Simple oxygraphic analysis for the presence of adenylate kinase 1 and 2 in normal and tumor cells

Abstract

The adenylate kinase (AK) isoforms network plays an important role in the intracellular energy transfer processes, the maintenance of energy homeostasis, and it is a major player in AMP metabolic signaling circuits in some highly-differentiated cells. For this purpose, a rapid and sensitive method was developed that enables to estimate directly and semi-quantitatively the distribution between cytosolic AK1 and mitochondrial AK2 localized in the intermembrane space, both in isolated cells and tissue samples (biopsy material). Experiments were performed on isolated rat mitochondria or permeabilized material, including undifferentiated and differentiated neuroblastoma Neuro-2a cells, HL-1 cells, isolated rat heart cardiomyocytes as well as on human breast cancer postoperative samples. In these samples, the presence of AK1 and AK2 could be detected by high-resolution respirometry due to the functional coupling of these enzymes with ATP synthesis. By eliminating extra-mitochondrial ADP with an excess of pyruvate kinase and its substrate phosphoenolpyruvate, the coupling of the AK reaction with mitochondrial ATP synthesis could be quantified for total AK and mitochondrial AK2 as a specific AK index. In contrast to the creatine kinase pathway, the AK phosphotransfer pathway is up-regulated in murine neuroblastoma and HL-1 sarcoma cells and in these malignant cells expression of AK2 is higher than AK1. Differentiated Neuro-2a neuroblastoma cells exhibited considerably higher OXPHOS capacity than undifferentiated cells, and this was associated with a remarkable decrease in their AK activity. The respirometric method also revealed a considerable difference in mitochondrial affinity for AMP between non-transformed cells and tumor cells.

Viability and mitochondrial bioenergetic functions in human colon cancer cells are not affected by treatment with peptides mtCPP1, UPF25, mtgCPP, and mtCPP1gHO

“…It is upregulated in human CRC [37] with the total AK activity about 2 times higher than surrounding normal tissue [10]. The same was also observed in tumor cells of other histological types [38]. The AK system could play an important role in the adaptation of cancer cells to an unfavorable microenvironment and tumor progression [39,40].…”

PrePhyloPro: phylogenetic profile-based prediction of whole proteome linkages

“…Moreover, PrePhyloPro predicted the high correlations (Jaccard similarity >0.95) between α∕β subunits and adenylate kinase isoforms (AKs) ( Table S7 ). Consistently, AKs maintain the cellular energy balance and collaborate in ATP synthesis, especially through coupling of the mitochondrial resident AK2 with the OXPHOS activity ( Klepinin et al, 2016 ). Additionally, the transcription of AK2 and α∕β subunit are both enhanced by triiodothyronine ( Severino et al, 2011 ).…”

Intracellular Energy-Transfer Networks and High-Resolution Respirometry: A Convenient Approach for Studying Their Function

“…Proteomics studies have revealed up-regulation of AK2 in human prostate and pancreatic cancer cells [ 75 , 76 ]. It was shown that AK2 can promote cell proliferation under normal circumstances and high expression of AK2 can be associated with poorly differentiated cells with high proliferative index, and that strong differences exist between highly differentiated and tumor cells in the affinity of their mitochondrial respiration for exogenous AMP [ 49 , 77 , 78 ]. The signaling function of AK realizes through the amplifying a small change in the ATP/ADP ratio into a much higher increase in the AMP/ATP ratio that in turn activates several cellular AMP-sensitive components, including those in the glycolytic and glycogenolytic pathways, and metabolic sensors and effectors such as ATP-sensitive potassium channels and AMP-activated protein kinase, which adjust energy state in the given tissue [ 1 , 49 , 79 ].…”

“…Critical steps: Cells with a low K m(ADP) should be measured at low (0.1 mM) ATP concentrations, while for cells with higher K m(ADP) vales the use of higher (2 mM) ATP concentrations is recommended [ 77 ]. Representative traces can be found in [ 30 , 77 ].…”