[Show abstract][Hide abstract] ABSTRACT: Detailed analysis of disease-affected tissue provides insight into molecular mechanisms contributing to pathogenesis. Substantia nigra, striatum, and cortex are functionally connected with increasing degrees of alpha-synuclein pathology in Parkinson's disease. We undertook functional and causal pathway analysis of gene expression and proteomic alterations in these three regions, and the data revealed pathways that correlated with disease progression. In addition, microarray and RNAseq experiments revealed previously unidentified causal changes related to oligodendrocyte function and synaptic vesicle release, and these and other changes were reflected across all brain regions. Importantly, subsets of these changes were replicated in Parkinson's disease blood; suggesting peripheral tissue may provide important avenues for understanding and measuring disease status and progression. Proteomic assessment revealed alterations in mitochondria and vesicular transport proteins that preceded gene expression changes indicating defects in translation and/or protein turnover. Our combined approach of proteomics, RNAseq and microarray analyses provides a comprehensive view of the molecular changes that accompany functional loss and alpha-synuclein pathology in Parkinson's disease, and may be instrumental to understand, diagnose and follow Parkinson's disease progression.
PLoS ONE 01/2014; 9(8):e102909. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Parkin is a RING-between-RING E3 ligase that functions in the covalent attachment of ubiquitin to specific substrates, and mutations in Parkin are linked to Parkinson's disease, cancer and mycobacterial infection. The RING-between-RING family of E3 ligases are suggested to function with a canonical RING domain and a catalytic cysteine residue usually restricted to HECT E3 ligases, thus termed 'RING/HECT hybrid' enzymes. Here we present the 1.58 Å structure of Parkin-R0RBR, revealing the fold architecture for the four RING domains, and several unpredicted interfaces. Examination of the Parkin active site suggests a catalytic network consisting of C431 and H433. In cells, mutation of C431 eliminates Parkin-catalysed degradation of mitochondria, and capture of an ubiquitin oxyester confirms C431 as Parkin's cellular active site. Our data confirm that Parkin is a RING/HECT hybrid, and provide the first crystal structure of an RING-between-RING E3 ligase at atomic resolution, providing insight into this disease-related protein.
[Show abstract][Hide abstract] ABSTRACT: The diversity of ubiquitin (Ub)-dependent signaling is attributed to the ability of this small protein to form different types of covalently linked polyUb chains and to the existence of Ub binding proteins that interpret this molecular syntax. We used affinity capture/mass spectrometry to identify ALIX, a component of the ESCRT pathway, as a Ub binding protein. We report that the V domain of ALIX binds directly and selectively to K63-linked polyUb chains, exhibiting a strong preference for chains composed of more than three Ub. Sequence analysis identified two potential Ub binding sites on a single α-helical surface within the coiled-coil region of the V domain. Mutation of these putative Ub binding sites inhibited polyUb binding to the isolated V domain in vitro and impaired budding of lentiviruses. These data reveal an important role for K63 polyUb binding by ALIX in retroviral release.
[Show abstract][Hide abstract] ABSTRACT: Degradation of folding- or assembly -defective proteins by the ER-associated degradation (ERAD) ubiquitin ligase, Hrd1 is facilitated by a process that involves recognition of demannosylated N-glycans by the lectins OS-9/XTP3-B via the adaptor protein SEL1L. Most of our knowledge of the machinery that commits proteins to this fate in metazoans comes from studies of overexpressed mutant proteins in heterologous cells. In this study we used mass spectrometry to identify core-glycoslyated CD147 (CD147(CG)) as an endogenous substrate of the ERAD system that accumulates in a complex with OS-9 following SEL1L depletion. CD147 is an obligatory assembly factor for monocarboxylate transporters. The majority of newly synthesized endogenous CD147(CG) was degraded by the proteasome in a Hrd1-dependent manner. CD147(CG) turnover was blocked by kifunensine, and interaction of OS-9 and XTP3-B with CD147(CG) was inhibited by mutations to conserved residues in their lectin domains. These data establish unassembled CD147(CG) as an endogenous, constitutive ERAD substrate of the OS-9/SEL1L/Hrd1 pathway.
