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1.
In vertebrates, the thyroglobulin (Tg) gene product must be exported to the lumen of thyroid follicles for thyroid hormone synthesis. In toto, Tg is composed of multiple type-1 repeats connected by linker and hinge (altogether considered as "region I," nearly 1,200 residues); regions II-III (~720 residues); and cholinesterase-like (ChEL) domain (~570 residues). Regions II-III and ChEL rapidly acquire competence for secretion, yet regions I-II-III require 20 min to become a partially mature disulfide isomer; stabilization of a fully oxidized form requires ChEL. Transition from partially mature to mature Tg occurs as a discrete "jump" in mobility by nonreducing SDS-PAGE, suggesting formation of at most a few final pairings of Cys residues that may be separated by significant intervening primary sequence. Using two independent approaches, we have investigated which portion of Tg is engaged in this late stage of its maturation. First, we demonstrate that this event is linked to oxidation involving region I. Introduction of the Tg-C1245R mutation in the hinge (identical to that causing human goitrous hypothyroidism) inhibits this maturation, although the Cys-1245 partner remains unidentified. Second, we find that Tg truncated after its fourth type-1 repeat is a fully independent secretory protein. Together, the data indicate that final acquisition of secretory competence includes conformational maturation in the interval between linker and hinge segments of region I. 相似文献
2.
Xiaofan Wang Jaemin Lee Bruno Di Jeso A. Sonia Treglia Davide Comoletti Noga Dubi Palmer Taylor Peter Arvan 《The Journal of biological chemistry》2010,285(23):17564-17573
Thyroglobulin (Tg, precursor for thyroid hormone synthesis) is a large secreted glycoprotein composed of upstream regions I-II-III, followed by the ∼570 residue cholinesterase-like (ChEL) domain. ChEL has two identified functions: 1) homodimerization, and 2) binding to I-II-III that facilitates I-II-III oxidative maturation required for intracellular protein transport. Like its homologs in the acetylcholinesterase (AChE) family, ChEL possesses two carboxyl-terminal α-helices. We find that a Tg-AChE chimera (swapping AChE in place of ChEL) allows for dimerization with monomeric AChE, proving exposure of the carboxyl-terminal helices within the larger context of Tg. Further, we establish that perturbing trans-helical interaction blocks homodimerization of the Tg ChEL domain. Additionally, ChEL can associate with neuroligins (a related family of cholinesterase-like proteins), demonstrating potential for Tg cross-dimerization between non-identical partners. Indeed, when mutant rdw-Tg (Tg-G2298R, defective for protein secretion) is co-expressed with wild-type Tg, the two proteins cross-dimerize and secretion of rdw-Tg is partially restored. Moreover, we find that AChE and soluble neuroligins also can bind to the upstream Tg regions I-II-III; however, they cannot rescue secretion, because they cannot facilitate oxidative maturation of I-II-III. These data suggest that specific properties of distinct Tg ChEL mutants may result in distinct patterns of Tg monomer folding, cross-dimerization with wild-type Tg, and variable secretion behavior in heterozygous patients. 相似文献
3.
