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1.
Rheumatol Int ; 38(1): 111-120, 2018 Jan.
Article in English | MEDLINE | ID: mdl-29159471

ABSTRACT

OBJECTIVE: Adult onset Still's disease (AOSD) is a severe, autoimmune disease that can be challenging to treat with conventional therapeutics and biologicals in a considerable number of cases. Therefore, there is a high need to understand its pathogenesis better. As major clinical symptoms overlap between AOSD and hereditary periodic fever syndromes (HPFS), we analysed four known HPFS genes in AOSD. METHODS: We performed Sanger sequencing and quantitative analysis of all coding regions of MEFV, TNFRSF1A, MVK and NLRP3 in 40 AOSD patients. All rare coding variants (n = 6) were evaluated for several aspects to classify them as benign to pathogenic variants. Statistical analysis was performed to analyse whether variants classified as (likely) pathogenic were associated with AOSD. RESULTS: We identified three rare variants in MEFV, one previously not described. Association to the three likely pathogenic MEFV variants was significant (p c = 2.34E- 03), and two of the three carriers had a severe course of disease. We observed strong evidence for significant association to mutations in TNFRSF1A (p c = 2.40E- 04), as 5% of patients (2/40) carried a (likely) pathogenic variant in this gene. Both of them received a biological for treatment. CONCLUSION: Our results indicate TNFRSF1A as a relevant gene in AOSD, especially in patients with a more challenging course of disease, while causal variants remain to be identified in the majority of patients.


Subject(s)
Genetic Predisposition to Disease , Hereditary Autoinflammatory Diseases/genetics , Mutation , Still's Disease, Adult-Onset/genetics , Adolescent , Adult , Aged , Child , Female , Humans , Male , Middle Aged , NLR Family, Pyrin Domain-Containing 3 Protein/genetics , Phosphotransferases (Alcohol Group Acceptor)/genetics , Pyrin/genetics , Receptors, Tumor Necrosis Factor, Type I/genetics , Young Adult
2.
Br J Dermatol ; 178(3): 740-748, 2018 03.
Article in English | MEDLINE | ID: mdl-28887889

ABSTRACT

BACKGROUND: Rare variants in the genes IL36RN, CARD14 and AP1S3 have been identified to cause or contribute to pustular skin diseases, primarily generalized pustular psoriasis (GPP). OBJECTIVES: To better understand the disease relevance of these genes, we screened our cohorts of patients with pustular skin diseases [primarily GPP and palmoplantar pustular psoriasis (PPP)] for coding changes in these three genes. Carriers of single heterozygous IL36RN mutations were screened for a second mutation in IL36RN. METHODS: Coding exons of IL36RN, CARD14 and AP1S3 were sequenced in 67 patients - 61 with GPP, two with acute generalized exanthematous pustulosis and four with acrodermatitis continua of Hallopeau. We screened IL36RN and AP1S3 for intragenic copy-number variants and 258 patients with PPP for coding changes in AP1S3. Eleven heterozygous IL36RN mutations carriers were analysed for a second noncoding IL36RN mutation. Genotype-phenotype correlations in carriers/noncarriers of IL36RN mutations were assessed within the GPP cohort. RESULTS: The majority of patients (GPP, 64%) did not carry rare variants in any of the three genes. Biallelic and monoallelic IL36RN mutations were identified in 15 and five patients with GPP, respectively. Noncoding rare IL36RN variants were not identified in heterozygous carriers. The only significant genotype-phenotype correlation observed for IL36RN mutation carriers was early age at disease onset. Additional rare CARD14 or AP1S3 variants were identified in 15% of IL36RN mutation carriers. CONCLUSIONS: The identification of IL36RN mutation carriers harbouring additional rare variants in CARD14 or AP1S3 indicates a more complex mode of inheritance of pustular psoriasis. Our results suggest that, in heterozygous IL36RN mutation carriers, there are additional disease-causing genetic factors outside IL36RN.


