Post-translational Transport Research Articles

Understanding fungal and plant active urea transport systems: Keys from Aspergillus nidulans and beyond

Urea is present in all ecosystems, as a result of the metabolism of different organisms and also of human activity, being the world's most common form of nitrogen fertilizer. Fungi and plants can use urea as a nitrogen source, taking it up from the environment through specialized active transport proteins. These proteins belong to a subfamily of urea/H+ symporters included in the Solute:Sodium Symporter (SSS) family of transporters. In this review we summarize the current knowledge on this group of transporters, based on our previous studies on Aspergillus nidulans UreA. We delve into its transcriptional and post-translational regulation, structure-function relationships, transport mechanism, and certain aspects of its biogenesis. Recent findings suggest that this urea transporter subfamily is more expanded than originally thought, with representatives found in organisms as diverse as Archaea and mollusks, which raises questions on evolutionary aspects. A. nidulans ureA knockout strains provide a valuable platform for expressing urea transporters from diverse sources, facilitating their characterization and functional analysis. In this context, given the close relationship between plant and fungal active urea transporters, this knowledge could serve to develop strategies to improve the efficiency of applied urea as fertilizer.

The role of chloroplast SRP54 domains and its C-terminal tail region in post- and co-translational protein transport in vivo.

In the chloroplast, the 54 kDa subunit of the signal recognition particle (cpSRP54) is involved in the post-translational transport of the light-harvesting chlorophyll a/b-binding proteins (LHCPs) and the co-translational transport of plastid-encoded subunits of the photosynthetic complexes to the thylakoid membrane. It forms a high-affinity complex with plastid-specific cpSRP43 for post-translational transport, while a ribosome-associated pool coordinates its co-translational function. CpSRP54 constitutes a conserved multidomain protein, comprising a GTPase (NG) and a methionine-rich (M) domain linked by a flexible region. It is further characterized by a plastid-specific C-terminal tail region containing the cpSRP43-binding motif. To characterize the physiological role of the various regions of cpSRP54 in thylakoid membrane protein transport, we generated Arabidopsis cpSRP54 knockout (ffc1-2) lines producing truncated cpSRP54 variants or a GTPase point mutation variant. Phenotypic characterization of the complementation lines demonstrated that the C-terminal tail region of cpSRP54 plays an important role exclusively in post-translational LHCP transport. Furthermore, we show that the GTPase activity of cpSRP54 plays an essential role in the transport pathways for both nuclear as well as plastid-encoded proteins. In addition, our data revealed that plants expressing cpSRP54 without the C-terminal region exhibit a strongly increased accumulation of a photosystem I assembly intermediate.

Multiomics-Based Biocargo Components Analysis in Enterococcus faecium Membrane Vesicles.

Enterococcus spp. have been shown to have gastrointestinal tract protective functions; our recent results suggest that membrane vesicles (MVs) play an important role in the gastric protection of Enterococcus faecium (E. faecium). The specific function is determined by molecular compositions of MVs. To resolve biocargo components in E. faecium MVs (EfmMVs), MVs were isolated from E. faecium culture. Transcriptomics, label-free quantitative proteomics, and untargeted metabolomics were performed to obtain information about the complexity of ribonucleic acids (RNAs), proteins, and metabolites biocargo they carry, respectively. RNA-sequencing identified a total of 2122 transcripts. The top 20 transcripts accounted for 27.63% of total counts, which, including enzymes, participate in glycolysis, ribosomal proteins, DNA-directed RNA polymerases, protein-synthesizing relative enzymes, molecules associated with protein post-translational processing and transport, and peptidoglycan lyases. Label-free quantitative proteomics analysis identified a total of 711 proteins. The top 20 proteins accounted for 48.02% of all identified proteins, which including ribosomal proteins, enzymes participate in glycolysis, DNA-directed RNA polymerases, protein-synthesizing relative enzymes, peptidoglycan lyases, and autolysin. Untargeted metabolomics analysis identified a total of 519 metabolites. The top 20 metabolites accounted for 79.55% of all identified metabolites, which included amino acids, substrates, or products in the metabolism of amino acids, natural organic acids, products in the metabolism of organic acids, ketone compounds, and two other compounds. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses indicated that the identified biocargo components enriched in metabolism, genetic, and environmental information processing. Overall, we hope that the current exploration of multiple "-omics" analyses of this EfmMVs will provide useful information and further groundwork for future studies on E. faecium application.

