Auxin-inducible degron technology allows rapid and controlled protein depletion. However, basal d... more Auxin-inducible degron technology allows rapid and controlled protein depletion. However, basal degradation without auxin and inefficient auxin-inducible depletion have limited its utility. We have identified a potent auxin-inducible degron system composed of auxin receptor F-box protein AtAFB2 and short degron miniIAA7. The system showed minimal basal degradation and enabled rapid auxin-inducible depletion of endogenous human transmembrane, cytoplasmic and nuclear proteins in 1 h with robust functional phenotypes. Loss-of-function methods for reducing target protein levels target DNA, RNA or protein 1 . Auxin-inducible degron (AID) technology allows rapid targeted protein depletion with the smallmolecule auxin 2 . To apply AID, an auxin-inducible destabilizing domain, or 'degron' , is fused to the target protein. In addition, an auxin receptor F-box protein TIR1/AFB is exogenously expressed, forming a chimeric SKP1-CUL1-F-Box (SCF) ubiquitin E3 ligase with endogenous components in eukaryotic cells. The chimeric E3 ligase recruits the degron in an auxin-dependent manner 3 . Addition of indole-3-acetic acid (IAA) or other small molecules of the auxin class to the culture medium thus induces the polyubiquitination and rapid proteasomal degradation of the degron-fused protein (Fig. ). This approach has been used as a powerful tool to acutely deplete target proteins in studying their functions . However, current AID systems can severely degrade target proteins before IAA addition (known as 'basal degradation') and suffer from inefficient auxin-inducible depletion in a context-and target-specific manner 2,8-10 . These pitfalls have substantially limited the applications of AID technology. An AID system with both minimal basal degradation and rapid auxin-inducible depletion would be desirable. In current AID systems, Oryza sativa TIR1 (OsTIR1) is the most commonly used auxin receptor F-box protein in combination with different degrons deriving from Arabidopsis thaliana IAA17 (AtIAA17) 2,4,5,7-14 . We initially aimed to acutely deplete endogenous human seipin, a transmembrane protein involved in lipid droplet (LD) biogenesis. However, seipin tagged with a degron termed miniAID (composed of AtIAA17 amino acid (aa) 68-132) was severely degraded in cells expressing OsTIR1 without IAA addition. Consequently, cells exhibited defective LD biogenesis already before IAA addition, resembling a seipin knockout phenotype 15 (Supplementary Fig. ). To search for an improved AID system, we established a pipeline in human A431 cells for screening various auxin receptor F-box proteins and degrons, through co-integration into the adenoassociated virus integration site 1 (AAVS1) safe harbor locus 16 (Fig. and Supplementary Note 1). We first compared OsTIR1 to five other auxin receptor F-box proteins, using miniAID as the degron. A. thaliana AFB2 (AtAFB2) was identified as the best hit: compared to OsTIR1, it displayed minimal basal depletion
Lipid droplets (LDs) are cellular organelles specialized in triacylglycerol (TG) storage undergoi... more Lipid droplets (LDs) are cellular organelles specialized in triacylglycerol (TG) storage undergoing homotypic clustering and fusion. In non-adipocytic cells with numerous LDs this is balanced by poorly understood droplet dissociation mechanisms. We identify non-muscle myosin IIa (NMIIa/MYH-9) and formin-like 1 (FMNL1) in the LD proteome. NMIIa and actin filaments concentrate around LDs, and form transient foci between dissociating LDs. NMIIa depletion results in decreased LD dissociations, enlarged LDs, decreased hydrolysis and increased storage of TGs. FMNL1 is required for actin assembly on LDs in vitro and for NMIIa recruitment to LDs in cells. We propose a novel acto-myosin structure regulating lipid storage: FMNL1-dependent assembly of myosin II-functionalized actin filaments on LDs facilitates their dissociation, thereby affecting LD surface-to-volume ratio and enzyme accessibility to TGs. In neutrophilic leucocytes from MYH9-related disease patients NMIIa inclusions are accompanied by increased lipid storage in droplets, suggesting that NMIIa dysfunction may contribute to lipid imbalance in man.
