RESUMO
BACKGROUND: Kinase D-interacting substrate of 220 kDa (KIDINS220) is a multifunctional scaffolding protein essential for neuronal development. It has been implicated in neurological diseases with either autosomal dominant (AD) or autosomal recessive (AR) inheritance patterns. The molecular mechanisms underlying the AR/AD dual nature of KIDINS220 remain elusive, posing challenges to genetic interpretation and clinical interventions. Moreover, increased KIDINS220 exhibited neurotoxicity, but its role in neurodevelopment remains unclear. OBJECTIVE: The aim was to investigate the genotype-phenotype correlations of KIDINS220 and elucidate its pathophysiological role in neuronal development. METHODS: Whole-exome sequencing was performed in a four-generation family with cerebral palsy. CRISPR/Cas9 was used to generate KIDINS220 mutant cell lines. In utero electroporation was employed to investigate the effect of KIDINS220 variants on neurogenesis in vivo. RESULTS: We identified in KIDINS220 a pathogenic nonsense variant (c.4177C > T, p.Q1393*) that associated with AD cerebral palsy. We demonstrated that the nonsense variants located in the terminal exon of KIDINS220 are gain-of-function (GoF) variants, which enable the mRNA to escape nonsense-mediated decay and produce a truncated yet functional KIDINS220 protein. The truncated protein exhibited significant resistance to calpain and consequently accumulated within cells, resulting in the hyperactivation of Rac1 and defects in neuronal development. CONCLUSIONS: Our findings demonstrate that the location of variants within KIDINS220 plays a crucial role in determining inheritance patterns and corresponding clinical outcomes. The proposed interaction between Rac1 and KIDINS220 provides new insights into the pathogenesis of cerebral palsy, implying potential therapeutic perspectives. © 2023 International Parkinson and Movement Disorder Society.
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Paralisia Cerebral , Neurônios , Humanos , Neurônios/metabolismo , Transdução de Sinais , Paralisia Cerebral/genética , Mutação com Ganho de Função , Neurogênese/genética , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/genéticaRESUMO
Among the main structural protein of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), nucleocapsid phosphoprotein (NP) exhibits high immunogenicity and is the most abundant viral protein produced and shed during infection. Detection of antibodies against NP may help assess the number of individuals exposed to SARS-COV-2 or vaccinated against it. Based on these findings and other structural and antigenic evaluations, we designed a recombinant truncated fusion NP-based protein for application in an immunoassay for detecting immunoglobulins in patients who have recovered from COVID-19. In this research, we aligned the NPs from SARS-CoV and SARS-CoV-2 and selected highly antigenic parts of the SARS-CoV-2 sequences based on in-silico studies. The protein was expressed under optimum conditions in the bacterial host BL21 and purified by nickel immobilized metal affinity chromatography. Moreover, the purity level was assessed by SDS-PAGE and Western blotting whereas the folding of the protein was evaluated by circular dichroism. Ultimately, we used the purified recombinant protein in ELISA development in which 42 samples from convalescent patients were compared with 20 samples of the past 2019 patients who had attended laboratories for various clinical check-ups. The sensitivity and specificity were determined as 71% and 90%, respectively, in the optimum cut-off point measured by the receiver operating characteristic curve.
