Characterization of a group I Nme protein of Capsaspora owczarzaki—a close unicellular relative of animals

Abstract

Nucleoside diphosphate kinases are enzymes present in all domains of life. In animals, they are called Nme or Nm23 proteins, and are divided into group I and II. Human Nme1 was the first protein identified as a metastasis suppressor. Because of its medical importance, it has been extensively studied. In spite of the large research effort, the exact mechanism of metastasis suppression remains unclear. It is unknown which of the biochemical properties or biological functions are responsible for the antimetastatic role of the mammalian Nme1. Furthermore, it is not clear at which point in the evolution of life group I Nme proteins acquired the potential to suppress metastasis, a process that is usually associated with complex animals. In this study we performed a series of tests and assays on a group I Nme protein from filasterean Capsaspora owczarzaki, a close unicellular relative of animals. The aim was to compare the protein to the wellknown human Nme1 and Nme2 homologs, as well as with the homolog from a simple animal-sponge (Porifera), in order to see how the proteins changed with the transition to multicellularity, and subsequently in the evolution of complex animals. We found that premetazoan-type protein is highly similar to the homologs from sponge and human, in terms of biochemical characteristics and potential biological functions. Like the human Nme1 and Nme2, it is able to diminish the migratory potential of human cancer cells in culture.

Structure and function of cancer-related developmentally regulated GTP-binding protein 1 (DRG1) is conserved between sponges and humans

“…DNA-binding assay. The DNA binding ability of EsuDRG1 and HsaDRG1 was assayed in vitro as described 30,67,68 . Briefly, the reactions contained 200 ng of single-stranded circular DNA of bacteriophage ΦX174 (NEB, #N3023S) or double-stranded covalently closed circular form of ΦX174 (NEB, # N3021S).…”

Extracellular Vesicle-Mediated Metastasis Suppressors NME1 and NME2 Modify Lipid Metabolism in Fibroblasts

“…As a model, we used the invasive, human triple negative breast carcinoma cell line, MDA-MB-231T, which was stably transfected either by FLAG::NME1 or MYC::NME2 or the control vector [ 40 ]. Our aim was to examine the distribution of NME1 and NME2 in the EV fractions isolated from the supernatant of the above described cell lines.…”

Inhibition of TFEB oligomerization by co‐treatment of melatonin with vorinostat promotes the therapeutic sensitivity in glioblastoma and glioma stem cells

“…To demonstrate the biological functions that TFEB undergoes oligomerization, forming tetramers for the role of transcription factor, we firstly assessed the oligomerization of TFEB in U87MG cells and inhibition by vorinostat (Figure G). Glutaraldehyde is a well‐known reagent in the study of structural and chemical‐related functions of proteins and cross‐linking reagents . In addition, to determine whether inhibition of TFEB oligomerization increased vorinostat sensitivity and induced apoptosis in GBM cells, we generated TFEB‐depleted U87MG cells using lentiviral TFEB shRNA (Figure H).…”

“…Glutaraldehyde is a well-known reagent in the study of structural and chemical-related functions of proteins and cross-linking reagents. [44][45][46] In addition, to determine whether inhibition of TFEB oligomerization increased vorinostat sensitivity and induced apoptosis in GBM cells, we generated TFEB-depleted U87MG cells using lentiviral TFEB shRNA Cell viability was determined by MTT assays. Each experiment was repeated at least three times, and the data were expressed as mean ± SE of the mean.…”