Sodium Dodecyl Sulfate Gel Electrophoresis Research Articles

Protective role of ascorbic acid in the decontamination of cow milk casein by gamma-irradiation

Purpose: The aim of this work was to investigate the protective role of ascorbic acid on irradiation-induced modification of casein.Materials and methods: Casein stock solutions were irradiated with increasing doses 2–10 kGy using 60Co Gamma rays at a dose rate D• = 136.73 Gy/min at room temperature. The total viable microorganism content of cow milk casein was evaluated by Plate Count Agar (PCA) incubation for 48 h at 37°C. Sodium dodecylsulfate gel electrophoresis (SDS-PAGE) and Matrix-Assisted Laser Desorption-Ionization Time-of-Flight mass spectrometry (MALDI-TOF-MS) analysis were used to evaluate the effect of gamma irradiation on casein integrity.Results: Gamma irradiation reduced the bacterial contamination of casein solutions at a lower irradiation dose when performed in the presence of ascorbic acid. The irradiation treatment of casein in the absence of ascorbic acid with a dose of 4 kGy could reduce 99% of the original amount of bacterial colonies. However, in the presence of ascorbic acid the irradiation treatment of casein with a dose lower than 2 kGy could reduce 99% of the original amount of bacterial colonies which suggested that the irradiation dose lower than 2 kGy achieved almost the entire decontamination result. SDS-PAGE and MALDI-TOF-MS analysis showed that ascorbic acid protected cow milk casein from degradation and subsequent aggregation probably by scavenging oxygen and protein radicals produced by the irradiation.Conclusions: It is demonstrated that the combination of gamma irradiation and ascorbic acid produce additive effects, providing acceptable hygienic quality of cow milk casein and protects caseins against Reactive Oxygen Species (ROS) generated, during the irradiation process.

Studying the Stability of S-Layer Protein of Lactobacillus Acidophilus ATCC 4356 in Simulated Gastrointestinal Fluids Using SDS-PAGE and Circular Dichroism.

Crystalline arrays of proteinaceous subunits forming surface layers (S-layers) are now recognized as one of the most common outermost cell envelope components of prokaryotic organisms. The surface layer protein of Lactobacillus acidophilus ATCC4356 is composed of a single species of protein of apparent molecular weight of 43-46 KDa. Considering the Lactobacillus acidophilus ATCC4356 having the S-layer is stable in harsh gastrointestinal (GI) conditions, a protective role against destructive GI factors which has been proposed for these nanostructures. It opens interesting perspectives in the using and development of this S-layer as a protective coat for oral administration of unstable drug nanocarriers. To achieve this goal, it is necessary to study the in-vitro stability of the S-layers in the simulated gastrointestinal fluids (SGIF). This study was planned to evaluate the in-vitro stability of the extracted S-layer protein of Lactobacillus acidophilus ATCC4356 in SGIF using it as a protective coat in oral drug delivery. Sodium dodecyl sulfate gel electrophoresis (SDS-PAGE) and circular dichroism (CD) spectroscopy were used to study the stability of the S-layer protein incubated in SGIF. Both the SDS-PAGE and CD spectra results showed that Lactobacillus acidophilus ATCC4356 S-layer protein is stable in simulated gastric fluid (SGF) with pH = 2 up to 5 min. It is stable in SGF pH = 3.2 and above it, with and without pepsin. It is also stable in all the simulated intestinal fluids. This S-layer is also stable in all of the simulated intestinal fluids.

Natural proteins: Sources, isolation, characterization and applications

Worldwide, plant protein contributes substantially as a food resource because it contains essential amino acids for meeting human physiological requirements. However, many versatile plant proteins are used as medicinal agents as they are produced by using molecular tools of biotechnology. Proteins can be obtained from plants, animals and microorganism cells. The abundant economical proteins can be obtained from plant seeds. These natural proteins are obtained by isolation procedures depending on the physicochemical properties of proteins. Isolation and purification of single protein from cells containing mixtures of unrelated proteins is achievable due to the physical and chemical attributes of proteins. The following characteristics are unique to each protein: Amino acid composition, sequence, subunit structures, size, shape, net charge, isoelectric point, solubility, heat stability and hydrophobicity. Based on these properties, various methods of isolation exist, like salting out and isoionic precipitation. Purification of proteins is quiet challenging and, therefore, several approaches like sodium dodecyl sulfate gel electrophoresis and chromatography are available. Characterization of proteins can be performed by mass spectrometry/liquid chromatography-mass spectrometry (LC-MS). The amino acid sequence of a protein can be detected by using tandem mass spectrometry. In this article, a review has been made on the sources, isolation, purification and characterization of natural proteins.