In this article, we summarize the present information related to the export of LDL-derived choles... more In this article, we summarize the present information related to the export of LDL-derived cholesterol from late endosomes, with a focus on Nieman-Pick disease, type C1 (NPC1) cholesterol delivery toward the endoplasmic reticulum (ER). We review data suggesting that several pathways may operate in parallel, including membrane transport routes and membrane contact sites (MCSs). There is increasing appreciation that MCSs provide an important mechanism for intermembrane lipid transfer. In late endosome-ER contacts, three protein bridges involving oxysterol binding protein related protein (ORP)1L-vesicle associated membrane protein-associated protein (VAP), steroidogenic acute regulatory protein (StAR)D3-VAP and ORP5-NPC1 proteins have been reported. How much they contribute to the flux of LDL-cholesterol to the ER is currently open. Studies for lipid transfer via MCSs have been most advanced in Saccharomyces cerevisiae. Recently, a new sterol-binding protein family conserved between yeast and man was identified. Its members localize at MCSs and were named lipid transfer protein anchored at membrane contact sites (Lam) proteins. In yeast, sterol transfer between the ER and the yeast lysosome may be facilitated by a Lam protein.
Background and aims: Among subjects with high-density-lipoprotein cholesterol (HDL-C) below the 1... more Background and aims: Among subjects with high-density-lipoprotein cholesterol (HDL-C) below the 1st percentile in the general population, we identified a heterozygous variant OSBPL1A p.C39X encoding a short truncated protein fragment that co-segregated with low plasma HDL-C. Methods: We investigated the composition and function of HDL from the carriers and non-carriers and studied the properties of the mutant protein in cultured hepatocytes. Results: Plasma HDL-C and apolipoprotein (apo) A-I were lower in carriers versus non-carriers, whereas the other analyzed plasma components or HDL parameters did not differ. Sera of the carriers displayed a reduced capacity to act as cholesterol efflux acceptors (p < 0.01), whereas the cholesterol acceptor capacity of their isolated HDL was normal. Fibroblasts from a p.C39X carrier showed reduced cholesterol efflux to lipid-free apoA-I but not to mature HDL particles, suggesting a specific defect in ABCA1mediated efflux pathway. In hepatic cells, GFP-OSBPL1A partially co-localized in endosomes containing fluorescent apoA-I, suggesting that OSBPL1A may regulate the intracellular handling of apoA-I. The GFP-OSBPL1A-39X mutant protein remained in the cytosol and failed to interact with Rab7, which normally recruits OSBPL1A to late endosomes/lysosomes, suggesting that this mutation represents a lossof-function. Conclusions: The present work represents the first characterization of a human OSBPL1A mutation. Our observations provide evidence that a familial loss-of-function mutation in OSBPL1A affects the first step of the reverse cholesterol transport process and associates with a low HDL-C phenotype. This suggests that rare mutations in OSBPL genes may contribute to dyslipidemias.
The mechanisms by which low-density lipoprotein (LDL)-cholesterol exits the endocytic circuits ar... more The mechanisms by which low-density lipoprotein (LDL)-cholesterol exits the endocytic circuits are not well understood. The process is defective in Niemann-Pick type C (NPC) disease in which cholesterol and sphingolipids accumulate in late endosomal compartments. This is accompanied by defective cholesterol esterification in the endoplasmic reticulum and impaired ATP-binding cassette transporter A1 (ABCA1)-dependent cholesterol efflux. We show here that overexpression of the recycling/exocytic Rab GTPase Rab8 rescued the late endosomal cholesterol deposition and sphingolipid mistrafficking in NPC fibroblasts. Rab8 redistributed cholesterol from late endosomes to the cell periphery and stimulated cholesterol efflux to the ABCA1-ligand apolipoprotein A-I (apoA-I) without increasing cholesterol esterification. Depletion of Rab8 from wild-type fibroblasts resulted in cholesterol deposition within late endosomal compartments. This cholesterol accumulation was accompanied by impaired clearance of LDL-cholesterol from endocytic circuits to apoA-I and could not be bypassed by liver X receptor activation. Our findings establish Rab8 as a key component of the regulatory machinery that leads to ABCA1-dependent removal of cholesterol from endocytic circuits.
