studies clearly demonstrate the roles of NMT1 in the regulation of the developmental processes of the leukocytes cells and provide a basis for further research with the aim of unraveling the roles of protein myristoylation in both cellular and physiological context.     

Processing of interleukin-1 in cells of monocytic lineage is differentiation-dependent.

The interleukin-1 (IL-1) alpha and beta precursor proteins are processed and released from several cell types in the absence of a canonical signal peptide. To gain some insight into the mechanisms that allow the production of IL-1 alpha and beta, we have investigated by immunoprecipitation the synthesis, their release and processing in a promyeloblastic cell line of tumoral origin, U937, and in peripheral blood monocytes. We show that U937 monocytic cells, on induction with a tumor-promoting agent, synthesize and release into the culture medium proIL-1 beta but do not process it. Similarly, peripheral blood monocytes left in adherence for 24 h or longer, prior to addition of lipopolysaccharide, synthesize and release proIL-1 alpha and beta without detectable processing of either cytokine. Processing and release of IL-1 alpha and beta by peripheral blood monocytes can be observed when monocytes are left to adhere for periods less than 15 h before lipopolysaccharide addition. IL-1 alpha and beta show similar kinetics of release from the cells, suggesting the existence of a common mechanism regulating their secretion. Since peripheral blood monocytes left in adherence in the presence of lipopolysaccharide differentiate into macrophages, we conclude that release and processing of IL-1 can occur independently and that processing depends on the stage of differentiation of monocytes, i.e. only the monocytes at an early stage of differentiation produce 17-kDa IL-1 alpha and beta.     

Endocytosis of α1-acid glycoprotein variants by human monocytic lineage cells

Summary— Human α₁-acid glycoprotein (AGP or orosomucoid) is a major glycoprotein of plasma. AGP can be separated on immobilized concanavalin A into three variants bearing none (AGP A), one (AGP B) or two (AGP C) biantennary glycans. In this paper, we show, using flow cytometry and confocal microscopy, that AGP C which is eluted from concanavalin A with mannose, binds to human monocytes, monocyte-derived macrophages as well as human promonocytic cell lines such as THP1 or U937. Conversely HL60, a promyelocytic cell line, does not express the surface AGP C binding protein. AGP C is internalized and degraded with an efficiency depending on the state of differentiation of these cells. In contrast, AGP A which is not recognized by concanavalin A, does not bind to any of these cells.     

Amyloid precursor protein mediates proinflammatory activation of monocytic lineage cells

Alzheimer's disease is a progressive neurodegenerative disorder characterized by extracellular deposition of beta-amyloid (Abeta) peptide containing neuritic plaques. Abeta peptides are proteolytically derived from the membrane-bound amyloid precursor protein (APP). Although the function of APP is not entirely clear, previous studies demonstrate that neuronal APP colocalizes with beta(1) integrin receptors at sites of focal adhesion, suggesting that APP is involved in mediating neuronal process adhesion. Integrin-dependent adhesion is also a well-characterized component of immune cell proinflammatory activation. Using primary mouse microglia and the human monocytic cell line, THP-1, we have begun investigating the role of APP in integrin-dependent activation. Co-immunoprecipitation studies demonstrate that APP is recruited into a multi-receptor signaling complex during beta(1) integrin-mediated adhesion of monocytes. Stimulation induces a subsequent, specific recruitment of tyrosine phosphorylated proteins to APP, including Lyn and Syk. Antibody cross-linking of cell surface APP leads to a similar response characterized by activation and recruitment of tyrosine kinases to APP as well as subsequent activation of mitogen-activated protein kinases and increased proinflammatory protein levels. These data demonstrate that APP can act as a proinflammatory receptor in monocytic lineage cells and provide insight into the contribution of this protein to the inflammatory conditions described in Alzheimer's disease.     