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COVID-19 , Coronavírus Relacionado à Síndrome Respiratória Aguda Grave , Humanos , Proteínas Recombinantes de Fusão/genética , Proteínas do Nucleocapsídeo/genética , COVID-19/diagnóstico , SARS-CoV-2/genética , Ensaio de Imunoadsorção Enzimática/métodos , Nucleocapsídeo , Anticorpos Antivirais , Sensibilidade e EspecificidadeRESUMO
BACKGROUND: Neurofibromatosis type 1 (NF1) is an autosomal dominant with haploinsufficient, and multisystemic disorder including patches of skin Café-au-lait spots, Lisch nodules in the iris, and tumors in the peripheral nervous systems or fibromatous skin. METHODS: Blood samples were collected and DNA was extracted from a large Chinese pedigree suffering from NF1 disease with three spontaneous abortions or death for proband. Analysis for whole exome sequencing (WES), Sanger sequencing, and co-segregation was carried out. Prenatal gene diagnosis was also carried out in amniotic fluid DNA. The expression of NF1 was conducted by bioinformatics. RESULTS: A large Chinese pedigree with NF1 was recruited and a novel, heterozygous, variant (c.4272delA: p.I1426Ffs*2) for the NF1 gene in the proband was identified. This variant of NF1 produced a truncated protein that losses half of NF1 protein at the C-terminus including the CRAL-TRIO lipid-binding domain, NLS, and a small portion of Ras-GAP domain, thus leading to pathogenicity (ACMG criteria: PVS1 + PM2). NF1 expressions in different human tissues showed low tissue specificity, which may affect multiple organs presenting different phenotypes. Moreover, prenatal gene diagnosis for NF1 showed both alleles as wild types in the fetus of the proband. CONCLUSION: We thus successfully identified a novel, pathogenic, heterozygous variant (c.4272delA:p.I1426Ffs*2) in the NF1 gene of NF1 disorder, expanding the NF1 mutation spectrum, that will help elucidate the molecular pathogenesis of NF1 disease and to contribute to the NF1 diagnosis, genetic counseling, clinical management in this large Chinese family.
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Neurofibromatose 1 , Humanos , Neurofibromatose 1/genética , Neurofibromatose 1/diagnóstico , Genes da Neurofibromatose 1 , População do Leste Asiático , Neurofibromina 1/genética , Manchas Café com Leite/genéticaRESUMO
Porcine epidemic diarrhea virus (PEDV) can infect pigs of all ages, especially piglets. PEDV has spread across Asia since the 1980s. The highly virulent variant PEDV broke out on a large scale and caused huge economic losses to the pig industry in late 2010 in China. Rapid detection methods with high specificity and sensitivity are urgently needed for the diagnosis and control of the disease. In this study, we divided the PEDV S1 gene into three segments and constructed the recombinant plasmids pFastBac1-S1T1 (aa 21-279), pFastBac1-S1T2 (aa 280-539) and pFastBac1-S1T3 (aa 540-788), which carry the different antigenic regions of the S1 gene. Truncated S1 proteins PEDV-S1T1/S1T2/S1T3 were obtained by a Bac-to-Bac expression system, with protein sizes of 36 kDa, 38 kDa and 38 kDa, respectively. Recombinant proteins presented high reactivity with the monoclonal antibody against PEDV and positive pig serum. Based on full-length S1 protein and these truncated proteins, we established indirect ELISA methods for the detection of PEDV IgA antibody. A total of 213 clinical serum samples were tested by the above indirect ELISA methods, and IFA was used as the gold standard. ROC curves revealed a significant correlation between S1-ELISA and S1T2-ELISA with a 0.9134 correlation coefficient and favourable sensitivity and specificity of S1-ELISA (93.24%, 95.68%) and S1T2-ELISA (89.33%, 94.16%). Our results also indicated that serum with higher neutralizing activity (SNT ≥ 40) had a higher IgA antibody level based on S1-ELISA, S1T1-ELISA and S1T2-ELISA. In conclusion, both S1-ELISA and S1T2-ELISA can be used as candidate systems for detecting anti-PEDV IgA antibody titers in serum, which can reflect the level of neutralizing activity in pigs after natural infection or vaccination. The above research results provide a basis for the prevention and control of PEDV and can be used in the detection of host anti-infective immunity and evaluation of vaccine immune effects.
