Biomedical subjects

R Aebersold

Publications and source records attributed to R Aebersold.

At least 37 records · Page 2Linked to original sources

PubMed2001

Identification of the target of monoclonal antibody A6H as dipeptidyl peptidase IV/CD26 by LC MS\MS.

The monoclonal antibody (MAb) A6H, originally developed to fetal renal tissues, was found to be highly reactive to renal cell carcinoma and was subsequently demonstrated to co-stimulate a subpopulation of T cells. The A6H antigen had not been identified heretofore. Antigen from detergent extracts of renal cell carcinoma cells (7860) was immunoabsorbed with A6H-agarose, and the resin-bound proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The antigen had a molecular weight of approximately 120 kDa as determined by Western blots. The 120-kDa protein band was excised and subjected to in-gel tryptic digestion, and the resulting peptides were separated and analyzed by liquid chromatography tandem mass spectrometry (LC MS\MS). The tandem mass spectra of the eluting peptides were used in combination with the SEQUEST computer program to search a human National Cancer Institute (NCI) protein database for the identity of the protein. The target antigen was shown to be dipeptidyl peptidase IV (DPP IV), which is also known as the cluster differentiation antigen CD26. Flow analysis of the expression of the A6H antigen and of CD26 on 7860 cells and on peripheral blood lymphocytes supported the identification of the A6H antigen as DPP IV. Recognition that the A6H antigen is DPP IV/CD26 afforded the opportunity to compare previous studies on A6H with those on other anti-CD26 antibodies in terms of expression in cancer cell lines and various tissues and as co-stimulators of T-cell activation.

Amino Acid Sequence↗

PubMed2001

Association of two novel proteins, TbMP52 and TbMP48, with the Trypanosoma brucei RNA editing complex.

RNA editing in kinetoplastid mitochondria inserts and deletes uridylates at multiple sites in pre-mRNAs as directed by guide RNAs. This occurs by a series of steps that are catalyzed by endoribonuclease, 3'-terminal uridylyl transferase, 3'-exouridylylase, and RNA ligase activities. A multiprotein complex that contains these activities and catalyzes deletion editing in vitro was enriched from Trypanosoma brucei mitochondria by sequential ion-exchange and gel filtration chromatography, followed by glycerol gradient sedimentation. The complex size is approximately 1,600 kDa, and the purified fraction contains 20 major polypeptides. A monoclonal antibody that was generated against the enriched complex reacts with an approximately 49-kDa protein and specifically immunoprecipitates in vitro deletion RNA editing activity. The protein recognized by the antibody was identified by mass spectrometry, and the corresponding gene, designated TbMP52, was cloned. Recombinant TbMP52 reacts with the monoclonal antibody. Another novel protein, TbMP48, which is similar to TbMP52, and its gene were also identified in the enriched complex. These results suggest that TbMP52 and TbMP48 are components of the RNA editing complex.

Amino Acid Sequence↗

PubMed2001

Four related proteins of the Trypanosoma brucei RNA editing complex.

RNA editing in kinetoplastid mitochondria occurs by a series of enzymatic steps that is catalyzed by a macromolecular complex. Four novel proteins and their corresponding genes were identified by mass spectrometric analysis of purified editing complexes from Trypanosoma brucei. These four proteins, TbMP81, TbMP63, TbMP42, and TbMP18, contain conserved sequences to various degrees. All four proteins have sequence similarity in the C terminus; TbMP18 has considerable sequence similarity to the C-terminal region of TbMP42, and TbMP81, TbMP63, and TbMP42 contain zinc finger motif(s). Monoclonal antibodies that are specific for TbMP63 and TbMP42 immunoprecipitate in vitro RNA editing activities. The proteins are present in the immunoprecipitates and sediment at 20S along with the in vitro editing, and RNA editing ligases TbMP52 and TbMP48. Recombinant TbMP63 and TbMP52 coimmunoprecipitate. These results indicate that these four proteins are components of the RNA editing complex and that TbMP63 and TbMP52 can interact.

Amino Acid Motifs↗

PubMed2000

Ser(2194) is a highly conserved major phosphorylation site of the hepatitis C virus nonstructural protein NS5A.