Jaemin Lee Xiaofan Wang Bruno Di Jeso Peter Arvan 《The Journal of biological chemistry》2009,284(19):12752-12761
The carboxyl-terminal cholinesterase-like (ChEL) domain of thyroglobulin
(Tg) has been identified as critically important in Tg export from the
endoplasmic reticulum. In a number of human kindreds suffering from congenital
hypothyroidism, and in the cog congenital goiter mouse and
rdw rat dwarf models, thyroid hormone synthesis is inhibited because
of mutations in the ChEL domain that block protein export from the endoplasmic
reticulum. We hypothesize that Tg forms homodimers through noncovalent
interactions involving two predicted α-helices in each ChEL domain that
are homologous to the dimerization helices of acetylcholinesterase. This has
been explored through selective epitope tagging of dimerization partners and
by inserting an extra, unpaired Cys residue to create an opportunity for
intermolecular disulfide pairing. We show that the ChEL domain is necessary
and sufficient for Tg dimerization; specifically, the isolated ChEL domain can
dimerize with full-length Tg or with itself. Insertion of an N-linked
glycan into the putative upstream dimerization helix inhibits homodimerization
of the isolated ChEL domain. However, interestingly, co-expression of upstream
Tg domains, either in cis or in trans, overrides the
dimerization defect of such a mutant. Thus, although the ChEL domain provides
a nidus for Tg dimerization, interactions of upstream Tg regions with the ChEL
domain actively stabilizes the Tg dimer complex for intracellular
transport.The synthesis of thyroid hormone in the thyroid gland requires secretion of
thyroglobulin (Tg)2 to
the apical luminal cavity of thyroid follicles
(1). Once secreted, Tg is
iodinated via the activity of thyroid peroxidase
(2). A coupling reaction
involving a quinol-ether linkage especially engages di-iodinated tyrosyl
residues 5 and 130 to form thyroxine within the amino-terminal portion of the
Tg polypeptide (3,
4). Preferential iodination of
Tg hormonogenic sites is dependent not on the specificity of the peroxidase
(5) but upon the native
structure of Tg (6,
7). To date, no other thyroidal
proteins have been shown to effectively substitute in this role for Tg.The first 80% of the primary structure of Tg (full-length murine Tg: 2,746
amino acids) involves three regions called I-II-III comprised of
disulfide-rich repeat domains held together by intradomain disulfide bonds
(8,
9). The final 581 amino acids
of Tg are strongly homologous to acetylcholinesterase
(10–12).
Rate-limiting steps in the overall process of Tg secretion involve its
structural maturation within the endoplasmic reticulum (ER)
(13). Interactions between
regions I-II-III and the cholinesterase-like (ChEL) domain have recently been
suggested to be important in this process, with ChEL functioning as an
intramolecular chaperone and escort for I-II-III
(14). In addition, Tg
conformational maturation culminates in Tg homodimerization
(15,
16) with progression to a
cylindrical, and ultimately, a compact ovoid structure
(17–19).In human congenital hypothyroidism with deficient Tg, the ChEL domain is a
commonly affected site of mutation, including the recently described A2215D
(20,
21), R2223H
(22), G2300D, R2317Q
(23), G2355V, G2356R, and the
skipping of exon 45 (which normally encodes 36 amino acids), as well as the
Q2638stop mutant (24) (in
addition to polymorphisms including P2213L, W2482R, and R2511Q that may be
associated with thyroid overgrowth
(25)). As best as is currently
known, all of the congenital hypothyroidism-inducing Tg mutants are defective
for intracellular transport
(26). A homozygous G2300R
mutation (equivalent to residue 2,298 of mouse Tg) in the ChEL domain is
responsible for congenital hypothyroidism in rdw rats
(27,
28), whereas we identified the
Tg-L2263P point mutation as the cause of hypothyroidism in the cog
mouse (29). Such mutations
perturb intradomain structure
(30), and interestingly, block
homodimerization (31).
Acquisition of quaternary structure has long been thought to be required for
efficient export from the ER
(32) as exemplified by
authentic acetylcholinesterase
(33,
34) in which dimerization
enhances protein stability and export
(35).Tg comprised only of regions I-II-III (truncated to lack the ChEL domain)
is blocked within the ER (30),
whereas a secretory version of the isolated ChEL domain of Tg devoid of
I-II-III undergoes rapid and efficient intracellular transport and secretion
(14). A striking homology
positions two predicted α-helices of the ChEL domain to the identical
relative positions of the dimerization helices in acetylcholinesterase. This
raises the possibility that ChEL may serve as a homodimerization domain for
Tg, providing a critical function in maturation for Tg transport to the site
of thyroid hormone synthesis
(1).In this study, we provide unequivocal evidence for homodimerization of the
ChEL domain and “hetero”-dimerization of that domain with
full-length Tg, and we provide significant evidence that the predicted ChEL
dimerization helices provide a nidus for Tg assembly. On the other hand, our
data also suggest that upstream Tg regions known to interact with ChEL
(14) actively stabilize the Tg
dimer complex. Together, I-II-III and ChEL provide unique contributions to the
process of intracellular transport of Tg through the secretory pathway. 相似文献
4.
