Biochemical characterization and functional analysis of the POU transcription factor POU-M2 of Bombyx mori

Liu, Lina; Li, Yu; Wang, Yejing; Zhao, Peng; Wei, Shuguang; Li, Zhenzhen; Chang, Huaipu; He, Huawei

doi:10.1016/j.ijbiomac.2016.02.016

Cited by 18 publications

(10 citation statements)

References 47 publications

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“…Bombyx mori strain P50 (Dazao) was reared as previously described [ 44 ]. Hemolymph was collected from female silkworm larvae on day 7 of the fifth instar.…”

Section: Methodsmentioning

confidence: 99%

LBD1 of Vitellogenin Receptor Specifically Binds to the Female-Specific Storage Protein SP1 via LBR1 and LBR3

et al. 2016

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Storage proteins are the major protein synthesized in the fat body, released into hemolymph and re-sequestered into the fat body before pupation in most insect species. Storage proteins are important amino acid and nutrition resources during the non-feeding pupal period and play essential roles for the metamorphosis and oogenesis of insects. The sequestration of storage protein is a selective, specific receptor-mediated process. However, to date, the potential receptor mediating the sequestration of storage protein has not been determined in Bombyx mori. In this study, we expressed and purified the first ligand binding domain of Bombyx mori vitellogenin receptor (BmVgR), LBD1, and found LBD1 could bind with an unknown protein from the hemolymph of the ultimate silkworm larval instar via pull-down assay. This unknown protein was subsequently identified to be the female-specific storage protein SP1 by mass spectrometry. Furthermore, far western blotting assay, immunoprecipitation and isothermal titration calorimetry analysis demonstrated LBD1 specifically bound with the female-specific SP1, rather than another unisex storage protein SP2. The specific binding of LBD1 with SP1 was dependent on the presence of Ca2+ as it was essential for the proper conformation of LBD1. Deletion mutagenesis and ITC analysis revealed the first and third ligand binding repeats LBR1 and LBR3 were indispensable for the binding of LBD1 with SP1, and LBR2 and LBR4 also had a certain contribution to the specific binding. Our results implied BmVgR may mediate the sequestration of SP1 from hemolymph into the fat body during the larval-pupal transformation of Bombyx mori.

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“…Bombyx mori strain P50 (Dazao) was reared as previously described [ 44 ]. Hemolymph was collected from female silkworm larvae on day 7 of the fifth instar.…”

Section: Methodsmentioning

confidence: 99%

LBD1 of Vitellogenin Receptor Specifically Binds to the Female-Specific Storage Protein SP1 via LBR1 and LBR3

et al. 2016

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“…In brief, DNA target probes including the POU elements were designed and labeled with biotin (synthesized by Sangon Biotech, Shanghai, China). Probes were annealed by incubating the complementary strands at 100 • C for 10 min, followed by slow cooling to 25 • C. To perform the EMSA, 2.5 µM of the annealed labeled probes were incubated with the supplied buffer, the purified POU-M2 protein (as described in [38]), and the nucleoproteins of POU-M2-overexpressing BmNs cells were extracted using a Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime, Beijing, China). Some treatments were also performed with a 50 µM unlabeled competitor probe, a 2.5 µM mutational probe and a rabbit polyclonal antibody against POU-M2 (anti-POU-M2) (0.85 µg/µL).…”

Section: Electrophoretic Mobility Shift Assay (Emsa)mentioning

confidence: 99%

The POU Transcription Factor POU-M2 Regulates Vitellogenin Receptor Gene Expression in the Silkworm, Bombyx mori

Shen

Chen

et al. 2020

Genes

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Vitellogenin receptors (VgRs) play critical roles in egg formation by transporting vitellogenin (Vg) into oocytes in insects. Although the function of VgR in insects is well studied, the transcriptional regulation of this gene is still unclear. Here, we cloned the promoter of the VgR gene from Bombyx mori (BmVgR), and predicted many POU cis-response elements (CREs) in its promoter. Electrophoretic mobility shift and chromatin immunoprecipitation assays showed that the POU transcription factor POU-M2 bound directly to the CREs of the promoter. Overexpression of POU-M2 in an ovarian cell line (BmNs) enhanced BmVgR transcription and promoter activity detected by quantitative reverse transcription PCR and luciferase reporter assays. Analyses of expression patterns indicated that POU-M2 was expressed in ovary at day two of wandering stage initially, followed by BmVgR. RNA interference of POU-M2 significantly reduced the transcription of BmVgR in ovary and egg-laying rate. Our results suggest a novel function for the POU factor in silkworm oogenesis by its involvement in BmVgR regulation and expands the understanding of POU factors in insect VgR expression.

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“…The cellular location of BmADK was determined as described by Liu [34]. BmE cells were grown on glass coverslips.…”

Section: Methodsmentioning

confidence: 99%

A Novel Adenosine Kinase from Bombyx mori: Enzymatic Activity, Structure, and Biological Function

Song

Liu

et al. 2019

IJMS

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Adenosine kinase (ADK) is the first enzyme in the adenosine remediation pathway that catalyzes adenosine phosphorylation into adenosine monophosphate, thus regulating adenosine homeostasis in cells. To obtain new insights into ADK from Bombyx mori (BmADK), we obtained recombinant BmADK, and analyzed its activity, structure, and function. Gel-filtration showed BmADK was a monomer with molecular weight of approximately 38 kDa. Circular dichroism spectra indicated BmADK had 36.8% α-helix and 29.9% β-strand structures, respectively. The structure of BmADK was stable in pH 5.0–11.0, and not affected under 30 °C. The melting temperature and the enthalpy and entropy changes in the thermal transition of BmADK were 46.51 ± 0.50 °C, 253.43 ± 0.20 KJ/mol, and 0.79 ± 0.01 KJ/(mol·K), respectively. Site-directed mutagenesis demonstrated G68, S201, E229, and D303 were key amino acids for BmADK structure and activity. In particular, S201A mutation significantly increased the α-helix content of BmADK and its activity. BmADK was located in the cytoplasm and highly expressed in the silk gland during the pre-pupal stage. RNA interference revealed the downregulation of BmADK decreased ATG-8, Caspase-9, Ec-R, E74A, and Br-C expression, indicating it was likely involved in 20E signaling, apoptosis, and autophagy to regulate silk gland degeneration and silkworm metamorphosis. Our study greatly expanded the knowledge on the activity, structure, and role of ADK.

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Biochemical characterization and functional analysis of the POU transcription factor POU-M2 of Bombyx mori

Cited by 18 publications

References 47 publications

LBD1 of Vitellogenin Receptor Specifically Binds to the Female-Specific Storage Protein SP1 via LBR1 and LBR3

LBD1 of Vitellogenin Receptor Specifically Binds to the Female-Specific Storage Protein SP1 via LBR1 and LBR3

The POU Transcription Factor POU-M2 Regulates Vitellogenin Receptor Gene Expression in the Silkworm, Bombyx mori

A Novel Adenosine Kinase from Bombyx mori: Enzymatic Activity, Structure, and Biological Function

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