Biomedical subjects

Christoph H Borchers

Publications and source records attributed to Christoph H Borchers.

At least 19 recordsLinked to original sources

PubMed2026

Elevated mitochondrial superoxide promotes longevity through a mitochondria-to-nucleus kinase signaling pathway.

The reactive oxygen species superoxide is generated by mitochondria during the process of producing energy. While superoxide can cause oxidative damage to the cell, we and others have shown that a mild increase in mitochondrial superoxide extends longevity in multiple model organisms. To elucidate the molecular mechanisms involved, we identified transcriptional changes in mitochondrial superoxide dismutase deletion mutants (sod-2 worms) using RNA sequencing. sod-2 mutants exhibit a number of changes in nuclear gene expression resulting from elevated mitochondrial superoxide suggesting that mitochondria-to-nucleus signaling is contributing to their longevity. Gene ontology enrichment analysis demonstrated that genes involved in innate immunity and cuticle formation are significantly upregulated in sod-2 worms. To identify kinases involved in this lifespan-extending pathway, we completed a targeted RNA interference screen to examine the contribution of selected kinases to sod-2 longevity. From this screen, we found 25 kinases which are required for the long lifespan of sod-2 mutants including mak-2, which has a role in a kinase signaling pathway involved in axon regeneration. Disruption of mak-2 specifically reduces sod-2 lifespan but not wild-type longevity and also decreases resistance to multiple exogenous stressors. In examining other genes that act with mak-2 in established signaling pathways, we identified a SEK-3/PMK-3/MAK-2/CEBP-1 signaling pathway that is specifically required for sod-2 longevity but not wild-type lifespan. Combined these results suggest a novel role for kinases with established roles in axon regeneration in promoting longevity through a mitochondria-to-nucleus signaling pathway.

Aging↗

PubMed2006

Inhibition of APCCdh1 activity by Cdh1/Acm1/Bmh1 ternary complex formation.

The anaphase-promoting complex (APC) is an essential E3 ubiquitin ligase responsible for catalyzing proteolysis of key regulatory proteins in the cell cycle. Cdh1 is a co-activator of the APC aiding in the onset and maintenance of G(1) phase, whereas phosphorylation of Cdh1 at the end of G(1) phase by cyclin-dependent kinases assists in the inactivation of APC(Cdh1). Here, we suggest additional components are involved in the inactivation of APC(Cdh1) independent of Cdh1 phosphorylation. We have identified proteins known as Acm1 and Bmh1, which bind and form a ternary complex with Cdh1. The presence of phosphorylated Acm1 is critical for the ternary complex formation, and Acm1 is predominantly expressed in S phase when APC(Cdh1) is inactive. The assembly of the ternary complex inhibits ubiquitination of Clb2 in vitro by blocking the interaction of Cdh1 with Clb2. In vivo, lethality caused by overexpression of constitutively active Cdh1 is rescued by overexpression of Acm1. Partially phosphorylated Cdh1 in the absence of ACM1 still binds to and activates the APC. However, the addition of Acm1 decreases Clb2 ubiquitination when using either phosphorylated or nonphosphorylated Cdh1. Taken together, our results suggest an additional inactivation mechanism exists for APC(Cdh1) that is independent of Cdh1 phosphorylation.

14-3-3 Proteins↗

PubMed2006

Combining fluorescence detection and mass spectrometric analysis for comprehensive and quantitative analysis of redox-sensitive cysteines in native membrane proteins.

