Publications by authors named "Yuriy Kit"

14 Publications

  • Page 1 of 1

Isolation and identification in human blood serum of the proteins possessing the ability to bind with 48 kDa form of unconventional myosin 1c and their possible diagnostic and prognostic value.

Biomed Chromatogr 2021 Apr 2;35(4):e5029. Epub 2020 Dec 2.

Institute of Cell Biology, Nationa Academy of Sciences of Ukraine, Drahomanov st., 14\16, Lviv, Ukraine.

We firstly identified 48 kDa molecular form of the unconventional myosin 1c (p48/Myo1C), and isolated it from blood serum of multiple sclerosis patients. The amount of p48/Myo1C in human blood serum correlated with some autoimmune, hemato-oncological and neurodegenerative diseases and thus may serve as a potential molecular biomarker. The biological functions of this protein in human blood remain unknown. Previously, we used the monodisperse magnetic poly (glycidyl methacrylate)(mag-PGMA-NH ) microspheres with immobilized 48/Myo1C and western-blot analysis, which allowed us to identify IgM and IgG immunoglobulins presenting an affinity to this protein. Here, we used mass spectrometry followed by the western blotting in order to identify other blood serum proteins with affinity to 48/Myo1C. The obtained data demonstrate that 48/Myo1C binds to component 3 of the complement and the antithrombin-III proteins. A combination of magnetic microparticle-based affinity chromatography with MALDI-TOF mass spectrometry and an in silico analysis provided an opportunity to identify the partners of interaction of 48/Myo1C with other proteins, in particular those participating in complement and coagulation cascades.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/bmc.5029DOI Listing
April 2021

The Recombinant Fragment of Human κ-Casein Induces Cell Death by Targeting the Proteins of Mitochondrial Import in Breast Cancer Cells.

Cancers (Basel) 2020 May 31;12(6). Epub 2020 May 31.

Translational Inflammation Research, Medical Faculty, Center of Dynamic Systems (CDS), Otto von Guericke University, 39120 Magdeburg, Germany.

Breast cancer is still one of the most common cancers for women. Specified therapeutics are indispensable for optimal treatment. In previous studies, it has been shown that RL2, the recombinant fragment of human κ-Casein, induces cell death in breast cancer cells. However, the molecular mechanisms of RL2-induced cell death remain largely unknown. In this study, mechanisms of RL2-induced cell death in breast cancer cells were systematically investigated. In particular, we demonstrate that RL2 induces loss of mitochondrial membrane potential and cellular ATP loss followed by cell death in breast cancer cells. The mass spectrometry-based screen for RL2 interaction partners identified mitochondrial import protein TOM70 as a target of RL2, which was subsequently validated. Further to this, we show that RL2 is targeted to mitochondria after internalization into the cells, where it can also be found in the dimeric form. The importance of TOM70 and RL2 interaction in RL2-induced reduction in ATP levels was validated by siRNA-induced downregulation of TOM70, resulting in the partial rescue of ATP production. Taken together, this study demonstrates that RL2-TOM70 interaction plays a key role in RL2-mediated cell death and targeting this pathway may provide new therapeutic options for treating breast cancer.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/cancers12061427DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7352597PMC
May 2020

The purification and identification of human blood serum proteins with affinity to the antitumor active RL2 lactaptin using magnetic microparticles.

Biomed Chromatogr 2019 Nov 18;33(11):e4647. Epub 2019 Aug 18.

Institute of Cell Biology NAS Ukraine, Lviv, Ukraine.

The cytopoxic effect of RL2 lactaptin (the recombinant analog of proteolytic fragment of human kappa-casein) toward tumor cells in vitro and in vivo presents it as a novel promising antitumor drug. The binding of any drug with serum proteins can affect their activity, distribution, rate of excretion and toxicity in the human body. Here, we studied the ability of RL2 to bind to various blood serum proteins. Using magnetic microparticles bearing by RL2 as an affinity matrix, in combination with mass spectrometry and western blot analysis, we found a number of blood serum proteins possessing affinity for RL2. Among them IgA, IgM and IgG subclasses of immunoglobulins, apolipoprotein A1 and various cortactin isoforms were identified. This data suggests that in the bloodstream RL2 lactaptin takes part in complicate protein-protein interactions, which can affect its activity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/bmc.4647DOI Listing
November 2019

Characteristics of Potential Protein Biomarkers Extracted with 10% TCA from Blood Serum of Non-Hodgkin's Lymphoma and Multiple Myeloma Patients.

