Differential Protein-protein Interactions Research Articles

The DifferentialNet database of differential protein-protein interactions in human tissues.

DifferentialNet is a novel database that provides users with differential interactome analysis of human tissues (http://netbio.bgu.ac.il/diffnet/). Users query DifferentialNet by protein, and retrieve its differential protein–protein interactions (PPIs) per tissue via an interactive graphical interface. To compute differential PPIs, we integrated available data of experimentally detected PPIs with RNA-sequencing profiles of tens of human tissues gathered by the Genotype-Tissue Expression consortium (GTEx) and by the Human Protein Atlas (HPA). We associated each PPI with a score that reflects whether its corresponding genes were expressed similarly across tissues, or were up- or down-regulated in the selected tissue. By this, users can identify tissue-specific interactions, filter out PPIs that are relatively stable across tissues, and highlight PPIs that show relative changes across tissues. The differential PPIs can be used to identify tissue-specific processes and to decipher tissue-specific phenotypes. Moreover, they unravel processes that are tissue-wide yet tailored to the specific demands of each tissue.

Abstract P3-05-03: A novel progesterone receptor (PR)-RNA polymerase III association represses estrogen-dependent growth in breast tumor patient-derived xenografts

Abstract Background: Progesterone (P) is an important hormone for development and normal function of the breast; however, its role in established breast cancers is less clear. P has been implicated in regulating tumor cell growth, signaling, differentiation state, and stem/progenitor properties in breast cancer cells. Progesterone receptors (PRs) are considered positive prognostic indicators yet potential targets for treatment, although the dilemma of positive or negative targeting persists. P can either inhibit or stimulate breast cancer cell growth in the absence of estrogens (E), and usually blocks E mediated growth. Recent studies using breast cancer cell lines have deciphered a mechanism by which P suppresses E dependent growth through modulation of the estrogen receptor (ER) cistrome through a physical ER/PR association. However, the ER/PR association is weak to undetectable in our ER+PR+ breast cancer patient-derived xenografts (PDX). We therefore hypothesize that PR can regulate E-dependent breast cancer growth independent of direct interference of ER transcription. The purpose of this study was to use unbiased genomics and proteomics of breast cancer PDX to uncover additional mechanisms of P repression of breast cancer growth. Methods: These studies used two luminal ER+PR+ PDX (UCD4 and UCD65) that contain high levels of ER and PR (>90%), and where P inhibits E-dependent growth. Tumors were grown in vivo in female NSG mice under continuous placebo, E (17b-estradiol), E plus P, or E plus the synthetic progestin medroxyprogesterone acetate (MPA) for 8-10 weeks. RNAseq, chromatin immunoprecipitation sequencing (ChIPseq), and rapid immunoprecipitation followed by mass spectrometry of endogenous proteins (RIME) were performed to analyze differential transcriptional, cistromic, and protein-protein interactions in E compared to E plus P or MPA treated tumors. Co-immunoprecipitation (co-IP) and ChIP were used to verify results. Results: Both P and MPA potently inhibited E-dependent growth of both tumor lines. Gene expression studies found that both hormones reversed transcription of over one third of the estrogen/ER transcriptome in both tumors. RIME for PR uncovered significant interactions between PR and multiple RNA polymerase III subunits (POLR3). Co-IP using POLR3A and POLR3B confirmed PR associates with the POLR3 holoenzyme. Furthermore, ChIPseq revealed that PR binds to one third of POLR3 regulated tRNA genes. PR also associated with Maf1 in one tumor, a known POLR3 suppressor. Conclusions: Here we describe a novel association of PR with POLR3 in two luminal breast cancer PDX, an interaction not described in breast cancer cell lines. Our data suggest this is a negative regulatory interaction that may occur through recruiting a POLR3 repressor. These data implicate multifaceted P control of E dependent tumor growth; in addition to antagonizing E regulated genes at the transcription level, P can regulate translation though depletion of amino acid carrying tRNAs, thus slowing protein synthesis. Identifying which tumors utilize this growth-suppressive mechanism may pinpoint appropriate candidates for progestin therapy and/or provide a prognostic tool for predicting tumor progression. Citation Format: Finlay-Schultz J, Gillen AE, Brechbuhl HM, Ivie J, Bentley DL, Kabos P, Sartorius CA. A novel progesterone receptor (PR)-RNA polymerase III association represses estrogen-dependent growth in breast tumor patient-derived xenografts [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P3-05-03.