Purification, properties and cDNA cloning of glutamate decarboxylase in germinated faba bean (Vicia faba L.)

Gamma-aminobutyric acid (GABA) is a non-protein amino acid with bioactive functions in humans. In this work, glutamate decarboxylase (EC 4.1.1.15, GAD) which is key in the GABA bioformation was purified from 5-day germinated faba beans and characterized. A single band was observed at 58kDa using sodium dodecyl sulphate gel electrophoresis. GAD optimal activity was at pH 6.0 at 40°C with a Km value for glutamic acid (Glu) of 2.63mM. The enzyme was inhibited significantly by Cu2+, Fe3+, Mg2+, Ba2+, aminoxyacetate, EGTA, Na2EDTA, l-cysteine and beta-mercaptoethanol; and activated at low Ca2+ 0.2mM. Using RT-PCR, the GAD cDNA was sequenced which indicated 1787bp long, containing a 1527bp open reading frame (ORF) that encoded 509 amino-acid peptides with a calculated molecular weight of 57.74kDa and a pI of 5.41 (GenBank accession number: JX444699).

High-throughput analysis of therapeutic and diagnostic monoclonal antibodies by multicapillary SDS gel electrophoresis in conjunction with covalent fluorescent labeling

Capillary gel electrophoresis (CGE) in the presence of sodium dodecyl sulfate (SDS) is a well-established and widely used protein analysis technique in the biotechnology industry, and increasingly becoming the method of choice that meets the requirements of the standards of International Conference of Harmonization (ICH). Automated single channel capillary electrophoresis systems are usually equipped with UV absorbance and/or laser-induced fluorescent (LIF) detection options offering general applicability and high detection sensitivity, respectively; however, with limited throughput. This shortcoming is addressed by the use of multicapillary gel electrophoresis (mCGE) systems with LED-induced fluorescent detection (LED-IF), also featuring automation and excellent detection sensitivity, thus widely applicable to rapid and large-scale analysis of biotherapeutics, especially monoclonal antibodies (mAb). The methodology we report in this paper is readily applicable for rapid purity assessment and subunit characterization of IgG molecules including detection of non-glycosylated heavy chains (NGHC) and separation of possible subunit variations such as truncated light chains (Pre-LC) or alternative splice variants. Covalent fluorophore derivatization and the mCGE analysis of the labeled IgG samples with multi-capillary gel electrophoresis are thoroughly described. Reducing and non-reducing conditions were both applied with and without peptide N-glycosidase F mediated deglycosylation.

Detection of EPO‐Fc fusion protein in human blood: Screening and confirmation protocols for sports drug testing

The neonatal Fc receptor (FcRn) has been under investigation for several years as a pharmaceutical drug target. Clinical studies have shown that fusion proteins consisting of human recombinant erythropoietin (rhEPO) and the Fc-part of IgG can be transported after pulmonary administration via FcRn across the airway epithelium to the blood stream. So far, no clinically approved pharmaceutical formulation of EPO-Fc is available. Since various forms of recombinant erythropoietins have been frequently misused by athletes as performance-enhancing agents, EPO-Fc might play a similar role in sports in the future. In order to investigate the detectability of EPO-Fc in human blood, different strategies were tested and developed. Only two of them fulfilled the necessary requirements regarding sensitivity and specificity. A rapid protocol useful for screening purposes first enriches EPO-Fc from human serum via high capacity protein A beads and subsequently detects EPO-Fc in the eluate with a commercial EPO ELISA kit. The limit of detection (LOD) of the method is about 5 pg (45 amol) EPO-Fc and is independent of the serum volume used. For screening and/or confirmation purposes a second protocol was evaluated, which consists of a fast EPO immunopurification step followed by sodium dodecyl sulfate or sarcosyl polyacrylamide gel electrophoresis (SDS-PAGE, SAR-PAGE) and Western double-blotting with chemiluminescence detection - a method already established in routine EPO anti-doping control. The latter strategy allows the detection of EPO-Fc in serum together with all other recombinant erythropoietins and with an identical LOD (5 pg/45 amol) as for the rapid screening protocol.

