Pkc1p modifies CPY* degradation in the ERAD pathway

The process of endoplasmic reticulum-associated degradation (ERAD) involved in the degradation of misfolded N-linked glycoproteins utilizes Cdc48p which extracts misfolded glycoproteins from the lumen to the cytosol to present them for deglycosylation and degradation. Pkc1p has been identified as a component of the ERAD pathway, because deletion of the pkc1 gene impairs ERAD and causes accumulation of CPY* in the lumen of the ER, most probably because of the mislocalization of Cdc48p. In addition, we show that Cdc48p interacts in the cytosol with the deglycosylation enzyme, PNGase, only when Cdc48p is associated with a misfolded glycoprotein.     

Intracellular degradation of unassembled asialoglycoprotein receptor subunits: a pre-Golgi, nonlysosomal endoproteolytic cleavage

The human asialoglycoprotein receptor is a heterooligomer of the two homologous subunits H1 and H2. As occurs for other oligomeric receptors, not all of the newly made subunits are assembled in the RER into oligomers and some of each chain is degraded. We studied the degradation of the unassembled H2 subunit in fibroblasts that only express H2 (45,000 mol wt) and degrade all of it. After a 30 min lag, H2 is degraded with a half-life of 30 min. We identified a 35-kD intermediate in H2 degradation; it is the COOH-terminal, exoplasmic domain of H2. After a 90-min chase, all remaining intact H2 and the 35- kD fragment were endoglycosidase H sensitive, suggesting that the cleavage generating the 35-kD intermediate occurs without translocation to the medial Golgi compartment. Treatment of cells with leupeptin, chloroquine, or NH4Cl did not affect H2 degradation. Monensin slowed but did not block degradation. Incubation at 18-20 degrees C slowed the degradation dramatically and caused an increase in intracellular H2, suggesting that a membrane trafficking event occurs before H2 is degraded. Immunofluorescence microscopy of cells with or without an 18 degrees C preincubation showed a colocalization of H2 with the ER and not with the Golgi complex. We conclude that H2 is not degraded in lysosomes and never reaches the medial Golgi compartment in an intact form, but rather degradation is initiated in a pre-Golgi compartment, possibly part of the ER. The 35-kD fragment of H2 may define an initial proteolytic cleavage in the ER.     

Nonlysosomal, pre-Golgi degradation of unassembled asialoglycoprotein receptor subunits: a TLCK- and TPCK-sensitive cleavage within the ER

The human asialoglycoprotein receptor subunit H2a is cotranslationally inserted into the ER membrane. When expressed together with subunit H1 in mouse fibroblasts part forms a hetero-oligomer that is transported to the cell surface, but when expressed alone it is all rapidly degraded. Degradation is insensitive to lysosomotropic agents and the undegraded precursor is last detected in the ER region of the cell. Small amounts of an intermediate 35-kD degradation product can be detected (Amara, J. F., G. Lederkremer, and H. F. Lodish. 1989. J. Cell Biol. 109:3315). We show here that the oligosaccharides on both precursor H2a and the 35-kD fragment are Man6-9GlcNAc2, structures typically found in pre-Golgi compartments. Subcellular fractionation shows that the intermediate degradation product does not cofractionate with the lysosomal enzyme beta-galactosidase, but is found in a part of the ER that contains ribosomes. Thus the intermediate degradation product is localized in the ER, indicating that the initial degradation event does take place in the ER. All degradation of H2a, including the initial endoproteolytic cleavage generating the 35-kD intermediate, is blocked by the protease inhibitors N-tosyl-L-lysine chloromethyl ketone and N-tosyl-L-phenylalanine chloromethyl ketone. These drugs do not inhibit ER-to-Golgi transport of H1. Depleting the cells of ATP or inhibiting protein synthesis allows the initial endoproteolytic cleavage to occur, but blocks further degradation of the 35-kD intermediate; thus we can convert all cellular H2 into the 35-kD intermediate. Approximately 50% of H2b, a splicing variant differing from H2a by a five amino acid deletion, can be transported to the cell surface, and the rest appears to be degraded by the same pathway as H2a, both when expressed alone in fibroblasts and together with H1 in HepG2 cells. Addition of N-tosyl-L-lysine chloromethyl ketone or N-tosyl-L-phenylalanine chloromethyl ketone blocks degradation of the approximately 50% that is not transported, but does not affect the fraction of H2b that moves to the Golgi region. Thus, a protein destined for degradation will not be transported to the Golgi region if degradation is inhibited.     

