The editors of BMC Biochemistry would like to thank all our reviewers who have contributed their time to the journal in Volume 15 (2014).
Baculoviruses are widely used for the production of recombinant proteins, biopesticides and as gene delivery systems. One of the viral forms called polyhedra has been recently exploited as a scaffold system to incorporate or encapsulate foreign proteins or peptide fragments. However, an efficient strategy for foreign protein incorporation has not been thoroughly studied.
Based on the crystal structure of polyhedrin, we conducted an in silico analysis of the baculovirus Autographa californica nucleopolyhedrovirus (AcMNPV) polyhedrin protein to select the minimum fragments of polyhedrin that could be incorporated into polyhedra. Using confocal and transmission electron microscopy we analyzed the expression and cellular localization of the different polyhedrin fragments fused to the green fluorescent protein (EGFP) used as reporter. The amino fragment 1–110 contains two repeats formed each of two β sheets followed by a α helix (amino acids 1–58 and 58–110) that are important for the formation and stability of polyhedra. These fragments 1–58, 58–110 and 1–110 could be incorporated into polyhedra. However, only fragments 1–110 and 58–110 can self-aggregate.
These results demonstrate that 58–110 is the minimum fragment that contributes to the assembly of the recombinant polyhedra via self-aggregation. This is the minimum sequence that can be used to efficiently incorporate foreign proteins into polyhedra.
Autographa Californica; Polyhedrin; Polyhedra; Baculovirus
Par14, a member of the parvulin family of peptidyl-prolyl cis-trans isomerases that is involved in rRNA processing, microtubule formation and the glucose metabolism and has been suggested to play a role in chromatin remodeling on basis of sequence and structural identities to HMG proteins. Par14 is enriched in the nucleus and binds to double-stranded DNA in vitro.
By means of sub-nuclear biochemical fractionations, we demonstrate that cellular Par14 is associated with chromatin 3-fold higher than with the nuclear matrix in vivo. Par14 is released from the chromatin fraction after treatment with DNase I and elutes at high NaCl concentrations from the nucleic acid-binding fraction. Using qRT-PCR and western blotting we demonstrate that Par14 is up-regulated during the S and G2/M phases in synchronised human foreskin fibroblasts cells.
In the light of our results, Par14 can be described as an endogenous non-histone chromatin protein, which binds DNA in vivo. We propose that Par14 is involved in a DNA-dependent activity such as transcription.
Par14; Non-histone protein; Chromatin; DNA-binding; Transcription
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°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.
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 ~10 ng/mL. Heat-treated gluten digest did not inhibit leptin binding.
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.
Gluten; Leptin; Leptin resistance; Obesity
Histone deacetylase inhibitors (HDACi’s) are emerging as promising anticancer drugs alone or in combination with chemotherapy or radiotherapy agents. Previous research suggests that HDACi’s have a high degree of selectivity for killing cancer cells, but little is known regarding the impact of different cellular contexts on HDACi treatment. It is likely that the molecular mechanisms of HDACi’s involve processes that depend on the chromatin template, such as DNA damage and repair. We sought to establish the connection between the HDACi sodium butyrate and DNA double-strand break (DSB) damage in human breast cancer MCF-7 and non-cancerous human embryonic kidney293 (HEK293) cells.
Sodium butyrate inhibited the proliferation of both HEK293 and MCF-7 cells in a dose- and time- dependent manner, but the effects on MCF-7 cells were more obvious. This differential effect on cell growth was not explained by differences in cell cycle arrest, as sodium butyrate caused an arrest in G1/G2 phase and a decrease in S phase for both cell lines. At high doses of sodium butyrate or in combination with etoposide, MCF-7 cells formed fewer colonies than HEK293 cells. Furthermore, sodium butyrate enhanced the formation of etoposide-induced γ-H2AX foci to a greater extent in MCF-7 than in HEK293 cells. The two cells also displayed differential patterns in the nuclear expression of DNA DSB repair proteins, which could, in part, explain the cytotoxic effects of sodium butyrate.
These studies suggest that sodium butyrate treatment leads to a different degree of chromatin relaxation in HEK293 and cancerous MCF-7 cells, which results in differential sensitivity to the toxic effects of etoposide in controlling damaged DNA repair.