Monobromobimane (MBB) is a lipophilic reagent that selectively modifies free cysteine residues in proteins. Because of its lipophilic character, MBB is capable of labeling cysteine residues in membrane proteins under native conditions. Reaction of MBB with the sulfhydryl groups of free cysteines leads to formation of highly fluorescent derivatives. Here we describe a procedure for the detection and relative quantitation of MBB-labeled cysteines using fluorescence and mass spectrometric analyses, which allow determination of free cysteine content and unambiguous identification of MBB-modified cysteine residues. We have applied this approach to the analysis of the free and redox-sensitive cysteine residues of a large membrane protein, the sarcoplasmic reticulum Ca2+ release channel with a molecular mass of 2.2 million Da. Labeling was performed under physiologic conditions where the channel complex is in its native environment and is functionally active. The purified MBB-labeled channel complex was enzymatically digested, and the resulting peptides were separated by reversed-phase high-performance chromatography. MBB-labeled peptides were detected by fluorescence and identified by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. Under MALDI conditions, partial photolytic fragmentation of the MBB-peptide bound occurred, thus allowing convenient screening for the MBB-modified peptides in the MS spectrum by detection of the specific mass increment of 190.07 Da for MBB-modified cysteine residues. Modification of the peptides was further confirmed by tandem mass spectrometric analysis, utilizing sequencing information and the presence of the specific immonium ion for the MBB-modified cysteine residues at m/z 266.6. Quantitative information was obtained by comparison of both fluorescence and MS signal intensities of MBB-modified peptides. Combination of fluorescence with MS detection and analysis of MBB-labeled peptides supported by a customized software program provides a convenient method for identifying and quantifying redox-sensitive cysteines in membrane proteins of native biological systems. Identification of one redox-sensitive cysteine (2327) in the native membrane-bound sarcoplasmic reticulum Ca2+ release channel is described.

Animals↗

PubMed2006

Cohesin protein SMC1 represses the nuclear receptor CAR-mediated synergistic activation of a human P450 gene by xenobiotics.

CAR (constitutive active/androstane receptor) regulates both the distal enhancer PBREM (phenobarbital-responsive enhancer module) and the proximal element OARE [OA (okadaic acid) response element] to synergistically up-regulate the endogenous CYP2B6 (where CYP is cytochrome P450) gene in HepG2 cells. In this up-regulation, CAR acts as both a transcription factor and a co-regulator, directly binding to and enhancing PBREM upon activation by xenobiotics such as TCPOBOP {1,4-bis-[2-(3,5-dichloropyridyloxy)]benzene} and indirectly associating with the OARE in response to OA [Swales, Kakizaki, Yamamoto, Inoue, Kobayashi and Negishi (2005) J. Biol. Chem. 280, 3458-3466]. We have now identified the cohesin protein SMC1 (structural maintenance of chromosomes 1) as a CAR-binding protein and characterized it as a negative regulator of OARE activity, thus repressing synergy. Treatment with SMC1 small interfering RNA augmented the synergistic up-regulation of CYP2B6 expression 20-fold in HepG2 cells, while transient co-expression of spliced form of SMC1 abrogated the synergistic activation of a 1.8 kb CYP2B6 promoter. SMC1 indirectly binds to a 19 bp sequence (-236/-217) immediately downstream from the OARE in the CYP2B6 promoter. Both DNA affinity and chromatin immunoprecipitation assays showed that OA treatment dissociates SMC1 from the CYP2B6 promoter, reciprocating the indirect binding of CAR to OARE. These results are consistent with the conclusion that SMC1 binding represses OARE activity and its dissociation allows the recruitment of CAR to the OARE, synergizing PBREM activity and the expression of the CYP2B6 gene.

Animals↗

PubMed2006

Discovery of a small-molecule HIV-1 integrase inhibitor-binding site.

Herein, we report the identification of a unique HIV-1 integrase (IN) inhibitor-binding site using photoaffinity labeling and mass spectrometric analysis. We chemically incorporated a photo-activatable benzophenone moiety into a series of coumarin-containing IN inhibitors. A representative of this series was covalently photo-crosslinked with the IN core domain and subjected to HPLC purification. Fractions were subsequently analyzed by using MALDI-MS and electrospray ionization (ESI)-MS to identify photo-crosslinked products. In this fashion, a single binding site for an inhibitor located within the tryptic peptide (128)AACWWAGIK(136) was identified. Site-directed mutagenesis followed by in vitro inhibition assays resulted in the identification of two specific amino acid residues, C130 and W132, in which substitutions resulted in a marked resistance to the IN inhibitors. Docking studies suggested a specific disruption in functional oligomeric IN complex formation. The combined approach of photo-affinity labeling/MS analysis with site-directed mutagenesis/molecular modeling is a powerful approach for elucidating inhibitor-binding sites of proteins at the atomic level. This approach is especially important for the study of proteins that are not amenable to traditional x-ray crystallography and NMR techniques. This type of structural information can help illuminate processes of inhibitor resistance and thereby facilitate the design of more potent second-generation inhibitors.