Molecular biology of the cell 10/2012; · 5.98 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Regulation of the microtubule- and actin-binding protein adenomatous polyposis coli (APC) is crucial for the formation of cell extensions in many cell types. This process requires inhibition of glycogen synthase kinase-3β (GSK-3β), which otherwise phosphorylates APC and decreases APC-mediated microtubule bundling. Although it is assumed, therefore, that APC phosphorylation is decreased during initiation of cell extensions, the phosphorylation state of APC has never been analyzed directly. We show here that NGF- and EGF-induced initial cell extensions result in APC phosphorylation by the MAPK/ERK pathway, which, in parallel with inhibition of GSK-3β, promotes localization of APC to the tip of cell extensions. Whereas GSK-3β inhibition promotes APC binding and stabilization of microtubules, we show that phosphorylation by ERK inhibits the interaction of APC with F-actin, and APC-mediated F-actin bundling, but not APC-mediated microtubule bundling, in vitro. These results identify a previously unknown APC regulatory pathway during growth-factor-induced cell extension, and indicate that the GSK-3β and ERK pathways act in parallel to regulate interactions between APC and the cytoskeleton during the formation of cell extensions.
[Show abstract][Hide abstract] ABSTRACT: Pathognomonic accumulation of ubiquitin (Ub) conjugates in human neurodegenerative diseases, such as Huntington's disease, suggests that highly aggregated proteins interfere with 26S proteasome activity. In this paper, we examine possible mechanisms by which an N-terminal fragment of mutant huntingtin (htt; N-htt) inhibits 26S function. We show that ubiquitinated N-htt-whether aggregated or not-did not choke or clog the proteasome. Both Ub-dependent and Ub-independent proteasome reporters accumulated when the concentration of mutant N-htt exceeded a solubility threshold, indicating that stabilization of 26S substrates is not linked to impaired Ub conjugation. Above this solubility threshold, mutant N-htt was rapidly recruited to cytoplasmic inclusions that were initially devoid of Ub. Although synthetically polyubiquitinated N-htt competed with other Ub conjugates for access to the proteasome, the vast majority of mutant N-htt in cells was not Ub conjugated. Our data confirm that proteasomes are not directly impaired by aggregated N-terminal fragments of htt; instead, our data suggest that Ub accumulation is linked to impaired function of the cellular proteostasis network.
The Journal of Cell Biology 03/2012; 196(5):573-87. · 10.82 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Proteins that fail to correctly fold or assemble into oligomeric complexes in the endoplasmic reticulum (ER) are degraded by a ubiquitin- and proteasome-dependent process known as ER-associated degradation (ERAD). Although many individual components of the ERAD system have been identified, how these proteins are organized into a functional network that coordinates recognition, ubiquitylation and dislocation of substrates across the ER membrane is not well understood. We have investigated the functional organization of the mammalian ERAD system using a systems-level strategy that integrates proteomics, functional genomics and the transcriptional response to ER stress. This analysis supports an adaptive organization for the mammalian ERAD machinery and reveals a number of metazoan-specific genes not previously linked to ERAD.
[Show abstract][Hide abstract] ABSTRACT: The protein ubiquitin is an important post-translational modifier that regulates a wide variety of biological processes. In cells, ubiquitin is apportioned among distinct pools, which include a variety of free and conjugated species. Although maintenance of a dynamic and complex equilibrium among ubiquitin pools is crucial for cell survival, the tools necessary to quantify each cellular ubiquitin pool have been limited. We have developed a quantitative mass spectrometry approach to measure cellular concentrations of ubiquitin species using isotope-labeled protein standards and applied it to characterize ubiquitin pools in cells and tissues. Our method is convenient, adaptable and should be a valuable tool to facilitate our understanding of this important signaling molecule.
