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1.
Front Immunol ; 13: 979491, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36091065

RESUMO

Hookworms infect more that 400 million people and cause significant socio-economic burden on endemic countries. The lack of efficient vaccines and the emergence of anthelminthic drug resistance are of major concern. Free-living hookworm larvae infect their hosts via the skin and live as adult worms in the small intestine where they feed on host tissue and blood. Excretory/secretory (E/S) products, released by helminths as they migrate through their host, are thought to play a key role in facilitating infection and successful establishment of parasitism. However, E/S products can also elicit protective immune responses that might be harnessed for vaccine development. By performing Western blots with serum of Nippostrongylus brasiliensis (Nb) infected mice as a model for human hookworm infection, we identified a largely overlapping set of IgG1- and IgE-reactive antigens in E/S from infective L3 stage larvae. Mass spectrometry analysis led to the identification of a new protein family with 6 paralogues in the Nb genome which we termed Nb-LSA1 for "Nippostrongylus brasiliensis larval secreted protein 1". The recombinantly expressed 17 kDa family member Nb-LSA1a was recognized by antibodies in the serum of Nb immune mice. Immunization of mice with Nb-LSA1a in alum elicited a strong IgG1 response but no detectable antigen-specific IgE. Most importantly, immunized mice were largely protected against a challenge Nb infection. This effect was dependent on the presence of basophils and occurred before the parasites reached the intestine. Therefore, basophils appear to play a critical role for rapid control of infection with L3 stage larvae in mice immunized with a single secreted larval protein. A better understanding of basophil-mediated protective immunity and identification of potent larval antigens of human hookworms could help to develop promising vaccination strategies.


Assuntos
Antígenos de Helmintos , Basófilos , Ancylostomatoidea , Animais , Humanos , Imunoglobulina E , Imunoglobulina G , Larva , Camundongos , Nippostrongylus
2.
Ann Neurol ; 2022 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-36073542

RESUMO

OBJECTIVE: Rare inherited missense variants in SLC32A1, the gene that encodes the vesicular gamma-aminobutyric acid (GABA) transporter, have recently been shown to cause genetic epilepsy with febrile seizures plus. We aimed to clarify if de novo missense variants in SLC32A1 can also cause epilepsy with impaired neurodevelopment. METHODS: Using exome sequencing, we identified four individuals with a developmental and epileptic encephalopathy and de novo missense variants in SLC32A1. To assess causality, we performed functional evaluation of the identified variants in a murine neuronal cell culture model. RESULTS: The main phenotype comprises moderate-to-severe intellectual disability, infantile-onset epilepsy within the first 18 months of life, and a choreiform, dystonic, or dyskinetic movement disorder. In silico modeling and functional analyses reveal that three of these variants, which are located in helices that line the putative GABA transport pathway, result in reduced quantal size, consistent with impaired filling of synaptic vesicles with GABA. The fourth variant, located in the vesicular gamma-aminobutyric acid N-terminus, does not affect quantal size, but increases presynaptic release probability, leading to more severe synaptic depression during high-frequency stimulation. Thus, variants in vesicular gamma-aminobutyric acid can impair GABAergic neurotransmission through at least two mechanisms, by affecting synaptic vesicle filling and by altering synaptic short-term plasticity. INTERPRETATION: This work establishes de novo missense variants in SLC32A1 as a novel cause of a developmental and epileptic encephalopathy. SUMMARY FOR SOCIAL MEDIA IF PUBLISHED: @platzer_k @lemke_johannes @RamiJamra @Nirgalito @GeneDx The SLC family 32 Member 1 (SLC32A1) is the only protein identified to date, that loads gamma-aminobutyric acid (GABA) and glycine into synaptic vesicles, and is therefore also known as the vesicular GABA transporter (VGAT) or vesicular inhibitory amino acid transporter (VIAAT). Rare inherited missense variants in SLC32A1, the gene that encodes VGAT/vesicular inhibitory amino acid transporter, have recently been shown to cause genetic epilepsy with febrile seizures plus. We aimed to clarify if de novo missense variants in SLC32A1 can also cause epilepsy with impaired neurodevelopment. We report on four individuals with de novo missense variants in SLC32A1 and a developmental and epileptic encephalopathy with infantile onset epilepsy. We establish causality of the variants via in silico modeling and their functional evaluation in a murine neuronal cell culture model. SLC32A1 variants represent a novel genetic etiology in neurodevelopmental disorders with epilepsy and a new GABA-related disease mechanism. ANN NEUROL 2022.

