| 1. |
- Wallin, Hanna, et al.
(författare)
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Externally added cystatin C reduces growth of A375 melanoma cells by increasing cell cycle time
- 2021
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Ingår i: FEBS Open Bio. - : Wiley. - 2211-5463. ; 11:6, s. 1645-1658
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Tidskriftsartikel (refereegranskat)abstract
- Some secreted cysteine protease inhibitors of the cystatin family appear to affect intracellular proteolysis and growth of human cells, as a result of internalization. Here, we studied the effects of external addition of the most abundant human cystatin, cystatin C, on viability and proliferation of cancer cells in culture. A dose-dependent decrease in viable cells was seen for A375 melanoma, MCF-7 breast cancer, and PC-3 prostate cancer cells cultured in 1–5 µm cystatin C after 24 h. Real-time assessment of growth rates in A375 cell cultures for 48 h by digital holographic microscopy showed an increased doubling time for cells cultured in the presence of 5 µm cystatin C (20.1 h) compared with control cells (14.7 h). A prolonged doubling time was already observed during the first 12 h, indicating a rapid general decrease in cell proliferation at the population level. Tracking of individual cells in phase holographic images showed that dividing cells incubated with 5 µm cystatin C underwent fewer mitoses during 48 h than control cells. In addition, the time between cell divisions was longer, especially for the first cell cycle. Incubation with the variant W106F-cystatin C (with high cellular uptake rate) resulted in a lower number of viable cells and a prolonged doubling time than when cells were incubated with wild-type cystatin C, but no effect was observed for (R24A,R25A)-cystatin C (low cellular uptake). Thus, cystatin C causes prolonged cell division leading to decreased proliferation of melanoma cells, and internalization seems to be a prerequisite for this effect.
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| 2. |
- Hunaiti, Samar, et al.
(författare)
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Secreted cystatins decrease proliferation and enhance apoptosis of human leukemic cells
- 2020
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Ingår i: FEBS Open Bio. - : Wiley. - 2211-5463. ; 10:10, s. 2166-2181
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Tidskriftsartikel (refereegranskat)abstract
- Cysteine proteases are implicated in proteolysis events favoring cancer cell growth, spread, and death by apoptosis. Herein, we have studied whether the net growth and survival of the leukemic cell lines Jurkat, U937, and HL-60 are affected by external addition of five proteins acting as natural cysteine protease inhibitors. None of the cystatins examined (A, C, D, and E/M) or chagasin showed consistent effects on Fas-induced apoptosis when evaluated at 1 µm. In contrast, when the intrinsic apoptosis pathway was activated by hydrogen peroxide, addition of cystatin D augmented caspase-3-like activity within all three cell lines. Flow cytometric analysis of U937 cells also showed increased numbers of annexin V-positive cells when hydrogen peroxide was used to initiate apoptosis and cells were cultured in the presence of cystatin D or C. Moreover, stimulation of hydrogen peroxide-induced apoptotic U937 cells with either cystatin C or D resulted in a dose-dependent decrease in the number of cells. Cell viability was also decreased when U937 cells were cultured in the presence of cystatin C or D (1–9 µm) only, demonstrating that these cystatins can reduce cell proliferation by themselves in addition to enhancing apoptosis induced by oxidative stress. These effects on U937 cells were paralleled by internalization of cystatins C and D, indicating these effects are caused by downregulation of intracellular proteolysis. External addition of cystatins C and D to HL-60 and Jurkat cells demonstrated similar degrees of cystatin D uptake and decreased viability as for U937 cells, indicating that these effects are general for leukemic cells.
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| 3. |
- Sakhnini, Laila I., et al.
(författare)
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Multimeric fusion single-chain variable fragments as potential novel high-capacity ligands
- 2020
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Ingår i: FEBS Open Bio. - : Wiley. - 2211-5463. ; 10:4, s. 507-514
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Tidskriftsartikel (refereegranskat)abstract
- In basic and applied biotechnology, design of affinity ligands has become essential for high-capacity applications such as affinity-based downstream processes for therapeutic molecules. Here, we established a proof-of-concept for the use of multimeric fusion single-chain variable fragment (scFvs) as high-capacity ligands in affinity adsorbents. Mono- and di/tri-scFvs separated by Pro-rich negatively charged linkers were designed, produced, and immobilized to 6% cross-linked agarose beads. Frontal binding experiments with a target protein of 50 kDa resulted in up to 20 mg·mL−1 and 82% in dynamic binding capacity and utilization yield, respectively, at 100% breakthrough. The utilization of the binding sites was impacted by the ligand format and ligand density, rather than limitation in pore size of adsorbent as previously suggested. Overall, we demonstrated that multimeric fusion scFvs can successfully be developed and used as high-capacity ligands in affinity adsorbents, enabling lean process design and alignment with process specifications.