The transcriptome response of Enterobacter sp. S-33 is modulated by low pH-stress.

Acidic environments naturally occur worldwide and uncontrolled use of agricultural practices may also cause acidification of soils. The development of acidic conditions disturbs the establishment of efficient microbial populations in their natural niches. The survival of Enterobacter species under acidic stress remains poorly understood. This study aimed to investigate the survival of an environmental isolate Enterobacter sp. S-33 under acidic stress and to identify the various genes involved in stress protection at the global gene transcription level. The obtained results provide new targets that will allow understanding the in-depth mechanisms involved in the adaptation of bacteria to environmental pH changes. We used the next-generation sequencing (NGS) method to analyze the expression (up-regulation & down-regulation) of genes under varying pH conditions. A total of 4214 genes were differentially expressed under acidic conditions (pH 5.0), with 294 up-regulated and 167 down-regulated. At pH 6.0, 50 genes were significantly expressed, of which 34 and 16 were identified as up-regulated and down-regulated, respectively. Many of the up-regulated genes were involved in carbohydrate metabolism, amino acid transport & metabolism, and the most down-regulated genes were related to post-translational modification, lipid transport & metabolism, etc. The observed transcriptomic regulation of genes and pathways identified that Enterobacter reduced its post-translational modification, lipid transport & metabolism, and increased carbohydrate metabolism, amino acid metabolism & transport, energy production & conversion to adapt and grow in acidic stress. The present work provides in-depth information on the characterization of genes associated with tolerance or adaptation to acidic stress of Enterobacter bacterium.

Proteomic analysis reveals the mechanism of green regulation in garlic puree induced by purple light stress.

Greening is an undesirable appearance in garlic puree during processing. Our previous study indicated that purple light could induce the greening changes in garlic. In order to investigate the mechanism of green regulation in garlic puree, purple light-induced greening and nongreening garlic puree were used as materials to investigate the differentially expressed proteins (DEPs) by sodium dodecyl-sulfate polyacrylamide gel electrophoresis and data-independent acquisition (DIA) technology. The results showed that a total of 186 DEPs were detected by DIA, with 73 DEPs were up-regulated in greening garlic puree and 113 of them were down-regulated in greening garlic puree. Most DEPs were belonged to 20 functional categories, and mainly participated in post-translational modification and transport of proteins, molecular chaperones (12.93%) and signal transduction mechanisms (10.20%), energy production and transformation (6.80%), carbohydrate transport and metabolism (5.44%) and amino acid transport and metabolism (4.08%), indicating that the biological metabolic pathway, metabolic direction, and metabolic strength efficiency significantly changed in garlic puree after greening. Besides, the physiological and biochemical experiments showed that purple light significantly induced the γ-glutathione transpeptidase activity and prompted the conversion of thiosulfinate into garlic green pigment. This study explained the general molecular mechanism of greening changes of garlic puree in response to purple light. Practical Application Greening is an undesirable appearance in garlic puree during processing, which deteriorate the qualities of garlic. This study provides a comprehensive understanding of green regulation in garlic puree based on proteomics analysis.

Role of the SEC62 gene in dermato-oncology - impact on tumor cell biology, prognostication, and personalized therapy management.

The SEC62 gene encodes for a transmembrane protein of the endoplasmic reticulum (ER). Sec62 protein is involved in the post-translational transport of secretory and membrane-bound proteins in eukaryotic cells, regulates intracellular calcium homeostasis through direct interaction with the Sec61 channel and makes a decisive contribution to the cellular compensation of ER stress in the context of recovER-phagy. A significantly increased expression of the SEC62 gene has already been demonstrated in various tumor entities. First approaches of a targeted therapy have been tested for various tumor entities in vitro and in vivo with promising results that motivate further preclinical and clinical studies. Nevertheless, many questions remain unanswered, in particular with regard to the molecular mechanisms underlying the observed clinical effects, and require further investigation in future studies. The protein also plays a relevant role in dermato-oncology. The overexpression of SEC62 in atypical fibroxanthomas and malignant melanomas has already been demonstrated and a correlation of SEC62 expression with various clinical and pathological features has been observed. Future studies, especially in vivo and clinical, will show whether Sec62 can be established as a prognostic marker in dermato-oncology and whether it can serve as a starting point for targeted therapy.

