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.     

Quantitative Proteomics and Differential Protein Abundance Analysis after Depletion of Putative mRNA Receptors in the ER Membrane of Human Cells Identifies Novel Aspects of mRNA Targeting to the ER

In human cells, one-third of all polypeptides enter the secretory pathway at the endoplasmic reticulum (ER). The specificity and efficiency of this process are guaranteed by targeting of mRNAs and/or polypeptides to the ER membrane. Cytosolic SRP and its receptor in the ER membrane facilitate the cotranslational targeting of most ribosome-nascent precursor polypeptide chain (RNC) complexes together with the respective mRNAs to the Sec61 complex in the ER membrane. Alternatively, fully synthesized precursor polypeptides are targeted to the ER membrane post-translationally by either the TRC, SND, or PEX19/3 pathway. Furthermore, there is targeting of mRNAs to the ER membrane, which does not involve SRP but involves mRNA- or RNC-binding proteins on the ER surface, such as RRBP1 or KTN1. Traditionally, the targeting reactions were studied in cell-free or cellular assays, which focus on a single precursor polypeptide and allow the conclusion of whether a certain precursor can use a certain pathway. Recently, cellular approaches such as proximity-based ribosome profiling or quantitative proteomics were employed to address the question of which precursors use certain pathways under physiological conditions. Here, we combined siRNA-mediated depletion of putative mRNA receptors in HeLa cells with label-free quantitative proteomics and differential protein abundance analysis to characterize RRBP1- or KTN1-involving precursors and to identify possible genetic interactions between the various targeting pathways. Furthermore, we discuss the possible implications on the so-called TIGER domains and critically discuss the pros and cons of this experimental approach.     

The aftermath of the interplay between the endoplasmic reticulum stress response and redox signaling

The endoplasmic reticulum (ER) is an essential organelle of eukaryotic cells. Its main functions include protein synthesis, proper protein folding, protein modification, and the transportation of synthesized proteins. Any perturbations in ER function, such as increased demand for protein folding or the accumulation of unfolded or misfolded proteins in the ER lumen, lead to a stress response called the unfolded protein response (UPR). The primary aim of the UPR is to restore cellular homeostasis; however, it triggers apoptotic signaling during prolonged stress. The core mechanisms of the ER stress response, the failure to respond to cellular stress, and the final fate of the cell are not yet clear. Here, we discuss cellular fate during ER stress, cross talk between the ER and mitochondria and its significance, and conditions that can trigger ER stress response failure. We also describe how the redox environment affects the ER stress response, and vice versa, and the aftermath of the ER stress response, integrating a discussion on redox imbalance-induced ER stress response failure progressing to cell death and dynamic pathophysiological changes.Subject terms: Mechanisms of disease, Cell biology     

Mechanism regulating cell surface expression and activation of Toll-like receptor 4

The Toll family of receptors senses microbial invasion and activates defense responses. Toll-like receptor 4 (TLR4) is a member of the Toll family that senses lipopolysaccharide (LPS), a principal membrane component from Gram-negative bacteria. LPS is known as an endotoxin that strongly activates immune cells such as macrophages and dendritic cells. LPS recognition by TLR4 requires an additional accessory molecule, MD-2. MD-2 is associated with the extracellular portion of TLR4, directly binds to LPS, and regulates subsequent LPS-induced TLR4 clustering. LPS recognition occurs on the cell surface. The subcellular distribution of TLR was shown to influence TLR responses. An endoplasmic reticulum (ER) chaperone, glycoprotein 96, is required for the stability of TLR4 and the formation of a TLR4/MD-2 complex in ER. MD-2 facilitates TLR4 glycosylation and its trafficking to the cell surface. Recently, another molecule, a protein associated with Toll-like receptor 4 (PRAT4A), was shown to play a critical role in cell surface expression of TLR4. These molecules control LPS responsiveness by regulating the subcellular distribution of TLR4.     

BODIPY/Nile‐Red‐Based Efficient FRET Pair: Selective Assay of Endoplasmic Reticulum Membrane Fluidity

We synthesized a boron‐dipyrromethene (BODIPY)/Nile Red hybrid probe capable of selectively recognizing fluidity changes in the endoplasmic reticulum (ER) membrane due to its preferential localization to the ER and strong energy transfer from BODIPY to the Nile Red moiety, emitting only in nonaqueous environments. ER membrane fluidity in HepG2 cells was markedly reduced by a cell model of metabolic syndrome.     