RECOVERY AND UTILIZATION OF PROTEIN DERIVED FROM SURIMI WASH‐WATER

ABSTRACTSurimi processors are committed to improve utilization of seafood resources, increase productivity and reduce organic matter discharged into the environment. The object of this study was to recover protein from pollock surimi processing wash‐water using membrane filtration and characterize properties of the recovered material. A pilot unit equipped with membrane elements concentrated protein from the surimi wash‐water. Membrane concentrate and control surimi samples were analyzed for proximate composition, lipid oxidation, color, sodium dodecyl sulfate gel electrophoresis, amino acids and minerals. Membrane concentrate, membrane concentrate plus surimi and control surimi were monitored for 180 days of storage at −20C. The membrane concentrate had significantly higher moisture and lipid, but lower protein content than surimi. As determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis, membrane concentrate proteins displayed a greater amount of lower molar mass molecules compared with surimi. The amino acid profile was comparable to control surimi and the recovered membrane concentrate proteins had similar nutritional values to that of surimi. The results indicate that the addition of 5% membrane concentrate to surimi will not adversely affect the storage at −20C and that the recovered wash‐water protein could be used to obtain a fish protein ingredient or added back at a low percentage to surimi products.PRACTICAL APPLICATIONSIn order to increase productivity and improve utilization of seafood resources, surimi processors are looking into alternative technologies to recover proteins and other material from the wastewater. Membrane filtration is a promising option for the concentration of wastewater. This study was conducted to determine the recovery and characterize the material recovered from surimi wash‐water using a commercial membrane filtration unit. It was demonstrated that the recoverable material is nutritionally similar to the final surimi product and that the overall yield can be increased using membrane technology. In addition to the benefit of recovering protein, the membrane filtration can reduce the amount of material in the waste stream.

The sclerostin-bone protein interactome

The secreted glycoprotein, sclerostin alters bone formation. To gain insights into the mechanism of action of sclerostin, we examined the interactions of sclerostin with bone proteins using a sclerostin affinity capture technique. Proteins from decalcified rat bone were captured on a sclerostin-maltose binding protein (MBP) amylose column, or on a MBP amylose column. The columns were extensively washed with low ionic strength buffer, and bound proteins were eluted with buffer containing 1M sodium chloride. Eluted proteins were separated by denaturing sodium-dodecyl sulfate gel electrophoresis and were identified by mass spectrometry. Several previously unidentified full-length sclerostin-interacting proteins such as alkaline phosphatase, carbonic anhydrase, gremlin-1, fetuin A, midkine, annexin A1 and A2, and collagen α1, which have established roles in bone formation or resorption processes, were bound to the sclerostin-MBP amylose resin but not to the MBP amylose resin. Other full-length sclerostin-interacting proteins such as casein kinase II and secreted frizzled related protein 4 that modulate Wnt signaling were identified. Several peptides derived from proteins such as Phex, asporin and follistatin that regulate bone metabolism also bound sclerostin. Sclerostin interacts with multiple proteins that alter bone formation and resorption and is likely to function by altering several biologically relevant pathways in bone.

P1-01-16: Detecting a Breast Carcinoma-Deriving B-Cell Response: An Immunoproteomics Biomarker Approach.

