Biochemistry and Molecular Biology

Cytochrome c (Cytc) and cytochrome c oxidase (COX) catalyze the terminal reaction of the mitochondrial electron transport chain (ETC), the reduction of oxygen to water. This irreversible step is highly regulated, as indicated by the presence of tissue-specific and developmentally expressed isoforms, allosteric regulation, and reversible phosphorylations, which are found in both Cytc and COX. The crucial role of the ETC in health and disease is obvious since it, together with ATP synthase, provides the vast majority of cellular energy, which drives all cellular processes. However, under conditions of stress, the ETC generates reactive oxygen species (ROS), which cause cell damage and trigger death processes. We here discuss current knowledge of the regulation of Cytc and COX with a focus on cell signaling pathways, including cAMP/protein kinase A and tyrosine kinase signaling. Based on the crystal structures we highlight all identified phosphorylation sites on Cytc and COX, and we present a new phosphorylation site, Ser126 on COX subunit II. We conclude with a model that links cell signaling with the phosphorylation state of Cytc and COX. This in turn regulates their enzymatic activities, the mitochondrial membrane potential, and the production of ATP and ROS. Our model is discussed through two distinct human pathologies, acute inflammation as seen in sepsis, where phosphorylation leads to strong COX inhibition followed by energy depletion, and ischemia/reperfusion injury, where hyperactive ETC complexes generate pathologically high mitochondrial membrane potentials, leading to excessive ROS production. Although operating at opposite poles of the ETC activity spectrum, both conditions can lead to cell death through energy deprivation or ROS-triggered apoptosis.

Recent advancements in isolation techniques for cytochrome c (Cytc) have allowed us to discover post-translational modifications of this protein. We previously identified two distinct tyrosine phosphorylated residues on Cytc in mammalian liver and heart that alter its electron transfer kinetics and the ability to induce apoptosis. Here we investigated the phosphorylation status of Cytc in ischemic brain and sought to determine if insulin-induced neuroprotection and inhibition of Cytc release was associated with phosphorylation of Cytc. Using an animal model of global brain ischemia, we found a ,50% decrease in neuronal death in the CA1 hippocampal region with post-ischemic insulin administration. This insulinmediated increase in neuronal survival was associated with inhibition of Cytc release at 24 hours of reperfusion. To investigate possible changes in the phosphorylation state of Cytc we first isolated the protein from ischemic pig brain and brain that was treated with insulin. Ischemic brains demonstrated no detectable tyrosine phosphorylation. In contrast Cytc isolated from brains treated with insulin showed robust phosphorylation of Cytc, and the phosphorylation site was unambiguously identified as Tyr97 by immobilized metal affinity chromatography/nano-liquid chromatography/ electrospray ionization mass spectrometry. We next confirmed these results in rats by in vivo application of insulin in the absence or presence of global brain ischemia and determined that Cytc Tyr97-phosphorylation is strongly induced under both conditions but cannot be detected in untreated controls. These data suggest a mechanism whereby Cytc is targeted for phosphorylation by insulin signaling, which may prevent its release from the mitochondria and the induction of apoptosis.

Intellectual disability (ID) is defined as significantly subaverage intellectual functioning with deficits in adaptive behavior. For ∼40% of individuals, cause for disability remains unknown and these are categorized as idiopathic ID (IID). Various behavioral problems co-occur with ID and thus serotonergic neurotransmission, known to control emotion, mood and drive, has received immense attention. Synaptic serotonin (5-HT) level is primarily maintained by metabolizing enzyme MAOA and serotonin transporter (SLC6A4) which helps in reuptake of the neurotransmitter. Since functional genetic polymorphisms have a potency to affect activities of these proteins, in the present investigation polymorphisms in these genes (MAOA-u VNTR, rs6323, 5-HTTLPR and STIN2) have been analyzed in IID individuals associated with various behavioral problems.Families (N=189) with IID probands were recruited following DSM-IV. After obtaining informed written consent for participation, peripheral blood was collected for isolation of genomic DNA used for PCR-based genotyping of target sites followed by family-based statistical analyses of data.Significant association of MAOA rs6323 “T” allele with female IID (P=0.016) and a trend towards association with female IID patients exhibiting behavioral problems (P=0.046) was noticed. Non significant over transmission of the 5-HTTLPR “L” allele was also observed in female IID probands with behavioral problems (P=0.076). Synergistic epistatic interaction, with a sex-bias, was noticed between MAOA and 5-HTT (P< 0.05).From the data obtained it could be summarized that serotonergic system may have some role in the etiology of behavioral problems of female IID individuals.