Elements of the C-terminal t peptide of acetylcholinesterase that determine amphiphilicity, homomeric and heteromeric associations, secretion and degradation.

The C-terminal t peptide (40 residues) of vertebrate acetylcholinesterase (AChE) T subunits possesses a series of seven conserved aromatic residues and forms an amphiphilic alpha-helix; it allows the formation of homo-oligomers (monomers, dimers and tetramers) and heteromeric associations with the anchoring proteins, ColQ and PRiMA, which contain a proline-rich motif (PRAD). We analyzed the influence of mutations in the t peptide of Torpedo AChE(T) on oligomerization and secretion. Charged residues influenced the distribution of homo-oligomers but had little effect on the heteromeric association with Q(N), a PRAD-containing N-terminal fragment of ColQ. The formation of homo-tetramers and Q(N)-linked tetramers required a central core of four aromatic residues and a peptide segment extending to residue 31; the last nine residues (32-40) were not necessary, although the formation of disulfide bonds by cysteine C37 stabilized T(4) and T(4)-Q(N) tetramers. The last two residues of the t peptide (EL) induced a partial intracellular retention; replacement of the C-terminal CAEL tetrapeptide by KDEL did not prevent tetramerization and heteromeric association with Q(N), indicating that these associations take place in the endoplasmic reticulum. Mutations that disorganize the alpha-helical structure of the t peptide were found to enhance degradation. Co-expression with Q(N) generally increased secretion, mostly as T(4)-Q(N) complexes, but reduced it for some mutants. Thus, mutations in this small, autonomous interaction domain bring information on the features that determine oligomeric associations of AChE(T) subunits and the choice between secretion and degradation.     

Conformational study of the threonine-rich C-terminal pentapeptide of peptide T

The conformation of the synthetic pentapeptide Thr-Thr-Asn-Tyr-Thr, the C-terminal part of peptide T has been studied using 2D NMR experiments. The nuclear Overhauser effects (NOESY) and the low temperature coefficients for two particular NH chemical shifts allow the proposal for two distinct beta-turn arrangements. This conformation is not in accordance with recent reports but is consistent with observed beta-bends in two sequences of ribonuclease A. The semi-rigid conformation found in the pentapeptide in which the hydroxyl groups are exposed at the periphery of the molecule could be a crucial feature to explain the ability of peptide T to bind to a specific receptor and to correlate with the observed biological activity against HIV.     

Acetylcholinesterase H and T dimers are associated through the same contact. Mutations at this interface interfere with the C-terminal T peptide, inducing degradation rather than secretion.

Acetylcholinesterase (AChE) exists as AChE(H) and AChE(T) subunits, which differ by their C-terminal H or T peptides, generating glycophosphatidylinositol-anchored dimers and various oligomers, respectively. We introduced mutations in the four-helix bundle interface of glycophosphatidylinositol-anchored dimers, and analyzed their effect on the production and oligomerization of AChE(H), of AChE(T), and of truncated subunits, AChE(C) (without H or T peptide). Dimerization was reduced for all types of subunits, showing that they interact through the same contact zone; the formation of amphiphilic tetramers (Torpedo AChE(T)) and 13.5 S oligomers (rat AChE(T)) was also suppressed. Oligomerization appeared totally blocked by introduction of an N-linked glycan on the surface of helix alpha(7,8). Other point mutations did not affect the synthesis or the catalytic properties of AChE but reduced or blocked the secretion of AChE(T) subunits. Secretion of AChE(T) was partially restored by co-expression with Q(N), a secretable protein containing a proline-rich attachment domain (PRAD); Q(N) organized PRAD-linked tetramers, except for the N-glycosylated mutants. Thus, the simultaneous presence of an abnormal four-helix bundle zone and an exposed T peptide targeted the enzyme toward degradation, indicating a cross-talk between the catalytic and tetramerization domains.     

