A Novel Mechanism for NF-κB-activation via IκB-aggregation: Implications for Hepatic Mallory-Denk-Body Induced Inflammation.

Mallory-Denk-bodies (MDBs) are hepatic protein aggregates associated with inflammation both clinically and in MDB-inducing models. Similar protein aggregation in neurodegenerative diseases also triggers inflammation and NF-κB activation. However, the precise mechanism that links protein aggregation to NF-κB-activation and inflammatory response remains unclear. Herein we find that treating primary hepatocytes with MDB-inducing agents (N-methylprotoporphyrin (NMPP), protoporphyrin IX (PPIX), or Zinc-protoporphyrin IX (ZnPP)) elicited an IκBα-loss with consequent NF-κB activation. Four known mechanisms of IκBα-loss i.e. the canonical ubiquitin-dependent proteasomal degradation (UPD), autophagic-lysosomal degradation, calpain degradation and translational inhibition, were all probed and excluded. Immunofluorescence analyses of ZnPP-treated cells coupled with 8 M urea/CHAPS-extraction revealed that this IκBα-loss was due to its sequestration along with IκBß into insoluble aggregates, thereby releasing NF-κB. Through affinity pulldown, proximity biotinylation by antibody recognition, and other proteomic analyses, we verified that NF-κB subunit p65, which stably interacts with IκBα under normal conditions, no longer binds to it upon ZnPP-treatment. Additionally, we identified 10 proteins that interact with IκBα under baseline conditions, aggregate upon ZnPP-treatment, and maintain the interaction with IκBα after ZnPP-treatment, either by cosequestering into insoluble aggregates or through a different mechanism. Of these 10 proteins, the nucleoporins Nup153 and Nup358/RanBP2 were identified through RNA-interference, as mediators of IκBα-nuclear import. The concurrent aggregation of IκBα, NUP153, and RanBP2 upon ZnPP-treatment, synergistically precluded the nuclear entry of IκBα and its consequent binding and termination of NF-κB activation. This novel mechanism may account for the protein aggregate-induced inflammation observed in liver diseases, thus identifying novel targets for therapeutic intervention. Because of inherent commonalities this MDB cell model is a bona fide protoporphyric model, making these findings equally relevant to the liver inflammation associated with clinical protoporphyria.

Substrates of IAP ubiquitin ligases identified with a designed orthogonal E3 ligase, the NEDDylator.

Inhibitors of Apoptosis Protein (IAPs) are guardian ubiquitin ligases that keep classic proapoptotic proteins in check. Systematic identification of additional IAP substrates is challenged by the heterogeneity and sheer number of ubiquitinated proteins (>5,000). Here we report a powerful catalytic tagging tool, the NEDDylator, which fuses a NEDD8 E2-conjugating enzyme, Ubc12, to the ubiquitin ligase, XIAP or cIAP1. This permits transfer of the rare ubiquitin homolog NEDD8 to the ubiquitin E3 substrates, allowing them to be efficiently purified for LC-MS/MS identification. We have identified >50 potential IAP substrates of both cytosolic and mitochondrial origin that bear hallmark N-terminal IAP binding motifs. These substrates include the recently discovered protein phosphatase PGAM5, which we show is proteolytically processed, accumulates in cytosol during apoptosis, and sensitizes cells to death. These studies reveal mechanisms and antagonistic partners for specific IAPs, and provide a powerful technology for labeling binding partners in transient protein-protein complexes.

Prediction of LC-MS/MS Properties of Peptides from Sequence by Deep Learning.