Abstract Background: Noninvasive biomarkers for the early detection of breast cancer are crucial due to the fact that the relapse risk of breast cancer is rising with the time point of its detection. Currently, none of the reported molecular biomarkers is established for the clinical use as a diagnostic tool. Previous proteomics-based studies showed the immunogenicity of breast carcinoma and the following B-cell mediated immune response. As a result, several autoantibodies against tumor proteins were detected in the sera of breast cancer patients. However, these putative biomarkers are still lacking of clinically reliable specificity and sensitivity, even of better discrimination of cancer patients when combining different biomarkers. The search for a new antibody biomarker signature remains very important as a potential cancer detection tool. For further investigations we analyzed the antibody pattern in serum samples of diseased patients and healthy controls after incubation with whole protein extract from a native carcinoma and identified the putative tumor-specific immunoreactive antigens. Materials and methods: For our de novo profiling of tumor antigens we used a protein extract from a primary invasive ductal carcinoma. Sera from 20 women, of which 19 were diagnosed with breast carcinoma and one with DCIS (CA), and 20 sera from age-matched healthy donors (CTRL) were obtained and pooled separately. For an optimal separation of tumor antigens a two-dimensional sodium dodecylsulfate gel electrophoresis (2D SDS-PAGE) was applied. Following immunoblots with each serum pool were performed and the immunospecific reactions visualized with a horseradish peroxidase-conjugated anti-IgG antibody. The relevant tumor antigens were identified via Matrix-Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry (MALDI TOF-TOF MS). Results: After the incubation of each serum pool with tumor antigens we obtained different extensive antibody profiles, whereas the visualized immunoreactive components varied in the signal intensity. We identified over 10 tumor antigens which reacted with the corresponding autoantibodies in CA or CTRL approach, showing again the complexity of immune response. Besides of already described breast carcinoma related antigens like alpha enolase, which showed immune reaction also with the healthy serum pool, we identified several potential antigens of interest like peroxiredoxin 6 which showed a strong immune response only after the incubation with cancer sera. Discussion: In our study we visualized a complex immune response pattern showing the autoantibody profiles in cancer and healthy sera against tumor-deriving antigens. Also some of identified breast carcinoma antigens were already described; other novel breast carcinoma-related antigens were detected too. Our next step in the intriguing search for cancer antibody biomarkers is the individual screening of sera to confirm the specificity of the tumor-deriving B-cell responses and the validation of these results by using an independent study population. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-01-16.

AFM study of Escherichia coli RNA polymerase σ 70 subunit aggregation

The self-assembly of Escherichia coli RNA polymerase σ 70 subunit was investigated using several experimental approaches. A novel rodlike shape was reported for σ 70 subunit aggregates. Atomic force microscopy reveals that these aggregates, or σ 70 polymers, have a straight rodlike shape 5.4 nm in diameter and up to 300 nm in length. Atomic force microscopy data, Congo red binding assay, and sodium dodecyl sulfate gel electrophoresis confirm the amyloid nature of observed aggregates. The process of formation of rodlike structures proceeds spontaneously under nearly physiological conditions. E. coli RNA polymerase σ 70 subunit may be an interesting object for investigation of amyloidosis as well as for biotechnological applications that exploit self-assembled bionanostructures. Polymerization of σ 70 subunit may be a competitive process with its three-dimensional crystallization and association with core RNA polymerase. From the Clinical Editor In this basic science study, the self-assembly of Escherichia coli RNA polymerase σ 70 subunit was investigated using atomic force microscopy and other complementary approaches.

Intrinsic protein kinase activity in mitochondrial oxidative phosphorylation complexes.

Mitochondrial protein phosphorylation is a well-recognized metabolic control mechanism, with the classical example of pyruvate dehydrogenase (PDH) regulation by specific kinases and phosphatases of bacterial origin. However, despite the growing number of reported mitochondrial phosphoproteins, the identity of the protein kinases mediating these phosphorylation events remains largely unknown. The detection of mitochondrial protein kinases is complicated by the low concentration of kinase relative to that of the target protein, the lack of specific antibodies, and contamination from associated, but nonmatrix, proteins. In this study, we use blue native gel electrophoresis (BN-PAGE) to isolate rat and porcine heart mitochondrial complexes for screening of protein kinase activity. To detect kinase activity, one-dimensional BN-PAGE gels were exposed to [γ-(32)P]ATP and then followed by sodium dodecyl sulfate gel electrophoresis. Dozens of mitochondrial proteins were labeled with (32)P in this setting, including all five complexes of oxidative phosphorylation and several citric acid cycle enzymes. The nearly ubiquitous (32)P protein labeling demonstrates protein kinase activity within each mitochondrial protein complex. The validity of this two-dimensional BN-PAGE method was demonstrated by detecting the known PDH kinases and phosphatases within the PDH complex band using Western blots and mass spectrometry. Surprisingly, these same approaches detected only a few additional conventional protein kinases, suggesting a major role for autophosphorylation in mitochondrial proteins. Studies on purified Complex V and creatine kinase confirmed that these proteins undergo autophosphorylation and, to a lesser degree, tenacious (32)P-metabolite association. In-gel Complex IV activity was shown to be inhibited by ATP, and partially reversed by phosphatase activity, consistent with an inhibitory role for protein phosphorylation in this complex. Collectively, this study proposes that many of the mitochondrial complexes contain an autophosphorylation mechanism, which may play a functional role in the regulation of these multiprotein units.

