Duc M. Duong

Quantitative Proteomics Reveals the Function of Unconventional Ubiquitin Chains in Proteasomal Degradation

All seven lysine residues in ubiquitin contribute to the synthesis of polyubiquitin chains on protein substrates. Whereas K48-linked chains are well established as mediators of proteasomal degradation, and K63-linked chains act in nonproteolytic events, the roles of unconventional polyubiquitin chains linked through K6, K11, K27, K29, or K33 are not well understood. Here, we report that the unconventional linkages are abundant in vivo and that all non-K63 linkages may target proteins for degradation. Ubiquitin with K48 as the single lysine cannot support yeast viability, and different linkages have partially redundant functions. By profiling both the entire yeast proteome and ubiquitinated proteins in wild-type and ubiquitin K11R mutant strains using mass spectrometry, we identified K11 linkage-specific substrates, including Ubc6, a ubiquitin-conjugating enzyme involved in endoplasmic reticulum-associated degradation (ERAD). Ubc6 primarily synthesizes K11-linked chains, and K11 linkages function in the ERAD pathway. Thus, unconventional polyubiquitin chains are critical for ubiquitin-proteasome system function.

Relative and Absolute Quantification of Postsynaptic Density Proteome Isolated from Rat Forebrain and Cerebellum

The postsynaptic density (PSD) of central excitatory synapses is essential for postsynaptic signaling, and its components are heterogeneous among different neuronal subtypes and brain structures. Here we report large scale relative and absolute quantification of proteins in PSDs purified from adult rat forebrain and cerebellum. PSD protein profiles were determined using the cleavable ICAT strategy and LC-MS/MS. A total of 296 proteins were identified and quantified with 43 proteins exhibiting statistically significant abundance change between forebrain and cerebellum, indicating marked molecular heterogeneity of PSDs between different brain regions. Moreover we utilized absolute quantification strategy, in which synthetic isotope-labeled peptides were used as internal standards, to measure the molar abundance of 32 key PSD proteins in forebrain and cerebellum. These data confirm the abundance of calcium/calmodulin-dependent protein kinase II and PSD-95 and reveal unexpected stoichiometric ratios between glutamate receptors, scaffold proteins, and signaling molecules in the PSD. Our data also demonstrate that the absolute quantification method is well suited for targeted quantitative proteomic analysis. Overall this study delineates a crucial molecular difference between forebrain and cerebellar PSDs and provides a quantitative framework for measuring the molecular stoichiometry of the PSD.

Polyubiquitin Linkage Profiles in Three Models of Proteolytic Stress Suggest the Etiology of Alzheimer Disease

Polyubiquitin chains on substrates are assembled through any of seven lysine residues or the N terminus of ubiquitin (Ub), generating diverse linkages in the chain structure. PolyUb linkages regulate the fate of modified substrates, but their abundance and function in mammalian cells are not well studied. We present a mass spectrometry-based method to measure polyUb linkages directly from total lysate of mammalian cells. In HEK293 cells, the level of polyUb linkages was found to be 52% (Lys48), 38% (Lys63), 8% (Lys29), 2% (Lys11), and 0.5% or less for linear, Lys6, Lys27, and Lys33 linkages. Tissue specificity of these linkages was examined in mice fully labeled by heavy stable isotopes (i.e. SILAC mice). Moreover, we profiled the Ub linkages in brain tissues from patients of Alzheimer disease with or without concurrent Lewy body disease as well as three cellular models of proteolytic stress: proteasome deficiency, lysosome deficiency, and heat shock. The data support that polyUb chains linked through Lys6, Lys11, Lys27, Lys29, and Lys48 mediate proteasomal degradation, whereas Lys63 chains are preferentially involved in the lysosomal pathway. Mixed linkages, including Lys48, may also contribute to lysosomal targeting, as both Lys63 and Lys48 linkages are colocalized in LC3-labeled autophagosomes. Interestingly, heat shock treatment augments Lys11, Lys48, and Lys63 but not Lys29 linkages, and this unique pattern is similar to that in the profiled neurodegenerative cases. We conclude that different polyUb linkages play distinct roles under the three proteolytic stress conditions, and protein folding capacity in the heat shock responsive pathway might be more affected in Alzheimer disease.