Double strand breaks; Histone deacetylase inhibitor; MCF-7; HEK293; Etoposide; Sodium butyrate
Mesenchymal stem cells (MSCs) can differentiate into chondroblasts, adipocytes, or osteoblasts under appropriate stimulation. Mechano-growth factor (MGF) reportedly displays a neuroprotective effect in cerebral regions that were exposed to ischemia and is expressed in stromal cells of the eutopic endometrium and in glandular cells of the ectopic endometrium.
This study sought to understand the potential involvement of phosphatidylinositol-3-kinase (PI3K)/protein kinase B (AKT) in MGF-induced growth of rabbit MSCs (rMSCs). We applied various concentrations of MGF to cultured rMSCs and observed the growth rate of the cells, the changes in the phosphorylation state of AKT and mammalian target of rapamycin (mTOR), and the expression levels of alkaline phosphatase and osteocalcin. We found that the growth and osteogenic differentiation of MGF-induced rMSCs were promoted primarily by phosphorylated AKT, and that this phosphorylation, as well mTOR phosphorylation, was mediated by the MGF receptor.
Our study suggests that MGF promotes the growth and osteogenic differentiation of rMSCs primarily through the PI3K/AKT pathway.
rMSCs; PI3K/AKT; MGF; Osteogenic differentiation
The Arabidopsis thaliana protein atTic20 is a key component of the protein import machinery at the inner envelope membrane of chloroplasts. As a component of the TIC complex, it is believed to form a preprotein-conducting channel across the inner membrane.
We report a method for producing large amounts of recombinant atTic20 using a codon-optimized strain of E. coli coupled with an autoinduction method of protein expression. This method resulted in the recombinant protein being directed to the bacterial membrane without the addition of a bacterial targeting sequence. Using biochemical and biophysical approaches, we were able to demonstrate that atTic20 homo-oligomerizes in vitro when solubilized in detergents or reconstituted into liposomes. Furthermore, we present evidence that the extramembranous N-terminus of the mature protein displays characteristics that are consistent with it being an intrinsically disordered protein domain.
Our work strengthens the hypothesis that atTic20 functions similarly to other small α-helical integral membrane proteins, such as Tim23, that are involved in protein transport across membranes.
Electronic supplementary material
The online version of this article (doi:10.1186/s12858-014-0029-y) contains supplementary material, which is available to authorized users.
Tic20; TIC complex; Protein self-assembly; Circular dichroism; Protein folding; Structure-function relationship; Protein reconstitution; Chloroplast membrane proteins
A shift between two dimer conformations has been proposed for the transcription factor STAT1 (signal transducer and activator of transcription 1) which links DNA binding of the parallel dimer to tyrosine dephosphorylation of the antiparallel dimer as two consecutive and important steps in interferon- γ (IFNγ)-mediated signalling. However, neither the kinetics nor the molecular mechanisms involved in this conformational transition have been determined so far.
Our results demonstrated that the dissociation of dimers into monomers and their subsequent re-association into newly formed tyrosine-phosphorylated dimers is a relatively slow process as compared to the fast release from high-affinity DNA-binding sites, termed GAS (gamma-activated sequence). In addition, we noted an inhibitory effect of GAS binding on the exchange rate of protomers, indicating that DNA binding substantially impedes the recombination of dimeric STAT1. Furthermore, we found that reciprocal aminoterminal interactions between two STAT1 molecules are not required for the interchange of protomers, as an oligomerization-deficient point mutant displayed similar interdimeric exchange kinetics as the wild-type molecule.
Our results demonstrate that DNA binding impairs the oscillation rate between STAT1 conformers. Furthermore, these data suggest that the rapid release from high-affinity GAS sites is not a rate-limiting step in IFNγ-mediated signal transduction. Further investigations are needed to decipher the physiological significance of the observed dissociation/re-association process of STAT1 dimers.
STAT; Dimerization; Interferon signalling; Transcriptional activation
The mitogen-activated protein kinases (MAPKs) pathway is critical for cellular signaling, and proteins such as phosphatases that regulate this pathway are important for normal tissue development. Based on our previous work on dual specificity phosphatase-5 (DUSP5), and its role in embryonic vascular development and disease, we hypothesized that mutations in DUSP5 will affect its function.
