Biomedical subjects

Carmela Galli

Publications and source records attributed to Carmela Galli.

8 recordsLinked to original sources

PubMed2005

Persistent glycoprotein misfolding activates the glucosidase II/UGT1-driven calnexin cycle to delay aggregation and loss of folding competence.

The UDP-glucose:glycoprotein glucosyltransferase (UGT) is a central player of glycoprotein quality control in the endoplasmic reticulum (ER). UGT reglucosylation of nonnative glycopolypeptides prevents their release from the calnexin cycle and secretion. Here, we compared the fate of a glycoprotein with a reversible, temperature-dependent folding defect in cells with and without UGT1. Upon persistent misfolding, tsO45 G was slowly released from calnexin and entered a second level of retention-based ER quality control by forming BiP/GRP78-associated disulfide-bonded aggregates. This correlated with loss in the ability to correct misfolding. Deletion of UGT1 did not affect the stringency of ER quality control. Rather, it accelerated release from calnexin and transfer to the second ER quality control level, but it did so after an unexpectedly long lag, showing that cycling in the calnexin chaperone system is not frenetic, as claimed by existing models, and is fully activated only upon persistent glycoprotein misfolding.

Animals↗

PubMed2005

The use of calnexin and calreticulin by cellular and viral glycoproteins.

Calnexin and calreticulin are homologous lectin chaperones that assist maturation of cellular and viral glycoproteins in the mammalian endoplasmic reticulum. Calnexin and calreticulin share the same specificity for monoglucosylated protein-bound N-glycans but associate with a distinct set of newly synthesized polypeptides. We report here that most calnexin substrates do not associate with calreticulin even upon selective calnexin inactivation, while BiP associates more abundantly with nascent polypeptides under these conditions. Calreticulin associated more abundantly with orphan calnexin substrates only in infected cells and preferentially with polypeptides of viral origin, showing stronger dependence of model viral glycoproteins on endoplasmic reticulum lectins. This may explain why inactivation of the calnexin cycle affects viral replication and infectivity but not viability of mammalian cells.

Amyloid Precursor Protein Secretases↗

PubMed2005

beta-site specific intrabodies to decrease and prevent generation of Alzheimer's Abeta peptide.

Endoproteolysis of the beta-amyloid precursor protein (APP) by beta- and gamma-secretases generates the toxic amyloid beta-peptide (Abeta), which accumulates in the brain of Alzheimer's disease (AD) patients. Here, we established a novel approach to regulate production of Abeta based on intracellular expression of single chain antibodies (intrabodies) raised to an epitope adjacent to the beta-secretase cleavage site of human APP. The intrabodies rapidly associated, within the endoplasmic reticulum (ER), with newly synthesized APP. One intrabody remained associated during APP transport along the secretory line, shielded the beta-secretase cleavage site and facilitated the alternative, innocuous cleavage operated by alpha-secretase. Another killer intrabody with an ER retention sequence triggered APP disposal from the ER. The first intrabody drastically inhibited and the second almost abolished generation of Abeta. Intrabodies association with specific substrates rather than with enzymes, may modulate intracellular processes linked to disease with highest specificity and may become instrumental to investigate molecular mechanisms of cellular events.

Alzheimer Disease↗

PubMed2004

A novel stress-induced EDEM variant regulating endoplasmic reticulum-associated glycoprotein degradation.

Proteins expressed in the endoplasmic reticulum (ER) are subjected to a tight quality control. Persistent association with ER-resident molecular chaperones prevents exit of misfolded or incompletely assembled polypeptides from the ER and forward transport along the secretory line. ER-associated degradation (ERAD) is in place to avoid ER constipation. Folding-incompetent products have to be identified to interrupt futile folding attempts and then targeted for unfolding and dislocation into the cytosol for proteasome-mediated destruction. These processes are better understood for N-glycosylated proteins that represent the majority of polypeptides expressed in the ER. EDEM, a mannosidase-like chaperone, regulates the extraction of misfolded glycoproteins from the calnexin cycle. Here we identify and characterize EDEM2, a novel, stress-regulated mannosidase-like protein that operates in the ER lumen. We show that transcriptional up-regulation of EDEM2 depends on the ER stress-activated transcription factor Xbp1, that EDEM2 up-regulation selectively accelerates ERAD of terminally misfolded glycoproteins by facilitating their extraction from the calnexin cycle, and that the previously characterized homolog EDEM is also a soluble protein of the ER lumen in HEK293 cells.