3.
Cell Mol Life Sci ; 79(9): 475, 2022 Aug 09.
Artigo em Inglês | MEDLINE | ID: mdl-35943635

RESUMO

In malignant melanoma, a highly aggressive form of skin cancer, many microRNAs are aberrantly expressed contributing to tumorigenesis and progression. Further, deregulation of microRNA processing enzymes, like the miRNA-binding protein Argonaute 2, significantly impacts microRNA function. This study characterizes a novel splice variant of Argonaut 2, AGO2-ex1/3. AGO2-ex1/3 is substantially expressed in different melanoma cell lines and patient-derived tissue samples. It is a mature mRNA, which is translated into an N-terminally truncated Argonaute 2 protein form. Molecular dynamics simulations show that the PAZ, MID, and PIWI domain largely retain their structure in AGO2-ex1/3 and that the truncation of the N-terminus leads to an increased interdomain flexibility. Expression of AGO2-ex1/3 provides a survival advantage for melanoma cells while the knockdown causes significantly reduced proliferation and increases apoptosis. RNA-sequencing revealed that in cells lacking AGO2-ex1/3 expression many miRNA target genes are deregulated, implicating a considerable role of AGO2-ex1/3 for miRNA function. This study inaugurates insights into an important role of a so far unknown splice variant of Argonaute 2 for the miRNA pathway as well as the mechanisms which drive growth and survival of melanoma cells. This knowledge provides the basis for potential new promising therapeutic targets focusing on small RNA-mediated gene regulation in melanoma.


Assuntos
Melanoma , MicroRNAs , Neoplasias Cutâneas , Apoptose/genética , Proteínas Argonauta/genética , Proteínas Argonauta/metabolismo , Humanos , Melanoma/genética , MicroRNAs/genética , MicroRNAs/metabolismo , Interferência de RNA , Neoplasias Cutâneas/genética
4.
Int J Mol Sci ; 23(15)2022 Jul 25.
Artigo em Inglês | MEDLINE | ID: mdl-35897753

RESUMO

Mutations in the spike protein of SARS-CoV-2 can lead to evasion from neutralizing antibodies and affect the efficacy of passive and active immunization strategies. Immunization of mice harboring an entire set of human immunoglobulin variable region gene segments allowed to identify nine neutralizing monoclonal antibodies, which either belong to a cluster of clonally related RBD or NTD binding antibodies. To better understand the genetic barrier to emergence of SARS-CoV-2 variants resistant to these antibodies, escape mutants were selected in cell culture to one antibody from each cluster and a combination of the two antibodies. Three independently derived escape mutants to the RBD antibody harbored mutations in the RBD at the position T478 or S477. These mutations impaired the binding of the RBD antibodies to the spike protein and conferred resistance in a pseudotype neutralization assay. Although the binding of the NTD cluster antibodies were not affected by the RBD mutations, the RBD mutations also reduced the neutralization efficacy of the NTD cluster antibodies. The mutations found in the escape variants to the NTD antibody conferred resistance to the NTD, but not to the RBD cluster antibodies. A variant resistant to both antibodies was more difficult to select and only emerged after longer passages and higher inoculation volumes. VOC carrying the same mutations as the ones identified in the escape variants were also resistant to neutralization. This study further underlines the rapid emergence of escape mutants to neutralizing monoclonal antibodies in cell culture and indicates the need for thorough investigation of escape mutations to select the most potent combination of monoclonal antibodies for clinical use.


Assuntos
Anticorpos Neutralizantes , COVID-19 , Animais , Anticorpos Monoclonais , Anticorpos Antivirais , Humanos , Camundongos , Mutação , Testes de Neutralização , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/química
5.
Clin Genet ; 2022 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-35908153

