scholarly journals Stabilization of the CD81 Large Extracellular Loop with De Novo Disulfide Bonds Improves Its Amenability for Peptide Grafting

Pharmaceutics ◽  
2018 ◽  
Vol 10 (3) ◽  
pp. 138 ◽  
Author(s):  
Stefan Vogt ◽  
Gerhard Stadlmayr ◽  
Katharina Stadlbauer ◽  
Flávio Sádio ◽  
Peter Andorfer ◽  
...  
Keyword(s):  
Disulfide Bonds ◽  
De Novo ◽  
Specific Antigen ◽  
Thermal Stabilization ◽  
Peptide Sequence ◽  
Size Exclusion ◽  
Extracellular Loop ◽  
Human Transferrin Receptor ◽  
Exclusion Chromatography ◽  
Large Extracellular Loop

Tetraspan proteins are significantly enriched in the membranes of exosomal vesicles (EVs) and their extracellular domains are attractive targets for engineering towards specific antigen recognition units. To enhance the tolerance of a tetraspanin fold to modification, we achieved significant thermal stabilization of the human CD81 large extracellular loop (hCD81 LEL) via de novo disulfide bonds. The best mutants were shown to exhibit a positive shift in the melting temperature (Tm) of up to 25 °C. The combination of two most potent disulfide bonds connecting different strands of the protein resulted in a mutant with a Tm of 109 °C, 43 °C over the Tm of the wild-type hCD81 LEL. A peptide sequence binding to the human transferrin receptor (hTfr) was engrafted into the D-segment of the hCD81 LEL, resulting in a mutant that still exhibited a compact fold. Grafting of the same peptide sequence between helices A and B resulted in a molecule with an aberrant profile in size exclusion chromatography (SEC), which could be improved by a de novo cysteine bond connecting both helices. Both peptide-grafted proteins showed an enhanced internalization into the cell line SK-BR3, which strongly overexpresses hTfr. In summary, the tetraspan LEL fold could be stabilized to enhance its amenability for engineering into a more versatile protein scaffold.

Characterization of the novelTrypanosoma bruceiinosine 5′-monophosphate dehydrogenase

Parasitology ◽  
2013 ◽  
Vol 140 (6) ◽  
pp. 735-745 ◽  
Author(s):  
TOMOAKI BESSHO ◽  
SHOKO MORII ◽  
TOSHIHIDE KUSUMOTO ◽  
TAKAHIRO SHINOHARA ◽  
MASANORI NODA ◽  
...  
Keyword(s):  
Analytical Ultracentrifugation ◽  
De Novo ◽  
Binding Mode ◽  
Nucleotide Metabolism ◽  
Sub Saharan Africa ◽  
Size Exclusion ◽  
Purine Nucleotides ◽  
Exclusion Chromatography ◽  
Sub Saharan ◽  
Monophosphate Dehydrogenase

SUMMARYThere is an alarming rate of human African trypanosomiasis recrudescence in many parts of sub-Saharan Africa. Yet, the disease has no successful chemotherapy.Trypanosomalacks the enzymatic machinery for thede novosynthesis of purine nucleotides, and is critically dependent on salvage mechanisms. Inosine 5′-monophosphate dehydrogenase (IMPDH) is responsible for the rate-limiting step in guanine nucleotide metabolism. Here, we characterize recombinantTrypanosoma bruceiIMPDH (TbIMPDH) to investigate the enzymatic differences between TbIMPDH and host IMPDH. Size-exclusion chromatography and analytical ultracentrifugation sedimentation velocity experiments reveal that TbIMPDH forms a heptamer, different from type 1 and 2 mammalian tetrameric IMPDHs. Kinetic analysis reveals calculatedKmvalues of 30 and 1300 μmfor IMP and NAD, respectively. The obtainedKmvalue of TbIMPDH for NAD is approximately 20–200-fold higher than that of mammalian enzymes and indicative of a different NAD binding mode between trypanosomal and mammalian IMPDHs. Inhibition studies showKivalues of 3·2 μm, 21 nM and 3·3 nM for ribavirin 5′-monophosphate, mycophenolic acid and mizoribine 5′-monophosphate, respectively. Our results show that TbIMPDH is different from its mammalian counterpart and thus may be a good target for further studies on anti-trypanosomal drugs.

