Ocular pterygium-digital keloid dysplasia (OPDKD) is a rare hereditary disease characterized by corneal ingrowth of vascularized conjunctival tissue early in life. Later, patients develop keloids... Show moreOcular pterygium-digital keloid dysplasia (OPDKD) is a rare hereditary disease characterized by corneal ingrowth of vascularized conjunctival tissue early in life. Later, patients develop keloids on fingers and toes but are otherwise healthy. In a recently described family with OPDKD, we report the presence of a de novo c.770C > T, p.(Thr257Ile) variant in PELI2 in the affected individual. PELI2 encodes for the E3 ubiquitin ligase Pellino-2. In transgenic U87MG cells overexpressing Pellino-2 with the p.(Thr257Ile) amino acid substitution, constitutive activation of the NLRP3 inflammasome was observed. However, the Thr257Ile variant did not affect Pellino-2 intracellular localization, its binding to known interaction partners, nor its stability. Our findings indicate that constitutive autoactivation of the NLRP3 inflammasome contributes to the development of PELI2-associated OPDKD. Show less
Glycosphingolipids (GSLs) fulfil diverse functions in cells. Abnormalities in their metabolism are associated with specific pathologies and, consequently, the pharmacological modulation of GSLs is... Show moreGlycosphingolipids (GSLs) fulfil diverse functions in cells. Abnormalities in their metabolism are associated with specific pathologies and, consequently, the pharmacological modulation of GSLs is considered a therapeutic avenue. The accurate measurement of in situ metabolism of GSLs and the modulatory impact of drugs is warranted. Employing synthesised sphingosine and sphinganine containing C-13 atoms, we developed a method to monitor the de novo synthesis of glucosylceramide, the precursor of complex GSLs, by the enzyme glucosylceramide synthase (GCS). We show that feeding cells with isotope-labelled precursor combined with liquid chromatography-mass spectrometry (MS)/MS analysis allows accurate determination of the IC50 values of therapeutically considered inhibitors (iminosugars and ceramide mimics) of GCS in cultured cells. Acquired data were comparable to those obtained with an earlier method using artificial fluorescently labelled ceramide to feed cells. Show less
Veseli, B.E.; Perrotta, P.; Wielendaele, P. van; Lambeir, A.M.; Abdali, A.; Bellosta, S.; ... ; Meyer, G.R.Y. de 2020
6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase isoform 3 (PFKFB3) is a key enzyme of the glycolytic pathway, and it plays an essential role in angiogenesis. 3-(3-Pyridinyl)-1-(4-pyridinyl)-2... Show more6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase isoform 3 (PFKFB3) is a key enzyme of the glycolytic pathway, and it plays an essential role in angiogenesis. 3-(3-Pyridinyl)-1-(4-pyridinyl)-2-propen-1-one (3PO) is frequently used as a glycolysis inhibitor and is thought to inhibit PFKFB3. However, this latter effect of 3PO has never been investigated in detail and was the aim of the present study. To demonstrate binding of 3PO to PFKFB3, we used isothermal titration calorimetry. However, 3PO did not bind to PFKFB3, even up to 750 µm, in contrast to 3 µm of AZ67, which is a potent and specific PFKFB3 inhibitor. Instead, 3PO accumulated lactic acid inside the cells, leading to a decrease in the intracellular pH and an inhibition of enzymatic reactions of the glycolytic pathway. Show less
Oculopharyngeal muscular dystrophy is caused by small alanine expansions in polyadenylate binding protein nuclear 1 (PABPN1) protein resulting in its intranuclear accumulation in skeletal muscle.... Show moreOculopharyngeal muscular dystrophy is caused by small alanine expansions in polyadenylate binding protein nuclear 1 (PABPN1) protein resulting in its intranuclear accumulation in skeletal muscle. 3F5 llama antibody specifically interferes with the PABPN1 aggregation process in vitro and in vivo. To understand the structural basis for its epitope recognition we mapped the binding interface of 3F5 with PABPN1 and provide a structural model of the 3F5-PABPN1 complex. We show that 3F5 complementarity determining regions create a cavity in which PABPN1 alpha-helix domain resides by involving critical residues previously implicated in the aggregation process. These results may increase our understanding of the PABPN1 aggregation mechanism and the therapeutic potential of 3F5. (C) 2010 Federation of European Biochemical Societies. Published by Elsevier B. V. All rights reserved. Show less
Krens, S.F.G.; Spaink, H.P.; Snaar-Jagalska, B.E. 2006
We have analyzed the ribosomal protein profile of Escherichia coli 30S subunits with the mutation C(18)A in the central pseudoknot of their 16S ribosomal RNA, This mutation was shown to inhibit... Show moreWe have analyzed the ribosomal protein profile of Escherichia coli 30S subunits with the mutation C(18)A in the central pseudoknot of their 16S ribosomal RNA, This mutation was shown to inhibit translational activity in vivo and to affect ribosome stability in vitro, The majority of the mutant 30S particles were present as free subunits in which a reproducible decrease in amount of proteins S1, S2, S18 and S21 was observed, The protein gels also showed the appearance of a satellite band nest to S5, This band reacted with anti-S5 antibodies and had a slightly increased positive charge, The simplest interpretation of these findings, also considering published data, is that the satellite band is S5 with a non-acetylated N-terminal alanine, Underacetylation of S5 due to mutations in the 16S rRNA implies that the modification is performed on the ribosome. Show less
Dennison, C.; Berg, A.; Vries, S. de; Canters, G.W. 1996
The dinuclear paramagnetic center of the soluble Cu-A domain of the cytochrome c oxidase from Bacillus subtilis has been studied using H-1 NMR. The spectrum possesses remarkably sharp shifted... Show moreThe dinuclear paramagnetic center of the soluble Cu-A domain of the cytochrome c oxidase from Bacillus subtilis has been studied using H-1 NMR. The spectrum possesses remarkably sharp shifted resonances, Comparison with the spectrum of the Cu-A amicyanin variant provides the spin density distribution in the Cu-A site of cytochrome c oxidase. This represents the first paramagnetic NMR study of the dinuclear Cu-A center from the soluble domain of subunit II of cytochrome c oxidase. Show less
Gorren, A.C.F.; Blaauwen, T. den; Canters, G.W.; Hopper, D.J.; Duine, J.A. 1996
The electron-transfer properties of H117G- and wild-type azurin were compared by applying both as electron accepters in the conversion of 1-ethylphenol by 4-ethylphenol methylenehydroxylase (4-EPMH... Show moreThe electron-transfer properties of H117G- and wild-type azurin were compared by applying both as electron accepters in the conversion of 1-ethylphenol by 4-ethylphenol methylenehydroxylase (4-EPMH). The reactivity of H117G-azurin was determined in the absence and presence of imidazoles, which can substitute the missing fourth ligand, In the absence of imidazoles, H117G-azurin reacted directly with 4-ethylphenol, this reaction was abolished in the presence of imidazoles, The enzymatic reduction of H117G-azurin by 4-EPMH was 40 times slower than that of wild-type azurin, The rate of this reaction was enhanced by some imidazoles, diminished by others. In all cases the reduction of H117G-azurin was irreversible. These results demonstrate that His117 is vital for electron transfer and effectively protects the copper site against aspecific reactions. Show less
