Нүклеозид дифосфат киназалары: құрылымы, функциясы және реттелуі

Class of enzymes

Nucleoside diphosphate kinases (NDPKs, also NDP kinase, (poly)nucleotide kinases and nucleoside diphosphokinases) are enzymes that catalyze the exchange of terminal phosphate between different nucleoside diphosphates (NDP) and triphosphates (NTP) in a reversible manner to produce nucleotide triphosphates. Many NDP serve as acceptor while NTP are donors of phosphate group. The general reaction via ping pong mechanism is as follows: XDP + YTP ←→ XTP + YDP (X and Y each represent different nitrogenous base). NDPK activities maintain an equilibrium between the concentrations of different nucleoside triphosphates such as, for example, when guanosine triphosphate (GTP) produced in the citric acid (Krebs) cycle is converted to adenosine triphosphate (ATP). Other activities include cell proliferation, differentiation and development, signal transduction, G protein coupled receptor, endocytosis, and gene expression.

NDPK are homohexameric proteins made up of monomers approximately 152 amino acids long with a theoretical weight of 17.17KDa. The complex is found in mitochondria and in the soluble cytoplasm of cells.

NDPK usually consumes ATP, the most abundant cellular nucleotide, and stores the nucleotides. However, consumption of ATP would definitely influence the cellular energy balance, which brings upon the regulation of AMP activated protein kinase (AMPK). AMPK acts as the energy sensor and regulates ATP pathways by turning the generating pathways or not. Because of such activity, AMPK could directly inhibit NDPK through phosphorylation. To be more specific, NDPK supports the production of nucleotides in high energy and low stress cellular states. However, this can only happen when AMPK is inactivated because low stress cellular states of ATP triggers the activation of AMPK, which eventually decreases NDPK activity by phosphorylating serine residues.

In most prokaryotes, the NDPK enzyme is tetrameric. It has been reported in a number of pathogens. NDPK function has been studied in Escherichia coli, Bacillus subtilis, Salmonella typhimurium, Microccocus luteus, and Myxococcus xanthus. Prokaryotic NDPK forms a functional homotetramer. Nucleoside diphosphate kinase activity involves the transfer of the γ phosphate of nucleoside triphosphate (NTP) to nucleoside diphosphate (NDP), where N1 and N2 can be ribo or deoxyribonucleosides. This is done via a high energy phosphohistidine intermediate. Besides involvement in the synthesis of pyrimidine nucleotides, prokaryotic NDPK is also involved in several metabolism cycles. NDPK has also been discovered to act as a protein histidine kinase, which involves a reversible histidine phosphorylation as a well known regulatory signal. However, in most prokaryotes, NDPK expression levels are involved in the cell growth, development and differentiation of the organism, especially bacteria.

In the (p)ppGpp biosynthesis cycle, NDPK serves an important role. When there is an absence of a charged tRNA in the A site of a ribosome, the ribosome will stall and trigger the synthesis of the guanosine pentaphosphate ((p)ppGpp) molecule. (p)ppGpp biosynthesis is a part of the purine metabolism pathway and coordinates a series of cellular activities in response to nutritional abundances. Synthesis of (p)ppGpp is triggered by carbon starvation, or the lack of carbon in the cell's environment, and causes the protein SpoT to activate. SpoT works in conjunction with NDPK and both serve as essential enzymes in the (p)ppGpp biosynthesis cycle. NDPK synthesizes the formation of GDP from GTP via dephosphorylation.

While the biomolecular mechanism by which the Nm23 gene works in cells is currently unknown, like in most prokaryotes, nucleoside diphosphate kinase (NDPK) expression levels determine cell growth and differentiation.

There are at least four enzymatically active isoforms of NDPK in humans: NDPK A, NDPK B, NDPK C and NDPK D. All four isoforms have very similar structures and can combine in any form to become functional NDPK hexamers. NDPK is suggested to participate in transmembrane signaling in eukaryotic cells.

