Mechanisms of insertion of tail-anchored proteins into the membrane of the endoplasmic reticulum

Tail-anchored proteins (TAPs) are a subclass of type II integral membrane proteins that carry out important and diverse functions within cells. Structurally, TAPs present an N-terminal domain exposed to the cytosol and a single transmembrane domain (TMD) close to the C-terminus, the latter is responsible for the targeting and insertion into the proper intracellular membrane (endoplasmic reticulum (ER), mitochondria, peroxisomes). Due to this particular topology, TAPs insert obligatorily into membranes by post-translational pathways and are excluded from the classical SRP dependent co-translational ER insertion. ER-targeted TAPs can follow two distinct ways of insertion according to the hydrophobicity of their TMD. In the "assisted" pathway, TAPs with more hydrophobic TMDs insert in the ER membrane with the requirement of energy and the involvement of proteinaceous component(s). By contrast neither energy, nor membrane or cytosolic proteins are necessary and do not even improve the "unassisted" insertion of TAPs with moderately hydrophobic TMDs. In this review, we discuss the most relevant recent data regarding the molecular mechanism that underlies these processes.     

Electrostatics and Intrinsic Disorder Drive Translocon Binding of the SRP Receptor FtsY

Integral membrane proteins in bacteria are co‐translationally targeted to the SecYEG translocon for membrane insertion via the signal recognition particle (SRP) pathway. The SRP receptor FtsY and its N‐terminal A domain, which is lacking in any structural model of FtsY, were studied using NMR and fluorescence spectroscopy. The A domain is mainly disordered and highly flexible; it binds to lipids via its N terminus and the C‐terminal membrane targeting sequence. The central A domain binds to the translocon non‐specifically and maintains disorder. Translocon targeting and binding of the A domain is driven by electrostatic interactions. The intrinsically disordered A domain tethers FtsY to the translocon, and because of its flexibility, allows the FtsY NG domain to scan a large area for binding to the NG domain of ribosome‐bound SRP, thereby promoting the formation of the quaternary transfer complex at the membrane.     

Roles of p97-Associated Deubiquitinases in Protein Quality Control at the Endoplasmic Reticulum

To maintain protein homeostasis in the ER, an ER protein quality control system retains unfolded polypeptides and misassembled membrane proteins, allowing only properly folded proteins to exit the ER. Misfolded proteins held in the ER are retrotranslocated into the cytosol, ubiquitinated, and degraded by the proteasome through the ER-associated degradation pathway (ERAD). By timely eliminating misfolded proteins, the ERAD system alleviates cytotoxic stress imposed by protein misfolding. It is well established that ER-associated ubiquitin ligases play pivotal roles in ERAD by assembling ubiquitin conjugates on retrotranslocation substrates, which serve as degradation signals for the proteasome. Surprisingly, recent studies have revealed an equally important function for deubiquitinases (DUBs), enzymes that disassemble ubiquitin chains, in ERAD. Intriguingly, many ERAD specific DUBs are physically associated with the retrotranslocation- driving ATPase p97. Here we discuss the potential functions of p97-associated DUBs including ataxin-3 and YOD1. Our goal is to integrate the emerging evidence into models that may explain how protein quality control could benefit from deubiquitination, a process previously deemed destructive for proteasomal degradation.     

Intracellular transport and localization of microsomal cytochrome P450

The cytochrome P450 (P450) enzymes are mainly localized to the endoplasmic reticulum (ER), where they function within catalytic complexes metabolizing xenobiotics and some endogenous substrates. However, certain members of families 1–3 were also found in other subcellular compartments, such as mitochondria, plasma membrane, and lysosomes. The physiological function of these enzymes in non-ER locations is not known, although plasma-membrane-associated P450s have been described to be catalytically active and to participate in immune-mediated reactions with autoantibody formation that can trigger drug-induced hepatitis. Several retention/retrieval mechanisms are active in the ER retention of the P450s and inverse integration of the translated P450 into the ER membrane appears to be responsible for transport to the plasma membrane. Furthermore, hydrophilic motifs in the NH₂-terminal part have been suggested to be important for mitochondrial import. Phosphorylation of P450s has been described to be important for increased rate of degradation as well as for targeting into mitochondria. It was also suggested that the mitochondria-targeted P450s from families 1–3 could be active in drug metabolism using an alternative electron transport chain. In this review we present an update of the field emphasizing studies concerning localization, posttranslational modification, such as phosphorylation, and intracellular transport of microsomal P450s.     