Deep learning models for prediction of three key LC-MS/MS properties from peptide sequences were developed. The LC-MS/MS properties or behaviors are indexed retention times (iRT), MS1 or survey scan charge state distributions, and sequence ion intensities of HCD spectra. A common core deep supervised learning architecture, bidirectional long-short term memory (LSTM) recurrent neural networks was used to construct the three prediction models. Two featurization schemes were proposed and demonstrated to allow for efficient encoding of modifications. The iRT and charge state distribution models were trained with on order of 105 data points each. An HCD sequence ion prediction model was trained with 2 × 106 experimental spectra. The iRT prediction model and HCD sequence ion prediction model provide improved accuracies over the start-of-the-art models available in literature. The MS1 charge state distribution prediction model offers excellent performance. The prediction models can be used to enhance peptide identification and quantification in data-dependent acquisition and data-independent acquisition (DIA) experiments as well as to assist MRM (multiple reaction monitoring) and PRM (parallel reaction monitoring) experiment design.

Data Dependent-Independent Acquisition (DDIA) Proteomics.

Data dependent acquisition (DDA) and data independent acquisition (DIA) are traditionally separate experimental paradigms in bottom-up proteomics. In this work, we developed a strategy combining the two experimental methods into a single LC-MS/MS run. We call the novel strategy data dependent-independent acquisition proteomics, or DDIA for short. Peptides identified from DDA scans by a conventional and robust DDA identification workflow provide useful information for interrogation of DIA scans. Deep learning based LC-MS/MS property prediction tools, developed previously, can be used repeatedly to produce spectral libraries facilitating DIA scan extraction. A complete DDIA data processing pipeline, including the modules for iRT vs RT calibration curve generation, DIA extraction classifier training, and false discovery rate control, has been developed. Compared to another spectral library-free method, DIA-Umpire, the DDIA method produced a similar number of peptide identifications, but nearly twice as many protein group identifications. The primary advantage of the DDIA method is that it requires minimal information for processing its data.

Repeat-Preserving Decoy Database for False Discovery Rate Estimation in Peptide Identification.

The sequence database searching method is widely used in proteomics for peptide identification. To control the false discovery rate (FDR) of the searching results, the target-decoy method generates and searches a decoy database together with the target database. A known problem is that the target protein sequence database may contain numerous repeated peptides. The structures of these repeats are not preserved by most existing decoy generation algorithms. Previous studies suggest that such discrepancy between the target and decoy databases may lead to an inaccurate FDR estimation. Based on the de Bruijn graph model, we propose a new repeat-preserving algorithm to generate decoy databases. We prove that this algorithm preserves the structures of the repeats in the target database to a great extent. The de Bruijn method has been compared with a few other commonly used methods and demonstrated superior FDR estimation accuracy and an improved number of peptide identification.

Evidence for sortilin modulating regional accumulation of human tau prions in transgenic mice.

Misfolding of tau proteins into prions and their propagation along neural circuits are thought to result in neurodegeneration causing Alzheimer's disease, progressive supranuclear palsy, chronic traumatic encephalopathy, and other tauopathies. Little is known about the molecular processes mediating tau prion replication and spreading in different brain regions. Using transgenic (Tg) mice with a neuronal promoter driving expression of human mutant (P301S) tau, we found that tau prion formation and histopathologic deposition is largely restricted to the hindbrain. Unexpectedly, tau mRNA and protein levels did not differ between the forebrain and hindbrain, suggesting that other factors modulating the conversion of tau into a prion exist and are region specific. Using a cell-based prion propagation assay, we discovered that tau prion replication is suppressed by forebrain-derived inhibitors, one of which is sortilin, a lysosomal sorting receptor. We also show that sortilin expression is higher in the forebrain than the hindbrain across the life span of the Tg mice, suggesting that sortilin, at least in part, inhibits forebrain tau prion replication in vivo. Our findings provide evidence for selective vulnerability in mice resulting in highly regulated levels of tau prion propagation, thus affording a model for identification of additional molecules that could mitigate the levels of tau prions in human tauopathies.

The CDG1 kinase mediates brassinosteroid signal transduction from BRI1 receptor kinase to BSU1 phosphatase and GSK3-like kinase BIN2.

