Search

Protein phosphorylation regulates brain development and neuronal activities; and dysregulation of phosphorylation contributes to neurobiological disorders. Phosphoproteomic analysis provides comprehensive modification maps for measuring protein activities in cellular pathways and biological processes. Here, we introduce a mass spectrometry (MS)-based protocol to quantitatively analyze the phosphoproteome of human postmortem brains of Alzheimer's disease. In this isobaric labeling protocol, up to ten brain samples are selected from control and diseased cases for comparison. Approximately 1 mg proteins per sample are extracted, digested, labeled, and then mixed at an equal ratio. To improve the coverage of phosphoproteome, the peptide mix is further fractionated by offline basic pH reversed-phase liquid chromatography (LC) with high-resolution power. Phosphopeptides in each fraction are then enriched by the titanium dioxide method and analyzed by online acidic pH reverse phase LC-MS/MS, leading to the analysis of tens of thousands of phosphorylation events. This protocol can also be adapted to profile phosphoproteome in other biological samples.

This paper considers the cooperative output synchronization of networked fractional-order single-machine infinite-bus (FOSMIB) power systems, in which the agent's dynamic is modeled by fractional-order derivative in spite of parametric uncertainties. Combining Backstepping approach together with Graph Theory, a distributed adaptive controller is designed recursively for each agent to drive its output synchronized to its neighbors in the network while other variables in the overall system maintain bounded. It is proved that under the topology condition that the graph is connected and at least one agent can obtain the leader's information, all agents outputs can be synchronized to the leader, i.e., cooperative output synchronization of the network is achieved. The distributed controller for each agent has three parts: state feedback of itself, neighborhood information obtained from its neighbors, and parameter updates for the unknown parameters in its dynamic. Simulation result has been presented to verify the effectiveness of the proposed controller.

Human Mas-related G protein-coupled receptor X1 (MRGPRX1) is a promising target for pain inhibition, mainly because of its restricted expression in nociceptors within the peripheral nervous system. However, constrained by species differences across Mrgprs, drug candidates that activate MRGPRX1 do not activate rodent receptors, leaving no responsive animal model to test the effect on pain in vivo. Here, we generated a transgenic mouse line in which we replaced mouse Mrgprs with human MrgprX1 This humanized mouse allowed us to characterize an agonist [bovine adrenal medulla 8-22 (BAM8-22)] and a positive allosteric modulator (PAM), ML382, of MRGPRX1. Cellular studies suggested that ML382 enhances the ability of BAM8-22 to inhibit high-voltage-activated Ca2+ channels and attenuate spinal nociceptive transmission. Importantly, both BAM8-22 and ML382 effectively attenuated evoked, persistent, and spontaneous pain without causing obvious side effects. Notably, ML382 by itself attenuated both evoked pain hypersensitivity and spontaneous pain in MrgprX1 mice after nerve injury without acquiring coadministration of an exogenous agonist. Our findings suggest that humanized MrgprX1 mice provide a promising preclinical model and that activating MRGPRX1 is an effective way to treat persistent pain.

Isobaric labeling quantification by mass spectrometry (MS) has emerged as a powerful technology for multiplexed large-scale protein profiling, but measurement accuracy in complex mixtures is confounded by the interference from co-isolated ions, resulting in ratio compression. Here we report that the ratio compression can be essentially resolved by the combination of pre-MS peptide fractionation, MS2-based interference detection and post-MS computational interference correction. To recapitulate the complexity of biological samples, we pooled tandem mass tag (TMT) labeled E. coli peptides at 1 : 3 : 10 ratios, and added in ∼20-fold more rat peptides as background, followed by the analysis of two dimensional liquid chromatography (LC)-MS/MS. Systematic investigation show that quantitative interference was impacted by LC fractionation depth, MS isolation window and peptide loading amount. Exhaustive fractionation (320 × 4 h) can nearly eliminate the interference and achieve results comparable to the MS3-based method. Importantly, the interference in MS2 scans can be estimated by the intensity of contaminated y1 product ions, and we thus developed an algorithm to correct reporter ion ratios of tryptic peptides. Our data indicate that intermediate fractionation (40 × 2 h) and y1 ion-based correction allow accurate and deep TMT profiling of more than 10,000 proteins, which represents a straightforward and affordable strategy in isobaric labeling proteomics.

Mass spectrometry-based proteomics has experienced an unprecedented advance in comprehensive analysis of proteins and posttranslational modifications, with particular technical progress in liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) and isobaric labeling multiplexing capacity. Here, we introduce a deep proteomics profiling protocol that combines 10-plex tandem mass tag (TMT) labeling with an optimized LC-MS/MS platform to quantitate whole proteome and phosphoproteome. The major steps include protein extraction and digestion, TMT labeling, two-dimensional liquid chromatography, TiO2-mediated phosphopeptide enrichment, high-resolution mass spectrometry, and computational data processing. This protocol routinely leads to confident quantification of more than 10,000 proteins and approximately 30,000 phosphosites in mammalian samples. Quality control steps are implemented for troubleshooting and evaluating experimental variation. Such a multiplexed robust method provides a powerful tool for dissecting proteomic signatures at the systems level in a variety of complex samples, ranging from cell culture, animal tissues to human clinical specimens.

Protein phosphorylation regulates brain development and neuronal activities; and dysregulation of phosphorylation contributes to neurobiological disorders. Phosphoproteomic analysis provides comprehensive modification maps for measuring protein activities in cellular pathways and biological processes. Here, we introduce a mass spectrometry (MS)-based protocol to quantitatively analyze the phosphoproteome of human postmortem brains of Alzheimer's disease. In this isobaric labeling protocol, up to ten brain samples are selected from control and diseased cases for comparison. Approximately 1 mg proteins per sample are extracted, digested, labeled, and then mixed at an equal ratio. To improve the coverage of phosphoproteome, the peptide mix is further fractionated by offline basic pH reversed-phase liquid chromatography (LC) with high-resolution power. Phosphopeptides in each fraction are then enriched by the titanium dioxide method and analyzed by online acidic pH reverse phase LC-MS/MS, leading to the analysis of tens of thousands of phosphorylation events. This protocol can also be adapted to profile phosphoproteome in other biological samples.

The 26S proteasome is ubiquitinated by proteasome-associating ubiquitination enzymes. Proteasome ubiquitination impairs proteasomal degradation and is regulated by deubiquitination, substrate binding, and cellular stress. It is proposed that in situ ubiquitination autoregulates proteasomal activity in cells., The 26S proteasome degrades ubiquitinated proteins, and proteasomal degradation controls various cellular events. Here we report that the human 26S proteasome is ubiquitinated, by which the ubiquitin receptors Adrm1 and S5a, the ATPase subunit Rpt5, and the deubiquitinating enzyme Uch37 are ubiquitinated in situ by proteasome-associating ubiquitination enzymes. Ubiquitination of these subunits significantly impairs the 26S proteasome's ability to bind, deubiquitinate, and degrade ubiquitinated proteins. Moreover, ubiquitination of the 26S proteasome can be antagonized by proteasome-residing deubiquitinating enzymes, by the binding of polyubiquitin chains, and by certain cellular stress, indicating that proteasome ubiquitination is dynamic and regulated in cells. We propose that in situ ubiquitination of the 26S proteasome regulates its activity, which could function to adjust proteasomal activity in response to the alteration of cellular ubiquitination levels.