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| 4. |
- Yao, Shuang, et al.
(författare)
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Apolipoprotein M promotes cholesterol uptake and efflux from mouse macrophages
- 2021
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Ingår i: FEBS Open Bio. - : Wiley. - 2211-5463. ; 11:6, s. 1607-1620
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Tidskriftsartikel (refereegranskat)abstract
- Apolipoprotein M (ApoM) exhibits various anti-atherosclerotic functions as a component of high-density lipoprotein (HDL) particles. Scavenger receptor class B type I (SR-BI) is a classic HDL receptor that mediates selective cholesterol uptake and enhances the efflux of cellular cholesterol to HDL. However, the effect of ApoM on cholesterol transport in macrophages remains unclear. In this study, we identified for the first time that ApoM is expressed in mouse macrophages and is involved in cholesterol uptake, similar to SR-BI. NBD-cholesterol uptake and efflux in cells were characterized using fluorescence spectrophotometry. The uptake ratios of cholesterol by macrophages from ApoM−/−SR-BI−/− mice were significantly lower than those from ApoM+/+SR-BI−/− and ApoM−/−SR-BI+/+ mice. Real-time fluorescence quantitative PCR was used to analyze the expression of cholesterol transport-related genes involved in cholesterol uptake. ApoM-enriched HDL (ApoM+HDL) facilitated more cholesterol efflux from murine macrophage Ana-1 cells than ApoM-free HDL (ApoM−HDL). However, recombinant human ApoM protein inhibited the ability of ApoM−HDL to induce cholesterol efflux. In conclusion, ApoM promotes cholesterol uptake and efflux in mouse macrophages. A better understanding of ApoM function may lead to the development of novel therapeutic strategies for treating atherosclerotic diseases.
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| 5. |
- Huang, Peng, et al.
(författare)
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The intracellular helical bundle of human glucose transporter GLUT4 is important for complex formation with ASPL
- 2023
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Ingår i: FEBS Open Bio. - 2211-5463. ; 13:11, s. 2094-2107
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Tidskriftsartikel (refereegranskat)abstract
- Glucose transporters (GLUTs) are responsible for transporting hexose molecules across cellular membranes. In adipocytes, insulin stimulates glucose uptake by redistributing GLUT4 to the plasma membrane. In unstimulated adipose-like mouse cell lines, GLUT4 is known to be retained intracellularly by binding to TUG protein, while upon insulin stimulation, GLUT4 dissociates from TUG. Here, we report that the TUG homolog in human, ASPL, exerts similar properties, i.e., forms a complex with GLUT4. We describe the structural details of complex formation by combining biochemical assays with cross-linking mass spectrometry and computational modeling. Combined, the data suggest that the intracellular domain of GLUT4 binds to the helical lariat of ASPL and contributes to the regulation of GLUT4 trafficking by cooperative binding.
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| 6. |
- Marnissi, Boutheina, et al.
(författare)
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Accurate detection of Newcastle disease virus using proximity-dependent DNA aptamer ligation assays
- 2021
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Ingår i: FEBS Open Bio. - : John Wiley & Sons. - 2211-5463. ; 11:4, s. 1122-1131
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Tidskriftsartikel (refereegranskat)abstract
- Detecting viral antigens at low concentrations in field samples can be crucial for early veterinary diagnostics. Proximity ligation assays (PLAs) in both solution and solid-phase formats are widely used for high-performance protein detection in medical research. However, the affinity reagents used, which are mainly poly- and monoclonal antibodies, play an important role in the performance of PLAs. Here, we have established the first homogeneous and solid-phase proximity-dependent DNA aptamer ligation assays for rapid and accurate detection of Newcastle disease virus (NDV). NDV is detected by a pair of extended DNA aptamers that, upon binding in proximity to proteins on the envelope of the virus, are joined by enzymatic ligation to form a unique amplicon that can be sensitively detected using real-time PCR. The sensitivity, specificity, and reproducibility of the assays were validated using 40 farm samples. The results demonstrated that the developed homogeneous and solid-phase PLAs, which use NDV-selective DNA aptamers, are more sensitive than the sandwich enzymatic-linked aptamer assay (ELAA), and have a comparable sensitivity to real-time reverse transcription PCR (rRT-PCR) as the gold standard detection method. In addition, the solid-phase PLA was shown to have a greater dynamic range with improved lower limit of detection, upper- and lower limit of quantification, and minimal detectable dose as compared with those of ELAA and rRT-PCR. The specificity of PLA is shown to be concordant with rRT-PCR.
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| 7. |
- Mette Frøbert, Anne, et al.