The brassinosteroid (BR) signaling pathway includes two receptor-like kinases (BRI1 and BAK1), a plasma membrane-associated kinase (BSK1), two phosphatases (BSU1 and PP2A), a GSK3-like kinase (BIN2), and two homologous transcription factors (BZR1 and BES1/BZR2). But the mechanisms of signal relay are not fully understood. Here, we show that a receptor-like cytoplasmic kinase named CDG1 mediates signal transduction from BRI1 to BSU1. Transgenic experiments confirm that CDG1 and its homolog CDL1 positively regulate BR signaling and plant growth. Mass spectrometry analysis identified BRI1 phosphorylation sites in CDG1 and CDG1 phosphorylation sites in BSU1. Mutations of these phosphorylation sites compromised the BR signaling functions. The results demonstrate that BRI1 phosphorylates S234 to activate CDG1 kinase, and CDG1 in turn phosphorylates S764 to activate BSU1, which inactivates BIN2 by dephosphorylating Y200 of BIN2. This study thus demonstrates a complete phosphorylation/dephosphorylation cascade linking a steroid-activated receptor kinase to a GSK3-like kinase in plants.

Characterization of Dynamic UbR-Proteasome Subcomplexes by In vivo Cross-linking (X) Assisted Bimolecular Tandem Affinity Purification (XBAP) and Label-free Quantitation.

Proteasomes are protein degradation machines that exist in cells as heterogeneous and dynamic populations. A group of proteins function as ubiquitin receptors (UbRs) that can recognize and deliver ubiquitinated substrates to proteasome complexes for degradation. Defining composition of proteasome complexes engaged with UbRs is critical to understand proteasome function. However, because of the dynamic nature of UbR interactions with the proteasome, it remains technically challenging to capture and isolate UbR-proteasome subcomplexes using conventional purification strategies. As a result, distinguishing the molecular differences among these subcomplexes remains elusive. We have developed a novel affinity purification strategy, in vivo cross-linking (X) assisted bimolecular tandem affinity purification strategy (XBAP), to effectively isolate dynamic UbR-proteasome subcomplexes and define their subunit compositions using label-free quantitative mass spectrometry. In this work, we have analyzed seven distinctive UbR-proteasome complexes and found that all of them contain the same type of the 26S holocomplex. However, selected UbRs interact with a group of proteasome interacting proteins that may link each UbR to specific cellular pathways. The compositional similarities and differences among the seven UbR-proteasome subcomplexes have provided new insights on functional entities of proteasomal degradation machineries. The strategy described here represents a general and useful proteomic tool for isolating and studying dynamic and heterogeneous protein subcomplexes in cells that have not been fully characterized.

Proteomic Analysis of Mouse Cortex Postsynaptic Density following Neonatal Brain Hypoxia-Ischemia.

Proteomics of the synapses and postsynaptic densities (PSDs) have provided a deep understanding of protein composition and signal networks in the adult brain, which underlie neuronal plasticity and neurodegenerative or psychiatric disorders. However, there is a paucity of knowledge about the architecture and organization of PSDs in the immature brain, and how it is modified by brain injury in an early developing stage. Mass spectrometry (MS)-based proteomic analysis was performed on PSDs prepared from cortices of postnatal day 9 naïve mice or pups which had suffered hypoxic-ischemic (HI) brain injury. 512 proteins of different functional groups were identified from PSDs collected 1 h after HI injury, among which 60 have not been reported previously. Seven newly identified proteins involved in neural development were highlighted. HI injury increased the yield of PSDs at early time points upon reperfusion, and multiple proteins were recruited into PSDs following the insult. Quantitative analysis was performed using spectral counting, and proteins whose relative expression was more than 50% up- or downregulated compared to the sham animals 1 h after HI insult were reported. Validation with Western blotting demonstrated changes in expression and phosphorylation of the N-methyl-D-aspartate receptor, activation of a series of postsynaptic protein kinases and dysregulation of scaffold and adaptor proteins in response to neonatal HI insult. This work, along with other recent studies of synaptic protein profiling in the immature brain, builds a foundation for future investigation on the molecular mechanisms underlying developing plasticity. Furthermore, it provides insights into the biochemical changes of PSDs following early brain hypoxia-ischemia, which is helpful for understanding not only the injury mechanisms, but also the process of repair or replenishment of neuronal circuits during recovery from brain damage.