scholarly journals Complex-centric proteome profiling by SEC-SWATH-MS for the parallel detection of hundreds of protein complexes

2019 ◽  
Author(s):  
Isabell Bludau ◽  
Moritz Heusel ◽  
Max Frank ◽  
George Rosenberger ◽  
Robin Hafen ◽  
...  
Keyword(s):  
Error Control ◽  
Complex Analysis ◽  
De Novo ◽  
Protein Complexes ◽  
Statistical Error ◽  
Size Exclusion ◽  
Spectrometric Analysis ◽  
Native Protein ◽  
Proteome Profiling ◽  
Exclusion Chromatography

Abstract Most catalytic, structural and regulatory functions of the cell are carried out by functional modules, typically complexes containing or consisting of proteins. The composition and abundance of these complexes and the quantitative distribution of specific proteins across different modules is therefore of major significance in basic and translational biology. To date, the systematic detection and quantification of protein complexes has remained technically challenging. The chromatographic separation of native protein complexes followed by the mass spectrometric analysis of the proteins contained in sequential fractions results in potentially thousands of protein elution profiles from which, in principle, the presence of specific complexes can be inferred. However, the de novo inference of protein complexes from such datasets has so far remained limited with regard to selectivity and the retrieval of quantitative information.We recently developed a variant of this strategy, complex-centric proteome profiling, which extends the concepts of targeted proteomics to the level of native protein complex analysis. The complex-centric workflow consists of size exclusion chromatography (SEC) to fractionate native protein complexes, DIA/SWATH mass spectrometry to precisely quantify the proteins in each SEC fraction based on a consistent set of peptides, and targeted, complex-centric analysis where prior information from generic protein interaction maps is used to detect and quantify protein complexes with high selectivity and statistical error control via the computational framework CCprofiler. Complex-centric proteome profiling captures the majority of proteins in complex-assembled state and reveals their organization into hundreds of complexes and complex variants observable in a given cellular state. The protocol is applicable to genetically unaltered tissue cultures and adaptable to primary tissue. At present it requires approximately 8 days of wet-lab work, 15 days of MS measurement time and 7 days of computational analysis.

Effect of the NaOH Concentration and Temperature on the Dissolution Mechanisms of ß-Lactoglobulin Gels in Alkali

2008 ◽  
Vol 4 (5) ◽  
Author(s):  
Ruben Mercadé-Prieto ◽  
D. I. Wilson ◽  
William R. Paterson
Keyword(s):  
Dissolution Rate ◽  
Disulfide Bonds ◽  
Size Exclusion ◽  
Dissolution Process ◽  
Dissolution Behavior ◽  
Exclusion Chromatography ◽  
Model Protein ◽  
Different Temperatures ◽  
Alkaline Conditions ◽  
Protein Gels

The dissolution mechanisms of model protein gels (made from a single protein, ß- lactoglobulin) in alkali were studied at different NaOH concentrations and temperatures. A recent study has shown that the free volume within the swollen gel is a key parameter that determines the dissolution rate (Food Hydrocolloids (2007) 21:1275). This suggests that proteins ‘diffuse’ in the swollen gel next to the solution before they are dissolved. We extend this hypothesis here by showing that only small oligomers (trimers and smaller), such as those formed after the cleavage of large aggregates, are likely to diffuse throughout the gel. Large aggregates (> 200 kDa) are considered to disentangle at the gel/solvent interface. Size exclusion chromatography has been used to determine the size distributions of the aggregates released from the gel matrix, and to follow the breakdown kinetics of soluble aggregates in alkaline conditions. Analysis of the dissolution process at different temperatures reveals that at low NaOH concentrations (< 0.1 M), the breakdown of large aggregates in alkali exhibits a temperature dependency different from that of the overall dissolution process. At high NaOH concentrations (> 0.3 M), where swelling is inhibited, the increase in the dissolution rate seems to be related to the destruction of cross-linked aggregates, in particular via the beta-elimination of intermolecular disulfide bonds. A previously unobserved type of dissolution behavior at 0.87 M NaOH and at high temperatures is reported.

scholarly journals Is the mammalian serine palmitoyltransferase a high-molecular-mass complex?