(författare)
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Characterization and comparison of recombinant full-length ursine and human sex hormone-binding globulin
- 2022
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Ingår i: FEBS Open Bio. - : John Wiley & Sons. - 2211-5463. ; 12:2, s. 362-378
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Tidskriftsartikel (refereegranskat)abstract
- Sex hormone-binding globulin (SHBG) regulates the bioavailability of sex steroid hormones in the blood. Levels of SHBG increase markedly in brown bears (Ursus arctos) during hibernation, suggesting that a key regulatory role of this protein is to quench sex steroid bioavailability in hibernation physiology. To enable characterization of ursine SHBG and a cross species comparison, we established an insect cell-based expression system for recombinant full-length ursine and human SHBG. Compared with human SHBG, we observed markedly lower secretion levels of ursine SHBG, resulting in a 10-fold difference in purified protein yield. Both human and ursine recombinant SHBG appeared as dimeric proteins in solution, with a single unfolding temperature of ~58 °C. The thermal stability of ursine and human SHBG increased 5.4 and 9.5 °C, respectively, in presence of dihydrotestosterone (DHT), suggesting a difference in affinity. The dissociation constants for [3 H]DHT were determined to 0.21±0.04 nM for human and 1.32±0.10 nM for ursine SHBG, confirming a lower affinity of ursine SHBG. A similarly reduced affinity, determined from competitive steroid binding, was observed for most steroids. Overall, we found that ursine SHBG had similar characteristics to human SHBG, specifically, being a homodimeric glycoprotein capable of binding steroids with high affinity. Therefore, ursine SHBG likely has similar biological functions to those known for human SHBG. The determined properties of ursine SHBG will contribute to elucidating its potential regulatory role in hibernation physiology.
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| 8. |
- Persson, Karina, et al.
(författare)
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Structural and functional characterization of a plant alpha-actinin
- 2021
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Ingår i: FEBS Open Bio. - : John Wiley & Sons. - 2211-5463. ; 11:8, s. 2198-2210
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Tidskriftsartikel (refereegranskat)abstract
- The Australian tree malletwood (Rhodamnia argentea) is unique. The genome of malletwood is the only known plant genome that contains a gene coding for an α-actinin-like protein. Several organisms predating the animal-plant bifurcation express an α-actinin or α-actinin-like protein. Therefore, it appears that plants in general, but not malletwood, have lost the α-actinin or α-actinin-like gene during evolution. In order to characterize its structure and function, we synthesized the gene and expressed the recombinant R. argentea protein. The results clearly show that this protein has all properties of genuine α-actinin. The N-terminal actin-binding domain (ABD), with two calponin homology motifs, is very similar to the ABD of any α-actinin. The C-terminal calmodulin-like domain, as well as the intervening rod domain, are also similar to the corresponding regions in other α-actinins. The R. argentea α-actinin-like protein dimerises in solution and thereby can cross-link actin filaments. Based on these results, we believe the R. argentea protein represents a genuine α-actinin, making R. argentea unique in the plant world.
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| 9. |
- Batool, Tahira, et al.
(författare)
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Overexpression of heparanase attenuated TGF-beta-stimulated signaling in tumor cells
- 2017
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Ingår i: FEBS Open Bio. - : Wiley. - 2211-5463. ; 7:3, s. 405-413
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Tidskriftsartikel (refereegranskat)abstract
- Heparan sulfate (HS) mediates the activity of various growth factors including TGF-beta. Heparanase is an endo-glucuronidase that specifically cleaves and modifies HS structure. In this study, we examined the effect of heparanase expression on TGF-beta 1-dependent signaling activities. We found that overexpression of heparanase in human tumor cells (i.e., Fadu pharyngeal carcinoma, MCF7 breast carcinoma) attenuated TGF-beta 1-stimulated Smad phosphorylation and led to a slower cell proliferation. TGF-beta 1-stimulated Akt and Erk phosphorylation was also affected in the heparanase overexpression cells. This effect involved the enzymatic activity of heparanase, as overexpression of mutant inactive heparanase did not affect TGF-beta 1 signaling activity. Analysis of HS isolated from Fadu cells revealed an increase in sulfation of the HS that had a rapid turnover in cells overexpressing heparanase. It appears that the structural alterations of HS affect the ability of TGF-beta 1 to signal via its receptors and elicit a growth response. Given that heparanase expression promotes tumor growth in most cancers, this finding highlights a crosstalk between heparanase, HS, and TGF-beta 1 function in tumorigenesis.
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| 10. |
- Bennett, Matthew, et al.