Location of sequences within rotavirus SA11 glycoprotein VP7 which direct it to the endoplasmic reticulum. 总被引:7,自引:0,他引:7
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P L Whitfeld C Tyndall S C Stirzaker A R Bellamy G W Both 《Molecular and cellular biology》1987,7(7):2491-2497
The Simian 11 rotavirus glycoprotein VP7 is directed to the endoplasmic reticulum (ER) of the cell and retained as an integral membrane protein. The gene coding for VP7 predicts two potential initiation codons, each of which precedes a hydrophobic region of amino acids (H1 and H2) with the characteristics of a signal peptide. Using the techniques of gene mutagenesis and expression, we have determined that either hydrophobic domain alone can direct VP7 to the ER. A protein lacking both hydrophobic regions was not transported to the ER. Some polypeptides were directed across the ER membrane and then into the secretory pathway of the cell. For a variant retaining only the H1 domain, secretion was cleavage dependent, since an amino acid change which prevented cleavage also stopped secretion. However, secretion of two other deletion mutants lacking H1 and expressing truncated H2 domains was unaffected by this mutation, suggesting that these proteins were secreted without cleavage of their NH2-terminal hydrophobic regions or secreted after cleavage at a site(s) not predicted by current knowledge. 相似文献
5.
The glycan domain of thrombopoietin (TPO) acts in trans to enhance secretion of the hormone and other cytokines 总被引:1,自引:0,他引:1
Thrombopoietin (TPO), the primary regulator of platelet production, is composed of an amino-terminal 152 amino acids, sufficient for activity, and a carboxyl-terminal region rich in carbohydrates (183 residues) that enhances secretion of the molecule. Full-length TPO is secreted at levels 10-20-fold greater than truncated TPO. By introducing into mammalian cells a novel cDNA encoding the TPO secretory leader linked to its carboxyl-terminal domain (TPO glycan domain (TGD)), we tested whether TGD could function in trans to enhance secretion of TPO. The artificial TGD was secreted, inactive in proliferation assays, and did not inhibit TPO activity. However, when co-transfected with a cDNA encoding truncated TPO, TGD enhanced secretion 4-fold, measured by specific bioassay and immunoassay. TGD also enhanced secretion of granulocyte monocyte colony-stimulating factor and stem cell factor but did not affect the production of erythropoietin, interleukin-3, growth hormone, or of full-length TPO. To localize TGD function, we added an endoplasmic reticulum (ER) retention signal to TGD and, separately, deleted the secretory leader. Deletion of the secretory leader attenuated the secretory function of TGD, whereas addition of the ER retention signal did not alter its function. To investigate the physiologic role of TGD in folding and proteasomal protection, we tested full-length and truncated TPO in assays of protein refolding, and we examined protein stability in the presence of proteasome inhibitors. We found that truncated TGD re-folds readily and that proteasome-mediated degradation contributes to the poor secretion of truncated TPO. We conclude that TGD enhances secretion of TPO and can additionally function as an inter-molecular chaperone, in part because of its ability to prevent degradation of the hormone. The cellular location of TGD action is likely to be within the ER or earlier in the secretory pathway. 相似文献
6.
Sakamoto K Bu G Chen S Takei Y Hibi K Kodera Y McCormick LM Nakao A Noda M Muramatsu T Kadomatsu K 《The Journal of biological chemistry》2011,286(10):8405-8413
Protein production within the secretory pathway is accomplished by complex but organized processes. Here, we demonstrate that the growth factor midkine interacts with LDL receptor-related protein 1 (LRP1) at high affinity (K(d) value, 2.7 nm) not only at the cell surface but also within the secretory pathway during biosynthesis. The latter premature ligand-receptor interaction resulted in aggregate formation and consequently suppressed midkine secretion and LRP1 maturation. We utilized an endoplasmic reticulum (ER) retrieval signal and an LRP1 fragment, which strongly bound to midkine and the LRP1-specialized chaperone receptor-associated protein (RAP), to construct an ER trapper. The ER trapper efficiently trapped midkine and RAP and mimicked the premature ligand-receptor interaction, i.e. suppressed maturation of the ligand and receptor. The ER trapper also diminished the inhibitory function of LRP1 on platelet-derived growth factor-mediated cell migration. Complementary to these results, an increased expression of RAP was closely associated with midkine expression in human colorectal carcinomas (33 of 39 cases examined). Our results suggest that the premature ligand-receptor interaction plays a role in protein production within the secretory pathway. 相似文献
7.
