Lobelia Samavati

The multiple functions of cytochrome c and their regulation in life and death decisions of the mammalian cell: From respiration to apoptosis

Cytochrome c (Cytc) is essential in mitochondrial electron transport and intrinsic type II apoptosis. Mammalian Cytc also scavenges reactive oxygen species (ROS) under healthy conditions, produces ROS with the co-factor p66(Shc), and oxidizes cardiolipin during apoptosis. The recent finding that Cytc is phosphorylated in vivo underpins a model for the pivotal role of Cytc regulation in making life and death decisions. An apoptotic sequence of events is proposed involving changes in Cytc phosphorylation, increased ROS via increased mitochondrial membrane potentials or the p66(Shc) pathway, and oxidation of cardiolipin by Cytc followed by its release from the mitochondria. Cytc regulation in respiration and cell death is discussed in a human disease context including neurodegenerative and cardiovascular diseases, cancer, and sepsis.

Regulation of mitochondrial respiration and apoptosis through cell signaling: cytochrome c oxidase and cytochrome c in ischemia/reperfusion injury and inflammation.

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.

Tumor necrosis factor alpha inhibits oxidative phosphorylation through tyrosine phosphorylation at subunit I of cytochrome c oxidase

Mitochondrial oxidative phosphorylation provides most cellular energy. As part of this process, cytochrome c oxidase (CcO) pumps protons across the inner mitochondrial membrane, contributing to the generation of the mitochondrial membrane potential, which is used by ATP synthase to produce ATP. During acute inflammation, as in sepsis, aerobic metabolism appears to malfunction and switches to glycolytic energy production. The pro-inflammatory cytokine tumor necrosis factor α (TNFα) has been shown to play a central role in inflammation. We hypothesized that TNFα-triggered cell signaling targets CcO, which is a central enzyme of the aerobic energy metabolism and can be regulated through phosphorylation. Using total bovine and murine hepatocyte homogenates TNFα treatment led to an ∼60% reduction in CcO activity. In contrast, there was no direct effect of TNFα on CcO activity using isolated mitochondria and purified CcO, indicating that a TNFα-triggered intracellular signaling cascade mediates CcO inhibition. CcO isolated after TNFα treatment showed tyrosine phosphorylation on CcO catalytic subunit I and was ∼50 and 70% inhibited at high cytochrome c concentrations in the presence of allosteric activator ADP and inhibitor ATP, respectively. CcO phosphorylation occurs on tyrosine 304 as demonstrated with a phosphoepitope-specific antibody. Furthermore, the mitochondrial membrane potential was decreased in H2.35 cells in response to TNFα. Concomitantly, cellular ATP was more than 35 and 64% reduced in murine hepatocytes and H2.35 cells. We postulate that an important contributor in TNFα-mediated pathologies, such as sepsis, is energy paucity, which parallels the poor tissue oxygen extraction and utilization found in such patients.

TLR4-Mediated AKT Activation Is MyD88/TRIF Dependent and Critical for Induction of Oxidative Phosphorylation and Mitochondrial Transcription Factor A in Murine Macrophages

Mitochondria play a critical role in cell survival and death. Mitochondrial recovery during inflammatory processes such as sepsis is associated with cell survival. Recovery of cellular respiration, mitochondrial biogenesis, and function requires coordinated expression of transcription factors encoded by nuclear and mitochondrial genes, including mitochondrial transcription factor A (T-fam) and cytochrome c oxidase (COX, complex IV). LPS elicits strong host defenses in mammals with pronounced inflammatory responses, but also triggers activation of survival pathways such as AKT pathway. AKT/PKB is a serine/threonine protein kinase that plays an important role in cell survival, protein synthesis, and controlled inflammation in response to TLRs. Hence we investigated the role of LPS-mediated AKT activation in mitochondrial bioenergetics and function in cultured murine macrophages (B6-MCL) and bone marrow-derived macrophages. We show that LPS challenge led to increased expression of T-fam and COX subunits I and IV in a time-dependent manner through early phosphorylation of the PI3K/AKT pathway. PI3K/AKT pathway inhibitors abrogated LPS-mediated T-fam and COX induction. Lack of induction was associated with decreased ATP production, increased proinflammatory cytokines (TNF-α), NO production, and cell death. The TLR4-mediated AKT activation and mitochondrial biogenesis required activation of adaptor protein MyD88 and Toll/IL-1R domain-containing adaptor-inducing IFN-β. Importantly, using a genetic approach, we show that the AKT1 isoform is pivotal in regulating mitochondrial biogenesis in response to TLR4 agonist.