The ribosomal protein (RP)-HDM2-p53 pathway has been shown to have key roles in oncogene-induced apoptosis and senescence, but the mechanism regulating this pathway remains elusive. The proline-rich Akt substrate of 40 kDa (PRAS40) has recently been identified as a binding partner and inhibitor of the mechanistic (formerly referred to as mammalian) target of rapamycin complex 1 (mTORC1). Although other inhibitors of mTORC1 are known tumor suppressors, PRAS40 promotes cell survival and tumorigenesis. Here we demonstrate that Akt- and mTORC1-mediated phosphorylation of PRAS40 at T246 and S221, respectively, promotes nuclear-specific association of PRAS40 with ribosomal protein L11 (RPL11). Importantly, silencing of PRAS40 induces upregulation of p53 in a manner dependent on RPL11. This effect is rescued by wild-type PRAS40, but not by the RPL11-binding-null PRAS40T246A mutant. We found that PRAS40 negatively regulates the RPL11-HDM2-p53 nucleolar stress response pathway and suppresses induction of p53-mediated cellular senescence. This work identifies nuclear PRAS40 as a dual-input signaling checkpoint that links cell growth and proliferation to inhibition of cellular senescence. These findings may help to explain the protumorigenic effect of PRAS40 and identify the PRAS40-RPL11 complex as a promising target for p53-restorative anticancer drug discovery.

Pregnane X receptor (PXR) is a xenobiotic sensor regulating the expression of genes involved in xenobiotic detoxification and elimination. Phosphorylation plays an important role in modulating PXR activity and several phosphorylation sites have been predicted and characterized in in vitro experiments. Although PXR has been shown to be a phosphoprotein in vivo, the exact residues that are phosphorylated remain elusive. Using mass spectrometry, we identified for the first time S114, T133/135, S167, and S200 residues that are phosphorylated in PXR following an in vitro kinase assay using cyclin-dependent kinase 2. We further found that the phosphorylation at S114, T133, and T135 occurred in PXR isolated from cells. We tested the phosphodeficient and phosphomimetic mutants corresponding to all the sites identified and determined that phosphorylation at S114 attenuates the transcriptional activity of PXR, consistent with the observation that the S114D mutant displayed reduced association with the PXR-targeted DNA response element. Phosphomimetic mutations at either T133 or T135 did not show a significant change in transcriptional activity however, the dual phosphomimetic mutant T133D/T135D displayed reduced transcriptional activity. Subcellular localization studies showed a varied distribution of the mutants suggesting that the regulation of PXR is much more complex than what we can observe by just overexpressing the mutants. Thus, our results provide the first direct evidence that PXR is phosphorylated at specific residues and suggest that further investigation is warranted to fully understand the regulation of PXR by phosphorylation.

We introduce a formula-based strategy and algorithm (JUMPm) for global metabolite identification and false discovery analysis in untargeted mass spectrometry-based metabolomics. JUMPm determines the chemical formulas of metabolites from unlabeled and stable-isotope labeled metabolome data, and derives the most likely metabolite identity by searching structure databases. JUMPm also estimates the false discovery rate (FDR) with a target-decoy strategy based on the octet rule of chemistry. With systematic stable isotope labeling of yeast, we identified 2,085 chemical formulas (10% FDR), 892 of which were assigned with metabolite structures. We evaluated JUMPm with a library of synthetic standards, and found that 96% of the formulas were correctly identified. We extended the method to mammalian cells with direct isotope labeling and by heavy yeast spike-in. This strategy and algorithm provide a powerful a practical solution for global identification of metabolites with a critical measure of confidence.

Ubiquitin-specific-processing protease 7 (USP7), a protein deubiquitinase, is one of the most frequently mutated genes (33%) in the TAL1 subtype of pediatric T-lineage acute lymphoblastic leukemia (T-ALL). However, the functional effect of USP7 haploinsufficiency on T-ALL pathogenesis remains elusive. To understand USP7 haploinsufficiency’s impact on T-ALL, we performed gene expression analysis on 42 non-early T-cell precursor T-ALL RNAseq samples downloaded from phs000218. The gene expression analysis suggested that USP7 haploinsufficiency (USP7-mut N = 12; USP7-wt N = 30) down-regulated the expression of T-cell maturation markers (e.g. RAG1, RAG2, and CD1B), which were negatively regulated by TAL1 [1]. RNAseq on a T-ALL cell line with USP7 knocked down by shRNA also found the same TAL1 negatively regulated gene set, further supporting an increase of TAL1 activity in the USP7 mutated T-ALLs. To examine whether the T-cell maturation was affected by USP7 heterozygous knockout, we generated a conditional knockout (cKO) mouse model by cross-breeding the transgenic vav1-cre mice with the USP7fl/fl mice to obtain heterozygous USP7fl/wt-vav1-cre. Thymocytes isolated from cKO mice were co-cultured with the OP9-Δ1 cells in the medium supplied with cytokines. The cell surface differentiation markers, CD4 and CD8 were stained for flow cytometry detection, and we observed an increase of double-negative cells and a decrease of double-positive cells in USP7-het-KO mice versus control (N = 4 in each group; p-value < 0.05), consolidating the disrupted T-cell development ex vivo. To further understand USP7’s mechanism to regulate TAL1, we analyzed proteins that interacted with USP7 by affinity purification-mass spectrometry (AP-MS) and immune-precipitated followed by western-blotting (IP/WB). AP-MS with the anti-USP7 antibody revealed that USP7 directly interacts with TAL1 in Jurkat cells; AP-MS with the anti-TAL1 antibody also reciprocated the USP7-TAL1 interactions. Whole proteome analysis, through tandem-mass-tag and two-dimensional liquid chromatography-tandem mass spectrometry, revealed that USP7 knockdown down-regulated TRIM27, a deubiquitin target of USP7. IP/WB further confirmed the interaction between USP7, TRIM27, and TAL1, suggesting a possible synergistic relationship between USP7 and TRIM27 to regulate TAL1. In conclusion, our finding demonstrates heterozygous loss of function of USP7 dysregulates T-cell maturation by enhancing TAL1 activity. Future work on TRIM27 could further shed light on the mechanism underlying USP7’s ability to regulate TAL1. Reference: [1] Sanda et al. Cancer Cell 22, 209 (2012). Citation Format: Timothy I. Shaw, Li Dong, Anthony High, Yu Liu, Bensheng Ju, Kanisha Kavdia, Vishwajeeth Pagala, Bridget Shaner, John Easton, Chenxi Qian, Jiyang Yu, Janke Janke, John Kim Choi, Junmin Peng, Wei Gu, James R. Downing, Jinghui Zhang. USP7 heterozygous loss-of-function affects T-cell differentiation in pediatric T-ALL [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3652.

N6-methyladenosine (m6A) is the most abundant internal modification in eukaryotic mRNA. Recent conceptual and technological advances have sparked extensive research interests in the functional roles of m6A. The writers, erasers and readers of m6A have been identified and characterized in a number of biological processes in recent years. However, the physiological roles of m6A and its effectors in lymphogenesis remained unknown. Here, we report that loss of METTL14, a core component of the m6A writer complex, in mouse B lineage cells blocked B cell development. Deletion of METTL14 in developing B cells caused dramatic reductions in the mRNA m6A level, together with an incomplete blockage during the transition from the quiescent pro-B stage to the proliferating large pre-B stage as well as a complete blockage during the proliferation-to-quiescence transition between large pre-B and small pre-B stages. We found that >67% transcripts contain m6A in developing B cells, suggesting that m6A might be widely involved in gene expression regulation. m6A controlled the quiescence-to-proliferation switch partially through its cytoplasmic reader YTHDF2, as deleting YTHDF2 partially mimicked the earlier incomplete blockage between pro-B and large pre-B stages due to METTL14 deficiency, but did not cause the later proliferation-to-quiescence blockage. Altogether, our data identifies m6A, which specifically controls transitions between quiescence and proliferation, as a critical new layer of gene expression regulation in B cell development.