Metabolic adaptation is increasingly recognized as a key factor in tumor progression, yet its involvement in metastatic bone disease is not understood. Bone is as an adipocyte-rich organ, and a major site of metastasis from prostate cancer. Bone marrow adipocytes are metabolically active cells capable of shaping tumor metabolism via lipolysis and lipid transfer. In this study, using in vitro and in vivo models of marrow adiposity, we demonstrate that marrow fat cells promote Warburg phenotype in metastatic prostate cancer cells. We show increased expression of glycolytic enzymes, increased lactate production, and decreased mitochondrial oxidative phosphorylation in tumor cells exposed to adipocytes that require paracrine signaling between the two cell types. We also reveal that prostate cancer cells are capable of inducing adipocyte lipolysis as a postulated mechanism of sustenance. We provide evidence that adipocytes drive metabolic reprogramming of tumor cells via oxygen-independent mechanism of HIF-1α activation that can be reversed by HIF-1α downregulation. Importantly, we also demonstrate that the observed metabolic signature in tumor cells exposed to adipocytes mimics the expression patterns seen in patients with metastatic disease. Together, our data provide evidence for a functional relationship between marrow adipocytes and tumor cells in bone that has likely implications for tumor growth and survival within the metastatic niche.

import complex TIM23 was just recently discovered: Mgr2. Our initial analysis of protein sequence showed that, like other TIM23 subunits, Mgr2 contained GXXXG motifs in certain trans membrane domains. These motifs were implicated in subunit oligomerization and pore stability. We hypothesized that Mgr2 was a structural component of the insertion pore, and that its deletion would impair peptide import and mitochondrial functioning. We analyzed protein import in Mgr2 deficient strains using fluorescein labeled synthetic import peptides. We also tested the effect of Mgr2 deletion on mitochondrial function by measuring accumulation of fluorescent membrane potential indicators and by determining respiratory activity using a Clark-type oxygen electrode. Moreover, TIM23 function was also examined in isolated mitochondria by patch clamp electrophysiology. The results suggested that Mgr2 depletion affected the basal membrane potential and the response to depolarizing and hyperpolarizing agents. Interestingly, patch clamp analysis suggested a decrease in pore size and gating properties. Further investigation will target the role of GXXXG motifs present in Mgr2 and other TIM23 components.

Blood-based bioenergetic profiling has promising applications as a minimally invasive biomarker of systemic bioenergetic capacity. In the present study, we examined peripheral blood mononuclear cell (PBMC) mitochondrial function and brain morphology in a cohort of African Americans with long-standing Type 2 diabetes. Key parameters of PBMC respiration were correlated with white matter, gray matter, and total intracranial volumes. Our analyses indicate that these relationships are primarily driven by the relationship of systemic bioenergetic capacity with total intracranial volume, suggesting that systemic differences in mitochondrial function may play a role in overall brain morphology.

Cytochromec(Cytc) is a multifunctional protein, acting as an electron carrier in the electron transport chain (ETC), where it shuttles electrons frombc1complex to cytochromecoxidase (COX), and as a trigger of type II apoptosis when released from the mitochondria. We previously showed that Cytcis regulated in a highly tissue-specific manner: Cytcisolated from heart, liver, and kidney is phosphorylated on Y97, Y48, and T28, respectively. Here, we have analyzed the effect of a new Cytcphosphorylation site, threonine 58, which we mapped in rat kidney Cytcby mass spectrometry. We generated and overexpressed wild-type, phosphomimetic T58E, and two controls, T58A and T58I Cytc; the latter replacement is found in human and testis-specific Cytc.In vitro, COX activity, caspase-3 activity, and heme degradation in the presence of H2O2were decreased with phosphomimetic Cytccompared to wild-type. Cytc-knockout cells expressing T58E or T58I Cytcshowed a reduction in intact cell respiration, mitoch...

