| 1. |
- Clauss, Adam, et al.
(författare)
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Three genes expressing Kunitz domains in the epididymis are related to genes of WFDC-type protease inhibitors and semen coagulum proteins in spite of lacking similarity between their protein products
- 2011
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Ingår i: BMC Biochemistry. - : Springer Science and Business Media LLC. - 1471-2091. ; 12:55
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Tidskriftsartikel (refereegranskat)abstract
- Background: We have previously identified a locus on human chromosome 20q13.1, encompassing related genes of postulated WFDC-type protease inhibitors and semen coagulum proteins. Three of the genes with WFDC motif also coded for the Kunitz-type protease inhibitor motif. In this report, we have reinvestigated the locus for homologous genes encoding Kunitz motif only. The identified genes have been analyzed with respect to structure, expression and function. Results: We identified three novel genes; SPINT3, SPINT4 and SPINT5, and the structure of their transcripts were determined by sequencing of DNA generated by rapid amplification of cDNA ends. Each gene encodes a Kunitz domain preceded by a typical signal peptide sequence, which indicates that the proteins of 7.6, 8.7, and 9.7 kDa are secreted. Analysis of transcripts in 26 tissues showed that the genes predominantly are expressed in the epididymis. The recombinantly produced proteins could not inhibit the amidolytic activity of trypsin, chymotrypsin, plasmin, thrombin, coagulation factor Xa, elastase, urokinase and prostate specific antigen, whereas similarly made bovine pancreatic trypsin inhibitor (BPTI) had the same bioactivity as the protein isolated from bovine pancreas. Conclusions: The similar organization, chromosomal location and site of expression, suggests that the novel genes are homologous with the genes of WFDC-type protease inhibitors and semen coagulum proteins, despite the lack of similarity in primary structure of their protein products. Their restricted expression to the epididymis suggests that they could be important for male reproduction. The recombinantly produced proteins are presumably bioactive, as demonstrated with similarly made BPTI, but may have a narrower spectrum of inhibition, as indicated by the lacking activity against eight proteases with differing specificity. Another possibility is that they have lost the protease inhibiting properties, which is typical of Kunitz domains, in favor of hitherto unknown functions.
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| 2. |
- Khan, Samiullah, et al.
(författare)
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Aglycone specificity of Thermotoga neapolitana beta-glucosidase 1A modified by mutagenesis, leading to increased catalytic efficiency in quercetin-3-glucoside hydrolysis
- 2011
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Ingår i: BMC Biochemistry. - : Springer Science and Business Media LLC. - 1471-2091. ; 12
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Tidskriftsartikel (refereegranskat)abstract
- Background: The thermostable beta-glucosidase (TnBgl1A) from Thermotoga neapolitana is a promising biocatalyst for hydrolysis of glucosylated flavonoids and can be coupled to extraction methods using pressurized hot water. Hydrolysis has however been shown to be dependent on the position of the glucosylation on the flavonoid, and e. g. quercetin-3-glucoside (Q3) was hydrolysed slowly. A set of mutants of TnBgl1A were thus created to analyse the influence on the kinetic parameters using the model substrate para-nitrophenyl-beta-D-glucopyranoside (pNPGlc), and screened for hydrolysis of Q3. Results: Structural analysis pinpointed an area in the active site pocket with non-conserved residues between specificity groups in glycoside hydrolase family 1 (GH1). Three residues in this area located on beta-strand 5 (F219, N221, and G222) close to sugar binding sub-site +2 were selected for mutagenesis and amplified in a protocol that introduced a few spontaneous mutations. Eight mutants (four triple: F219L/P165L/M278I, N221S/P165L/M278I, G222Q/P165L/M278I, G222Q/V203M/K214R, two double: F219L/K214R, N221S/P342L and two single: G222M and N221S) were produced in E. coli, and purified to apparent homogeneity. Thermostability, measured as T-m by differential scanning calorimetry (101.9 degrees C for wt), was kept in the mutated variants and significant decrease (Delta T of 5 -10 degrees C) was only observed for the triple mutants. The exchanged residue(s) in the respective mutant resulted in variations in K-M and turnover. The K-M-value was only changed in variants mutated at position 221 (N221S) and was in all cases monitored as a 2-3 x increase for pNPGlc, while the K-M decreased a corresponding extent for Q3. Turnover was only significantly changed using pNPGlc, and was decreased 2-3 x in variants mutated at position 222, while the single, double and triple mutated variants carrying a mutation at position 221 (N221S) increased turnover up to 3.5 x compared to the wild type. Modelling showed that the mutation at position 221, may alter the position of N291 resulting in increased hydrogen bonding of Q3 (at a position corresponding to the +1 subsite) which may explain the decrease in K-M for this substrate. Conclusion: These results show that residues at the +2 subsite are interesting targets for mutagenesis and mutations at these positions can directly or indirectly affect both K-M and turnover. An affinity change, leading to a decreased K-M, can be explained by an altered position of N291, while the changes in turnover are more difficult to explain and may be the result of smaller conformational changes in the active site.