Disturbances in autophagy and stress granule dynamics have been implicated as potential mechanisms underlying inclusion body myopathy (IBM) and related disorders. Yet the roles of core autophagy proteins in IBM and stress granule dynamics remain poorly characterized. Here, we demonstrate that disrupted expression of the core autophagy proteins ULK1 and ULK2 in mice causes a vacuolar myopathy with ubiquitin and TDP-43-positive inclusions; this myopathy is similar to that caused by VCP/p97 mutations, the most common cause of familial IBM. Mechanistically, we show that ULK1/2 localize to stress granules and phosphorylate VCP, thereby increasing VCP's activity and ability to disassemble stress granules. These data suggest that VCP dysregulation and defective stress granule disassembly contribute to IBM-like disease in Ulk1/2-deficient mice. In addition, stress granule disassembly is accelerated by an ULK1/2 agonist, suggesting ULK1/2 as targets for exploiting the higher-order regulation of stress granules for therapeutic intervention of IBM and related disorders.

SUMMARY Skeletal muscle cell (myofiber) atrophy is a detrimental component of aging and cancer that primarily results from muscle protein degradation via the proteasome and ubiquitin ligases. Transcriptional upregulation of some ubiquitin ligases contributes to myofiber atrophy, but little is known about the role that most other ubiquitin ligases play in this process. To address this question, we have used RNAi screening in Drosophila to identify the function of > 320 evolutionarily conserved ubiquitin ligases in myofiber size regulation in vivo. We find that whereas RNAi for some ubiquitin ligases induces myofiber atrophy, loss of others (including the N-end rule ubiquitin ligase UBR4) promotes hypertrophy. In Drosophila and mouse myofibers, loss of UBR4 induces hypertrophy via decreased ubiquitination and degradation of a core set of target proteins, including the HAT1/RBBP4/RBBP7 histone-binding complex. Together, this study defines the repertoire of ubiquitin ligases that regulate myofiber size and the role of UBR4 in myofiber hypertrophy., Graphical Abstract, In Brief Hunt et al. use the fruit fly Drosophila to identify ubiquitin-related enzymes that regulate skeletal muscle cell (myofiber) size, including the ubiquitin ligase UBR4. Loss of UBR4 promotes myofiber hypertrophy in Drosophila and mice via decreased ubiquitination and degradation of a core set of target proteins.

In this study, we have considered the cooperative output synchronisation problem of non-linear multi-agent system. The dynamic of each agent in the network is a class of genuinely non-linear system, named as high-order power integrator that may be uncontrollable after linearisation around the origin. Disturbance is considered in the input channel of each agent as well. Combined Graph theory with the method of adding a power integrator, a distributed controller is designed recursively for each agent to achieve the control objective. The distributed controller of each agent has three parts: state feedback of itself, output information of its neighbours and an adaptive disturbance compensator. It is proved that when the undirected graph is connected, all agents’ outputs in the network can be synchronised, that is, cooperative output synchronisation is achieved. Simulation result is presented to verify its effectiveness.

Hallmarks of Alzheimer’s disease (AD), a progressive neurodegenerative disease causing dementia, include protein aggregates such as amyloid beta plaques and tau neurofibrillary tangles in a patient’s brain. Understanding the complete composition and structure of protein aggregates in AD can shed light on the as-yet unidentified underlying mechanisms of AD development and progression. Biochemical isolation of aggregates coupled with mass spectrometry (MS) provides a comprehensive proteomic analysis of aggregates in AD. Dissection of these AD-specific aggregate components, such as U1 small nuclear ribonucleoprotein complex (U1 snRNP), provides novel insights into the deregulation of RNA splicing in the disease. In this review, we summarize the methodologies of laser capture microdissection (LCM) and differential extraction to analyze the aggregated proteomes in AD samples, and discuss the derived novel insights that may contribute to AD pathogenesis.

The 26S proteasome is the major proteolytic machine for breaking down cytosolic and nuclear proteins in eukaryotes. Due to the lack of a suitable assay, it is difficult to measure routinely and quantitatively the breakdown of proteins by the 26S proteasome in vitro. In the present study, we developed an assay to monitor proteasome-mediated protein degradation. Using this assay, we discovered that epidithiodiketopiperazine (ETPs) blocked the degradation of our model substrate in vitro. Further characterization revealed that ETPs inhibited proteasome function by targeting the essential proteasomal deubiquitinase Rpn11 (POH1/PSMD14). ETPs also inhibited other JAMM (JAB1/MPN/Mov34 metalloenzyme) proteases such as Csn5 and AMSH. An improved ETP with fewer non-specific effects, SOP11, stabilized a subset of proteasome substrates in cells, induced the unfolded protein response, and led to cell death. SOP11 represents a class of Rpn11 inhibitor and provides an alternative route to develop proteasome inhibitors. [Abstract copyright: Copyright © 2018 Elsevier Ltd. All rights reserved.]

SummaryTank-binding kinase 1 (TBK1) is a serine/threonine protein-kinase mediating innate antimicrobial immunity. TBK1 is involved in the signaling of TLRs, RLRs, and STING-mediated sensing of cytosolic DNA. Stimulation of these receptors results in the activation of TBK1, which phosphorylates interferon regulatory factor (IRF)-3. Phosphorylated IRF-3 translocates into the nucleus to initiate the transcription of the interferon (IFN)-β gene. Here, we show that TBK1 is activated by autophosphorylation at residue Ser172. Structures of TBK1 bound to two inhibitors showed that TBK1 has the IκB kinase fold with three distinct domains: the kinase domain, the ubiquitin-like domain, and the scaffold and dimerization domain. However, the overall structures of the TBK1 monomer and its dimer are different from IKKβ in the arrangements of the three domains and in dimer formation. Phosphorylation of IRF-3 by TBK1 in vitro results in its oligomerization, and phosphorylation of residue Ser386 plays a key role in IRF-3 activation.

SummaryIn eukaryotic cells a molecular chaperone network associates with translating ribosomes, assisting the maturation of emerging nascent polypeptides. Hsp70 is perhaps the major eukaryotic ribosome-associated chaperone and the first reported to bind cotranslationally to nascent chains. However, little is known about the underlying principles and function of this interaction. Here, we use a sensitive and global approach to define the cotranslational substrate specificity of the yeast Hsp70 SSB. We find that SSB binds to a subset of nascent polypeptides whose intrinsic properties and slow translation rates hinder efficient cotranslational folding. The SSB-ribosome cycle and substrate recognition is modulated by its ribosome-bound cochaperone, RAC. Deletion of SSB leads to widespread aggregation of newly synthesized polypeptides. Thus, cotranslationally acting Hsp70 meets the challenge of folding the eukaryotic proteome by stabilizing its longer, more slowly translated, and aggregation-prone nascent polypeptides.

Expansion of a hexanucleotide repeat GGGGCC (G4C2) in C9ORF72 is the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Transcripts carrying (G4C2) expansions undergo unconventional, non-ATG-dependent translation, generating toxic dipeptide repeat (DPR) proteins thought to contribute to disease. Here, we identify the interactome of all DPRs and find that arginine-containing DPRs, polyGly-Arg (GR) and polyPro-Arg (PR), interact with RNA-binding proteins and proteins with low complexity sequence domains (LCDs) that often mediate the assembly of membrane-less organelles. Indeed, most GR/PR interactors are components of membrane-less organelles such as nucleoli, the nuclear pore complex and stress granules. Genetic analysis in Drosophila demonstrated the functional relevance of these interactions to DPR toxicity. Furthermore, we show that GR and PR altered phase separation of LCD-containing proteins, insinuating into their liquid assemblies and changing their material properties, resulting in perturbed dynamics and/or functions of multiple membrane-less organelles.

In this paper, the leader-following consensus of multi-agent system in noisy environment under fixed and switching topology is investigated. Based on the optimal regulator, a distributed consensus controller is designed, using the stochastic analysis and algebraic graph theory, a sufficient condition that under which the consensus error (disagreement vector) can be exponentially stable in mean square with the perturbation of noise in environment is obtained. Moreover the result is extended to switching topology case. Numerical examples are provided in this paper to illustrate the effectiveness of the proposed consensus controller.

