植物内质网胁迫研究进展

内质网是蛋白质折叠和蛋白质糖基化修饰的重要场所。在内质网中存在多种调控机制来确保其中的蛋白质被正确地折叠、修饰和组装,以维持内质网稳态,这对于细胞正常的生理活动十分重要。然而,多种物理、化学因素均可使内质网稳态失衡,即在应激条件下,错误折叠和未折叠蛋白质的大量积累将导致内质网胁迫(endoplasmic reticulum stress, ERS),进而会引起未折叠蛋白质响应(unfolded protein response, UPR),极端情况下还会启动细胞程序性死亡(program cell death, PCD)。目前,植物内质网胁迫方面的研究较酵母和动物滞后,因此,从内质网质量控制系统和未折叠蛋白质响应2个方面对植物内质网胁迫现有研究进行了综述,以期为进一步理解内质网胁迫与植物逆境胁迫的关系提供参考。     

Lysyl oxidase: properties,specificity, and biological roles inside and outside of the cell

Lysyl oxidase (LO) plays a critical role in the formation and repair of the extracellular matrix (ECM) by oxidizing lysine residues in elastin and collagen, thereby initiating the formation of covalent crosslinkages which stabilize these fibrous proteins. Its catalytic activity depends upon both its copper cofactor and a unique carbonyl cofactor and has been shown to extend to a variety of basic globular proteins, including histone H1. Although the three-dimensional structure of LO has yet to be determined, the present treatise offers hypotheses based upon its primary sequence, which may underlie the prominent electrostatic component of its unusual substrate specificity as well as the catalysis-suppressing function of the propeptide domain of prolysyl oxidase. Recent studies have demonstrated that LO appears to function within the cell in a manner, which strongly modifies cellular activity. Newly discovered LO-like proteins also likely play unique roles in biology.     

The ER‐resident ubiquitin‐specific protease 19 participates in the UPR and rescues ERAD substrates

Ubiquitination regulates membrane events such as endocytosis, membrane trafficking and endoplasmic‐reticulum‐associated degradation (ERAD). Although the involvement of membrane‐associated ubiquitin‐conjugating enzymes and ligases in these processes is well documented, their regulation by ubiquitin deconjugases is less well understood. By screening a database of human deubiquitinating enzymes (DUBs), we have identified a putative transmembrane domain in ubiquitin‐specific protease (USP)19. We show that USP19 is a tail‐anchored ubiquitin‐specific protease localized to the ER and is a target of the unfolded protein response. USP19 rescues the ERAD substrates cystic fibrosis transmembrane conductance regulator (CFTR)ΔF508 and T‐cell receptor‐α (TCRα) from proteasomal degradation. A catalytically inactive USP19 was still able to partly rescue TCRα but not CFTRΔF508, suggesting that USP19 might also exert a non‐catalytic function on specific ERAD substrates. Thus, USP19 is the first example of a membrane‐anchored DUB involved in the turnover of ERAD substrates.     

Endoplasmic reticulum degradation. Reverse protein transport and its end in the proteasome

Degradation of misfolded or unassembled proteins of the secretory pathway is an essential function of the quality control system of the Endoplasmic Reticulum (ER). Using yeast as a model organism we show that a mutated and therefore misfolded soluble lumenal protein carboxypeptidase yscY (CPY*), and a polytopic membrane protein, the ATP-binding cassette transporter Pdr5 (Pdr5*), are retrograde transported out of the ER and degraded via the cytoplasmic ubiquitin-proteasome system. Retrograde transport depends on an intact Sec61 translocon. Complete import of CPY* into the lumen of the ER requests a new targeting mechanism for retrograde transport of the malfolded enzyme through the Sec61 channel to occur. For soluble CPY*, but not for the polytopic membrane protein Pdr5* action of the ER-lumenal Hsp70 chaperone Kar2 is necessary to deliver the protein to the ubiquitin-proteasome machinery. Polyubiquitination of CPY* and Pdr5* by the ubiquitin conjugating enzymes Ubc6 and Ubc7 is crucial for degradation to occur. Also transport of CPY* out of the ER-lumen depends on ubiquitination. Newly discovered proteins of the ER membrane, Der1, Der3/Hrd1, and Hrd3 are specifically involved in the retrograde transport processes.     