Exploring the Potential of Chemical Inhibitors for Targeting Post-translational Glycosylation of Coronavirus (SARS-CoV-2).

The Spike (S) protein of SARS-CoV-2 expressed on the viral cell surface is of particular importance as it facilitates viral entry into the host cells. The S protein is heavily glycosylated with 22 N-glycosylation sites and a few N-glycosylation sites. During the viral surface protein synthesis via the host ribosomal machinery, glycosylation is an essential step in post-translational modifications (PTMs) and consequently vital for its life cycle, structure, immune evasion, and cell infection. Interestingly, the S protein of SARS-CoV-2 and the host receptor protein, ACE2, are also extensively glycosylated and these surface glycans are critical for the viral–host cell interaction for viral entry. The glycosylation pathway of both virus (hijacked from the host biosynthetic machinery) and target cells crucially affect SARS-CoV-2 infection at different levels. For example, the glycosaminoglycans (GAGs) of host cells serve as a cofactor as they interact with the receptor-binding domain (RBD) of S-glycoprotein and play a protective role in host immune evasion via masking the viral peptide epitopes. Hence, the post-translational glycan biosynthesis, processing, and transport events could be potential targets for developing therapeutic drugs and vaccines. Especially, inhibition of the N-glycan biosynthesis pathway amplifies S protein proteolysis and, thus, blocks viral entry. The chemical inhibitors of SARS-CoV-2 glycosylation could be evaluated for Covid-19. In this review, we discuss the current status of the chemical inhibitors (both natural and synthetically designed inhibitors) of viral glycosylation for Covid-19 and provide a future perspective. It could be an important strategy in targeting the various emerging SARS-CoV-2 variants of concern (VOCs), as these inhibitors are postulated to aid in reducing the viral load as well as infectivity.

Mitochondrial-derived compartments facilitate cellular adaptation to amino acid stress

Amino acids are essential building blocks of life. However, increasing evidence suggests that elevated amino acids cause cellular toxicity associated with numerous metabolic disorders. How cells cope with elevated amino acids remains poorly understood. Here, we show that a previously identified cellular structure, the mitochondrial-derived compartment (MDC), functions to protect cells from amino acid stress. In response to amino acid elevation, MDCs are generated from mitochondria, where they selectively sequester and deplete SLC25A nutrient carriers and their associated import receptor Tom70 from the organelle. Generation of MDCs promotes amino acid catabolism, and their formation occurs simultaneously with transporter removal at the plasma membrane via the multivesicular body (MVB) pathway. The combined loss of vacuolar amino acid storage, MVBs, and MDCs renders cells sensitive to high amino acid stress. Thus, we propose that MDCs operate as part of a coordinated cell network that facilitates amino acid homeostasis through post-translational nutrient transporter remodeling.

Stepwise gating of the Sec61 protein-conducting channel by Sec63 and Sec62.

Many proteins are transported into the endoplasmic reticulum by the universally conserved Sec61 channel. Post-translational transport requires two additional proteins, Sec62 and Sec63, but their functions are poorly defined. Here, we determined cryo-EM structures of several variants of Sec61–Sec62–Sec63 complexes from Saccharomyces cerevisiae and Thermomyces lanuginosus and show that Sec62 and Sec63 induce opening of the Sec61 channel. Without Sec62, the translocation pore of Sec61 remains closed by the plug domain, rendering the channel inactive. We further show that the lateral gate of Sec61 must first be partially opened by interactions between Sec61 and Sec63 in cytosolic and lumenal domains, a simultaneous disruption of which completely closes the channel. The structures and molecular dynamics simulations suggest that Sec62 may also prevent lipids from invading the channel through the open lateral gate. Our study shows how Sec63 and Sec62 work together in a hierarchical manner to activate Sec61 for post-translational protein translocation.

