Degradation of a Short-lived Glycoprotein from the Lumen of the Endoplasmic Reticulum: The Role of N-linked Glycans and the Unfolded Protein Response

We are studying endoplasmic reticulum–associated degradation (ERAD) with the use of a truncated variant of the type I ER transmembrane glycoprotein ribophorin I (RI). The mutant protein, RI332, containing only the N-terminal 332 amino acids of the luminal domain of RI, has been shown to interact with calnexin and to be a substrate for the ubiquitin-proteasome pathway. When RI332 was expressed in HeLa cells, it was degraded with biphasic kinetics; an initial, slow phase of ∼45 min was followed by a second phase of threefold accelerated degradation. On the other hand, the kinetics of degradation of a form of RI332 in which the single used N-glycosylation consensus site had been removed (RI332-Thr) was monophasic and rapid, implying a role of the N-linked glycan in the first proteolytic phase. RI332 degradation was enhanced when the binding of glycoproteins to calnexin was prevented. Moreover, the truncated glycoprotein interacted with calnexin preferentially during the first proteolytic phase, which strongly suggests that binding of RI332 to the lectin-like protein may result in the slow, initial phase of degradation. Additionally, mannose trimming appears to be required for efficient proteolysis of RI332. After treatment of cells with the inhibitor of N-glycosylation, tunicamycin, destruction of the truncated RI variants was severely inhibited; likewise, in cells preincubated with the calcium ionophore A23187, both RI332 and RI332-Thr were stabilized, despite the presence or absence of the N-linked glycan. On the other hand, both drugs are known to trigger the unfolded protein response (UPR), resulting in the induction of BiP and other ER-resident proteins. Indeed, only in drug-treated cells could an interaction between BiP and RI332 and RI332-Thr be detected. Induction of BiP was also evident after overexpression of murine Ire1, an ER transmembrane kinase known to play a central role in the UPR pathway; at the same time, stabilization of RI332 was observed. Together, these results suggest that binding of the substrate proteins to UPR-induced chaperones affects their half lives.

Deletion of Yeast p24 Genes Activates the Unfolded Protein Response

Yeast cells lacking a functional p24 complex accumulate a subset of secretory proteins in the endoplasmic reticulum (ER) and increase the extracellular secretion of HDEL-containing ER residents such as Kar2p/BiP. We report that a loss of p24 function causes activation of the unfolded protein response (UPR) and leads to increased KAR2 expression. The HDEL receptor (Erd2p) is functional and traffics in p24 deletion strains as in wild-type strains, however the capacity of the retrieval pathway is exceeded. Other conditions that activate the UPR and elevate KAR2 expression also lead to extracellular secretion of Kar2p. Using an in vitro assay that reconstitutes budding from the ER, we detect elevated levels of Kar2p in ER-derived vesicles from p24 deletion strains and from wild-type strains with an activated UPR. Silencing the UPR by IRE1 deletion diminished Kar2p secretion under these conditions. We suggest that activation of the UPR plays a major role in extracellular secretion of Kar2p.

The ricinosomes of senescing plant tissue bud from the endoplasmic reticulum

The ricinosome (synonym, precursor protease vesicle) is a novel organelle, found so far exclusively in plant cells. Electron microscopic studies suggest that it buds off from the endoplasmic reticulum in senescing tissues. Biochemical support for this unusual origin now comes from the composition of the purified organelle, which contains large amounts of a 45-kDa cysteine endoprotease precursor with a C-terminal KDEL motif and the endoplasmic reticulum lumen residents BiP (binding protein) and protein disulfide isomerase. Western blot analysis, peptide sequencing, and mass spectrometry demonstrate retention of KDEL in the protease proform. Acidification of isolated ricinosomes causes castor bean cysteine endopeptidase activation, with cleavage of the N-terminal propeptide and the C-terminal KDEL motif. We propose that ricinosomes accumulate during senescence by programmed cell death and are activated by release of protons from acidic vacuoles.