Proteomics reveal a link between the endoplasmic reticulum and lipid secretory mechanisms in mammary epithelial cells

The synthesis and secretion of lipids by mammary epithelial cells is a highly ordered process that involves several distinct steps. Triacylglycerols are synthesized in the endoplasmic reticulum and incorporated into microlipid droplets which coalesce into cytoplasmic lipid droplets. These are vectorially transported to the apical plasma membrane where they are secreted into the milk surrounded by a membrane bilayer. The origin of this membrane as well as the mechanism by which cytoplasmic lipid droplets form and become surrounded by membrane is poorly understood. Proteomic analysis of the protein composition of milk fat globules and cytoplasmic lipid droplet has revealed that the endoplasmic reticulum is not only involved in the synthesis of the lipid but also potentially contributes to the membrane component of milk fat globules. The proteins identified suggest possible mechanisms of multiple steps during this process. Completion of the proteome of milk fat globule membranes and cytoplasmic lipid droplets will provide the necessary reporter molecules to follow and dissect the mechanisms of the sorting and ultimate secretion of cytoplasmic lipid droplets.     

硒蛋白S的生物学功能

硒蛋白S是一种新发现的内质网和细胞膜驻留硒蛋白。以往的研究结果揭示硒蛋白S可以保护细胞拮抗氧化损伤及内质网应激诱导的细胞凋亡;参与脂蛋白代谢、精子发育过程、炎症反应及将错误折叠蛋白从内质网腔逆向转移到细胞质中然后降解的过程(即内质网相关蛋白降解)。硒蛋白S基因多态性与糖尿病、冠状动脉心脏病或先兆子痫等疾病密切相关。本文结合本课题组的工作对硒蛋白S的最新研究进展,尤其是硒蛋白S功能的研究成果作了较为详细的介绍,并对未来的研究方向作了展望。     

Ultraviolet Light,Unfolded Protein Response and Autophagy†

The endoplasmic reticulum (ER) plays an important role in the regulation of protein synthesis. Alterations in the folding capacity of the ER induce stress, which activates three ER sensors that mediate the unfolded protein response (UPR). Components of the pathways regulated by these sensors have been shown to regulate autophagy. The last corresponds to a mechanism of self-eating and recycling important for proper cell maintenance. Ultraviolet radiation (UV) is an external damaging stimulus that is known for inducing oxidative stress, and DNA, lipid and protein damage. Many controversies exist regarding the role of UV-inducing ER stress or autophagy. However, a connection between the three of them has not been addressed. In this review, we will discuss the contradictory theories regarding the relationships between UV radiation with the induction of ER stress and autophagy, as well as hypothetic connections between UV, ER stress and autophagy.     

ER-associated CTRP1 regulates mitochondrial fission via interaction with DRP1

C1q/TNF-related protein 1 (CTRP1) is a CTRP family member that has collagenous and globular C1q-like domains. The secreted form of CTRP1 is known to be associated with cardiovascular and metabolic diseases, but its cellular roles have not yet been elucidated. Here, we showed that cytosolic CTRP1 localizes to the endoplasmic reticulum (ER) membrane and that knockout or depletion of CTRP1 leads to mitochondrial fission defects, as demonstrated by mitochondrial elongation. Mitochondrial fission events are known to occur through an interaction between mitochondria and the ER, but we do not know whether the ER and/or its associated proteins participate directly in the entire mitochondrial fission event. Interestingly, we herein showed that ablation of CTRP1 suppresses the recruitment of DRP1 to mitochondria and provided evidence suggesting that the ER–mitochondrion interaction is required for the proper regulation of mitochondrial morphology. We further report that CTRP1 inactivation-induced mitochondrial fission defects induce apoptotic resistance and neuronal degeneration, which are also associated with ablation of DRP1. These results demonstrate for the first time that cytosolic CTRP1 is an ER transmembrane protein that acts as a key regulator of mitochondrial fission, providing new insight into the etiology of metabolic and neurodegenerative disorders.Subject terms: Endoplasmic reticulum, Mitochondria     

Upregulation of the SERCA-type Ca2+ pump activity in response to endoplasmic reticulum stress in PC12 cells

