Biomedical subjects

A Helenius

Publications and source records attributed to A Helenius.

At least 19 recordsLinked to original sources

PubMed2001

Quaternary and domain structure of glycoprotein processing glucosidase II.

Glucose trimming from newly synthesized glycoproteins regulates their interaction with the calnexin/calreticulin chaperone system. We have recently proposed that glucosidase II consisted of two different subunits, alpha and beta. The alpha subunit is the catalytic component, and deletion of its homologue in yeast obliterates glucosidase II activity. Deletion of the homologue of the noncatalytic beta subunit in Schizosaccharomices pombe drastically reduces glucosidase II activity, but the role of the beta subunit in glucosidase II activity has not been established. Furthermore, a direct interaction between alpha and beta subunits has not been demonstrated. Using chemical cross-linking and hydrodynamic analysis by analytical ultracentrifugation, we found that the two subunits form a defined complex, composed of one catalytic subunit and one accessory subunit (alpha(1)beta(1)) with a molecular mass of 161 kDa. The complex had an s value of 6.3 S, indicative of a highly nonglobular shape. The asymmetric shape of the alpha(1)beta(1) complex was confirmed by its high susceptibility to proteases. The beta subunit could be proteolytically removed from the alpha(1)beta(1) complex without affecting catalysis, demonstrating that it is not required for glucosidase II activity in vitro. Furthermore, we isolated a monomeric C-terminal fragment of the alpha subunit, which retained full glucosidase activity. We conclude that the catalytic core of glucosidase II resides in a globular domain of the alpha subunit, which can function independently of the beta subunit, while the complete alpha and beta subunits assemble in a defined heterodimeric complex with a highly extended conformation, which may favor interaction with other proteins in the endoplasmic reticulum (ER). Through its C-terminal HDEL signal, the beta subunit may retain the complete alpha(1)beta(1) complex in the ER.

Amino Acid Sequence↗

PubMed2001

Intracellular functions of N-linked glycans.

N-linked oligosaccharides arise when blocks of 14 sugars are added cotranslationally to newly synthesized polypeptides in the endoplasmic reticulum (ER). These glycans are then subjected to extensive modification as the glycoproteins mature and move through the ER via the Golgi complex to their final destinations inside and outside the cell. In the ER and in the early secretory pathway, where the repertoire of oligosaccharide structures is still rather small, the glycans play a pivotal role in protein folding, oligomerization, quality control, sorting, and transport. They are used as universal "tags" that allow specific lectins and modifying enzymes to establish order among the diversity of maturing glycoproteins. In the Golgi complex, the glycans acquire more complex structures and a new set of functions. The division of synthesis and processing between the ER and the Golgi complex represents an evolutionary adaptation that allows efficient exploitation of the potential of oligosaccharides.

Animals↗

PubMed2001

NMR structure of the calreticulin P-domain.

The NMR structure of the rat calreticulin P-domain, comprising residues 189-288, CRT(189-288), shows a hairpin fold that involves the entire polypeptide chain, has the two chain ends in close spatial proximity, and does not fold back on itself. This globally extended structure is stabilized by three antiparallel beta-sheets, with the beta-strands comprising the residues 189-192 and 276-279, 206-209 and 262-265, and 223-226 and 248-251, respectively. The hairpin loop of residues 227-247 and the two connecting regions between the beta-sheets contain a hydrophobic cluster, where each of the three clusters includes two highly conserved tryptophyl residues, one from each strand of the hairpin. The three beta-sheets and the three hydrophobic clusters form a repeating pattern of interactions across the hairpin that reflects the periodicity of the amino acid sequence, which consists of three 17-residue repeats followed by three 14-residue repeats. Within the global hairpin fold there are two well-ordered subdomains comprising the residues 219-258, and 189-209 and 262-284, respectively. These are separated by a poorly ordered linker region, so that the relative orientation of the two subdomains cannot be precisely described. The structure type observed for CRT(189-288) provides an additional basis for functional studies of the abundant endoplasmic reticulum chaperone calreticulin.

Amino Acid Sequence↗

PubMed2001

Quality control in the secretory assembly line.

As a rule, only proteins that have reached a native, folded and assembled structure are transported to their target organelles and compartments within the cell. In the secretory pathway of eukaryotic cells, this type of sorting is particularly important. A variety of molecular mechanisms are involved that distinguish between folded and unfolded proteins, modulate their intracellular transport, and induce degradation if they fail to fold. This phenomenon, called quality control, occurs at several levels and involves different types of folding sensors. The quality control system provides a stringent and versatile molecular sorting system that guaranties fidelity of protein expression in the secretory pathway.

Endoplasmic Reticulum↗

PubMed2001

Three-dimensional structure topology of the calreticulin P-domain based on NMR assignment.