4-Hydroxycoumarins↗

PubMed2006

Combined top-down and bottom-up proteomics identifies a phosphorylation site in stem-loop-binding proteins that contributes to high-affinity RNA binding.

The stem-loop-binding protein (SLBP) is involved in multiple aspects of histone mRNA metabolism. To characterize the modification status and sites of SLBP, we combined mass spectrometric bottom-up (analysis of peptides) and top-down (analysis of intact proteins) proteomic approaches. Drosophilia SLBP is heavily phosphorylated, containing up to seven phosphoryl groups. Accurate M(r) determination by Fourier transform ion cyclotron resonance (FTICR)-MS and FTICR-MS top-down experiments using a variety of dissociation techniques show there is removal of the initiator methionine and acetylation of the N terminus in the baculovirus-expressed protein, and that T230 is stoichiometrically phosphorylated. T230 is highly conserved; we have determined that this site is also completely phosphorylated in baculovirus-expressed mammalian SLBP and extensively phosphorylated in both Drosophila and mammalian cultured cells. Removal of the phosphoryl group from T230 by either dephosphorylation or mutation results in a 7-fold reduction in the affinity of SLBP for the stem-loop RNA.

Amino Acid Motifs↗

PubMed2006

Photoaffinity labeling combined with mass spectrometric approaches as a tool for structural proteomics.

Protein chemistry, such as crosslinking and photoaffinity labeling, in combination with modern mass spectrometric techniques, can provide information regarding protein-protein interactions beyond that normally obtained from protein identification and characterization studies. While protein crosslinking can make tertiary and quaternary protein structure information available, photoaffinity labeling can be used to obtain structural data about ligand-protein interaction sites, such as oligonucleotide-protein, drug-protein and protein-protein interaction. In this article, we describe mass spectrometry-based photoaffinity labeling methodologies currently used and discuss their current limitations. We also discuss their potential as a common approach to structural proteomics for providing 3D information regarding the binding region, which ultimately will be used for molecular modeling and structure-based drug design.

Azides↗

PubMed2005

Histone demethylation by a family of JmjC domain-containing proteins.

Covalent modification of histones has an important role in regulating chromatin dynamics and transcription. Whereas most covalent histone modifications are reversible, until recently it was unknown whether methyl groups could be actively removed from histones. Using a biochemical assay coupled with chromatography, we have purified a novel JmjC domain-containing protein, JHDM1 (JmjC domain-containing histone demethylase 1), that specifically demethylates histone H3 at lysine 36 (H3-K36). In the presence of Fe(ii) and alpha-ketoglutarate, JHDM1 demethylates H3-methyl-K36 and generates formaldehyde and succinate. Overexpression of JHDM1 reduced the level of dimethyl-H3-K36 (H3K36me2) in vivo. The demethylase activity of the JmjC domain-containing proteins is conserved, as a JHDM1 homologue in Saccharomyces cerevisiae also has H3-K36 demethylase activity. Thus, we identify the JmjC domain as a novel demethylase signature motif and uncover a protein demethylation mechanism that is conserved from yeast to human.

Amino Acid Motifs↗

PubMed2005

Phosphorylation of the RGS protein Sst2 by the MAP kinase Fus3 and use of Sst2 as a model to analyze determinants of substrate sequence specificity.

Previously, we used mass spectrometry to demonstrate pheromone-stimulated phosphorylation of Ser-539 in Sst2, a regulator of G protein signaling in yeast Saccharomyces cerevisiae [Garrison, T. R., et al. (1999) J. Biol. Chem. 274, 36387-36391]. Here, we show that Sst2 phosphorylation is mediated by the mitogen-activated protein (MAP) kinase Fus3. Phosphorylation occurs within a canonical MAP kinase phosphorylation site (Pro-X-Ser/Thr-Pro, where "X" at the -1 position can be any amino acid), a consensus sequence deduced earlier from analysis of synthetic peptide substrates. In a direct test of the model, we compared Sst2 phosphorylation following systematic substitution of the -1 residue His-538. Each of the substitution mutants was suitable as a MAP kinase substrate, as shown by phosphorylation-dependent mobility shifts in vivo and/or by direct phosphorylation in vitro followed by peptide mapping and mass spectrometry sequencing. This analysis documents phosphorylation of Sst2 by Fus3 and demonstrates that the prevailing model for MAP kinase recognition is valid for a native substrate protein in vivo as well as for small synthetic peptides tested in vitro.

