Faculty Bibliography

In recent years, stable isotope labeling by amino acids in cell culture (SILAC) has become increasingly popular as a quantitative proteomic method. In SILAC experiments, proteins are metabolically labeled by culturing cells in media containing normal and heavy isotope amino acids. This makes proteins from the light and heavy cells distinguishable by mass spectrometry (MS) after the cell lysates are mixed and the proteins separated and/or enriched. SILAC is a powerful tool for the study of intracellular signal transduction. In particular, it has been very popular and successful in quantitative analysis of phosphoty-rosine (pTyr) proteomes to characterize pTyr-dependent signaling pathways. In this chapter, we describe the SILAC procedure and use EphB signaling pathway as an example to illustrate the use of SILAC to investigate such pathways

In comparative proteomic studies, it is important to know the variability associated with sample preparation. In this study, we report the strategy of using SILAC (stable isotope labeling by amino acids in cell culture) to evaluate the effect of the variation in sample preparation for quantitative proteomics. Variability can be measured when equal amounts of light and heavy SILAC samples undergo the same sample preparation procedures in parallel, and the two samples are mixed for relative protein quantitation by mass spectrometry. The high quantitative accuracy of SILAC allows for characterization of small variations. First, the reproducibility of immunoprecipitation (IP) and in-gel digestion was evaluated, and the impact of replicate number on quantitative accuracy was characterized. Second, we evaluated the overall variation in a comparative workflow involving three sequential sample preparation steps: IP, SDS-PAGE fractionation, and in-gel digestion. The evaluation of individual sample preparation steps was very valuable for experimental design: the optimal number of replicates for each step could be readily determined and the overall variation of the workflow could be predicted from the variation of the individual steps involved. By using informed experimental design, we demonstrated that the error associated with multiple steps of sample preparation in a comparative experiment can be limited to a reasonably low level

CIRL (the calcium-independent receptor of alpha-latrotoxin), a neuronal cell surface receptor implicated in the regulation of exocytosis, is a member of the GPS family of chimeric cell adhesion/G protein-coupled receptors. The predominant form of CIRL is a membrane-bound complex of two subunits, p120 and p85. Extracellularly oriented p120 contains hydrophilic cell adhesion domains, whereas p85 is a heptahelical membrane protein. Both subunits are encoded by the same gene and represent products of intracellular proteolytic processing of the CIRL precursor. In this study, we demonstrate that a soluble form of CIRL also exists in vitro and in vivo. It results from the further cleavage of CIRL by a second protease. The site of the second cleavage is located in the short N-terminal extracellular tail of p85, between the GPS domain and the first transmembrane segment of CIRL. Thus, the soluble form of CIRL represents a complex of p120 noncovalently bound to a 15 amino acid residue N-terminal peptide fragment of p85. We have previously shown that mutations of CIRL in the GPS domain inhibit intracellular proteolytic processing and also result in the absence of the receptors from the cell surface. Our current data suggest that although CIRL trafficking to the cell membrane is impaired by mutations in the GPS region, it is not blocked completely. However, at the cell surface, the noncleaved mutants are preferentially targeted by the second protease that sheds the extracellular subunit. Therefore, the two-step proteolytic processing may represent a regulatory mechanism that controls cell surface expression of membrane-bound and soluble forms of CIRL

Perturbation of the cytoplasmic protein folding environment by exposure to oxidative stress-inducing As(III)-containing compounds challenges the ubiquitin-proteasome system. Here we report on mass spectrometric analysis of As(III)-induced changes in the proteasome's composition in samples prepared by stable isotope labeling with amino acids in cell culture, using mammalian cells in which TRP32 (thioredoxin-related protein of 32 kDa; also referred to as TXNL1) was identified as a novel subunit of the 26 S proteasome. Quantitative genetic interaction mapping, using the epistatic miniarray profiling approach, identified a functional connection between TRP32 and the proteasome. Deletion of txl1, the Schizosaccharomyces pombe homolog of TRP32, results in a slow growth phenotype when combined with deletion of cut8, a gene required for normal proteasome localization. Deletion analysis in vivo, chemical cross-linking, and manipulation of the ATP concentration in vitro during proteasome immunopurification revealed that the C-terminal domain of mammalian TRP32 binds the 19 S regulatory particle in proximity to the proteasome substrate binding site. Thiol modification with polyethylene glycol-maleimide showed disulfide bond formation at the active site of TRP32 in cells exposed to As(III). Pulse-chase labeling showed that TRP32 is a stable protein whose half-life of >6 h is surprisingly reduced to 1 h upon exposure of cells to As(III). These findings reveal a previously undescribed thiol reductase at the proteasome's regulatory particle

