| 1931. |
- Nicolaes, Gerry A. F., et al.
(författare)
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Inhibition of Thrombin Formation by Active Site Mutated (S360A) Activated Protein C
- 2010
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Ingår i: Journal of Biological Chemistry. - 1083-351X. ; 285:30, s. 22888-22898
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Tidskriftsartikel (refereegranskat)abstract
- Activated protein C (APC) down-regulates thrombin formation through proteolytic inactivation of factor Va (FVa) by cleavage at Arg(506) and Arg(306) and of factor VIIIa (FVIIIa) by cleavage at Arg(336) and Arg(562). To study substrate recognition by APC, active site-mutated APC (APC(S360A)) was used, which lacks proteolytic activity but exhibits anticoagulant activity. Experiments in model systems and in plasma show that APC(S360A), and not its zymogen protein C(S360A), expresses anticoagulant activities by competing with activated coagulation factors X and IX for binding to FVa and FVIIIa, respectively. APC(S360A) bound to FVa with a K-D of 0.11 +/- 0.05 nM and competed with active site-labeled Oregon Green activated coagulation factor X for binding to FVa. The binding of APC(S360A) to FVa was not affected by protein S but was inhibited by prothrombin. APC(S360A) binding to FVa was critically dependent upon the presence of Arg(506) and not Arg(306) and additionally required an active site accessible to substrates. Inhibition of FVIIIa activity by APC(S360A) was > 100-fold less efficient than inhibition of FVa. Our results show that despite exosite interactions near the Arg(506) cleavage site, binding of APC(S360A) to FVa is almost completely dependent on Arg(506) interacting with APC(S360A) to form a nonproductive Michaelis complex. Because docking of APC to FVa and FVIIIa constitutes the first step in the inactivation of the cofactors, we hypothesize that the observed anticoagulant activity may be important for in vivo regulation of thrombin formation.
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| 1932. |
- Nicolaes, GAF, et al.
(författare)
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Altered inactivation pathway of factor Va by activated protein C in the presence of heparin
- 2004
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Ingår i: European Journal of Biochemistry. - : Wiley. - 0014-2956 .- 1432-1033. ; 271:13, s. 2724-2736
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Tidskriftsartikel (refereegranskat)abstract
- Inactivation of factor Va (FVa) by activated protein C (APC) is a predominant mechanism in the down-regulation of thrombin generation. In normal FVa, APC-mediated inactivation occurs after cleavage at Arg306 (with corresponding rate constant k'(306)) or after cleavage at Arg506 (k(506)) and subsequent cleavage at Arg306 (k(306)). We have studied the influence of heparin on APC-catalyzed FVa inactivation by kinetic analysis of the time courses of inactivation. Peptide bond cleavage was identified by Western blotting using FV-specific antibodies. In normal FVa, unfractionated heparin (UFH) was found to inhibit cleavage at Arg506 in a dose-dependent manner. Maximal inhibition of k(506) by UFH was 12-fold, with the secondary cleavage at Arg306 (k(306)) being virtually unaffected. In contrast, UFH stimulated the initial cleavage at Arg306 (k'(306)) two- to threefold. Low molecular weight heparin (Fragmin(R)) had the same effects on the rate constants of FVa inactivation as UFH, but pentasaccharide did not inhibit FVa inactivation. Analysis of these data in the context of the 3D structures of APC and FVa and of simulated APC-heparin and FVa-APC complexes suggests that the heparin-binding loops 37 and 70 in APC complement electronegative areas surrounding the Arg506 site, with additional contributions from APC loop 148. Fewer contacts are observed between APC and the region around the Arg306 site in FVa. The modeling and experimental data suggest that heparin, when bound to APC, prevents optimal docking of APC at Arg506 and promotes association between FVa and APC at position Arg306.