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Infecções por Coronavirus , Vírus da Diarreia Epidêmica Suína , Doenças dos Suínos , Animais , Anticorpos Antivirais , Infecções por Coronavirus/diagnóstico , Infecções por Coronavirus/veterinária , Ensaio de Imunoadsorção Enzimática/métodos , Ensaio de Imunoadsorção Enzimática/veterinária , Imunoglobulina A , Vírus da Diarreia Epidêmica Suína/genética , SuínosRESUMO
CpdB is a 3'-nucleotidase/2'3'-cyclic nucleotide phosphodiesterase, active also with reasonable efficiency on cyclic dinucleotides like c-di-AMP (3',5'-cyclic diadenosine monophosphate) and c-di-GMP (3',5'-cyclic diadenosine monophosphate). These are regulators of bacterial physiology, but are also pathogen-associated molecular patterns recognized by STING to induce IFN-ß response in infected hosts. The cpdB gene of Gram-negative and its homologs of gram-positive bacteria are virulence factors. Their protein products are extracytoplasmic enzymes (either periplasmic or cell-wall anchored) and can hydrolyze extracellular cyclic dinucleotides, thus reducing the innate immune responses of infected hosts. This makes CpdB(-like) enzymes potential targets for novel therapeutic strategies in infectious diseases, bringing about the necessity to gain insight into the molecular bases of their catalytic behavior. We have dissected the two-domain structure of Escherichia coli CpdB to study the role of its N-terminal and C-terminal domains (CpdB_Ndom and CpdB_Cdom). The specificity, kinetics and inhibitor sensitivity of point mutants of CpdB, and truncated proteins CpdB_Ndom and CpdB_Cdom were investigated. CpdB_Ndom contains the catalytic site, is inhibited by phosphate but not by adenosine, while CpdB_Cdom is inactive but contains a substrate-binding site that determines substrate specificity and adenosine inhibition of CpdB. Among CpdB substrates, 3'-AMP, cyclic dinucleotides and linear dinucleotides are strongly dependent on the CpdB_Cdom binding site for activity, as the isolated CpdB_Ndom showed much-diminished activity on them. In contrast, 2',3'-cyclic mononucleotides and bis-4-nitrophenylphosphate were actively hydrolyzed by CpdB_Ndom, indicating that they are rather independent of the CpdB_Cdom binding site.
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2',3'-Nucleotídeo Cíclico Fosfodiesterases/química , 2',3'-Nucleotídeo Cíclico Fosfodiesterases/metabolismo , Adenosina/metabolismo , Biocatálise , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Fosfatos/metabolismo , 2',3'-Nucleotídeo Cíclico Fosfodiesterases/genética , Sítios de Ligação , Domínio Catalítico , Proteínas de Escherichia coli/genética , Histidina/metabolismo , Hidrólise , Cinética , Modelos Moleculares , Mutação Puntual/genética , Domínios Proteicos , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
A heterozygous frameshift PRRT2 mutation (c.649_650InsC) has been identified as the major causative mutation in several paroxysmal disorders, including paroxysmal kinesigenic dyskinesia (PKD). Since PKD is an autosomal dominant disorder and since the frameshift mutations of PRRT2 may create a truncated protein, it remains unclear whether this mutation causes toxic gain of function or loss of function. By generating Prrt2 knock-in (KI) mice that express human PRRT2 with the c.649_650InsC mutation and by comparing the phenotypes of Prrt2 KI mice with knockout (KO) mice, we find that both KI and KO mice show the same extents of impaired rotarod and balance beam performance as well as the same sensitivity to seizure induction. Both KI and KO mice show altered formation of SNARE complex and number of synaptic vesicles. In addition, western blotting of KI mouse brain tissues could not detect truncated PRRT2 protein that might be generated by the c.649_650InsC mutation. Moreover, the level of PRRT2 mRNA in KI mice is significantly decreased, recapitulating the reduction of PRRT2 mRNA reported in PKD patients. Furthermore, mutant PRRT2 mRNA is unstable and showed shortened half-life than wild-type PRRT2 mRNA. Our studies suggest that PRRT2 frameshift mutation leads to the loss of function by affecting its mRNA stability, a mechanism that is different from haploinsufficiency due to dysfunctional protein or gain of function caused by truncated protein.
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Distonia/genética , Mutação da Fase de Leitura , Proteínas de Membrana/genética , Proteínas do Tecido Nervoso/genética , RNA Mensageiro/genética , Animais , Distonia/patologia , Técnicas de Introdução de Genes , Humanos , Mutação com Perda de Função , Camundongos , Camundongos Knockout , RNA Mensageiro/químicaRESUMO
Marine luciferases are regularly employed as useful reporter molecules across a range of various applications. However, attempts to transition expression from their native eukaryotic environment into a more economical prokaryotic, i.e. bacterial, expression system often presents several challenges. Specifically, bacterial protein expression inherently lacks chaperone proteins to aid in the folding process, while Escherichia coli presents a reducing cytoplasmic environment in. These conditions contribute to the inhibition of proper folding of cysteine-rich proteins, leading to incorrect tertiary structure and ultimately inactive and potentially insoluble protein. Vargula luciferase (Vluc) is a cysteine-rich marine luciferase that exhibits glow-type bioluminescence through a reaction between its unique native substrate and molecular oxygen. Because most other commonly used bioluminescent proteins exhibit flash-type emission kinetics, this emission characteristic of Vluc is desirable for high-throughput applications where stability of emission is required for the duration of data collection. A truncated form of Vluc that retains considerable bioluminescence activity (55%) compared to the native full-length protein has been reported in the literature. However, expression and purification of this luciferase from bacterial systems has proven difficult. Herein, we demonstrate the expression and purification of a truncated form of Vluc from E. coli. This truncated Vluc (tVluc) was subsequently characterized in terms of both its biophysical and bioluminescence properties.
