3 - Overview of Reactive Oxygen Species and Nitrogen Pathways Flashcards
Reactive oxygen species (ROS) production:
- O2-dependent killing of bacteria by phagocytes
- Key enzymes: NADPH oxidase and myeloperoxidase (MPO)
Reactive nitrogen species (RNS) production:
- Nitric oxide (NO) and other intermediates generated which can act as signalling molecules and in interactions with ROS
- Key enzyme: Nitric oxide synthase (NOS)
What problems are there with ROS and RNS?
Problems associated with deficiency/excess of each type
Mechanism of ROS production
- activation of membrane bound NADPH oxidase during uptake
- enzyme activation leads to generation of toxic oxygen metabolites
- toxic metabolites catalysed to harmless products by antioxidant enzymes e.g., superoxide dismutase, catalase etc.
- oxygen metabolites include: superoxide, hydrogen peroxide, hydroxyl radicals
Haber-Weiss
Reaction of O2- with H2O2 producing O2, hydroxyl radicals
- iron catalysed
Fenton
Formation of hydroxyl radicals and Fe3+ from non enzymatic reaction of Fe2 with H2O2
Superoxide and H2O2 production
Superoxide (O2˙̄) and hydrogen peroxide (H2O2) are produced by leaks of electrons from donor redox centers of the mitochondrial electron transport chain and associated metabolic enzymes to cause either one-electron or two-electron reduction of oxygen
Production of ROS
- Produced mainly by phagocytes, generated by activation of membrane
bound enzyme NADPH oxidase - Enzyme comprises several sub-units, membrane and cytosolic
- Catalyzes cytoplasmic NADPH into NADP+
- Assembled on receipt of signal, e.g. internalization of phagocyte receptors, chemoattractants, chemokines
The phagocyte NADPH oxidase
- The enzyme responsible for O2−. production has been called the respiratory burst oxidase, the phagocyte NADPH oxidase, or the leucocyte NADPH oxidase
- It is a multicomponent enzyme system, which in its active state is composed of several proteins
- The cytochrome b558 is a heterodimer comprising two transmembrane proteins, that is gp91phox (phox: phagocyte oxidase) and p22phox, while four proteins, p47phox, p67phox, p40phox and Rac2 (in neutrophils) are recruited from the cytosol11, 12
gp91
contains FAD (flavine adenine dinucleotide) co-factor, plus 2 heme groups, necessary for electron transport
p22^phox
Acts as a docking site for cytosolic subunits
Resting phagocytes
cytosolic complex exists of 3 pox subunits
p47^phox
- phosphorylation essential
- directs complex to membrane and promotes binding of complex
p67
binds GTP-rac on activation, promoting e- flow
rac
- in resting cells, associated with GDI (guanine nucleotide dissociation inhibitor
- translocates independently on activation
Involvement of NADPH oxidase/MPO in successful defence -
demonstrated using transgenic mice
Defective element (a) Infection consequences (b)
- (a) gp47 (b) Lethal - Staphylococcus, fungi Mycobacteriosis in spleen & lung, listeriosis in liver and spleen
- (a) gp91 (b) Lethal – commensal bacteria, Listeria, Systemic infection from Salmonella
- (a) Rac 2 (b) Lethal – Aspergillosis
- (a) MPO (b) Peritonitis from S. aureus, pneumonia, candidiasis
ROS in immune defence
ROS: direct killing via oxidation. chlorination, nitrosylation, nitrosylation of microbial molecules
What are ROS essential for in immune defence?
- Essential for killing S. aureus, Burkholderia cepacia, Aspergillus fumigatus
- Can neutralize virulence factors, e.g. Leukotoxin, pneumlysin
- Essential for extracellular trap formation
What are important features of ROS in immune defence
- Oxidation & chlorination important
- Highly reactive with sulphur: oxidizes methionine residues in bacterial
cytosol and inner membrane - Bacterial proteins oxidized include those involved in synthesis
Chronic granulomatous disease (CGD)
- Inherited immunodeficiency, can cause fatal septicaemia in infancy
- Phagocytes able to ingest microorganisms (reduced rate), but unable to produce
- ROS, resulting in microbial survival
- Neutrophils from CGD patients: unable to expose phosphatidylserine residues, essential for apoptosis of redundant cells
- Recurrent infections, e.g. Aspergillus sp. S. aureus, Burkholderia cepacia (especially catalase- positive bacteria)
- Inflammatory granulomas common often cause obstruction/disruption of organ function
Diseases linked to ROS
Acute respiratory distress syndrome, ageing, Alzheimer, atherosclerosis, cancer, cardiovascular disease, diabetes, inflammation, inflammatory joint disease, obesity, Parkinson, pulmonary fibrosis, rheumatoid arthritis, vascular disease
Cancers linked to ROS
Bladder, brain tumour, breast, cervical, gastric, liver, lung, melanoma, multiple myeloma, leukaemia, lymphoma, oral, ovarian, pancreatic, prostate, sarcoma
Is NADPH oxidase just for phagocytes?
No -
- Conventionally – NADPH oxidase and its functions regarded as solely
present in phagocytes
- Now established that NADPH oxidase family exists – Nox
- 6 Nox2 homologues found in non-phagocytes: 3 main groups
Isoforms of nitric oxide synthase
Nitric oxide synthase: 3 isoforms
- neuronal (ncNOS) (cytosol)
- endothelial (ecNOS) (membrane)
- macrophage (macNOS or iNOS – inducible NOS) (cytosol)
Role of NO
- diffuses across the plasma membrane
- reacts with ROS
- acts as a signalling molecule
Bactericidal effects of MPO, iNOs and NADPH oxidase
MPO - oxidation, chlorination, tyrosine nitration
iNOS - S-nitrosylation, disruption of Fe-S- or ZnS clusters
NADPH oxidase - oxidation
Examples of RNS in disease (excess RNS)
- atherosclerosis
- asthma
- systemic lupus erythematosus (SLE)
Atherosclerosis
-Vascular disease, fatty material collects along artery walls, forming plaques
- iNOS present in plaques, even at early stages. Correlated with lipid oxidation
- Formation of peroxynitrite thought to contribute to hypertension
- NO as signalling molecule thought to contribute generally to vascular diseases
Asthma
NO: exhaled levels correlate with clinical symptoms
Systemic lupus erythematosus (SLE):
iNOS activity increases with disease progression
Risk of excess peroxynitrite formation by combination of NO and ROI
Peroxynitrite: modifies self antigens, lipid peroxidation
What are ROS are responsible for?
ROS are responsible for O2-dependent killing of bacteria by phagocytes through the activity of NADPH oxidase and myeloperoxidase (MPO)
What is known about NADPH oxidase?
NADPH oxidase now known to comprise several homologs and is referred to as NOX, with NOX2 being the phagocyte oxidase
What does ROS include?
ROS include superoxide, hydrogen peroxide, hydroxyl radicals, which are catalysed by enzymes such as superoxide dismutase and catalase or removed by scavangers such as mannitol
What does RNS do?
RNS include nitric oxide and nitrogen dioxide and act as signalling
molecules as well as combining with ROS to form compounds such as
peroxynitrite
What do deficiencies result in?
Deficiencies results in increased risk of infections, excess associated with numerous diseases and cancers