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| 1933. |
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| 1934. |
- Nielsen, Lars Bo
(författare)
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Lipoprotein production by the heart: a novel pathway of triglyceride export from cardiomyocytes
- 2002
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Ingår i: Scandinavian Journal of Clinical & Laboratory Investigation. - : Informa UK Limited. - 1502-7686 .- 0036-5513. ; 62:7, s. 35-40
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Tidskriftsartikel (refereegranskat)abstract
- The current knowledge on lipoprotein secretion from the heart is examined in this article. The ability of cells to secrete apolipoprotein B (apo-B) containing lipoproteins depends on expression of the apo-B and microsomal triglyceride transfer protein (MTP) genes. Initially, it was shown that both genes are expressed in cardiac myocytes of mice and humans. Electron microscopy of human cardiac myocytes revealed lipoproteins in the secretory pathway and metabolic labelling studies demonstrated the secretion of LDL-like lipoproteins from minced heart biopsies. To examine the role of lipoprotein formation in the heart, we tested whether overexpression of a human apo-B transgene in the heart affects cardiac triglyceride accumulation. In wild-type mice, diabetes conferred an increase in heart triglycerides. In apo-B transgenic mice, diabetes did not affect heart triglycerides. Also, apo-B overexpression prevents fasting-induced heart triglyceride accumulation, whereas inhibition of MTP expression increases heart triglycerides in mice. In hypoxic human hearts, MTP mRNA expression was negatively associated with triglyceride contents. These findings suggest that lipoprotein formation rates affect cardiac triglyceride stores. The MTP mRNA levels are similar to 2-fold higher in hypoxic compared with normoxic human myocardium and in diabetic compared with non-diabetic mouse hearts. In both hypoxia and diabetes, the delivery of triglycerides to the heart exceeds their utilization for beta-oxidation. Thus, endogenous lipoprotein secretion rates might be upregulated to remove surplus fat from the heart. Diabetes negatively affected indexes of systolic and diastolic function in wild-type mice. However, the diabetogenic effects on the heart were absent or much less pronounced in apo-B transgenic mice. This suggests that accelerated lipoprotein formation by the heart attenuates development of diabetic cardiomyopathy in mice. In conclusion, current evidence suggests that lipoprotein secretion from the heart plays an integrated role in cardiac lipid homeostasis and that it can affect the biomechanical function of the heart.
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| 1935. |
- Niemela, Tuomas Aarni Akseli, et al.
(författare)
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Mechanistic Insights into the Activation of Lecithin-Cholesterol Acyltransferase in Therapeutic Nanodiscs Composed of Apolipoprotein A-I Mimetic Peptides and Phospholipids
- 2023
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Ingår i: European Biophysics Journal. - 1432-1017 .- 0175-7571. ; 52:SUPPL 1, s. S98-S98
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Konferensbidrag (övrigt vetenskapligt/konstnärligt)abstract
- Lecithin-cholesterol acyltransferase (LCAT) maturates high density lipoprotein (HDL) particles enabling reverse cholesterol transport (RCT). Its primary cofactor on HDL is apolipoprotein A1 (apoA1). In order to boost RCT efficiency to combat atherosclerotic cardiovascular diseases, apoA1 mimetic peptides have been developed, such as 22 amino acid long 22A. Despite being ~10 % the size of apoA1, when 22A is formulated to a synthetic HDL particle, called a nanodisc, it is able to activate LCAT as well as apoA1 based HDL. However, the mechanism of how 22A, or apoA1, activates LCAT is not known. We set out to explain this mechanism with a combination of coarse-grained molecular dynamics simulations, LCAT activity assays and electron microscopy (EM). The computational data matched the experimental data in two ways. First, EM imaging showed that LCAT binds to the perimeter of 22A based nanodiscs, which was replicated by the simulations. The overall binding pose was also consistent. Second, by simulating and assaying a set of 22A mutants, we were able to see a correlation between the in silico binding kinetics of a peptide to aspecific site in LCAT and how well the peptide activates LCAT in vitro. Taken together, we’ve shown that these simulated systems can be exploited for designing peptides with increased efficacy. A more detailed description is found in our similarly titled article in Mol. Pharmaceutics 19: 4135 -4148, 2022.
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| 1936. |
- Nilson, B H, et al.
