Metastases suppressor NM23-H2 interaction with G-quadruplex DNA within c-MYC promoter nuclease hypersensitive element induces c-MYC expression

Regulatory influence of the G-quadruplex or G4 motif present within the nuclease hypersensitive element (NHE) in the promoter of c-MYC has been noted. On the other hand, association of NM23-H2 to the NHE leads to c-MYC activation. Therefore, NM23-H2 interaction with the G4 motif within the c-MYC NHE presents an interesting mechanistic possibility. Herein, using luciferase reporter assay and chromatin immunoprecipitation we show NM23-H2 mediated c-MYC activation involves NM23-H2-G4 motif binding within the c-MYC NHE. G4 motif complex formation with recombinant NM23-H2 was independently confirmed using fluorescence energy transfer, which also indicated that the G4 motif was resolved to an unfolded state within the protein-bound complex. Taken together, this supports transcriptional role of NM23-H2 via a G4 motif

Clinical Significance of Serum Biomarkers in Pediatric Solid Mediastinal and Abdominal Tumors

Childhood cancer is the leading cause of death by disease among U.S. children between infancy and age 15. Despite successes in treating solid tumors such as Wilms tumor, disappointments in the outcomes of high-risk solid tumors like neuroblastoma have precipitated efforts towards the early and accurate detection of these malignancies. This review summarizes available solid tumor serum biomarkers with a special focus on mediastinal and abdominal cancers in children

A Unique Mode of Coenzyme A Binding to the Nucleotide Binding Pocket of Human Metastasis Suppressor NME1

Coenzyme A (CoA) is a key cellular metabolite which participates in diverse metabolic pathways, regulation of gene expression and the antioxidant defense mechanism. Human NME1 (hNME1), which is a moonlighting protein, was identified as a major CoA-binding protein. Biochemical studies showed that hNME1 is regulated by CoA through both covalent and non-covalent binding, which leads to a decrease in the hNME1 nucleoside diphosphate kinase (NDPK) activity. In this study, we expanded the knowledge on previous findings by focusing on the non-covalent mode of CoA binding to the hNME1. With X-ray crystallography, we solved the CoA bound structure of hNME1 (hNME1-CoA) and determined the stabilization interactions CoA forms within the nucleotide-binding site of hNME1. A hydrophobic patch stabilizing the CoA adenine ring, while salt bridges and hydrogen bonds stabilizing the phosphate groups of CoA were observed. With molecular dynamics studies, we extended our structural analysis by characterizing the hNME1-CoA structure and elucidating possible orientations of the pantetheine tail, which is absent in the X-ray structure due to its flexibility. Crystallographic studies suggested the involvement of arginine 58 and threonine 94 in mediating specific interactions with CoA. Site-directed mutagenesis and CoA-based affinity purifications showed that arginine 58 mutation to glutamate (R58E) and threonine 94 mutation to aspartate (T94D) prevent hNME1 from binding to CoA. Overall, our results reveal a unique mode by which hNME1 binds CoA, which differs significantly from that of ADP binding: the α- and β-phosphates of CoA are oriented away from the nucleotide-binding site, while 3′-phosphate faces catalytic histidine 118 (H118). The interactions formed by the CoA adenine ring and phosphate groups contribute to the specific mode of CoA binding to hNME1

Sponges: A Reservoir of Genes Implicated in Human Cancer

Recently, it was shown that the majority of genes linked to human diseases, such as cancer genes, evolved in two major evolutionary transitions—the emergence of unicellular organisms and the transition to multicellularity. Therefore, it has been widely accepted that the majority of disease-related genes has already been present in species distantly related to humans. An original way of studying human diseases relies on analyzing genes and proteins that cause a certain disease using model organisms that belong to the evolutionary level at which these genes have emerged. This kind of approach is supported by the simplicity of the genome/proteome, body plan, and physiology of such model organisms. It has been established for quite some time that sponges are an ideal model system for such studies, having a vast variety of genes known to be engaged in sophisticated processes and signalling pathways associated with higher animals. Sponges are considered to be the simplest multicellular animals and have changed little during evolution. Therefore, they provide an insight into the metazoan ancestor genome/proteome features. This review compiles current knowledge of cancer-related genes/proteins in marine sponges

Subcellular localisation of the NME6 protein and anti-NME6 antibody testing

Nukleozid-difosfatske kinaze (NDPK/Nm23/NME) čine obitelj evolucijski očuvanih enzima, koji prenose fosfatnu skupinu γ s andenozin trifosfata na nukleozid difosfate i na taj način održavaju ravnotežu nukleotida u stanci, a smatra se da imaju i neke druge biokemijske funkcije. Nukleozid-difosfatske kinaze sudjeluju u brojnim biološkim procesima kao što su proliferacija, diferencijacija i razvoj, a imaju značajnu ulogu i u razvoju tumora i metastaziranju. Unatoč brojnim istraživanjima, njihova biološka funkcija nije do danas u potpunosti razjašnjena. Deset članova obitelji proteina NME (od NME1 do NME10) podijeljeni su u dvije skupine. Skupinu I čine proteini od NME1 do NME4 i svi posjeduju aktivnost kinaze NDP, dok u skupinu II ubrajamo NME5 do NME10, od kojih većina ne pokazuje aktivnost kinaze NDP. Strukturno, NME6 dijeli svega 34-41% homologije s proteinima iz prve skupine, dok je njegov izražaj relativno nizak u većini tkiva. Malobrojna istraživanja upućuju na to da je NME6 smješten u mitohondrijima i u citoplazmi. Cilj diplomskog rada bio je odrediti lokalizaciju proteina NME6 u humanim tumorskim stanicama HeLa uz pomoć fluorescentnog reporterskog sustava, testirati antitijelo anti-NME6 te optimizirati metodu Western blot. U tu svrhu, koristili smo metode transformacije, transfekcije, fluorescentnu imunocitokemiju, analizu Western blot te konfokalnu mikroskopiju.Nucleoside diphosphate kinases are evolutionary conserved enzymes which transfer the γ phosphate from adenosine triphosphates to nucleoside diphosphates consequently maintaining the nucleotide homeostasis in the cell. Additionally, they are also considered to have other biochemical functions. NDP kinases are involved in several biological processes such as proliferation, differentiation and development. Moreover, they are proven to have a significant role in tumour development and metastasis. However, despite intensive research in the field, all of their biological functions still aren’t completely resolved. The NME family consists of 10 genes/proteins (from NME1 to NME10), divided into two groups. Group I encompasses proteins from NME1 to NME4 which all exhibit the NDP kinase. Group II consists of proteins from NME5 to NME10 most of which show no NDP kinase activity. Structurally, NME6 shares only 34-41% homology with Group I proteins, while its expression is relatively low in most tissues. Scarce data on NME6 indicates that it is located in the mitochondria and the cytoplasm. The aim of this thesis was to determine the localization of the NME6 protein in HeLa tumour cells using GFP reporter system, to test the anti-NME6 antibody, and optimise the Western blot method. For that purpose, we used transformation, transfection and fluorescent immunocytochemistry methods, Western blot analysis and confocal microscopy