Refined measurement of SecA-driven protein secretion reveals that translocation is indirectly coupled to ATP turnover

The universally conserved Sec system is the primary method cells utilize to transport proteins across membranes. Until recently, measuring the activity-a prerequisite for understanding how biological systems work-has been limited to discontinuous protein transport assays with poor time resolution or reported by large, nonnatural tags that perturb the process. The development of an assay based on a split superbright luciferase (NanoLuc) changed this. Here, we exploit this technology to unpick the steps that constitute posttranslational protein transport in bacteria. Under the conditions deployed, the transport of a model preprotein substrate (proSpy) occurs at 200 amino acids (aa) per minute, with SecA able to dissociate and rebind during transport. Prior to that, there is no evidence for a distinct, rate-limiting initiation event. Kinetic modeling suggests that SecA-driven transport activity is best described by a series of large (∼30 aa) steps, each coupled to hundreds of ATP hydrolysis events. The features we describe are consistent with a nondeterministic motor mechanism, such as a Brownian ratchet.

Dynamic Localization of Hepatocellular Transporters: Role in Biliary Excretion and Impairment in Cholestasis.

Bile flow generation is driven by the vectorial transfer of osmotically active compounds from sinusoidal blood into a confined space, the bile canaliculus. Hence, localization of hepatocellular transporters relevant to bile formation is crucial for bile secretion. Hepatocellular transporters are localized either in the plasma membrane or in recycling endosomes, from where they can be relocated to the plasma membrane on demand, or endocytosed when the demand decreases. The balance between endocytic internalization/ exocytic targeting to/from this recycling compartment is therefore the main determinant of the hepatic capability to generate bile, and to dispose endo- and xenobiotics. Furthermore, the exacerbated endocytic internalization is a common pathomechanisms in both experimental and human cholestasis; this results in bile secretory failure and, eventually, posttranslational transporter downregulation by increased degradation. This review summarizes the proposed structural mechanisms accounting for this pathological condition (e.g., alteration of function, localization or expression of F-actin or F-actin/transporter cross-linking proteins, and switch to membrane microdomains where they can be readily endocytosed), and the mediators implicated (e.g., triggering of "cholestatic" signaling transduction pathways). Lastly, we discussed the efficacy to counteract the cholestatic failure induced by transporter internalization of a number of therapeutic experimental approaches based upon the use of compounds that trigger exocytic targetting of canalicular transporters (e.g., cAMP, tauroursodeoxycholate). This therapeutics may complement treatments aimed to transcriptionally improve transporter expression, by affording proper localization and membrane stability to the de novo synthesized transporters.

Cerebellar Neurodegeneration and Neuronal Circuit Remodeling in Golgi pH Regulator-Deficient Mice.

The Golgi apparatus plays an indispensable role in posttranslational modification and transport of proteins to their target destinations. Although it is well established that the Golgi apparatus requires an acidic luminal pH for optimal activity, morphological and functional abnormalities at the neuronal circuit level because of perturbations in Golgi pH are not fully understood. In addition, morphological alteration of the Golgi apparatus is associated with several neurodegenerative diseases, including Parkinson’s disease, Alzheimer’s disease, and amyotrophic lateral sclerosis. Here, we used anatomical and electrophysiological approaches to characterize morphological and functional abnormalities of neuronal circuits in Golgi pH regulator (GPHR) conditional knock-out mice. Purkinje cells (PCs) from the mutant mice exhibited vesiculation and fragmentation of the Golgi apparatus, followed by axonal degeneration and progressive cell loss. Morphological analysis provided evidence for the disruption of basket cell (BC) terminals around PC soma, and electrophysiological recordings showed selective loss of large amplitude responses, suggesting BC terminal disassembly. In addition, the innervation of mutant PCs was altered such that climbing fiber (CF) terminals abnormally synapsed on the somatic spines of mutant PCs in the mature cerebellum. The combined results describe an essential role for luminal acidification of the Golgi apparatus in maintaining proper neuronal morphology and neuronal circuitry.