Pathways to Paraptosis After ER Photodamage in OVCAR‐5 Cells

A death mode termed paraptosis is initiated by photodamage to the endoplasmic reticulum (ER). This is characterized by an extensive pattern of cytoplasmic vacuole formation and leads to a gradual loss of cytoplasm and of viability. Many photosensitizers in clinical use target sub‐cellular organelles that include the ER and can, therefore, invoke paraptosis as well as apoptosis. In this study, we explore pathways to paraptosis in OVCAR‐5, an ovarian cancer cell line of human origin. At low PDT doses, the route to paraptosis follows the pattern that occurs after chemotherapy, that is, a pattern of vacuole formation that can be suppressed by MAPK antagonists or inhibition of new protein synthesis. At PDT doses clinically pertinent for tumor eradication, the pathway to paraptosis appears to be independent of these factors. In addition to morphologic changes, translocation of the nuclear protein HMGB1(high mobility group protein B1) to the cell periphery has been described as a potential marker for drug‐induced paraptosis. This occurs to only a very minor extent after a lethal PDT dose that targets the ER. It appears that a lethal level of ER photodamage can initiate a different pathway to paraptosis, perhaps associated with ER protein cross‐linking.     

不同的引导肽对丝状噬菌体基因8蛋白展示外源多肽的影响

为了研究不同的引导肽对在基因8蛋白上展示外源多肽的影响,利用基因工程方法将一短肽插入基因8蛋白的N端,并将引导多肽去除或用基因3蛋白的引导肽序列替代基因8的引导肽序列.采用放射性脉冲追踪技术检测不同的引导肽对在基因8蛋白上展示外源多肽的作用.结果表明,无引导肽序列的引导,蛋白前体无法向成熟蛋白转化.基因3蛋白的引导肽可以引导展示有外源多肽的基因8蛋白,完成从前体向成熟蛋白的转化,但转化率明显下降.研究结果对阐明噬菌体外壳蛋白在大肠杆菌中的跨膜机制具有重要的意义.     

Proteome analysis of tunicamycin-induced ER stress

Endoplasmic reticulum (ER) stress occurs upon increased levels of unfolded proteins and results in activation of cellular responses such as the unfolded protein response (UPR) and ER-associated protein degradation (ERAD). To examine ER stress, we performed a quantitative proteome analysis of human neuroblastoma cells using stable isotope labeling with amino acids in cell culture (SILAC) in combination with SDS-PAGE and LC-MS/MS. Proteins associated with the ER were overrepresented in the dataset of altered proteins. In particular, ER chaperones responsible for protein folding were significantly upregulated in response to ER stress. The important ER stress regulator 78 kDa glucose-regulated protein (GRP-78 or BiP) was highly upregulated together with several proteins that have been found to form a multiprotein complex with BiP including cyclophilin B, DnaJ homolog subfamily B member 11, endoplasmin, hypoxia upregulated protein 1, protein disulfide isomerase and protein disulfide isomerase A4 upon tunicamycin-induced ER stress. Furthermore, seven aminoacyl-tRNA synthetases and five proteins belonging to the Sec61 complex were increased in response to tunicamycin-induced ER stress.     

Optimized orbitrap HCD for quantitative analysis of phosphopeptides

Despite the tremendous commercial success of radio frequency quadrupole ion traps for bottom-up proteomics studies, there is growing evidence that peptides decorated with labile post-translational modifications are less amenable to low-energy, resonate excitation MS/MS analysis. Moreover, multiplexed stable isotope reagents designed for MS/MS-based quantification of peptides rely on accurate and robust detection of low-mass fragments for all precursors. Collectively these observations suggest that beam-type or tandem in-space MS/MS measurements, such as that available on traditional triple quadrupole mass spectrometers, may provide beneficial figures of merit for quantitative proteomics analyses. The recent introduction of a multipole collision cell adjacent to an Orbitrap mass analyzer provides for higher energy collisionally activated dissociation (HCD) with efficient capture of fragment ions over a wide mass range. Here we describe optimization of various instrument and post-acquisition parameters that collectively provide for quantification of iTRAQ-labeled phosphorylated peptides isolated from complex cell lysates. Peptides spanning a concentration dynamic range of 100:1 are readily quantified. Our results indicate that appropriate parameterization of collision energy as a function of precursor m/z and z provides for optimal performance in terms of peptide identification and relative quantification by iTRAQ. Using this approach, we readily identify activated signaling pathways downstream of oncogenic mutants of Flt-3 kinase in a model system of human myeloid leukemia.     