[Show abstract][Hide abstract] ABSTRACT: Genetic ablation of autophagy in mice leads to liver and brain degeneration accompanied by the appearance of ubiquitin (Ub) inclusions, which has been considered to support the hypothesis that ubiquitination serves as a cis-acting signal for selective autophagy. We show that tissue-specific disruption of the essential autophagy genes Atg5 and Atg7 leads to the accumulation of all detectable Ub-Ub topologies, arguing against the hypothesis that any particular Ub linkage serves as a specific autophagy signal. The increase in Ub conjugates in Atg7(-/-) liver and brain is completely suppressed by simultaneous knockout of either p62 or Nrf2. We exploit a novel assay for selective autophagy in cell culture, which shows that inactivation of Atg5 leads to the selective accumulation of aggregation-prone proteins, and this does not correlate with an increase in substrate ubiquitination. We propose that protein oligomerization drives autophagic substrate selection and that the accumulation of poly-Ub chains in autophagy-deficient circumstances is an indirect consequence of activation of Nrf2-dependent stress response pathways.
The Journal of Cell Biology 11/2010; 191(3):537-52. · 10.82 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Terminally misfolded or unassembled proteins in the early secretory pathway are degraded by a ubiquitin- and proteasome-dependent process known as ER-associated degradation (ERAD). How substrates of this pathway are recognized within the ER and delivered to the cytoplasmic ubiquitin-conjugating machinery is unknown. We report here that OS-9 and XTP3-B/Erlectin are ER-resident glycoproteins that bind to ERAD substrates and, through the SEL1L adaptor, to the ER-membrane-embedded ubiquitin ligase Hrd1. Both proteins contain conserved mannose 6-phosphate receptor homology (MRH) domains, which are required for interaction with SEL1L, but not with substrate. OS-9 associates with the ER chaperone GRP94 which, together with Hrd1 and SEL1L, is required for the degradation of an ERAD substrate, mutant alpha(1)-antitrypsin. These data suggest that XTP3-B and OS-9 are components of distinct, partially redundant, quality control surveillance pathways that coordinate protein folding with membrane dislocation and ubiquitin conjugation in mammalian cells.
[Show abstract][Hide abstract] ABSTRACT: Huntington's disease (HD) is a dominantly inherited neurodegenerative disorder caused by expansion of CAG triplet repeats in the huntingtin (HTT) gene (also called HD) and characterized by accumulation of aggregated fragments of polyglutamine-expanded HTT protein in affected neurons. Abnormal enrichment of HD inclusion bodies with ubiquitin, a diagnostic characteristic of HD and many other neurodegenerative disorders including Alzheimer's and Parkinson's diseases, has suggested that dysfunction in ubiquitin metabolism may contribute to the pathogenesis of these diseases. Because modification of proteins with polyubiquitin chains regulates many essential cellular processes including protein degradation, cell cycle, transcription, DNA repair and membrane trafficking, disrupted ubiquitin signalling is likely to have broad consequences for neuronal function and survival. Although ubiquitin-dependent protein degradation is impaired in cell-culture models of HD and of other neurodegenerative diseases, it has not been possible to evaluate the function of the ubiquitin-proteasome system (UPS) in HD patients or in animal models of the disease, and a functional role for UPS impairment in neurodegenerative disease pathogenesis remains controversial. Here we exploit a mass-spectrometry-based method to quantify polyubiquitin chains and demonstrate that the abundance of these chains is a faithful endogenous biomarker of UPS function. Lys 48-linked polyubiquitin chains accumulate early in pathogenesis in brains from the R6/2 transgenic mouse model of HD, from a knock-in model of HD and from human HD patients, establishing that UPS dysfunction is a consistent feature of HD pathology. Lys 63- and Lys 11-linked polyubiquitin chains, which are not typically associated with proteasomal targeting, also accumulate in the R6/2 mouse brain. Thus, HD is linked to global changes in the ubiquitin system to a much greater extent than previously recognized.