Subject(s)
Interleukins/genetics , Mutation/genetics , Psoriasis/genetics , Adult , CARD Signaling Adaptor Proteins/genetics , Female , Genetic Predisposition to Disease/genetics , Genetic Testing , Guanylate Cyclase/genetics , Heterozygote , Humans , Male , Membrane Proteins/genetics , Middle Aged , Vesicular Transport Proteins/genetics
3.
Antiviral Res ; 134: 130-143, 2016 10.
Article in English | MEDLINE | ID: mdl-27515131

ABSTRACT

HCMV is a member of the family Herpesviridae and represents a worldwide distributed pathogen with seropositivity rates in the adult population ranging between 40% and 90%. Notably, HCMV infection is a serious, sometimes life-threatening medical problem for newborns and immunosuppressed individuals, including transplant recipients and patients under antitumoral chemotherapy. Current standard therapy with valganciclovir has the disadvantage of inducing drug-resistant virus mutants and toxicity-related side effects. Our analysis stresses the earlier finding that kinase inhibitors of the quinazoline class exert an antiviral response by targeting the viral protein kinase pUL97 without inducing resistance. Therefore, quinazolines have been used as a core structure to gain insight in the mode of inhibitor-kinase interaction. Here, we demonstrate that (i) the novel quinazolines Vi7392 and Vi7453 are highly active against HCMV laboratory and clinically relevant strains including maribavir- and ganciclovir-resistant variants, (ii) antiviral activity is not cell-type specific and was also detected in a placental explant tissue model using a genetically intact HCMV strain (iii) the viral kinase pUL97 represents a target of the anticytomegaloviral activity of these compounds, (iv) induction of pUL97-conferring drug resistance was not detectable under single-step selection, thus differed from the induction of ganciclovir resistance, and (v) pUL97 drug docking simulations enabled detailed insights into specific drug-target binding properties providing a promising basis for the design of optimized kinase inhibitors. These novel findings may open new prospects for the future medical use of quinazoline drug candidates and the use of drug-target dynamic simulations for rational design of antivirals.


Subject(s)
Cytomegalovirus/drug effects , Drug Design , Protein Kinase Inhibitors/pharmacology , Quinazolines/pharmacology , Antiviral Agents/chemistry , Antiviral Agents/isolation & purification , Antiviral Agents/pharmacology , Cells, Cultured , Cytomegalovirus/chemistry , Cytomegalovirus/enzymology , Drug Resistance, Viral , Female , Fibroblasts/virology , Humans , Models, Molecular , Molecular Docking Simulation , Placenta/cytology , Pregnancy , Protein Kinase Inhibitors/chemistry , Quinazolines/chemistry , Quinazolines/classification , Viral Proteins/chemistry , Viral Proteins/drug effects , Virus Replication/drug effects
4.
Gene ; 568(1): 76-80, 2015 Aug 15.
Article in English | MEDLINE | ID: mdl-25967385

ABSTRACT

We report the identification of a novel mutation in the fork-head box C1 (FOXC1) gene which occurred de novo in an Italian patient with unrecognized Axenfeld-Rieger syndrome. He was previously diagnosed as having late recognized primary congenital glaucoma at the age of 14 years and was subsequently subjected to multiple surgical interventions due to uncontrolled intraocular pressure and progressive visual field loss. After exclusion of mutations in CYP1B1 and MYOC, trio-whole-exome sequencing revealed de novo in frame deletion in the coding region of the FOXC1 gene (c.407_409delGTC, p.V137del) leading to a deletion of the evolutionary conserved amino acid Valine at position 137 of the protein. Molecular modeling predicted that Val137 deletion impairs FOXC1 DNA-binding capacity and transcriptional activation. Since loss-of-function mutations in FOXC1 are associated with Axenfeld-Rieger syndrome, the genetic findings in combination with re-evaluation of the patient's clinical data resulted in a corrected diagnosis of Axenfeld-Rieger syndrome with developmental glaucoma. We therefore suggest that in addition to CYP1B1 and MYOC, FOXC1 should be included in the genetic analysis of cases with unclear glaucomatous phenotypes to ensure proper diagnosis, adequate treatment and appropriate genetic counseling.


Subject(s)
Anterior Eye Segment/abnormalities , Eye Abnormalities/diagnosis , Forkhead Transcription Factors/genetics , Glaucoma/diagnosis , Adult , Amino Acid Sequence , Base Sequence , Binding Sites , DNA Mutational Analysis , Delayed Diagnosis , Exome/genetics , Eye Abnormalities/genetics , Eye Diseases, Hereditary , Forkhead Transcription Factors/chemistry , Glaucoma/genetics , Humans , Male , Models, Molecular , Molecular Sequence Data , Protein Binding , Protein Structure, Secondary
5.
Curr Mol Med ; 12(2): 199-205, 2012 Feb.
Article in English | MEDLINE | ID: mdl-22280357