Proceedings of the National Academy of Sciences of the United States of America, Apr 15, 1997
Cerebral plaques containing -amyloid (A4) represent an invariant pathological feature of Alzhei... more Cerebral plaques containing -amyloid (A4) represent an invariant pathological feature of Alzheimer disease (AD). A4 is proteolytically generated from its parent molecule, amyloid precursor protein (APP). In nonneuronal cells A4 has been shown to be secreted via a pH-sensitive and endocytosis-dependent pathway, and this process, when occurring in the brain, is considered to play an important role in AD. In neurons the mechanisms of A4 production are not known. Here we have analyzed these mechanisms by expressing human APP and its mutant versions in hippocampal neurons using the Semliki forest virus system. We show that these cells initially generate two pools of A4, an extracellular and an intracellular, and only the extracellular pool is produced via a pH-sensitive and endocytosis-dependent pathway. Thus, hippocampal neurons are able to utilize an alternate pathway to produce intracellular A4. We also show that a common feature of two types of APP mutations (''Swedish'' and ''London'') implicated in earlyonset AD is their increased production of C-terminally elongated A4 ( 42 ), both intra-and extracellularly. Since neurons are the only cells that produce substantial levels of intracellular A4 and also the main victims in AD, these findings may provide an important link between A4 and neurodegeneration.
Mammalian cells acquire cholesterol, a major membrane constituent, via low-density lipoprotein (L... more Mammalian cells acquire cholesterol, a major membrane constituent, via low-density lipoprotein (LDL) uptake. However, the mechanisms by which LDL cholesterol reaches the plasma membrane (PM) have remained obscure. Here, we applied LDL labeled with BODIPY cholesteryl linoleate to identify this pathway in living cells. The egress of BODIPY cholesterol (BC) from late endosomal (LE) organelles was dependent on acid lipase and Niemann-Pick C1 (NPC1) protein, as for natural cholesterol. We show that NPC1 was needed to recruit Rab8a to BC-containing LEs, and Rab8a enhanced the motility and segregation of BC-and CD63-positive organelles from lysosomes. The BC carriers docked to the cortical actin by a Rab8a-and Myosin5b (Myo5b)dependent mechanism, typically in the proximity of focal adhesions (FAs). LDL increased the number and dynamics of FAs and stimulated cell migration in an acid lipase, NPC1, and Rab8a-dependent fashion, providing evidence that this cholesterol delivery route to the PM is important for cell movement. We labeled human LDL with BC LN (Figures and). A431 cells were preloaded with dextran to visualize LE/LY, and then
The Journal of Steroid Biochemistry and Molecular Biology, Feb 1, 2022
Oxysterol-binding protein (OSBP) homologues, designated ORP or OSBPL proteins, constitute one of ... more Oxysterol-binding protein (OSBP) homologues, designated ORP or OSBPL proteins, constitute one of the largest families of intracellular lipid-binding/transfer proteins (LTP). This review summarizes the mounting evidence that several members of this family participate in the machinery facilitating cholesterol trafficking in the late endocytic pathway. There are indications that OSBP, besides acting as a cholesterol/phosphatidylinositol 4-phosphate (PI4P) exchanger at the endoplasmic reticulum (ER)-trans-Golgi network (TGN) membrane contact sites (MCS), also exchanges these lipids at ER-lysosome (Lys) contacts, increasing Lys cholesterol content. The long isoform of ORP1 (ORP1 L), which also targets ER-late endosome (LE)/Lys MCS, has the capacity to mediate cholesterol transport either from ER to LE or in the opposite direction. Moreover, it regulates the motility, positioning and fusion of LE as well as autophagic flux. ORP2, the closest relative of ORP1, is mainly cytosolic, but also targets PI(4,5)P2-rich endosomal compartments. Our latest data suggest that ORP2 transfers cholesterol from LE to recycling endosomes (RE) in exchange for PI(4,5)P2, thus stimulating the recruitment of focal adhesion kinase (FAK) on the RE and cell adhesion. FAK activates phosphoinositide kinase on the RE to enhance PI(4,5)P2 synthesis. ORP2 in turn transfers PI(4,5)P2 from RE to LE, thus regulating LE tubule formation and transport activity.
