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1.
Nat Commun ; 11(1): 687, 2020 Feb 04.
Artigo em Inglês | MEDLINE | ID: mdl-32019926

RESUMO

Glutamine fructose-6-phosphate amidotransferase (GFAT) is the key enzyme in the hexosamine pathway (HP) that produces uridine 5'-diphospho-N-acetyl-D-glucosamine (UDP-GlcNAc), linking energy metabolism with posttranslational protein glycosylation. In Caenorhabditis elegans, we previously identified gfat-1 gain-of-function mutations that elevate UDP-GlcNAc levels, improve protein homeostasis, and extend lifespan. GFAT is highly conserved, but the gain-of-function mechanism and its relevance in mammalian cells remained unclear. Here, we present the full-length crystal structure of human GFAT-1 in complex with various ligands and with important mutations. UDP-GlcNAc directly interacts with GFAT-1, inhibiting catalytic activity. The longevity-associated G451E variant shows drastically reduced sensitivity to UDP-GlcNAc inhibition in enzyme activity assays. Our structural and functional data point to a critical role of the interdomain linker in UDP-GlcNAc inhibition. In mammalian cells, the G451E variant potently activates the HP. Therefore, GFAT-1 gain-of-function through loss of feedback inhibition constitutes a potential target for the treatment of age-related proteinopathies.

2.
FEBS Open Bio ; 2020 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-32031736

RESUMO

Type VII collagen is an extracellular matrix protein which is important for skin stability; however, detailed information at the molecular level is scarce. The second vWFA (von-Willebrand-factor type A) domain of type VII collagen mediates important interactions, and immunization of mice induces skin blistering in certain strains. To understand vWFA2 function and the pathophysiological mechanisms leading to skin blistering, we structurally characterized this domain by X-ray crystallography and NMR-spectroscopy. Cell adhesion assays identified two new interactions: one with ß1 integrin via its RGD motif and one with laminin-332. The latter interaction was confirmed by surface plasmon resonance with a KD of about 1 mM. These data show that vWFA2 has additional functions in the extracellular matrix besides interacting with type I collagen.

3.
Artigo em Inglês | MEDLINE | ID: mdl-31944532

RESUMO

Collagen model peptides (CMPs) serve as tools for understanding stability and function of the collagen triple helix and have a potential for biomedical applications. In the past, interstrand cross-linking or conformational preconditioning of proline units through stereoelectronic effects have been utilized in the design of stabilized CMPs. To further study the effects determining collagen triple helix stability we investigated a series of CMPs containing synthetic diproline-mimicking modules (ProMs), which were preorganized in a PPII-helix-type conformation by a functionalizable intrastrand C2 bridge. Results of CD-based denaturation studies were correlated with calculated (DFT) conformational preferences of the ProM units, revealing that the relative helix stability is mainly governed by an interplay of main-chain preorganization, ring-flip preference, adaptability, and steric effects. Triple helix integrity was proven by crystal structure analysis and binding to HSP47.

4.
Stem Cell Res ; 43: 101713, 2020 Jan 17.
Artigo em Inglês | MEDLINE | ID: mdl-32000109

RESUMO

Chronic mucocutaneous candidiasis (CMC) is a disease that is characterized by susceptibility to chronic or recurrent infections with Candida spp. due to mutations affecting mainly the IL-17 signaling of T-Cells. The most common etiologies of CMC are gain-of-function (GOF) mutations in the STAT1 gene. In this paper we report the generation of a hiPSC line from a patient suffering from CMC due to a heterozygous GOF STAT1 p.R274Q mutation which can be used for disease modeling purposes.

5.
Clin Immunol ; 210: 108269, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31683054

RESUMO

Genetic studies have led to identification of an increasing number of monogenic primary immunodeficiency disorders. Monoallelic pathogenic gain-of-function (GOF) variants in NFKBIA, the gene encoding IκBα, result in an immunodeficiency disorder, typically accompanied by anhidrotic ectodermal dysplasia (EDA). So far, 14 patients with immunodeficiency due to NFKBIA GOF mutations have been reported. In this study we report three patients from the same family with immunodeficiency, presenting with recurrent respiratory tract infections, bronchiectasis and viral skin conditions due to a novel pathogenic NFKBIA variant (c.106 T > G, p.Ser36Ala), which results in reduced IκBα degradation. Immunological investigations revealed inadequate antibody responses against vaccine antigens, despite hypergammaglobulinemia. Interestingly, none of the studied patients displayed features of EDA. Therefore, missense NFKBIA variants substituting serine 36 of IκBα, differ from the rest of pathogenic GOF NFKBIA variants in that they cause combined immunodeficiency, even in the absence of EDA.

