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| 2. |
- Inatomi, Teppei, et al.
(författare)
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TFB2M and POLRMT are essential for mammalian mitochondrial DNA replication
- 2022
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Ingår i: Biochimica et Biophysica Acta. Molecular Cell Research. - : Elsevier. - 0167-4889 .- 1879-2596. ; 1869:1
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Tidskriftsartikel (refereegranskat)abstract
- Two classes of replication intermediates have been observed from mitochondrial DNA (mtDNA) in many mammalian tissue and cells with two-dimensional agarose gel electrophoresis. One is assigned to leading-strand synthesis in the absence of synchronous lagging-strand synthesis (strand-asynchronous replication), and the other has properties of coupled leading- and lagging-strand synthesis (strand-coupled replication). While strand-asynchronous replication is primed by long noncoding RNA synthesized from a defined transcription initiation site, little is known about the commencement of strand-coupled replication. To investigate it, we attempted to abolish strand-asynchronous replication in cultured human cybrid cells by knocking out the components of the transcription initiation complexes, mitochondrial transcription factor B2 (TFB2M/mtTFB2) and mitochondrial RNA polymerase (POLRMT/mtRNAP). Unexpectedly, removal of either protein resulted in complete mtDNA loss, demonstrating for the first time that TFB2M and POLRMT are indispensable for the maintenance of human mtDNA. Moreover, a lack of TFB2M could not be compensated for by mitochondrial transcription factor B1 (TFB1M/mtTFB1). These findings indicate that TFB2M and POLRMT are crucial for the priming of not only strand-asynchronous but also strand-coupled replication, providing deeper insights into the molecular basis of mtDNA replication initiation.
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| 3. |
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| 4. |
- Mahato, Dhani Ram, et al.
(författare)
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Dynamic lipid interactions in the plasma membrane Na+,K+-ATPase
- 2023
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Ingår i: Biochimica et Biophysica Acta. Molecular Cell Research. - : Elsevier. - 0167-4889 .- 1879-2596. ; 1870:7
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Tidskriftsartikel (refereegranskat)abstract
- The function of ion-transporting Na+,K+-ATPases depends on the surrounding lipid environment in biological membranes. Two established lipid-interaction sites A and B within the transmembrane domain have been observed to induce protein activation and stabilization, respectively. In addition, lipid-mediated inhibition has been assigned to a site C, but with the exact location not experimentally confirmed. Also, possible effects on lipid interactions by disease mutants dwelling in the membrane-protein interface remain relatively uncharacterized. We simulated human Na+,K+-ATPase α1β1FXYD homology models in E1 and E2 states in an asymmetric, multicomponent plasma membrane to determine both wild-type and disease mutant lipid-protein interactions. The simulated wild-type lipid interactions at the established sites A and B were in agreement with experimental results thereby confirming the membrane-protein model system. The less well-characterized, proposed inhibitory site C was dominated by lipids lacking inhibitory properties. Instead, two sites hosting inhibitory lipids were identified at the extracellular side and also a cytoplasmic CHL-binding site that provide putative alternative locations of Na+,K+-ATPase inhibition. Three disease mutations, Leu302Arg, Glu840Arg and Met859Arg resided in the lipid-protein interface and caused drastic changes in the lipid interactions. The simulation results show that lipid interactions to the human Na+,K+-ATPase α1β1FXYD protein in the plasma membrane are highly state-dependent and can be disturbed by disease mutations located in the lipid interface, which can open up for new venues to understand genetic disorders.
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| 5. |
- Smoler, M., et al.