Assuntos
Proteínas de Artrópodes , Crustáceos/genética , Luciferases , Animais , Proteínas de Artrópodes/biossíntese , Proteínas de Artrópodes/química , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/isolamento & purificação , Crustáceos/enzimologia , Luciferases/biossíntese , Luciferases/química , Luciferases/genética , Luciferases/isolamento & purificação , Domínios Proteicos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , SolubilidadeRESUMO
In this study, we successfully produced VLPs derived from full-length or chimeric VP1 of a documented GII.6 strain. Trypsin digestion of purified VLPs led to total cleavage of VP1, while the integrity of assembled VLPs was not affected. In vitro VLP-histo-blood group antigen (HBGA) binding and binding blockade assays indicated that trypsin digestion enhanced the binding of GII.6 VLPs to salivary HBGAs and that this binding could only be blocked by serum produced against a homologous strain. The data regarding the assembly, morphology and binding patterns of GII.6 NoV VLPs presented here might be useful for further study of GII.6 NoVs.
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Proteínas do Capsídeo/genética , Norovirus/genética , Virossomos/genética , Virossomos/metabolismo , Antígenos de Grupos Sanguíneos/metabolismo , Humanos , Multimerização Proteica , Virossomos/ultraestrutura , Montagem de Vírus , Ligação ViralRESUMO
α-Thalassemia (α-thal) is a common genetic disorder in Iran and many parts of the world. Genetic defects on the α-globin gene cluster can result in α-thal that may develop a clinical phenotype varying from almost asymptomatic to a lethal hemolytic anemia. In the present study, four Iranian individuals with hypochromic microcytic anemia, who revealed none of the known mutations responsible for α-thal, were subjected for further investigations. The thalassemic phenotype of these patients resulted from abnormal RNA splicing sites owing to a missense at the splice donor site, a truncated protein or hemoglobin (Hb) variants as a result of two different substitutions on the α1-globin gene. The clinical presentation of mild anemia in these individuals showed the contribution of these novel mutations in α-thal in spite of the dominantly expressed α2-globin gene. This study describes hematological manifestations of subjects carrying some novel mutations comparable to the reported phenotype of α(+)-thal trait.
Assuntos
Anemia Hipocrômica/genética , Hemoglobinas Glicadas/genética , Mutação , Sítios de Splice de RNA , Talassemia alfa/genética , Adulto , Sequência de Bases , Feminino , Genes Dominantes , Humanos , Masculino , Dados de Sequência Molecular , alfa-Globinas/genéticaRESUMO
Dysferlin protein deficiency can cause neuromuscular dysfunction, resulting in autosomal recessive dysferlinopathy, which is caused by DYSF gene mutation. Dysferlin proteins belongs to the Ferlin1-like protein family and are associated with muscle membrane repair and regeneration. In China, pathogenic mutations of the protein often result in two clinical phenotypes of Miyoshi muscular or limb band muscular dystrophy type 2B. It is clinically characterized by progressive muscle weakness and elevated serum creatine kinase. The data of the child were collected, blood samples of the child and his family members were collected, and whole exome sequencing (WES) was performed. The recombinant expression vector was constructed, the function of the mutation was verified by minigene, and the pathogenicity of the mutation was further analyzed by combining with biological information analysis. The patient initially presented with asymptomatic elevation of serum creatine kinaseï¼CKï¼. Then progressive lower limb weakness, mainly distal limb weakness. Large amounts of scattered necrosis, myogenic lesions, and complete deletion of dysferlin protein were observed under muscle biopsy, which further improved genetic detection. Whole exome sequencing showed compound mutations (c.1397 + 1_1397 + 3del and c.1375dup p.M459Nfs*15) in DYSF gene. c.1375dup p.M459Nfs*15 have been reported. The other mutation is the deletion of c.1397 + 1_1397 + 3 in Intron15, which is an intron mutation that may affect splicing and the pathogenesis is still unknown. Minigene splicing assay verified that c.1397 + 1_1397 + 3del resulted in exon15 skipping and produced a premature termination codon. We report a novel pathogenic mutation in DYSF gene with Miyoshi myopathy and demonstrate this variant causes skipping of exon15 by minigene splicing assay. We point out the need of conducting functional analysis to verify the pathogenicity of intronic mutation. The finding enriches the mutation spectrum of DYSF gene and laid a foundation for future studies on the correlation between genotype and phenotype.
