PMCC PMCC

Search tips
Search criteria

Advanced
Results 1-25 (510)
 

Clipboard (0)
None

Select a Filter Below

Journals
Year of Publication
more »
1.  Altered activity patterns of transcription factors induced by endoplasmic reticulum stress 
BMC Biochemistry  2016;17:8.
Background
The endoplasmic-reticulum (ER) responds to the burden of unfolded proteins in its lumen by activating intracellular signal transduction pathways, also known as the unfolded protein response (UPR). Many signal transduction events and transcription factors have been demonstrated to be associated with ER stress. The process in which ER stress affects or interacts with other pathways is still a progressing topic that is not completely understood. Identifying new transcription factors associated with ER stress pathways provides a platform to comprehensively characterize mechanism and functionality of ER.
Methods
We utilized a transcription factor (TF) activation plate array to profile the TF activities which were affected by ER stress induced by pharmacological agents, thapsigargin (TG) and tunicamycin (TM) at 1 h, 4 h, 8 h and 16 h respectively, in MiaPACA2 cells. The altered activity patterns were analyzed and validated using gel shift assays and cell-based luciferase reporter assay.
Results
The study has not only confirmed previous findings, which the TFs including ATF4, ATF6, XBP, NFkB, CHOP and AP1, were activated by ER stress, but also found four newly discovered TFs, NFAT, TCF/LEF were activated, and PXR was repressed in response of ER stress. Different patterns of TF activities in MiaPaCa2 were demonstrated upon TM or TG treatment in the time course experiments. The altered activities of TFs were confirmed using gel shift assays and luciferase reporter vectors.
Conclusion
This study utilized a TF activation array technology to identify four new TFs, HIF, NFAT, TCF/LEF and PXR that were changed in their activity as a result of ER stress induced by TG and TM. The TF activity patterns were demonstrated to be diverse in response to the duration of TG or TM treatment. These new findings will facilitate further unveiling the complex mechanisms of the ER stress process and associated diseases.
doi:10.1186/s12858-016-0060-2
PMCID: PMC4806502  PMID: 27009139
ER stress; UPR; TF; Plate array; Activation and Signal pathways
2.  Conserved motif VIII of murine DNA methyltransferase Dnmt3a is essential for methylation activity 
BMC Biochemistry  2016;17:7.
Background
Dnmt3a is a DNA methyltransferase that establishes de novo DNA methylation in mammals. The structure of the Dnmt3a C-terminal domain is similar to the bacterial M. HhaI enzyme, a well-studied prokaryotic DNA methyltransferase. No X-ray structure is available for the complex of Dnmt3a with DNA and the mechanistic details of DNA recognition and catalysis by mammalian Dnmts are not completely understood.
Results
Mutant variants of the catalytic domain of the murine Dnmt3a carrying substitutions of highly conserved N167, R200, and R202 have been generated by site directed mutagenesis and purified. Their methylation activity, DNA binding affinity, ability to flip the target cytosine out of the DNA double helix and covalent complex formation with DNA have been examined. Substitutions of N167 lead to reduced catalytic activity and reduced base flipping. Catalytic activity, base flipping, and covalent conjugate formation were almost completely abolished for the mutant enzymes with substitutions of R200 or R202.
Conclusions
We conclude that R202 plays a similar role in catalysis in Dnmt3a-CD as R232 in M.SssI and R165 in M.HhaI, which could be positioning of the cytosine for nucleophilic attack by a conserved Cys. R200 of Dnmt3a-CD is important in both catalysis and cytosine flipping. Both conserved R200 and R202 are involved in creating and stabilizing of the transient covalent intermediate of the methylation reaction. N167 might contribute to the positioning of the residues from the motif VI, but does not play a direct role in catalysis.
doi:10.1186/s12858-016-0064-y
PMCID: PMC4802922  PMID: 27001594
Dnmt3a DNA methyltransferase; DNA methylation; DNA binding; Dnmt3a mutants; Catalytic domain
3.  Linalool isomerase, a membrane-anchored enzyme in the anaerobic monoterpene degradation in Thauera linaloolentis 47Lol 
BMC Biochemistry  2016;17:6.
Background
Thauera linaloolentis 47Lol uses the tertiary monoterpene alcohol (R,S)-linalool as sole carbon and energy source under denitrifying conditions. The conversion of linalool to geraniol had been observed in carbon-excess cultures, suggesting the presence of a 3,1-hydroxyl-Δ1-Δ2-mutase (linalool isomerase) as responsible enzyme. To date, only a single enzyme catalyzing such a reaction is described: the linalool dehydratase/isomerase (Ldi) from Castellaniella defragrans 65Phen acting only on (S)-linalool.
Results
The linalool isomerase activity was located in the inner membrane. It was enriched by subcellular fractionation and sucrose gradient centrifugation. MALDI-ToF MS analysis of the enriched protein identified the corresponding gene named lis that codes for the protein in the strain with the highest similarity to the Ldi. Linalool isomerase is predicted to have four transmembrane helices at the N-terminal domain and a cytosolic domain. Enzyme activity required a reductant for activation. A specific activity of 3.42 ± 0.28 nkat mg * protein−1 and a kM value of 455 ± 124 μM were determined for the thermodynamically favored isomerization of geraniol to both linalool isomers at optimal conditions of pH 8 and 35 °C.
Conclusion
The linalool isomerase from T. linaloolentis 47Lol represents a second member of the enzyme class 5.4.4.4, next to the linalool dehydratase/isomerase from C. defragrans 65Phen. Besides considerable amino acid sequence similarity both enzymes share common characteristics with respect to substrate affinity, pH and temperature optima, but differ in the dehydratase activity and the turnover of linalool isomers.
Electronic supplementary material
The online version of this article (doi:10.1186/s12858-016-0062-0) contains supplementary material, which is available to authorized users.
doi:10.1186/s12858-016-0062-0
PMCID: PMC4791888  PMID: 26979141
Linalool; Geraniol; Thauera; Isomerase; Allyl alcohol; Monoterpene
4.  1,2-Dichlorobenzene affects the formation of the phosphoenzyme stage during the catalytic cycle of the Ca2+-ATPase from sarcoplasmic reticulum 
BMC Biochemistry  2016;17:5.