Segmental allergen challenge alters multimeric structure and function of surfactant protein D in humans.

Surfactant protein D (SP-D), a 43-kD collectin, is synthesized and secreted by airway epithelia as a dodecamer formed by assembly of four trimeric subunits. We have previously shown that the quaternary structure of SP-D can be altered during inflammatory lung injury through its modification by S-nitrosylation, which in turn alters its functional behavior producing a proinflammatory response in effector cells. We hypothesized that alterations in structure and function of SP-D may occur in humans with acute allergic inflammation. Bronchoalveolar lavage (BAL) fluid was collected from 15 nonsmoking patients with mild intermittent allergic asthma before and 24 hours after segmental provocation with saline, allergen, LPS, and mixtures of allergen and LPS. Structural modifications of SP-D were analyzed by native and sodium dodecyl sulfate gel electrophoresis. The multimeric structure of native SP-D was found to be disrupted after provocation with allergen or a mixture of allergen and LPS. Interestingly, under reducing conditions, sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated that 7 of 15 patients with asthma developed an abnormal cross-linked SP-D band after segmental challenge with either allergen or a mixture of allergen with LPS but not LPS alone. Importantly, patients with asthma with cross-linked SP-D demonstrated significantly higher levels of BAL eosinophils, nitrogen oxides, IL-4, IL-5, IL-13, and S-nitrosothiol-SP-D compared with patients without cross-linked SP-D. We conclude that segmental allergen challenge results in changes of SP-D multimeric structure and that these modifications are associated with an altered local inflammatory response in the distal airways.

Method development and validation of capillary sodium dodecyl sulfate gel electrophoresis for the characterization of a monoclonal antibody

A capillary sodium dodecyl sulfate gel electrophoresis (cSDS) method has been developed and qualified for purity and impurity analysis of monoclonal antibodies. This method was optimized and qualified for the analysis of monoclonal antibody (mAb1) under reduced and non-reduced conditions. Some of the sample preparation parameters including sample buffer pH, incubation temperature and duration, alkylation conditions with iodoacetamide (IAM), and reduction conditions with 2-mercaptoethanol (2-ME) were optimized. It was observed that under slightly acidic conditions (pH 5.5–6.5) the thermally induced fragmentation of non-reduced mAb1 was greatly decreased. As such, a citrate–phosphate buffer at pH 6.5 was used for sample preparation to replace the original Beckman sample buffer (pH 9.0). The optimal sample preparation conditions were found to be as follows: (1) incubation temperature and duration (reduced and non-reduced), 65 °C for 5 min; (2) alkylation condition, 10 μL of 0.25 M IAM; (3) reduction condition, 10 μL of 5-fold diluted 2-ME. The method was qualified by evaluating specificity, accuracy, precision, limit of quantitation (LOQ), and linearity. The method exhibited no interference from sample buffer matrix. The method was found to be linear, accurate, and precise in the range of 0.25–3.0 mg/mL protein concentration. The LOQ of the method was determined to be 0.02 mg/mL for reduced and non-reduced mAb1. In addition, some aspects of sample stability were examined during qualification.

Deletion of a terminal residue disrupts oligomerization of a transmembrane α-helixThis paper is one of a selection of papers published in this special issue entitled “Canadian Society of Biochemistry, Molecular & Cellular Biology 52nd Annual Meeting — Protein Folding: Principles and Diseases” and has undergone the Journal's usual peer review process.

In studies of the structural biology of membrane proteins, the success of strategies based on the "divide and conquer" approach, where peptides are used to model the individual transmembrane (TM) alpha-helices of membrane proteins, depends on the correct identification of the membrane-embedded TM alpha-helix amino acid sequence within the full-length protein. In the present work, we examine the effects of excluding or including TM boundary residues on the intrinsic properties of a Lys-tagged TM2 alpha-helix of myelin proteolipid protein (PLP), of parent sequence KKKK-66AFQYVIYGTASFFFLYGALLLAEG89-KKKK along with analogs containing an additional wild type Phe-90, Phe-90 and Tyr-91, and of a hydrophobic mutant Leu-90. Using protein gel electrophoresis, circular dichroism, and fluorescence resonance energy transfer in the membrane-mimetic detergent sodium dodecylsulfate (SDS), we demonstrate that the removal of a single amino acid from the C-terminus of this TM segment is enough to change its intrinsic properties, with TM2 66-89 displaying only a monomeric form, but with dimers arising for the other 3 peptides. A novel use of trifluoroethanol (TFE) as a maximal helix-supporting solvent demonstrated that peptides containing residues at positions 90 and (or) 90-91 displayed significantly increased helical content vs. the TM2 parent peptide. The findings suggest that deletion of critical C-terminal residue(s) tends to reposition the helix terminus toward the membrane-aqueous interface. Our overall results emphasize the potential influence of boundary residues on TM properties when using peptides as models for TM alpha-helices, and may implicate a role for these residues in membrane protein folding and assembly.

