| 1. |
- Altgärde, Noomi, 1983, et al.
(författare)
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Mucin-like region of herpes simplex virus type 1 attachment protein gC modulates the virus-glycosaminoglycan interaction.
- 2015
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 290:35, s. 21473-21485
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Tidskriftsartikel (refereegranskat)abstract
- Glycoprotein C (gC) mediates the attachment of herpes simplex virus type 1 (HSV-1) to susceptible host cells by interacting with glycosaminoglycans (GAGs) on the cell surface. gC contains a mucin-like region located near the GAG-binding site, which may affect the binding activity. Here, we address this issue by studying an HSV-1 mutant lacking the mucin- like domain in gC and the corresponding purified mutant protein (gCΔmuc), in cell culture and GAG-binding assays, respectively. The mutant virus exhibited two functional alterations as compared to native HSV-1, i.e. decreased sensitivity to GAG-based inhibitors of virus attachment to cells, and reduced release of viral particles from the surface of infected cells. Kinetic and equilibrium binding characteristics of purified gC were assessed using surface plasmon resonance-based sensing together with a surface platform consisting of end-on immobilized GAGs. Both native gC and gCΔmuc bound via the expected binding region to chondroitin sulfate and sulfated hyaluronan but not to the non-sulfated hyaluronan, confirming binding specificity. In contrast to native gC, gCΔmuc exhibited a decreased affinity for GAGs and a slower dissociation, indicating that once formed, the gCΔmuc-GAG complex is more stable. It was also found that a larger number of gCΔmuc bound to a single GAG chain, compared to native gC. Taken together, our data suggest that the mucin-like region of HSV-1 gC is involved in the modulation of the GAG-binding activity, a feature of importance both for unrestricted virus entry into the cells and release of newly produced viral particles from infected cells.
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| 2. |
- Andersen, T. C. B., et al.
(författare)
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The SH3 domains of the protein kinases ITK and LCK compete for adjacent sites on T cell?specific adapter protein
- 2019
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Ingår i: Journal of Biological Chemistry. - 0021-9258. ; 294:42, s. 15480-15494
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Tidskriftsartikel (refereegranskat)abstract
- T-cell activation requires stimulation of specific intracellular signaling pathways in which protein-tyrosine kinases, phosphatases, and adapter proteins interact to transmit signals from the T-cell receptor to the nucleus. Interactions of LCK proto-oncogene, SRC family tyrosine kinase (LCK), and the IL-2?inducible T cell kinase (ITK) with the T cell-specific adapter protein (TSAD) promotes LCK-mediated phosphorylation and thereby ITK activation. Both ITK and LCK interact with TSAD's proline-rich region (PRR) through their Src homology 3 (SH3) domains. Whereas LCK may also interact with TSAD through its SH2 domain, ITK interacts with TSAD only through its SH3 domain. To begin to understand on a molecular level how the LCK SH3 and ITK SH3 domains interact with TSAD in human HEK293T cells, here we combined biochemical analyses with NMR spectroscopy. We found that the ITK and LCK SH3 domains potentially have adjacent and overlapping binding sites within the TSAD PRR amino acids (aa) 239?274. Pulldown experiments and NMR spectroscopy revealed that both domains may bind to TSAD aa 239?256 and aa 257?274. Co-immunoprecipitation experiments further revealed that both domains may also bind simultaneously to TSAD aa 242?268. Accordingly, NMR spectroscopy indicated that the SH3 domains may compete for these two adjacent binding sites. We propose that once the associations of ITK and LCK with TSAD promote the ITK and LCK interaction, the interactions among TSAD, ITK, and LCK are dynamically altered by ITK phosphorylation status.
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| 3. |
- Andersson, Christian X, 1973, et al.