Background
1,2-Dichlorobenzene (1,2-DCB) is a benzene-derived molecule with two Cl atoms that is commonly utilized in the synthesis of pesticides. 1,2-DCB can be absorbed by living creatures and its effects on naturally-occurring enzymatic systems, including the effects on Ca2+-ATPases, have been poorly studied. Therefore, we aimed to study the effect of 1,2-DCB on the Ca2+-ATPase from sarcoplasmic reticulum (SERCA), a critical regulator of intracellular Ca2+ concentration.
Results
Concentrations of 0.05–0.2 mM of 1,2-DCB were able to stimulate the hydrolytic activity of SERCA in a medium-containing Ca2+-ionophore. At higher concentrations (0.25–0.75 mM), 1,2-DCB inhibited the ATP hydrolysis to ~80 %. Moreover, ATP hydrolysis and Ca2+ uptake in a medium supported by K-oxalate showed that starting at 0.05 mM,1,2-DCB was able to uncouple the ratio of hydrolysis/Ca2+ transported. The effect of this compound on the integrity of the SR membrane loaded with Ca2+ remained unaffected. Finally, the analysis of phosphorylation of SERCA by [γ-32P]ATP, starting under different conditions at 0° or 25 °C showed a reduction in the phosphoenzyme levels by 1,2-DCB, mostly at 0 °C.
Conclusions
The temperature-dependent decreased levels of phosphoenzyme by 1,2-DCB could be due to the acceleration of the dephosphorylation mechanism – E2P · Ca2 state to E2 and Pi, which explains the uncoupling of the ATP hydrolysis from the Ca2+ transport.
doi:10.1186/s12858-016-0061-1
PMCID: PMC4788898  PMID: 26968444
Ca2+-ATPase; Sarcoplasmic reticulum; Dichlorobenzene; Phosphoenzyme
5.  Substrate specificity and function of acetylpolyamine amidohydrolases from Pseudomonas aeruginosa 
BMC Biochemistry  2016;17:4.
Background
Pseudomonas aeruginosa, a Gram-negative, aerobic coccobacillus bacterium is an opportunistic human pathogen and worldwide the fourth most common cause of hospital-acquired infections which are often high mortality such as ventilator-associated pneumoniae. The polyamine metabolism of P. aeruginosa and particularly the deacetylation of acetylpolyamines has been little studied up to now. Results with other bacterial pathogens e.g., Y. pestis suggest that polyamines may be involved in the formation of biofilms or confer resistance against certain antibiotics.
Results
To elucidate the role of acetylpolyamines and their enzymatic deacetylation in more detail, all three putative acetylpolyamine amidohydrolases (APAHs) from P. aeruginosa have been expressed in enzymatic active form. The APAHs PA0321 and PA1409 are shown to be true polyamine deacetylases, whereas PA3774 is not able to deacetylate acetylated polyamines. Every APAH can hydrolyze trifluoroacetylated lysine-derivatives, but only PA1409 and much more efficiently PA3774 can also process the plain acetylated lysine substrate. P. aeruginosa is able to utilize acetylcadaverine and acetylputrescine as a carbon source under glucose starvation. If either the PA0321 or the PA1409 but not the PA3774 gene is disrupted, the growth of P. aeruginosa is reduced and delayed. In addition, we were able to show that the APAH inhibitors SAHA and SATFMK induce biofilm formation in both PA14 and PAO1 wildtype strains.
Conclusions
P. aeruginosa has two functional APAHs, PA0321 and PA1409 which enable the utilization of acetylpolyamines for the metabolism of P. aeruginosa. In contrast, the physiological role of the predicted APAH, PA3774, remains to be elucidated. Its ability to deacetylate synthetic acetylated lysine substrates points to a protein deacetylation functionality with yet unknown substrates.
Electronic supplementary material
The online version of this article (doi:10.1186/s12858-016-0063-z) contains supplementary material, which is available to authorized users.
doi:10.1186/s12858-016-0063-z
PMCID: PMC4784309  PMID: 26956223
Acetylpolyamine amidohydrolases; Pseudomonas aeruginosa; Substrate specificity; Acetylpolyamines; Polyamine metabolism
7.  Characterization of a cold-active and salt tolerant esterase identified by functional screening of Arctic metagenomic libraries 
BMC Biochemistry  2016;17:1.
Background
The use of metagenomics in enzyme discovery constitutes a powerful approach to access to genomes of unculturable community of microorganisms and isolate novel valuable biocatalysts for use in a wide range of biotechnological and pharmaceutical fields.
Results
Here we present a novel esterase gene (lip3) identified by functional screening of three fosmid metagenomic libraries, constructed from three marine sediment samples. The sequenced positive fosmid revealed an enzyme of 281 amino acids with similarity to class 3 lipases. The 3D modeling of Lip3 was generated by homology modeling on the basis of four lipases templates [PDB ID: 3O0D, 3NGM, 3G7N, 2QUB] to unravel structural features of this novel enzyme. The catalytic triad of Lip3 was predicted to be Asp207, His267 and the catalytic nucleophile Ser150 in a conserved pentapeptide (GXSXG). The 3D model highlighted the presence of a one-helix lid able to regulate the access of the substrate to the active site when the enzyme binds a hydrophobic interface. Moreover an analysis of the external surface of Lip3 model showed that the majority of the surface regions were hydrophobic (59.6 %) compared with homologous lipases (around 35 %) used as templates. The recombinant Lip3 esterase, expressed and purified from Escherichia coli, preferentially hydrolyzed short and medium length p-nitrophenyl esters with the best substrate being p-nitrophenyl acetate. Further characterization revealed a temperature optimum of 35 °C and a pH optimum of 8.0. Lip3 exhibits a broad temperature stability range and tolerates the presence of DTT, EDTA, PMSF, β-mercaptoethanol and high concentrations of salt. The enzyme was also highly activated by NaCl.
