| 1. |
- Doyle, Siamsa, et al.
(författare)
-
Trafficking modulator TENin1 inhibits endocytosis, causes endomembrane protein accumulation at the pre-vacuolar compartment and impairs gravitropic response in Arabidopsis thaliana
- 2014
-
Ingår i: Biochemical Journal. - 0264-6021 .- 1470-8728. ; 460, s. 177-185
-
Tidskriftsartikel (refereegranskat)abstract
- Auxin gradients are established and maintained by polarized distribution of auxin transporters that undergo constitutive endocytic recycling from the PM (plasma membrane) and are essential for the gravitropic response in plants. The present study characterizes an inhibitor of endomembrane protein trafficking, TE1 (trafficking and endocytosis inhibitor 1/TENin1) that reduces gravitropic root bending in Arabidopsis thaliana seedlings. Short-term TE1 treatment causes accumulation of PM proteins, including the BR (brassinosteroid) receptor BRI1 (BR insensitive 1), PIP2a (PM intrinsic protein 2a) and the auxin transporter PIN2 (PIN-FORMED 2) in a PVC (pre-vacuolar related compartment), which is sensitive to BFA (Brefeldin A). This compound inhibits endocytosis from the PM and promotes trafficking to the vacuole, consistent with inhibition of retrieval of proteins to the TGN (trans-Golgi network) from the PVC and the PM. However, trafficking of newly synthesized proteins to the PM is unaffected. The short-term protein trafficking inhibition and long-term effect on plant growth and survival caused by TE1 were fully reversible upon drug washout. Structure activity relationship studies revealed that only minor modifications were possible without loss of biological activity. Diversity in Arabidopsis ecotypes was also exploited to identify two Arabidopsis accessions that display reduced sensitivity to TE1. This compound and the resistant Arabidopsis accessions may be used as a resource in future studies to better understand endomembrane trafficking in plants.
|
|
| 2. |
- Johansson, Jan, et al.
(författare)
-
BRICHOS binds to a designed amyloid-forming beta-protein and reduces proteasomal inhibition and aggresome formation
- 2016
-
Ingår i: Biochemical Journal. - 0264-6021 .- 1470-8728. ; 473, s. 167-178
-
Tidskriftsartikel (refereegranskat)abstract
- The BRICHOS domain is associated with proliferative, degenerative and amyloid diseases, and it has been shown to inhibit fibril formation and toxicity of the Alzheimer's disease-associated amyloid beta-peptide. ProSP-C (prosurfactant protein C) BRICHOS binds to stretches of hydrophobic amino acid residues, which are unfolded or in beta-strand conformation, suggesting that it may have broad anti-amyloid activity. We have studied the effect of the proSP-C BRICHOS domain on the designed amyloidogenic beta-sheet proteins beta 17 and beta 23. beta 17 expressed in the secretory pathway of HEK (human embryonic kidney)-293 cells forms intracellular inclusions, whereas beta 23 is rapidly degraded. Co-expression of BRICHOS leads to a reduction in beta 17 inclusion size and increased levels of soluble beta 17 and beta 23. Furthermore, BRICHOS interacts with the beta-proteins intracellularly, reduces their ubiquitination and decreases aggresome formation and proteasomal inhibition. Collectively, these data suggest that BRICHOS is capable of delaying the aggregation process and toxicity of amyloidogenic proteins in a generic manner.
|
|
| 3. |
- Johansson, Jan, et al.