Background  

Ca²⁺-ATPases of endoplasmic reticulum (SERCAs) are responsible for maintenance of the micro- to millimolar Ca²⁺ ion concentrations within the endoplasmic reticulum (ER) of eukaryotic cells. This intralumenal Ca²⁺ storage is important for the generation of Ca²⁺ signals as well as for the correct folding and posttranslational processing of proteins entering ER after synthesis. ER perturbations such as depletion of Ca²⁺ or abolishing the oxidative potential, inhibition of glycosylation, or block of secretory pathway, activate the Unfolded Protein Response, consisting of an upregulation of a number of ER-resident chaperones/stress proteins in an effort to boost the impaired folding capacity.     

Approaches toward High‐Mannose‐Type Glycan Libraries

Asparagine‐linked (N‐linked) sugar chains are widely found in the rough endoplasmic reticulum (ER), which has attracted renewed attention because of its participation in the glycoprotein quality control process. In the ER, newly formed glycoproteins are properly folded to higher‐order structures by the action of a variety of lectin chaperones and processing enzymes and are transported into the Golgi, while terminally misfolded glycoproteins are carried into the cytosol for degradation. A group of proteins related to this system are known to recognize subtle differences in the high‐mannose‐type oligosaccharide structures of glycoproteins; however, their molecular foundations are still unclear. In order to gain a more precise understanding, our group has established a strategy for the systematic synthesis of high‐mannose‐type glycans. More recently, we have developed “top‐down” chemoenzymatic approaches that allow expeditious access to theoretically all types of high‐mannose glycans. This strategy comprehensively delivered 37 high‐mannose‐type glycans, including G1M9–M3 glycans, and opened up the possibility of the elucidation of structure–function relationships with a series of high‐mannose‐type glycans.     

Synthesis of adenylyl-(3'----5')-guanosine and some analogues as probes to explore the molecular mechanism of stimulation of influenza virus RNA polymerase

Influenza virus mRNA synthesis is primed by a capped oligonucleotide which is cleaved off from a cellular mRNA by a viral protein. The dinucleotide A3'p5'G can be used as a primer for the viral RNA polymerase mediated RNA synthesis in a cell-free system. Analogues of A3'p5'G have therefore been synthesized using the phosphotriester approach, and their priming ability for the influenza virus mRNA synthesis has been determined. An absence of the 2'-hydroxyl function in the guanosine residue in the dinucleotide, as in A3'p5'dG, drastically decreased its priming ability. Similarly, an alteration of the 3'----5' phosphate linkage to a 2'----5' phosphodiester linkage affected the priming ability quite severely. However a dinucleotide, with the 2'-hydroxyl function omitted in the adenosine moiety, as in dA3'p5'G, could still stimulate the mRNA synthesis. None of the modified dinucleotides inhibited A3'p5'G or globin mRNA primed influenza mRNA synthesis.     

Effects of glycosylation on peptide conformation: a synergistic experimental and computational study

Asparagine-linked glycosylation, the co-translational covalent attachment of carbohydrates to asparagine side chains, has a major effect on the folding, stability, and function of many proteins. The carbohydrate composition in mature glycoproteins is heterogeneous due to modification of the initial oligosaccharide by glycosidases and glycosyltransferases during the glycoprotein passage through the endoplasmic reticulum and Golgi apparatus. Despite the diversity of carbohydrate structures, the core beta-D-(GlcNAc)(2) remains conserved in all N-linked glycoproteins. Previously, results from our laboratory showed that the molecular composition of the core disaccharide has a critical and unique conformational effect on the peptide backbone. Herein, we employ a synergistic experimental and computational approach to study the effect of the stereochemistry of the carbohydrate--peptide linkage on glycopeptide structure. A glycopeptide derived from a hemagglutinin protein fragment was synthesized, with the carbohydrate attached to the peptide with an alpha-linked stereochemistry. Computational and biophysical analyses reveal that the conformations of the peptide and alpha- and beta-linked glycopeptides are uniquely influenced by the attached saccharide. The value of computational approaches for probing the influence of attached saccharides on polypeptide conformation is highlighted.     