Conclusions
The biochemical characterization and homology model reveals a novel esterase originating from the marine Arctic metagenomics libraries with features of a cold-active, relatively thermostable and highly halotolerant enzyme. Taken together, these results suggest that this esterase could be a highly valuable candidate for biotechnological applications such as organic synthesis reactions and cheese ripening processes.
Electronic supplementary material
The online version of this article (doi:10.1186/s12858-016-0057-x) contains supplementary material, which is available to authorized users.
doi:10.1186/s12858-016-0057-x
PMCID: PMC4717575  PMID: 26782782
Metagenomics libraries; Cold-active esterase; Salt; Homology modeling; Biotechnological applications
8.  A comparison of the enzymatic properties of three recombinant isoforms of thrombolytic and antibacterial protein—Destabilase-Lysozyme from medicinal leech 
BMC Biochemistry  2015;16:27.
Background
Destabilase-Lysozyme (mlDL) is a multifunctional i-type enzyme that has been found in the secretions from the salivary glands of medicinal leeches. mlDL has been shown to exhibit isopeptidase, muramidase and antibacterial activity. This enzyme attracts interest because it expresses thrombolytic activity through isopeptidolysis of the ε-(γ-Glu)-Lys bonds that cross-link polypeptide chains in stabilised fibrin. To date, three isoforms of mlDL have been identified.
The enzymatic properties of pure mlDL isoforms have not yet been described because only destabilase complexes containing other proteins could be isolated from the salivary gland secretion and because low product yield from the generation of recombinant proteins has made comprehensive testing difficult.
Results
In the present study, we optimised the procedures related to the expression, isolation and purification of active mlDL isoforms (mlDL-Ds1, mlDL-Ds2, mlDL-Ds3) using an Escherichia coli expression system, and we detected and compared their muramidase, lytic, isopeptidase and antimicrobial activities. After optimisation, the product yield was 30 mg per litre of culture. The data obtained in our study led to the suggestion that the recombinant mlDL isoforms isolated from inclusion bodies form stable oligomeric complexes. Analyses of the tested activities revealed that all isoforms exhibited almost identical patterns of pH and ionic strength effects on the activities. We determined that mlDL-Ds1, 2, 3 possessed non-enzymatic antibacterial activity independent of their muramidase activity. For the first time, we demonstrated the fibrinolytic activity of the recombinant mlDL and showed that only intact proteins possessed this activity, suggesting their enzymatic nature.
Conclusions
The recombinant Destabilase-Lysozyme isoforms obtained in our study may be considered potential thrombolytic agents that act through a mechanism different from that of common thrombolytics.
Electronic supplementary material
The online version of this article (doi:10.1186/s12858-015-0056-3) contains supplementary material, which is available to authorized users.
doi:10.1186/s12858-015-0056-3
PMCID: PMC4654880  PMID: 26589324
Destabilase-Lysozyme; Recombinant protein; Thrombolysis; Isopeptidase; Antimicrobial activity
9.  The GH5 1,4-β-mannanase from Bifidobacterium animalis subsp. lactis Bl-04 possesses a low-affinity mannan-binding module and highlights the diversity of mannanolytic enzymes 
BMC Biochemistry  2015;16:26.
Background
β-Mannans are abundant and diverse plant structural and storage polysaccharides. Certain human gut microbiota members including health-promoting Bifidobacterium spp. catabolize dietary mannans. Little insight is available on the enzymology of mannan deconstruction in the gut ecological niche. Here, we report the biochemical properties of the first family 5 subfamily 8 glycoside hydrolase (GH5_8) mannanase from the probiotic bacterium Bifidobacterium animalis subsp. lactis Bl-04 (BlMan5_8).
Results
BlMan5_8 possesses a novel low affinity carbohydrate binding module (CBM) specific for soluble mannan and displays the highest catalytic efficiency reported to date for a GH5 mannanase owing to a very high kcat (1828 ± 87 s-1) and a low Km (1.58 ± 0.23 g · L-1) using locust bean galactomannan as substrate. The novel CBM of BlMan5_8 mediates increased binding to soluble mannan based on affinity electrophoresis. Surface plasmon resonance analysis confirmed the binding of the CBM10 to manno-oligosaccharides, albeit with slightly lower affinity than the catalytic module of the enzyme. This is the first example of a low-affinity mannan-specific CBM, which forms a subfamily of CBM10 together with close homologs present only in mannanases. Members of this new subfamily lack an aromatic residue mediating binding to insoluble cellulose in canonical CBM10 members consistent with the observed low mannan affinity.
Conclusion
BlMan5_8 is evolved for efficient deconstruction of soluble mannans, which is reflected by an exceptionally low Km and the presence of an atypical low affinity CBM, which increases binding to specifically to soluble mannan while causing minimal decrease in catalytic efficiency as opposed to enzymes with canonical mannan binding modules. These features highlight fine tuning of catalytic and binding properties to support specialization towards a preferred substrate, which is likely to confer an advantage in the adaptation to competitive ecological niches.
Electronic supplementary material
The online version of this article (doi:10.1186/s12858-015-0055-4) contains supplementary material, which is available to authorized users.
doi:10.1186/s12858-015-0055-4
PMCID: PMC4642672  PMID: 26558435
Bifidobacterium; Carbohydrate-binding module; Gut microbiota; Mannan; Probiotic bacteria; Surface plasmon resonance
10.  The laforin/malin E3-ubiquitin ligase complex ubiquitinates pyruvate kinase M1/M2 
BMC Biochemistry  2015;16:24.
Background
Lafora disease (LD, OMIM 254780) is a fatal neurodegenerative disorder produced mainly by mutations in two genes: EPM2A, encoding the dual specificity phosphatase laforin, and EPM2B, encoding the E3-ubiquitin ligase malin. Although it is known that laforin and malin may form a functional complex, the underlying molecular mechanisms of this pathology are still far from being understood.
Methods
In order to gain information about the substrates of the laforin/malin complex, we have carried out a yeast substrate-trapping screening, originally designed to identify substrates of protein tyrosine phosphatases.
Results
Our results identify the two muscular isoforms of pyruvate kinase (PKM1 and PKM2) as novel interaction partners of laforin.
