Redundant and Antagonistic Roles of XTP3B and OS9 in Decoding Glycan and Non-glycan Degrons in ER-Associated Degradation.

Glycoproteins engaged in unproductive folding in the ER are marked for degradation by a signal generated by progressive demannosylation of substrate N-glycans that is decoded by ER lectins, but how the two lectins, OS9 and XTP3B, contribute to non-glycosylated protein triage is unknown. We generated cell lines with homozygous deletions of both lectins individually and in combination. We found that OS9 and XTP3B redundantly promote glycoprotein degradation and stabilize the SEL1L/HRD1 dislocon complex, that XTP3B profoundly inhibits the degradation of non-glycosylated proteins, and that OS9 antagonizes this inhibition. The relative expression of OS9 and XTP3B and the distribution of glycan and non-glycan degrons within the same protein contribute to the fidelity and processivity of glycoprotein triage and, therefore, determine the fates of newly synthesized proteins in the early secretory pathway.

Characterization of protein complexes of the endoplasmic reticulum-associated degradation E3 ubiquitin ligase Hrd1.

Hrd1 is the core structural component of a large endoplasmic reticulum membrane-embedded protein complex that coordinates the destruction of folding-defective proteins in the early secretory pathway. Defining the composition, dynamics, and ultimately, the structure of the Hrd1 complex is a crucial step in understanding the molecular basis of glycoprotein quality control but has been hampered by the lack of suitable techniques to interrogate this complex under native conditions. In this study we used genome editing to generate clonal HEK293 (Hrd1.KI) cells harboring a homozygous insertion of a small tandem affinity tag knocked into the endogenous Hrd1 locus. We found that steady-state levels of tagged Hrd1 in these cells are indistinguishable from those of Hrd1 in unmodified cells and that the tagged variant is functional in supporting the degradation of well characterized luminal and membrane substrates. Analysis of detergent-solubilized Hrd1.KI cells indicates that the composition and stoichiometry of Hrd1 complexes are strongly influenced by Hrd1 expression levels. Analysis of affinity-captured Hrd1 complexes from these cells by size-exclusion chromatography, immunodepletion, and absolute quantification mass spectrometry identified two major high-molecular-mass complexes with distinct sets of interacting proteins and variable stoichiometries, suggesting a hitherto unrecognized heterogeneity in the functional units of Hrd1-mediated protein degradation.

USP8 regulates mitophagy by removing K6-linked ubiquitin conjugates from parkin.

Mutations in the Park2 gene, encoding the E3 ubiquitin-ligase parkin, are responsible for a familial form of Parkinson's disease (PD). Parkin-mediated ubiquitination is critical for the efficient elimination of depolarized dysfunctional mitochondria by autophagy (mitophagy). As damaged mitochondria are a major source of toxic reactive oxygen species within the cell, this pathway is believed to be highly relevant to the pathogenesis of PD. Little is known about how parkin-mediated ubiquitination is regulated during mitophagy or about the nature of the ubiquitin conjugates involved. We report here that USP8/UBPY, a deubiquitinating enzyme not previously implicated in mitochondrial quality control, is critical for parkin-mediated mitophagy. USP8 preferentially removes non-canonical K6-linked ubiquitin chains from parkin, a process required for the efficient recruitment of parkin to depolarized mitochondria and for their subsequent elimination by mitophagy. This work uncovers a novel role for USP8-mediated deubiquitination of K6-linked ubiquitin conjugates from parkin in mitochondrial quality control.

Mitogen-activated protein kinase (MAPK/ERK) regulates adenomatous polyposis coli during growth-factor-induced cell extension.

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-3ß (GSK-3ß), 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-3ß, promotes localization of APC to the tip of cell extensions. Whereas GSK-3ß 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-3ß and ERK pathways act in parallel to regulate interactions between APC and the cytoskeleton during the formation of cell extensions.

Protein standard absolute quantification (PSAQ) method for the measurement of cellular ubiquitin pools.

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.