ABSTRACT

In patients with congenital heart defects, chromosomal anomalies are 100 times more frequent than in control subjects. Coarctation of the aorta can be detected in 15-20% of patients with Ullrich-Turner syndrome. By extensively reviewing literature involving breakpoint analysis of gonosomal deletions in Ullrich- Turner syndrome patients with and without coarctation of the aorta, we identified several gonosomal homolgous gene pairs of interest. Four of these homologous gene pairs were investigated by standard DNA sequencing in a cohort of 83 patients with non-syndromic coarctation of the aorta. Subsequently stability of mutant RNA and protein was analyzed to verify functional relevance of detected mutations. We identified two unreported missense mutations in Exon 8 (p.D69H) and 9 (p.R176W) of TBL1Y. Bioinformatic analysis and 3D modelling predicted that both mutations lead to TBL1Y loss of function. In RT-PCR and Western blot analyses of HEK293 cells transfected with a vector carrying the full-length TBL1Y (wild-type and mutant), we documented the predicted protein instability by showing protein decay for both mutant proteins. TBL1Y is similar to its gonosomal homologue, TBL1X, and its autosomal homologue, TBLR1, on chromosome 3. Both genes are part of co-repressor machineries and required for transcriptional activation by transcription factors that involve CtBP1/2, which contributes to Notch signaling. Several studies have shown that Notch signalling is important for proper development of the left ventricular outflow tract. Our findings suggest that TBL1Y is involved in the genesis of non-syndromic coarctation of the aorta.


Subject(s)
Aortic Coarctation/genetics , Genetic Predisposition to Disease , Mutation , Sex Chromosomes , Transducin/genetics , Adolescent , Adult , Aortic Coarctation/metabolism , Base Sequence , Cell Line , Child , Child, Preschool , Female , Gene Expression , Gene Order , Humans , Infant , Male , Models, Molecular , Protein Stability , Protein Structure, Secondary , Transducin/chemistry , Transducin/metabolism , Young Adult
6.
J Neurogenet ; 25(4): 182-8, 2011 Dec.
Article in English | MEDLINE | ID: mdl-22091729

ABSTRACT

Mutations in the Dynamin 2 gene (DNM2) cause autosomal dominant centronuclear myopathy or autosomal dominant (AD) Charcot-Marie-Tooth (CMT) disease. Here the authors report one large Czech family with 15 members affected with an AD CMT phenotype of extraordinary variability. Genetic linkage analysis using SNP arrays revealed a locus of about 9.6 Mb on chromosome 19p13.1-13.2. In this critical interval, 373 genes were located. The only gene herein known to be associated with an intermediate type of CMT was Dynamin 2 (DNM2). Subsequent sequence analysis of the DNM2 gene in the index patient revealed a novel missense mutation p.Met580Thr. This missense mutation segregated with the neuropathy, indicating the causal character of this mutation. The phenotype of CMT in this family shows mild to moderate impairment with relatively preserved upper limbs and a very broad range of the onset of clinical symptoms from an early onset around the age of 12 to the late onset during the fifth decade. Electrophysiology showed an intermediate type of peripheral neuropathy. The motor median nerve conduction velocity varied from 36 m/s to normal values with signs of asymmetrical affection of peripheral nerves. No additional symptoms such as cranial nerve involvement, cataract, and signs of neutropenia or myopathy syndrome were observed in any member of the family yet. The progression was slow with no loss of ambulation. The authors suggest that the characterization of clinical variability in a single family may help to direct the genetic analysis directly to the rarely observed DNM2 mutations.


Subject(s)
Charcot-Marie-Tooth Disease/genetics , Dynamin II/deficiency , Dynamin II/genetics , Genetic Predisposition to Disease/genetics , Adolescent , Adult , Charcot-Marie-Tooth Disease/diagnosis , Charcot-Marie-Tooth Disease/metabolism , Child , Child, Preschool , Czechoslovakia , Female , Genetic Predisposition to Disease/ethnology , Humans , Infant , Infant, Newborn , Male , Middle Aged , Mutation, Missense/genetics , Pedigree , Phenotype , Young Adult
7.
Mol Syndromol ; 1(3): 99-112, 2010 Sep.
Article in English | MEDLINE | ID: mdl-21031079

