Biochemistry (Moscow) (v.78, #11)

Intratumor heterogeneity: Nature and biological significance by T. S. Gerashchenko; E. V. Denisov; N. V. Litviakov; M. V. Zavyalova; S. V. Vtorushin; M. M. Tsyganov; V. M. Perelmuter; N. V. Cherdyntseva (1201-1215).
Intratumor heterogeneity inherent in the majority of human cancers is a major obstacle for a highly efficient diagnosis and successful prognosis and treatment of these diseases. Being a result of clonal diversity within the same tumor, intratumor heterogeneity can be manifested in variability of genetic and epigenetic status, gene and protein expression, morphological structure, and other features of the tumor. It is most likely that the appearance of this diversity is a source for the adaptation of the tumor to changes in microenvironmental conditions and/or a tool for changing its malignant potential. In any case, both processes result in the appearance of cell clones with different undetermined sets of hallmarks. In this review, we describe the heterogeneity of molecular disorders in various human tumors and consider modern viewpoints of its development including genetic and non-genetic factors of heterogeneity origin and the role of cancer stem cells and clonal evolution. We also systematize data on the contribution of tumor diversity to progression of various tumors and the efficiency of their treatment. The main problems are indicated in the diagnosis and therapy of malignant tumors caused by intratumor heterogeneity and possible pathways for their solution. Moreover, we also suggest the key goals whose achievement promises to minimize the problem of intratumor heterogeneity and to identify new prognostic, predictive, and target markers for adequate and effective treatment of cancer.
Keywords: intratumor heterogeneity; clonal diversity; tumor evolution; cancer stem cell; tumor progression; cancer prognosis; cancer therapy

Experimental and theoretical studies of mechanical unfolding of different proteins by A. V. Glyakina; N. K. Balabaev; O. V. Galzitskaya (1216-1227).
Mechanical properties of proteins are important for a wide range of biological processes including cell adhesion, muscle contraction, and protein translocation across biological membranes. It is necessary to reveal how proteins achieve their required mechanical stability under natural conditions in order to understand the biological processes and also to use the knowledge for constructing new biomaterials for medical and industrial purposes. In this connection, it is important to know how a protein will behave in response to various impacts. Theoretical and experimental works on mechanical unfolding of globular proteins will be considered in detail in this review.
Keywords: stability; protein; atomic force microscope; denaturant

Malignant melanoma and melanocortin 1 receptor by A. A. Rosenkranz; T. A. Slastnikova; M. O. Durymanov; A. S. Sobolev (1228-1237).
The conventional chemotherapeutic treatment of malignant melanoma still remains poorly efficient in most cases. Thus the use of specific features of these tumors for development of new therapeutic modalities is highly needed. Melanocortin 1 receptor (MC1R) overexpression on the cell surface of the vast majority of human melanomas, making MC1R a valuable marker of these tumors, is one of these features. Naturally, MC1R plays a key role in skin protection against damaging ultraviolet radiation by regulating eumelanin production. MC1R activation is involved in regulation of melanocyte cell division. This article reviews the peculiarities of regulation and expression of MC1R, melanocytes, and melanoma cells, along with the possible connection of MC1R with signaling pathways regulating proliferation of tumor cells. MC1R is a cell surface endocytic receptor, thus considered perspective for diagnostics and targeted drug delivery. A number of new therapeutic approaches that utilize MC1R, including endoradiotherapy with Auger electron and α- and β-particle emitters, photodynamic therapy, and gene therapy are now being developed.
Keywords: melanoma; melanocortin 1 receptor; α-melanocyte-stimulating hormone; receptor; endocytosis; cancer therapy; diagnostics

Biological photoreceptors of light-dependent regulatory processes by G. Ya. Fraikin; M. G. Strakhovskaya; A. B. Rubin (1238-1253).
Progress in understanding primary mechanisms of light reception in photoregulatory processes is achieved through discovering new biological photoreceptors, chiefly the regulatory sensors of blue/UV-A light. Among them are LOV domain-containing proteins and DNA photolyase-like cryptochromes, which constitute two widespread groups of photoreceptors that use flavin cofactors (FMN or FAD) as the photoactive chromophores. Bacterial LOV domain modules are connected in photoreceptor proteins with regulatory domains such as diguanylate cyclases/phosphodiesterases, histidine kinases, and DNA-binding domains that are activated by photoconversions of flavin. Identification of red/far-red light sensors in chemotrophic bacteria (bacteriophytochromes) and crystal structures of their photosensor module with bilin chromophore are significant for decoding the mechanisms of phytochrome receptor photoconversion and early step mechanisms of phytochrome-mediated signaling. The only UV-B regulatory photon sensor, UVR8, recently identified in plants, unlike other photoreceptors functions without a prosthetic chromophore: tryptophans of the unique UVR8 protein structure provide a “UV-B antenna”. Our analysis of new data on photosensory properties of the identified photoreceptors in conjunction with their structure opens insight on the influence of the molecular microenvironment on light-induced chromophore reactions, the mechanisms by which the photoactivated chromophores trigger conformational changes in the surrounding protein structure, and structural bases of propagation of these changes to the interacting effector domains/proteins.
Keywords: phytochromes; cryptochromes; LOV-domain photosensors; chromophores; structure; signalin

