scholarly journals Point Mutations of Nicotinic Receptor α1 Subunit Reveal New Molecular Features of G153S Slow-Channel Myasthenia

Molecules ◽  
2021 ◽  
Vol 26 (5) ◽  
pp. 1278
Author(s):  
Denis Kudryavtsev ◽  
Anastasia Isaeva ◽  
Daria Barkova ◽  
Ekaterina Spirova ◽  
Renata Mukhutdinova ◽  
...  
Keyword(s):  
Hydrogen Bonds ◽  
Binding Site ◽  
Molecular Mechanisms ◽  
Genetic Diseases ◽  
Point Mutations ◽  
Targeted Mutagenesis ◽  
Decay Kinetics ◽  
Molecular Features ◽  
Slow Channel ◽  
Patch Clamp Electrophysiology

Slow-channel congenital myasthenic syndromes (SCCMSs) are rare genetic diseases caused by mutations in muscle nicotinic acetylcholine receptor (nAChR) subunits. Most of the known SCCMS-associated mutations localize at the transmembrane region near the ion pore. Only two SCCMS point mutations are at the extracellular domains near the acetylcholine binding site, α1(G153S) being one of them. In this work, a combination of molecular dynamics, targeted mutagenesis, fluorescent Ca2+ imaging and patch-clamp electrophysiology has been applied to G153S mutant muscle nAChR to investigate the role of hydrogen bonds formed by Ser 153 with C-loop residues near the acetylcholine-binding site. Introduction of L199T mutation to the C-loop in the vicinity of Ser 153 changed hydrogen bonds distribution, decreased acetylcholine potency (EC50 2607 vs. 146 nM) of the double mutant and decay kinetics of acetylcholine-evoked cytoplasmic Ca2+ rise (τ 14.2 ± 0.3 vs. 34.0 ± 0.4 s). These results shed light on molecular mechanisms of nAChR activation-desensitization and on the involvement of such mechanisms in channelopathy genesis.

scholarly journals Nuclear proteins that bind the human gamma-globin gene promoter: alterations in binding produced by point mutations associated with hereditary persistence of fetal hemoglobin.

1988 ◽  
Vol 8 (12) ◽  
pp. 5310-5322 ◽  
Author(s):  
D L Gumucio ◽  
K L Rood ◽  
T A Gray ◽  
M F Riordan ◽  
C I Sartor ◽  
...  
Keyword(s):  
Binding Site ◽  
Molecular Mechanisms ◽  
Globin Gene ◽  
Point Mutations ◽  
Fetal Hemoglobin ◽  
Nuclear Proteins ◽  
Proximal Promoter ◽  
Gamma Globin ◽  
Hereditary Persistence ◽  
Globin Promoter

The molecular mechanisms responsible for the human fetal-to-adult hemoglobin switch have not yet been elucidated. Point mutations identified in the promoter regions of gamma-globin genes from individuals with nondeletion hereditary persistence of fetal hemoglobin (HPFH) may mark cis-acting sequences important for this switch, and the trans-acting factors which interact with these sequences may be integral parts in the puzzle of gamma-globin gene regulation. We have used gel retardation and footprinting strategies to define nuclear proteins which bind to the normal gamma-globin promoter and to determine the effect of HPFH mutations on the binding of a subset of these proteins. We have identified five proteins in human erythroleukemia cells (K562 and HEL) which bind to the proximal promoter region of the normal gamma-globin gene. One factor, gamma CAAT, binds the duplicated CCAAT box sequences; the -117 HPFH mutation increases the affinity of interaction between gamma CAAT and its cognate site. Two proteins, gamma CAC1 and gamma CAC2, bind the CACCC sequence. These proteins require divalent cations for binding. The -175 HPFH mutation interferes with the binding of a fourth protein, gamma OBP, which binds an octamer sequence (ATGCAAAT) in the normal gamma-globin promoter. The HPFH phenotype of the -175 mutation indicates that the octamer-binding protein may play a negative regulatory role in this setting. A fifth protein, EF gamma a, binds to sequences which overlap the octamer-binding site. The erythroid-specific distribution of EF gamma a and its close approximation to an apparent repressor-binding site suggest that it may be important in gamma-globin regulation.