(författare)
-
Control of amyloid assembly by autoregulation
- 2012
-
Ingår i: Biochemical Journal. - 0264-6021 .- 1470-8728. ; 447, s. 185-192
-
Forskningsöversikt (refereegranskat)abstract
- The assembly of proteins into amyloid fibrils can be an element of both protein aggregation diseases and a functional unit in healthy biological pathways. In both cases, it must be kept under tight control to prevent undesired aggregation. In normophysiology, proteins can self-chaperone amyloidogenic segments by restricting their conformational flexibility in an overall stabilizing protein fold. However, some aggregation-prone segments cannot be controlled in this manner and require additional regulatory elements to limit fibrillation. The present review summarizes different molecular mechanisms that proteins use to control their own assembly into fibrils, such as the inclusion of a chaperoning domain or a blocking segment in the proform, the controlled release of an amyloidogenic region from the folded protein, or the adjustment of fibrillation propensity according to pH. Autoregulatory elements can control disease-related as well as functional fibrillar protein assemblies and distinguish a group of self-regulating amyloids across a wide range of biological functions and organisms.
|
|
| 4. |
- Krus, Ulrika, et al.
(författare)
-
Pyruvate dehydrogenase kinase 1 controls mitochondrial metabolism and insulin secretion in INS-1 832/13 clonal beta-cells
- 2010
-
Ingår i: Biochemical Journal. - 0264-6021 .- 1470-8728. ; 429, s. 205-213
-
Tidskriftsartikel (refereegranskat)abstract
- Tight coupling between cytosolic and mitochondrial metabolism is key for GSIS (glucose-stimulated insulin secretion). In the present study we examined the regulatory contribution of PDH (pyruvate dehydrogenase) kinase 1, a negative regulator of PDH, to metabolic coupling in 832/13 clonal beta-cells. Knockdown of PDH kinase 1 with siRNA (small interfering RNA) reduced its mRNA (>80 %) and protein level (>40 %) after 72 h. PDH activity, glucose-stimulated cellular oxygen consumption and pyruvate-stimulated mitochondrial oxygen consumption increased 1.7- (P < 0.05), 1.6- (P < 0.05) and 1.6-fold (P < 0.05) respectively. Gas chromatography/MS revealed an altered metabolite profile upon silencing of PDH kinase 1, determined by increased levels of the tricarboxylic acid cycle intermediates malate, fumarate and alpha-ketoglutarate. These metabolic alterations were associated with exaggerated GSIS (5-fold compared with 3.1-fold in control cells; P < 0.01). Insulin secretion, provoked by leucine and dimethylsuccinate, which feed into the tricarboxylic acid cycle bypassing PDH, was unaffected. The oxygen consumption and metabolic data strongly suggest that knockdown of PDH kinase 1 in beta-cells permits increased metabolic flux of glucose-derived carbons into the tricarboxylic acid cycle via PDH. Enhanced insulin secretion is probably caused by increased generation of tricarboxylic acid cycle-derived reducing equivalents for mitochondrial electron transport to generate ATP and/or stimulatory metabolic intermediates. On the basis of these findings, we suggest that PDH kinase 1 is an important regulator of PDH in clonal beta-cells and that PDH kinase 1 and PDH are important for efficient metabolic coupling. Maintaining low PDH kinase I expression/activity, keeping PDH in a dephosphorylated and active state, may be important for beta-cells to achieve the metabolic flux rates necessary for maximal GSIS.
|
|
| 5. |
|
|
| 6. |
- Reyhani, Vahid, et al.
(författare)
-
Fibrin binds to collagen and provides a bridge for alpha V beta 3 integrin-dependent contraction of collagen gels
- 2014
-
Ingår i: Biochemical Journal. - 0264-6021 .- 1470-8728. ; 462, s. 113-123
-
Tidskriftsartikel (refereegranskat)abstract
- The functional significance of fibrin deposits typically seen in inflammatory lesions, carcinomas and in healing wounds is not fully understood. In the present study, we demonstrate that fibrinogen/fibrin specifically bound to native Col I (collagen type I) and used the Col I fibre network as a base to provide a functional interface matrix that connects cells to the Col I fibres through alpha V beta 3 integrins. This allowed murine myoblast C2C12 cells to contract the collagenous composite gel via alpha V beta 3 integrin. We show that fibrinogen specifically bound to immobilized native Col I at the site known to bind matrix metalloproteinase-1, discoidin domain receptor-2 and fibronectin, and that binding had no effect on Col I fibrillation. A specific competitive inhibitor blocking the Col-I-binding site for fibrinogen abolished the organization of fibrin into discernable fibrils, as well as the C2C12-mediated contraction of Col I gels. Our data show that fibrin can function as a linkage protein between Col I fibres and cells, and suggest that fibrin at inflammatory sites indirectly connects alpha V beta 3 integrins to Col I fibres and thereby promotes cell-mediated contraction of collagenous tissue structures.
|
|
| 7. |
- Selles, Benjamin, et al.
