SARS-COV-2 Coronavirus Papain-like Protease PLpro as an Antiviral Target for Inhibitors of Active Site and Protein-Protein Interactions.

The papain-like protease PLpro of the SARS-CoV-2 coronavirus is a multifunctional enzyme that catalyzes the proteolytic processing of two viral polyproteins, pp1a and pp1ab. PLpro also cleaves peptide bonds between host cell proteins and ubiquitin (or ubiquitin-like proteins), which is associated with a violation of immune processes. Nine structures of the most effective inhibitors of the PLpro active center were prioritized according to the parameters of biochemical (IC 50) and cellular tests to assess the suppression of viral replication (EC 50) and cytotoxicity (CC 50). A literature search has shown that PLpro can interact with at least 60 potential protein partners in cells, 23 of which are targets for other viral proteins (human papillomavirus and Epstein-Barr virus). The analysis of protein-protein interactions showed that the proteins USP3, UBE2J1, RCHY1, and FAF2 involved in deubiquitinylation and ubiquitinylation processes contain the largest number of bonds with other proteins; the interaction of viral proteins with them can affect the architecture of the entire network of protein-protein interactions. Using the example of a spatial model of the PLpro/ubiquitin complex and a set of 154 naturally occurring compounds with known antiviral activity, 13 compounds (molecular masses in the range of 454-954 Da) were predicted as potential PLpro inhibitors. These compounds bind to the "hot" amino acid residues of the protease at the positions Gly163, Asp164, Arg166, Glu167, and Tyr264 involved in the interaction with ubiquitin. Thus, pharmacological effects on peripheral PLpro sites, which play important roles in binding protein substrates, may be an additional target-oriented antiviral strategy.

[A pseudoallergic reaction involved in developing bullous oral lichen planus in patients with increased gastric acid-producing function]. / Rol' psevdoallergicheskoi reaktsii v razvitii bulleznoi formy krasnogo ploskogo lishaya slizistoi obolochki rta u bol'nykh s povyshennoi kislotoobrazuyushchei funktsiei zheludka.

Lichen planus of the oral mucosa (LPOOR) is a chronic autoimmune disease. With a complicated course of the disease, other clinical and pathomorphological signs are added to the clinical picture of the reticular form, including blisters in the bullous form of the disease. It is known that LPOOR develops mainly by the mechanism of delayed-type hypersensitivity reaction, and in complicated forms - with the addition of a true allergic reaction of immediate-type hypersensitivity. OBJECTIVE: To determine the role of pseudo-allergic reaction in the development of the bullous form of LPR in patients with increased acid-forming function of the stomach. MATERIAL AND METHODS: The level of histamine in blood was studied by high-performance liquid chromatography and blood IgE by the immunochemiluminescent method in 38 patients with the bullous form of LPOOR against the background of acid-dependent diseases and 14 patients with the reticular form of LP RR with the absence of hyperchlorhydria. For the treatment of patients with the bullous form of LPOOR, drugs were used - H1 and H2 receptor blockers and proton pump inhibitors. RESULTS: A positive result was obtained in 68.4% of cases. CONCLUSION: Thus, a pseudoallergic component has been established in the development of the bullous form of the LP of ROS against the background of increased acid-forming function of the stomach.

Methods of Computational Interactomics for Investigating Interactions of Human Proteoforms.

Human genome contains ca. 20,000 protein-coding genes that could be translated into millions of unique protein species (proteoforms). Proteoforms coded by a single gene often have different functions, which implies different protein partners. By interacting with each other, proteoforms create a network reflecting the dynamics of cellular processes in an organism. Perturbations of protein-protein interactions change the network topology, which often triggers pathological processes. Studying proteoforms is a relatively new research area in proteomics, and this is why there are comparatively few experimental studies on the interaction of proteoforms. Bioinformatics tools can facilitate such studies by providing valuable complementary information to the experimental data and, in particular, expanding the possibilities of the studies of proteoform interactions.

[Direct Molecular Fishing of Zinc-Dependent Protein Partners of Amyloid-beta 1-16 with the Taiwan (D7H) Mutation and Phosphorylated Ser8 Residue].

