| 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. |
- Arruda, Lucas C. M., et al.
(författare)
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A novel CD34-specific T-cell engager efficiently depletes acute myeloid leukemia and leukemic stem cells in vitro and in vivo
- 2022
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Ingår i: Haematologica. - : Ferrata Storti Foundation (Haematologica). - 0390-6078 .- 1592-8721. ; 107:8, s. 1786-1795
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Tidskriftsartikel (refereegranskat)abstract
- Less than a third of patients with acute myeloid leukemia (AML) are cured by chemotherapy and/or hematopoietic stem cell transplantation, highlighting the need to develop more efficient drugs. The low efficacy of standard treatments is associated with inadequate depletion of CD34(+) blasts and leukemic stem cells, the latter a drug-resistant subpopulation of leukemia cells characterized by the CD34(+)CD38(-) phenotype. To target these drug-resistant primitive leukemic cells better, we have designed a CD34/CD3 bi-specific T-cell engager (BTE) and characterized its anti-leukemia potential in vitro, ex vivo and in vivo. Our results show that this CD34-specific BTE induces CD34-dependent T-cell activation and subsequent leukemia cell killing in a dose-dependent manner, further corroborated by enhanced T-cell-mediated killing at the singlecell level. Additionally, the BTE triggered efficient T-cell-mediated depletion of CD34(+) hematopoietic stem cells from peripheral blood stem cell grafts and CD34(+) blasts from AML patients. Using a humanized AML xenograft model, we confirmed that the CD34-specific BTE had in vivo efficacy by depleting CD34(+) blasts and leukemic stem cells without side effects. Taken together, these data demonstrate that the CD34-specific BTE has robust antitumor effects, supporting development of a novel treatment modality with the aim of improving outcomes of patients with AML and myelodysplastic syndromes.
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| 3. |
- De Oliveira, Danilo Hirabae, et al.
(författare)
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Silk Assembly against Hydrophobic Surfaces?Modeling and Imaging of Formation of Nanofibrils
- 2023
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Ingår i: ACS Applied Bio Materials. - : AMER CHEMICAL SOC. - 2576-6422. ; 6:3, s. 1011-1018
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Tidskriftsartikel (refereegranskat)abstract
- A detailed insight about the molecular organization behind spider silk assembly is valuable for the decoding of the unique properties of silk. The recombinant partial spider silk protein 4RepCT contains four poly-alanine/glycine-rich repeats followed by an amphiphilic C-terminal domain and has shown the capacity to self-assemble into fibrils on hydrophobic surfaces. We herein use molecular dynamic simulations to address the structure of 4RepCT and its different parts on hydrophobic versus hydrophilic surfaces. When 4RepCT is placed in a wing arrangement model and periodically repeated on a hydrophobic surface, fi-sheet structures of the poly-alanine repeats are preserved, while the CT part is settled on top, presenting a fibril with a height of similar to 7 nm and a width of similar to 11 nm. Both atomic force microscopy and cryo-electron microscopy imaging support this model as a possible fibril formation on hydrophobic surfaces. These results contribute to the understanding of silk assembly and alignment mechanism onto hydrophobic surfaces.
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| 4. |
- de Rivero Vaccari, J. P., et al.
(författare)
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Mechanism of action of IC 100, a humanized IgG4 monoclonal antibody targeting apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC)
- 2023
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Ingår i: Translational Research. - : Elsevier BV. - 1931-5244 .- 1878-1810. ; 251, s. 27-40
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Tidskriftsartikel (refereegranskat)abstract
- Inflammasomes are multiprotein complexes of the innate immune response that recognize a diverse range of intracellular sensors of infection or cell damage and recruit the adaptor protein apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) into an inflammasome signaling complex. The recruitment, polymerization and cross-linking of ASC is upstream of caspase-1 activation and interleukin-1β release. Here we provide evidence that IC 100, a humanized IgG4κ monoclonal antibody against ASC, is internalized into the cell and localizes with endosomes, while another part is recycled and redistributed out of the cell. IC 100 binds intracellular ASC and blocks interleukin-1β release in a human whole blood cell inflammasome assay. In vitro studies demonstrate that IC 100 interferes with ASC polymerization and assembly of ASC specks. In vivo bioluminescence imaging showed that IC 100 has broad tissue distribution, crosses the blood brain barrier, and readily penetrates the brain and spinal cord parenchyma. Confocal microscopy of fluorescent-labeled IC 100 revealed that IC 100 is rapidly taken up by macrophages via a mechanism utilizing the Fc region of IC 100. Coimmunoprecipitation experiments and confocal immunohistochemistry showed that IC 100 binds to ASC and to the atypical antibody receptor Tripartite motif-containing protein-21 (TRIM21). In A549 WT and TRIM21 KO cells treated with either IC 100 or IgG4κ isotype control, the levels of intracellular IC 100 were higher than in the IgG4κ-treated controls at 2 hours, 1 day and 3 days after administration, indicating that IC 100 escapes degradation by the proteasome. Lastly, electron microscopy studies demonstrate that IC 100 binds to ASC filaments and alters the architecture of ASC filaments. Thus, IC 100 readily penetrates a variety of cell types, and it binds to intracellular ASC, but it is not degraded by the TRIM21 antibody-dependent intracellular neutralization pathway.
