Faculty Bibliography

Being gated by high-energy nucleotides, cardiac ATP-sensitive potassium (K(ATP)) channels are exquisitely sensitive to changes in cellular energy metabolism. An emerging view is that proteins associated with the K(ATP) channel provide an additional layer of regulation. Using putative sulfonylurea receptor (SUR) coiled-coil domains as baits in a 2-hybrid screen against a rat cardiac cDNA library, we identified glycolytic enzymes (GAPDH and aldolase A) as putative interacting proteins. Interaction between aldolase and SUR was confirmed using GST pulldown assays and coimmunoprecipitation assays. Mass spectrometry of proteins from K(ATP) channel immunoprecipitates of rat cardiac membranes identified glycolysis as the most enriched biological process. Coimmunoprecipitation assays confirmed interaction for several glycolytic enzymes throughout the glycolytic pathway. Immunocytochemistry colocalized many of these enzymes with K(ATP) channel subunits in rat cardiac myocytes. The catalytic activities of aldolase and pyruvate kinase functionally modulate K(ATP) channels in patch-clamp experiments, whereas d-glucose was without effect. Overall, our data demonstrate close physical association and functional interaction of the glycolytic process (particularly the distal ATP-generating steps) with cardiac K(ATP) channels.-Hong, M., Kefaloyianni, E., Bao, L., Malester, B., Delaroche, D., Neubert, T. A., Coetzee, W. A. Cardiac ATP-sensitive K(+) channel associates with the glycolytic enzyme complex

We previously reported that expression of NRIF3 (nuclear receptor interacting factor-3) rapidly and selectively leads to apoptosis of breast cancer cells. DIF-1 (also known as interferon regulatory factor-2 binding protein 2 [IRF-2BP2]), the cellular target of NRIF3, was identified as a transcriptional repressor, and DIF-1 knockdown leads to apoptosis of breast cancer cells but not other cell types. Here, we identify IRF-2BP1 and EAP1 (enhanced at puberty 1) as important components of the DIF-1 complex mediating both complex stability and transcriptional repression. This interaction of DIF-1, IRF-2BP1, and EAP1 occurs through the conserved C4 zinc fingers of these proteins. Microarray studies were carried out in breast cancer cell lines engineered to conditionally and rapidly increase the levels of the death domain (DD1) region of NRIF3. The DIF-1 complex was found to repress FASTKD2, a putative proapoptotic gene, in breast cancer cells and to bind to the FASTKD2 gene by chromatin immunoprecipitation. FASTKD2 knockdown prevents apoptosis of breast cancer cells from NRIF3 expression or DIF-1 knockdown, while expression of FASTKD2 leads to apoptosis of both breast and nonbreast cancer cells. Thus, regulation of FASTKD2 by NRIF3 and the DIF-1 complex acts as a novel death switch that selectively modulates apoptosis in breast cancer

Receptor tyrosine kinases (RTKs) are proteins that upon ligand stimulation undergo dimerization and autophosphorylation. Eph receptors (EphRs) are RTKs that are found in different cell types, from both tissues that are developing and from mature tissues, and play important roles in the development of the central nervous system and peripheral nervous system. EphRs also play roles in synapse formation, neural crest formation, angiogenesis and in remodeling the vascular system. Interaction of EphRs with their ephrin ligands lead to activation of signal transduction pathways and formation of many transient protein-protein interactions that ultimately leads to cytoskeletal remodeling. However, the sequence of events at the molecular level is not well understood. We used blue native PAGE and MS to analyze the transient protein-protein interactions that resulted from the stimulation of EphB2 receptors by their ephrinB1-Fc ligands. We analyzed the phosphotyrosine-containing protein complexes immunoprecipitated from the cell lysates of both unstimulated (-) and ephrinB1-Fc-stimulated (+) NG108 cells. Our experiments allowed us to identify many signaling proteins, either known to be part of EphB2 signaling or new for this pathway, which are involved in transient protein-protein interactions upon ephrinB1-Fc stimulation. These data led us to investigate the roles of proteins such as FAK, WAVEs and Nischarin in EphB2 signaling