In this study, we tested this hypothesis by generating full-length glutathione-S-transferase-tagged DUSP5 and serine 147 proline mutant (S147P) proteins from bacteria. Light scattering analysis, circular dichroism, enzymatic assays and molecular modeling approaches have been performed to extensively characterize the protein form and function. We demonstrate that both proteins are active and, interestingly, the S147P protein is hypoactive as compared to the DUSP5 WT protein in two distinct biochemical substrate assays. Furthermore, due to the novel positioning of the S147P mutation, we utilize computational modeling to reconstruct full-length DUSP5 and S147P to predict a possible mechanism for the reduced activity of S147P.
Taken together, this is the first evidence of the generation and characterization of an active, full-length, mutant DUSP5 protein which will facilitate future structure-function and drug development-based studies.
Electronic supplementary material
The online version of this article (doi:10.1186/s12858-014-0027-0) contains supplementary material, which is available to authorized users.
DUSP5; Mutation; Vascular anomalies; Protein purification; Molecular modeling
Cholix toxin is an ADP-ribosyltransferase found in non-O1/non-O139 strains of Vibrio cholera. The catalytic fragment of cholix toxin was characterized as a diphthamide dependent ADP-ribosyltransferase.
Our studies on the enzymatic activity of cholix toxin catalytic fragment show that the transfer of ADP-ribose to toxin takes place by a predominantly intramolecular mechanism and results in the preferential alkylation of arginine residues proximal to the NAD+ binding pocket. Multiple arginine residues, located near the catalytic site and at distal sites, can be the ADP-ribose acceptor in the auto-reaction. Kinetic studies of a model enzyme, M8, showed that a diffusible intermediate preferentially reacted with arginine residues in proximity to the NAD+ binding pocket. ADP-ribosylarginine activity of cholix toxin catalytic fragment could also modify exogenous substrates. Auto-ADP-ribosylation of cholix toxin appears to have negatively regulatory effect on ADP-ribosylation of exogenous substrate. However, at the presence of both endogenous and exogenous substrates, ADP-ribosylation of exogenous substrates occurred more efficiently than that of endogenous substrates.
We discovered an ADP-ribosylargininyl activity of cholix toxin catalytic fragment from our studies in auto-ADP-ribosylation, which is mediated through diffusible intermediates. The lifetime of the hypothetical intermediate exceeds recorded and predicted lifetimes for the cognate oxocarbenium ion. Therefore, a diffusible strained form of NAD+ intermediate was proposed to react with arginine residues in a proximity dependent manner.
Electronic supplementary material
The online version of this article (doi:10.1186/s12858-014-0026-1) contains supplementary material, which is available to authorized users.
Cholix toxin; ADP-ribosylation; Auto-ADP-ribosylation; Arginine
Staphyloccocal nuclease domain-containing protein 1 (SND1) is involved in the regulation of gene expression and RNA protection. While numerous studies have established that SND1 protein expression is modulated by cellular stresses associated with tumor growth, hypoxia, inflammation, heat-shock and oxidative conditions, little is known about the factors responsible for SND1 expression. Here, we have approached this question by analyzing the transcriptional response of human SND1 gene to pharmacological endoplasmic reticulum (ER) stress in liver cancer cells.
We provide first evidence that SND1 promoter activity is increased in human liver cancer cells upon exposure to thapsigargin or tunicamycin or by ectopic expression of ATF6, a crucial transcription factor in the unfolded protein response triggered by ER stress. Deletion analysis of the 5’-flanking region of SND1 promoter identified maximal activation in fragment (-934, +221), which contains most of the predicted ER stress response elements in proximal promoter. Quantitative real-time PCR revealed a near 3 fold increase in SND1 mRNA expression by either of the stress-inducers; whereas SND1 protein was maximally upregulated (3.4-fold) in cells exposed to tunicamycin, a protein glycosylation inhibitor.
Promoter activity of the cell growth- and RNA-protection associated SND1 gene is up-regulated by ER stress in human hepatoma cells.