Evolutionary and Functional Diversification of the Vitamin D Receptor-Lithocholic Acid Partnership.

The evolution, molecular behavior, and physiological function of nuclear receptors are of particular interest given their diverse roles in regulating essential biological processes. The vitamin D receptor (VDR) is well known for its canonical roles in calcium homeostasis and skeletal maintenance. Additionally, VDR has received an increased amount of attention due to the discovery of numerous non-calcemic functions, including the detoxification of lithocholic acid. Lithocholic acid is a toxic metabolite of chenodeoxycholic acid, a primary bile acid. The partnership between the VDR and lithocholic acid has been hypothesized to be a recent adaptation that evolved to mediate the detoxification and elimination of lithocholic acid from the gut. This partnership is speculated to be limited to higher vertebrates (birds and mammals), as lower vertebrates do not synthesize the parent compound of lithocholic acid. However, the molecular functions associated with the observed insensitivity of basal VDRs to lithocholic acid have not been explored. Here we characterize canonical nuclear receptor functions of VDRs from select species representing key nodes in vertebrate evolution and span a range of bile salt phenotypes. Competitive ligand binding assays revealed that the receptor’s affinity for lithocholic acid is highly conserved across species, suggesting that lithocholic acid affinity is an ancient and non-adaptive trait. However, transient transactivation assays revealed that lithocholic acid-mediated VDR activation might have evolved more recently, as the non-mammalian receptors did not respond to lithocholic acid unless exogenous coactivator proteins were co-expressed. Subsequent functional assays indicated that differential lithocholic acid-mediated receptor activation is potentially driven by differential protein-protein interactions between VDR and nuclear receptor coregulator proteins. We hypothesize that the vitamin D receptor-lithocholic acid partnership evolved as a by-product of natural selection on the ligand-receptor partnership between the vitamin D receptor and the native VDR ligand: 1α,25-dihydroxyvitamin D3, the biologically active metabolite of vitamin D3.

Comprehensive Analysis of the Structural, Biochemical and Signaling Differences of the p210 and p185 Isoforms of Bcr-Abl in CML and B-ALL

Distinct chromosomal translocation breakpoints result in the expression of two major isoforms of Bcr-Abl - p210 and p185 - from the Philadelphia (Ph)-chromosome. While p210 is the molecular hallmark of Chronic Myeloid Leukemia (CML), in Ph-positive B-cell Acute Lymphoblastic Leukemia (B-ALL), ~1/3 of the patients express p210 and ~2/3 express p185. In mouse models, under certain experimental conditions, expression of p185 causes disease with a shorter latency and more B-ALL than CML-like leukemias as did p210. Ph-positive B-ALL patients show a poorer response and frequent resistance to tyrosine kinase inhibitor (TKI) therapy as compared to CML patients. Specific intrinsic differences in p185 and p210 Bcr-Abl signaling have long been hypothesized, but have never been studied in a comparative, unbiased and comprehensive manner. We have now:(i) Mapped the differences in the p210 and p185 protein interaction network: We performed an unbiased proteomics analysis of the composition of the endogenous p210 and p185 protein complexes in murine cell lines using SILAC (stable isotope labeling with amino acids in cell culture) quantification. The interactome analysis confirmed previously reported p210 interactors in human CML cells and identified unexpectedly many interactors that preferentially associate with either p185 or p210, including an endocytosis-regulating complex (enriched in p185) and a phosphatase complex (enriched in p210). Several differential interactors were validated in a panel of nine human CML and B-ALL cell lines.(ii) Performed a comprehensive phospho-proteomics study in p210 and p185 cell lines: Using phospho-tyrosine, as well as general phospho-peptide enrichment strategies coupled to SILAC quantification, we mapped >800 phosphorylation sites and identified differentially activated signaling by p210 and p185, including differential activation of the STAT5 and STAT3 transcription factors, SRC kinases and ERK1/2 signaling. These findings were validated in a panel of nine human CML and B-ALL cell lines.(iii) Determined the atomic structures of the Dbl-homology (DH) and pleckstrin homology (PH) domains of p210: The DH-PH domain unit of p210 is the only structural difference to p185 and has remained the only structurally uncharacterized region of Bcr-Abl. We solved different structures of these domains by NMR spectroscopy, X-ray crystallography, as well as Small-Angle X-ray scattering, alone and in complex with an engineered monobody inhibitor targeting the DH domain. While the DH-PH structural motif is found in Rho guanine nucleotide exchange factors (Rho GEF), our results have not provided evidence that the p210 DH-PH domain acts as a Rho GEF or binds Rho GTPases, in contrast to previous reports. On the other hand, we found the PH domain to be important for specific membrane binding of p210 and we determined the binding specificity of the PH domain to certain phospho-inositide lipids.Our results provide the first systematic insight showing critical differences in protein-protein interactions, signaling and structure of p210 and p185 Bcr-Abl. This will contribute to a better understanding of Bcr-Abl dependent signaling, mechanisms of oncogenic transformation, resulting disease pathobiology and responses to TKIs. DisclosuresHantschel:Ariad Pharmaceuticals: Honoraria.