Cleavage of tau by asparagine endopeptidase mediates the neurofibrillary pathology in Alzheimer's disease

Neurofibrillary tangles (NFTs), composed of truncated and hyperphosphorylated tau, are a common feature of numerous aging-related neurodegenerative diseases, including Alzheimers disease (AD). However, the molecular mechanisms mediating tau truncation and aggregation during aging remain elusive. Here we show that asparagine endopeptidase (AEP), a lysosomal cysteine proteinase, is activated during aging and proteolytically degrades tau, abolishes its microtubule assembly function, induces tau aggregation and triggers neurodegeneration. AEP is upregulated and active during aging and is activated in human AD brain and tau P301S–transgenic mice with synaptic pathology and behavioral impairments, leading to tau truncation in NFTs. Tau P301S–transgenic mice with deletion of the gene encoding AEP show substantially reduced tau hyperphosphorylation, less synapse loss and rescue of impaired hippocampal synaptic function and cognitive deficits. Mice infected with adeno-associated virus encoding an uncleavable tau mutant showed attenuated pathological and behavioral defects compared to mice injected with adeno-associated virus encoding tau P301S. Together, these observations indicate that AEP acts as a crucial mediator of tau-related clinical and neuropathological changes. Inhibition of AEP may be therapeutically useful for treating tau-mediated neurodegenerative diseases.

U1 small nuclear ribonucleoprotein complex and RNA splicing alterations in Alzheimer’s disease

Deposition of insoluble protein aggregates is a hallmark of neurodegenerative diseases. The universal presence of β-amyloid and tau in Alzheimer’s disease (AD) has facilitated advancement of the amyloid cascade and tau hypotheses that have dominated AD pathogenesis research and therapeutic development. However, the underlying etiology of the disease remains to be fully elucidated. Here we report a comprehensive study of the human brain-insoluble proteome in AD by mass spectrometry. We identify 4,216 proteins, among which 36 proteins accumulate in the disease, including U1-70K and other U1 small nuclear ribonucleoprotein (U1 snRNP) spliceosome components. Similar accumulations in mild cognitive impairment cases indicate that spliceosome changes occur in early stages of AD. Multiple U1 snRNP subunits form cytoplasmic tangle-like structures in AD but not in other examined neurodegenerative disorders, including Parkinson disease and frontotemporal lobar degeneration. Comparison of RNA from AD and control brains reveals dysregulated RNA processing with accumulation of unspliced RNA species in AD, including myc box-dependent-interacting protein 1, clusterin, and presenilin-1. U1-70K knockdown or antisense oligonucleotide inhibition of U1 snRNP increases the protein level of amyloid precursor protein. Thus, our results demonstrate unique U1 snRNP pathology and implicate abnormal RNA splicing in AD pathogenesis.

Systematical Optimization of Reverse-Phase Chromatography for Shotgun Proteomics

We report the optimization of a common LC-MS/MS platform to maximize the number of proteins identified from a complex biological sample. The platform uses digested yeast lysate on a 75 microm internal diameter x 12 cm reverse-phase column that is combined with an LTQ-Orbitrap mass spectrometer. We first generated a yeast peptide mix that was quantified by multiple methods including the strategy of stable isotope labeling with amino acids in cell culture (SILAC). The peptide mix was analyzed on a highly reproducible, automated nanoLC-MS/MS system with systematic adjustment of loading amount, flow rate, elution gradient range and length. Interestingly, the column was found to be almost saturated by loading approximately 1 microg of the sample. Whereas the optimal flow rate ( approximately 0.2 microL/min) and elution buffer range (13-32% of acetonitrile) appeared to be independent of the loading amount, the best gradient length varied according to the amount of samples: 160 min for 1 microg of the peptide mix, but 40 min for 10 ng of the same sample. The effect of these parameters on elution peptide peak width is evaluated. After full optimization, 1012 proteins (clustered in 806 groups) with an estimated protein false discovery rate of approximately 3% were identified in 1 microg of yeast lysate in a single 160-min LC-MS/MS run.

Proteomic identification of novel proteins associated with Lewy bodies.

The manifestation of Lewy bodies (LB) in the brain is a hallmark of Parkinsons disease. Here, we present a comprehensive analysis of protein elements in Lewy bodies by comparative mass spectrometry. Cortical LB inclusions were enriched by sucrose gradient centrifugation from postmortem brains, and a negative control sample was prepared from specimen without LB pathology. Whereas approximately 550 proteins were identified in the LB-enriched sample by mass spectrometry, quantitative comparison with the control sample revealed that approximately 40 proteins were co-enriched with alpha-synuclein, the major component in Lewy bodies. As expected, the list of proteins included previously reported constituents, such as those involved in protein folding, membrane trafficking and oxidative stress. More interestingly, we discovered in the LB-enriched sample several kinases (MAPKK1/MEK1, protein kinase C, and doublecortin-like kinase), a novel deubiquitinating enzyme (otubain 1), and numerous ubiquitin ligases (KPC and SCF). The proteomic studies provide enzyme candidates to investigate the regulation of alpha-synuclein and/or other LB proteins, which may contribute to the formation of Lewy bodies and the toxicity of alpha-synuclein in the related neurodegenerative disorders.