(författare)
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Crystal structure of the essential biotin-dependent carboxylase AccA3 from Mycobacterium tuberculosis
- 2017
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Ingår i: FEBS Open Bio. - : Wiley. - 2211-5463. ; 7:5, s. 620-626
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Tidskriftsartikel (refereegranskat)abstract
- Biotin-dependent acetyl-CoA carboxylases catalyze the committed step in type II fatty acid biosynthesis, the main route for production of membrane phospholipids in bacteria, and are considered a key target for antibacterial drug discovery. Here we describe the first structure of AccA3, an essential component of the acetyl-CoA carboxylase system in Mycobacterium tuberculosis (MTb). The structure, sequence comparisons, and modeling of ligand-bound states reveal that the ATP cosubstrate-binding site shows distinct differences compared to other bacterial and eukaryotic biotin carboxylases, including all human homologs. This suggests the possibility to design MTb AccA3 subtype-specific inhibitors.
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| 11. |
- Bergkvist, Liza, et al.
(författare)
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Mapping pathogenic processes contributing to neurodegeneration in Drosophila models of Alzheimers disease
- 2020
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Ingår i: FEBS Open Bio. - : WILEY. - 2211-5463. ; 10:3, s. 338-350
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Tidskriftsartikel (refereegranskat)abstract
- Alzheimers disease (AD) is the most common form of dementia, affecting millions of people and currently lacking available disease-modifying treatments. Appropriate disease models are necessary to investigate disease mechanisms and potential treatments. Drosophila melanogaster models of AD include the A beta fly model and the A beta PP-BACE1 fly model. In the A beta fly model, the A beta peptide is fused to a secretion sequence and directly overexpressed. In the A beta PP-BACE1 model, human A beta PP and human BACE1 are expressed in the fly, resulting in in vivo production of A beta peptides and other A beta PP cleavage products. Although these two models have been used for almost two decades, the underlying mechanisms resulting in neurodegeneration are not yet clearly understood. In this study, we have characterized toxic mechanisms in these two AD fly models. We detected neuronal cell death and increased protein carbonylation (indicative of oxidative stress) in both AD fly models. In the A beta fly model, this correlates with high A beta(1-42) levels and down-regulation of the levels of mRNA encoding lysosomal-associated membrane protein 1, lamp1 (a lysosomal marker), while in the A beta PP-BACE1 fly model, neuronal cell death correlates with low A beta(1-42) levels, up-regulation of lamp1 mRNA levels and increased levels of C-terminal fragments. In addition, a significant amount of A beta PP/A beta antibody (4G8)-positive species, located close to the endosomal marker rab5, was detected in the A beta PP-BACE1 model. Taken together, this study highlights the similarities and differences in the toxic mechanisms which result in neuronal death in two different AD fly models. Such information is important to consider when utilizing these models to study AD pathogenesis or screening for potential treatments.
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| 12. |
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| 13. |
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| 14. |
- Davies, Jonathan R., et al.
(författare)
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High-resolution crystal structures of the botulinum neurotoxin binding domains from subtypes A5 and A6
- 2020
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Ingår i: FEBS Open Bio. - : Wiley. - 2211-5463. ; 10:8, s. 1474-1481
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Tidskriftsartikel (refereegranskat)abstract
- Clostridium botulinumneurotoxins (BoNTs) cause flaccid paralysis through inhibition of acetylcholine release from motor neurons; however, at tiny doses, this property is exploited for use as a therapeutic. Each member of the BoNT family of proteins consists of three distinct domains: a binding domain that targets neuronal cell membranes (H-C), a translocation domain (H-N) and a catalytic domain (LC). Here, we present high-resolution crystal structures of the binding domains of BoNT subtypes/A5 (H-C/A5) and/A6 (H-C/A6). These structures show that the core fold identified in other subtypes is maintained, but with subtle differences at the expected receptor-binding sites.
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| 15. |
- Kasaragod, Prasad, et al.
(författare)
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Structural enzymology comparisons of multifunctional enzyme, type-1 (MFE1) : the flexibility of its dehydrogenase part
- 2017
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Ingår i: FEBS Open Bio. - : Wiley. - 2211-5463. ; 7:12, s. 1830-1842
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Tidskriftsartikel (refereegranskat)abstract
- Multifunctional enzyme, type-1 (MFE1) is a monomeric enzyme with a 2E-enoyl-CoA hydratase and a 3S-hydroxyacyl-CoA dehydrogenase (HAD) active site. Enzyme kinetic data of rat peroxisomal MFE1 show that the catalytic efficiencies for converting the short-chain substrate 2E-butenoyl-CoA into acetoacetyl-CoA are much lower when compared with those of the homologous monofunctional enzymes. The mode of binding of acetoacetyl-CoA (to the hydratase active site) and the very similar mode of binding of NAD + and NADH (to the HAD part) are described and compared with those of their monofunctional counterparts. Structural comparisons suggest that the conformational flexibility of the HAD and hydratase parts of MFE1 are correlated. The possible importance of the conformational flexibility of MFE1 for its biocatalytic properties is discussed.
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| 16. |
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| 17. |
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| 18. |
- Leiros, H. K. S., et al.