Deficiency of the endoplasmic reticulum (ER) protein seipin results in generalized lipodystrophy ... more Deficiency of the endoplasmic reticulum (ER) protein seipin results in generalized lipodystrophy by incompletely understood mechanisms. Here, we report mitochondrial abnormalities in seipin-deficient patient cells. A subset of seipin is enriched at ER-mitochondria contact sites (MAMs) in human and mouse cells and localizes in the vicinity of calcium regulators SERCA2, IP3R, and VDAC. Seipin association with MAM calcium regulators is stimulated by fasting-like stimuli, while seipin association with lipid droplets is promoted by lipid loading. Acute seipin removal does not alter ER calcium stores but leads to defective mitochondrial calcium import accompanied by a widespread reduction in Krebs cycle metabolites and ATP levels. In mice, inducible seipin deletion leads to mitochondrial dysfunctions preceding the development of metabolic complications. Together, these data suggest that seipin controls mitochondrial energy metabolism by regulating mitochondrial calcium influx at MAMs. In seipin-deficient adipose tissue, reduced ATP production compromises adipocyte properties, contributing to lipodystrophy pathogenesis.
Auxin-inducible degron technology allows rapid and controlled protein depletion. However, basal d... more Auxin-inducible degron technology allows rapid and controlled protein depletion. However, basal degradation without auxin and inefficient auxin-inducible depletion have limited its utility. We have identified a potent auxin-inducible degron system composed of auxin receptor F-box protein AtAFB2 and short degron miniIAA7. The system showed minimal basal degradation and enabled rapid auxin-inducible depletion of endogenous human transmembrane, cytoplasmic and nuclear proteins in 1 h with robust functional phenotypes. Loss-of-function methods for reducing target protein levels target DNA, RNA or protein 1 . Auxin-inducible degron (AID) technology allows rapid targeted protein depletion with the smallmolecule auxin 2 . To apply AID, an auxin-inducible destabilizing domain, or 'degron' , is fused to the target protein. In addition, an auxin receptor F-box protein TIR1/AFB is exogenously expressed, forming a chimeric SKP1-CUL1-F-Box (SCF) ubiquitin E3 ligase with endogenous components in eukaryotic cells. The chimeric E3 ligase recruits the degron in an auxin-dependent manner 3 . Addition of indole-3-acetic acid (IAA) or other small molecules of the auxin class to the culture medium thus induces the polyubiquitination and rapid proteasomal degradation of the degron-fused protein (Fig. ). This approach has been used as a powerful tool to acutely deplete target proteins in studying their functions . However, current AID systems can severely degrade target proteins before IAA addition (known as 'basal degradation') and suffer from inefficient auxin-inducible depletion in a context-and target-specific manner 2,8-10 . These pitfalls have substantially limited the applications of AID technology. An AID system with both minimal basal degradation and rapid auxin-inducible depletion would be desirable. In current AID systems, Oryza sativa TIR1 (OsTIR1) is the most commonly used auxin receptor F-box protein in combination with different degrons deriving from Arabidopsis thaliana IAA17 (AtIAA17) 2,4,5,7-14 . We initially aimed to acutely deplete endogenous human seipin, a transmembrane