内质网应激反应与衰老相关疾病

内质网是真核细胞合成膜蛋白和分泌蛋白的主要场所,当细胞经历缺氧、钙离子稳态失衡、糖基化异常或内质网内蛋白合成急剧增加时,就会造成腔内未折叠蛋白聚集体的形成,引发细胞毒性.这时便会激活一系列信号通路,通过增加内质网中分子伴侣的数量、降低蛋白合成速率、加快未折叠蛋白降解来保护细胞,当刺激严重或时间过长则会引起细胞凋亡.这种反应就称为内质网应激反应,也叫未折叠蛋白反应.正常人体细胞随着分裂次数增加或受外界因素诱导逐渐进入一种不可逆的细胞周期阻滞,即细胞衰老.细胞衰老会伴随着各种生理生化的变化,如内质网的结果和功能的改变.内质网应激反应会随着细胞衰老而发生一些改变,与衰老相关疾病密切相关而备受关注.因此深入研究内质网应激反应对于揭示衰老及衰老相关疾病的分子机制具有重要的科学意义.     

Sequence and topology of a model intracellular membrane protein, E1 glycoprotein, from a coronavirus

In the eukaryotic cell, both secreted and plasma membrane proteins are synthesized at the endoplasmic reticulum, then transported, via the Golgi complex, to the cell surface. Each of the compartments of this transport pathway carries out particular metabolic functions, and therefore presumably contains a distinct complement of membrane proteins. Thus, mechanisms must exist for localizing such proteins to their respective destinations. However, a major obstacle to the study of such mechanisms is that the isolation and detailed analysis of such internal membrane proteins pose formidable technical problems. We have therefore used the E1 glycoprotein from coronavirus MHV-A59 as a viral model for this class of protein. Here we present the primary structure of the protein, determined by analysis of cDNA clones prepared from viral mRNA. In combination with a previous study of its assembly into the endoplasmic reticulum membrane, the sequence reveals several unusual features of the protein which may be related to its intracellular localization.     

Assembly of yeast Sec proteins involved in translocation into the endoplasmic reticulum into a membrane-bound multisubunit complex

Secretory-protein translocation into the endoplasmic reticulum (ER) is thought to be catalysed by integral membrane proteins. Genetic selections uncovered three Saccharomyces cerevisiae genes (SEC61, SEC62 and SEC63), mutations in which block import of precursor proteins into the ER lumen in vivo and in vitro. The DNA sequences of SEC62 and SEC63 predict multispanning membrane proteins, and biochemical characterization of the SEC62 protein (Sec62) confirms that it is an integral ER membrane protein. Here we show that Sec61, Sec62 and Sec63 are assembled with two additional proteins into a multisubunit membrane-associated complex. These results confirm previous predictions, based upon genetic interactions between the SEC genes, that Sec61, Sec62 and Sec63 act together to facilitate protein translocation into the ER.     

A substrate-specific inhibitor of protein translocation into the endoplasmic reticulum

The segregation of secretory and membrane proteins to the mammalian endoplasmic reticulum is mediated by remarkably diverse signal sequences that have little or no homology with each other. Despite such sequence diversity, these signals are all recognized and interpreted by a highly conserved protein-conducting channel composed of the Sec61 complex. Signal recognition by Sec61 is essential for productive insertion of the nascent polypeptide into the translocation site, channel gating and initiation of transport. Although subtle differences in these steps can be detected between different substrates, it is not known whether they can be exploited to modulate protein translocation selectively. Here we describe cotransin, a small molecule that inhibits protein translocation into the endoplasmic reticulum. Cotransin acts in a signal-sequence-discriminatory manner to prevent the stable insertion of select nascent chains into the Sec61 translocation channel. Thus, the range of substrates accommodated by the channel can be specifically and reversibly modulated by a cell-permeable small molecule that alters the interaction between signal sequences and the Sec61 complex.     

Post-translational insertion of a fragment of the glucose transporter into microsomes requires phosphoanhydride bond cleavage

Most eukaryotic secretory and membrane proteins insert co-translationally into the membrane of the rough endoplasmic reticulum (RER), and are targeted there by one or more NH2-terminal or internal signal sequences. However, little is known about the actual translocation and membrane integration processes. In particular, any energy requirements for targeting and integration have remained obscure because of the inability to uncouple the processes from concomitant protein synthesis. We recently showed that the human glucose transporter (GT), an integral membrane glycoprotein, can insert post-translationally into dog pancreatic microsomes with low but demonstrable efficiency in vitro, and that a fragment corresponding to the NH2-terminal 340 amino acids and 8 of the 12 membrane-spanning alpha-helixes of GT (GT-N) can insert with significantly greater efficiency. We report here that post-translational insertion of GT-N into pancreatic microsomes requires energy in the form of a phosphodiester bond, and suggest that co-translational insertion of proteins into the RER may also require energy independent of that used for polypeptide synthesis.     

