Biomedical subjects

Robert J Chalkley

Publications and source records attributed to Robert J Chalkley.

16 recordsLinked to original sources

PubMed2006

Keratin 20 serine 13 phosphorylation is a stress and intestinal goblet cell marker.

Keratin polypeptide 20 (K20) is an intermediate filament protein with preferential expression in epithelia of the stomach, intestine, uterus, and bladder and in Merkel cells of the skin. K20 expression is used as a marker to distinguish metastatic tumor origin, but nothing is known regarding its regulation and function. We studied K20 phosphorylation as a first step toward understanding its physiologic role. K20 phosphorylation occurs preferentially on serine, with a high stoichiometry as compared with keratin polypeptides 18 and 19. Mass spectrometry analysis predicted that either K20 Ser(13) or Ser(14) was a likely phosphorylation site, and Ser(13) was confirmed as the phospho-moiety using mutation and transfection analysis and generation of an anti-K20-phospho-Ser(13) antibody. K20 Ser(13) phosphorylation increases after protein kinase C activation, and Ser(13)-to-Ala mutation interferes with keratin filament reorganization in transfected cells. In physiological contexts, K20 degradation and associated Ser(13) hyperphosphorylation occur during apoptosis, and chemically induced mouse colitis also promotes Ser(13) phosphorylation. Among mouse small intestinal enterocytes, K20 Ser(13) is preferentially phosphorylated in goblet cells and undergoes dramatic hyperphosphorylation after starvation and mucin secretion. Therefore, K20 Ser(13) is a highly dynamic protein kinase C-related phosphorylation site that is induced during apoptosis and tissue injury. K20 Ser(13) phosphorylation also serves as a unique marker of small intestinal goblet cells.

Animals↗

PubMed2006

O-linked N-acetylglucosamine proteomics of postsynaptic density preparations using lectin weak affinity chromatography and mass spectrometry.

O-GlcNAc is a widespread dynamic carbohydrate modification of cytosolic and nuclear proteins with features analogous to phosphorylation. O-GlcNAc acts critically in many cellular processes, including signal transduction, protein degradation, and regulation of gene expression. However, the study of its specific regulatory functions has been limited by difficulties in mapping sites of O-GlcNAc modification. We report methods for direct enrichment and identification of in vivo O-GlcNAc-modified peptides through lectin weak affinity chromatography (LWAC) and mass spectrometry. The effectiveness of this strategy on complex peptide mixtures was demonstrated through enrichment of 145 unique O-GlcNAc-modified peptides from a postsynaptic density preparation. 65 of these O-GlcNAc-modified peptides were sequenced and belonged to proteins with diverse functions in synaptic transmission. Beta-elimination/Michael addition, MS(3) on O-GlcNAc neutral loss ions, and electron capture dissociation were shown to facilitate analysis of O-GlcNAc-modified peptides/sites from lectin weak affinity chromatography enriched postsynaptic density samples. Bassoon and Piccolo, proteins critical to synapse assembly and vesicle docking, were extensively modified by O-GlcNAc. In some cases, O-GlcNAc was mapped to peptides previously identified as phosphorylated, indicating potential interplay between these modifications. Shared substrate amino acid context was apparent in subsets of O-GlcNAc-modified peptides, including "PVST" and a novel "TTA" motif (two hydroxyl-containing amino acids adjacent to an alanine). The results suggest specific roles for O-GlcNAc modification in synaptic transmission, establish a basis for site-specific regulatory studies, and provide methods that will facilitate O-GlcNAc proteome analysis across a wide variety of cells and tissues.

Acetylglucosamine↗

PubMed2005

Mapping post-translational modifications of the histone variant MacroH2A1 using tandem mass spectrometry.

