S. V. Kulemzin, V. V. Kuznetsova, M. Mamonkin, A. V. Taranin, А. A. Gorchakov
Engineering Chimeric Antigen Receptors
Chimeric antigen receptors (CARs) are recombinant protein molecules that redirect cytotoxic lymphocytes toward malignant and other target cells. The high feasibility of manufacturing CAR-modified lymphocytes for the therapy of cancer has spurred the development and optimization of new CAR T cells directed against a broad range of target antigens. In this review, we describe the main structural and functional elements constituting a CAR, discuss the roles of these elements in modulating the anti-tumor activity of CAR T cells, and highlight alternative approaches to CAR engineering.
adoptive immunotherapy, cancer, chimeric antigen receptor, T cells
The physiology of Mycobacterium tuberculosis, the causative agent of tuberculosis, is being studied with intensity. However, despite the genomic and transcriptomic data available today, the pathogenic potential of these bacteria remains poorly understood. Therefore, proteomic approaches seem relevant in studying mycobacteria. This review covers the main stages in the proteomic analysis methods used to study mycobacteria. The main achievements in the area of M. tuberculosis proteomics are described in general. Special attention is paid to the proteomic features of the Beijing family, which is widespread in Russia. Considering that the proteome is a set of all the proteins in the cell, post-translational modifications of mycobacterium proteins are also described.
Cholesterol in the Pathogenesis of Alzheimer’s, Parkinson’s Diseases and Autism: Link to Synaptic Dysfunction
In our previous review, we described brain cholesterol metabolism in control conditions and in the case of some rare neurological pathologies linked to defects in the genes which are directly involved in the synthesis and/or traffic of cholesterol. Here, we have analyzed disruptions in cholesterol homeostasis in widespread neurodegenerative diseases (Alzheimer’s and Parkinson’s diseases) and autism spectrum disorders. We particularly focused on the synaptic dysfunctions that could arise from changes in both membrane cholesterol availability and oxysterol production. Notably, alterations in the brain cholesterol metabolism and neurotransmission occur in the early stages of these pathologies and the polymorphism of the genes associated with cholesterol homeostasis and synaptic communication affects the risk of onset and severity of these diseases. In addition, pharmacological and genetic manipulations of brain cholesterol homeostasis in animal models frequently have marked effects on the progression of neurodegenerative diseases. Thus, the development of Alzheimer’s, Parkinson’s and autism spectrum disorders may be partially associated with an imbalance of cholesterol homeostasis that leads to changes in the membrane cholesterol and oxysterol levels that, in turn, modulates key steps in the synaptic transmission.
I. V. Balalaeva, E. A. Sokolova, A. D. Puzhikhina, A. A. Brilkina, S. M. Deyev
Spheroids of HER2-Positive Breast Adenocarcinoma for Studying Anticancer Immunotoxins In Vitro
Tumor response to therapeutic treatment is largely determined by its heterogeneity and the presence of intercellular junctions, hindering the penetration of large molecules deep into the three-dimensional structure of the tumor. In that context, 3D in vitro tumor models such as cancer cell spheroids are becoming increasingly popular. We obtained spheroids of human breast adenocarcinoma SKBR-3 overexpressing the HER2 cancer marker. The toxicity of HER2-targeted immunotoxin 4D5scFv-PE40 against spheroids was shown to be several orders of magnitude lower compared to a monolayer cell culture. The significant difference in the severity of the immunotoxin effect can be explained by the fact that it ineffectively penetrates the spheroid and predominantly influences the cells of the outer layers. The resulting tumor spheroid model can be used in development of drugs for targeted therapy as well as to study ways to improve the efficiency of anticancer agents by targeting cell–cell contacts.
4D5scFv-PE40 immunotoxin, cancer marker HER2, drug penetration into tumor, spheroids, targeted therapy
Building a Full-Atom Model of L,Dtranspeptidase 2 from Mycobacterium tuberculosis for Screening New Inhibitors
L,D-transpeptidase 2 from Mycobacterium tuberculosis plays a key role in the formation of the cell wall of a pathogen and catalyzes the cross-linking of growing peptidoglycan chains by non-classical 3-3 bonds, which causes resistance to a broad spectrum of penicillins. Molecular modeling of enzyme interactions with the N- and C-terminal tetrapeptide fragments of growing peptidoglycan chains has been performed for the first time and has allowed us to highlight the peculiarities of their binding at the formation of 3-3 cross-linkages, as well as to build a full-atom model of L,D-transpeptidase 2 for the screening and optimizing of inhibitors’ structures.