(författare)
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Insulin antagonizes interleukin-6 signaling and is anti-inflammatory in 3T3-L1 adipocytes
- 2007
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Ingår i: J Biol Chem. - 0021-9258. ; 282:13, s. 9430-5
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Tidskriftsartikel (refereegranskat)abstract
- Adipose tissue secretes different adipokines, including interleukin-6 (IL-6), that have been implicated in the insulin resistance and inflammatory state characterizing obesity. We examined the putative cross-talk between insulin and IL-6 in adipose cells and found that insulin exerts an inhibitory effect on the IL-6 signaling pathway by altering the post-translational modifications of the signal transducer and activator of transcription 3 (STAT3). Insulin reduces the tyrosine phosphorylation and increases the serine phosphorylation of STAT3, thereby reducing its nuclear localization and transcriptional activity. Signaling through the MEK/MAPK pathway plays an important role as treatment with the MEK inhibitor PD98059 reduces the effects of insulin on IL-6 signaling. We also show that the protein tyrosine phosphatase SHP2 is activated upon insulin signaling and is required for the dephosphorylation of STAT3 and that insulin exerts a synergistic effect with IL-6 on suppressor of cytokine signaling 3 expression. As a consequence, the IL-6-induced expression of the inflammatory markers serum amyloid A 3 and haptoglobin are significantly decreased in cells incubated with both IL-6 and insulin. Thus, insulin exerts an important anti-inflammatory effect in adipose cells by impairing the IL-6 signal at several levels.
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| 4. |
- Andersson, Fredrik, 1977, et al.
(författare)
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Cyanobacterial ClpC/HSP100 protein displays intrinsic chaperone activity
- 2006
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Ingår i: Journal of Biological Chemistry. - 0021-9258. ; 281:9, s. 5468-5475
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Tidskriftsartikel (refereegranskat)abstract
- HSP100 proteins are molecular chaperones that belong to the broader family of AAA+ proteins ( ATPases associated with a variety of cellular activities) known to promote protein unfolding, disassembly of protein complexes and translocation of proteins across membranes. The ClpC form of HSP100 is an essential, highly conserved, constitutively expressed protein in cyanobacteria and plant chloroplasts, and yet little is known regarding its specific activity as a molecular chaperone. To address this point, ClpC from the cyanobacterium Synechococcus elongatus (SyClpC) was purified using an Escherichia coli-based overexpression system. Recombinant SyClpC showed basal ATPase activity, similar to that of other types of HSP100 protein in non-photosynthetic organisms but different to ClpC in Bacillus subtilis. SyClpC also displayed distinct intrinsic chaperone activity in vitro, first by preventing aggregation of unfolded polypeptides and second by resolubilizing and refolding aggregated proteins into their native structures. The refolding activity of SyClpC was enhanced 3-fold in the presence of the B. subtilis ClpC adaptor protein MecA. Overall, the distinctive ClpC protein in photosynthetic organisms indeed functions as an independent molecular chaperone, and it is so far unique among HSP100 proteins in having both "holding" and disaggregase chaperone activities without the need of other chaperones or adaptor proteins.
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| 5. |
- Andersson, Fredrik, 1977, et al.
(författare)
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Structure and function of a novel type of ATP-dependent Clp protease.
- 2009
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Ingår i: The Journal of biological chemistry. - 0021-9258 .- 1083-351X. ; 284:20, s. 13519-32
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Tidskriftsartikel (refereegranskat)abstract
- The Clp protease is conserved among eubacteria and most eukaryotes, and uses ATP to drive protein substrate unfolding and translocation into a chamber of sequestered proteolytic active sites. The main constitutive Clp protease in photosynthetic organisms has evolved into a functionally essential and structurally intricate enzyme. The model Clp protease from the cyanobacterium Synechococcus consists of the HSP100 molecular chaperone ClpC and a mixed proteolytic core comprised of two distinct subunits, ClpP3 and ClpR. We have purified the ClpP3/R complex, the first for a Clp proteolytic core comprised of heterologous subunits. The ClpP3/R complex has unique functional and structural features, consisting of twin heptameric rings each with an identical ClpP3(3)ClpR(4) configuration. As predicted by its lack of an obvious catalytic triad, the ClpR subunit is shown to be proteolytically inactive. Interestingly, extensive modification to ClpR to restore proteolytic activity to this subunit showed that its presence in the core complex is not rate-limiting for the overall proteolytic activity of the ClpCP3/R protease. Altogether, the ClpP3/R complex shows remarkable similarities to the 20 S core of the proteasome, revealing a far greater degree of convergent evolution than previously thought between the development of the Clp protease in photosynthetic organisms and that of the eukaryotic 26 S proteasome.
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| 6. |
- Andersson, Mats X., 1977, et al.