Proteogenomics is an emerging approach to improve gene annotation and interpretation of proteomics data. Here we present JUMPg, an integrative proteogenomics pipeline including customized database construction, tag-based database search, peptide-spectrum match filtering, and data visualization. JUMPg creates multiple databases of DNA polymorphisms, mutations, splice junctions, partially trypticity, as well as protein fragments translated from the whole transcriptome in all six frames upon RNA-seq de novo assembly. We use a multistage strategy to search these databases sequentially, in which the performance is optimized by re-searching only unmatched high-quality spectra and reusing amino acid tags generated by the JUMP search engine. The identified peptides/proteins are displayed with gene loci using the UCSC genome browser. Then, the JUMPg program is applied to process a label-free mass spectrometry data set of Alzheimer's disease postmortem brain, uncovering 496 new peptides of amino acid substitutions, alternative splicing, frame shift, and "non-coding gene" translation. The novel protein PNMA6BL specifically expressed in the brain is highlighted. We also tested JUMPg to analyze a stable-isotope labeled data set of multiple myeloma cells, revealing 991 sample-specific peptides that include protein sequences in the immunoglobulin light chain variable region. Thus, the JUMPg program is an effective proteogenomics tool for multiomics data integration.

This paper focuses on a robust cooperative output tracking problem of networked power integrator systems. The dynamics of each system is considered as a nonlinear high-order power integrator whose linearized model is uncontrollable around its origin. It is proven via Lyapunov Theory that under some mild assumptions and graph structural properties, all agents' outputs in the network can be synchronized to a desired trajectory with a bounded error in presence of external disturbances as well as model uncertainties. Moreover, the tracking performance can be tuned by appropriately choosing parameters within the controller. The proposed controller for each agent is in essence constructed via backstepping technique consisting of three components: the state feedback of its own, the outputs of its neighbors and the information of the desired trajectory if connected, and thus in a distributed manner.

TAR DNA-binding protein 43 (TDP-43) is a nuclear protein involved in RNA splicing and a major protein component in ubiquitin-positive, tau-negative inclusions of frontotemporal lobar degeneration and amyotrophic lateral sclerosis. Under disease conditions, TDP-43 redistributes to the cytoplasm where it can be phosphorylated, ubiquitinated, and proteolytically cleaved. Enzymes responsible for TDP-43 proteolytic processing in brain remain largely unreported. Using a MS approach, we identified two truncated TDP-43 peptides, terminating C-terminal to asparagines 291 (N291) and 306 (N306). The only documented mammalian enzyme capable of cleaving C-terminal to asparagine is asparaginyl endopeptidase (AEP). TDP-43-immunoreactive fragments (~35 and 32 kDa) predicted to be generated by AEP cleavage at N291 and N306 were observed by Western blot analyses of postmortem frontotemporal lobar degeneration brain tissue and cultured human cells over-expressing TDP-43. Studies in vitro determined that AEP can directly cleave TDP-43 at seven sites, including N291 and N306. Western blots of brain homogenates isolated from AEP-null mice and wild-type littermate controls revealed that TDP-43 proteolytic fragments were substantially reduced in the absence of AEP in vivo. Taken together, we conclude that TDP-43 is cleaved by AEP in brain. Moreover, these data highlight the utility of combining proteomic strategies in vitro and in vivo to provide insight into TDP-43 biology that will fuel the design of more detailed models of disease pathogenesis.

Summary Endogenous 24-hour rhythms are generated by circadian clocks located in most tissues. The molecular clock mechanism is based on feedback loops involving clock genes and their protein products. Post-translational modifications, including ubiquitination, are important for regulating the clock feedback mechanism. Previous work has focused on the role of ubiquitin ligases in the clock mechanism. Here we show a role for the rhythmically-expressed deubiquitinating enzyme ubiquitin specific peptidase 2 (USP2) in clock function. Mice with a deletion of the Usp2 gene (Usp2 KO) display a longer free-running period of locomotor activity rhythms and altered responses of the clock to light. This was associated with altered expression of clock genes in synchronized Usp2 KO mouse embryonic fibroblasts and increased levels of clock protein PERIOD1 (PER1). USP2 can be coimmunoprecipitated with several clock proteins but directly interacts specifically with PER1 and deubiquitinates it. Interestingly, this deubiquitination does not alter PER1 stability. Taken together, our results identify USP2 as a new core component of the clock machinery and demonstrate a role for deubiquitination in the regulation of the circadian clock, both at the level of the core pacemaker and its response to external cues.

A hallmark of neurodegeneration is the aggregation of disease related proteins that are resistant to detergent extraction. In the major pathological subtype of frontotemporal lobar degeneration (FTLD), modified TAR-DNA binding protein 43 (TDP-43), including phosphorylated, ubiquitinated and proteolytically cleaved forms, is enriched in detergent-insoluble fractions from post-mortem brain tissue. Additional proteins that accumulate in the detergent-insoluble FTLD brain proteome remain largely unknown. In this study, we used proteins from stable isotope-labeled (SILAC) human embryonic kidney 293 cells (HEK293) as internal standards for peptide quantitation across control and FTLD insoluble brain proteomes. Proteins were identified and quantified by liquid-chromatography coupled with tandem mass spectrometry (LC-MS/MS) and twenty-one proteins were determined to be enriched in FTLD using SILAC internal standards. In parallel, label free quantification of only the unlabeled brain derived peptides by spectral counts (SC) and G-test analysis identified additional brain-specific proteins significantly enriched in disease. Several proteins determined to be enriched in FTLD using SILAC internal standards were not considered significant by G-test due to their low total number of SC. However immunoblotting of FTLD and control samples confirmed enrichment of these proteins, highlighting the utility of SILAC internal standard to quantify low abundance proteins in brain. Of these, the RNA binding protein PTB-associated splicing factor (PSF) was further characterized because of structural and functional similarities to TDP-43. Full-length PSF and shorter molecular weight fragments, likely resulting from proteolytic cleavage, were enriched in FTLD cases. Immunohistochemical analysis of PSF revealed predominately nuclear localization in control and FTLD brain tissue and was not associated with phosphorylated pathologic TDP-43 neuronal inclusions. However, in a subset of FTLD cases, PSF was aberrantly localized to the cytoplasm of oligodendrocytes. These data raise the possibility that PSF directed RNA processes in oligodendrocytes are altered in neurodegenerative disease.

Posttranslational protein modification by the ubiquitin-like SUMO protein is critical to eukaryotic cell regulation, but much remains unknown regarding its operation and substrates. Here we report that specific mutations in the Saccharomyces cerevisiae Ulp1 SUMO protease, including its coiled-coil (CC) domain, lead to the accumulation of distinct sumoylated proteins in vivo. A prominent ∼50-kDa sumoylated protein accumulates in a Ulp1 CC mutant. The protein was identified as Scs2, an endoplasmic reticulum (ER) membrane protein that regulates phosphatidylinositol synthesis and lipid trafficking. Mutation of lysine 180 of Scs2 abolishes its sumoylation. Notably, impairment of either cellular sumoylation or cellular desumoylation mechanisms inhibits cell growth in the absence of inositol and exacerbates the inositol auxotrophy caused by deletion of SCS2. Mutants lacking the Ulp2 SUMO protease are the most severely affected, and this defect was traced to the mutants' impaired ability to induce transcription of INO1, which encodes the rate-limiting enzyme of inositol biosynthesis. Conversely, inositol starvation induces a striking change in the profiles of total cellular SUMO conjugates. These results provide the first evidence of cross-regulation between the SUMO and inositol pathways, including the sumoylation of an ER membrane protein central to phospholipid synthesis and phosphoinositide signaling.

