Dissociation of mitochondrial HK-II elicits mitophagy and confers cardioprotection against ischemia

Abstract

Preservation of mitochondrial integrity is critical for maintaining cellular homeostasis. Mitophagy is a mitochondria-specific type of autophagy which eliminates damaged mitochondria thereby contributing to mitochondrial quality control. Depolarization of the mitochondrial membrane potential is an established mechanism for inducing mitophagy, mediated through PINK1 stabilization and Parkin recruitment to mitochondria. Hexokinase-II (HK-II) which catalyzes the first step in glucose metabolism, also functions as a signaling molecule to regulate cell survival, and a significant fraction of cellular HK-II is associated with mitochondria (mitoHK-II). We demonstrate here that pharmacological interventions and adenoviral expression of a mitoHK-II dissociating peptide which reduce mitoHK-II levels lead to robust increases in mitochondrial Parkin and ubiquitination of mitochondrial proteins in cardiomyocytes and in a human glioblastoma cell line 1321N1, independent of mitochondrial membrane depolarization or PINK1 accumulation. MitoHK-II dissociation-induced mitophagy was demonstrated using Mito-Keima in cardiomyocytes and in 1321N1 cells. Subjecting cardiomyocytes or the in vivo heart to ischemia leads to modest dissociation of mitoHK-II. This response is potentiated by expression of the mitoHK-II dissociating peptide, which increases Parkin recruitment to mitochondria and, importantly, provides cardioprotection against ischemic stress. These results suggest that mitoHK-II dissociation is a physiologically relevant cellular event that is induced by ischemic stress, the enhancement of which protects against ischemic damage. The mechanism which underlies the effects of mitoHK-II dissociation can be attributed to the ability of Bcl2-associated athanogene 5 (BAG5), an inhibitor of Parkin, to localize to mitochondria and form a molecular complex with HK-II. Overexpression of BAG5 attenuates while knockdown of BAG5 sensitizes the effect of mitoHK-II dissociation on mitophagy. We suggest that HK-II, a glycolytic molecule, can function as a sensor for metabolic derangements at mitochondria to trigger mitophagy, and modulating the intracellular localization of HK-II could be a novel way of regulating mitophagy to prevent cell death induced by ischemic stress.

Pleiotropic role of melatonin in brain mitochondria of obese mice

“…Although there is debate regarding the metabolic effects of melatonin, our data strongly support that its effect could be mediated by HKII reduction, which includes either mitoHKII or cytosolic HKII (25). Dissociation mitoHKII is a relevant cellular event usually induced by adverse conditions, such as ischemia or cancer; this can have protective actions into the cell, triggering mitophagy (26) and increasing NADPH levels in the cytosol with measured antioxidant consequences (75). In the subjective situation of our leptin-deficient animals, anchored to a permanent starvation feeling, cytosolic HKII facilitated autophagy during starvation (25), which would aggravate the situation.…”

“…In support of this, Bcl-2 proteins have been implicated in mitoHKII dissociation and mitophagy activation (26,76). This is supported by accumulated evidence which reveals that HKII is not only a molecule involved with glycolysis, but also it has a crucial role in cellular protection by preventing the mitochondrial death pathway (25).…”

“…These three molecules, i.e., HKII, leptin and melatonin, collaborate in multiple cellular processes. Thus, HKII also to plays a crucial role in cell survival (24,25,31) as a protective molecule against oxidative stress (26) and the induction of autophagy (24), including mitophagy, a selective mitochondrial autophagy, that occurs when oxidative stress increases or when mitochondrial dysfunction is exacerbated. Likewise, HKII is also involved in the regulation of apoptotic processes; several studies have demonstrated that HKII competes with apoptotic B-cell lymphoma 2 protein (Bcl-2) family proteins, such as Bax, to prevent mitochondrial outer membrane rupture (36,37).…”

Hexokinase II dissociation alone cannot account for changes in heart mitochondrial function, morphology and sensitivity to permeability transition pore opening following ischemia

“…We believe this protocol to be preferable to the use of synthetic peptides mimicking the HK2 binding peptide since these might be carried through into the assay and mimic the bound HK2 in its effects on the mPTP. Indeed, isolated cardiac mitochondria of mice overexpressing GFP fused to this peptide show successful dissociation of mt-HK2 but retention of the recombinant protein [37].…”

BAG5 Promotes Alpha-Synuclein Oligomer Formation and Functionally Interacts With the Autophagy Adaptor Protein p62

“…The inhibition of Hsp70, parkin, and CHIP by BAG5 disrupts proteostasis, mitophagy (De Snoo et al, 2019), and promotes the formation of alpha-synuclein oligomers, as well as other protein aggregates, which contribute to neuronal death (Kalia et al, 2013). Consistent with these findings, BAG5 was found to promote dopaminergic neuron death in the substantia nigra in rodent models of PD (Kalia et al, 2004) and interact with other PD relevant proteins including LRRK2, PINK1, and DJ-1 (Beilina et al, 2014;Wang et al, 2014;Qin et al, 2017;Tan et al, 2019;De Snoo et al, 2019).…”