Int J Mol Cell Med 2017 11;6(4):235-238. Epub 2017 Nov 11.

Department of Regulation of Cell Proliferation and Apoptosis, Institute of Cell Biology, National Academy of Sciences of Ukraine, Lviv, Ukraine .

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.22088/BUMS.6.4.235DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6004295PMC
November 2017

Monodisperse magnetic poly(glycidyl methacrylate) microspheres for isolation of autoantibodies with affinity for the 46 kDa form of unconventional Myo1C present in autoimmune patients.

Mikrochim Acta 2018 04 23;185(5):262. Epub 2018 Apr 23.

Department of Polymer Particles, Institute of Macromolecular Chemistry, Academy of Sciences of the Czech Republic, Heyrovského nám. 2, 162 06, Prague 6, Czech Republic.

Monodisperse nonmagnetic macroporous poly(glycidyl methacrylate) (PGMA) microspheres were synthesized by multistep swelling polymerization of glycidyl methacrylate, ethylene dimethacrylate and 2-[(methoxycarbonyl)methoxy]ethyl methacrylate (MCMEMA). This was followed (a) by ammonolysis to modify the microspheres with amino groups, and (b) by incorporation of iron oxide (γ-FeO) into the pores to render the particles magnetic. The resulting porous and magnetic microspheres were characterized by scanning and transmission electron microscopy (SEM and TEM), atomic absorption and Fourier transform infrared spectroscopy (AAS and FTIR), elemental analysis, vibrating magnetometry, mercury porosimetry and Brunauer-Emmett-Teller adsorption/desorption isotherms. The microspheres are meso- and macroporous, typically 5 μm in diameter, contain 0.9 mM · g of amino groups and 14 wt.% of iron according to elemental analysis and AAS, respectively. The particles were conjugated to p46/Myo1C protein, a potential biomarker of autoimmune diseases, to isolate specific autoantibodies in the blood of patients suffering from multiple sclerosis (MS). The p46/Myo1C loaded microspheres are shown to enable the preconcentration of minute quantities of specific immunoglobulins prior to their quantification via SDS-PAGE. The immunoglobulin M (IgM) with affinity to Myo1C was detected in MS patients. Graphical abstract Monodisperse magnetic poly(glycidyl methacrylate) microspheres were synthesized, conjugated with 46 kDa form of unconventional Myo1C protein (p46/Myo1C) via carbodiimide (DIC) chemistry, and specific autoantibodies isolated from blood of multiple sclerosis (MS) patients; immunoglobulin M (IgM) level increased in MS patients.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s00604-018-2807-5DOI Listing
April 2018

Identification of SER-PRO-CYS Peptide in Blood Serum of Multiple Sclerosis Patients.

Protein Pept Lett 2016 ;23(9):808-11

Institute of Cell Biology, National Academy of Sciences of Ukraine, Drahomanov St., 14/16, 79005, Lviv, Ukraine.

Monitoring of multiple sclerosis (MS) requires additional molecular markers. Recently, we used original TCA-precipitation/extraction approach in combination with MALDI TOF/TOF mass-spectrometry and identified earlier unknown 48 kDa form of the unconventional myosin IC isoform b (Myo1C) in blood serum of the MS patients. Further examination of TCA-extracted fraction of blood serum of these patients by means of thin-layer chromatography and HPLC gel-filtration allowed detecting 300-500 Da peptides. MALDI TOF/TOF massspectrometry of these peptides showed that they contain Ser-Pro-Cys amino acid sequence. We discussed potential mechanisms of a release of these peptides that were earlier unknown in blood serum of the MS patients.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.2174/0929866523666160622215628DOI Listing
March 2017

Identification of a 48 kDa form of unconventional myosin 1c in blood serum of patients with autoimmune diseases.

