Amyotrophic lateral sclerosis (ALS) is a clinically heterogeneous disorder characterized by degeneration of upper motor neurons in the brainstem and lower motor neurons in the spinal cord. Multiple mechanisms of motor neuron injury have been implicated, including more than 20 different genetic factors. The pathogenesis of ALS consists of two stages: an early neuroprotective stage and a later neurotoxic. During early phases of disease progression, the immune system through glial and T cell activities provides anti-inflammatory factors that sustain motor neuron viability. As the disease progresses and motor neuron injury accelerates, a rapidly succeeding neurotoxic phase develops. A well-orchestrated purine-mediated dialog among motor neurons, surrounding glia and immune cells control the beneficial and detrimental activities occurring in the nervous system. In general, low adenosine triphosphate (ATP) concentrations protect cells against excitotoxic stimuli through purinergic P2X4 receptor, whereas high concentrations of ATP trigger toxic P2X7 receptor activation. Finally, adenosine is also involved in ALS progression since A2A receptor antagonists prevent motor neuron death. Given the complex cellular cross-talk occurring in ALS and the recognized function of extracellular nucleotides and adenosine in neuroglia communication, the comprehensive understanding of purinome dynamics might provide new research perspectives to decipher ALS and help to design more efficient and targeted drugs. This review will focus on the purinergic players involved in ALS etiology and disease progression and current therapeutic strategies to enhance neuroprotection and suppress neurotoxicity.
Growing murine mesenchymal stem cells (mMSCs) from mouse bone marrow decreased their rate of proliferation in the presence of benzoylbenzoyl-ATP persistently, but the inhibitory effect of ATP was strong only in a concentration of 50 μmol·L(-1) and lasted for 48 h in culture. These results hinted at ATP hydrolysis by the cell surface enzymes at the lower concentrations and thus it may be not able to inhibit MSCs. By using ATP, ADP, or AMP as substrates, we tested the ectonucleotidase activity on the surface of undifferentiated MSCs and MSC-derived osteoblasts. Here, we report that although nucleoside triphosphate diphosphohydrolase (NTPDase)1 and NTPDase8 are engaged in the metabolism of ATP in MSC-derived osteoblasts, NTPDase3 is responsible for its metabolism in undifferentiated MSCs. In this study, we also realized that osteoblasts effectively metabolize ADP to ATP and AMP. The enzymatic activity of adenylate kinase (AK) is consistent with the high expression level of the AK gene. Therefore, it was tempting to suggest that this enzyme, together with NTPDase1 and NTPDase8, assume the role of specific markers that allowed distinction between differentiated osteoblasts and early undifferentiated MSCs. Additionally, unlike osteoblasts, undifferentiated MSCs demonstrated the activity of 5'-nucleotidase (CD73). However, the expression analysis of CD73 mRNA did not show any differences; CD73 mRNA was expressed in both kinds of cells to the same extent.
Extracellular purines, principally adenosine triphosphate and adenosine, are among the oldest evolutionary and widespread chemical messengers. The integrative view of purinergic signaling as a multistage coordinated cascade involves the participation of nucleotides/nucleosides, their receptors, enzymes metabolizing extracellular nucleosides and nucleotides as well as several membrane transporters taking part in the release and/or uptake of these molecules. In view of the emerging data, it is evident and widely accepted that an extensive network of diverse enzymatic activities exists in the extracellular space. The enzymes regulate the availability of nucleotide and adenosine receptor agonists, and consequently, the course of signaling events. The current data indicate that mesenchymal stem cells (MSCs) and cells induced to differentiate exhibit different sensitivity to purinergic ligands as well as a distinct activity and expression profiles of ectonucleotidases than mature cells. In the proposed review, we postulate for a critical role of these enzymatic players which, by orchestrating a fine-tune regulation of nucleotides concentrations, are integrally involved in modulation and diversification of purinergic signals. This specific hallmark of the MSC purinome should be linked with cell-specific biological potential and capacity for tissue regeneration. We anticipate this publication to be a starting point for scientific discussion and novel approach to the in vitro and in vivo regulation of the MSC properties.
Background: Pulmonary hypertension (PH) is a life-threatening disease, characterized by excessive pulmonary vascular remodeling, leading to elevated pulmonary arterial pressure and right heart hypertrophy. PH can be caused by chronic hypoxia, leading to hyper-proliferation of pulmonary arterial smooth muscle cells (PASMC) and apoptosis-resistant pulmonary microvascular endothelial cells (PMVEC). Upon re-exposure to normoxia, chronic hypoxia-induced PH in mice is reversible. In this study, we aim to identify novel candidate genes involved in pulmonary vascular remodeling specifically in the pulmonary vasculature. Methods: Following a microarray analysis, we assessed the role of secreted protein acidic and rich in cysteine (SPARC) in PH using lung tissue from idiopathic pulmonary arterial hypertension (IPAH) patients as well as from chronic hypoxic mice. In vitro studies were conducted in primary human PASMC and PMVEC. In vivo function of SPARC was proven in chronic hypoxia-induced PH in mice by using an AAV-mediated Sparc knockdown approach. Results: C57BL/6J mice were exposed to normoxia, chronic hypoxia, or chronic hypoxia with subsequent re-exposure to normoxia for different time points. Microarray analysis of the pulmonary vascular compartment after laser microdissection identified Sparc as one of the genes down-regulated at all re-oxygenation time points investigated. Intriguingly, SPARC was vice versa upregulated in lungs during development of hypoxia-induced PH in mice as well as in IPAH, although SPARC plasma levels were not elevated in PH. Transforming growth factor (TGF)-β1 or hypoxia-inducible factor (HIF)-2A signaling pathways induced SPARC expression in human PASMC. In loss of function studies, SPARC silencing enhanced apoptosis and reduced proliferation. In gain of function studies, elevated SPARC levels induced PASMC but not PMVEC proliferation. Co-culture and conditioned medium experiments revealed that PMVEC-secreted SPARC acts as a paracrine factor triggering PASMC proliferation. Against our expectations, in vivo congenital Sparc knockout mice were not protected from hypoxia-induced PH, most probably due to counter-regulatory pro-proliferative signaling. However, AAV-mediated Sparc knockdown in adult mice significantly improved hemodynamic and cardiac function in PH mice. Conclusions: Our study provided evidence for the involvement of SPARC in the pathogenesis of human PH and chronic hypoxia-induced PH in mice, most probably by affecting vascular cell function.