Optimization of Aryl Amides that Extend Survival in Prion-Infected Mice.

Developing therapeutics for neurodegenerative diseases (NDs) prevalent in the aging population remains a daunting challenge. With the growing understanding that many NDs progress by conformational self-templating of specific proteins, the prototypical prion diseases offer a platform for ND drug discovery. We evaluated high-throughput screening hits with the aryl amide scaffold and explored the structure-activity relationships around three series differing in their N-aryl core: benzoxazole, benzothiazole, and cyano. Potent anti-prion compounds were advanced to pharmacokinetic studies, and the resulting brain-penetrant leads from each series, together with a related N-aryl piperazine lead, were escalated to long-term dosing and efficacy studies. Compounds from each of the four series doubled the survival of mice infected with a mouse-passaged prion strain. Treatment with aryl amides altered prion strain properties, as evidenced by the distinct patterns of neuropathological deposition of prion protein and associated astrocytic gliosis in the brain; however, none of the aryl amide compounds resulted in drug-resistant prion strains, in contrast to previous studies on compounds with the 2-aminothiazole (2-AMT) scaffold. As seen with 2-AMTs and other effective anti-prion compounds reported to date, the novel aryl amides reported here were ineffective in prolonging the survival of transgenic mice infected with human prions. Most encouraging is our discovery that aryl amides show that the development of drug resistance is not an inevitable consequence of efficacious anti-prion therapeutics.

Modulation of Creutzfeldt-Jakob disease prion propagation by the A224V mutation.

OBJECTIVE: Mutations in the gene encoding the prion protein (PrP) are responsible for approximately 10 to 15% of cases of prion disease in humans, including Creutzfeldt-Jakob disease (CJD). Here, we report on the discovery of a previously unreported C-terminal PrP mutation (A224V) in a CJD patient exhibiting a disease similar to the rare VV1 subtype of sporadic (s) CJD and investigate the role of this mutation in prion replication and transmission. METHODS: We generated transgenic (Tg) mice expressing human PrP with the V129 polymorphism and A224V mutation, denoted Tg(HuPrP,V129,A224V) mice, and inoculated them with different subtypes of sCJD prions. RESULTS: Transmission of sCJD VV2 or MV2 prions was accelerated in Tg(HuPrP,V129,A224V) mice, compared to Tg(HuPrP,V129) mice, with incubation periods of ∼110 and ∼210 days, respectively. In contrast, sCJD MM1 prions resulted in longer incubation periods in Tg(HuPrP,V129,A224V) mice, compared to Tg(HuPrP,V129) mice (∼320 vs. ∼210 days). Prion strain fidelity was maintained in Tg(HuPrP,V129,A224V) mice inoculated with sCJD VV2 or MM1 prions, despite the altered replication kinetics. INTERPRETATION: Our results suggest that A224V is a risk factor for prion disease and modulates the transmission behavior of CJD prions in a strain-specific manner, arguing that residues near the C-terminus of PrP are important for controlling the kinetics of prion replication.

Multisite light-induced phosphorylation of the transcription factor PIF3 is necessary for both its rapid degradation and concomitant negative feedback modulation of photoreceptor phyB levels in Arabidopsis.