Nuclear proteins that bind the human gamma-globin gene promoter: alterations in binding produced by point mutations associated with hereditary persistence of fetal hemoglobin

1988 ◽  
Vol 8 (12) ◽  
pp. 5310-5322
Author(s):  
D L Gumucio ◽  
K L Rood ◽  
T A Gray ◽  
M F Riordan ◽  
C I Sartor ◽  
...  
Keyword(s):  
Binding Site ◽  
Molecular Mechanisms ◽  
Globin Gene ◽  
Point Mutations ◽  
Fetal Hemoglobin ◽  
Nuclear Proteins ◽  
Proximal Promoter ◽  
Gamma Globin ◽  
Hereditary Persistence ◽  
Globin Promoter

The molecular mechanisms responsible for the human fetal-to-adult hemoglobin switch have not yet been elucidated. Point mutations identified in the promoter regions of gamma-globin genes from individuals with nondeletion hereditary persistence of fetal hemoglobin (HPFH) may mark cis-acting sequences important for this switch, and the trans-acting factors which interact with these sequences may be integral parts in the puzzle of gamma-globin gene regulation. We have used gel retardation and footprinting strategies to define nuclear proteins which bind to the normal gamma-globin promoter and to determine the effect of HPFH mutations on the binding of a subset of these proteins. We have identified five proteins in human erythroleukemia cells (K562 and HEL) which bind to the proximal promoter region of the normal gamma-globin gene. One factor, gamma CAAT, binds the duplicated CCAAT box sequences; the -117 HPFH mutation increases the affinity of interaction between gamma CAAT and its cognate site. Two proteins, gamma CAC1 and gamma CAC2, bind the CACCC sequence. These proteins require divalent cations for binding. The -175 HPFH mutation interferes with the binding of a fourth protein, gamma OBP, which binds an octamer sequence (ATGCAAAT) in the normal gamma-globin promoter. The HPFH phenotype of the -175 mutation indicates that the octamer-binding protein may play a negative regulatory role in this setting. A fifth protein, EF gamma a, binds to sequences which overlap the octamer-binding site. The erythroid-specific distribution of EF gamma a and its close approximation to an apparent repressor-binding site suggest that it may be important in gamma-globin regulation.

scholarly journals The Phosphatidylserine Receptor TIM-1 Enhances Authentic Chikungunya Virus Cell Entry

Cells ◽  
2021 ◽  
Vol 10 (7) ◽  
pp. 1828
Author(s):  
Jared Kirui ◽  
Yara Abidine ◽  
Annasara Lenman ◽  
Koushikul Islam ◽  
Yong-Dae Gwon ◽  
...  
Keyword(s):  
Chikungunya Virus ◽  
Molecular Mechanisms ◽  
Rna Virus ◽  
Ectopic Expression ◽  
Point Mutations ◽  
Cell Entry ◽  
Target Cells ◽  
Human Hepatoma Cells ◽  
Chikv Infection ◽  
Phosphatidylserine Receptor

Chikungunya virus (CHIKV) is a re-emerging, mosquito-transmitted, enveloped positive stranded RNA virus. Chikungunya fever is characterized by acute and chronic debilitating arthritis. Although multiple host factors have been shown to enhance CHIKV infection, the molecular mechanisms of cell entry and entry factors remain poorly understood. The phosphatidylserine-dependent receptors, T-cell immunoglobulin and mucin domain 1 (TIM-1) and Axl receptor tyrosine kinase (Axl), are transmembrane proteins that can serve as entry factors for enveloped viruses. Previous studies used pseudoviruses to delineate the role of TIM-1 and Axl in CHIKV entry. Conversely, here, we use the authentic CHIKV and cells ectopically expressing TIM-1 or Axl and demonstrate a role for TIM-1 in CHIKV infection. To further characterize TIM-1-dependent CHIKV infection, we generated cells expressing domain mutants of TIM-1. We show that point mutations in the phosphatidylserine binding site of TIM-1 lead to reduced binding, entry, and infection of CHIKV. Ectopic expression of TIM-1 renders immortalized keratinocytes permissive to CHIKV, whereas silencing of endogenously expressed TIM-1 in human hepatoma cells reduces CHIKV infection. Altogether, our findings indicate that, unlike Axl, TIM-1 readily promotes the productive entry of authentic CHIKV into target cells.

scholarly journals Degenerative Cervical Myelopathy: Insights into Its Pathobiology and Molecular Mechanisms

2021 ◽  
Vol 10 (6) ◽  
pp. 1214
Author(s):  
Ji Tu ◽  
Jose Vargas Castillo ◽  
Abhirup Das ◽  
Ashish D. Diwan
Keyword(s):  
Cervical Myelopathy ◽  
Molecular Mechanisms ◽  
Current Knowledge ◽  
Imaging Techniques ◽  
Disease Process ◽  
Classification Systems ◽  
Molecular Features ◽  
Formidable Challenge ◽  
Long Term Treatment ◽  
Degenerative Cervical Myelopathy

