15.12
糙面内质网中的蛋白质修饰
进入糙面内质网的分泌蛋白和跨膜蛋白的修饰起始于内质网腔。这些修饰有助于蛋白质的折叠，并使其能够稳定的得到三级结构。糙面内质网中的蛋白质修饰同时发生在蛋白质折叠的不同阶段。
总体上，这些修饰能够被分为四大类 —— 糖基化、二硫键的形成、蛋白质亚基的组装以及特定的蛋白水解切割（例如信号序列的去除）。
蛋…
N-连接糖基化和二硫键形成是内质网中发生的两种重要蛋白质修饰。
在糖基化过程中，寡糖基转移酶复合物会将支链寡糖分子添加到新进入的多肽链的某些特定天冬酰胺残基上。
相比之下，二硫键的形成发生在同一或多肽链上两个相邻的半胱氨酸残基之间。
该过程涉及两个关键组分——蛋白质二硫键异构酶（protein disulfide isomerase，PDI）和内质网氧化还原酶I（ER oxidoreductase I，Ero1）。
PDI 分子呈马蹄形，其活性位点含有二硫键。
氧化态的PDI具有开放构象，并可结合未折叠的多肽。首先，多肽的还原态半胱氨酸残基与酶的活性位点形成二硫键。
该中间产物随后与多肽链上另一个相邻的半胱氨酸相互作用，导致两个残基之间形成二硫键。
随后，PDI 转变为闭合构象，释放多肽链。
Ero1 将还原态的 PDI 重新转化为氧化态，使其能够进行下一轮反应。
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Q1: What is N-linked glycosylation and why does it occur in the ER?
N-linked glycosylation is the covalent attachment of branched oligosaccharide molecules to asparagine residues on incoming polypeptide chains, catalyzed by the oligosaccharyltransferase complex. This modification improves protein folding kinetics, increases stability by masking cleavage sites, and allows ER chaperones to assess proper protein folding before the protein exits the ER.
Q2: How does protein disulfide isomerase facilitate disulfide bond formation?
Protein disulfide isomerase (PDI) resembles a horseshoe with disulfide bonds in its active site. Oxidized PDI binds unfolded polypeptides and forms a disulfide link between a reduced cysteine on the polypeptide and the enzyme's active site. This intermediate then interacts with another nearby cysteine, creating a disulfide bond between the two residues before PDI releases the polypeptide.
Q3: What role does Ero1 play in maintaining the ER redox environment?
ER oxidoreductase 1 (Ero1) recycles reduced protein disulfide isomerase back to its oxidized state, preparing it for additional rounds of disulfide bond formation. Ero1 utilizes a significant fraction of molecular oxygen available in the cell and generates hydrogen peroxide, maintaining redox homeostasis inside the ER while supporting oxidative protein folding.
Q4: Which amino acid sequences are recognized for N-linked glycosylation?
N-linked glycosylation occurs at asparagine residues within the tripeptide sequences Asn-X-Ser and Asn-X-Thr, where X represents any amino acid except proline. The oligosaccharyltransferase complex recognizes these specific motifs and adds oligosaccharides during both cotranslational and post-translational protein translocation in the ER lumen.
Q5: How does PDI function as both an enzyme and a proofreader for disulfide bonds?
Oxidized PDI catalyzes disulfide bond formation between cysteine residues, while reduced PDI acts as a proofreader by correcting inappropriately paired cysteines through rearranging disulfide linkages. This dual function ensures accurate disulfide bond formation and proper protein folding in the oxidizing ER environment.
Q6: What physical properties of proteins are altered by N-linked glycosylation?
N-linked glycosylation improves the thermodynamic kinetics of protein folding, allowing glycosylated proteins to fold better than their non-glycosylated counterparts. Glycosylation also increases protein stability by masking hydrophobic stretches and cleavage sites, protecting the protein from degradation and enhancing its structural integrity.
Q7: Where do disulfide bonds predominantly form in the cell?
Disulfide bonds form predominantly in the rough ER lumen, which provides an oxidizing environment favorable for their formation. A small fraction of disulfide bonds can also form in the mitochondrial intermembrane space, but the ER remains the primary site for this modification of secretory and transmembrane proteins.