Phosphorylation of the nonstructural NS5A protein is highly conserved among hepatitis C virus (HCV) genotypes. However, the precise site or sites of phosphorylation of NS5A have not been determined, and the functional significance of phosphorylation remains unknown. Here, we showed by two-dimensional phosphopeptide mapping that a protein kinase or kinases present in yeast, insect, and mammalian cells phosphorylated a highly purified HCV genotype 1b NS5A from insect cells on identical serine residues. We identified a major phosphopeptide (corresponding to amino acids 2193-2212 of the HCV 1b polyprotein) by using negative-ion electrospray ionization-microcapillary high performance liquid chromatography-mass spectrometry. The elution time of the phosphopeptide determined by negative-ion electrospray ionization-mass spectrometry corresponded with the elution time of the majority of (32)P-label that was incorporated into the phosphopeptide by an in vitro kinase reaction. Subsequent analysis of the peak fraction by automated positive-ion electrospray ionization-tandem mass spectrometry revealed that Ser(2194) was the major phosphorylated residue on the phosphopeptide GpSPPSLASSSASQLSAPSLK. Substitution for Ser(2194) with Ala resulted in the concomitant disappearance of major in vivo phosphorylated peptides. Ser(2194) and surrounding amino acids are highly conserved in all HCV genotypes, suggesting NS5A phosphorylation at Ser(2194) may be an important mechanism for modulating NS5A biological functions.

Amino Acid Sequence↗

PubMed2000

A role for poly(ADP-ribose) polymerase in the transcriptional regulation of the melanoma growth stimulatory activity (CXCL1) gene expression.

The melanoma growth stimulatory activity/growth-regulated protein, CXCL1, is constitutively expressed at high levels during inflammation and progression of melanocytes into malignant melanoma. It has been shown previously that CXCL1 overexpression in melanoma cells is due to increased transcription as well as stability of the CXCL1 message. The transcription of CXCL1 is regulated through several cis-acting elements including Sp1, NF-kappaB, HMGI(Y), and the immediate upstream region (IUR) element (nucleotides -94 to -78), which lies immediately upstream to the nuclear factor kappaB (NF-kappaB) element. Previously, it has been shown that the IUR is necessary for basal and cytokine-induced transcription of the CXCL1 gene. UV cross-linking and Southwestern blot analyses indicate that the IUR oligonucleotide probe selectively binds a 115-kDa protein. In this study, the IUR element has been further characterized. We show here that proximity of the IUR element to the adjacent NF-kappaB element is critical to its function as a positive regulatory element. Using binding site oligonucleotide affinity chromatography, we have selectively purified the 115-kDa IUR-F. Mass spectrometry/mass spectrometry/matrix-assisted laser desorption ionization/time of flight spectroscopy and amino acid analysis as well as microcapillary reverse phase chromatography electrospray ionization tandem mass spectrometry identified this protein as the 114-kDa poly(ADP-ribose) polymerase (PARP1). Furthermore, 3-aminobenzamide, an inhibitor of PARP-specific ADP-ribosylation, inhibits CXCL1 promoter activity and reduces levels of CXCL1 mRNA. The data point to the possibility that PARP may be a coactivator of CXCL1 transcription.

Adenosine Diphosphate Ribose↗

PubMed2000

Regulation of the meiosis-inhibited protein kinase, a p38(MAPK) isoform, during meiosis and following fertilization of seastar oocytes.

A p38(MAPK) homolog Mipk (meiosis-inhibited protein kinase) was cloned from seastar oocytes. This 40-kDa protein shares approximately 65% amino acid identity with mammalian p38-alpha isoforms. Mipk was one of the major tyrosine-phosphorylated proteins in immature oocytes arrested at the G(2)/M transition of meiosis I. The tyrosine phosphorylation of Mipk was increased in response to anisomycin, heat, and osmotic shock of oocytes. During 1-methyladenine-induced oocyte maturation, Mipk underwent tyrosine dephosphorylation and remained dephosphorylated in mature oocytes and during the early mitotic cell divisions until approximately 12 h after fertilization. At the time of differentiation and acquisition of G phases in the developing embryos, Mipk was rephosphorylated on tyrosine. In oocytes that were microinjected with Mipk antisense oligonucleotides and subsequently were allowed to mature and become fertilized, differentiation was blocked. Because MipK antisense oligonucleotides and a dominant-negative (K62R)Mipk when microinjected into immature oocytes failed to induce germinal vesicle breakdown, inhibition of Mipk function was not sufficient by itself to cause oocyte maturation. These findings point to a putative role for Mipk in cell cycle control as a G-phase-promoting factor.