Selection of peptide inhibitors for double-stranded RNA-dependent protein kinase PKR by M. -J. Du; H. -K. Zhang; A. -J. He; Y. -S. Chang; Y. Yang; Y. Wang; C. -Z. Zhang; Y. Cao (1254-1262).
Protein kinase inhibitors have been developed and applied as antitumor drugs. The majority of these inhibitors are derived from ATP analogs with limited specificity towards the kinase target. Here we present our proof-of-principle study on peptide inhibitors for kinases. Two peptides were selected by phage display against double-stranded RNA-dependent protein kinase (PKR). In vitro assay revealed that these peptides exhibit an inhibitory effect on PKR-catalyzed phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF2α). The peptides also interrupt PKR activity in cells infected by viruses, as PKR activation is one of the hallmarks of host response to viral infection. Kinetic study revealed that one of the peptides, named P1, is a competitive inhibitor for PKR, while the other, named P2, exhibits a more complicated pattern of inhibition on PKR activity. Fragment-based docking of the PKR-peptide complex suggests that P1 occupies the substrate pocket of PKR and thus inhibits the binding between PKR and eIF2α, whereas P2 sits near the substrate pocket. The computational model of PKR-peptide complex agrees with their kinetic behavior. We surmise that peptide inhibitors for kinases have higher specificity than ATP analogs, and that they provide promising leads for the optimization of kinase inhibitors.
Keywords: fragment-based docking; kinase inhibitors; PKR; phage display; peptides

Bacterial and cell-free production of APP671-726 containing amyloid precursor protein transmembrane and metal-binding domains by O. V. Bocharova; A. S. Urban; K. D. Nadezhdin; E. V. Bocharov; A. S. Arseniev (1263-1271).
More than half of the mutations associated with familiar Alzheimer’s disease have been found in the transmembrane domain of amyloid precursor protein (APP). These pathogenic mutations presumably influence the APP transmembrane domain structural and dynamic properties and result in its conformational change or/and lateral dimerization. Despite much data about the pathogenesis of Alzheimer’s disease, the initial steps of the pathogenesis remain unclear so far. For the investigation of the molecular basis of Alzheimer’s disease, we selected amyloid precursor protein fragment APP671-726 containing the transmembrane and metal-binding domains. This fragment is the substrate of the γ-secretase complex whose abnormal activity leads to the formation of amyloidogenic Aβ42 peptides. This work for the first time describes a highly effective cell-free APP671-726 production method and improved method of bacterial synthesis. Both methods yield milligram quantities of isotope-labeled protein for structural study by high resolution NMR spectroscopy in membrane mimicking milieus.
Keywords: amyloid precursor protein; transmembrane domain; recombinant protein; NMR spectroscopy

The gene encoding the family 6 carbohydrate-binding module (CtCBM6A) from Clostridium thermocellum, cloned in pET-21a(+) expression vector, was overexpressed using Escherichia coli BL-21(DE3) cells and purified by immobilized metal-ion affinity chromatography. SDS-PAGE analysis of the recombinant CtCBM6A showed molecular size of approximately 15 kDa. Ligand-binding analysis of CtCBM6A with rye arabinoxylan and oat spelt xylan by affinity gel electrophoresis showed low affinity for these ligands (K a of 40 and 26 liter/g, respectively), and analysis by fluorescence spectroscopy (K a of 33 and 15 liter/g, respectively) corroborated lower binding affinity with the above soluble ligands. However, CtCBM6A displayed significantly higher ligand-binding affinity with insoluble wheat arabinoxylan with equilibrium association constant K a of 230 M−1 and binding capacity (N 0) of 11 μmole/g. The protein melting curve of CtCBM6A displayed a peak shift from 53 to 58°C in the presence of Ca2+, indicating that Ca2+ imparts thermal stability to the CtCBM6A structure. Homology modeling of CtCBM6A revealed a characteristic β-sandwich core structure. The Ramachandran plot of CtCBM6A showed 89% of the residues in the most favorable region, 10% in additionally favored region, and 1% in generously allowed region, indicating that CtCBM6A has a stable conformation.
Keywords: CtCBM6A; insoluble wheat arabinoxylan; oat spelt xylan; homology modeling; affinity gel electrophoresis

