1. |
- Jonsson, Magnus, et al.
(författare)
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Semenogelins I and II bind zinc and regulate the activity of prostate-specific antigen
- 2005
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Ingår i: Biochemical Journal. - 0264-6021. ; 387:Part 2, s. 447-453
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Tidskriftsartikel (refereegranskat)abstract
- In semen. the gel proteins SgI and SgII (semenogelins I and II) are digested by PSA (prostate-specific antigen), resulting in liquefaction and release of motile spermatozoa. Semen contains a high concentration of Zn2+, which is known to inhibit the protease activity of PSA. We characterized the binding of Zn2+ to SgI and SgII and found evidence that these proteins are involved in regulating the activity of PSA. Intact SgI and SgII and synthetic semenogelin peptides were used in the experiments. Binding of Zn2+ was studied by radioligand blotting, titration with a zinc (II) fluorophore chelator and NMR analysis. A chromogenic substrate was used to measure the enzymatic activity of PSA. SgI and SgII bound Zn2+ with a stoichiometry of at least 10 cool (mol of protein)(-1) and with an average dissociation constant of approx. 5 mu M per site. Moreover, Zn2+-inhibited PSA was activated by exposure to SgI or SgII. Since both proteins have high affinity for Zn2+ and are the dominating proteins in semen, they probably represent the major Zn2+ binders in semen, one function of which may be to regulate the activity of PSA. The system is self-regulating, and PSA is maintained in an active state by its substrate.
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2. |
- Kreida, Stefan, et al.
(författare)
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The role of phosphorylation in calmodulin-mediated gating of human AQP0
- 2024
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Ingår i: The Biochemical journal. - 0264-6021. ; 481:1, s. 17-32
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Tidskriftsartikel (refereegranskat)abstract
- Aquaporin-0 (AQP0) is the main water channel in the mammalian lens and is involved in accommodation and maintaining lens transparency. AQP0 binds the Ca2+-sensing protein calmodulin (CaM) and this interaction is believed to gate its water permeability by closing the water-conducting pore. Here, we express recombinant and functional human AQP0 in Pichia pastoris and investigate how phosphorylation affects the interaction with CaM in vitro as well as the CaM-dependent water permeability of AQP0 in proteoliposomes. Using microscale thermophoresis and surface plasmon resonance technology we show that the introduction of the single phospho-mimicking mutations S229D and S235D in AQP0 reduces CaM binding. In contrast, CaM interacts with S231D with similar affinity as wild type, but in a different manner. Permeability studies of wild-type AQP0 showed that the water conductance was significantly reduced by CaM in a Ca2+-dependent manner, whereas AQP0 S229D, S231D and S235D were all locked in an open state, insensitive to CaM. We propose a model in which phosphorylation of AQP0 control CaM-mediated gating in two different ways (1) phosphorylation of S229 or S235 abolishes binding (the pore remains open) and (2) phosphorylation of S231 results in CaM binding without causing pore closure, the functional role of which remains to be elucidated. Our results suggest that site-dependent phosphorylation of AQP0 dynamically controls its CaM-mediated gating. Since the level of phosphorylation increases towards the lens inner cortex, AQP0 may become insensitive to CaM-dependent gating along this axis.
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3. |
- O'Malley, Tiernan T., et al.
(författare)
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A beta dimers differ from monomers in structural propensity, aggregation paths and population of synaptotoxic assemblies
- 2014
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Ingår i: Biochemical Journal. - 0264-6021. ; 461, s. 413-426
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Tidskriftsartikel (refereegranskat)abstract
- Dimers of A beta (amyloid beta-protein) are believed to play an important role in Alzheimer's disease. In the absence of sufficient brain-derived dimers, we studied one of the only possible dimers that could be produced in vivo, [A beta](DiY) (dityrosine cross-linked A beta). For comparison, we used the A beta monomer and a design dimer cross-linked by replacement of Ser(26) with cystine [A beta S26C](2). We showed that similar to monomers, unaggregated dimers lack appreciable structure and fail to alter long-term potentiation. Importantly, dimers exhibit subtly different structural propensities from monomers and each other, and can self-associate to form larger assemblies. Although [A beta](DiY) and [A beta S26C](2) have distinct aggregation pathways, they both populate bioactive soluble assemblies for longer durations than A beta monomers. Our results indicate that the link between A beta dimers and Alzheimer's disease results from the ability of dimers to further assemble and form synaptotoxic assemblies that persist for long periods of time.
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4. |
- Rogstam, Annika, et al.
(författare)
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Binding of calcium ions and SNAP-25 to the hexa EF-hand protein secretagogin
- 2007
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Ingår i: Biochemical Journal. - 0264-6021. ; 401:Pt 1, s. 353-363
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Tidskriftsartikel (refereegranskat)abstract
- Secretagogin is a hexa EF-hand protein, which has been identified as a novel potential tumour marker. In the present study, we show that secretagogin binds four Ca2+ ions (log K-1 = 7.1 +/- 0.4, log K-2 = 4.7 +/- 0.6, log K-3 = 3.6 +/- 0.7 and log K-4 = 4.6 +/- 0.6 in physiological salt buffers) with a [Ca2+](0.5) of approx. 25 mu M. The tertiary structure of secretagogin changes significantly upon Ca2+ binding, but not upon Mg2+ binding, and the amount of exposed hydrophobic surface in secretagogin increases upon Ca2+ binding, but not upon Mg2+ binding. These properties suggest that secretagogin belongs to the 'sensor' family of Ca2+-binding proteins. However, in contrast with the prototypical Ca2+ sensor calmodulin, which interacts with a very large number of proteins, secretagogin is significantly less promiscuous. Only one secretagogin-interacting protein was reproducibly identified from insulinoma cell lysates and from bovine and mouse brain homogenates. This protein was identified as SNAP-25 (25 kDa synaptosome-associated protein), a protein involved in Ca2+-induced exocytosis in neurons and in neuroendocrine cells. K-d was determined to be 1.2 x 10(-7) M in the presence of Ca2+ and 1.5 x 10(-6) M in the absence of Ca2+. The comparatively low Ca2+ affinity for secretagogin and the fact that it undergoes Ca2+-induced conformational changes and interacts with SNAP-25 raise the possibility that secretagogin may link Ca2+ signalling to exocytotic processes.
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