Degradation of Misfolded Endoplasmic Reticulum Glycoproteins in Saccharomyces cerevisiae Is Determined by a Specific Oligosaccharide Structure

In Saccharomyces cerevisiae, transfer of N-linked oligosaccharides is immediately followed by trimming of ER-localized glycosidases. We analyzed the influence of specific oligosaccharide structures for degradation of misfolded carboxypeptidase Y (CPY). By studying the trimming reactions in vivo, we found that removal of the terminal α1,2 glucose and the first α1,3 glucose by glucosidase I and glucosidase II respectively, occurred rapidly, whereas mannose cleavage by mannosidase I was slow. Transport and maturation of correctly folded CPY was not dependent on oligosaccharide structure. However, degradation of misfolded CPY was dependent on specific trimming steps. Degradation of misfolded CPY with N-linked oligosaccharides containing glucose residues was less efficient compared with misfolded CPY bearing the correctly trimmed Man₈GlcNAc₂ oligosaccharide. Reduced rate of degradation was mainly observed for mis- folded CPY bearing Man₆GlcNAc₂, Man₇GlcNAc₂ and Man₉GlcNAc₂ oligosaccharides, whereas Man₈GlcNAc₂ and, to a lesser extent, Man₅GlcNAc₂ oligosaccharides supported degradation. These results suggest a role for the Man₈GlcNAc₂ oligosaccharide in the degradation process. They may indicate the presence of a Man₈GlcNAc₂-binding lectin involved in targeting of misfolded glycoproteins to degradation in S. cerevisiae.     

ERp44 and ERGIC‐53 Synergize in Coupling Efficiency and Fidelity of IgM Polymerization and Secretion

Owing to the quality control mechanisms operating in the early secretory compartment, only native proteins are secreted. Despite the difficulties in assembling planar immunoglobulin M (IgM) polymers, antibody‐secreting cells can release up to thousands of IgM per second. The finding that secretory μ (μ_s) chains bind to ERGIC‐53, a lectin transporter that cycles in the early secretory compartment, suggested that ERGIC‐53 hexamers could provide a polymerization platform. Here, we show that ERGIC‐53 binds to the conserved Asn563 glycan in the C‐terminal μ_s tailpiece (μ_stp). Removal of this glycan inhibits ERGIC‐53 binding and results in the rapid formation of larger polymeric assemblies. In contrast, removal of the Asn402 oligosaccharides prevents both polymerization and secretion. ERp44, a chaperone that interacts with ERGIC‐53, binds to Cys575 in the μ_stp, providing a fail‐safe mechanism that retrieves unpolymerized IgM subunits and promotes polymerization. The coordinated action of ERGIC‐53 and ERp44 provides a way to improve the efficiency of IgM secretion without perturbing its fidelity.     

神经变性构象病及其分子基础

构象病的概念被广泛用于命名与蛋白质的构象异常相关的疾病。随着生命科学的进步,人们对神经变性疾病发病的分子机制有了较好的认识,发现几乎所有的此类疾病,诸如阿尔采末病（AD）、帕金森病（PD）、亨廷顿病（HD）以及朊蛋白病（PrD）等都具有一个共同的特征,即病变细胞中蓄积有大量错误折叠并易于聚合的蛋白质,这符合构象病的特点,所以又派生了神经变性构象病的新概念。近年来,人们在神经变性构象病的蛋白质错误折叠和聚合以及其细胞毒性方面的认识越来越走向深入,这将对寻找有效的治疗方法起到极大的推动作用。     

Protein quality control in the early secretory pathway

Eukaryotic cells are able to discriminate between native and non-native polypeptides, selectively transporting the former to their final destinations. Secretory proteins are scrutinized at the endoplasmic reticulum (ER)-Golgi interface. Recent findings reveal novel features of the underlying molecular mechanisms, with several chaperone networks cooperating in assisting the maturation of complex proteins and being selectively induced to match changing synthetic demands. 'Public' and 'private' chaperones, some of which enriched in specializes subregions, operate for most or selected substrates, respectively. Moreover, sequential checkpoints are distributed along the early secretory pathway, allowing efficiency and fidelity in protein secretion.     

