Variability of haptoglobin beta-chain proteoforms.

Existing knowledge on changes of the haptoglobin (Hp) molecule suggests that it may exist in multiple proteoforms, which obviously exhibit different functions. Using two-dimensional electrophoresis (2DE) in combination with mass spectrometry and immunodetection, we have analyzed blood plasma samples from both healthy donors and patients with primary grade IV glioblastoma (GBM), and obtained a detailed composite 2DE distribution map of ß-chain proteoforms, as well as the full-length form of Hp (zonulin). Although the total level of plasma Hp exceeded normal values in cancer patients (especially patients with GBM), the presence of particuar proteoforms, detected by their position on the 2DE map, was very individual. Variability was found in both zonulin and the Hp ß-chain. The presence of an alkaline form of zonulin in plasma can be considered a conditional, but insufficient, GBM biomarker. In other words, we found that at the level of minor proteoforms of Hp, even in normal conditions, there was a high individual variability. On the one hand, this raises questions about the reasons for such variability, if it is present not only in Hp, but also in other proteins. On the other hand, this may explain the discrepancy between the number of experimentally detected proteoforms and the theoretically possible ones not only in Hp, but also in other proteins.

[Zonulin - regulation of tight contacts in the brain and intestine - facts and hypotheses]. / Zonulin ­ reguliatsiia plotnykh kontaktov v mozge i kishechnike ­ fakty i gipotezy.

In recent years, the interrelationship between the brain and the gut has become an area of high scientific interest. The intestine is responsible not only for digestion, as it contains millions of neurons, its own immune system, and affects the emotional and cognitive processes. The relationship between the gut and the brain suggests that the processes carried out by the gut microbiota play a significant role in the regulation of brain function, and vice versa. A special role here is played by intercellular tight junctions (TJ), where the zonulin protein holds an important place. Zonulin, an unprocessed precursor of mature haptoglobin, is the only physiological modulator of intercellular TJ that can reversibly regulate the permeability of the intestinal (IB) and blood-brain (BBB) barriers in the human body. BBB disruption and altered microbiota composition are associated with many diseases, including neurological disorders and neuroinflammation. That is, there is a gut-brain axis (GBA) - a communication system through which the brain modulates the functions of the gastrointestinal tract (GIT) and vice versa. GBA is based on neuronal, endocrine, and immunological mechanisms that are interconnected at the organismal, organ, cellular, and molecular levels.

[Haptoglobin as a biomarker]. / Gaptoglobin kak biomarker.

Haptoglobin (Hp) is a blood plasma glycoprotein that binds free hemoglobin (Hb) and plays a critical role in tissue protection and the prevention of oxidative damage. In addition, it has a number of regulatory functions. Haptoglobin is an acute phase protein, its concentration in plasma changes in pathology, and the test for its concentration is part of normal clinical practice. Haptoglobin is a conservative protein synthesized mainly in the liver and lungs and is the subject of research as a potential biomarker of many diseases, including various forms of malignant neoplasms. Haptoglobin has several unique biophysical characteristics. Only in humans, the Hp gene is polymorphic, has three structural alleles that control the synthesis of three major phenotypes of Hp, homozygous Hp1-1 and Hp2-2, and heterozygous Hp2-1, determined by a combination of allelic variants that are inherited. Numerous studies indicate that the phenotype of haptoglobin can be used to judge the individual's predisposition to various diseases. In addition, Hp undergoes various post-translational modifications (PTMs). These are structural transformations (removal of the signal peptide, cutting of the Pre-Hp precursor molecule into two subunits, α and ß, limited proteolysis of α-chains, formation of disulfide bonds, multimerization), as well as chemical modifications of α-chains and glycosylation of the ß-chain. Glycosylation of the ß-chain of haptoglobin at four Asn sites is the most important variable PTM that regulates the structure and function of the glycoprotein. The study of modified oligosaccharides of the Hp ß-chain has become the main direction in the study of pathological processes, including malignant neoplasms. Many studies are focused on the identification of PTM and changes in the level of the α2-chain of this protein in pathology. These characteristics of Hp indicate the possibility of the existence of this protein as different proteoforms, probably with different functions. This review is devoted to the description of the structural and functional diversity of Hp and its potential use as a biomarker of various pathologies.

[Structural-functional diversity of p53 proteoforms]. / Strukturno-funktsional'noe mnogoobrazie proteoform belka r53.

