AdipoR2 recruits protein interactors to promote fatty acid elongation and membrane fluidity
2023; Elsevier BV; Volume: 299; Issue: 6 Linguagem: Inglês
10.1016/j.jbc.2023.104799
ISSN1083-351X
AutoresMario Ruiz, Ranjan Devkota, Delaney Kaper, Hanna Ruhanen, Kiran Busayavalasa, Uroš Radović, Marcus Henricsson, Reijo Käkelä, Jan Borén, Marc Pilon,
Tópico(s)Pancreatic function and diabetes
ResumoThe human AdipoR2 and its Caenorhabditis elegans homolog PAQR-2 are multipass plasma membrane proteins that protect cells against membrane rigidification. However, how AdipoR2 promotes membrane fluidity mechanistically is not clear. Using 13C-labeled fatty acids, we show that AdipoR2 can promote the elongation and incorporation of membrane-fluidizing polyunsaturated fatty acids into phospholipids. To elucidate the molecular basis of these activities, we performed immunoprecipitations of tagged AdipoR2 and PAQR-2 expressed in HEK293 cells or whole C. elegans, respectively, and identified coimmunoprecipitated proteins using mass spectrometry. We found that several of the evolutionarily conserved AdipoR2/PAQR-2 interactors are important for fatty acid elongation and incorporation into phospholipids. We experimentally verified some of these interactions, namely, with the dehydratase HACD3 that is essential for the third of four steps in long-chain fatty acid elongation and ACSL4 that is important for activation of unsaturated fatty acids and their channeling into phospholipids. We conclude that AdipoR2 and PAQR-2 can recruit protein interactors to promote the production and incorporation of unsaturated fatty acids into phospholipids. The human AdipoR2 and its Caenorhabditis elegans homolog PAQR-2 are multipass plasma membrane proteins that protect cells against membrane rigidification. However, how AdipoR2 promotes membrane fluidity mechanistically is not clear. Using 13C-labeled fatty acids, we show that AdipoR2 can promote the elongation and incorporation of membrane-fluidizing polyunsaturated fatty acids into phospholipids. To elucidate the molecular basis of these activities, we performed immunoprecipitations of tagged AdipoR2 and PAQR-2 expressed in HEK293 cells or whole C. elegans, respectively, and identified coimmunoprecipitated proteins using mass spectrometry. We found that several of the evolutionarily conserved AdipoR2/PAQR-2 interactors are important for fatty acid elongation and incorporation into phospholipids. We experimentally verified some of these interactions, namely, with the dehydratase HACD3 that is essential for the third of four steps in long-chain fatty acid elongation and ACSL4 that is important for activation of unsaturated fatty acids and their channeling into phospholipids. We conclude that AdipoR2 and PAQR-2 can recruit protein interactors to promote the production and incorporation of unsaturated fatty acids into phospholipids. In animals, most fatty acids in the body, including in cell membranes, are of dietary origin. In human, de novo lipogenesis (mostly from liver) accounts for only ∼10% of fatty acids in circulating very-low-density lipoproteins, meaning that ∼90% is diet derived (1Hodson L. Gunn P.J. The regulation of hepatic fatty acid synthesis and partitioning: the effect of nutritional state.Nat. Rev. Endocrinol. 