Calreticulin (CRT) is an abundant molecular chaperone of the endoplasmic reticulum. Its central, proline-rich P-domain, comprising residues 189-288, contains three copies of each of two repeat sequences (types 1 and 2), which are arranged in a characteristic '111222' pattern. Here we show that the three-dimensional structure of CRT(189-288) contains a single hairpin fold formed by the entire polypeptide chain. The loop at the bottom of the hairpin consists of residues 227-247, and is closed by an anti-parallel beta-sheet of residues 224-226 and 248-250. Two additional beta-sheets contain residues 207-209 and 262-264, and 190-192 and 276-278. The 17-residue spacing of the beta-strands in the N-terminal part of the hairpin and the 14-residue spacing in the C-terminal part reflect the length of the type 1 and type 2 sequence repeats. As a consequence of this topology the peptide segments separating the beta-strands in the N-terminal part of the hairpin are likely to form bulges to accommodate the extra residues. These results are based on nearly complete sequence-specific NMR assignments for CRT(189-288), which were obtained using standard NMR techniques with the (13)C/(15)N-labeled protein, and collection of nuclear Overhauser enhancement upper distance constraints.

Amino Acid Sequence↗

PubMed2001

ER quality control: towards an understanding at the molecular level.

The process of 'quality control' in the endoplasmic reticulum (ER) involves a variety of mechanisms that collectively ensure that only correctly folded, assembled and modified proteins are transported along the secretory pathway. In contrast, non-native proteins are retained and eventually targeted for degradation. Recent work provides the first structural insights into the process of glycoprotein folding in the ER involving the lectin chaperones calnexin and calreticulin. Underlying principles governing the choice of chaperone system engaged by different proteins have also been discovered.

Animals↗

PubMed2001

Caveolar endocytosis of simian virus 40 reveals a new two-step vesicular-transport pathway to the ER.

Simian virus 40 (SV40) is unusual among animal viruses in that it enters cells through caveolae, and the internalized virus accumulates in a smooth endoplasmic reticulum (ER) compartment. Using video-enhanced, dual-colour, live fluorescence microscopy, we show the uptake of individual virus particles in CV-1 cells. After associating with caveolae, SV40 leaves the plasma membrane in small, caveolin-1-containing vesicles. It then enters larger, peripheral organelles with a non-acidic pH. Although rich in caveolin-1, these organelles do not contain markers for endosomes, lysosomes, ER or Golgi, nor do they acquire ligands of clathrin-coated vesicle endocytosis. After several hours in these organelles, SV40 is sorted into tubular, caveolin-free membrane vesicles that move rapidly along microtubules, and is deposited in perinuclear, syntaxin 17-positive, smooth ER organelles. The microtubule-disrupting agent nocodazole inhibits formation and transport of these tubular carriers, and blocks viral infection. Our results demonstrate the existence of a two-step transport pathway from plasma-membrane caveolae, through an intermediate organelle (termed the caveosome), to the ER. This pathway bypasses endosomes and the Golgi complex, and is part of the productive infectious route used by SV40.

Animals↗

PubMed2001

Folding of hepatitis C virus E1 glycoprotein in a cell-free system.

The hepatitis C virus (HCV) envelope proteins, E1 and E2, form noncovalent heterodimers and are leading candidate antigens for a vaccine against HCV. Studies in mammalian cell expression systems have focused primarily on E2 and its folding, whereas knowledge of E1 folding remains fragmentary. We used a cell-free in vitro translation system to study E1 folding and asked whether the flanking proteins, Core and E2, influence this process. We translated the polyprotein precursor, in which the Core is N-terminal to E1, and E2 is C-terminal, and found that when the core protein was present, oxidation of E1 was a slow, E2-independent process. The half-time for E1 oxidation was about 5 h in the presence or absence of E2. In contrast with previous reports, analysis of three constructs of different lengths revealed that the E2 glycoprotein undergoes slow oxidation as well. Unfolded or partially folded E1 bound to the endoplasmic reticulum chaperones calnexin and (with lower efficiency) calreticulin, whereas no binding to BiP/GRP78 or GRP94 could be detected. Release from calnexin and calreticulin was used to assess formation of mature E1. When E1 was expressed in the absence of Core and E2, its oxidation was impaired. We conclude that E1 folding is a process that is affected not only by E2, as previously shown, but also by the Core. The folding of viral proteins can thus depend on complex interactions between neighboring proteins within the polyprotein precursor.

Cell-Free System↗

PubMed2000

Chaperone selection during glycoprotein translocation into the endoplasmic reticulum.