Biochem Biophys Rep 2016 Mar 3;5:175-179. Epub 2015 Dec 3.

Institute of Cell Biology, National Academy of Sciences of Ukraine, Drahomanova Street 14/16, 79005 Lviv, Ukraine.

We searched for protein markers present in blood serum of multiple sclerosis (MS), rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) patients in comparison to healthy human individuals. We used precipitation/extraction methods and MALDI TOF/TOF mass spectrometry, and identified a protein with Mr ~46 kDa as a fragment of human unconventional myosin IC isoform b (Myo1C). Western blotting with specific anti-human Myo1C antibodies confirmed the identity. Screening of blood serum samples from different autoimmune patients for the presence of Myo1c revealed its high level in MS and RA patients, relatively low level in SLE patients, and undetected in healthy donors. These data are suggesting that the level of p46 Myo1C in blood serum is a potential marker for testing of autoimmune diseases.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.bbrep.2015.12.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5600340PMC
March 2016

Detection of novel auto-antigens in patients with recurrent miscarriage: description of an approach and preliminary findings.

Croat Med J 2014 Jun;55(3):259-64

Rostyslav Stoika, Department of Regulation of Cell Proliferation and Apoptosis, Institute of Cell Biology, National Academy of Science of Ukraine, Drahomanov Str. 14/16, 79005, Lviv, Ukraine,

Aim: To develop and test a protocol for isolation of potential auto-antigens from chorionic tissue that may be linked to recurrent miscarriage (RM).

Methods: The strategy included: 1) isolation of IgGs tightly bound to chorionic tissue of RM patients by protein G chromatography; 2) construction of affinity columns using the chorionic antibodies for isolation of auto-antigens; 3) enrichment of auto-antigens from detergent extracted solution of chorionic proteins by affinity chromatography; 4) separation by dodecyl sulfate-electrophoresis followed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry identification.

Results: Five potential auto-antigens were detected: neutral alpha-glucosidase AB, endoplasmin, transitional endoplasmic reticulum ATPase, putative endoplasmin-like protein, and cytoplasmic actin 2.

Conclusions: We developed a strategy for identification of auto-antigens in the chorionic tissue of women with RM, which could be of diagnostic and prognostic value.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3325/cmj.2014.55.259DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4049207PMC
June 2014

Macrophages discriminate glycosylation patterns of apoptotic cell-derived microparticles.

J Biol Chem 2012 Jan 10;287(1):496-503. Epub 2011 Nov 10.

Department of Internal Medicine-3, Institute for Clinical Immunology and Rheumatology, University of Erlangen-Nuremberg, 91054 Erlangen, Germany. Electronic address:

Inappropriate clearance of apoptotic remnants is considered to be the primary cause of systemic autoimmune diseases, like systemic lupus erythematosus. Here we demonstrate that apoptotic cells release distinct types of subcellular membranous particles (scMP) derived from the endoplasmic reticulum (ER) or the plasma membrane. Both types of scMP exhibit desialylated glycotopes resulting from surface exposure of immature ER-derived glycoproteins or from surface-borne sialidase activity, respectively. Sialidase activity is activated by caspase-dependent mechanisms during apoptosis. Cleavage of sialidase Neu1 by caspase 3 was shown to be directly involved in apoptosis-related increase of surface sialidase activity. ER-derived blebs possess immature mannosidic glycoepitopes and are prioritized by macrophages during clearance. Plasma membrane-derived blebs contain nuclear chromatin (DNA and histones) but not components of the nuclear envelope. Existence of two immunologically distinct types of apoptotic blebs may provide new insights into clearance-related diseases.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M111.273144DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3249103PMC
January 2012

Antibody-mediated sialidase activity in blood serum of patients with multiple myeloma.

J Mol Recognit 2011 Jul-Aug;24(4):576-84. Epub 2010 Nov 16.

Institute of Cell Biology, National Academy of Sciences of Ukraine, Lviv 79005, Ukraine.