乙型肝炎病毒小表面抗原(small hepatitis B virus surface antigen,SHB)在细胞内质网上表达,沿着细胞分泌途径分泌到胞外。为系统分析SHB拓扑结构对SHB表达和分泌的影响,首先通过生物信息学预测临床病毒株HBV C8和8种基因型(A~H)代表株的SHB拓扑结构,发现这些SHB均为四次跨膜蛋白,拥有基本相同的拓扑结构。相对内质网膜而言,SHB的拓扑结构拥有3个内质网腔内区段(Inside1~Inside3)、4个跨膜螺旋区(Tmhelix1~Tmhelix4)和2个内质网膜外区段(Outside1和Outside2)。6种基因型(基因型A、B、C、D、E和G)代表株与病毒株C8的SHB拓扑结构预测结果完全相同,而基因型F和H的SHB有4个区段与C8等不完全一致。通过对C8的SHB拓扑结构各区段进行缺失突变研究,发现Inside1区段不是SHB表达和分泌所必需的;Outside1、Tmhelix2和Inside2区段是SHB表达和分泌所必需的;Tmhelix1和Outside2不是SHB表达所必需的,但为SHB分泌所必需;Tmhelix3和Tmhelix4对SHB表达有重要影响,也是SHB分泌所必需的。进一步对Outside1和Outside2进行小片段(6个氨基酸)的缺失突变研究,发现小片段缺失基本不显著影响SHB的表达,但Outside1的氨基酸55~78及Outside2是SHB分泌所必需的。本研究首次系统性分析了SHB的拓扑结构各区段对SHB表达和分泌的影响,为深入探索SHB结构与功能的关系提供了线索。 相似文献
8.
Calnexin and BiP act as sequential molecular chaperones during thyroglobulin folding in the endoplasmic reticulum 总被引:16,自引:2,他引:14
《The Journal of cell biology》1995,128(1):29-38
Before secretion, newly synthesized thyroglobulin (Tg) folds via a series of intermediates: disulfide-linked aggregates and unfolded monomers-->folded monomers-->dimers. Immediately after synthesis, very little Tg associated with calnexin (a membrane-bound molecular chaperone in the ER), while a larger fraction bound BiP (a lumenal ER chaperone); dissociation from these chaperones showed superficially similar kinetics. Calnexin might bind selectively to carbohydrates within glycoproteins, or to hydrophobic surfaces of secretory proteins while they form proper disulfide bonds (Wada, I., W.-J. Ou, M.-C. Liu, and G. Scheele, J. Biol. Chem. 1994. 269:7464-7472). Because Tg has multiple disulfides, as well as glycans, we tested a brief exposure of live thyrocytes to dithiothreitol, which resulted in quantitative aggregation of nascent Tg, as analyzed by SDS-PAGE of cells lysed without further reduction. Cells lysed in the presence of dithiothreitol under non-denaturing conditions caused Tg aggregates to run as reduced monomers. For cells lysed either way, after in vivo reduction, Tg coprecipitated with calnexin. After washout of dithiothreitol, nascent Tg aggregates dissolved intracellularly and were secreted ultimately. 1 h after washout, > or = 92% of labeled Tg was found to dissociate from calnexin, while the fraction of labeled Tg bound to BiP rose from 0 to approximately 40%, demonstrating a "precursor-product" relationship. Whereas intralumenal reduction was essential for efficient Tg coprecipitation with calnexin, Tg glycosylation was not required. These data are among the first to demonstrate sequential chaperone function involved in conformational maturation of nascent secretory proteins within the ER. 相似文献
9.