6.
Scand J Immunol ; 91(1): e12811, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31378960

RESUMO

Hypomorphic mutations in the gene encoding Bruton tyrosine kinase (BTK) may result in milder phenotypes and delayed diagnosis of B-cell related immunodeficiencies due to residual BTK function. Newborn screening for kappa-deleting-recombination-excision circles (KRECs) reliably identifies classical X-linked agammaglobulinaemia (XLA) patients with profound B-cell lymphopenia at birth but has not been evaluated in patients with residual BTK function. We aimed to evaluate clinical findings, BTK function and KREC copy numbers in three patients with BTK mutations presenting with impaired polysaccharide responsiveness without agammaglobulinaemia. One patient had an invasive pneumococcal infection at the age of 4 years. All three patients (two brothers) had visible tonsils, normal to slightly decreased immunoglobulin G levels, undetectable pneumococcal antibodies despite pneumococcal conjugate vaccinations, no antibody response after a diagnostic polysaccharide vaccination as well as profound B-cell lymphopenia with residual B-cell differentiation. BTK mutations were identified by Sanger sequencing. BTK staining and phosphorylation assays were performed on peripheral B cells. KREC copy numbers were determined from dried blood spots obtained within the first week of life as well as once at the age of 8, 6 and 3 years, respectively. BTK staining showed residual protein expression. Also, residual BTK activity could be demonstrated. KREC copy numbers from dried blood spots were above the threshold set for detection of patients with profound B-cell lymphopenia. Male patients with impaired polysaccharide responsiveness should be evaluated for B-cell lymphopenia followed by BTK analyses irrespective of immunoglobulin levels or tonsil size.


Assuntos
Tirosina Quinase da Agamaglobulinemia/genética , Agamaglobulinemia/diagnóstico , Agamaglobulinemia/etiologia , Mutação , Fenótipo , Polissacarídeos/imunologia , Biomarcadores , Criança , Pré-Escolar , Variações do Número de Cópias de DNA , Análise Mutacional de DNA , Seguimentos , Genótipo , Humanos , Imunofenotipagem , Recém-Nascido , Masculino , Triagem Neonatal , /etiologia , Avaliação de Sintomas
7.
Toxins (Basel) ; 11(11)2019 Nov 12.
Artigo em Inglês | MEDLINE | ID: mdl-31718085

RESUMO

RTX proteins are a large family of polypeptides of mainly Gram-negative origin that are secreted into the extracellular medium by a type I secretion system featuring a non-cleavable C-terminal secretion signal, which is preceded by a variable number of nine-residue tandem repeats. The three-dimensional structure forms a parallel ß-roll, where ß-strands of two parallel sheets are connected by calcium-binding linkers in such a way that a right-handed spiral is built. The Ca2+ ions are an integral part of the structure, which cannot form without them. The structural determinants of this unique architecture will be reviewed with its conservations and variations together with the implication for secretion and folding of these proteins. The general purpose of the RTX domains appears to act as an internal chaperone that keeps the polypeptide unfolded in the calcium-deprived cytosol and triggers folding in the calcium-rich extracellular medium. A rather recent addition to the structural biology of the RTX toxin is a variant occurring in a large RTX adhesin, where this non-canonical ß-roll binds to ice and diatoms.

8.
Ann Hum Genet ; 2019 Oct 09.
Artigo em Inglês | MEDLINE | ID: mdl-31596515

RESUMO

Wilson's disease is an autosomal recessive disorder resulting from copper excess. Some patients with clinical Wilson's disease symptoms exhibit no or only heterozygous pathogenic variants in the coding region of the disease-causing ATP7B gene. Therefore, the ATP7B promoter region is of special interest. Metal-responsive elements (MREs) located in the ATP7B promoter are promising motifs in modulating the ATP7B expression. We studied protein interaction of MREe, MREc, and MREd by electrophoretic mobility shift assays and revealed specific interactions for all MREs. We further narrowed down the specific binding site. Proteins potentially binding to the three MREs were identified by MatInspector analyses. Metal regulatory transcription factor 1 (MTF1) could be validated to bind to MREe by electrophoretic mobility shift assays. ATP7B promoter-driven reporter gene expression was significantly increased because of this interaction. MTF1 is a strong candidate in regulating the ATP7B expression through MREe binding.