RESUMO

TCF4 haploinsufficiency by deletions, truncating variants or loss-of-function missense variants within the DNA-binding and protein interacting bHLH domain causes Pitt-Hopkins syndrome (PTHS). This neurodevelopmental disorder (NDD) is characterized by severe intellectual disability (ID), epilepsy, hyperbreathing and a typical facial gestalt. Only few aberrations of the N-terminus of TCF4 were associated with milder or atypical phenotypes. By personal communication and searching databases we assembled six cases with the novel, recurrent, de novo missense variant c.1165C > T, p.(Arg389Cys) in TCF4. This variant was identified by diagnostic exome or panel sequencing and is located upstream of the bHLH domain. All six individuals presented with moderate to severe ID with language impairment. Microcephaly occurred in two individuals, epilepsy only in one, and no breathing anomalies or myopia were reported. Facial gestalt showed some aspects of PTHS but was rather non-specific in most individuals. Interestingly, the variant is located within the AD2 activation domain next to a highly conserved coactivator-recruitment motif and might alter interaction with coactivator proteins independently from the bHLH domain. Our findings of a recurrent missense variant outside the bHLH domain in six individuals with an ID phenotype overlapping with but not typical for PTHS delineate a novel genotype-phenotype correlation for TCF4-related NDDs.

6.
Am J Hum Genet ; 109(8): 1421-1435, 2022 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-35830857

RESUMO

PPFIBP1 encodes for the liprin-ß1 protein, which has been shown to play a role in neuronal outgrowth and synapse formation in Drosophila melanogaster. By exome and genome sequencing, we detected nine ultra-rare homozygous loss-of-function variants in 16 individuals from 12 unrelated families. The individuals presented with moderate to profound developmental delay, often refractory early-onset epilepsy, and progressive microcephaly. Further common clinical findings included muscular hyper- and hypotonia, spasticity, failure to thrive and short stature, feeding difficulties, impaired vision, and congenital heart defects. Neuroimaging revealed abnormalities of brain morphology with leukoencephalopathy, ventriculomegaly, cortical abnormalities, and intracranial periventricular calcifications as major features. In a fetus with intracranial calcifications, we identified a rare homozygous missense variant that by structural analysis was predicted to disturb the topology of the SAM domain region that is essential for protein-protein interaction. For further insight into the effects of PPFIBP1 loss of function, we performed automated behavioral phenotyping of a Caenorhabditis elegans PPFIBP1/hlb-1 knockout model, which revealed defects in spontaneous and light-induced behavior and confirmed resistance to the acetylcholinesterase inhibitor aldicarb, suggesting a defect in the neuronal presynaptic zone. In conclusion, we establish bi-allelic loss-of-function variants in PPFIBP1 as a cause of an autosomal recessive severe neurodevelopmental disorder with early-onset epilepsy, microcephaly, and periventricular calcifications.


Assuntos
Epilepsia , Microcefalia , Malformações do Sistema Nervoso , Transtornos do Neurodesenvolvimento , Acetilcolinesterase/genética , Animais , Drosophila melanogaster/genética , Epilepsia/genética , Perda de Heterozigosidade , Microcefalia/genética , Transtornos do Neurodesenvolvimento/genética , Linhagem
7.
NPJ Genom Med ; 7(1): 45, 2022 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-35906228

RESUMO

The magnitude of clinical utility of preconception expanded carrier screening (ECS) concerning its potential to reduce the risk of affected offspring is unknown. Since neurodevelopmental disorders (NDDs) in their offspring is a major concern of parents-to-be, we addressed the question of residual risk by assessing the risk-reduction potential for NDDs in a retrospective study investigating ECS with different criteria for gene selection and definition of pathogenicity. We used exome sequencing data from 700 parents of children with NDDs and blindly screened for carrier-alleles in up to 3046 recessive/X-linked genes. Depending on variant pathogenicity thresholds and gene content, NDD-risk-reduction potential was up to 43.5% in consanguineous, and 5.1% in nonconsanguineous couples. The risk-reduction-potential was compromised by underestimation of pathogenicity of missense variants (false-negative-rate 4.6%), inherited copy-number variants and compound heterozygosity of one inherited and one de novo variant (0.9% each). Adherence to the ACMG recommendations of restricting ECS to high-frequency genes in nonconsanguineous couples would more than halve the detectable inherited NDD-risk. Thus, for optimized clinical utility of ECS, screening in recessive/X-linked genes regardless of their frequency (ACMG Tier-4) and sensible pathogenicity thresholds should be considered for all couples seeking ECS.