2007 ◽  
Vol 405 (1) ◽  
pp. 157-164 ◽  
Author(s):  
Thorsten Hornemann ◽  
Yu Wei ◽  
Arnold von Eckardstein
Keyword(s):  
Molecular Mass ◽  
De Novo ◽  
Size Exclusion ◽  
Published Data ◽  
Serine Palmitoyltransferase ◽  
Native Page ◽  
Tissue Specific ◽  
Dynamic Composition ◽  
293 Cells ◽  
Exclusion Chromatography

SPT (serine palmitoyltransferase) catalyses the rate-limiting step for the de novo synthesis of sphingolipids. Mammalian SPT is believed to be a heterodimer composed of two subunits, SPTLC1 and SPTLC2. We reported previously the identification of a new third SPT subunit, SPTLC3. In the present study, we have investigated the structure of the SPT complex in more detail. Pull-down assays with antibodies against SPTLC3 concomitantly co-precipitated SPTLC1 and SPTLC2 in human placenta extracts and SPTLC3 overexpressing human embryonic kidney-293 cells. By size exclusion chromatography, we determined the molecular mass of the functional SPT complex to be approx. 480 kDa. By Blue-native-PAGE experiments we demonstrated that all three SPT subunits (SPTLC1–3) are co-localized within a single SPT complex. On the basis of these results we conclude that the functional SPT is not a dimer, but a higher organized complex, composed of three distinct subunits (SPTLC1, SPTLC2 and SPTLC3) with a molecular mass of 480 kDa. The stoichiometry of SPTLC2 and SPTLC3 in this complex seems not to be fixed and is probably changed dynamically in dependence of the tissue specific SPTLC2 and SPTLC3 expression levels. Based on our own and earlier published data we propose a model of an octameric SPT structure. The observed dynamic composition of the SPT complex could provide a cellular mechanism to adjust SPT activity to tissue specific requirements in sphingolipid synthesis.

scholarly journals New Insight into the Octamer of Thymidylate Synthetase Stabilized by Intermolecular Cys43-Disulfide

2018 ◽  
Author(s):  
Dan Xie ◽  
Lulu Wang ◽  
Qi Xiao ◽  
Xiaoyan Wu ◽  
Lin Zhang ◽  
...  
Keyword(s):  
Drug Targets ◽  
Molecular Mechanisms ◽  
De Novo ◽  
Physical Structure ◽  
Size Exclusion ◽  
Primary Target ◽  
Sds Page ◽  
Exclusion Chromatography ◽  
Essential Enzyme ◽  
Insight Into

Thymidylate synthase (TYMS) is an essential enzyme for the de novo synthesis of dTMP and has been a primary target for cancer chemotherapy. Although the physical structure of TYMS and the molecular mechanisms of TYMS catalyzing the conversion of dUMP to dTMP have been conducted thorough studies, oligomeric structure remains unclear. Here, we show that human TYMS not only exists in dimer but also octamer by intermolecular Cys43-disulfide formation. We optimize the expression condition of recombinant human TYMS using Escherichia coli system. Using HPLC-MS/MS, we show that purified TYMS has catalytic activity for producing dTMP. In the absence of reductant &beta;-mercaptoethanol, SDS-PAGE and size exclusion chromatography (SEC) showed size of TYMS protein is about 35 KDa, 70 KDa, and 280 KDa. While the Cys43 was mutated to Gly, the band of ~280 KDa and the peak of octamer disappeared. Therefore, TYMS was determined to form octamer, dependent on the presence of Cys43-disulfide. By measuring Steady-State Parameters for monomer, dimer and octamer, we found the kcat of octamer is increased slightly than monomer. On the basis of these findings, we suggest that octamer in the active state might have a potential influence on the design of new drug targets.

scholarly journals PRESENTATION OF RECEPTOR-CONTACTING LOOP OF HUMAN IGE ON THE HBCAG PARTICLES