Plants constantly monitor informational light signals using sensory photoreceptors, which include the phytochrome (phy) family (phyA to phyE), and adjust their growth and development accordingly. Following light-induced nuclear translocation, photoactivated phy molecules bind to and induce rapid phosphorylation and degradation of phy-interacting basic Helix Loop Helix (bHLH) transcription factors (PIFs), such as PIF3, thereby regulating the expression of target genes. However, the mechanisms underlying the signal-relay process are still not fully understood. Here, using mass spectrometry, we identify multiple, in vivo, light-induced Ser/Thr phosphorylation sites in PIF3. Using transgenic expression of site-directed mutants of PIF3, we provide evidence that a set of these phosphorylation events acts collectively to trigger rapid degradation of the PIF3 protein in response to initial exposure of dark-grown seedlings to light. In addition, we show that phyB-induced PIF3 phosphorylation is also required for the known negative feedback modulation of phyB levels in prolonged light, potentially through codegradation of phyB and PIF3. This mutually regulatory intermolecular transaction thus provides a mechanism with the dual capacity to promote early, graded, or threshold regulation of the primary, PIF3-controlled transcriptional network in response to initial light exposure, and later, to attenuate global sensitivity to the light signal through reductions in photoreceptor levels upon prolonged exposure.

A new in vivo cross-linking mass spectrometry platform to define protein-protein interactions in living cells.

Protein-protein interactions (PPIs) are fundamental to the structure and function of protein complexes. Resolving the physical contacts between proteins as they occur in cells is critical to uncovering the molecular details underlying various cellular activities. To advance the study of PPIs in living cells, we have developed a new in vivo cross-linking mass spectrometry platform that couples a novel membrane-permeable, enrichable, and MS-cleavable cross-linker with multistage tandem mass spectrometry. This strategy permits the effective capture, enrichment, and identification of in vivo cross-linked products from mammalian cells and thus enables the determination of protein interaction interfaces. The utility of the developed method has been demonstrated by profiling PPIs in mammalian cells at the proteome scale and the targeted protein complex level. Our work represents a general approach for studying in vivo PPIs and provides a solid foundation for future studies toward the complete mapping of PPI networks in living systems.

Syntaxin13 expression is regulated by mammalian target of rapamycin (mTOR) in injured neurons to promote axon regeneration.

Injured peripheral neurons successfully activate intrinsic signaling pathways to enable axon regeneration. We have previously shown that dorsal root ganglia (DRG) neurons activate the mammalian target of rapamycin (mTOR) pathway following injury and that this activity enhances their axon growth capacity. mTOR plays a critical role in protein synthesis, but the mTOR-dependent proteins enhancing the regenerative capacity of DRG neurons remain unknown. To identify proteins whose expression is regulated by injury in an mTOR-dependent manner, we analyzed the protein composition of DRGs from mice in which we genetically activated mTOR and from mice with or without a prior nerve injury. Quantitative label-free mass spectrometry analyses revealed that the injury effects were correlated with mTOR activation. We identified a member of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) family of proteins, syntaxin13, whose expression was increased by injury in an mTOR-dependent manner. Increased syntaxin13 levels in injured nerves resulted from local protein synthesis and not axonal transport. Finally, knockdown of syntaxin13 in cultured DRG neurons prevented axon growth and regeneration. Together, these data suggest that syntaxin13 translation is regulated by mTOR in injured neurons to promote axon regeneration.

Deconvolution method for specific and nonspecific binding of ligand to multiprotein complex by native mass spectrometry.

In native mass spectrometry, it has been difficult to discriminate between specific bindings of a ligand to a multiprotein complex target from the nonspecific interactions. Here, we present a deconvolution model that consists of two levels of data reduction. At the first level, the apparent association binding constants are extracted from the measured intensities of the target/ligand complexes by varying ligand concentration. At the second level, two functional forms representing the specific and nonspecific binding events are fit to the apparent binding constants obtained from the first level of modeling. Using this approach, we found that a power-law distribution described nonspecific binding of α-amanitin to yeast RNA polymerase II. Moreover, treating the concentration of the multiprotein complex as a fitting parameter reduced the impact of inaccuracies in this experimental measurement on the apparent association constants. This model improves upon current methods for separating specific and nonspecific binding to large, multiprotein complexes in native mass spectrometry, by modeling nonspecific binding with a power-law function.