A major unmet need in LC-MS/MS-based proteomics analyses is a set of tools for quantitative assessment of system performance and evaluation of technical variability. Here we describe 46 system performance metrics for monitoring chromatographic performance, electrospray source stability, MS1 and MS2 signals, dynamic sampling of ions for MS/MS, and peptide identification. Applied to data sets from replicate LC-MS/MS analyses, these metrics displayed consistent, reasonable responses to controlled perturbations. The metrics typically displayed variations less than 10% and thus can reveal even subtle differences in performance of system components. Analyses of data from interlaboratory studies conducted under a common standard operating procedure identified outlier data and provided clues to specific causes. Moreover, interlaboratory variation reflected by the metrics indicates which system components vary the most between laboratories. Application of these metrics enables rational, quantitative quality assessment for proteomics and other LC-MS/MS analytical applications.

Activation of the c-JUN N-terminal kinase (JNK) pathway is implicated in a number of important physiological processes, from embryonic morphogenesis to cell survival and apoptosis. JNK stimulatory phosphatase 1 (JSP1) is a member of the dual-specificity phosphatase subfamily of protein tyrosine phosphatases. In contrast to other dual-specificity phosphatases that catalyze the inactivation of mitogen-activated protein kinases, expression of JSP1 activates JNK-mediated signaling. JSP1 and its relative DUSP15 are unique among members of the protein tyrosine phosphatase family in that they contain a potential myristoylation site at the N-terminus (MGNGMXK). In this study, we investigated whether JSP1 was myristoylated and examined the functional consequences of myristoylation. Using mass spectrometry, we showed that wild-type JSP1, but not a JSP1 mutant in which Gly2 was mutated to Ala (JSP1-G2A), was myristoylated in cells. Although JSP1 maintained intrinsic phosphatase activity in the absence of myristoylation, the subcellular localization of the enzyme was altered. Compared with the wild type, the ability of nonmyristoylated JSP1 to induce JNK activation and phosphorylation of the transcription factor c-JUN was attenuated. Upon expression of wild-type JSP1, a subpopulation of cells, with the highest levels of the phosphatase, was induced to float off the dish and undergo apoptosis. In contrast, cells expressing similar levels of JSP1-G2A remained attached, further highlighting that the myristoylation mutant was functionally compromised.

Genetic analyses previously implicated the matrix-localized protease ClpP in signaling the stress of protein misfolding in the mitochondrial matrix to activate nuclear-encoded mitochondrial chaperone genes in C. elegans (UPR(mt)). Here, we report that haf-1, a gene encoding a mitochondria-localized ATP-binding cassette protein, is required for signaling within the UPR(mt) and for coping with misfolded protein stress. Peptide efflux from isolated mitochondria was ATP dependent and required HAF-1 and the protease ClpP. Defective UPR(mt) signaling in the haf-1-deleted worms was associated with failure of the bZIP protein, ZC376.7, to localize to nuclei in worms with perturbed mitochondrial protein folding, whereas zc376.7(RNAi) strongly inhibited the UPR(mt). These observations suggest a simple model whereby perturbation of the protein-folding environment in the mitochondrial matrix promotes ClpP-mediated generation of peptides whose haf-1-dependent export from the matrix contributes to UPR(mt) signaling across the mitochondrial inner membrane