protein involved in lipid droplet (LD) biogenesis. However, seipin tagged with a degron termed miniAID (composed of AtIAA17 amino acid (aa) 68-132) was severely degraded in cells expressing OsTIR1 without IAA addition. Consequently, cells exhibited defective LD biogenesis already before IAA addition, resembling a seipin knockout phenotype 15 (Supplementary Fig. ). To search for an improved AID system, we established a pipeline in human A431 cells for screening various auxin receptor F-box proteins and degrons, through co-integration into the adenoassociated virus integration site 1 (AAVS1) safe harbor locus 16 (Fig. and Supplementary Note 1). We first compared OsTIR1 to five other auxin receptor F-box proteins, using miniAID as the degron. A. thaliana AFB2 (AtAFB2) was identified as the best hit: compared to OsTIR1, it displayed minimal basal depletion
Lipid droplets (LDs) are cellular organelles specialized in triacylglycerol (TG) storage undergoi... more Lipid droplets (LDs) are cellular organelles specialized in triacylglycerol (TG) storage undergoing homotypic clustering and fusion. In non-adipocytic cells with numerous LDs this is balanced by poorly understood droplet dissociation mechanisms. We identify non-muscle myosin IIa (NMIIa/MYH-9) and formin-like 1 (FMNL1) in the LD proteome. NMIIa and actin filaments concentrate around LDs, and form transient foci between dissociating LDs. NMIIa depletion results in decreased LD dissociations, enlarged LDs, decreased hydrolysis and increased storage of TGs. FMNL1 is required for actin assembly on LDs in vitro and for NMIIa recruitment to LDs in cells. We propose a novel acto-myosin structure regulating lipid storage: FMNL1-dependent assembly of myosin II-functionalized actin filaments on LDs facilitates their dissociation, thereby affecting LD surface-to-volume ratio and enzyme accessibility to TGs. In neutrophilic leucocytes from MYH9-related disease patients NMIIa inclusions are accompanied by increased lipid storage in droplets, suggesting that NMIIa dysfunction may contribute to lipid imbalance in man.
In this article, we summarize the present information related to the export of LDL-derived choles... more In this article, we summarize the present information related to the export of LDL-derived cholesterol from late endosomes, with a focus on Nieman-Pick disease, type C1 (NPC1) cholesterol delivery toward the endoplasmic reticulum (ER). We review data suggesting that several pathways may operate in parallel, including membrane transport routes and membrane contact sites (MCSs). There is increasing appreciation that MCSs provide an important mechanism for intermembrane lipid transfer. In late endosome-ER contacts, three protein bridges involving oxysterol binding protein related protein (ORP)1L-vesicle associated membrane protein-associated protein (VAP), steroidogenic acute regulatory protein (StAR)D3-VAP and ORP5-NPC1 proteins have been reported. How much they contribute to the flux of LDL-cholesterol to the ER is currently open. Studies for lipid transfer via MCSs have been most advanced in Saccharomyces cerevisiae. Recently, a new sterol-binding protein family conserved between yeast and man was identified. Its members localize at MCSs and were named lipid transfer protein anchored at membrane contact sites (Lam) proteins. In yeast, sterol transfer between the ER and the yeast lysosome may be facilitated by a Lam protein.