Degradation of unassembled soluble Ig subunits by cytosolic proteasomes: evidence that retrotranslocation and degradation are coupled events.

Many aberrant or unassembled proteins synthesized in the endoplasmic reticulum (ER) are degraded by cytosolic proteasomes. To investigate how soluble glycoproteins destined for degradation are retrotranslocated across the ER membrane, we analyzed the fate of two IgM subunits, mu and J, retained in the ER by myeloma cells that do not synthesize light chains. Degradation of mu and J is prevented by proteasome inhibitors, suggesting that both chains are retrotranslocated to be disposed of by proteasomes. Indeed, when proteasomes are inhibited, some deglycosylated J chains that no longer contain intrachain disulfide bonds accumulate in the cytosol. However, abundant glycosylated J chains are still present in the ER at time points in which degradation would have been almost complete in the absence of proteasome inhibitors, suggesting that retrotranslocation and degradation are coupled events. This was confirmed by protease protection and cell fractionation assays, which revealed that virtually all mu chains are retained in the ER lumen in a glycosylated state when proteasomes are inhibited. Association with calnexin correlated with the failure of mu chains to dislocate to the cytosol. Taken together, these results suggest that active proteasomes are required for the extraction of Ig subunits from the ER, though the requirements for retrotranslocation may differ among individual substrates.     

C-terminal region of the active domain enhances enzymatic activity in dinoflagellate luciferase.

The dinoflagellate luciferase of Lingulodinium polyedrum has three catalytic domains in its single polypeptide chain (M(r) = 137 kDa), and each 42 kDa domain is enzymatically active. Deletion mutants for N- or C-terminal regions of domain 3 of the luciferase, ranging from 29 to 38 kDa, were constructed and expressed in E. coli cells. The activities of N-terminal deleted mutants were above 20% of wild type, but showed different pH-activity profiles. By contrast, the activities of C-terminal deleted mutants decreased drastically to below 1% of wild type, although their pH-activity profiles and spectra were identical to those of wild type L. polyedrum luciferase. These results indicate that the C-terminal region of this enzyme could be important for the bioluminescence reaction, although based on crystal structure of the luciferase domain, this region does not contain active or regulatory sites.     

Amyloid-beta peptide enhances tumor necrosis factor-alpha-induced iNOS through neutral sphingomyelinase/ceramide pathway in oligodendrocytes

Although accumulating evidence demonstrates that white matter degeneration contributes to pathology in Alzheimer's disease (AD), the underlying mechanisms are unknown. In order to study the roles of the amyloid-beta peptide in inducing oxidative stress damage in white matter of AD, we investigated the effects of amyloid-beta peptide 25-35 (Abeta) on proinflammatory cytokine tumor necrosis factor-alpha (TNF-alpha)-induced inducible nitric oxide synthase (iNOS) in cultured oligodendrocytes (OLGs). Although Abeta 25-35 by itself had little effect on iNOS mRNA, protein, and nitrite production, it enhanced TNF-alpha-induced iNOS expression and nitrite generation in OLGs. Abeta, TNF-alpha, or the combination of both, increased neutral sphingomyelinase (nSMase) activity, but not acidic sphingomyelinase (aSMase) activity, leading to ceramide accumulation. Cell permeable C2-ceramide enhanced TNF-alpha-induced iNOS expression and nitrite generation. Moreover, the specific nSMase inhibitor, 3-O-methyl-sphingomyelin (3-OMS), inhibited iNOS expression and nitrite production induced by TNF-alpha or by the combination of TNF-alpha and Abeta. Overexpression of a truncated mutant of nSMase with a dominant negative function inhibited iNOS mRNA production. 3-OMS also inhibited nuclear factor kappaB (NF-kappaB) binding activity induced by TNF-alpha or by the combination of TNF-alpha and Abeta. These results suggest that neutral sphingomyelinase/ceramide pathway is required but may not be sufficient for iNOS expression induced by TNF-alpha and the combination of TNF-alpha and Abeta.     