Aspergillus Lung Disease in Patients with Sarcoidosis: A Case Series and Review of the Literature

Chronic cavitary pulmonary aspergillosis (CCPA) has been associated with advanced lung diseases. Pulmonary sarcoidosis, a granulomatous inflammatory disorder, is associated with CCPA. We identified CCPA in 2% of cases in a large cohort of sarcoidosis patients. We found a lack of response to medical treatment and poor outcome in this subgroup.

Chapter 11 Isolation of Regulatory‐Competent, Phosphorylated Cytochrome c Oxidase

The role of posttranslational modifications, specifically reversible phosphorylation as a regulatory mechanism operating in the mitochondria, is a novel research direction. The mitochondrial oxidative phosphorylation system is a particularly interesting unit because it is responsible for the production of the vast majority of cellular energy in addition to free radicals, two factors that are aberrant in numerous human diseases and that may be influenced by reversible phosphorylation of the oxidative phosphorylation complexes. We here describe a detailed protocol for the isolation of mammalian liver and heart mitochondria and subsequently cytochrome c oxidase (CcO) under conditions maintaining the physiological phosphorylation state. The protocol employs the use of activated vanadate, an unspecific tyrosine phosphatase inhibitor, fluoride, an unspecific serine/threonine phosphatase inhibitor, and EGTA, a calcium chelator to prevent the activation of calcium-dependent protein phosphatases. CcO purified without manipulation of signaling pathways shows strong tyrosine phosphorylation on subunits II and IV, whereas tyrosine phosphorylation of subunit I can be induced by the cAMP- and TNFalpha-dependent pathways in liver. Using our protocol on cow liver tissue we further show the identification of a new phosphorylation site on CcO subunit IV tyrosine 11 of the mature protein (corresponding to tyrosine 33 of the precursor peptide) via immobilized metal affinity chromatography/nano-liquid chromatography/electrospray ionization mass spectrometry (IMAC/nano-LC/ESI-MS). This phosphorylation site is located close to the ATP and ADP binding site, which adjusts CcO activity to cellular energy demand, and we propose that phosphorylation of tyrosine 11 enables allosteric regulation.

Dysregulation of p38 and MKP-1 in Response to NOD1/TLR4 Stimulation in Sarcoid Bronchoalveolar Cells

RATIONALE Sarcoidosis is a systemic inflammatory disorder characterized by distinct up-regulation of Th1 cytokines, such as tumor necrosis factor (TNF)-α and IL-12. The mechanism underlying this up-regulation remains unclear. Recognition of microbial moieties through Toll-like or Nod-like receptors evokes sequential activation of mitogen-activated protein kinases (MAPKs), which plays a role in Th1-immune response. OBJECTIVES To test the hypothesis that dysregulation in MAPK signaling in response to microbial stimulation is important in mediating Th1 response in sarcoidosis. METHODS Ex vivo cultured bronchoalveolar lavage (BAL) cells isolated from patients with sarcoidosis and control subjects were stimulated with low-dose Toll-like receptor 4 (TLR4) and nucleotide-binding oligomerization domain 1 (NOD1) ligands as a model of microbial stimulation, and MAPK signaling and inflammatory response were analyzed. MEASUREMENTS AND MAIN RESULTS BAL cells from patients with sarcoidosis exhibited higher basal p38 activity, greater p38 phosphorylation, and more robust production of TNF-α and IL-12/IL-23p40 on stimulation with NOD1 and TLR4 agonists than cells isolated from control subjects. In contrast, control BAL cells had greater basal extracellular signal-regulated kinase (ERK) activity and NOD1 and TLR4 agonists preferentially activated the ERK pathway. Inhibition of p38, but not ERK, attenuated production of both IL12/IL23p40 and TNF-α. Interestingly, stimulation of cells from patients with sarcoidosis with either NOD1 or TLR4 ligand failed to induce MAPK phosphatase 1 (MKP-1). Adenovirus-mediated overexpression of MKP-1 attenuated p38 activation and decreased the production of IL12/IL23p40 and TNF-α in sarcoid BAL cells. CONCLUSIONS Our results suggest that enhanced p38 signaling in response to microbial products is caused by abnormal regulation of MKP-1 and contributes to heightened inflammation in sarcoidosis.