(författare)
-
Hydroperoxide and peroxynitrite reductase activity of poplar thioredoxin-dependent glutathione peroxidase 5 : kinetics, catalytic mechanism and oxidative inactivation.
- 2012
-
Ingår i: Biochemical Journal. - 0264-6021 .- 1470-8728. ; 442, s. 369-380
-
Tidskriftsartikel (refereegranskat)abstract
- Glutathione peroxidases constitute a family of peroxidases, including selenocysteine- or cysteine-containing isoforms ((SeCys- or Cys-Gpxs) which are regenerated by glutathione or thioredoxins, (Trxs) respectively. We present here new data concerning the substrates of poplar Gpx5 and the residues involved in its catalytic mechanism. This study establishes the capacity of this Cys-Gpx to reduce peroxynitrite with a catalytic efficiency of 106 M-1 s-1. In PtGpx5, Glu79, which replaces the Gln usually found in Gpx catalytic tetrad, is likely involved in substrate selectivity. Although the redox midpoint potential of the Cys44-Cys92 disulfide and the pKa of Cys44 are not modified in the E79Q variant, it exhibited significantly improved kinetic parameters (Kperoxide and kcat) with tert-butyl hydroperoxide. The characterization of the monomeric Y151R variant demonstrated that PtGpx5 is not an obligate homodimer. Also, we show that the conserved Phe90 is important for Trx recognition and that Trx-mediated recycling of PtGpx5 occurs via the formation of a transient disulfide between the Trx catalytic cysteine and the Gpx5 resolving cysteine. Finally, we demonstrate that the conformational changes observed during the transition from the reduced to the oxidized form of PtGpx5 are primarily determined by the oxidation of the peroxidatic cysteine into sulfenic acid. Besides, mass spectrometry analysis of in vitro oxidized PtGpx5 demonstrated that the peroxidatic cysteine can be over-oxidized into sulfinic or sulfonic acids. This suggests that some isoforms could have dual functions potentially acting as hydrogen peroxide- and peroxynitrite-scavenging systems and/or as mediators of peroxide signalling as proposed for 2-Cys peroxiredoxins.
|
|
| 8. |
- Shaikhali, Jehad, et al.
(författare)
-
Biochemical and redox characterization of the mediator complex and its associated transcription factor GeBPL, a GLABROUS1 enhancer binding protein
- 2015
-
Ingår i: Biochemical Journal. - 0264-6021 .- 1470-8728. ; 468:3, s. 385-400
-
Tidskriftsartikel (refereegranskat)abstract
- The eukaryotic mediator integrates regulatory signals from promoter-bound transcription factors (TFs) and transmits them to RNA polymerase II (Pol II) machinery. Although redox signalling is important in adjusting plant metabolism and development, nothing is known about a possible redox regulation of mediator. In the present study, using pull-down and yeast two-hybrid assays, we demonstrate the association of mediator (MED) subunits MED10a, MED28 and MED32 with the GLABROUS1 (GL1) enhancer-binding protein-like (GeBPL), a plant-specific TF that binds a promoter containing cryptochrome 1 response element 2 (CryR2) element. All the corresponding recombinant proteins form various types of covalent oligomers linked by intermolecular disulfide bonds that are reduced in vitro by the thioredoxin (TRX) and/or glutathione/glutaredoxin (GRX) systems. The presence of recombinant MED10a, MED28 and MED32 subunits or changes of its redox state affect the DNA-binding capacity of GeBPL suggesting that redox-driven conformational changes might modulate its activity. Overall, these results advance our understanding of how redox signalling affects transcription and identify mediator as a novel actor in redox signalling pathways, relaying or integrating redox changes in combinationwith specific TFs as GeBPL.
|
|
| 9. |
- Sörensson, Carolin, et al.