ABSTRACT

The etiology of non-syndromic hydrocephalus is poorly understood. Via positional cloning in a consanguineous family with autosomal recessive hydrocephalus we have now identified a homozygous splice site mutation in the CCDC88C gene as a novel cause of a complex hydrocephalic brain malformation. The only living patient showed normal psychomotor development at the age of 3 years and 3 months and her deceased aunt, who was assumed to suffer from the same condition, had mild mental retardation. The mutation in the affected patients, a homozygous substitution in the donor splice site of intron 29, resulted in a shorter transcript due to exclusion of exon 29 and loss of functional protein, as shown by Western blotting (p.S1591HfsX7). In normal human tissue panels, we found CCDC88C ubiquitously expressed, but most prominently in the fetal brain, especially in pons and cerebellum, while expression in the adult brain appeared to be restricted to cortex and medulla oblongata. CCDC88C encodes DAPLE (HkRP2), a Hook-related protein with a binding domain for the central Wnt signalling pathway protein Dishevelled. Targeted quantitative RT-PCR and expression profiling of 84 genes from the Wnt signalling pathway in peripheral blood from the index patient and her healthy mother revealed increased mRNA levels of CCDC88C indicating transcriptional upregulation. Due to loss of CCDC88C function ß-catenin (CTNNB1) and the downstream target LEF1 showed increased mRNA levels in the patient, but many genes from the Wnt pathway and transcriptional target genes showed reduced expression, which might be explained by a complex negative feedback loop. We have thus identified a further essential component of the Wnt signalling pathway in human brain development.

8.
Br J Pharmacol ; 159(3): 698-708, 2010 Feb 01.
Article in English | MEDLINE | ID: mdl-20100277

ABSTRACT

BACKGROUND AND PURPOSE: Organic anion transporting polypeptide 1B3 (OATP1B3) (SLCO1B3) mediates the uptake of endogenous substrates (e.g. estrone-3-sulphate) and drugs (e.g. pravastatin) from blood into hepatocytes. Structure-based modelling of OATP1B3 suggested that a pore with a positive electrostatic potential contributes to the transport mechanism. Therefore, we investigated the role of conserved positively charged amino acids for OATP1B3-mediated uptake of sulphobromophthalein (BSP) and pravastatin. EXPERIMENTAL APPROACH: Residues Lys28, Lys41 and Arg580 in OATP1B3 were substituted by alanine, arginine, glutamine, glycine or lysine. Using immunofluorescence, immunoblot analysis and cellular uptake assays, the effect of these mutations on protein expression and transport activity was investigated. KEY RESULTS: Immunofluorescence revealed that all mutants were localized in the plasma membrane with partial intracellular retention of the Arg580>Ala and Arg580>Lys mutants. Lys41>Ala, Lys41>Gln, Lys41>Gly, Arg580>Gly and Arg580>Lys showed significantly reduced transport for BSP and pravastatin. Kinetic analyses of BSP transport revealed a significant reduction of V(max) normalized to cell surface protein expression for Lys41>Ala (wild type: 190 +/- 8, Lys41>Ala:16 +/- 4 pmol (mg protein)(-1) min(-1), P < 0.001), whereas V(max) of Lys41>Arg and Arg580>Lys (103 +/- 8 and 123 +/- 14 pmol (mg protein)(-1) min(-1), P > 0.05) did not change significantly. This suggests that the positive charges at positions 41 and 580 are important for transport activity of BSP. Structural modelling indicated that the positively charged side chain of Lys41 is flexible within the pore. The orientation of Arg580 is defined by adjacent residues Glu74 and Asn77, which was confirmed by kinetic analysis of Glu74>Ala. CONCLUSIONS AND IMPLICATIONS: We demonstrated that the conserved positively charged amino acids Lys41 and Arg580 are pivotal to the transport activity of OATP1B3.


Subject(s)
Hepatocytes/metabolism , Organic Anion Transporters/metabolism , Alanine/genetics , Alanine/metabolism , Arginine/genetics , Arginine/metabolism , Biological Transport/drug effects , Biological Transport, Active , Cell Membrane/metabolism , Cellular Structures/metabolism , Estrone/analogs & derivatives , Glycine/genetics , Glycine/metabolism , Humans , Kinetics , Lysine/genetics , Lysine/metabolism , Organic Anion Transporters/chemistry , Pharmaceutical Preparations/metabolism , Pravastatin/metabolism , Protein Structure, Secondary , Sulfobromophthalein/metabolism
9.
J Med Genet ; 45(11): 738-44, 2008 Nov.
Article in English | MEDLINE | ID: mdl-18728071

ABSTRACT

BACKGROUND: Haploinsufficiency of the gene encoding for transcription factor 4 (TCF4) was recently identified as the underlying cause of Pitt-Hopkins syndrome (PTHS), an underdiagnosed mental-retardation syndrome characterised by a distinct facial gestalt, breathing anomalies and severe mental retardation. METHODS: TCF4 mutational analysis was performed in 117 patients with PTHS-like features. RESULTS: In total, 16 novel mutations were identified. All of these proven patients were severely mentally retarded and showed a distinct facial gestalt. In addition, 56% had breathing anomalies, 56% had microcephaly, 38% had seizures and 44% had MRI anomalies. CONCLUSION: This study provides further evidence of the mutational and clinical spectrum of PTHS and confirms its important role in the differential diagnosis of severe mental retardation.


