| 1. |
- Alastair, Kerr, et al.
(författare)
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The long noncoding RNA ADIPINT is a gatekeeper of pyruvate carboxylasefunction regulating human fat cell metabolism
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- The pleiotropic function of long noncoding RNAs (lncRNAs) is well recognized,but their direct role in governingmetabolic homeostasis is less understood. Herein,we describe a human adipocyte-specific lncRNA, ADIPINT, that regulatespyruvate carboxylase (PC) an enzyme pivotal to energy metabolism. With a novelapproach, Targeted RNA-protein identification using Orthogonal Organic PhaseSeparation (TROOPS) and validation with electron microscopy, we show thatADIPINT binds to PC. ADIPINT knockdown alters the interactome anddecreases the mitochondrial abundance and enzymatic activty of PC. Decreases inADIPINT or PC expression reduce adipocyte lipid synthesis, breakdown and lipidcontent. In human white adipose tissue, ADIPINT expression is increased inobesity, linked to fat cell size, adipose insulin resistance and PC activity. Thus, weidentify ADIPINT as a regulator of lipid metabolism in human white adipocytes,which at least in part is mediated through its interaction with PC.
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| 2. |
- Kerr, A. G., et al.
(författare)
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The long noncoding RNA ADIPINT regulates human adipocyte metabolism via pyruvate carboxylase
- 2022
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Ingår i: Nature Communications. - : Springer Nature. - 2041-1723. ; 13:1
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Tidskriftsartikel (refereegranskat)abstract
- The pleiotropic function of long noncoding RNAs is well recognized, but their direct role in governing metabolic homeostasis is less understood. Here, we describe a human adipocyte-specific lncRNA, ADIPINT, that regulates pyruvate carboxylase, a pivotal enzyme in energy metabolism. We developed an approach, Targeted RNA-protein identification using Orthogonal Organic Phase Separation, which identifies that ADIPINT binds to pyruvate carboxylase and validated the interaction with electron microscopy. ADIPINT knockdown alters the interactome and decreases the abundance and enzymatic activity of pyruvate carboxylase in the mitochondria. Reduced ADIPINT or pyruvate carboxylase expression lowers adipocyte lipid synthesis, breakdown, and lipid content. In human white adipose tissue, ADIPINT expression is increased in obesity and linked to fat cell size, adipose insulin resistance, and pyruvate carboxylase activity. Thus, we identify ADIPINT as a regulator of lipid metabolism in human white adipocytes, which at least in part is mediated through its interaction with pyruvate carboxylase.
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| 3. |
- Kerr, Alastair, et al.
(författare)
-
The long noncoding RNA ADIPINT is a gatekeeper of pyruvate carboxylase function regulating human fat cell metabolism
-
Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- The pleiotropic function of long noncoding RNAs (lncRNAs) is well recognized, but their direct role in governing metabolic homeostasis is less understood. Herein, we describe a human adipocyte-specific lncRNA, ADIPINT, that regulates pyruvate carboxylase (PC) an enzyme pivotal to energy metabolism. With a novel approach, Targeted RNA-protein identification using Orthogonal Organic Phase Separation (TROOPS) and validation with electron microscopy, we show that ADIPINT binds to PC. ADIPINT knockdown alters the interactome and decreases the mitochondrial abundance and enzymatic activty of PC. Decreases in ADIPINT or PC expression reduce adipocyte lipid synthesis, breakdown and lipid content. In human white adipose tissue, ADIPINT expression is increased in obesity, linked to fat cell size, adipose insulin resistance and PC activity. Thus, we identify ADIPINT as a regulator of lipid metabolism in human white adipocytes, which at least in part is mediated through its interaction with PC.
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| 4. |
- Gowrisankaran, Sindhuja, et al.
(författare)
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Cells Control BIN1-Mediated Membrane Tubulation by Altering the Membrane Charge
- 2020
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Ingår i: Journal of Molecular Biology. - : ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD. - 0022-2836 .- 1089-8638. ; 432:4, s. 1235-1250
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Tidskriftsartikel (refereegranskat)abstract
- The Bridging integrator 1 (BIN1)/Amphiphysin/Rvs (BAR) protein family is an essential part of the cell's machinery to bend membranes. BIN1 is a muscle-enriched BAR protein with an established role in muscle development and skeletal myopathies. Here, we demonstrate that BIN1, on its own, is able to form complex interconnected tubular systems in vitro, reminiscent of t-tubule system in muscle cells. We further describe how BIN1's electrostatic interactions regulate membrane bending: the ratio of negatively charged lipids in the bilayer altered membrane bending and binding properties of BIN1 and so did the manipulation of BIN1's surface charge. We show that the electrostatically mediated BIN1 membrane binding depended on the membrane curvature-it was less affected in liposomes with high curvature. Curiously, BIN1 membrane binding and bending was diminished in cells where the membrane's charge was experimentally reduced. Membrane bending was also reduced in BIN1 mutants where negative or positive charges in the BAR domain have been eliminated. This phenotype, characteristic of BIN1 mutants linked to myopathies, was rescued when the membrane charge was made more negative. The latter findings also show that cells can control tubulation at their membranes by simply altering the membrane charge and through it, the recruitment of BAR proteins and their interaction partners (e.g. dynamin).
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| 5. |
- Wang, Zuoneng, et al.