Network-Guided Key Gene Discovery for a Given Cellular Process.

Identification of key genes for a given physiological or pathological process is an essential but still very challenging task for the entire biomedical research community. Statistics-based approaches, such as genome-wide association study (GWAS)- or quantitative trait locus (QTL)-related analysis have already made enormous contributions to identifying key genes associated with a given disease or phenotype, the success of which is however very much dependent on a huge number of samples. Recent advances in network biology, especially network inference directly from genome-scale data and the following-up network analysis, opens up new avenues to predict key genes driving a given biological process or cellular function. Here we review and compare the current approaches in predicting key genes, which have no chances to stand out by classic differential expression analysis, from gene-regulatory, protein-protein interaction, or gene expression correlation networks. We elaborate these network-based approaches mainly in the context of immunology and infection, and urge more usage of correlation network-based predictions. Such network-based key gene discovery approaches driven by information-enriched 'omics' data should be very useful for systematic key gene discoveries for any given biochemical process or cellular function, and also valuable for novel drug target discovery and novel diagnostic, prognostic and therapeutic-efficiency marker prediction for a specific disease or disorder.

Differential Signaling through p190 and p210 Forms of BCR-ABL Fusion Proteins Revealed By Proteomic Analysis

Chromosomal translocations involving chromosome 9q34 and 22q11 generate the BCR-ABL1 fusion gene. The location of the translocation within the BCR gene dictates which exons are excluded or included in the resulting fusion with the ABL1 gene. The most common translocations produce three main protein products with molecular weights of 190, 210, and 230 kD. Interestingly, the p190 and p210 BCR-ABL1 forms are associated with different clinical characteristics. Specifically, BCR-ABL1+ acute lymphoblastic leukemia (ALL) cases typically harbor the p190 form, whereas chronic myelogenous leukemia (CML) cases typically harbor the p210 form. Despite a great deal of study over many decades devoted to the BCR-ABL1 molecule, neither phosphorylation of putative target proteins at site-specific resolution nor protein-protein interaction differences between the p190 and the p210 forms is well understood, preventing thorough understanding of the signaling programs employed by these variants.Therefore, to examine signaling differences between p190 and p210 we chose a homogeneous system to investigate global phosphorylation of tyrosine residues. Ba/F3 cell lines, which are normally dependent on IL-3 for growth, were transduced with either the p190 or p210 form of BCR-ABL1. Subsequently, these cell lines were selected for successful growth in cytokine free media, indicating that the BCR-ABL1 fusions were signaling and inducing aberrant growth. These two BCR-ABL1 cell lines, as well as the parental Ba/F3 line, were grown in SILAC (stable isotope labeling by amino acids in cell culture) media and lysates were enriched for phosphotyrosine (pTyr) peptides and submitted for mass spectrometric analysis. The resulting 3-state SILAC dataset includes quantitation on 355 pTyr sites in three pairwise comparisons; p190 vs. p210 as well as each BCR-ABL1 cell line vs. its parental control. Many of the known BCR-ABL1 associated phosphorylation sites are recapitulated in our dataset. To our knowledge we report the first data on pTyr site-specific differences between p190 and p210 forms within in the same cell type. Surprisingly, 78 pTyr sites are