Metabolic Labeling Reveals Proteome Dynamics of Mouse Mitochondria

Mitochondrial dysfunction is associated with many human diseases. Mitochondrial damage is exacerbated by inadequate protein quality control and often further contributes to pathogenesis. The maintenance of mitochondrial functions requires a delicate balance of continuous protein synthesis and degradation, i.e. protein turnover. To understand mitochondrial protein dynamics in vivo, we designed a metabolic heavy water (²H₂O) labeling strategy customized to examine individual protein turnover in the mitochondria in a systematic fashion. Mice were fed with ²H₂O at a minimal level (<5% body water) without physiological impacts. Mitochondrial proteins were analyzed from 9 mice at each of the 13 time points between 0 and 90 days (d) of labeling. A novel multiparameter fitting approach computationally determined the normalized peak areas of peptide mass isotopomers at initial and steady-state time points and permitted the protein half-life to be determined without plateau-level ²H incorporation. We characterized the turnover rates of 458 proteins in mouse cardiac and hepatic mitochondria and found median turnover rates of 0.0402 d⁻¹ and 0.163 d⁻¹, respectively, corresponding to median half-lives of 17.2 d and 4.26 d. Mitochondria in the heart and those in the liver exhibited distinct turnover kinetics, with limited synchronization within functional clusters. We observed considerable interprotein differences in turnover rates in both organs, with half-lives spanning from hours to months (∼60 d). Our proteomics platform demonstrates the first large-scale analysis of mitochondrial protein turnover rates in vivo, with potential applications in translational research.Mitochondrial dysfunctions are observed in disorders such as neurodegeneration, cardiovascular diseases, and aging (1–3). It is postulated that the failure to contain or replenish mitochondrial proteins damaged by reactive oxygen species directly underlies many pathological phenotypes (4). The development of effective treatments for these diseases therefore relies on understanding the molecular basis of protein dynamics. Outstanding questions are how the processes of mitochondrial proteome dynamics are regulated in different systems, and how their perturbations could progress to pathological remodeling of the organelle. Thus far, quantitative proteomics efforts have been predominated by steady-state measurements, which often provide fragmentary snapshots of the proteome that are difficult to comprehend in the context of other cellular events.To further understand mitochondrial dynamics in vivo, we examined the turnover rates of individual heart and liver mitochondrial proteins on a proteome scale. Both the liver and the heart contain large numbers of mitochondria, but cardiac and hepatic mitochondria differ in their protein composition, oxygen consumption, substrate utilization, and disease manifestation. However, these differences are often interpreted only by protein compositions and steady-state abundance, without the consideration of protein kinetics in the temporal dimension. Abnormal protein kinetics may indicate dysfunctions in protein quality control, the accumulation of damaged proteins, misfolding, or other proteinopathies. Protein dynamics itself is an important intrinsic property of the proteome, the disruption of which could be causal of cellular etiologies.At minimum, a kinetic definition of the proteome requires knowledge of the rate at which individual proteins are being replaced. Isotope tracers are particularly useful for tracking such continual renewal of the proteome in living systems, because they allow differentiation between preexisting and newly synthesized proteins (5). Among the available stable isotope precursors, heavy water (²H₂O) labeling offers several advantages with respect to safety, labeling kinetics, and cost (6, 7). First, ²H₂O administration to animals and humans at low enrichment levels is safe for months or even years (8). Second, maintaining constant ²H enrichment levels in body water following the initial intake of ²H₂O is easily achieved, because administrated ²H₂O rapidly equilibrates over all tissues but decays slowly (9, 10). Third, ²H₂O labeling is more cost effective than other stable isotope labeling methods. Importantly, ²H₂O intake induces universal ²H incorporation into biomolecules. Systematic insights into protein turnover in vivo could therefore be correlated to that of nucleic acids, carbohydrates, or lipids, enabling broad applications for this technology in studying mammalian systems, including humans.A variety of methodologies have been developed to analyze the extent of ²H incorporation in proteins following ²H₂O labeling, including GC-MS measurements of hydrolyzed target proteins (11–14) and peptide analysis in MALDI-TOF MS (15) and LC-MS (16, 17). More recently, Price et al. described an approach for measuring protein turnover by calculating the theoretical number of ²H-labeling sites on a peptide sequence (18) and reported the turnover rates of ∼100 human plasma proteins. Here we describe another novel strategy to determine protein turnover rates on a proteomic scale using ²H₂O labeling. By computing the parameters needed to deduce fractional protein synthesis using software we developed, we were able to obtain protein half-life data without relying on the asymptotic isotopic abundance of peptide ions. Our approach also has the unique benefit of automating all steps of isotopomer quantification and postcollection data analysis, and it does not require knowledge of the exact precursor enrichment or labeling sites of peptides. We observed diverse kinetics from 458 liver and heart mitochondrial proteins that inform essential characteristics of mitochondrial dynamics and intragenomic differences between the two organs.     