SND1 transcriptional activity; Tudor and nuclease domain containing protein 1; Tudor-SN; ER stress response; ATF6
Hydroxyl radical that has the highest reactivity among reactive oxygen species (ROS) is generated through l-tyrosine-tyrosinase reaction. Thus, the melanogenesis might induce oxidative stress in the skin. Arbutin (p-hydroxyphenyl-β-d-glucopyranoside), a well-known tyrosinase inhibitor has been widely used for the purpose of skin whitening. The aim of the present study was to examine if arbutin could suppress the hydroxyl radical generation via tyrosinase reaction with its substrates, l-tyrosine and l-DOPA.
The hydroxyl radical, which was determined by an electron spin resonance-spin trapping technique, was generated by the addition of not only l-tyrosine but l-DOPA to tyrosinase in a concentration dependent manner. Arbutin could inhibit the hydroxyl radical generation in the both reactions.
It is presumed that arbutin could alleviate oxidative stress derived from the melanogenic pathway in the skin in addition to its function as a whitening agent in cosmetics.
Hydroxyl radical; Tyrosinase reaction; Arbutin
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.
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.
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.
Previous screening of the substrate repertoires and substrate specificity profiles of granzymes resulted in long substrate lists highly likely containing bystander substrates. Here, a recently developed degradomics technology that allows distinguishing efficiently from less efficiently cleaved substrates was applied to study the degradome of mouse granzyme B (mGrB).
In vitro kinetic degradome analysis resulted in the identification of 37 mGrB cleavage events, 9 of which could be assigned as efficiently targeted ones. Previously, cleavage at the IEAD75 tetrapeptide motif of Bid was shown to be efficiently and exclusively targeted by human granzyme B (hGrB) and thus not by mGrB. Strikingly, and despite holding an identical P4-P1 human Bid (hBid) cleavage motif, mGrB was shown to efficiently cleave the BCL2/adenovirus E1B 19 kDa protein-interacting protein 2 or BNIP-2 at IEAD28. Like Bid, BNIP-2 represents a pro-apoptotic Bcl-2 protein family member and a potential regulator of GrB induced cell death. Next, in vitro analyses demonstrated the increased efficiency of human and mouse BNIP-2 cleavage by mGrB as compared to hGrB indicative for differing Bid/BNIP-2 substrate traits beyond the P4-P1 IEAD cleavage motif influencing cleavage efficiency. Murinisation of differential primed site residues in hBNIP-2 revealed that, although all contributing, a single mutation at the P3′ position was found to significantly increase the mGrB/hGrB cleavage ratio, whereas mutating the P1′ position from I29 > T yielded a 4-fold increase in mGrB cleavage efficiency. Finally, mutagenesis analyses revealed the composite BNIP-2 precursor patterns to be the result of alternative translation initiation at near-cognate start sites within the 5′ leader sequence (5′UTR) of BNIP-2.
Despite their high sequence similarity, and previously explained by their distinct tetrapeptide specificities observed, the substrate repertoires of mouse and human granzymes B only partially overlap. Here, we show that the substrate sequence context beyond the P4-P1 positions can influence orthologous granzyme B cleavage efficiencies to an unmatched extent. More specifically, in BNIP-2, the identical and hGrB optimal IEAD tetrapeptide substrate motif is targeted highly efficiently by mGrB, while this tetrapeptide motif is refractory towards mGrB cleavage in Bid.
BNIP-2; Bid; Granzyme B; Extended substrate specificity; N-terminal COFRADIC; Near-cognate translation initiation; Degradomics
Defects in skeletal muscle fatty acid oxidation have been implicated in the etiology of insulin resistance. Malonyl-CoA decarboxylase (MCD) has been a target of investigation because it reduces the concentration of malonyl-CoA, a metabolite that inhibits fatty acid oxidation. The in vivo role of muscle MCD expression in the development of insulin resistance remains unclear.