Post-Translational Protein Transport by the Sec Complex

Although it has been studied for 30 years, the mechanism by which secretory proteins are transported post-translationally into the endoplasmic reticulum (ER) has not yet been fully resolved. Recently published structures (Itskanov and Park, Science 2019;363:84-87; Wu, X. et al. Nature 2019;566:136-139) of the heptameric secretory (Sec) complex which mediates post-translational import into the yeast ER shed new light on the process.

Quantitative Proteomic Analysis of Castor (Ricinus communis L.) Seeds During Early Imbibition Provided Novel Insights into Cold Stress Response.

Early planting is one of the strategies used to increase grain yield in temperate regions. However, poor cold tolerance in castor inhibits seed germination, resulting in lower seedling emergence and biomass. Here, the elite castor variety Tongbi 5 was used to identify the differential abundance protein species (DAPS) between cold stress (4 °C) and control conditions (30 °C) imbibed seeds. As a result, 127 DAPS were identified according to isobaric tag for relative and absolute quantification (iTRAQ) strategy. These DAPS were mainly involved in carbohydrate and energy metabolism, translation and posttranslational modification, stress response, lipid transport and metabolism, and signal transduction. Enzyme-linked immunosorbent assays (ELISA) demonstrated that the quantitative proteomics data collected here were reliable. This study provided some invaluable insights into the cold stress responses of early imbibed castor seeds: (1) up-accumulation of all DAPS involved in translation might confer cold tolerance by promoting protein synthesis; (2) stress-related proteins probably protect the cell against damage caused by cold stress; (3) up-accumulation of key DAPS associated with fatty acid biosynthesis might facilitate resistance or adaptation of imbibed castor seeds to cold stress by the increased content of unsaturated fatty acid (UFA). The data has been deposited to the ProteomeXchange with identifier PXD010043.

Structure of the posttranslational Sec protein-translocation channel complex from yeast.

The Sec61 protein-conducting channel mediates transport of many proteins, such as secretory proteins, across the endoplasmic reticulum (ER) membrane during or after translation. Posttranslational transport is enabled by two additional membrane proteins associated with the channel, Sec63 and Sec62, but its mechanism is poorly understood. We determined a structure of the Sec complex (Sec61-Sec63-Sec71-Sec72) from Saccharomyces cerevisiae by cryo-electron microscopy (cryo-EM). The structure shows that Sec63 tightly associates with Sec61 through interactions in cytosolic, transmembrane, and ER-luminal domains, prying open Sec61's lateral gate and translocation pore and thus activating the channel for substrate engagement. Furthermore, Sec63 optimally positions binding sites for cytosolic and luminal chaperones in the complex to enable efficient polypeptide translocation. Our study provides mechanistic insights into eukaryotic posttranslational protein translocation.

Post-translational processing and membrane translocation of the yeast regulatory Mid1 subunit of the Cch1/VGCC/NALCN cation channel family

Saccharomyces cerevisiae Mid1 is composed of 548 amino acids and a regulatory subunit of Cch1, a member of the eukaryotic pore-forming, four-domain cation channel family. The amino acid sequence and voltage insensitivity of Cch1 are more similar to those of Na+ leak channel non-selective (NALCN) than to the α1 subunit of voltage-gated Ca2+ channels (VGCCs). Despite a lack in overall primary sequence similarity, Mid1 resembles in some aspects VGCC α2/δ regulatory subunits and NALCN-associated proteins. Unlike animal α2/δ subunits, Mid1 and NALCN-associated proteins are essential for the function of the pore-forming subunit. We herein investigated the processing and membrane translocation of Mid1. Mid1 was found to have a 20-amino-acid-long N-terminal signal peptide and appeared to be entirely localized extracellularly. A signal peptide-deleted Mid1 protein, Mid1ΔN23, was N-glycosylated and retained Ca2+ influx activity through Cch1. Moreover, an N-terminal truncation analysis revealed that even truncated Mid1 lacking 209 N-terminal amino acid residues was N-glycosylated and maintained Ca2+ influx activity. A 219-amino-acid-truncated Mid1 protein lost this activity but was still N-glycosylated. In the sec71Δ and sec72Δ single mutants defective in the post-translational protein transport into the endoplasmic reticulum (ER), Mid1ΔN23 could not mediate Ca2+ influx and did not undergo N-glycosylation, whereas wild-type Mid1 exhibited normal Ca2+ influx activity and N-glycosylation in these mutants. Therefore, the signal peptide-lacking Mid1ΔN23 protein may be translocated to the ER exclusively through the post-translational protein translocation, which typically requires an N-terminal signal peptide. Mid1 may provide a tool for studying mechanisms of protein translocation into the ER.