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| 3. |
- Sengottaiyan, Palanivelu, et al.
(författare)
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The intrinsic GTPase activity of the Gtr1 protein from Saccharomyces cerevisiae
- 2012
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Ingår i: BMC Biochemistry. - : BioMed Central (BMC). - 1471-2091. ; 13
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Tidskriftsartikel (refereegranskat)abstract
- BackgroundThe Gtr1 protein of Saccharomyces cerevisiae is a member of the RagA subfamily of the Ras-like small GTPase superfamily. Gtr1 has been implicated in various cellular processes. Particularly, the Switch regions in the GTPase domain of Gtr1 are essential for TORC1 activation and amino acid signaling [R. Gong, L. Li, Y. Liu, P. Wang, H. Yang, L. Wang, J. Cheng, K.L. Guan, Y. Xu, Genes Dev. 25 (2011) 1668–1673]. Therefore, knowledge about the biochemical activity of Gtr1 is required to understand its mode of action and regulation.ResultsBy employing tryptophan fluorescence analysis and radioactive GTPase assays, we demonstrate that Gtr1 can adopt two distinct GDP- and GTP-bound conformations, and that it hydrolyses GTP much slower than Ras proteins. Using cysteine mutagenesis of Arginine-37 and Valine-67, residues at the Switch I and II regions, respectively, we show altered GTPase activity and associated conformational changes as compared to the wild type protein and the cysteine-less mutant.ConclusionsThe extremely low intrinsic GTPase activity of Gtr1 implies requirement for interaction with activating proteins to support its physiological function. These findings as well as the altered properties obtained by mutagenesis in the Switch regions provide insights into the function of Gtr1 and its homologues in yeast and mammals.
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| 4. |
- Biester, EM, et al.
(författare)
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Identification of avian wax synthases
- 2012
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Ingår i: BMC biochemistry. - : Springer Science and Business Media LLC. - 1471-2091. ; 13, s. 4-
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Tidskriftsartikel (refereegranskat)
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| 5. |
- Hellenbrand, J, et al.
(författare)
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Fatty acyl-CoA reductases of birds
- 2011
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Ingår i: BMC biochemistry. - : Springer Science and Business Media LLC. - 1471-2091. ; 12, s. 64-
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Tidskriftsartikel (refereegranskat)
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| 6. |
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| 7. |
- Johansson, Nina, 1983, et al.
(författare)
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Comparative sequence analysis and mutagenesis of Ethylene Forming Enzyme (EFE) 2-oxoglutarate/Fe(II)-dependent dioxygenase homologs
- 2014
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Ingår i: BMC Biochemistry. - : Springer Science and Business Media LLC. - 1471-2091. ; 15:1, s. Art. no. 22-
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Tidskriftsartikel (refereegranskat)abstract
- Background: Ethylene is one of the most used chemical monomers derived from non-renewable sources and we are investigating the possibility of producing it in yeast via the ethylene forming enzyme (EFE) from Pseudomonas syringae. To enable engineering strategies to improve the enzyme, it is necessary to identify the regions and amino acid residues involved in ethylene formation.Results: We identified the open reading frame for the EFE homolog in Penicillium digitatum and also showed its capability of mediating ethylene production in yeast. The sequence of the EFE homologs from P. digitatum and P. syringae was compared to that of the non-functional EFE-homolog from Penicillium chrysogenum and ten amino acids were found to correlate with ethylene production. Several of these amino acid residues were found to be important for ethylene production via point mutations in P. syringae EFE. The EFE homolog from P. chrysogenum was engineered at 10 amino acid residues to mimic the P. syringae EFE, but this did not confer ethylene producing capability. Furthermore, we predicted the structure of EFE by homology to known structures of 2-oxoglutarate/Fe(II) dependent dioxygenases. Three of the amino acids correlating with ethylene production are located in the predicted 2 oxoglutarate binding domain. A protein domain specific for the EFE class was shown to be essential for activity. Based on the structure and alanine substitutions, it is likely that amino acids (H189, D191 and H268) are responsible for binding the Fe(II) ligand.Conclusion: We provide further insight into the structure and function of the ethylene forming (EFE) - subclass of 2-oxoglutarate/Fe(II) dependent dioxygenases. We conclude that residues in addition to the 10 identified positions implicated in ethylene production by sequence comparison, are important for determining ethylene formation. We also demonstrate the use of an alternative EFE gene. The data from this study will provide the basis for directed protein engineering to enhance the ethylene production capability and properties of EFE.