To determine the role of MCD in skeletal muscle of diet induced obese and insulin resistant mouse models we generated mice expressing a muscle specific transgene for MCD (Tg-fMCDSkel) stabilized posttranslationally by the small molecule, Shield-1. Tg-fMCDSkel and control mice were placed on either a high fat or low fat diet for 3.5 months. Obese and glucose intolerant as well as lean control Tg-fMCDSkel and nontransgenic control mice were treated with Shield-1 and changes in their body weight and insulin sensitivity were determined upon induction of MCD. Inducing MCD activity >5-fold in skeletal muscle over two weeks did not alter body weight or glucose intolerance of obese mice. MCD induction further potentiated the defects in insulin signaling of obese mice. In addition, key enzymes in fatty acid oxidation were suppressed following MCD induction.
Acute induction of MCD in the skeletal muscle of obese and glucose intolerant mice did not improve body weight and decreased insulin sensitivity compared to obese nontransgenic controls. Induction of MCD in skeletal muscle resulted in a suppression of mitochondrial oxidative genes suggesting a redundant and metabolite driven regulation of gene expression.
Diabetes; Metabolism; Insulin resistance; Fatty acid oxidation; Chemical-genetics
Dioscorea is a genus of flowering plants, and some Dioscorea species are known and used as a source for the steroidal sapogenin diosgenin. To screen potential resource from Dioscorea species and related medicinal plants for diosgenin extraction, a rapid method to compare the contents of diosgenin in various plants is crucial.
An ultra-performance liquid chromatography (UPLC) coupled with diode array detection (DAD) and electrospray ionization mass spectrometry (ESI-MS) method was developed for identification and determination of diosgenin in various plants. A comprehensive validation of the developed method was conducted. Twenty-four batches of plant samples from four Dioscorea species, one Smilax species and two Heterosmilax species were analyzed by using the developed method.
The present method presented good sensitivity, precision and accuracy. Diosgenin was found in three Dioscorea species and one Heterosmilax species, namely D. zingiberensis, D. septemloba, D. collettii and H. yunnanensis.
The method is suitable for the screening of diosgenin resources from plants. D. zingiberensis is an important resource for diosgenin harvesting.
Diosgenin; UPLC-DAD-MS; Dioscorea; Medicinal plants; Quality evaluation
The cosmetics market has rapidly increased over the last years. For example, in 2011 it reached 242.8 billion US dollars, which was a 3.9% increase compared to 2010. There have been many recent trials aimed at finding the functional ingredients for new cosmetics. Gallic acid is a phytochemical derived from various herbs, and has anti-fungal, anti-viral, and antioxidant properties. Although phytochemicals are useful as cosmetic ingredients, they have a number of drawbacks, such as thermal stability, residence time in the skin, and permeability through the dermal layer. To overcome these problems, we considered conjugation of gallic acid with a peptide.
We synthesized galloyl-RGD, which represents a conjugate of gallic acid and the peptide RGD, purified it by HPLC and characterized by MALDI-TOF with the aim of using it as a new cosmetic ingredient. Thermal stability of galloyl-RGD was tested at alternating temperatures (consecutive 4°C, 20°C, or 40°C for 8 h each) on days 2, 21, 41, and 61. Galloyl-RGD was relatively safe to HaCaT keratinocytes, as their viability after 48 h incubation with 500 ppm galloyl-RGD was 93.53%. In the group treated with 50 ppm galloyl-RGD, 85.0% of free radicals were removed, whereas 1000 ppm galloyl-RGD suppressed not only L-DOPA formation (43.8%) but also L-DOPA oxidation (54.4%).
Galloyl-RGD is a promising candidate for a cosmetic ingredient.
Human telomerase reverse transcriptase (hTERT), the catalytic subunit of telomesase, is responsible for telomere maintenance and its reactivation is implicated in almost 90% human cancers. Recent evidences show that hTERT is essential for neoplastic transformation independent of its canonical function. However, the roles of hTERT in the process remain elusive. In the current work, we explore the extra-telomeric role of hTERT in the neoplastic transformation of fibroblast IMR90.
