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Results for “proteome stability”

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At least 289 records · Page 16Linked to original sources
PubMed2003

Fluorobodies combine GFP fluorescence with the binding characteristics of antibodies.

The difficulty of deriving binding ligands to targets identified by genomic sequencing has led to a bottleneck in genomic research. By inserting diverse antibody binding loops into four of the exposed loops at one end of green fluorescent protein (GFP), we have mimicked the natural antibody binding footprint to create robust binding ligands that combine the advantages of antibodies (high affinity and specificity) with those of GFP (intrinsic fluorescence, high stability, expression and solubility). These 'fluorobodies' have been used effectively in enzyme-linked immunosorbent assays (ELISAs), flow cytometry, immuno-fluorescence, arrays and gel shift assays, and show affinities as high as antibodies. Furthermore, the intrinsic fluorescence of fluorobodies correlates with binding activity, allowing the rapid determination of functionality, concentration and affinity. These properties render them especially suitable for the high-throughput genomic scale selections required in proteomics, as well as in diagnostics, target validation and drug development.

Antigen-Antibody Complex↗
PubMed2004

Defining the SUMO-modified proteome by multiple approaches in Saccharomyces cerevisiae.

SUMO, or Smt3 in Saccharomyces cerevisiae, is a ubiquitin-like protein that is post-translationally attached to multiple proteins in vivo. Many of these substrate modifications are cell cycle-regulated, and SUMO conjugation is essential for viability in most eukaryotes. However, only a limited number of SUMO-modified proteins have been definitively identified to date, and this has hampered study of the mechanisms by which SUMO ligation regulates specific cellular pathways. Here we use a combination of yeast two-hybrid screening, a high copy suppressor selection with a SUMO isopeptidase mutant, and tandem mass spectrometry to define a large set of proteins (>150) that can be modified by SUMO in budding yeast. These three approaches yielded overlapping sets of proteins with the most extensive set by far being those identified by mass spectrometry. The two-hybrid data also yielded a potential SUMO-binding motif. Functional categories of SUMO-modified proteins include SUMO conjugation system enzymes, chromatin- and gene silencing-related factors, DNA repair and genome stability proteins, stress-related proteins, transcription factors, proteins involved in translation and RNA metabolism, and a variety of metabolic enzymes. The results point to a surprisingly broad array of cellular processes regulated by SUMO conjugation and provide a starting point for detailed studies of how SUMO ligation contributes to these different regulatory mechanisms.

Amino Acid Motifs↗
PubMed2005

Prediction of collagen stability from amino acid sequence.

An algorithm was derived to relate the amino acid sequence of a collagen triple helix to its thermal stability. This calculation is based on the triple helical stabilization propensities of individual residues and their intermolecular and intramolecular interactions, as quantitated by melting temperature values of host-guest peptides. Experimental melting temperature values of a number of triple helical peptides of varying length and sequence were successfully predicted by this algorithm. However, predicted T(m) values are significantly higher than experimental values when there are strings of oppositely charged residues or concentrations of like charges near the terminus. Application of the algorithm to collagen sequences highlights regions of unusually high or low stability, and these regions often correlate with biologically significant features. The prediction of stability from sequence indicates an understanding of the major forces maintaining this protein motif. The use of highly favorable KGE and KGD sequences is seen to complement the stabilizing effects of imino acids in modulating stability and may become dominant in the collagenous domains of bacterial proteins that lack hydroxyproline. The effect of single amino acid mutations in the X and Y positions can be evaluated with this algorithm. An interactive collagen stability calculator based on this algorithm is available online.

Algorithms↗
PubMed2005

Intracellular inhibitory effects of Velcade correlate with morphoproteomic expression of phosphorylated-nuclear factor-kappaB and p53 in breast cancer cell lines.

