| 1. |
- Georgieva, Polina, et al.
(författare)
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The reaction mechanism of phenylethanolamine N-methyltransferase : A density functional theory study
- 2009
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Ingår i: Biochimica et Biophysica Acta - Proteins and Proteomics. - : Elsevier BV. - 1570-9639 .- 1878-1454. ; 1794:12, s. 1831-1837
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Tidskriftsartikel (refereegranskat)abstract
- Hybrid density functional theory methods were used to investigate the reaction mechanism of human phenylethanolamine N-methyltransferase (hPNMT). This enzyme catalyzes the S-adenosyl-L-methionine-dependent conversion of norepinephrine to epinephrine, which constitutes the terminal step in the catecholamine biosynthesis. Several models of the active site were constructed based on the X-ray structure. Geometries of the stationary points along the reaction path were optimized and the reaction barrier and energy were calculated and compared to the experimental values. The calculations demonstrate that the reaction takes place via an S(N)2 mechanism with methyl transfer being rate-limiting, a suggestion supported by mutagenesis studies. Optimal agreement with experimental data is reached using a model in which both active site glutamates; are protonated. Overall, the mechanism of hPNMT is more similar to those of catechol O-methyltransferase and glycine N-methyltransferase than to that of guanidinoacetate N-methyltransferase in which methyl transfer is coupled to proton transfer.
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| 2. |
- Karlsson, Oskar, et al.
(författare)
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MALDI imaging delineates hippocampal glycosphingolipid changes associated with neurotoxin induced proteopathy following neonatal BMAA exposure.
- 2017
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Ingår i: Biochimica et Biophysica Acta. - : Elsevier BV. - 0006-3002 .- 1878-2434. ; 1865:7, s. 740-746
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Tidskriftsartikel (refereegranskat)abstract
- The environmental toxin β-N-methylamino-L-alanine (BMAA) has been proposed to contribute to neurodegenerative diseases. We have previously shown that neonatal exposure to BMAA results in dose-dependent cognitive impairments, proteomic alterations and progressive neurodegeneration in the hippocampus of adult rats. A high BMAA dose (460mg/kg) also induced intracellular fibril formation, increased protein ubiquitination and enrichment of proteins important for lipid transport and metabolism. The aim of this study was therefore to elucidate the role of neuronal lipids in BMAA-induced neurodegeneration. By using matrix assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS), we characterized the spatial lipid profile in the hippocampus of six month-old rats that were treated neonatally (postnatal days 9-10) with 460mg/kg BMAA. Multivariate statistical analysis revealed long-term changes in distinct ganglioside species (GM, GD, GT) in the dentate gyrus. These changes could be a consequence of direct effects on ganglioside biosynthesis through the b-series (GM3-GD3-GD2-GD1b-GT1b) and may be linked to astrogliosis. Complementary immunohistochemistry experiments towards GFAP and S100β further verified the role of increased astrocyte activity in BMAA-induced brain damage. This highlights the potential of imaging MS for probing chemical changes associated with neuropathological mechanisms in situ. This article is part of a Special Issue entitled: MALDI Imaging, edited by Dr. Corinna Henkel and Prof. Peter Hoffmann.
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| 3. |
- Lohkamp, Bernhard, et al.
(författare)
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Insights into the mechanism of dihydropyrimidine dehydrogenase from site-directed mutagenesis targeting the active site loop and redox cofactor coordination
- 2010
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Ingår i: Biochimica et Biophysica Acta - Proteins and Proteomics. - : Elsevier BV. - 1570-9639 .- 1878-1454. ; 1804:12, s. 2198-2206
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Tidskriftsartikel (refereegranskat)abstract
- In mammals, the pyrimidines uracil and thymine are metabolised by a three-step reductive degradation pathway. Dihydropyrimidine dehydrogenase (DPD) catalyses its first and rate-limiting step, reducing uracil and thymine to the corresponding 5,6-dihydropyrimidines in an NADPH-dependent reaction. The enzyme is an adjunct target in cancer therapy since it rapidly breaks down the anti-cancer drug 5-fluorouracil and related compounds. Five residues located in functionally important regions were targeted in mutational studies to investigate their role in the catalytic mechanism of dihydropyrimidine dehydrogenase from pig. Pyrimidine binding to this enzyme is accompanied by active site loop closure that positions a catalytically crucial cysteine (C671) residue. Kinetic characterization of corresponding enzyme mutants revealed that the deprotonation of the loop residue H673 is required for active site closure, while S670 is important for substrate recognition. Investigations on selected residues involved in binding of the redox cofactors revealed that the first FeS cluster, with unusual coordination, cannot be reduced and displays no activity when Q156 is mutated to glutamate, and that R235 is crucial for FAD binding.
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| 4. |
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| 5. |
- Carlsson, Karin, et al.