Voltage-gated sodium channels (Nav) produce sodium currents that underlie the initiation and propagation of action potentials in nerve and muscle cells. Fibroblast growth factor homologous factors (FHFs) bind to the intracellular C-terminal region of the Nav alpha subunit to modulate fast inactivation of the channel. In this study we solved the crystal structure of a 149-residue-long fragment of human FHF2A which unveils the structural features of the homology core domain of all 10 human FHF isoforms. Through analysis of crystal packing contacts and site-directed mutagenesis experiments we identified a conserved surface on the FHF core domain that mediates channel binding in vitro and in vivo. Mutations at this channel binding surface impaired the ability of FHFs to co-localize with Navs at the axon initial segment of hippocampal neurons. The mutations also disabled FHF modulation of voltage-dependent fast inactivation of sodium channels in neuronal cells. Based on our data, we propose that FHFs constitute auxiliary subunits for Navs

We investigated the formation of hydroxyl radical (OH(*)) and H(2)O(2) mediated oxidation products of a synthetic peptide, HCSAGIGRS, which is an active site sequence motif of protein tyrosine phosphatase 1B (PTP1B). We determined that a novel cysteine sulfinamide HC[S(O)N]SAGIGRS is produced in the oxidation reaction by Fenton reagents (Fe(+2)/H(2)O(2)) as well as by H(2)O(2). These products were characterized by tandem mass spectrometry experiments on both singly and doubly charged precursor ions. MS(3) experiments using an ion trap instrument as well as LC-MS/MS experiments using a quadrupole time-of-flight (Q-TOF) instrument demonstrated that HC[S(O)N]SAGIGRS is not a water loss product of cysteine sulfinic acid [HC(SO(2)H)SAGIGRS]. We also obtained data from tandem mass spectrometry experiments that provided evidence for the existence of stable cysteine sulfenic acid [HC(SOH)SAGIGRS] in solution. A mechanism for the formation of the cysteine sulfinamide product is proposed based on the above experimental results. The preparation and identification of cysteine sulfinamide in this study may provide insight into the mechanism of both OH(*) and H(2)O(2) induced oxidation reactions of protein tyrosine phosphatases

BACKGROUND: Calmodulin (CaM) activation by Ca(2+), its translocation to the nucleus, and stimulation of phosphorylation of cyclic adenosine monophosphate response element-binding protein (CREB) (P-CREB) are necessary for new gene expression and have been linked to long-term potentiation, a process important in memory formation. Because isoflurane affects memory, we tested whether isoflurane interfered with the translocation of CaM to the neuronal cell nucleus and attenuated the formation P-CREB. METHODS: SH-SY5Y cells, a human neuroblastoma cell line, were cultured. Cells were depolarized with KCl and the phosphorylation of CREB examined by Western blotting, enzyme-linked immunosorbant assay, and immunocytochemistry. The translocation of CaM from the cytosol to the nucleus was also examined after depolarization. Cells were depolarized and lysed and fractionated by centrifugation to determine the amount of CaM translocated to the nucleus. CaM was localized by immunocytochemistry and quantitated by Western blotting and imaging. Before and during KCl depolarization, cells were exposed to isoflurane, isoflurane plus Bay K 8644, nitrendipine, and omega-conotoxin GVIa, respectively. RESULTS: P-CREB increased after KCl depolarization. The increase of P-CREB peaked at depolarization duration of 30 s. The increase in P-CREB formation was inhibited by nitrendipine, but not omega-conotoxin, and by isoflurane in a concentration-dependent fashion. Pretreatment with the L-type Ca(2+) channel agonist, Bay K 8644, attenuated the inhibition of P-CREB formation by isoflurane. CaM presence in the nucleus occurred after KCl depolarization. CaM translocation was inhibited by nitrendipine and attenuated by isoflurane. Bay K 8644 pretreatment decreased the isoflurane inhibition of CaM translocation to the nucleus. CONCLUSIONS: Our data demonstrate that isoflurane inhibits CaM translocation and P-CREB formation. This most likely occurs through isoflurane inhibition of Ca(2+)entry through L-type Ca(2+) channels