(författare)
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Optical manipulation reveals strong attracting forces at membrane contact sites between endoplasmic reticulum and chloroplasts
- 2007
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Ingår i: Journal of Biological Chemistry. - 0021-9258. ; 282:2, s. 1170-1174
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Tidskriftsartikel (refereegranskat)abstract
- Eukaryote cells depend on membrane lipid trafficking from biogenic membranes, like the endoplasmic reticulum (ER), to other membranes in the cell. Two major routes for membrane lipid transport are recognized: vesicular trafficking and lipid transfer at zones of close contact between membranes. Specific ER regions involved in such membrane contact sites (MCSs) have been isolated, and lipid transfer at MCSs as well as protein-protein interactions between the partaking membranes have been demonstrated (reviewed by Holthuis, J. C. M., and Levine, T. P. (2005) Nat. Rev. 6, 209–220). Here we present the first demonstration of the physical association between membranes involved in MCSs: by using optical imaging and manipulation, strong attracting forces between ER and chloroplasts are revealed. We used Arabidopsis thaliana expressing green fluorescent protein in the ER lumen and observed leaf protoplasts by confocal microscopy. The ER network was evident, with ER branch end points apparently localized at chloroplast surfaces. After rupture of a protoplast using a laser scalpel, the cell content was released. ER fragments remained attached to the released chloroplasts and could be stretched out by optical tweezers. The applied force, 400 pN, could not drag a chloroplast free from its attached ER, which could reflect protein-protein interactions at the ER-chloroplast MCSs. As chloroplasts rely on import of ER-synthesized lipids, we propose that lipid transfer occurs at these MCSs. We suggest that lipid transfer at the MCSs also occurs in the opposite direction, for example to channel plastid-synthesized acyl groups to supply substrates for ER-localized synthesis of membrane and storage lipids.
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| 7. |
- Andersson, Mats X., 1977, et al.
(författare)
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Oxylipin profiling of the hypersensitive response in Arabidopsis thaliana - Formation of a novel oxo-phytodienoic acid-containing galactolipid, arabidopside E
- 2006
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Ingår i: JOURNAL OF BIOLOGICAL CHEMISTRY. - 0021-9258. ; 281:42, s. 31528-31537
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Tidskriftsartikel (refereegranskat)abstract
- Oxidation products of unsaturated fatty acids, collectively known as oxylipins, function as signaling molecules in plants during development, wounding, and insect and pathogen attack. Certain oxylipins are also known to have direct cytotoxic effects on pathogens. We used inducible expression of bacterial avirulence proteins in planta to study the involvement of oxylipins in race-specific defense against bacterial pathogens. We demonstrate that recognition of the Pseudomonas syringae avirulence protein AvrRpm1 induces 9- and 13-lipoxygenase-dependent oxylipin synthesis in Arabidopsis thaliana. The major oxylipins accumulated were jasmonic acid, 12-oxo-phytodienoic acid, and dinor-oxo-phytodienoic acid. The majority of the newly formed oxylipins (> 90%) was found to be esterified to glycerolipids, whereby 12-oxo-phytodienoic acid and dinor-oxo-phytodienoic acid were found to be esterified to a novel galactolipid. The structure of the substance was determined as a monogalactosyldiacylglycerol containing two 12-oxo-phytodienoic acids and one dinor-oxo-phytodienoic acid acyl chain and was given the trivial name arabidopside E. This substance accumulated to surprisingly high levels, 7 - 8% of total lipid content, and was shown to inhibit growth of a bacterial pathogen in vitro. Arabidopside E was formed also after recognition of the avirulence protein AvrRpt2, suggesting that this could be a conserved feature of defense reactions against bacterial pathogens. In conclusion, the data presented suggest a role of enzymatically formed oxylipins, especially the octadecanoids and arabidopside E in race-specific resistance against bacterial pathogens.
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| 8. |
- Asin-Cayuela, Jorge, et al.
(författare)
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The human mitochondrial transcription termination factor (mTERF) is fully active in vitro in the non-phosphorylated form.