Alanine↗

PubMed2005

Isotopically coded cleavable cross-linker for studying protein-protein interaction and protein complexes.

An emerging approach for studying protein-protein interaction in complexes is the combination of chemical cross-linking and mass spectrometric analysis of the cross-linked peptides (cross-links) obtained after proteolysis of the complex. This approach, however, has several challenges and limitations, including the difficulty of detecting the cross-links, the potential interference from non-informative "cross-linked peptides" (dead end and intrapeptide cross-links), and unambiguous identification of the cross-links by mass spectrometry. Thus, we have synthesized an isotopically coded ethylene glycol bis(succinimidylsuccinate) derivate (D12-EGS), which contains 12 deuterium atoms for easy detection of cross-links when applied in a 1:1 mixture with its H12 counterpart and is also cleavable for releasing the cross-linked peptides allowing unambiguous identification by MS sequencing. Moreover, hydrolytic cleavage permits rapid distinguishing between different types of cross-links. Cleavage of a dead end cross-link produces a doublet with peaks 4.03 Da apart, with the lower peak appearing at a molecular mass 162 Da lower than the mass of the H12 form of the original cross-linked peptide. Cleavage of an intrapeptide cross-link leads to a doublet 8.05 Da apart and 62 Da lower than the molecular mass of the H12 form of the original cross-linked peptide. Cleavage of an interpeptide cross-link forms a pair of 4.03-Da doublets, with the lower mass member of each pair each shifted up from its unmodified molecular weight by 82 Da because of the attached portion of the cross-linker. All of this information has been incorporated into a software algorithm allowing automatic screening and detection of cross-links and cross-link types in matrix-assisted laser desorption/ionization mass spectra. In summary, the ease of detection of these species through the use of an isotopically coded cleavable cross-linker and our software algorithm, followed by mass spectrometric sequencing of the cross-linked peptides after cleavage, has been shown to be a powerful tool for studies of multi-component protein complexes.

Algorithms↗

PubMed2005

Electrospray ionization tandem mass spectrometry of model peptides reveals diagnostic fragment ions for protein ubiquitination.

In this work, synthetic peptides were used to determine the fragmentation behavior of ubiquitinated peptides and to find ions diagnostic for peptide ubiquitination. The ubiquitin-calmodulin peptide1 was chosen as the model peptide for naturally occurring ubiquitinated proteins cleaved with endoproteinase gluC. In addition, the fragmentation behavior of model ubiquitinated peptides produced by tryptic digestion was also of great interest since the standard protocols for proteomics-based protein identification use trypsin as the protease. Attachment of ubiquitin to a target protein results in a branched structure, but only ions from the ubiquitin side chain (and the lysine to which it is attached) can be used as diagnostic ions, since fragment ions that contain other amino acids from the parent protein will vary in mass. Characteristic b-type fragment ions from the gluC cleavage of the ubiquitin side chain (designated as b ions) were found which involve only the ubiquitin tail (b2, b3, b4, b5 and b6 ions at m/z 189.06, 302.12, 439.18, 552.30 and 651.30, respectively). Maximum production of these ions occurred at a collision energy of 45 eV in a Q-TOF instrument. Although a non-ubiquitinated peptide may produce isobaric fragment ions, it is unlikely that it can produce these ions in combination. With liquid chromatography/tandem mass spectrometry (LC/MS/MS) experiments, ubiquitinated peptides can readily be determined by surveying the reconstructed or extracted ion chromatograms of the diagnostic fragment ions for common peaks. Characteristic ions resulting from tryptic cleavage of the side chain were found in cleavage products with a missed cleavage, resulting in a LRGG- tag instead of a GG- tag. For the LRGG-tagged peptide, diagnostic MS/MS fragment ions (at m/z 270.17 and 384.21) from the ubiquitin tail (b2 and b4, respectively) were found, along with an internal fragment ion (LRGGK-28) at m/z 484.30. These ions should prove useful in precursor-ion scanning experiments for identifying peptides modified by attachment of ubiquitin, and for locating the site of ubiquitin attachment.

