Human ABCC1 interacts and colocalizes with ATP synthase α, revealed by interactive proteomics analysis.

Human ABCC1 is a member of the ATP-binding cassette (ABC) transporter superfamily, and its overexpression has been shown to cause multidrug resistance by active efflux of a wide variety of anticancer drugs. ABCC1 has been shown to exist and possibly function as a homodimer. However, a possible heterocomplex involving ABCC1 has been indicated. In this study, we performed an interactive proteomics study to examine proteins that bind to and form heterocomplexes with ABCC1 using coimmunoprecipitation and tandem mass spectrometry (MS/MS) analyses. We found that ATP synthase α binds to ABCC1 in plasma membranes with a ratio of 2:1. The ATP synthase α binding site in ABCC1 is located in the linker domain at the carboxyl core of ABCC1, and phosphorylation of the linker domain at the protein kinase A site enhances ATP synthase α binding. The interaction between ABCC1 and ATP synthase α in a heterocomplex may indicate a novel function of ABCC1 in regulating extracellular ATP level and purinergic signaling cascade.

Identification of markers for nipple epidermis: changes in expression during pregnancy and lactation.

In vertebrates, specific regions of skin crucial for interaction with and manipulation of elements in the environment are characterized by specialized epidermis. Regions of specialized epidermis show distinct patterns of cellular differentiation and express specific keratins that provide an increased ability to withstand mechanical strain. The nipple, which must endure the mechanical strain of nursing, is a type of specialized epidermis. The entire ventral skin of the keratin 14 promoter driven PTHrP mouse provides a model for nipple development. To identify novel markers for this specialized epidermis, we have used two-dimensional (2-D) gels, mass spectrometric protein identification, Western blotting and immunohistochemistry to compare intermediate filament preparations from the nipple-like K14-PTHrP ventral skin to that of wild-type littermates. We identified 64 spots on 2-D gels that were increased in expression in the nipple-like skin of the female K14-PTHrP mouse and 11 spots that were elevated in the wild type. Microsequencing suggested that K17 and epiplakin were among the proteins with the greatest increase in expression in the K14-PTHrP ventral skin. Using Western blots and immunohistochemistry, we evaluated the expression of these proteins as well as K6 in the wild-type nipple, K14-PTHrP ventral skin and wild-type ventral skin. In addition, we found that the expression of K6 was minimally changed in the pregnant and lactating nipple, but the expression of a previously identified marker, K2e, was reduced during lactation. Using a model of the mechanical strain induced by nursing, we found that K2e but not K6 expression was responsive to this condition. The identification of epidermal markers and their expression patterns will provide insight into the cellular differentiation patterns of the nipple and the underlying epidermal-mesenchymal interactions that direct this differentiation.

Changes in liver protein abundance in inbred alcohol-preferring rats due to chronic alcohol exposure, as measured through a proteomics approach.

This study compares the total liver proteome of inbred alcohol-preferring line (iP) rats exposed to alcohol with iP rats without alcohol experience. Rat liver proteins were extracted using a three-step procedure. Each of the three solutions solubilizes a different set of proteins. The extracted proteins were separated by 2-DE. Scanned gels of two sample groups, alcohol-exposed iP and alcohol-naïve iP, were compared, revealing many protein spots with significantly higher or lower densities. These spots were cut from the gel, destained, and subjected to trypsin digestion and subsequent identification by LC-MS/MS. Twenty-four individual rats, 12 alcohol-naïve, and 12 alcohol-exposed, were used in this study. Two groups, each containing six naïve and six exposed animals, were created for statistical comparison. For the first group, 64 spots were observed to have statistically significant intensity differences upon alcohol exposure across all three extracts while 118 such spots were found in the second group. There were 113 unique proteins in both groups together. The majority of these proteins were enzymes. Significant changes are observed for three major metabolic pathways: glycolysis, gluconeogenesis, and fatty acid beta-oxidation. In addition, enzymes involved in protein synthesis and antioxidant activity show significant changes in abundance in response to alcohol exposure.

Modulation of differentiation-related gene 1 expression by cell cycle blocker mimosine, revealed by proteomic analysis.

