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The glomerulosclerosis gene Mpv17 encodes a peroxisomal protein producing reactive oxygen species
(1994)
MOTIVATION: The genome projects produce a wealth of protein sequences. Theoretical methods to predict possible structures and functions are needed for screening purposes, large-scale comparisons and in-depth analysis to identify worthwhile targets for further experimental research. Sequence-structure alignment is a basic tool for the identification of model folds for protein sequences and the construction of crude structural models. Empirical contact potentials (potentials of mean force) are used to optimize and evaluate such alignments. RESULTS: We propose new scoring schemes based on a contact definition derived from Voronoi decompositions of the three-dimensional coordinates of protein structures. We demonstrate that Voronoi potentials are superior to pure distance-based contact potentials with respect to recognition rate and significance for native folds. Moreover, the scoring scheme has the potential to provide a reasonable balance of detail and ion such that it is also useful for the recognition of distantly related (both homologous and non-homologous) proteins. This is demonstrated here on a set of structural alignments showing much better correspondence of native and model scores for the Voronoi potentials as compared to conventional distance-based potentials.
Recently, we discovered a cholinergic mechanism that inhibits the adenosine triphosphate (ATP)-dependent release of interleukin-1 beta (IL-1 beta) by human monocytes via nicotinic acetylcholine receptors (nAChRs) composed of alpha 7, alpha 9 and/or alpha 10 subunits. Furthermore, we identified phosphocholine (PC) and dipalmitoylphosphatidylcholine (DPPC) as novel nicotinic agonists that elicit metabotropic activity at monocytic nAChR. Interestingly, PC does not provoke ion channel responses at conventional nAChRs composed of subunits alpha 9 and alpha 10. The purpose of this study is to determine the composition of nAChRs necessary for nicotinic signaling in monocytic cells and to test the hypothesis that common metabolites of phosphatidylcholines, lysophosphatidylcholine (LPC) and glycerophosphocholine (G-PC), function as nAChR agonists. In peripheral blood mononuclear cells from nAChR gene-deficient mice, we demonstrated that inhibition of ATP-dependent release of IL-1 beta by acetylcholine (ACh), nicotine and PC depends on subunits alpha 7, alpha 9 and alpha 10. Using a panel of nAChR antagonists and siRNA technology, we confirmed the involvement of these subunits in the control of IL-1 beta release in the human monocytic cell line U937. Furthermore, we showed that LPC (C16:0) and G-PC efficiently inhibit ATP-dependent release of IL-1 beta. Of note, the inhibitory effects mediated by LPC and G-PC depend on nAChR subunits alpha 9 and alpha 10, but only to a small degree on alpha 7. In Xenopus laevis oocytes heterologously expressing different combinations of human alpha 7, alpha 9 or alpha 10 subunits, ACh induced canonical ion channel activity, whereas LPC, G-PC and PC did not. In conclusion, we demonstrate that canonical nicotinic agonists and PC elicit metabotropic nAChR activity in monocytes via interaction of nAChR subunits alpha 7, alpha 9 and alpha 10. For the metabotropic signaling of LPC and G-PC, nAChR subunits alpha 9 and alpha 10 are needed, whereas alpha 7 is virtually dispensable. Furthermore, molecules bearing a PC group in general seem to regulate immune functions without perturbing canonical ion channel functions of nAChR.
The epithelial sodium channel (ENaC) is a critical regulator of vertebrate electrolyte homeostasis. ENaC is the only constitutively open ion channel in the degenerin/ENaC protein family, and its expression, membrane abundance, and open probability therefore are tightly controlled. The canonical ENaC is composed of three subunits (, , and ), but a fourth -subunit may replace and form atypical -ENaCs. Using Xenopus laevis as a model, here we found that mRNAs of the - and -subunits are differentially expressed in different tissues and that -ENaC predominantly is present in the urogenital tract. Using whole-cell and single-channel electrophysiology of oocytes expressing Xenopus - or -ENaC, we demonstrate that the presence of the -subunit enhances the amount of current generated by ENaC due to an increased open probability, but also changes current into a transient form. Activity of canonical ENaCs is critically dependent on proteolytic processing of the - and -subunits, and immunoblotting with epitope-tagged ENaC subunits indicated that, unlike -ENaC, the -subunit does not undergo proteolytic maturation by the endogenous protease furin. Furthermore, currents generated by -ENaC were insensitive to activation by extracellular chymotrypsin, and presence of the -subunit prevented cleavage of -ENaC at the cell surface. Our findings suggest that subunit composition constitutes an additional level of ENaC regulation, and we propose that the Xenopus -ENaC subunit represents a functional example that demonstrates the importance of proteolytic maturation during ENaC evolution.