Target-selective one-way membrane fusion system based on a pH-responsive coiled coil assembly at the interface of liposomal vesicles

The coiled coil trimer structure is a common motif observed in membrane fusion processes of specific fusion proteins such as the hemagglutinin glycoprotein. The HA2 subunit in the hemagglutinin changes its conformation or geometry to be favorable to membrane fusion in response to endosomal weakly acidic pH. This pH responsiveness is indispensable to an artificial polypeptide-triggered delivery system as well as the membrane fusion reaction in biology. In this study, we have constructed an AAB-type coiled coil heteroassembled system that is sensitive to weakly acidic pH. The heterotrimer is formed from two kinds of polypeptides containing an Ala or a Trp residue at a hydrophobic a position, and it was observed that the Glu residue at the other a position induced an acidic pH-dependent conformational change. On the basis of this pH-responsive coiled coil heteroassembled system, a boronic acid coupled working polypeptide for the combination of an intervesicular complex with a sugarlike compound on the surface of the target liposome, and a supporting polypeptide for the construction of a pH-responsive heterotrimer with the working polypeptide were designed and synthesized. The process of membrane fusion was characterized by lipid-mixing, inner-leaflet lipid-mixing, and content-mixing assays. The target selective vesicle fusion is clearly observed at a weakly acidic pH, where the working polypeptides form a heterotrimeric coiled coil with the supporting polypeptides in a 1:2 binding stoichiometry and the surfaces between pilot and target vesicles come into close proximity to each other.     

Paraptosis and Photodynamic Therapy: A Progress Report

Photodamage to the endoplasmic reticulum (ER) can initiate a death pathway termed paraptosis. The “canonical” model of paraptosis, initiated by certain drugs and other stimuli, requires a brief interval of protein synthesis, involves the action of MAP kinases and can be followed by biochemical markers. The latter include changes in expression of AIP-1/Alix and IGF-1R proteins and translocation of HMGB-1 from nucleus to plasma membrane. There is also a report indicating that an enhanced level of autophagy can impair death by paraptosis. The pathway to paraptosis follows the canonical pathway when ER photodamage is minor (<LD₅₀). When the extent of ER photodamage approaches LD₉₀ levels, there are deviations from the “canonical” pathway: interfering with protein synthesis does not prevent paraptosis nor does a brief chilling of cells after irradiation, MAP kinases are not involved, and stimulation of autophagy was not cytoprotective. We had previously speculated that ER protein cross-linking might potentiate paraptosis (Photochem. Photobiol. 95, 2019, 1239) but this appears to be incorrect. At higher PDT doses, substantial cross-linking of a typical ER protein (BiP, binding immunoglobin protein, an HSP chaperone) was detected and paraptosis was impaired. This may relate to decreased mobility of cross-linked proteins. Other pathways to cell death were then observed.     

Synthesis of Fluorophores that Target Small Molecules to the Endoplasmic Reticulum of Living Mammalian Cells

The endoplasmic reticulum (ER) plays critical roles in the processing of secreted and transmembrane proteins. To deliver small molecules to this organelle, we synthesized fluorinated hydrophobic analogues of the fluorophore rhodol. These cell‐permeable fluorophores are exceptionally bright, with quantum yields of around 0.8, and they were found to specifically accumulate in the ER of living HeLa cells, as imaged by confocal laser scanning microscopy. To target a biological pathway controlled by the ER, we linked a fluorinated hydrophobic rhodol to 5‐nitrofuran‐2‐acrylaldehyde. In contrast to an untargeted nitrofuran warhead, delivery of this electrophilic nitrofuran to the ER by the rhodol resulted in cytotoxicity comparable to the ER‐targeted cytotoxin eeyarestatin I, and specifically inhibited protein processing by the ubiquitin–proteasome system. Fluorinated hydrophobic rhodols are outstanding fluorophores that enable the delivery of small molecules for targeting ER‐associated proteins and pathways.     

Therapeutic Targeting of the NRF2 Signaling Pathway in Cancer

Cancer is one of the most fatal diseases with an increasing incidence and mortality all over the world. Thus, there is an urgent need for novel therapies targeting major cancer-related pathways. Nuclear factor-erythroid 2-related factor 2 (NRF2) and its major negative modulator Kelch-like ECH-associated protein 1 (KEAP1) are main players of the cellular defense mechanisms against internal and external cell stressors. However, NRF2/KEAP1 signaling pathway is dysregulated in various cancers, thus promoting tumor cell survival and metastasis. In the present review, we discuss the mechanisms of normal and deregulated NRF2 signaling pathway focusing on its cancer-related functions. We further explore activators and inhibitors of this pathway as cancer targeting drug candidates in order to provide an extensive background on the subject.     