The complexity of proteomic instrumentation for LC-MS/MS introduces many possible sources of variability. Data-dependent sampling of peptides constitutes a stochastic element at the heart of discovery proteomics. Although this variation impacts the identification of peptides, proteomic identifications are far from completely random. In this study, we analyzed interlaboratory data sets from the NCI Clinical Proteomic Technology Assessment for Cancer to examine repeatability and reproducibility in peptide and protein identifications. Included data spanned 144 LC-MS/MS experiments on four Thermo LTQ and four Orbitrap instruments. Samples included yeast lysate, the NCI-20 defined dynamic range protein mix, and the Sigma UPS 1 defined equimolar protein mix. Some of our findings reinforced conventional wisdom, such as repeatability and reproducibility being higher for proteins than for peptides. Most lessons from the data, however, were more subtle. Orbitraps proved capable of higher repeatability and reproducibility, but aberrant performance occasionally erased these gains. Even the simplest protein digestions yielded more peptide ions than LC-MS/MS could identify during a single experiment. We observed that peptide lists from pairs of technical replicates overlapped by 35-60%, giving a range for peptide-level repeatability in these experiments. Sample complexity did not appear to affect peptide identification repeatability, even as numbers of identified spectra changed by an order of magnitude. Statistical analysis of protein spectral counts revealed greater stability across technical replicates for Orbitraps, making them superior to LTQ instruments for biomarker candidate discovery. The most repeatable peptides were those corresponding to conventional tryptic cleavage sites, those that produced intense MS signals, and those that resulted from proteins generating many distinct peptides. Reproducibility among different instruments of the same type lagged behind repeatability of technical replicates on a single instrument by several percent. These findings reinforce the importance of evaluating repeatability as a fundamental characteristic of analytical technologies

Work from multiple laboratories has clarified how the structural domains of botulinum neurotoxin A (BoNT/A) disable neuronal exocytosis, but important questions remain unanswered. Because BoNT/A intoxication disables its own uptake, light chain (LC) does not accumulate in neurons at detectable levels. We have therefore designed, expressed and purified a series of BoNT/A atoxic derivatives (ad) that retain the wild type features required for native trafficking. BoNT/A1ad(ek) and BoNT/A1ad(tev) are full length derivatives rendered atoxic through double point mutations in the LC protease (E(224)>A; Y(366)>A). DeltaLC-peptide-BoNT/A(tev) and DeltaLC-GFP-BoNT/A(tev) are derivatives wherein the catalytic portion of the LC is replaced with a short peptide or with GFP plus the peptide. In all four derivatives, we have fused the S6 peptide sequence GDSLSWLLRLLN to the N-terminus of the proteins to enable site-specific attachment of cargo using Sfp phosphopantetheinyl transferase. Cargo can be attached in a manner that provides a homogeneous derivative population rather than a polydisperse mixture of singly and multiply-labeled molecular species. All four derivatives contain an introduced cleavage site for conversion into disulfide-bonded heterodimers. These constructs were expressed in a baculovirus system and the proteins were secreted into culture medium and purified to homogeneity in yields ranging from 1 to 30 mg per liter. These derivatives provide unique tools to study toxin trafficking in vivo, and to assess how the structure of cargo linked to the heavy chain (HC) influences delivery to the neuronal cytosol. Moreover, they create the potential to engineer BoNT-based molecular vehicles that can target therapeutic agents to the neuronal cytoplasm

Mutations within the amyloid-beta (Abeta) sequence, especially those clustered at residues 21-23, which are linked to early onset familial Alzheimer's disease (AD), are primarily associated with cerebral amyloid angiopathy (CAA). The basis for this predominant vascular amyloid burden and the differential clinical phenotypes of cerebral hemorrhage/stroke in some patients and dementia in others remain unknown. The AbetaD23N Iowa mutation is associated with progressive AD-like dementia, often without clinically manifested intracerebral hemorrhage. Neuropathologically, the disease is characterized by predominant preamyloid deposits, severe CAA, and abundant neurofibrillary tangles in the presence of remarkably few mature plaques. Biochemical analyses using a combination of immunoprecipitation, mass spectrometry, amino acid sequence, and Western blot analysis performed after sequential tissue extractions to separately isolate soluble components, preamyloid, and fibrillar amyloid species indicated that the Iowa deposits are complex mixtures of mutated and nonmutated Abeta molecules. These molecules exhibited various degrees of solubility, were highly heterogeneous at both the N- and C-termini, and showed partial aspartate isomerization at positions 1, 7, and 23. This collection of Abeta species-the Iowa brain Abeta peptidome-contained clear imprints of amyloid clearance mechanisms yet highlighted the unique neuropathological features shared by a non-Abeta cerebral amyloidosis, familial Danish dementia, in which neurofibrillary tangles coexist with extensive pre-amyloid deposition in the virtual absence of fibrillar lesions. These data therefore challenge the importance of neuritic plaques as the sole contributors for the development of dementia