Amino Acid Sequence↗

PubMed2000

Simultaneous detection and identification of O-GlcNAc-modified glycoproteins using liquid chromatography-tandem mass spectrometry.

Glycoproteins carrying O-linked N-acetylglucosamine (O-GlcNAc) modifications have been isolated from a wide range of organisms ranging from trypanosomes to humans. Interest in this modification is increasing as evidence accumulates that it is an abundant and transient modification that is dynamic and responsive to cellular stimuli. Concurrent advances in biological mass spectrometry (MS) have facilitated high-sensitivity protein identification by tandem MS. In this study, we show that the lability of the O-GlcNAc moiety to low-energy collision in tandem MS offers a means of distinguishing such peptides from others that are not modified. The differential between the energy required to remove the O-GlcNAc group and the energy required to fragment the peptide chain allows the O-GlcNAc group to be detected and the peptide sequence, and therefore the protein, to be identified. This technique thus allows the simultaneous detection and identification of O-GlcNAc-modified peptides, even when present at low levels in complex mixtures. The method was initially developed and validated using a synthetic O-GlcNAc-modified peptide and then applied to the detection of an extremely low abundance O-GlcNAc-modified peptide from bovine alpha-crystallin. We believe that with further development this assay system may prove to be a useful tool for the direct investigation of intracellular O-GlcNAc levels, thus providing valuable insights into the physiological role of O-GlcNAc modified proteins.

Acetylglucosamine↗

PubMed2000

CArG binding factor A (CBF-A) is involved in transcriptional regulation of the rat Ha-ras promoter.

In the present study we identified a positive transcriptional element within the rat Ha-ras promoter previously known as Ha-ras response element (HRE) and identified a trans-acting factor that binds HRE sequences in rat mammary cells. To identify the binding protein we employed sequence specific DNA affinity chromatography. Amino acid sequence analysis of the affinity-purified proteins was performed by tandem mass spectroscopy. The results unexpectedly demonstrated that in rat mammary cells CArG box-binding factor A (CBF-A) is the major protein species that bind specifically to the rat and human HRE sequences with high affinity. The affinity of CBF-A binding to HRE was significantly higher than to the CArG box described as a recognition sequence for CBF-A protein. Transient transfection assays using reporter plasmids verified that mutations within the HRE that disrupt binding of CBF-A also reduced the activity of the rat Ha-ras promoter. Despite the fact that the HRE within the Ha-ras promoter resembles a binding site for Ets transcription factors, we did not detect the binding of Ets-related proteins to the rat HRE in BICR-M1Rk cells. We further demonstrated a correlation between the presence of HRE binding activity and induction of Ha-ras mRNA expression following serum stimulation in the mammary carcinoma cell line. Taken together, our results suggest that CBF-A may play an important role in transcriptional regulation of Ha-ras promoter activity during normal mammary cell growth and carcinogenesis.

Animals↗

PubMed2000

Nicastrin modulates presenilin-mediated notch/glp-1 signal transduction and betaAPP processing.

Nicastrin, a transmembrane glycoprotein, forms high molecular weight complexes with presenilin 1 and presenilin 2. Suppression of nicastrin expression in Caenorhabditis elegans embryos induces a subset of notch/glp-1 phenotypes similar to those induced by simultaneous null mutations in both presenilin homologues of C. elegans (sel-12 and hop-1). Nicastrin also binds carboxy-terminal derivatives of beta-amyloid precursor protein (betaAPP), and modulates the production of the amyloid beta-peptide (A beta) from these derivatives. Missense mutations in a conserved hydrophilic domain of nicastrin increase A beta42 and A beta40 peptide secretion. Deletions in this domain inhibit A beta production. Nicastrin and presenilins are therefore likely to be functional components of a multimeric complex necessary for the intramembranous proteolysis of proteins such as Notch/GLP-1 and betaAPP.

