Evolutionary Origin of Insulin-Degrading Enzyme and Its Subcellular Localization and Secretion Mechanism: A Study in Microglial Cells

Abstract

The insulin-degrading enzyme (IDE) is a zinc-dependent metalloendopeptidase that belongs to the M16A metalloprotease family. IDE is markedly expressed in the brain, where it is particularly relevant due to its in vitro amyloid beta (Aβ)-degrading activity. The subcellular localization of IDE, a paramount aspect to understand how this enzyme can perform its proteolytic functions in vivo, remains highly controversial. In this work, we addressed IDE subcellular localization from an evolutionary perspective. Phylogenetic analyses based on protein sequence and gene and protein structure were performed. An in silico analysis of IDE signal peptide suggests an evolutionary shift in IDE exportation at the prokaryote/eukaryote divide. Subcellular localization experiments in microglia revealed that IDE is mostly cytosolic. Furthermore, IDE associates to membranes by their cytoplasmatic side and further partitions between raft and non-raft domains. When stimulated, microglia change into a secretory active state, produces numerous multivesicular bodies and IDE associates with their membranes. The subsequent inward budding of such membranes internalizes IDE in intraluminal vesicles, which later allows IDE to be exported outside the cells in small extracellular vesicles. We further demonstrate that such an IDE exportation mechanism is regulated by stimuli relevant for microglia in physiological conditions and upon aging and neurodegeneration.

The Role of Microglia in Alzheimer’s Disease From the Perspective of Immune Inflammation and Iron Metabolism

“…The deficiency of CCL2 and CCR2 is followed by reduced Aβ phagocytosis and amyloid clearance (Jorda et al, 2021). In the early stage of AD pathology, microglia also release insulin-degrading enzyme (IDE), proteins of the matrix metalloproteinase 9 (MMP9), plasminogen and neprilysin, which are involved in Aβ degradation and induce Aβ clearance (Gao et al, 2018;Corraliza-Gómez et al, 2022). Another study with AD mouse models through high-resolution confocal and in vivo two-photon imaging found that microglia appear to have another neuroprotective mechanism, namely Aβ plaques are tightly wrapped by microglial processes, constituting a barrier that prevents the outward expansion of the plaque and the formation of neurotoxic fibril Aβ 42 around the plaques (Condello et al, 2015).…”

Altered Surface Expression of Insulin-Degrading Enzyme on Monocytes and Lymphocytes from COVID-19 Patients Both at Diagnosis and after Hospital Discharge

“…Additionally, the potential export/secretion mechanism of IDE is still obscure because the lack of a signal peptide argues against IDE’s association to the extracellular side of a plasma membrane. In this line of evidence, Corraliza-Gómez and collaborators have shown that stimulated microglia allow IDE to be exported outside of cells in small extracellular vesicles that have arisen from multivesicular bodies, albeit, in their study, it was associated with plasma membranes exclusively at the cytoplasmic side [ 2 ]. Thus, it is plausible to hypothesize that IDE might be secreted from monocytes and lymphocytes via non-conventional secretory pathways.…”

“…Insulin-degrading enzyme (IDE) is a ubiquitously expressed metallo-endopeptidase characterized by a zinc-binding consensus sequence (HxxEH) that is inverted with respect to the sequence in most conventional metalloproteases (HExxH). IDE is evolutionary and ancient, with homologs and paralogs highly conserved and present in phylogenetically diverse organisms ranging from viruses to humans, suggesting that it has proteolytic and non-proteolytic functions [ 1 , 2 ]. The subcellular localization of IDE is primarily cytosolic, albeit it has also been reported in intracellular organelles and extracellular vesicles [ 1 ].…”

Insulin-degrading enzyme (IDE) as a modulator of microglial phenotypes in the context of Alzheimer’s disease and brain aging

“…The insulin-degrading enzyme (IDE) is an evolutionarily conserved zinc-dependent metalloendopeptidase [ 22 ], named after its ability to strongly bind and degrade insulin in tissue extracts [ 23 ]. Besides insulin, many other different short peptides with similar conformation have been reported to be degraded by IDE in vitro [ 24 ].…”

“…However, brain insulin levels are much lower than the Michaelis constant (Km) of insulin for IDE, and thus unlikely to competitively inhibit Aβ degradation [ 43 ]. Furthermore, we recently described that in microglia IDE associates with membranes by their cytosolic side and is exported outside the cell inside extracellular vesicles [ 22 ]. On the contrary, both Aβ and insulin would be outside these vesicles in the extracellular compartment or inside endocytic vesicles within the cell.…”