2019; 15: 689-700Google Scholar, 2Wallace M. Metallo C.M. Tracing insights into de novo lipogenesis in liver and adipose tissues.Semin. Cell Dev. Biol. 2020; 108: 65-71Google Scholar). Similarly, in the nematode Caenorhabditis elegans, >75% of phospholipid fatty acids are obtained from the diet (3Dancy B.C. Chen S.W. Drechsler R. Gafken P.R. Olsen C.P. 13C- and 15N-labeling strategies combined with mass spectrometry comprehensively quantify phospholipid dynamics in C. elegans.PLoS One. 2015; 10e0141850Google Scholar). Critically, local de novo lipogenesis and lipid remodeling within each cell can compensate for the varied mixture of fatty acids imported from the circulation. This explains the robustness of membrane composition despite extremely varied diets (4Abbott S.K. Else P.L. Atkins T.A. Hulbert A.J. Fatty acid composition of membrane bilayers: importance of diet polyunsaturated fat balance.Biochim. Biophys. Acta. 2012; 1818: 1309-1317Google Scholar, 5Field C.J. Angel A. Clandinin M.T. Relationship of diet to the fatty acid composition of human adipose tissue structural and stored lipids.Am. J. Clin. Nutr. 1985; 42: 1206-1220Google Scholar, 6Levental K.R. Malmberg E. Symons J.L. Fan Y.Y. Chapkin R.S. Ernst R. et al.Lipidomic and biophysical homeostasis of mammalian membranes counteracts dietary lipid perturbations to maintain cellular fitness.Nat. Commun. 2020; 11: 1339Google Scholar). This homeostasis is necessary since the vital properties of cell membranes, such as packing density, lateral mobility, curvature, and permeability, are greatly influenced by the phospholipid composition (7Mouritsen O.G. Lipidology and lipidomics--quo vadis? A new era for the physical chemistry of lipids.Phys. Chem. Chem. Phys. 2011; 13: 19195-19205Google Scholar, 8Harayama T. Riezman H. Understanding the diversity of membrane lipid composition.Nat. Rev. Mol. Cell Biol. 2018; 19: 281-296Google Scholar, 9Renne M.F. de Kroon A. The role of phospholipid molecular species in determining the physical properties of yeast membranes.FEBS Lett. 2018; 592: 1330-1345Google Scholar, 10Sezgin E. Levental I. Mayor S. Eggeling C. The mystery of membrane organization: composition, regulation and roles of lipid rafts.Nat. Rev. Mol. Cell Biol. 2017; 18: 361-374Google Scholar). Phospholipids with saturated fatty acids (SFAs) pack more densely than those with unsaturated fatty acids (UFAs) of the same length and consequently form more rigid and thicker membranes. Although polyunsaturated fatty acids (PUFAs) are long (18 carbons or more), their multiple double bonds introduce kinks that weaken lateral van der Waals attraction of the acyl chains in phospholipids, thus greatly increasing membrane fluidity. The hydrophilic head groups also impact membrane properties (11Somerharju P. Virtanen J.A. Cheng K.H. Hermansson M. The superlattice model of lateral organization of membranes and its implications on membrane lipid homeostasis.Biochim. Biophys. Acta. 2009; 1788: 12-23Google Scholar). For example, phospholipids with a large choline head group have nearly cylindrical shapes and thus with a small share of the small-head phosphatidylethanolamine can naturally form flat membranes. In contrast, phospholipids with the small ethanolamine head group are conical in shape and therefore form curved membranes. How do cells robustly maintain their membrane geometry and viscosity given the varied and unpredictable mixture of fatty acids supplied through the circulation? In molecular terms, this robustness depends on protein sensors that monitor membrane properties and can signal to fatty acid synthesis/remodeling enzymes that rectify phospholipid composition and contribute to maintaining the viscosity gradient from the nucleus to the plasma membrane (12de Mendoza D. Pilon M. Control of membrane lipid homeostasis by lipid-bilayer associated sensors: a mechanism conserved from bacteria to humans.Prog. Lipid Res. 2019; 76100996Google Scholar, 13Radanovic T. Reinhard J. Ballweg S. Pesek K. Ernst R. An emerging group of membrane property sensors controls the physical state of organellar membranes to maintain their identity.Bioessays. 