scholarly journals Creating a ‘Molecular Band-Aid’; Blocking an Exposed Protease Target Site in Desmoplakin

2021 ◽  
Vol 11 (5) ◽  
pp. 401
Author(s):  
Catherine A. Hoover ◽  
Kendahl L. Ott ◽  
Heather R. Manring ◽  
Trevor Dew ◽  
Maegen A. Borzok ◽  
...  
Keyword(s):  
Hot Spot ◽  
Single Point ◽  
Md Simulations ◽  
Amino Acid Position ◽  
Target Site ◽  
Molecular Band ◽  
Single Point Mutation ◽  
Enzymatic Assays ◽  
Protein Levels ◽  
Clinical Variants

Desmoplakin (DSP) is a large (~260 kDa) protein found in the desmosome, a subcellular complex that links the cytoskeleton of one cell to its neighbor. A mutation ‘hot-spot’ within the NH2-terminal third of the DSP protein (specifically, residues 299–515) is associated with both cardiomyopathies and skin defects. In select DSP variants, disease is linked specifically to the uncovering of a previously-occluded calpain target site (residues 447–451). Here, we partially stabilize these calpain-sensitive DSP clinical variants through the addition of a secondary single point mutation—tyrosine for leucine at amino acid position 518 (L518Y). Molecular dynamic (MD) simulations and enzymatic assays reveal that this stabilizing mutation partially blocks access to the calpain target site, resulting in restored DSP protein levels. This ‘molecular band-aid’ provides a novel way to maintain DSP protein levels, which may lead to new strategies for treating this subset of DSP-related disorders.

Repeated Application of Sequence-Specific SiRNA Molecules Leads to an Effective Downmodulation of All Clinically Relevant bcr-abl Gene Variants.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4319-4319 ◽  
Author(s):  
Lara Wohlbold ◽  
Heiko van der Kuip ◽  
Alexandra Moehring ◽  
Galit Granot ◽  
Moshe Oren ◽  
...  
Keyword(s):  
Single Point ◽  
K562 Cells ◽  
Single Point Mutation ◽  
Single Treatment ◽  
Repeated Application ◽  
Hairpin Rna ◽  
Sirna Sequence ◽  
Knock Down ◽  
Protein Levels ◽  
Optimized Protocol

Abstract Fusion transcripts such as bcr-abl encoding pathological oncogenic proteins represent ideal targets for a tumor-specific RNA interference (RNAi) approach. The aim of the present study was to optimize the efficacy of bcr-abl RNAi. We evaluated several synthetic siRNAs targeting the fusion sites of all common bcr-abl transcript variants (e14a2, e13a2, or e1a2). Significant knock-down of p210Bcr-abl and p190Bcr-abl fusion proteins was successfully achieved in bcr-abl expressing 32D cells and human leukemic K562 and MEG-01 cells. Repeated application of siRNA proved to be significantly more efficient compared to single treatment. The optimized protocol led to a total decrease of up to 75–90% in Bcr-abl protein levels, which was accompanied by a loss of viability of up to 90%. The target specificity of the siRNA was high, and even a single point mutation in the siRNA-sequence led to significant, albeit not complete, loss of siRNA efficacy. To expend the duration of the knock-down effect, we explored the use of plasmids driving the stable expression of short hairpin RNA (shRNA). Efficient downregulation of Bcr-abl protein was achieved in K562 cells transfected with a pSUPER-based plasmid encoding bcr-abl shRNA.

scholarly journals Contribution of a mutational hot spot to hemoglobin adaptation in high-altitude Andean house wrens

2015 ◽  
Vol 112 (45) ◽  
pp. 13958-13963 ◽  
Author(s):  
Spencer C. Galen ◽  
Chandrasekhar Natarajan ◽  
Hideaki Moriyama ◽  
Roy E. Weber ◽  
Angela Fago ◽  
...  
Keyword(s):  
High Altitude ◽  
Hot Spot ◽  
Globin Gene ◽  
Single Point ◽  
Evolutionary Genetics ◽  
Single Point Mutation ◽  
Phenotypic Evolution ◽  
House Wren ◽  
House Wrens ◽  
Low Altitude