8.
Int J Mol Sci ; 23(11)2022 Jun 04.
Artigo em Inglês | MEDLINE | ID: mdl-35682988

RESUMO

Based on the structure of a de novo designed miniprotein (LCB1) in complex with the receptor binding domain (RBD) of the SARS-CoV-2 spike protein, we have generated and characterized truncated peptide variants of LCB1, which present only two of the three LCB1 helices, and which fully retained the virus neutralizing potency against different SARS-CoV-2 variants of concern (VOC). This antiviral activity was even 10-fold stronger for a cyclic variant of the two-helix peptides, as compared to the full-length peptide. Furthermore, the proteolytic stability of the cyclic peptide was substantially improved, rendering it a better potential candidate for SARS-CoV-2 therapy. In a more mechanistic approach, the peptides also served as tools to dissect the role of individual mutations in the RBD for the susceptibility of the resulting virus variants to neutralization by the peptides. As the peptides reported here were generated through chemical synthesis, rather than recombinant protein expression, they are amenable to further chemical modification, including the incorporation of a wide range of non-proteinogenic amino acids, with the aim to further stabilize the peptides against proteolytic degradation, as well as to improve the strength, as well the breadth, of their virus neutralizing capacity.


Assuntos
COVID-19 , SARS-CoV-2 , Enzima de Conversão de Angiotensina 2 , Anticorpos Neutralizantes , Anticorpos Antivirais , Humanos , Peptídeos/genética , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus
9.
Clin Genet ; 102(3): 182-190, 2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-35662002

RESUMO

While inherited hemizygous variants in PHF6 cause X-linked recessive Borjeson-Forssman-Lehmann syndrome (BFLS) in males, de novo heterozygous variants in females are associated with an overlapping but distinct phenotype, including moderate to severe intellectual disability, characteristic facial dysmorphism, dental, finger and toe anomalies, and linear skin pigmentation. By personal communication with colleagues, we assembled 11 additional females with BFLS due to variants in PHF6. We confirm the distinct phenotype to include variable intellectual disability, recognizable facial dysmorphism and other anomalies. We observed skewed X-inactivation in blood and streaky skin pigmentation compatible with functional mosaicism. Variants occurred de novo in 10 individuals, of whom one was only mildly affected and transmitted it to her more severely affected daughter. The mutational spectrum comprises a two-exon deletion, five truncating, one splice-site and three missense variants, the latter all located in the PHD2 domain and predicted to severely destabilize the domain structure. This observation supports the hypothesis of more severe variants in females contributing to gender-specific phenotypes in addition to or in combination with effects of X-inactivation and functional mosaicism. Therefore, our findings further delineate the clinical and mutational spectrum of female BFLS and provide further insights into possible genotype-phenotype correlations between females and males.


Assuntos
Hipogonadismo , Deficiência Intelectual , Retardo Mental Ligado ao Cromossomo X , Anormalidades Musculoesqueléticas , Proteínas Repressoras , Epilepsia , Face/anormalidades , Feminino , Dedos/anormalidades , Transtornos do Crescimento , Humanos , Hipogonadismo/genética , Deficiência Intelectual/complicações , Masculino , Retardo Mental Ligado ao Cromossomo X/genética , Anormalidades Musculoesqueléticas/complicações , Obesidade , Proteínas Repressoras/genética
10.
Genet Med ; 24(8): 1761-1773, 2022 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-35511136

RESUMO

PURPOSE: The study aimed to investigate the role of PABPC1 in developmental delay (DD). METHODS: Children were examined by geneticists and pediatricians. Variants were identified using exome sequencing and standard downstream bioinformatics pipelines. We performed in silico molecular modeling and coimmunoprecipitation to test if the variants affect the interaction between PABPC1 and PAIP2. We performed in utero electroporation of mouse embryo brains to enlighten the function of PABPC1. RESULTS: We describe 4 probands with an overlapping phenotype of DD, expressive speech delay, and autistic features and heterozygous de novo variants that cluster in the PABP domain of PABPC1. Further symptoms were seizures and behavioral disorders. Molecular modeling predicted that the variants are pathogenic and would lead to decreased binding affinity to messenger RNA metabolism-related proteins, such as PAIP2. Coimmunoprecipitation confirmed this because it showed a significant weakening of the interaction between mutant PABPC1 and PAIP2. Electroporation of mouse embryo brains showed that Pabpc1 knockdown decreases the proliferation of neural progenitor cells. Wild-type Pabpc1 could rescue this disturbance, whereas 3 of the 4 variants did not. CONCLUSION: Pathogenic variants in the PABP domain lead to DD, possibly because of interference with the translation initiation and subsequently an impaired neurogenesis in cortical development.


