Faculty Bibliography
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215
Guidelines for reporting the use of mass spectrometry in proteomics [Letter]
PMID: 18688232
ISSN: 1546-1696
CID: 96817
Stable isotopic labeling of amino acids in cultured primary neurons: Application to BDNF-dependent phosphotyrosine-associated signaling
Cultured primary neurons are a well established model for the study of neuronal function in vitro. Here we have demonstrated that Stable Isotope Labeling by Amino Acids in Cell culture (SILAC) can be applied to a differentiated, non-dividing cell type such as primary neurons, and we have applied this technique to assess changes in the neuronal phosphotyrosine proteome in response to stimulation by BDNF (brain derived neurotrophic factor), an important molecule for the development and regulation of neuronal connections. We found that 13 proteins had SILAC ratios above 1.50 or below 0.67 in phosphotyrosine immunoprecipitates (pY IPs) comparing BDNF-treated and control samples, and an additional 18 proteins had ratios above 1.25 or below 0.80. These proteins include TrkB, the receptor tyrosine kinase (RTK) for BDNF, and others such as Hrs (hepatocyte growth factor-regulated tyrosine kinase substrate) and STAM (signal-transducing adaptor molecule), which are proteins known to regulate intracellular trafficking of RTKs. These results demonstrate that the combination of primary neuronal cell culture and SILAC can be a powerful tool for the study of the proteomes of neuronal molecular and cellular dynamics
PMCID:2424194
PMID: 18256212
ISSN: 1535-9476
CID: 76648
Proceedings of the National Academy of Sciences of the United States of America (PNAS). 2008:105(19):7094-9.DOI: 10.1073/pnas.0707025105
Proteasomal adaptation to environmental stress links resistance to proteotoxicity with longevity in Caenorhabditis elegans
The burden of protein misfolding is believed to contribute to aging. However, the links between adaptations to conditions associated with protein misfolding and resistance to the time-dependent attrition of cellular function remain poorly understood. We report that worms lacking aip-1, a homologue of mammalian AIRAP (arsenic-inducible proteasomal 19S regulatory particle-associated protein), are not only impaired in their ability to resist exposure to arsenite but also exhibit shortened lifespan and hypersensitivity to misfolding-prone proteins under normal laboratory conditions. Mammals have a second, constitutively expressed AIRAP-like gene (AIRAPL) that also encodes a proteasome-interacting protein, which shares with AIRAP the property of enhancing peptide accessibility to the proteasome's active site. Genetic rescue experiments suggest that features common to the constitutively expressed worm AIP-1 and mammalian AIRAPL (but missing in the smaller, arsenite-inducible AIRAP) are important to lifespan extension. In worms, a single AIRAP-related protein links proteasomal adaptation to environmental stress with resistance to both proteotoxic insults and maintenance of animal life span under normal conditions
PMCID:2383958
PMID: 18467495
ISSN: 1091-6490
CID: 94504
Phosphorylation of liver X receptor alpha selectively regulates target gene expression in macrophages
Dysregulation of liver X receptor alpha (LXRalpha) activity has been linked to cardiovascular and metabolic diseases. Here, we show that LXRalpha target gene selectivity is achieved by modulation of LXRalpha phosphorylation. Under basal conditions, LXRalpha is phosphorylated at S198; phosphorylation is enhanced by LXR ligands and reduced both by casein kinase 2 (CK2) inhibitors and by activation of its heterodimeric partner RXR with 9-cis-retinoic acid (9cRA). Expression of some (AIM and LPL), but not other (ABCA1 or SREBPc1) established LXR target genes is increased in RAW 264.7 cells expressing the LXRalpha S198A phosphorylation-deficient mutant compared to those with WT receptors. Surprisingly, a gene normally not expressed in macrophages, the chemokine CCL24, is activated specifically in cells expressing LXRalpha S198A. Furthermore, inhibition of S198 phosphorylation by 9cRA or by a CK2 inhibitor similarly promotes CCL24 expression, thereby phenocopying the S198A mutation. Thus, our findings reveal a previously unrecognized role for phosphorylation in restricting the repertoire of LXRalpha-responsive genes
PMCID:2293109
PMID: 18250151
ISSN: 1098-5549
CID: 76646
Calsyntenins Are Secretory Granule Proteins in Anterior Pituitary Gland and Pancreatic Islet {alpha} Cells
Calsyntenins are members of the cadherin superfamily of cell adhesion molecules. They are present in postsynaptic membranes of excitatory neurons and in vesicles in transit to neuronal growth cones. In the current study, calsyntenin-1 (CST-1) and calsyntenin-3 (CST-3) were identified by mass spectrometric analysis (LC-MS/MS) of integral membrane proteins from highly enriched secretory granule preparations from bovine anterior pituitary gland. Immunofluorescence microscopy on thin frozen sections of rat pituitary revealed that CST-1 was present only in gonadotropes where it colocalized with follicle-stimulating hormone in secretory granules. In contrast, CST-3 was present not only in gonadotrope secretory granules but also in those of somatotropes and thyrotropes. Neither protein was detected in mammatropes. In addition, CST-1 was also localized to the glucagon-containing secretory granules of alpha cells in the pancreatic islets of Langerhans. Results indicate that calsyntenins function outside the nervous system and potentially are modulators of endocrine function