Velcade, a proteasome inhibitor, has been shown to inhibit DNA binding activity of nuclear factor-kappaB (NF-kappaB) and to stabilize p53 in vitro. But its impact, in the context of activated (phosphorylated and translocated) NF-kappaB and the expression of p53, has not been studied in breast cancer. It would be desirable to determine whether or not the immunohistochemical (IHC) expressions of activated NF-kappaB and of p53 can predict the effects of Velcade in viable tumor cells. To answer these questions, we selected 3 breast cancer cell lines (SKBR-3, MDA-175, and MDA-231), which are negative for hormonal receptors, but differ in HER-2/neu expression (strong, mild, and minimal, respectively). The 3 cell lines showed different expressions of phosphorylated (p)- NF-kappaB and p53, as evaluated using immunohistochemistry with visual quantification by brightfield microscopy. After being treated with Velcade for 2 days, MDA-231 cells showed markedly reduced proliferation, followed by SKBR-3 cells, and then by MDA-175 cells. There was strong correlation between the nuclear expression of either p-NF-kappaB or p53 and the inhibitory rate of Velcade in the 3 cell lines (r = 0.987 and 0.807, respectively). Western blotting showed an increase in inhibitor-kappaB (I-kappaB) expression in nuclei of MDA-231 and SKBR-3 cells, but not in MDA-175 cells, following exposure to Velcade. Velcade treatment resulted in cleaved caspase-3 expression in MDA-231 cells and in the overexpression of p53 and p21WAF1 in all 3 cell lines, as evaluated using Western blotting. In summary, morphoproteomic analysis of p-NF-kappaB and p53 can be correlated with the inhibitory effect of Velcade in vitro. We propose that this proliferative inhibition is variably associated with blocking p-NF-kappaB function by upregulation of nuclear I-kappaB, stabilization of p53, and induction of p21WAF1.

Antineoplastic Agents↗
PubMed2005

Human Ran cysteine 112 oxidation by pervanadate regulates its binding to keratins.

We used a proteomic approach to identify proteins that associate with keratins 8 or 18 (K8/K18) in a pervanadate-dependent manner. Pervanadate triggers Ran-K8/K18 binding and a gel-migration-shift of Ran from 25 to 27 kDa, which does not occur upon exposure to H2O2 or vanadate or if pervanadate is excluded during cell solubilization. Generation of 27-kDa Ran is not related to hyperphosphorylation, is heat-insensitive, but occurs upon conversion of Ran cysteines to cysteic acid. The pervanadate-mediated Ran cysteine --> cysteic acid oxidation and its related gel migration shift affects other proteins including actin. Mutation of the three Ran cysteines (Cys-85, -112, and -120) showed that Ran Cys-112 oxidation generates 27-kDa Ran and accounts for its keratin binding. Proteasome inhibition accentuates Ran-keratin binding after cell exposure to pervanadate. Therefore, cell-free exposure to pervanadate causes cysteine to cysteic acid oxidation of Ran and several other proteins and Ran-K8/K18 association. In cells, stabilization of oxidized Ran by proteasome inhibition promotes Ran-keratin interaction. Keratin sequestration of oxidized Ran may provide a back-up protective mechanism in some cases of oxidative injury.

Actins↗
PubMed2002

Myocardial proteome analysis reveals reduced NOS inhibition and enhanced glycolytic capacity in areas of low local blood flow.

In the heart, in situ local myocardial blood flow (MBF) varies greater than 10-fold between individual areas and displays a spatially heterogeneous pattern. To analyze its molecular basis, we analyzed protein expression of low and high flow samples (300 mg, <50% or >150% of mean MBF, each n=30) of six beagle dogs by 2-D polyacrylamide gel electrophoresis (380 +/- 78 spots/gel). In low flow samples, dimethylarginine dimethylaminohydrolase (DDAH1) was increased greatly (+377%, compared with high flow samples). This increase resulted in a 75% reduction of asymmetric dimethylarginine (ADMA), the potent endogenous inhibitor of NO synthase, whereas eNOS showed no difference. Low flow samples exhibited enhanced expression of GAPDH (+89%) and phosphoglycerate kinase (+100%), whereas hydroxyacyl-CoA dehydrogenase, electron transfer flavoprotein, myoglobin, and desmin were decreased. Assessing local MBF on different days within 2 weeks revealed a high degree of MBF stability (r2 > 0.79). Thus, stable differences in local MBF are associated with significant differences in local gene and protein expression. In low flow areas, the increased DDAH1 reduces ADMA concentration and NOS inhibition, which strongly suggests enhanced NO formation. Low flow areas are also characterized by a higher glycolytic and a lower fatty acid oxidation capacity. Both the shift in substrate utilization and the rise in NO may contribute to the known lower oxygen consumption in these areas.

Amidohydrolases↗
PubMed2003

Comparative proteomic analysis of proliferating and functionally differentiated mammary epithelial cells.

