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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.

SUBMITTER: Trychta KA

PROVIDER: S-EPMC8276033 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:OBJECTIVE:Single-cell RNA sequencing studies have revealed that the type-2 diabetes associated two-pore domain K+ (K2P) channel TALK-1 is abundantly expressed in somatostatin-secreting ?-cells. However, a physiological role for TALK-1 in ?-cells remains unknown. We previously determined that in ?-cells, K+ flux through endoplasmic reticulum (ER)-localized TALK-1 channels enhances ER Ca2+ leak, modulating Ca2+ handling and insulin secretion. As glucose amplification of islet somatostatin release relies on Ca2+-induced Ca2+ release (CICR) from the ?-cell ER, we investigated whether TALK-1 modulates ?-cell Ca2+ handling and somatostatin secretion. METHODS:To define the functions of islet ?-cell TALK-1 channels, we generated control and TALK-1 channel-deficient (TALK-1 KO) mice expressing fluorescent reporters specifically in ?- and ?-cells to facilitate cell type identification. Using immunofluorescence, patch clamp electrophysiology, Ca2+ imaging, and hormone secretion assays, we assessed how TALK-1 channel activity impacts ?- and ?-cell function. RESULTS:TALK-1 channels are expressed in both mouse and human ?-cells, where they modulate glucose-stimulated changes in cytosolic Ca2+ and somatostatin secretion. Measurement of cytosolic Ca2+ levels in response to membrane potential depolarization revealed enhanced CICR in TALK-1 KO ?-cells that could be abolished by depleting ER Ca2+ with sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) inhibitors. Consistent with elevated somatostatin inhibitory tone, we observed significantly reduced glucagon secretion and ?-cell Ca2+ oscillations in TALK-1 KO islets, and found that blockade of ?-cell somatostatin signaling with a somatostatin receptor 2 (SSTR2) antagonist restored glucagon secretion in TALK-1 KO islets. CONCLUSIONS:These data indicate that TALK-1 reduces ?-cell cytosolic Ca2+ elevations and somatostatin release by limiting ?-cell CICR, modulating the intraislet paracrine signaling mechanisms that control glucagon secretion.

Project description:Ildr2, a modifier of diabetes susceptibility in obese mice, is expressed in most organs, including islets and hypothalamus, with reduced levels in livers of diabetes-susceptible B6.DBA mice congenic for a 1.8 Mb interval of Chromosome 1. In hepatoma and neuronal cells, ILDR2 is primarily located in the endoplasmic reticulum membrane. We used adenovirus vectors that express shRNA or are driven by the CMV promoter, respectively, to knockdown or overexpress Ildr2 in livers of wild type and ob/ob mice. Livers in knockdown mice were steatotic, with increased hepatic and circulating triglycerides and total cholesterol. Increased circulating VLDL, without reduction in triglyceride clearance suggests an effect of reduced hepatic ILDR2 on hepatic cholesterol clearance. In animals that overexpress Ildr2, hepatic triglyceride and total cholesterol levels were reduced, and strikingly so in ob/ob mice. There were no significant changes in body weight, energy expenditure or glucose/insulin homeostasis in knockdown or overexpressing mice. Knockdown mice showed reduced expression of genes mediating synthesis and oxidation of hepatic lipids, suggesting secondary suppression in response to increased hepatic lipid content. In Ildr2-overexpressing ob/ob mice, in association with reduced liver fat content, levels of transcripts related to neutral lipid synthesis and cholesterol were increased, suggesting "relief" of the secondary suppression imposed by lipid accumulation. Considering the fixed location of ILDR2 in the endoplasmic reticulum, we investigated the possible participation of ILDR2 in ER stress responses. In general, Ildr2 overexpression was associated with increases, and knockdown with decreases in levels of expression of molecular components of canonical ER stress pathways. We conclude that manipulation of Ildr2 expression in liver affects both lipid homeostasis and ER stress pathways. Given these reciprocal interactions, and the relatively extended time-course over which these studies were conducted, we cannot assign causal primacy to either the effects on hepatic lipid homeostasis or ER stress responses.

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

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