Hsp70 Molecular Chaperone Facilitates Endoplasmic Reticulum-associated Protein Degradation of Cystic Fibrosis Transmembrane Conductance Regulator in Yeast

Membrane and secretory proteins fold in the endoplasmic reticulum (ER), and misfolded proteins may be retained and targeted for ER-associated protein degradation (ERAD). To elucidate the mechanism by which an integral membrane protein in the ER is degraded, we studied the fate of the cystic fibrosis transmembrane conductance regulator (CFTR) in the yeast Saccharomyces cerevisiae. Our data indicate that CFTR resides in the ER and is stabilized in strains defective for proteasome activity or deleted for the ubiquitin-conjugating enzymes Ubc6p and Ubc7p, thus demonstrating that CFTR is a bona fide ERAD substrate in yeast. We also found that heat shock protein 70 (Hsp70), although not required for the degradation of soluble lumenal ERAD substrates, is required to facilitate CFTR turnover. Conversely, calnexin and binding protein (BiP), which are required for the proteolysis of ER lumenal proteins in both yeast and mammals, are dispensable for the degradation of CFTR, suggesting unique mechanisms for the disposal of at least some soluble and integral membrane ERAD substrates in yeast.

Accumulation of Mutant Huntingtin Fragments in Aggresome-like Inclusion Bodies as a Result of Insufficient Protein Degradation

The huntingtin exon 1 proteins with a polyglutamine repeat in the pathological range (51 or 83 glutamines), but not with a polyglutamine tract in the normal range (20 glutamines), form aggresome-like perinuclear inclusions in human 293 Tet-Off cells. These structures contain aggregated, ubiquitinated huntingtin exon 1 protein with a characteristic fibrillar morphology. Inclusion bodies with truncated huntingtin protein are formed at centrosomes and are surrounded by vimentin filaments. Inhibition of proteasome activity resulted in a twofold increase in the amount of ubiquitinated, SDS-resistant aggregates, indicating that inclusion bodies accumulate when the capacity of the ubiquitin–proteasome system to degrade aggregation-prone huntingtin protein is exhausted. Immunofluorescence and electron microscopy with immunogold labeling revealed that the 20S, 19S, and 11S subunits of the 26S proteasome, the molecular chaperones BiP/GRP78, Hsp70, and Hsp40, as well as the RNA-binding protein TIA-1, the potential chaperone 14–3-3, and α-synuclein colocalize with the perinuclear inclusions. In 293 Tet-Off cells, inclusion body formation also resulted in cell toxicity and dramatic ultrastructural changes such as indentations and disruption of the nuclear envelope. Concentration of mitochondria around the inclusions and cytoplasmic vacuolation were also observed. Together these findings support the hypothesis that the ATP-dependent ubiquitin–proteasome system is a potential target for therapeutic interventions in glutamine repeat disorders.

A Novel Quality Control Compartment Derived from the Endoplasmic Reticulum

Degradation of proteins that, because of improper or suboptimal processing, are retained in the endoplasmic reticulum (ER) involves retrotranslocation to reach the cytosolic ubiquitin-proteasome machinery. We found that substrates of this pathway, the precursor of human asialoglycoprotein receptor H2a and free heavy chains of murine class I major histocompatibility complex (MHC), accumulate in a novel preGolgi compartment that is adjacent to but not overlapping with the centrosome, the Golgi complex, and the ER-to-Golgi intermediate compartment (ERGIC). On its way to degradation, H2a associated increasingly after synthesis with the ER translocon Sec61. Nevertheless, it remained in the secretory pathway upon proteasomal inhibition, suggesting that its retrotranslocation must be tightly coupled to the degradation process. In the presence of proteasomal inhibitors, the ER chaperones calreticulin and calnexin, but not BiP, PDI, or glycoprotein glucosyltransferase, concentrate in the subcellular region of the novel compartment. The “quality control” compartment is possibly a subcompartment of the ER. It depends on microtubules but is insensitive to brefeldin A. We discuss the possibility that it is also the site for concentration and retrotranslocation of proteins that, like the mutant cystic fibrosis transmembrane conductance regulator, are transported to the cytosol, where they form large aggregates, the “aggresomes.”

Perturbations in maturation of secretory proteins and their association with endoplasmic reticulum chaperones in a cell culture model for epithelial ischemia.