Conclusions
We present evidence indicating that the laforin/malin complex is able to interact with and ubiquitinate both PKM1 and PKM2. This post-translational modification, although it does not affect the catalytic activity of PKM1, it impairs the nuclear localization of PKM2.
doi:10.1186/s12858-015-0053-6
PMCID: PMC4619252  PMID: 26493215
Laforin; Malin; Ubiquitination; Pyruvate kinase; Nuclear localization
11.  The eukaryotic translation initiation factor 3f (eIF3f) interacts physically with the alpha 1B-adrenergic receptor and stimulates adrenoceptor activity 
BMC Biochemistry  2015;16:25.
Background
eIF3f is a multifunctional protein capable of interacting with proteins involved in different cellular processes, such as protein synthesis, DNA repair, and viral mRNA edition. In human cells, eIF3f is related to cell cycle and proliferation, and its deregulation compromises cell viability.
Results
We here report that, in native conditions, eIF3f physically interacts with the alpha 1B-adrenergic receptor, a plasma membrane protein considered as a proto-oncogene, and involved in vasoconstriction and cell proliferation. The complex formed by eIF3f and alpha 1B-ADR was found in human and mouse cell lines. Upon catecholamine stimulation, eIF3f promotes adrenoceptor activity in vitro, independently of the eIF3f proline- and alanine-rich N-terminal region.
Conclusions
The eIF3f/alpha adrenergic receptor interaction opens new insights regarding adrenoceptor-related transduction pathways and proliferation control in human cells. The eIf3f/alpha 1B-ADR complex is found in mammals and is not tissue specific.
doi:10.1186/s12858-015-0054-5
PMCID: PMC4619320  PMID: 26497985
eIF3f; Alpha 1B-ADR; Gαq/11; Adrenoceptors; Protein-protein interaction
12.  Evaluating the role of a trypsin inhibitor from soap nut (Sapindus trifoliatus L. Var. Emarginatus) seeds against larval gut proteases, its purification and characterization 
BMC Biochemistry  2015;16:23.
Background
The defensive capacities of plant protease Inhibitors (PI) rely on inhibition of proteases in insect guts or those secreted by microorganisms; and also prevent uncontrolled proteolysis and offer protection against proteolytic enzymes of pathogens.
Methods
An array of chromatographic techniques were employed for purification, homogeneity was assessed by electrophoresis. Specificity, Ki value, nature of inhibition, complex formation was carried out by standard protocols. Action of SNTI on insect gut proteases was computationally evaluated by modeling the proteins by threading and docking studies by piper using Schrodinger tools.
Results
We have isolated and purified Soap Nut Trypsin Inhibitor (SNTI) by acetone fractionation, ammonium sulphate precipitation, ion exchange and gel permeation chromatography. The purified inhibitor was homogeneous by both gel filtration and polyacrylamide gel electrophoresis (PAGE). SNTI exhibited a molecular weight of 29 kDa on SDS-PAGE, gel filtration and was negative to Periodic Acid Schiff’s stain. SNTI inhibited trypsin and pronase of serine class. SNTI demonstrated non-competitive inhibition with a Ki value of 0.75 ± 0.05×10-10 M. The monoheaded inhibitor formed a stable complex in 1:1 molar ratio. Action of SNTI was computationally evaluated on larval gut proteases from Helicoverpa armigera and Spodoptera frugiperda. SNTI and larval gut proteases were modeled and docked using Schrodinger software. Docking studies revealed strong hydrogen bond interactions between Lys10 and Pro71, Lys299 and Met80 and Van Der Waals interactions between Leu11 and Cys76amino acid residues of SNTI and protease from H. Armigera. Strong hydrogen bonds were observed between SNTI and protease of S. frugiperda at positions Thr79 and Arg80, Asp90 and Gly73, Asp2 and Gly160 respectively.
Conclusion
We conclude that SNTI potentially inhibits larval gut proteases of insects and the kinetics exhibited by the protease inhibitor further substantiates its efficacy against serine proteases.
doi:10.1186/s12858-015-0052-7
PMCID: PMC4618930  PMID: 26489418
Ki; Monoheaded inhibitor; PAGE; SNTI; Specificity; Gut proteases; Protein-protein docking
13.  Respiration and substrate transport rates as well as reactive oxygen species production distinguish mitochondria from brain and liver 
BMC Biochemistry  2015;16:22.
Background
Aberrant mitochondrial function, including excessive reactive oxygen species (ROS) production, has been implicated in the pathogenesis of human diseases. The use of mitochondrial inhibitors to ascertain the sites in the electron transport chain (ETC) resulting in altered ROS production can be an important tool. However, the response of mouse mitochondria to ETC inhibitors has not been thoroughly assessed. Here we set out to characterize the differences in phenotypic response to ETC inhibitors between the more energetically demanding brain mitochondria and less energetically demanding liver mitochondria in commonly utilized C57BL/6J mice.
Results
We show that in contrast to brain mitochondria, inhibiting distally within complex I or within complex III does not increase liver mitochondrial ROS production supported by complex I substrates, and liver mitochondrial ROS production supported by complex II substrates occurred primarily independent of membrane potential. Complex I, II, and III enzymatic activities and membrane potential were equivalent between liver and brain and responded to ETC. inhibitors similarly. Brain mitochondria exhibited an approximately two-fold increase in complex I and II supported respiration compared with liver mitochondria while exhibiting similar responses to inhibitors. Elevated NADH transport and heightened complex II–III coupled activity accounted for increased complex I and II supported respiration, respectively in brain mitochondria.
Conclusions
We conclude that important mechanistic differences exist between mouse liver and brain mitochondria and that mouse mitochondria exhibit phenotypic differences compared with mitochondria from other species.
Electronic supplementary material
The online version of this article (doi:10.1186/s12858-015-0051-8) contains supplementary material, which is available to authorized users.
doi:10.1186/s12858-015-0051-8
PMCID: PMC4564979  PMID: 26358560
14.  Complex kinetics and residual structure in the thermal unfolding of yeast triosephosphate isomerase 
BMC Biochemistry  2015;16:20.