The limited sodium availability of freshwater and terrestrial environments was a major physiological challenge during vertebrate evolution. The epithelial sodium channel (ENaC) is present in the apical membrane of sodium-absorbing vertebrate epithelia and evolved as part of a machinery for efficient sodium conservation. ENaC belongs to the degenerin/ENaC protein family and is the only member that opens without an external stimulus. We hypothesized that ENaC evolved from a proton-activated sodium channel present in ionocytes of freshwater vertebrates and therefore investigated whether such ancestral traits are present in ENaC isoforms of the aquatic pipid frog Xenopus laevis. Using whole-cell and single-channel electrophysiology of Xenopus oocytes expressing ENaC isoforms assembled from alpha beta gamma- or delta beta gamma-subunit combinations, we demonstrate that Xenopus delta beta gamma-ENaC is profoundly activated by extracellular acidification within biologically relevant ranges (pH 8.0-6.0). This effect was not observed in Xenopus alpha beta gamma-ENaC or human ENaC orthologs. We show that protons interfere with allosteric ENaC inhibition by extracellular sodium ions, thereby increasing the probability of channel opening. Using homology modeling of ENaC structure and site-directed mutagenesis, we identified a cleft region within the extracellular loop of the delta-subunit that contains several acidic amino acid residues that confer proton-sensitivity and enable allosteric inhibition by extracellular sodium ions. We propose that Xenopus delta beta gamma-ENaC can serve as a model for investigating ENaC transformation from a proton-activated toward a constitutively-active ion channel. Such transformation might have occurred during the evolution of tetrapod vertebrates to enable bulk sodium absorption during the water-to-land transition.
Wesch D, Althaus M, Miranda P, Cruz-Muros I, Fronius M, Gonzalez-Hernandez T, Clauss WG, de la Rosa DA, Giraldez T. Differential N termini in epithelial Na+ channel delta-subunit isoforms modulate channel trafficking to the membrane. Am J Physiol Cell Physiol 302: C868-C879, 2012. First published December 7, 2011; doi: 10.1152/ajpcell.00255.2011.-The epithelial Na+ channel (ENaC) is a heteromultimeric ion channel that plays a key role in Na+ reabsorption across tight epithelia. The canonical ENaC is formed by three analogous subunits, alpha, beta, and gamma. A fourth ENaC subunit, named delta, is expressed in the nervous system of primates, where its role is unknown. The human delta-ENaC gene generates at least two splice isoforms, delta(1) and delta(2), differing in the N-terminal sequence. Neurons in diverse areas of the human and monkey brain differentially express either delta(1) or delta(2), with few cells coexpressing both isoforms, which suggests that they may play specific physiological roles. Here we show that heterologous expression of delta(1) in Xenopus oocytes and HEK293 cells produces higher current levels than delta(2). Patch-clamp experiments showed no differences in single channel current magnitude and open probability between isoforms. Steady-state plasma membrane abundance accounts for the dissimilarity in macroscopic current levels. Differential trafficking between isoforms is independent of beta- and gamma-subunits, PY-motif-mediated endocytosis, or the presence of additional lysine residues in delta(2)-N terminus. Analysis of delta(2)-N terminus identified two sequences that independently reduce channel abundance in the plasma membrane. The delta(1) higher abundance is consistent with an increased insertion rate into the membrane, since endocytosis rates of both isoforms are indistinguishable. Finally, we conclude that delta-ENaC undergoes dynamin-independent endocytosis as opposed to alpha beta gamma-channels.
Two distinct sequence elements mediate retroviral gene expression in embryonal carcinoma cells
(1987)
Striated muscle contraction is regulated by the translocation of troponin-tropomyosin strands over the thin filament surface. Relaxation relies partly on highly-favorable, conformation-dependent electrostatic contacts between actin and tropomyosin, which position tropomyosin such that it impedes actomyosin associations. Impaired relaxation and hypercontractile properties are hallmarks of various muscle disorders. The α-cardiac actin M305L hypertrophic cardiomyopathy-causing mutation lies near residues that help confine tropomyosin to an inhibitory position along thin filaments. Here, we investigate M305L actin in vivo, in vitro, and in silico to resolve emergent pathological properties and disease mechanisms. Our data suggest the mutation reduces actin flexibility and distorts the actin-tropomyosin electrostatic energy landscape that, in muscle, result in aberrant contractile inhibition and excessive force. Thus, actin flexibility may be required to establish and maintain interfacial contacts with tropomyosin as well as facilitate its movement over distinct actin surface features and is, therefore, likely necessary for proper regulation of contraction.