The role of circulating serotonin in the development of chronic obstructive pulmonary disease

Background

Cigarette smoking is a major risk factor in the development of age-related chronic obstructive pulmonary disease (COPD). The serotonin transporter (SERT) gene polymorphism has been reported to be associated with COPD, and the degree of cigarette smoking has been shown to be a significant mediator in this relationship. The interrelation between circulating serotonin (5-hydroxytyptamine, 5-HT), cigarette smoking and COPD is however largely unknown. The current study aimed at investigating the mediation effects of plasma 5-HT on cigarette smoking-induced COPD and the relation between plasma 5-HT levels and age.

Methods

The association between plasma 5-HT, age and COPD was analyzed in a total of 62 COPD patients (ever-smokers) and 117 control subjects (healthy non-smokers and ever-smokers). Plasma 5-HT levels were measured by enzyme-linked immuno assay (EIA).

Results

The elevated plasma 5-HT levels were significantly associated with increased odds for COPD (OR = 1.221, 95% CI = 1.123 to 1.319, p<0.0001). The effect remained significant after being adjusted for age and pack-years smoked (OR = 1.271, 95% CI = 1.134 to 1.408, p = 0.0003). Furthermore, plasma 5-HT was found to mediate the relation between pack-years smoked and COPD. A positive correlation (r = 0.303, p = 0.017) was found between plasma 5-HT levels and age in COPD, but not in the control subjects (r = −0.149, p = 0.108).

Conclusion

Our results suggest that cigarette smoke-induced COPD is partially mediated by the plasma levels of 5-HT, and that these become elevated with increased age in COPD. The elevated plasma 5-HT levels in COPD might contribute to the pathogenesis of this disease.     

First Discovery of Two Polyketide Synthase Genes for Mitorubrinic Acid and Mitorubrinol Yellow Pigment Biosynthesis and Implications in Virulence of Penicillium marneffei

Background

The genome of P. marneffei, the most important thermal dimorphic fungus causing respiratory, skin and systemic mycosis in China and Southeast Asia, possesses 23 polyketide synthase (PKS) genes and 2 polyketide synthase nonribosomal peptide synthase hybrid (PKS-NRPS) genes, which is of high diversity compared to other thermal dimorphic pathogenic fungi. We hypothesized that the yellow pigment in the mold form of P. marneffei could also be synthesized by one or more PKS genes.

Methodology/Principal Findings

All 23 PKS and 2 PKS-NRPS genes of P. marneffei were systematically knocked down. A loss of the yellow pigment was observed in the mold form of the pks11 knockdown, pks12 knockdown and pks11pks12 double knockdown mutants. Sequence analysis showed that PKS11 and PKS12 are fungal non-reducing PKSs. Ultra high performance liquid chromatography-photodiode array detector/electrospray ionization-quadruple time of flight-mass spectrometry (MS) and MS/MS analysis of the culture filtrates of wild type P. marneffei and the pks11 knockdown, pks12 knockdown and pks11pks12 double knockdown mutants showed that the yellow pigment is composed of mitorubrinic acid and mitorubrinol. The survival of mice challenged with the pks11 knockdown, pks12 knockdown and pks11pks12 double knockdown mutants was significantly better than those challenged with wild type P. marneffei (P<0.05). There was also statistically significant decrease in survival of pks11 knockdown, pks12 knockdown and pks11pks12 double knockdown mutants compared to wild type P. marneffei in both J774 and THP1 macrophages (P<0.05).

Conclusions/Significance

The yellow pigment of the mold form of P. marneffei is composed of mitorubrinol and mitorubrinic acid. This represents the first discovery of PKS genes responsible for mitorubrinol and mitorubrinic acid biosynthesis. pks12 and pks11 are probably responsible for sequential use in the biosynthesis of mitorubrinol and mitorubrinic acid. Mitorubrinol and mitorubrinic acid are virulence factors of P. marneffei by improving its intracellular survival in macrophages.     