A key question in evolutionary genetics is why certain mutations or certain types of mutation make disproportionate contributions to adaptive phenotypic evolution. In principle, the preferential fixation of particular mutations could stem directly from variation in the underlying rate of mutation to function-altering alleles. However, the influence of mutation bias on the genetic architecture of phenotypic evolution is difficult to evaluate because data on rates of mutation to function-altering alleles are seldom available. Here, we report the discovery that a single point mutation at a highly mutable site in the βA-globin gene has contributed to an evolutionary change in hemoglobin (Hb) function in high-altitude Andean house wrens (Troglodytes aedon). Results of experiments on native Hb variants and engineered, recombinant Hb mutants demonstrate that a nonsynonymous mutation at a CpG dinucleotide in the βA-globin gene is responsible for an evolved difference in Hb–O2 affinity between high- and low-altitude house wren populations. Moreover, patterns of genomic differentiation between high- and low-altitude populations suggest that altitudinal differentiation in allele frequencies at the causal amino acid polymorphism reflects a history of spatially varying selection. The experimental results highlight the influence of mutation rate on the genetic basis of phenotypic evolution by demonstrating that a large-effect allele at a highly mutable CpG site has promoted physiological differentiation in blood O2 transport capacity between house wren populations that are native to different elevations.

scholarly journals COMBINED TARGET SITE VGSC MUTATIONS PLAY A PRIMARY ROLE IN PYRETHROID RESISTANT PHENOTYPES OF Aedes aegypti AS DENGUE VECTOR FROM PALU CITY, CENTRAL SULAWESI

2019 ◽  
Vol 7 (5) ◽  
pp. 93
Author(s):  
Sitti Rahmah Umniyati
Keyword(s):  
Aedes Aegypti ◽  
Point Mutation ◽  
Gene Mutation ◽  
Single Point ◽  
Pcr Primers ◽  
Gene Bank ◽  
Target Site ◽  
Single Point Mutation ◽  
Primary Role ◽  
Endemic Areas

It has been reported that Aedes aegypti mosquitoes in Palu City had been resistant to cypermethrin insecticide but the resistance mechanism is not well known. This study aimed to determine the resistance status of Ae. aegypti to cypermethrin and whether the mutation of voltage-gated sodium channel (VGSC) was associated with pyretroid resistance in high and low dengue endemic areas in Palu City. Aedes aegypti collected from each village was reared to adult and assayed for susceptibility test to cypermethrin using the CDC bottle bioassay method. PCR primers of AaSCF1 and AaSCR4 were used for screening of IIS6 VGSC gene mutation. PCR primers of AaSCF7 and AaSCR7 were used for screening of IIIS6 VGSC gene mutation. For an identification of mutation sites were sequenced and aligned to Gene bank (access No. AB914689 and AB914690) for IIS6 VGSC and Gene bank (access No. AB914687 and AB914688) for IIIS6 VGSC gene mutation. The susceptibility status of Ae. aegypti to cypermethrin was resistant in high dengue endemic areas and moderately resistant in low dengue endemic areas. It was found double point mutation at S989P and V1016G in Ae. aegypti from high and low dengue endemic areas in Palu City and there was a single point mutation only in high dengue endemic area at target site V1016G. Aedes aegypti from both high and low dengue endemic areas were resistant to cyperpethrinn and the two alleles had a major role in the occurrence of cypermethrin resistance in Palu City.

