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Arefi'eva TI Mukhina SA Poliakov AA Stepanova VV Minashkin MM Gurskiĭ IaG Domogatskiĭ SP Krasnikova TL 《Rossi?skii fiziologicheski? zhurnal imeni I.M. Sechenova / Rossi?skaia akademiia nauk》1998,84(12):1432-1437
Urokinase activates adhesion of monocytic U937 cells to fibrinogen-coated surface. This effect is due to urokinase proteolytic activity and does not depend on the urokinase binding to its receptor. 相似文献
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Kapustin A Stepanova V Aniol N Cines DB Poliakov A Yarovoi S Lebedeva T Wait R Ryzhakov G Parfyonova Y Gursky Y Yanagisawa H Minashkin M Beabealashvilli R Vorotnikov A Bobik A Tkachuk V 《The Biochemical journal》2012,443(2):491-503
uPA (urokinase-type plasminogen activator) stimulates cell migration through multiple pathways, including formation of plasmin and extracellular metalloproteinases, and binding to the uPAR (uPA receptor; also known as CD87), integrins and LRP1 (low-density lipoprotein receptor-related protein 1) which activate intracellular signalling pathways. In the present paper we report that uPA-mediated cell migration requires an interaction with fibulin-5. uPA stimulates migration of wild-type MEFs (mouse embryonic fibroblasts) (Fbln5+/+ MEFs), but has no effect on fibulin-5-deficient (Fbln5-/-) MEFs. Migration of MEFs in response to uPA requires an interaction of fibulin-5 with integrins, as MEFs expressing a mutant fibulin-5 incapable of binding integrins (Fbln(RGE/RGE) MEFs) do not migrate in response to uPA. Moreover, a blocking anti-(human β1-integrin) antibody inhibited the migration of PASMCs (pulmonary arterial smooth muscle cells) in response to uPA. Binding of uPA to fibulin-5 generates plasmin, which excises the integrin-binding N-terminal cbEGF (Ca2+-binding epidermal growth factor)-like domain, leading to loss of β1-integrin binding. We suggest that uPA promotes cell migration by binding to fibulin-5, initiating its cleavage by plasmin, which leads to its dissociation from β1-integrin and thereby unblocks the capacity of integrin to facilitate cell motility. 相似文献
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Ya. G. Gurskii M. M. Minashkin E. S. Feoktistova A. V. Skamrov N. A. Skrypina R. Sh. Bibilashvili 《Applied Biochemistry and Microbiology》2010,46(8):776-780
A recombinant plasmid carrying a modified gene of human plasminogen (mini-plasminogen), lacking four kringle domains and an
amino terminal fragment, and containing an additional oligopeptide of six N-terminal histidine residues has been constructed.
The plasmid was used for transformation of E. coli JM 109 cells to obtain a strain producing a recombinant modified human plasminogen. The target protein is superexpressed
in a form of inclusion bodies and is composed of more than 50% insoluble protein. The renaturated and chromatographically
purified protein exhibits amidolytic activity specific for plasminogen proenzyme in a fibrinolytic system. 相似文献
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A A Poliakov S A Mukhina D O Traktouev R S Bibilashvily Y G Gursky M M Minashkin V V Stepanova V A Tkachuk 《Journal of receptor and signal transduction research》1999,19(6):939-951
Urokinase type plasminogen activator (uPA) converts plasminogen to plasmin and is highly chemotactic for many cell types. We examined, using recombinant wild type and mutated forms of uPA, the extent to which its proteolytic properties, its growth-like domain (GFD) and/or interactions with the specific receptor (uPAR) contribute to the chemotactic activity towards vascular smooth muscle cells (SMC). Recombinant wild type uPA (r-uPA) stimulated cell migration nearly 5.8-fold, inactive r-uPA, with a mutation in the catalitic domain (r-uPA(H/Q)), 3-fold, uPA without growth factor like domain (r-uPA(GFD )), 2.6-fold, and a form containing both mutations (r-uPA(H/Q, GFD ), 3.3-fold. All recombinant forms of uPA, wild type and those with mutations were equally and highly effective (IC50 approximately 20 nM) in displacing 125I-r-uPA bound to SMC. These results indicate that additional mechanisms, not dependent on uPA's proteolytic activity or the binding ability of its GFD to uPAR, are the major contributors to its chemotactic action on SMC. 相似文献