[Show abstract][Hide abstract] ABSTRACT: A method is presented to quantify intermediate-abundance proteins in human serum using a single-quadrupole linear ion trap mass spectrometer-in contrast, for example, to a triple-quadrupole mass spectrometer. Stable-isotope-labeled (tryptic) peptides are spiked into digested protein samples as internal standards, aligned with the traditional isotope dilution approach. As a proof-of-concept experiment, four proteins of intermediate abundance were selected, coagulation factor V, adiponectin, C-reactive protein (CRP), and thyroxine binding globulin. Stable-isotope-labeled peptides were synthesized with one tryptic sequence from each of these proteins. The normal human serum concentration ranges of these proteins are from 1 to 30 microg/mL (or 20 to 650 pmol/mL). These labeled peptides and their endogenous counterparts were analyzed by LC-MS/MS using multiple reaction monitoring, a multiplexed form of the selected reaction monitoring technique. For these experiments, only one chromatographic dimension (on-line reversed-phase capillary column) was used. Improved limits of detection will result with multidimensional chromatographic methods utilizing more material per sample. Standard curves of the spiked calibrants were generated with concentrations ranging from 3 to 700 pmol/mL using both neat solutions and peptides spiked into the complex matrix of digested serum protein solution where ion suppression effects and interferences are common. Endogenous protein concentrations were determined by comparing MS/MS peak areas of the endogenous peptides to the isotopically labeled internal calibrants. The derived concentrations from a normal human serum pool (neglecting loss of material during sample processing) were 9.2, 110, 120, and 246 pmol/mL for coagulation factor V, adiponectin, CRP, and thyroxine binding globulin, respectively. These concentrations generally agree with the reported normal ranges for these proteins. As a measure of analytical reproducibility of this single-quadrupole assay, the coefficients of variance based on 12 repeated measurements for each of the endogenous tryptic peptides were 17.0, 25.4, 24.2, and 14.0% for coagulation factor V, adiponectin, CRP, and thyroxine binding globulin, respectively.
[Show abstract][Hide abstract] ABSTRACT: A new method is presented for quantifying proteomic and metabolomic profile data by liquid chromatography-mass spectrometry (LC-MS) with electrospray ionization. This biotechnology provides differential expression measurements and enables the discovery of biological markers (biomarkers). Work presented here uses human serum but is applicable to any fluid or tissue. The approach relies on linearity of signal versus molecular concentration and reproducibility of sample processing. There is no use of isotopic labeling or chemically similar standard materials. Linear standard curves are reported for a variety of compounds introduced into human serum. As a measure of analytical reproducibility for proteome and metabolome sampling, median coefficients of variation of 25.7 and 23.8%, respectively, were determined for approximately 3400 molecular ions (not counting their numerous isotopes) from 25 independently processed human serum samples, corresponding to a total of 85000 individual molecular ion measurements.
[Show abstract][Hide abstract] ABSTRACT: The high frequency of single nucleotide polymorphisms (SNPs) in the human genome makes them a valuable source of genetic markers for identity testing, genome mapping, and medical diagnostics. Conventional technologies for detecting SNPs are laborious and time-consuming, often prohibiting large-scale analysis. A rapid, accurate, and cost-effective method is needed to meet the demands of a high-throughput DNA assay. We demonstrate here that analysis of these genetic markers can now be performed routinely in a rapid, automated, and high-throughput fashion using time-of-flight mass spectrometry and a primer extension assay with a novel cleavable primer. SNP genotyping by mass spectrometry involves detection of single-base extension products of a primer immediately adjacent to the SNP site. Measurement of the mass difference between the SNP primer and the extension peak reveals which nucleotide is present at the polymorphic site. The primer is designed such that its extension products can be purified and chemically released from the primer in an automated format. The reduction in size of the products as a result of this chemical cleavage allows more accurate identification of the polymorphic base, especially in samples from a heterozygotic population. All six possible heterozygotes are resolved unambiguously, including an A/T heterozygote with extension products differing by only 9 Da. Multiplex SNP determination is demonstrated by simultaneously probing multiple SNP sites from a single polymerase chain reaction (PCR) product as well as from multiplexed PCR amplicons. Samples are processed in parallel on a robotic workstation, and analyzed serially in an automated mass spectrometer with analysis times of only a few seconds per sample, making it possible to process thousands of samples per day.