The C-terminal t peptide of acetylcholinesterase forms an alpha helix that supports homomeric and heteromeric interactions.

Acetylcholinesterase subunits of type T (AChET) possess an alternatively spliced C-terminal peptide (t peptide) which endows them with amphiphilic properties, the capacity to form various homo-oligomers and to associate, as a tetramer, with anchoring proteins containing a proline rich attachment domain (PRAD). The t peptide contains seven conserved aromatic residues. By spectroscopic analyses of the synthetic peptides covering part or all of the t peptide of Torpedo AChET, we show that the region containing the aromatic residues adopts an alpha helical structure, which is favored in the presence of lipids and detergent micelles: these residues therefore form a hydrophobic cluster in a sector of the helix. We also analyzed the formation of disulfide bonds between two different AChET subunits, and between AChET subunits and a PRAD-containing protein [the N-terminal fragment of the ColQ protein (QN)] possessing two cysteines upstream or downstream of the PRAD. This shows that, in the complex formed by four T subunits with QN (T4-QN), the t peptides are not folded on themselves as hairpins but instead are all oriented in the same direction, antiparallel to that of the PRAD. The formation of disulfide bonds between various pairs of cysteines, introduced by mutagenesis at various positions in the t peptides, indicates that this complex possesses a surprising flexibility.     

Glycoprotein quality control in the endoplasmic reticulum. Mannose trimming by endoplasmic reticulum mannosidase I times the proteasomal degradation of unassembled immunoglobulin subunits

Quality control in the endoplasmic reticulum must discriminate nascent proteins in their folding process from terminally unfolded molecules, selectively degrading the latter. Unassembled Ig-mu and J chains, two glycoproteins with five N-linked glycans and one N-linked glycan, respectively, are degraded by cytosolic proteasomes after a lag from synthesis, during which glycan trimming occurs. Inhibitors of mannosidase I (kifunensine), but not of mannosidase II (swainsonine), prevent the degradation of mu chains. Kifunensine also inhibits J chain dislocation and degradation, without inhibiting secretion of IgM polymers. In contrast, glucosidase inhibitors do not significantly affect the kinetics of mu and J degradation. These results suggest that removal of the terminal mannose from the central branch acts as a timer in dictating the degradation of transport-incompetent, glycosylated Ig subunits in a calnexin-independent way. Kifunensine does not inhibit the degradation of an unglycosylated substrate (lambda Ig light chains) or of chimeric mu chains extended with the transmembrane region of the alpha T cell receptor chain, implying the existence of additional pathways for extracting proteins from the endoplasmic reticulum lumen for proteasomal degradation.     

Role of the endoplasmic reticulum-associated degradation (ERAD) pathway in degradation of hepatitis C virus envelope proteins and production of virus particles

Viral infections frequently cause endoplasmic reticulum (ER) stress in host cells leading to stimulation of the ER-associated degradation (ERAD) pathway, which subsequently targets unassembled glycoproteins for ubiquitylation and proteasomal degradation. However, the role of the ERAD pathway in the viral life cycle is poorly defined. In this paper, we demonstrate that hepatitis C virus (HCV) infection activates the ERAD pathway, which in turn controls the fate of viral glycoproteins and modulates virus production. ERAD proteins, such as EDEM1 and EDEM3, were found to increase ubiquitylation of HCV envelope proteins via direct physical interaction. Knocking down of EDEM1 and EDEM3 increased the half-life of HCV E2, as well as virus production, whereas exogenous expression of these proteins reduced the production of infectious virus particles. Further investigation revealed that only EDEM1 and EDEM3 bind with SEL1L, an ER membrane adaptor protein involved in translocation of ERAD substrates from the ER to the cytoplasm. When HCV-infected cells were treated with kifunensine, a potent inhibitor of the ERAD pathway, the half-life of HCV E2 increased and so did virus production. Kifunensine inhibited the binding of EDEM1 and EDEM3 with SEL1L, thus blocking the ubiquitylation of HCV E2 protein. Chemical inhibition of the ERAD pathway neither affected production of the Japanese encephalitis virus (JEV) nor stability of the JEV envelope protein. A co-immunoprecipitation assay showed that EDEM orthologs do not bind with JEV envelope protein. These findings highlight the crucial role of the ERAD pathway in the life cycle of specific viruses.     