Dihydropyrimidine dehydrogenase (DPD) deficiency is an infrequently described autosomal recessive disorder of the pyrimidine degradation pathway and can lead to mental and motor retardation and convulsions. DPD deficiency is also known to cause a potentially lethal toxicity following administration of the antineoplastic agent 5-fluorouracil. In an ongoing study of 72 DPD deficient patients, we analysed the molecular background of 5 patients in more detail in whom initial sequence analysis did not reveal pathogenic mutations. In three patients, a 13.8 kb deletion of exon 12 was found and in one patient a 122 kb deletion of exon 14–16 of DPYD. In the fifth patient, a c.299_302delTCAT mutation in exon 4 was found and also loss of heterozygosity of the entire DPD gene. Further analysis demonstrated a de novo deletion of approximately 14 Mb of chromosome 1p13.3–1p21.3, which includes DPYD. Haploinsufficiency of NTNG1, LPPR4, GPSM2, COL11A1 and VAV3 might have contributed to the severe psychomotor retardation and unusual craniofacial features in this patient. Our study showed for the first time the presence of genomic deletions affecting DPYD in 7% (5/72) of all DPD deficient patients. Therefore, screening of DPD deficient patients for genomic deletions should be considered.
Dihydropyrimidinase deficiency: Phenotype, genotype and structural consequences in 17 patients
(2010)
Dihydropyrimidine Dehydrogenase Deficiency Caused by a Novel Genomic Deletion c.505_513del of DPYD
(2010)
Gene expression and cell growth rely on the intracellular concentration of amino acids, which in metazoans depends on extracellular amino acid availability and transmembrane transport. To investigate the impact of extracellular amino acid concentrations on the expression of a concentrative amino acid transporter, we overexpressed the main kidney proximal tubule luminal neutral amino acid transporter B0AT1-collectrin (SLC6A19-TMEM27) in MDCK cell epithelia. Exogenously expressed proteins co-localized at the luminal membrane and mediated neutral amino acid uptake. However, the transgenes were lost over few cell culture passages. In contrast, the expression of a control transgene remained stable. To test whether this loss was due to inappropriately high amino acid uptake, freshly transduced MDCK cell lines were cultivated either with physiological amounts of amino acids or with the high concentration found in standard cell culture media. Expression of exogenous transporters was unaffected by physiological amino acid concentration in the media. Interestingly, mycoplasma infection resulted in a significant increase in transgene expression and correlated with the rapid metabolism of L-arginine. However, L-arginine metabolites were shown to play no role in transgene expression. In contrast, activation of the GCN2 pathway revealed by an increase in eIF2α phosphorylation may trigger transgene derepression. Taken together, high extracellular amino acid concentration provided by cell culture media appears to inhibit the constitutive expression of concentrative amino acid transporters whereas L-arginine depletion by mycoplasma induces the expression of transgenes possibly via stimulation of the GCN2 pathway.
We propose a new alignment procedure that is capable of aligning protein sequences and structures in a unified manner. Recursive dynamic programming (RDP) is a hierarchical method which, on each level of the hierarchy, identifies locally optimal solutions and assembles them into partial alignments of sequences and/or structures. In contrast to classical dynamic programming, RDP can also handle alignment problems that use objective functions not obeying the principle of prefix optimality, e.g.\ scoring schemes derived from energy potentials of mean force. For such alignment problems, RDP aims at computing solutions that are near-optimal with respect to the involved cost function and biologically meaningful at the same time. Towards this goal, RDP maintains a dynamic balance between different factors governing alignment fitness such as evolutionary relationships and structural preferences. As in the RDP method gaps are not scored explicitly, the problematic assignment of gap cost parameters is circumvented. In order to evaluate the RDP approach we analyse whether known and accepted multiple alignments based on structural information can be reproduced with the RDP method. For this purpose, we consider the family of ferredoxins as our prime example. Our experiments show that, if properly tuned, the RDP method can outperform methods based on classical sequence alignment algorithms as well as methods that take purely structural information into account.