Post-translational histone modifications modulate chromatin-templated processes and therefore affect cellular proliferation, growth, and development. Although post-translational modifications on the core histones have been under intense investigation for several years, the modifications on variant histones are poorly understood. We used tandem mass spectrometry to identify covalent modifications on a histone H2A variant, macroH2A1.2. MacroH2A1.2 can be monoubiquitinated; however, the site of monoubiquitination has not been documented. In this study we used green fluorescent protein-tagged macroH2A1.2 to determine that Lys(115) is a site of ubiquitination. In addition, we found that this variant H2A is methylated on the epsilon amino group of lysine residues Lys(17), Lys(122), and Lys(238) and phosphorylated on Thr(128). Three of these modifications were also found to be present in the endogenous protein by mass spectrometric analysis. These results provide the first direct evidence that multiple post-translational modifications are imposed on macroH2A1.2, suggesting that, like canonical H2A, this variant H2A is subject to regulation by combinatorial use of covalent modifications.

Histones↗

PubMed2005

A combinatorial code for the interaction of alpha-synuclein with membranes.

Considerable genetic and pathological evidence has implicated the small, soluble protein alpha-synuclein in the pathogenesis of familial and sporadic forms of Parkinsons disease (PD). However, the precise role of alpha-synuclein in the disease process as well as its normal function remain poorly understood. We recently found that an interaction with lipid rafts is crucial for the normal, pre-synaptic localization of alpha-synuclein. To understand how alpha-synuclein interacts with lipid rafts, we have now developed an in vitro binding assay to rafts purified from native membranes. Recapitulating the specificity observed in vivo, recombinant wild type but not PD-associated A30P mutant alpha-synuclein binds to lipid rafts isolated from cultured cells and purified synaptic vesicles. Proteolytic digestion of the rafts does not disrupt the binding of alpha-synuclein, indicating an interaction with lipid rather than protein components of these membranes. We have also found that alpha-synuclein binds directly to artificial membranes whose lipid composition mimics that of lipid rafts. The binding of alpha-synuclein to these raft-like liposomes requires acidic phospholipids, with a preference for phosphatidylserine (PS). Interestingly, a variety of synthetic PS with defined acyl chains do not support binding when used individually. Rather, the interaction with alpha-synuclein requires a combination of PS with oleic (18:1) and polyunsaturated (either 20:4 or 22:6) fatty acyl chains, suggesting a role for phase separation within the membrane. Furthermore, alpha-synuclein binds with higher affinity to artificial membranes with the PS head group on the polyunsaturated fatty acyl chain rather than on the oleoyl side chain, indicating a stringent combinatorial code for the interaction of alpha-synuclein with membranes.

Amino Acid Substitution↗

PubMed2005

Comprehensive analysis of a multidimensional liquid chromatography mass spectrometry dataset acquired on a quadrupole selecting, quadrupole collision cell, time-of-flight mass spectrometer: II. New developments in Protein Prospector allow for reliable and comprehensive automatic analysis of large datasets.

A thorough analysis of the protein interaction partners of the yeast GTPase Gsp1p was carried out by a multidimensional chromatography strategy of strong cation exchange fractionation of peptides followed by reverse phase LC-ESI-MSMS using a QSTAR instrument. This dataset was then analyzed using the latest developmental version of Protein Prospector. The Prospector search results were also compared with results from the search engine "Mascot" using a new results comparison program within Prospector named "SearchCompare." The results from this study demonstrate that the high quality data produced on a quadrupole selecting, quadrupole collision cell, time-of-flight (QqTOF) geometry instrument allows for confident assignment of the vast majority of interpretable spectra by current search engines.

Autoanalysis↗

PubMed2005

Comprehensive analysis of a multidimensional liquid chromatography mass spectrometry dataset acquired on a quadrupole selecting, quadrupole collision cell, time-of-flight mass spectrometer: I. How much of the data is theoretically interpretable by search engines?

An in-depth analysis of a multidimensional chromatography-mass spectrometry dataset acquired on a quadrupole selecting, quadrupole collision cell, time-of-flight (QqTOF) geometry instrument was carried out. A total of 3269 CID spectra were acquired. Through manual verification of database search results and de novo interpretation of spectra 2368 spectra could be confidently determined as predicted tryptic peptides. A detailed analysis of the non-matching spectra was also carried out, highlighting what the non-matching spectra in a database search typically are composed of. The results of this comprehensive dataset study demonstrate that QqTOF instruments produce information-rich data of which a high percentage of the data is readily interpretable.