A variety of molecular chaperones and folding enzymes assist the folding of newly synthesized proteins in the endoplasmic reticulum. Here we investigated why some glycoproteins interact with the molecular chaperone BiP, and others with the calnexin/calreticulin pathway. The folding of Semliki forest virus glycoproteins and influenza hemagglutinin was studied in living cells. The initial choice of chaperone depended on the location of N-linked glycans in the growing nascent chain. Direct interaction with calnexin and calreticulin without prior interaction with BiP occurred if glycans were present within about 50 residues of the protein's NH2-terminus.

Animals↗

PubMed2000

Conformational requirements for glycoprotein reglucosylation in the endoplasmic reticulum.

Newly synthesized glycoproteins interact during folding and quality control in the ER with calnexin and calreticulin, two lectins specific for monoglucosylated oligosaccharides. Binding and release are regulated by two enzymes, glucosidase II and UDP-Glc:glycoprotein:glycosyltransferase (GT), which cyclically remove and reattach the essential glucose residues on the N-linked oligosaccharides. GT acts as a folding sensor in the cycle, selectively reglucosylating incompletely folded glycoproteins and promoting binding of its substrates to the lectins. To investigate how nonnative protein conformations are recognized and directed to this unique chaperone system, we analyzed the interaction of GT with a series of model substrates with well defined conformations derived from RNaseB. We found that conformations with slight perturbations were not reglucosylated by GT. In contrast, a partially structured nonnative form was efficiently recognized by the enzyme. When this form was converted back to a nativelike state, concomitant loss of recognition by GT occurred, reproducing the reglucosylation conditions observed in vivo with isolated components. Moreover, fully unfolded conformers were poorly recognized. The results indicated that GT is able to distinguish between different nonnative conformations with a distinct preference for partially structured conformers. The findings suggest that discrete populations of nonnative conformations are selectively reglucosylated to participate in the calnexin/calreticulin chaperone pathway.

Amino Acid Sequence↗

PubMed2000

Recognition of local glycoprotein misfolding by the ER folding sensor UDP-glucose:glycoprotein glucosyltransferase.

The endoplasmic reticulum (ER) contains a stringent quality control system that ensures the correct folding of newly synthesized proteins to be exported via the secretory pathway. In this system UDP-Glc:glycoprotein glucosyltransferase (GT) serves as a glycoprotein specific folding sensor by specifically glucosylating N-linked glycans in misfolded glycoproteins thus retaining them in the calnexin/calreticulin chaperone cycle. To investigate how GT senses the folding status of glycoproteins, we generated RNase B heterodimers consisting of a folded and a misfolded domain. Only glycans linked to the misfolded domain were found to be glucosylated, indicating that the enzyme recognizes folding defects at the level of individual domains and only reglucosylates glycans directly attached to a misfolded domain. The result was confirmed with complexes of soybean agglutinin and misfolded thyroglobulin.

Alkylation↗

PubMed2000

Role of ribosome and translocon complex during folding of influenza hemagglutinin in the endoplasmic reticulum of living cells.

Protein folding in the living cell begins cotranslationally. To analyze how it is influenced by the ribosome and by the translocon complex during translocation into the endoplasmic reticulum, we expressed a mutant influenza hemagglutinin (a type I membrane glycoprotein) with a C-terminal extension. Analysis of the nascent chains by two-dimensional SDS-PAGE showed that ribosome attachment as such had little effect on ectodomain folding or trimer assembly. However, as long as the chains were ribosome bound and inside the translocon complex, formation of disulfides was partially suppressed, trimerization was inhibited, and the protein protected against aggregation.

Animals↗

PubMed2000

Role of the influenza virus M1 protein in nuclear export of viral ribonucleoproteins.

The protein kinase inhibitor H7 blocks influenza virus replication, inhibits production of the matrix protein (M1), and leads to a retention of the viral ribonucleoproteins (vRNPs) in the nucleus at late times of infection (K. Martin and A. Helenius, Cell 67:117-130, 1991). We show here that production of assembled vRNPs occurs normally in H7-treated cells, and we have used H7 as a biochemical tool to trap vRNPs in the nucleus. When H7 was removed from the cells, vRNP export was specifically induced in a CHO cell line stably expressing recombinant M1. Similarly, fusion of cells expressing recombinant M1 from a Semliki Forest virus vector allowed nuclear export of vRNPs. However, export was not rescued when H7 was present in the cells, implying an additional role for phosphorylation in this process. The viral NS2 protein was undetectable in these systems. We conclude that influenza virus M1 is required to induce vRNP nuclear export but that cellular phosphorylation is an additional factor.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed2000

Herpes simplex virus type 1 entry into host cells: reconstitution of capsid binding and uncoating at the nuclear pore complex in vitro.