Blood-based bioenergetic profiling has promising applications as a minimally invasive biomarker of systemic bioenergetic capacity. In this study, we examined peripheral blood mononuclear cell (PBMC) mitochondrial function and brain morphology in a cohort of African Americans with longstanding Type 2 diabetes. Key parameters of PBMC respiration were correlated with white matter, gray matter, and total intracranial volumes. Our analyses indicate that these relationships are primarily driven by the relationship of systemic bioenergetic capacity with total intracranial volume, suggesting that systemic differences in mitochondrial function may play a role in overall brain morphology.

cytochrome c (cytc) is a multifunctional protein, acting as an electron carrier in the electron transport chain (ETC), where it shuttles electrons from bc 1 complex to cytochrome c oxidase (COX), and as a trigger of type II apoptosis when released from the mitochondria. We previously showed that cytc is regulated in a highly tissue-specific manner: Cytc isolated from heart, liver, and kidney is phosphorylated on Y97, Y48, and T28, respectively. Here, we have analyzed the effect of a new Cytc phosphorylation site, threonine 58, which we mapped in rat kidney Cytc by mass spectrometry. We generated and overexpressed wild-type, phosphomimetic T58E, and two controls, T58A and T58I cytc; the latter replacement is found in human and testis-specific Cytc. In vitro, COX activity, caspase-3 activity, and heme degradation in the presence of H 2 o 2 were decreased with phosphomimetic Cytc compared to wild-type. Cytc-knockout cells expressing T58E or T58I Cytc showed a reduction in intact cell respiration, mitochondrial membrane potential (∆Ψ m), ROS production, and apoptotic activity compared to wild-type. We propose that, under physiological conditions, Cytc is phosphorylated, which controls mitochondrial respiration and apoptosis. Under conditions of stress Cytc phosphorylations are lost leading to maximal respiration rates, ∆Ψ m hyperpolarization, ROS production, and apoptosis. Cytochrome c (Cytc) is a small, globular nuclear-encoded protein with a covalently attached heme group. It is located at the mitochondrial intermembrane space (IMS) as a mobile single electron carrier between Complexes III (bc 1 complex) and IV (cytochrome c oxidase, COX) of the electron transport chain (ETC). Cytc null mice die at midgestation 1 when energy metabolism switches from mainly glycolytic to mainly aerobic 2 , indicating that Cytc is an essential protein for the development of organisms and indispensable for mitochondrial ATP production. In addition to functioning in mitochondrial respiration, under stress conditions Cytc serves as a proapoptotic signal 3. Upon its release from the mitochondria into the cytosol, Cytc interacts with apoptotic protease-activating factor 1 (Apaf-1) to form the apoptosome, leading to activation of caspase-9 and the downstream caspase cascade 3,4. Beyond these two major functions, Cytc acts as a peroxidase that oxidizes the mitochondrial membrane lipid cardiolipin at earlier stages of apoptosis, thus promoting Cytc release from the IMS into the cytosol 5. In addition, Cytc can function as both a reactive oxygen species (ROS) scavenger and producer via reduction of p66 shc , a protein involved in ROS generation and apoptosis 6. Furthermore, Cytc serves as the electron acceptor in the Erv1-Mia40 redox relay system involved in mitochondrial IMS protein import 7. Considering the multiple functions that Cytc plays in different pathophysiological conditions, it is imperative that Cytc is tightly regulated. Earlier, the study of regulatory mechanisms of Cytc mainly focused on allosteric regulation by binding of ATP and the expressions of the tissue-specific (somatic and testis) isoforms in mammals 8,9. Recently, a novel regulatory mechanism has emerged, and we reported that Cytc can be phosphorylated