Chromatography, Liquid↗

PubMed2005

Quantitative analysis of both protein expression and serine / threonine post-translational modifications through stable isotope labeling with dithiothreitol.

While phosphorylation and O-GlcNAc (cytoplasmic and nuclear glycosylation) are linked to normal and pathological changes in cell states, these post-translational modifications have been difficult to analyze in proteomic studies. We describe advances in beta-elimination / Michael addition-based approaches which allow for mass spectrometry-based identification and comparative quantification of O-phosphate or O-GlcNAc-modified peptides, as well as cysteine-containing peptides for expression analysis. The method (BEMAD) involves differential isotopic labeling through Michael addition with normal dithiothreitol (DTT) (d0) or deuterated DTT (d6), and enrichment of these peptides by thiol chromatography. BEMAD was comparable to isotope-coded affinity tags (ICAT; a commercially available differential isotopic quantification technique) in protein expression analysis, but also provided the identity and relative amounts of both O-phosphorylation and O-GlcNAc modification sites. Specificity of O-phosphate vs. O-GlcNAc mapping is achieved through coupling enzymatic dephosphorylation or O-GlcNAc hydrolysis with differential isotopic labeling. Blocking of cysteine labeling by prior oxidation of a cytosolic lysate from mouse brain allowed specific targeting of serine / threonine post-translational modifications as demonstrated through identification of 21 phosphorylation sites (5 previously reported) in a single mass spectrometry analysis. These results demonstate BEMAD is suitable for large-scale quantitative analysis of both protein expression and serine / threonine post-translational modifications.

Animals↗

PubMed2005

Mass spectrometric analysis of histone posttranslational modifications.

The uses of tandem and Fourier transform mass spectrometric methodologies for assignment of the posttranslational sites and occupancies of histones and their isoforms is described employing several illustrative examples. A comparison of information that can be obtained from intact protein sequencing and proteolytic digestion is presented.

Acetylation↗

PubMed2005

Bioinformatic methods to exploit mass spectrometric data for proteomic applications.

The new technologies in mass spectrometric analysis of peptides and proteins necessary to accommodate proteomics-scale analyses require, in turn, concomitant development of informatics technologies suitable for very large-scale data handling and analysis. This chapter focuses on the data analysis tools available to the community for analysis of mass spectrometric proteomics data. Different database searching strategies are discussed for peptide and protein identification, and approaches and tools available for comparative quantitative analysis of samples are outlined.

Animals↗

PubMed2004

The urinary proteome in Fanconi syndrome implies specificity in the reabsorption of proteins by renal proximal tubule cells.

Polypeptides present in the glomerular filtrate are almost completely reabsorbed in the first segment of the proximal tubule by receptor-mediated endocytosis; in renal Fanconi syndrome (FS), there is failure to reabsorb many of these polypeptides. We have compared the urinary proteomes in patients with Dent's disease (due to a CLC5 mutation), a form of FS, with normal subjects using three different proteomic methods. No differences in the levels of several plasma proteins were detected when standardized to total protein amounts. In contrast, several vitamin and prosthetic group carrier proteins were found in higher amounts in Dent's urine (with respect to total protein). Similarly, complement components, apolipoproteins, and some cytokines represented a larger proportion of the Dent's urinary proteome, suggesting that such proteins are reabsorbed more efficiently than other classes of proteins. Conversely, proteins of renal origin were found in proportionately higher amounts in normal urine. Thus the uptake of filtered vitamins, which are normally bound to their respective carrier proteins to prevent urinary losses, seems a key function of the proximal tubule; in addition, this nephron segment may also play a critical role in reabsorbing potentially cytotoxic polypeptides of plasma origin, preventing them from acting at more distal nephron sites.

Adolescent↗

PubMed2004

Two distinct coactivators, DRIP/mediator and SRC/p160, are differentially involved in VDR transactivation during keratinocyte differentiation.

