Purification of an insect juvenile hormone receptor complex enables insights into its post-translational phosphorylation

Kód výsledku v IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F60077344%3A_____%2F21%3A00549785" target="_blank" >RIV/60077344:_____/21:00549785 - isvavai.cz</a>

Výsledek na webu

<a href="https://www.sciencedirect.com/science/article/pii/S0021925821011935/pdfft?md5=ca6e964441ca194640b8b079e570c909&pid=1-s2.0-S0021925821011935-main.pdf" target="_blank" >https://www.sciencedirect.com/science/article/pii/S0021925821011935/pdfft?md5=ca6e964441ca194640b8b079e570c909&pid=1-s2.0-S0021925821011935-main.pdf</a>

DOI - Digital Object Identifier

<a href="http://dx.doi.org/10.1016/j.jbc.2021.101387" target="_blank" >10.1016/j.jbc.2021.101387</a>

Jazyk výsledku

angličtina

Název v původním jazyce

Purification of an insect juvenile hormone receptor complex enables insights into its post-translational phosphorylation

Popis výsledku v původním jazyce

Juvenile hormone (JH) plays vital roles in insect reproduction, development, and in many aspects of physiology. JH primarily acts at the gene-regulatory level through interaction with an intracellular receptor (JH receptor [JHR]), a ligand-activated complex of transcription factors consisting of the JH-binding protein methoprene-tolerant (MET) and its partner taiman (TAI). Initial studies indicated significance of post-transcriptional phosphorylation, subunit assembly, and nucleocytoplasmic transport of JHR in JH signaling. However, our knowledge of JHR regulation at the protein level remains rudimentary, partly because of the difficulty of obtaining purified and functional JHR proteins. Here, we present a method for high-yield expression and purification of JHR complexes from two insect species, the beetle T. castaneum and the mosquito Aedes aegypti. Recombinant JHR subunits from each species were coexpressed in an insect cell line using a baculovirus system. MET–TAI complexes were purified through affinity chromatography and anion exchange columns to yield proteins capable of binding both the hormonal ligand (JH III) and DNA bearing cognate JH-response elements. We further examined the beetle JHR complex in greater detail. Biochemical analyses and MS confirmed that T. castaneum JHR was a 1:1 heterodimer consisting of MET and Taiman proteins, stabilized by the JHR agonist ligand methoprene. Phosphoproteomics uncovered multiple phosphorylation sites in the MET protein, some of which were induced by methoprene treatment. Finally, we report a functional bipartite nuclear localization signal, straddled by phosphorylated residues, within the disordered C-terminal region of MET. Our present characterization of the recombinant JHR is an initial step toward understanding JHR structure and function.

Název v anglickém jazyce

Purification of an insect juvenile hormone receptor complex enables insights into its post-translational phosphorylation

Popis výsledku anglicky

Juvenile hormone (JH) plays vital roles in insect reproduction, development, and in many aspects of physiology. JH primarily acts at the gene-regulatory level through interaction with an intracellular receptor (JH receptor [JHR]), a ligand-activated complex of transcription factors consisting of the JH-binding protein methoprene-tolerant (MET) and its partner taiman (TAI). Initial studies indicated significance of post-transcriptional phosphorylation, subunit assembly, and nucleocytoplasmic transport of JHR in JH signaling. However, our knowledge of JHR regulation at the protein level remains rudimentary, partly because of the difficulty of obtaining purified and functional JHR proteins. Here, we present a method for high-yield expression and purification of JHR complexes from two insect species, the beetle T. castaneum and the mosquito Aedes aegypti. Recombinant JHR subunits from each species were coexpressed in an insect cell line using a baculovirus system. MET–TAI complexes were purified through affinity chromatography and anion exchange columns to yield proteins capable of binding both the hormonal ligand (JH III) and DNA bearing cognate JH-response elements. We further examined the beetle JHR complex in greater detail. Biochemical analyses and MS confirmed that T. castaneum JHR was a 1:1 heterodimer consisting of MET and Taiman proteins, stabilized by the JHR agonist ligand methoprene. Phosphoproteomics uncovered multiple phosphorylation sites in the MET protein, some of which were induced by methoprene treatment. Finally, we report a functional bipartite nuclear localization signal, straddled by phosphorylated residues, within the disordered C-terminal region of MET. Our present characterization of the recombinant JHR is an initial step toward understanding JHR structure and function.

Druh

J_imp - Článek v periodiku v databázi Web of Science

CEP obor

—

OECD FORD obor

10605 - Developmental biology

Projekt

<a href="/cs/project/GX20-05151X" target="_blank" >GX20-05151X: Nalezení chybějících genetických komponent a chemických regulátorů signalizace juvenilního hormonu</a><br>

Návaznosti

P - Projekt vyzkumu a vyvoje financovany z verejnych zdroju (s odkazem do CEP)

Rok uplatnění

2021

Kód důvěrnosti údajů

S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů

Název periodika

Journal of Biological Chemistry

ISSN

0021-9258

e-ISSN

1083-351X

Svazek periodika

297

Číslo periodika v rámci svazku

6

Stát vydavatele periodika

US - Spojené státy americké

Počet stran výsledku

21

Strana od-do

101387

Kód UT WoS článku

000748084900001

EID výsledku v databázi Scopus

2-s2.0-85111949580