We previously showed that the metal-binding domain 1-16 of intact amyloid-beta (Aß) is involved in interactions with a number of proteins from the cytosolic fraction of SK-N-SH human neuroblastoma cells in a zinc-dependent manner only. It is known that hereditary mutations in the Aß metal-binding domain (Aß(1-16)), which accelerate the development of Alzheimer's disease and post-translational modifications of amino acid residues, can significantly affect the domain's structure in the presence of zinc ions. In this work, using the molecular fishing methodology for Aß(l-16) isoforms with the Taiwanese mutation (D7H) and a phosphorylated Ser8 residue, proteins from the cytosol of SK-N-SH cells were found that are able to form zinc-dependent non-covalent complexes with these domains. The partner proteins identified for these isoforms differed from those for intact Aß(1-16). In contrast, the Aß(1-16) isoform with the English mutation (H6R) and the Aß(1-16) isoform containing both an isomerized Asp7 residue and phosphorylated Ser8 residue did not interact with cytosolic proteins. The results are useful for developing methods for rational modulation of protein-protein interactions involving natural isoforms of beta-amyloid, and also indicate the possible role of beta-amyloid with phosphorylated Ser8 as a molecule involved in normal physiological processes.

New Targets of Kunitz-Type Peptide from Sea Anemone Heteractis magnifica.

The interaction of Kunitz-type peptide, HMIQ3c1, from the sea anemone Heteractis magnifica with several serine proteases, including inflammatory proteases, was investigated using the surface plasmon resonance approach. We showed that the recombinant analog of HMIQ3c1 forms sufficiently strong complexes with trypsin (KD = 1.07 × 10-9 М) and chymotrypsin (KD = 4.70 × 10-8 М). Analysis of thermodynamic parameters of HMIQ3c1/chymotrypsin revealed significant contribution of the entropic factor to the complex formation. The formation of specific complexes of HMIQ3c1 with the kallikrein (KD = 2.81 × 10-8 М) and neutrophil elastase (KD = 1.11 × 10-7 М) indicates its anti-inflammatory activity and makes prospects to use the peptide as a potential therapeutic agent.

[The effectiveness of dentoalveolar anomalies treatment in children during early mixed dentition period]. / Éffektivnost' lecheniia zubocheliustnykh anomalii u detei v period rannego smennogo prikusa.

AIM: To analyze the effectiveness of orthodontic treatment in 7-9 years old children, dental status in children residents of Moscow was compared with 15-17 years adolescents with completed bite formation. MATERIAL AND METHODS: The adolescents were divided into two groups: those who received orthodontic treatment during the mixed dentition period and those who did not receive the treatment. A WHO survey chart was used to assess the dental status of children (WHO, 2013) with additional section 'Dentoalveolar anomalies'. DMFT and DMFT+dft, OHI-S, CPI, PAR, Little irregularity index and DAI were assessed in all groups. The need for dental and orthodontic treatment was calculated. RESULTS: Dental morbidity in Moscow children during the mixed dentition period was characterized by a high prevalence of caries (92.0%) with an intensity of 1.67±0.03, the 17.0% prevalence of premature teeth loss and dentoalveolar anomalies in 73.9% of children with a combination of anomalies in 60.8% of children. Orthodontic treatment in the mixed dentition period is associated with systematic professional control and by the age of 15 years results in two-fold reduction in the intensity of caries and periodontal disease, proper detection of poor hygiene and the lower prevalence of abnormal positioning of the teeth (crowding and misalignment of teeth) and the improvement of dental arches ratio (completely eliminating cross-bite). At the same time early orthodontic treatment has no significant effect on the prevalence of tooth rotation, interdental spaces, deep, open, distal, mesial occlusion and displacement of dental arches. CONCLUSION: Orthodontic treatment of children during the mixed dentition period is indicated in cases of crowding and displacement of teeth, as well as cross-bite; with respect to other types of dentoalveolar anomalies, early orthodontic treatment is only justified by the severe impact of dentoalveolar anomalies on psychological and functional indicators.

[Enalaprilat Inhibits Zinc-Dependent Oligomerization of Metal-Binding Domain of Amyloid-beta Isoforms and Protects Human Neuroblastoma Cells from Toxic Action of these Isoforms].

