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ER is the primary site for the maturation and folding of soluble and transmembrane secretory proteins. The calnexin cycle is a specific chaperone system that folds and assesses the confirmation of N-glycosylated proteins before they can exit the ER lumen. The primary players of this quality check pipeline are the lectins, ER-resident chaperones, and a glucosyl transferase enzyme. In case the calnexin system in the lumen fails to salvage a misfolded protein, it is transported to the cytoplasm...
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In the secretory pathway, vesicles transport proteins from one cellular compartment to another in forward transport to deliver the protein to its correct location. Occasionally, misfolded proteins and incorrect proteins escape their original compartments, and a retrieval pathway is used to return the escaped proteins to their original compartment.
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Tail-anchoring of Proteins in the ER Membrane01:45

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Tail-anchored, or TA, proteins are estimated to make up to 3-5% of membrane proteins found in the eukaryotic cell. Such proteins have a single transmembrane domain located approximately 30 amino acid residues upstream from the C-terminal end. As a result, the signal recognition particle (SRP) cannot guide a TA protein to the ER membrane for cotranslational insertion. Hence, they are integrated into the ER membrane post-translationally using their C-terminal end as the anchor. TA proteins...
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After folding, the ER assesses the quality of secretory and membrane proteins. The correctly folded proteins are cleared by the calnexin cycle for transport to their final destination, while misfolded proteins are held back in the ER lumen. The ER chaperones attempt to unfold and refold the misfolded proteins but sometimes fail to achieve the correct native conformation. Such terminally misfolded proteins are then exported to the cytosol by ER-associated degradation or ERAD pathway for...
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Many proteins’ biological role depends on their interactions with their ligands, small molecules that bind to specific locations on the protein known as ligand-binding sites. Ligand-binding sites are often conserved among homologous proteins as these sites are critical for protein function.
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Modification of secretory and transmembrane proteins entering the rough ER begins in the ER lumen. These modifications aid in protein folding and stabilize the acquired tertiary structure. Protein modifications in the rough ER co-occur at different stages of protein folding.
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Mapping human calreticulin regions important for structural stability.

Evaldas Čiplys1, Tautvydas Paškevičius2, Eimantas Žitkus1

  • 1Institute of Biotechnology, Life Sciences Center, Vilnius University, Saulėtekio 7, LT-10257 Vilnius, Lithuania.

Biochimica Et Biophysica Acta. Proteins and Proteomics
|August 6, 2021
PubMed
Summary
This summary is machine-generated.

Researchers identified key residues, Tyr172 and Asp187, critical for calreticulin (CALR) protein stability. Mutations in these residues impact CALR

Keywords:
CalreticulinMutantsSecretionStructural stabilityThermal stability

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Protein Chemistry

Background:

  • Calreticulin (CALR) is a conserved chaperone protein in the endoplasmic reticulum regulating calcium homeostasis.
  • CALR also performs critical functions on the cell surface and in the extracellular space.
  • Understanding CALR's structural stability is crucial for elucidating its diverse roles.

Purpose of the Study:

  • To identify specific amino acid residues and regions essential for the structural stability of human calreticulin (CALR).
  • To investigate the impact of mutations on CALR's secretion, thermal stability, and oligomeric state.
  • To explore potential correlations between CALR structural integrity and associated diseases like myeloproliferative neoplasms (MPN) and sudden unexpected death (SUD).

Main Methods:

  • Utilized a Saccharomyces cerevisiae expression system to produce 50 human CALR mutants.
  • Analyzed mutants for secretion titer, melting temperature (Tm), overall stability, and oligomeric state.
  • Focused on characterizing the contribution of conserved surface residues (amino acids 166-187) to CALR structure.

Main Results:

  • Identified a conserved surface residue patch (amino acids 166-187, "cluster 2") as vital for CALR structural stability.
  • Found that Tyr172 and Asp187 are critical residues for maintaining CALR's native structure.
  • Observed that mutant D187A exhibited significantly reduced secretion, thermal instability, degradation susceptibility, and altered oligomerization.
  • Demonstrated Tyr172's importance for thermal stability, noting its interaction with Cys163, suggesting a unique stabilizing unit involving Asp187, Tyr172, and Cys163.

Conclusions:

  • Asp187, Tyr172, and Cys163 form a critical structural unit contributing to CALR's unusual thermal stability.
  • Specific CALR mutations are linked to altered protein stability and potentially to diseases such as MPN and SUD.
  • This study provides key insights into the structure-function relationships of calreticulin.