9.
Hum Gene Ther ; 30(10): 1297-1305, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31502485

RESUMO

Adeno-associated virus (AAV) vector-mediated gene therapy is currently evaluated as a potential treatment for Crigler-Najjar syndrome (CN) (NCT03466463). Pre-existing immunity to AAV is known to hinder gene transfer efficacy, restricting enrollment of seropositive subjects in ongoing clinical trials. We assessed the prevalence of anti-AAV serotype 8 (AAV8) neutralizing antibodies (NAbs) in subjects affected by CN and investigated the impact of low NAb titers (<1:5) on liver gene transfer efficacy in an in vivo passive immunization model. A total of 49 subjects with a confirmed molecular diagnosis of CN were included in an international multicenter study (NCT02302690). Pre-existing NAbs against AAV8 were detected in 30.6% (15/49) of screened patients and, in the majority of positive cases, cross-reactivity to AAV2 and AAV5 was detected. To investigate the impact of low NAbs on AAV vector-mediated liver transduction efficiency, adult wild-type C57BL/6 mice were passively immunized with pooled human donor-derived immunoglobulins to achieve titers of up to 1:3.16. After immunization, animals were injected with different AAV8 vector preparations. Hepatic vector gene copy number was unaffected by low anti-AAV8 NAb titers when column-purified AAV vector batches containing both full and empty capsids were used. In summary, although pre-existing anti-AAV8 immunity can be found in about a third of subjects affected by CN, low anti-AAV8 NAb titers are less likely to affect liver transduction efficiency when using AAV vector preparations manufactured to contain both full and empty capsids. These findings have implications for the design of liver gene transfer clinical trials and for the definition of inclusion criteria related to seropositivity of potential participants.

10.
Front Immunol ; 10: 1272, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31379802

RESUMO

Introduction: The German PID-NET registry was founded in 2009, serving as the first national registry of patients with primary immunodeficiencies (PID) in Germany. It is part of the European Society for Immunodeficiencies (ESID) registry. The primary purpose of the registry is to gather data on the epidemiology, diagnostic delay, diagnosis, and treatment of PIDs. Methods: Clinical and laboratory data was collected from 2,453 patients from 36 German PID centres in an online registry. Data was analysed with the software Stata® and Excel. Results: The minimum prevalence of PID in Germany is 2.72 per 100,000 inhabitants. Among patients aged 1-25, there was a clear predominance of males. The median age of living patients ranged between 7 and 40 years, depending on the respective PID. Predominantly antibody disorders were the most prevalent group with 57% of all 2,453 PID patients (including 728 CVID patients). A gene defect was identified in 36% of patients. Familial cases were observed in 21% of patients. The age of onset for presenting symptoms ranged from birth to late adulthood (range 0-88 years). Presenting symptoms comprised infections (74%) and immune dysregulation (22%). Ninety-three patients were diagnosed without prior clinical symptoms. Regarding the general and clinical diagnostic delay, no PID had undergone a slight decrease within the last decade. However, both, SCID and hyper IgE- syndrome showed a substantial improvement in shortening the time between onset of symptoms and genetic diagnosis. Regarding treatment, 49% of all patients received immunoglobulin G (IgG) substitution (70%-subcutaneous; 29%-intravenous; 1%-unknown). Three-hundred patients underwent at least one hematopoietic stem cell transplantation (HSCT). Five patients had gene therapy. Conclusion: The German PID-NET registry is a precious tool for physicians, researchers, the pharmaceutical industry, politicians, and ultimately the patients, for whom the outcomes will eventually lead to a more timely diagnosis and better treatment.