Intact amyloid-ß peptides (Aß) may undergo prion-like aggregation when they interact with chemically or structurally modified variants of Aß present in extracellular pathohistological inclusions (amyloid plaques). This aggregation is regarded as one of the key molecular mechanisms of Alzheimer's disease (AD) pathogenesis. Zinc ions are involved in the pathological dimerization and oligomerization of natural Aß isoforms, and zinc-induced oligomers can also initiate the pathological aggregation of Aß. Based on the earlier found molecular mechanism of zinc-dependent oligomerization of Aß, it has been suggested that the targeted inhibition of the 11EVHH14 site in one Aß molecule from zinc-mediated interactions with the same site of another Aß molecule can effectively inhibit the oligomerization and aggregation of Aß. Taking into account the similarity in the structural organization of zinc-binding sites within Aß and angiotensin-converting enzyme (ACE), we hypothesized that inhibitors of the ACE active sites could specifically interact with the 11EVHH14 site of Aß. Using a surface plasmon resonance biosensor and nuclear magnetic resonance spectroscopy, we have found that the ACE inhibitor enalaprilat effectively inhibits zinc-dependent dimerization of the metal-binding domains of intact Aß and Aß with isomerized Asp7 (isoAß). We have also found that enalaprilat protects SH-SY5Y human neuroblastoma cells from the toxic effects of Aß(1-42) and isoAß(1-42), which are among the most common components of amyloid plaques. The results confirm the role of zincdependent oligomerization of Aß in AD pathogenesis and make it possible one to consider enalaprilat as a prototype of antiaggregation agents for treating AD.

Quantitative Affinity Interaction of Ubiquitinated and Non-ubiquitinated Proteins with Proteasome Subunit Rpn10.

Recent proteomic profiling of mouse brain preparations using the ubiquitin receptor, Rpn10 proteasome subunit, as an affinity ligand revealed a representative group of proteins bound to this sorbent (Medvedev, A. E., et al. (2017) Biochemistry (Moscow), 82, 330-339). In the present study, we investigated interaction of the Rpn10 subunit of proteasomes with some of these identified proteins: glyceraldehyde-3-phosphate dehydrogenase (GAPDH), pyruvate kinase, and histones H2A and H2B. The study revealed: (i) quantitative affinity interaction of the proteasome subunit immobilized on a Biacore-3000 optical biosensor cuvette with both the GAPDH (Kd = 2.4·10-6 M) and pyruvate kinase (Kd = 2.8·10-5 M); (ii) quantitative high-affinity interaction of immobilized histones H2A and H2B with the Rpn10 subunit (Kd values of 6.5·10-8 and 3.2·10-9 M, respectively). Mass spectrometric analysis revealed the presence of the ubiquitin signature (GG) only in a highly purified preparation of GAPDH. We suggest that binding (especially high-affinity binding) of non-ubiquitinated proteins to the Rpn10 proteasome subunit can both regulate the functioning of this proteasomal ubiquitin receptor (by competing with ubiquitinated substrates) and promote activation of other pathways for proteolytic degradation of proteins destined to the proteasome.

[Use of stents with bioactive coating in treatment of patients with lesions of the superficial femoral artery]. / Primenenie stentov s bioaktivnym pokrytiem v lechenii bol'nykh s porazheniem poverkhnostnoi bedrennoi arterii.