2015 ◽  
Vol 3 ◽  
pp. 390-397
Author(s):  
Aliya Zhanatayevna Baltabekova ◽  
Alexander Vyacheslavovich Shustov
Keyword(s):  
De Novo ◽  
Core Protein ◽  
Primary Objective ◽  
Size Exclusion ◽  
Expression Vectors ◽  
Therapeutic Vaccines ◽  
Ige Antibodies ◽  
Genes Encoding ◽  
Exclusion Chromatography ◽  
Ige Epitope

Immunoglobulin of class E(IgE) plays a central role in allergies and allergic asthma. Therapeutic anti-IgE vaccine is expected to elicit autologous antibodies targeting the endogenous IgE molecules and neutralize allergic mechanism. Virus-like particles (VLPs) are a form of immunogens capable of breaking self-tolerance and represent an attractive platform for designing the therapeutic vaccines against self-antigens, such as the IgE. We designed VLPsfrom the core protein of hepatitis B virus (HBcAg). Arrays of the receptor-contacting epitopes of the human IgE were engineered on the VLP surfaces.Objectives: Primary objective of this study was to develop IgE-epitope antigen that induces antibodies against the receptor-contacting epitope on the human IgE molecule.Methods: Genes encoding the empty carrier and HBcAg with insertion of IgE epitope were constructed de novo and cloned into pET expression vectors. Ultracentrifugation in sucrose gradients and size exclusion chromatography (SEC) were used to purify VLPs.Electron microscopy and dynamic light scattering were used for proof of the VLPs appearance. Mice were immunized, andwestern blotswere conducted with immune sera to reveal the presence of the induced anti-(human)IgE antibodies. Titers of the anti-IgE were measured in ELISA.Results: Two types ofplasmid constructs for expression of the VLPs carrying IgEepitope arrays were produced. One typerepresents aninsertion of an immunogenic peptide into HBcAg by construction of a contiguous fusion protein in which the peptide is flanked with sequences of the carrier. The other type is different in that the carrier protein is “split” into two polypeptide chains which upon expression remain associated in a stable VLP-forming subunit. The splitting was achieved by construction of a bicistronic RNA (s.c. “SplitCore” technology). The obtained IgE-epitope antigens with contiguous sequences appeared to be incapable of formation of VLPs. On the contrary, the split core protein efficiently formed VLPs. Immunization of mice with the VLPs presenting the IgE Cε3 FG loop resulted in the development of high titers of antibodies-recognizing human IgE in ELISA.Conclusion: Recombinantly expressed VLPs presenting the IgE Cε3 FG loop elicitthe anti-IgE antibodies upon immunization. Application of the SplitCore technology to construct IgE-epitope antigens can result in a pan-allergy anti-IgE vaccine.

scholarly journals Selenosugars Are the Major Selenometabolites in Liver of Turkeys Fed High Selenium

2020 ◽  
Vol 4 (Supplement_2) ◽  
pp. 1840-1840
Author(s):  
Roger Sunde ◽  
Katarzyna Bierla ◽  
Rachel Taylor ◽  
Joanna Szpunar ◽  
Ryszard Lobinski
Keyword(s):  
De Novo ◽  
Water Soluble ◽  
Size Exclusion ◽  
Deficient Diet ◽  
Funding Sources ◽  
Mixed Disulfide ◽  
Turkey Poults ◽  
Nutrition Research ◽  
Exclusion Chromatography ◽  
Se Status