The tafazzin gene encodes a phospholipid-lysophospholipid transacylase involved in cardiolipin metabolism, but it is not known why it forms multiple transcripts as a result of alternative splicing. Here we studied the intracellular localization, enzymatic activity, and metabolic function of four isoforms of human tafazzin and three isoforms of Drosophila tafazzin upon expression in different mammalian and insect systems. When expressed in HeLa cells, all isoforms were localized in mitochondria except for the B-form of Drosophila tafazzin, which was associated with multiple intracellular membranes. Among the human isoforms, only full-length tafazzin (FL) and tafazzin lacking exon 5 (Delta5) had transacylase activity, and only these two isoforms were able to restore a normal cardiolipin pattern, normal respiratory activity of mitochondria, and male fertility in tafazzin-deficient flies. Both FL and Delta5 were associated with large protein complexes in 293T cell mitochondria, but treatment with alkali and proteinase K suggested that the Delta5 isoform was more integrated into the hydrophobic core of the membrane than the FL isoform. Although all Drosophila isoforms showed transacylase activity in vitro, only the A-form supported cardiolipin remodeling in flies. The data suggest that humans express two mitochondrial isoenzymes of tafazzin that have similar transacylase activities but different membrane topologies. Furthermore, the data show that the expression of human tafazzin in flies creates cardiolipin with a Drosophila pattern, suggesting that the characteristic fatty acid profile of cardiolipin is not determined by the substrate specificity of tafazzin

The NSD (nuclear receptor SET domain-containing) family of histone lysine methyltransferases is a critical participant in chromatin integrity as evidenced by the number of human diseases associated with the aberrant expression of its family members. Yet, the specific targets of these enzymes are not clear, with marked discrepancies being reported in the literature. We demonstrate that NSD2 can exhibit disparate target preferences based on the nature of the substrate provided. The NSD2 complex purified from human cells and recombinant NSD2 both exhibit specific targeting of histone H3 lysine 36 (H3K36) when provided with nucleosome substrates, but histone H4 lysine 44 is the primary target in the case of octamer substrates, irrespective of the histones being native or recombinant. This disparity is negated when NSD2 is presented with octamer targets in conjunction with short single- or double-stranded DNA. Although the octamers cannot form nucleosomes, the target is nonetheless nucleosome-specific as is the product, dimethylated H3K36. This study clarifies in part the previous discrepancies reported with respect to NSD targets. We propose that DNA acts as an allosteric effector of NSD2 such that H3K36 becomes the preferred target

Receptor tyrosine kinases (RTK) are proteins that undergo dimerization and/or multimerization and autophosphorylation in response to ligand stimulation. Members of the RTK family are receptors for a series of growth factors that. upon stimulation, are able to start signaling events that promote cell growth and differentiation. A class of RTKs, the Eph receptors (EphRs), are found in a variety of cell types and play important roles in patterning the central and peripheral nervous systems, as well as in synapse and neural crest formation. Interaction of Eph receptors with their ephrin ligands activates signal transduction pathways that lead to cytoskeletal remodeling through formation of many stable or transient protein-protein interactions. However, these intracellular signal transduction pathways that lead to cytoskeletal remodeling are not well understood. Here, we combined Blue Native PAGE (BN-PAGE) and mass spectrometry (MS) to analyze protein-protein interactions as a result of ephrin stimulation. We analyzed both lysates and phosphotyrosine immunoprecipitate (pY99-IP) of unstimulated and ephrin-stimulated cells. Our experiments allowed us to characterize many constitutive homo- and hetero-protein complexes from the cell lysate. Furthermore, BN-PAGE and MS of the pY99-IPs from both unstimulated and stimulated cells allowed us to analyze protein-protein interactions that resulted upon ephrin stimulation. Combination of BN-PAGE and MS also has the potential for the analysis of stable and transient protein-protein interactions in other ligand-stimulated RTK-dependent signal transduction pathways.