(författare)
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Structural insights into the enhanced carbapenemase efficiency of OXA-655 compared to OXA-10
- 2020
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Ingår i: FEBS Open Bio. - : Wiley. - 2211-5463. ; 10:9, s. 1821-1832
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Tidskriftsartikel (refereegranskat)abstract
- Carbapenemases are the main cause of carbapenem resistance in Gram-negative bacteria. How beta-lactamases with weak carbapenemase activity, such as the OXA-10-type class D beta-lactamases, contribute to anti-bacterial drug resistance is unclear. OXA-655 is a T26M and V117L OXA-10 variant, recently identified from hospital wastewater. Despite exhibiting stronger carbapenemase activity towards ertapenem (ETP) and meropenem (MEM) inEscherichia coli, OXA-655 exhibits reduced activity towards oxyimino-substituted beta-lactams like ceftazidime. Here, we have solved crystal structures of OXA-10 in complex with imipenem (IPM) and ETP, and OXA-655 in complex with MEM in order to unravel the structure-function relationship and the impact of residue 117 in enzyme catalysis. The new crystal structures show that L117 is situated at a critical position with enhanced Van der Waals interactions to L155 in the omega loop. This restricts the movements of L155 and could explain the reduced ability for OXA-655 to bind a bulky oxyimino group. The V117L replacement in OXA-655 makes the active site S67 and the carboxylated K70 more water exposed. This could affect the supply of new deacylation water molecules required for hydrolysis and possibly the carboxylation rate of K70. But most importantly, L117 leaves more space for binding of the hydroxyethyl group in carbapenems. In summary, the crystal structures highlight the importance of residue 117 in OXA-10 variants for carbapenemase activity. This study also illustrates the impact of a single amino acid substitution on the substrate profile of OXA-10 and the evolutionary potential of new OXA-10 variants.
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| 19. |
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| 20. |
- Tashakor, A., et al.
(författare)
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A new split-luciferase complementation assay identifies pentachlorophenol as an inhibitor of apoptosome formation
- 2019
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Ingår i: Febs Open Bio. - : Wiley. - 2211-5463. ; 9:7, s. 1194-1203
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Tidskriftsartikel (refereegranskat)abstract
- The expense and time required for in vivo reproductive and developmental toxicity studies have driven the development of in vitro alternatives. Here, we used a new in vitro split luciferase-based assay to screen a library of 177 toxicants for inhibitors of apoptosome formation. The apoptosome contains seven Apoptotic Protease-Activating Factor-1 (Apaf-1) molecules and induces cell death by activating caspase-9. Apaf-1-dependent caspase activation also plays an important role in CNS development and spermatogenesis. In the in vitro assay, Apaf-1 fused to an N-terminal fragment of luciferase binds to Apaf-1 fused to a C-terminal fragment of luciferase and reconstitutes luciferase activity. Our assay indicated that pentachlorophenol (PCP) inhibits apoptosome formation, and further investigation revealed that PCP binds to cytochrome c. PCP is a wood preservative that reduces male fertility by ill-defined mechanisms. Although the data show that PCP inhibited apoptosome formation, the concentration required suggests that other mechanisms may be more important for PCP's effects on spermatogenesis. Nonetheless, the data demonstrate the utility of the new assay in identifying apoptosome inhibitors, and we suggest that the assay may be useful in screening for reproductive and developmental toxicants.
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| 21. |
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| 22. |
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| 23. |
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| 24. |
- Colicchia, Valeria, et al.
(författare)
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New regulators of the tetracycline‐inducible gene expression system identified by chemical and genetic screens
- 2022
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Ingår i: FEBS Open Bio. - : Wiley-Blackwell. - 2211-5463. ; 12:10, s. 1896-1908
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Tidskriftsartikel (refereegranskat)abstract
- The tetracycline repressor (tetR)-regulated system is a widely used tool to specifically control gene expression in mammalian cells. Based on this system, we generated a human osteosarcoma cell line, which allows for the inducible expression of an EGFP fusion of the TAR DNA-binding protein 43 (TDP-43), which has been linked to neurodegenerative diseases. Consistent with previous findings, TDP-43 overexpression led to the accumulation of aggregates and limited the viability of U2OS. Using this inducible system, we conducted a chemical screen with a library that included FDA-approved drugs. While the primary screen identified several compounds that prevented TDP-43 toxicity, further experiments revealed that these chemicals abrogated the doxycycline-dependent TDP-43 expression. This antagonistic effect was observed with both doxycycline and tetracycline, and in several Tet-On cell lines expressing different genes, confirming the general effect of these compounds as inhibitors of the tetR system. Using the same cell line, a genome-wide CRISPR/Cas9 screen identified epigenetic regulators such as the G9a methyltransferase and TRIM28 as potential modifiers of TDP-43 toxicity. Yet again, further experiments revealed that G9a inhibition or TRIM28 loss prevented doxycycline-dependent expression of TDP-43. In summary, we have identified new chemical and genetic regulators of the tetR system, thereby raising awareness of the limitations of this approach to conduct chemical or genetic screening in mammalian cells.
