Dataset Information

Functional Identification of a Nuclear Localization Signal of MYB2 Protein in Giardia lamblia.

ABSTRACT: MYB2 protein was identified as a transcription factor that showed encystation-induced expression in Giardia lamblia. Although nuclear import is essential for the functioning of a transcription factor, an evident nuclear localization signal (NLS) of G. lamblia MYB2 (GlMYB2) has not been defined. Based on putative GlMYB2 NLSs predicted by 2 programs, a series of plasmids expressing hemagglutinin (HA)-tagged GlMYB2 from the promoter of G. lamblia glutamate dehydrogenase were constructed and transfected into Giardia trophozoites. Immunofluorescence assays using anti-HA antibodies indicated that GlMYB2 amino acid sequence #507-#530 was required for the nuclear localization of GlMYB2, and this sequence was named as NLSGlMYB2. We further verified this finding by demonstrating the nuclear location of a protein obtained by the fusion of NLSGlMYB2 and G. lamblia glyceraldehyde 3-phosphate dehydrogenase, a non-nuclear protein. Our data on GlMYB2 will expand our understanding on NLSs functioning in G. lamblia.

SUBMITTER: Kim J

PROVIDER: S-EPMC7806428 | biostudies-literature | 2020 Dec

REPOSITORIES: biostudies-literature

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Functional Identification of a Nuclear Localization Signal of MYB2 Protein in Giardia lamblia.

Kim Juri J Shin Mee Young MY Park Soon-Jung SJ

The Korean journal of parasitology 20201229 6

MYB2 protein was identified as a transcription factor that showed encystation-induced expression in Giardia lamblia. Although nuclear import is essential for the functioning of a transcription factor, an evident nuclear localization signal (NLS) of G. lamblia MYB2 (GlMYB2) has not been defined. Based on putative GlMYB2 NLSs predicted by 2 programs, a series of plasmids expressing hemagglutinin (HA)-tagged GlMYB2 from the promoter of G. lamblia glutamate dehydrogenase were constructed and transfe ...[more]

PMID: 33412772

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Project description:BackgroundEncystation is one of the two processes comprising the life cycle of Giardia lamblia, a protozoan pathogen with tetraploid genome. Giardia lamblia Myb2 (GlMyb2) is a distinct encystation-induced transcription factor whose binding sites are found in the promoter regions of many encystation-induced genes, including its own.MethodsTwo sequential CRISPR/Cas9 experiments were performed to remove four glmyb2 alleles. The expression level of G. lamblia cyst wall protein 1 (GlCWP1), a well-known target gene of GlMyb2, was measured via western blotting and immunofluorescence assays. Chromatin immunoprecipitation experiments using anti-GlMyb2 antibodies were performed on the encysting G. lamblia cells. Quantitative real-time PCR was performed to confirm an expression of candidate GlMyb2-regulated genes by comparing the transcript level for each target candidate in wild-type and knockout mutant Giardia. The promoter region of glcwp1 was analyzed via deletion and point mutagenesis of the putative GlMyb2 binding sites in luciferase reporters.ResultsCharacterization of the null glmyb2 mutant indicated loss of functions related to encystation, i.e. cyst formation, and expression of GlCWP1. The addition of the wild-type glmyb2 gene to the null mutant restored the defects in encystation. Chromatin immunoprecipitation experiments revealed dozens of target genes. Nineteen genes were confirmed as GlMyb2 regulons, which include the glmyb2 gene, six for cyst wall proteins, five for signal transduction, two for transporter, two for metabolic enzymes, and three with unknown functions. Detailed analysis on the promoter region of glcwp1 defined three GlMyb2 binding sites important in its encystation-induced expression.ConclusionsOur data confirm that GlMyb2 acts as a transcription activator especially during encystation by comparing the glmyb2 knockout mutant with the wild type. Further investigation using glmyb2 null mutant will provide knowledge regarding transcriptional apparatus required for the encystation process of G. lamblia.

Project description:The deep-branching protozoan parasite Giardia lamblia is the causative agent of the intestinal disease giardiasis. Consistent with its proposed evolutionary position, many pathways are minimalistic or divergent, including its actin cytoskeleton. Giardia is the only eukaryote known to lack all canonical actin-binding proteins. Previously, our lab identified a number of noncanonical Giardia lamblia actin (GlActin) interactors; however, these proteins appeared to interact only with monomeric or globular actin (G-actin) rather than with filamentous actin (F-actin). To identify F-actin interactors, we used a chemical cross-linker to preserve native interactions followed by an anti-GlActin antibody, protein A affinity chromatography, and liquid chromatography coupled to mass spectrometry. We found 46 putative actin interactors enriched under the conditions favoring F-actin. Data are available via ProteomeXchange with identifier PXD026067. None of the proteins identified contain known actin-interacting motifs, and many lacked conserved domains. Each potential interactor was then tagged with the fluorescent protein mNeonGreen and visualized in live cells. We categorized the proteins based on their primary localization; localizations included ventral disc, marginal plate, nuclei, flagella, plasma membrane, and internal membranes. One protein from each of the six categories was colocalized with GlActin using immunofluorescence microscopy. We also co-immunoprecipitated one protein from each category and confirmed three of the six potential interactions. Most of the localization patterns are consistent with previously demonstrated GlActin functions, but the ventral disc represents a new category of actin interactor localization. These results suggest a role for GlActin in ventral disc function, which has previously been controversial. IMPORTANCE Giardia lamblia is an intestinal parasite that colonizes the small intestine and causes diarrhea, which can lead to dehydration and malnutrition. Giardia actin (GlActin) has a conserved role in Giardia cells, despite being a highly divergent protein with none of the conserved regulators found in model organisms. Here, we identify and localize 46 interactors of polymerized actin. These putative interactors localize to a number of places in the cell, underlining GlActin's importance in multiple cellular processes. Surprisingly, eight of these proteins localize to the ventral disc, Giardia's host attachment organelle. Since host attachment is required for infection, proteins involved in this process are an appealing target for new drugs. While treatments for Giardia exist, drug resistance is becoming more common, resulting in a need for new treatments. Giardia and human systems are highly dissimilar, thus drugs specifically tailored to Giardia proteins would be less likely to have side effects.

Dataset Information

Functional Identification of a Nuclear Localization Signal of MYB2 Protein in Giardia lamblia.

Publications

Functional Identification of a Nuclear Localization Signal of MYB2 Protein in Giardia lamblia.

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