Here we established transformed IMR90 cells by co-expression of three oncogenic factors, namely, H-Ras, SV40 Large-T antigen and hTERT (RSH). The RSH-transformed cells acquired hallmarks of cancer, such as they can grow under anchorage independent conditions; self-sufficient in growth signals; attenuated response to apoptosis; and possessed recurrent chromosomal abnormalities. Furthermore, the RSH-transformed cells showed enhanced migration capability which was also observed in IMR90 cells expressing hTERT alone, indicating that hTERT plays a role in cell migration, and thus possibly contribute to their metastatic potential during tumor transformation. This notion was further supported by our microarray analysis. In addition, we found that Ku70 were exclusively upregulated in both RSH-transformed IMR90 cells and hTERT-overexpressing IMR90 cells, suggesting the potential role of hTERT in DNA damage response (DDR).
Collectively, our study revealed the extra-telomeric effects of hTERT in cell migration and DDR during neoplastic transformation.
Extra-telomeric; hTERT; Neoplastic transformation; Cell migration; DNA damage response
Cytochrome c (Cyt c) is an apoptosis-initiating protein when released into the cytoplasm of eukaryotic cells and therefore a possible cancer drug candidate. Although proteins have been increasingly important as pharmaceutical agents, their chemical and physical instability during production, storage, and delivery remains a problem. Chemical glycosylation has been devised as a method to increase protein stability and thus enhance their long-lasting bioavailability.
Three different molecular weight glycans (lactose and two dextrans with 1 kD and 10 kD) were chemically coupled to surface exposed Cyt c lysine (Lys) residues using succinimidyl chemistry via amide bonds. Five neo-glycoconjugates were synthesized, Lac4-Cyt-c, Lac9-Cyt-c, Dex5(10kD)-Cyt-c, Dex8(10kD)-Cyt-c, and Dex3(1kD)-Cyt-c. Subsequently, we investigated glycoconjugate structure, activity, and stability. Circular dichroism (CD) spectra demonstrated that Cyt c glycosylation did not cause significant changes to the secondary structure, while high glycosylation levels caused some minor tertiary structure perturbations. Functionality of the Cyt c glycoconjugates was determined by performing cell-free caspase 3 and caspase 9 induction assays and by measuring the peroxidase-like pseudo enzyme activity. The glycoconjugates showed ≥94% residual enzyme activity and 86 ± 3 to 95 ± 1% relative caspase 3 activation compared to non-modified Cyt c. Caspase 9 activation by the glycoconjugates was with 92 ± 7% to 96 ± 4% within the error the same as the caspase 3 activation. There were no major changes in Cyt c activity upon glycosylation. Incubation of Dex3(1 kD)-Cyt c with mercaptoethanol caused significant loss in the tertiary structure and a drop in caspase 3 and 9 activation to only 24 ± 8% and 26 ± 6%, respectively. This demonstrates that tertiary structure intactness of Cyt c was essential for apoptosis induction. Furthermore, glycosylation protected Cyt c from detrimental effects by some stresses (i.e., elevated temperature and humidity) and from proteolytic degradation. In addition, non-modified Cyt c was more susceptible to denaturation by a water-organic solvent interface than its glycoconjugates, important for the formulation in polymers.
The results demonstrate that chemical glycosylation is a potentially valuable method to increase Cyt c stability during formulation and storage and potentially during its application after administration.
Apoptosis; Chemical glycosylation; Drug delivery; Pharmaceutical protein; Protein formulation; Protein stability
Urease, one of the highly efficient known enzymes, catalyzes the hydrolysis of urea into ammonia and carbon dioxide. The present study aimed to extract urease from pea seeds (Pisum Sativum L). The enzyme was then purified in three consequence steps: acetone precipitation, DEAE-cellulose ion-exchange chromatography, and gel filtration chromatography (Sephacryl S-200 column).
The purification fold was 12.85 with a yield of 40%. The molecular weight of the isolated urease was estimated by chromatography to be 269,000 Daltons. Maximum urease activity (190 U/g) was achieved at the optimum conditions of 40°C and pH of 7.5 after 5 min of incubation. The kinetic parameters, K
, were estimated by Lineweaver-Burk fits and found to be 500 mM and 333.3 U/g, respectively. The thermodynamic constants of activation, ΔH, E
, and ΔS, were determined using Arrhenius plot and found to be 21.20 kJ/mol, 23.7 kJ/mol, and 1.18 kJ/mol/K, respectively.