2018; 40e1700250Google Scholar). Many of these proteins are evolutionarily conserved and include PCYT1A in the inner nuclear membrane where it promotes phosphatidylcholine synthesis in response to loosening membrane packing (14Haider A. Wei Y.C. Lim K. Barbosa A.D. Liu C.H. Weber U. et al.PCYT1A regulates phosphatidylcholine homeostasis from the inner nuclear membrane in response to membrane stored curvature elastic stress.Dev. Cell. 2018; 45: 481-495.e8Google Scholar) and IRE1 in the endoplasmic reticulum (ER) that activates lipogenic pathways in response to thick domains rich in SFAs (15Cho H. Stanzione F. Oak A. Kim G.H. Yerneni S. Qi L. et al.Intrinsic structural features of the human IRE1alpha transmembrane domain sense membrane lipid saturation.Cell Rep. 2019; 27: 307-320.e5Google Scholar, 16Halbleib K. Pesek K. Covino R. Hofbauer H.F. Wunnicke D. Hanelt I. et al.Activation of the unfolded protein response by lipid bilayer stress.Mol. Cell. 2017; 67: 673-684.e8Google Scholar). Human AdipoR2 and its C. elegans homolog PAQR-2 are also critical for membrane homeostasis (17Svensk E. Stahlman M. Andersson C.H. Johansson M. Boren J. Pilon M. PAQR-2 regulates fatty acid desaturation during cold adaptation in C. elegans.PLoS Genet. 2013; 9e1003801Google Scholar, 18Svensk E. Biermann J. Hammarsten S. Magnusson F. Pilon M. Leveraging the withered tail tip phenotype in C. elegans to identify proteins that influence membrane properties.Worm. 2016; 5e1206171Google Scholar, 19Svensk E. Devkota R. Stahlman M. Ranji P. Rauthan M. Magnusson F. et al.Caenorhabditis elegans PAQR-2 and IGLR-2 protect against glucose toxicity by modulating membrane lipid composition.PLoS Genet. 2016; 12e1005982Google Scholar, 20Devkota R. Svensk E. Ruiz M. Stahlman M. Boren J. Pilon M. The adiponectin receptor AdipoR2 and its Caenorhabditis elegans homolog PAQR-2 prevent membrane rigidification by exogenous saturated fatty acids.PLoS Genet. 2017; 13e1007004Google Scholar, 21Bodhicharla R. Devkota R. Ruiz M. Pilon M. Membrane fluidity is regulated cell nonautonomously by Caenorhabditis elegans PAQR-2 and its mammalian homolog AdipoR2.Genetics. 2018; 210: 189-201Google Scholar, 22Ruiz M. Bodhicharla R. Svensk E. Devkota R. Busayavalasa K. Palmgren H. et al.Membrane fluidity is regulated by the C. elegans transmembrane protein FLD-1 and its human homologs TLCD1/2.Elife. 2018; 7e40686Google Scholar, 23Ruiz M. Bodhicharla R. Stahlman M. Svensk E. Busayavalasa K. Palmgren H. et al.Evolutionarily conserved long-chain Acyl-CoA synthetases regulate membrane composition and fluidity.Elife. 2019; 8e47733Google Scholar, 24Ruiz M. Stahlman M. Boren J. Pilon M. AdipoR1 and AdipoR2 maintain membrane fluidity in most human cell types and independently of adiponectin.J. Lipid Res. 2019; 60: 995-1004Google Scholar, 25Busayavalasa K. Ruiz M. Devkota R. Stahlman M. Bodhicharla R. Svensk E. et al.Leveraging a gain-of-function allele of Caenorhabditis elegans paqr-1 to elucidate membrane homeostasis by PAQR proteins.PLoS Genet. 2020; 16e1008975Google Scholar, 26Pilon M. Paradigm shift: the primary function of the "adiponectin receptors" is to regulate cell membrane composition lipids.Health Dis. 2021; 20: 43Google Scholar). AdipoR2 and PAQR-2 have seven transmembrane domains, with a short extracellular C terminus and a large cytoplasmic N-terminal domain that may regulate access to a hydrolase activity present within a large cytoplasmic-facing cavity (25Busayavalasa K. Ruiz M. Devkota R. Stahlman M. Bodhicharla R. Svensk E. et al.Leveraging a gain-of-function allele of Caenorhabditis elegans paqr-1 to elucidate membrane homeostasis by PAQR proteins.PLoS Genet. 2020; 16e1008975Google Scholar, 27Tanabe H. Fujii Y. Okada-Iwabu M. Iwabu M. Nakamura Y. Hosaka T. et al.Crystal structures of the human adiponectin receptors.Nature. 