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| 2. |
- Zhu, Changlian, 1964, et al.
(författare)
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Cyclophilin A participates in the nuclear translocation of apoptosis-inducing factor in neurons after cerebral hypoxia-ischemia
- 2007
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Ingår i: J Exp Med. - : Rockefeller University Press. - 0022-1007. ; 204:8, s. 1741-8
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Tidskriftsartikel (refereegranskat)abstract
- Upon cerebral hypoxia-ischemia (HI), apoptosis-inducing factor (AIF) can move from mitochondria to nuclei, participate in chromatinolysis, and contribute to the execution of cell death. Previous work (Cande, C., N. Vahsen, I. Kouranti, E. Schmitt, E. Daugas, C. Spahr, J. Luban, R.T. Kroemer, F. Giordanetto, C. Garrido, et al. 2004. Oncogene. 23:1514-1521) performed in vitro suggests that AIF must interact with cyclophilin A (CypA) to form a proapoptotic DNA degradation complex. We addressed the question as to whether elimination of CypA may afford neuroprotection in vivo. 9-d-old wild-type (WT), CypA(+/-), or CypA(-/-) mice were subjected to unilateral cerebral HI. The infarct volume after HI was reduced by 47% (P = 0.0089) in CypA(-/-) mice compared with their WT littermates. Importantly, CypA(-/-) neurons failed to manifest the HI-induced nuclear translocation of AIF that was observed in WT neurons. Conversely, CypA accumulated within the nuclei of damaged neurons after HI, and this nuclear translocation of CypA was suppressed in AIF-deficient harlequin mice. Immunoprecipitation of AIF revealed coprecipitation of CypA, but only in injured, ischemic tissue. Surface plasmon resonance revealed direct molecular interactions between recombinant AIF and CypA. These data indicate that the lethal translocation of AIF to the nucleus requires interaction with CypA, suggesting a model in which two proteins that normally reside in separate cytoplasmic compartments acquire novel properties when moving together to the nucleus.
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| 3. |
- Björquist, P, et al.
(författare)
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Plasminogen activator inhibitor type-1 interacts exclusively with the proteinase domain of tissue plasminogen activator.
- 1994
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Ingår i: Biochimica et Biophysica Acta. - 0006-3002 .- 1878-2434. ; 1209:2, s. 191-202
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Tidskriftsartikel (refereegranskat)abstract
- Two different techniques have been used to study the complex formation of recombinant human plasminogen activator inhibitor type-1, PAI-1, with either recombinant human two-chain tissue plasminogen activator, tc tPA (EC 3.4.21.68), or the tPA deletion variants tc K2P, containing the kringle 2 domain and the proteinase domain, and P, containing only the proteinase domain. The same value for Kon, 2.10(7) M-1s-1 for binding of PAI-1 was found for the three tPA forms by direct detection of the complex formation in real time by surface plasmon resonance, BIAcore, or indirectly by monitoring the time course of the inhibition of tPA using the chromogenic substrate N-methylsulfonyl-D-Phe-Gly-Arg-4-pNA-acetate. Apparently, no conformational change is involved in the rate-limiting step, since the kon value was found to be independent of the temperature from 20 to 35 degrees C. By the BIAcore technique, it was found that the complex between PAI-1 and tPA covalently coupled to the surface, was stable at 25 degrees C, since no dissociation was seen in buffer. However, extended treatment with 1 M NH4OH destroyed the complex with t 1/2 = 5 h. The same kon values and complex composition were found by measuring either the binding of tPA to PAI-1 captured on the monoclonal antibody MAI-11 or the binding of PAI-1 to tPA captured on the monoclonal antibody 2:2 B10. Quantification of the complex composition between PAI-1 captured on the monoclonal antibody MAI-11 with either tPA, K2P or P gave a one-to-one ratio with the fraction of active PAI-1, consistent with the results from SDS-PAGE and the specific activity of PAI-1. The complexes of the three tPA forms with PAI-1 captured on a large surface of MAI-11 dissociated more rapidly from MAI-11, with the same apparent koff, kdis, = 2.10(-3) s-1, compared with 0.7-10(-3) s-1 for the dissociation of PAI-1 alone. In consistance, the Kd, calculated from the direct determination of the kon and koff for the association of different form of PAI-1 to a small surface of MAI-11, was found to be higher for PAI-1 in complex with tPA than for free active PAI-1. Apparently, upon complex formation, a change is induced in PAI-1 at the binding epitope for MAI-11.(ABSTRACT TRUNCATED AT 400 WORDS)
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| 5. |
- Lincoln, Per, 1958, et al.