- 2005
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Ingår i: The Journal of biological chemistry. - 0021-9258. ; 280:27, s. 25499-505
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Tidskriftsartikel (refereegranskat)abstract
- The human mitochondrial transcription termination factor (mTERF) is a 39-kDa protein that terminates transcription at the 3'-end of the 16 S rRNA gene and thereby controls expression of the ribosomal transcription unit of mitochondrial DNA. The transcription termination activity of human mTERF has been notoriously difficult to study in vitro, and it has been suggested that the activity of the protein is regulated by posttranslational modifications or by protein polymerization. We here characterize the activity of recombinant human mTERF expressed in insect cells. We observed that mTERF efficiently promotes sequence-specific termination in a completely recombinant and highly purified in vitro system for mitochondrial transcription. The termination activity has a distinct polarity, and we observed complete transcription termination when the mTERF-binding site is oriented in a forward position relative the heavy strand promoter but only partial transcription termination when the binding site is in the reverse position. We analyzed the biochemical characteristics of the active mTERF protein and found that it is a stable monomer at physiological salt concentration. Structural analysis, including phosphostaining, two-dimensional electrophoresis, and electrospray mass spectrometry, detected no evidence of phosphorylation. We conclude that the monomeric human mTERF is fully active in its non-phosphorylated form and that the protein does not require additional cellular factors to terminate mitochondrial transcription in vitro.
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| 9. |
- Asker, Noomi, 1968, et al.
(författare)
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Dimerization of the human MUC2 mucin in the endoplasmic reticulum is followed by a N-glycosylation-dependent transfer of the mono- and dimers to the Golgi apparatus.
- 1998
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Ingår i: The Journal of biological chemistry. - 0021-9258. ; 273:30, s. 18857-63
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Tidskriftsartikel (refereegranskat)abstract
- Pulse-chase experiments in the colon cell line LS 174T combined with subcellular fractionation by sucrose density gradient centrifugation showed that the initial dimerization of the MUC2 apomucin started directly after translocation of the apomucin into the rough endoplasmic reticulum as detected by calnexin reactivity. As the mono- and dimers were chased, O-glycosylated MUC2 mono- and dimers were precipitated using an O-glycosylation-insensitive antiserum against the N-terminal domain of the MUC2 mucin. These O-glycosylated species were precipitated from the fractions that comigrated with the galactosyltransferase activity during the subcellular fractionation, indicating that not only MUC2 dimers but also a significant amount of monomers are transferred into the Golgi apparatus. Inhibition of N-glycosylation with tunicamycin treatment slowed down the rate of dimerization and introduced further oligomerization of the MUC2 apomucin in the endoplasmic reticulum. Results of two-dimensional gel electrophoresis demonstrated that these oligomers (putative tri- and tetramers) were stabilized by disulfide bonds. The non-N-glycosylated species of the MUC2 mucin were retained in the endoplasmic reticulum because no O-glycosylated species were precipitated after inhibition by tunicamycin. This suggests that N-glycans of MUC2 are necessary for the correct folding and dimerization of the MUC2 mucin.
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| 10. |
- Aslani, Alireza, 1965, et al.
(författare)
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A novel conformation of the herpes simplex virus origin of DNA replication recognized by the origin binding protein.
- 2000
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Ingår i: The Journal of biological chemistry. - 0021-9258. ; 275:8, s. 5880-7
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Tidskriftsartikel (refereegranskat)abstract
- The Herpes simplex virus type I origin binding protein (OBP) is a sequence-specific DNA-binding protein and a dimeric DNA helicase encoded by the UL9 gene. It is required for the activation of the viral origin of DNA replication oriS. Here we demonstrate that the linear double-stranded form of oriS can be converted by heat treatment to a stable novel conformation referred to as oriS*. Studies using S1 nuclease suggest that oriS* consists of a central hairpin with an AT-rich sequence in the loop. Single-stranded oligonucleotides corresponding to the upper strand of oriS can adopt the same structure. OBP forms a stable complex with oriS*. We have identified structural features of oriS* recognized by OBP. The central oriS palindrome as well as sequences at the 5' side of the oriS palindrome were required for complex formation. Importantly, we found that mutations that have been shown to reduce oriS-dependent DNA replication also reduce the formation of the OBP-oriS* complex. We suggest that oriS* serves as an intermediate in the initiation of DNA replication providing the initiator protein with structural information for a selective and efficient assembly of the viral replication machinery.
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