Protein p53 is one of the most studied proteins. This attention is primarily due to its key role in the cellular mechanisms associated with carcinogenesis. Protein p53 is a transcription factor involved in a wide variety of processes: cell cycle regulation and apoptosis, signaling inside the cell, DNA repair, coordination of metabolic processes, regulation of cell interactions, etc. This multifunctionality is apparently determined by the fact that p53 is a vivid example of how the same protein can be represented by numerous proteoforms bearing completely different functional loads. By alternative splicing, using different promoters and translation initiation sites, the TP53 gene gives rise to at least 12 isoforms, which can additionally undergo numerous (>200) post-translational modifications. Proteoforms generated due to numerous point mutations in the TP53 gene are adding more complexity to this picture. The proteoforms produced are involved in various processes, such as the regulation of p53 transcriptional activity in response to various factors. This review is devoted to the description of the currently known p53 proteoforms, as well as their possible functionality.

Comprehensive Analysis of Carbohydrate-Active Enzymes from the Filamentous Fungus Scytalidium candidum 3C.

Complete enzymatic degradation of plant polysaccharides is a result of combined action of various carbohydrate-active enzymes (CAZymes). In this paper, we demonstrate the potential of the filamentous fungus Scytalidium candidum 3C for processing of plant biomass. Structural annotation of the improved assembly of S. candidum 3C genome and functional annotation of CAZymes revealed putative gene sequences encoding such proteins. A total of 190 CAZyme-encoding genes were identified, including 104 glycoside hydrolases, 52 glycosyltransferases, 28 oxidative enzymes, and 6 carbohydrate esterases. In addition, 14 carbohydrate-binding modules were found. Glycoside hydrolases secreted during the growth of S. candidum 3C in three media were analyzed with a variety of substrates. Mass spectrometry analysis of the fungal culture liquid revealed the presence of peptides identical to 36 glycoside hydrolases, three proteins without known enzymatic function belonging to the same group of families, and 11 oxidative enzymes. The activity of endo-hemicellulases was determined using specially synthesized substrates in which the glycosidic bond between monosaccharide residues was replaced by a thio-linkage. During analysis of the CAZyme profile of S. candidum 3C, four ß-xylanases from the GH10 family and two ß-glucanases from the GH7 and GH55 families were detected, partially purified, and identified.

[Multiomics study of HepG2 cell line proteome]. / Mul'tiomnaia strategiia issledovaniia proteoma kletochnoi linii gepatotselliuliarnoi kartsinomy HepG2.

Current proteomic studies are generally focused on the most abundant proteoforms encoded by canonical nucleic sequences. Transcriptomic and proteomic data, accumulated in a variety of postgenome sources and coupled with state-of-art analytical technologies, allow to start the identification of aberrant (non-canonical) proteoforms. The main sources of aberrant proteoforms are alternative splicing, single nucleotide polymorphism, and post-translational modifications. The aim of this work was to estimate the heterogeneity of HepG2 proteome. We suggested multiomics approach, which combines transcriptomic (RNAseq) and proteomic (2DE-MS/MS) methods, as a promising strategy to explore the proteome.

[Quaternary structures of human cytoplasmic and nuclear PCNA are the same]. / Chetvertichnaia struktura iadernogo antigena deliashchikhsia kletok (PCNA) v tsitoplazme i iadre kletok cheloveka organizovana odinakovo.

Properties and mechanisms of PCNA (proliferating cell nuclear antigen) functions have been investigated for a long time and are studied in great detail. As follows from its name, most known PCNA functions (DNA replication, DNA repair, DNA recombination and others) are connected with cell proliferation and localization of this protein in nuclei. In addition, there is good reason to believe that PCNA also performs some functions in the cytoplasm. However, the possible role and mechanisms of PCNA action in the cytoplasm require careful study and clarification. Interestingly, such cells as neutrophils differ in that they are non-dividing on one hand and on the other hand contain a rather large amount of PCNA, which is localized only in the cytoplasm, that is, they are an ideal model for the study of cytoplasmic PCNA. Using cross-linkages with formaldehyde, we showed that this cytoplasmic PCNA is cross-linked in a similar way, that is, organized in the same way as the nuclear PCNA that is present in the proliferating cells. Previously, we showed that PCNA in such cells is organized into a dynamic complex of double trimer on the basis of the back-to-back principle (Naryzhny S.N. et al. (2005) J. Biol. Chem., 280, 13888). Apparently, such organization of this hub-protein allows it to better coordinate the processes taking place in the cytoplasm as well.

AGGREGATION BY LECTIN-METHODICAL APPROACH FOR EFFECTIVE ISOLATION OF EXOSOMES FROM CELL CULTURE SUPERNATANT FOR PROTEOME PROFILING.