Many animal tissues/cells are photosensitive, yet only two types of photoreceptors (opsins and cryptochromes) have been discovered in metazoans. The question arises as to whether unknown types of photoreceptors exist in the animal kingdom. LITE-1, a seven-transmembrane gustatory receptor (GR) homolog, mediates UV light-induced avoidance behavior in C. elegans. However, whether LITE-1 functions as a chemoreceptor or photoreceptor has not been determined. Here, we show that LITE-1 directly absorbs both UVA and UVB light with an extinction coefficient 10–100 times that of opsins and cryptochromes, indicating that LITE-1 is highly efficient in capturing photons. Unlike typical photoreceptor proteins employing a prosthetic chromophore to capture photons, LITE-1 strictly depends on its protein conformation for photon absorption. We further identified two tryptophan residues critical for LITE-1 function. Interestingly, unlike GPCRs, LITE-1 adopts a reversed membrane topology. Thus, LITE-1, a taste receptor homolog, represents a distinct type of photoreceptor in the animal kingdom.

Gliomas are a group of aggressive brain tumors that diffusely infiltrate adjacent brain tissues, rendering them largely incurable, even with multiple treatment modalities and agents. Mostly asymptomatic at early stages, they present in several subtypes with astrocytic or oligodendrocytic features and invariably progress to malignant forms. Gliomas are difficult to classify precisely because of interobserver variability during histopathologic grading. Identifying biological signatures of each glioma subtype through protein biomarker profiling of tumor or tumor-proximal fluids is therefore of high priority. Such profiling not only may provide clues regarding tumor classification but may identify clinical biomarkers and pathologic targets for the development of personalized treatments. In the past decade, differential proteomic profiling techniques have utilized tumor, cerebrospinal fluid, and plasma from glioma patients to identify the first candidate diagnostic, prognostic, predictive, and therapeutic response markers, highlighting the potential for glioma biomarker discovery. The number of markers identified, however, has been limited, their reproducibility between studies is unclear, and none have been validated for clinical use. Recent technological advancements in methodologies for high-throughput profiling, which provide easy access, rapid screening, low sample consumption, and accurate protein identification, are anticipated to accelerate brain tumor biomarker discovery. Reliable tools for biomarker verification forecast translation of the biomarkers into clinical diagnostics in the foreseeable future. Herein we update the reader on the recent trends and directions in glioma proteomics, including key findings and established and emerging technologies for analysis, together with challenges we are still facing in identifying and verifying potential glioma biomarkers.

The mechanisms by which ubiquitin ligases are regulated remain poorly understood. Here we describe a series of molecular events that coordinately regulate CHIP, a neuroprotective E3 implicated in protein quality control. Through their opposing activities, the initiator E2, Ube2w, and the specialized deubiquitinating enzyme (DUB), ataxin-3, participate in initiating, regulating, and terminating the CHIP ubiquitination cycle. Monoubiquitination of CHIP by Ube2w stabilizes the interaction between CHIP and ataxin-3, which through its DUB activity limits the length of chains attached to CHIP substrates. Upon completion of substrate ubiquitination, ataxin-3 deubiquitinates CHIP, effectively terminating the reaction. Our results suggest that functional pairing of E3s with ataxin-3 or similar DUBs represents an important point of regulation in ubiquitin-dependent protein quality control. In addition, the results shed light on disease pathogenesis in SCA3, a neurodegenerative disorder caused by polyglutamine expansion in ataxin-3.

The processes that trigger severe muscle atrophy and loss of myosin in critical illness myopathy (CIM) are poorly understood. It has been reported that muscle disuse alters Ca2+handling by the sarcoplasmic reticulum. Since inactivity is an important contributor to CIM, this finding raises the possibility that elevated levels of the proteins involved in Ca2+handling might contribute to development of CIM. CIM was induced in 3- to 5-mo-old rats by sciatic nerve lesion and infusion of dexamethasone for 1 wk. Western blot analysis revealed increased levels of ryanodine receptor (RYR) isoforms-1 and -2 as well as the dihydropyridine receptor/voltage-gated calcium channel type 1.1 (DHPR/CaV1.1). Immunostaining revealed a subset of fibers with elevation of RYR1 and CaV1.1 that had severe atrophy and disorganization of sarcomeres. These findings suggest increased Ca2+release from the sarcoplasmic reticulum may be an important contributor to development of CIM. To assess the endogenous functional effects of increased intracellular Ca2+in CIM, proteolysis of α-fodrin, a well-known target substrate of Ca2+-activated proteases, was measured and found to be 50% greater in CIM. There was also selective degradation of myosin heavy chain relative to actin in CIM muscle. Taken together, our findings suggest that increased Ca2+release from the sarcoplasmic reticulum may contribute to pathology in CIM.

SET domain containing 6 (SETD6) monomethylates the RelA subunit of nuclear factor kappa B (NF-κB). The ankyrin repeats of G9a-like protein (GLP) recognizes RelA monomethylated at Lys310. Adjacent to Lys310 is Ser311, a known phosphorylation site of RelA. Ser311 phosphorylation inhibits Lys310 methylation by SETD6 as well as binding of Lys310me1 by GLP. The structure of SETD6 in complex with RelA peptide containing the methylation site, in the presence of S-adenosyl-l-methionine, reveals a V-like protein structure and suggests a model for NF-κB binding to SETD6. In addition, structural modeling of the GLP ankyrin repeats bound to Lys310me1 peptide provides insight into the molecular basis for inhibition of Lys310me1 binding by Ser311 phosphorylation. Together, these findings provide a structural explanation for a key cellular signaling pathway centered on RelA Lys310 methylation, which is generated by SETD6 and recognized by GLP, and incorporate a methylation–phosphorylation switch of adjacent lysine and serine residues. Finally, SETD6 is structurally similar to the Rubisco large subunit methyltransferase. Given the restriction of Rubisco to plant species, this particular appearance of the protein lysine methyltransferase has been evolutionarily well conserved.

TAR DNA-binding protein 43 (TDP-43) is associated with a spectrum of neurodegenerative diseases. Although TDP-43 resembles heterogeneous nuclear ribonucleoproteins, its RNA targets and physiological protein partners remain unknown. Here we identify RNA targets of TDP-43 from cortical neurons by RNA immunoprecipitation followed by deep sequencing (RIP-seq). The canonical TDP-43 binding site (TG)(n) is 55.1-fold enriched, and moreover, a variant with adenine in the middle, (TG)(n)TA(TG)(m), is highly abundant among reads in our TDP-43 RIP-seq library. TDP-43 RNA targets can be divided into three different groups: those primarily binding in introns, in exons, and across both introns and exons. TDP-43 RNA targets are particularly enriched for Gene Ontology terms related to synaptic function, RNA metabolism, and neuronal development. Furthermore, TDP-43 binds to a number of RNAs encoding for proteins implicated in neurodegeneration, including TDP-43 itself, FUS/TLS, progranulin, Tau, and ataxin 1 and -2. We also identify 25 proteins that co-purify with TDP-43 from rodent brain nuclear extracts. Prominent among them are nuclear proteins involved in pre-mRNA splicing and RNA stability and transport. Also notable are two neuron-enriched proteins, methyl CpG-binding protein 2 and polypyrimidine tract-binding protein 2 (PTBP2). A PTBP2 consensus RNA binding motif is enriched in the TDP-43 RIP-seq library, suggesting that PTBP2 may co-regulate TDP-43 RNA targets. This work thus reveals the protein and RNA components of the TDP-43-containing ribonucleoprotein complexes and provides a framework for understanding how dysregulation of TDP-43 in RNA metabolism contributes to neurodegeneration.