2015; 520: 312-316Google Scholar, 28Vasiliauskaite-Brooks I. Sounier R. Rochaix P. Bellot G. Fortier M. Hoh F. et al.Structural insights into adiponectin receptors suggest ceramidase activity.Nature. 2017; 544: 120-123Google Scholar, 29Volkmar N. Gawden-Bone C.M. Williamson J.C. Nixon-Abell J. West J.A. St George-Hyslop P.H. et al.Regulation of membrane fluidity by RNF145-triggered degradation of the lipid hydrolase ADIPOR2.EMBO J. 2022; 41e110777Google Scholar). Genetic screens in C. elegans indicate that a primary function of PAQR-2 is to respond to membrane rigidification by promoting desaturase expression and increasing the PUFA content of phospholipids and that this function is conserved in the human AdipoR2 (17Svensk E. Stahlman M. Andersson C.H. Johansson M. Boren J. Pilon M. PAQR-2 regulates fatty acid desaturation during cold adaptation in C. elegans.PLoS Genet. 2013; 9e1003801Google Scholar, 18Svensk E. Biermann J. Hammarsten S. Magnusson F. Pilon M. Leveraging the withered tail tip phenotype in C. elegans to identify proteins that influence membrane properties.Worm. 2016; 5e1206171Google Scholar, 19Svensk E. Devkota R. Stahlman M. Ranji P. Rauthan M. Magnusson F. et al.Caenorhabditis elegans PAQR-2 and IGLR-2 protect against glucose toxicity by modulating membrane lipid composition.PLoS Genet. 2016; 12e1005982Google Scholar, 22Ruiz M. Bodhicharla R. Svensk E. Devkota R. Busayavalasa K. Palmgren H. et al.Membrane fluidity is regulated by the C. elegans transmembrane protein FLD-1 and its human homologs TLCD1/2.Elife. 2018; 7e40686Google Scholar, 23Ruiz M. Bodhicharla R. Stahlman M. Svensk E. Busayavalasa K. Palmgren H. et al.Evolutionarily conserved long-chain Acyl-CoA synthetases regulate membrane composition and fluidity.Elife. 2019; 8e47733Google Scholar, 26Pilon M. Paradigm shift: the primary function of the "adiponectin receptors" is to regulate cell membrane composition lipids.Health Dis. 2021; 20: 43Google Scholar, 30Devkota R. Henricsson M. Boren J. Pilon M. The C. elegans PAQR-2 and IGLR-2 membrane homeostasis proteins are uniquely essential for tolerating dietary saturated fats.Biochim. Biophys. Acta Mol. Cell Biol. Lipids. 2021; 1866158883Google Scholar). At least part of the AdipoR2/PAQR-2 downstream cascade relies on a protein-intrinsic ceramidase activity that produces a sphingosine-1 phosphate (S1P) signal that activates the SREBF1/SBP-1 and PPARγ/NHR-49-dependent transcription of desaturases (SBP-1 and NHR-49 are C. elegans homologs of SREBF1 and PPARγ, respectively) (31Holland W.L. Miller R.A. Wang Z.V. Sun K. Barth B.M. Bui H.H. et al.Receptor-mediated activation of ceramidase activity initiates the pleiotropic actions of adiponectin.Nat. Med. 2011; 17: 55-63Google Scholar, 32Ruiz M. Devkota R. Panagaki D. Bergh P.O. Kaper D. Henricsson M. et al.Sphingosine 1-phosphate mediates adiponectin receptor signaling essential for lipid homeostasis and embryogenesis.Nat. Commun. 2022; 13: 7162Google Scholar). How AdipoR2/PAQR-2 also promotes PUFA incorporation into phospholipids has not been directly investigated and remains, however, undefined. This is an intriguing question given that fatty acid elongation and desaturation, as well as phospholipid synthesis, take place in the ER while most reports emphasize that AdipoR2/PAQR-2 are present on the plasma membrane, although they are also found in the ER where they could play important roles (29Volkmar N. Gawden-Bone C.M. Williamson J.C. Nixon-Abell J. West J.A. St George-Hyslop P.H. et al.Regulation of membrane fluidity by RNF145-triggered degradation of the lipid hydrolase ADIPOR2.EMBO J. 2022; 41e110777Google Scholar, 33Almabouada F. Diaz-Ruiz A. Rabanal-Ruiz Y. Peinado J.R. Vazquez-Martinez R. Malagon M.M. Adiponectin receptors form homomers and heteromers exhibiting distinct ligand binding and intracellular signaling