Cell programs such as proliferation and differentiation involve the sequential activation and repression of gene expression. Vitamin D, via its active metabolite 1,25-dihydroxyvitamin D (1,25(OH)(2)D(3)), controls the proliferation and differentiation of a number of cell types, including keratinocytes, by directly regulating transcription. Two classes of coactivators, the Vitamin D receptor (VDR) interacting proteins (DRIP/mediator) and the p160 steroid receptor coactivator family (SRC/p160), control the actions of nuclear hormone receptors, including the Vitamin D receptor. However, the relationship between these two classes of coactivators is not clear. Using GST-VDR affinity beads, we have identified the DRIP/mediator complex as the major VDR binding complex in proliferating keratinocytes. After the cells differentiated, members of the SRC/p160 family were identified in the complex but not major DRIP subunits. Both DRIP205 and SRC-3 potentiated Vitamin D-induced transcription in proliferating cells, but during differentiation, DRIP205 was no longer effective. These results indicate that these two distinct coactivators are differentially involved in Vitamin D regulation of gene transcription during keratinocyte differentiation, suggesting that these coactivators are part of the means by which the temporal sequence of gene expression is regulated during the differentiation process.

Animals↗

PubMed2003

Regulated expression of apolipoprotein E by human retinal pigment epithelial cells.

In early age-related macular degeneration (AMD), lipid-containing deposits (drusen) accumulate in Bruch's membrane underlying the retinal pigment epithelium (RPE). Recent studies indicate that apolipoprotein E (apoE) may play a role in lipid trafficking in AMD. Compared with the apoE3 allele, the apoE4 and apoE2 alleles are associated with decreased and increased risk for AMD, respectively; drusen contain high levels of apoE, and apoE null mice develop lipid deposits in Bruch's membrane similar to those observed in AMD. Primary cultures of human RPE cells expressing the apoE3 allele were grown on Transwell culture plates. Western blotting, ELISA assay, and mass spectrometry confirmed that apoE3 was secreted into the apical and basal chambers and that secretion was upregulated by thyroid hormone, 9-cis-retinoic acid, and 22(R)-hydroxycholesterol. In addition, basally secreted apoE associated with exogenously added HDL. These results indicate that apoE secretion can be regulated by specific hormones and that apoE associates with HDL. The findings are consistent with a role for apoE in lipid trafficking through Bruch's membrane and may be relevant to AMD.

Adult↗

PubMed2003

Two distinct coactivators, DRIP/mediator and SRC/p160, are differentially involved in vitamin D receptor transactivation during keratinocyte differentiation.

Cell programs such as proliferation and differentiation involve the sequential activation and repression of gene expression. Vitamin D, via its active metabolite 1,25-dihydroxyvitamin D [1,25-(OH)2D3)], controls the proliferation and differentiation of a number of cell types, including keratinocytes, by directly regulating transcription. Two classes of coactivators, the vitamin D receptor (VDR)-interacting proteins (DRIP/mediator) and the p160 steroid receptor coactivator family (SRC/p160), control the actions of nuclear hormone receptors, including the VDR. However, the relationship between these two classes of coactivators is not clear. Using glutathione-S-transferase-VDR affinity beads, we have identified the DRIP/mediator complex as the major VDR binding complex in proliferating keratinocytes. After the cells differentiated, members of the SRC/p160 family were identified in the complex but not major DRIP subunits. Both DRIP and SRC proteins were expressed in keratinocytes. DRIP205 expression decreased during differentiation, although SRC-3 levels increased. Both DRIP205 and SRC-3 potentiated vitamin D-induced transcription in proliferating cells, but during differentiation, DRIP205 was no longer effective. These results indicate that these two distinct coactivators are sequentially involved in vitamin D regulation of gene transcription during keratinocyte differentiation, suggesting that these coactivators are part of the means by which the temporal sequence of gene expression is regulated during the differentiation process.

Amino Acid Sequence↗

PubMed2003

Mass spectrometric analysis of protein mixtures at low levels using cleavable 13C-isotope-coded affinity tag and multidimensional chromatography.

