[DNA Probes for Analysis of the Activity of Key Enzymes of the Base Excision DNA Repair Pathway in Human Cells].

The important role of DNA damage in the occurrence of various diseases, including cancer, has led to study of the mechanisms of genetic information stability, that have been carried out since the discovery of DNA repair systems. The question of the relationship between the accumulation of DNA damage, disorders in DNA repair pathways, and increased risk of disease development is still relevant. Over the past few years, significant efforts have been made to develop methods for analyzing the activity of DNA repair enzymes in human cells. In this work, we developed fluorescent DNA probes that allow us to determine the activity of key enzymes of base excision DNA repair in cell extracts, namely the DNA glycosylases UNG2, SMUG1, MBD4, TDG, AAG, NEIL1, NTHL1, and OGG1 and the AP endonuclease APE1. The sensitivity of DNA probes was determined on pure enzyme preparations. Determination of the activity of repair enzymes in cell extracts of the human ovarian tumor lines TOV112, 79, OVCAR3, MESOV, SCOV3, and TOV21 revealed significant variability in the level of enzyme activity in these cell lines. These results may become a test system platform for analyzing the activity of the base excision DNA repair system in the human body.

Differences in Protein Secretion by Multipotent Mesenchymal Stromal Cells Effective and Ineffective in the Prevention of Acute Graft-Versus-Host Disease after Allogeneic Hematopoietic Stem Cell Transplantation.

Multipotent mesenchymal stromal cells (MSC) were administered to patients after allogeneic hematopoietic stem cell transplantation to prevent the development of acute graft-versus- host disease (GVHD). The injection of MSC did not always prevent the development of GVHD. The aim of the work was to compare the secretome of MSC effective and ineffective in the prevention of GVHD. MSC were obtained from the bone marrow of hematopoietic stem cells donors. The secretome was studied using a TripleTOF 5600+ mass spectrometer with a NanoSpray III ion source coupled to a NanoLC Ultra 2D Plus nano-HPLC System. A total of 1,965 proteins were analyzed. Analysis of the secretome of effective and ineffective MSC samples revealed significant differences in the secretion of 1,119 proteins associated with ribosomes, exosomes, focal contacts, and others. Analysis of proteins secreted by MSC can be used to identify prognostically effective samples.

Changes in the serum protein composition in mice with transplanted Ehrlich's carcinoma.

Injection of blood serum from mice with Ehrlich's carcinoma stimulates the growth of transplanted tumor, which proves the presence of tumor-specific factors in the serum. Experiments on (CBA×C57Bl/6)F1 male mice with transplanted Ehrlich's carcinoma demonstrated the appearance of new proteins in the serum, some of them are identified. The authors suggest continuing the search for tumor-associated factor by combining proteomic analytical methods and testing of identified candidate proteins for their effects on tumor growth.

Identification of Novel Interaction Partners of AIF Protein on the Outer Mitochondrial Membrane.

In response to the wide variety of external and internal signals, mammalian cells undergo apoptosis, programmed cell death. Dysregulation of apoptosis is involved in multiple human diseases, including cancer, autoimmunity, and ischemic injuries. Two types of apoptosis have been described: the caspase-dependent one, leading to digestion of cellular proteins, and caspase-independent apoptosis, resulting in DNA fragmentation. The latter type of apoptosis is executed by AIF protein and is believed to have appeared first during evolution. The key step in the caspase-independent apoptosis program is the dissociation of AIF from the outer mitochondrial membrane (OMM). However, the molecular mechanism of interaction between AIF and OMM remains poorly understood. In this study, we demonstrated that AIF can bind to OMM via mortalin protein. We confirmed interaction between AIF and mortalin both in vitro and in vivo and mapped the amino acid sequences that are important for the binding of these proteins. Next, we showed that apoptosis induction by chemotherapy leads to downregulation of AIF-mortalin interaction and dissociation of AIF from the OMM. Finally, a bioinformatic analysis demonstrated that a high level of mortalin expression correlates with a worse survival prognosis for glioma patients. Altogether, our data revealed that mortalin plays an important role in the regulation of the caspase-independent apoptotic pathway and allowed us to speculate that inhibition of AIF-mortalin interaction may induce a dissociation of AIF from the OMM and subsequent apoptosis of cancer cells.

Expression and Intracellular Localization of Paraoxonase 2 in Different Types of Malignancies.

PON2 belongs to the paraoxonase protein family that consists of lactone hydrolyzing enzymes with different substrate specificities. Unlike other members of the family, PON2 exhibits substantial antioxidant activity, is localized predominantly inside the cell, and is ubiquitously expressed in all human tissues. Previously, it was proffered that defense against pathogens, such as Pseudomonas aeruginosa, is the main function of paraoxonases. However, recent findings have highlighted the important role played by PON2 in protection against oxidative stress, inhibition of apoptosis, and progression of various types of malignancies. In the current study, we performed a bioinformatic analysis of RNA and DNA sequencing data extracted from tumor samples taken from more than 10,000 patients with 31 different types of cancer and determined expression levels and mutations in the PON2 gene. Next, we investigated the intracellular localization of PON2 in multiple cancer cell lines and identified the proteins interacting with PON2 using the LC-MS/MS technique. Our data indicate that a high PON2 expression level correlates with a worse prognosis for patients with multiple types of solid tumors and suggest that PON2, when localized on the nuclear envelope and endoplasmic reticulum, may protect cancer cells against unfavorable environmental conditions and chemotherapy.