N. S. Bondarenko, A. N. Shneiderman, A. A. Guseva, B. A. Umarova
Prolyl-glycyl-proline (PGP) Peptide Prevents an Increase in Vascular Permeability in Inflammation
This study was aimed at investigating the effect of prolyl-glycyl-proline (PGP) tripeptide on vascular permeability in rats with an inflammation. It was found that the peptide reduces the rat paw edema induced by a subcutaneous administration of histamine to the same extent as the conventional anti-inflammatory agent diclofenac. However, an assessment of the relative expression level of the cox-2 gene at the inflammation focus using real-time PCR showed that, in contrast to diclofenac, PGP does not affect the cox-2 gene expression. This is indicative of the fact that they have different mechanisms of action. We used the model of acute peritonitis induced by an intraperitoneal injection of thioglycolate to demonstrate that the inflammatory response of an organism is accompanied by increased vascular permeability in the tissues of the stomach and small intestine. Pre-administration (30 minutes before the induction of the inflammation) of PGP prevented this increase, whereby the level of vascular permeability, exudate volume in the peritoneal cavity, and the amount of the Evans Blue dye in this exudate remained at the control level. Therefore, these results suggest that the anti-inflammatory action of PGP is based on its ability to prevent an increase in vascular permeability.
Intratumoral Morphological Heterogeneity of Breast Cancer as an Indicator of the Metastatic Potential and Tumor Chemosensitivity
Breast cancer (BC) demonstrates considerable intratumoral morphological heterogeneity. The aim of this work was to evaluate the relationship among different morphological structures, the rate of metastasis, and efficacy of neoadjuvant chemotherapy (NAC) in NAC-treated (n = 427) and NAC-naïve (n = 249) BC patients. We also studied the involvement of an epithelial-mesenchymal transition (EMT) in the development of the intratumoral morphological heterogeneity of BC. We found a significant association between the intratumoral morphological heterogeneity and the rate of BC metastasis and response to NAC, which, in most cases, correlated with the presence of alveolar and trabecular structures. In particular, the rate of lymph node metastasis in tumors containing alveolar and trabecular structures was higher compared to that in tumors lacking such structures. NAC-treated patients with alveolar and trabecular structures had a high distant metastasis rate and a low metastasis-free survival rate. Furthermore, alveolar and trabecular structures were found to be associated with a lack of response to NAC. Interestingly, the association between alveolar structures and a high distant metastasis rate was found only in NAC-unresponsive patients, whereas the association between trabecular structures and an increased distant metastasis was revealed in responders. Alveolar structures were associated with chemoresistance only in patients with lymph node metastases, whereas trabecular structures were associated with chemoresistance only in patients without lymph node metastases. In general, increased intratumoral morphological diversity correlated with considerable chemoresistance and a high metastasis rate of BC. We found variable expressions of epithelial (EPCAM and CDH1) and mesenchymal (ITGA5, ITGB5, CDH2, CDH11, TGFb2, ZEB1, MMP2, DCN, MST1R) markers and, thus, different EMT manifestations in different morphological structures. Therefore, intratumoral morphological heterogeneity of BC may serve as an indicator of the metastatic potential and tumor chemosensitivity.
intratumoral heterogeneity, invasion, metastasis, breast cancer, chemotherapy, epithelial-mesenchymal transition
NAC – neoadjuvant chemotherapy; BC – breast cancer; EMT – epithelial-mesenchymal transition.
E. S. Philonenko, M. V. Shutova, Е. А. Khomyakova, Е. М. Vassina, О. S. Lebedeva, S. L. Kiselev, М. А. Lagarkova
Differentiation of Human Pluripotent Stem Cells into Mesodermal and Ectodermal Derivatives Is Independent of the Type of Isogenic Reprogrammed Somatic Cells
Induced pluripotent stem cells (iPSCs) have the capacity to unlimitedly proliferate and differentiate into all types of somatic cells. This capacity makes them a valuable source of cells for research and clinical use. However, the type of cells to be reprogrammed, the selection of clones, and the various genetic manipulations during reprogramming may have an impact both on the properties of iPSCs and their differentiated derivatives. To assess this influence, we used isogenic lines of iPSCs obtained by reprogramming of three types of somatic cells differentiated from human embryonic stem cells. We showed that technical manipulations in vitro, such as cell sorting and selection of clones, did not lead to the bottleneck effect, and that isogenic iPSCs derived from different types of somatic cells did not differ in their ability to differentiate into the hematopoietic and neural directions. Thus, the type of somatic cells used for the generation of fully reprogrammed iPSCs is not important for the practical and scientific application of iPSCs.
Voltage-Dependent Interaction of Capsaicine and Protons on TRPV1- Receptors
The interaction of TRPV1-receptors agonists (capsaicin and protons) has been studied on cultured CHO cells transfected by TRPV1-receptors. Using the whole-cell patch-clamp approach, it was shown that summation of the currents induced by agonist application was dependent on the membrane potential. The TRPV1-mediated currents induced by the pH and Capsaicin demonstrated arithmetical summation at potentials between 40–-40 mV, while they were potentiated at potentials below -40 mV. Currents induced by the pH and Capsaicin combined were higher in comparison with the arithmetic sum of the currents induced by the pH and Capsaicin applied separately at such potentials. Such a potential dependence seems to be a base of the sensitization that is induced by inflammation or pain, when concentrations of proinflammatory mediators acting as TRPV1 agonists are increasing. Further depolarization induced by TRPV1 activation doesn’t generate potentiation, which might serve as a protective mechanism to restrict their activity.