Transgenic rescue demonstrates involvement of the Ian5 gene in T cell development in the rat

2004 ◽  
Vol 19 (2) ◽  
pp. 228-232 ◽  
Author(s):  
Mieczyslaw Michalkiewicz ◽  
Teresa Michalkiewicz ◽  
Ruth A. Ettinger ◽  
Elizabeth A. Rutledge ◽  
Jessica M. Fuller ◽  
...  
Keyword(s):  
T Cell ◽  
Single Point ◽  
T Cell Development ◽  
Artificial Chromosome ◽  
Single Point Mutation ◽  
Wild Type ◽  
Protein Levels ◽  
Novel Gene ◽  
Definitive Method ◽  
T Cell Lymphopenia

A single point mutation in a novel immune-associated nucleotide gene 5 ( Ian5) coincides with severe T cell lymphopenia in BB rats. We used a transgenic rescue approach in lymphopenic BB-derived congenic F344. lyp/ lyp rats to determine whether this mutation is responsible for lymphopenia and to establish the functional importance of this novel gene. A 150-kb P1 artificial chromosome (PAC) transgene harboring a wild-type allele of the rat Ian5 gene restored Ian5 transcript and protein levels, completely rescuing the T cell lymphopenia in the F344. lyp/ lyp rats. This successful complementation provides direct functional evidence that the Ian5 gene product is essential for maintaining normal T cell levels. It also demonstrates that transgenic rescue in the rat is a practical and definitive method for revealing the function of a novel gene.

scholarly journals Widespread Mutations in Voltage-Gated Sodium Channel Gene of Cimex lectularius (Hemiptera: Cimicidae) Populations in Paris

2021 ◽  
Vol 18 (2) ◽  
pp. 407
Author(s):  
Mohammad Akhoundi ◽  
Dahlia Chebbah ◽  
Denis Sereno ◽  
Anthony Marteau ◽  
Julie Jan ◽  
...  
Keyword(s):  
Single Point ◽  
Cimex Lectularius ◽  
Single Point Mutation ◽  
Homozygous Mutation ◽  
Knockdown Resistance ◽  
Bed Bugs ◽  
Bed Bug ◽  
Channel Gene ◽  
Blood Sucking ◽  
The Status

Bed bugs, Cimex lectularius and C. hemipterus, are common blood-sucking ectoparasites of humans with a large geographical distribution, worldwide. In France, little is known about the status of bed bugs’ infestation and their resistance to insecticides, particularly, pyrethroids. Here, we aimed to find mutations in the kdr gene, known to be involved in resistance to insecticides. We gathered bed bugs from various infested locations, including 17 private houses, 12 HLM building complex, 29 apartments, 2 EHPAD, and 2 immigrants’ residences. A total of 1211 bed bugs were collected and morphologically identified as C. lectularius. Two fragments of the kdr gene, encompassing codons V419L and L925I, were successfully amplified for 156 specimens. We recorded sense mutation in the first amplified fragment (kdr1) in 89 out of 156 (57%) samples, in which in 61 out of 89 (68.5%) sequences, a change of valine (V) into leucine (L) V419L was observed. Within the second fragment (kdr2), a homozygous mutation was recorded in 73 out of 156 (46.7%) specimens at the codon 925. At this position, 43 out of 73 (58.9%) specimens had a sense mutation leading to the replacement of leucine (L) by isoleucine (I). Among 162 mutant sequences analyzed (89 for the kdr1 fragment and 73 for the kdr2 one), we detected single point mutation in 26.6%, while 73.4% presented the mutation in both kdr1 and kdr2 fragments. All modifications recorded in bed bug populations of Paris are described to be involved in the knockdown resistance (kdr) against pyrethroids.

scholarly journals A Fungal Defensin Targets the SARS−CoV−2 Spike Receptor−Binding Domain

2021 ◽  
Vol 7 (7) ◽  
pp. 553
Author(s):  
Bin Gao ◽  
Shunyi Zhu
Keyword(s):  
Receptor Binding ◽  
Viral Entry ◽  
Single Point ◽  
Binding Domain ◽  
Host Cells ◽  
Single Point Mutation ◽  
Binding Motif ◽  
Microsporum Canis ◽  
Receptor Binding Domain ◽  
Fungal Defensin