The effects of ischemia on the maturation of secretory proteins are not well understood. Among several events that occur during ischemia-reperfusion are a rapid and extensive decrease in ATP levels and an alteration of cellular oxidative state. Since the normal folding and assembly of secretory proteins are mediated by endoplasmic reticulum (ER) molecular chaperones, the function of which depends on ATP and maintenance of an appropriate redox environment, ischemia might be expected to perturb folding of secretory proteins. In this study, whole animal and cultured cell models for the epithelial ischemic state were used to examine this possibility. After acute kidney ischemia, marked increases in the mRNA levels of the ER chaperones glucose-regulated protein (grp)78/immunoglobulin-binding protein (BiP), grp94, and ER protein (ERp)72 were noted. Likewise, when cellular ATP was depleted to less than 10% of control with antimycin A, mRNA levels of BiP, ERp72, and grp94 were increased in kidney and thyroid epithelial cell culture models. Since the signal for the up-regulation of these stress proteins is believed to be the accumulation of misfolded/misassembled secretory proteins in the ER, their induction after ischemia in vivo and antimycin treatment of cultured cells suggests that maturation of secretory proteins in the ER lumen might indeed be perturbed. To analyze the effects of antimycin A on the maturation of secretory proteins, we studied the fate of thyroglobulin (Tg), a large oligomeric secretory glycoprotein, the folding and assembly of which seems to require a variety of ER chaperones. Treatment of cultured thyroid epithelial cells with antimycin A greatly inhibited ( > 90%) the secretion of Tg. Sucrose density gradient analysis revealed that in antimycin A-treated cells Tg associates into large macromolecular complexes which, by immunofluorescence, appeared to localize to the ER. Furthermore, coimmunoprecipitation studies after antimycin A treatment demonstrated that Tg stably associates with BiP, grp94, and ERp72. Together, our results suggest that a key cellular lesion in ischemia is the misfolding of secretory proteins as they transit the ER, and this leads not only to increased expression of ER chaperones but also to their stable association with and the subsequent retention of at least some misfolded secretory proteins.

Roles of heavy and light chains in IgM polymerization.

IgM antibodies are secreted as multisubunit polymers that consist of as many as three discrete polypeptides: mu heavy chains, light (L) chains, and joining (J) chains. We wished to determine whether L chains that are required to confer secretory competence on immunoglobulin molecules must be present for IgM to polymerize--that is, for intersubunit disulfide bonds to form between mu chains. Using a L-chain-loss variant of an IgM-secreting hybridoma, we demonstrated that mu chains were efficiently polymerized independent of L chains, in a manner similar to that observed for conventional microL complexes, and that the mu polymers incorporated J chain. These mu polymers were not secreted but remained associated with the endoplasmic reticulum-resident chaperone BiP (GRP78). This finding is consistent with the endoplasmic reticulum being the subcellular site of IgM polymerization. We conclude that mu chain alone has the potential to direct the polymerization of secreted IgM, a process necessary but not sufficient for IgM to attain secretory competence.

Stathmin interaction with a putative kinase and coiled-coil-forming protein domains.

Stathmin is a ubiquitous, cytosolic 19-kDa protein, which is phosphorylated on up to four sites in response to many regulatory signals within cells. Its molecular characterization indicates a functional organization including an N-terminal regulatory domain that bears the phosphorylation sites, linked to a putative alpha-helical binding domain predicted to participate in coiled-coil, protein-protein interactions. We therefore proposed that stathmin may play the role of a relay integrating diverse intracellular regulatory pathways; its action on various target proteins would be a function of its combined phosphorylation state. To search for such target proteins, we used the two-hybrid screen in yeast, with stathmin as a "bait." We isolated and characterized four cDNAs encoding protein domains that interact with stathmin in vivo. One of the corresponding proteins was identified as BiP, a member of the hsp70 heat-shock protein family. Another is a previously unidentified, putative serine/threonine kinase, KIS, which might be regulated by stathmin or, more likely, be part of the kinases controlling its phosphorylation state. Finally, two clones code for subdomains of two proteins, CC1 and CC2, predicted to form alpha-helices participating in coiled-coil interacting structures. Their isolation by interaction screening further supports our model for the regulatory function of stathmin through coiled-coil interactions with diverse downstream targets via its presumed alpha-helical binding domain. The molecular and biological characterization of KIS, CC1, and CC2 proteins will give further insights into the molecular functions and mechanisms of action of stathmin as a relay of integrated intracellular regulatory pathways.