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| 8. |
- Lönneborg, Rosa, 1979-, et al.
(författare)
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Factors that influence the response of the LysR type transcriptional regulators to aromatic compounds
- 2011
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Ingår i: BMC Biochemistry. - : BioMed Central (BMC). - 1471-2091. ; 12:49
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Tidskriftsartikel (refereegranskat)abstract
- Background: The transcriptional regulators DntR, NagR and NtdR have a high sequence identity and belong to the large family of LysR type transcriptional regulators (LTTRs). These three regulators are all involved in regulation of genes identified in pathways for degradation of aromatic compounds. They activate the transcription of these genes in the presence of an inducer, but the inducer specificity profiles are different. Results: The results from this study show that NtdR has the broadest inducer specificity, responding to several nitro-aromatic compounds. Mutational studies of residues that differ between DntR, NagR and NtdR suggest that a number of specific residues are involved in the broader inducer specificity of NtdR when compared to DntR and NagR. The inducer response was also investigated as a function of the experimental conditions and a number of parameters such as the growth media, plasmid arrangement of the LTTR-encoding genes, promoter and gfp reporter gene, and the presence of a His(6) tag were shown to affect the inducer response in E. coli DH5 alpha. Furthermore, the response upon addition of both salicylate and 4-nitrobenzoate to the growth media was larger than the sum of responses upon addition of each of the compounds, which suggests the presence of a secondary binding site, as previously reported for other LTTRs. Conclusions: Optimization of the growth conditions and gene arrangement resulted in improved responses to nitro-aromatic inducers. The data also suggests the presence of a previously unknown secondary binding site in DntR, analogous to that of BenM.
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| 9. |
- Seibert, Volker, et al.
(författare)
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Combinatorial diversity of fission yeast SCF ubiquitin ligases by homo- and heterooligomeric assemblies of the F-box proteins Pop1p and Pop2p
- 2002
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Ingår i: BMC Biochemistry. - London, United Kingdom : BioMed Central (BMC). - 1471-2091. ; 3, s. 1-15
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Tidskriftsartikel (refereegranskat)abstract
- Background: SCF ubiquitin ligases share the core subunits cullin 1, SKP1, and HRT1/RBX1/ROC1, which associate with different F-box proteins. F-box proteins bind substrates following their phosphorylation upon stimulation of various signaling pathways. Ubiquitin-mediated destruction of the fission yeast cyclin-dependent kinase inhibitor Rum1p depends on two heterooligomerizing F-box proteins, Pop1p and Pop2p. Both proteins interact with the cullin Pcu1p when overexpressed, but it is unknown whether this reflects their co-assembly into bona fide SCF complexes.Results: We have identified Psh1p and Pip1p, the fission yeast homologues of human SKP1 and HRT1/RBX1/ROC1, and show that both associate with Pop1p, Pop2p, and Pcu1p into a ~500 kDa SCFPop1p-Pop2p complex, which supports polyubiquitylation of Rum1p. Only the F-box of Pop1p is required for SCFPop1p-Pop2p function, while Pop2p seems to be attracted into the complex through binding to Pop1p. Since all SCFPop1p-Pop2p subunits, except for Pop1p, which is exclusively nuclear, localize to both the nucleus and the cytoplasm, the F-box of Pop2p may be critical for the assembly of cytoplasmic SCFPop2p complexes. In support of this notion, we demonstrate individual SCFPop1p and SCFPop2p complexes bearing ubiquitin ligase activity.Conclusion: Our data suggest that distinct homo- and heterooligomeric assemblies of Pop1p and Pop2p generate combinatorial diversity of SCFPop function in fission yeast. Whereas a heterooligomeric SCFPop1p-Pop2p complex mediates polyubiquitylation of Rum1p, homooligomeric SCFPop1p and SCFPop2p complexes may target unknown nuclear and cytoplasmic substrates.
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| 10. |
- Frisoni, G. B., et al.