differentially regulated between p190 and p210, with notable examples including pTyr sites on TEC, SHC1, PEAK1, PTPN6 and LYN. Additionally, sites in the kinase domain of JAK2 and the DNA binding domain of STAT6 showed a reciprocal relationship in p190 vs. p210, suggesting the differential involvement of JAK-STAT pathway. Notably, 28 pTyr sites were differentially regulated exclusively in either p190 or p210 BCR-ABL1 Ba/F3 cells, suggesting that there may be signaling pathways unique to p190 and p210, respectively.To attempt to identify direct substrates of BCR-ABL1 and detect differential binding of p190 and p210, we used the BioID system (Roux et al., JBC 2012). In this system, a specialized biotin ligase (BirA) is used to label adjacent or interacting proteins with a biotin moiety on lysine residues. Specifically, Ba/F3 cells transduced with BirA-tagged p190 or untagged p190 were grown in SILAC media and whole cell lysates were subjected streptavidin immunoprecipitation followed by mass spectrometry. Forty-four proteins were enriched over the control and known interactors such as SHC1, DOK2, and GAB1 were detected, along with several putative novel interactors. Combining both the phosphorylation and interactome datasets revealed all these proteins are hyper-phosphorylated compared to parental cell lines, suggesting these are direct substrates or in proximal complexes with p190 BCR-ABL1. A direct comparison of p190 and p210 interactomes using BioID is pending, and will further elucidate potential differences between these important fusion protein isoforms. In sum, our data suggests that p190 and p210 have at least partially independent signaling cascades that are mediated by differential protein-protein interactions, which may help explain the observed association of p190 with BCR-ABL1+ ALL and p210 with CML. DisclosuresNo relevant conflicts of interest to declare.

Identification of network-based biomarkers of cardioembolic stroke using a systems biology approach with time series data.

BackgroundMolecular signaling of angiogenesis begins within hours after initiation of a stroke and the following regulation of endothelial integrity mediated by growth factor receptors and vascular growth factors. Recent studies further provided insights into the coordinated patterns of post-stroke gene expressions and the relationships between neurodegenerative diseases and neural function recovery processes after a stroke.ResultsDifferential protein-protein interaction networks (PPINs) were constructed at 3 post-stroke time points, and proteins with a significant stroke relevance value (SRV) were discovered. Genes, including UBC, CUL3, APP, NEDD8, JUP, and SIRT7, showed high associations with time after a stroke, and Ingenuity Pathway Analysis results showed that these post-stroke time series-associated genes were related to molecular and cellular functions of cell death, cell survival, the cell cycle, cellular development, cellular movement, and cell-to-cell signaling and interactions. These biomarkers may be helpful for the early detection, diagnosis, and prognosis of ischemic stroke.ConclusionsThis is our first attempt to use our theory of a systems biology framework on strokes. We focused on 3 key post-stroke time points. We identified the network and corresponding network biomarkers for the 3 time points, further studies are needed to experimentally confirm the findings and compare them with the causes of ischemic stroke. Our findings showed that stroke-associated biomarker genes at different time points were significantly involved in cell cycle processing, including G2-M, G1-S and meiosis, which contributes to the current understanding of the etiology of stroke. We hope this work helps scientists reveal more hidden cellular mechanisms of stroke etiology and repair processes.