Mecanismo de interacción, calcio dependiente, entre Calcineurina y PERK, involucrado en el Estrés de Retículo Endoplásmico

El Estrés de Retículo Endoplásmico (RE) es inducido por la acumulación de proteínas sin plegar en el lumen de la organela. Esto se puede observar en diversas situaciones fisio-patológicas como durante una infección viral o en proceso isquémico. Además, contribuye a la base molecular de numerosas enfermedades ya sea índole metabólico (Fibrosis quística o Diabetes Miellitus) o neurodegenerativas como mal de Alzheimer o Parkinson (Mutat Res, 2005, 569). Para restablecer la homeostasis en la organela se activa una señal de transducción (UPR), cuya respuesta inmediata es la atenuación de la síntesis de proteína debido a la fosforilación de subunidad alpha del factor eucariótico de iniciación de translación (eIF2α) vía PERK. Esta es una proteína de membrana de RE que detecta estrés. Bajo condiciones normales, PERK está inactiva debido a la asociación de su dominio luminar con la chaperona BIP (Nat Cell Biol, 2000, 2: 326). Frente a una situación de estrés, la chaperona se disocia causando desinhibición. Recientemente, (Plos One 5: e11925) se observó, bajo condiciones de estrés, un aumento de Ca2+ citosólico y un rápido incremento de la expresión de calcineurina (CN), una fosfatasa citosólica dependiente de calcio, heterodimérica formada por una subunidad catalítica (CN-A) y una regulatoria (CN-B). Además, CN interacciona, sin intermediarios, con el dominio citosólico de PERK favoreciendo su trans-autofosforilación. Resultados preliminares indican que, astrocitos CNAβ-/- exhibieron, en condiciones basales, un mayor número de células muertas y de niveles de eIF2α fosforilado que los astrocitos CNAα-/-. Hipótesis: CNAβ/B interacciona con PERK cuando el Ca2+ citosólico esta incrementado luego de haberse inducido Estrés de RE, lo cual promueve dimerización y auto-fosforilación de la quinasa, acentuándose así la fosforilación de eIF2α e inhibición de la síntesis de proteínas. Esta activación citosólica de PERK colaboraría con la ya descrita, desinhibición luminal llevada cabo por BIP. Cuando el Ca2+ citosólico retorna a los niveles basales, PERK fosforila a CN, reduciendo su afinidad de unión y disociándose el complejo CN/PERK. Objetivo general: Definir las condiciones por las cuales CN interacciona con PERK y regula la fosforilación de eIF2α e inhibición de la síntesis de proteína. Objetivos específicos: I-Estudiar la diferencia de afinidades y dependencia de Ca2+, de las dos isoformas de CN (α y β) en su asociación con PERK. Además verificar la posible participación de la subunidad B de CN en esta interacción. II-Determinar si la auto-fosforilación de PERK es diferencialmente regulada por las dos isoformas de CN. III-Discernir la relación del estado de fosforilación de CN con su unión a PERK. IV-Determinar efectos fisiológicos de la interacción de CN-PERK durante la respuesta de Estrés de RE. Para llevar a cabo este proyecto se realizarán experimentos de biología molecular, interacción proteína-proteína, ensayos de fosforilación in vitro y un perfil de polisoma con astrocitos CNAβ-/- , CNA-/- y astrocitos controles. Se espera encontrar una mayor afinidad de unión a PERK de la isoforma β de CN y en condiciones donde la concentración de Ca2+ sea del orden micromolar e imite niveles del ión durante un estrés. Con respecto al estado de fosforilación de CN, debido a los resultados preliminares, donde solo se la encontró fosforilada en condiciones basales, se piensa que CN podría interactuar con mayor afinidad con PERK cuando CN se encuentre desfosforilada. Por último, se espera encontrar un aumento de eIF2α fosforilado y una acentuación de la atenuación de la síntesis de proteína como consecuencia de la mayor activación de PERK por su asociación con la isoforma β de CN en astrocitos donde el Estrés de RE se indujo por privación de oxigeno y glucosa. Estos experimentos permitirán avanzar en el estudio de una nueva función citoprotectora de CN recientemente descrita por nuestro grupo de trabajo y sus implicancias en un modelo de isquemia.

