Secretion of thiols and disulfide bond formation: retraction.
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Retraction Notice↗
Biomedical subjects
R Sitia
Publications and source records attributed to R Sitia.
KIF3C, a novel member of the kinesin superfamily: sequence, expression, and mapping to human chromosome 2 at 2p23.
Kinesins are microtubule-dependent molecular motors involved in intracellular transport and mitosis. Here, we report the cloning, sequencing, mapping, and expression of a novel member of the kinesin superfamily. The sequence of this newly identified human cDNA reveals an open reading frame encoding a putative protein of 792 residues. Based on its high sequence similarity to the kinesin-like molecule KIF3B, we named this protein KIF3C. KIF3C is encoded by transcripts that are distinct from the KIF3B mRNA in human, rat, and mouse and is preferentially expressed in the brain. Fluorescence in situ hybridization reveals that, in the human genome, the KIF3C gene maps to chromosome 2 at 2p23. The sequence of KIF3C predicts an unusually long insertion in the proximity of L11, a region thought to mediate microtubule binding. Taken together, these findings suggest that KIF3C is a novel kinesin-like protein that might be specifically involved in microtubule-based transport in neuronal cells.
Amino Acid Sequence↗
Biogenesis and function of IgM: the role of the conserved mu-chain tailpiece glycans.
The tailpiece of secretory Ig-mu-chains (mu(s)tp) is highly conserved throughout evolution: in particular, a carboxy-terminal cysteine residue (Cys575) and a glycan linked to Asn563 are found in all species sequenced so far. Here we show that the mu(s)tp oligosaccharide moieties are important for the binding of J-chains and for the process of IgM polymerization. In the absence of the mu(s)tp glycans, pentamers cannot be assembled and polymers containing six or more subunits are secreted. Despite their increased valency, these molecules have a lower association rate with antigen than wild-type polymers. Unexpectedly, the C-terminal oligosaccharides also affect kinetic parameters on unpolymerized subunits. Thus, monomers lacking the C-terminal sugars because of either site-directed mutagenesis or selective enzymatic deglycosylation with endoglycosidase H, have a lower k(on) for the antigen. Taken together, our results indicate that the C-terminal mu-chain glycans can shape the structure of mu(s2)L2 subunits and their further assembly into polymers.
Animals↗
HIV-I Tat: a polypeptide for all seasons.
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Gene Products, tat↗
Differential expression of Galalpha1,3Gal epitope in polymeric and monomeric IgM secreted by mouse myeloma cells deficient in alpha2, 6-sialyltransferase.
IgM are glycoproteins secreted by plasma cells as (mu2L2)5+J or (mu2L2)6 polymers. In most species, mu- and J-chains bear five and one N -glycans, respectively. Here we compare the terminal glycosylation patterns of 4-hydroxy-3-nitrophenylacetyl (NP)-specific IgM secreted by transfectants of the J558L mouse myeloma deficient in the alpha2,6 sialyltransferase [alpha2,6ST(N)] or by a hybridoma expressing this enzyme (B1.8 cells). The absence of alpha2,6-sialylation results in an increased addition of alpha1, 3-galactosyl residues to mu- and J-chain N-glycans. Since alpha1, 3-galactosyltransferase (alpha1,3Gal-T) is similarly expressed in the two cell lines, these results indicate that a competition reaction occurs in vivo between alpha2,6ST(N) and alpha1,3Gal-T. In the alpha2,6ST(N) deficient transfectants, mu-chains lacking the C-subterminal Cys575 residue, which are secreted mainly in the form of mu2L2 monomers, are more efficiently capped by alpha1, 3-galactosyl residues, confirming that polymerization significantly reduces the accessibility of mu-chain glycans to the Golgi processing enzymes involved in the biogenesis of antennary sugars. Functional assays indicate that IgM sialylation affects antigen-binding and complement-dependent hemolysis of haptenated red blood cells.