Our study aims to establish whether CXorf67 has a role in the pathogenesis of posterior fossa (PF) ependymoma. Among molecular groups of ependymoma defined by DNA methylation profiling, PF type-A (PFA) is the commonest. PFA ependymomas present mainly in infants and are difficult to treat, having a 10-year overall survival of approximately 40%. Published genomic studies have not identified a recurrent driver mutation in PFA ependymomas, but they do show widespread epigenetic alterations, including global loss of histone H3 K27-trimethlyation (H3K27-me3). Another childhood PF tumor, the diffuse pontine glioma (DPG), also shows loss of H3K27-me3. In DPGs, loss of H3K27-me3 is associated with H3 K27M mutation, but the mechanism in PFA ependymomas is not yet established. We reexamined published PF ependymoma sequencing data and discovered recurrent mutations in a novel gene, CXorf67. Targeted sequencing in a series of PFA ependymomas revealed CXorf67 mutations in 22/234 (9.4%). CXorf67 is a single-exon gene of unknown function. Its protein product is predicted to be “disordered,” apart from one region towards the N terminus. Mutations in PFA ependymomas are missense, and there is a mutation hotspot in the “ordered” region. CXorf67 mutations are not present in other molecular groups of ependymoma and are rare in other cancers. Analyzing the DNA methylation profiles of 675 PFA ependymomas, we have discovered two subgroups, PFA-1 and PFA-2, and nine subtypes among these two subgroups. All PFA subtypes harbor CXorf67 mutations with the exception of PFA-1f and PFA-2c. Targeted sequencing in our tumor series also revealed H3 K27M mutations in PFA ependymomas at a frequency of 3.9%. Two thirds of mutations in HIST1H3B, HIST1H3C, and H3F3A were found in PFA-1f ependymomas, among which H3 mutations were present at a frequency of 35%. Mutations in H3 genes and CXorf67 were mutually exclusive across our series of PFA ependymomas. We used Affymetrix u133v2 arrays to establish that CXorf67 is expressed at high levels in PFA ependymomas, in contrast to low levels in other ependymomas from the PF and supratentorial compartments. A mechanism for CXorf67 overexpression was revealed in a similar comparative analysis of CpG island methylation profiles, which showed that the promoter region of CXorf67 is hypomethylated in PFA tumors, but not in other ependymomas. Using immunohistochemical preparations, we detected expression of CXorf67 at the protein level in the nuclei of PFA ependymomas; PFB and supratentorial tumors were immunonegative. CXorf67 overexpression is found in all PFA molecular subtypes, except PFA-1f, which shows similar levels to those in supratentorial and PFB ependymomas. CXorf67 expression is unrelated to mutation status. Elevated CXorf67 expression is found in the Daoy and U2-OS cancer cell lines. We used immunoprecipitation (IP)/mass spectrometry (MS) to study proteins bound to CXorf67 in Daoy and U2-OS. Analysis of enriched peptides following immunoprecipitation of CXorf67 indicated that it binds EZH2, SUZ12, and EED, three core components of the PRC2 complex. Complementary immunoprecipitation of SUZ12 detected CXorf67. Detecting CXorf67 mutations in almost 10% of PFA ependymomas led to the discovery of overexpression related to promoter region hypomethylation in these tumors. Overexpression of CXorf67 was detected in all PFA ependymoma subtypes except PFA-1f, 35% of which harbor an H3 K27M mutation. The protein product of CXorf67 is found in tumor cell nuclei, where our IP/MS data suggest it is bound to PRC2. Our findings suggest that global loss of H3K27-me3 in PFA ependymomas could be related to overexpression of CXorf67 and its interaction with PRC2, except in tumors where alterations in histone H3 genes are responsible. However, the selective advantage of CXorf67 mutation is yet to be explained. Citation Format: Ji Wen, Jens-Martin Hübner, Wilda Orisme, Gang Wu, Bo Tang, Sujuan Jia, John Easton, Kelly Haupfear, Brian D. Freibaum, Hong Joo Kim, Anthony High, Junmin Peng, Ruth G. Tatevossian, J. Paul Taylor, Stefan M. Pfister, Jinghui Zhang, Kristian W. Pajtler, Marcel Kool, David W. Ellison. Overexpression and mutations of CXorf67 in “infant-type” posterior fossa type-A ependymomas [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr PR08.

Genes encoding epigenetic regulators are commonly mutated at relapse in acute lymphoblastic leukemia, but the way in which such mutations promote drug resistance and their potential for therapeutic targeting is largely unknown. We previously identified mutations in CREBBP, encoding CREBBP binding protein, a histone and non-histone acetylase and transcriptional coregulator in relapsed ALL and non-Hodgkin’s lymphoma. In view of the known role of CREBBP mediation of the transcriptional response to glucocorticoids, we hypothesized that CREBBP mutations promote glucocorticoid resistance, and sought to comprehensively define the prevalence and spectrum of mutations in relapsed ALL, and elucidate their role in resistance. In studies of childhood ALL from St. Jude and the Children’s Oncology Group, CREBBP mutations were observed in 37 (21.2%) of patients, representing the most common target of sequence mutation at relapse. The mutations were either preserved from diagnosis to relapse, or evolved as new mutations or from minor clones to become clonal mutations at relapse. There were hotspots of mutation at R1446 and between P1488-P1494 in the histone acetyltransferase domain, residues critical for recognition of acetylation substrates, and are known or predicted to impair histone acetylation in biochemical assays and in Crebbp-/-;Ep300-/- mouse embryonic fibroblasts reconstituted with mutant CREBBP. To examine the role of CREBBP mutations in drug resistance, we generated isogenic CREBBP wild-type and mutant human ALL cell lines, and conventional and conditional Crebbp Q1501P (=human Q1500) and R1447H (=human R1446) mouse strains. Mice bearing germline Q1501P or R1447H alleles exhibited dysmorphology, reduced fertility, lymphoproliferation, and reduced lifespan. This phenotype was most pronounced in Crebbp R1447 mice, preventing establishment of a stable line; thus, we generated a conditional Crebbp-R1447H knockin strain (CREBBP-R1447H-CKI) that expresses CREBBP R1447H following Cre-mediated recombination. We generated BCR-ABL1 pre-B ALL models by transducing E13.5 fetal liver cells with BCR-ABL1 p185 retrovirus. Consistently, leukemias derived from heterozygous or homozygous CREBBP-Q1501P or heterozygous CREBBP-R1447H-CKI mice showed profound and selective resistance to prednisolone and dexamethasone. Similarly, NALM6 (DUX4/ERG) human ALL cells harboring CREBBP mutations induced by genome editing (NALM6-C001) also showed complete resistance to glucocorticoids, without change in expression of the glucocorticoid receptor (GR). Chromatin immunoprecipitation and sequencing showed that this was accompanied by marked attenuation of recruitment of GR to chromatin, attenuation of histone 3 lysine 18 (H3K18) and H3K27 acetylation, and reduced expression of H3K18Ac-marked genes following dexamethasone treatment. Global acetylome profiling showed that CREBBP mutations resulted in perturbed acetylation of multiple histone and non-histone targets, including CREBBP itself, IKZF1, and MYC, with reciprocal increase in EP300 acetylation. To identify agents that kill CREBBP mutant leukemic cells and that may reverse steroid resistance, we screened NALM6-C001 with a collection of 11,293 chemicals (5,832 unique compounds enriched for FDA-approved drugs) dosed at 10 mM in the presence or absence of dexamethasone for 48 or 96 hrs. 223 active compounds and 220 compounds targeting epigenetic modifications were tested in 10-point dose response screen. Seventeen compounds with IC50 These results demonstrate the high prevalence of CREBBP in relapsed ALL, and show that these mutations have a central role in driving glucocorticoid resistance. These data also suggest that these mutations create a therapeutic vulnerability through EP300 acetylation that may be exploited by bromodomain inhibition. Citation Format: Yunchao Chang, David W. Woessner, Wenwei Lin, Taosheng Chen, Beisi Xu, Yiping Fan, Haiyan Tan, Junmin Peng, Lawryn Kasper, Michelle L. Churchman, Daniela S. Gerhard, Mignon L. Loh, Stephen P. Hunger, Aman Seth, Karen Gascoigne, Charles G. Mullighan. Modeling and targeting CREBBP mutations in relapsed acute lymphoblastic leukemia [abstract]. In: Proceedings of the AACR Special Conference: Pediatric Cancer Research: From Basic Science to the Clinic; 2017 Dec 3-6; Atlanta, Georgia. Philadelphia (PA): AACR; Cancer Res 2018;78(19 Suppl):Abstract nr IA12.