(författare)
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Site-directed fluorescence probing to dissect the calcium-dependent association between soluble tissue factor and factor VIIa domains
- 2003
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Ingår i: Biochimica et Biophysica Acta - Proteins and Proteomics. - 1570-9639 .- 1878-1454. ; 1648:1-2, s. 12-16
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Tidskriftsartikel (refereegranskat)abstract
- We have used the site-directed labeling approach to study the Ca 2+-dependent docking of factor VIIa (FVIIa) to soluble tissue factor (sTF). Nine Ca2+ binding sites are located in FVIIa and even though their contribution to the overall binding between TF and FVIIa has been thoroughly studied, their importance for local protein-protein interactions within the complex has not been determined. Specifically we have monitored the association of the ?-carboxyglutamic acid (Gla), the first EGF-like (EGF1), and the protease domains (PD) of FVIIa to sTF. Our results revealed that complex formation between sTF and FVIIa during Ca2+ titration is initiated upon Ca2+ binding to EGF1, the domain containing the site of highest Ca2+ affinity. Besides we showed that a Ca 2+-loaded Gla domain is required for an optimal association of all domains of FVIIa to sTF. Ca2+ binding to the PD seems to be of some importance for the docking of this domain to sTF. © 2003 Elsevier Science B.V. All rights reserved.
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| 6. |
- Chirica, Laura C., et al.
(författare)
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Expression and localization of α- and β-carbonic anhydrase in Helicobacter pylori
- 2002
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Ingår i: Biochimica et Biophysica Acta - Proteins and Proteomics. - 1570-9639 .- 1878-1454. ; 1601:2, s. 192-199
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Tidskriftsartikel (refereegranskat)abstract
- Helicobacter pylori, the causative agent of peptic ulcer disease, expresses two different forms of the zinc-containing enzyme carbonic anhydrase (CA) (α and β), catalyzing the reversible hydration of CO2. Presumably, the high CO2 requirement of H. pylori implies an important role for this enzyme in the bacterial physiology. In this paper, expression of the CAs has been analyzed in three different strains of the bacterium, 26695, J99 and 17.1, and appears to be independent of CO2 concentration in the investigated range (0.1–10%). Presence of the potent and highly specific CA inhibitor, acetazolamide, in the medium does not seem to inhibit bacterial growth at the given sulfonamide concentration. Moreover, the localization and distribution of the α-CA was analyzed by immunonegative staining, while SDS-digested freeze-fracture immunogold labelling was used for the β-form of the enzyme. The latter method has the advantage of allowing assessment of protein localization to distinct cell compartments and membrane structures. The resulting electron microscopy images indicate a localization of the β-CA in the cytosol, on the cytosolic side of the inner membrane and on the outer membrane facing the periplasmic space. The α-enzyme was found attached to the surface of the bacterium.
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| 7. |
- Lindholm, D, et al.
(författare)
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Neuronal apoptosis inhibitory protein : structural requirements for hippocalcin binding and effects on survival of NGF-denendent sympathetic neurons
- 2002
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Ingår i: Biochimica et Biophysica Acta - Proteins and Proteomics. - : ELSEVIER SCIENCE BV. - 1570-9639 .- 1878-1454. ; 1600:1-2, s. 138-147
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Tidskriftsartikel (refereegranskat)abstract
- Neuronal apoptosis inhibitory protein (NAIP) has been linked to the inherited disease, spinal muscular atrophy (SMA), which occurs in children with degeneration of the motorneurons. In the nervous system, NAIP is expressed by specific classes of neurons including spinal -motorneurons. Recently, NAIP was shown to interact with hippocalcin, which belongs to the neuronal calcium sensor (NCS) protein family. Here we-have studied this interaction in more detail, using deletions and a mutagenesis of the third baculovirus inhibitory repeat (BIR) motif in NAIP, and functional assays for neuronal death. The results showed that specific amino acids and the zinc finger domain in BIR3 are needed for efficient interaction of NAIP with hippocalcin. Cotransfections of NAIP-BIR3 and hippocalcin resulted in translocation and colocalisation of the two proteins in neuroblastoma cells. This was accompanied by an enhanced resistance towards cell death induced by high levels of calcium. In contrast, expression of NAIP-BIR3 and hippocalcin in sympathetic neurons did not protect against death induced by nerve growth factor (NGF) withdrawal. The results demonstrate a functional interaction of hippocalcin with NAIP-BIR3, which in neuroblastoma cells leads to rescue of cells after high intracellular calcium, but which in sympathetic neurons had no significant effect. The results indicate that NAIP in conjunction with hippocalcin can affect the survival of some, but not all neural cells, and this interaction may play a role in the neurodegenerative processes in SMA, and possible other human disorders. (C) 2002 Elsevier Science B.V All rights reserved.