(författare)
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Protein L from Peptostreptococcus magnus binds to the kappa light chain variable domain
- 1992
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Ingår i: Journal of Biological Chemistry. - 0021-9258. ; 267:4, s. 9-2234
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Tidskriftsartikel (refereegranskat)abstract
- Protein L is an immunoglobulin light chain-binding protein expressed by some strains of the anaerobic bacterial species Peptostreptococcus magnus. The major variable region subgroups of human kappa and lambda light chains were tested for protein L binding; V kappa I, V kappa III, and V kappa IV bound protein L, whereas no binding occurred with proteins of the V kappa II subgroup or with any lambda light chain subgroups. Studies of the protein L binding capacity of naturally occurring VL fragments, and VL- and CL-related trypsin- and pepsin-derived peptides prepared from a kappa I light chain, localized the site of interaction to the VL domain. The affinity constant for the binding to an isolated V kappa I fragment was comparable to that for the native protein (Ka 0.9 x 10(9) M-1 and Ka 1.5 x 10(9) M-1, respectively). No binding occurred with CL-related fragments. Extensive reduction and alkylation of the V kappa fragment or the native kappa chain resulted in complete loss of protein L binding. Although it is possible, from comparative amino acid sequence data, to identify certain VL-framework region residues that account for the selective binding of protein L by kappa I, kappa III, and kappa IV proteins, our studies indicate that this interaction is essentially dependent upon the tertiary structural integrity of the kappa chain VL domain.
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| 1937. |
- Nilsson, Ola, 1970-
(författare)
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Behandling av akondroplasi är på väg – kan ge nya möjligheter : [New treatments for achondroplasia may be efficacious in other forms of short stature]
- 2021
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Ingår i: Läkartidningen. - : Läkartidningen Förlag AB. - 0023-7205 .- 1652-7518. ; 118
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Tidskriftsartikel (refereegranskat)abstract
- Achondroplasia is the most common form of monogenic skeletal dysplasia and is caused by activating mutations in fibroblast growth factor receptor 3 (FGFR3) resulting in exaggerated MAPK signaling in the growth cartilage chondrocytes and thus reduced growth. C-type natriuretic peptide (CNP) stimulates growth by inhibiting MAPK signaling in growth plate cartilage. Several new treatments that target these pathways are currently in clinical trials for the treatment of achondroplasia. A recent phase 3 clinical trial confirmed near normal growth rate in children with achondroplasia during treatment with vosoritide, a CNP analogue with a prolonged half-life. Vosoritide is currently being evaluated for approval by the EMA. Treatments that enable normalization of growth in children with achondroplasia may soon be available. It is likely that some of these treatments also will increase growth in other forms of short stature.
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| 1938. |
- Nilsson, Sara, et al.
(författare)
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Analysis of binding sites on complement factor I that are required for its activity.
- 2010
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Ingår i: Journal of Biological Chemistry. - 1083-351X. ; 285, s. 6235-6245
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Tidskriftsartikel (refereegranskat)abstract
- The central complement inhibitor factor I (FI) degrades activated complement factors C4b and C3b in the presence of cofactors such as C4b-binding protein, factor H, complement receptor 1 and membrane cofactor protein. FI is a serine protease composed of two chains; the light chain comprises the serine protease domain, while the heavy chain contains several domains: the FI and membrane attack complex domain (FIMAC), CD5, low density lipoprotein receptor 1 (LDLr1) and LDLr2 domains. In order to understand better how FI acts as a complement inhibitor, we used homology-based models of FI domains to predict potential binding sites. Specific amino acids were then mutated to yield 16 well-expressed mutants, which were then purified from media of eukaryotic cells for functional analyses. The Michaelis constant (Km) of all FI mutants towards a small substrate was not altered while some mutants showed increased maximum initial velocity (Vmax). All the mutations in the FIMAC domain affected the ability of FI to degrade C4b and C3b irrespective of the cofactor used whereas only some mutations in the CD5 and LDLr1/2 domains had similar effect. These same mutants also showed impaired binding to C3met. In conclusion, the FIMAC domain appears to harbor the main binding sites important for the ability of FI to degrade C4b and C3b.
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| 1939. |
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| 1940. |
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