[Show abstract][Hide abstract] ABSTRACT: DNA separations which traditionally have been performed by slab gel or capillary electrophoresis, may now be conducted via time-of-flight mass spectrometry (TOF-MS). The advantages of using a mass spectrometry approach for short tandem repeat (STR) characterization include a dramatic increase in both the speed of analysis and the accuracy of mass measurements. We report here typing of the STR loci TH01, TPOX, and CSF1PO as well as the sex-typing marker amelogenin using TOF-MS. Allelic ladders, which are typically used with electrophoretic separation systems to correct for mobility differences of DNA fragments under various conditions, are not needed for accurate genotyping with TOF-MS. A mass precision of 0.1% RSD, which corresponds to approximately 0.1 nucleotide, was routinely observed. Mass accuracies were better than a fraction of a single nucleotide when a daily mass calibration was used. STR microvariants, such as the TH01 allele 9.3, could be detected and resolved from alleles which differ by as little as a single base. In addition, the smaller PCR product sizes (55-125 bp) examined in this study have the potential advantage of being more successful when amplifying forensic samples with degraded DNA.
Deutsche Zeitschrift für die Gesamte Gerichtliche Medizin 02/1999; 112(1):45-9. · 2.69 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The effect of impurities on the analysis of single-stranded DNA oligomers by the technique of matrix-assisted laser desorption/ionization with time-of-flight mass spectrometry has been studied using the matrix 3-hydroxypicolinic acid and 355-nm pulsed light. By mixing the DNA oligomers with different concentrations of impurities and recording mass spectra, limits are set on the tolerable level of a given impurity in a sample. The tolerance limits for sodium chloride, potassium chloride, sodium acetate, sodium fluoride, sodium dodecyl sulfate (SDS), and manganese(II) chloride were found to be approximately 10(-2) M. It was found that magnesium salts degraded the mass spectrum at much lower levels of 10(-4) M. The organic compounds tris(hydroxymethyl)aminomethane (Tris), urea, dithiothreitol (DTT), glycerol, and ethylenediaminetetraacetic acid (EDTA), when present as its ammonium salt, were tolerable at concentrations into the range of 0.25-0.5 M, while the organic polyamine compound spermine substantially degraded the mass spectrum at concentrations above 10(-2) M. When comparing these results for DNA analysis with previously reported limits for protein analysis, large differences are seen for some of the impurities tested.
[Show abstract][Hide abstract] ABSTRACT: The products from base-specific, dideoxy-nucleotide chain-termination DNA sequencing reactions catalyzed by the modified T7 DNA polymerase have been analyzed by using the technique of matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry. Preliminary experiments were performed to determine detection limits for a synthetic mixture of mixed-base single-stranded DNA which contained a 14-mer, a 21-mer, and a 41-mer; acceptable spectra, showing peaks for each component, were obtainable for samples that contained as little as 5 fmol per component. Initial sequencing reactions were therefore carried out on 2-pmol amounts of a short synthetic template that was 45 nucleotides in length, employing 2 pmol of 12-mer as the primer strand. This provided readable sequence information out to the 19th base past the primer. Using a 21-mer primer, nearly the entire sequence of the template could be read.
Rapid Communications in Mass Spectrometry 02/1995; 9(10):942-7. · 2.51 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Matrix-assisted laser desorption with concomitant ionization (MALDI) in conjunction with time-of-flight mass spectrometry (TOF-MS) has been used to analyze underivatized random-base single-stranded DNA (ssDNA) oligomers ranging from 10 to 89 nucleotides in length by embedding them in a solid matrix of 3-hydroxypicolinic acid. At 355-nm desorption wavelength, mass spectra of positive and negative ions measured by reflecting and linear time-of-flight mass spectrometers are compared. Results from the linear system show the ionization yield is approximately equal for each polarity. Metastable ion decay is significant for the larger ssDNA oligomer ions, which results in a decrease in signal intensity and the broadening of mass peaks. In order to obtain an acceptable signal-to-noise ratio on a reflecting TOF system, a higher laser irradiance is needed, which consequently causes further degradation of mass resolution. With the apparent advantages of better sensitivity and mass resolution, it is concluded that a linear TOF system is better suited for the mass spectrometric analysis of ssDNA oligomers larger than about a 25-mer. The current system permits one-base resolution up to about a 40-mer. Mass accuracy for a 20-mer or smaller is within +/- 0.05%. Comparison of mass spectra from 5-ns and 35-ps pulse widths at the same energy density shows no significant differences. Mechanisms for oligonucleotide ion production in these experiments are discussed.