Exosomes are small membrane vesicles secreted by most cell types in vivo and in vitro. Exosomes are found in cell culture media, blood, urine, amniotic fluid, malignant ascite fluids and contain distinct subsets of microRNAs and proteins depending upon the tissue from which they are secreted. Thus exosomes constitute potential biomarkers of human diseases, such as cancer. A major bottleneck in the development of exosome-based diagnostic assays is the challenging purification of these vesicles; this requires time-consuming and instrument-based procedures. Isolation of exosomes can be a tedious, non-specific, and difficult process. Here, we provide a preparative technique for isolation of exosomes based on their ability to aggregate in the presence of lectins. The new method for lectin-based isolation of exosomes from cell culture media was developed as a sample preparation step for exosome-based protein biomarker research.

[Experimental estimation of proteome size for cells and human plasma].

Huge range of concentrations of different protein and insufficient sensitivity of methods for detection of proteins at a single molecule level does not yet allow obtaining the whole image of 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 2-DE after staining by protein dyes with different sensitivities. The function representing the dependence of the number of protein spots on 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 70,000 proteoforms, and plasma--1.5 mln. Utilization of this approach to other, smaller proteomes showed the competency of this extrapolation. For instance, the size of mycoplas ma (Acholeplasma laidlawii) was estimated in 1100 proteoforms, yeast (Saccharomyces cerevisiae)--40,000, E. coli--6200, P. furiosus--3400. In hepatocytes, the amount of proteoforms was the same as in HepG2--70,000. Significance of obtained data is in possibilities to estimating the proteome organization and planning next steps in its study.

The immortalizing and transforming ability of two common human papillomavirus 16 E6 variants with different prevalences in cervical cancer.

Persistent infection with high-risk human papillomaviruses (HPVs), especially type 16 has been undeniably linked to cervical cancer. The Asian-American (AA) variant of HPV16 is more common in the Americas than the prototype in cervical cancer. The different prevalence is based on three amino acid changes within the E6 protein denoted Q14H/H78Y/L83V. To investigate the mechanism(s) behind this observation, both E6 proteins, in the presence of E7, were evaluated for their ability to extend the life span of and transform primary human foreskin keratinocytes (PHFKs). Long-term cell culture studies resulted in death at passage 9 of vector-transduced PHFKs (negative control), but survival of both E6 PHFKs to passage 65 (and beyond). Compared with E6/E7 PHFKs, AA/E7 PHFKs were significantly faster dividing, developed larger cells in monolayer cultures, showed double the epithelial thickness and expressed cytokeratin 10 when grown as organotypic raft cultures. Telomerase activation and p53 inactivation, two hallmarks of immortalization, were not significantly different between the two populations. Both were resistant to anoikis at later passages, but only AA/E7 PHFKs acquired the capacity for in vitro transformation. Proteomic analysis revealed markedly different protein patterns between E6/E7 and AA/E7, particularly with respect to key cellular metabolic enzymes. Our results provide new insights into the reasons underlying the greater prevalence of the AA variant in cervical cancer as evidenced by characteristics associated with higher oncogenic potential.

Proliferating cell nuclear antigen: a proteomics view.

Proliferating cell nuclear antigen (PCNA), a cell cycle marker protein, is well known as a DNA sliding clamp for DNA polymerase delta and as an essential component for eukaryotic chromosomal DNA replication and repair. Due to its mobility inside nuclei, PCNA is dynamically presented in a soluble or chromatin-associated form. The heterogeneity and specific modifications of PCNA may reflect its multiple functions and the presence of many binding partners in the cell. The recent proteomics approaches applied to characterizing PCNA interactions revealed multiple PCNA partners with a wide spectrum of activity and unveiled the possible existence of new PCNA functions. Since more than 100 PCNA-interacting proteins and several PCNA modifications have already been reported, a proteomics point of view seems exactly suitable to better understand the role of PCNA in cellular functions.

Protein profiles of the Chinese hamster ovary cells in the resting and proliferating stages.

Identification and characterization of the proteins that regulate the transition from the resting stage (G0) through G1 to S phase of the cell cycle are of central importance to understand the control of cell proliferation and chromosome replication. Unlike in lower organisms, where relatively small numbers of key factors are involved in this process, the factors involved in the same control mechanisms in mammalian systems are much more complex. Furthermore, accumulating lines of evidence now suggest that the nuclear matrix and chromatin organization also play an essential role for the cell cycle control in mammalian cells. To gain a better understanding of the overall dynamics and changes of the protein factors in the context of matrix/chromatin organization, we examined the protein profiles of the Chinese hamster ovary (CHO) cells in different cell cycle compartments. The methods used in this study included subcellular fractionations (cytosol, nuclear extraction, chromatin, and nuclear matrix), two-dimensional polyacrylamide gel electrophoresis (2-D PAGE), silver staining, and immunoblotting. As expected, significant changes of protein profiles were observed when cells entered into proliferating stages from G0. Among approximately 1200 protein spots analyzed by 2-D PAGE, at least 12 showed marked increase or decrease at this transitional period. Further cell-cycle progression from G1 to S phase showed less dramatic changes of overall protein protile. However, the profile of certain proteins showed rather dramatic changes of their subcellular localization during this transitional period. In particular, the levels of proliferating cell nuclear antigen (PCNA) in the nuclear matrix and chromatin dramatically increased in mid-G1 and in the beginning of S phase, respectively, while the overall PCNA level was relatively constant throughout the cell cycle.