Subject(s)
Apnea , DNA Mutational Analysis , DNA-Binding Proteins/genetics , Face/abnormalities , Hyperventilation , Intellectual Disability/genetics , Transcription Factors/genetics , Adolescent , Apnea/diagnosis , Apnea/genetics , Apnea/pathology , Basic Helix-Loop-Helix Leucine Zipper Transcription Factors , Child , Child, Preschool , Face/pathology , Female , Genotype , Humans , Hyperventilation/diagnosis , Hyperventilation/genetics , Hyperventilation/pathology , Infant , Intellectual Disability/diagnosis , Intellectual Disability/pathology , Male , Microcephaly , Phenotype , Syndrome , Transcription Factor 4 , Young Adult
10.
J Pept Res ; 63(1): 36-47, 2004 Jan.
Article in English | MEDLINE | ID: mdl-14984572

ABSTRACT

The CCL15 is a human CC chemokine that activates the receptors, CCR1 and CCR3. Unlike other chemokines, it contains an unusually long N-terminal domain of 31 amino acids preceding the first cysteine residue and a third disulfide bond. To elucidate the functional role of distinct structural determinants, a series of sequential amino-terminal truncated and point-mutated CCL15 derivatives as well as mutants lacking the third disulfide bond and the carboxy-terminal alpha-helix were synthesized using 9-fluorenylmethoxycarbonyl (Fmoc) chemistry. We demonstrate that a truncation of 24 amino acid residues (delta24-CCL15) converts the slightly active 92-residue delta0-CCL15 into a potent agonist of CC chemokine receptor 1 (CCR1) and a weak agonist of CCR3 in cell-based assays. The biological activity decreases from delta24-CCL15 to delta29-CCL15, and re-increases from delta29-CCL15 to delta30-CCL15. Thus, an exocyclic N-terminal region of only one amino acid residue is sufficient for efficient CCR1 activation. As none of the peptides investigated except for delta24-CCL15 activates CCR3, we suggest that CCR1 is the major receptor for CCL15 in vivo. Further we demonstrate that the third disulfide bond of CCL15 and an exchange of tyrosine in position 70 by a leucine residue, which is conserved in CXC chemokines, do not alter the interaction with CCR1. In contrast, a CCL15 derivative lacking the carboxy-terminal alpha-helix exhibits a complete loss of tertiary structure and hence loss of CCR1 agonistic and binding activity. This study demonstrates that specific protein residues in chemokines, which contribute to receptor-ligand interaction, vary significantly between chemokines and cannot be extrapolated using data from functionally related chemokines.


Subject(s)
Monokines/chemistry , Peptides/chemical synthesis , Peptides/pharmacology , Receptors, Chemokine/agonists , Amino Acid Sequence , Animals , Binding, Competitive , CHO Cells , Calcium/metabolism , Cell Line, Tumor , Chemokines, CC , Chemotaxis, Leukocyte/drug effects , Cricetinae , Cricetulus , Heparin/metabolism , Humans , Ligands , Macrophage Inflammatory Proteins , Molecular Sequence Data , Monocytes/immunology , Monokines/antagonists & inhibitors , Nuclear Magnetic Resonance, Biomolecular , Peptides/chemistry , Protein Structure, Tertiary , Radioligand Assay , Receptors, CCR1 , Receptors, Chemokine/chemistry , Receptors, Chemokine/metabolism , Structure-Activity Relationship
11.
Protein Sci ; 10(12): 2470-9, 2001 Dec.
Article in English | MEDLINE | ID: mdl-11714914