Proliferation and differentiation of mammary epithelial cells are governed by hormonal stimuli, cell-cell, and cell-matrix interactions. Terminal differentiation of mammary epithelial cells depends upon the action of the lactogenic hormones, insulin, glucocorticoids, and prolactin that enable them to synthesize and secrete milk proteins. These differentiated cells are polarized and carry out vectorial transport of milk constituents across the apical plasma membrane. To gain additional insights into the mechanisms governing differentiation of mammary epithelial cells, we identified proteins whose expression distinguishes proliferating from differentiated mammary epithelial cells. For this purpose we made use of the HC11 mammary epithelial line, which is capable of differentiation in response to lactogenic hormones. Using two-dimensional gel electrophoresis and mass spectrometry, we found about 60 proteins whose expression levels changed in between these two differentiation states. Bioinformatic analysis revealed differential expression of cytoskeletal components, molecular chaperones and regulators of protein folding and stability, calcium-binding proteins, and components of RNA-processing pathways. The actin cytoskeleton is asymmetrically distributed in differentiated epithelial cells, and the identification of proteins involved in mRNA binding and localization suggests that asymmetry might in part be achieved by controlling cellular localization of mRNAs. The proteins identified provide insights into the differentiation of mammary epithelial cells and the regulation of this process.

Animals↗
PubMed2005

A "solvated rotamer" approach to modeling water-mediated hydrogen bonds at protein-protein interfaces.

Water-mediated hydrogen bonds play critical roles at protein-protein and protein-nucleic acid interfaces, and the interactions formed by discrete water molecules cannot be captured using continuum solvent models. We describe a simple model for the energetics of water-mediated hydrogen bonds, and show that, together with knowledge of the positions of buried water molecules observed in X-ray crystal structures, the model improves the prediction of free-energy changes upon mutation at protein-protein interfaces, and the recovery of native amino acid sequences in protein interface design calculations. We then describe a "solvated rotamer" approach to efficiently predict the positions of water molecules, at protein-protein interfaces and in monomeric proteins, that is compatible with widely used rotamer-based side-chain packing and protein design algorithms. Finally, we examine the extent to which the predicted water molecules can be used to improve prediction of amino acid identities and protein-protein interface stability, and discuss avenues for overcoming current limitations of the approach.

Algorithms↗
PubMed2004

Pentamerization of single-domain antibodies from phage libraries: a novel strategy for the rapid generation of high-avidity antibody reagents.

We describe a novel type of molecule in which single-domain antibodies (sdAbs) isolated from a nai;ve llama single domain antibody library are linked to an oligomerization domain to generate high-avidity, antigen-binding reagents. An sdAb is fused to the B-subunit of Escherichia coli verotoxin, or shiga-like toxin, which self-assembles to form a homopentamer and results in simultaneous sdAb pentamerization and introduction of avidity. Molecular modeling indicated that this fusion protein (PDB: 1OJF), termed pentabody, has structural flexibility for binding to surface-presented antigen. In the instance of an sdAb specific for a peptide antigen, pentamerization resulted in a dramatic increase in functional affinity for immobilized antigen. The pentabody was expressed in high yield in E.coli in a non-aggregated state, and exhibited excellent thermostability and protease resistance. This technology provides a relatively rapid means of generating novel antigen-binding molecules that bind strongly to immobilized antigen. It is expected that pentavalent sdAbs will have general applicability in proteomics, immunochemical staining, cancer diagnosis and other applications in which antigens are presented multivalently.

Animals↗
PubMed2024

Nanobodies: From High-Throughput Identification to Therapeutic Development.

The camelid single-domain antibody fragment, commonly referred to as a nanobody, achieves the targeting power of conventional monoclonal antibodies (mAbs) at only a fraction of their size. Isolated from camelid species (including llamas, alpacas, and camels), their small size at &#x223c;15&#xa0;kDa, low structural complexity, and high stability compared with conventional antibodies have propelled nanobody technology into the limelight of biologic development. Nanobodies are proving themselves to be a potent complement to traditional mAb therapies, showing success in the treatment of, for example, autoimmune diseases and cancer, and more recently as therapeutic options to treat infectious diseases caused by rapidly evolving biological targets such as the SARS-CoV-2 virus. This review highlights the benefits of applying a proteomic approach to identify diverse nanobody sequences against a single antigen. This proteomic approach coupled with conventional yeast/phage display methods enables the production of highly diverse repertoires of nanobodies able to bind the vast epitope landscape of an antigen, with epitope sampling surpassing that of mAbs. Additionally, we aim to highlight recent findings illuminating the structural attributes of nanobodies that make them particularly amenable to comprehensive antigen sampling and to synergistic activity-underscoring the powerful advantage of acquiring a large, diverse nanobody repertoire against a single antigen. Lastly, we highlight the efforts being made in the clinical development of nanobodies, which have great potential as powerful diagnostic reagents and treatment options, especially when targeting infectious disease agents.

Animals↗
PubMed2005

DNA-protein crosslinks: their induction, repair, and biological consequences.