Assuntos
Deficiência Intelectual , Transtornos do Neurodesenvolvimento , Proteína I de Ligação a Poli(A)/metabolismo , Animais , Criança , Deficiências do Desenvolvimento/genética , Heterozigoto , Humanos , Deficiência Intelectual/genética , Camundongos , Transtornos do Neurodesenvolvimento/genética , Proteína I de Ligação a Poli(A)/química , RNA Mensageiro , Proteínas de Ligação a RNA/genética , Sequenciamento Completo do Exoma
11.
Viruses ; 14(5)2022 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-35632762

RESUMO

The nucleo-cytoplasmic capsid egress of herpesviruses is a unique regulated process that ensures the efficiency of viral replication and release. For human cytomegalovirus (HCMV), the core of the nuclear egress complex (NEC) consists of the pUL50-pUL53 heterodimer that is able to oligomerize and thus to build hexameric lattices. These structures determine capsid binding and multicomponent protein interaction including NEC-associated host factors. The underlying characteristic of the core NEC formation is based on the N-terminal hook structure of pUL53 that binds into an alpha-helical groove of pUL50, and is thus described as a hook-into-groove interaction. This central regulatory element has recently been validated as a target of antiviral strategies, and first NEC-targeted prototypes of inhibitory small molecules were reported by our previous study. Here, we further analyzed the oligomerization properties of the viral NEC through an approach of chemical protein cross-linking. Findings were as follows: (i) a cross-link approach demonstrated the oligomeric state of the HCMV core NEC using material from HCMV-infected or plasmid-transfected cells, (ii) a Western blot-based identification of NEC-associated kinases using the cross-linked multicomponent NECs was successful, and (iii) we demonstrated the NEC-inhibitory and antiviral activity of specific inhibitors directed to these target kinases. Combined, the results strongly underline the functional importance of the oligomerization of the HCMV-specific NEC that is both phosphorylation-dependent and sensitive to antiviral kinase inhibitors.


Assuntos
Antivirais , Citomegalovirus , Antivirais/metabolismo , Antivirais/farmacologia , Citomegalovirus/metabolismo , Humanos , Recém-Nascido , Proteínas Virais/metabolismo , Liberação de Vírus , Replicação Viral
12.
J Control Release ; 347: 115-126, 2022 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-35489547

RESUMO

3D printing of biomaterials enables spatial control of drug incorporation during automated manufacturing. This study links bioresponsive release of the anabolic biologic, insulin-like growth factor-I (IGF-I) in response to matrix metalloproteinases (MMP) to 3D printing using the block copolymer of poly(2-methyl-2-oxazoline) and thermoresponsive poly(2-n-propyl-2-oxazine) (POx-b-POzi). For that, a chemo-enzymatic synthesis was deployed, ligating IGF-I enzymatically to a protease sensitive linker (PSL), which was conjugated to a POx-b-POzi copolymer. The product was blended with the plain thermogelling POx-b-POzi hydrogel. MMP exposure of the resulting hydrogel triggered bioactive IGF-I release. The bioresponsive IGF-I containing POx-b-POzi hydrogel system was further detailed for shape control and localized incorporation of IGF-I via extrusion 3D printing for future applications in biomedicine and biofabrication.


Assuntos
Hidrogéis , Fator de Crescimento Insulin-Like I , Materiais Biocompatíveis/metabolismo , Hidrogéis/metabolismo , Metaloproteinases da Matriz/metabolismo , Polímeros , Impressão Tridimensional
13.
Am J Hum Genet ; 109(5): 944-952, 2022 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-35358416

RESUMO

Calcium (Ca2+) is a universal second messenger involved in synaptogenesis and cell survival; consequently, its regulation is important for neurons. ATPase plasma membrane Ca2+ transporting 1 (ATP2B1) belongs to the family of ATP-driven calmodulin-dependent Ca2+ pumps that participate in the regulation of intracellular free Ca2+. Here, we clinically describe a cohort of 12 unrelated individuals with variants in ATP2B1 and an overlapping phenotype of mild to moderate global development delay. Additional common symptoms include autism, seizures, and distal limb abnormalities. Nine probands harbor missense variants, seven of which were in specific functional domains, and three individuals have nonsense variants. 3D structural protein modeling suggested that the variants have a destabilizing effect on the protein. We performed Ca2+ imaging after introducing all nine missense variants in transfected HEK293 cells and showed that all variants lead to a significant decrease in Ca2+ export capacity compared with the wild-type construct, thus proving their pathogenicity. Furthermore, we observed for the same variant set an incorrect intracellular localization of ATP2B1. The genetic findings and the overlapping phenotype of the probands as well as the functional analyses imply that de novo variants in ATP2B1 lead to a monogenic form of neurodevelopmental disorder.