Contribution of a pyrene fluorescence probe to the aggregation propensity of polypeptides

[structure: see text] Two synthetic polypeptides, TT1p and TT1b, have been used in comparative aggregation equilibrium studies. The findings reveal that a single alkylpyrene moiety in TT1p contributes about 30% of the polypeptide dimerization energy in aqueous media. This result not only is informative with regard to the aggregation properties of these particular photoactive polypeptides but also provides a quantitative understanding of the limitations on the use of pyrene chromophores as emission probes.     

Current Status of Endoplasmic Reticulum Stress in Type II Diabetes

The endoplasmic reticulum (ER) plays a multifunctional role in lipid biosynthesis, calcium storage, protein folding, and processing. Thus, maintaining ER homeostasis is essential for cellular functions. Several pathophysiological conditions and pharmacological agents are known to disrupt ER homeostasis, thereby, causing ER stress. The cells react to ER stress by initiating an adaptive signaling process called the unfolded protein response (UPR). However, the ER initiates death signaling pathways when ER stress persists. ER stress is linked to several diseases, such as cancer, obesity, and diabetes. Thus, its regulation can provide possible therapeutic targets for these. Current evidence suggests that chronic hyperglycemia and hyperlipidemia linked to type II diabetes disrupt ER homeostasis, thereby, resulting in irreversible UPR activation and cell death. Despite progress in understanding the pathophysiology of the UPR and ER stress, to date, the mechanisms of ER stress in relation to type II diabetes remain unclear. This review provides up-to-date information regarding the UPR, ER stress mechanisms, insulin dysfunction, oxidative stress, and the therapeutic potential of targeting specific ER stress pathways.     

Detection of polypeptides associated with the cold acclimation process in spinach

Exposure of spinach (Spinacia oleracea) seedlings to 5 degrees C for several days has previously been shown to induce a greater tolerance to the stresses of extracellular freezing. Associated with this response to low temperature, termed cold acclimation, was a subtle shift in protein synthesis and altered polypeptide composition. Two-dimensional gel electrophoresis was used to study the changes in spinach leaf tissue protein synthesis in an effort to identify polypeptides that may play a central role in the induction of greater freezing tolerance. Through a combination of silver staining, in vivo labeling, and in vitro translation of mRNAs, we identified several high molecular weight polypeptides whose synthesis and presence in spinach leaf tissue were highly correlated with freezing tolerance. Synthesis of these polypeptides was elevated or induced during cold acclimation when freezing tolerance increases, but was rapidly reduced or halted during deacclimation when freezing tolerance declines. The close association of the synthesis of these polypeptides with the induction and loss of freezing tolerance suggested that they could play a role in cold tolerance mechanisms of spinach.     

Proteins of the hepatoma tissue culture cell plasma membrane.

The specificity of lactoperoxidase-catalyzed iodination for the proteins of the hepatoma tissue culture cell plasma membrane was examined by histochemical, biochemical, and cell fractionation techniques. Light microscope autoradiography of sectioned cells shows the incorporated label to be localized primarily at the periphery of the cell. Most of this label can be released from the cell by trypsin but not by collagenase or hyaluronidase. The label is recovered from the cells as either monoiodotyrosine or diiodotyrosine after hydrolysis of cell extracts with a mixture of proteolytic enzymes. The label co-purifies during cell fractionation with an authentic liver cell plasma membrane marker enzyme, 5'-nucleotidase. Thus, the incorporated iodide is itself a valid marker for those membrane polypeptides having tyrosine residues accessible to the lactoperoxidase. The polypeptide complexity of the purified plasma membrane was examined by high resolution dodecyl sulfate-polyacrylamide gel electrophoresis. At least 50 polypeptides in the membrane are accessible to iodination. These polypeptides probably represent the bulk of the protein mass of the membrane and iodinating them does not affect cell viability, growth rate, or cell function. Labeling experiments with fucose and glucosamine show that at least nine of the iodinated peptides may be glycoproteins.     