properties.J. Biol. Chem. 2013; 288: 3112-3125Google Scholar). Forward genetic screens have important limitations, including the fact that identification of essential genes or genes that are functionally redundant for an important biological process is difficult through screening strategies. Having already performed several forward genetics screens in C. elegans to define the PAQR-2 pathway, we felt that other strategies should be deployed. Here, we used a proteomics approach to identify novel PAQR-2 and AdipoR2 interaction partners and discovered that they recruit an evolutionarily conserved fatty acid elongation complex that can channel polyunsaturated fatty acids into phospholipids and in this way contribute to membrane homeostasis. To test whether AdipoR2 influences fatty acid elongation, desaturation, and incorporation of PUFAs into phospholipids, we treated human HEK293 cells with either nontarget siRNA (negative control) or AdipoR2 siRNA, then incubated the cells in the presence of 13C-labeled oleic acid (OA; 18:1n-9) or linoleic acid (LA; 18:2n-6) for 6 h. Samples were then harvested, and their phospholipid composition was determined by mass spectrometry, which allowed detection of 13C-labeled species. In agreement with earlier studies, we found that AdipoR2 siRNA-treated cells have an excess of SFAs in unlabeled phospholipids (see, e.g., PC30:0 and 32:0 in Table S1). We also found that AdipoR2 silencing reduced the levels of 13C-labeled OA and its derivatives in phosphatidylcholines (PCs) and phosphatidylethanolamines (PEs) (Fig. 1A) but had no effect on the total levels of 13C-labeled LA in PCs or PEs (Fig. 1B). AdipoR2 silencing may therefore reduce the total uptake or incorporation of exogenous OA, but not of LA, into phospholipids. The levels of 13C-labeled LA and its derivates in specific PC and PE species were significantly changed in the AdipoR2 siRNA-treated cells, with a marked excess of short, less desaturated PCs and PEs at the expense of longer more desaturated species derived from exogenously supplied 13C-labeled LA (Fig. 1, C and D). No such bias was seen when following the fate of 13C-labeled OA: AdipoR2 siRNA-treated cells showed uniformly reduced levels of 13C-labeled species of all lengths and desaturation (Table S1 and Fig. S1). This likely reflects the fact that mammalian cells lack a desaturase capable of introducing a second double bond at the Δ12 position of a fatty acid, and thus neither control nor AdipoR2 siRNA-treated cells can further desaturate OA to LA. Altogether, these results suggest that AdipoR2 contributes to the elongation and further desaturation of LA and/or the incorporation of the resulting long-chain PUFAs into phospholipids. We next sought to identify molecular interactors that could explain how AdipoR2/PAQR-2 influences fatty acid elongation and incorporation into phospholipids. An unbiased approach to identify novel protein interactors is purification by immunoprecipitation of protein complexes containing the query protein from mildly lysed cell extracts. We performed immunoprecipitations using an anti-HA antibody from lysates of worms carrying HA::PAQR-2, with a HA tag at the N-terminal end of the endogenous PAQR-2 locus, or of stable clones of HEK293 cells carrying a HA::AdipoR2 expression construct that were generated in a previous study (34Ruiz M. Henricsson M. Boren J. Pilon M. Palmitic acid causes increased dihydroceramide levels when desaturase expression is directly silenced or indirectly lowered by silencing AdipoR2.Lipids Health Dis. 2021; 20: 173Google Scholar). Proteins present in the immunoprecipitated complexes were identified by mass spectrometry. Only proteins that reproducibly and specifically interacted with HA::PAQR-2 or HA::AdipoR2, i.e., proteins that were not present in worms or cells that do not express HA-tagged proteins, were further investigated. These proteins are listed in Table 1 (human proteins) and Table 2 (worm proteins); the complete proteomics data are publicly available at ProteomeXchange (http://www.proteomexchange.org) with identifier PXD031395. The STRING database (35Szklarczyk D. Gable A.L. Nastou K.C. Lyon D. Kirsch R. Pyysalo S. et al.The STRING database in 2021: customizable protein-protein networks, and functional characterization of user-uploaded gene/measurement sets.Nucleic Acids Res. 2021; 49: D605-D612Google Scholar), which mines many sources of information for proteins, was then used to analyze the identified proteins and assign them into clusters (Fig. 2). Three main conclusions can be drawn from the analysis of the AdipoR2/PAQR-2 interactors. First, IGLR-2 was reproducibly detected as a PAQR-2 interaction partner in C. elegans, in agreement with our previous genetic interaction studies (19Svensk E. Devkota R. Stahlman M. Ranji P. Rauthan M. Magnusson F. et al.Caenorhabditis elegans PAQR-2 and IGLR-2 protect against glucose toxicity by modulating membrane lipid composition.PLoS Genet. 2016; 12e1005982Google Scholar, 25Busayavalasa K. Ruiz M. Devkota R. Stahlman M. Bodhicharla R. Svensk E. et al.Leveraging a gain-of-function allele of Caenorhabditis elegans paqr-1 to elucidate membrane homeostasis by PAQR proteins.PLoS Genet. 2020; 16e1008975Google Scholar), bifluorescence complementation (BiFC) (19Svensk E. Devkota R. Stahlman M. Ranji P. Rauthan M. Magnusson F. et al.Caenorhabditis elegans PAQR-2 and IGLR-2 protect against glucose toxicity by modulating membrane lipid composition.PLoS Genet. 2016; 12e1005982Google Scholar), and fluorescence resonance energy transfer experiments (30Devkota R. Henricsson M. Boren J. Pilon M. The C. elegans PAQR-2 and IGLR-2 membrane homeostasis proteins are uniquely essential for tolerating dietary saturated fats.Biochim. Biophys. Acta Mol. Cell Biol. Lipids. 2021; 1866158883Google Scholar). No IGLR-2 mammalian ortholog has yet been identified, and no IGLR-2 homologs were identified in the AdipoR2 immunoprecipitations. Second, several complexes important for the life cycle of transmembrane proteins were found associated with both HA::AdipoR2 and HA::PAQR-2. The fact that the HA tag is present at the N terminus of AdipoR2 and PAQR-2 means that even nascent polypeptides being synthesized can be purified. Indeed, HA::AdipoR2 and HA::PAQR-2 were associated with multiple proteins involved in RNA processing, translation, protein modification (glycosylation), degradation (proteasome components), and coatomer-dependent trafficking. Third, and most interestingly, we found that HA::PAQR-2 and/or HA::AdipoR2 were associated with fatty acid metabolism enzymes including the multifunctional fatty acid synthetase FASN-1 (homologous to human FASN), the elongase ELO-2 (homologous to human ELOVL3/6), the dehydrogenase LET-767 (homologous to human HSD17B12) and fatty acid CoA synthetase ACS-4 (homologous to human ACSL4) in C. elegans samples, and the fatty acid binding protein FABP4 (homologous to worm LBP-5/6) and the very-long-chain (3R)-3-hydroxyacyl-CoA dehydratase 3 HACD3 (homologous to C. elegans HPO-8) in both the worm and HEK293 samples.Table 1Human proteomics summaryNoHumanDescription of human proteins1AdipoR2Adiponectin receptor protein 22ALDOCFructose-bisphosphate aldolase C3ARPC2Actin-related protein 2/3 complex subunit 24BAG2BAG family molecular chaperone regulator 25CASC3Protein CASC36CDC42Cell division control protein 42 homolog7COX4I1Cytochrome c oxidase subunit 4 isoform 1, mitochondrial8DARSAspartate–tRNA ligase, cytoplasmic9DDOSTDolichyl-diphosphooligosaccharide–protein glycosyltransferase10DYNLL1Dynein