For overproduction of recombinant proteins both eukaryotic and prokaryotic expression systems are used. Choosing the right system depends, among other things, on the growth rate and culture of host cells, level of the target gene expression and posttranslational processing of the synthesized protein. Regardless of the type of expression system, its basic elements are the vector and the expression host. The most widely used system for protein overproduction, both on a laboratory and industrial scale, is the prokaryotic system. This system is based primarily on the bacteria E. coli, although increasingly often Bacillus species are used. The prokaryotic system allows one to obtain large quantities of recombinant proteins in a short time. A simple and inexpensive bacterial cell culture and well-known mechanisms of transcription and translation facilitate the use of these microorganisms. The simplicity of genetic modifications and the availability of many bacterial mutants are additional advantages of the prokaryotic system. In this article we characterize the structural elements of prokaryotic expression vectors. Also strategies for preparation of the target protein gene that increase productivity, facilitate detection and purification of recombinant protein and provide its activity are discussed. Bacterial strains often used as host cells in expression systems as well as the potential location of heterologous proteins are characterized. Knowledge of the basic elements of the prokaryotic expression system allows for production of biologically active proteins in a short time and in satisfactory quantities.
Increased proliferation of pulmonary arterial smooth muscle cells (PASMCs) in response to chronic hypoxia contributes to pulmonary vascular remodeling in pulmonary hypertension (PH). PH shares numerous similarities with cancer, including a metabolic shift towards glycolysis. In lung cancer, adenylate kinase 4 (AK4) promotes metabolic reprogramming and metastasis. Against this background, we show that AK4 regulates cell proliferation and energy metabolism of primary human PASMCs. We demonstrate that chronic hypoxia upregulates AK4 in PASMCs in a hypoxia-inducible factor-1α (HIF-1α)-dependent manner. RNA interference of AK4 decreases the viability and proliferation of PASMCs under both normoxia and chronic hypoxia. AK4 silencing in PASMCs augments mitochondrial respiration and reduces glycolytic metabolism. The observed effects are associated with reduced levels of phosphorylated protein kinase B (Akt) as well as HIF-1α, indicating the existence of an AK4-HIF-1α feedforward loop in hypoxic PASMCs. Finally, we show that AK4 levels are elevated in pulmonary vessels from patients with idiopathic pulmonary arterial hypertension (IPAH), and AK4 silencing decreases glycolytic metabolism of IPAH-PASMCs. We conclude that AK4 is a new metabolic regulator in PASMCs interacting with HIF-1α and Akt signaling pathways to drive the pro-proliferative and glycolytic phenotype of PH.
Adenylate kinase (AK, EC 2.7.4.3) is a ubiquitous phosphotransferase which catalyzes the reversible transfer of high-energy β - and γ-phosphate groups between nucleotides. All classified AKs show a similar structure: they contain a large central CORE region, nucleoside monophosphate and triphosphate binding domains (NMPbd and NTPbd) and the LID domain. Analysis of amino acid sequence similarity revealed the presence of as many as nine human AK isoenzymes, which demonstrate different organ-tissue and intercellular localization. Among these kinases, only two, AK1 and AK2, fulfill the structural and functional criterion by the highest affinity for adenine nucleotides and the utilization of only AMP or dAMP as phosphate acceptors. Human AK isoenzymes are involved in nucleotide homeostasis and monitor disturbances of cell energy charge. Participating in large regulatory protein complexes, AK supplies high energy substrates for controlling the functions of channels and transporters as well as ligands for extracellular P2 nucleotide receptors. In pathological conditions AK can take over the function of other kinases, such as creatine kinase in oxygen-depleted myocardium. Directed mutagenesis and genetic studies of diseases (such as aleukocytosis, hemolytic anemia, primary ciliary dyskinesia (PCD)) link the presence and activity of AK with etiology of these disturbances. Moreover, AK participates in regulation of differentiation and maturation of cells as well as in apoptosis and oncogenesis. Involvement of AK in a wide range of processes and the correlation between AK and etiology of diseases support the medical potential for the use of adenylate kinases in the diagnosis and treatment of certain diseases. This paper summarizes the current knowledge on the structure, properties and functions of human adenylate kinase.
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