《Molecular membrane biology》2013,30(8):412-427
AbstractThe endoplasmic reticulum (ER) is a highly organized and specialized organelle optimized for the production of proteins. It is comprised of a highly interconnected network of tubules that contain a large set of resident proteins dedicated to the maturation and processing of proteins that traverse the eukaryotic secretory pathway. As protein maturation is an imperfect process, frequently resulting in misfolding and/or the formation of aggregates, proteins are subjected to a series of evaluation processes within the ER. Proteins deemed native are sorted for anterograde trafficking, while immature or non-native proteins are initially retained in the ER in an attempt to rescue the aberrant products. Terminally misfolded substrates are eventually targeted for turnover through the ER-associated degradation or ERAD pathway to protect the cell from the release of a defective product. A clearer picture of the identity of the machinery involved in these quality control evaluation processes and their mechanisms of actions has emerged over the past decade. 相似文献
10.
Evidence that luminal ER proteins are sorted from secreted proteins in a post-ER compartment. 总被引:41,自引:7,他引:34
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H R Pelham 《The EMBO journal》1988,7(4):913-918
Several soluble proteins that reside in the lumen of the ER contain a specific C-terminal sequence (KDEL) which prevents their secretion. This sequence may be recognized by a receptor that either immobilizes the proteins in the ER, or sorts them from other proteins at a later point in the secretory pathway and returns them to their normal location. To distinguish these possibilities, I have attached an ER retention signal to the lysosomal protein cathepsin D. The oligosaccharide side chains of this protein are normally modified sequentially by two enzymes to form mannose-6-phosphate residues; these enzymes do not act in the ER, but are thought to be located in separate compartments within (or near) the Golgi apparatus. Cathepsin D bearing the ER signal accumulates within the ER, but continues to be modified by the first of the mannose-6-phosphate forming enzymes. Modification is strongly temperature-dependent, which is also a feature of ER-to-Golgi transport. These results support the idea that luminal ER proteins are continuously retrieved from a post-ER compartment, and that this compartment contains N-acetylglucosaminyl-1-phosphotransferase activity. 相似文献
11.
Crystal structure of the complex of plasminogen activator inhibitor 2 with a peptide mimicking the reactive center loop 总被引:1,自引:0,他引:1
Jankova L Harrop SJ Saunders DN Andrews JL Bertram KC Gould AR Baker MS Curmi PM 《The Journal of biological chemistry》2001,276(46):43374-43382
The structure of the serpin, plasminogen activator inhibitor type-2 (PAI-2), in a complex with a peptide mimicking its reactive center loop (RCL) has been determined at 1.6-A resolution. The structure shows the relaxed state serpin structure with a prominent six-stranded beta-sheet. Clear electron density is seen for all residues in the peptide. The P1 residue of the peptide binds to a well defined pocket at the base of PAI-2 that may be important in determining the specificity of protease inhibition. The stressed-to-relaxed state (S --> R) transition in PAI-2 can be modeled as the relative motion between a quasirigid core domain and a smaller segment comprising helix hF and beta-strands s1A, s2A, and s3A. A comparison of the Ramachandran plots of the stressed and relaxed state PAI-2 structures reveals the location of several hinge regions connecting these two domains. The hinge regions cluster in three locations on the structure, ensuring a cooperative S --> R transition. We hypothesize that the hinge formed by the conserved Gly(206) on beta-strand s3A in the breach region of PAI-2 effects the S --> R transition by altering its backbone torsion angles. This torsional change is due to the binding of the P14 threonine of the RCL to the open breach region of PAI-2. 相似文献
12.
Lysosomal storage disorders are often caused by mutations that destabilize native folding and impair trafficking of secretory proteins. We demonstrate that endoplasmic reticulum (ER)-associated degradation (ERAD) prevents native folding of mutated lysosomal enzymes in patient-derived fibroblasts from two clinically distinct lysosomal storage disorders, namely Gaucher and Tay-Sachs disease. Prolonging ER retention via ERAD inhibition enhanced folding, trafficking, and activity of these unstable enzyme variants. Furthermore, combining ERAD inhibition with enhancement of the cellular folding capacity via proteostasis modulation resulted in synergistic rescue of mutated enzymes. ERAD inhibition was achieved by cell treatment with small molecules that interfere with recognition (kifunensine) or retrotranslocation (eeyarestatin I) of misfolded substrates. These different mechanisms of ERAD inhibition were shown to enhance ER retention of mutated proteins but were associated with dramatically different levels of ER stress, unfolded protein response activation, and unfolded protein response-induced apoptosis. 相似文献
13.