In order to identify and compare the protein content of very low quantity samples of high complexity, a protocol has been established that combines the differential profiling strength of a new cleavable 13C isotope-coded affinity tag (cICAT) reagent with the high sequence coverage provided by multidimensional liquid chromatography and two modes of tandem mass spectrometry. Major objectives during protocol optimization were to minimize sample losses and establish a robust procedure that employs volatile buffer systems that are highly compatible with mass spectrometry. Cleavable ICAT-labeled tryptic peptides were separated from nonlabeled peptides by avidin affinity chromatography. Subsequently, peptide samples were analyzed by nanoflow liquid chromatography electrospray ionization tandem mass spectrometry and liquid chromatography matrix-assisted laser desorption/ionization tandem mass spectrometry. The use of two ionization/instrumental configurations led to complementary peptide identifications that increased the confidence of protein assignments. Examples that illustrate the power of this strategy are taken from two different projects: i) immunoaffinity purified complexes containing the prion protein from the murine brain, and ii) human tracheal epithelium gland secretions. In these studies, a large number of novel proteins were identified using stringent match criteria, in addition to many that had been identified in previous experiments. In the latter case, the ICAT method produced significant new information on changes that occur in protein expression levels in a patient suffering from cystic fibrosis.

Amino Acid Sequence↗

PubMed2003

Identification of novel sites of O-N-acetylglucosamine modification of serum response factor using quadrupole time-of-flight mass spectrometry.

The addition of a single N-acetylglucosamine moiety O-linked to serine and threonine residues of nuclear and cytoplasmic proteins is a widespread post-translational modification. The conventional method for detecting and locating sites of modification is through a multistep radioactivity-based approach. We have recently shown that sites of O-GlcNAc modification can be determined using quadrupole time-of-flight tandem mass spectrometry (Chalkley, R. J., and Burlingame, A. L. (2001) Identification of GlcNAcylation sites of peptides and alpha-crystallin using Q-TOF mass spectrometry. J. Am. Soc. Mass Spectrom. 12, 1106-1113). In this work utilization of this new approach has revealed previously undetected sites of O-GlcNAc modification of the transcription factor serum response factor.

Acetylglucosamine↗

PubMed2002

Deciphering networks of protein interactions at the nuclear pore complex.

The nuclear pore complex (NPC) gates the only known conduit for molecular exchange between the nucleus and cytoplasm of eukaryotic cells. Macromolecular transport across the NPC is mediated by nucleocytoplasmic shuttling receptors termed karyopherins (Kaps). Kaps interact with NPC proteins (nucleoporins) that contain FG peptide repeats (FG Nups) and altogether carry hundreds of different cargoes across the NPC. Previously we described a biochemical strategy to identify proteins that interact with individual components of the nucleocytoplasmic transport machinery. We used bacterially expressed fusions of glutathione S-transferase with nucleoporins or karyopherins as bait to capture interacting proteins from yeast extracts. Forty-five distinct proteins were identified as binding to one or several FG Nups and Kaps. Most of the detected interactions were expected, such as Kap-Nup interactions, but others were unexpected, such as the interactions of the multisubunit Nup84p complex with several of the FG Nups. Also unexpected were the interactions of various FG Nups with the nucleoporins Nup2p and Nup133p, the Gsp1p-GTPase-activating protein Rna1p, and the mRNA-binding protein Pab1p. Here we resolve how these interactions occur. We show that Pab1p associates nonspecifically with immobilized baits via RNA. More interestingly, we demonstrate that the Nup84p complex contains Nup133p as a subunit and binds to the FG repeat regions of Nups directly via the Nup85p subunit. Binding of Nup85p to the GLFG region of Nup116p was quantified in vitro (K(D) = 1.5 micro M) and was confirmed in vivo using the yeast two-hybrid assay. We also demonstrate that Nup2p and Rna1p can be tethered directly to FG Nups via the importin Kap95p-Kap60p and the exportin Crm1p, respectively. We discuss possible roles of these novel interactions in the mechanisms of nucleocytoplasmic transport.

Active Transport, Cell Nucleus↗