Amino Acid Sequence↗

PubMed2000

Peptide electroextraction for direct coupling of in-gel digests with capillary LC-MS/MS for protein identification and sequencing.

An electrophoretic method has been developed for the extraction of peptides following in-gel digests of SDS-PAGE separated proteins. During electroextraction, the peptides are trapped on a strong cation-exchange microcartridge, before analysis by capillary LC--ESI-tandem mass spectrometry. The spectra obtained by tandem mass spectrometry are searched directly against a protein database for identification of the protein from which the peptide originated. By minimizing surface exposure of the peptides during electroextraction, a reduction of the detection limits for protein identification is realized. The performance of the peptide electroextraction was compared directly with the standard extraction method for in-gel protein digests, using a standard dilution series of phosphorylase B and carbonic anhydrase, separated by SDS-PAGE. The lowest gel loading in which phosphorylase B was identified using the standard extraction method was 2.5 ng or 25 fmol, and the lowest gel loading in which phosphorylase B was identified using electroextraction was 1.25 ng or 12.5 fmol. The design of the microextraction cartridge allows for direct interfacing with capillary LC, which is crucial for maintaining low detection limits. Furthermore, this method can be used for high-throughput proteomics since it can be easily multiplexed and requires only voltage control and low pressures (approximately 15 psi) for operation. We believe that peptide electroextraction is a significant advance for identification of proteins separated by one-dimensional or two-dimensional gel electrophoresis, as it can be easily automated and requires less protein than conventional methods.

Amino Acid Sequence↗

PubMed2000

Evaluation of two-dimensional gel electrophoresis-based proteome analysis technology.

Proteome analysis is most commonly accomplished by a combination of two-dimensional gel electrophoresis (2DE) to separate and visualize proteins and mass spectrometry (MS) for protein identification. Although this technique is powerful, mature, and sensitive, questions remain concerning its ability to characterize all of the elements of a proteome. In the current study, more than 1,500 features were visualized by silver staining a narrow pH range (4.9-5. 7) 2D gel in which 0.5 mg of total soluble yeast protein was separated. Fifty spots migrating to a region of 4 cm(2) were subjected to MS protein identification. Despite the high sample load and extended electrophoretic separation, proteins from genes with codon bias values of <0.1 (lower abundance proteins) were not found, even though fully one-half of all yeast genes fall into that range. Proteins from genes with codon bias values of <0.1 were found, however, if protein amounts exceeding the capacity of 2DE were fractionated and analyzed. We conclude that the large range of protein expression levels limits the ability of the 2DE-MS approach to analyze proteins of medium to low abundance, and thus the potential of this technique for proteome analysis is likewise limited.

Amino Acid Sequence↗

PubMed2000

Structural organization of distinct domains within the non-collagenous N-terminal region of collagen type XI.

Collagen XI is a heterotrimeric molecule found predominantly in heterotypic cartilage fibrils, where it is involved in the regulation of fibrillogenesis. This function is thought to involve the complex N-terminal domain. The goal of this current study was to examine its structural organization to further elucidate the regulatory mechanism. The amino-propeptide (alpha1-Npp) alone or with isoforms of the variable region were recombinantly expressed and purified by affinity and molecular sieve chromatography. Cys-1-Cys-4 and Cys-2-Cys-3 disulfide bonds were detected by liquid chromatography-tandem mass spectrometry. This pattern is identical to the homologous alpha2-Npp, indicating that the recombinant proteins were folded correctly. Anomalous elution on molecular sieve chromatography suggested that the variable region was extended, which was confirmed using rotary shadowing; the alpha1-Npp formed a globular "head" and the variable region an extended "tail." Circular dichroism spectra analysis determined that the alpha1-Npp comprised 33% beta-sheet, whereas the variable region largely comprised non-periodic structure. Taken together, these results imply that the alpha1-Npp cannot be accommodated within the core of the fibril and that the variable region and/or minor helix facilitates its exclusion to the fibril surface. This provides further support for regulation of fibril diameter by steric hindrance or by interactions with other matrix components that affect fibrillogenesis.

