The role of Neuroglobin in hematopoietic system

Hemoglobin and Myoglobin are proteins that belong to the globins family and are able to deliver and conserve oxygen. In the 2000 a new component of this family has been identified and named Neuroglobin (Ngb). Ngb is mainly expressed in the nervous system, from which it takes its name, but it is located also in the gastrointestinal tract, in the endocrine tissue and in the retina. Ngb is a monometric protein formed by 151 aminoacids (17 KDa) and has a high affinity for oxygen. The human Ngb gene is located in the chromosome 14q24. Over the evolution, the Ngb gene has been conserved and this suggests its relevant role in biological systems. Ngb, as all the globins familyࢠs members, is an heme protein that shows the heme prostetic group, by which can reversibly bind oxygen and other ligands. The iron atom in the prostetic group can have the oxidative state +2 (ferrous) or +3 (ferric). Both in the ferrous and in the ferric state the Ngb is hexacoordinated with other endogenous ligands (proximal and distal hystidine) and O2 or CO crowd-out the distal hystidine to link themselves to the protein. Even though many studies have been conducted, it is not completely clear which functions the Ngb has. It is known that the Ngb plays a protective role in neurons under conditions of hypoxia and hyschemia. Moreover it plays, too, a protective function during the oxidative stress and lipidic peroxidation. However the mechanism by which Ngb can protect neurons has not been completely elucidated yet. Several studies have shown which mechanisms this function can be linked to: 1) the Ngb ability to scavenge reactive oxygen and nitrogen species; 2) its high affinity for oxygen that allows the organism to have a bulk of oxygen during hypoxia conditions; 3) its role in the signal transduction. In fact, the body of literature suggests that Ngb, in its ferrous state and linking oxygen, is quickly converted in the ferric state and only the latter is able to bind the G? subunit of the G protein, that in turn is bound to the GDP. The link between the Ngb in the ferric state and the G protein does not allow the release of the GDP and consequently the activation of the G protein itself. This way Ngb halts the signal transduction that leads to cell death and guarantees the survival of the cells. Additionally, in the ferric state Ngb can bind Flotillin-1, a protein mainly express in rafts, small membrane domains, rich in sfingolipids e cholesterol, that function as platforms to concentrate and segregate signal molecules during cellular processes. Thus in rafts there are several proteins involved in the signal transduction, among which G proteins. Flotillin is an integral membrane protein that plays a key role in assembling rafts and in the signal transduction. Two different isiform of Flotillin are known: Flotillin-1 and Flotillin-2. The Flotillin-1 gene is localized in the chromosome 6 and code for a 46 KDa protein, particularly expressed in neurons, but also present in the fagosomes membrane, in endosomes and in the nucleus. The function of Flotillin is associated with its localization in rafts and it is involved in many cellular processes: it participates in the glucose income in insulin-stimulated cells; it induces the neuronal regeneration; it participates in the fagosome maturation; it drives the proliferation of prostatic cancer cells and finally it defines an endocytic Clatrin-dependent pathway. According to this information, the aim of the project is to evaluate either the possible expression of Ngb in myeloid cells, and to analyze its function in this specific cell system, and to find a functional relationship between Ngb and Flotillin-1. In this project, a significant expression of Ngb has been individuated in myeloid cells (THP1, U937 and HL60) and in lung cancer cells (H1299, H1650 and H1975). According to these findings, the expression of Ngb during the myeloid differentiation has been analyzed. The expression of Ngb, as well as of Flotillin, increases during the differentiation. Additionally, Ngb is mainly expressed in macrophagic cells. Because of the neuroprotective role of Ngb during the oxidative stress, its expression has been evaluated during a specific condition of stress that occurs in myeloid cells, that is the fagocytosis. The Ngb level improves during the fagocytic process, as well as Flotillin-1, and increases even more during the fagocytosis realized by mature macrophages, which have a basic role in this process. These results suggest a possible involvement of these two proteins in this mechanism. Moreover, the Ngb expression has been analyzed in specific conditions that could have induced cellular stress and apoptosis, particularly during serum deprivation (by testing concentrations of serum lower than the concentration used in standard conditions) and after treatment with hydroxyurea (a DNA synthesis inhibitor). In both these situations there was not a modification in the Ngb expression, suggesting that probably the protein is not involved. Conversely, a remarkable increase of the Ngb expression in myeloid cells has been observed after H2O2 treatment, inducing oxidative stress. This result could suggest that, during oxidative stress conditions, Ngb may play a protective role in myeloid cells as well as shown in neurons. Different drugs can induce oxidative stress, such as Isoniazid (Inh), a first line anti-tubercolar drug. Inh is able to reversibly bind both the ferric and ferrous iron of the heme group in the truncated hemoglobin of the Mycobacterium Tuberculosis. This link modify the heme group properties, making the hemoglobin unable to perform its normal functions. The Ngb, too, has an heme group whose ferrous iron is converted in ferric iron under oxidative stress conditions to protect neurons. Thus, the Ngb expression has been evaluated after treatment with different concentrations of Inh in this cellular system and consequently a dose-dependent increase of Ngb expression has been observed. Afterwards, combining the myeloid differentiation with oxidative stress conditions (H2O2-induced), a higher Ngb level has been measured in mature myeloid cells compared to the Ngb expression in undifferentiated cells. Finally the functional relationship between Ngb and Flotillin-1 in the hematopoietic system has been studied. First of all the possible influence of Flotilin-1 on Ngb has been evaluated. According to this purpose, over-expressing Flotillin-1 cells and Flotillin-1 knockdown cells have been generated. In the first condition no variations in the expression of Ngb have been observed, in the latter a remarkable decrease in the protein have been noticed. The lack of Flotillin-1 prevent Ngb from increasing also in H2O2-induced oxidative stress conditions. This data suggests a possible protective and stabilizing function of Flotillin-1 on Ngb. To confirm this hypothesis, the physical interaction between these two proteins has been investigated by setting up a co-immunoprecipitation assay with an antibody anti-Flotillin-1, and then a Western Blotting with an antibody anti-Ngb, in myeloid cells both in basal conditions and after an H2O2-induced oxidative stress treatment. The results obtained showed that, in myeloid cells, the physical interaction occurs only after H2O2 treatment, thus in conditions of oxidative stress. In conclusions, to sum the data collected during this project: Ngb is expressed in myeloid cells and in lung cancer cells, too; its expression increases during the myeloid differentiation, the fagocytosis and the oxidative stress; additionally, its level tends to diminish in Flotillin-1 knockdown cells; and finally, Ngb interacts with Flotillin-1 during the oxidative stress. According to these results, further studies could be conducted to elucidate and better understand the functions of Ngb in different cellular systems. As examples, the role of Ngb could be analyzed in myeloid progenitors treated with growth factors and induced to differentiate in granulocytes or monocytes. Moreover, as a support to the hypothesis that Ngb has a potential protective effect in myeloid cells under oxidative stress conditions, consequences of the silencing or over-expression of Ngb could be evaluated.