The covalent crosslinking of proteins to DNA presents a major physical challenge to the DNA metabolic machinery. DNA-protein crosslinks (DPCs) are induced by a variety of endogenous and exogenous agents (including, paradoxically, agents that are known to cause cancer as well as agents that are used to treat cancer), and yet they have not received as much attention as other types of DNA damage. This review summarizes the current state of knowledge of DPCs in terms of their induction, structures, biological consequences and possible mechanisms of repair. DPCs can be formed through several different chemistries, which is likely to affect the stability and repair of these lesions, as well as their biological consequences. The considerable discrepancy in the DPC literature reflects both the varying chemistries of this heterogeneous group of lesions and the fact that a number of different methods have been used for their analysis. In particular, research in this area has long been hampered by the inability to chemically define these lesions in intact cells and tissues. However, the emergence of proteomics as a tool for identifying specific proteins that become crosslinked to DNA has heralded a new era in our ability to study these lesions. Although there are still many unanswered questions, the identification of specific proteins crosslinked to DNA should facilitate our understanding of the down-stream effects of these lesions.

DNA↗
PubMed2005

Chaotic tumors and 2 mistakes of molecular oncologists.

The structure of DNA is wondrously appealing in its elegant simplicity combined with its captivating power. The power of DNA derives directly from, and solely from, the fact that it represents digital information. However, this delightful digital determinism does not carry over into the multitude of cellular processes, especially in a cancer cell. This means that the sequencing of the human genome has some limitations regarding furthering the understanding of details of cancer cell biochemistry. The fading of digital control in cells, however, seems not well comprehended by most. This factor, together with the loss of DNA stability that is an underpinning of cancer, virtually guarantees that chaotic dynamics are very important in cancer cells. Yet, chaos is very underappreciated in oncology. Failure to appreciate the significance of the dissolution of digital control in cell dynamics and the lack of perception of the importance of chaotic dynamics in the cancer cell are 2 errors that will tend to enhance the frequency of mistakes made in analyzing information from gene expression microarrays, proteomic microarrays, and other advances in molecular oncology. This article explains how these mistakes will come about and why, in some instances, they could result in adverse effects in patient therapy as the goals for targeted, individualized, and molecular-based treatments are sought.

DNA, Neoplasm↗
PubMed2002

Manipulation of the cell cycle by human cytomegalovirus.

The human cytomegalovirus-induced changes to the transcriptome and proteome of infected cells in many ways resemble an abortive mitogenic response. The virus induces quiescent cells to re-enter the cell cycle, but they are prevented from entering the S phase, where the synthesis of the cellular genome would compete with that of the virus for the available precursors for DNA replication. The mechanisms of these cell cycle alterations include transcriptional induction and repression, post-translational modifications and changes in protein stability. Essentially every class of cell cycle regulators is affected, and some of the key proteins are targeted by multiple different mechanisms. While the effects on cell cycle progression of viral infection, and of individual viral genes outside the context of viral infection have been described, it is now important to synthesize these two experimental approaches to gain a more complete understanding of how and why human cytomegalovirus infection affects cell cycle progression.

Animals↗
PubMed2005

Proteomic analysis of the venom and characterization of toxins specific for Na+ - and K+ -channels from the Colombian scorpion Tityus pachyurus.

The Colombian scorpion Tityus pachyurus is toxic to humans and is capable of producing fatal accidents, but nothing is known about its venom components. This communication reports the separation of at least 57 fractions from the venom by high performance liquid chromatography. From these, at least 104 distinct molecular weight compounds were identified by mass spectrometry analysis. The complete amino acid sequences of three peptides were determined and the partial sequences of three others were also identified. Electrophysiological experiments conducted with ion-channels expressed heterologously on Sf9 cells showed the presence of a potent Shaker B K(+)-channel blocker. This peptide (trivial name Tpa1) contains 23 amino acid residues closely packed by three disulfide bridges with a molecular mass of 2,457 atomic mass units. It is the third member of the sub-family 13, for which the systematic name is proposed to be alpha-KTx13.3. The mice assay showed clearly the presence of toxic peptides to mammals. One of them named Tpa2, containing 65 amino acid residues with molecular mass of 7,522.5 atomic mass units, is stabilized by four disulfide bridges. It was shown to modify the Na(+)-currents of F-11 and TE671 cells in culture, similar to the beta scorpion toxins. These results demonstrate the presence of toxic peptides in the venom of T. pachyurus and confirm that accidents with this species of scorpion should be considered an important human hazard in Colombia.

Amino Acid Sequence↗
PubMed2005

Roles of plasma proteins in the formation of silicotic nodules in rats.