Summary Personalized cancer therapy targeting somatic mutations in patient tumors is increasingly being incorporated into practice. Other therapeutic vulnerabilities resulting from changes in gene expression due to tumor specific epigenetic perturbations are progressively being recognized. These genomic and epigenomic changes are ultimately manifest in the tumor proteome and phosphoproteome. We integrated transcriptomic, epigenomic, and proteomic/phosphoproteomic data to elucidate the cellular origins and therapeutic vulnerabilities of rhabdomyosarcoma (RMS). We discovered that alveolar RMS occurs further along the developmental program than embryonal RMS. We also identified deregulation of the RAS/MEK/ERK/CDK4/6, G2/M, and unfolded protein response pathways through our integrated analysis. Comprehensive preclinical testing revealed that targeting the WEE1 kinase in the G2/M pathway is the most effective approach in vivo for high-risk RMS.

The discovery of biomarkers for neurodegenerative diseases will have a major impact on the efficiency of therapeutic clinical trials and may be important for understanding basic pathogenic mechanisms. We have approached the discovery of protein biomarkers for amyotrophic lateral sclerosis (ALS) by profiling affected tissues in a relevant animal model and then validating the findings in human tissues. Ventral roots from SOD1(G93A) "ALS" mice were analyzed by label-free quantitative mass spectrometry, and the resulting data were compared with data for matched samples from nontransgenic littermates and transgenic mice carrying wild-type human SOD1 (SOD1(WT)). Of 1299 proteins, statistical inference of the data in the three groups identified 14 proteins that were dramatically altered in the ALS mice compared with the two control groups. The protein galectin-3 emerged as a lead biomarker candidate on the basis of its differential expression as assessed by immunoblot and immunocytochemistry in SOD1(G93A) mice as compared to controls and because it is a secreted protein that could potentially be measured in human biofluids. Spinal cord tissue from ALS patients also exhibited increased levels of galectin-3 when compared to controls. Further measurement of galectin-3 in cerebrospinal fluid samples showed that ALS patients had approximately twice as much galectin-3 as normal and disease controls. These results provide the proof of principle that biomarker identification in relevant and well-controlled animal models can be translated to human disease. The challenge is to validate our biomarker candidate proteins as true biomarkers for ALS that will be useful for diagnosis and/or monitoring disease activity in future clinical trials.

Innate immune responses are initiated by recognition of pathogen-associated molecular patterns (PAMPs) via pattern recognition receptors (PRRs) in both animals and plants. In animal, perception of bacteria flagellin by TLR5 transduces signal through MyD88-IRAK1-IRAK4 to ubiquitin E3 ligase TRAF6. TRAF6 coordinates with UBC13 to produce K63-linked polyubiquitination chains serving as scaffold to mediate downstream signaling. In plant, flagellin is recognized by receptor-like kinase FLS2 and co-receptor BAK1. Upon flagellin perception, BAK1 directly phosphorylates an IRAK-like cytoplasmic kinase BIK1, leading to BIK1 dissociation from the FLS2-BAK1 complex to relay signal. How BIK1 activation is regulated and how it transduces signal to downstream remain largely elusive. We report here that flagellin perception triggers rapid monoubiquitination of BIK1 in planta. Time course and mutational analysis suggested that flagellin-induced BIK1 phosphorylation precedes its monoubiquitination. Using a yeast-two-hybrid screen we identified a RING type E3 ligase LUCKY that interacts with BIK1 and monoubiquitinates multiple residues of BIK1. Mass spectrometry analysis of in vitro-ubiquitinated BIK1 revealed nine lysine residues and the BIK19KR mutant with all nine lysine-to-arginine substitutions blocked flagellin-triggered BIK1 ubiquitination. Transgenic complementation assays indicated that BIK19KR was unable to execute normal BIK1 functions. Moreover, we found that monoubiquitination of BIK1 controls ligand-induced BIK1 dissociation from receptor FLS2. Our study revealed that the ligand-induced monoubiquitination of a key PRR complex component positively regulates innate immunity.

During autophagy, the microtubule-associated protein light chain 3 (LC3), a specific autophagic marker in mammalian cells, is processed from the cytosolic form (LC3-I) to the membrane-bound form (LC3-II). In HEK293 cells stably expressing FLAG-tagged LC3, activation of protein kinase C inhibited the autophagic processing of LC3-I to LC3-II induced by amino acid starvation or rapamycin. PKC inhibitors dramatically induced LC3 processing and autophagosome formation. Unlike autophagy induced by starvation or rapamycin, PKC inhibitor-induced autophagy was not blocked by the PI-3 kinase inhibitor wortmannin. Using orthophosphate metabolic labeling, we found that LC3 was phosphorylated in response to the PKC activator PMA or the protein phosphatase inhibitor calyculin A. Furthermore, bacterially expressed LC3 was directly phosphorylated by purified PKC in vitro. The sites of phosphorylation were mapped to T6 and T29 by nanoLC-coupled tandem mass spectrometry. Mutations of these residues significantly reduced LC3 phosphorylation by purified PKC in vitro. However, in HEK293 cells stably expressing LC3 with these sites mutated either singly or doubly to Ala, Asp or Glu, autophagy was not significantly affected, suggesting that PKC regulates autophagy through a mechanism independent of LC3 phosphorylation.

The maturation of young neurons is regulated by complex mechanisms and dysregulation of this process is frequently found in neurodevepmental disorders. MicroRNAs have been implicated in several steps of neuronal maturation including dendritic and axonal growth, spine development, and synaptogenesis. We demonstrate that one brain-enriched microRNA, miR-137, has a significant role in regulating neuronal maturation. Overexpression of miR-137 inhibits dendritic morphogenesis, phenotypic maturation, and spine development both in brain and cultured primary neurons. On the other hand, a reduction in miR-137 had opposite effects. We further show that miR-137 targets the Mind bomb one (Mib1) protein through the conserved target site located in the 3′ untranslated region of Mib1 messenger RNA. Mib1 is an ubiquitin ligase known to be important for neurodevelopment. We show that exogenously expressed Mib1 could partially rescue the phenotypes associated with miR-137 overexpression. These results demonstrate a novel miRNA-mediated mechanism involving miR-137 and Mib1 that function to regulate neuronal maturation and dendritic morphogenesis during development.

Ufd2 is the founding member of E4 enzymes that are specifically involved in ubiquitin chain elongation but whose roles in proteolysis remain scarce. Here, using a genome-wide screen, we identified one cellular target of yeast Ufd2 as the membrane protein Pex29. The ubiquitin chains assembled on Pex29 in vivo by Ufd2 mainly contain Lys-48 linkages. We found that the ubiquitin-protein E3 ligase for overexpressed Pex29 is Doa10, which is known to be involved in protein quality control. Interestingly, not all Doa10 substrates are regulated by Ufd2, suggesting that E4 involvement is not specific to a particular E3, but may depend on the spatial arrangement of the E3-substrate interaction. Cells lacking UFD2 elicit an unfolded protein response, expanding the physiological function of Ufd2. Our results lead to novel insights into the biological role of Ufd2 and further underscore the significance of Ufd2 in proteolysis.