Eva C. Ennemann Karthikeyan Radhakrishnan Malaiyalam Mariappan Michaela Wachs Thomas H. Pringle Bernhard Schmidt Thomas Dierks 《The Journal of biological chemistry》2013,288(8):5828-5839
Formylglycine-generating enzyme (FGE) post-translationally converts a specific cysteine in newly synthesized sulfatases to formylglycine (FGly). FGly is the key catalytic residue of the sulfatase family, comprising 17 nonredundant enzymes in human that play essential roles in development and homeostasis. FGE, a resident protein of the endoplasmic reticulum, is also secreted. A major fraction of secreted FGE is N-terminally truncated, lacking residues 34–72. Here we demonstrate that this truncated form is generated intracellularly by limited proteolysis mediated by proprotein convertase(s) (PCs) along the secretory pathway. The cleavage site is represented by the sequence RYSR72↓, a motif that is conserved in higher eukaryotic FGEs, implying important functionality. Residues Arg-69 and Arg-72 are critical because their mutation abolishes FGE processing. Furthermore, residues Tyr-70 and Ser-71 confer an unusual property to the cleavage motif such that endogenous as well as overexpressed FGE is only partially processed. FGE is cleaved by furin, PACE4, and PC5a. Processing is disabled in furin-deficient cells but fully restored upon transient furin expression, indicating that furin is the major protease cleaving FGE. Processing by endogenous furin occurs mostly intracellularly, although also extracellular processing is observed in HEK293 cells. Interestingly, the truncated form of secreted FGE no longer possesses FGly-generating activity, whereas the unprocessed form of secreted FGE is active. As always both forms are secreted, we postulate that furin-mediated processing of FGE during secretion is a physiological means of higher eukaryotic cells to regulate FGE activity upon exit from the endoplasmic reticulum. 相似文献
14.
Glew MD Veith PD Peng B Chen YY Gorasia DG Yang Q Slakeski N Chen D Moore C Crawford S Reynolds EC 《The Journal of biological chemistry》2012,287(29):24605-24617
Protein substrates of a novel secretion system of Porphyromonas gingivalis contain a conserved C-terminal domain (CTD) of ~70-80 amino acid residues that is essential for their secretion and attachment to the cell surface. The CTD itself has not been detected in mature substrates, suggesting that it may be removed by a novel signal peptidase. More than 10 proteins have been shown to be essential for the proper functioning of the secretion system, and one of these, PG0026, is a predicted cysteine proteinase that also contains a CTD, suggesting that it may be a secreted component of the secretion system and a candidate for being the CTD signal peptidase. A PG0026 deletion mutant was constructed along with a PG0026C690A targeted mutant encoding an altered catalytic Cys residue. Analysis of clarified culture fluid fractions by SDS-PAGE and mass spectrometry revealed that the CTD was released intact into the surrounding medium in the wild type strain, but not in the PG0026 mutant strains. Western blot experiments revealed that the maturation of a model substrate was stalled at the CTD-removal step specifically in the PG0026 mutants, and whole cell ELISA experiments demonstrated partial secretion of substrates to the cell surface. The CTD was also shown to be accessible at the cell surface in the PG0026 mutants, suggesting that the CTD was secreted but could not be cleaved. The data indicate that PG0026 is responsible for the cleavage of the CTD signal after substrates are secreted across the OM. 相似文献
15.