Coronavirus Disease 2019 (COVID−19) elicited by the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS−CoV−2) is calling for novel targeted drugs. Since the viral entry into host cells depends on specific interactions between the receptor−binding domain (RBD) of the viral Spike protein and the membrane−bound monocarboxypeptidase angiotensin converting enzyme 2 (ACE2), the development of high affinity RBD binders to compete with human ACE2 represents a promising strategy for the design of therapeutics to prevent viral entry. Here, we report the discovery of such a binder and its improvement via a combination of computational and experimental approaches. The binder micasin, a known fungal defensin from the dermatophytic fungus Microsporum canis with antibacterial activity, can dock to the crevice formed by the receptor−binding motif (RBM) of RBD via an extensive shape complementarity interface (855.9 Å2 in area) with numerous hydrophobic and hydrogen−bonding interactions. Using microscale thermophoresis (MST) technique, we confirmed that micasin and its C−terminal γ−core derivative with multiple predicted interacting residues exhibited a low micromolar affinity to RBD. Expanding the interface area of micasin through a single point mutation to 970.5 Å2 accompanying an enhanced hydrogen bond network significantly improved its binding affinity by six−fold. Our work highlights the naturally occurring fungal defensins as an emerging resource that may be suitable for the development into antiviral agents for COVID−19.

scholarly journals Biochemical and molecular characterization of Alternaria alternata isolates highly resistant to procymidone from broccoli and cabbage

2021 ◽  
Vol 3 (1) ◽  
Author(s):  
Bingran Wang ◽  
Tiancheng Lou ◽  
Lingling Wei ◽  
Wenchan Chen ◽  
Longbing Huang ◽  
...  
Keyword(s):  
Alternaria Alternata ◽  
Single Point ◽  
Sodium Dodecyl ◽  
Cross Resistance ◽  
Molecular Characteristics ◽  
Single Point Mutation ◽  
Wide Range ◽  
Significant Difference ◽  
Leaf Blights ◽  
High Level

AbstractAlternaria alternata, a causal agent of leaf blights and spots on a wide range of hosts, has a high risk of developing resistance to fungicides. Procymidone, a dicarboximide fungicide (DCF), has been widely used in controlling Alternaria leaf blights in China for decades. However, the resistance of A. alternata against DCFs has rarely been reported from crucifer plants. A total of 198 A. alternata isolates were collected from commercial fields of broccoli and cabbage during 2018–2019, and their sensitivities to procymidone were determined. Biochemical and molecular characteristics were subsequently compared between the high-level procymidone-resistant (ProHR) and procymidone-sensitive (ProS) isolates, and also between ProHR isolates from broccoli and cabbage. Compared with ProS isolates, the mycelial growth rate, sporulation capacity and virulence of most ProHR isolates were reduced; ProHR isolates displayed an increased sensitivity to osmotic stresses and a reduced sensitivity to sodium dodecyl sulfate (SDS); all ProHR isolates showed a reduced sensitivity to hydrogen peroxide (H2O2) except for the isolate B102. Correlation analysis revealed a positive cross-resistance between procymidone and iprodione, or fludioxonil. When treated with 10 μg/mL of procymidone, both mycelial intracellular glycerol accumulations (MIGAs) and relative expression of AaHK1 in ProS isolates were higher than those in ProHR isolates. Sequence alignment of AaHK1 from ten ProHR isolates demonstrated that five of them possessed a single-point mutation (P94A, V612L, E708K or Q924STOP), and four isolates had an insertion or a deletion in their coding regions. No significant difference in biochemical characteristics was observed among ProHR isolates from two different hosts, though mutations in AaHK1 of the cabbage-originated ProHR isolates were distinct from those of the broccoli-originated ProHR isolates.

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