(författare)
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Coming of Age: Cryo-Electron Tomography as a Versatile Tool to Generate High-Resolution Structures at Cellular/Biological Interfaces
- 2021
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Ingår i: International Journal of Molecular Sciences. - : MDPI. - 1661-6596 .- 1422-0067. ; 22:12, s. 6177-6177
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Forskningsöversikt (övrigt vetenskapligt/konstnärligt)abstract
- Over the last few years, cryo electron microscopy has become the most important methodin structural biology. While 80% of deposited maps are from single particle analysis, electrontomography has grown to become the second most important method. In particular sub-tomogramaveraging has matured as a method, delivering structures between 2 and 5 Å from complexes in cellsas well as in vitro complexes. While this resolution range is not standard, novel developments pointtoward a promising future. Here, we provide a guide for the workflow from sample to structure togain insight into this emerging field.
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| 6. |
- Wang, Zuoneng, et al.
(författare)
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CryoEM reveals BIN1 (isoform 8) does not bind to single actinfilaments in vitro
- 2021
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Ingår i: microPublication biology. - : Caltech Library. - 2578-9430.
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- Cells change their appearance by a concerted action of the cytoskeleton and the plasma membrane. The machinery thatbends the membrane includes Bin/Amphiphysin/Rvs (BAR) domain proteins. Recently BAR domain proteins garneredattention as actin regulators, either by recruiting actin regulating proteins or through binding to actin directly. BIN1 (animportant protein in Alzheimer’s Disease, heart disease and cancer) is one of the few BAR proteins that bind to actindirectly. Here, we imaged a complex of BIN1 and actin with cryoEM. Our results reveal that BIN1 cannot be found onsingle actin filaments.
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| 7. |
- Wang, Zuoneng, 1991-, et al.
(författare)
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Optimizing purification of the peripheral membrane protein FAM92A1 fused to a modified spidroin tag
- 2022
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Ingår i: Protein Expression and Purification. - : Elsevier. - 1046-5928 .- 1096-0279. ; 189, s. 105992-105992
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- Cryo-electron microscopy has revolutionized structural biology. In particular structures of proteins at themembrane interface have been a major contribution of cryoEM. Yet, visualization and characterization of peripheralmembrane proteins remains challenging; mostly because there is no unified purification strategy forthese proteins. FAM92A1 is a novel peripheral membrane protein that binds to the mitochondrial inner membrane.There, FAM92A1 dimers bind to the membrane and play an essential role in regulating the mitochondrialultrastructure. Curiously, FAM92A1 has also an important function in ciliogenesis. FAM92A1 is part of themembrane bending Bin1/Amphiphsyin/RVS (BAR) domain protein family. Currently, there is no structure ofFAM92A1, mostly because FAM92A1 is unstable and insoluble at high concentrations, like many BAR domainproteins. Yet, pure and concentrated protein is a necessity for screening to generate samples suitable for structuredetermination. Here, we present an optimized purification and expression strategy for dimeric FAM92A1. To ourknowledge, we are the first to use the spidroin tag NT* to successfully purify a peripheral membrane protein. Ourresults show that NT* not only increases solubility but stabilizes FAM92A1 as a dimer. FAM92A1 fused to NT* isactive because it is able to efficiently bend membranes. Taken together, our strategy indicates that this is apossible avenue to express and purify other challenging BAR domain proteins.
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| 8. |
- Wang, Zuoneng, 1991-
(författare)
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Structure studies of membrane associated proteins by transmission electron microscopy
- 2021
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Cell membranes need to change their shapes during many cellular processeslike protein trafficking, cytokinesis and membrane homeostasis. The lattershuttles lipids, synthesized in the endoplasmic reticulum, to all membranouscompartments. Bin/Amphiphysin/Rvs (BAR) proteins are peripheralmembrane proteins (PMP) and play an important role in sculpturingmembranes and in the regulation of actin dynamics. Cryo-electronmicroscopy (cryoEM) has emerged as a powerful tool to visualize proteinsat the membrane interface. Here, we employed transmission electronmicroscopy and other biophysical methods to elucidate how BAR domainproteins steer processes at the membrane.In this work we studied the BAR protein bridging integrator 1 (BIN1), whichhas an established role in cancer, Alzheimer’s disease and skeletalmyopathies. To obtain information about BIN1’s interaction with themembrane in near native environments, we used artificial lipid systems suchas liposomes and lipids nanotubes.First, we have shown that electrostatic interactions are more important forBIN1 when binding to membranes with low curvature. At high curvature,binding is likely driven by non-polar interactions. The formation ofinvaginations (or tubules) is regulated by the composition of negativecharged lipids in membrane bilayer or electrostatic residues on the BARdomain. Therefore electrostatic interactions regulate recruitment andcrowding of BIN1; and consequently membrane deformation.Second, we clarified BIN1’s role in actin dynamics. CryoEM reveals that themuscular BIN1 isoform does not bind to single actin filaments, althoughBIN1 can be co-sedimented with actin after polymerization of actin. Thisimplies that BIN1 rather bundles actin than decorates single filaments.Third, we explored a strategy to purify an aggregation prone BAR protein.Aggregation is a property common in Peripheral Membrane Proteins. Thenovel NT* tag is derived from a spider silk protein and was reported to be apromising fusion tag for protein purification. We showed that the NT* tagimproves the solubility and reduces the aggregation of the BAR proteinFAM92A1. The activity of purified FAM92A1-NT* was verified bynegative stain EM.IIFourth, we were interested in the regulation of the lipid metabolism. PyruvateCarboxylase (PC) is a pivotal enzyme to generate lipid precursors. Cellbiological assays identified a long non-coding (lnc) RNA that regulates theactivity of PC. We studied the interaction between the lnc RNA and PC bybiophysical techniques. Size exclusion chromatography confirmed thepresence lncRNA-PC complex in vitro.
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