Glutamate Dehydrogenase 1 Signals through Antioxidant Glutathione Peroxidase 1 to Regulate Redox Homeostasis and Tumor Growth

How mitochondrial glutaminolysis contributes to redox homeostasis in cancer cells remains unclear. Here we report that the mitochondrial enzyme glutamate dehydrogenase 1 (GDH1) is commonly upregulated in human cancers. GDH1 is important for redox homeostasis in cancer cells by controlling the intracellular levels of its product alpha-ketoglutarate and subsequent metabolite fumarate. Mechanistically, fumarate binds to and activates a reactive oxygen species scavenging enzyme glutathione peroxidase 1. Targeting GDH1 by shRNA or a small molecule inhibitor R162 resulted in imbalanced redox homeostasis, leading to attenuated cancer cell proliferation and tumor growth.

Phosphoproteomic analysis of human brain by calcium phosphate precipitation and mass spectrometry

Alzheimers disease (AD), the most common form of dementia, is manifested in the brain by the aggregation of amyloid plaques and neurofibrillary tangles. The tangles are primarily composed of microtubule-associated protein tau that is aberrantly hyperphosphorylated, suggesting that deregulated phosphorylation may contribute to AD pathogenesis. However, systematic analysis of the phosphoproteome in AD brain tissues has not been reported. We used calcium phosphate precipitation to analyze an AD postmortem brain, followed by liquid chromatography-tandem mass spectrometry. The protein sample was first resolved by one-dimensional polyacrylamide gel electrophoresis and subjected to gel excision and in-gel digestion. Phosphopeptides in the resulting peptide mixtures were enriched in a single step of calcium phosphate precipitation, and then analyzed by the LC-MS/MS approach. After database search, stringent filtering, and manual validation of neutral loss in the MS/MS spectra, a total of 466 phosphorylation sites on 185 proteins including tau were identified. A majority of sites were not described previously. This study demonstrates the feasibility of combining calcium phosphate precipitation with mass spectrometry for phosphoproteome analysis of postmortem human brain tissue.

Neuronal Morphogenesis Is Regulated by the Interplay between Cyclin-Dependent Kinase 5 and the Ubiquitin Ligase Mind Bomb 1

Neuronal communication requires the coordinated assembly of polarized structures including axons, dendrites, and synapses. Here, we report the identification of a ubiquitin ligase mind bomb 1 (Mib1) in the postsynaptic density and the characterization of its role in neuronal morphogenesis. Expression of Mib1 inhibits neurite outgrowth in cell culture and its gene deletion enhances synaptic growth at the neuromuscular junction in Drosophila. The analysis of Mib1 interactome by mass spectrometry revealed that Mib1 primarily interacts with membrane trafficking proteins [e.g., EEA1 (early endosomal antigen 1), Rab11-interacting proteins, and SNAP25 (synaptosomal-associated protein of 25 kDa)-like protein] and cell adhesion components (e.g., catenin, coronin, dystrobrevin, and syndecan), consistent with its previously reported function in protein sorting. More interestingly, Mib1 is associated with deubiquitinating enzymes, BRCC36 and the mammalian ortholog of fat facets, and a number of kinases, such as casein kinase II, MARK (microtubule affinity regulating kinase)/PAR1, and cyclin-dependent kinase 5 (CDK5). Further characterization of the Mib1-CDK5 interaction indicated that the N-terminal domain of Mib1 directly binds to the regulatory subunit p35 of the CDK5 complex. In cell culture, Mib1 induces the relocalization of p35/CDK5 without affecting its degradation. Surprisingly, p35/CDK5 downregulates the protein level of Mib1 by its kinase activity, and completely rescues the Mib1-induced inhibitory effect on neurite morphology. p35/CDK5 also genetically interacts with Mib1 in the fly according to the rough-eye phenotype. The data strongly support that the negative interplay between Mib1 and p35/CDK5 may integrate the activities of multiple pathways during neuronal development.