Abstract 108: Glycolysis, Cardioprotection and Their Modulation by Differential Interactions of Hexokinase I and II with Mitochondria

Rationale: Neonatal heart is more glycolytically active and less susceptible to anoxia/reoxygenation injury than adult heart. Hexokinase (HK) is the first step in glycolysis and its binding to mitochondria is known to be cardioprotective. Since neonatal cardiac myocytes mainly express the HKI isoform, whereas HKII predominates in adults, we explored the functional consequences of such difference. Objective: To investigate whether differential interactions of HKI and HKII with mitochondria in neonatal (NRVM) and adult rat ventricular myocytes (ARVM) account for their different metabolic profiles and susceptibility to anoxia/reoxygenation injury. Methods and Results: We performed real-time imaging in isolated NRVM and ARVM expressing the genetically-encoded biosensors FLIPglu-600µM to track intracellular glucose metabolism and YFP-tagged HK constructs to track subcellular HK localization, together with a propidium iodide-based cell death assay. We show that HKI remains bound to mitochondria in both NRVM and ARVM, while HKII distributes between the mitochondria and cytoplasm. Extracellular glucose removal displaced HKII from mitochondria in ARVM, but not in NRVM, whereas iodoacetate (IAA) displaces HKII in both. These differences were associated with a 35-fold greater glycolytic activity and markedly enhanced resistance to anoxia/reoxygenation injury in NVRM. NRVM became comparably susceptible to anoxia/reoxygenation injury only when treated with IAA to suppress HK interaction with mitochondria (whereas glucose removal alone had no effect). Conclusions: HKs exhibit stronger interaction with mitochondria in NRVM, accounting for their enhanced glycolytic activity and increased resistance to anoxia/reoxygenation injury. HKI may be superior to HKII in protecting the heart because of its stronger interaction with mitochondria.

E1∧E4 Protein of Human Papillomavirus Type 16 Associates with Mitochondria

ABSTRACT The human papillomavirus (HPV) E1∧E4 protein is the most abundantly expressed viral protein in HPV-infected epithelia. It possesses diverse activities, including the ability to bind to the cytokeratin network and to DEAD-box proteins, and in some cases induces the collapse of the former. E1∧E4 is also able to prevent the progression of cells into mitosis by arresting them in the G2 phase of the cell cycle. In spite of these intriguing properties, the role of this protein in the life cycle of the virus is not clear. Here we report that after binding to and collapsing the cytokeratin network, the HPV type 16 E1∧E4 protein binds to mitochondria. When cytokeratin is not present in the cell, E1∧E4 appears associated with mitochondria soon after its synthesis. The leucine cluster within the N-terminal portion of the E1∧E4 protein is pivotal in mediating this association. After the initial binding to mitochondria, the E1∧E4 protein induces the detachment of mitochondria from microtubules, causing the organelles to form a single large cluster adjacent to the nucleus. This is followed by a severe reduction in the mitochondrial membrane potential and an induction of apoptosis. HPV DNA replication and virion production occur in terminally differentiating cells which are keratin-rich, rigid squamae that exfoliate after completion of the differentiation process. Perturbation of the cytokeratin network and the eventual induction of apoptotic properties are processes that could render these unyielding cells more fragile and ease the exit of newly synthesized HPVs for subsequent rounds of infection.