(författare)
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The probabilistic model of Alzheimer disease: the amyloid hypothesis revised
- 2022
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Ingår i: Nature Reviews Neuroscience. - : Springer Science and Business Media LLC. - 1471-003X .- 1471-0048. ; 23, s. 53-66
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Tidskriftsartikel (refereegranskat)abstract
- The amyloid hypothesis has been the dominant model for the pathogenesis of Alzheimer disease for several decades. In this Perspective, Giovanni Frisoni and colleagues examine evidence for and against this hypothesis before outlining an alternative model, the probabilistic model of Alzheimer disease. The current conceptualization of Alzheimer disease (AD) is driven by the amyloid hypothesis, in which a deterministic chain of events leads from amyloid deposition and then tau deposition to neurodegeneration and progressive cognitive impairment. This model fits autosomal dominant AD but is less applicable to sporadic AD. Owing to emerging information regarding the complex biology of AD and the challenges of developing amyloid-targeting drugs, the amyloid hypothesis needs to be reconsidered. Here we propose a probabilistic model of AD in which three variants of AD (autosomal dominant AD, APOE epsilon 4-related sporadic AD and APOE epsilon 4-unrelated sporadic AD) feature decreasing penetrance and decreasing weight of the amyloid pathophysiological cascade, and increasing weight of stochastic factors (environmental exposures and lower-risk genes). Together, these variants account for a large share of the neuropathological and clinical variability observed in people with AD. The implementation of this model in research might lead to a better understanding of disease pathophysiology, a revision of the current clinical taxonomy and accelerated development of strategies to prevent and treat AD.
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| 11. |
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| 12. |
- Bonzom, Cyrielle, 1987, et al.
(författare)
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Feruloyl esterase immobilization in mesoporous silica particles and characterization in hydrolysis and transesterification
- 2018
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Ingår i: BMC Biochemistry. - : Springer Science and Business Media LLC. - 1471-2091. ; 19:1
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Tidskriftsartikel (refereegranskat)abstract
- Background: Enzymes display high reactivity and selectivity under natural conditions, but may suffer from decreased efficiency in industrial applications. A strategy to address this limitation is to immobilize the enzyme. Mesoporous silica materials offer unique properties as an immobilization support, such as high surface area and tunable pore size. Results: The performance of a commercially available feruloyl esterase, E-FAERU, immobilized on mesoporous silica by physical adsorption was evaluated for its transesterification ability. We optimized the immobilization conditions by varying the support pore size, the immobilization buffer and its pH. Maximum loading and maximum activity were achieved at different pHs (4.0 and 6.0 respectively). Selectivity, shown by the transesterification/hydrolysis products molar ratio, varied more than 3-fold depending on the reaction buffer used and its pH. Under all conditions studied, hydrolysis was the dominant activity of the enzyme. pH and water content had the greatest influence on the enzyme selectivity and activity. Determined kinetic parameters of the enzyme were obtained and showed that Km was not affected by the immobilization but kcat was reduced 10-fold when comparing the free and immobilized enzymes. Thermal and pH stabilities as well as the reusability were investigated. The immobilized biocatalyst retained more than 20% of its activity after ten cycles of transesterification reaction. Conclusions: These results indicate that this enzyme is more suited for hydrolysis reactions than transesterification despite good reusability. Furthermore, it was found that the immobilization conditions are crucial for optimal enzyme activity as they can alter the enzyme performance.
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| 13. |
- Holmgren, Christian, et al.
(författare)
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Molecular characterization of protein kinase C delta (PKCδ)-Smac interactions
- 2016
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Ingår i: BMC Biochemistry. - : Springer Science and Business Media LLC. - 1471-2091. ; 17:1
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Tidskriftsartikel (refereegranskat)abstract
- Background: Protein kinase C o (PKCo) is known to be an important regulator of apoptosis, having mainly pro-but also anti-Apoptotic effects depending on context. In a previous study, we found that PKCo interacts with the pro-Apoptotic protein Smac. Smac facilitates apoptosis by suppressing inhibitor of apoptosis proteins (IAPs). We previously established that the PKCo-Smac complex dissociates during induction of apoptosis indicating a functional importance. Because the knowledge on the molecular determinants of the interaction is limited, we aimed at characterizing the interactions between PKCo and Smac. Results: We found that PKCo binds directly to Smac through its regulatory domain. The interaction is enhanced by the PKC activator TPA and seems to be independent of PKCo catalytic activity since the PKC kinase inhibitor GF109203X did not inhibit the interaction. In addition, we found that C1 and C2 domains from several PKC isoforms have Smac-binding capacity. Conclusions: Our data demonstrate that the Smac-PKCo interaction is direct and that it is facilitated by an open conformation of PKCo. The binding is mediated via the PKCo regulatory domain and both the C1 and C2 domains have Smac-binding capacity. With this study we thereby provide molecular information on an interaction between two apoptosis-regulating proteins.
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| 14. |
- Jönsson, Tommy, et al.