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| 5. |
- Farsi, Zohreh, et al.
(författare)
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Clathrin coat controls synaptic vesicle acidification by blocking vacuolar ATPase activity
- 2018
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Ingår i: eLIFE. - : ELIFE SCIENCES PUBLICATIONS LTD. - 2050-084X. ; 7
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Tidskriftsartikel (refereegranskat)abstract
- Newly-formed synaptic vesicles (SVs) are rapidly acidified by vacuolar adenosine triphosphatases (vATPases), generating a proton electrochemical gradient that drives neurotransmitter loading. Clathrin-mediated endocytosis is needed for the formation of new SVs, yet it is unclear when endocytosed vesicles acidify and refill at the synapse. Here, we isolated clathrin-coated vesicles (CCVs) from mouse brain to measure their acidification directly at the single vesicle level. We observed that the ATP-induced acidification of CCVs was strikingly reduced in comparison to SVs. Remarkably, when the coat was removed from CCVs, uncoated vesicles regained ATP-dependent acidification, demonstrating that CCVs contain the functional vATPase, yet its function is inhibited by the clathrin coat. Considering the known structures of the vATPase and clathrin coat, we propose a model in which the formation of the coat surrounds the vATPase and blocks its activity. Such inhibition is likely fundamental for the proper timing of SV refilling.
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| 6. |
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| 7. |
- 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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| 8. |
- 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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| 9. |
- 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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| 10. |
- Mamand, Doste R., et al.
(författare)
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Extracellular vesicles originating from melanoma cells promote dysregulation in haematopoiesis as a component of cancer immunoediting
- 2024
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Ingår i: Journal of Extracellular Vesicles. - : Wiley. - 2001-3078. ; 13:7
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Tidskriftsartikel (refereegranskat)abstract
- Haematopoiesis dysregulation with the presence of immature myeloid and erythroid immunosuppressive cells are key characteristics of the immune escape phase of tumour development. Here, the role of in vitro generated B16F10 tumour cell-derived extracellular vesicles (tEVs) as indirect cellular communicators, participating in tumour-induced dysregulation of haematopoiesis, was explored. The isolated tEVs displayed features of small EVs with a size range of 100-200 nm, expressed the common EV markers CD63, CD9, and Alix, and had a spherical shape with a lipid bilayer membrane. Proteomic profiling revealed significant levels of angiogenic factors, particularly vascular endothelial growth factor (VEGF), osteopontin, and tissue factor, associated with the tEVs. Systemic administration of these tEVs in syngeneic mice induced splenomegaly and disrupted haematopoiesis, leading to extramedullary haematopoiesis, expansion of splenic immature erythroid progenitors, reduced bone marrow cellularity, medullary expansion of granulocytic myeloid suppressor cells, and the development of anaemia. These effects closely mirrored those observed in tumour-bearing mice and were not seen after heat inactivating the tEVs. In vitro studies demonstrated that tEVs independently induced the expansion of bone marrow granulocytic myeloid suppressor cells and B cells while reducing the frequency of cells in the erythropoietic lineage. These effects of tEVs were significantly abrogated by the blockade of VEGF or heat inactivation. Our findings underscore the important role of tEVs in dysregulating haematopoiesis during the immune escape phase of cancer immunoediting, suggesting their potential as targets for addressing immune evasion and reinstating normal hematopoietic processes.
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