The authors analysed the immediate outcomes of implanting stents with bioactive coating in treatment of patients presenting with atherosclerotic lesions of the superficial femoral artery. Over the period from January 2014 to December 2015, endovascular interventions on the superficial femoral artery were carried out in a total of 18 patients (mean age 61.3±9.2 years). The implants inserted were stents with bioactive coating based on titanium oxinitride, measuring 6 to 8 mm in diameter and being from 50 to 200 mm long. Prior to operation and 7 days after implantation of the stent, the immunoenzymatic assay was used to determine the level of nitrogen nitric oxide (NO) in blood. The stents' patency was assessed by the findings of ultrasound duplex scanning performed at 30 days, and then 6 and 12 months after the intervention. There were no complications either during the operation or in the early postoperative period. An increase in the ankle-brachial index was observed in all patients: with the average value prior to treatment amounting to 0.4±0.3 and equalling 1.1±0.2 after stenting (p<0.0001). Normalization of the blood NO level was revealed (was noted to normalize): the mean value prior to operation amounted to 18.9±2.3 µmol/L and after operation to 28.9±4.1 µmol/L (p<0.05). Primary patency rate of the stents was 100% at 30 days, 94.5% (1 occlusion) at 6 months and 88.8% (1 restenosis and 1 occlusion) at 12 months. The patients with occlusion or restenosis were subjected to repeat endovascular interventions, with restoration of patency of the construction (with the construction's patency restored). By now all the 18 patients show preserved patency (currently patency was preserved) of lower-limb arteries, with no evidence of restenosis in the zones of operations. It was concluded that using stents with bioactive coating based on titanium oxinitride results in normalization of the level of NO in blood, which may contribute favourably to prolongation of the period of functioning of endovascular constructions. The first data concerning primary patency of stents of this type make it possible to count on betterment of the remote results of treatment of patients presenting with atherosclerotic lesions of the superficial femoral artery.

Anion Photoelectron Spectroscopy and CASSCF/CASPT2/RASSI Study of Lan(-) (n = 1, 3-7).

We present a combined experimental and theoretical study of small lanthanum clusters. The experimental photoelectron spectra of Lan(-) (n = 1, 3-7) were obtained using negative ion photoelectron spectroscopy. Electron affinities for these clusters were found to be in a range of 0.49 eV (La) to 1.5 eV (La7). Our computational tour de force in exploring the electronic structure and its consequences for the lanthanum atom and its anion as well as for lanthanum trimer and its anion shows the multiconfigurational method and large basis set with spin-orbit corrections: CASSCF/CASPT2/RASSI/ANO-RCC-L level of theory is needed to reproduce experimental accuracy. The most stable structure for La3(-) was established to be an equilateral triangle ((1)A1'). Chemical bonding analysis of the La3(-) global minimum reveals that this is the first experimentally observed species with d-AO double σ and π aromaticity.

[Effect of mutations and modifications of amino acid residues on zinc-induced interaction of the metal-binding domain of ß-amyloid with DNA].

Interaction of intranuclear ß-amyloid with DNA is considered to be a plausible mechanism of Alzheimer's disease pathogenesis. The interaction of single- and double-stranded DNA with synthetic peptides was analyzed using surface plasmon resonance. The peptides represent the metal-binding domain of ß-amyloid (amino acids 1-16) and its variants with chemical modifications and point substitutions of amino acid residues which are associated with enhanced neurotoxicity of ß-amyloid in cell tests. It has been shown that the presence of zinc ions is necessary for the interaction of the peptides with DNA in solution. H6R substitution has remarkably reduced the ability of domain 1-16 to bind DNA. This is in accordance with the supposition that the coordination of a zinc ion by amino acid residues His6, Glu11, His13, and His14 of the ß-amyloid metal-binding domain results in the occurrence of an anion-binding site responsible for the interaction of the domain with DNA. Zinc-induced dimerization and oligomerization of domain 1-16 associated with phosphorylation of Ser8 and the presence of unblocked amino- and carboxy-terminal groups have resulted in a decrease of peptide concentrations required for detection of the peptide-DNA interaction. The presence of multiple anion-binding sites on the dimers and oligomers is responsible for the enhancement of the peptide-DNA interaction. A substitution of the negatively charged residue Asp7 for the neutral residue Asn in close proximity to the anion-binding site of the domain 1-16 of Aß facilitates the electrostatic interaction between this site and phosphates of a polynucleotide chain, which enhances zinc-induced binding to DNA.

[ENDOVASCULAR ABDOMINAL AORTIC ANEURISM REPAIR].

The authors analyzed the single-center experience of treatment of 72 patients with abdominal aortic aneurisms and severe accompanied pathology. The aneurisms were repaired by stent-grafts. All the patients had abdominal aortic aneurisms with the diameters from 41 to 84 mm against the background of severe somatic pathology. It was a contraindication to planned open surgery. An installation of stent-graft was successful in all 72 follow-ups. It wasn't necessary to use a conversion to open surgery. The follow-up period consisted of 44,6?2,1 months. Control ultrasound and computer tomography studies hadn't revealed an increase of aneurism sack sizes or "eakages". A reduction of abdominal aortic aneurism sizes was noted in 37 patients on 4-5% during first year after operation. The stent-graft implantation extends the possibilities of abdominal aortic aneurism treatment for patients from a high surgical risk group.

