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- Björkegren Sjögren, Camilla
(författare)
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Profilin in cell motility and signal transduction studies using site-specific mutagenesis
- 1997
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- When a cell is stimulated by signalling molecules such as growth factors, one immediate response is an increased motile activity of the cell. Membrane lamella and spikes appear at the cell surface and perform undulating movements. This is caused by the activation and reorganisation of the highly dynamic microfilament system, present beneath the cell membrane. The major component of the microfilament system is actin, a protein that can polymerise into filaments -microfilaments-, and several actin-binding proteins which act in concert to organise actin into different supramolecular arrangements. To understand how a signal is transmitted form the exterior of the cell to the microfilament system, it is important to reveal how the activities of different actin-binding proteins are regulated.This thesis focus on the actin-binding protein profilin, which binds actin monomers and regulates actin polymerisation. Profilin also interacts with polyphosphoinositides, which are important signalling molecules, and this association appears to regulate both the function of profilin and the turnover of polyphosphoinositides. Furthermore, profilin binds poly(L-proline) and proline-rich proteins. The latter interaction seems to be involved in the positioning of profilin within a cell, and may also be important for signal transduction-related processes. In the current investigation site-directed mutagenesis was combined with biochemical methods to further investigate the function of profilin.The replacement of two amino acid residues located in a hydrophobic patch on the surface of the profilin molecule was shown to abolish the interaction with poly(L-proline), and enabled the localisation of the poly(L-proline) binding-site to this part of profilin. A new purification method was developed for these profilin mutants and further biochemical characterisation showed that the two mutations also decreased the affinity of profilin for actin. As the poly(L-proline)-binding site is separated from the actin-binding surface of profilin this showed that these amino acid replacements in the poly(L-proline)-binding site also introduced long-range alterations in the profilin molecule.Profilin was shown to be phosphorylated on a serine and a tyrosine residue by an epidermal growth factor receptor complex isolated from stimulated cells. The phosphorylated residues were located to the poly(L-proline) binding site of profilin, and the phosphorylation interfered with the binding of profilin to poly(L-proline). This suggests that this phosphorylation regulate interactions between profilin and proline-rich proteins.The simultaneous deletion of two amino acid residues adjacent to the actin-binding site of profilin was shown to reduce the affinity of profilin for actin, without affecting the interaction of profilin with other ligands. The effect of this mutant profilin on the organisation of the microfilament system in living cells was compared to that of wild type profilin in microinjection experiments. While wild type profilin caused disruption of actin bundles within the cell, the profilin mutant had no apparent effect, showing that the derangement of the actin bundles by profilin is dependent on the stability of the profilin:actin complex.
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| 2. |
- Karlberg, Tobias, et al.
(författare)
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14-3-3 proteins activate Pseudomonas exotoxins-S and -T by chaperoning a hydrophobic surface
- 2018
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Ingår i: Nature Communications. - : Nature Publishing Group. - 2041-1723. ; 9
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Tidskriftsartikel (refereegranskat)abstract
- Pseudomonas are a common cause of hospital-acquired infections that may be lethal. ADP-ribosyltransferase activities of Pseudomonas exotoxin-S and -T depend on 14-3-3 proteins inside the host cell. By binding in the 14-3-3 phosphopeptide binding groove, an amphipathic C-terminal helix of ExoS and ExoT has been thought to be crucial for their activation. However, crystal structures of the 14-3-3 beta: ExoS and -ExoT complexes presented here reveal an extensive hydrophobic interface that is sufficient for complex formation and toxin activation. We show that C-terminally truncated ExoS ADP-ribosyltransferase domain lacking the amphipathic binding motif is active when co-expressed with 14-3-3. Moreover, swapping the amphipathic C-terminus with a fragment from Vibrio Vis toxin creates a 14-3-3 independent toxin that ADP-ribosylates known ExoS targets. Finally, we show that 14-3-3 stabilizes ExoS against thermal aggregation. Together, this indicates that 14-3-3 proteins activate exotoxin ADP-ribosyltransferase domains by chaperoning their hydrophobic surfaces independently of the amphipathic C-terminal segment.
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