(författare)
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CONFORMATION OF THIOCOLCHICINE AND 2 B-RING-MODIFIED ANALOGS BOUND TO TUBULIN STUDIED WITH OPTICAL SPECTROSCOPY
- 1991
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 1520-4995 .- 0006-2960. ; 30:5, s. 1179-1187
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Tidskriftsartikel (refereegranskat)abstract
- The interaction of tubulin with thiocolchicine and two thiocolchicine analogues, one lacking the B ring and one with a six-membered B ring, has been studied by using near-UV and CD spectroscopies. Rapid, reversible binding of the latter analogue to tubulin demonstrates the ability of the colchicine binding site to accommodate the phenyltropone system with a more coplanar conformation than is present in free colchicine. There is no evidence, however, that bound thiocolchicine should have a much less twisted conformation than free thiocolchicine. Thiocolchicine and the bicyclic analogue appear to have approximately the same conformation of the phenyltropone system, in both the free and the bound states, suggesting that this conformation has an optimal arrangement of the phenyl and tropone rings for binding to tubulin. In contrast to colchicine and related derivatives, the three thiocolchicine analogues show pronounced near-UV CD bands upon association to tubulin. No simple relation could be found between the sign pattern of the CD components in the near-UV band of the thiocolchicinoid chromophore and its axial chirality.
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| 6. |
- Rylander, A. C. J., et al.
(författare)
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Fibrinolysis inhibitors in plaque stability: a morphological association of PAI-1 and TAFI in advanced carotid plaque
- 2017
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Ingår i: Journal of Thrombosis and Haemostasis. - : Elsevier BV. - 1538-7933 .- 1538-7836. ; 15:4, s. 758-769
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Tidskriftsartikel (refereegranskat)abstract
- Background: Fibrinolysis plays an important role in destabilization of atherosclerotic plaques and is tightly regulated by specific inhibitors. Objective: The fibrinolysis inhibitors plasminogen activator inhibitor type-1 (PAI-1) and thrombin-activatable fibrinolysis inhibitor (TAFI) were quantified and described in the morphological context of advanced carotid plaques American Heart Association VI-VIII to elucidate their role in plaque stability. Methods: Immunohistochemistry in serial sections along the longitudinal axis of endarterectomies from patients with symptomatic carotid stenosis (n = 19) were studied using an antibody specific for free PAI-1 (I205), an antibody with high affinity for TAFI/TAFIa (CP17) and established antibodies for smooth muscle cells (alpha-actin), endothelial cells (von Willebrand factor [VWF]), macrophages (CD68) and platelets (CD42). Results: PAI-1 and TAFI show a specific distribution in these advanced plaques with a maximum corresponding to the internal carotid artery (ICA). Free PAI-1 was mainly detected in macrophages and in intravascular thrombi, and TAFI in endothelial cells (ECs) but also macrophages. The one-way ANOVA analysis with Bonferroni's correction showed a significant increase of macrophages and ECs, TAFI and PAI-1 in areas with high neovascularization in endarterectomy sections corresponding to ICA. High Spearman factors for TAFI, PAI-1 and VWF indicate neovascularization as the main source of plasma proteins, transported by platelets into the atheroma (PAI-1) or expressed by ECs (TAFI). CD68 was highly associated with VWF, PAI-1 and especially TAFI, underlining the role of macrophages in fibrinolytic activity and inflammation. Conclusion: The abundance of free PAI-1 and TAFI in the plaque may inhibit plasmin generation and thereby counteract plaque destabilization by fibrinolysis, cell migration and inflammation.