(författare)
-
Determination of primary sequence specificity of Arabidopsis MAPKs MPK3 and MPK6 leads to identification of new substrates
- 2012
-
Ingår i: Biochemical Journal. - 0264-6021 .- 1470-8728. ; 446, s. 271-278
-
Tidskriftsartikel (refereegranskat)abstract
- MAPKs (mitogen-activated protein kinases) are signalling components highly conserved among eukaryotes. Their diverse biological functions include cellular differentiation and responses to different extracellular stress stimuli. Although some substrates of MAPKs have been identified in plants, no information is available about whether amino acids in the primary sequence other than proline-directed phosphorylation (pS-P) contribute to kinase specificity towards substrates. in the present study, we used a random positional peptide library to search for consensus phosphorylation sequences for Arabidopsis MAPKs MPK3 and MPK6. These experiments indicated a preference towards the sequence L/P-P/X-S-P-R/K for both kinases. After bioinformatic processing, a number of novel candidate MAPK substrates were predicted and subsequently confirmed by in vitro kinase assays using bacterially expressed native Arabidopsis proteins as substrates. MPK3 and MPK6 phosphorylated all proteins tested more efficiently than did another MAPK, MPK4. These results indicate that the amino acid residues in the primary sequence surrounding the phosphorylation site of Arabidopsis MAPK substrates can contribute to MAPK specificity. Further characterization of one of these new substrates confirmed that AtIg80180.1 was phosphorylated in planta in a MAPK-dependent manner. Phenotypic analyses of Arabidopsis expressing phosphorylation site mutant forms of AtIg80180.1 showed clustered stomata and higher stomatal index in cotyledons expressing the phosphomimetic form of AtIg80180.1, providing a link between this new MAPK substrate and the defined role for MPK3 and MPK6 in stomatal patterning.
|
|
| 10. |
- Zavialov, Anton
(författare)
-
Caf1A usher possesses a Caf1 subunit-like domain that is crucial for Caf1 fibre secretion
- 2009
-
Ingår i: Biochemical Journal. - 0264-6021 .- 1470-8728. ; 418, s. 541-551
-
Tidskriftsartikel (refereegranskat)abstract
- The chaperone/usher pathway controls assembly of fibres of adhesive organelles of Gram-negative bacteria. The final steps of fibre assembly and fibre translocation to the cell surface are co-ordinated by the outer membrane proteins, ushers. Ushers consist of several soluble periplasmic domains and a single transmembrane beta-barrel. Here we report isolation and structural/functional characterization of a novel middle domain of the Caf1A usher from Yersinia pestis. The isolated UMD (usher middle domain) is a highly soluble monomeric protein capable of autonomous folding. A 2.8 angstrom (1 angstrom = 0.1 nm) resolution crystal structure of UMD revealed that this domain has an immunoglobulin-like fold similar to that of donor-strand-complemented Caf1 fibre subunit. Moreover, these proteins displayed significant structural similarity. Although UMD is in the middle of the predicted amphipathic beta-barrel of Caf1A, the usher still assembled in the membrane in the absence of this domain. UMD did not bind Caf1M-Caf1 complexes, but its presence was shown to be essential for Caf1 fibre secretion. The study suggests that UMD may play the role of a subunit-substituting protein (dummy subunit), plugging or priming secretion through the channel in the Caf1A usher. Comparison of isolated UMD with the recent strcture of the corresponding domain of PapC usher revealed high similarity of the core structures, suggesting a universal structural adaptation of FGL (F(1)G(1) long) and FGS (F(1)G(1) short) chaperone/usher pathways for the secretion of different types of fibres. The functional role of two topologically different states of this plug domain suggested by structural and biochemical results is discussed.
|
|