X-ray fluorescence scannings, diatoms, and dinocyst of ODP Site 189-1172

Eocene sediments drilled at the East Tasman Plateau (ETP) exhibit well-defined cycles, high-resolution magnetic stratigraphy, and environmentally-controlled dinoflagellate and diatom distribution patterns. We derive a cyclostratigraphy from the spectral analysis of high-resolution elemental concentration records (Ca, Fe) for this shallow marine time series spanning the middle to early late Eocene (C16n.2n - C21). Changes in carbonate content, the ratio between Gonyaulacoid and Peridinioid dinocysts, and relative abundance of "oligotrophic" diatoms serve as proxies for a high-resolution climatic and sea-level history with high values representing high sea-level stands and decreased eutrophy of surface waters. Changing ratios between high latitude dinocysts versus cosmopolitan species provide clues on sea surface temperature trends and water mass exchange. Our results show that the relatively shallow-water middle Eocene environments of the ETP are influenced by orbitally-forced climatic cycles superimposed on third order relative sea-level changes. Changes in the dominance of Milankovitch frequency at ~38.6 Ma (late Eocene) is related to an initial deepening-step within the Tasmanian Gateway prior to the major deepening during the middle late Eocene (~35.5 Ma). Decreasing sedimentation rates at 38 Ma and 37.2 Ma reflect winnowing associated with sea-level fall. This episode is followed by renewed transgression. Dinocyst distribution patterns indicate high latitude, probably cool temperate surface water conditions throughout, with the exception of a sudden surge in cosmopolitan species near the base of subchron C18.2r, at ~41 Ma; this event is tentatively correlated to the Middle Eocene Climatic Optimum.

Comparative proteomic analysis of GS-NSO murine myeloma cell lines with varying recombinant monoclonal antibody production rate

We have employed an inverse engineering strategy based on quantitative proteome analysis to identify changes in intracellular protein abundance that correlate with increased specific recombinant monoclonal antibody production (qMab) by engineered murine myeloma (NSO) cells. Four homogeneous NSO cell lines differing in qMab were isolated from a pool of primary transfectants. The proteome of each stably transfected cell line was analyzed at mid-exponential growth phase by two-dimensional gel electrophoresis (2D-PAGE) and individual protein spot volume data derived from digitized gel images were compared statistically. To identify changes in protein abundance associated with qMab clatasets were screened for proteins that exhibited either a linear correlation with cell line qMab or a conserved change in abundance specific only to the cell line with highest qMab. Several proteins with altered abundance were identified by mass spectrometry. Proteins exhibiting a significant increase in abundance with increasing qMab included molecular chaperones known to interact directly with nascent immunoglobulins during their folding and assembly (e.g., BiP, endoplasmin, protein disulfide isomerase). 2D-PAGE analysis showed that in all cell lines Mab light chain was more abundant than heavy chain, indicating that this is a likely prerequisite for efficient Mab production. In summary, these data reveal both the adaptive responses and molecular mechanisms enabling mammalian cells in culture to achieve high-level recombinant monoclonal antibody production. (C) 2004 Wiley Periodicals, Inc.

Ein multisektoraler Sammelindikator für die Schweizer Konjunktur

Der multisektorale Sammelindikator für die Schweizer Gesamtkonjunktur weist gegenüber eine Reihe von methodischen Innovationen auf und berücksichtigt eine vergleichsweise grosse Anzahl von Indikatorreihen. Für den Stützbereich von 1991 bis 2002 erhalten wir auf Quartalsbasis einen stabilen Vorlauf von zwei Quartalen vor der Referenzreihe Vorjahreswachstumsrate des BIP, und auch die Niveaus der Wachstumsrate werden gut getroffen. Der neue Sammelindikator zeigt auch gute "out of sample" Prognoseeigenschaften, und zwar sowohl bezüglich des Vorlaufs als auch hinsichtlich der Niveaus der Referenzreihe.

The impact of the New University Code on the physical education curriculum of four-year colleges and universities in Taiwan, Republic of China

With the establishment of The New University Code in 1994, Taiwan's colleges and universities were mandated to shift physical education from a required course to an elective. The four-year colleges and universities are now responsible for either developing new physical education programs or removing existing programs from their curriculum. Planned change and curriculum leadership are considered in light of policy changes regarding required physical education programs enacted by the Ministry of Education. ^ This study compared the organizational structure and the curriculum of physical education at accredited colleges and universities in Taiwan. Chairpersons of physical education departments from 60 four-year colleges and universities were surveyed using a modified version of the Hensley's Basic Instruction Program (BIP) Questionnaire. Results were analyzed using analysis of variance (ANOVA) and crosstabs. The findings confirmed that physical education programs were effected by declining enrollment and administrative decisions to eliminate them. However, at the same time, chairpersons expressed strong support for the maintenance of the traditional physical education curriculum. ^

Étude du rôle de la calnexine de Schizosaccharomyces pombe dans le repliement et la sécrétion de protéines

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.