Electrophoresis today and tomorrow: Helping biologists' dreams come true

Intensive research and development of electrophoresis methodology and instrumentation during past decades has resulted in unique methods widely implemented in bioanalysis. While two-dimensional electrophoresis and denaturing polyacrylamide gel electrophoresis in sodium dodecylsulfate are still the most frequently used electrophoretic methods applied to analyses of proteins, new miniaturized capillary and microfluidic versions of electromigrational methods have been developed. High-throughput electrophoretic instruments with hundreds of capillaries for parallel separations and laser-induced fluorescence detection of labeled DNA strands have been of key importance for the scientific and commercial success of the Human Genome Project. Another powerful method, capillary isoelectric focusing with pressurized and pH-driven mobilization, provides efficient separations and on-line sensitive detection of proteins, bacteria and viruses. Electrophoretic microfluidic devices can integrate single-cell injection, cell lysis, separation of its components and fluorescence or mass spectrometry detection. These miniaturized devices also proved the capability of single-molecule detection.

Response of Cyprinus carpio to copper exposure: alterations in reduced glutathione, catalase and proteins electrophoretic patterns

This study was conducted to characterize the alterations in reduced glutathione (GSH) level, catalase (CAT) activity and proteins electrophoretic patterns in response to sublethal copper (Cu) exposure in Cyprinus carpio and to determine whether these responses are related to Cu accumulation in gills, chosen as target tissue. Fish were exposed to 0.1 and 1.0mg/l Cu for 10 and 20days. There were increasing level of Cu in the gill with increasing concentrations of metal in the exposure medium, and with increasing duration of exposure. GSH level and CAT activity increased in fish exposed to 1.0mg/l Cu for both exposure periods, while no change was detected at the lower Cu concentration. Electrophoretic patterns of gill proteins by sodium dodecyl sulphate gel electrophoresis (SDS-PAGE) consist of 25-, 26-, 30-, 44- and 48-kDA medium molecular weight proteins (MMP) for five bands and 64-, 72-, 90- and 101-kDA high molecular weight proteins (HMP) for four bands in both control and treatment groups. The levels of 25-, 26- and 30-kDA MMP and 72- and 90-kDA HMP increased in response to Cu exposure. The present study demonstrated that Cu caused stress in fish gills and an acclimation with induction of GSH, CAT, MMP and HMP, which were important in the protection against metal damage, was observed.

Oligomerization of Transmembrane Alpha-Helices Modulated By C-terminal Boundary Residues

In studies of the structural biology of membrane proteins, the success of strategies based on the “divide-and-conquer” approach, where peptides are used to model the individual transmembrane (TM) α-helices of membrane proteins, depends upon the correct identification of the membrane-embedded TM α-helix amino acid sequence within the full-length protein. In the present work, we examine the effects of excluding or including TM boundary residues on the intrinsic properties of the TM2 α-helix of myelin proteolipid protein (PLP). Using protein gel electrophoresis, circular dichroism, and fluorescence resonance energy transfer in the membrane-mimetic detergent sodium dodecylsulfate (SDS) to study parent sequence KKKK-61AFQYVIYGTASFFFLYGALL-LAEG89-KKKK - along with analogs containing an additional wild type Phe-90, Phe-90 and Tyr-91, and a hydrophobic mutant Leu-90 - we demonstrate that the removal of a single amino acid from the C-terminus of this TM segment is sufficient to change its intrinsic properties, with TM2 61-89 displaying only a monomeric form, but with principally dimers arising for the other three peptides. The findings suggest that deletion of critical C-terminal residue(s) tends to re-position the helix terminus toward the membrane-aqueous interface, and emphasize the potential influence of boundary residues on TM properties when utilizing peptides as models for TM α-helices. These finding may implicate a role for such residues in membrane protein folding and assembly.