Hafnium oxide and zirconium oxide atomic layer deposition: initial precursor and potential side-reaction product pathways with H/Si(100)-2 x 1

Hybrid density functional calculations have been carried out using cluster models of the H/Si(100)-2 x 1 surface to investigate the mechanistic details of the initial surface reactions occurring in the atomic layer deposition of hafnium and zirconium oxides (HfO2 and ZrO2). Reaction pathways involving the metal precursors ZrCl4, Zr(CH3)4, HfCl4, and Hf(CH3)4 have been examined. Pathways leading to the formation of a Zr-Si or Hf-Si linkage show a significant sensitivity to the identity of the leaving group, with chloride loss reactions being both kinetically and thermodynamically less favorable than reactions leading to the loss of a methyl group. The energetics of the Zr(CH3)4 and Hf(CH3)4 reactions are similar with an overall exothermicity of 0.3-0.4 eV and a classical barrier height of 1.1-1.2 eV. For the reaction between H2O and the H/Si(100)-2 x 1 surface, the activation energy and overall reaction enthalpy are 1.6 and -0.8 eV, respectively. Due to contamination, trace amounts of H2O may be encountered by metal precursors, leading to the formation of minor species that can lead to unanticipated side-reaction pathways. Such gas-phase reactions between the halogenated and alkylated metal precursors and H2O are exothermic with small or no reaction barriers, allowing for the possibility of metal precursor hydroxylation before the H/Si surface is encountered. Of the contaminant surface reaction pathways, the most kinetically favorable corresponds to the surface -OH deposition. Interestingly, for the hydroxylated metal precursors, a unique reaction pathway resulting in the direct formation of Si-O-Zr and Si-O-Hf linkages has been identified and found to be the most thermodynamically stable pathway available, being exothermic by approximately 1.0 eV.     

Assignment of human plasma polypeptides on a nondenaturing 2-D gel using MALDI-MS and PMF and comparisons with the results of intact protein mapping

Human plasma proteins were separated by 2-DE under nondenaturing conditions followed by the assignment of the CBB-stained spots using MALDI-MS and PMF, aiming to correlate the information of intact proteins with that of constituent polypeptides. A microgel system was employed to facilitate the analysis. Totally 157 spots on a nondenaturing micro-2-DE gel were numbered, the spots were excised, the proteins in the gel pieces were subjected to in-gel digestion with trypsin followed by polypeptide analysis using MALDI-MS and PMF. Two PMF algorithms, MASCOT (with Swiss-Prot database) and ProFound (with NCBInr database) were employed. A total of 153 spots out of the 157 provided significant match (p <0.05) with polypeptides in databases. Eighty spots were assigned to contain multiple (2-4) polypeptides, suggesting (i) noncovalent interaction between proteins/polypeptides, (ii) disulfide bonding of polypeptides, or (iii) overlapping of the protein locations on the gel. The results of polypeptide assignment coincided very well with the results of protein mapping previously reported, in which 33 plasma proteins were identified using blotting-immunochemical staining (Manabe, T., Takahashi, Y., Higuchi, N., Okuyama, T., Electrophoresis 1985, 6, 462-467). Further, 19 polypeptides in 25 spots were newly assigned. These results demonstrate that the techniques of MALDI-MS and PMF can be applied for analysis of proteins separated on nondenaturing 2-DE gels, providing information on their polypeptide structure. The integrated information on proteins and polypeptides would help the comprehensive understanding on the functions of complex protein systems.     

Modular pathway engineering of diterpenoid synthases and the mevalonic acid pathway for miltiradiene production

Microbial production can be advantageous over the extraction of phytoterpenoids from natural plant sources, but it remains challenging to rationally and rapidly access efficient pathway variants. Previous engineering attempts mainly focused on the mevalonic acid (MVA) or methyl-d-erythritol phosphate (MEP) pathways responsible for the generation of precursors for terpenoids biosynthesis, and potential interactions between diterpenoids synthases were unexplored. Miltiradiene, the product of the stepwise conversion of (E,E,E)-geranylgeranyl diphosphate (GGPP) catalyzed by diterpene synthases SmCPS and SmKSL, has recently been identified as the precursor to tanshionones, a group of abietane-type norditerpenoids rich in the Chinese medicinal herb Salvia miltiorrhiza . Here, we present the modular pathway engineering (MOPE) strategy and its application for rapid assembling synthetic miltiradiene pathways in the yeast Saccharomyces cerevisiae . We predicted and analyzed the molecular interactions between SmCPS and SmKSL, and engineered their active sites into close proximity for enhanced metabolic flux channeling to miltiradiene biosynthesis by constructing protein fusions. We show that the fusion of SmCPS and SmKSL, as well as the fusion of BTS1 (GGPP synthase) and ERG20 (farnesyl diphosphate synthase), led to significantly improved miltiradiene production and reduced byproduct accumulation. The MOPE strategy facilitated a comprehensive evaluation of pathway variants involving multiple genes, and, as a result, our best pathway with the diploid strain YJ2X reached miltiradiene titer of 365 mg/L in a 15-L bioreactor culture. These results suggest that terpenoids synthases and the precursor supplying enzymes should be engineered systematically to enable an efficient microbial production of phytoterpenoids.