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Biomedical subjects

Maurizio Molinari

Publications and source records attributed to Maurizio Molinari.

At least 19 recordsLinked to original sources
PubMed2006

N-glycan processing in ER quality control.

Glycosylation of asparagine residues in Asn-x-Ser/Thr motifs is a common covalent modification of proteins in the lumen of the endoplasmic reticulum (ER). By substantially contributing to the overall hydrophilicity of the polypeptide, pre-assembled core glycans inhibit possible aggregation caused by the inevitable exposure of hydrophobic patches on the as yet unstructured chains. Thereafter, N-glycans are modified by ER-resident enzymes glucosidase I (GI), glucosidase II (GII), UDP-glucose:glycoprotein glucosyltransferase (UGT) and mannosidase(s) and become functional appendices that determine the fate of the associated polypeptide. Recent work has improved our understanding of how the removal of terminal glucose residues from N-glycans allows newly synthesized proteins to access the calnexin chaperone system; how substrate retention in this specialized chaperone system is regulated by de-/re-glucosylation cycles catalyzed by GII and UGT1; and how acceleration of N-glycan dismantling upon induction of EDEM variants promotes ER-associated degradation (ERAD) under conditions of ER stress. In particular, characterization of cells lacking certain ER chaperones has revealed important new information on the mechanisms regulating protein folding and quality control. Tight regulation of N-glycan modifications is crucial to maintain protein quality control, to ensure the synthesis of functional polypeptides and to avoid constipation of the ER with folding-defective polypeptides.

Animals↗
PubMed2006

EDEM1 regulates ER-associated degradation by accelerating de-mannosylation of folding-defective polypeptides and by inhibiting their covalent aggregation.

Proteins expressed in the endoplasmic reticulum (ER) are covalently modified by co-translational addition of pre-assembled core glycans (glucose(3)-mannose(9)-N-acetylglucosamine(2)) to asparagines in Asn-X-Ser/Thr motifs. N-Glycan processing is essential for protein quality control in the ER. Cleavages and re-additions of the innermost glucose residue prolong folding attempts in the calnexin cycle. Progressive loss of mannoses is a symptom of long retention in the ER and elicits preparation of terminally misfolded polypeptides for dislocation into the cytosol and proteasome-mediated degradation. The ER stress-induced protein EDEM1 regulates disposal of folding-defective glycoproteins and has been described as a mannose-binding lectin. Here we show that elevation of the intralumenal concentration of EDEM1 accelerates ER-associated degradation (ERAD) by accelerating de-mannosylation of terminally misfolded glycoproteins and by inhibiting formation of covalent aggregates upon release of terminally misfolded ERAD candidates from calnexin. Acceleration of Man(9) or Man(5)N-glycans dismantling upon overexpression was fully blocked by substitution in EDEM1 of one catalytic residue conserved amongst alpha1,2-mannosidases, thus suggesting that EDEM1 is an active mannosidase. This mutation did not affect the chaperone function of EDEM1.

Animals↗
PubMed2006

N-linked glycan recognition and processing: the molecular basis of endoplasmic reticulum quality control.

Nascent polypeptides emerging into the lumen of the endoplasmic reticulum (ER) are N-glycosylated on asparagines in Asn-Xxx-Ser/Thr motifs. Processing of the core oligosaccharide eventually determines the fate of the associated polypeptide by regulating entry into and retention by the calnexin chaperone system, or extraction from the ER folding environment for disposal. Recent advances have shown that at least two N-glycans are necessary for protein access to the calnexin chaperone system and that polypeptide cycling in the system is a rather rare event, which, for folding-defective polypeptides, is activated only upon persistent misfolding. Additionally, dismantling of the polypeptide-bound N-glycan interrupts futile folding attempts, and elicits preparation of the misfolded chain for dislocation into the cytosol and degradation.

Animals↗
PubMed2006

Consequences of ERp57 deletion on oxidative folding of obligate and facultative clients of the calnexin cycle.

Members of the protein-disulfide isomerase superfamily catalyze the formation of intra- and intermolecular disulfide bonds, a rate-limiting step of protein folding in the endoplasmic reticulum (ER). Here we compared maturation of one obligate and two facultative calnexin substrates in cells with and without ERp57, the calnexin-associated, glycoprotein-specific oxidoreductase. ERp57 deletion did not prevent the formation of disulfide bonds during co-translational translocation of nascent glycopolypeptides in the ER. It affected, however, the post-translational phases of oxidative influenza virus hemagglutinin (HA) folding, resulting in significant loss of folding efficiency for this obligate calnexin substrate. Without ERp57, HA also showed reduced capacity to recover from an artificially induced aberrant conformation, thus revealing a crucial role of ERp57 during post-translational reshuffling to the native set of HA disulfides. ERp57 deletion did not affect maturation of the model facultative calnexin substrates E1 and p62 (and of most cellular proteins, as shown by lack of induction of ER stress). ERp72 was identified as one of the ER-resident oxidoreductases associating with the orphan ERp57 substrates to maintain their folding competence.