(författare)
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Digested wheat gluten inhibits binding between leptin and its receptor
- 2015
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Ingår i: BMC Biochemistry. - : Springer Science and Business Media LLC. - 1471-2091. ; 16
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Tidskriftsartikel (refereegranskat)abstract
- Background: Leptin resistance is considered a primary risk factor for obesity. It has been hypothesized that dietary cereal grain protein could cause leptin resistance by preventing leptin from binding to its receptor. Non-degraded dietary wheat protein has been found in human serum at a mean level of 41 ng/mL. Here, we report our findings from testing whether enzymatically digested gluten from wheat prevents leptin from binding to the leptin receptor in vitro. Gluten from wheat was digested with pepsin and trypsin under physiological conditions. Pepsin and trypsin activity was removed from the gluten digest with a 10 kDa spin-filter or by heat treatment at 100 degrees C for 30 min. Binding to the leptin receptor of leptin mixed with gluten digest at a series of concentrations was measured using surface plasmon resonance technology. Results: Binding of the gluten digest to the leptin receptor was not detected. Spin-filtered gluten digest inhibited binding of leptin to the leptin receptor, with 50% inhibition at a gluten digest concentration of similar to 10 ng/mL. Heat-treated gluten digest did not inhibit leptin binding. Conclusions: Digested wheat gluten inhibits binding of leptin to the leptin receptor, with half-maximal inhibition at 10 ng/mL. The inhibition is significant at clinically relevant concentrations and could therefore serve as a novel pathway to investigate to understand the molecular basis of leptin resistance, obesity and associated disorders.
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| 15. |
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| 16. |
- Östberg, Linus J., et al.
(författare)
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Computational studies of human class V alcohol dehydrogenase - the odd sibling
- 2016
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Ingår i: BMC Biochemistry. - : Springer Science and Business Media LLC. - 1471-2091. ; 17
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Tidskriftsartikel (refereegranskat)abstract
- Background: All known attempts to isolate and characterize mammalian class V alcohol dehydrogenase (class V ADH), a member of the large ADH protein family, at the protein level have failed. This indicates that the class V ADH protein is not stable in a non-cellular environment, which is in contrast to all other human ADH enzymes. In this report we present evidence, supported with results from computational analyses performed in combination with earlier in vitro studies, why this ADH behaves in an atypical way. Results: Using a combination of structural calculations and sequence analyses, we were able to identify local structural differences between human class V ADH and other human ADHs, including an elongated beta-strands and a labile a-helix at the subunit interface region of each chain that probably disturb it. Several amino acid residues are strictly conserved in class I-IV, but altered in class V ADH. This includes a for class V ADH unique and conserved Lys51, a position directly involved in the catalytic mechanism in other ADHs, and nine other class V ADH-specific residues. Conclusions: In this study we show that there are pronounced structural changes in class V ADH as compared to other ADH enzymes. Furthermore, there is an evolutionary pressure among the mammalian class V ADHs, which for most proteins indicate that they fulfill a physiological function. We assume that class V ADH is expressed, but unable to form active dimers in a non-cellular environment, and is an atypical mammalian ADH. This is compatible with previous experimental characterization and present structural modelling. It can be considered the odd sibling of the ADH protein family and so far seems to be a pseudoenzyme with another hitherto unknown physiological function.
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| 17. |
- Hein, Catherine L., et al.
(författare)
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Fish introductions reveal the temperature dependence of species interactions
- 2014
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Ingår i: Proceedings of the Royal Society of London. Biological Sciences. - : The Royal Society. - 0962-8452 .- 1471-2954. ; 281:1775, s. 20132641-
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Tidskriftsartikel (refereegranskat)abstract
- A major area of current research is to understand how climate change will impact species interactions and ultimately biodiversity. A variety of environmental conditions are rapidly changing owing to climate warming, and these conditions often affect both the strength and outcome of species interactions. We used fish distributions and replicated fish introductions to investigate environmental conditions influencing the coexistence of two fishes in Swedish lakes: brown trout (Salmo trutta) and pike (Esox lucius). A logistic regression model of brown trout and pike coexistence showed that these species coexist in large lakes (more than 4.5 km(2)), but not in small, warm lakes (annual air temperature more than 0.9-1.5 degrees C). We then explored how climate change will alter coexistence by substituting climate scenarios for 2091-2100 into our model. The model predicts that brown trout will be extirpated from approximately half of the lakes where they presently coexist with pike and from nearly all 9100 lakes where pike are predicted to invade. Context dependency was critical for understanding pike-brown trout interactions, and, given the widespread occurrence of context-dependent species interactions, this aspect will probably be critical for accurately predicting climate impacts on biodiversity.
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