11.
Nucleic Acids Res ; 47(14): 7363-7379, 2019 08 22.
Artigo em Inglês | MEDLINE | ID: mdl-31184713

RESUMO

LeuO is a pleiotropic LysR-type transcriptional regulator (LTTR) and co-regulator of the abundant nucleoid-associated repressor protein H-NS in Gammaproteobacteria. As other LTTRs, LeuO is a tetramer that is formed by dimerization of the N-terminal DNA-binding domain (DBD) and C-terminal effector-binding domain (EBD). To characterize the Escherichia coli LeuO protein, we screened for LeuO mutants that activate the cas (CRISPR-associated/Cascade) promoter more effectively than wild-type LeuO. This yielded nine mutants carrying amino acid substitutions in the dimerization interface of the regulatory EBD, as shown by solving the EBD's crystal structure. Superimposing of the crystal structures of LeuO-EBD and LeuO-S120D-EBD suggests that the Ser120 to Asp substitution triggers a structural change that is related to effector-induced structural changes of LTTRs. Corresponding functional analyses demonstrated that LeuO-S120D has a higher DNA-binding affinity than wild-type LeuO. Further, a palindromic DNA-binding core-site and a consensus sequence were identified by DNase I footprinting with LeuO-S120D as well as with the dimeric DBD. The data suggest that LeuO-S120D mimics an effector-induced form of LeuO regulating a distinct set of target loci. In general, constitutive mutants and determining the DNA-binding specificity of the DBD-dimer are feasible approaches to characterize LTTRs of unknown function.


Assuntos
Proteínas de Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Pleiotropia Genética/genética , Regiões Promotoras Genéticas/genética , Fatores de Transcrição/genética , DNA/química , DNA/genética , DNA/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Modelos Moleculares , Mutação , Conformação de Ácido Nucleico , Ligação Proteica , Domínios Proteicos , Multimerização Proteica , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo
12.
J Biol Chem ; 294(30): 11525-11535, 2019 Jul 26.
Artigo em Inglês | MEDLINE | ID: mdl-31182482

RESUMO

Pro-Pro endopeptidase-1 (PPEP-1) is a secreted metalloprotease from the bacterial pathogen Clostridium difficile that cleaves two endogenous adhesion proteins. PPEP-1 is therefore important for bacterial motility and hence for efficient gut colonization during infection. PPEP-1 exhibits a unique specificity for Pro-Pro peptide bonds within the consensus sequence VNP↓PVP. In this study, we combined information from crystal and NMR structures with mutagenesis and enzyme kinetics to investigate the mechanism and substrate specificity of PPEP-1. Our analyses revealed that the substrate-binding cleft of PPEP-1 is shaped complementarily to the major conformation of the substrate in solution. We found that it possesses features that accept a tertiary amide and help discriminate P1' residues by their amide hydrogen bond-donating potential. We also noted that residues Lys-101, Trp-103, and Glu-184 are crucial for proteolytic activity. Upon substrate binding, these residues position a flexible loop over the substrate-binding cleft and modulate the second coordination sphere of the catalytic zinc ion. On the basis of these findings, we propose an induced-fit model in which prestructured substrates are recognized followed by substrate positioning within the active-site cleft and a concomitant increase in the Lewis acidity of the catalytic Zn2+ ion. In conclusion, our findings provide detailed structural and mechanistic insights into the substrate recognition and specificity of PPEP-1 from the common gut pathogen C. difficile.

13.
Respir Res ; 20(1): 99, 2019 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-31118031

RESUMO

BACKGROUND: Recurrent and persistent infections are known to affect airways of patients with Primary Immunodeficiency despite appropriate replacement immunoglobulin serum levels. Interestingly, patients with Chronic Obstructive Pulmonary Disease or with non-CF bronchiectasis also show similar susceptibility to such infections. This may be due to the limited availability of immunoglobulins from the systemic circulation in the conductive airways, resulting in local immunodeficiency. Topical application of nebulized plasma-derived immunoglobulins may represent a means to address this deficiency. In this study, we assessed the feasibility of nebulizing plasma-derived immunoglobulins and delivering them into the airways of rats and non-human primates. METHODS: Distinct human plasma-derived immunoglobulin isotype preparations were nebulized with an investigational eFlow® nebulizer and analyzed in vitro or deposited into animals. Biochemical and immunohistological analysis of nebulized immunoglobulins were then performed. Lastly, efficacy of topically applied human plasma-derived immunoglobulins was assessed in an acute Streptococcus pneumoniae respiratory infection in mice. RESULTS: Characteristics of the resulting aerosols were comparable between preparations, even when using solutions with elevated viscosity. Neither the structural integrity nor the biological function of nebulized immunoglobulins were compromised by the nebulization process. In animal studies, immunoglobulins levels were assessed in plasma, broncho-alveolar lavages (BAL) and on lung sections of rats and non-human primates in samples collected up to 72 h following application. Nebulized immunoglobulins were detectable over 48 h in the BAL samples and up to 72 h on lung sections. Immunoglobulins recovered from BAL fluid up to 24 h after inhalation remained structurally and functionally intact. Importantly, topical application of human plasma-derived immunoglobulin G into the airways of mice offered significant protection against acute pneumococcal pneumonia. CONCLUSION: Taken together our data demonstrate the feasibility of topically applying plasma-derived immunoglobulins into the lungs using a nebulized liquid formulation. Moreover, topically administered human plasma-derived immunoglobulins prevented acute respiratory infection.