Context-dependent effects of asparagine glycosylation on Pin WW folding kinetics and thermodynamics

Asparagine glycosylation is one of the most common and important post-translational modifications of proteins in eukaryotic cells. N-glycosylation occurs when a triantennary glycan precursor is transferred en bloc to a nascent polypeptide (harboring the N-X-T/S sequon) as the peptide is cotranslationally translocated into the endoplasmic reticulum (ER). In addition to facilitating binding interactions with components of the ER proteostasis network, N-glycans can also have intrinsic effects on protein folding by directly altering the folding energy landscape. Previous work from our laboratories (Hanson et al. Proc. Natl. Acad. Sci. U.S.A. 2009, 109, 3131-3136; Shental-Bechor, D.; Levy, Y. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 8256-8261) suggested that the three sugar residues closest to the protein are sufficient for accelerating protein folding and stabilizing the resulting structure in vitro; even a monosaccharide can have a dramatic effect. The highly conserved nature of these three proximal sugars in N-glycans led us to speculate that introducing an N-glycosylation site into a protein that is not normally glycosylated would stabilize the protein and increase its folding rate in a manner that does not depend on the presence of specific stabilizing protein-saccharide interactions. Here, we test this hypothesis experimentally and computationally by incorporating an N-linked GlcNAc residue at various positions within the Pin WW domain, a small β-sheet-rich protein. The results show that an increased folding rate and enhanced thermodynamic stability are not general, context-independent consequences of N-glycosylation. Comparison between computational predictions and experimental observations suggests that generic glycan-based excluded volume effects are responsible for the destabilizing effect of glycosylation at highly structured positions. However, this reasoning does not adequately explain the observed destabilizing effect of glycosylation within flexible loops. Our data are consistent with the hypothesis that specific, evolved protein-glycan contacts must also play an important role in mediating the beneficial energetic effects on protein folding that glycosylation can confer.     

New fluorescent probes reveal that flippase-mediated flip-flop of phosphatidylinositol across the endoplasmic reticulum membrane does not depend on the stereochemistry of the lipid

Glycerophospholipid flip-flop across biogenic membranes such as the endoplasmic reticulum (ER) is a fundamental feature of membrane biogenesis. Flip-flop requires the activity of specific membrane proteins called flippases. These proteins have yet to be identified in biogenic membranes and the molecular basis of their action is unknown. It is generally believed that flippase-facilitated glycerophospholipid flip-flop across the ER is governed by the stereochemistry of the glycerolipid, but this important issue has not been resolved. Here we investigate whether the ER flippase stereochemically recognizes the glycerophospholipids that it transports. To address this question we selected phosphatidylinositol (PI), a biologically important molecule with chiral centres in both its myo-inositol headgroup and its glycerol-lipid tail. The flip-flop of PI across the ER has not been previously reported. We synthesized fluorescence-labeled forms of all four diastereoisomers of PI and evaluated their flipping in rat liver ER vesicles, as well as in flippase-containing proteoliposomes reconstituted from a detergent extract of ER. Our results show that the flippase is able to translocate all four PI isomers and that both glycerol isomers of PI flip-flop across the ER membrane at rates similar to that measured for fluorescence-labeled phosphatidylcholine. Our data have important implications for recent hypotheses concerning the evolution of distinct homochiral glycerophospholipid membranes during the speciation of archaea and bacteria/eukarya from a common cellular ancestor.     

In Vitro Synthesis of the E. coli Sec Translocon from DNA

Difficulties in constructing complex lipid/protein membranes have severely limited the development of functional artificial cells endowed with vital membrane‐related functions. The Sec translocon membrane channel, which mediates the insertion of membrane proteins into the plasma membrane, was constructed in the membrane of lipid vesicles through in vitro expression of its component proteins. The components of the Sec translocon were synthesized from their respective genes in the presence of liposomes, thereby bringing about a functional complex. The synthesized E. coli Sec translocon mediated the membrane translocation of single‐ and multi‐span membrane proteins. The successful translocation of a functional peptidase into the liposome lumen further confirmed the proper insertion of the translocon complex. Our results demonstrate the feasible construction of artificial cells, the membranes of which can be functionalized by directly decoding genetic information into membrane functions.     