Glembotski CC Thuerauf DJ Huang C Vekich JA Gottlieb RA Doroudgar S 《The Journal of biological chemistry》2012,287(31):25893-25904
The endoplasmic reticulum (ER) stress protein mesencephalic astrocyte-derived neurotrophic factor (MANF) has been reported to protect cells from stress-induced cell death before and after its secretion; however, the conditions under which it is secreted are not known. Accordingly, we examined the mechanism of MANF release from cultured ventricular myocytes and HeLa cells, both of which secrete proteins via the constitutive pathway. Although the secretion of proteins via the constitutive pathway is not known to increase upon changes in intracellular calcium, MANF secretion was increased within 30 min of treating cells with compounds that deplete sarcoplasmic reticulum (SR)/ER calcium. In contrast, secretion of atrial natriuretic factor from ventricular myocytes was not increased by SR/ER calcium depletion, suggesting that not all secreted proteins exhibit the same characteristics as MANF. We postulated that SR/ER calcium depletion triggered MANF secretion by decreasing its retention. Consistent with this were co-immunoprecipitation and live cell, zero distance, photo affinity cross-linking, demonstrating that, in part, MANF was retained in the SR/ER via its calcium-dependent interaction with the SR/ER-resident protein, GRP78 (glucose-regulated protein 78 kDa). This unusual mechanism of regulating secretion from the constitutive secretory pathway provides a potentially missing link in the mechanism by which extracellular MANF protects cells from stresses that deplete SR/ER calcium. Consistent with this was our finding that administration of recombinant MANF to mice decreased tissue damage in an in vivo model of myocardial infarction, a condition during which ER calcium is known to be dysregulated, and MANF expression is induced. 相似文献
16.
The endoplasmic reticulum (ER) contains a family of luminal proteins (reticuloplasmins) that are normally excluded from the secretory pathway. However, reticuloplasmins are efficiently secreted when murine fibroblasts are treated with calcium ionophores. The secreted and cellular forms of endoplasmin are clearly distinguishable on the basis of gel mobility and endoglycosidase H sensitivity. Reticuloplasmin secretion leads to the depletion of the proteins from the ER and their accumulation in the Golgi apparatus. The stress response to calcium ionophore induces reaccumulation of reticuloplasmins in the ER and suppresses their secretion. Secretion is also associated with changes in the structure and distribution of the ER. These observations show that perturbation of cellular calcium levels leads to the breakdown of the mechanism for ER retention of reticuloplasmins and suggest a role for calcium ions in their sorting from secretory proteins. 相似文献
17.
《Biochimica et Biophysica Acta (BBA)/Molecular and Cell Biology of Lipids》2020,1865(6):158660
Proprotein convertase subtilisin/kexin type 9 (PCSK9) is a secretory protein that promotes low-density lipoprotein receptor (LDLR) degradation and thereby regulating plasma levels of LDL cholesterol. Previous studies have revealed the role of the C-terminal domain (CTD) of PCSK9 in its secretion, however, how CTD regulates PCSK9 secretion is not completely understood. Additionally, SEC24A, the cargo adaptor protein of the coat protein complex II, has been implicated in the secretion of mouse PCSK9. Here, we investigated how CTD and SEC24 regulated PCSK9 secretion in humans. We found that mutant PCSK91–528, in which amino acids from 529 to the end (amino acid 692) were deleted, was maturated and secreted from cells as effectively as the wild-type protein. On the other hand, lacking amino acids 454 to 692 in mutant PCSK91–453 significantly reduced its maturation and secretion, but to a lesser extent when compared to mutants PCSK91–446, PCSK91–445 and PCSK91–444, that all markedly impaired PCSK9 maturation. However, mutant PCSK91–444 virtually eliminated PCSK9 secretion while PCSK91–446 and PCSK91–445 could still be adequately detected in culture medium. Interestingly, mutation of Pro445 to other amino acid residues considerably impaired the secretion of mutant PCSK91–445 but not the full-length protein. We also found that natural variants in CTD including S462P, S465L, E482G, R495Q and A522T impaired PCSK9 secretion. Further, the knockdown of SEC24A, SEC24B, SEC24C but not SEC24D reduced secretion of the full-length PCSK9 but not mutant PCSK91–446. Therefore, SEC24A, SEC24B, and SEC24C facilitate endogenous PCSK9 secretion from cultured human hepatocytes, that are most likely mediated by the CTD of PCSK9. Our studies also indicate that the CTD of PCSK9 may allosterically and independently modulate the stability of the hinge region. Collectively, these data revealed that the CTD of PCSK9 and the hinge region play a critical role in PCSK9 maturation and secretion. 相似文献
18.