[WAYS OF IMPROVEMENT OF LOOP ENDARTERECTOMY RESULTS FROM ILIAC-FEMORAL SEGMENT].

The article presents an analysis of treatment results of 91 patients with iliac-femoral segment artery occlusion at the period from 2008 to 2014. Patients were divided into 2 groups: main group (n=30) consisted of patients who undergoing a half-closed loop endarterectomy with following implantation of stent-grafts in this area and control group (n=61) had patients whom were performed routine half-closed loop endarterectomy. The II degree of ischemia of lower extremities was in 88 (96,7%) patients and III degree had 3 (3,3%) patients. The areas of abnormalities of intravascular pattern were detected in 100% of cases in intraoperative angiography. They were modified using stent-grafts. A primary vascular patency was 100% in the first group and it numbered 65% in the second group during 5 years. The intraoperative angiography control with stent-graft implantation to the area of endarterectomy allowed reliable improvement of treatment results.

[Medico-social and psychosomatic status of gerontostomatologic patients].

Assessment of medico-social and psychosomatic status of patients of the advanced and senile age having dental diseases was carried out. The results of the study show that the level of stomatological morbidity in this contingent of patients is rather high, and the treatment must be carried out with obligatory participation of doctors-interns and taking into account mental health of a person.

[Interactomics of CXXC proteins involved in epigenetic regulation of gene expression]. / Interaktomika CXXC-belkov, uchastvuiushchikh v épigeneticheskoi reguliatsii gennoi ékspressii.

Regulation of gene expression is an extremely complex and multicomponent biological phenomenon. Proteins containing the CXXC-domain "zinc fingers" (CXXC-proteins) are master regulators of expression of many genes and have conserved functions of methylation of DNA bases and histone proteins. CXXC proteins function as a part of multiprotein complexes, which indicates the fundamental importance of studying post-translational regulation through modulation of the protein-protein interaction spectrum (PPI) in both normal and pathological conditions. In this paper we discuss general aspects of the involvement of CXXC proteins and their protein partners in neoplastic processes, both from the literature data and our own studies. Special attention is paid to recent data on the particular interactomics of the CFP1 protein encoded by the CXXC1 gene located on the human chromosome 18. CFP1 is devoid of enzymatic activity and implements epigenetic regulation of expression through binding to chromatin and a certain spectrum of PPIs.

[Adrenodoxins and their role in the cytochrome P450 systems]. / Adrenodoksiny i ikh rol' v sistemakh tsitokhroma R450.

The role of partner proteins in the formation of functional complexes in cytochrome P450 systems was investigated by means of optical biosensor technique. Kinetic constants and equilibrium dissociation constants of complexes of cytochrome CYP11A1 (P450scc) with wild-type adrenodoxin (Adx WT) and mutant forms of adrenodoxin R106D and D109R were determined using an optical biosensor. Wild-type adrenodoxin (Kd = (1.23±0.09)⋅10⁻6 M) and mutant D109R (Kd = (2.37±0.09)⋅10⁻8 M) formed complexes with cytochrome P450scc. For the R106D mutant, no complex formation was detected. To investigate the possibility of the participation of adrenodoxins and their mutant variants in the process of electron transfer as electron donors in mitochondrial cytochrome P450 systems, the electrochemical properties of these iron-sulfur proteins Adx WT and mutant forms of adrenodoxins were studied. Adx WT, mutant forms R106D and D109R have redox potentials E1/2 significantly more negative than cytochromes P450 (-579±10 mV, -590±15 mV, and -528±10 mV, respectively). These results suggest that Adx WT and mutant forms may be electron donors in the cytochrome P450 systems.

[Comparative analysis of proteins associated with 26S and 20S proteasomes isolated from rabbit brain and liver]. / Sravnitel'nyi analiz belkov, assotsiirovannykh s 26S i 20S proteasomami mozga i pecheni krolika.