Degenerative cervical myelopathy (DCM), earlier referred to as cervical spondylotic myelopathy (CSM), is the most common and serious neurological disorder in the elderly population caused by chronic progressive compression or irritation of the spinal cord in the neck. The clinical features of DCM include localised neck pain and functional impairment of motor function in the arms, fingers and hands. If left untreated, this can lead to significant and permanent nerve damage including paralysis and death. Despite recent advancements in understanding the DCM pathology, prognosis remains poor and little is known about the molecular mechanisms underlying its pathogenesis. Moreover, there is scant evidence for the best treatment suitable for DCM patients. Decompressive surgery remains the most effective long-term treatment for this pathology, although the decision of when to perform such a procedure remains challenging. Given the fact that the aged population in the world is continuously increasing, DCM is posing a formidable challenge that needs urgent attention. Here, in this comprehensive review, we discuss the current knowledge of DCM pathology, including epidemiology, diagnosis, natural history, pathophysiology, risk factors, molecular features and treatment options. In addition to describing different scoring and classification systems used by clinicians in diagnosing DCM, we also highlight how advanced imaging techniques are being used to study the disease process. Last but not the least, we discuss several molecular underpinnings of DCM aetiology, including the cells involved and the pathways and molecules that are hallmarks of this disease.

scholarly journals Knock-in and precise nucleotide substitution using near-PAMless engineered Cas9 variants in Dictyostelium discoideum

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Yuu Asano ◽  
Kensuke Yamashita ◽  
Aoi Hasegawa ◽  
Takanori Ogasawara ◽  
Hoshie Iriki ◽  
...  
Keyword(s):  
Genome Editing ◽  
Dictyostelium Discoideum ◽  
Streptococcus Pyogenes ◽  
Nucleotide Substitution ◽  
Point Mutations ◽  
Targeted Mutagenesis ◽  
Single Amino Acid ◽  
Specific Gene ◽  
Knock Out ◽  
Protospacer Adjacent Motif

AbstractThe powerful genome editing tool Streptococcus pyogenes Cas9 (SpCas9) requires the trinucleotide NGG as a protospacer adjacent motif (PAM). The PAM requirement is limitation for precise genome editing such as single amino-acid substitutions and knock-ins at specific genomic loci since it occurs in narrow editing window. Recently, SpCas9 variants (i.e., xCas9 3.7, SpCas9-NG, and SpRY) were developed that recognise the NG dinucleotide or almost any other PAM sequences in human cell lines. In this study, we evaluated these variants in Dictyostelium discoideum. In the context of targeted mutagenesis at an NG PAM site, we found that SpCas9-NG and SpRY were more efficient than xCas9 3.7. In the context of NA, NT, NG, and NC PAM sites, the editing efficiency of SpRY was approximately 60% at NR (R = A and G) but less than 22% at NY (Y = T and C). We successfully used SpRY to generate knock-ins at specific gene loci using donor DNA flanked by 60 bp homology arms. In addition, we achieved point mutations with efficiencies as high as 97.7%. This work provides tools that will significantly expand the gene loci that can be targeted for knock-out, knock-in, and precise point mutation in D. discoideum.

scholarly journals Molecular mechanisms in nickel carcinogenesis: modeling Ni(II) binding site in histone H4.

2002 ◽  
Vol 110 (suppl 5) ◽  
pp. 719-723 ◽  
Author(s):  
Maria Antonietta Zoroddu ◽  
Laura Schinocca ◽  
Teresa Kowalik-Jankowska ◽  
Henryk Kozlowski ◽  
Konstantin Salnikow ◽  
...  
Keyword(s):  
Binding Site ◽  
Molecular Mechanisms ◽  
Histone H4

scholarly journals RNA Editing as a Therapeutic Approach for Retinal Gene Therapy Requiring Long Coding Sequences

2020 ◽  
Vol 21 (3) ◽  
pp. 777 ◽  
Author(s):  
Lewis E. Fry ◽  
Caroline F. Peddle ◽  
Alun R. Barnard ◽  
Michelle E. McClements ◽  
Robert E. MacLaren
Keyword(s):  
Gene Therapy ◽  
Rna Editing ◽  
Therapeutic Approach ◽  
Genetic Diseases ◽  
Point Mutations ◽  
Transcript Level ◽  
Gene Replacement ◽  
Promising Therapeutic Approach ◽  
Pairing Site ◽  
Large Genes

RNA editing aims to treat genetic disease through altering gene expression at the transcript level. Pairing site-directed RNA-targeting mechanisms with engineered deaminase enzymes allows for the programmable correction of G>A and T>C mutations in RNA. This offers a promising therapeutic approach for a range of genetic diseases. For inherited retinal degenerations caused by point mutations in large genes not amenable to single-adeno-associated viral (AAV) gene therapy such as USH2A and ABCA4, correcting RNA offers an alternative to gene replacement. Genome editing of RNA rather than DNA may offer an improved safety profile, due to the transient and potentially reversible nature of edits made to RNA. This review considers the current site-directing RNA editing systems, and the potential to translate these to the clinic for the treatment of inherited retinal degeneration.

scholarly journals Transcription of alpha-specific genes in Saccharomyces cerevisiae: DNA sequence requirements for activity of the coregulator alpha 1.