Networks: Elucidating Experimental Data by Differential Protein-Protein Interactions

Recently, the pathogenesis of coronary atherosclerosis in a swine model has been investigated by considering in particular the effects of a high cholesterol diet lasting 8 or 16 weeks. The proposed systems medicine approach integrating data of plasma adhesion molecules, cytokines, lipoproteins, tissue proteins and histology allowed for an assessment of their relationships with artery-specific proteins.

Protein kinase A activation enhances β-catenin transcriptional activity through nuclear localization to PML bodies.

The Protein Kinase A (PKA) and Wnt signaling cascades are fundamental pathways involved in cellular development and maintenance. In the osteoblast lineage, these pathways have been demonstrated functionally to be essential for the production of mineralized bone. Evidence for PKA-Wnt crosstalk has been reported both during tumorigenesis and during organogenesis, and the nature of the interaction is thought to rely on tissue and cell context. In this manuscript, we analyzed bone tumors arising from mice with activated PKA caused by mutation of the PKA regulatory subunit Prkar1a. In primary cells from these tumors, we observed relocalization of β-catenin to intranuclear punctuate structures, which were identified as PML bodies. Cellular redistribution of β-catenin could be recapitulated by pharmacologic activation of PKA. Using 3T3-E1 pre-osteoblasts as a model system, we found that PKA phosphorylation sites on β-catenin were required for nuclear re-localization. Further, β-catenin's transport to the nucleus was accompanied by an increase in canonical Wnt-dependent transcription, which also required the PKA sites. PKA-Wnt crosstalk in the cells was bi-directional, including enhanced interactions between β-catenin and the cAMP-responsive element binding protein (CREB) and transcriptional crosstalk between the Wnt and PKA signaling pathways. Increases in canonical Wnt/β-catenin signaling were associated with a decrease in the activity of the non-canonical Wnt/Ror2 pathway, which has been shown to antagonize canonical Wnt signaling. Taken together, this study provides a new understanding of the complex regulation of the subcellular distribution of β-catenin and its differential protein-protein interaction that can be modulated by PKA signaling.

VDR mutant M1T protein has diminished protein‐protein interactions

The Vitamin D3 Receptor (VDR) is a steroid hormone receptor protein that forms homodimers and heterodimers. The Retinoid X Receptor‐alpha receptor (RXRa) protein is a heterodimerization binding partner for VDR. A start codon polymorphism in the VDR gene, known as M1T VDR, has been reported. This VDR gene variant has been associated with increased risk for some human cancers. The mutant M1T VDR mRNA initiates protein translation at an internal Met codon and produces M1T VDR protein lacking the first three amino acids present in full length VDR protein. The goal of this work was to detect any differences in protein‐protein interactions formed by M1T VDR with VDR, M1T VDR or RXRa. We have constructed six mammalian expression vectors that produces either untagged VDR, M1T VDR, or RXRa, as well as, N‐terminally epitope‐tagged myc‐VDR, myc‐M1T VDR or myc‐ RXRa. The experimental approached employed in this study involves transient co‐transfection of H1299 cells with combinations of untagged and myc‐tagged expression vectors, followed by co‐immunoprecipitation (Co‐IP) assays and Western blotting for VDR, M1T VDR or RXRa. Our data are consistent with at least a 50% reduction in the ability of M1T VDR to form homodimers and heterodimers. These data suggest a potential molecular etiology for increased cancer risks associated with the M1T VDR mutant gene.

Differential Regulation of RasGAPs in Cancer

Ever since their discovery as cellular counterparts of viral oncogenes more than 25 years ago, much progress has been made in understanding the complex networks of signal transduction pathways activated by oncogenic Ras mutations in human cancers. The activity of Ras is regulated by nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs), and much emphasis has been put into the biochemical and structural analysis of the Ras/GAP complex. The mechanisms by which GAPs catalyze Ras-GTP hydrolysis have been clarified and revealed that oncogenic Ras mutations confer resistance to GAPs and remain constitutively active. However, it is yet unclear how cells coordinate the large and divergent GAP protein family to promote Ras inactivation and ensure a certain biological response. Different domain arrangements in GAPs to create differential protein-protein and protein-lipid interactions are probably key factors determining the inactivation of the 3 Ras isoforms H-, K-, and N-Ras and their effector pathways. In recent years, in vitro as well as cell- and animal-based studies examining GAP activity, localization, interaction partners, and expression profiles have provided further insights into Ras inactivation and revealed characteristics of several GAPs to exert specific and distinct functions. This review aims to summarize knowledge on the cell biology of RasGAP proteins that potentially contributes to differential regulation of spatiotemporal Ras signaling.

Differential Modulation of TCF/LEF-1 Activity by the Soluble LRP6-ICD

The canonical Wnt/β-catenin (Wnt) pathway is a master transcriptional regulatory signaling pathway that controls numerous biological processes including proliferation and differentiation. As such, transcriptional activity of the Wnt pathway is tightly regulated and/or modulated by numerous proteins at the level of the membrane, cytosol and/or nucleus. In the nucleus, transcription of Wnt target genes by TCF/LEF-1 is repressed by the long Groucho/TLE co-repressor family. However, a truncated member of the Groucho/TLE family, amino terminal enhancer of Split (AES) can positively modulate TCF/LEF-1 activity by antagonizing long Groucho/TLE members in a dominant negative manner. We have previously shown the soluble intracellular domain of the LRP6 receptor, a receptor required for activation of the Wnt pathway, can positively regulate transcriptional activity within the Wnt pathway. In the current study, we show the soluble LRP6 intracellular domain (LRP6-ICD) can also translocate to the nucleus in CHO and HEK 293T cells and in contrast to cytosolic LRP6-ICD; nuclear LRP6-ICD represses TCF/LEF-1 activity. In agreement with previous reports, we show AES enhances TCF/LEF-1 mediated reporter transcription and further we demonstrate that AES activity is spatially regulated in HEK 293T cells. LRP6-ICD interacts with AES exclusively in the nucleus and represses AES mediated TCF/LEF-1 reporter transcription. These results suggest that LRP6-ICD can differentially modulate Wnt pathway transcriptional activity depending upon its subcellular localization and differential protein-protein interactions.

Long- and Short-Range Electrostatic Interactions Affect the Rheology of Highly Concentrated Antibody Solutions

To explain the differences in protein-protein interactions (PPI) of concentrated versus dilute formulations of a model antibody. High frequency rheological measurements from pH 3.0 to 12.0 quantitated viscoelasticity and PPI at high concentrations. Dynamic light scattering (DLS) characterized PPI in dilute solutions. For concentrated solutions at low ionic strength, the storage modulus, a viscosity component and a measure of PPI, is highest at the isoelectric point (pH 9.0) and lowest at pH 5.4. This profile flattens at higher ionic strength but not completely, indicating PPI consist of long-range electrostatics and other short-range attractions. At low concentrations, PPI are near zero at pI but become repulsive as the pH is shifted. Higher salt concentrations completely flatten this profile to zero, indicating that these PPI are mainly electrostatic. This discrepancy occurs because long-range interactions are significant at low concentrations, whereas both long- and short-range interactions are significant at higher concentrations. Computer modeling was used to calculate antibody properties responsible for long- and short-range interactions, i.e. net charge and dipole moment. Charge-charge interactions are repulsive while dipole-dipole interactions are attractive. Their net effect correlated with the storage modulus profile. However, only charge-charge repulsions correlated with PPI determined by DLS.

Chapter 2 Biological and Biophysical Properties of Vascular Connexin Channels

Intercellular channels formed by connexin proteins play a pivotal role in the direct movement of ions and larger cytoplasmic solutes between vascular endothelial cells, between vascular smooth muscle cells, and between endothelial and smooth muscle cells. Multiple genetic and epigenetic factors modulate connexin expression levels and/or channel function, including cell-type-independent and cell-type-specific transcription factors, posttranslational modifications, and localized membrane targeting. Additionally, differences in protein-protein interactions, including those between connexins, significantly contribute to both vascular homeostasis and disease progression. The biophysical properties of the connexin channels identified in the vasculature, those formed by Cx37, Cx40, Cx43 and/or Cx45 proteins, are discussed in this chapter in the physiological and pathophysiological context of vessel function.

Proteomics Analysis Identifies Phosphorylation-dependent α-Synuclein Protein Interactions

Mutations and copy number variation in the SNCA gene encoding the neuronal protein alpha-synuclein have been linked to familial Parkinson disease (Thomas, B., and Beal, M. F. (2007) Parkinson's disease. Hum. Mol. Genet. 16, R183-R194). The carboxyl terminus of alpha-synuclein can be phosphorylated at tyrosine 125 and serine 129, although only a small fraction of the protein is phosphorylated under normal conditions (Okochi, M., Walter, J., Koyama, A., Nakajo, S., Baba, M., Iwatsubo, T., Meijer, L., Kahle, P. J., and Haass, C. (2000) Constitutive phosphorylation of the Parkinson's disease associated alpha-synuclein. J. Biol. Chem. 275, 390-397). Under pathological conditions, such as in Parkinson disease, alpha-synuclein is a major component of Lewy bodies, a pathological hallmark of Parkinson disease, and is mostly phosphorylated at Ser-129 (Anderson, J. P., Walker, D. E., Goldstein, J. M., de Laat, R., Banducci, K., Caccavello, R. J., Barbour, R., Huang, J. P., Kling, K., Lee, M., Diep, L., Keim, P. S., Shen, X. F., Chataway, T., Schlossmacher, M. G., Seubert, P., Schenk, D., Sinha, S., Gai, W. P., and Chilcote, T. J. (2006) Phosphorylation of Ser-129 is the dominant pathological modification of alpha-synuclein in familial and sporadic Lewy body disease. J. Biol. Chem. 281, 29739-29752). Controversy exists over the extent to which phosphorylation of alpha-synuclein and/or the visible protein aggregation in Lewy bodies are steps in disease pathogenesis, are protective, or are neutral markers for the disease process. Here we used the combination of peptide pulldown assays and mass spectrometry to identify and compare protein-protein interactions of phosphorylated and non-phosphorylated alpha-synuclein. We showed that non-phosphorylated alpha-synuclein carboxyl terminus pulled down protein complexes that were highly enriched for mitochondrial electron transport proteins, whereas alpha-synuclein carboxyl terminus phosphorylated on either Ser-129 or Tyr-125 did not. Instead the set of proteins pulled down by phosphorylated alpha-synuclein was highly enriched in certain cytoskeletal proteins, in vesicular trafficking proteins, and in a small number of enzymes involved in protein serine phosphorylation. This targeted comparative proteomics approach for unbiased identification of protein-protein interactions suggests that there are functional consequences when alpha-synuclein is phosphorylated.

Activity of Hypoxia-inducible Factor 2α Is Regulated by Association with the NF-κB Essential Modulator

The hypoxia-inducible factors 1alpha (HIF-1alpha) and 2alpha (HIF-2alpha) are key regulators of the transcriptional response to low oxygen and are closely related in domain architecture, DNA binding, and activation mechanisms. Despite these similarities, targeted disruption of the HIF-alpha genes in mice results in distinctly different phenotypes demonstrating nonredundancy of function, although the underlying mechanisms remain unclear. Here we report on the novel and specific interaction of HIF-2alpha, but not HIF-1alpha, with the NF-kappaB essential modulator (NEMO) using immunoprecipitation, mammalian two-hybrid, and in vitro protein interaction assays. Reporter gene assays demonstrate that this interaction specifically enhances normoxic HIF-2alpha transcriptional activity, independently of the HIF-2alpha transactivation domain, consistent with a model by which NEMO aids CBP/p300 recruitment to HIF-2alpha. In contrast, HIF-2alpha overexpression does not alter NF-kappaB signaling, suggesting that the functional consequence of the HIF-2alpha/NEMO interaction is limited to the HIF pathway. The specificity of NEMO for HIF-2alpha represents one of the few known differential protein-protein interactions between the HIF-alpha proteins, which has important implications for the activity of HIF-2alpha and is also the first postulated NF-kappaB-independent role for NEMO.

Regulation of sodium transporters in the thick ascending limb of rat kidney: response to angiotensin II.

The effect of ANG II treatment of rats for 7 days was examined with respect to the abundance and subcellular localization of key thick ascending limb (TAL) Na+ transporters. Rats were on a fixed intake of Na+ and water and treated with 0, 12.5, 25, 50 (ANG II-50), 100 (ANG II-100), and 200 (ANG II-200) ng x min(-1) x kg(-1) ANG II (sc). Semiquantitative immunoblotting revealed that Na+/H+ exchanger 3 (NHE3) abundance in the inner stripe of the outer medulla (ISOM) of ANG II-treated rats was significantly increased: 179 +/- 28 (ANG II-50, n = 5), 166 +/- 23 (ANG II-100, n = 7), and 167 +/- 19% (ANG II-200, n = 4) of control levels (n = 6, P < 0.05), whereas lower doses of ANG II were ineffective. The abundance of the bumetanide-sensitive Na(+)-K(+)-2Cl(-) cotransporter (BSC-1) in the ISOM was also increased to 187 +/- 28 (ANG II-50), 162 +/- 23 (ANG II-100), and 166 +/- 19% (ANG II-200) of control levels (P < 0.05), but there were no changes in the abundance of Na(+)-K(+)-ATPase and the electroneutral Na(+)-HCO3 cotransporter NBCn1. Immunocytochemistry confirmed the increase in NHE3 and BSC-1 labeling in medullary TAL (mTAL). In the cortex and the outer strip of the outer medulla, NHE3 abundance was unchanged, whereas immunocytochemistry revealed markedly increased NHE3 labeling of the proximal tubule brush border, suggesting subcellular redistribution of NHE3 or differential protein-protein interaction. Despite this, ANG II-treated rats (50 ng x min(-1) x kg(-1) for 5 days, n = 6) had a higher urinary pH compared with controls. NH4Cl loading completely blocked all effects of ANG II infusion on NHE3 and BSC-1, suggesting a potential role of pH as a mediator of these effects. In conclusion, increased abundance of NHE3 and BSC-1 in mTAL cells as well as increased NHE3 in the proximal tubule brush border may contribute to enhanced renal Na+ and HCO3 reabsorption in response to ANG II.

Phosphorylation-dephosphorylation differentially affects activities of splicing factor ASF/SF2.

SR proteins are a conserved family of splicing factors that function in both constitutive and activated splicing. We reported previously that phosphorylation of the SR protein ASF/SF2 enhances its interaction with the U1 snRNP-specific 70K protein and is required for the protein to function in splicing, while other studies have provided evidence that subsequent dephosphorylation can also be required for SR protein function, at least in constitutive splicing. We now show that the phosphorylation status of ASF/SF2 can differentially affect several properties of the protein. In keeping with a dynamic cycle of phosphorylation-dephosphorylation during splicing, ASF/SF2 phosphorylation was found to affect interaction with several putative protein targets in different ways: positively, negatively or not at all. Extending these results, we also show that, in contrast to constitutive splicing, dephosphorylation is not required for ASF/SF2 to function as a splicing activator. We discuss these results with respect to the differential protein-protein interactions that must occur during constitutive and activated splicing.

Electrophoretic migration of adenovirus hexon under non-denaturing conditions

While SDS-PAGE/ immunoblotting is a valuable approach for the characterization of monoclonal antibodies, the denaturing conditions involved can compromise the recognition of conformational epitopes. This report demonstrates that a group specific epitope on adenovirus hexon can be recognized by immunoblotting following SDS-PAGE provided that samples are not boiled prior to electrophoresis. Under these conditions, multiple bands corresponding to native forms of hexon were detected above the position of the denatured hexon monomer. Among representative serotypes of subgroups A, B and F, two predominant bands, corresponding to hexon trimers and ‘group of nine’ hexons (GONs), were routinely observed. In contrast, higher order structures, in addition to trimers and GONs, were characteristic of subgroup C adenoviruses. These serotypic differences in stability of hexon structures may reflect differences in protein-protein interactions within the corresponding virions.