Urease was purified from germinating Pisum Sativum L. seeds. The purification fold, yield, and molecular weight were determined. The effects of pH, concentration of enzyme, temperature, concentration of substrate, and storage period on urease activity were examined. This may provide an insight on the various aspects of the property of the enzyme. The significance of extracting urease from different sources could play a good role in understanding the metabolism of urea in plants.
Urease; Enzyme activity; Enzyme purification; Pisum Sativum L; Pea seeds
Secreted luciferases are highly useful bioluminescent reporters for cell-based assays and drug discovery. A variety of secreted luciferases from marine organisms have been described that harbor an N-terminal signal peptide for release along the classical secretory pathway. Here, we have characterized the secretion of Gaussia luciferase in more detail.
We describe three basic mechanisms by which GLUC can be released from cells: first, classical secretion by virtue of the N-terminal signal peptide; second, internal signal peptide-mediated secretion and third, non-conventional secretion in the absence of an N-terminal signal peptide. Non-conventional release of dNGLUC is not stress-induced, does not require autophagy and can be enhanced by growth factor stimulation. Furthermore, we have identified the golgi-associated, gamma adaptin ear containing, ARF binding protein 1 (GGA1) as a suppressor of release of dNGLUC.
Due to its secretion via multiple secretion pathways GLUC can find multiple applications as a research tool to study classical and non-conventional secretion. As GLUC can also be released from a reporter construct by internal signal peptide-mediated secretion it can be incorporated in a novel bicistronic secretion system.
Non-conventional secretion; Gaussia luciferase; GGA1; Bicistronic expression; Signal peptide
During EGFR internalization CIN85 bridges EGFR-Cbl complex, endocytic machinery and fusible membrane through the interactions of CIN85 with c-Cbl, endophilins and phosphatidic acid. These protein-protein and protein-lipid interactions are mediated or regulated by the positively charged C-terminal coiled-coil domain of CIN85. However, the details of CIN85-lipid interaction remain unknown. The present study suggested a possible electric interaction between the negative charge of phosphatidic acid and the positive charge of basic amino acids in coiled-coil domain.
Mutations of the basic amino acids in the coiled-coil domain, especially K645, K646, R648 and R650, into neutral amino acid alanine completely blocked the interaction of CIN85 with c-Cbl or phosphatidic acid. However, they did not affect CIN85-endophilin interaction. In addition, CIN85 was found to associate with the internalized EGFR endosomes. It interacted with several ESCRT (Endosomal Sorting Complex Required for Transport) component proteins for ESCRT assembly on endosomal membrane. Mutations in the coiled-coil domain (deletion of the coiled-coil domain or point mutations of the basic amino acids) dissociated CIN85 from endosomes. These mutants bound the ESCRT components in cytoplasm to prevent them from assembly on endosomal membrane and inhibited EGFR sorting for degradation.
As an adaptor protein, CIN85 interacts with variety of partners through several domains. The positive charges of basic amino acids in the coiled-coil domain are not only involved in the interaction with phosphatidic acid, but also regulate the interaction of CIN85 with c-Cbl. CIN85 also interacts with ESCRT components for protein sorting in endosomes. These CIN85-protein and CIN85-lipid interactions enable CIN85 to link EGFR-Cbl endocytic complex with fusible membrane during EGFR endocytosis and subsequently to facilitate ESCRT formation on endosomal membrane for EGFR sorting and degradation.
CIN85; EGFR endocytosis; Phosphatidic acid; Coiled-coil; ESCRT
Trichomonas vaginalis, a flagellated protozoan, is the agent responsible for trichomoniasis, the most common nonviral sexually transmitted infection worldwide. A reported 200 million cases are documented each year with far more cases going unreported. However, T. vaginalis is disproportionality under studied, especially considering its basic metabolism. It has been reported that T. vaginalis does not grow on sucrose. Nevertheless, the T. vaginalis genome contains some 11 putative sucrose transporters and a putative β-fructofuranosidase (invertase). Thus, the machinery for both uptake and cleavage of sucrose appears to be present.