The contribution of plasma protein(s) to the stabilization of fibroids formed in rat lungs exposed to acute silica dust inhalation was examined. Antibodies against component proteins of the nodules remaining insoluble in 2% SDS, 10M urea and 40 mM sulfhydryl reagents under prolonged boiling conditions were raised in rabbits and used to capture plasma proteins, which were identified by 2D-gel electrophoresis and MALDI-TOF analysis. The silica particles were encapsulated with extracellular protein composites whose amino acid compositions showed high levels of alanine, i.e., above those of glycine and proline, a building block of collagen. Antibody-captured plasma proteins showed the dominant presences of fibrinogen, albumin, and prealbumin (transthyretin), and other minor proteins, which included alpha-1-protease inhibitor, contraspin-like protease inhibitor, cathepsin B, etc. The presence of the N(epsilon)-(gamma-glutamyl) lysine isopeptide bond in the nodules was evidenced by direct chemical methods and by immunoreactivity for anti-isopeptide bonds. Immunostaining of affected lung tissue and of the fibroid regions showed elevated levels of transglutaminase (TGase) E and plasma factor XIII (F-XIII), but showed no reactivity towards other TGases. These findings suggest that the silica encapsulated nodules are a mixture of extracellular proteins that include collagen type I, fibrin and transthyretin, which is stabilized by TGase catalyzed crosslinking between plasma and extracellular proteins during fibrosis to eventually form insoluble nodules.

Air Pollutants, Occupational↗
PubMed2003

Nonsense-mediated decay breaks the circle?

This Commentary discusses new data from the laboratory of Matthias Hentze (Schell et al., in this issue) that begins to dissect the RNA-protein and protein-protein interactions important for the process of nonsense-mediated decay (NMD). Schell and co-workers have developed a novel tool for analysis of such interactions in vivo. Using a proteomics approach, they have identified two different protein-RNA complexes that contain human Upf (up-frameshift) proteins central to the NMD process. Intriguingly, they identified a poly[A]-binding protein associated with these complexes.

Codon, Nonsense↗
PubMed2002

Physiological analysis of the role of truB in Escherichia coli: a role for tRNA modification in extreme temperature resistance.

The truB gene of Escherichia coli encodes the pseudouridine-55 (psi55) synthase and is responsible for modifying all tRNA molecules in the cell at the U55 position. A truB null mutant grew normally on all growth media tested, but exhibited a competitive disadvantage in extended co-culture with its wild-type progenitor. The mutant phenotype could be complemented by both the cloned truB gene and by a D48C, catalytically inactive allele of truB. The truB mutant also exhibited a defect in survival of rapid transfer from 37 to 50 degrees C. This mutant phenotype could be complemented by the cloned truB gene but not by a D48C, catalytically inactive allele of truB. The temperature sensitivity of truB mutants could be enhanced by combination with a mutation in the trmA gene, encoding an m(5)U-methyltransferase, modifying the universal U54 tRNA nucleoside, but not by mutations in trmH, encoding the enzyme catalysing the formation of Gm18. The truB mutant proteome contained altered levels of intermediates involved in biogenesis of the outer-membrane proteins OmpA and OmpX. The truB mutation also reduced the basal expression from two sigma(E) promoters, degP and rpoHP3. Three novel aspects to the phenotype of truB mutants were identified. Importantly the data support the hypothesis that TruB-effected psi55 modification of tRNA is not essential, but contributes to thermal stress tolerance in E. coli, possibly by optimizing the stability of the tRNA population at high temperatures.

Bacterial Outer Membrane Proteins↗
PubMed2006

On-chip protein synthesis for making microarrays.

Protein microarrays are a miniaturized format for displaying in close spatial density hundreds or thousands of purified proteins that provide a powerful platform for the high-throughput assay of protein function. The traditional method of producing them requires the high-throughput production and printing of proteins, a laborious method that raises concerns about the stability of the proteins and the shelf life of the arrays. A novel method of producing protein microarrays, called nucleic acid programmable protein array (NAPPA), overcomes these limitations by synthesizing proteins in situ. NAPPA entails spotting plasmid DNA encoding the relevant proteins, which are then simultaneously transcribed and translated by a cell-free system. The expressed proteins are captured and oriented at the site of expression by a capture reagent that targets a fusion protein on either the N- or C-terminus of the protein. Using a mammalian extract, NAPPA expresses and captures 1000-fold more protein per feature than conventional protein-printing arrays. Moreover, this approach minimizes concerns about protein stability and integrity, because proteins are produced just in time for assaying. NAPPA has already proven to be a robust tool for protein functional assays.

Animals↗