Publications by authors named "Najla M Albishi"

2 Publications

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Identification of juvenile hormone-induced posttranslational modifications of methoprene tolerant and Kr├╝ppel homolog 1 in the yellow fever mosquito, Aedes aegypti.

J Proteomics 2021 06 4;242:104257. Epub 2021 May 4.

Department of Entomology, College of Agriculture, Food and Environment, University of Kentucky, S-225 Agricultural Science Center North Lexington, KY 40546-0091, United States. Electronic address:

Recent studies reported that JH-regulated phosphorylation status of the JH-receptor complex contributes to its transcription activity in Aedes aegypti. However, phosphorylation sites of these proteins have not yet been identified. In this study, we found that the fusion of an EGFP tag to Ae. aegypti Kr-h1 (AaKr-h1) and Met (AaMet) improved their stability in mosquito Aag-2 cells, which allowed their purification. The liquid chromatography and tandem mass spectrometry analysis of the purified AaKr-h1 showed that the phosphoserine residue at position 694, located in the evolutionarily conserved SVIQ motif, is dephosphorylated when the cells are exposed to JH. The AaKr-h1 dephosphorylation mutant (S694V) showed significantly higher activity in inducing the luciferase gene regulated by JH response elements. The phosphorylation profile of Met also changed after exposing Aag-2 cells to JH III. The Ser-77 and Ser-710 residues of Met were phosphorylated after JH III treatment. In contrast, the two phosphoserine residues at positions 73 and 747 were dephosphorylated after JH III treatment. JH exposure also induced transient and reversible phosphorylation of Thr-664 and Ser-723 residues. Overall, these data show that JH induces changes in post-translational modifications of AaMet and AaKr-h1. SIGNIFICANCE: Female Aedes aegypti mosquitoes are known to vector many disease agents, including Zika virus, dengue virus chikungunya virus, and Mayaro and yellow fever virus. In the present study, we developed an efficient method to prepare Ae. aegypti Met and Kr-h1, which are typically difficult to produce and purify, using a mosquito cell line expression system. A liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based approaches were utilized to map the phosphorylation profiles of the isolated proteins. We then monitored the changes induced by JH activation in the phosphorylation profiles to check if the JH modulates post-translation modification of its key transcription factors. We found that the JH induced alterations in the phosphorylation profiles of the multiple residues of AaMet. In contrast, activation of the JH signaling pathway was accompanied by dephosphorylation of AaKr-h1 at phosphoserine-694, increasing its transcriptional activity. In addition, S694 of AaKr-h1 was located in the RMSSVIQYA motif highly conserved in orthologous proteins from other insect species. These results can help us further understand how JH modulates its key transcription factors and provide a basis for the development of novel insect control strategies.
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http://dx.doi.org/10.1016/j.jprot.2021.104257DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8218339PMC
June 2021

Expanding the Toolkit for Genome Editing in a Disease Vector, Transgenic Lines Expressing Cas9 and Single Guide RNA Induce Efficient Mutagenesis.

CRISPR J 2021 Jan 15. Epub 2021 Jan 15.

Department of Entomology, College of Agriculture, Food and Environment, University of Kentucky, Lexington, Kentucky, USA.

CRISPR-Cas9 mediated genome editing methods are being used for the analysis of gene function. However, it is hard to identify gene knockout mutants for genes whose knockout does not cause distinct phenotypes. To overcome this issue in the disease vector, , a transgenic Cas9/single guide RNA (sgRNA) method, was used to knock out the eye marker gene, (), and the juvenile hormone receptor, (). PiggyBac transformation vectors were prepared to express sgRNAs targeting and under the control of the U6 promoter. Transgenic expressing -sgRNA or -sgRNA under the control of the U6 promoter and enhanced green fluorescent protein (eGFP) under the control of the hr5ie1 promoter were produced. The U6-sgRNA adults were mated with AAEL010097-Cas9 adults. The progeny were screened, and the insects expressing eGFP and DsRed were selected and evaluated for mutations in target genes. About 77% and 78% of the progeny that were positive for both eGFP and DsRed in -sgRNA and -sgRNA groups, respectively, showed mutations in their target genes.
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http://dx.doi.org/10.1089/crispr.2020.0052DOI Listing
January 2021