L-mimosine, a plant amino acid, can reversibly block mammalian cells at late G1 phase and has been found to affect translation of mRNAs of the cyclin-dependent kinase inhibitor p27, eIF3a (eIF3 p170), and ribonucleotide reductase M2. The effect of mimosine on the expression of these genes may be essential for the G1 phase arrest. To determine additional genes that may be early respondents to the mimosine treatment, we performed two-dimensional gel electrophoretic analysis of [35S]methionine-labeled cell lysates followed by identification of the altered protein spots by LC-tandem mass spectrometry. In this study, the synthesis of two protein spots (MIP42 and MIP17) was found to be enhanced by mimosine, whereas the formation of another protein spot (MSP17) was severely blocked following mimosine treatment. These protein spots, MIP42, MIP17, and MSP17, were identified to be differentiation-related gene 1 (Drg-1; also called RTP, cap43, rit42, Ndrg-1, and PROXY-1), deoxyhypusine-containing eIF5A intermediate, and mature hypusine-containing eIF5A, respectively. The effect of mimosine on eIF5A maturation was due to inhibition of deoxyhypusine hydroxylase, the enzyme catalyzing the final step of hypusine biosynthesis in eIF5A. The mimosine-induced expression of Drg-1 was mainly attributable to increased transcription likely by the c-Jun/AP-1 transcription factor. Because induction of Drg-1 is an early event after mimosine treatment and is observed before a notable reduction in the steady-state level of mature eIF5A, eIF5A does not appear to be involved in the modulation of Drg-1 expression.

A proteomic survey of rat cerebral cortical synaptosomes.

Previous findings from our laboratory and others indicate that two-dimensional gel electrophoresis (2-DE) can be used to study protein expression in defined brain regions, but mainly the proteins which are present in high abundance in glia are readily detected. The current study was undertaken to determine the protein profile in a synaptosomal subcellular fraction isolated from the cerebral cortex of the rat. Both 2-DE and liquid chromatography - tandem mass spectrometry (LC-MS/MS) procedures were used to isolate and identify proteins in the synaptosomal fraction and accordingly >900 proteins were detected using 2-DE; the 167 most intense gel spots were isolated and identified with matrix-assisted laser desorption/ionization - time of flight peptide mass fingerprinting or LC-MS/MS. In addition, over 200 proteins were separated and identified with the LC-MS/MS "shotgun proteomics" technique, some in post-translationally modified form. The following classes of proteins associated with synaptic function were detected: (a) proteins involved in synaptic vesicle trafficking-docking (e.g., SNAP-25, synapsin I and II, synaptotagmin I, II, and V, VAMP-2, syntaxin 1A and 1B, etc.); (b) proteins that function as transporters or receptors (e.g., excitatory amino acid transporters 1 and 2, GABA transporter 1); (c) proteins that are associated with the synaptic plasma membrane (e.g., post-synaptic density-95/synapse-associated protein-90 complex, neuromodulin (GAP-43), voltage-dependent anion-selective channel protein (VDACs), sodium-potassium ATPase subunits, alpha 2 spectrin, septin 7, etc.); and (d) proteins that mediate intracellular signaling cascades that modulate synaptic function (e.g., calmodulin, calcium-calmodulin-dependent protein kinase subunits, etc.). Other identified proteins are associated with mitochondrial or general cytosolic function. Of the two proteins identified as endoplasmic reticular, both interact with the synaptic SNARE complex to regulate vesicle trafficking. Taken together, these results suggest that the integrity of the synaptosomes was maintained during the isolation procedure and that this subcellular fractionation technique enables the enrichment of proteins associated with synaptic function. The results also suggest that this experimental approach can be used to study the differential expression of multiple proteins involved in alterations of synaptic function.

Fast proteolytic digestion coupled with organelle enrichment for proteomic analysis of rat liver.

The use of an acid-labile surfactant as an alternative to urea denaturation allows for same-day proteolytic digestion and fast cleanup of cellular lysate samples. Homogenized rat liver tissue was separated into four fractions enriched in nuclei, mitochondria, microsomes (remaining organelles), and cytosol. Each subcellular fraction was then subjected to proteolytic digestion with trypsin for 2 h after denaturing with an acid-labile surfactant (ALS), separated by nanoflow reversed phase HPLC, and mass analyzed by tandem mass spectrometry in a 3-D ion trap. The results obtained from ALS denaturation for both organelle enrichment and whole cell lysate samples were comparable to those obtained from aliquots of the same samples treated by reduction, alkylation, and urea denaturation. Each method resulted in a similar number of peptides (694 for urea, 674 for ALS) and proteins (225 for urea, 229 for ALS) identified, with generally the same proteins (47% overlap) identified. As expected, organelle enrichment enabled the identification of more proteins (66% more with urea, 60% more with ALS) compared to a whole cell lysate. With organelle enrichment, the number of proteins with equal or increased sequence coverage went up by 73% with urea and 67% with ALS compared to the whole cell lysate. Additional information regarding the subcellular location of many proteins is obtained by organelle enrichment. While organelle enrichment is demonstrated with a bottom-up proteomics approach, it should be easily amenable to top-down proteomics approaches.