Inhibitors of Protein Translocation Across the ER Membrane

Protein translocation into the endoplasmic reticulum (ER) constitutes the first step of protein secretion. ER protein import is essential in all eukaryotic cells and is particularly critical in fast‐growing tumour cells. Thus, the process can serve as target both for potential cancer drugs and for bacterial virulence factors. Inhibitors of protein transport across the ER membrane range from broad‐spectrum to highly substrate‐specific and can interfere with virtually any stage of this multistep process, and even with transport of endocytosed antigens into the cytosol for cross‐presentation.      

N‐linked glycosylation of a subunit isoforms is critical for vertebrate vacuolar H+‐ATPase (V‐ATPase) biosynthesis

The a subunit of the V₀ membrane‐integrated sector of human V‐ATPase has four isoforms, a1‐a4, with diverse and crucial functions in health and disease. They are encoded by four conserved paralogous genes, and their vertebrate orthologs have positionally conserved N‐glycosylation sequons within the second extracellular loop, EL2, of the a subunit membrane domain. Previously, we have shown directly that the predicted sequon for the a4 isoform is indeed N‐glycosylated. Here we extend our investigation to the other isoforms by transiently transfecting HEK 293 cells to express cDNA constructs of epitope‐tagged human a1‐a3 subunits, with or without mutations that convert Asn to Gln at putative N‐glycosylation sites. Expression and N‐glycosylation were characterized by immunoblotting and mobility shifts after enzymatic deglycosylation, and intracellular localization was determined using immunofluorescence microscopy. All unglycosylated mutants, where predicted N‐glycosylation sites had been eliminated by sequon mutagenesis, showed increased relative mobility on immunoblots, identical to what was seen for wild‐type a subunits after enzymatic deglycosylation. Cycloheximide‐chase experiments showed that unglycosylated subunits were turned over at a higher rate than N‐glycosylated forms by degradation in the proteasomal pathway. Immunofluorescence colocalization analysis showed that unglycosylated a subunits were retained in the ER, and co‐immunoprecipitation studies showed that they were unable to associate with the V‐ATPase assembly chaperone, VMA21. Taken together with our previous a4 subunit studies, these observations show that N‐glycosylation is crucial in all four human V‐ATPase a subunit isoforms for protein stability and ultimately for functional incorporation into V‐ATPase complexes.     

Distinct retrieval and retention mechanisms are required for the quality control of endoplasmic reticulum protein folding.

Proteins destined for the secretory pathway must first fold and assemble in the lumen of endoplasmic reticulum (ER). The pathway maintains a quality control mechanism to assure that aberrantly processed proteins are not delivered to their sites of function. As part of this mechanism, misfolded proteins are returned to the cytosol via the ER protein translocation pore where they are ubiquitinated and degraded by the 26S proteasome. Previously, little was known regarding the recognition and targeting of proteins before degradation. By tracking the fate of several mutant proteins subject to quality control, we demonstrate the existence of two distinct sorting mechanisms. In the ER, substrates are either sorted for retention in the ER or are transported to the Golgi apparatus via COPII-coated vesicles. Proteins transported to the Golgi are retrieved to the ER via the retrograde transport system. Ultimately, both retained and retrieved proteins converge at a common machinery at the ER for degradation. Furthermore, we report the identification of a gene playing a novel role specific to the retrieval pathway. The gene, BST1, is required for the transport of misfolded proteins to the Golgi, although dispensable for the transport of many normal cargo proteins.     

