Computational Modeling of C-Terminal Tails to Predict the Calcium-Dependent Secretion of Endoplasmic Reticulum Resident Proteins

Abstract

The lumen of the endoplasmic reticulum (ER) has resident proteins that are critical to perform the various tasks of the ER such as protein maturation and lipid metabolism. These ER resident proteins typically have a carboxy-terminal ER retention/retrieval sequence (ERS). The canonical ERS that promotes ER retrieval is Lys-Asp-Glu-Leu (KDEL) and when an ER resident protein moves from the ER to the Golgi, KDEL receptors (KDELRs) in the Golgi recognize the ERS and return the protein to the ER lumen. Depletion of ER calcium leads to the mass departure of ER resident proteins in a process termed exodosis, which is regulated by KDELRs. Here, by combining computational prediction with machine learning-based models and experimental validation, we identify carboxy tail sequences of ER resident proteins divergent from the canonical “KDEL” ERS. Using molecular modeling and simulations, we demonstrated that two representative non-canonical ERS can stably bind to the KDELR. Collectively, we developed a method to predict whether a carboxy-terminal sequence acts as a putative ERS that would undergo secretion in response to ER calcium depletion and interacts with the KDELRs. The interaction between the ERS and the KDELR extends beyond the final four carboxy terminal residues of the ERS. Identification of proteins that undergo exodosis will further our understanding of changes in ER proteostasis under physiological and pathological conditions where ER calcium is depleted.

Identification of ER/SR resident proteins as biomarkers for ER/SR calcium depletion in skeletal muscle cells

“…In addition to ERS proteins, several non-ERS proteins were also released from the cell in response to ER/SR calcium depletion. Of note, selenoprotein N (SEPN1) was the most abundant non-ERS extracellular protein detected based on previously established ERS criteria [ 8 , 9 ] (Fig. 2 ).…”

“…Of the 4,465 extracellular proteins identified, 1,280 proteins significantly changed following Tg treatment. 54 corresponded to ERS proteins [ 8 , 9 ], with 33 ERS proteins significantly increased after Tg (Fig. 2 ; Additional file 1 : Fig.…”

“…Depletion of ER/SR calcium causes the secretion of ER/SR resident protein in phenomenon described as exodosis [ 8 ]. Under physiological conditions, resident proteins are retained within ER/SR via a carboxy-terminal ER retention/retrieval sequence (ERS) that interacts with the KDEL receptor retrieval pathway [ 9 ]. Retention of ERS proteins is vital to cellular function, as many ERS proteins contribute to proper protein folding, modification, and trafficking [ 9 ].…”

“…Under physiological conditions, resident proteins are retained within ER/SR via a carboxy-terminal ER retention/retrieval sequence (ERS) that interacts with the KDEL receptor retrieval pathway [ 9 ]. Retention of ERS proteins is vital to cellular function, as many ERS proteins contribute to proper protein folding, modification, and trafficking [ 9 ]. Using a bioluminescent reporter protein, exodosis was recently shown to occur in skeletal muscle cells in response to pharmacologically induced ER/SR calcium depletion [ 10 ].…”

KDEL Receptors: Pathophysiological Functions, Therapeutic Options, and Biotechnological Opportunities

“…As mentioned earlier, many variants of the tetrapeptide binding motif are present on human ER-resident proteins [ 17 , 44 ]. Structural and functional studies have shown that the −1 and −2 positions within the canonical binding sequence are the most important for interaction with the KDELR, likely because the −1 and −2 positions can interact with as many as 10 and 6 amino acids of the receptor, respectively [ 47 ]. On the other hand, the −3 and −4 positions of the motif contact only 3 and 4 amino acids of the KDELR, and, indeed, the substitution of these amino acids produces less dramatic effects on receptor binding [ 47 ].…”