RNA Wars During RNA interference (RNAi), the Dicer endonuclease generates small interfering (si)RNAs that, with the help of the protein R2D2, are loaded into the siRNA-induced silencing complex (RISC). Using siRNAs as guides, RISC, and specifically its Argonaute subunit, targets complementary RNAs for destruction. In order to identify other components of the RISC complex, Liu et al. (p. 750 ) reconstituted the core RISC activity, using purified Drosophila Dicer, R2D2, and Ago-2. The protein C3PO (component 3 promoter of RISC), which consists of heterodimer of Translin and Translin-associated factor X (Trax), was found to enhance RISC activity in this system, and in vivo, with the Trax endonuclease activity activating RISC through the removal of siRNA passenger strand cleavage products.

Argonaute proteins are a part of a larger RNA interference (RNAi) complex, RISC, in which they mediate cleavage of target mRNAs. In this study, Shi and colleagues show that Ago2 interacts with and is hydroxylated by type I collagen prolyl-4-hydroxylase. When this activity is depleted in human or mouse cells, the level of Ago2 is reduced and siRISC activity mediated by the let-7 miRNA is affected. These results suggest that hydroxylation of Ago2, by affecting its stability, can influence the efficiency of RNAi. Human Argonaute (Ago) proteins are essential components of the RNA-induced silencing complexes (RISCs). Argonaute 2 (Ago2) has a P-element-induced wimpy testis (PIWI) domain, which folds like RNase H and is responsible for target RNA cleavage in RNA interference1. Proteins such as Dicer, TRBP, MOV10, RHA, RCK/p54 and KIAA1093 associate with Ago proteins and participate in small RNA processing, RISC loading and localization of Ago proteins in the cytoplasmic messenger RNA processing bodies1,2. However, mechanisms that regulate RNA interference remain obscure. Here we report physical interactions between Ago2 and the α-(P4H-α(I)) and β-(P4H-β) subunits of the type I collagen prolyl-4-hydroxylase (C-P4H(I)). Mass spectrometric analysis identified hydroxylation of the endogenous Ago2 at proline 700. In vitro, both Ago2 and Ago4 seem to be more efficiently hydroxylated than Ago1 and Ago3 by recombinant human C-P4H(I). Importantly, human cells depleted of P4H-α(I) or P4H-β by short hairpin RNA and P4H-α(I) null mouse embryonic fibroblast cells showed reduced stability of Ago2 and impaired short interfering RNA programmed RISC activity. Furthermore, mutation of proline 700 to alanine also resulted in destabilization of Ago2, thus linking Ago2 P700 and hydroxylation at this residue to its stability regulation. These findings identify hydroxylation as a post-translational modification important for Ago2 stability and effective RNA interference.

Joubert syndrome is an autosomal recessive disorder characterized by congenital malformation of the cerebellum and brainstem, with abnormal decussation in the brain. Mutations in the Abelson helper integration site 1 gene, which encodes the protein AHI1, have been shown to cause Joubert syndrome. In this study, we found that mouse Ahi1 formed a stable complex with huntingtin-associated protein 1 (Hap1), which is critical for neonatal development and involved in intracellular trafficking. Hap1-knockout mice showed significantly reduced Ahi1 levels, defective cerebellar development, and abnormal axonal decussation. Suppression of Ahi1 also decreased the level of Hap1; and truncated Ahi1, which corresponds to the mutations in Joubert syndrome, inhibited neurite outgrowth in neuronal culture. Reducing Hap1 expression suppressed the level and internalization of TrkB, a neurotrophic factor receptor that mediates neurogenesis and neuronal differentiation, which led to decreased TrkB signaling. These findings provide insight into the pathogenesis of Joubert syndrome and demonstrate the critical role of the Ahi1-Hap1 complex in early brain development.

Summary Estrogen receptor α (ER) is a ligand-dependent transcription factor. Upon binding estrogen, ER recruits coactivator complexes with histone acetyltransferase or methyltransferase activities to activate downstream target genes. In addition to histones, coactivators can modify ER itself and other proteins in the transactivation complex. Here, we show that ER is directly methylated at lysine 302 (K302) by the SET7 methyltransferase. SET7-mediated methylation stabilizes ER and is necessary for the efficient recruitment of ER to its target genes and for their transactivation. The SET7-ER complex structure reveals the molecular basis for ER peptide recognition and predicts that modifications or mutations of nearby residues would affect K302 methylation. Indeed, a breast cancer-associated mutation at K303 (K303R) alters methylation at K302 in vitro and in vivo. These findings raise the possibility that generation, recognition, and removal of modifications within the ER hinge region generate "ER modification cassettes" that yield distinct patterns for signaling downstream events.

Protein ubiquitination plays an essential regulatory role within all eukaryotes. Large-scale analyses of ubiquitinated proteins are usually performed by combining affinity purification strategies with mass spectrometry. However, there is no reliable method to systematically differentiate ubiquitinated species from copurified unmodified components. Here we report a simple strategy for the large-scale validation of ubiquitination by reconstructing virtual Western blots for proteins analyzed by gel electrophoresis and mass spectrometry. Because protein ubiquitination, especially polyubiquitination, causes a dramatic shift of molecular weight, the difference between experimental and expected molecular weight was used to confirm the status of ubiquitination. Experimental molecular weight of putative yeast ubiquitin-conjugates was computed from the value and distribution of spectral counts in the gel using a Gaussian curve fitting approach. Unmodified proteins in yeast cell lysate were also analyzed as a control to assess the accuracy of the method. Multiple thresholds that incorporated the mass of ubiquitin and/or experimental variations were evaluated with respect to sensitivity and specificity. Ultimately, only approximately 30% of the candidate ubiquitin-conjugates were accepted based on the stringent filtering criteria, although they were purified under denaturing conditions. These accepted conjugates had an estimated false discovery rate of approximately 8% and primarily consisted of proteins larger than 100 kDa. Compared with another validation method (i.e., identification of ubiquitinated lysine sites), approximately 95% of the proteins with defined modification sites showed a convincing increase in molecular weight on the virtual Western blots. A second independent analysis indicated that the method can be simplified by excising fewer than ten gel bands. Therefore, this strategy establishes criteria necessary for the interpretation of ubiquitinated proteins.

Ubiquitin is an essential, highly-conserved small regulatory protein in eukaryotic cells. It covalently modifies a wide variety of targeted proteins in the forms of monomer and polymers, altering the conformation and binding properties of the proteins and thus regulating proteasomal delivery, protein activities and localization. Mass spectrometry has emerged as an indispensable tool for in-depth characterization of protein ubiquitination. Ubiquitinated proteins in cell lysates are usually enriched by affinity chromatography and subsequently analyzed by mass spectrometry for identification and quantification. Ubiquitin-conjugated amino acid residues can be determined by unique mass shift caused by the modification. Moreover, the complex structure of polyubiquitin chains on substrates can be dissected by bottom-up and middle-down mass spectrometric approaches, revealing potential novel functions of polyubiquitin linkages. Here I review the advances and caveats of these strategies, emphasizing caution in the validation of ubiquitinated proteins and in the interpretation of raw data.

Ischemia and seizure cause excessive neuronal excitation that is associated with brain acidosis and neuronal cell death. However, the molecular mechanism of acidification-triggered neuronal injury is incompletely understood. Here, we show that Asparagine Endopeptidase (AEP) is activated under acidic condition and cuts SET, an inhibitor of Dnase, and triggers DNA damage in brain, which is inhibited by PIKE-L. SET, a substrate of caspases, was cleaved by acidic cytosolic extract independent of caspase activation. Fractionation of the acidic cellular extract yielded AEP that is required for SET cleavage. We found that kainate provoked AEP activation and SET cleavage at N175, triggering DNA nicking in wild-type but not AEP-null mice. PIKE-L strongly bound SET and prevented its degradation by AEP, leading to resistance of neuronal cell death. Moreover, AEP also mediated stroke-provoked SET cleavage and cell death in brain. Thus, AEP might be one of the proteinases activated by acidosis triggering neuronal injury during neuroexcitotoxicity or ischemia.