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| 25. |
- Hellsten, Sofie V., et al.
(författare)
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The gene expression of numerous SLC transporters is altered in the immortalized hypothalamic cell line N25/2 following amino acid starvation
- 2017
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Ingår i: FEBS Open Bio. - : Wiley. - 2211-5463. ; 7:2, s. 249-264
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Tidskriftsartikel (refereegranskat)abstract
- Amino acids are known to play a key role in gene expression regulation,and in mammalian cells, amino acid signaling is mainly mediated via twopathways, the mammalian target of rapamycin complex 1 (mTORC1) pathwayand the amino acid responsive (AAR) pathway. It is vital for cells tohave a system to sense amino acid levels, in order to control protein andamino acid synthesis and catabolism. Amino acid transporters are crucialin these pathways, due to both their sensing and transport functions. Inthis large-scale study, an immortalized mouse hypothalamic cell line (N25/2)was used to study the gene expression changes following 1, 2, 3, 5 or 16 hof amino acid starvation. We focused on genes encoding solute carriers(SLCs) and putative SLCs, more specifically on amino acid transporters.The microarray contained 28 270 genes and 86.2% of the genes wereexpressed in the cell line. At 5 h of starvation, 1001 genes were upregulatedand 848 genes were downregulated, and among these, 47 genes from theSLC superfamily or atypical SLCs were found. Of these, 15 were genesencoding amino acid transporters and 32 were genes encoding other SLCsor atypical SLCs. Increased expression was detected for genes encodingamino acid transporters from system A, ASC, L, N, T, xc-, and y+. UsingGO annotations, genes involved in amino acid transport and amino acidtransmembrane transporter activity were found to be most upregulated at3 h and 5 h of starvation.
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| 26. |
- Hellsten, Sofie V., et al.
(författare)
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The neuronal and astrocytic protein SLC38A10 transports glutamine, glutamate, and aspartate, suggesting a role in neurotransmission
- 2017
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Ingår i: FEBS Open Bio. - : WILEY. - 2211-5463. ; 7:6, s. 730-746
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Tidskriftsartikel (refereegranskat)abstract
- In brain cells, glutamine transporters are vital to monitor and control the levels of glutamate and GABA. There are 11 members of the SLC38 family of amino acid transporters of which eight have been functionally characterized. Here, we report the first histological and functional characterization of the previously orphan member, SLC38A10. We used pairwise global sequence alignments to determine the sequence identity between the SLC38 family members. SLC38A10 was found to share 20-25% transmembrane sequence identity with several family members, and was predicted to have 11 transmembrane helices. SLC38A10 immunostaining was abundant in mouse brain using a custom-made anti-SLC38A10 antibody and colocalization of SLC38A10 immunoreactivity with markers for neurons and astrocytes was detected. Using Xenopus laevis oocytes overexpressing SLC38A10, we show that SLC38A10 mediates bidirectional transport of L-glutamine, L-alanine, L-glutamate, and D-aspartate, and efflux of L-serine. This profile mostly resembles system A members of the SLC38 family. In conclusion, the bidirectional transport of glutamine, glutamate, and aspartate by SLC38A10, and the immunostaining detected in neurons and astrocytes, suggest that SLC38A10 plays a role in pathways involved in neurotransmission.
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| 27. |
- Okmane, Laura, et al.
(författare)
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The first crystal structure of a family 45 glycoside hydrolase from a brown-rot fungus, Gloeophyllum trabeum GtCel45A
- 2024
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Ingår i: FEBS open bio. - 2211-5463. ; 14, s. 505-514
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Tidskriftsartikel (refereegranskat)abstract
- Here we describe the first crystal structure of a beta-1,4-endoglucanase from a brown-rot fungus, Gloeophyllum trabeum GtCel45A, which belongs to subfamily C of glycoside hydrolase family 45 (GH45). GtCel45A is similar to 18 kDa in size and the crystal structure contains 179 amino acids. The structure is refined at 1.30 angstrom resolution and R-free 0.18. The enzyme consists of a single catalytic module folded into a six-stranded double-psi beta-barrel domain surrounded by long loops. GtCel45A is very similar in sequence (82% identity) and structure to PcCel45A from the white-rot fungus Phanerochaete chrysosporium. Surprisingly though, initial hydrolysis of barley beta-glucan was almost twice as fast in GtCel45A as compared to PcCel45A.
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| 28. |
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| 29. |
- Sridhar, Shruthi, et al.