Global changes to the ubiquitin system in Huntington's disease.

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.

Indirect inhibition of 26S proteasome activity in a cellular model of Huntington's disease.

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.

ALIX is a Lys63-specific polyubiquitin binding protein that functions in retrovirus budding.

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.

Unassembled CD147 is an endogenous endoplasmic reticulum-associated degradation substrate.

Degradation of folding- or assembly-defective proteins by the endoplasmic reticulum-associated degradation (ERAD) ubiquitin ligase, Hrd1, is facilitated by a process that involves recognition of demannosylated N-glycans by the lectin 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.

Defining human ERAD networks through an integrative mapping strategy.

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.

Ubiquitin accumulation in autophagy-deficient mice is dependent on the Nrf2-mediated stress response pathway: a potential role for protein aggregation in autophagic substrate selection.

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.

OS-9 and GRP94 deliver mutant alpha1-antitrypsin to the Hrd1-SEL1L ubiquitin ligase complex for ERAD.

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.

Quantification of intermediate-abundance proteins in serum by multiple reaction monitoring mass spectrometry in a single-quadrupole ion trap.

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.

Capture of peptides with N-terminal serine and threonine: a sequence-specific chemical method for Peptide mixture simplification.

The objective of this study was to evaluate a sequence-specific chemistry for the ability to specifically capture peptides that contain N-terminal serine or threonine residues from mixtures. The first step is the oxidation of the 1,2-amino alcohol structure -CH(NH(2))CH(OH)- of peptides containing N-terminal serine or threonine with periodate. The newly formed aldehyde reacts with a labeling reagent containing a hydrazide, RCONHNH(2), to form a hydrazone-peptide conjugate, RCONHN=CH-peptide. Biotin-labeled conjugates can then be isolated by affinity purification with streptavidin. The method described in this report can be useful in simplifying the complex mixtures of peptides that are generated in typical proteomic analysis, where proteins are digested with trypsin and analyzed using liquid chromatography mass spectrometry data. The sequence-specific peptide selection not only reduces the complexity of digest mixtures, but also provides additional information for peptide identification. The targeted peptides are those that have either serine or threonine adjacent to a protease cleavage site. The sequence information should greatly aid in both database matching for protein identification and for de novo sequence determination.

Identification and relative quantitation of protein mixtures by enzymatic digestion followed by capillary reversed-phase liquid chromatography-tandem mass spectrometry.

In this report, we describe an approach for identification and relative quantitation of individual proteins within mixtures using LC/MS/MS analysis of protein digests. First, the proteins are automatically identified by correlating the tandem mass spectra with peptide sequences from a database. Then, peak areas of identified peptides from one protein are added together to define the total reconstructed peak area of the protein digest. The total reconstructed peak area is further normalized to the peak area of an internal standard protein digest present in the mixture at a constant level. The method was illustrated using digested mixtures of five standard proteins as follows. One protein was gradually diluted while the other four components were present in the mixtures at constant level. This study revealed that relative peak area of the variable protein increased linearly (trend line R2 = 0.9978) with increasing amount from 10 to 1000 fmol, while relative peak areas of four constant proteins remained approximately the same (within 20% relative standard deviation). To further evaluate the applicability of this method for the quantitation of proteins from complex mixtures, human plasma protein digest was spiked with 200 and 400 fmol of myoglobin digest. Total peak area of myoglobin peptides was normalized to the total peak area of apolipoprotein A-I peptides from human plasma, which played the role of an internal standard. The myoglobin/apolipoprotein A-I peak area ratio was 2 times larger for the human plasma digest spiked with a double amount of myoglobin. After several repetitions, the error of the relative peak area measurements remained below 11%, suggesting that the method described here can be used for relative concentration measurements of proteins in the complex biological mixtures. In the presented method, chemical derivatization steps are not needed to create an internal standard, as in isotope-coded affinity tag or similar methods.

Quantification of proteins and metabolites by mass spectrometry without isotopic labeling or spiked standards.

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.