Abstract
ERp57, a member of the protein disulfide isomerase family, is a ubiquitous disulfide catalyst that functions in the oxidative folding of various clients in the mammalian endoplasmic reticulum (ER). In concert with ER lectin-like chaperones calnexin and calreticulin (CNX/CRT), ERp57 functions in virtually all folding stages from co-translation to post-translation, and thus plays a critical role in maintaining protein homeostasis, with direct implication for pathology. Here, we present mechanisms by which Ca2+ regulates the formation of the ERp57-calnexin complex. Biochemical and isothermal titration calorimetry analyses revealed that ERp57 strongly interacts with CNX via a non-covalent bond in the absence of Ca2+. The ERp57-CNX complex not only promoted the oxidative folding of human leukocyte antigen heavy chains, but also inhibited client aggregation. These results suggest that this complex performs both enzymatic and chaperoning functions under abnormal physiological conditions, such as Ca2+ depletion, to effectively guide proper oxidative protein folding. The findings shed light on the molecular mechanisms underpinning crosstalk between the chaperone network and Ca2+.
Highlights
The endoplasmic reticulum (ER) harbors specific enzymes and associated factors that both assist productive protein folding and eliminate the risk of protein aggregation to ensure protein homeostasis in the ER [1,2,3,4]
Complex Formation between ERp57 and CNX Is Modulated by Ca2+
The two bands around 66 kDa and 57 kDa are consistent with monomeric CNX and ERp57, respectively, the upper band around 146 kDa was observed in the presence of both proteins (Figure 1a, lanes 3 and 6)
Summary
The endoplasmic reticulum (ER) harbors specific enzymes and associated factors that both assist productive protein folding and eliminate the risk of protein aggregation to ensure protein homeostasis in the ER [1,2,3,4]. Most PDIs contain the Cys-X-X-Cys motif within the redox active site of their thioredoxin (Trx)-like domain(s) that catalyze disulfide introduction, isomerization, and reduction in substrates [5]. The newly synthesized HLA heavy chain (HC) interacts with the CNX-ERp57 complex during the early folding step, and natively folded HLA-HC interacts with the CRT-ERp57 complex for the assembly of HLA heterodimer with β2m and PLC [21]. The ERp57-CNX complex promotes the oxidative folding of HLA-HC, and inhibits client aggregation. These findings shed light on the molecular mechanism underpinning crosstalk in chaperoneregulated protein homeostasis and Ca2+
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