Assuntos
Imunoglobulina A/administração & dosagem , Imunoglobulina G/administração & dosagem , Imunoglobulina M/administração & dosagem , Pulmão/efeitos dos fármacos , Nebulizadores e Vaporizadores/tendências , Administração Tópica , Animais , Relação Dose-Resposta a Droga , Humanos , Imunoglobulina A/metabolismo , Imunoglobulina G/metabolismo , Pulmão/metabolismo , Macaca fascicularis , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Primatas , Ratos , Ratos Sprague-Dawley , Especificidade da Espécie
15.
Diseases ; 7(2)2019 Apr 04.
Artigo em Inglês | MEDLINE | ID: mdl-30987377

RESUMO

Immunodeficiency, centromeric instability and facial anomalies syndrome 2 (ICF2) is a rare autosomal recessive primary immunodeficiency disorder. So far, 27 patients have been reported. Here, we present three siblings with ICF2 due to a homozygous ZBTB24 gene mutation (c.1222 T>G, p. (Cys408Gly)). Immune deficiency in these patients ranged from late-onset combined immunodeficiency (CID) with severe respiratory tract infections and recurrent shingles to asymptomatic selective antibody deficiency. Evident clinical heterogeneity manifested despite a common genetic background, suggesting the pathogenic relevance of epigenetic modification. Immunological follow-up reveals a previously unidentified gradual depletion of B and CD4⁺ T cells in all three presented patients with transition of a common variable immunodeficiency (CVID)-like disease to late-onset-CID in one of them. Considering all previously published cases with ICF2, we identify inadequate antibody responses to vaccines and reduction in CD27⁺ memory B cells as prevalent immunological traits. High mortality among ICF2 patients (20%) together with the progressive course of immunodeficiency suggest that hematopoietic stem cell transplantation (HSCT) should be considered as a treatment option in due time.

16.
J Hepatol ; 70(5): 822-823, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30979394
18.
Photochem Photobiol Sci ; 18(6): 1398-1407, 2019 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-30924488

RESUMO

In photopharmacology, photoswitchable compounds including azobenzene or other diarylazo moieties exhibit bioactivity against a target protein typically in the slender E-configuration, whereas the rather bulky Z-configuration usually is pharmacologically less potent. Herein we report the design, synthesis and photochemical/inhibitory characterization of new photoswitchable kinase inhibitors targeting p38α MAPK and CK1δ. A well characterized inhibitor scaffold was used to attach arylazo- and diazocine moieties. When the isolated isomers, or the photostationary state (PSS) of isomers, were tested in commonly used in vitro kinase assays, however, only small differences in activity were observed. X-ray analyses of ligand-bound p38α MAPK and CK1δ complexes revealed dynamic conformational adaptations of the protein with respect to both isomers. More importantly, irreversible reduction of the azo group to the corresponding hydrazine was observed. Independent experiments revealed that reducing agents such as DTT (dithiothreitol) and GSH (glutathione) that are typically used for protein stabilization in biological assays were responsible. Two further sources of error are the concentration dependence of the E-Z-switching efficiency and artefacts due to incomplete exclusion of light during testing. Our findings may also apply to a number of previously investigated azobenzene-based photoswitchable inhibitors.


Assuntos
Azocinas/farmacologia , Caseína Quinase Idelta/antagonistas & inibidores , Imidazóis/farmacologia , Proteína Quinase 14 Ativada por Mitógeno/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Tiazóis/farmacologia , Azocinas/química , Caseína Quinase Idelta/metabolismo , Relação Dose-Resposta a Droga , Imidazóis/química , Ligantes , Proteína Quinase 14 Ativada por Mitógeno/metabolismo , Modelos Moleculares , Estrutura Molecular , Processos Fotoquímicos , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Relação Estrutura-Atividade , Tiazóis/química
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