Animals↗
Formation of one or more intrachain disulphide bonds is required for the intracellular processing and transport of CD36.
In monocytes/macrophages, CD36 is thought to have a role as a scavenger receptor, mediating the phagocytosis of apoptotic cells and the endocytic uptake of oxidized low-density lipoproteins and fatty acids. The proposed topology of CD36 predicts that, of ten cysteine residues, six lie in the extracellular domain, whereas four are equally distributed in the two short terminal tails flanking the N-terminal and C-terminal hydrophobic stretches. Here we investigate the formation of intrachain disulphide bonds, on the basis of the assumption that the cysteine residues present in the luminal domains are generally oxidized, whereas those in the cytosol are reduced. As revealed by gel mobility-shift assays, disulphide bonds are present in the extracellular domain of the CD36 molecule. The formation of these bonds is required for the transport of CD36 from endoplasmic reticulum to Golgi. Furthermore reactive thiol groups are present in the CD36 sequence, which upon lysis form an intrachain extra loop as an artifact. This disulphide bond is not formed in either (1) truncated CD36 lacking the two C-terminal cysteine residues or (2) Triton X-100-insoluble wild-type CD36 molecules, suggesting that, in this fraction, the C-terminal thiol groups are modified.
Biological Transport↗
Cysteine and glutathione secretion in response to protein disulfide bond formation in the ER.
Protein folding in the endoplasmic reticulum (ER) often involves the formation of disulfide bonds. The oxidizing conditions required within this organelle were shown to be maintained through the release of small thiols, mainly cysteine and glutathione. Thiol secretion was stimulated when proteins rich in disulfide bonds were translocated into the ER, and secretion was prevented by the inhibition of protein synthesis. Endogenously generated cysteine and glutathione counteracted thiol-mediated retention in the ER and altered the extracellular redox. The secretion of thiols might link disulfide bond formation in the ER to intra- and intercellular redox signaling.
Animals↗
The association of HIV-1 Tat with nuclei is regulated by Ca2+ ions and cytosolic factors.
Human immunodeficiency virus-1 (HIV-1) Tat, a nuclear transcription factor, has been shown to function extracellularly, implying that some Tat molecules escape nuclear import and are secreted. This raises the question of what regulates, in HIV-1-infected cells, the nuclear targeting of the polypeptide. Here we show that cytosolic components activated by Ca2+ ions are required to reveal the karyophilic properties of Tat: in vitro translated Tat molecules do not associate with isolated nuclei unless preincubated with Ca2+. Moreover, Ca2+ ions induce karyophilicity of chemically synthesized Tat molecules only upon addition of cytosolic extracts. The Ca2+-induced karyophilicity is prevented by inhibitors of either tyrosine kinases (herbimycin A and genistein) or tyrosine phosphatases (vanadate), suggesting the involvement of Ca2+-dependent phosphorylation/dephosphorylation events. In line with these observations, the transcriptional activity of Tat is inhibited by treatment with either vanadate or genistein. The same occurs with Tat mutants lacking either one or both the two tyrosine residues (positions 26 and 47). Hence, Ca2+-dependent tyrosine kinase(s) and phosphatase(s) act on accessory cellular protein(s), which in turn are responsible of Tat karyophilicity.
Animals↗
Stringent thiol-mediated retention in B lymphocytes and Xenopus oocytes correlates with inefficient IgM polymerization.