Assuntos
Creatina Quinase , Miopatias Distais , Atrofia Muscular , Criança , Humanos , Disferlina/genética , Fenótipo , Genótipo , Creatina Quinase/genéticaRESUMO
To analyse the genetic aetiology of a child with oculocutaneous albinism and to explore the effects of two mutation sites on the function of the OCA2 protein at the mRNA and protein levels via the use of recombinant carriers in vitro. Whole-exome sequencing (WES) and Sanger sequencing were used to analyse the pathogenic genes of the child and validate the mutations in the parents. pEGFP and phage vectors carrying wild-type and mutant OCA2 were constructed using the coding DNA sequence (CDS) of the whole gene-synthesized OCA2 as a template and transfected into HEK293T cells, after which expression analysis was performed. The child in this study was born with white skin, hair, eyelashes, and eyebrows and exhibited nystagmus. Genetic analysis indicated that the child carried two heterozygous mutations: c.1079C > T (p.Ser360Phe) of maternal origin and c.1095_1103delAGCACTGGC (p.Ala366_Ala368del) of paternal origin, conforming to an autosomal recessive inheritance pattern. In vitro analysis showed that the expression of the c.1079C > T (p.Ser360Phe) mutant did not significantly change at the mRNA level but did increase at the protein level, suggesting that the mutation may lead to enhanced protein stability, and the c.1095_1103delAGCACTGGC (p.Ala366_Ala368del) mutation resulted in the loss of three amino acids in exon 10, producing a truncated protein. In vitro expression analysis also revealed that the expression of the mutant gene was significantly downregulated at both the mRNA and protein levels, suggesting that the mutation can simultaneously produce truncated proteins and lead to protein degradation. This case study enriches the phenotypic spectrum of OCA2 gene disease. In vitro expression analysis confirmed that both mutations affect protein expression, providing a theoretical basis for analysing the pathogenicity of these two mutations.
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Albinismo Oculocutâneo , Proteínas de Membrana Transportadoras , Mutação , Humanos , Células HEK293 , Albinismo Oculocutâneo/genética , Proteínas de Membrana Transportadoras/genética , Sequenciamento do Exoma , Feminino , Masculino , Linhagem , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Alternative splicing plays a crucial role in increasing the diversity of mRNAs expressed in the genome. Serine/arginine-rich splicing factor 3 (SRSF3) is responsible for regulating the alternative splicing of its own mRNA and ensuring that its expression is balanced to maintain homeostasis. Moreover, the exon skipping of SRSF3 leads to the production of a truncated protein instead of a frameshift mutation that generates a premature termination codon (PTC). However, the precise regulatory mechanism involved in the splicing of SRSF3 remains unclear. In this study, we first established a platform for coexpressing full-length SRSF3 (SRSF3-FL) and SRSF3-PTC and further identified a specific antibody against the SRSF3-FL and truncated SRSF3 (SRSF3-TR) proteins. Next, we found that exogenously overexpressing SRSF3-FL or SRSF3-PTC failed to reverse the effects of digoxin, caffeine, or both in combination on this molecule and its targets. Endoplasmic reticulum-related pathways, transcription factors, and chemicals such as palmitic acid and phosphate were found to be involved in the regulation of SRSF3 expression. The downregulation of SRSF3-FL by palmitic acid and phosphate was mediated via different regulatory mechanisms in HeLa cells. In summary, we provide new insights into the altered expression of the SRSF3-FL and SRSF3-TR proteins for the identification of the functions of SRSF3 in cells.