vii 2.2.25 Cytochrome c oxidase (CcO) activity measurements……………....26 2.2.26 Caspase 3 activation by Cytc variants…………………………….....27 2.2.27 Measurement of Cytc redox potential……………………………......28 2.2.28 Measurement of rates of oxidation and reduction……………………………………………….…………………….....28 2.2.29 Heme degradation assay…………………………………………..…29 2.2.30 Measurement of mitochondrial oxygen consumption rate tissue homogenates.

vii 2.2.25 Cytochrome c oxidase (CcO) activity measurements……………....26 2.2.26 Caspase 3 activation by Cytc variants…………………………….....27 2.2.27 Measurement of Cytc redox potential……………………………......28 2.2.28 Measurement of rates of oxidation and reduction……………………………………………….…………………….....28 2.2.29 Heme degradation assay…………………………………………..…29 2.2.30 Measurement of mitochondrial oxygen consumption rate tissue homogenates.

Prostate cancer is the second leading cause of cancer-related death in men. Two classic cancer hallmarks are a metabolic switch from oxidative phosphorylation (OxPhos) to glycolysis, known as the Warburg effect, and resistance to cell death. Cytochrome c (Cytc) is at the intersection of both pathways, as it is essential for electron transport in mitochondrial respiration and a trigger of intrinsic apoptosis when released from the mitochondria. However, its functional role in cancer has never been studied. Our data show that Cytc is acetylated on lysine 53 in both androgen hormone-resistant and -sensitive human prostate cancer xenografts. To characterize the functional effects of K53 modification in vitro, K53 was mutated to acetylmimetic glutamine (K53Q), and to arginine (K53R) and isoleucine (K53I) as controls. Cytochrome c oxidase (COX) activity analyzed with purified Cytc variants showed reduced oxygen consumption with acetylmimetic Cytc compared to the non-acetylated Cytc (WT), ...

Systemic mitochondrial dysfunction is reported with AD progression, suggesting that peripheral blood cells may be used to investigate systemic mitochondrial alterations related to cognitive decline. We aimed to identify bioenergetic signatures associated with AD-related dementia and differences in insulin sensitivity associated with AD risk. We analyzed mitochondrial bioenergetics in peripheral blood cells collected from 365 older adults with varying cognitive status (normal, mild cognitive impairment, and AD) and insulin sensitivity. Normoglycemic individuals exhibited lower maximal bioenergetic capacity with AD (PBMCs: 239.6 pmol·min−1, p = 0.02; Platelets: 151.7 pmol·min−1, p = 0.06) compared to normal cognition (PBMCs: 271.5 pmol·min−1; Platelets: 171.7 pmol·min−1). Individuals with impaired insulin sensitivity exhibited lower maximal bioenergetic capacity in platelets with AD (171.6 pmol·min−1, p = 0.008) compared to normal cognition (210.6 pmol.min−1). Participants with impair...

Mitochondrial bioenergetic alterations occur in the brain and peripheral cells of patients with Alzheimer’s disease (AD). This study focuses on plasma circulating factors, namely lipids, as mediators of systemic bioenergetic differences in participants with normal cognition (NC), mild cognitive impairment (MCI), and dementia due to probable AD (DEM). We examined bioenergetic differences across cognitive groups by measuring the mitochondrial respiration of peripheral blood mononuclear cells (PBMCs) from 37 participants (12 NC, 12 MCI, 13 DEM). PBMC bioenergetics were lower in the DEM group compared to the NC group. To determine whether circulating factors can mediate bioenergetic differences according to cognitive status, we exposed naïve neuronal Neuro-2a (N2a) cells to plasma from each participant in vitro. N2a bioenergetics were lower following plasma exposure from DEM compared to NC group participants. Notably, PBMC Max and N2a Max positively correlated, suggesting that circulati...