TRPV1, Capsaicin, pH, Agonists of TRPV1-receptors, Interaction of agonists of TRPV1-receptors
CHO – Chinese Hamster Ovary (cells of connective tissue of Chinese hamster ovary); TRPV1 – Transient Receptor Potential channel subfamily V member 1 (vanilloid receptor, type 1); GFP – Green Fluorescent Protein; MP –membrane potential.
O.V. Chertkov, G.A. Armeev, I.V. Uporov, S.A. Legotsky, N.N. Sykilinda, A.K. Shaytan, N.L. Klyachko, and K.A. Miroshnikov
Dual Active Site in the Endolytic Transglycosylase gp144 of Bacteriophage phiKZ
Lytic transglycosylases are abundant peptidoglycan lysing enzymes that degrade the heteropolymers of bacterial cell walls in metabolic processes or in the course of a bacteriophage infection. The conventional catalytic mechanism of transglycosylases involves only the Glu or Asp residue. Endolysin gp144 of Pseudomonas aeruginosa bacteriophage phiKZ belongs to the family of Gram-negative transglycosylases with a modular composition and C-terminal location of the catalytic domain. Glu115 of gp144 performs the predicted role of a catalytic residue. However, replacement of this residue does not completely eliminate the activity of the mutant protein. Site-directed mutagenesis has revealed the participation of Tyr197 in the catalytic mechanism, as well as the presence of a second active site involving Glu178 and Tyr147. The existence of the dual active site was supported by computer modeling and monitoring of the molecular dynamics of the changes in the conformation and surface charge distribution as a consequence of point mutations.
D. A. Yakovlev, A. A. Kuznetsova, O. S. Fedorova and N. A. Kuznetsov
Search for Modified DNA Sites with the Human Methyl-CpG-Binding Enzyme MBD4
The MBD4 enzyme initiates the process of DNA demethylation by the excision of modified DNA bases, resulting in the formation of apurinic/apyrimidinic sites. MBD4 contains a methyl-CpG-binding domain which provides the localization of the enzyme at the CpG sites, and a DNA glycosylase domain that is responsible for the catalytic activity. The aim of this work was to clarify the mechanisms of specific site recognition and formation of catalytically active complexes between model DNA substrates and the catalytic N-glycosylase domain MBD4cat. The conformational changes in MBD4cat and DNA substrates during their interaction were recorded in real time by stopped-flow detection of the fluorescence of tryptophan residues in the enzyme and fluorophores in DNA. A kinetic scheme of MBD4cat interaction with DNA was proposed, and the rate constants for the formation and decomposition of transient reaction intermediates were calculated. Using DNA substrates of different lengths, the formation of the catalytically active complex was shown to follow the primary DNA binding step which is responsible for the search and recognition of the modified base. The results reveal that in the primary complex of MBD4cat with DNA containing modified nucleotides, local melting and bending of the DNA strand occur. On the next step, when the catalytically competent conformation of the enzyme-substrate complex is formed, the modified nucleotide is everted from the double DNA helix into the active center and the void in the helix is filled by the enzyme’s amino acids.
MBD4, DNA demethylation, DNA repair, pre-steady-state kinetics, conformational dynamics
L. S. Melnikova, Е. A. Pomerantseva, V.V. Molodina, P. G. Georgiev
Mapping the D.melanogaster En1A Enhancer Modules Responsible for Transcription Activation and Long- Distance Enhancer-Promoter Interactions
The structure of the new enhancer En1A of the 1A region of the X chromosome of D. melanogaster was investigated. Two distinct regulatory elements were found. The first element is responsible for transcription activation, and the second element provides specific interaction with the promoter of the yellow gene. The findings support the hypothesis of a modular structure for enhancers, including certain sequences that bind transcription activators and special communication elements providing long-distance enhancer-promoter interaction.
TF – transcription factors; Еn1А (Еnhancer 1А) – enhancer of the 1A region of the X chromosome; Cm1A (Сommunicator 1А) – communication element of the En1A enhancer; TE (tethering element) – a regulatory element in the promoter responsible for long-distance enhancer-promoter interactions.
Antiretroviral Activity Of a Novel Pyrimidyl-Di(Diazaspiroalkane) Derivative
A novel compound, 3,3’-(5-nitropyrimidine-4,6-diyl)bis-3,12-diaza-6,9-diazoniadispiro[220.127.116.11]hexadecane tetrachloride dihydrochloride, was synthesized. The compound was found to inhibit the replication of various viral families by blocking specific heparan sulfate receptors on the host cell’s surface. In experiments, the compound was found to be highly effective against several strains of HIV retroviruses.
antiviral activity, dispiro compounds, heparan sulfate, HIV