O-acetylation of the serine-rich repeat glycoprotein GspB is coordinated with accessory Sec transport.

The serine-rich repeat (SRR) glycoproteins are a family of adhesins found in many Gram-positive bacteria. Expression of the SRR adhesins has been linked to virulence for a variety of infections, including streptococcal endocarditis. The SRR preproteins undergo intracellular glycosylation, followed by export via the accessory Sec (aSec) system. This specialized transporter is comprised of SecA2, SecY2 and three to five accessory Sec proteins (Asps) that are required for export. Although the post-translational modification and transport of the SRR adhesins have been viewed as distinct processes, we found that Asp2 of Streptococcus gordonii also has an important role in modifying the SRR adhesin GspB. Biochemical analysis and mass spectrometry indicate that Asp2 is an acetyltransferase that modifies N-acetylglucosamine (GlcNAc) moieties on the SRR domains of GspB. Targeted mutations of the predicted Asp2 catalytic domain had no effect on transport, but abolished acetylation. Acetylated forms of GspB were only detected when the protein was exported via the aSec system, but not when transport was abolished by secA2 deletion. In addition, GspB variants rerouted to export via the canonical Sec pathway also lacked O-acetylation, demonstrating that this modification is specific to export via the aSec system. Streptococci expressing GspB lacking O-acetylated GlcNAc were significantly reduced in their ability bind to human platelets in vitro, an interaction that has been strongly linked to virulence in the setting of endocarditis. These results demonstrate that Asp2 is a bifunctional protein involved in both the post-translational modification and transport of SRR glycoproteins. In addition, these findings indicate that these processes are coordinated during the biogenesis of SRR glycoproteins, such that the adhesin is optimally modified for binding. This requirement for the coupling of modification and export may explain the co-evolution of the SRR glycoproteins with their specialized glycan modifying and export systems.

Host Cellular Protein TRAPPC6AΔ Interacts with Influenza A Virus M2 Protein and Regulates Viral Propagation by Modulating M2 Trafficking.

ABSTRACTInfluenza A virus (IAV) matrix protein 2 (M2) plays multiple roles in the early and late phases of viral infection. Once synthesized, M2 is translocated to the endoplasmic reticulum (ER), travels to the Golgi apparatus, and is sorted at the trans-Golgi network (TGN) for transport to the apical plasma membrane, where it functions in virus budding. We hypothesized that M2 trafficking along with its secretory pathway must be finely regulated, and host factors could be involved in this process. However, no studies examining the role of host factors in M2 posttranslational transport have been reported. Here, we used a yeast two-hybrid (Y2H) system to screen for host proteins that interact with the M2 protein and identified transport protein particle complex 6A (TRAPPC6A) as a potential binding partner. We found that both TRAPPC6A and its N-terminal internal-deletion isoform, TRAPPC6A delta (TRAPPC6AΔ), interact with M2. Truncation and mutation analyses showed that the highly conserved leucine residue at position 96 of M2 is critical for mediating this interaction. The role of TRAPPC6AΔ in the viral life cycle was investigated by the knockdown of endogenous TRAPPC6AΔ with small interfering RNA (siRNA) and by generating a recombinant virus that was unable to interact with TRAPPC6A/TRAPPC6AΔ. The results indicated that TRAPPC6AΔ, through its interaction with M2, slows M2 trafficking to the apical plasma membrane, favors viral replication in vitro, and positively modulates virus virulence in mice.IMPORTANCE The influenza A virus M2 protein regulates the trafficking of not only other proteins but also itself along the secretory pathway. However, the host factors involved in the regulation of the posttranslational transport of M2 are largely unknown. In this study, we identified TRAPPC6A and its N-terminal internal-deletion isoform, TRAPPC6AΔ, as interacting partners of M2. We found that the leucine (L) residue at position 96 of M2 is critical for mediating this interaction, which leads us to propose that the high level of conservation of 96L is a consequence of M2 adaptation to its interacting host factor TRAPPC6A/TRAPPC6AΔ. Importantly, we discovered that TRAPPC6AΔ can positively regulate viral replication in vitro by modulating M2 trafficking to the plasma membrane.