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| 8. |
- Ahmad, Shabbir, et al.
(författare)
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Trimeric microsomal glutathione transferase 2 displays one third of the sites reactivity
- 2015
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Ingår i: Biochimica et Biophysica Acta - Proteins and Proteomics. - : Elsevier BV. - 1570-9639 .- 1878-1454. ; 1854:1010 Pt A, s. 1365-1371
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Tidskriftsartikel (refereegranskat)abstract
- Human microsomal glutathione transferase 2 (MGST2) is a trimeric integral membrane protein that belongs to the membrane-associated proteins in eicosanoid and glutathione metabolism (MAPEG) family. The mammalian MAPEG family consists of six members where four have been structurally determined. MGST2 activates glutathione to form a thiolate that is crucial for GSH peroxidase activity and GSH conjugation reactions with electrophilic substrates, such as 1-chloro-2,4-dinitrobenzene (CDNB). Several studies have shown that MGST2 is able to catalyze a GSH conjugation reaction with the epoxide LTA(4) forming the pro-inflammatory LTC4. Unlike its closest homologue leukotriene C-4 synthase (LTC4S), MGST2 appears to activate its substrate GSH using only one of the three potential active sites [Ahmad S, et al. (2013) Biochemistry. 52, 1755-1764]. In order to demonstrate and detail the mechanism of one-third of the sites reactivity of MGST2, we have determined the enzyme oligomeric state, by Blue native PAGE and Differential Scanning Calorimetry, as well as the stoichiometty of substrate and substrate analog inhibitor binding to MGST2, using equilibrium dialysis and Isothermal Titration Calorimetry, respectively. Global simulations were used to fit kinetic data to determine the catalytic mechanism of MGST2 with GSH and CDNB (1-chloro-2,4-dinitrobenzene) as substrates. The best fit was observed with 1/3 of the sites catalysis as compared with a simulation where all three sites were active. In contrast to LTC4S, MGST2 displays a 1/3 the sites reactivity, a mechanism shared with the more distant family member MGST1 and recently suggested also for microsomal prostaglandin E synthase-1.
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| 9. |
- Blokzijl, Andries, et al.
(författare)
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Protein biomarker validation via proximity ligation assays
- 2014
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Ingår i: Biochimica et Biophysica Acta - Proteins and Proteomics. - : Elsevier BV. - 1570-9639 .- 1878-1454. ; 1844:5, s. 933-939
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Forskningsöversikt (refereegranskat)abstract
- The ability to detect minute amounts of specific proteins or protein modifications in blood as biomarkers for a plethora of human pathological conditions holds great promise for future medicine. Despite a large number of plausible candidate protein biomarkers published annually, the translation to clinical use is impeded by factors such as the required size of the initial studies, and limitations of the technologies used. The proximity ligation assay (PLA) is a versatile molecular tool that has the potential to address some obstacles, both in validation of biomarkers previously discovered using other techniques, and for future routine clinical diagnostic needs. The enhanced specificity of PIA extends the opportunities for large-scale, high-performance analyses of proteins. Besides advantages in the form of minimal sample consumption and an extended dynamic range, the PLA technique allows flexible assay reconfiguration. The technology can be adapted for detecting protein complexes, proximity between proteins in extracellular vesicles or in circulating tumor cells, and to address multiple post-translational modifications in the same protein molecule. We discuss herein requirements for biomarker validation, and how PLA may play an increasing role in this regard. We describe some recent developments of the technology, including proximity extension assays, the use of recombinant affinity reagents suitable for use in proximity assays, and the potential for single cell proteomics. This article is part of a Special Issue entitled: Biomarkers: A Proteomic Challenge. (C) 2013 Elsevier B.V. All rights reserved.
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| 10. |
- Cordeiro, Yraima, et al.
(författare)
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Modulation of p53 and prion protein aggregation by RNA
- 2019
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Ingår i: Biochimica et Biophysica Acta - Proteins and Proteomics. - : Elsevier BV. - 1570-9639 .- 1878-1454. ; 1867:10, s. 933-940
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Forskningsöversikt (refereegranskat)abstract
- Several RNA-binding proteins undergo reversible liquid-liquid phase transitions, which, in pathological conditions, might evolve into transitions to solid-state phases, giving rise to amyloid structures. Amyloidogenic and prion-like proteins, such as the tumor suppressor protein p53 and the mammalian prion protein (PrP), bind RNAs specifically or nonspecifically, resulting in changes in their propensity to undergo aggregation. Mutant p53 aggregation seems to play a crucial role in cancer through loss of function, negative dominance and gain of function. PrP conversion modulated by RNA results in highly toxic aggregates. Here, we review data on the modulatory action of RNAs on the aggregation of both proteins.
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