Biochemistry (Moscow), Supplement Series B: Biomedical Chemistry (v.9, #4)

Experimental estimation of proteome size for cells and human plasma by S. N. Naryzhny; V. G. Zgoda; M. A. Maynskova; N. L. Ronzhina; N. V. Belyakova; O. K. Legina; A. I. Archakov (305-311).
Huge range of concentrations of different proteoforms and insufficient sensitivity of methods for detection of proteins at a single molecule level does not yet allow obtaining the whole image of the human proteome. In our investigations, we tried to evaluate the size of different proteomes (cells and plasma). The approach used is based on detection of protein spots in two-dimensional electrophoresis (2-DE) after protein staining by dyes with different sensitivities. The functional dependence of the number of detected protein spots from sensitivity of protein dyes was generated. Next, by extrapolation of this function curve to theoretical point of the maximum sensitivity (detection of a single smallest polypeptide) it was calculated that a single human cell (HepG2) may contain minimum 70000 proteoforms, human plasma—1.5 million. Utilization of this approach to other, smaller proteomes, showed the competency of this extrapolation. For instance, the size of mycoplasma (Acholeplasma laidlawii) was estimated in 1100 proteoforms, yeast (Saccharomyces cerevisiae)—40000, Escherichia coli—6200, Pyrococcus furiosus—3400. In hepatocytes, the amount of proteoforms was the same as in HepG2–70000. Significance of obtained data is in possibilities to estimating the proteome organization and planning next steps in its study.
Keywords: proteome; proteoforms; 2-DE

Molecular mechanisms of antitumor activity of niclosamide by E. Yu. Moskaleva; V. G. Perevozchikova; A. S. Zhirnik; S. E. Severin (312-324).
The review summarizes literature data on the antitumor activity of niclosamide and its molecular mechanisms. Earlier niclosamide was used for treatment of intestinal parasitic infections. Recent screening of various drugs and chemical compounds, aimed at identifying putative agents with high antitumor activity, revealed that it possesses such activity. Niclosamide not only inhibits such signaling pathways as Wnt/β-cate-nin, mTORC1, Stat3, NF-κB and Notch, but also damages tumor cell mitochondria, inhibits proliferation and induces apoptosis. The effectiveness of niclosamide has been demonstrated in vitro experiments as well as in vivo models using xenotransplantation of human tumors to immunodeficient mice. Niclosamide was active not only against tumor cells but also cancer stem cells. Normal cells are resistant to niclosamide. The accumulated experimental data suggest that niclosamide is a promising pharmacological agent for the treatment of various types of cancer.
Keywords: niclosamide; antitumor agents; cancer stem cells; signaling pathways; mitochondrial damage

Bacterial recombinant L-asparaginases: Properties, structure, and anti-proliferative activity by N. N. Sokolov; M. A. Eldarov; M. V. Pokrovskaya; S. S. Aleksandrova; O. Yu. Abakumova; O. V. Podobed; N. S. Melik-Nubarov; E. V. Kudryashova; D. V. Grishin; A. I. Archakov (325-338).
For more than 40 years L-asparaginases are used in combined therapy of acute lymphoblastic leukemias in children and the range of tumors sensitive to these enzymes constantly extends. This review summarizes results of studies aimed at creation of new systems for heterological expression of bacterial L-asparaginases in Erwinia carotovora (EwA), Helicobacter pylori (HpA), Yersinia pseudotuberculosis (YpA), and Rhodospirillum rubrum (RrA). Special attention is paid to isolation of purified enzymes and their crystallization, as well as physico-chemical, kinetic and structural properties. The resultant recombinant L-asparaginases (EwA, YpA, HpA, and RrA) demonstrate reasonable cytotoxic action on the human leukemia cells comparable to the pharmacologically available Escherichia coli L-asparaginase EcA and represent practical interest in the context of creation of effective new antitumor agents. Further prospects of studies on bacterial L-asparaginases are associated with development of analogues of Rhodospirillum rubrum L-asparaginase (RrA) by means of directed changes in the protein structure using genetic engineering, development of L-asparaginase preparations modified by polyethylene glycol and chitosan for improvement of their pharmacokinetic characteristics.
Keywords: L-asparaginase; L-glutaminase; recombinant proteins; leukemia; PEGylated L-asparaginase; chito-PEGylation

