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- Ariöz, Candan, 1983-, et al.
(author)
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Anionic Lipid Binding to the Foreign Protein MGS Provides a Tight Coupling between Phospholipid Synthesis and Protein Overexpression in Escherichia coli
- 2013
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In: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 52:33, s. 5533-5544
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Journal article (peer-reviewed)abstract
- Certain membrane proteins involved in lipid synthesis can induce formation of new intracellular membranes in Escherichia coli, i.e., intracellular vesicles. Among those, the foreign monotopic glycosyltransferase MGS from Acholeplasma laidlawii triggers such massive lipid synthesis when overexpressed. To examine the mechanism behind the increased lipid synthesis, we investigated the lipid binding properties of MGS in vivo together with the correlation between lipid synthesis and MGS overexpression levels. A good correlation between produced lipid quantities and overexpressed MGS protein was observed when standard LB medium was supplemented with four different lipid precursors that have significant roles in the lipid biosynthesis pathway. Interestingly, this correlation was highest concerning anionic lipid production and at the same time dependent on the selective binding of anionic lipid molecules by MGS. A selective interaction with anionic lipids was also observed in vitro by P-31 NMR binding studies using bicelles prepared with E. coli lipids. The results clearly demonstrate that the discriminative withdrawal of anionic lipids, especially phosphatidylglycerol, from the membrane through MGS binding triggers an in vivo signal for cells to create a feed-forward stimulation of lipid synthesis in E. coil. By this mechanism, cells can produce more membrane surface in order to accommodate excessively produced MGS molecules, which results in an interdependent cycle of lipid and MGS protein synthesis.
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| 2. |
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| 3. |
- Bakali, Amin, et al.
(author)
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Crystal structure of YegS, a homologue to the mammalian diacylglycerol kinases, reveals a novel regulatory metal binding site
- 2007
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In: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 282:27, s. 19644-19652
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Journal article (peer-reviewed)abstract
- The human lipid kinase family controls cell proliferation, differentiation, and tumorigenesis and includes diacylglycerol kinases, sphingosine kinases, and ceramide kinases. YegS is an Escherichia coli protein with significant sequence homology to the catalytic domain of the human lipid kinases. We have solved the crystal structure of YegS and shown that it is a lipid kinase with phosphatidylglycerol kinase activity. The crystal structure reveals a two-domain protein with significant structural similarity to a family of NAD kinases. The active site is located in the interdomain cleft formed by four conserved sequence motifs. Surprisingly, the structure reveals a novel metal binding site composed of residues conserved in most lipid kinases.
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| 4. |
- Bakali, Amin, 1971-
(author)
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Structural studies of three cell signaling proteins : crystal structures of EphB1, PTPA, and YegS
- 2007
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Doctoral thesis (other academic/artistic)abstract
- Kinases and phosphatases are key regulatory proteins in the cell. The disruption of their activities leads ultimately to the abolishment of the homeostasis of the cell, and is frequently correlated with cancer. EphB1 is a member of the largest family of receptor tyrosine kinases. It is associated with neurogenesis, angiogenesis, and cancer. The cytosolic part of the human EphB1 receptor is composed of two domains. Successful generation of soluble constructs, using a novel random construct screening approach, led to the structure determination of the kinase domain of this receptor. The native structure and the complex structure with an ATP analogue revealed novel features in the regulation of the Eph family of kinases. The structure of PTPA, an activator of protein phosphatase 2 A, a tumor suppressor and a key phosphatase in the cell was solved. The structure revealed a novel fold containing a conserved cleft predicted to be involved in interaction with PP2A. Finally, the structure of YegS, an Escherichia coli protein annotated as a putative diacylglycerol kinase, has been determined. Beside the elucidation of its atomic structure, a phosphatidylglycerol (PG) kinase activity, never seen before, has been assigned to YegS based on biochemical studies. The YegS structure shows resemblance to the fold previously seen in NAD kinases. The structure also revealed the existence of a novel metal site that could potentially play a regulatory role. The YegS structure has important implications for understanding related proteins in pathogenic organisms and is the first homologue of a human lipid kinase for which the structure has been elucidated.
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| 7. |
- Kmiec, Beata, et al.
(author)
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Organellar oligopeptidase (OOP) provides a complementary pathway for targeting peptide degradation in mitochondria and chloroplasts
- 2013
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In: Proceedings of the National Academy of Sciences of the United States of America. - : Proceedings of the National Academy of Sciences. - 0027-8424 .- 1091-6490. ; 110:40, s. E3761-E3769
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Journal article (peer-reviewed)abstract
- Both mitochondria and chloroplasts contain distinct proteolytic systems for precursor protein processing catalyzed by the mitochondrial and stromal processing peptidases and for the degradation of targeting peptides catalyzed by presequence protease. Here, we have identified and characterized a component of the organellar proteolytic systems in Arabidopsis thaliana, the organellar oligopeptidase, OOP (At5g65620). OOP belongs to the M3A family of peptide-degrading metalloproteases. Using two independent in vivo methods, we show that the protease is dually localized to mitochondria and chloroplasts. Furthermore, we localized the OPP homolog At5g10540 to the cytosol. Analysis of peptide degradation by OOP revealed substrate size restriction from 8 to 23 aa residues. Short mitochondrial targeting peptides (presequence of the ribosomal protein L29 and presequence of 1-aminocyclopropane-1-carboxylic acid deaminase 1) and N- and C-terminal fragments derived from the presequence of the ATPase beta subunit ranging in size from 11 to 20 aa could be degraded. MS analysis showed that OOP does not exhibit a strict cleavage pattern but shows a weak preference for hydrophobic residues (F/L) at the P1 position. The crystal structures of OOP, at 1.8-1.9 angstrom, exhibit an ellipsoidal shape consisting of two major domains enclosing the catalytic cavity of 3,000 angstrom(3). The structural and biochemical data suggest that the protein undergoes conformational changes to allow peptide binding and proteolysis. Our results demonstrate the complementary role of OOP in targeting-peptide degradation in mitochondria and chloroplasts.
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| 10. |
- Muheim, Claudio, et al.
(author)
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Identification of a Fragment-Based Scaffold that Inhibits the Glycosyltransferase WaaG from Escherichia coli
- 2016
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In: Antibiotics. - : MDPI AG. - 2079-6382. ; 5:1
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Journal article (peer-reviewed)abstract
- WaaG is a glycosyltransferase that is involved in the biosynthesis of lipopolysaccharide in Gram-negative bacteria. Inhibitors of WaaG are highly sought after as they could be used to inhibit the biosynthesis of the core region of lipopolysaccharide, which would improve the uptake of antibiotics. Herein, we establish an activity assay for WaaG using C-14-labeled UDP-glucose and LPS purified from a increment waaG strain of Escherichia coli. We noted that addition of the lipids phosphatidylglycerol (PG) and cardiolipin (CL), as well as the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) increased activity. We then use the assay to determine if three molecular scaffolds, which bind to WaaG, could inhibit its activity in vitro. We show that 4-(2-amino-1,3-thiazol-4-yl)phenol inhibits WaaG (IC50 1.0 mM), but that the other scaffolds do not. This study represents an important step towards an inhibitor of WaaG by fragment-based lead discovery.
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| 11. |
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