3 - Overview of Reactive Oxygen Species and Nitrogen Pathways

Question 1

Reactive oxygen species (ROS) production:

Answer 1

• O2-dependent killing of bacteria by phagocytes
• Key enzymes: NADPH oxidase and myeloperoxidase (MPO)

Question 2

Reactive nitrogen species (RNS) production:

Answer 2

• Nitric oxide (NO) and other intermediates generated which can act as signalling molecules and in interactions with ROS
• Key enzyme: Nitric oxide synthase (NOS)

Question 3

What problems are there with ROS and RNS?

Answer 3

Problems associated with deficiency/excess of each type

Question 4

Mechanism of ROS production

Answer 4

• 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

Question 5

Haber-Weiss

Answer 5

Reaction of O2- with H2O2 producing O2, hydroxyl radicals
- iron catalysed

Question 6

Fenton

Answer 6

Formation of hydroxyl radicals and Fe3+ from non enzymatic reaction of Fe2 with H2O2

Question 7

Superoxide and H2O2 production

Answer 7

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

Question 8

Production of ROS

Answer 8

• 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

Question 9

The phagocyte NADPH oxidase

Answer 9

• 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

Question 10

gp91

Answer 10

contains FAD (flavine adenine dinucleotide) co-factor, plus 2 heme groups, necessary for electron transport

Question 11

p22^phox

Answer 11

Acts as a docking site for cytosolic subunits

Question 12

Resting phagocytes

Answer 12

cytosolic complex exists of 3 pox subunits

Question 13

p47^phox

Answer 13

• phosphorylation essential
• directs complex to membrane and promotes binding of complex

Question 14

p67

Answer 14

binds GTP-rac on activation, promoting e- flow

Question 15

rac

Answer 15

• in resting cells, associated with GDI (guanine nucleotide dissociation inhibitor
• translocates independently on activation

Question 16

Involvement of NADPH oxidase/MPO in successful defence -
demonstrated using transgenic mice

Answer 16

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

Question 17

ROS in immune defence

Answer 17

ROS: direct killing via oxidation. chlorination, nitrosylation, nitrosylation of microbial molecules

Question 18

What are ROS essential for in immune defence?

Answer 18

• Essential for killing S. aureus, Burkholderia cepacia, Aspergillus fumigatus
• Can neutralize virulence factors, e.g. Leukotoxin, pneumlysin
• Essential for extracellular trap formation

Question 19

What are important features of ROS in immune defence

Answer 19

• