In the sympatho‐adrenal system, angiotensin II (Ang II) acts as a key neuromodulatory component. At sympathetic nerve terminals, Ang II influences sympathetic transmission by enhancing norepinephrine ...(NE) synthesis, facilitating NE release and inhibiting NE uptake. Previously, it was demonstrated that tyrosine hydroxylase (TH) mRNA is trafficked to the distal axons of primary superior cervical ganglia (SCG) neurons, directed by a cis‐acting regulatory element (i.e. zipcode) located in the 3'UTR of the transcript. Results of metabolic labeling studies established that the mRNA is locally translated. It was further shown that the axonal trafficking of the mRNA encoding the enzyme plays an important role in mediating dopamine (DA) and NE synthesis and may facilitate the maintenance of axonal catecholamine levels. In the present study, the hypothesis was tested that Ang II induces NE synthesis in rat primary SCG neurons via the modulation of the trafficking of the mRNAs encoding the catecholamine synthesizing enzymes TH and dopamine β‐hydroxylase (DBH). Treatment of SCG neurons with the Ang II receptor type 1 (AT1R) agonist, L‐162,313, increases the axonal levels of TH and DBH mRNA and protein and results in elevated NE levels. Conversely, treatment of rat SCG neurons with the AT1R antagonist, Eprosartan, abolished the L‐162,313‐mediated increase in axonal levels of TH and DBH mRNA and protein. In a first attempt to identify the proteins involved in the Ang II‐mediated axonal transport of TH mRNA, we used a biotinylated 50‐nucleotide TH RNA zipcode as bait in the affinity purification of TH zipcode‐associated proteins. Mass spectrometric analysis of the TH zipcode ribonucleoprotein (RNP) complex immune‐purified from SCG neurons led to the identification of 163 somal and 127 axonal proteins functionally involved in binding nucleic acids, the translational machinery or acting as subunits of cytoskeletal and motor proteins. Surprisingly, immune‐purification of the TH axonal trafficking complex, results in the acquisition of DBH mRNA, suggesting that these mRNAs maybe transported to the axon together, possibly in the same RNP complex. Taken together, our results point to a novel mechanism by which Ang II participates in the regulation of axonal synthesis of NE by modulating the local trafficking and expression of TH and DBH, two key enzymes involved in the catecholamine biosynthetic pathway.
Angiotensin II (Ang II) acts as a key neuromodulatory component, influencing sympathetic transmission by enhancing norepinephrine (NE) synthesis and facilitating NE release. Here, we tested the hypothesis that Ang II regulates the axonal levels of the mRNAs encoding tyrosine hydroxylase (TH) and Dopamine β‐Hydroxylase (DBH), resulting in the modulation of catecholamine synthesis in sympathetic neurons. The outcome of our studies demonstrates that treatment of superior cervical ganglia (SCG) neurons with a selective Ang II agonist increases the axonal levels of TH and DBH mRNA and protein and results in elevated presynaptic NE synthesis and release in primary rat sympathetic neurons.
Open Mass Spectrometry Search Algorithm Geer, Lewis Y; Markey, Sanford P; Kowalak, Jeffrey A ...
Journal of proteome research,
09/2004, Letnik:
3, Številka:
5
Journal Article
Recenzirano
Odprti dostop
Large numbers of MS/MS peptide spectra generated in proteomics experiments require efficient, sensitive and specific algorithms for peptide identification. In the Open Mass Spectrometry Search ...Algorithm (OMSSA), specificity is calculated by a classic probability score using an explicit model for matching experimental spectra to sequences. At default thresholds, OMSSA matches more spectra from a standard protein cocktail than a comparable algorithm. OMSSA is designed to be faster than published algorithms in searching large MS/MS datasets. Keywords: protein identification • algorithm • bioinformatics • mass spectrometry • proteomics • significance testing
Abstract
Atypical Teratoid Rhabdoid Tumor (AT/RT) is a rare pediatric central nervous system cancer often characterized by deletion or mutation of
SMARCB1
, a tumor suppressor gene. In this study, we ...found that SMARCB1 regulates Human Endogenous Retrovirus K (HERV-K, subtype HML-2) expression. HML-2 is a repetitive element scattered throughout the human genome, encoding several intact viral proteins that have been associated with stem cell maintenance and tumorigenesis. We found HML-2 env expression in both the intracellular and extracellular compartments in all AT/RT cell lines (n = 4) and in 95% of AT/RT patient tissues (n = 37) evaluated.
SMARCB1
knock-down in neural stem cells (NSCs) led to an upregulation of HML-2 transcription. We found that SMARCB1 binds adjacent to the HML-2 promoter, repressing its transcription via chromatin immunoprecipitation; restoration of
SMARCB1
expression in AT/RT cell lines significantly downregulated HML-2 expression. Further, targeted downregulation of HML-2 transcription via CRISPR-dCas9 coupled with suppressor proteins led to cellular dispersion, decreased proliferation
,
and cell death in vitro. HML-2 knock-down with shRNA, siRNA, and CRISPR-dCas9 significantly decreased Ras expression as measured by qRT-PCR, suggesting that HML-2 modulates
MAPK/ERK
signaling in AT/RT cells. Overexpression of
NRAS
was sufficient to restore cellular proliferation, and MYC, a transcription factor downstream of
NRAS
, was bound to the HERV-K LTR significantly more in the absence of
SMARCB1
expression in AT/RT cells. We show a mechanism by which these undifferentiated tumors remain pluripotent, and we demonstrate that their formation is aided by aberrant HML-2 activation, which is dependent on
SMARCB1
and its interaction with MYC.
Senescent Ccl2(-/-) mice are reported to develop cardinal features of human age-related macular degeneration (AMD). Loss-of-function single-nucleotide polymorphisms within CX3CR1 are also found to be ...associated with AMD. The authors generated Ccl2(-/-)/Cx3cr1(-/-) mice to establish a more characteristic and reproducible AMD model.
Single Ccl2- and Cx3cr1-deficient mice were crossbred to obtain Ccl2(-/-)/Cx3cr1(-/-) mice. Funduscopy, histopathology, retinal A2E quantification, proteomics, RT-PCR gene expression assay, immunochemistry, and Western blotting were used to examine the retina and to evaluate gene expression within the retinal tissue.
By 6 weeks of age, all Ccl2(-/-)/Cx3cr1(-/-) mice developed AMD-like retinal lesions, including drusen, retinal pigment epithelium alteration, and photoreceptor degeneration. Furthermore, choroidal neovascularization occurred in 15% of the mice. These degenerative lesions progressed with age. A2E, a major lipofuscin fluorophore that accumulated during AMD progression, was significantly higher in the Ccl2(-/-)/Cx3cr1(-/-) retina than in the wild-type retina. Complement cofactor was higher in the Ccl2(-/-)/Cx3cr1(-/-) RPE. Proteomics data indicated that four proteins were differentially expressed in Ccl2(-/-)/Cx3cr1(-/-) retina compared with control. One of these proteins, ERp29, an endoplasmic reticulum protein, functions as an escort chaperone and in protein folding.
The authors concluded that Ccl2(-/-)/Cx3cr1(-/-) mice develop a broad spectrum of AMD abnormalities with early onset and high penetrance. These observations implicate certain chemokines and endoplasmic reticulum proteins in AMD pathogenesis. Similar to the mechanism of neurodegeneration caused by dysfunction of endoplasmic reticulum proteins, decreased chaperoning may cause misfolded protein accumulation, leading to drusen formation and retinal degeneration.
MicroRNAs (miRNAs) selectively localize to subcompartments of the neuron, such as dendrites, axons, and presynaptic terminals, where they regulate the local protein synthesis of their putative target ...genes. In addition to mature miRNAs, precursor miRNAs (pre-miRNAs) have also been shown to localize to somatodendritic and axonal compartments. miRNA-338 (miR-338) regulates the local expression of several nuclear-encoded mitochondrial mRNAs within axons of sympathetic neurons. Previous work has shown that precursor miR-338 (pre-miR-338) introduced into the axon can locally be processed into mature miR-338, where it can regulate local ATP synthesis. However, the mechanisms underlying the localization of pre-miRNAs to the axonal compartment remain unknown. In this study, we investigated the axonal localization of pre-miR-338. Using proteomic and biochemical approaches, we provide evidence for the localization of pre-miR-338 to distal neuronal compartments and identify several constituents of the pre-miR-338 ribonucleoprotein complex. Furthermore, we found that pre-miR-338 is associated with the mitochondria in axons of superior cervical ganglion (SCG) neurons. The maintenance of mitochondrial function within axons requires the precise spatiotemporal synthesis of nuclear-encoded mRNAs, some of which are regulated by miR-338. Therefore, the association of pre-miR-338 with axonal mitochondria could serve as a reservoir of mature, biologically active miRNAs, which could coordinate the intra-axonal expression of multiple nuclear-encoded mitochondrial mRNAs.
In previous studies, we identified a putative 38-nucleotide stem-loop structure (zipcode) in the 3′ untranslated region of the cytochrome c oxidase subunit IV (COXIV) mRNA that was necessary and ...sufficient for the axonal localization of the message in primary superior cervical ganglion (SCG) neurons. However, little is known about the proteins that interact with the COXIV-zipcode and regulate the axonal trafficking and local translation of the COXIV message. To identify proteins involved in the axonal transport of the COXIV mRNA, we used the biotinylated 38-nucleotide COXIV RNA zipcode as bait in the affinity purification of COXIV zipcode binding proteins. Gel-shift assays of the biotinylated COXIV zipcode indicated that the putative stem-loop structure functions as a nucleation site for the formation of ribonucleoprotein complexes. Mass spectrometric analysis of the COXIV zipcode ribonucleoprotein complex led to the identification of a large number RNA binding proteins, including fused in sarcoma/translated in liposarcoma (FUS/TLS), and Y-box protein 1 (YB-1). Validation experiments, using western analyses, confirmed the presence of the candidate proteins in the COXIV zipcode affinity purified complexes obtained from SCG axons. Immunohistochemical studies show that FUS, and YB-1 are present in SCG axons. Importantly, RNA immunoprecipitation studies show that FUS, and YB-1 interact with endogenous axonal COXIV transcripts. siRNA-mediated downregulation of the candidate proteins FUS and YB-1 expression in the cell-bodies diminishes the levels of COXIV mRNA in the axon, suggesting functional roles for these proteins in the axonal trafficking of COXIV mRNA.
•COXIV mRNA zipcode is a nucleation site for formation of axonal trafficking RNPs.•Zipcode-associated proteins were identified using a proteomics-based approach.•Approximately 53 proteins are associated with the full-length zipcode.•FUS and YB-1 were among the zipcode-associated proteins.•Silencing the expression of FUS and YB-1 reduced axonal levels of COXIV mRNA.
The determination of differences in relative protein abundance is a critical aspect of proteomics research that is increasingly used to answer diverse biological questions. The Association of ...Biomolecular Resource Facilities Proteomics Research Group 2006 study was a quantitative proteomics project in which the aim was to determine the identity and the relative amounts of eight proteins in two mixtures. There are numerous methodologies available to study the relative abundance of proteins between samples, but to date, there are few examples of studies that have compared these different approaches. For the 2006 Proteomics Research Group study, there were 52 participants who used a wide variety of gel electrophoresis-, HPLC-, and mass spectrometry-based methods for relative quantitation. The quantitative data arising from this study were evaluated along with several other experimental details relevant to the methodologies used.
Alleles at NACP-Rep1, the polymorphic microsatellite repeat located ∼10 kb upstream of the α-synuclein gene (
SNCA), are associated, in some reports, with differing risks of sporadic Parkinson ...disease (PD). We showed previously that NACP-Rep1 acts as a negative modulator of
SNCA transcription, with an effect that varied threefold among different NACP-Rep1 alleles. Given that duplications and triplications of
SNCA have been implicated in familial Parkinson disease (PD), even a 1.5–2-fold increase in α-synuclein expression may, over many decades, contribute to PD. Thus, the association of different NACP-Rep1 alleles with PD may be a consequence of polymorphic differences in transcriptional regulation of
SNCA. Here we aimed to identify the factor(s) that bind to NACP-Rep1 and potentially contribute to
SNCA transcriptional modulation, by pulling down proteins that bind to NACP-Rep1 and identifying them by mass spectrometry. One of these proteins was poly-(ADP-ribose) transferase/polymerase-1 (PARP-1), a DNA-binding protein and transcriptional regulator. Electrophoresis mobility shift and chromatin immunoprecipitation assays showed specific binding of PARP-1 to NACP-Rep1. Inhibition of PARP-1's catalytic domain increased the endogenous
SNCA mRNA levels in cultured SH-SY5Y cells. Furthermore, PARP-1 binding to NACP-Rep1 specifically reduced the transcriptional activity of the
SNCA promoter/enhancer in luciferase reporter assays. This down-regulation effect of PARP-1 depended on NACP-Rep1 being present in the construct and was abrogated by inhibiting PARP-1's catalytic activity with 3-aminobenzamide. The association of different NACP-Rep1 alleles with PD may be mediated, in part, by the effect of PARP-1, as well as other factors, on
SNCA expression.
The bacterial ribosomal protein S12 contains a universally conserved D88 residue on a loop region thought to be critically involved in translation due to its proximal location to the A site of the ...30S subunit. While D88 mutants are lethal this residue has been found to be post-translationally modified to β-methylthioaspartic acid, a post-translational modification (PTM) identified in S12 orthologs from several phylogenetically distinct bacteria. In a previous report focused on characterizing this PTM, our results provided evidence that this conserved loop region might be involved in forming multiple proteins-protein interactions ( Strader M. B. ; Costantino N. ; Elkins C. A. ; Chen C. Y. ; Patel I. ; Makusky A. J. ; Choy J. S. ; Court D. L. ; Markey S. P. ; Kowalak J. A. A proteomic and transcriptomic approach reveals new insight into betamethylthiolation of Escherichia coli ribosomal protein S12. Mol. Cell. Proteomics 2011, 10, M110 005199 ). To follow-up on this study, the D88 containing loop was probed to identify candidate binders employing a two-step complementary affinity purification strategy. The first involved an endogenously expressed S12 protein containing a C-terminal tag for capturing S12 binding partners. The second strategy utilized a synthetic biotinylated peptide representing the D88 conserved loop region for capturing S12 loop interaction partners. Captured proteins from both approaches were detected by utilizing SDS-PAGE and one-dimensional liquid chromatography–tandem mass spectrometry. The results presented in this report revealed proteins that form direct interactions with the 30S subunit and elucidated which are likely to interact with S12. In addition, we provide evidence that two proteins involved in regulating ribosome and/or mRNA transcript levels under stress conditions, RNase R and Hfq, form direct interactions with the S12 conserved loop, suggesting that it is likely part of a protein binding interface.
β-methylthiolation is a novel post-translational modification mapping to a universally conserved Asp 88 of the bacterial ribosomal protein S12. This S12 specific modification has been identified on ...orthologs from multiple bacterial species. The origin and functional significance was investigated with both a proteomic strategy to identify candidate S12 interactors and expression microarrays to search for phenotypes that result from targeted gene knockouts of select candidates. Utilizing an endogenous recombinant E. coli S12 protein with an affinity tag as bait, mass spectrometric analysis identified candidate S12 binding partners including RimO (previously shown to be required for this post-translational modification) and YcaO, a conserved protein of unknown function. Transcriptomic analysis of bacterial strains with deleted genes for RimO and YcaO identified an overlapping transcriptional phenotype suggesting that YcaO and RimO likely share a common function. As a follow up, quantitative mass spectrometry additionally indicated that both proteins dramatically impacted the modification status of S12. Collectively, these results indicate that the YcaO protein is involved in β-methylthiolation of S12 and its absence impairs the ability of RimO to modify S12. Additionally, the proteomic data from this study provides direct evidence that the E. coli specific β-methylthiolation likely occurs when S12 is assembled as part of a ribosomal subunit.
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