Non-typable Haemophilus influenzae is a common cause of human disease and initiates infection by colonizing the upper respiratory tract. The non-typable H. influenzae HMW1 and HMW2 adhesins mediate attachment to human epithelial cells, an essential step in the process of colonization. HMW1 and HMW2 have an unusual N-terminus and undergo cleavage of a 441-amino-acid N-terminal fragment during the course of their maturation. Following translocation across the outer membrane, they remain loosely associated with the bacterial surface, except for a small amount that is released extracellularly. In the present study, we localized the signal sequence to the first 68 amino acids, which are characterized by a highly charged region from amino acids 1-48, followed by a more typical signal peptide with a predicted leader peptidase cleavage site after the amino acid at position 68. Additional experiments established that the SecA ATPase and the SecE translocase are essential for normal export and demonstrated that maturation involves cleavage first between residues 68 and 69, via leader peptidase, and next between residues 441 and 442. Site-directed mutagenesis revealed that HMW1 processing, secretion and extracellular release are dependent on amino acids in the region between residues 150 and 166 and suggested that this region interacts with the HMW1B outer membrane translocator. Deletion of the C-terminal end of HMW1 resulted in augmented extracellular release and elimination of HMW1-mediated adherence, arguing that the C-terminus may serve to tether the adhesin to the bacterial surface. These observations suggest that the HMW proteins are secreted by a variant form of the general secretory pathway and provide insight into the mechanisms of secretion of a growing family of Gram-negative bacterial exoproteins. 相似文献
19.
Corinne Lambert-Buisine Eve Willery Camille Locht & Françoise Jacob-Dubuisson 《Molecular microbiology》1998,28(6):1283-1293
The major adhesin of Bordetella pertussis , filamentous haemagglutinin (FHA), is produced and secreted at high levels by the bacterium. Mature FHA derives from a large precursor, FhaB, that undergoes several post-translational maturations. In this work, we demonstrate by site-directed mutagenesis that the N-terminal signal peptide of FHA is composed of 71 amino acids, including a 22-residue-long 'N-terminal extension' sequence. This sequence, although highly conserved in various other secretory proteins, does not appear to play an essential part in FHA secretion, as shown by deletion mutagenesis. The entire N-terminal signal region of FhaB is removed in the course of secretion by proteolytic cleavage at a site that corresponds to a Lep signal peptidase recognition sequence. After this maturation, the N-terminal glutamine residue is modified to a pyroglutamate residue. This modification is not crucial for heparin binding, haemagglutination or secretion. Interestingly, however, the modification is absent from Escherichia coli secreted FHA derivatives. In addition, it is dependent in B. pertussis on the presence of all three cysteines contained in the signal peptide of FhaB. These observations suggest that it does not occur spontaneously but perhaps requires a specific enzymatic machinery. 相似文献
20.
Tomonori Ueno Keisuke Tanaka Keiko Kaneko Yuki Taga Tetsutaro Sata Shinkichi Irie Shunji Hattori Kiyoko Ogawa-Goto 《The Journal of biological chemistry》2010,285(39):29941-29950
A coiled-coil microtubule-bundling protein, p180, was originally reported as a ribosome-binding protein on the rough endoplasmic reticulum (ER) and is highly expressed in secretory tissues. Recently, we reported a novel role for p180 in the trans-Golgi network (TGN) expansion following stimulated collagen secretion. Here, we show that p180 plays a key role in procollagen biosynthesis and secretion in diploid fibroblasts. Depletion of p180 caused marked reductions of secreted collagens without significant loss of the ER membrane or mRNA. Metabolic labeling experiments revealed that the procollagen biosynthetic activity was markedly affected following p180 depletion. Moreover, loss of p180 perturbs ascorbate-stimulated de novo biosynthesis mainly in the membrane fraction with a preferential secretion defect of large proteins. At the EM level, one of the most prominent morphological features of p180-depleted cells was insufficient ribosome association on the ER membranes. In contrast, the ER of control cells was studded with numerous ribosomes, which were further enhanced by ascorbate. Similarly biochemical analysis confirmed that levels of membrane-bound ribosomes were altered in a p180-dependent manner. Taken together, our data suggest that p180 plays crucial roles in enhancing collagen biosynthesis at the entry site of the secretory compartments by a novel mechanism that mainly involves facilitating ribosome association on the ER. 相似文献