Animals↗
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↗
PubMed2005

Degradation of trafficking-defective long QT syndrome type II mutant channels by the ubiquitin-proteasome pathway.

Mutations in the human ether-a-go-go-related gene (hERG) cause chromosome 7-linked long QT syndrome type II (LQT2). We have shown previously that LQT2 mutations lead to endoplasmic reticulum (ER) retention and rapid degradation of mutant hERG proteins. In this study we examined the role of the ubiquitin-proteasome pathway in the degradation of the LQT2 mutation Y611H. We showed that proteasome inhibitors N-acetyl-L-leucyl-L-leucyl-L-norleucinal and lactacystin but not lysosome inhibitor leupeptin inhibited the degradation of Y611H mutant channels. In addition, ER mannosidase I inhibitor kifunensine and down-regulation of EDEM (ER degradation-enhancing alpha-mannosidase-like protein) also suppressed the degradation of Y611H mutant channels. Proteasome inhibition but not mannosidase inhibition led to the accumulation of full-length hERG protein in the cytosol. The hERG protein accumulated in the cytosol was deglycosylated. Proteasome inhibition also resulted in the accumulation of polyubiquitinated hERG channels. These results suggest that the degradation of LQT2 mutant channels is mediated by the cytosolic proteasome in a process that involves mannose trimming, polyubiquitination, and deglycosylation of mutant channels.

Acetylcysteine↗
PubMed2005

The glycan code of the endoplasmic reticulum: asparagine-linked carbohydrates as protein maturation and quality-control tags.

The majority of proteins that traverse the secretory pathway receive asparagine (Asn)-linked glycosylations. Glycans are bulky hydrophilic modifications that serve a variety of structural and functional roles within the cell. Here, we review the recent growing knowledge on the role of Asn-linked glycans as maturation and quality-control protein tags in the early secretory pathway. The carbohydrate composition encodes crucial information about the structure, localization and age of glycoproteins. The "glycan code" is encoded by a series of glycosidases and carbohydrate transferases that line the secretory pathway. This code is deciphered by carbohydrate-binding proteins that possess distinct carbohydrate binding properties and act as molecular chaperones or sorting receptors. These glycosidases and transferases work in concert with resident secretory pathway carbohydrate-binding proteins to form a network that assists in the maturation and trafficking of both native and aberrant glycoproteins within the cell.

Animals↗
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↗
PubMed2002

Folding of viral glycoproteins in the endoplasmic reticulum.

The endoplasmic reticulum (ER) is a subcellular compartment specialized in folding and assembly of newly synthesized polypeptides. The polypeptides expressed in the ER include all secretory proteins produced in the cell, lumenal or membrane-bound proteins of the endocytic/vacuolar and secretory compartments and transmembrane proteins that operate at the plasma membrane. In the lumen of the ER, molecular chaperones and folding factors facilitate the maturation of newly synthesized proteins. In a process defined as ER-quality control, they also warrant that only properly structured and assembled products leave the ER and are transported to their target organelles and compartments. If proper maturation fails, the aberrant products are degraded. Quality control in the ER is essential to prevent exit of improperly regulated or not-functional products that could lead to harmful effects. The mechanisms of protein folding and quality control in the ER are far from being fully understood. They are fundamental for the life of cells and organisms, but they are also linked to important human hereditary diseases in which mutated gene products are retained in the ER and degraded (e.g., cystic fibrosis and hereditary lung emphysema).

Endoplasmic Reticulum↗
PubMed2002

The disulphide bonds in the catalytic domain of BACE are critical but not essential for amyloid precursor protein processing activity.

beta-Site APP-cleaving enzyme (BACE) initiates the processing of the amyloid precursor protein (APP) leading to the generation of beta-amyloid, the main component of Alzheimer's disease senile plaques. BACE (Asp2, memapsin 2) is a type I transmembrane aspartic protease responsible for the beta-secretase cleavage of APP producing a soluble form of the ectodomain (sAPPbeta) and the membrane-bound, carboxy-terminal intermediates C99 and C89. BACE maturation involves cysteine bridge formation, N -glycosylation and propeptide removal. We investigated variants of BACE in which the disulphide bonds of the catalytic domain spanning between Cys216/Cys420, Cys278/Cys443 and Cys330/Cys380 were removed by mutagenesis. When transfected in cultured cells, these mutants showed impaired maturation. Nevertheless, a fraction of mutated protein retained both the competence to mature as well as the activity to process APP. For the generation of a functional enzyme the conserved Cys330/Cys380 bond was the most critical, whereas the two bonds between Cys216/Cys420 and Cys278/Cys443, which are typical for the membrane-bound BACE, appeared to be less important.

Alzheimer Disease↗