Thiol-dependent retention mechanisms involving the microsecond chain Cys575 ensure that only polymeric IgM are secreted. B lymphocytes are unable to polymerize IgM and degrade unpolymerized precursors intracellularly. Since several non-lymphoid transfectants secrete hexameric IgM, specific mechanism(s) inhibiting IgM polymerization/secretion may be active in B cells. Here, we show that Xenopus laevis oocytes are also unable to polymerize IgM and retain this isotype via Cys575 as efficiently as B cells. The mechanisms and the hierarchy of the thiol-dependent pre-Golgi retention are conserved in amphibian oocytes, as indicated by the efficient retention of secretory IgA and the slow secretion of unassembled J558 lambda chains. We also show that B cells do not lack any structural component necessary to polymerize IgM: after retention has been weakened by 2-mercaptoethanol, polymerization can occur if oxidizing conditions are restored. Since release from retention can result in polymerization, stringent retention in B cells and oocytes might be at the basis of their common inability to polymerize secretory IgM. Our findings suggest that disulfide interchange reactions in the exocytic compartment can be modulated during B cell differentiation to control IgM secretion.
Animals↗
Exposed thiols confer localization in the endoplasmic reticulum by retention rather than retrieval.
The cysteine present in the Ig micro chain tailpiece (microtp) prevents the secretion of unpolymerized IgM intermediates and causes their accumulation in the endoplasmic reticulum (ER). In principle, this can be the consequence of actual retention in this organelle or of retrieval from the Golgi. To determine which of the two mechanisms underlies the cysteine-dependent ER localization, we analyze here the post-translational modifications of suitably engineered cathepsin D (CD) molecules. The glycans of this protease are phosphorylated by post-ER phosphotransferases and further modified in the trans-Golgi to generate a mannose 6-phosphate lysosome targeting signal. Only trace amounts of the mutp-tagged CD (CDM&mutpCys) are phosphorylated, unless retention is reversed by exogenous reducing agents or the critical cysteine mutated (CDMmutpSer). In contrast, a KDEL-tagged CD, that is retrieved from the Golgi into the ER, acquires phosphates, though mainly resistant to alkaline phosphatase. Similarly to CDMmutpSer, the few CDMmutpCys molecules that escape retention and acquire phosphates in the cis-Golgi are transported beyond the KDEL retrieval compartment, as indicated by their sensitivity to alkaline phosphatase. These results demonstrate that the thiol-dependent ER localization arises primarily from true retention, without recycling through the Golgi.
Animals↗
Formation of reversible disulfide bonds with the protein matrix of the endoplasmic reticulum correlates with the retention of unassembled Ig light chains.
Exposed thiols act as intracellular retention elements for unassembled secretory molecules. Yet, some free Ig lambda light chains are secreted despite the presence of an unpaired cysteine (Cys214). This is due largely to the presence of a flanking acidic residue: substitution of Asp213 for Gly or Lys increases pre-Golgi retention and degradation of free lambda. Secretion is restored by exogenous reducing agents or by assembly with heavy chains. In the endoplasmic reticulum (ER), lambda chains form covalent complexes with many proteins through Cys214. These complexes are absent from the Golgi. They are more abundant in transfectants expressing the lambdaGly2I3 and lambdaLys213 mutants that are poorly secreted. Radioactive N-ethylmaleimide labels some monomeric lambda chains isolated from the ER, but not from the Golgi or from the medium, indicating that the Cys214 thiol is masked during ER-Golgi transport. Mass spectrometry reveals the presence of a free cysteine residue disulfide-linked to Cys214. We suggest that thiol-mediated retention involves the formation of reversible disulfide bonds with the protein matrix of the ER. The presence of an acidic residue next to the critical cysteine may allow the masking of the thiol and transport to the Golgi.
Base Sequence↗
Synthesis, processing, and intracellular transport of CD36 during monocytic differentiation.