Background
Saccharomyces cerevisiae triosephosphate isomerase (yTIM) is a dimeric protein that shows noncoincident unfolding and refolding transitions (hysteresis) in temperature scans, a phenomenon indicative of the slow forward and backward reactions of the native-unfolded process. Thermal unfolding scans suggest that no stable intermediates appear in the unfolding of yTIM. However, reported evidence points to the presence of residual structure in the denatured monomer at high temperature.
Results
Thermally denatured yTIM showed a clear trend towards the formation of aggregation-prone, β-strand-like residual structure when pH decreased from 8.0 to 6.0, even though thermal unfolding profiles retained a simple monophasic appearance regardless of pH. However, kinetic studies performed over a relatively wide temperature range revealed a complex unfolding mechanism comprising up to three observable phases, with largely different time constants, each accompanied by changes in secondary structure. Besides, a simple sequential mechanism is unlikely to explain the observed variation of amplitudes and rate constants with temperature. This kinetic complexity is, however, not linked to the appearance of residual structure. Furthermore, the rate constant for the main unfolding phase shows small, rather unvarying values in the pH region where denatured yTIM gradually acquires a β-strand-like conformation. It appears, therefore, that the residual structure has no influence on the kinetic stability of the native protein. However, the presence of residual structure is clearly associated with increased irreversibility.
Conclusions
The slow temperature-induced unfolding of yeast TIM shows three kinetic phases. Rather than a simple sequential pathway, a complex mechanism involving off-pathway intermediates or even parallel pathways may be operating. β-strand-type residual structure, which appears below pH 8.0, is likely to be associated with increased irreversible aggregation of the unfolded protein. However, this denatured form apparently accelerates the refolding process.
Electronic supplementary material
The online version of this article (doi:10.1186/s12858-015-0049-2) contains supplementary material, which is available to authorized users.
doi:10.1186/s12858-015-0049-2
PMCID: PMC4558838  PMID: 26334568
15.  Purification and characterization of a cytochrome c with novel caspase-3 activation activity from the pathogenic fungus Rhizopus arrhizus 
BMC Biochemistry  2015;16:21.
Background
Members of Rhizopus species are the most common cause of mucormycosis, a rare but often fatal fungal infection. Host induced pathogen apoptosis and pathogen induced host cell apoptosis are often involved in fungal infections. In many organisms, the release of mitochondrial cytochrome c can trigger apoptosis by activating caspase proteases, but the role of fungal cytochrome c in apoptosis remains unknown.
Results
DNA sequence encoding Rhizopus arrhizus cytochrome c was cloned and expressed in E. coli. Both native and recombinant cytochrome c were purified using ion exchange followed by gel filtration chromatography. The identities of purified proteins were confirmed by MALDI-MS and UV-Visible spectroscopy. For the first time, we demonstrated that Rhizopus arrhizus cytochrome c could activate human capspase-3 in HeLa cell extracts. We also found that Rhizopus arrhizus cytochrome c has redox potential, peroxidase activity, and spectral properties similar to human and horse cytochrome c proteins.
Conclusions
Rhizopus arrhizus cytochrome c can activate human caspase-3 in HeLa cell extracts and it possesses similar physical and spectral properties as human and horse cytochrome c. This protein was found to have a previously unknown potential to activate human caspase-3, an important step in the apoptosis cascade.
Electronic supplementary material
The online version of this article (doi:10.1186/s12858-015-0050-9) contains supplementary material, which is available to authorized users.
doi:10.1186/s12858-015-0050-9
PMCID: PMC4559206  PMID: 26334686
16.  Identification of inhibitors that target dual-specificity phosphatase 5 provide new insights into the binding requirements for the two phosphate pockets 
BMC Biochemistry  2015;16:19.
Background
Dual-specificity phosphatase-5 (DUSP5) plays a central role in vascular development and disease. We present a p-nitrophenol phosphate (pNPP) based enzymatic assay to screen for inhibitors of the phosphatase domain of DUSP5.
Methods
pNPP is a mimic of the phosphorylated tyrosine on the ERK2 substrate (pERK2) and binds the DUSP5 phosphatase domain with a Km of 7.6 ± 0.4 mM. Docking followed by inhibitor verification using the pNPP assay identified a series of polysulfonated aromatic inhibitors that occupy the DUSP5 active site in the region that is likely occupied by the dual-phosphorylated ERK2 substrate tripeptide (pThr-Glu-pTyr). Secondary assays were performed with full length DUSP5 with ERK2 as substrate.
Results
The most potent inhibitor has a naphthalene trisulfonate (NTS) core. A search for similar compounds in a drug database identified suramin, a dimerized form of NTS. While suramin appears to be a potent and competitive inhibitor (25 ± 5 μM), binding to the DUSP5 phosphatase domain more tightly than the monomeric ligands of which it is comprised, it also aggregates. Further ligand-based screening, based on a pharmacophore derived from the 7 Å separation of sulfonates on inhibitors and on sulfates present in the DUSP5 crystal structure, identified a disulfonated and phenolic naphthalene inhibitor (CSD3_2320) with IC50 of 33 μM that is similar to NTS and does not aggregate.
Conclusions
The new DUSP5 inhibitors we identify in this study typically have sulfonates 7 Å apart, likely positioning them where the two phosphates of the substrate peptide (pThr-Glu-pTyr) bind, with one inhibitor also positioning a phenolic hydroxyl where the water nucleophile may reside. Polysulfonated aromatic compounds do not commonly appear in drugs and have a tendency to aggregate. One FDA-approved polysulfonated drug, suramin, inhibits DUSP5 and also aggregates. Docking and modeling studies presented herein identify polysulfonated aromatic inhibitors that do not aggregate, and provide insights to guide future design of mimics of the dual-phosphate loops of the ERK substrates for DUSPs.
Electronic supplementary material
The online version of this article (doi:10.1186/s12858-015-0048-3) contains supplementary material, which is available to authorized users.
doi:10.1186/s12858-015-0048-3
PMCID: PMC4545774  PMID: 26286528
DUSP5; Phosphatase; Drug discovery; Docking; Suramin; Vascular anomalies
17.  Design of symmetric TIM barrel proteins from first principles 
BMC Biochemistry  2015;16:18.