Assuntos
Deficiência Intelectual , Malformações do Sistema Nervoso , Transtornos do Neurodesenvolvimento , Células HEK293 , Humanos , Deficiência Intelectual/diagnóstico , Deficiência Intelectual/genética , Mutação de Sentido Incorreto/genética , Transtornos do Neurodesenvolvimento/genética , Fenótipo , ATPases Transportadoras de Cálcio da Membrana Plasmática/genética
14.
Brain ; 145(6): 1916-1923, 2022 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-35202461

RESUMO

The Kennedy pathways catalyse the de novo synthesis of phosphatidylcholine and phosphatidylethanolamine, the most abundant components of eukaryotic cell membranes. In recent years, these pathways have moved into clinical focus because four of ten genes involved have been associated with a range of autosomal recessive rare diseases such as a neurodevelopmental disorder with muscular dystrophy (CHKB), bone abnormalities and cone-rod dystrophy (PCYT1A) and spastic paraplegia (PCYT2, SELENOI). We identified six individuals from five families with bi-allelic variants in CHKA presenting with severe global developmental delay, epilepsy, movement disorders and microcephaly. Using structural molecular modelling and functional testing of the variants in a cell-based Saccharomyces cerevisiae model, we determined that these variants reduce the enzymatic activity of CHKA and confer a significant impairment of the first enzymatic step of the Kennedy pathway. In summary, we present CHKA as a novel autosomal recessive gene for a neurodevelopmental disorder with epilepsy and microcephaly.


Assuntos
Colina Quinase , Epilepsia , Microcefalia , Malformações do Sistema Nervoso , Transtornos do Neurodesenvolvimento , Alelos , Colina Quinase/genética , Epilepsia/genética , Humanos , Microcefalia/complicações , Microcefalia/genética , Malformações do Sistema Nervoso/genética , Transtornos do Neurodesenvolvimento/genética
15.
Int J Mol Sci ; 23(3)2022 Jan 26.
Artigo em Inglês | MEDLINE | ID: mdl-35163341

RESUMO

The histamine H1 receptor (H1R) is a G protein-coupled receptor (GPCR) and represents a main target in the treatment of allergic reactions as well as inflammatory reactions and depressions. Although the overall effect of antagonists on H1 function has been extensively investigated, rather little is known about the potential modulatory effect of ions or sequence variants on antagonist binding. We investigated the dynamics of a phosphate ion present in the crystal structure and of a sodium ion, for which we determined the position in the allosteric pocket by metadynamics simulations. Both types of ions exhibit significant dynamics within their binding site; however, some key contacts remain stable over the simulation time, which might be exploited to develop more potent drugs targeting these sites. The dynamics of the ions is almost unaffected by the presence or absence of doxepin, as also reflected in their small effect (less than 1 kcal·mol-1) on doxepin binding affinity. We also examined the effect of four H1R sequence variants observed in the human population on doxepin binding. These variants cause a reduction in doxepin affinity of up to 2.5 kcal·mol-1, indicating that personalized medical treatments that take into account individual mutation patterns could increase precision in the dosage of GPCR-targeting drugs.


Assuntos
Doxepina , Histamina , Sítios de Ligação , Doxepina/química , Doxepina/metabolismo , Histamina/metabolismo , Antagonistas dos Receptores Histamínicos H1 , Humanos , Íons , Receptores Acoplados a Proteínas G/metabolismo , Receptores Histamínicos/metabolismo , Receptores Histamínicos H1/metabolismo
16.
Int J Biochem Cell Biol ; 145: 106185, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-35219876