Effects of HIV-1 Nef on human N-myristoyltransferase 1

The HIV-1 accessory protein Nef is N-terminally myristoylated, and this post-translational modification is essential for Nef function in AIDS progression. Transfer of a myristate group from myristoyl coenzyme A to Nef occurs cotranslationally and is catalyzed by human N-myristoyltransferase 1 (NMT). To investigate the conformational effects of myristoylation on Nef structure as well as to probe the nature of the Nef:NMT complex, we investigated various forms of Nef with hydrogen exchange mass spectrometry. Conformational changes in Nef were not detected as a result of myristoylation, and NMT had no effect on deuterium uptake by Nef in a myrNef:NMT complex. However, myrNef binding did have an effect on NMT deuterium uptake. Major HX differences in NMT were primarily located around the active site, with more subtle differences, at the longer time points, across the structure. At the shortest time point, significant differences between the two states were observed in two regions which interact strongly with the phosphate groups of coenzyme A. On the basis of our results, we propose a model of the Nef:NMT complex in which only the myristoyl moiety holds the two proteins together in complex and speculate that perhaps NMT chaperones Nef to the membrane and thereby protects the myristic acid group from the cytosol rather than Nef operating through a myristoyl switch mechanism.     

Glycosphingolipids of the globo-series are associated with the monocytic lineage of human myeloid cells

Neutral glycosphingolipids (neutral GSLs) of the human myeloid leukemia cell lines ML-2, ML-3, HL-60 and THP-1-0 were metabolically labeled with [3H]galactose and [3H]glucosamine, and analyzed by high-performance liquid chromatography. They were compared with unlabeled neutral GSLs from purified human granulocytes and monocytes. Neutral GSLs were identified by retention times and the structures were further confirmed by degradation with specific exoglycosidases. Two neutral GSLs of the globoseries, globotetraosylceramide and globotriaosylceramide were found in monocytes and the monoblastic leukemia line THP-1-0. The leukemia-derived cell-lines, ML-3 and HL-60, representing successively earlier stages of myeloid differentiation, contained respectively less neutral GSLs of the globoseries and an increasing proportion of (neo)lacto neutral GSLs. Granulocytes and the cell line ML-2 contained almost exclusively neutral GSLs of the (neo)lacto series.     

Cell lineage in plant development.

Lineage analyses in several plant species demonstrate that meristematic cells proliferate in a predictable manner to form the differentiated tissues of the mature shoot system. These studies also demonstrate, however, that the fates of meristematic cells are not absolutely dependent on their lineage. This variability indicates that interactions between cells must play a role in morphogenesis.     

Cell lineage in plant development.

Lineage analyses in several plant species demonstrate that meristematic cells proliferate in a predictable manner to form the differentiated tissues of the mature shoot system. These studies also demonstrate, however, that the fates of meristematic cells are not absolutely dependent on their lineage. This variability indicates that interactions between cells must play a role in morphogenesis.     

The oligodendrocyte lineage genes Olig1 and Olig2 in CNS development

After contusion-derived spinal cord injury there is localized tissue disruption and energy failure that results in early necrosis and delayed apoptosis, events that contribute to chronic central pain in a majority of patients. We assessed mechanisms of contusion-induced apoptosis of neurons and glia in a known central pain signalling pathway, the spinothalamic tract (STT), which may be a contributor to SCI-induced pain. Twenty-four hours after injury there was demonstrable apoptosis among neurons of the spinothalamic tract. Apoptosis in the injured spinal cord correlated well with prompt decreases in Bcl-xL and Bcl-xL/Bax protein ratios at the contusion site. There was definitive triggering of the inflammatory cytokine cascade with IL-1b being most robust and prompt in responding. Clearly, a better understanding of inflammatory processes, especially the role of cytokines after nerve injury, can lead to the development of new therapies that may prevent, and not just treat chronic central pain. Intervention in the inflammatory cascade had beneficial effects with confounds, which were mostly assessed by cDNA microarray analyses. We interpret these results as evidence that regulation of Bcl-xL and other genes that determine cell death outcomes may play a role in the inflammatory response to spinal injury and pain signalling function.
Acknowledgements:   Supported in part by NINDS and Mission Connect.     