Global profiling of SRP interaction with nascent polypeptides.

Signal recognition particle (SRP) is a universally conserved protein-RNA complex that mediates co-translational protein translocation and membrane insertion by targeting translating ribosomes to membrane translocons. The existence of parallel co- and post-translational transport pathways, however, raises the question of the cellular substrate pool of SRP and the molecular basis of substrate selection. Here we determine the binding sites of bacterial SRP within the nascent proteome of Escherichia coli at amino acid resolution, by sequencing messenger RNA footprints of ribosome-nascent-chain complexes associated with SRP. SRP, on the basis of its strong preference for hydrophobic transmembrane domains (TMDs), constitutes a compartment-specific targeting factor for nascent inner membrane proteins (IMPs) that efficiently excludes signal-sequence-containing precursors of periplasmic and outer membrane proteins. SRP associates with hydrophobic TMDs enriched in consecutive stretches of hydrophobic and bulky aromatic amino acids immediately on their emergence from the ribosomal exit tunnel. By contrast with current models, N-terminal TMDs are frequently skipped and TMDs internal to the polypeptide sequence are selectively recognized. Furthermore, SRP binds several TMDs in many multi-spanning membrane proteins, suggesting cycles of SRP-mediated membrane targeting. SRP-mediated targeting is not accompanied by a transient slowdown of translation and is not influenced by the ribosome-associated chaperone trigger factor (TF), which has a distinct substrate pool and acts at different stages during translation. Overall, our proteome-wide data set of SRP-binding sites reveals the underlying principles of pathway decisions for nascent chains in bacteria, with SRP acting as the dominant triaging factor, sufficient to separate IMPs from substrates of the SecA-SecB post-translational translocation and TF-assisted cytosolic protein folding pathways.

Prodrugs Bioactivated to Quinones Target NF-κB and Multiple Protein Networks: Identification of the Quinonome.

Electrophilic reactive intermediates resulting from drug metabolism have been associated with toxicity and off-target effects and in some drug discovery programs trigger NO-GO decisions. Many botanicals and dietary supplements are replete with such reactive electrophiles, notably Michael acceptors, which have been demonstrated to elicit chemopreventive mechanisms; and Michael acceptors are gaining regulatory approval as contemporary cancer therapeutics. Identifying protein targets of these electrophiles is central to understanding potential therapeutic benefit and toxicity risk. NO-donating NSAID prodrugs (NO-NSAIDs) have been the focus of extensive clinical and preclinical studies in inflammation and cancer chemoprevention and therapy: a subset exemplified by pNO-ASA, induces chemopreventive mechanisms following bioactivation to an electrophilic quinone methide (QM) Michael acceptor. Having previously shown that these NO-independent, QM-donors activated Nrf2 via covalent modification of Keap-1, we demonstrate that components of canonical NF-κB signaling are also targets, leading to the inhibition of NF-κB signaling. Combining bio-orthogonal probes of QM-donor ASA prodrugs with mass spectrometric proteomics and pathway analysis, we proceeded to characterize the quinonome: the protein cellular targets of QM-modification by pNO-ASA and its ASA pro-drug congeners. Further comparison was made using a biorthogonal probe of the "bare-bones", Michael acceptor, and clinical anti-inflammatory agent, dimethyl fumarate, which we have shown to inhibit NF-κB signaling. Identified quinonome pathways include post-translational protein folding, cell-death regulation, protein transport, and glycolysis; and identified proteins included multiple heat shock elements, the latter functionally confirmed by demonstrating activation of heat shock response.