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Processamento Alternativo , Fatores de Processamento de Serina-Arginina , Fatores de Processamento de Serina-Arginina/metabolismo , Fatores de Processamento de Serina-Arginina/genética , Humanos , Células HeLa , Estabilidade Proteica , Regulação Neoplásica da Expressão Gênica , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Objective: To report two cases of congenital myasthenic syndromes (CMS) in a Chinese family with mutations in the COLQ gene and to prove the consequence defect of the ColQ protein. Method: Clinical characteristics of the two children from the same family were described. Next-generation sequencing (NGS) and sanger sequencing was performed on the proband and family members. The consequence of the mutation was predicted by 3D protein structure prediction using I-TASSER. The wild type and mutant were transfected to 293T cells, and ColQ protein was detected by Western Blot. Results: The diagnosis of CMS was based on a symptom combination of fatigable muscle weakness, ptosis, scoliosis, and hypotonia, aggravation of muscle weakness after the neostigmine test, and a 46% decrement in repetitive nerve stimulation. A muscle biopsy was performed on the proband, revealing mild variation in the myofiber size. NGS data revealed two compound heterozygous mutations at c.173delC (p.Pro58Hisfs*22) and c.C706T (p.R236X) in the COLQ gene, where the former was a novel mutation. A 3D structure prediction showed two truncated ColQ proteins with 78aa and 235aa, respectively. The truncated ColQ protein was proved in 293T cells transfected with c.173delC or c.C706T mutants by Western Blot. Conclusions: The mutations of c.173delC and c.C706T in the COLQ gene led to truncated ColQ protein and contributed to the pathogenesis of CMS in this Chinese family.
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Primary ciliary dyskinesia (PCD) is a genetically heterogeneous disorder caused by defects in motile ciliary function and/or structure. Outer dynein arm docking complex subunit 1 (ODAD1) is an important component of the outer dynein arm docking complex (ODA-DC). To date, 13 likely pathogenic mutations of ODAD1 have been reported. However, the pathogenesis of ODAD1 mutations remains elusive. To investigate the pathogenesis of splice-site mutations in ODAD1 discovered in this study and those reported previously, molecular and functional analyses were performed. Whole-exome sequencing revealed a compound mutation in ODAD1 (c.71-2A>C; c.598-2A>C) in a patient with PCD, with c.598-2A>C being a novel mutation that resulted in two mutant transcripts. The compound mutation in ODAD1 (c.71-2A>C; c.598-2A>C) led to aberrant splicing that resulted in the absence of the wild-type ODAD1 and defects of the outer dynein arm in ciliary axonemes, causing a decrease in ciliary beat frequency. Furthermore, we demonstrated that the truncated proteins resulting from splice-site mutations in ODAD1 could retain partial function and inhibit the interaction between wild-type ODAD1 and ODAD3. The results of this study expand the mutational and clinical spectrum of PCD, provide more evidence for genetic counseling, and offer new insights into gene-based therapeutic strategies for PCD.
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BACKGROUND: Acephalic spermatozoa (AS) is a serious but rare reproductive genetic disorder that causes infertility in men. To date, only a few genes associated with AS defects have been identified, including the polyamine modulated factor 1 binding protein 1 (PMFBP1) gene. Consistent with this, PMFBP1 localizes to the head-neck connection, which bridges the implantation fossa and basal body. METHODS: A male patient was diagnosed as having an AS defect. Blood samples from all family members and a sample of the patient's semen were collected to determine the genetic causes of his infertility. RESULTS: Compound heterozygote mutation in the PMFBP1 gene, which is associated with AS defects in the present case: two loss-of-function mutations, with one a nonsense mutation c.361C > T p.Gln121Ter, and another a splice donor mutation c.414 + 1G > T. The current study, together with previous studies, suggests that the nonsense mutation is responsible for a truncated PMFBP1 protein during its formation; a splice donor mutation c.414 + 1G > T might lead to new open reading frames, from which the dysfunction of an abnormal PMFBP1 protein might be predicted. Additionally, the expression of outer dense fiber 1 (ODF1) and ODF2 proteins has been experimentally shown to be regulated by the truncated PMFBP1 protein. CONCLUSION: We herein present a case with AS defects associated with heterozygote mutations of PMFBP1, which have been shown to be rare and pathogenic; the association with an AS defect is a monogenic disorder with a recessive inherited pattern in the patient's family.