Amino Acid Sequence↗

PubMed2000

Protein identification with a single accurate mass of a cysteine-containing peptide and constrained database searching.

A method for rapid and unambiguous identification of proteins by sequence database searching using the accurate mass of a single peptide and specific sequence constraints is described. Peptide masses were measured using electrospray ionization-Fourier transform ion cyclotron resonance mass spectrometry to an accuracy of 1 ppm. The presence of a cysteine residue within a peptide sequence was used as a database searching constraint to reduce the number of potential database hits. Cysteine-containing peptides were detected within a mixture of peptides by incorporating chlorine into a general alkylating reagent specific for cysteine residues. Secondary search constraints included the specificity of the protease used for protein digestion and the molecular mass of the protein estimated by gel electrophoresis. The natural isotopic distribution of chlorine encoded the cysteine-containing peptide with a distinctive isotopic pattern that allowed automatic screening of mass spectra. The method is demonstrated for a peptide standard and unknown proteins from a yeast lysate using all 6118 possible yeast open reading frames as a database. As judged by calculation of codon bias, low-abundance proteins were identified from the yeast lysate using this new method but not by traditional methods such as tandem mass spectrometry via data-dependent acquisition or mass mapping.

Amino Acid Sequence↗

PubMed2000

Role of tyrosine phosphorylation in the regulation of the interaction of heterogenous nuclear ribonucleoprotein K protein with its protein and RNA partners.

The heterogeneous nuclear ribonucleoprotein K protein recruits a diversity of molecular partners and may act as a docking platform involved in such processes as transcription, RNA processing, and translation. We show that K protein is tyrosine-phosphorylated in vitro by Src and Lck. Treatment with H(2)O(2)/Na(3)VO(4), which induces oxidative stress, stimulated tyrosine phosphorylation of K protein in cultured cells and in intact livers. Tyrosine phosphorylation increased binding of Lck and the proto-oncoprotein Vav to K protein in vitro. Oxidative stress increased the association of K protein with Lck and Vav, suggesting that tyrosine phosphorylation regulates the ability of K protein to recruit these effectors in vivo. Translation-based assay showed that K protein is constitutively bound to many mRNAs in vivo. Native immunoprecipitated K protein-mRNA complexes were disrupted by tyrosine phosphorylation, suggesting that the in vivo binding of K protein to mRNA may be responsive to the extracellular signals that activate tyrosine kinases. This study shows that tyrosine phosphorylation of K protein regulates K protein-protein and K protein-RNA interactions. These data are consistent with a model in which functional interaction of K protein is responsive to changes in the extracellular environment. Acting as a docking platform, K protein may bridge signal transduction pathways to sites of nucleic acid-dependent process such as transcription, RNA processing, and translation.

HeLa Cells↗

PubMed2000

Serum-stimulated, rapamycin-sensitive phosphorylation sites in the eukaryotic translation initiation factor 4GI.

The eukaryotic translation initiation factor 4G (eIF4G) proteins play a critical role in the recruitment of the translational machinery to mRNA. The eIF4Gs are phosphoproteins. However, the location of the phosphorylation sites, how phosphorylation of these proteins is modulated and the identity of the intracellular signaling pathways regulating eIF4G phosphorylation have not been established. In this report, two-dimensional phosphopeptide mapping demonstrates that the phosphorylation state of specific eIF4GI residues is altered by serum and mitogens. Phosphopeptides resolved by this method were mapped to the C-terminal one-third of the protein. Mass spectrometry and mutational analyses identified the serum-stimulated phosphorylation sites in this region as serines 1108, 1148 and 1192. Phosphoinositide-3-kinase (PI3K) inhibitors and rapamycin, an inhibitor of the kinase FRAP/mTOR (FKBP12-rapamycin-associated protein/mammalian target of rapamycin), prevent the serum-induced phosphorylation of these residues. Finally, the phosphorylation state of N-terminally truncated eIF4GI proteins acquires resistance to kinase inhibitor treatment. These data suggest that the kinases phosphorylating serines 1108, 1148 and 1192 are not directly downstream of PI3K and FRAP/mTOR, but that the accessibility of the C-terminus to kinases is modulated by this pathway(s).