Cell surface sialylation is known to be tightly connected with tumorigenicity, invasiveness, metastatic potential, clearance of aged cells, while the sialylation of IgG molecules determines their anti-inflammatory properties. Four sialidases - hydrolytic enzymes responsible for cleavage of sialic residues - were described in different cellular compartments. However, sialidases activity in body fluids, and specifically in blood serum, remains poorly studied. Here, we characterize first known IgG antibodies possessing sialidase-like activity in blood serum of multiple myeloma (MM) patients. Ig fractions were precipitated with ammonium sulfate (50% of saturation) from blood serum of 12 healthy donors and 14 MM patients, and screened for the presence of sialidase activity by using 4-MUNA (2'-(4-methylumbelliferyl)-α-D-N-acetylneuraminic acid) as substrate. High level of sialidase activity was detected in the MM patients, but not in healthy donors. Subsequent antibody purification by protein-G affinity chromatography and HPLC size exclusion chromatography at acidic conditions demonstrated that sialidase activity was attributable to IgG molecules. Sialidase activity was also specific for (Fab)(2) fragment of IgG and blocked by sialidase inhibitor DANA. Sialidase activity of IgG molecule was also confirmed by in gel assay for cleavage of sialidase substrate. Kinetic parameters of the catalysis reaction were described by Michaelis-Menten equation with K(m)  = 44.4-108 µM and k(cat) = 2.7-23.1 min(-1). The action of IgG possessing sialidase-like activity towards human red blood cells resulted in a subsequent increase in their agglutination by the peanut agglutinin, that confirms their desialylation by the studied IgG. This is the first demonstration of the intrinsic sialidase activity of IgG isolated from blood serum of MM patients.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/jmr.1071DOI Listing
September 2011

AMID: new insights on its intracellular localization and expression at apoptosis.

Apoptosis 2008 May;13(5):729-32

Department of Regulation of Cell Proliferation and Apoptosis, Institute of Cell Biology, National Academy of Sciences of Ukraine, Drahomanov Street 14/16, Lviv, 79005, Ukraine.

AMID (apoptosis-inducing factor (AIF)-like mitochondrion-associated inducer of death) is a poorly studied member of the AIF family; despite the given name AMID, predicting its association with mitochondria, its real cellular localization, as well as its role and changes during apoptosis are currently unclear. By means of MALDI-TOF mass spectrometry, we have identified as AMID (accession number AAH38129, sequence coverage 31%) the protein isolated by Pisum sativum lectin-affinity chromatography from the plasma membrane fraction of apoptotic murine leukemia L1210 cells, lacking in the intact cells. The obtained results suggest its possible glycosylation that was further suggested by finding N-glycosylation sequon in the signal peptide of AMID protein (in silica), and by predicting transmembrane localization of its N-terminal part. Using monoclonal antibodies to AMID, we demonstrated an increased expression of AMID in human leukemia Jurkat T-cells after apoptosis induction. Immunocytochemical study suggested its association to the plasma membrane.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s10495-008-0198-5DOI Listing
May 2008

In vivo expression and characteristics of novel alpha-D-mannose-rich glycoprotein markers of apoptotic cells.

Cell Biol Int 2005 Nov 20;29(11):920-8. Epub 2005 Oct 20.

Institute of Cell Biology, National Academy of Sciences of Ukraine, Drahomanov Street 14/16, Lviv 79005, Ukraine.