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Códon sem Sentido , Teratozoospermia , Proteínas de Choque Térmico/genética , Humanos , Masculino , Mutação , Poliaminas/metabolismo , Proteínas/genética , Espermatozoides/metabolismo , Teratozoospermia/genéticaRESUMO
BACKGROUND: Premature ovarian insufficiency (POI) is a highly heterogeneous disease, and up to 25% of cases can be explained by genetic causes. The transcription factor WT1 has long been reported to play a crucial role in ovary function. Wt1-mutated female mice exhibited POI-like phenotypes. METHODS AND RESULTS: In this study, whole exome sequencing (WES) was applied to find the cause of POI in Han Chinese women. A nonsense variant in the WT1 gene: NM_024426.6:c.1387C>T(p.R463*) was identified in a non-syndromic POI woman. The variant is a heterozygous de novo mutation that is very rare in the human population. The son of the patient inherited the mutation and developed Wilms' tumor and urethral malformation at the age of 7. According to the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG/AMP) guidelines, the novel variant is categorized as pathogenic. Western blot analysis further demonstrated that the WT1 variant could produce a truncated WT1 isoform in vitro. CONCLUSIONS: A rare heterozygous nonsense WT1 mutant is associated with non-syndromic POI and Wilms' tumor. Our finding characterized another pathogenic WT1 variant, providing insight into genetic counseling.
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Menopausa Precoce , Insuficiência Ovariana Primária , Tumor de Wilms , Feminino , Heterozigoto , Humanos , Masculino , Insuficiência Ovariana Primária/genética , Insuficiência Ovariana Primária/patologia , Proteínas WT1/genética , Sequenciamento do Exoma , Tumor de Wilms/genéticaRESUMO
BACKGROUND: Krabbe disease, also called globoid cell leukodystrophy, is an autosomal recessive disease caused by a deficiency of lysosomal galactocerebrosidase. Infantile Krabbe occurring before 12 months of age accounts for most cases. Typical clinical features include irritability, seizures, peripheral neuropathy, and progressive neurodegeneration. METHODS: We collected and summarized the clinical and genetic data of an 8-month-old boy who demonstrated Krabbe disease onset at around 6 months. Potential pathogenic variants were screened by whole exome sequencing, and effects of candidate variants on alternative transcript and truncated protein were further validated at the RNA and protein level. RESULTS: Galactocerebrosidase activity was nearly absent in his blood, and whole exome sequencing revealed compound heterozygous variants [NM_000153.4: (c.658C>T); (c.328+5G>T)] in galactosylceramidase (GALC). The variant c.328+5G>T was predicted to alter splicing, and the abnormal isoform transcript was validated by observation of abnormal RNA isoforms. The variant c.658C>T was predicted to cause truncation of the protein, which was validated by western blotting. CONCLUSIONS: Our findings revealed compound heterozygous variants with solid experimental results for Krabbe disease and provides strong evidence for further Krabbe disease screening and clinical consulting. As a rare inherited systemic disorder, genetic variants in Krabbe disease should be investigated, as experimental validation for clinical diagnosis is needed.
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BACKGROUND: Vip3Aa is a vegetative insecticidal protein produced by Bacillus thuringiensis. The protein is produced as an 88-kDa protoxin that could be processed by insect gut proteases into a 22-kDa N-terminal and a 66-kDa C-terminal fragments. The C-terminal part could bind to a specific receptor while the N-terminal part is required for toxicity and structural stability. OBJECTIVE: To demonstrate the antagonistic effect of truncated fragments on the insecticidal activity of the full-length Vip3Aa. METHODS: The full-length protein (Vip3Aa), a 66-kDa C-terminal fragment (Vip3Aa-D199) and a predicted carbohydrate binding module (CBM) were produced in Escherichia coli. Purified proteins were mixed at different ratios and fed to Spodoptera litura and Spodoptera exigua larvae. Mortality was recorded and compared between larvae fed with individual toxin and mixtures of the full-length and truncated toxins. RESULTS: Production level of the Vip3Aa-D199 was significantly decreased comparing to that of the full-length protein. Vip3Aa-D199 and CBM fragment were not toxic to insect larvae whereas Vip3Aa showed high toxicity with LC50 about 200 ng/cm2. Feeding the larvae with mixtures of the Vip3Aa and Vip3Aa-D199 at different ratios revealed antagonistic effect of the Vip3Aa-D199 on the toxicity of Vip3Aa. Results showed that the lethal time (LT 50 and LT 95) of larvae fed the mixture toxins was longer than those fed the Vip3Aa alone. In addition, a CBM fragment could inhibit toxicity of the full-length Vip3Aa. CONCLUSION: Our results demonstrated that the Vip3Aa-D199 and a CBM fragment could complete for the membrane binding thus rendering activity of the full-length Vip3Aa.