ABSTRACT

Defensins are cationic and cysteine-rich peptides that play a crucial role in the host defense against microorganisms of many organisms by their capability to permeabilize bacterial membranes. The low sequence similarity among the members of the large mammalian beta-defensin family suggests that their antimicrobial activity is largely independent of their primary structure. To investigate to what extent these defensins share a similar fold, the structures of the two human beta-defensins, hBD-1 and hBD-2, as well as those of two novel murine defensins, termed mBD-7 and mBD-8, were determined by nuclear magnetic resonance spectroscopy. All four defensins investigated share a striking similarity on the level of secondary and tertiary structure including the lack of a distinct hydrophobic core, suggesting that the fold is mainly stabilized by the presence of three disulfide bonds. In addition to the overall shape of the molecules, the ratio of solvent-exposed polar and hydrophobic side chains is also very similar among the four defensins investigated. It is significant that beta-defensins do not exhibit a common pattern of charged and hydrophobic residues on the protein surface and that the beta-defensin-specific fold appears to accommodate a wide range of different amino acids at most sequence positions. In addition to the implications for the mode of biological defensin actions, these findings are of particular interest because beta-defensins have been suggested as lead compounds for the development of novel peptide antibiotics for the therapy of infectious diseases.


Subject(s)
beta-Defensins/chemistry , Amino Acid Sequence , Animals , Chromatography , Conserved Sequence , Crystallography, X-Ray , Disulfides , Humans , Magnetic Resonance Spectroscopy , Mice , Models, Molecular , Molecular Sequence Data , Peptides/chemistry , Protein Conformation , Protein Folding , Protein Structure, Secondary , Protein Structure, Tertiary , Sequence Homology, Amino Acid
12.
J Biol Chem ; 276(34): 32064-70, 2001 Aug 24.
Article in English | MEDLINE | ID: mdl-11356847

ABSTRACT

Nun protein from coliphage HK022 binds to phage boxB RNA and functions, in contrast to phage lambda N protein, as a transcriptional terminator. The basic Nun-(10-44) peptide contains the boxB RNA binding arginine rich motif, ARM. The peptide binds boxB RNA and competes with the phage lambda ARM peptide N-(1-36) as indicated by nuclear magnetic resonance (NMR) spectroscopy titrations. In two-dimensional nuclear Overhauser enhancement spectroscopy experiments boxB RNA in complex with Nun-(20-44) exhibits the same pattern of resonances as it does in complex with N peptides containing the ARM, and we could show that Nun-(20-44) forms a bent alpha-helix upon binding to the boxB RNA. The structure of the boxB RNA-bound Nun-(20-44) was determined on the basis of 191 intra- and 30 intermolecular distance restraints. Ser-24 is anchored to the lower RNA stem, and stacking of Tyr-39 and A7 is clearly experimentally indicated. Arg-28 shows numerous contacts to the RNA stem. Leu-22, Ile-30, Trp-33, Ile-37, and Leu-41 form a hydrophobic surface, which could be a recognition site for additional host factors such as NusG. Such a hydrophobic surface area is not present in N-(1-36) bound to boxB RNA.


Subject(s)
Bacteriophage lambda/chemistry , Coliphages/chemistry , RNA, Viral/chemistry , Transcription Factors/chemistry , Transcription, Genetic , Viral Proteins/chemistry , Amino Acid Sequence , Base Sequence , Models, Molecular , Molecular Sequence Data , Molecular Structure , Nuclear Magnetic Resonance, Biomolecular , Sequence Homology, Amino Acid , Templates, Genetic
13.
J Biol Chem ; 276(25): 22756-63, 2001 Jun 22.
Article in English | MEDLINE | ID: mdl-11287426

ABSTRACT

Birch pollinosis is often accompanied by hypersensitivity to fruit as a consequence of the cross-reaction of pollen allergen-specific IgE antibodies with homologous food proteins. To provide a basis for examining the cross-reactivity on a structural level, we used heteronuclear multidimensional NMR spectroscopy to determine the high-resolution three-dimensional structure of the major cherry allergen, Pru av 1, in solution. Based on a detailed comparison of the virtually identical structures of Pru av 1 and Bet v 1, the major birch pollen allergen, we propose an explanation for a significant aspect of the observed cross-reactivity pattern among the family of allergens under consideration. The large hydrophobic cavity expected to be important for the still unknown physiological function of Bet v 1 is conserved in Pru av 1. Structural homology to a domain of human MLN64 associated with cholesterol transport suggests phytosteroids as putative ligands for Pru av 1. NMR spectroscopy provides experimental evidence that Pru av 1 interacts with phytosteroids, and molecular modeling shows that the hydrophobic cavity is large enough to accommodate two such molecules.