Endocrine disruptors are exogenous substances that enter the body and exhibit hormone-like action thus disrupting the homeostatic action of endogenous hormones. The most wide-spread disruptor is dichlorodiphenyltrichloroethane (DDT). The aim of this study was to investigate changes in thyroid hormone production induced by prolonged exposure to low doses of DDT. The experiment was performed on male Wistar rats treated with daily doses of DDT 1.89 ± 0.86 μg/kg and 7.77 ± 0.17 μg/kg for six and ten weeks. After six weeks there was a dose dependent increase of serum total thyroxine, total triiodthyronine, and thyroid peroxidase. Subsequently, concentration of free thyroxine decreased. These data indicate that impaired production of thyroxine by follicular thyrocytes, rather than decreased deiodinase activity and blood transport proteins is the major cause of altered thyroid status induced by DDT.
Keywords: endocrine disruptors; thyroid gland; thyroid hormones; DDT

Preparation of a peptide template for the SRM method by means of in vitro phosphorylation by M. G. Zavialova; V. G. Zgoda; O. N. Kharybin; E. N. Nikolayev (343-350).
Phosphorylation is one of the most common and the most important posttranslational modifications (PTM) of proteins. The study of protein phosphoforms is complicated by their low abundance in analyzed biological samples compared with the abundance of corresponding unmodified proteins. The method of selected reactions monitoring (SRM) allows to perform very sensitive and selective PTM analysis with high specificity and sensitivity. Using myelin basic protein (MBP) as a model we have developed a method for phosphoprotein detection by SRM. The method is based on obtaining phosphoproteins in a reconstituted kinase system and following usage of tryptic fragments of the phosphorylated protein as a template for the development of the SRM method. The developed method has been successfully applied for detection of phosphopeptides of myelin basic protein in human brain glioma samples.
Keywords: proteomics; selected reaction monitoring (SRM); post-translational modifications (PTM); protein phosphorylation

Membrane type 1 matrix metalloproteinase (MT1-MMP) is one of matrix metalloproteinases (MMP), which play a key role in tumor invasion and metastasis, thus determining tumor malignancy. The aim of this study was to elucidate the peculiarities of expression of MT1-MMP and endogenous regulators of its activity (the activator furin and the inhibitor TIMP-2), as invasive factors of squamous cell cervical carcinomas (SCC). The study was carried out using 11 paired carcinoma specimens, including SCC and morphologically normal tissue adjacent to the tumor. It was shown that the increase in MT1-MMP and furin expression and low expression of TIMP-2 make the major contribution to the destructive (invasive) potential of SCC. The substantial expression of MT1-MMP registered in the morphologically normal tissue adjacent to the tumor appears to make an additional contribution to the destructive potential of the cervical tumor.
Keywords: membrane type 1 matrix metalloproteinase (MT1-MMP); MMP activator—furin; tissue inhibitor of MMPs—TIMP-2; cervical squamous cell carcinoma

An injection model of the pre-clinical-stage of Alzheimer’s disease has been reproduced in rats. Experiments have shown that a decrease in the latent period of the conditioned avoidance response (CAR) is accompanied by the increase in endogenous β-amyloid peptide 1–40 and activation of inflammatory cytokines (IL-1β, TNFα, IL-6, IL-10) in the cerebral cortex, hippocampus and blood serum of experimental animals. We do believe that the changes detected at the biochemical level are an important precondition for modulating neuronal function in rats induced by administration of human β-amyloid peptide 1–40. The toxic effects of the exogenous of β-amyloid peptide 1–40 homoaggregates induced intensification of the cytokine response, while the liposomal form of this peptide determined a mild signal causes to the activation of innate immunity. TNFα and endogenous β-amyloid peptide 1–40 servers as the most informative markers for the presence neuroinflammation and amyloidogenic status, respectively.
Keywords: β-amyloid 1–40 peptide; cytokines; neuroinflammation; Alzheimer’s disease