1993 ◽  
Vol 13 (11) ◽  
pp. 6866-6875 ◽  
Author(s):  
D C Hagen ◽  
L Bruhn ◽  
C A Westby ◽  
G F Sprague
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Binding Site ◽  
Dna Sequences ◽  
Point Mutations ◽  
Transcription Activation ◽  
Specific Gene ◽  
Binding Assays ◽  
Weak Binding

Transcription activation of alpha-specific genes in Saccharomyces cerevisiae is regulated by two proteins, MCM1 and alpha 1, which bind to DNA sequences, called P'Q elements, found upstream of alpha-specific genes. Neither MCM1 nor alpha 1 alone binds efficiently to P'Q elements. Together, however, they bind cooperatively in a manner that requires both the P' sequence, which is a weak binding site for MCM1, and the Q sequence, which has been postulated to be the binding site for alpha 1. We analyzed a collection of point mutations in the P'Q element of the STE3 gene to determine the importance of individual base pairs for alpha-specific gene transcription. Within the 10-bp conserved Q sequence, mutations at only three positions strongly affected transcription activation in vivo. These same mutations did not affect the weak binding to P'Q displayed by MCM1 alone. In vitro DNA binding assays showed a direct correlation between the ability of the mutant sequences to form ternary P'Q-MCM1-alpha 1 complexes and the degree to which transcription was activated in vivo. Thus, the ability of alpha 1 and MCM1 to bind cooperatively to P'Q elements is critical for activation of alpha-specific genes. In all natural alpha-specific genes the Q sequence is adjacent to the degenerate side of P'. To test the significance of this geometry, we created several novel juxtapositions of P, P', and Q sequences. When the Q sequence was opposite the degenerate side, the composite QP' element was inactive as a promoter element in vivo and unable to form stable ternary QP'-MCM1-alpha 1 complexes in vitro. We also found that addition of a Q sequence to a strong MCM1 binding site allows the addition of alpha 1 to the complex. This finding, together with the observation that Q-element point mutations affected ternary complex formation but not the weak binding of MCM1 alone, supports the idea that the Q sequence serves as a binding site for alpha 1.

GATA and Ets cis-acting sequences mediate megakaryocyte-specific expression

1993 ◽  
Vol 13 (1) ◽  
pp. 668-676
Author(s):  
V Lemarchandel ◽  
J Ghysdael ◽  
V Mignotte ◽  
C Rahuel ◽  
P H Roméo
Keyword(s):  
Binding Site ◽  
Consensus Sequence ◽  
Mrna Level ◽  
Point Mutations ◽  
Specific Gene ◽  
Cell Type Specificity ◽  
Specific Expression ◽  
Platelet Factor ◽  
Cis Acting ◽  
Dna Fragment

The human glycoprotein IIB (GPIIB) gene is expressed only in megakaryocytes, and its promoter displays cell type specificity. We show that this specificity involved two cis-acting sequences. The first one, located at -55, contains a GATA binding site. Point mutations that abolish protein binding on this site decrease the activity of the GPIIB promoter but do not affect its tissue specificity. The second one, located at -40, contains an Ets consensus sequence, and we show that Ets-1 or Ets-2 protein can interact with this -40 GPIIB sequence. Point mutations that impair Ets binding decrease the activity of the GPIIB promoter to the same extent as do mutations that abolish GATA binding. A GPIIB 40-bp DNA fragment containing the GATA and Ets binding sites can confer activity to a heterologous promoter in megakaryocytic cells. This activity is independent of the GPIIB DNA fragment orientation, and mutations on each binding site result in decreased activity. Using cotransfection assays, we show that c-Ets-1 and human GATA1 can transactive the GPIIB promoter in HeLa cells and can act additively. Northern (RNA) blot analysis indicates that the ets-1 mRNA level is increased during megakaryocyte-induced differentiation of erythrocytic/megakaryocytic cell lines. Gel retardation assays show that the same GATA-Ets association is found in the human GPIIB enhancer and the rat platelet factor 4 promoter, the other two characterized regulatory regions of megakaryocyte-specific genes. These results indicate that GATA and Ets cis-acting sequences are an important determinant of megakaryocytic specific gene expression.

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