Abstract Objectives Liver and other tissues accumulate selenium (Se) when animals are supplemented with high dietary Se as inorganic Se. Because the nature of this accumulated Se is not well characterized, we studied selenometabolomics in Se-deficient, Se-adequate, and high-Se liver. Methods Turkey poults were fed 0, 0.4, and 5 μg Se/g diet as Na2SeO3 in a Se-deficient (0.005 μg Se/g) diet for 28 days, and the effects of Se status determined using HPLC-ICP-MS and HPLC-ESI-MS/MS. Results In liver from turkeys fed a true Se-deficient diet and supplemented with inorganic Se (selenite), no selenomethionine (SeMet) was detected showing that the turkey cannot synthesize SeMet de novo from inorganic Se. Selenocysteine (Sec) was also below the level of detection in Se-deficient turkey liver, as expected in animals with negligible selenoprotein levels. Sec content in liver of turkeys fed high Se only doubled compared with Se-adequate liver, indicating that the 6-fold incryscease in liver Se in these birds was not due to increases in selenoproteins. What increased dramatically in high Se liver were the low molecular weight (MW) selenometabolites, glutathione-, cysteine- and methyl- conjugates of the selenosugar, seleno-N-acetyl galactosamine (SeGalNac). In addition, size-exclusion chromatography and followup analysis demonstrated that a substantial amount of Se in Se-adequate liver was present as selenosugars decorating general proteins via mixed-disulfide links, in addition to Sec-containing selenoproteins. In high-Se liver, these “selenosugar-decorated” proteins comprised ∼50% of the Se in the water-soluble fraction, in addition to the low MW selenometabolites. Conclusions In Se-adequate liver, far more Se is present as the selenosugar moiety, mostly decorating general proteins, than is present as Sec in selenoproteins. With high Se supplementation, increased selenosugar formation occurs, further increasing selenosugar-decorated proteins, but also increasing selenosugar linked to low MW thiols, leading to the formation of methyl-SeGalNac. This suggested pathway, underlying adaptation to high Se status in animals, needs further investigation including study of the potential of selenosugar compounds as biomarkers of high Se status. Funding Sources USDA, Hatch project 1,004,389, and by the Wisconsin Alumni Foundation, Selenium Nutrition Research Fund 12,046,295.

mTOR inhibition with temsirolimus causes acute increases in glomerular permeability, but inhibits the dynamic permeability actions of puromycin aminonucleoside

2015 ◽  
Vol 308 (10) ◽  
pp. F1056-F1064 ◽  
Author(s):  
Josefin Axelsson ◽  
Anna Rippe ◽  
Bengt Rippe
Keyword(s):  
High Performance ◽  
De Novo ◽  
Mammalian Target Of Rapamycin ◽  
Size Exclusion ◽  
Ang Ii ◽  
Puromycin Aminonucleoside ◽  
Glomerular Permeability ◽  
Mtor Inhibition ◽  
Transplant Patients ◽  
Exclusion Chromatography

Inhibitors of the mammalian target of rapamycin (mTORi) can produce de novo proteinuria in kidney transplant patients. On the other hand, mTORi has been shown to suppress disease progression in several animal models of kidney disease. In the present study, we investigated whether glomerular permeability can be acutely altered by the mTORi temsirolimus and whether mTORi can affect acute puromycin aminonucleoside (PAN) or angiotensin II (ANG II)-induced glomerular hyperpermeability. In anesthetized Wistar rats, the left ureter was cannulated for urine collection, while simultaneously blood access was achieved. Temsirolimus was administered as a single intravenous dose 30 min before the start of the experiments in animals infused with PAN or ANG II or in nonexposed animals. Polydispersed FITC-Ficoll-70/400 (molecular radius 10–80 Å) and 51Cr-EDTA infusion was given during the whole experiment. Measurements of Ficoll in plasma and urine were performed sequentially before the temsirolimus injection (baseline) and at 5, 15, 30, 60, and 120 min after the start of the experiments. Urine and plasma samples were analyzed by high-performance size-exclusion chromatography (HPSEC) to assess glomerular sieving coefficients (θ) for Ficoll10-80Å. Temsirolimus per se increased baseline glomerular permeability to Ficoll50-80Å 45 min after its administration, a reactive oxygen species (ROS)-dependent phenomenon. PAN caused a rapid and reversible increase in glomerular permeability, peaking at 5 min, and again at 60–120 min, which could be blocked by the ROS scavenger tempol. mTORi abrogated the second permeability peak induced by PAN. However, it had no effect on the immediate ANG II- or PAN-induced increases in glomerular permeability.

scholarly journals Identification of Two Catalases in Azotobacter vinelandii: a KatG Homologue and a Novel Bacterial Cytochrome c Catalase, CCCAv