Different 2-Aminothiazole Therapeutics Produce Distinct Patterns of Scrapie Prion Neuropathology in Mouse Brains.

Because no drug exists that halts or even slows any neurodegenerative disease, developing effective therapeutics for any prion disorder is urgent. We recently reported two compounds (IND24 and IND81) with the 2-aminothiazole (2-AMT) chemical scaffold that almost doubled the incubation times in scrapie prion-infected, wild-type (wt) FVB mice when given in a liquid diet. Remarkably, oral prophylactic treatment with IND24 beginning 14 days prior to intracerebral prion inoculation extended survival from ∼120 days to over 450 days. In addition to IND24, we evaluated the pharmacokinetics and efficacy of five additional 2-AMTs; one was not followed further because its brain penetration was poor. Of the remaining four new 2-AMTs, IND114338 doubled and IND125 tripled the incubation times of RML-inoculated wt and Tg4053 mice overexpressing wt mouse prion protein (PrP), respectively. Neuropathological examination of the brains from untreated controls showed a widespread deposition of self-propagating, ß-sheet-rich "scrapie" isoform (PrP(Sc)) prions accompanied by a profound astrocytic gliosis. In contrast, mice treated with 2-AMTs had lower levels of PrP(Sc) and associated astrocytic gliosis, with each compound resulting in a distinct pattern of deposition. Notably, IND125 prevented both PrP(Sc) accumulation and astrocytic gliosis in the cerebrum. Progressive central nervous system dysfunction in the IND125-treated mice was presumably due to the PrP(Sc) that accumulated in their brainstems. Disappointingly, none of the four new 2-AMTs prolonged the lives of mice expressing a chimeric human/mouse PrP transgene inoculated with Creutzfeldt-Jakob disease prions.

Hepatic cytochrome P450 ubiquitination: conformational phosphodegrons for E2/E3 recognition?

Hepatic endoplasmic reticulum (ER) integral cytochromes P450 (P450s) are monooxygenases engaged in the biotransformation and elimination of endo- as well as xenobiotics. Of the human liver P450s, CYP3A4 is the major and most dominant catalyst responsible for the biotransformation of over 50% of clinically prescribed drugs. CYP2E1 metabolizes smaller molecular weight compounds (EtOH), carcinogens, environmental toxins, and endobiotics, and is justly implicated in various toxigenic/pathogenic mechanisms of human disease. Both P450s are notorious for their potential to generate pathogenic reactive oxygen species (ROS) during futile oxidative cycling and/or oxidative uncoupling. Such ROS not only oxidatively damage the P450 catalytic cage, but on their escape into the cytosol, also the P450 outer surface and any surrounding cell organelles. Given their ER-monotopic topology coupled with this high potential to acquire oxidative lesions in their cytosolic (C) domain, not surprisingly these P450 proteins exhibit shorter lifespans and are excellent prototype substrates of ER-associated degradation ("ERAD-C") pathway. Indeed, we have shown that both CYP3A4 and CYP2E1 incur ERAD-C, during which they are first phosphorylated by protein kinases A and C, which greatly enhance/accelerate their ubiquitination by UBC7/gp78 and UbcH5a/CHIP/Hsp70/Hsp40 E2/E3 ubiquitin ligase complexes. Such P450 phosphorylation occurs on Ser/Thr residues within linear sequences as well as spatially clustered acidic (Asp/Glu) residues. We propose that such S/T phosphorylation within these clusters creates negatively charged patches or conformational phosphodegrons for interaction with positively charged E2/E3 domains. Such P450 S/T phosphorylation we posit serves as a molecular switch to turn on its ubiquitination and ERAD-C.

Multisite phosphorylation of human liver cytochrome P450 3A4 enhances Its gp78- and CHIP-mediated ubiquitination: a pivotal role of its Ser-478 residue in the gp78-catalyzed reaction.