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Mukhina S Stepanova V Traktouev D Poliakov A Beabealashvilly R Gursky Y Minashkin M Shevelev A Tkachuk V 《The Journal of biological chemistry》2000,275(22):16450-16458
Urokinase plasminogen activator (uPA) is thought to exert its effects on cell growth, adhesion, and migration by mechanisms involving proteolysis and interaction with its cell surface receptor (uPAR). The functional properties of uPA and the significance of its various domains for chemotactic activity were analyzed using human airway smooth muscle cells (hAWSMC). The wild-type uPA (r-uPAwt), inactive urokinase with single mutation (His(204) to Gln) (r-uPA(H/Q)), urokinase with mutation of His(204) to Gln together with a deletion of growth factor-like domain (r-uPA(H/Q)-GFD), the catalytic domain of urokinase (r-uPA(LMW)), and its kringle domain (r-KD) were expressed in Escherichia coli. We demonstrate that glycosylated uPA, r-uPAwt, r-uPA(H/Q), and r-uPA(H/Q)-GFD elicited similar chemotactic effects. Half-maximal chemotaxis (EC(50)) were apparent at approximately 2 nm with all the uPA variants. The kringle domain induced cell migration with an EC(50) of about 6 nm, whereas the denaturated r-KD and r-uPA(LMW) were without effect. R-uPAwt-induced chemotaxis was dependent on an association with uPAR and a uPA-kringle domain-binding site, determined using a monoclonal uPAR antibody to prevent the uPA-uPAR interaction, and a monoclonal antibody to the uPA-kringle domain. The binding of iodinated r-uPAwt with hAWSMC was due to interaction with a high affinity binding site on the uPAR, and a lower affinity binding site on an unidentified cell surface target, which was mediated exclusively through the kringle domain of urokinase. Specific binding of r-uPA(H/Q)-GFD to hAWSMC involved an interaction with a single site whose characteristics were similar to those of the low affinity site of r-uPAwt binding to hAWSMC. uPAR-deficient HEK 293 cells specifically bound r-uPAwt and r-uPA(H/Q)-GFD via a single, similar type of binding site. These cells migrated when stimulated by r-uPA(H/Q)-GFD and uPAwt, but not r-uPA(LMW). HEK 293 cells transfected with the uPAR cDNA expressed two classes of sites that bound r-uPAwt; however, only a single site was responsible for the binding of r-uPA(H/Q)-GFD. Together, these findings indicate that uPA-induced chemotaxis is dependent on the binding of the uPA-kringle to the membrane surface of cells and the association of uPA with uPAR. 相似文献
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Yaroslav Gursky Robert Bibilashvili Mikchail Minashkin Alex Krasnov Alex Deikin Tatyana Ermolkevich Andrey Popov Lilia Verbovaya Nicolai Rutkevich Alexsander Shevelev Sofia Georgieva Sergey V. Razin Igor Goldman Elena Sadchikova 《Transgenic research》2009,18(5):747-756
Human pro-urokinase expressed in the mammary glands of transgenic animals is quickly activated and converted to urokinase
by proteases that are present in the milk. Thus, it is nearly impossible to isolate full-sized pro-urokinase from the milk
of transgenic animals. To solve this problem, we constructed transgenic mice that express human pro-urokinase and modified
ecotin, which is a potent serine protease inhibitor from E. coli, in their mammary glands. The gene encoding ecotin was modified so as to enhance its specificity for the human urokinase-type
plasminogen activator. Co-expression of modified ecotin and human pro-urokinase in the mammary glands allows for purification
of full-length human pro-urokinase from these transgenic mice. The results described here suggest a general way of preventing
the activation of zymogens that are expressed in the mammary glands of transgenic animals by co-expression of a zymogen along
with a protease inhibitor. 相似文献
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