(författare)
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Structural enzymology studies with the substrate3S-hydroxybutanoyl-CoA : bifunctional MFE1 is a less efficient dehydrogenase than monofunctional HAD
- 2024
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Ingår i: FEBS Open Bio. - : John Wiley & Sons. - 2211-5463. ; 14:4, s. 655-674
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Tidskriftsartikel (refereegranskat)abstract
- Multifunctional enzyme, type-1 (MFE1) catalyzes the second and third step of the β-oxidation cycle, being, respectively, the 2E-enoyl-CoA hydratase (ECH) reaction (N-terminal part, crotonase fold) and the NAD+-dependent, 3S-hydroxyacyl-CoA dehydrogenase (HAD) reaction (C-terminal part, HAD fold). Structural enzymological properties of rat MFE1 (RnMFE1) as well as of two of its variants, namely the E123A variant (a glutamate of the ECH active site is mutated into alanine) and the BCDE variant (without domain A of the ECH part), were studied, using as substrate 3S-hydroxybutanoyl-CoA. Protein crystallographic binding studies show the hydrogen bond interactions of 3S-hydroxybutanoyl-CoA as well as of its 3-keto, oxidized form, acetoacetyl-CoA, with the catalytic glutamates in the ECH active site. Pre-steady state binding experiments with NAD+ and NADH show that the kon and koff rate constants of the HAD active site of monomeric RnMFE1 and the homologous human, dimeric 3S-hydroxyacyl-CoA dehydrogenase (HsHAD) for NAD+ and NADH are very similar, being the same as those observed for the E123A and BCDE variants. However, steady state and pre-steady state kinetic data concerning the HAD-catalyzed dehydrogenation reaction of the substrate 3S-hydroxybutanoyl-CoA show that, respectively, the kcat and kchem rate constants for conversion into acetoacetyl-CoA by RnMFE1 (and its two variants) are about 10 fold lower as when catalyzed by HsHAD. The dynamical properties of dehydrogenases are known to be important for their catalytic efficiency, and it is discussed that the greater complexity of the RnMFE1 fold correlates with the observation that RnMFE1 is a slower dehydrogenase than HsHAD.
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| 36. |
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| 38. |
- Edwin, Aaron, 1983-, et al.
(författare)
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Calcium binding by the PKD1 domain regulates interdomain flexibility in Vibrio cholerae metalloprotease PrtV
- 2013
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Ingår i: FEBS Open Bio. - : Elsevier. - 2211-5463. ; 3, s. 263-270
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Tidskriftsartikel (refereegranskat)abstract
- Vibrio cholerae, the causative agent of cholera, releases several virulence factors including secreted proteases when it infects its host. These factors attack host cell proteins and break down tissue barriers and cellular matrix components such as collagen, laminin, fibronectin, keratin, elastin, and they induce necrotic tissue damage. The secreted protease PrtV constitutes one virulence factors of V. cholerae. It is a metalloprotease belonging to the M6 peptidase family. The protein is expressed as an inactive, multidomain, 102 kDa pre-pro-protein that undergoes several N- and C-terminal modifications after which it is secreted as an intermediate variant of 81 kDa. After secretion from the bacteria, additional proteolytic steps occur to produce the 55 kDa active M6 metalloprotease. The domain arrangement of PrtV is likely to play an important role in these maturation steps, which are known to be regulated by calcium. However, the molecular mechanism by which calcium controls proteolysis is unknown. In this study, we report the atomic resolution crystal structure of the PKD1 domain from V. cholera PrtV (residues 755–838) determined at 1.1 Å. The structure reveals a previously uncharacterized Ca2+-binding site located near linker regions between domains. Conformational changes in the Ca2+-free and Ca2+-bound forms suggest that Ca2+-binding at the PKD1 domain controls domain linker flexibility, and plays an important structural role, providing stability to the PrtV protein.
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| 39. |
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| 40. |
- Ha, V. T., et al.