CYP3A4, an integral endoplasmic reticulum (ER)-anchored protein, is the major human liver cytochrome P450 enzyme responsible for the disposition of over 50% of clinically relevant drugs. Alterations of its protein turnover can influence drug metabolism, drug-drug interactions, and the bioavailability of chemotherapeutic drugs. Such CYP3A4 turnover occurs via a classical ER-associated degradation (ERAD) process involving ubiquitination by both UBC7/gp78 and UbcH5a/CHIP E2-E3 complexes for 26 S proteasomal targeting. These E3 ligases act sequentially and cooperatively in CYP3A4 ERAD because RNA interference knockdown of each in cultured hepatocytes results in the stabilization of a functionally active enzyme. We have documented that UBC7/gp78-mediated CYP3A4 ubiquitination requires protein phosphorylation by protein kinase (PK) A and PKC and identified three residues (Ser-478, Thr-264, and Ser-420) whose phosphorylation is required for intracellular CYP3A4 ERAD. We document herein that of these, Ser-478 plays a pivotal role in UBC7/gp78-mediated CYP3A4 ubiquitination, which is accelerated and enhanced on its mutation to the phosphomimetic Asp residue but attenuated on its Ala mutation. Intriguingly, CYP3A5, a polymorphically expressed human liver CYP3A4 isoform (containing Asp-478) is ubiquitinated but not degraded to a greater extent than CYP3A4 in HepG2 cells. This suggests that although Ser-478 phosphorylation is essential for UBC7/gp78-mediated CYP3A4 ubiquitination, it is not sufficient for its ERAD. Additionally, we now report that CYP3A4 protein phosphorylation by PKA and/or PKC at sites other than Ser-478, Thr-264, and Ser-420 also enhances UbcH5a/CHIP-mediated ubiquitination. Through proteomic analyses, we identify (i) 12 additional phosphorylation sites that may be involved in CHIP-CYP3A4 interactions and (ii) 8 previously unidentified CYP3A4 ubiquitination sites within spatially associated clusters of Asp/Glu and phosphorylatable Ser/Thr residues that may serve to engage each E2-E3 complex. Collectively, our findings underscore the interplay between protein phosphorylation and ubiquitination in ERAD and, to our knowledge, provide the very first example of gp78 substrate recognition via protein phosphorylation.

Mapping the structural topology of the yeast 19S proteasomal regulatory particle using chemical cross-linking and probabilistic modeling.

Structural characterization of proteasome complexes is an essential step toward understanding the ubiquitin-proteasome system. Currently, high resolution structures are not available for the 26S proteasome holocomplex as well as its subcomplex, the 19S regulatory particle (RP). Here we have employed a novel integrated strategy combining chemical cross-linking with multistage tandem mass spectrometry to define the proximity of subunits within the yeast 19S RP to elucidate its topology. This has resulted in the identification of 174 cross-linked peptides of the yeast 19S RP, representing 43 unique lysine-lysine linkages within 24 nonredundant pair-wise subunit interactions. To map the spatial organization of the 19S RP, we have developed and utilized a rigorous probabilistic framework to derive maximum likelihood (ML) topologies based on cross-linked peptides determined from our analysis. Probabilistic modeling of the yeast 19S AAA-ATPase ring (i.e., Rpt1-6) has produced an ML topology that is in excellent agreement with known topologies of its orthologs. In addition, similar analysis was carried out on the 19S lid subcomplex, whose predicted ML topology corroborates recently reported electron microscopy studies. Together, we have demonstrated the effectiveness and potential of probabilistic modeling for unraveling topologies of protein complexes using cross-linking data. This report describes the first study of the 19S RP topology using a new integrated strategy combining chemical cross-linking, mass spectrometry, and probabilistic modeling. Our results have provided a solid foundation to advance our understanding of the 19S RP architecture at peptide level resolution. Furthermore, our methodology developed here is a valuable proteomic tool that can be generalized for elucidating the structures of protein complexes.