Active dissociation of the fluorescent dye Hoechst 33342 from DNA in a living cell: who could do it?

It is assumed that DNA in mammalian cells is a dynamic conformationally unstable system. This instability provides the cell with a mechanism for dissociating a large number of substances that bind tightly but not covalently to DNA. Among these is the fluorescent dye Hoechst 33342, which binds to DNA in the minor groove. We have selected cell lines with a high capability for active dissociation of Hoechst 33342. Comparative protein analysis of these lines by means of two-dimensional (2-D) electrophoresis was performed. Cell and nuclear proteins were analyzed from these and normal strains. A few proteins with significantly changed quantities have been found. The preliminary search of the 2-D database allowed us to identity some known and unknown cellular proteins that could participate in active dissociation of the dye from DNA.

"Active" two-dimensional electrophoresis of rat liver DNA-polymerase alpha.

The approach of so-called active gel analysis was used to determine the position and appearance of the catalytic subunit of rat liver DNA polymerase alpha on a two-dimensional (2-D) electrophoretic map. In this case a polyacrylamide gel containing DNA was used for the second dimension. DNA presence does not change the 2-D protein pattern but makes it possible to conduct a polymerase reaction directly in the gel after separation. A crude extract of rat liver nuclei was used for analysis. The extract is quickly isolated and contains mainly DNA polymerase alpha activity. It was shown that this enzyme restores its activity after 2-D electrophoresis and sodium dodecyl sulfate (SDS) elution. After polymerase reaction with labeled dNTPs and autoradiography, the catalytic polypeptide or, rather, polypeptide cluster is revealed as chains of spots (possibly because of the presence of different hydrolyzed and phosphorylated forms). These spots are located on the 2-D electrophoretic map in the region corresponding to molecular masses of 160, 140, and 130 kDa and pI 5.5-6.2.

Upside-down stopped-flow electrofractionation of complex protein mixtures.

The excellent resolution of SDS-PAGE in protein analysis stimulated the creation of various preparative devices. The main approach used in these devices is the construction of a elution chamber in the lower end of the polyacrylamide gel cylinder or plate. Although this continuous lower buffer flow electrofractionation system serves as an acceptable preparative electrophoresis, some limitations to this approach exist. There is strong dilution of protein zones by the eluting buffer, which drastically restricts the sensitivity of the determination of minor proteins, and the restricted current flow caused by electric resistance arising from the column holder prevents application to purification of complex protein mixtures. To overcome these problems, the upside-down stopped-flow electrofractionation system (UDSFE) was designed. The necessary quantity of fraction is drawn with a pipet in a small volume from just above the gel cylinder. This invention improves the possibility of electrofractionation of deluted complex protein mixtures. The efficiency of this technique is demonstrated by purification a protein kinase from rat liver. The method has also been successfully used for purification of error-correcting 3'-5' exonuclease.

Proof-reading 3'-->5' exonucleases isolated from rat liver nuclei.

Mammalian nuclear DNA polymerases alpha and beta are known to be devoid of the editing 3'-->5' exonucleolytic activity. Presumably this activity could be effected by the exonucleases non-associated covalently with DNA polymerases. Two 3'-->5' exonucleases of 40 kDa and 50 kDa (exo-40 and exo-5) have been isolated from rat liver nuclei and purified to near homogeneity. They are shown to excise mismatched nucleotides from poly[d(A-T)] template, respectively, 10-fold and 2-fold faster than the matched ones. Upon addition of either of these exonucleases to the DNA polymerase alpha from rat liver or calf thymus, the fidelity of in-vitro reproduction of the primed DNA from bacteriophage phi X174 amber 3 is increased 5-10-fold, levels of exonuclease and DNA-polymerase activities being similar. Extrapolation of in-vitro DNA-replication fidelity to the cellular levels of activities of the exonucleases and the alpha-polymerase suggests that exonucleolytic proof-reading augments the accuracy of DNA synthesis by 2-3 orders of magnitude.