Localization of unassembled subunits of the mitochondrial ATPase in an assembly-defective yeast nuclear mutant

The yeast nuclear mutant, pet 936, has previously been shown to be defective in the assembly of a functional mitochondrial ATPase (Todd, R. D., McAda, P. C., and Douglas, M. G. (1979) J. Biol. Chem. 254, 11134-11141). In the present report, trypsin degradation and subunit-specific antibody binding have been used to localize subunits 1, 2, and 3 external to or associated with the outer aspect of the inner mitochondrial membrane in the mutant strain. A similar population of unassembled subunits was found in the parental strain as well. Isotope dilution experiments are compatible with those unassembled subunits being normal intermediates in the assembly pathway of the ATPase complex which are blocked from transport across the inner mitochondrial membrane in the mutant, pet 936.     

Non-hydrolytic functions of acetylcholinesterase. The significance of C-terminal peptides

This review explores the possibility that acetylcholinesterase may play a pivotal, non-hydrolytic role in neurodegeneration. More specifically, C-terminal sequences of acetylcholinesterase may act as signalling molecules in key brain regions characteristically vulnerable to Alzheimer's, Parkinson's and motor neuron disease.     

Glutamine degradation through the omega-amidase pathway in Saccharomyces cerevisiae

A glutamine transaminase activity has been identified in Saccharomyces cerevisiae, and the existence of the omega-amidase activity previously described in this yeast has been confirmed. The glutamine transaminase utilizes different 2-oxo acids as substrates, including pyruvate and glyoxylate, and is regulated by the available nitrogen source. The glutamine transaminase activity decreases when lysine or glycine is added to the medium; the inhibition by lysine diminishes under microaerophilic culture conditions.     

Determinants of the t peptide involved in folding, degradation, and secretion of acetylcholinesterase

The C-terminal 40-residue t peptide of acetylcholinesterase (AChE) forms an amphiphilic alpha helix with a cluster of seven aromatic residues. It allows oligomerization and induces a partial degradation of AChE subunits through the endoplasmic reticulum-associated degradation pathway. We show that the t peptide induces the misfolding of a fraction of AChE subunits, even when mutations disorganized the cluster of aromatic residues or when these residues were replaced by leucines, indicating that this effect is due to hydrophobic residues. Mutations in the aromatic-rich region affected the cellular fate of AChE in a similar manner, with or without mutations that prevented dimerization. Degradation was decreased and secretion was increased when aromatic residues were replaced by leucines, and the opposite occurred when the amphiphilic alpha helix was disorganized. The last two residues (Asp-Leu) somewhat resembled an endoplasmic reticulum retention signal and caused a partial retention but only in mutants possessing aromatic residues in their t peptide. Our results suggested that several "signals" in the catalytic domain and in the t peptide act cooperatively for AChE quality control.     

Aberrant C-terminal domain of polymerase η targets the functional enzyme to the proteosomal degradation pathway