BACKGROUND
Activator protein-2 (AP-2) transcription factors are critically involved in a variety of fundamental cellular processes such as proliferation, differentiation and apoptosis and have also been implicated in carcinogenesis. Expression of the family members AP-2alpha and AP-2gamma is particularly well documented in malignancies of the female breast. Despite increasing evaluation of single AP-2 isoforms in mammary tumors the functional role of concerted expression of multiple AP-2 isoforms in breast cancer remains to be elucidated. AP-2 proteins can form homo- or heterodimers, and there is growing evidence that the net effect whether a cell will proliferate, undergo apoptosis or differentiate is partly dependent on the balance between different AP-2 isoforms.
METHODS
We simultaneously interfered with all AP-2 isoforms expressed in ErbB-2-positive murine N202.1A breast cancer cells by conditionally over-expressing a dominant-negative AP-2 mutant.
RESULTS
We show that interference with AP-2 protein function lead to reduced cell number, induced apoptosis and increased chemo- and radiation-sensitivity. Analysis of global gene expression changes upon interference with AP-2 proteins identified 139 modulated genes (90 up-regulated, 49 down-regulated) compared with control cells. Gene Ontology (GO) investigations for these genes revealed Cell Death and Cell Adhesion and Migration as the main functional categories including 25 and 12 genes, respectively. By using information obtained from Ingenuity Pathway Analysis Systems we were able to present proven or potential connections between AP-2 regulated genes involved in cell death and response to chemo- and radiation therapy, (i.e. Ctgf, Nrp1, Tnfaip3, Gsta3) and AP-2 and other main apoptosis players and to create a unique network.
CONCLUSIONS
Expression of AP-2 transcription factors in breast cancer cells supports proliferation and contributes to chemo- and radiation-resistance of tumor cells by impairing the ability to induce apoptosis. Therefore, interference with AP-2 function could increase the sensitivity of tumor cells towards therapeutic intervention.
Possible genotype-phenotype correlations in children with mild clinical course of Canavan disease
(2005)
The Olig3 gene encodes a bHLH factor that is expressed in the ventricular zone of the dorsal alar plate of the hindbrain. We found that the Olig3(+) progenitor domain encompassed subdomains that co-expressed Math1, Ngn1, Mash1 and Ptf1a. Olig3(+) cells give rise to neuronal types in the dorsal alar plate that we denote as class A neurons. We used genetic lineage tracing to demonstrate that class A neurons contribute to the nucleus of the solitary tract and to precerebellar nuclei. The fate of class A neurons was not correctly determined in Olig3 mutant mice. As a consequence, the nucleus of the solitary tract did not form, and precerebellar nuclei, such as the inferior olivary nucleus, were absent or small. At the expense of class A neurons, ectopic Lbx1(+) neurons appeared in the alar plate in Olig3 mutant mice. By contrast, electroporation of an Olig3 expression vector in the chick hindbrain suppressed the emergence of Lbx1(+) neurons. Climbing fiber neurons of the inferior olivary nucleus express Foxd3 and require Olig3 as well as Ptf1a for the determination of their fate. We observed that electroporation of Olig3 and Ptf1a expression vectors, but not either alone, induced Foxd3. We therefore propose that Olig3 can cooperate with Ptf1a to determine the fate of climbing fiber neurons of the inferior olivary nucleus.
TNF-related activation-induced cytokine (TRANCE), also known as receptor activator of NF-kappaB ligand (RANKL), is the key molecule responsible for the bone loss observed in osteoporosis. Passive administration of osteoprotegerin, the soluble decoy receptor of TRANCE/RANKL, is efficient in blocking disease progression, but may not find widespread clinical use due to patient compliance problems and the expected high costs. In this study, we describe an efficient, safe, and potentially cost-effective active immunization strategy against TRANCE/RANKL. We show in mice that immunization with TRANCE/RANKL covalently linked to virus-like particles can overcome the natural tolerance of the immune system toward self proteins and produce high levels of specific Abs without the addition of any adjuvant. Serum Abs of immunized mice neutralized TRANCE/RANKL activity in vitro and were highly active in preventing bone loss in a mouse model of osteoporosis. Active immunization against TRANCE/RANKL was essentially reversible and did not produce any measurable immunosuppressive side effects, underscoring its potential as a new therapeutic approach to the treatment of human bone-degenerative disorders.