Xenopus oocytes can secrete bacterial beta-lactamase

Most secretory proteins are synthesized as precursor polypeptides carrying N-terminal, hydrophobic sequences which, by means of a signal recognition particle (SRP), trigger the membrane transfer of the polypeptide and are subsequently cleaved off. The signal sequences appear to be interchangeable between prokaryotes and eukaryotes. In bacteria, secretion only involves the crossing of a membrane, whereas in eukaryotes the secretory process can be separated into two distinct phases: translocation across the membrane of the rough endoplasmic reticulum and subsequent intraluminal transport by processes involving vesicle budding and fusion. Since secretory proteins must be distinguished from other soluble proteins destined for various sites in the reticular system, it is conceivable that eukaryotic secretory proteins possess additional markers distinct from the signal peptide to guide the polypeptide after its transfer through the membrane. Proteins are secreted at different rates from a eukaryotic cell, suggesting a role in intracellular transport for receptors with differing affinities for some topogenic features in secretory proteins. We have tested this possibility by introducing into the lumen of eukaryotic rough endoplasmic reticulum a prokaryotic protein which, by virtue of its origin, had not been adapted to the eukaryotic secretory pathway. We reasoned that secretion of the bacterial protein would indicate that after membrane transfer no topogenic signal(s) and corresponding recognition system(s) are required. We report here that this is indeed the case.     

Identification by anti-idiotype antibodies of an intracellular membrane protein that recognizes a mammalian endoplasmic reticulum retention signal

Monoclonal antibodies were raised against antibodies to distinct carboxy-terminal KDEL sequences of two soluble, resident endoplasmic reticulum proteins. These anti-idiotype reagents recognize an intrinsic membrane protein with characteristics expected of a receptor responsible for the recognition and return of resident proteins to the endoplasmic reticulum.     

Evidence for the involvement of ATP in co-translational protein translocation

Identification of the source of energy for protein translocation across biological membranes is important in understanding the mechanism of this process. In eukaryotic cells, the tight coupling between translation and translocation and firm attachment of the secreting ribosomes to membranes, as well as theoretical calculations, have led to the suggestion that energy derived from protein synthesis is sufficient for protein translocation. On the other hand, in bacterial systems neither the attachment of ribosomes to membrane (other than nascent chains) nor tight coupling of translocation to translocation has been observed. Moreover, certain proteins can be translocated across membranes either at the time of, or after, translation. The separation of protein translocation from translation has made possible the demonstration that ATP hydrolysis is essential for post-translational protein translocation across bacterial membranes and, more recently, also across canine and yeast endoplasmic reticulum membranes. Here we report that certain ATP analogues inhibit co-translational protein translocation at concentrations that do not interfere with protein synthesis, suggesting that ATP is also required for co-translational protein translocation.     

X-ray structure of a bacterial oligosaccharyltransferase

Asparagine-linked glycosylation is a post-translational modification of proteins containing the conserved sequence motif Asn-X-Ser/Thr. The attachment of oligosaccharides is implicated in diverse processes such as protein folding and quality control, organism development or host-pathogen interactions. The reaction is catalysed by oligosaccharyltransferase (OST), a membrane protein complex located in the endoplasmic reticulum. The central, catalytic enzyme of OST is the STT3 subunit, which has homologues in bacteria and archaea. Here we report the X-ray structure of a bacterial OST, the PglB protein of Campylobacter lari, in complex with an acceptor peptide. The structure defines the fold of STT3 proteins and provides insight into glycosylation sequon recognition and amide nitrogen activation, both of which are prerequisites for the formation of the N-glycosidic linkage. We also identified and validated catalytically important, acidic amino acid residues. Our results provide the molecular basis for understanding the mechanism of N-linked glycosylation.     

Recognition of transmembrane helices by the endoplasmic reticulum translocon

Membrane proteins depend on complex translocation machineries for insertion into target membranes. Although it has long been known that an abundance of nonpolar residues in transmembrane helices is the principal criterion for membrane insertion, the specific sequence-coding for transmembrane helices has not been identified. By challenging the endoplasmic reticulum Sec61 translocon with an extensive set of designed polypeptide segments, we have determined the basic features of this code, including a 'biological' hydrophobicity scale. We find that membrane insertion depends strongly on the position of polar residues within transmembrane segments, adding a new dimension to the problem of predicting transmembrane helices from amino acid sequences. Our results indicate that direct protein-lipid interactions are critical during translocon-mediated membrane insertion.     

Action of brefeldin A blocked by activation of a pertussis-toxin-sensitive G protein.

In many mammalian cells brefeldin A interferes with mechanisms that keep the Golgi appartus separate from the endoplasmic reticulum. The earliest effect of brefeldin A is release of the coat protein beta-COP from the Golgi. This release is blocked by pretreatment with GTP-gamma S or AlF4- (ref. 12). The AlF4- ion activates heterotrimeric G proteins but not proteins of the ras superfamily, suggesting that a heterotrimeric G protein might control membrane transfer from the endoplasmic reticulum to the Golgi. We report here that mastoparan, a peptide that activates heterotrimeric G proteins, promotes binding of beta-COP to Golgi membranes in vitro and antagonizes the effect of brefeldin A on beta-COP in perforated cells and on isolated Golgi membranes. This inhibition is greatly diminished if cells are pretreated with pertussis toxin before perforation. Thus, a heterotrimeric G protein of the Gi/Go subfamily regulates association of coat components with Golgi membranes.     