Xeroderma pigmentosum variant (XP-V) is a rare genetic disease, characterized by sunlight sensitivity and predisposition to cutaneous malignancies. XP-V is caused by a deficiency in DNA polymerase eta (Polη) that plays a pivotal role in translesion synthesis by bypassing UV-induced pyrimidine dimers. Previously we identified a new Polη variant containing two missense mutations, one mutation within the bipartite NLS (T692A) and a second mutation on the stop codon (X714W) leading to a longer protein with an extra 8 amino acids (721 instead of 713 AA). First biochemical analysis revealed that this Polη missense variant was barely detectable by western blot. As this mutant is extremely unstable and is nearly undetectable, a definitive measure of its functional deficit in cells has not been explored. Here we report the molecular and cellular characterization of this missense variant. In cell free extracts, the extra 8 amino acids in the C-terminal of Polη⁷²¹ only slightly reduce the bypass efficiency through CPD lesions. In vivo, Polη⁷²¹ accumulates in replication factories and interacts with mUb-PCNA albeit at lower level than Polη^wt. XP-V cells overexpressing Polη⁷²¹ were only slightly UV-sensitive. Altogether, our data strongly suggest that Polη⁷²¹ is functional and that the patient displays a XP-V phenotype because the mutant protein is excessively unstable. We then investigated the molecular mechanisms involved in this excessive proteolysis. We showed that Polη⁷²¹ is degraded by the proteasome in an ubiquitin-dependent manner and that this proteolysis is independent of the E3 ligases, CRL4^cdt2 and Pirh2, reported to promote Polη degradation. We then demonstrated that the extra 8 amino acids of Polη⁷²¹ do not act as a degron but rather induce a conformational change of the Polη C-terminus exposing its bipartite NLS as well as a sequence close to its UBZ to the ubiquitin/proteasome system. Interestingly we showed that the clinically approved proteasome inhibitor, Bortezomib restores the levels of Polη⁷²¹ suggesting that this might be a therapeutic approach to preventing tumor development in certain XP-V patients harboring missense mutations.     

Functionalization with C-terminal cysteine enhances transfection efficiency of cell-penetrating peptides through dimer formation

Cell-penetrating peptides have the ability to stimulate uptake of macromolecular cargo in mammalian cells in a non-toxic manner and therefore hold promise as efficient and well tolerated gene delivery vectors. Non-covalent peptide-DNA complexes ("peptiplexes") enter cells via endocytosis, but poor peptiplex stability and endosomal entrapment are considered as main barriers to peptide-mediated delivery. We explore a simple, yet highly efficient, strategy to improve the function of peptide-based vectors, by adding one terminal cysteine residue. This allows the peptide to dimerize by disulfide bond formation, increasing its affinity for nucleic acids by the "chelate effect" and, when the bond is reduced intracellularly, letting the complex dissociate to deliver the nucleic acid. By introducing a single C-terminal cysteine in the classical CPP penetratin and the penetratin analogs PenArg and EB1, we show that this minor modification greatly enhances the transfection capacity for plasmid DNA in HEK293T cells. We conclude that this effect is mainly due to enhanced thermodynamic stability of the peptiplexes as endosome-disruptive chloroquine is still required for transfection and the effect is more pronounced for peptides with lower inherent DNA condensation capacity. Interestingly, for EB1, addition of one cysteine makes the peptide able to mediate transfection in absence of chloroquine, indicating that dimerisation can also improve endosomal escape properties. Further, the cytotoxicity of EB1 peptiplexes is considerably reduced, possibly due to lower concentration of free peptide dimer resulting from its stronger binding to DNA.     

A truncated isoform of c-Mpl with an essential C-terminal peptide targets the full-length receptor for degradation

Thrombopoietin and its cognate receptor c-Mpl are the primary regulators of megakaryopoiesis and platelet production. They also play an important role in the maintenance of hematopoietic stem cells. Here, we have analyzed the function of a truncated Mpl receptor isoform (Mpl-tr), which results from alternative splicing. The mpl-tr variant is the only alternate mpl isoform conserved between mouse and humans, suggesting a relevant function in regulating Mpl signaling. Despite the presence of a signal peptide and the lack of a transmembrane domain, Mpl-tr is retained intracellularly. Our results provide evidence that Mpl-tr exerts a dominant-negative effect on thrombopoietin-dependent cell proliferation and survival. We demonstrate that this inhibitory effect is due to down-regulation of the full-length Mpl protein. The C terminus of Mpl-tr, consisting of 30 amino acids of unique sequence, is essential for the suppression of thrombopoietin-dependent proliferation and Mpl protein down-regulation. Cathepsin inhibitor-1 (CATI-1), an inhibitor of cathepsin-like cysteine proteases, counteracts the effect of Mpl-tr on Mpl protein expression, suggesting that Mpl-tr targets Mpl for lysosomal degradation. Together, these data suggest a new paradigm for the regulation of cytokine receptor expression and function through a proteolytic process directed by a truncated isoform of the same receptor.