We amplified the β-fructofuranosidase from T. vaginalis cDNA and cloned it into an Escherichia coli expression system. The expressed, purified protein was found to behave similarly to other known β-fructofuranosidases. The enzyme exhibited maximum activity at pH close to 5.0, with activity falling off rapidly at increased or decreased pH. It had a similar Km and Vmax to previously characterized enzymes using sucrose as a substrate, was also active towards raffinose, but had no detectable activity towards inulin.
T. vaginalis has the coding capacity to produce an active β-fructofuranosidase capable of hydrolyzing di- and trisaccharides containing a terminal, non-reducing fructose residue. Since we cloned this enzyme from cDNA, we know that the gene in question is transcribed. Furthermore, we could detect β-fructofuranosidase activity in T. vaginalis cell lysates. Therefore, the inability of the organism to utilize sucrose as a carbon source cannot be explained by an inability to degrade sucrose.
Trichomonas vaginalis; Carbohydrate utilization; Invertase; Purification
Organic solute carrier partner 1 (OSCP1) is known to facilitate the transport of various organic solutes into cells and reported to play a role in cell growth and cell differentiation. Moreover, OSCP1 is known as a tumor suppressor gene that is frequently down-expressed in nasopharyngeal carcinomas and acute myeloid leukemia. However, the underlying mechanisms of action remain unclear and the subcellular localization of OSCP1 has yet to be determined in detail.
Drosophila contains a single orthologue of OSCP1 (dOSCP1) that shares 58% homology with its human counterpart. To study the expression pattern and subcellular localization of dOSCP1, we prepared a specific antibody. Subcellular localization analyses of dOSCP1 with these revealed localization in the plasma membrane, endoplasmic reticulum, Golgi apparatus and mitochondria, but no detection in cytosol. dOSCP1 signals were also detected in the nucleus, although at weaker intensity than in plasma membranes and subcellular organelles. In addition, native polyacrylamide gel electrophoresis analysis with and without β-mercaptoethanol treatment revealed that recombinant dOSCP1 forms dimers and trimers in solution. The dimer form of dOSCP1 could also be detected by Western immunoblot analyses in third instar larval extracts.
The data revealed that dOSCP1 localizes not only in the plasma membrane but also in the nucleus, ER, Golgi apparatus and mitochondria. It is therefore conceivable that this protein may interact with various partners or form multimeric complexes with other proteins to play multiple roles in cells, providing clues to understanding the functions of dOSCP1 during Drosophila development.
Organic solute carrier partner 1; Drosophila; Subcellular organelle
In order to understand the effects of FeS cluster attachment in [NiFe] hydrogenase, we undertook a study to substitute all 12 amino acid positions normally ligating the three FeS clusters in the hydrogenase small subunit. Using the hydrogenase from Alteromonas macleodii “deep ecotype” as a model, we substituted one of four amino acids (Asp, His, Asn, Gln) at each of the 12 ligating positions because these amino acids are alternative coordinating residues in otherwise conserved-cysteine positions found in a broad survey of NiFe hydrogenase sequences. We also hoped to discover an enzyme with elevated hydrogen evolution activity relative to a previously reported “G1” (H230C/P285C) improved enzyme in which the medial FeS cluster Pro and the distal FeS cluster His were each substituted for Cys.
Among all the substitutions screened, aspartic acid substitutions were generally well-tolerated, and examination suggests that the observed deficiency in enzyme activity may be largely due to misprocessing of the small subunit of the enzyme. Alignment of hydrogenase sequences from sequence databases revealed many rare substitutions; the five substitutions present in databases that we tested all exhibited measurable hydrogen evolution activity. Select substitutions were purified and tested, supporting the results of the screening assay. Analysis of these results confirms the importance of small subunit processing. Normalizing activity to quantity of mature small subunit, indicative of total enzyme maturation, weakly suggests an improvement over the “G1” enzyme.
We have comprehensively screened 48 amino acid substitutions of the hydrogenase from A. macleodii “deep ecotype”, to understand non-canonical ligations of amino acids to FeS clusters and to improve hydrogen evolution activity of this class of hydrogenase. Our studies show that non-canonical ligations can be functional and also suggests a new limiting factor in the production of active enzyme.
[NiFe] hydrogenase; Iron-sulfur cluster; Alteromonas macleodii deep ecotype