A Role for the DnaJ Homologue Scj1p in Protein Folding in the Yeast Endoplasmic Reticulum

Members of the eukaryotic heat shock protein 70 family (Hsp70s) are regulated by protein cofactors that contain domains homologous to bacterial DnaJ. Of the three DnaJ homologues in the yeast rough endoplasmic reticulum (RER; Scj1p, Sec63p, and Jem1p), Scj1p is most closely related to DnaJ, hence it is a probable cofactor for Kar2p, the major Hsp70 in the yeast RER. However, the physiological role of Scj1p has remained obscure due to the lack of an obvious defect in Kar2p-mediated pathways in scj1 null mutants. Here, we show that the Δscj1 mutant is hypersensitive to tunicamycin or mutations that reduce N-linked glycosylation of proteins. Although maturation of glycosylated carboxypeptidase Y occurs with wild-type kinetics in Δscj1 cells, the transport rate for an unglycosylated mutant carboxypeptidase Y (CPY) is markedly reduced. Loss of Scj1p induces the unfolded protein response pathway, and results in a cell wall defect when combined with an oligosaccharyltransferase mutation. The combined loss of both Scj1p and Jem1p exaggerates the sensitivity to hypoglycosylation stress, leads to further induction of the unfolded protein response pathway, and drastically delays maturation of an unglycosylated reporter protein in the RER. We propose that the major role for Scj1p is to cooperate with Kar2p to mediate maturation of proteins in the RER lumen.     

Pathogenic mutation of UBQLN2 impairs its interaction with UBXD8 and disrupts endoplasmic reticulum‐associated protein degradation

Protein aggregation is a common feature of several neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration. How protein aggregates are formed and contribute to neurodegeneration, however, is not clear. Mutation of Ubiquilin 2 (UBQLN2) has recently been linked to ALS and frontotemporal lobar degeneration. Therefore, we examined the effect of ALS‐linked UBQLN2 mutation on endoplasmic reticulum‐associated protein degradation (ERAD). Compared to its wild‐type counterpart, mutated UBQLN2 caused greater accumulation of the ERAD substrate Hong Kong variant of α‐1‐antitrypsin, although ERAD was disturbed by both UBQLN2 over‐expression and knockdown. Also, UBQLN2 interacted with ubiquitin regulatory X domain‐containing protein 8 (UBXD8) in vitro and in vivo, and this interaction was impaired by pathogenic mutation of UBQLN2. As UBXD8 is an endoplasmic membrane protein involved in the translocation of ubiquitinated ERAD substrates, UBQLN2 likely cooperates with UBXD8 to transport defective proteins from the endoplasmic reticulum to the cytosol for degradation, and this cell‐protective function is disturbed by pathogenic mutation of UBQLN2.

     

Medicago falcata MfSTMIR,an E3 ligase of endoplasmic reticulum‐associated degradation,is involved in salt stress response

Recent studies on E3 of endoplasmic reticulum (ER)‐associated degradation (ERAD) in plants have revealed homologs in yeast and animals. However, it remains unknown whether the plant ERAD system contains a plant‐specific E3 ligase. Here, we report that MfSTMIR, which encodes an ER‐membrane‐localized RING E3 ligase that is highly conserved in leguminous plants, plays essential roles in the response of ER and salt stress in Medicago. MfSTMIR expression was induced by salt and tunicamycin (Tm). mtstmir loss‐of‐function mutants displayed impaired induction of the ER stress‐responsive genes BiP1/2 and BiP3 under Tm treatment and sensitivity to salt stress. MfSTMIR promoted the degradation of a known ERAD substrate, CPY*. MfSTMIR interacted with the ERAD‐associated ubiquitin‐conjugating enzyme MtUBC32 and Sec61‐translocon subunit MtSec61γ. MfSTMIR did not affect MtSec61γ protein stability. Our results suggest that the plant‐specific E3 ligase MfSTMIR participates in the ERAD pathway by interacting with MtUBC32 and MtSec61γ to relieve ER stress during salt stress.     