2007 ◽  
Vol 190 (3) ◽  
pp. 954-962 ◽  
Author(s):  
James R. Sandercock ◽  
William J. Page
Keyword(s):  
Azotobacter Vinelandii ◽  
Residual Activity ◽  
Peptide Sequence ◽  
Size Exclusion ◽  
Proteinase K ◽  
Exponential Growth Phase ◽  
Binding Motif ◽  
Fold Reduction ◽  
Exclusion Chromatography ◽  
Liquid Chromatography Tandem Mass

ABSTRACT Azotobacter vinelandii produces two detectable catalases during growth on minimal medium. The heat-labile catalase expressed during exponential growth phase was identified as a KatG homologue by liquid chromatography-tandem mass spectrometry (LC-MS/MS) using a mixed protein sample. The second catalase was heat resistant and had substantial residual activity after treatment at 90°C. This enzyme was purified by anion-exchange and size exclusion chromatography and was found to exhibit strong absorption at 407 nm, which is often indicative of associated heme moieties. The purified protein was fragmented by proteinase K and identified by LC-MS/MS. Some identity was shared with the MauG/bacterial cytochrome c peroxidase (BCCP) protein family, but the enzyme exhibited a strong catalase activity never before observed in this family. Because two putative c-type heme sites (CXXCH) were predicted in the peptide sequence and were demonstrated experimentally, the enzyme was designated a cytochrome c catalase (CCC Av ). However, the local organization of the CCC Av heme motifs differed significantly from that of the BCCPs as the sites were confined to the C-terminal half of the catalase. A possible Ca2+ binding motif, previously described in the BCCPs, is also present in the CCC Av peptide sequence. Some instability in the presence of EGTA was observed. Expression of the catalase was abolished in cccA mutants, resulting in a nearly 8,700-fold reduction in peroxide resistance in stationary phase.

scholarly journals A Comparative Proteomic Analysis of Extracellular Vesicles Associated With Lipotoxicity

2021 ◽  
Vol 9 ◽  
Author(s):  
Yasuhiko Nakao ◽  
Masanori Fukushima ◽  
Amy S. Mauer ◽  
Chieh-Yu Liao ◽  
Anya Ferris ◽  
...  
Keyword(s):  
Size Exclusion Chromatography ◽  
Extracellular Vesicles ◽  
Proteomic Analysis ◽  
De Novo ◽  
Size Exclusion ◽  
Dependent Manner ◽  
Exclusion Chromatography ◽  
Acid Labile Subunit ◽  
Molecular Patterns ◽  
Gradient Separation

Extracellular vesicles (EVs) are emerging mediators of intercellular communication in nonalcoholic steatohepatitis (NASH). Palmitate, a lipotoxic saturated fatty acid, activates hepatocellular endoplasmic reticulum stress, which has been demonstrated to be important in NASH pathogenesis, including in the release of EVs. We have previously demonstrated that the release of palmitate-stimulated EVs is dependent on the de novo synthesis of ceramide, which is trafficked by the ceramide transport protein, STARD11. The trafficking of ceramide is a critical step in the release of lipotoxic EVs, as cells deficient in STARD11 do not release palmitate-stimulated EVs. Here, we examined the hypothesis that protein cargoes are trafficked to lipotoxic EVs in a ceramide-dependent manner. We performed quantitative proteomic analysis of palmitate-stimulated EVs in control and STARD11 knockout hepatocyte cell lines. Proteomics was performed on EVs isolated by size exclusion chromatography, ultracentrifugation, and density gradient separation, and EV proteins were measured by mass spectrometry. We also performed human EV proteomics from a control and a NASH plasma sample, for comparative analyses with hepatocyte-derived lipotoxic EVs. Size exclusion chromatography yielded most unique EV proteins. Ceramide-dependent lipotoxic EVs contain damage-associated molecular patterns and adhesion molecules. Haptoglobin, vascular non-inflammatory molecule-1, and insulin-like growth factor-binding protein complex acid labile subunit were commonly detected in NASH and hepatocyte-derived ceramide-dependent EVs. Lipotoxic EV proteomics provides novel candidate proteins to investigate in NASH pathogenesis and as diagnostic biomarkers for hepatocyte-derived EVs in NASH patients.

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