Background
Computational protein design is a rapidly maturing field within structural biology, with the goal of designing proteins with custom structures and functions. Such proteins could find widespread medical and industrial applications. Here, we have adapted algorithms from the Rosetta software suite to design much larger proteins, based on ideal geometric and topological criteria. Furthermore, we have developed techniques to incorporate symmetry into designed structures. For our first design attempt, we targeted the (α/β)8 TIM barrel scaffold. We gained novel insights into TIM barrel folding mechanisms from studying natural TIM barrel structures, and from analyzing previous TIM barrel design attempts.
Methods
Computational protein design and analysis was performed using the Rosetta software suite and custom scripts. Genes encoding all designed proteins were synthesized and cloned on the pET20-b vector. Standard circular dichroism and gel chromatographic experiments were performed to determine protein biophysical characteristics. 1D NMR and 2D HSQC experiments were performed to determine protein structural characteristics.
Results
Extensive protein design simulations coupled with ab initio modeling yielded several all-atom models of ideal, 4-fold symmetric TIM barrels. Four such models were experimentally characterized. The best designed structure (Symmetrin-1) contained a polar, histidine-rich pore, forming an extensive hydrogen bonding network. Symmetrin-1 was easily expressed and readily soluble. It showed circular dichroism spectra characteristic of well-folded alpha/beta proteins. Temperature melting experiments revealed cooperative and reversible unfolding, with a Tm of 44 °C and a Gibbs free energy of unfolding (ΔG°) of 8.0 kJ/mol. Urea denaturing experiments confirmed these observations, revealing a Cm of 1.6 M and a ΔG° of 8.3 kJ/mol. Symmetrin-1 adopted a monomeric conformation, with an apparent molecular weight of 32.12 kDa, and displayed well resolved 1D-NMR spectra. However, the HSQC spectrum revealed somewhat molten characteristics.
Conclusions
Despite the detection of molten characteristics, the creation of a soluble, cooperatively folding protein represents an advancement over previous attempts at TIM barrel design. Strategies to further improve Symmetrin-1 are elaborated. Our techniques may be used to create other large, internally symmetric proteins.
Electronic supplementary material
The online version of this article (doi:10.1186/s12858-015-0047-4) contains supplementary material, which is available to authorized users.
doi:10.1186/s12858-015-0047-4
PMCID: PMC4531894  PMID: 26264284
18.  Structural plasticity of green fluorescent protein to amino acid deletions and fluorescence rescue by folding-enhancing mutations 
BMC Biochemistry  2015;16:17.
Background
Green fluorescent protein (GFP) and its derivative fluorescent proteins (FPs) are among the most commonly used reporter systems for studying gene expression and protein interaction in biomedical research. Most commercially available FPs have been optimized for their oligomerization state to prevent potential structural constraints that may interfere with the native function of fused proteins. Other approach to reducing structural constraints may include minimizing the structure of GFPs. Previous studies in an enhanced GFP variant (EGFP) identified a series of deletions that can retain GFP fluorescence. In this study, we interrogated the structural plasticity of a UV-optimized GFP variant (GFPUV) to amino acid deletions, characterized the effects of deletions and explored the feasibility of rescuing the fluorescence of deletion mutants using folding-enhancing mutations.
Methods
Transposon mutagenesis was used to screen amino acid deletions in GFP that led to fluorescent and nonfluorescent phenotypes. The fluorescent GFP mutants were characterized for their whole-cell fluorescence and fraction soluble. Fluorescent GFP mutants with internal deletions were purified and characterized for their spectral and folding properties. Folding-ehancing mutations were introduced to deletion mutants to rescue their compromised fluorescence.
Results
We identified twelve amino acid deletions that can retain the fluorescence of GFPUV. Seven of these deletions are either at the N- or C- terminus, while the other five are located at internal helices or strands. Further analysis suggested that the five internal deletions diminished the efficiency of protein folding and chromophore maturation. Protein expression under hypothermic condition or incorporation of folding-enhancing mutations could rescue the compromised fluorescence of deletion mutants. In addition, we generated dual deletion mutants that can retain GFP fluorescence.
Conclusion
Our results suggested that a “size-minimized” GFP may be developed by iterative incorporation of amino acid deletions, followed by fluorescence rescue with folding-enhancing mutations.
Electronic supplementary material
The online version of this article (doi:10.1186/s12858-015-0046-5) contains supplementary material, which is available to authorized users.
doi:10.1186/s12858-015-0046-5
PMCID: PMC4513630  PMID: 26206151
Green fluorescent protein (GFP); Transposon mutagenesis; Amino acid deletions; Protein folding; Chromophore maturation
19.  Kv1.3 contains an alternative C-terminal ER exit motif and is recruited into COPII vesicles by Sec24a 
BMC Biochemistry  2015;16:16.
Background
Potassium channels play a fundamental role in resetting the resting membrane potential of excitable cells. Determining the intracellular trafficking and localization mechanisms of potassium channels provides a platform to fully characterize their maturation and functionality. Previous investigations have discovered residues or motifs that exist in their primary structure, which directly promote anterograde trafficking of nascent potassium channels. Recently, a non-conical di-acidic motif (E483/484) has been discovered in the C-terminus of the mammalian homologue of the Shaker voltage-gated potassium channel subfamily member 3 (Kv1.3), and was shown to disrupt the anterograde trafficking of Kv1.3.
Results
We have further investigated the intracellular trafficking requirements of Kv1.3 both in vivo and in vitro. First, three alternative C-terminal acidic residues, E443, E445, E447 were probed for their involvement within the early secretory pathway of Kv1.3. Single point (E443A, E445A, and E447A) and double point (E443A-E445A, E445A-E447A) mutations exhibited no significant changes in their endoplasmic reticulum (ER) retention. The triple point mutant E443A-E445A-E447A displayed a modest ER retention while deletion of the C-terminus showed dramatic ER retention. Second, we demonstrate in vivo the requirement for the Sec24a isoform to confer anterograde trafficking using a siRNA knockdown assay. Third, we show in vitro the association of recombinantly expressed Kv1.3 and Sec24a proteins.