The P2 of Wheat yellow mosaic virus rearranges the endoplasmic reticulum and recruits other viral proteins into replication‐associated inclusion bodies

Viruses commonly modify host endomembranes to facilitate biological processes in the viral life cycle. Infection by viruses belonging to the genus Bymovirus (family Potyviridae) has long been known to induce the formation of large membranous inclusion bodies in host cells, but their assembly and biological roles are still unclear. Immunoelectron microscopy of cells infected with the bymovirus Wheat yellow mosaic virus (WYMV) showed that P1, P2 and P3 are the major viral protein constituents of the membranous inclusions, whereas NIa‐Pro (nuclear inclusion‐a protease) and VPg (viral protein genome‐linked) are probable minor components. P1, P2 and P3 associated with the endoplasmic reticulum (ER), but only P2 was able to rearrange ER and form large aggregate structures. Bioinformatic analyses and chemical experiments showed that P2 is an integral membrane protein and depends on the active secretory pathway to form aggregates of ER membranes. In planta and in vitro assays demonstrated that P2 interacts with P1, P3, NIa‐Pro or VPg and recruits these proteins into the aggregates. In vivo RNA labelling using WYMV‐infected wheat protoplasts showed that the synthesis of viral RNAs occurs in the P2‐associated inclusions. Our results suggest that P2 plays a major role in the formation of membranous compartments that house the genomic replication of WYMV.     

Zonal changes in the rat liver following an acute dose of phenobarbitone: An ultrastructural,morphometric and biochemical correlation

Alterations in the liver of rats 6 h after a dose of phenobarbitone have been studied by subcellular fractionation, conventional electron microscopy and morphometric analysis. The area immediately surrounding the central vein was the only area to undergo any alterations. There was a morphometrically measurable but not observable cellular hypertrophy of 71% whilst the hepatocyte complement of rough endoplasmic reticulum (RER) and smooth endoplasmic reticulum (SER) was increased by 72% and 93% respectively. The increases in RER and SER were not apparent by observation and it is assumed that they have been diluted by the cell hypertrophy to 1% and 22% which must be below the threshold for detection by subjective observation. Following subcellular fractionation and measurement of microsomal protein, there was no significant difference in the level of microsomes isolated from control or treated rats. Therefore, the morphometrically measured increase in RER and SER would appear to be restricted to a relatively small population of hepatocytes adjacent to the central vein. Such an increase would represent only a small percentage of total microsomes in a homogenate and would almost certainly be masked by variation in animals and techniques. Disruption of RER was also observed in hepatocytes that would proliferate their SER should phenobarbitone treatment have been continued. Therefore this RER disruption would seem in no way to interfere with the process of membrane and enzyme synthesis.     

The nucleotide exchange factor activity of Grp170 may explain the non-lethal phenotype of loss of Sil1 function in man and mouse

Recent genetic work characterized homozygous mutations in the SIL1 gene as cause for the neurodegeneration that is associated with Marinesco-Sj?gren syndrome in man and the woozy mouse mutant. All reported mutations were expected to result in loss of Sil1 function. Sil1 has previously been shown to act as nucleotide exchange factor for the molecular chaperone immunoglobulin heavy chain binding protein (BiP) in the lumen of the endoplasmic reticulum (ER). In the yeast ER Lhs1p was shown to be able to substitute for Sil1p and to represent an alternative nucleotide exchange activity. Therefore, by analogy the mammalian ortholog of Lhs1p, Grp170, was suggested to be able to compensate for the loss of Sil1 function in many mammalian organs. Here we characterize mammalian Grp170 as alternative nucleotide exchange factor for BiP, thus providing a likely explanation for the non-lethal phenotype of the homozygous human and murine SIL1 mutations.