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| 7. |
- Brogren, Helén, 1977, et al.
(författare)
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Platelets retain high levels of active plasminogen activator inhibitor 1
- 2011
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Ingår i: PloS one. - : Public Library of Science (PLoS). - 1932-6203. ; 6:11
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Tidskriftsartikel (refereegranskat)abstract
- The vascular fibrinolytic system is crucial for spontaneous lysis of blood clots. Plasminogen activator inhibitor 1 (PAI-1), the principal inhibitor of the key fibrinolytic enzyme tissue-type plasminogen activator (tPA), is present in platelets at high concentrations. However, the majority of PAI-1 stored in platelets has been considered to be inactive. Our recent finding (Brogren H, et al. Blood 2004) that PAI-1 de novo synthesized in platelets remained active for over 24 h, suggested that PAI-1 stored in the alpha-granules might be active to a larger extent than previously reported. To re-evaluate this issue, we performed experiments where the fraction of active PAI-1 was estimated by analyzing the tPA-PAI-1 complex formation. In these experiments platelets were lysed with Triton X-100 in the presence of serial dilutions of tPA and subsequently the tPA-PAI-1 complex was evaluated by Western blot. Also, using a non-immunologic assay, tPA was labeled with (125)I, and (125)I-tPA and (125)I-tPA-PAI-1 was quantified by scintigraphy. Interestingly, both methods demonstrated that the majority (>50%) of platelet PAI-1 is active. Further analyses suggested that pre-analytical procedures used in previous studies (sonication or freezing/thawing) may have substantially reduced the activity of platelet PAI-1, which has lead to an underestimation of the proportion of active PAI-1. Our in vitro results are more compatible with the role of PAI-1 in clot stabilization as demonstrated in physiological and pathophysiological studies.
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| 8. |
- Fridén, B, et al.
(författare)
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The effect of estramustine derivatives on microtubule assembly in vitro depends on the charge of the substituent.
- 1991
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Ingår i: Biochemical pharmacology. - 0006-2952. ; 42:5, s. 997-1006
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Tidskriftsartikel (refereegranskat)abstract
- Estramustine, and derivatives of estramustine with a charged substituent at position 17 on the estrogen moiety, have been investigated for their effects on bovine brain microtubules in vitro. The negatively charged estramustine phosphate has been found previously to be a microtubule-associated protein (MAP)-dependent microtubule inhibitor [Wallin M, Deinum J and Fridén B, FEBS Lett 179: 289-293, 1985]. In the present study the binding of estramustine phosphate to MAP2 and tau was investigated. Both these MAPs were found to have two to three binding sites for estramustine phosphate which is compatible with the reported number of basic amino acid repeats of these MAPs, considered to be the ultimate tubulin binding domains. The Kd for the binding of estramustine phosphate to MAP2 was estimated to be 20 microM at 4 degrees, and for the binding of tau, 200 microM. The rate of dissociation was very low (T1/2 greater than 2 hr), which indicates that the binding of estramustine phosphate may stabilize the protein-drug complex by changing the protein conformation. Two new negatively charged estramustine derivatives, estramustine sulphate and estramustine glucuronide, were found to be similar MAP-dependent microtubule inhibitors. The concentration for 50% inhibition of assembly was 100 microM for the sulphate derivative, the same as found previously for estramustine phosphate, and 250 microM for the more bulky estramustine glucuronide. A positively charged derivative, estramustine sarcosinate, did not inhibit microtubule assembly or alter the composition of the coassembled MAPs. The morphology of the microtubules was, however, affected. The uncharged estramustine bound to both tubulin and MAPs, but no effects were seen on microtubule assembly, the composition of coassembled MAPs or the microtubule morphology. Our results suggest that only negatively charged estramustine derivatives have a MAP-dependent microtubule inhibitory effect. The two new negatively charged derivatives could therefore be valuable tools in the study of tubulin-MAP interactions. The results also confirm that these interactions between tubulin and MAPs are mainly electrostatic.