Conclusion
These results expand upon previous studies aimed at deciphering the Kv1.3 secretory trafficking mechanisms and further show in vitro evidence of the association between Kv1.3 and the COPII cargo adaptor subunit isoform Sec24a.
Electronic supplementary material
The online version of this article (doi:10.1186/s12858-015-0045-6) contains supplementary material, which is available to authorized users.
doi:10.1186/s12858-015-0045-6
PMCID: PMC4497498  PMID: 26156069
Early secretory pathway; Anterograde intracellular trafficking; COPII; Endoplasmic reticulum; Voltage-gated potassium channel; Di-acidic motif; siRNA
20.  Enzyme assays for synthesis and degradation of 2-5As and other 2′-5′ oligonucleotides 
BMC Biochemistry  2015;16:15.
Background
The 5′-triphosphorylated, 2′-5′-linked oligoadenylate polyribonucleotides (2-5As) are central to the interferon-induced antiviral 2-5A system. The 2-5As bind and activate the RNase L, an endoRNase degrading viral and cellular RNA leading to inhibition of viral replication. The 2-5A system is tightly controlled by synthesis and degradation of 2-5As. Whereas synthesis is mediated by the 2′-5′ oligoadenylate synthetase family of enzymes, degradation seems to be orchestrated by multiple enzyme nucleases including phosphodiesterase 12, the ectonucleotide pyrophosphatase/phosphodiesterase 1 and the A-kinase anchoring protein 7.
Results
Here we present assay tools for identification and characterization of the enzymes regulating cellular 2-5A levels. A procedure is described for the production of 2′-5′ oligoadenylates, which are then used as substrates for development and demonstration of enzyme assays measuring synthetase and nuclease activities, respectively. The synthetase assays produce only a single reaction product allowing for very precise kinetic assessment of the enzymes. We present an assay using dATP and the A(pA)3 tetramer core as substrates, which requires prior isolation of A(pA)3. A synthetase assay using either of the dNTPs individually together with NAD+ as substrates is also presented. The nuclease reactions make use of the isolated 2′-5′ oligoadenylates in producing a mixture of shorter reaction products, which are resolved by ion-exchange chromatography to determine the enzyme activities. A purified human 2′-5′ oligoadenylate synthetase and a purified human phosphodiesterase 12 along with crude extracts expressing those proteins, are used to demonstrate the assays.
Conclusions
This paper comprises an assay toolbox for identification and characterization of the synthetases and nucleases regulating cellular 2-5A levels. Assays are presented for both enzyme families. The assays can also be used to address a broader cellular role of the OAS enzymes, based on the multiple substrate specificity intrinsic to these proteins.
Electronic supplementary material
The online version of this article (doi:10.1186/s12858-015-0043-8) contains supplementary material, which is available to authorized users.
doi:10.1186/s12858-015-0043-8
PMCID: PMC4481073  PMID: 26113370
2′-5′ oligoadenylate synthetase (OAS); 2′-5′ oligoadenylate nuclease; 5′-triphosphorylated, 2′-5′-linked oligoadenylate polyribonucleotides (2-5As); 5′-dephosphorylated forms of 2-5As (2-5A core molecules); Enzyme assays
21.  Human POLD1 modulates cell cycle progression and DNA damage repair 
BMC Biochemistry  2015;16:14.
Background
The activity of eukaryotic DNA polymerase delta (Pol δ) plays an essential role in genome stability through its effects on DNA replication and repair. The p125 catalytic subunit of Pol δ is encoded by POLD1 gene in human cells. To clarify biological functions of POLD1, we investigated the effects of POLD1 overexpression or downregulation on cell proliferation, cell cycle progression, DNA synthesis and oxidative DNA damage induced by H2O2.
Methods
HEK293 cells were transfected with POLD1 expression plasmid or shRNA, cell proliferation, cell cycle progression, and DNA synthesis in HEK293 cells were analyzed.
Results
HEK293 cells were transfected with POLD1 expression plasmid or shRNA. POLD1 downregulation by shRNA suppressed cell proliferation, cell cycle progression, and DNA synthesis in HEK293 cells. However, POLD1 overexpression had no significant effects on these processes. Finally, comet assay showed that POLD1 downregulation led to increased DNA damage.
Conclusions
Our results suggest that human POLD1 plays important role in the regulation of cell cycle progression and DNA damage repair.
doi:10.1186/s12858-015-0044-7
PMCID: PMC4471906  PMID: 26087769
DNA polymerase delta; POLD1; Cell proliferation; Cell cycle; DNA damage
22.  The androgen receptor plays a suppressive role in epithelial- mesenchymal transition of human prostate cancer stem progenitor cells 
BMC Biochemistry  2015;16:13.
Background
To investigate the roles of androgen receptor (AR) in epithelial- mesenchymal transition (EMT) in human prostate cancer stem progenitor (S/P) cells isolated from LNCaP cell line.
Methods
The S/P cells were obtained from LNCaP cell line through florescence-activated cell sorting (FACS). AR was overexpressed in S/P cells through lentivirus. Western blot assay was used to detect the EMT markers expression, such as E Cadherin, N Cadherin, Vimentin and Snail. MTT assay, soft agar colony formation assay, sphere formation assay and migration assay were used to investigate AR’s roles in EMT of S/P cells. Cell signaling pathways associated with proliferation and apoptosis of S/P cells were detected simultaneously. And S/P cells were treated with in vitro combinatory use of LY 294002 (inhibitor of AKT signaling molecules) with γ-TT and/or 5-AZA.
Results
Our data showed that S/P cells from LNCaP had high EMT markers expression, more tumorigenesis and strong migration ability. And in S/P cells overexpressed with AR, the expression of EMT markers decreased. In addition, these cells had less proliferation ability, tumorigenesis ability, self-renewal and migration ability. At the same time, targeting S/P cells with AKT signaling pathway inhibitor LY29004 andγ-TT and/or 5-AZA could inhibit S/P cell’s proliferation and tumorigenesis.
