Dongmei Cheng

A proteomics approach to understanding protein ubiquitination.

There is a growing need for techniques that can identify and characterize protein modifications on a large or global scale. We report here a proteomics approach to enrich, recover, and identify ubiquitin conjugates from Saccharomyces cerevisiae lysate. Ubiquitin conjugates from a strain expressing 6xHis-tagged ubiquitin were isolated, proteolyzed with trypsin and analyzed by multidimensional liquid chromatography coupled with tandem mass spectrometry (LC/LC-MS/MS) for amino acid sequence determination. We identified 1,075 proteins from the sample. In addition, we detected 110 precise ubiquitination sites present in 72 ubiquitin-protein conjugates. Finally, ubiquitin itself was found to be modified at seven lysine residues providing evidence for unexpected diversity in polyubiquitin chain topology in vivo. The methodology described here provides a general tool for the large-scale analysis and characterization of protein ubiquitination.

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.

Autophosphorylated CaMKIIα Acts as a Scaffold to Recruit Proteasomes to Dendritic Spines

The molecular mechanisms regulating the ubiquitin proteasome system (UPS) at synapses are poorly understood. We report that CaMKIIalpha-an abundant postsynaptic protein kinase-mediates the activity-dependent recruitment of proteasomes to dendritic spines in hippocampal neurons. CaMKIIalpha is biochemically associated with proteasomes in the brain. CaMKIIalpha translocation to synapses is required for activity-induced proteasome accumulation in spines, and is sufficient to redistribute proteasomes to postsynaptic sites. CaMKIIalpha autophosphorylation enhances its binding to proteasomes and promotes proteasome recruitment to spines. In addition to this structural role, CaMKIIalpha stimulates proteasome activity by phosphorylating proteasome subunit Rpt6 on Serine 120. However, CaMKIIalpha translocation, but not its kinase activity, is required for activity-dependent degradation of polyubiquitinated proteins in spines. Our findings reveal a scaffolding role of postsynaptic CaMKIIalpha in activity-dependent proteasome redistribution, which is commensurate with the great abundance of CaMKIIalpha in synapses.

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.

A novel transferrin/TfR2-mediated mitochondrial iron transport system is disrupted in Parkinson's disease

More than 80 years after iron accumulation was initially described in the substantia nigra (SN) of Parkinsons disease (PD) patients, the mechanisms responsible for this phenomenon are still unknown. Similarly, how iron is delivered to its major recipients in the cell - mitochondria and the respiratory complexes - has yet to be elucidated. Here, we report a novel transferrin/transferrin receptor 2 (Tf/TfR2)-mediated iron transport pathway in mitochondria of SN dopamine neurons. We found that TfR2 has a previously uncharacterized mitochondrial targeting sequence that is sufficient to import the protein into these organelles. Importantly, the Tf/TfR2 pathway can deliver Tf bound iron to mitochondria and to the respiratory complex I as well. The pathway is redox-sensitive and oxidation of Tf thiols to disulfides induces release from Tf of highly reactive ferrous iron, which contributes to free radical production. In the rotenone model of PD, Tf accumulates in dopamine neurons, with much of it accumulating in the mitochondria. This is associated with iron deposition in SN, similar to what occurs in PD. In the human SN, TfR2 is also found in mitochondria of dopamine neurons, and in PD there is a dramatic increase of oxidized Tf in SN. Thus, we have discovered a novel mitochondrial iron transport system that goes awry in PD, and which may provide a new target for therapeutic intervention.

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.

Loss of LR11/SORLA enhances early pathology in a mouse model of amyloidosis: evidence for a proximal role in Alzheimer's disease.

Alzheimers disease (AD) is the most prevalent form of dementia, resulting in progressive neuronal death and debilitating damage to brain loci that mediate memory and higher cognitive function. While pathogenic genetic mutations have been implicated in ∼2% of AD cases, the proximal events that underlie the common, sporadic form of the disease are incompletely understood. Converging lines of evidence from human neuropathology, basic biology, and genetics have implicated loss of the multifunctional receptor LR11 (also known as SORLA and SORL1) in AD pathogenesis. Cell-based studies suggest that LR11 reduces the formation of β-amyloid (Aβ), the molecule believed to be a primary toxic species in AD. Recently, mutant mice deficient in LR11 were shown to upregulate murine Aβ in mouse brain. In the current study, LR11-deficient mice were crossed with transgenic mice expressing autosomal-dominant human AD genes, presenilin-1 (PS1ΔE9) and amyloid precursor protein (APPswe). Here, we show that LR11 deficiency in this AD mouse model significantly increases Aβ levels and exacerbates early amyloid pathology in brain, causing a forward shift in disease onset that is LR11 gene dose-dependent. Loss of LR11 increases the processing of the APP holo-molecule into α-, β-, and γ-secretase derived metabolites. We propose that LR11 regulates APP processing and Aβ accumulation in vivo and is of proximal importance to the cascade of pathological amyloidosis. The results of the current study support the hypothesis that control of LR11 expression may exert critical effects on Alzheimers disease susceptibility in humans.

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.

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.