Alkylphenols from the Roots of <i>Ardisia brevicaulis</i> Induce G1 Arrest and Apoptosis through Endoplasmic Reticulum Stress Pathway in Human Non-small-cell Lung Cancer Cells

From the roots of Ardisia brevicaulis DIELS, two new alkylphenol derivatives, named ardisiphenol E (2) and F (3), have been isolated together with a known alkylphenol, ardisiphenol D (1). The structures of 1-3 were elucidated by chemical and spectroscopic techniques. Compounds 1 and 2 exhibited strong cytotoxicities on two human non-small-cell lung cancer cell lines (H1299 and A549). We found that compounds 1 and 2 upregulated mRNA and protein expressions of endoplasmic reticulum (ER) stress markers including C/EBP homologous protein (CHOP), binding immunoglobulin protein (Bip) and inositol-requiring enzyme 1 (IRE1) indicating 1 and 2 are novel natural ER stress inducers. Treatments with 1 and 5?μM of 1 or 2 triggered G1 arrest in H1299 and A549 cells with concomitant downregulation of ubiquitin fusion degradation protein 1 (Ufd1) and S-phase kinase-associated protein 2 (Skp2) proteins and the accumulation of p27, the key axes of ER stress-mediated G1 arrest. Compounds 1 and 2 also induced apoptosis at high concentrations (10, 20?μM) which was shown to be coupled with the upregulation of CHOP and Bim, the activation of caspase-9, caspase-3 and poly(ADP-ribose) polymerase (PARP) cleavage. These results indicate that compounds 1 and 2 induce ER stress that subsequently causes G1 arrest and apoptosis in human non-small-cell lung cancer cells and they may have potential anticancer effects.     

Fluorescence Quenching‐based Assay for Measuring Golgi endo‐α‐Mannosidase

Golgi endo‐α‐mannosidase (G‐EM) catalyzes an alternative deglucosylation process for N‐glycans and plays important roles in the post‐endoplasmic reticulum (ER) quality control pathway. To understand the post‐ER quality control mechanism, we synthesized a tetrasaccharide probe for the detection of the hydrolytic activity of G‐EM based on a fluorescence quenching assay. The probe was labeled with an N‐methylanthraniloyl group as a reporter dye at the non‐reducing end and a 2,4‐dinitrophenyl group as a quencher at the reducing end. This probe is hydrolyzed to disaccharide derivatives by G‐EM, resulting in increased fluorescence intensity. Thus, the fluorescence signal is directly proportional to the amount of disaccharide derivative present, allowing the G‐EM activity to be evaluated easily and quantitatively.     

A mononuclear copper(II) complex containing benzimidazole and pyridyl ligands: Synthesis,characterization, and antiproliferative activity against human cancer cells

Metal complexes are recently being hybridized with different moieties to discover new drugs due to their advantageous attributes. Among the metals, copper is a good one to synthesize a metal complex due to its being endogenous, redox and DNA cleavage potential, reported anti-cancer efficacies and selective permeability for cancer cells. In this study, first we synthesized a new copper (II) complex and determined its toxic doses on NIH/3T3 normal fibroblast cells, SPC212 mesothelioma and DU145 prostate cancer cells. Then, we ascertained anti-proliferative, apoptotic, morphological, oxidative and endoplasmic reticulum (ER) stress inducing effects of these newly synthesized compounds on DU145 prostate cancer cells. A novel Copper(II)/1-(4-(trifluoromethyl)benzyl)-1H-benzimidazole/2,2′-bipyridyl complex was synthesized and mainly characterized by single crystal X-ray diffraction analysis. Anti-proliferative effect of copper(II) complex was gauged by MTT. Oxidative and ER stress were evaluated by ELISA and Western blot. The morphological effect was examined by microscope analysis. Besides, immunocytochemistry of Bax, a pro-apoptotic protein and PCNA, a proliferation marker protein was performed. As a result, the inhibitory effect of newly synthesized substance was superior to the chemicals from which it was synthesized. Its IC₅₀s against DU145 were 37.0, 21.1 and 10.0 µM for 24, 48 and 72 h-treatments, respectively. Oxidative and ER stress increased after treatment. In microscopy, we observed apoptotic hallmarks like nuclear condensation, cellular shrinkage and membrane blebbings. In immunochemistry, increased Bax and decreased PCNA were apparent. Copper(II) complex with its relatively low IC₅₀ can also be tested on other cancer and normal cell lines.     

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.