Background and aims: Among subjects with high-density-lipoprotein cholesterol (HDL-C) below the 1... more Background and aims: Among subjects with high-density-lipoprotein cholesterol (HDL-C) below the 1st percentile in the general population, we identified a heterozygous variant OSBPL1A p.C39X encoding a short truncated protein fragment that co-segregated with low plasma HDL-C. Methods: We investigated the composition and function of HDL from the carriers and non-carriers and studied the properties of the mutant protein in cultured hepatocytes. Results: Plasma HDL-C and apolipoprotein (apo) A-I were lower in carriers versus non-carriers, whereas the other analyzed plasma components or HDL parameters did not differ. Sera of the carriers displayed a reduced capacity to act as cholesterol efflux acceptors (p < 0.01), whereas the cholesterol acceptor capacity of their isolated HDL was normal. Fibroblasts from a p.C39X carrier showed reduced cholesterol efflux to lipid-free apoA-I but not to mature HDL particles, suggesting a specific defect in ABCA1mediated efflux pathway. In hepatic cells, GFP-OSBPL1A partially co-localized in endosomes containing fluorescent apoA-I, suggesting that OSBPL1A may regulate the intracellular handling of apoA-I. The GFP-OSBPL1A-39X mutant protein remained in the cytosol and failed to interact with Rab7, which normally recruits OSBPL1A to late endosomes/lysosomes, suggesting that this mutation represents a lossof-function. Conclusions: The present work represents the first characterization of a human OSBPL1A mutation. Our observations provide evidence that a familial loss-of-function mutation in OSBPL1A affects the first step of the reverse cholesterol transport process and associates with a low HDL-C phenotype. This suggests that rare mutations in OSBPL genes may contribute to dyslipidemias.
The mechanisms by which low-density lipoprotein (LDL)-cholesterol exits the endocytic circuits ar... more The mechanisms by which low-density lipoprotein (LDL)-cholesterol exits the endocytic circuits are not well understood. The process is defective in Niemann-Pick type C (NPC) disease in which cholesterol and sphingolipids accumulate in late endosomal compartments. This is accompanied by defective cholesterol esterification in the endoplasmic reticulum and impaired ATP-binding cassette transporter A1 (ABCA1)-dependent cholesterol efflux. We show here that overexpression of the recycling/exocytic Rab GTPase Rab8 rescued the late endosomal cholesterol deposition and sphingolipid mistrafficking in NPC fibroblasts. Rab8 redistributed cholesterol from late endosomes to the cell periphery and stimulated cholesterol efflux to the ABCA1-ligand apolipoprotein A-I (apoA-I) without increasing cholesterol esterification. Depletion of Rab8 from wild-type fibroblasts resulted in cholesterol deposition within late endosomal compartments. This cholesterol accumulation was accompanied by impaired clearance of LDL-cholesterol from endocytic circuits to apoA-I and could not be bypassed by liver X receptor activation. Our findings establish Rab8 as a key component of the regulatory machinery that leads to ABCA1-dependent removal of cholesterol from endocytic circuits.
Proceedings of the National Academy of Sciences of the United States of America, Apr 15, 1997
Cerebral plaques containing -amyloid (A4) represent an invariant pathological feature of Alzhei... more Cerebral plaques containing -amyloid (A4) represent an invariant pathological feature of Alzheimer disease (AD). A4 is proteolytically generated from its parent molecule, amyloid precursor protein (APP). In nonneuronal cells A4 has been shown to be secreted via a pH-sensitive and endocytosis-dependent pathway, and this process, when occurring in the brain, is considered to play an important role in AD. In neurons the mechanisms of A4 production are not known. Here we have analyzed these mechanisms by expressing human APP and its mutant versions in hippocampal neurons using the Semliki forest virus system. We show that these cells initially generate two pools of A4, an extracellular and an intracellular, and only the extracellular pool is produced via a pH-sensitive and endocytosis-dependent pathway. Thus, hippocampal neurons are able to utilize an alternate pathway to produce intracellular A4. We also show that a common feature of two types of APP mutations (''Swedish'' and ''London'') implicated in earlyonset AD is their increased production of C-terminally elongated A4 ( 42 ), both intra-and extracellularly. Since neurons are the only cells that produce substantial levels of intracellular A4 and also the main victims in AD, these findings may provide an important link between A4 and neurodegeneration.