RESUMO

Viroporins are indispensable for viral replication. As intracellular ion channels they disturb pH gradients of organelles and allow Ca2+ flux across ER membranes. Viroporins interact with numerous intracellular proteins and pathways and can trigger inflammatory responses. Thus, they are relevant targets in the search for antiviral drugs. Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) underlies the world-wide pandemic of COVID-19, where an effective therapy is still lacking despite impressive progress in the development of vaccines and vaccination campaigns. Among the 29 proteins of SARS-CoV-2, the E- and ORF3a proteins have been identified as viroporins that contribute to the massive release of inflammatory cytokines observed in COVID-19. Here, we describe structure and function of viroporins and their role in inflammasome activation and cellular processes during the virus replication cycle. Techniques to study viroporin function are presented, with a focus on cellular and electrophysiological assays. Contributions of SARS-CoV-2 viroporins to the viral life cycle are discussed with respect to their structure, channel function, binding partners, and their role in viral infection and virus replication. Viroporin sequences of new variants of concern (α-ο) of SARS-CoV-2 are briefly reviewed as they harbour changes in E and 3a proteins that may affect their function.


Assuntos
COVID-19 , Humanos , Estágios do Ciclo de Vida , SARS-CoV-2 , Proteínas Viroporinas
17.
Alzheimers Res Ther ; 14(1): 15, 2022 01 21.
Artigo em Inglês | MEDLINE | ID: mdl-35063014

RESUMO

BACKGROUND: Alzheimer's disease (AD), the most common form of dementia, is a progressive neurodegenerative disorder that mainly affects older adults. One of the pathological hallmarks of AD is abnormally aggregated Tau protein that forms fibrillar deposits in the brain. In AD, Tau pathology correlates strongly with clinical symptoms, cognitive dysfunction, and neuronal death. METHODS: We aimed to develop novel therapeutic D-amino acid peptides as Tau fibrillization inhibitors. It has been previously demonstrated that D-amino acid peptides are protease stable and less immunogenic than L-peptides, and these characteristics may render them suitable for in vivo applications. Using a phage display procedure against wild type full-length Tau (TauFL), we selected a novel Tau binding L-peptide and synthesized its D-amino acid version ISAD1 and its retro inversed form, ISAD1rev, respectively. RESULTS: While ISAD1rev inhibited Tau aggregation only moderately, ISAD1 bound to Tau in the aggregation-prone PHF6 region and inhibited fibrillization of TauFL, disease-associated mutant full-length Tau (TauFLΔK, TauFL-A152T, TauFL-P301L), and pro-aggregant repeat domain Tau mutant (TauRDΔK). ISAD1 and ISAD1rev induced the formation of large high molecular weight TauFL and TauRDΔK oligomers that lack proper Thioflavin-positive ß-sheet conformation even at lower concentrations. In silico modeling of ISAD1 Tau interaction at the PHF6 site revealed a binding mode similar to those known for other PHF6 binding peptides. Cell culture experiments demonstrated that ISAD1 and its inverse form are taken up by N2a-TauRDΔK cells efficiently and prevent cytotoxicity of externally added Tau fibrils as well as of internally expressed TauRDΔK. CONCLUSIONS: ISAD1 and related peptides may be suitable for therapy development of AD by promoting off-pathway assembly of Tau, thus preventing its toxicity.


Assuntos
Doença de Alzheimer , Peptídeos , Proteínas tau , Idoso , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/patologia , Aminoácidos/uso terapêutico , Células Cultivadas , Humanos , Peptídeos/uso terapêutico , Conformação Proteica em Folha beta , Proteínas tau/metabolismo , Proteínas tau/toxicidade
18.
J Biol Chem ; 298(3): 101625, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-35074430

RESUMO

Varicella-zoster virus (VZV) is a human pathogen from the α-subfamily of herpesviruses. The VZV Orf24-Orf27 complex represents the essential viral core nuclear egress complex (NEC) that orchestrates the egress of the preassembled virus capsids from the nucleus. While previous studies have primarily emphasized that the architecture of core NEC complexes is highly conserved among herpesviruses, the present report focuses on subfamily-specific structural and functional features that help explain the differences in the autologous versus nonautologous interaction patterns observed for NEC formation across herpesviruses. Here, we describe the crystal structure of the Orf24-Orf27 complex at 2.1 Å resolution. Coimmunoprecipitation and confocal imaging data show that Orf24-Orf27 complex formation displays some promiscuity in a herpesvirus subfamily-restricted manner. At the same time, analysis of thermodynamic parameters of NEC formation of three prototypical α-, ß-, and γ herpesviruses, i.e., VZV, human cytomegalovirus (HCMV), and Epstein-Barr virus (EBV), revealed highly similar binding affinities for the autologous interaction with specific differences in enthalpy and entropy. Computational alanine scanning, structural comparisons, and mutational data highlight intermolecular interactions shared among α-herpesviruses that are clearly distinct from those seen in ß- and γ-herpesviruses, including a salt bridge formed between Orf24-Arg167 and Orf27-Asp126. This interaction is located outside of the hook-into-groove interface and contributes significantly to the free energy of complex formation. Combined, these data explain distinct properties of specificity and permissivity so far observed in herpesviral NEC interactions. These findings will prove valuable in attempting to target multiple herpesvirus core NECs with selective or broad-acting drug candidates.