Calmodulin↗

PubMed2005

Identification of components of protein complexes.

Protocols are given for a variety of techniques used in protein identification of complexes, including identification of in-gel separated proteins and LC-MS/MS. Gels, staining procedures, and peptide extraction protocols that are compatible with mass spectrometry are described. The detection limits of the various staining procedures and their compatibility with mass spectrometry are discussed. The various mass spectrometric techniques used (MALDI-MS, MALDI-MS/MS, nanospray, and ESI/LC-MS/MS) are also described, along with an indication of the advantages and disadvantages of each, and when they would most appropriately be used. Common pitfalls associated with database searching are also discussed.

Animals↗

PubMed2005

Mass spectrometric determination of protein ubiquitination.

Mass spectrometric methods of determining protein ubiquitination are described. Characteristic mass shifts and fragment ions indicating ubiquitinated lysine residues in tryptic and gluC digests are discussed. When a ubiquitinated protein is enzymatically digested, a portion of the ubiquitin side chain remains attached to the modified lysine. The ubiquitinated peptide thus has two N-termini- one from the original peptide and one from the ubiquitin side chain. Thus, it is possible to have two series of b ions and y ions, the additional series is the one that includes fragments containing portions of the ubiquitin side chain. Any diagnostic ions for the modification must include portions of this side chain. Fragment ions involving any part of the "normal" peptide will vary in mass according to the peptide being modified and will therefore not be of general diagnostic use. These diagnostic ions, found through examination of the MS/MS spectra of model ubiquitinated tryptic and gluC peptides, can be used to trigger precursor ion scanning in automated MS/MS data acquisition scanning modes.

Animals↗

PubMed2005

Absolute quantitation of cancer-related proteins using an MS-based peptide chip.

New technologies are needed that can diagnose cancer more rapidly and accurately. These technologies must also have the ability to identify the particular cellular abnormalities contributing to the malignancy, thus directing the appropriate treatments. Such technologies should permit absolute quantitation of specific tumor biomarkers and their level of posttranslational modifications. Quantitative molecular profiling of cancer signaling networks would provide a more detailed understanding of the contribution of protein expression and posttranslational modification levels to tumorigenesis. We have developed a unique approach for absolute quantitation of protein expression that integrates affinity capture of proteolytic peptides with mass spectrometry and thus provides detection, identification, and quantitation of their cognate proteins. We have previously shown the high sensitivity and specificity of this approach. Here we demonstrate the absolute quantitation of a model peptide using our technology. We have used this approach to capture epitope-containing peptides from proteolytically digested target proteins, including p53, epidermal growth factor receptor (EGFR), and prostate-specific antigen (PSA). Our technology can easily be extended to the absolute quantitation of protein modification levels, in addition to the determination of protein expression levels, and can be readily adapted for use in a microarray format. This method offers an improved approach to protein chip technology that should prove useful for clinical diagnosis and drug development applications.

Antibodies, Monoclonal↗

PubMed2004

Fourier transform ion cyclotron resonance MS reveals the presence of a water molecule in an enzyme transition-state analogue complex.

The structures of several powerful inhibitors of hydrolytic enzymes resemble that of the altered substrate in the transition state, except that a hydrogen atom replaces one substituent (typically the leaving group). To test the hypothesis that a water molecule might be present in the gap resulting from this replacement, we examined a transition-state analogue complex formed by Escherichia coli cytidine deaminase by Fourier transform ion cyclotron resonance MS in electrospray mode. Upon nebularization from aqueous solution under conditions (pH 5.6) where the enzyme is active, cytidine deaminase remains dimeric in the vapor phase. In the presence of inhibitor, the enzyme's exact mass can be used to infer the presence at each active site of zinc, 5-fluoro-3,4-dihydrouridine, and a single water molecule.

Cyclotrons↗

PubMed2004

Multi-kinase phosphorylation of the APC/C activator Cdh1 revealed by mass spectrometry.