Subject(s)
Allergens , Cross Reactions , Hypersensitivity/immunology , Plant Proteins/chemistry , Amino Acid Sequence , Antigens, Plant , Humans , Hypersensitivity/therapy , Immunotherapy , Models, Molecular , Molecular Sequence Data , Nuclear Magnetic Resonance, Biomolecular , Plant Proteins/immunology , Protein Conformation , Recombinant Proteins/chemistry , Recombinant Proteins/immunology , Sequence Homology, Amino Acid
16.
Protein Sci ; 9(8): 1474-86, 2000 Aug.
Article in English | MEDLINE | ID: mdl-10975569

ABSTRACT

The rubredoxin from the cryptomonad Guillardia theta is one of the first examples of a rubredoxin encoded in a eukaryotic organism. The structure of a soluble zinc-substituted 70-residue G. theta rubredoxin lacking the membrane anchor and the thylakoid targeting sequence was determined by multidimensional heteronuclear NMR, representing the first three-dimensional (3D) structure of a eukaryotic rubredoxin. For the structure calculation a strategy was applied in which information about hydrogen bonds was directly inferred from a long-range HNCO experiment, and the dynamics of the protein was deduced from heteronuclear nuclear Overhauser effect data and exchange rates of the amide protons. The structure is well defined, exhibiting average root-mean-square deviations of 0.21 A for the backbone heavy atoms and 0.67 A for all heavy atoms of residues 7-56, and an increased flexibility toward the termini. The structure of this core fold is almost identical to that of prokaryotic rubredoxins. There are, however, significant differences with respect to the charge distribution at the protein surface, suggesting that G. theta rubredoxin exerts a different physiological function compared to the structurally characterized prokaryotic rubredoxins. The amino-terminal residues containing the putative signal peptidase recognition/cleavage site show an increased flexibility compared to the core fold, but still adopt a defined 3D orientation, which is mainly stabilized by nonlocal interactions to residues of the carboxy-terminal region. This orientation might reflect the structural elements and charge pattern necessary for correct signal peptidase recognition of the G. theta rubredoxin precursor.


Subject(s)
Eukaryota/enzymology , Rubredoxins/chemistry , Zinc/chemistry , Amino Acid Sequence , Crystallography, X-Ray , Cysteine/chemistry , Data Interpretation, Statistical , Eukaryota/chemistry , Eukaryota/metabolism , Hydrogen Bonding , Iron/chemistry , Mathematics , Models, Molecular , Molecular Sequence Data , Nuclear Magnetic Resonance, Biomolecular , Oxidation-Reduction , Protein Conformation , Protein Structure, Secondary , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Rubredoxins/genetics , Sequence Homology, Amino Acid , Solutions
17.
J Biol Chem ; 275(39): 30058-63, 2000 Sep 29.
Article in English | MEDLINE | ID: mdl-10878021

ABSTRACT

We analyzed a eukaryotically encoded rubredoxin from the cryptomonad Guillardia theta and identified additional domains at the N- and C-termini in comparison to known prokaryotic paralogous molecules. The cryptophytic N-terminal extension was shown to be a transit peptide for intracellular targeting of the protein to the plastid, whereas a C-terminal domain represents a membrane anchor. Rubredoxin was identified in all tested phototrophic eukaryotes. Presumably facilitated by its C-terminal extension, nucleomorph-encoded rubredoxin (nmRub) is associated with the thylakoid membrane. Association with photosystem II (PSII) was demonstrated by co-localization of nmRub and PSII membrane particles and PSII core complexes and confirmed by comparative electron paramagnetic resonance measurements. The midpoint potential of nmRub was determined as +125 mV, which is the highest redox potential of all known rubredoxins. Therefore, nmRub provides a striking example of the ability of the protein environment to tune the redox potentials of metal sites, allowing for evolutionary adaption in specific electron transport systems, as for example that coupled to the PSII pathway.