Assuntos
Herpesvirus Humano 3 , Membrana Nuclear , Proteínas Virais , Cristalografia por Raios X , Herpesvirus Humano 3/química , Herpesvirus Humano 3/genética , Humanos , Membrana Nuclear/química , Membrana Nuclear/genética , Proteínas Virais/química , Proteínas Virais/genética , Liberação de Vírus
19.
Eur J Immunol ; 52(5): 770-783, 2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-34355795

RESUMO

TRIANNI mice carry an entire set of human immunoglobulin V region gene segments and are a powerful tool to rapidly isolate human monoclonal antibodies. After immunizing these mice with DNA encoding the spike protein of SARS-CoV-2 and boosting with spike protein, we identified 29 hybridoma antibodies that reacted with the SARS-CoV-2 spike protein. Nine antibodies neutralize SARS-CoV-2 infection at IC50 values in the subnanomolar range. ELISA-binding studies and DNA sequence analyses revealed one cluster of three clonally related neutralizing antibodies that target the receptor-binding domain and compete with the cellular receptor hACE2. A second cluster of six clonally related neutralizing antibodies bind to the N-terminal domain of the spike protein without competing with the binding of hACE2 or cluster 1 antibodies. SARS-CoV-2 mutants selected for resistance to an antibody from one cluster are still neutralized by an antibody from the other cluster. Antibodies from both clusters markedly reduced viral spread in mice transgenic for human ACE2 and protected the animals from SARS-CoV-2-induced weight loss. The two clusters of potent noncompeting SARS-CoV-2 neutralizing antibodies provide potential candidates for therapy and prophylaxis of COVID-19. The study further supports transgenic animals with a human immunoglobulin gene repertoire as a powerful platform in pandemic preparedness initiatives.


Assuntos
COVID-19 , Glicoproteína da Espícula de Coronavírus , Animais , Anticorpos Monoclonais , Anticorpos Neutralizantes , Anticorpos Antivirais , Humanos , Camundongos , SARS-CoV-2
20.
Hum Mol Genet ; 31(3): 440-454, 2022 02 03.
Artigo em Inglês | MEDLINE | ID: mdl-34505148

RESUMO

Recently, others and we identified de novo FBXO11 (F-Box only protein 11) variants as causative for a variable neurodevelopmental disorder (NDD). We now assembled clinical and mutational information on 23 additional individuals. The phenotypic spectrum remains highly variable, with developmental delay and/or intellectual disability as the core feature and behavioral anomalies, hypotonia and various facial dysmorphism as frequent aspects. The mutational spectrum includes intragenic deletions, likely gene disrupting and missense variants distributed across the protein. To further characterize the functional consequences of FBXO11 missense variants, we analyzed their effects on protein expression and localization by overexpression of 17 different mutant constructs in HEK293 and HeLa cells. We found that the majority of missense variants resulted in subcellular mislocalization and/or reduced FBXO11 protein expression levels. For instance, variants located in the nuclear localization signal and the N-terminal F-Box domain lead to altered subcellular localization with exclusion from the nucleus or the formation of cytoplasmic aggregates and to reduced protein levels in western blot. In contrast, variants localized in the C-terminal Zn-finger UBR domain lead to an accumulation in the cytoplasm without alteration of protein levels. Together with the mutational data, our functional results suggest that most missense variants likely lead to a loss of the original FBXO11 function and thereby highlight haploinsufficiency as the most likely disease mechanism for FBXO11-associated NDDs.


Assuntos
Proteínas F-Box , Deficiência Intelectual , Transtornos do Neurodesenvolvimento , Proteínas F-Box/genética , Células HEK293 , Células HeLa , Humanos , Deficiência Intelectual/genética , Mutação de Sentido Incorreto/genética , Transtornos do Neurodesenvolvimento/genética , Proteína-Arginina N-Metiltransferases/genética
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