Cdh1 contributes to proper exit from mitosis and maintenance of G(1) phase in eukaryotic cells by activating a large ubiquitin ligase called the anaphase-promoting complex, or cyclosome (APC/C). At the end of G(1), APC/C(Cdh1) is inhibited by cyclin-dependent kinase (CDK) phosphorylation of Cdh1. The specific Cdh1 phosphorylation sites used to regulate APC/C(Cdh1) activity have not been directly identified. Here, we used a mass spectrometric approach to identify the in vivo phosphorylation sites on yeast Cdh1. Surprisingly, in addition to several expected CDK phosphorylation sites, we discovered numerous nonCDK phosphorylation sites. In total, at least 19 serine and threonine residues on Cdh1 are phosphorylated in vivo. Seventeen of these sites are located in the N-terminal half of Cdh1, outside the highly conserved WD40 repeats. The pattern of phosphorylation was the same when Cdh1 was purified from yeast cultures arrested in S, early M and late M. Mutation of CDK consensus sequences eliminated detectable phosphorylation at many of the nonCDK sites. In contrast, mutation of nonCDK sites had no significant effect on CDK phosphorylation. We conclude that phosphorylation of CDK sites promotes the subsequent recognition of Cdh1 by at least one additional kinase. The function of nonCDK phosphorylation may differ from CDK phosphorylation because mutation of nonCDK sites did not result in constitutive activation of APC and consequent cell cycle arrest. These results suggest that phosphoregulation of APC/C(Cdh1) activity is much more complex than previously thought.

Amino Acid Sequence↗

PubMed2004

Development of a protein chip: a MS-based method for quantitation of protein expression and modification levels using an immunoaffinity approach.

Protein chip technology permits analysis of the expression and modification status of numerous targeted proteins within a single experiment, mainly through the use of antibody-based microarrays. Despite recent improvements in these protein chips, their applications are still limited for a variety of reasons, which include technical challenges in fabrication of the antibody chips as well as the very low specificity achieved by current detection methods. We have developed a unique approach for relative and/or absolute quantitation of protein expression and modification based on the capture of epitope peptides on affinity beads, which can be used to develop a mass-spectrometry-based protein chip technology. This new method, which utilizes antibodies immobilized on beads for the capture of target peptides, instead of proteins, eliminates many of the problems previously associated with protein chips. We present here several proof-of-principle experiments examining model peptides by this technique. These experiments show that the method is capable of (i). detecting peptides bound to a single antibody bead, (ii). detecting peptides at low (fmol) levels, (iii). producing MS/MS data of suitable quality for protein identification via database searching or de novo sequencing, (iv). quantitating peptides affinity-bound to antibody beads, (v). specifically detecting target peptides in complex mixtures over wide dynamic ranges, and (vi) is compatible with a microarray format for high-throughput analysis. Because our novel method uses antibody beads instead of a derivatized capture surface, and peptides instead of proteins for affinity capture, it can overcome many of the pitfalls of previous protein chip fabrications. Therefore, this method offers an improved approach to protein chip technology that should prove useful for diagnostics and drug development applications.

Antibodies↗

PubMed2004

Phosphorylation of paxillin by p38MAPK is involved in the neurite extension of PC-12 cells.

Cell adhesions play an important role in neurite extension. Paxillin, a focal adhesion adaptor protein involved in focal adhesion dynamics, has been demonstrated to be required for neurite outgrowth. However, the molecular mechanism by which paxillin regulates neurite outgrowth is unknown. Here, we show that paxillin is phosphorylated by p38MAPK in vitro and in nerve growth factor (NGF)-induced PC-12 cells. Ser 85 (Ser 83 for endogenous paxillin) is identified as one of major phosphorylation sites by phosphopeptide mapping and mass spectrometry. Moreover, expression of the Ser 85 --> Ala mutant of paxillin (paxS85A) significantly inhibits NGF-induced neurite extension of PC-12 cells, whereas expression of wild-type (wt) paxillin does not influence neurite outgrowth. Further experiments indicate that cells expressing paxS85A exhibit small, clustered focal adhesions which are not normally seen in cells expressing wt paxillin. Although wt paxillin and paxS85A have the same ability to bind vinculin and focal adhesion kinase, wt paxillin more efficiently associates with Pyk2 than paxS85A. Thus, phosphorylation of paxillin is involved in NGF-induced neurite extension of PC-12 cells, probably through regulating focal adhesion organization.

Animals↗