PMCID:2326105
PMID: 18158283
ISSN: 0022-1554
CID: 76650
Structural and biochemical characterization of the KRLB region in insulin receptor substrate-2
Insulin receptor substrates 1 and 2 (IRS1 and -2) are crucial adaptor proteins in mediating the metabolic and mitogenic effects of insulin and insulin-like growth factor 1. These proteins consist of a pleckstrin homology domain, a phosphotyrosine binding domain and a C-terminal region containing numerous sites of tyrosine, serine and threonine phosphorylation. Previous yeast two-hybrid studies identified a region unique to IRS2, termed the kinase regulatory-loop binding (KRLB) region, which interacts with the tyrosine kinase domain of the insulin receptor. Here we present the crystal structure of the insulin receptor kinase in complex with a 15-residue peptide from the KRLB region. In the structure, this segment of IRS2 is bound in the kinase active site with Tyr628 positioned for phosphorylation. Although Tyr628 was phosphorylated by the insulin receptor, its catalytic turnover was poor, resulting in kinase inhibition. Our studies indicate that the KRLB region functions to limit tyrosine phosphorylation of IRS2
PMID: 18278056
ISSN: 1545-9985
CID: 76468
Human Proteinpedia enables sharing of human protein data [Letter]
PMID: 18259167
ISSN: 1546-1696
CID: 76647
Use of DNA ladders for reproducible protein fractionation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (sds-page) for quantitative proteomics
In proteomics, one-dimensional (1D) sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) is widely used for protein fractionation prior to mass spectrometric analysis to enhance the dynamic range of analysis and to improve the identification of low-abundance proteins. Such protein prefractionation works well for quantitation strategies if the proteins are labeled prior to separation. However, because of the poor reproducibility of cutting gel slices, especially when small amounts of samples are analyzed, its application in label-free and peptide-labeling quantitative proteomics methods has been greatly limited. To overcome this limitation, we developed a new strategy in which a DNA ladder is mixed with the protein sample before PAGE separation. After PAGE separation, the DNA ladder is stained to allow for easy, precise, and reproducible gel cutting. To this end, a novel visible DNA-staining method was developed. This staining method is fast, sensitive, and compatible with mass spectrometry. To evaluate the reproducibility of DNA-ladder-assisted gel cutting for quantitative protein fractionation, we used stable isotope labeling with amino acids in cell culture (SILAC). Our results show that the quantitative error associated with fractionation can be minimized using the DNA-assisted fractionation and multiple replicates of gel cutting. In conclusion, 1D PAGE fractionation in combination with DNA ladders can be used for label-free comparative proteomics without compromising quantitation
PMCID:2667379
PMID: 18189343
ISSN: 1535-3893
CID: 76649
Analysis of electroblotted proteins by mass spectrometry: protein identification after Western blotting
We describe a new approach for the identification and characterization by mass spectrometry of proteins that have been electroblotted onto nitrocellulose. Using this method (Blotting and Removal of Nitrocellulose (BARN)), proteins can be analyzed either as intact proteins for molecular weight determination or as peptides generated by on-membrane proteolysis. Acetone is used to dissolve the nitrocellulose and to precipitate the adsorbed proteins/peptides, thus removing the nitrocellulose which can interfere with MS analysis. This method offers improved protein coverage, especially for membrane proteins, such as uroplakins, because the extraction step after in-gel digestion is avoided. Moreover, removal of nitrocellulose from the sample solution allows sample analysis by both MALDI- and (LC) ESI-based mass spectrometers. Finally, we demonstrate the utility of BARN for the direct identification of soluble and membrane proteins after Western blotting, obtaining comparable or better results than with in-gel digestion
PMCID:2667373
PMID: 17938404
ISSN: 1535-9484
CID: 76651
NATO Science for Peace & Security. Series A: Chemistry & Biology. 2008:3-22.DOI: 10.1007/978-1-4020-8811-7_1
BLUE NATIVE PAGE AND MASS SPECTROMETRY ANALYSIS OF EPHRIN STIMULATION-DEPENDENT PROTEIN-PROTEIN INTERACTIONS IN NG108-EPHB2 CELLS [Meeting Abstract]
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.
ISI:000259998700001
ISSN: 1871-4641
CID: 2337802
