#ER stress

Protein Sorting and Trafficking

The unfolded protein response (UPR) is triggered by:

Unfolded protein response is evolutionarily conserved stress signaling program activated when concentration of unfolded polypeptides in ER lumen exceeds buffering capacity of BiP and other chaperones, threatening proteostasis. Under basal conditions, BiP binds lumenal domains of three ER transmembrane sensors keeping them inactive: IRE1 bifunctional kinase-endoribonuclease that upon dimerization autophosphorylates and splices XBP1 mRNA removing 26 nucleotide intron producing active transcription factor inducing chaperones, lipid synthesis and ERAD genes; PERK kinase that phosphorylates eIF2α attenuating global translation initiation while allowing selective ATF4 translation controlling amino acid import, redox defense and pro-apoptotic CHOP; and ATF6 bZIP factor that upon BiP release travels in COPII vesicles to Golgi where Site-1 and Site-2 proteases liberate cytosolic fragment activating folding enzymes and ER expansion genes. Accumulation of misfolded proteins titrates BiP away permitting sensor oligomerization and activation. Lipid bilayer stress and calcium depletion also activate UPR via same sensors. Initial response aims at adaptation restoring homeostasis, but prolonged unresolved stress switches UPR to apoptotic program eliminating compromised cells via CHOP mediated pathways and caspase activation and inflammatory signaling.

Ref: Walter & Ron, Science 334: 2011, UPR Triggered by Misfolded Proteins.

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Protein Sorting and Trafficking

What happens when a protein fails to fold correctly in the ER?

Inability to achieve native conformation triggers layered proteostasis network aimed at restoring balance and if impossible eliminating dangerous species. Initially, chaperone BiP binds exposed hydrophobic stretches, UGGT reglucosylates non-native N-glycoproteins returning them to calnexin-calreticulin cycle for additional attempts at oxidative folding assisted by PDI family, ERp57, Ero1 and peptidyl-prolyl isomerases. Kinetic competition with slow mannosidases EDEM1-3 acts as timer: prolonged residence leads to progressive removal of terminal mannoses preventing reglucosylation and generating degradation signal with exposed α1-6 mannose. Degradation lectins OS-9 and XTP3-B recognize trimmed glycans and deliver clients to Sel1L-Hrd1 and Doa10 ubiquitin ligase complexes. Retrotranslocation via Derlin1-3 or Hrd1 channels, polyubiquitination by Ubc6/7 E2 enzymes, extraction by hexameric ATPase Cdc48/p97 using ATP hydrolysis and degradation by cytosolic 26S proteasome removes client. Persistent overload saturates ERAD activating unfolded protein response sensors IRE1, PERK and ATF6 that upregulate chaperones, expand ER volume, attenuate translation and may induce apoptosis via CHOP transcription factor when stress remains unresolved and adaptation fails to restore homeostasis. Additional coordination with cellular stress pathways ensures fidelity, prevents aggregation, and links trafficking to growth control and proteostasis maintenance across diverse cell types and developmental stages.

Ref: Walter & Ron, Science 334: 2011, Handling of Misfolded Proteins and UPR.

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Protein Sorting and Trafficking

Misfolded proteins in the ER are eventually:

When attempts at native conformation repeatedly fail despite chaperone assistance including BiP binding and calnexin-mediated retention plus UGGT reglucosylation cycles, terminally misfolded proteins must be removed to prevent proteotoxic aggregation, ER stress and activation of apoptotic pathways. Lumenal quality control uses mannose timer: ER mannosidase I and EDEM1-3 remove specific α1-2 mannose residues from core glycan converting Man9GlcNAc2 to Man7-6 isoforms with reduced affinity for folding lectins and increased affinity for degradation lectins. Trimmed glycans recognized by MRH domains of OS-9 and XTP3-B lectins deliver clients to membrane-embedded ubiquitin ligase complex centered on Sel1L adaptor and Hrd1 RING E3 ligase. Retrotranslocation through Derlin1-3 or Hrd1 channel itself moves polypeptide to cytosol where E2 conjugases Ubc6 and Ubc7 add K48-linked polyubiquitin chains, AAA ATPase p97/VCP with cofactors Ufd1-Npl4 provides mechanical pulling force extracting substrate, and 26S proteasome degrades it. Non-glycosylated misfolded proteins recognized via BiP and similar ERAD adaptors and ubiquitin ligase Doa10, maintaining proteome integrity and freeing chaperone capacity for new synthesis and folding attempts and organismal homeostasis.

Ref: Ruggiano et al., Nature Rev Mol Cell Biol 15: 2014, ERAD of Misfolded Proteins.

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Protein Sorting and Trafficking

Which molecular chaperone is responsible for stabilizing unfolded proteins in the ER?

Stabilization of unfolded secretory proteins inside endoplasmic reticulum oxidizing environment depends on lectin chaperone system that monitors glycosylation status and provides time for folding. Calreticulin, soluble paralog of membrane-bound calnexin sharing lectin domain but lacking transmembrane anchor, resides in ER lumen at millimolar concentration. It binds specifically monoglucosylated Glc1Man9GlcNAc2 N-glycans generated after glucosidase I and II trimming of precursor added en bloc. Its long proline-rich P-domain arm extends to recruit oxidoreductase ERp57 forming mixed disulfides with client to catalyze correct disulfide pairing while holding client soluble preventing aggregation. Substrates include MHC class I heavy chains before beta2-microglobulin association, influenza hemagglutinin, CFTR and many hormones. Unlike cytosolic Hsp90, KDEL receptor that retrieves escaped ER proteins via COPI, or Rab GTPases regulating vesicle docking and tethering, calreticulin directly holds folding intermediates through glycan recognition and acts also as major calcium buffer storing up to fifty percent of ER calcium via acidic C-domain, linking calcium homeostasis to proteostasis and antigen presentation efficiency and developmental processes. Additional coordination with cellular stress pathways ensures fidelity, prevents aggregation, and links trafficking to growth control and proteostasis maintenance across diverse cell types and developmental stages.

Ref: Braakman & Hebert, Cold Spring Harb Perspect Biol 5: 2013, Calreticulin Chaperone Role.

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