Subject(s)
Cell Nucleus/genetics , Chloroplasts/chemistry , Photosynthetic Reaction Center Complex Proteins/chemistry , Rubredoxins/isolation & purification , Biological Transport , Cell Compartmentation , Chloroplasts/ultrastructure , Eukaryota/chemistry , Eukaryota/ultrastructure , Eukaryotic Cells , Pisum sativum , Photosystem II Protein Complex , Protein Sorting Signals , Rubredoxins/metabolism
19.
FEBS Lett ; 471(2-3): 191-6, 2000 Apr 14.
Article in English | MEDLINE | ID: mdl-10767421

ABSTRACT

We have identified an open reading frame with homology to prokaryotic rubredoxins (rds) on a nucleomorph chromosome of the cryptomonad alga Guillardia theta. cDNA analysis let us propose that the rd preprotein has an NH(2)-terminal extension that functions as a transit peptide for import into the plastid. Compared to rds found in non-photosynthetic prokaryotes or found in bacteria that exhibit an anoxigenic photosynthesis apparatus, nucleomorph rd has a COOH-terminal extension, which shows high homology exclusively to the COOH-termini of cyanobacterial rds as well as to a hypothetical rd in the Arabidopsis genome. This extension can be divided into a putative membrane anchor and a stretch of about 20 amino acids with unknown function linking the common rd fold to this anchor. Overexpression of nucleomorph rd in Escherichia coli using a T7 RNA polymerase/promotor system resulted in a mixture of iron-containing holorubredoxin and zinc-substituted protein. Preliminary spectroscopic studies of the iron form of nucleomorph rd suggest the existence of a native rd-type iron site. One-dimensional nuclear magnetic resonance spectroscopy of recombinant Zn-rd suggests the presence of a stable tertiary fold similar to that of other rd structures determined previously.


Subject(s)
Eukaryota/cytology , Eukaryota/genetics , Eukaryotic Cells/cytology , Organelles/genetics , Rubredoxins/genetics , Amino Acid Sequence , Binding Sites , Biological Transport , Cell Nucleus/genetics , Cloning, Molecular , Eukaryota/metabolism , Iron/metabolism , Molecular Sequence Data , Open Reading Frames/genetics , Plastids/metabolism , Prokaryotic Cells/chemistry , Protein Conformation , Protein Precursors/chemistry , Protein Precursors/genetics , Protein Precursors/isolation & purification , Protein Precursors/metabolism , Protein Sorting Signals/genetics , Protein Sorting Signals/physiology , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Rubredoxins/chemistry , Rubredoxins/isolation & purification , Rubredoxins/metabolism , Sequence Alignment , Sequence Homology, Amino Acid , Spectrum Analysis , Symbiosis/genetics , Zinc/metabolism
20.
Eur J Biochem ; 267(8): 2397-408, 2000 Apr.
Article in English | MEDLINE | ID: mdl-10759866

ABSTRACT

The solution structure of a 15-mer nutRboxB RNA hairpin complexed with the 36-mer N-terminal peptide of the N protein (N36) from bacteriophage lambda was determined by 2D and 3D homonuclear and heteronuclear magnetic resonance spectroscopy. These 36 amino acids include the arginine-rich motif of the N protein involved in transcriptional antitermination of phage lambda. Upon complex formation with boxB RNA, the synthetic N36 peptide binds tightly to the major groove of the boxB hairpin through hydrophobic and electrostatic interactions forming a bent alpha helix. Four nucleotides of the GAAAA pentaloop of the boxB RNA adopt a GNRA-like tetraloop fold in the complex. The formation of a GAAA tetraloop involves a loop-closing sheared base pair (G6-A10), base stacking of three adenines (A7, A8, and A10), and extrusion of one nucleotide (A9) from the loop, as observed previously for the complex of N(1-22) peptide and the nutLboxB RNA [Legault, P., Li, J., Mogridge, J., Kay, L.E. & Greenblatt, J. (1998) Cell 93, 289-299]. Stacking of the bases is extended by the indole-ring of Trp18 which also forms hydrophobic contacts to the side-chains of Leu24, Leu25, and Val26. Based on the structure of the complex, three mutant peptides were synthesized and investigated by CD and NMR spectroscopy in order to determine the role of particular residues for complex formation. These studies revealed very distinct amino-acid requirements at positions 3, 4, and 8, while replacement of Trp18 with tyrosine did not result in any gross structural changes.


Subject(s)
Peptide Fragments/chemistry , RNA-Binding Proteins/chemistry , Ribonucleoproteins/chemistry , Amino Acid Sequence , Bacterial Proteins/chemistry , Circular Dichroism , Escherichia coli/metabolism , Gene Expression Regulation, Viral , Magnetic Resonance Spectroscopy , Models, Molecular , Molecular Sequence Data , Mutation , Nucleic Acid Conformation , Oligoribonucleotides/chemistry , Protein Structure, Secondary , RNA, Viral/chemistry , Viral Proteins/chemistry , Viral Regulatory and Accessory Proteins
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