CD36 is an integral membrane glycoprotein expressed by several cell types, including endothelial cells of the microvasculature, erythrocytes, platelets, and monocytes. In the monocytic lineage, CD36 is expressed during the late stages of differentiation in the bone marrow, in circulating monocytes, and in some tissue resident macrophages, and it is thought to mediate the phagocytosis of apoptotic cells and the endocytic uptake of modified lipoproteins. Here we analyze the synthesis, processing, and intracellular transport of CD36 in U937 and THP-1, two human cell lines representing different stages of monocytic maturation. In both cell lines, phorbol 12-myristate 13-acetate induces the expression of CD36. A 74-kDa intracellular precursor is first synthesized that has the hallmarks of a resident protein of the endoplasmic reticulum. The precursor protein is later processed into a mature form of 90-105 kDa which is transported to the cell surface. The kinetics of processing differ significantly in U937 and THP-1. These differences are specific for the CD36, as two unrelated proteins (CD11b and CD45R) are processed and transported to the surface at similar rates in the two cell lines. A 33-kDa endoglycosidase H-sensitive glycoprotein specifically associates with the 74-kDa precursor. Coprecipitation of gp33 correlates with slow processing of CD36 precursor, suggesting that gp33 may play a role in regulating the intracellular transport of CD36, during monocyte maturation.
Antibodies, Monoclonal↗
IgM polymerization inhibits the Golgi-mediated processing of the mu-chain carboxy-terminal glycans.
Secreted glycoproteins generally contain oligosaccharides of the complex type. However, several molecules have been described in which individual glycans are processed differently from one another. Folding, assembly and oligomerization could affect the maturation of certain glycans by hindering them to the Golgi processing machinery. We have tested this possibility by analysing a panel of engineered murine mu chains secreted as mu2L2 monomers or as polymers, and having or not the carboxy-terminal glycan (Asn563). In secreted IgM polymers, Asn563 bears high-mannose oligosaccharides, typical of endoplasmic reticulum resident proteins, while complex sugars are found at the other four sites (Brenckle and Kornfeld, 1980 Arch. Biochem. Biophys. 243, 605-618). Polymeric and monomeric IgM contain mu chains whose glycans are processed differently. We show here that this is mainly due to the differential processing at the Asn563 glycan, which undergoes Golgi-mediated processing when IgM are secreted in the monomeric form. These results indicate that the oligomerization-dependent accessibility to the sugar modifying enzymes can be one of the key features that dictate the extent of oligosaccharide processing in multimeric glycoproteins. The presence of high mannose glycans at Asn563 implies that IgM polymerization takes place before encountering mannosidase II, likely in a pre-Golgi compartment.
Amino Acid Sequence↗
Monitoring dynamic changes in free Ca2+ concentration in the endoplasmic reticulum of intact cells.
Direct monitoring of the free Ca2+ concentration in the lumen of the endoplasmic reticulum (ER) is an important but still unsolved experimental problem. We have shown that a Ca(2+)-sensitive photoprotein, aequorin, can be addressed to defined subcellular compartments by adding the appropriate targeting sequences. By engineering a new aequorin chimera with reduced Ca2+ affinity, retained in the ER lumen via interaction of its N-terminus with the endogenous resident protein BiP, we show here that, after emptying the ER, Ca2+ is rapidly re-accumulated up to concentrations of > 100 microM, thus consuming most of the reporter photoprotein. An estimate of the steady-state Ca2+ concentration was obtained using Sr2+, a well-known Ca2+ surrogate which elicits a significantly slower rate of aequorin consumption. Under conditions in which the rate and extent of Sr2+ accumulation in the ER closely mimick those of Ca2+, the steady-state mean lumenal Sr2+ concentration ([Sr2+]er) was approximately 2 mM. Receptor stimulation causes, in a few seconds, a 3-fold decrease of the [Sr2+]er, whereas specific inhibition of the ER Ca2+ ATPase leads to an approximately 10-fold drop in a few minutes.
Aequorin↗
High rates of thioredoxin secretion correlate with growth arrest in hepatoma cells.
Thioredoxin (TRX), a disulfide-reducing intracellular dithiol enzyme, is synthesized by both normal liver cells and the hepatocarcinoma cell line HepG2. Only the former, however, secrete abundant TRX extracellularly. When cultured in mild reducing conditions, HepG2 cells but not normal hepatocytes increase the rate of TRX secretion and undergo growth inhibition accompanied by morphological changes. Also, recombinant TRX inhibits proliferation of HepG2 cells. In contrast, exogenous thiols and TRX stimulate proliferation of a B-cell lymphoma line, indicating that different cell types respond differently to variations in the extracellular redox potential.