We recently established that an increased expression of alpha-D-mannose (Man)- and beta-D-galactose-rich plasma membrane glycoproteins (GPs) is characteristic for apoptotic cells in vitro [Bilyy, R.O., Stoika, R.S., 2003. Lectinocytochemical detection of apoptotic murine leukemia L1210 cells. Cytometry 56A, 89-95]. It was independent of cell line or apoptosis-inducing agent, and can therefore be considered as a selective marker for identification and isolation of apoptotic cells [Bilyy, R.O., Antonyuk, V.O., Stoika, R.S., 2004. Cytochemical study of role of alpha-D-mannose- and beta-D-galactose-containing glycoproteins in apoptosis. J. Mol. Histol. 35, 829-838]. The main goals of the present study were: (1) to determine whether an increased expression of specific GPs also takes place after apoptosis induction in vivo; and (2) to identify additional characteristics of the membrane GP markers of the apoptotic cells. To reach these goals, we studied the expression of alpha-Man-rich membrane GPs in murine leukemia L1210 cells inoculated into abdominal cavities of mice which were then subjected to the action of apoptosis inducer doxorubicin. Another experimental model used in the present work was splenocytes obtained from mice treated with dexamethasone. Lectin-affinity chromatography and PAGE electrophoresis, or PAGE electrophoresis and lectinoblot analysis were applied for isolation of plasma membrane GPs (34 kDa, and high M(W) of approximately 600 and 800 kDa) whose expressions were increased during apoptosis. Triton X-114 treatment of cell membrane samples showed that the apoptotic cell-specific GPs were localized in the peripheral and integral compartments of plasma membrane. Apoptosis in vitro and in vivo was accompanied by an increased expression of the same GP, identified by MALDI-TOF MS analysis as the microtubule-actin cross-linking factor 1. Other GPs, whose expressions were also increased at apoptosis, were similarly identified as G-protein beta-subunit like (Acc# BAA06185.1) and dystonin isoform beta.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.cellbi.2005.08.003DOI Listing
November 2005

Peroxisome division in the yeast Yarrowia lipolytica is regulated by a signal from inside the peroxisome.

J Cell Biol 2003 Sep 22;162(7):1255-66. Epub 2003 Sep 22.

Department of Biology, Concordia University, Montreal, Quebec H4B 1R6, Canada.

We describe an unusual mechanism for organelle division. In the yeast Yarrowia lipolytica, only mature peroxisomes contain the complete set of matrix proteins. These mature peroxisomes assemble from several immature peroxisomal vesicles in a multistep pathway. The stepwise import of distinct subsets of matrix proteins into different immature intermediates along the pathway causes the redistribution of a peroxisomal protein, acyl-CoA oxidase (Aox), from the matrix to the membrane. A significant redistribution of Aox occurs only in mature peroxisomes. Inside mature peroxisomes, the membrane-bound pool of Aox interacts with Pex16p, a membrane-associated protein that negatively regulates the division of early intermediates in the pathway. This interaction inhibits the negative action of Pex16p, thereby allowing mature peroxisomes to divide.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1083/jcb.200305055DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2173948PMC
September 2003

Phosphorylation of Minor Lipids of Human Milk Tightly Bound to Secretory Immunoglobulin A.

Russ J Immunol 2000 Oct;5(3):267-278

Institute of Bioorganic Chemistry, Siberian Branch of Russian Academy of Sciences, Novosibirsk, Russia.

Catalytic antibodies or abzymes possessing the different catalytic activities were detected in the sera of patients with various autoimmune pathologies, where their presence is most probably associated with autoimmunization. Normal humans are generally considered to have no abzymes, since no obvious immunizing factors are present. The ability of small fraction of sIgA from human milk to phosphorylate selectively casein in the presence of gamma(32)P-ATP was previously shown to be a property of the Abs. Here we revealed for the first time that the same small fraction of sIgA contains Ab subfraction, which is tightly bound to unusual minor lipids of human milk. The lipids may be phosphorylated in the presence of gamma(32)P-ATP and remain partially bound to Abs after purification of polyclonal sIgA by several sequential chromatographies. These lipids may be effectively separated from sIgA only by gel filtration in a buffer containing 5% dioxane or by extraction of the sIgA solutions with chloroform-methanol mixture. It has been shown that two minor milk phospholipids, similarly to the previously described GM1, GM3 and GD3 gangliosides contain residue of sialic acid, but in conrast to the latter lipids, they can not be oxidized with sodium periodate; alkaline methanolysis of them results in formation of 4 and 5 products respectively, while hydrolysis of the gangliosides gives only one or two products. All the data obtained give an evidence in favor of that the minor lipids tightly bound to sIgA may be considered as new lipids of human milk. It was suggested that phosphorylation of the lipids is catalyzed by sIgA antibodies.
View Article and Find Full Text PDF

Download full-text PDF

Source
October 2000
-->