Expression of Aeromonas caviae polyhydroxyalkanoate synthase gene in Burkholderia sp. USM (JCM15050) enables the biosynthesis of SCL-MCL PHA from palm oil products

AIMS: Burkholderia sp. USM (JCM15050) isolated from oil-polluted wastewater is capable of utilizing palm oil products and glycerol to synthesize poly(3-hydroxybutyrate) [P(3HB)]. To confer the ability to produce polymer containing 3-hydroxyhexanoate (3HHx), plasmid (pBBREE32d13) harbouring the polyhydroxyalkanoate (PHA) synthase gene of Aeromonas caviae (phaC(Ac)) was transformed into this strain. Methods and Results: The resulting transformant incorporated approximately 1 ± 0·3 mol% of 3HHx in the polymer when crude palm kernel oil (CPKO) or palm kernel acid oil was used as the sole carbon source. In addition, when the transformed strain was cultivated in the mixtures of CPKO and sodium valerate, PHA containing 69 mol% 3HB, 30 mol% 3-hydroxyvalerate and 1 mol% 3HHx monomers was produced. Batch feeding of carbon sources with 0·5% (v/v) CPKO at 0 h and 0·25% (w/v) sodium valerate at 36 h yielded 6 mol% of 3HHx monomer by controlled-feeding strategies. CONCLUSIONS: Burkholderia sp. USM (JCM15050) has the metabolic pathways to supply both the short-chain length (SCL) and medium-chain length (MCL) PHA monomers. By transforming the strain with the Aer. caviae PHA synthase with broader substrate specificity, SCL-MCL PHA was produced. Significance and Impact of the Study: This is the first study demonstrating the ability of transformant Burkholderia to produce P(3HB-co-3HHx) from a single carbon source.     

Bronchioalveolar stem cells increase after mesenchymal stromal cell treatment in a mouse model of bronchopulmonary dysplasia

Bronchopulmonary dysplasia (BPD) remains a major complication of prematurity resulting in significant morbidity and mortality. The pathology of BPD is multifactorial and leads to alveolar simplification and distal lung injury. Previous studies have shown a beneficial effect of systemic treatment with bone marrow-derived mesenchymal stromal cells (MSCs) and MSC-conditioned media (MSC-CM) leading to amelioration of the lung parenchymal and vascular injury in vivo in the hyperoxia murine model of BPD. It is possible that the beneficial response from the MSCs is at least in part due to activation of endogenous lung epithelial stem cells. Bronchioalveolar stem cells (BASCs) are an adult lung stem cell population capable of self-renewal and differentiation in culture, and BASCs proliferate in response to bronchiolar and alveolar lung injury in vivo. Systemic treatment of neonatal hyperoxia-exposed mice with MSCs or MSC-CM led to a significant increase in BASCs compared with untreated controls. Treatment of BASCs with MSC-CM in culture showed an increase in growth efficiency, indicating a direct effect of MSCs on BASCs. Lineage tracing data in bleomycin-treated adult mice showed that Clara cell secretory protein-expressing cells including BASCs are capable of contributing to alveolar repair after lung injury. MSCs and MSC-derived factors may stimulate BASCs to play a role in the repair of alveolar lung injury found in BPD and in the restoration of distal lung cell epithelia. This work highlights the potential important role of endogenous lung stem cells in the repair of chronic lung diseases.     

Apocynin attenuates oxidative stress and hypertension in young spontaneously hypertensive rats independent of ADMA/NO pathway

Both NADPH oxidase-derived reactive oxygen species (ROS) and asymmetric dimethylarginine (ADMA) are increased in hypertension. Apocynin, an NADPH oxidase inhibitor, could inhibit ROS, thus we tested whether apocynin can block NADPH oxidase and prevent increases of ADMA and blood pressure (BP) in spontaneously hypertensive rats (SHRs). SHRs and Wistar Kyoto (WKY) rats, aged 4 weeks, were assigned to four groups: untreated SHRs and WKY rats, SHRs and WKY rats that received 2.5 mM apocynin for 8 weeks. BP was significantly higher in SHRs compared to WKY rats, which was attenuated by apocynin. Apocynin prevented p47phox translocation in SHR kidneys, but not the increase of superoxide and H(2)O(2). Additionally, apocynin did not protect SHRs against increased ADMA. Apocynin blocks NADPH oxidase to attenuate hypertension, but has little effect on the ADMA/nitric oxide (NO) pathway in young SHRs. The reduction of ROS and the preservation of NO simultaneously might be a better approach to restoring ROS-NO balance to prevent hypertension.     