Rapamycin Induces Mitogen-activated Protein (MAP) Kinase Phosphatase-1 (MKP-1) Expression through Activation of Protein Kinase B and Mitogen-activated Protein Kinase Kinase Pathways

Background: MAP kinase phosphatase-1 (MKP-1) plays a critical role in regulating inflammation in innate and adaptive immunity. Results: mTOR inhibition leads to induction of MKP-1 through the activation of AKT1 and MEK1/MEK2 pathways. Rapamycin pretreatment of macrophages inhibits LPS-mediated p38 activation and IL-6 and nitric oxide production. Conclusion: Both AKT1 and MEK1/2 regulate rapamycin-mediated MKP-1 induction. Significance: mTORC1 inhibition regulates immunity through MKP-1 induction. Mitogen-activated protein kinase phosphatase-1 (MKP-1), also known as dual specificity phosphatase-1 (DUSP-1), plays a crucial role in the deactivation of MAPKs. Several drugs with immune-suppressive properties modulate MKP-1 expression as part of their mechanism of action. We investigated the effect of mTOR inhibition through rapamycin and a dual mTOR inhibitor (AZD2014) on MKP-1 expression. Low dose rapamycin led to a rapid activation of both AKT and ERK pathways with a subsequent increase in MKP-1 expression. Rapamycin treatment led to phosphorylation of CREB, transcription factor 1 (ATF1), and ATF2, three transcription factors that bind to the cyclic AMP-responsive elements on the Mkp-1 promoter. Inhibition of either the MEK/ERK or the AKT pathway attenuated rapamycin-mediated MKP-1 induction. AZD2014 did not activate AKT but activated the ERK pathway, leading to a moderate MKP-1 induction. Using bone marrow-derived macrophages (BMDMs) derived from wild-type (WT) mice or mice deficient in AKT1 and AKT2 isoforms or BMDM from targeted deficiency in MEK1 and MEK2, we show that rapamycin treatment led to an increased MKP1 expression in BMDM from WT but failed to do so in BMDMs lacking the AKT1 isoform or MEK1 and MEK2. Importantly, rapamycin pretreatment inhibited LPS-mediated p38 activation and decreased nitric oxide and IL-6 production. Our work provides a conceptual framework for the observed immune modulatory effect of mTOR inhibition.

Distance saturation product predicts health–related quality of life among sarcoidosis patients

BackgroundSarcoidosis is a chronic disease with different phenotypic manifestations. Health-related quality of life is an important aspect in sarcoidosis, yet difficult to measure. The objective of this study was to identify clinical markers predictive of poor quality of life in sarcoidosis patients that can be followed over time and targeted for intervention.MethodsWe assessed the quality of life of 162 patients with confirmed sarcoidosis in a prospective, cross-sectional study using the Sarcoidosis Health Questionnaire (SHQ) and Short Form-36 Health Survey (SF-36). We evaluated the validity of these questionnaires and sought to identify variables that would best explain the performance scores of the patients.ResultsOn multivariate regression analyses, the very best composite model to predict total scores from both surveys was a model containing the distance-saturation product and Borg Dyspnea Scale score at the end of a 6-min walk test. This model could better predict SF-36 scores (R2 = 0.33) than SHQ scores (R2 = 0.24). Substitution of distanced walked in 6 min for the distance-saturation product in this model resulted in a lesser ability to predict both scores (R2 = 0.26 for SF-36; R2 = 0.22 for SHQ).ConclusionsBoth the SHQ and SF-36 surveys are valuable tools in the assessment of health-related quality of life in sarcoidosis patients. The best model to predict quality of life among these patients, as determined by regression analyses, included the distance-saturation product and Borg score after the 6-min walk test. Both variables represent easily obtainable clinical parameters that can be followed over time and targeted for intervention.

Dual Inhibition of Rip2 and IRAK1/4 regulates IL-1β and IL-6 in sarcoidosis alveolar macrophages and peripheral blood mononuclear cells

Sarcoidosis is a multisystem granulomatous disease of unknown etiology that primarily affects the lungs. Our previous work indicates that activation of p38 plays a pivotal role in sarcoidosis inflammatory response. Therefore, we investigated the upstream kinase responsible for activation of p38 in sarcoidosis alveolar macrophages (AMs) and PBMCs. We identified that sustained p38 phosphorylation in sarcoidosis AMs and PBMCs is associated with active MAPK kinase 4 but not with MAPK kinase 3/6. Additionally, we found that sarcoidosis AMs exhibit a higher expression of IRAK1, IRAK-M, and receptor interacting protein 2 (Rip2). Surprisingly, ex vivo treatment of sarcoidosis AMs or PBMCs with IRAK1/4 inhibitor led to a significant increase in IL-1β mRNA expression both spontaneously and in response to TLR2 ligand. However, a combination of Rip2 and IRAK-1/4 inhibitors significantly decreased both IL-1β and IL-6 production in sarcoidosis PBMCs and moderately in AMs. Importantly, a combination of Rip2 and IRAK-1/4 inhibitors led to decreased IFN-γ and IL-6 and decreased percentage of activated CD4+CD25+ cells in PBMCs. These data suggest that in sarcoidosis, both pathways, namely IRAK and Rip2, are deregulated. Targeted modulation of Rip2 and IRAK pathways may prove to be a novel treatment for sarcoidosis.