Amino Acid Sequence↗

PubMed2000

Purification and identification of secreted oxidative stress-induced factors from vascular smooth muscle cells.

Reactive oxygen species have been implicated in the pathogenesis of atherosclerosis and hypertension, in part by promoting vascular smooth muscle cell (VSMC) growth. We have previously shown that LY83583, a generator of O-(2), activated extracellular signal-regulated kinases (ERK1/2) with early (10 min) and late (2 h) peaks and stimulated VSMC growth. To investigate whether secreted oxidative stress-induced factors (termed SOXF) from VSMC were responsible for late ERK1/2 activation in response to LY83583, we purified putative SOXF proteins from conditioned medium (2 h of LY83583 exposure) by sequential chromatography based on activation of ERK1/2. Proteins identified by capillary chromatography, electrospray ionization tandem mass spectrometry, and data base searching included heat shock protein 90-alpha (HSP90-alpha) and cyclophilin B. Western blot analysis of conditioned medium showed specific secretion of HSP90-alpha but not HSP90-beta. Immunodepletion of HSP90-alpha from conditioned medium significantly inhibited conditioned medium-induced ERK1/2 activation. Human recombinant HSP90-alpha reproduced the effect of conditioned medium on ERK1/2 activation. These results show that brief oxidative stress causes sustained release of protein factors from VSMC that can stimulate ERK1/2. These factors may be important mediators for the effects of reactive oxygen species on vascular function.

1,2-Dihydroxybenzene-3,5-Disulfonic Acid Disodium ↗

PubMed2000

Quantitative in vitro kinase reaction as a guide for phosphoprotein analysis by mass spectrometry.

A simple calculation using the radioactive decay of (32)P incorporated into a protein during in vitro kinase reactions is described that allows the overall stoichiometry of phosphorylation for the substrate protein or peptide to be calculated. Prior to using techniques such as diagnostic ion scanning to identify the molecular weight of an unknown phosphopeptide in a complex mixture followed by tandem mass spectrometry (MS/MS) to locate the phosphorylated residue within the phosphopeptide, such calculations are predictive of the chances for successful characterization by these methods. An example of estimating the stoichiometry of peptide phosphorylation will be presented along with calculations that predict when adequate phosphopeptide is present in any given spot on the thin-layer chromatography (TLC) plates used for two-dimensional phosphopeptide (2DPP) mapping to allow extraction and complete characterization by MS/MS.

Adenosine Triphosphate↗

PubMed2000

Comprehensive analyses of prostate gene expression: convergence of expressed sequence tag databases, transcript profiling and proteomics.

Several methods have been developed for the comprehensive analysis of gene expression in complex biological systems. Generally these procedures assess either a portion of the cellular transcriptome or a portion of the cellular proteome. Each approach has distinct conceptual and methodological advantages and disadvantages. We have investigated the application of both methods to characterize the gene expression pathway mediated by androgens and the androgen receptor in prostate cancer cells. This pathway is of critical importance for the development and progression of prostate cancer. Of clinical importance, modulation of androgens remains the mainstay of treatment for patients with advanced disease. To facilitate global gene expression studies we have first sought to define the prostate transcriptome by assembling and annotating prostate-derived expressed sequence tags (ESTs). A total of 55000 prostate ESTs were assembled into a set of 15953 clusters putatively representing 15953 distinct transcripts. These clusters were used to construct cDNA microarrays suitable for examining the androgen-response pathway at the level of transcription. The expression of 20 genes was found to be induced by androgens. This cohort included known androgen-regulated genes such as prostate-specific antigen (PSA) and several novel complementary DNAs (cDNAs). Protein expression profiles of androgen-stimulated prostate cancer cells were generated by two-dimensional electrophoresis (2-DE). Mass spectrometric analysis of androgen-regulated proteins in these cells identified the metastasis-suppressor gene NDKA/nm23, a finding that may explain a marked reduction in metastatic potential when these cells express a functional androgen receptor pathway.

DNA, Complementary↗