Cell Line↗

PubMed2004

EDEM contributes to maintenance of protein folding efficiency and secretory capacity.

A stringent quality control process selects misfolded polypeptides generated in the endoplasmic reticulum (ER) for ER-associated degradation (ERAD). Here we assessed the maintenance of efficient glycoprotein folding in cells with defective ERAD caused by lack of adaptation of the intralumenal level of ER degradation-enhancing alpha-mannosidase-like protein (EDEM) to an increase in the ER cargo load. When these cells were converted into factories for production of high levels of human beta-secretase, maturation of this N-glycosylated aspartic protease progressed as in wild-type cells initially to gradually become less efficient. Up-regulation of EDEM to strengthen the ERAD machinery (but not up-regulation of calnexin to reinforce the folding machinery) was instrumental in maintaining folding efficiency and secretory capacity. Our data underscore the important role that the degradation machinery plays in maintaining a functional folding environment in the ER.

Animals↗

PubMed2004

Contrasting functions of calreticulin and calnexin in glycoprotein folding and ER quality control.

Calreticulin and calnexin are homologous lectins that serve as molecular chaperones for glycoproteins in the endoplasmic reticulum of eukaryotic cells. Here we show that calreticulin depletion specifically accelerates the maturation of cellular and viral glycoproteins with a modest decrease in folding efficiency. Calnexin depletion prevents proper maturation of some proteins such as influenza hemagglutinin but does not interfere appreciably with the maturation of several others. A dramatic loss of stringency in the ER quality control with transport at the cell surface of misfolded glycoprotein conformers is only observed when substrate access to both calreticulin and calnexin is prevented. Although not fully interchangeable during assistance of glycoprotein folding, calreticulin and calnexin may work, independently, as efficient and crucial factors for retention in the ER of nonnative polypeptides.

Animals↗

PubMed2003

Role of EDEM in the release of misfolded glycoproteins from the calnexin cycle.

The mechanisms that determine how folding attempts are interrupted to target folding-incompetent proteins for endoplasmic reticulum-associated degradation (ERAD) are poorly defined. Here the alpha-mannosidase I-like protein EDEM was shown to extract misfolded glycoproteins, but not glycoproteins undergoing productive folding, from the calnexin cycle. EDEM overexpression resulted in faster release of folding-incompetent proteins from the calnexin cycle and earlier onset of degradation, whereas EDEM down-regulation prolonged folding attempts and delayed ERAD. Up-regulation of EDEM during ER stress may promote cell recovery by clearing the calnexin cycle and by accelerating ERAD of terminally misfolded polypeptides.

Aspartic Acid Endopeptidases↗

PubMed2002

Sequential assistance of molecular chaperones and transient formation of covalent complexes during protein degradation from the ER.

BACE457 is a recently identified pancreatic isoform of human beta-secretase. We report that this membrane glycoprotein and its soluble variant are characterized by inefficient folding in the ER, leading to proteasome-mediated ER-associated degradation (ERAD). Dissection of the degradation process revealed that upon release from calnexin, extensively oxidized BACE457 transiently entered in disulfide-bonded complexes associated with the lumenal chaperones BiP and protein disulfide isomerase (PDI) before unfolding and dislocation into the cytosol for degradation. BACE457 and its lumenal variant accumulated in disulfide-bonded complexes, in the ER lumen, also when protein degradation was inhibited. The complexes were disassembled and the misfolded polypeptides were cleared from the ER upon reactivation of the degradation machinery. Our data offer new insights into the mechanism of ERAD by showing a sequential involvement of the calnexin and BiP/PDI chaperone systems. We report the unexpected transient formation of covalent complexes in the ER lumen during the ERAD process, and we show that PDI participates as an oxidoreductase and a redox-driven chaperone in the preparation of proteins for degradation from the mammalian ER.

Amyloid Precursor Protein Secretases↗