TY - JOUR
T1 - ORP2 interacts with phosphoinositides and controls the subcellular distribution of cholesterol
AU - Koponen, Annika
AU - Arora, Amita
AU - Takahashi, Kohta
AU - Kentala, Henriikka
AU - Kivelä, Annukka M.
AU - Jääskeläinen, Eeva
AU - Peränen, Johan
AU - Somerharju, Pentti
AU - Ikonen, Elina
AU - Viitala, Tapani
AU - Olkkonen, Vesa M.
N1 - Funding Information:
This study was supported by grants from the Academy of Finland ( 285223 to V.M.O.), the University of Helsinki Doctoral Programme in Biomedicine (H.K.), the Finnish Concordia Fund (H.K.), the Ida Montin Foundation (H.K), the Finnish-Norwegian Medical Foundation (H.K.), the Aarne Koskelo Foundation (H.K.), the Orion Research Foundation (H.K.), the Päivikki and Sakari Sohlberg Foundation (H.K.), the Sigrid Juselius Foundation , the Magnus Ehrnrooth Foundation , and the Finnish Foundation for Cardiovascular Research (V.M.O.). The funding bodies played no role in the study design, analysis or interpretation of the data, writing of the report or the decision to submit the article for publication.
Funding Information:
This study was supported by grants from the Academy of Finland (285223 to V.M.O.), the University of Helsinki Doctoral Programme in Biomedicine (H.K.), the Finnish Concordia Fund (H.K.), the Ida Montin Foundation (H.K), the Finnish-Norwegian Medical Foundation (H.K.), the Aarne Koskelo Foundation (H.K.), the Orion Research Foundation (H.K.), the Päivikki and Sakari Sohlberg Foundation (H.K.), the Sigrid Juselius Foundation, the Magnus Ehrnrooth Foundation, and the Finnish Foundation for Cardiovascular Research (V.M.O.). The funding bodies played no role in the study design, analysis or interpretation of the data, writing of the report or the decision to submit the article for publication.
Publisher Copyright:
© 2018 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM)
PY - 2019/3
Y1 - 2019/3
N2 - ORP2 is a sterol-binding protein with documented functions in lipid and glucose metabolism, Akt signaling, steroidogenesis, cell adhesion, migration and proliferation. Here we investigate the interactions of ORP2 with phosphoinositides (PIPs) by surface plasmon resonance (SPR), its affinity for cholesterol with a pull-down assay, and its capacity to transfer sterol in vitro. Moreover, we determine the effects of wild-type (wt) ORP2 and a mutant with attenuated PIP binding, ORP2(mHHK), on the subcellular distribution of cholesterol, and analyze the interaction of ORP2 with the related cholesterol transporter ORP1L. ORP2 showed specific affinity for PI(4,5)P 2 , PI(3,4,5)P 3 and PI(4)P, with suggestive K d values in the μM range. Also binding of cholesterol by ORP2 was detectable, but a K d could not be determined. Wt ORP2 was in HeLa cells mainly detected in the cytosol, ER, late endosomes, and occasionally on lipid droplets (LDs), while ORP2(mHHK) displayed an enhanced LD localization. Overexpression of wt ORP2 shifted the D4H cholesterol probe away from endosomes, while ORP2(mHHK) caused endosomal accumulation of the probe. Although ORP2 failed to transfer dehydroergosterol in an in vitro assay where OSBP is active, its knock-down resulted in the accumulation of cholesterol in late endocytic compartments, as detected by both D4H and filipin probes. Interestingly, ORP2 was shown to interact and partially co-localize on late endosomes with ORP1L, a cholesterol transporter/sensor at ER-late endosome junctions. Our data demonstrates that ORP2 binds several phosphoinositides, both PI(4)P and multiply phosphorylated species. ORP2 regulates the subcellular distribution of cholesterol dependent on its PIP-binding capacity. The interaction of ORP2 with ORP1L suggests a concerted action of the two ORPs.
AB - ORP2 is a sterol-binding protein with documented functions in lipid and glucose metabolism, Akt signaling, steroidogenesis, cell adhesion, migration and proliferation. Here we investigate the interactions of ORP2 with phosphoinositides (PIPs) by surface plasmon resonance (SPR), its affinity for cholesterol with a pull-down assay, and its capacity to transfer sterol in vitro. Moreover, we determine the effects of wild-type (wt) ORP2 and a mutant with attenuated PIP binding, ORP2(mHHK), on the subcellular distribution of cholesterol, and analyze the interaction of ORP2 with the related cholesterol transporter ORP1L. ORP2 showed specific affinity for PI(4,5)P 2 , PI(3,4,5)P 3 and PI(4)P, with suggestive K d values in the μM range. Also binding of cholesterol by ORP2 was detectable, but a K d could not be determined. Wt ORP2 was in HeLa cells mainly detected in the cytosol, ER, late endosomes, and occasionally on lipid droplets (LDs), while ORP2(mHHK) displayed an enhanced LD localization. Overexpression of wt ORP2 shifted the D4H cholesterol probe away from endosomes, while ORP2(mHHK) caused endosomal accumulation of the probe. Although ORP2 failed to transfer dehydroergosterol in an in vitro assay where OSBP is active, its knock-down resulted in the accumulation of cholesterol in late endocytic compartments, as detected by both D4H and filipin probes. Interestingly, ORP2 was shown to interact and partially co-localize on late endosomes with ORP1L, a cholesterol transporter/sensor at ER-late endosome junctions. Our data demonstrates that ORP2 binds several phosphoinositides, both PI(4)P and multiply phosphorylated species. ORP2 regulates the subcellular distribution of cholesterol dependent on its PIP-binding capacity. The interaction of ORP2 with ORP1L suggests a concerted action of the two ORPs.
KW - Cholesterol
KW - Lipid transfer
KW - OSBP-related protein
KW - Phosphoinositide
KW - Surface plasmon resonance
UR - http://www.scopus.com/inward/record.url?scp=85059310150&partnerID=8YFLogxK
U2 - 10.1016/j.biochi.2018.12.013
DO - 10.1016/j.biochi.2018.12.013
M3 - Article
C2 - 30590084
AN - SCOPUS:85059310150
SN - 0300-9084
VL - 158
SP - 90
EP - 101
JO - Biochimie
JF - Biochimie
ER -