In Eukaryotic systems, the role of the NDK is to synthesize nucleoside triphosphates other than ATP. The ATP gamma phosphate is transferred to the NDP beta phosphate via a ping pong mechanism, using a phosphorylated active site intermediate, and synthesize products such as UTP. NDK possesses nucleoside diphosphate kinase, serine/threonine specific protein kinase, geranyl and farnesyl pyrophosphate kinase, histidine protein kinase, and 3' 5' exonuclease activities. Its processes are involved with cell proliferation, differentiation and development, and gene expression in human cells. It is also part of the neural development process, which includes neural patterning and cell fate determination. Furthermore, NDPK is involved with the signal transduction processes and G protein coupled receptor endocytosis as it transfers a phosphate group onto the G β subunits and convert GDP to GTP. This increase in GTP concentration near G protein α subunits causes activation of G protein α subunits for G protein signaling. In addition to signaling, NDPK is involved in controlling K+ channels, cell secretion, and cellular energy production.

The biochemical reactions catalyzed by NDP kinase in plants are analogous to activities described in humans as autophosphorylation activity takes place from ATP and GTP. In addition to this, plants have four types of NDPK isoforms. Cytosolic type I NDPK is involved in metabolism, growth, and stress responses in plants. Type II NDPK is concentrated in the chloroplast and it is believed to be involved in the photosynthesis process and the oxidative stress management, but its function is not yet known clearly.

Ten paralogous genes code for the proteins NDPKs, which are separated into two groups. The first group encodes proteins with NDPK functions. The other group genes code for other various proteins that display low or no NDPK activities. In the first group, one of the genes named NM23 was identified as the first metastasis suppressor protein and its gene Nm23 was less activated in metastatic cells. In a different experiment, human Nm23 was cultured with cancer cells and showed inhibition of metastasis. The level of NM23 protein was inversely proportional to the metastatic potential for human solid tumors. However, other tumor types such as ovarian cancers, neuroblastoma and hematological malignancies displayed upregulated NM23 levels in patient samples. Therefore, understanding the biological basis of the Nm23 gene family is necessary to have a firm knowledge of its diverse results.

Nme2, one of the NDPK genes, has been associated with cardiovascular functions. Nme2 gene is known to form a complex with the beta subunit of the heterotrimetric G protein in heart cells and regulates the contractility of heart. There are two functions of Nme2 that allow for such regulation; one is the histidine kinase activity, which is the phosphorylation of the channels to regulate what goes through and the other is a scaffold function of the formation of caveolae. The depletion of Nme2/caveolin interaction exhibited a decreased rate of cardiac contractility. Furthermore, more studies with zebra fish revealed that the NDPK depletion has a detrimental effect on heart functioning.

There was a lot of debate on whether NM23 gene is responsible for suppressing or activating metastasis. The two contradicting sides on this subject remained ambiguous and undefined throughout the course of NDPK studies. However, recent experiments began to show evidence for NM23 being a suppressor of metastasis. Nme2 was tagged as an anti metastasis gene, using the tissue chip technology and immunohistochemistry. When Nme2 gene products were over produced in gastric cancer cells, there was a decrease in proliferation, migration, and invasion of such cancer cells. The cell cultures revealed that Nme2 impacts gastric cancer cells, but the question still remains about what regulates Nme2 activities among various cancer types. Nme1 was found in great number in poorly metastatic sublines of melanoma cells. Also, the transfection of Nme1 into a highly metastatic melanoma line significantly reduced metastasis. This theory has been tested with mice as well; the Nme1 deficient mice formed greater lung metastases than wild type mice, showing that this gene has suppressing activity. Invasion of cancer occurs due to changes in cell adhesion and it is caused by gene expression changes in the epithelial mesenchymal transition (EMT). Surprisingly, there are many adhesion molecules, motility factors, signaling pathways, proteolytic events, EMT hallmarks, and other transcriptional programs that have been linked to the Nme1 proteins. These proteins go about interrupting metastasis by binding metastasis promoting proteins. The Nme1 proteins bind to viral proteins, oncogenes, and other metastasis promoting factors. The binding may be indirect by using the signaling complex.