Organotypic culture of neural retina as a research model of neurodegeneration of ganglion cells by O. S. Gancharova; V. N. Manskikh; A. A. Zamyatnin Jr.; P. P. Philippov (1280-1286).
Organotypic models deserve special attention among the large variety of methods of vertebrate retina cultivation. The purpose of this study was to make a detailed qualitative and quantitative characterization of a model employing roller organotypic cultivation of the neural retina of rat eye posterior segment, with special attention to morphological and functional characteristics of retinal ganglion cells. The study included morphological analysis of retina histological preparations as well as estimation of RNA synthesis and evaluation of neuron survival by the Brachet and TUNEL methods, respectively. Retina has been shown to display normal morphofunctional characteristics for the first 12 h of cultivation. After 24 h, a substantial number of ganglion cells underwent pyknosis and stopped RNA synthesis. Almost all the cells of the retinal ganglion layer became apoptotic by 3–4 days in vitro. In the course of cultivation, neural retina is detached from the underlying layers of the posterior eye segment and undergoes significant cytoarchitectonic changes. The causes of ganglion cell death during organotypic cultivation of eye posterior segment are discussed. This method can serve as a suitable model for the screening of new retinoprotectors and for research on ganglion cell death resulting from retina degenerative diseases, e.g. glaucoma.
Keywords: retina; ganglion cells; apoptosis; organotypic roller culture

Construction and functional analysis of novel dominant-negative mutant of human SOX18 protein by M. Milivojevic; I. Petrovic; N. Kovacevic-Grujicic; J. Popovic; M. Mojsin; M. Stevanovic (1287-1292).
SOX18 transcription factor plays important roles in a range of biological processes such as vasculogenesis, hair follicle development, lymphangiogenesis, atherosclerosis, and angiogenesis. In this paper we present the generation of a novel SOX18 dominant-negative mutant (SOX18DN) encoding truncated SOX18 protein that lacks a trans-activation domain. We show that both wild-type SOX18 (SOX18wt) and truncated human SOX18 proteins are able to bind to their consensus sequence in vitro. Functional analysis confirmed that SOX18wt has potent trans-activation properties, while SOX18DN displays dominant-negative effect. We believe that these SOX18wt and SOX18DN expression constructs could be successfully used for further characterization of the function of this protein.
Keywords: SOX18 ; dominant-negative; transcription factor; truncated protein

Previously, we developed a method to monitor the development of oxidative stress in isolated liver mitochondria. The method is based on recording of membrane potential changes in response to sequential introduction of low concentrations (5–20 μM) of tert-butyl hydroperoxide (tBHP). It allows monitoring of the extent of amplification or attenuation of oxidative stress caused by external influences (changes in incubation conditions, additions of biologically active substances). Based on this method, we created a mitochondrial model for the study and improvement of treatment of pathologies associated with oxidative stress. The following two processes were simulated in the experiments: 1) introduction of desferal for treatment of serious diseases caused by cell overload with iron (high desferal concentrations were shown to suppress mitochondrial energetics); 2) efficiency of alkalization to reduce mitochondrial damage induced by oxidative stress. The experiments have shown that even a small increase in pH (alkalization) increases the amount of tBHP that can be added to mitochondria before the MPTP (“mitochondrial permeability transition pore”) is induced. The effect of alkalization was shown to be close to the effect of cyclosporin A in the pH range 7.2–7.8. The mechanism of the similarities of these effects in the organism and in mitochondrial suspensions is explained by the increase in toxic reactive oxygen species in both systems under oxidative stress.
Keywords: oxidative stress; mitochondria; tert-butyl hydroperoxide; cyclosporin A-sensitive pore; desferal; alkalization

Identification of novel acyl-ACP thioesterase gene ClFATB1 from Cinnamomum longepaniculatum by Na Lin; Tao-bo Ai; Ji-hai Gao; Lin-hong Fan; Sheng-hua Wang; Fang Chen (1298-1303).
A putative fatty acyl-acyl carrier protein (acyl-ACP) thioesterase (thioesterase) full-length cDNA sequence named as ClFATB1 was obtained from the seed cDNA library of Cinnamomum longepaniculatum by the SMART-RACE method. The novel gene encodes a protein of 382 amino acid residues with close homology to fatty acid thioesterase type B (FATB) enzymes of other plants, with two essential residues (His285 and Cys320) for thioesterase catalytic activity. The gene was transcribed in all tissues of C. longepaniculatum, the highest being in seeds. Recombinant ClFATB1 in Escherichia coli had higher specific activities against saturated 16:0- and 18:0-ACPs than on unsaturated 18:1-ACP. Overexpression of ClFATB1 in transgenic tobaccos upregulated thioesterase activities of crude proteins against 16:0-ACP and 18:0-ACP by 20.3 and 5.7%, respectively, and resulted in an increase in the contents of palmitic and stearic acids by 15.4 and 10.5%, respectively. However, ectopic expression of this gene decreased the substrate specificities of crude proteins to unsaturated 18:1-ACP by 12.7% in transgenic tobacco and lowered the contents of oleic, linoleic, and linolenic acids in transgenic leaves. So ClFATB1 would potentially upregulate the synthesis of saturated fatty acids and downregulate unsaturated ones in the fatty acid synthesis pathway of plants.
Keywords: acyl-ACP thioesterase; Cinnamomum longepaniculatum ; expression property; fatty acid; transgenic tobacco