Diamide Triggers Mainly S Thiolations in the Cytoplasmic Proteomes of Bacillus subtilis and Staphylococcus aureus

Glutathione constitutes a key player in the thiol redox buffer in many organisms. However, the gram-positive bacteria Bacillus subtilis and Staphylococcus aureus lack this low-molecular-weight thiol. Recently, we identified S-cysteinylated proteins in B. subtilis after treatment of cells with the disulfide-generating electrophile diamide. S cysteinylation is thought to protect protein thiols against irreversible oxidation to sulfinic and sulfonic acids. Here we show that S thiolation occurs also in S. aureus proteins after exposure to diamide. We further analyzed the formation of inter- and intramolecular disulfide bonds in cytoplasmic proteins using diagonal nonreducing/reducing sodium dodecyl sulfate gel electrophoresis. However, only a few proteins were identified that form inter- or intramolecular disulfide bonds under control and diamide stress conditions in B. subtilis and S. aureus. Depletion of the cysteine pool was concomitantly measured in B. subtilis using a metabolomics approach. Thus, the majority of reversible thiol modifications that were previously detected by two-dimensional gel fluorescence-based thiol modification assay are most likely based on S thiolations. Finally, we found that a glutathione-producing B. subtilis strain which expresses the Listeria monocytogenes gshF gene did not show enhanced oxidative stress resistance compared to the wild type.

Glycoengineered Acid α-Glucosidase With Improved Efficacy at Correcting the Metabolic Aberrations and Motor Function Deficits in a Mouse Model of Pompe Disease

Improving the delivery of therapeutics to disease-affected tissues can increase their efficacy and safety. Here, we show that chemical conjugation of a synthetic oligosaccharide harboring mannose 6-phosphate (M6P) residues onto recombinant human acid alpha-glucosidase (rhGAA) via oxime chemistry significantly improved its affinity for the cation-independent mannose 6-phosphate receptor (CI-MPR) and subsequent uptake by muscle cells. Administration of the carbohydrate-remodeled enzyme (oxime-neo-rhGAA) into Pompe mice resulted in an approximately fivefold higher clearance of lysosomal glycogen in muscles when compared to the unmodified counterpart. Importantly, treatment of immunotolerized Pompe mice with oxime-neo-rhGAA translated to greater improvements in muscle function and strength. Treating older, symptomatic Pompe mice also reduced tissue glycogen levels but provided only modest improvements in motor function. Examination of the muscle pathology suggested that the poor response in the older animals might have been due to a reduced regenerative capacity of the skeletal muscles. These findings lend support to early therapeutic intervention with a targeted enzyme as important considerations in the management of Pompe disease.

Gab2-Mediated Signaling Promotes Melanoma Metastasis

Metastatic melanoma is a disease with a poor prognosis that currently lacks effective treatments. Critical biological features of metastasis include acquisition of migratory competence, growth factor independence, and invasive potential. In an attempt to identify genes that contribute to melanoma pathogenesis, a genome-wide search using bacterial artificial chromosome array comparative genomic hybridization and single nucleotide polymorphism arrays in a series of 64 metastatic melanoma samples and 20 melanoma cell lines identified increased copy numbers of Gab2 located on 11q14.1. Gab2 is an adaptor protein that potentiates the activation of the Ras-Erk and PI3K-Akt pathways and has recently been implicated in human cancer; however, its role in melanoma has not been explored. In this study, we found that Gab2 was either amplified (approximately 11%) and/or overexpressed (approximately 50%) in melanoma. Gab2 protein expression correlated with clinical melanoma progression, and higher levels of expression were seen in metastatic melanomas compared with primary melanoma and melanocytic nevi. We found that overexpression of Gab2 potentiates, whereas silencing of Gab2 reduces, migration and invasion of melanoma cells. Gab2 mediated the hyperactivation of Akt signaling in the absence of growth factors, whereas inhibition of the PI3K-Akt pathway decreased Gab2-mediated tumor cell migration and invasive potential. Gab2 overexpression resulted in enhanced tumor growth and metastatic potential in vivo. These studies demonstrate a previously undefined role for Gab2 in melanoma tumor progression and metastasis.