During entry, herpes simplex virus type 1 (HSV-1) releases its capsid and the tegument proteins into the cytosol of a host cell by fusing with the plasma membrane. The capsid is then transported to the nucleus, where it docks at the nuclear pore complexes (NPCs), and the viral genome is rapidly released into the nucleoplasm. In this study, capsid association with NPCs and uncoating of the viral DNA were reconstituted in vitro. Isolated capsids prepared from virus were incubated with cytosol and purified nuclei. They were found to bind to the nuclear pores. Binding could be inhibited by pretreating the nuclei with wheat germ agglutinin, anti-NPC antibodies, or antibodies against importin beta. Furthermore, in the absence of cytosol, purified importin beta was both sufficient and necessary to support efficient capsid binding to nuclei. Up to 60 to 70% of capsids interacting with rat liver nuclei in vitro released their DNA if cytosol and metabolic energy were supplied. Interaction of the capsid with the nuclear pore thus seemed to trigger the release of the viral genome, implying that components of the NPC play an active role in the nuclear events during HSV-1 entry into host cells.

Animals↗

PubMed2000

Viral entry into the nucleus.

Because many viruses replicate in the nucleus of their host cells, they must have ways of transporting their genome and other components into and out of this compartment. For the incoming virus particle, nuclear entry is often one of the final steps in a complex transport and uncoating program. Typically, it involves recognition by importins (karyopherins), transport to the nucleus, and binding to nuclear pore complexes. Although all viruses take advantage of cellular signals and factors, viruses and viral capsids vary considerably in size, structure, and in how they interact with the nuclear import machinery. Influenza and adenoviruses undergo extensive disassembly prior to genome import; herpesviruses release their genome into the nucleus without immediate capsid disassembly. Polyoma viruses, parvoviruses, and lentivirus preintegration complexes are thought to enter in intact form, whereas the corresponding complexes of onco-retroviruses have to wait for mitosis because they cannot infect interphase nuclei.

Animals↗

PubMed1999

Setting the standards: quality control in the secretory pathway.

A variety of quality control mechanisms operate in the endoplasmic reticulum and in downstream compartments of the secretory pathway to ensure the fidelity and regulation of protein expression during cell life and differentiation. As a rule, only proteins that pass a stringent selection process are transported to their target organelles and compartments. If proper maturation fails, the aberrant products are degraded. Quality control improves folding efficiency by retaining proteins in the special folding environment of the endoplasmic reticulum, and it prevents harmful effects that could be caused by the deployment of incompletely folded or assembled proteins.

Animals↗

PubMed1999

Glycoproteins form mixed disulphides with oxidoreductases during folding in living cells.

The formation of intra- and interchain disulphide bonds constitutes an integral part of the maturation of most secretory and membrane-bound proteins in the endoplasmic reticulum. Evidence indicates that members of the protein disulphide isomerase (PDI) superfamily are part of the machinery needed for proper oxidation and isomerization of disulphide bonds. Models based on in vitro studies predict that the formation of mixed disulphide bonds between oxidoreductase and substrate is intermediate in the generation of the native intrachain disulphide bond in the substrate polypeptide. Whether this is how thiol oxidoreductases work inside the endoplasmic reticulum is not clear. Nor has it been established which of the many members of the PDI superfamily interacts directly with newly synthesized substrate proteins, because transient mixed disulphides have never been observed in the mammalian endoplasmic reticulum during oxidative protein folding. Here we describe the mechanisms involved in co- and post-translational protein oxidation in vivo. We show that the endoplasmic-reticulum-resident oxidoreductases PDI and ERp57 are directly involved in disulphide oxidation and isomerization, and, together with the lectins calnexin and calreticulin, are central in glycoprotein folding in the endoplasmic reticulum of mammalian cells.

Alkylating Agents↗

PubMed1999

Glycoprotein reglucosylation and nucleotide sugar utilization in the secretory pathway: identification of a nucleoside diphosphatase in the endoplasmic reticulum.

UDP is generated in the lumen of the endoplasmic reticulum (ER) as a product of the UDP-glucose-dependent glycoprotein reglucosylation in the calnexin/calreticulin cycle. We describe here the identification, purification and characterization of an ER enzyme that hydrolyzes UDP to UMP. This nucleoside diphosphatase is a ubiquitously expressed, soluble 45 kDa glycoprotein devoid of transmembrane domains and KDEL-related ER localization sequences. It requires divalent cations for activity and hydrolyzes UDP, GDP and IDP but not any other nucleoside di-, mono- or triphosphates, nor thiamine pyrophosphate. By eliminating UDP, which is an inhibitory product of the UDP-Glc:glycoprotein glucosyltransferase, it is likely to promote reglucosylation reactions involved in glycoprotein folding and quality control in the ER.

Acid Anhydride Hydrolases↗