Cajaninstilbene Acid Relaxes Rat Renal Arteries: Roles of Ca2+ Antagonism and Protein Kinase C-Dependent Mechanism

Cajaninstilbene acid (CSA) is a major active component present in the leaves of Cajanus cajan (L.) Millsp. The present study explores the underlying cellular mechanisms for CSA-induced relaxation in rat renal arteries. Vascular reactivity was examined in arterial rings that were suspended in a Multi Myograph System and the expression of signaling proteins was assessed by Western blotting method. CSA (0.1–10 µM) produced relaxations in rings pre-contracted by phenylephrine, serotonin, 9, 11-dideoxy-9α, 11α-epoxymethanoprostaglandin F_2α (U46619), and 60 mM KCl. CSA-induced relaxations did not show difference between genders and were unaffected by endothelium denudation, nor by treatment with N^G-nitro-L-arginine methyl ester, indomethacin, ICI-182780, tetraethylammonium ion, BaCl₂, glibenclamide, 4-aminopyridine or propranolol. CSA reduced contraction induced by CaCl₂ (0.01–5 mM) in Ca²⁺-free 60 mM KCl solution and by 30 nM (−)-Bay K8644 in 15 mM KCl solution. CSA inhibited 60 mM KCl-induced Ca²⁺ influx in smooth muscle of renal arteries. In addition, CSA inhibited contraction evoked by phorbol 12-myristate 13-acetate (PMA, protein kinase C agonist) in Ca²⁺-free Krebs solution. Moreover, CSA reduced the U46619- and PMA-induced phosphorylation of myosin light chain (MLC) at Ser19 and myosin phosphatase target subunit 1 (MYPT1) at Thr853 which was associated with vasoconstriction. CSA also lowered the phosphorylation of protein kinase C (PKCδ) at Thr505. In summary, the present results suggest that CSA relaxes renal arteries in vitro via multiple cellular mechanisms involving partial inhibition of calcium entry via nifedipine-sensitive calcium channels, protein kinase C and Rho kinase.     

Erythroid promoter confines FGF2 expression to the marrow after hematopoietic stem cell gene therapy and leads to enhanced endosteal bone formation

Fibroblast growth factor-2 (FGF2) has been demonstrated to be a promising osteogenic factor for treating osteoporosis. Our earlier study shows that transplantation of mouse Sca-1(+) hematopoietic stem/progenitor cells that are engineered to express a modified FGF2 leads to considerable endosteal/trabecular bone formation, but it also induces adverse effects like hypocalemia and osteomalacia. Here we report that the use of an erythroid specific promoter, β-globin, leads to a 5-fold decrease in the ratio of serum FGF2 to the FGF2 expression in the marrow cavity when compared to the use of a ubiquitous promoter spleen focus-forming virus (SFFV). The confined FGF2 expression promotes considerable trabeculae bone formation in endosteum and does not yield anemia and osteomalacia. The avoidance of anemia in the mice that received Sca1(+) cells transduced with FGF2 driven by the β-globin promoter is likely due to attenuation of high-level serum FGF2-mediated stem cell mobilization observed in the SFFV-FGF2 animals. The prevention of osteomalacia is associated with substantially reduced serum Fgf23/hypophosphatemia, and less pronounced secondary hyperparathyroidism. Our improved stem cell gene therapy strategy represents one step closer to FGF2-based clinical therapy for systemic skeletal augmentation.     

Organizational Determinants of Interprofessional Collaboration in Integrative Health Care: Systematic Review of Qualitative Studies

Context

Inteprofessional collaboration (IPC) between biomedically trained doctors (BMD) and traditional, complementary and alternative medicine practitioners (TCAMP) is an essential element in the development of successful integrative healthcare (IHC) services. This systematic review aims to identify organizational strategies that would facilitate this process.

Methods

We searched 4 international databases for qualitative studies on the theme of BMD-TCAMP IPC, supplemented with a purposive search of 31 health services and TCAM journals. Methodological quality of included studies was assessed using published checklist. Results of each included study were synthesized using a framework approach, with reference to the Structuration Model of Collaboration.

Findings

Thirty-seven studies of acceptable quality were included. The main driver for developing integrative healthcare was the demand for holistic care from patients. Integration can best be led by those trained in both paradigms. Bridge-building activities, positive promotion of partnership and co-location of practices are also beneficial for creating bonding between team members. In order to empower the participation of TCAMP, the perceived power differentials need to be reduced. Also, resources should be committed to supporting team building, collaborative initiatives and greater patient access. Leadership and funding from central authorities are needed to promote the use of condition-specific referral protocols and shared electronic health records. More mature IHC programs usually formalize their evaluation process around outcomes that are recognized both by BMD and TCAMP.

Conclusions

The major themes emerging from our review suggest that successful collaborative relationships between BMD and TCAMP are similar to those between other health professionals, and interventions which improve the effectiveness of joint working in other healthcare teams with may well be transferable to promote better partnership between the paradigms. However, striking a balance between the different practices and preserving the epistemological stance of TCAM will remain the greatest challenge in successful integration.