Identification of a ribosome receptor in the rough endoplasmic reticulum

Attachment of ribosomes to the membrane of the endoplasmic reticulum is one of the crucial first steps in the transport and secretion of intracellular proteins in mammalian cells. The process is mediated by an integral membrane protein of relative molecular mass 180,000 (Mr 180K), having a large (at least 160K) cytosolic domain that, when proteolytically detached from the membrane, can competitively inhibit the binding of ribosomes to intact membranes. Isolation of this domain has led to the identification, purification and characterization of the intact ribosome receptor, as well as its functional reconstitution into lipid vesicles.     

From endoplasmic-reticulum stress to the inflammatory response

The endoplasmic reticulum is responsible for much of a cell's protein synthesis and folding, but it also has an important role in sensing cellular stress. Recently, it has been shown that the endoplasmic reticulum mediates a specific set of intracellular signalling pathways in response to the accumulation of unfolded or misfolded proteins, and these pathways are collectively known as the unfolded-protein response. New observations suggest that the unfolded-protein response can initiate inflammation, and the coupling of these responses in specialized cells and tissues is now thought to be fundamental in the pathogenesis of inflammatory diseases. The knowledge gained from this emerging field will aid in the development of therapies for modulating cellular stress and inflammation.     

Model for signal sequence recognition from amino-acid sequence of 54K subunit of signal recognition particle

Protein targeting to the endoplasmic reticulum in mammalian cells is catalysed by signal recognition particle (SRP). Cross-linking experiments have shown that the subunit of relative molecular mass 54,000 (Mr 54K; SRP54) interacts directly with signal sequences as they emerge from the ribosome. Here we present the sequence of a complementary DNA clone of SRP54 which predicts a protein that contains a putative GTP-binding domain and an unusually methionine-rich domain. The properties of this latter domain suggest that it contains the signal sequence binding site. A previously uncharacterized Escherichia coli protein has strong homology to both domains. Closely homologous GTP-binding domains are also found in the alpha-subunit of the SRP receptor (SR alpha, docking protein) in the endoplasmic reticulum membrane and in a second E. coli protein, ftsY, which resembles SR alpha. Recent work has shown that SR alpha is a GTP-binding protein and that GTP is required for the release of SRP from the signal sequence and the ribosome on targeting to the endoplasmic reticulum membrane. We propose that SRP54 and SR alpha use GTP in sequential steps of the targeting reaction and that essential features of such a pathway are conserved from bacteria to mammals.     

莲子贮存蛋白基因的表达与子叶叶肉细胞的变构

莲子贮存蛋白基因（ＳＰＧ）的表达受发育调控。子叶叶肉细胞变构早于ＳＰＧ的开始表达,随发育过程加剧。在贮存组织中细胞变构的发生有严格的时空顺序性。变构主要表现在细胞核体积增大,外形变不规则,染色质分布不均匀化,核膜局部消失。     

A protein of the endoplasmic reticulum involved early in polypeptide translocation.

To identify components of the mammalian endoplasmic reticulum involved in the translocation of secretory proteins, crosslinking and reconstitution methods were combined. A multispanning abundant membrane glycoprotein was found which is in proximity to nascent chains early in translocation. In reconstituted proteoliposomes, this protein is stimulatory or required for the translocation of secretory proteins.     

Location of MHC-encoded transporters in the endoplasmic reticulum and cis-Golgi.

Immune recognition of intracellular proteins is mediated by major histocompatibility complex (MHC) class I molecules that present short peptides to cytotoxic T cells. Evidence suggests that peptides arise by cleavage of proteins in the cytoplasm and are transported by a signal-independent mechanism into a pre-Golgi region of the cell, where they take part in the assembly of class I heavy chains with beta 2-microglobulin (reviewed in refs 5-7). Analysis of cells that have defects in class I molecule assembly and antigen presentation has shown that this phenotype can result from mutations in either of the two ABC transporter genes located in the class II region of the MHC. This suggested that the protein complex encoded by these two genes transports peptides from the cytosol into the endoplasmic reticulum. Here we report additional evidence by showing that the transporter complex is located in the endoplasmic reticulum membrane and is probably oriented with its ATP-binding domains in the cytosol.     

Membrane curvature and mechanisms of dynamic cell membrane remodelling

Membrane curvature is no longer seen as a passive consequence of cellular activity but an active means to create membrane domains and to organize centres for membrane trafficking. Curvature can be dynamically modulated by changes in lipid composition, the oligomerization of curvature scaffolding proteins and the reversible insertion of protein regions that act like wedges in membranes. There is an interplay between curvature-generating and curvature-sensing proteins during vesicle budding. This is seen during vesicle budding and in the formation of microenvironments. On a larger scale, membrane curvature is a prime player in growth, division and movement.