Acetylcysteine↗
Entry of exogenous polypeptides into the nucleus of living cells: facts and speculations.
Although the plasma membrane acts as an impermeable barrier to most macromolecules, some exogenous proteins (for example fibroblast growth factor, HIV-1 Tat and lactoferrin) can gain access into the cytosol and reach the nucleus of living cells. How are these exogenous polypeptides selected over and above other extracellular proteins? How and where do they cross the cell membrane? Why do cells need to take up exogenous transcription factors when sophisticated signal-transduction pathways are available? Here, we review the current knowledge on these issues and discuss some mechanistic and physiological implications of this unconventional and direct way of taking messages to the nucleus.
Journal Article↗
Nuclear translocation of an exogenous fusion protein containing HIV Tat requires unfolding.
OBJECTIVE: To characterize the transcellular transport of HIV-1 Tat. HIV-1 Tat contains a putative localization signal and no leader peptide; however, it can be released from virus-infected cells and taken up by uninfected cells. DESIGN AND METHODS: We constructed a chimeric protein between Tat and dihydrofolate reductase (DHFR), a cytosolic enzyme that binds tightly to the folate analogue methotrexate (MTX). As confirmed by protease sensitivity assays, binding to MTX results in stabilization of the three-dimensional structure of the DHFR domain. The nuclear translocation of recombinant proteins was monitored by both functional [transcellular transactivation of a long terminal repeat-chloramphenicol acetyl transferase (LTR-CAT) reporter gene] and biochemical (subcellular localization in HeLa cells of exogenous radiolabelled proteins) assays and the effects of MTX-induced stabilization were evaluated. RESULTS: When in vitro translated proteins are added to HeLa cells in culture, both wild-type Tat and the chimeric protein Tat-DHFR are taken up by target cells and accumulate in the nucleus, unlike wild-type DHFR. Cells transfected with Tat-DHFR, when co-cultured with cells harbouring a LTR-CAT gene, induce transactivation of the reporter gene to the same extent as cells expressing wild-type Tat. These findings indicate that Tat can mediate the internalization of unrelated polypeptides. Pre-treatment of Tat-DHFR with MTX blocks the nuclear translocation of the chimeric protein. MTX has no effect on wild-type Tat. CONCLUSION: HIV-1 Tat can act as a vector to drive polypeptides into the nucleoplasm of living cells. The inhibitor effects of MTX on the nuclear localization of Tat-DHFR suggest that an unfolding step is required for the internalization of exogenous Tat.
Cell Nucleus↗
The efficiency of cysteine-mediated intracellular retention determines the differential fate of secretory IgA and IgM in B and plasma cells.
Previous studies on IgM secretion demonstrated a role for the mu chain C-terminal cysteine (Cys575) in preventing the transport of unpolymerized subunits along the secretory pathway. The sequence homology between the C-terminal tailpieces of mu and alpha heavy chains prompted us to investigate the role of cysteine-mediated, retention in the control of IgA secretion during B cell development. Similar to IgM, IgA are not secreted by B lymphocytes: the retention mechanism can be reversed by the reducing agent 2-mercaptoethanol, suggesting that disulfide interchange reactions are involved in the quality control of both IgM and IgA. Yet, alpha 2L2 subunits, but not mu2L2, are secreted constitutively by plasma cells. We demonstrate that the differential retention of IgM and IgA subunits by myeloma transfectants is mainly due to the presence of an acidic residue upstream the alpha chain C-terminal cysteine. The regulation of polymeric Ig secretion during B cell development provides an example of how thiol-mediated quality control can be modulated according to the aminoacidic context surrounding the critical cysteine and to the cell type.
Amino Acid Sequence↗