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| 9. |
- Nilebäck, Erik, 1984, et al.
(författare)
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Viscoelastic Sensing of Conformational Changes in Plasminogen Induced upon Binding of Low Molecular Weight Compounds
- 2010
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Ingår i: Analytical Chemistry. - : American Chemical Society (ACS). - 0003-2700 .- 1520-6882. ; 82:20, s. 8374-8376
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Tidskriftsartikel (refereegranskat)abstract
- Plasminogen is a precursor to the fibrinolytic enzyme plasmin and is known to undergo large conformational changes when subjected to low molecular lysine analogues such as tranexamic acid (TA) or epsilon-amino-n-caproic acid (EACA). Here, we demonstrate how well-controlled surface immobilization of biotinylated plasminogen allows for monitoring of the interaction between TA and EACA with plasminogen. The interaction was studied by the quartz crystal microbalance with dissipation monitoring (QCM-D) technique as well as by surface plasmon resonance (SPR) based sensing. QCM-D measures changes in acoustically coupled mass (by detection of changes in the resonance frequency of the crystal, Delta f) and is sensitive to changes in mass adsorbed on the sensor surface including how liquid medium is associated with this material. Through the dissipation factor (i.e., changes in the energy dissipation of the crystal oscillation, Delta D), QCM-D is also sensitive to the viscoelastic properties of material adsorbed to the sensor surface. Upon binding of TA or EACA, changes in the plasminogen structure were recorded as distinct, although small, Delta D responses which were used to determine affinity constants. By comparing native and truncated plasminogen, we conclude that the observed dissipation shifts were caused by conformational changes in the proteins leading to changes in the viscoelastic properties of the protein layer on the surface. These results demonstrate a novel application of the QCM-D technique, paving the way for a whole new approach to screening of this target for novel lead structures.
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| 10. |
- Nilsson, T, et al.
(författare)
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The mechanism of binding of low-molecular-weight active site inhibitors to human alpha-thrombin.
- 1998
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Ingår i: Journal of enzyme inhibition. - 8755-5093. ; 13:1, s. 11-29
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Tidskriftsartikel (refereegranskat)abstract
- The thrombin inhibitors argatroban, efegatran, NAPAP, CH 1091, CH 248, inogatran and melagatran have been characterised with respect to their mechanism of binding to human alpha-thrombin. Stopped-flow spectrophotometry was used to follow thrombin-catalysed hydrolysis of the chromogenic substrate S-2238 in the presence of inhibitors. The rate of onset or decay of inhibition was evaluated using progress curve analysis. It was possible to obtain apparent association and dissociation rate constants from the dependence of the rates on the inhibitor concentrations. Inhibition constants calculated from the association and dissociation rate constants were in good agreement with those calculated from steady-state rates. The binding of 6 inhibitors was also monitored directly using stopped-flow spectrofluorimetry when two kinetic components were found with all inhibitors. The faster component accounted for the largest part of the change in the intrinsic fluorescence of thrombin induced by inhibitor binding and was dependent on the inhibitor concentration. The slower component was independent of the concentration of the inhibitor. The concentration dependence of the faster component was linear with the compounds argatroban, NAPAP, CH 1091 and melagatran and hyperbolic with the compounds CH 248 and inogatran. The values of the apparent second-order rate constants at pH 7.4 and 37 degrees C range from slow to rapid binding in the interval 16-78 x 10(6) M-1 s-1, which is somewhat higher than 1-34 x 10(6)M-1 s-1 obtained from progress curve analysis of the onset of inhibition. The present results support a mechanism that includes rearrangement of a weak initial thrombin-inhibitor complex towards a tighter complex. Moreover, at least one additional step is required in the mechanism. In this model, the rate-limiting step for the binding of the inhibitor at concentrations in the nanomolar range depends on the primary interaction between the inhibitor and native thrombin.
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