Conclusions
Our data suggest that AR played a negative role in EMT of PCa S/P cells, by regulating AKT cell signaling pathway, which could be a new strategy to treat castration resistant prostate cancer (CRPC).
doi:10.1186/s12858-015-0042-9
PMCID: PMC4430921  PMID: 25943311
Prostatic neoplasms; Stem progenitor cell; Epithelial-mesenchymal transition; Androgen receptor
23.  Alternative divalent cations (Zn2+, Co2+, and Mn2+) are not mutagenic at conditions optimal for HIV-1 reverse transcriptase activity 
BMC Biochemistry  2015;16:12.
Background
Fidelity of DNA polymerases can be influenced by cation co-factors. Physiologically, Mg2+ is used as a co-factor by HIV reverse transcriptase (RT) to perform catalysis; however, alternative cations including Mn2+, Co2+, and Zn2+ can also support catalysis. Although Zn2+ supports DNA synthesis, it inhibits HIV RT by significantly modifying RT catalysis. Zn2+ is currently being investigated as a component of novel treatment options against HIV and we wanted to investigate the fidelity of RT with Zn2+.
Methods
We used PCR-based and plasmid-based alpha complementation assays as well as steady-state misinsertion and misincorporation assays to examine the fidelity of RT with Mn2+, Co2+, and Zn2+.
Results
The fidelity of DNA synthesis by HIV-1 RT was approximately 2.5 fold greater in Zn2+ when compared to Mg2+ at cation conditions optimized for nucleotide catalysis. Consistent with this, RT extended primers with mismatched 3′ nucleotides poorly and inserted incorrect nucleotides less efficiently using Zn2+ than Mg2+. In agreement with previous literature, we observed that Mn2+ and Co2+ dramatically decreased the fidelity of RT at highly elevated concentrations (6 mM). However, surprisingly, the fidelity of HIV RT with Mn2+ and Co2+ remained similar to Mg2+ at lower concentrations that are optimal for catalysis.
Conclusion
This study shows that Zn2+, at optimal extension conditions, increases the fidelity of HIV-1 RT and challenges the notion that alternative cations capable of supporting polymerase catalysis are inherently mutagenic.
doi:10.1186/s12858-015-0041-x
PMCID: PMC4472245  PMID: 25934642
24.  Sigma-1 receptor directly interacts with Rac1-GTPase in the brain mitochondria 
BMC Biochemistry  2015;16:11.
Background
Small Rho-GTPases are critical mediators of neuronal plasticity and are involved in the pathogenesis of several psychiatric and neurological disorders. Rac-GTPase forms a multiprotein complex with upstream and downstream regulators that are essential for the spatiotemporal transmission of Rac signaling. The sigma-1 receptor (Sig1R) is a ligand-regulated membrane protein chaperone, and multiprotein complex assembly is essential to sigma-receptor function.
Results
Using immunoprecipitation techniques, we have shown that in mitochondrial membranes Sig1R could directly interact with Rac1. Besides Rac1, the Sig1R forms complexes with inositol 1,4,5-trisphosphate receptor and Bcl2, suggesting that mitochondrial associated membranes (MAM) are involved in this macromolecular complex formation. Assembly of this complex is ligand-specific and depends on the presence of sigma agonist/antagonist, as well as on the presence of GTP/GDP. Treatment of mitochondrial membranes with (+)-pentazocine leads to the (+)-pentazocine-sensitive phosphorylation of Bad and the pentazocine-sensitive NADPH-dependent production of ROS.
Conclusion
We suggest that Sig1R through Rac1 signaling induces mild oxidative stress that possibly is involved in the regulation of neuroplasticity, as well as in the prevention of apoptosis and autophagy.
doi:10.1186/s12858-015-0040-y
PMCID: PMC4430930  PMID: 25924612
Mitochondria; Mitochondria-associated membranes; Sigma-receptor; Rac1; ROS
25.  Nickel quercetinase, a “promiscuous” metalloenzyme: metal incorporation and metal ligand substitution studies 
BMC Biochemistry  2015;16:10.
Background
Quercetinases are metal-dependent dioxygenases of the cupin superfamily. While fungal quercetinases are copper proteins, recombinant Streptomyces quercetinase (QueD) was previously described to be capable of incorporating Ni2+ and some other divalent metal ions. This raises the questions of which factors determine metal selection, and which metal ion is physiologically relevant.
Results
Metal occupancies of heterologously produced QueD proteins followed the order Ni > Co > Fe > Mn. Iron, in contrast to the other metals, does not support catalytic activity. QueD isolated from the wild-type Streptomyces sp. strain FLA contained mainly nickel and zinc. In vitro synthesis of QueD in a cell-free transcription-translation system yielded catalytically active protein when Ni2+ was present, and comparison of the circular dichroism spectra of in vitro produced proteins suggested that Ni2+ ions support correct folding. Replacement of individual amino acids of the 3His/1Glu metal binding motif by alanine drastically reduced or abolished quercetinase activity and affected its structural integrity. Only substitution of the glutamate ligand (E76) by histidine resulted in Ni- and Co-QueD variants that retained the native fold and showed residual catalytic activity.
Conclusions
Heterologous formation of catalytically active, native QueD holoenzyme requires Ni2+, Co2+ or Mn2+, i.e., metal ions that prefer an octahedral coordination geometry, and an intact 3His/1Glu motif or a 4His environment of the metal. The observed metal occupancies suggest that metal incorporation into QueD is governed by the relative stability of the resulting metal complexes, rather than by metal abundance. Ni2+ most likely is the physiologically relevant cofactor of QueD of Streptomyces sp. FLA.
Electronic supplementary material
The online version of this article (doi:10.1186/s12858-015-0039-4) contains supplementary material, which is available to authorized users.
doi:10.1186/s12858-015-0039-4
PMCID: PMC4416304  PMID: 25903361
Dioxygenase; Flavonol; Metalloprotein; Nickel; Coordination geometry; Cell-free protein synthesis

Results 1-25 (510)