(författare)
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Synergy between 15-lipoxygenase and secreted PLA2 promotes inflammation by formation of TLR4 agonists from extracellular vesicles
- 2021
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Ingår i: FEBS Open Bio. - : John Wiley & Sons. - 2211-5463. ; 11:Suppl. 1, s. 480-480
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Tidskriftsartikel (övrigt vetenskapligt/konstnärligt)abstract
- Damage assoiated molecular patterns (DAMPs) are endogenous ligands that induce innate immune response, thus promoting sterile inflammation. During oxidative stress, stress-derived EVs (stressEVs) were found to activate Toll-like receptor 4 (TLR4), but the activating ligands were not fully determined. Additionally, several enzymes such as 15-lipoxygenase (15-LO) and secreted phospholipase A2 (sPLA2) are induced during inflammation and were suggested to promote DAMP formation. Stress-EVs were produced from HEK293 exposed to 10uM A23187 and isolated with ultracentrifugation. 20:4 lysoPI was oxidized for 10 min with 15-LO. SynEVs were prepared from phospholipids (PLs), oxidized with 15-LO and hydrolyzed with sPLA2. Activity was measured by qPCR and ELISA on wt and KO cells. Ox 20:4 lysoPI was analyzed by MS. sPLA2 activity was measured in synovial fluid from patients using fluorometric assay. K/BxN serum transfer induced arthritis model on wt and TLR4 KO mice(C57Bl/6 mice) with sPLA2-IIA injection was performed. StressEVs released after oxidative stress were found to activate TLR4with a gene profile different from agonist lipopolysaccharide. StressEVs, 15-LO oxidized synEVs, but only 15-LO oxidized lysoPLs activated cytokine expression through TLR4/MD-2.Hydroxy, hydroperoxy and keto products of 20:4 lysoPI oxidation were determined by MS and they activated the same gene pattern as stressEVs. Furthermore, sPLA2 activity, which we detected in the SF from patients, promoted formation of TLR4 agonists after 15-LO oxidation. Injection of sPLA2-IIA into mice promoted K/BxN serum induced arthritis in TLR4-dependent manner. Both 15-LO and sPLA2 are induced during inflammation, therefore these results imply the role of oxidized lysoPLs in stressEVs in promoting sterile inflammation through TLR4 signaling. The formation of TLR4 agonists is enzyme driven so it provides an opportunity for therapy without compromising innate immunity against pathogens (Ha VT. et al., PNAS 2020).
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| 41. |
- Hall, Michael, et al.
(författare)
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Structure of the C-terminal domain of AspA (antigen I/II-family) protein from Streptococcus pyogenes
- 2014
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Ingår i: FEBS Open Bio. - : Elsevier. - 2211-5463. ; 4, s. 283-289
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Tidskriftsartikel (refereegranskat)abstract
- The pathogenic bacteria Streptococcus pyogenes can cause an array of diseases in humans, including moderate infections such as pharyngitis (strep throat) as well as life threatening conditions such as necrotizing fasciitis and puerperal fever. The antigen I/II family proteins are cell wall anchored adhesin proteins found on the surfaces of most oral streptococci and are involved in host colonization and biofilm formation. In the present study we have determined the crystal structure of the C2–3-domain of the antigen I/II type protein AspA from S. pyogenes M type 28. The structure was solved to 1.8 Å resolution and shows that the C2–3-domain is comprised of two structurally similar DEv-IgG motifs, designated C2 and C3, both containing a stabilizing covalent isopeptide bond. Furthermore a metal binding site is identified, containing a bound calcium ion. Despite relatively low sequence identity, interestingly, the overall structure shares high similarity to the C2–3-domains of antigen I/II proteins from Streptococcus gordonii and Streptococcus mutans, although certain parts of the structure exhibit distinct features. In summary this work constitutes the first step in the full structure determination of the AspA protein from S. pyogenes.
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| 42. |
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| 43. |
- Hassan, Noor, et al.
(författare)
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Crystal structures of Phanerochaete chrysosporium pyranose 2-oxidase suggest that the N-terminus acts as a propeptide that assists in homotetramer assembly
- 2013
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Ingår i: FEBS Open Bio. - : Wiley. - 2211-5463. ; 3, s. 496-504
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Tidskriftsartikel (refereegranskat)abstract
- The flavin-dependent homotetrameric enzyme pyranose 2-oxidase (P2O) is found mostly, but not exclusively, in lignocellulose-degrading fungi where it catalyzes the oxidation of β-. d-glucose to the corresponding 2-keto sugar concomitantly with hydrogen peroxide formation during lignin solubilization. Here, we present crystal structures of P2O from the efficient lignocellulolytic basidiomycete Phanerochaete chrysosporium. Structures were determined of wild-type PcP2O from the natural fungal source, and two variants of recombinant full-length PcP2O, both in complex with the slow substrate 3-deoxy-3-fluoro-. β-. d-glucose. The active sites in PcP2O and P2O from Trametes multicolor (TmP2O) are highly conserved with identical substrate binding. Our structural analysis suggests that the 17°C higher melting temperature of PcP2O compared to TmP2O is due to an increased number of intersubunit salt bridges. The structure of recombinant PcP2O expressed with its natural N-terminal sequence, including a proposed propeptide segment, reveals that the first five residues of the propeptide intercalate at the interface between A and B subunits to form stabilizing, mainly hydrophobic, interactions. In the structure of mature PcP2O purified from the natural source, the propeptide segment in subunit A has been replaced by a nearby loop in the B subunit. We propose that the propeptide in subunit A stabilizes the A/B interface of essential dimers in the homotetramer and that, upon maturation, it is replaced by the loop in the B subunit to form the mature subunit interface. This would imply that the propeptide segment of PcP2O acts as an intramolecular chaperone for oligomerization at the A/B interface of the essential dimer.
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