(författare)
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Apparent stiffness of vimentin intermediate filaments in living cells and its relation with other cytoskeletal polymers
- 2020
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Ingår i: Biochimica et Biophysica Acta. Molecular Cell Research. - : Elsevier. - 0167-4889 .- 1879-2596. ; 1867:8
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Tidskriftsartikel (refereegranskat)abstract
- The cytoskeleton is a complex network of interconnected biopolymers intimately involved in the generation and transmission of forces. Several mechanical properties of microtubules and actin filaments have been extensively explored in cells. In contrast, intermediate filaments (IFs) received comparatively less attention despite their central role in defining cell shape, motility and adhesion during physiological processes as well as in tumor progression. Here, we explored relevant biophysical properties of vimentin IFs in living cells combining confocal microscopy and a filament tracking routine that allows localizing filaments with ~20 nm precision. A Fourier-based analysis showed that IFs curvatures followed a thermal-like behavior characterized by an apparent persistence length (lp*) similar to that measured in aqueous solution. Additionally, we determined that certain perturbations of the cytoskeleton affect lp* and the lateral mobility of IFs as assessed in cells in which either the microtubule dynamic instability was reduced or actin filaments were partially depolymerized. Our results provide relevant clues on how vimentin IFs mechanically couple with microtubules and actin filaments in cells and support a role of this network in the response to mechanical stress.
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| 6. |
- Fredriksson, Johanna, et al.
(författare)
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GRK2 selectively attenuates the neutrophil NADPH-oxidase response triggered by beta-arrestin recruiting GPR84 agonists
- 2022
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Ingår i: Biochimica Et Biophysica Acta-Molecular Cell Research. - : Elsevier BV. - 0167-4889. ; 1869:7
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Tidskriftsartikel (refereegranskat)abstract
- In order to avoid a prolonged pro-inflammatory neutrophil response, signaling downstream of an agonist-activated G protein-coupled receptor (GPCR) has to be rapidly terminated. Among the family of GPCR kinases (GRKs) that regulate receptor phosphorylation and signaling termination, GRK2, which is highly expressed by immune cells, plays an important role. The medium chain fatty acid receptor GPR84 as well as formyl peptide receptor 2 (FPR2), receptors expressed in neutrophils, play a key role in regulating inflammation. In this study, we investigated the effects of GRK2 inhibitors on neutrophil functions induced by GPR84 and FPR2 agonists. GRK2 was shown to be expressed in human neutrophils and analysis of subcellular fractions revealed a cytosolic localization. The GRK2 inhibitors enhanced and prolonged neutrophil production of reactive oxygen species (ROS) induced by GPR84-but not FPR2-agonists, suggesting a receptor selective function of GRK2. This suggestion was supported by beta-arrestin recruitment data. The ROS production induced by a non beta-arrestin recruiting GPR84 agonist was not affected by the GRK2 inhibitor. Termination of this beta-arrestin independent response relied, similar to the response induced by FPR2 agonists, primarily on the actin cytoskeleton. In summary, we show that GPR84 utilizes GRK2 in concert with beta-arrestin and actin cytoskeleton dependent processes to fine-tune the activity of the ROS generating NADPH-oxidase in neutrophils.
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| 7. |
- Lange, Stephan, et al.
(författare)
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The M-band: The underestimated part of the sarcomere
- 2020
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Ingår i: Biochimica et Biophysica Acta - Molecular Cell Research. - : Elsevier BV. - 0167-4889. ; 1867:3
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Tidskriftsartikel (refereegranskat)abstract
- The sarcomere is the basic unit of the myofibrils, which mediate skeletal and cardiac Muscle contraction. Two transverse structures, the Z-disc and the M-band, anchor the thin (actin and associated proteins) and thick (myosin and associated proteins) filaments to the elastic filament system composed of titin. A plethora of proteins are known to be integral or associated proteins of the Z-disc and its structural and signalling role in muscle is better understood, while the molecular constituents of the M-band and its function are less well defined. Evidence discussed here suggests that the M-band is important for managing force imbalances during active muscle contraction. Its molecular composition is fine-tuned, especially as far as the structural linkers encoded by members of the myomesin family are concerned and depends on the specific mechanical characteristics of each particular muscle fibre type. Muscle activity signals from the M-band to the nucleus and affects transcription of sarcomeric genes, especially via serum response factor (SRF). Due to its important role as shock absorber in contracting muscle, the M-band is also more and more recognised as a contributor to muscle disease. © 2019
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| 8. |
- Lind, Simon, 1993, et al.