We have isolated fractions of 26S and 20S proteasomes were from the rabbit liver and the brain. According to mass spectrometric (MS) analysis, the 26S proteasome fractions from these organs contained catalytic and regulatory subunits characteristic of the proteasome core and regulatory subunits. The 20S fractions of brain and liver proteasomes contained only catalytic proteasome subunits. In addition to proteasome subunits, the isolated fractions contained components of the ubiquitin-proteasome system, ubiquitinated proteins, enzymes that play an important role in metabolic processes, cytoskeletal components, signaling, regulatory, and protective proteins, as well as proteins regulating gene expression, cell division, and differentiation. The abundance of a number of proteasome-associated proteins was comparable or exceeded the abundance of intrinsic proteasome components. About a third of the proteins common to all studied fractions (26S and 20S of brain and liver proteasomes) belong to the group of multifunctional proteins. Selective biosensor validation confirmed the affinity binding of proteins (aldolase, phosphoglycerate kinase) identified during MS analysis to the brain 20S proteasome. Comparison of the subproteomes of the 26S and 20S brain proteasomes showed that removal of components of the regulatory (19S) subparticles caused almost two-fold increase in the total number of individual proteins associated with the core part of the proteasome (20S). In the liver, the number of proteins associated with the core part of the proteasome remained basically unchanged after the removal of the components of the regulatory (19S) subparticles. This indicates that in the brain and, possibly, in other organs, proteins of the regulatory (19S) subunit play an important role in the formation of the proteasome interactome.

[Protein interactomics technologies].

The analysis of protein-protein interactions has the growing importance in modern proteomics. It is caused by necessity of in-depth functional study of proteins which, as a rule, operate in living systems due to each other interaction in the stable or dynamic protein complexes. In this review, the brief description and a comparative evaluation of most frequently used approaches in existing variety of protein interactomics methods are given.

Renal inflammatory myofibroblastic tumor: A case report.

Inflammatory myofibroblastic tumor (IMT) is an extremely rare disease composed of myofibroblast cells and inflammatory infiltrates. There are different sites of the urogenital system affected by IMT-bladder, prostate and kidney. We report a case of a 59-year-old male patient presented with abdominal pain, gross hematuria and a renal mass treated with partial nephrectomy. The final diagnosis was renal inflammatory myofibroblastic tumor. Despite recent improvements in imaging technology, preoperative diagnosis of IMT remains a dilemma. It is therefore mandatory to carry out clinical interpretation, careful histologic examination, and immunohistochemical studies which will generally determine the appropriate diagnosis and patient management.

[Screening of potential non-azole inhibitors of lanosterol14-alpha demethylase (CYP51) of Candida fungi]. / Skrining potentsial'nykh neazol'nykh ingibitorov 14-al'fademetilazy (CYP51) gribov roda Candida.

Currently, opportunistic fungi of the genus Candida are the main causative agents of mycoses, which are especially severe upon condition of acquired immunodeficiency. The main target for the development of new antimycotics is the cytochrome P450 51 (CYP51) of the pathogenic fungus. Due to the widespread distribution of Candida strains resistancy to inhibitors of the azole class, the screening for CYP51 inhibitors both among non-azole compounds and among clinically used drugs repurposing as antimycotics is becoming urgent. To identify potential inhibitors from the non-azole group, an integrated approach was applied, including bioinformatics analysis, computer molecular modeling, and a surface plasmon resonance (SPR) technology. Using in silico modeling, the binding sites for acetylsalicylic acid, ibuprofen, chlorpromazine and haloperidol (this compounds, according to the literature, showed antimycotic activity) were predicted in the active site of CYP51 of Candida albicans and Candida glabrata. The Kd values of molecular complexes of acetylsalicylic acid, ibuprofen and haloperidol with CYP51, determined by SPR analysis, ranged from 18 µM to 126 µM. It was also shown that structural derivatives of haloperidol, containing various substituents, could be positioned in the active site of CYP51 of Candida albicans with the possible formation of coordination bonds between the hydroxyl groups of the derivatives and the iron atom in the heme of CYP51. Thus, the potential basic structures of non-azole compounds have been proposed, which can be used for the design of new CYP51 inhibitors of Candida fungi.