The role of cell lineage in development

Studies of the role of cell lineage in development began in the latter part of the 19th century, fell into decline in the early part of the 20th, and were revived about 20 years ago. This recent revival was accompanied by the introduction of new and powerful analytical techniques. Concepts of importance for cell lineage studies include the principal division modes by which a cell may give rise to its descendant clone (proliferative, stem cell and diversifying); developmental determinacy, or indeterminacy, which refer to the degree to which the normal cleavage pattern of the early embryo and the developmental fate of its individual cells is, or is not, the same in specimen after specimen; commitment, which refers to the restriction of the developmental potential of a pluripotent embryonic cell; and equivalence group, which refers to two or more equivalently pluripotent cell clones that normally take on different fates but of which under abnormal conditions one clone can take on the fate of another. Cell lineage can be inferred to have a causative role in developmental cell fate in embryos in which induced changes in cell division patterns lead to changes in cell fate. Moreover, such a causative role of cell lineage is suggested by cases where homologous cell types characteristic of a symmetrical and longitudinally metameric body plan arise via homologous cell lineages. The developmental pathways of commitment to particular cell fates proceed according to a mixed typologic and topographic hierarchy, which appears to reflect an evolutionary compromise between maximizing the ease of ordering the spatial distribution of the determinants of commitment and minimizing the need for migration of differentially committed embryonic cells. Comparison of the developmental cell lineages in leeches and insects indicates that the early course of embryogenesis is radically different in these phyletically related taxa. This evolutionary divergence of the course of early embryogenesis appears to be attributable to an increasing prevalence of polyclonal rather than monoclonal commitment in the phylogenetic line leading from an annelid-like ancestor to insects.     

Potential role of N-myristoyltransferase in cancer

Colorectal cancer is the second leading cause of malignant death, and better preventive strategies are needed. The treatment of colonic cancer remains difficult because of the lack of effective chemotherapeutic agents; therefore it is important to continue to search for cellular functions that can be disrupted by chemotherapeutic drugs resulting in the inhibition of the development and progression of cancer. The current knowledge of the modification of proteins by myristoylation involving myristoyl-CoA: protein N-myristoyltransferase (NMT) is in its infancy. This process is involved in the pathogenesis of cancer. We have reported for the first time that NMT activity and protein expression were higher in human colorectal cancer, gallbladder carcinoma and brain tumors. In addition, an increase in NMT activity appeared at an early stage in colonic carcinogenesis. It is conceivable therefore that NMT can be used as a potential marker for the early detection of cancer. These observations lead to the possibility of developing NMT specific inhibitors, which may be therapeutically useful. We proposed that HSC70 and/or enolase could be used as an anticancer therapeutic target. This review summarized the status of NMT in cancer which has been carried in our laboratory.     

ISG15 modulates development of the erythroid lineage

Activation of erythropoietin receptor allows erythroblasts to generate erythrocytes. In a search for genes that are up-regulated during this differentiation process, we have identified ISG15 as being induced during late erythroid differentiation. ISG15 belongs to the ubiquitin-like protein family and is covalently linked to target proteins by the enzymes of the ISGylation machinery. Using both in vivo and in vitro differentiating erythroblasts, we show that expression of ISG15 as well as the ISGylation process related enzymes Ube1L, UbcM8 and Herc6 are induced during erythroid differentiation. Loss of ISG15 in mice results in decreased number of BFU-E/CFU-E in bone marrow, concomitant with an increased number of these cells in the spleen of these animals. ISG15(-/-) bone marrow and spleen-derived erythroblasts show a less differentiated phenotype both in vivo and in vitro, and over-expression of ISG15 in erythroblasts is found to facilitate erythroid differentiation. Furthermore, we have shown that important players of erythroid development, such as STAT5, Globin, PLC γ and ERK2 are ISGylated in erythroid cells. This establishes a new role for ISG15, besides its well-characterized anti-viral functions, during erythroid differentiation.     

Cell lineage and gene expression in the development of polychaetes

The developmental strategies of embryos within the various taxa of polychaetes are designed to set up the fate of the cell lines. Some of these traits of pattern formation are considered to be ancestral, but we also find a number of derived developmental characteristics, some of which might be useful indicators for phylogenetic relationships. A combination of ooplasmic segregation and anisotropic cleavage rapidly establishes the fate of several larval cell lines in the polychaete embryo. The setting-up of the primary trochoblasts basically concerns the same cell line (la²–ld²) in polychaetes and even in molluscs. Such mechanisms may thus be regarded as ancestral. The determination of the mesoderm precursor occurs very late in both equally cleaving annelids and mollusks, indicating that an equal cleavage pattern may be regarded as an ancestral trait. Since both disproportionate cytoplasmic distribution (either by spindle shift or polar lobe formation) and cell cycle asynchronies appear to speed up the development of the mesoderm-forming cell line, these strategies represent derived traits. An analysis of these derived traits of early development is given and is discussed in the light of the phylogenetic relationships among the polychaetes. These data are extended by an analysis of some of the postlarval structures in polychaetes and the molecular developmenta1 circuitry involved.