(författare)
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Interdependent allosteric free fatty acid receptor 2 modulators synergistically induce functional selective activation and desensitization in neutrophils
- 2020
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Ingår i: Biochimica Et Biophysica Acta-Molecular Cell Research. - : Elsevier BV. - 0167-4889. ; 1867:6
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Tidskriftsartikel (refereegranskat)abstract
- The non-activating allosteric modulator AZ1729, specific for free fatty acid receptor 2 (FFAR2), transfers the orthosteric FFAR2 agonists propionate and the P2Y(2)R specific agonist ATP into activating ligands that trigger an assembly of the neutrophil superoxide generating NADPH-oxidase. The homologous priming effect on the propionate response and the heterologous receptor cross-talk sensitized ATP response mediated by AZ1729 are functional characteristics shared with Cmp58, another non-activating allosteric FFAR2 modulator. In addition, AZ1729 also turned Cmp58 into a potent activator of the superoxide generating neutrophil NADPH-oxidase, and in agreement with the allosteric modulation concept, the effect was reciprocal in that Cmp58 turned AZ1729 into a potent activating allosteric agonist. The activation signals down-stream of FFAR2 when stimulated by the two interdependent allosteric modulators were biased in that, unlike for orthosteric agonists, the two complementary modulators together triggered an activation of the NADPH-oxidase, but not any transient rise in the cytosolic concentration of free calcium ions (Ca2+). Furthermore, following AZ1729/Cmp58 activation, the signaling by the desensitized FFAR2s was functionally selective in that the orthosteric agonist propionate could still induce a transient rise in intracellular Ca2+. The novel neutrophil activation and receptor down-stream signaling pattern mediated by the two cross-sensitizing allosteric FFAR2 modulators represent a new regulatory mechanism that controls receptor signaling.
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| 9. |
- Sundqvist, Martina, et al.
(författare)
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Barbadin selectively modulates FPR2-mediated neutrophil functions independent of receptor endocytosis
- 2020
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Ingår i: Biochimica Et Biophysica Acta-Molecular Cell Research. - : Elsevier BV. - 0167-4889. ; 1867:12
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Tidskriftsartikel (refereegranskat)abstract
- FPR2, a member of the family of G protein-coupled receptors (GPCRs), mediates neutrophil migration, a response that has been linked to beta-arrestin recruitment. beta-Arrestin regulates GPCR endocytosis and can also elicit non-canonical receptor signaling. To determine the poorly understood role of beta-arrestin in FPR2 endocytosis and in NADPH-oxidase activation in neutrophils, Barbadin was used as a research tool in this study. Barbadin has been shown to bind the clathrin adaptor protein (AP2) and thereby prevent beta-arrestin/AP2 interaction and beta-arrestin-mediated GPCR endocytosis. In agreement with this, AP2/beta-arrestin interaction induced by an FPR2-specific agonist was inhibited by Barbadin. Unexpectedly, however, Barbadin did not inhibit FPR2 endocytosis, indicating that a mechanism independent of beta-arrestin/AP2 interaction may sustain FPR2 endocytosis. This was confirmed by the fact, that FPR2 also underwent agonist-promoted endocytosis in beta-arrestin deficient cells, albeit at a diminished level as compared to wild type cells. Dissection of the Barbadin effects on FPR2-mediated neutrophil functions including NADPH-oxidase activation mediated release of reactive oxygen species (ROS) and chemotaxis revealed that Barbadin had no effect on chemotactic migration whereas the release of ROS was potentiated/primed. The effect of Barbadin on ROS production was reversible, independent of beta-arrestin recruitment, and similar to that induced by latrunculin A. Taken together, our data demonstrate that endocytic uptake of FPR2 occurs independently of beta-arrestin, while Barbadin selectively augments FPR2-mediated ROS production independently of receptor endocytosis. Given that Barbadin binds to AP2 and prevents the AP2/beta-arrestin interaction, our results indicate a role for AP2 in FPR2-mediated ROS release from neutrophils.
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