light chain 1, cytoplasmic11FABP4Fatty acid–binding protein, adipocyte12GDPD3Glycerophosphodiester phosphodiesterase domain-containing protein 313GGT7Gamma-glutamyltransferase 714GNAT1Guanine nucleotide-binding protein G(t) subunit alpha-115HACD3Very-long-chain (3R)-3-hydroxyacyl-CoA dehydratase 3 (aka PTPLAD1)16HARS2Probable histidine–tRNA ligase, mitochondrial17HMGA2High mobility group protein HMGI-C18HNRNPCL2Heterogeneous nuclear ribonucleoprotein C-like 219HNRNPUHeterogeneous nuclear ribonucleoprotein U20HSPH1Heat shock protein 105 kDa21IMPA2Inositol monophosphatase 222KLK10Kallikrein-1023LAMP1Lysosome-associated membrane glycoprotein 124LAMP2Lysosome-associated membrane glycoprotein 225LGALS3Galectin-3 OS=Homo sapiens26PCMTD1Protein-L-isoaspartate O-methyltransferase domain-containing protein 127PLBD1Phospholipase B-like 128PSAPL1Proactivator polypeptide-like 129PSMA6Proteasome subunit alpha type 630PSMB1Proteasome subunit beta type 131PSMB7Proteasome subunit beta type 732PTGES3Prostaglandin E synthase 333RAP1BRas-related protein Rap-1b34RPL2260S ribosomal protein L2235RPN1Dolichyl-diphosphooligosaccharide–protein glycosyltransferase subunit 136RPS2740S ribosomal protein S2737RTN1Reticulon-138RTN4Reticulon-439SCGB2A2Mammaglobin-A40SDR9C7Short-chain dehydrogenase/reductase family 9C member 741SHMT2Serine hydroxymethyltransferase, mitochondrial42SNRPBSmall nuclear ribonucleoprotein-associated proteins B and B′43SNRPD2Small nuclear ribonucleoprotein Sm D244SRP14Signal recognition particle 14-kDa protein45SRP9Signal recognition particle 9-kDa protein46SRSF3Serine/arginine-rich splicing factor 347TNNI3Troponin I, cardiac muscle48USMG5Upregulated during skeletal muscle growth protein 549VDAC2Voltage-dependent anion-selective channel protein 250XRCC5X-ray repair cross-complementing protein 5The human proteins listed here were found with a false discovery rate <1% in at least 2/4 HA::AdipoR2 IPs in HEK293 cells (and never in control cells), or were proteins where the homolog was identified at least once in worms and at least once in human. Open table in a new tab Table 2C. elegans proteomics summaryNoWorm (human)Description of worm proteins1ACDH-8 (ACADM)Acyl CoA Dehydrogenase2ACS-4 (ACSL4)Fatty acid CoA synthetase family3ALDO-1 (ALDOC)Fructose-bisphosphate aldolase 14ASP-2 (CTSE)Aspartyl protease5B0491.5 (−)Uncharacterized protein6C14B9.10 (−)Uncharacterized protein7CEY-3 (YBX1)C. elegans Y-box8CGH-1 (DDX6)ATP-dependent RNA helicase cgh-19COPB-2 (COPB2)Probable coatomer subunit beta'10COPE-1 (COPE)Coatomer subunit epsilon11COPG-1 (COPG1)Probable coatomer subunit gamma12CYN-3 (PPIF)Peptidyl-prolyl cis-trans isomerase 313DBT-1 (DBT)Lipoamide acyltransferase14DHS-3 (SDR9C7)Dehydrogenases, short chain15EARS-1 (EPRS)Glutamyl(E) amino-acyl tRNA synthetase16ELO-2 (ELOVL3/6)Elongation of very-long-chain fatty acids protein17EXC-15 (AKR1A1)alditol:NADP+ 1-oxidoreductase activity18F20G2.2 (HSD17B6)Similar to hydroxysteroid 17-beta dehydrogenase 619F42G8.10 (−)Uncharacterized protein20FASN-1 (FASN)Fatty acid synthase21GST-7 (HPGDS)Probable glutathione S-transferase 722GTBP-1 (G3BP1)Ras-GTPase-activating protein SH3 (three) domain-binding protein23HPO-8 (HACD3)Very-long-chain (3R)-3-hydroxyacyl-CoA dehydratase hpo-824HRPR-1 (HNRNPR)HnRNP A1 homolog25IGLR-2 (LGR4)Immunoglobulin domain and leucine-rich repeat–containing protein 226LBP-5 (FABP4)Fatty acid–binding protein homolog 527LBP-6 (FABP4)Fatty acid–binding protein homolog 628LEC-1 (LGALS3)32-kDa beta-galactoside-binding lectin29LET-767 (HSD17B12)Very-long-chain 3-oxooacyl-coA reductase let-76730LPD-1 (SCCPDH)Lipid droplet localized protein31MEL-32 (SHMT2)Serine hydroxymethyltransferase32OATR-1 (OAT)Probable ornithine aminotransferase, mitochondrial33OSTB-1 (DDOST)Dolichyl-diphosphooligosaccharide–protein glycosyltransferase 48-kDa subunit34PAQR-2 (AdipoR2)Progestin and AdipoQ Receptor family35PUD-1.2 (−)Protein upregulated in Daf-2(Gf)36PUD-3 (−)Protein upregulated in Daf-2(Gf)37RIBO-1 (RPN1)Dolichyl-diphosphooligosaccharide–protein glycosyltransferase subunit 138RPL-36.A (RPL36A)Ribosomal protein rpl-4139RPL-43 (RPL7A)60S ribosomal protein L37a40RPS-27 (RPS27)40S ribosomal protein S2741UBA-1 (UBA1)UBA (human ubiquitin) related42UNC-52 (HSPG2)Extracellular matrix protein related to heparan sulfate proteoglycan 243VGLN-1 (HDLBP)ViGiLN homolog44W08E12.7 (PA2G4)Protein with nucleic acid binding activity similar to proliferation associated 2G445Y48A6B.3 (NHP2)Putative H/ACA ribonucleoprotein complex subunit 2-like proteinThe worm proteins listed here were found with a false discovery rate <1% in 2/2 HA::PAQR-2 immunoprecipitations or were proteins where the homolog was identified at least once in worms and at least once in human. The closest human homolog as per WormBase is listed in parenthesis. Open table in a new tab The human proteins listed here were found with a false discovery rate <1% in at least 2/4 HA::AdipoR2 IPs in HEK293 cells (and never in control cells), or were proteins where the homolog was identified at least once in worms and at least once in human. The worm proteins listed here were found with a false discovery rate <1% in 2/2 HA::PAQR-2 immunoprecipitations or were proteins where the homolog was identified at least once in worms and at least once in human. The closest human homolog as per WormBase is listed in parenthesis. De novo synthesis of long-chain fatty acids in animals begins with the cytoplasmic enzyme FASN that produces palmitate (16:0) from malonyl-CoA and acetyl-CoA via seven cycles through its multiple enzymatic sites (36Smith S. The animal fatty acid synthase: one gene, one polypeptide, seven enzymes.FASEB J. 1994; 8: 1248-1259Google Scholar). Palmitate is then delivered to an ER complex of four enzymes responsible for the four steps of fatty acid elongation: the rate-limiting fatty acid elongase (one of ELOVL1-7 in human), a 3-ketoacyl-CoA reductase (e.g., HSD17B12 in human), a 3-hydroxyacyl-CoA dehydratase (one of HACD1-4 in human), and a 2,3-trans-enoyl-CoA reductase (TECR in human) (37Kihara A. Very long-chain fatty acids: elongation, physiology and related disorders.J. Biochem. 2012; 152: 387-395Google Scholar, 38Ikeda M. Kanao Y. Yamanaka M. Sakuraba H. Mizutani Y. Igarashi Y. et al.Characterization of four mammalian 3-hydroxyacyl-CoA dehydratases involved in very long-chain fatty acid synthesis.FEBS Lett. 2008; 582: 2435-2440Google Scholar, 39Denic V. Weissman J.S. A molecular caliper mechanism for determining very long-chain fatty acid length.Cell. 2007; 130: 663-677Google Scholar, 40Riezman H. The long and short of fatty acid synthesis.Cell. 2007; 130: 587-588Google Scholar, 41Sawai M. Uchida Y. Ohno Y. Miyamoto M. Nishioka C. Itohara S. et al.The 3-hydroxyacyl-CoA dehydratases HACD1 and HACD2 exhibit functional redundancy and are active in a wide range of fatty acid elongation pathways.J. Biol. Chem. 2017; 292: 15538-15551Google Scholar). Strikingly, three of the four enzymes coimmunoprecipitated with AdipoR2 and/or PAQR-2, namely, ELOVL3/6 (ELO-2 in worms), HSD17B12 (LET-767 in worms), and HACD3 (HPO-8 in worms) (Tables 1 and 2; Fig. 2). This strongly suggests that recruitment of this complex contributes to AdipoR2/PAQR-2-mediated membrane homeostasis. Here, we further characterized HACD3 and its worm homolog HPO-8 because they were found in immunoprecipitations of both HA::AdipoR2 and of HA::PAQR-2, respectively (Tables 1 and 2). We verified the PAQR-2/HPO-8 interaction using two methods: first, reverse immunoprecipitation from
Referência(s)