Assuntos
Bacillus thuringiensis/metabolismo , Proteínas de Bactérias/toxicidade , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Inseticidas/toxicidade , Larva/efeitos dos fármacos , Spodoptera/efeitos dos fármacos , Animais , Proteínas de Bactérias/metabolismo , Inseticidas/metabolismo , Larva/crescimento & desenvolvimento , Larva/metabolismo , Controle Biológico de Vetores , Spodoptera/crescimento & desenvolvimento , Spodoptera/metabolismoRESUMO
Ribulose 5-phosphate (Ru5P) and guanosine 5'-triphosphate (GTP) are two key precursors of riboflavin, whereby Ru5P is also a precursor of GTP. Ribulose 5-phosphate 3-epimerase (Rpe) catalyzes the conversion of ribulose 5-phosphate into xylulose 5-phosphate. Inactivation of Rpe can reduce the consumption of Ru5P, enhancing the carbon flux toward riboflavin biosynthesis. Here we investigated the effect of mutation of rpe and other related genes on riboflavin production, physiological and metabolic phenotypes in Bacillus subtilis LY (BSLY). Introducing single nucleotide deletion (generated BSR) or nonsense mutation (generated BSRN) on the genomic copy of rpe, resulting in more than fivefold increase of riboflavin production over the parental strain. BSR process 62% Rpe activity, while BSRN lost the entire Rpe activity and had a growth defect compared with the parent strain. BSR and BSRN exhibited increases of the inosine and guanine titers, in addition, BSRN exhibited an increase of inosine 5'-monophosphate titer in fermentation. The transcription levels of most oxidative pentose phosphate pathway and purine synthesis genes were unchanged in BSR, except for the levels of zwf and ndk, which were higher than in BSLY. The production of riboflavin was increased to 479.90 ± 33.21 mg/L when ribA was overexpressed in BSR. The overexpression of zwf, gntZ, prs, and purF also enhanced the riboflavin production. Finally, overexpression of the rib operon by the pMX45 plasmid and mutant gnd by pHP03 plasmid in BSR led to a 3.05-fold increase of the riboflavin production (977.29 ± 63.44 mg/L), showing the potential for further engineering of this strain.
RESUMO
A large variety of fusion tags have been developed to improve protein expression, solubilization, and purification. Nevertheless, these tags have been combined in a rather limited number of composite tags and usually these composite tags have been dictated by traditional commercially-available expression vectors. Moreover, most commercially-available expression vectors include either N- or C-terminal fusion tags but not both. Here, we introduce TSGIT, a fusion-tag system composed of both N- and a C-terminal composite fusion tags. The system includes two affinity tags, two solubilization tags and two cleavable tags distributed at both termini of the protein of interest. Therefore, the N- and the C-terminal composite fusion tags in TSGIT are fully orthogonal in terms of both affinity selection and cleavage. For using TSGIT, we streamlined the cloning, expression, and purification procedures. Each component tag is selected to maximize its benefits toward the final construct. By expressing and partially purifying the protein of interest between the components of the TSGIT fusion, the full-length protein is selected over truncated forms, which has been a long-standing problem in protein purification. Moreover, due to the nature of the cleavable tags in TSGIT, the protein of interest is obtained in its native form without any additional undesired N- or C-terminal amino acids. Finally, the resulting purified protein is ready for efficient ligation with other proteins or peptides for downstream applications. We demonstrate the use of this system by purifying a large amount of native fluorescent mRuby3 protein and bacteriophage T7 gp2.5 ssDNA-binding protein.