Mammalian cells acquire cholesterol, a major membrane constituent, via low-density lipoprotein (L... more Mammalian cells acquire cholesterol, a major membrane constituent, via low-density lipoprotein (LDL) uptake. However, the mechanisms by which LDL cholesterol reaches the plasma membrane (PM) have remained obscure. Here, we applied LDL labeled with BODIPY cholesteryl linoleate to identify this pathway in living cells. The egress of BODIPY cholesterol (BC) from late endosomal (LE) organelles was dependent on acid lipase and Niemann-Pick C1 (NPC1) protein, as for natural cholesterol. We show that NPC1 was needed to recruit Rab8a to BC-containing LEs, and Rab8a enhanced the motility and segregation of BC-and CD63-positive organelles from lysosomes. The BC carriers docked to the cortical actin by a Rab8a-and Myosin5b (Myo5b)dependent mechanism, typically in the proximity of focal adhesions (FAs). LDL increased the number and dynamics of FAs and stimulated cell migration in an acid lipase, NPC1, and Rab8a-dependent fashion, providing evidence that this cholesterol delivery route to the PM is important for cell movement. We labeled human LDL with BC LN (Figures and). A431 cells were preloaded with dextran to visualize LE/LY, and then
The Journal of Steroid Biochemistry and Molecular Biology, Feb 1, 2022
Oxysterol-binding protein (OSBP) homologues, designated ORP or OSBPL proteins, constitute one of ... more Oxysterol-binding protein (OSBP) homologues, designated ORP or OSBPL proteins, constitute one of the largest families of intracellular lipid-binding/transfer proteins (LTP). This review summarizes the mounting evidence that several members of this family participate in the machinery facilitating cholesterol trafficking in the late endocytic pathway. There are indications that OSBP, besides acting as a cholesterol/phosphatidylinositol 4-phosphate (PI4P) exchanger at the endoplasmic reticulum (ER)-trans-Golgi network (TGN) membrane contact sites (MCS), also exchanges these lipids at ER-lysosome (Lys) contacts, increasing Lys cholesterol content. The long isoform of ORP1 (ORP1 L), which also targets ER-late endosome (LE)/Lys MCS, has the capacity to mediate cholesterol transport either from ER to LE or in the opposite direction. Moreover, it regulates the motility, positioning and fusion of LE as well as autophagic flux. ORP2, the closest relative of ORP1, is mainly cytosolic, but also targets PI(4,5)P2-rich endosomal compartments. Our latest data suggest that ORP2 transfers cholesterol from LE to recycling endosomes (RE) in exchange for PI(4,5)P2, thus stimulating the recruitment of focal adhesion kinase (FAK) on the RE and cell adhesion. FAK activates phosphoinositide kinase on the RE to enhance PI(4,5)P2 synthesis. ORP2 in turn transfers PI(4,5)P2 from RE to LE, thus regulating LE tubule formation and transport activity.
Deficiency of the endoplasmic reticulum (ER) protein seipin results in generalized lipodystrophy ... more Deficiency of the endoplasmic reticulum (ER) protein seipin results in generalized lipodystrophy by incompletely understood mechanisms. Here, we report mitochondrial abnormalities in seipin-deficient patient cells. A subset of seipin is enriched at ER-mitochondria contact sites (MAMs) in human and mouse cells and localizes in the vicinity of calcium regulators SERCA2, IP3R, and VDAC. Seipin association with MAM calcium regulators is stimulated by fasting-like stimuli, while seipin association with lipid droplets is promoted by lipid loading. Acute seipin removal does not alter ER calcium stores but leads to defective mitochondrial calcium import accompanied by a widespread reduction in Krebs cycle metabolites and ATP levels. In mice, inducible seipin deletion leads to mitochondrial dysfunctions preceding the development of metabolic complications. Together, these data suggest that seipin controls mitochondrial energy metabolism by regulating mitochondrial calcium influx at MAMs. In seipin-deficient adipose tissue, reduced ATP production compromises adipocyte properties, contributing to lipodystrophy pathogenesis.
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Papers by Elina Ikonen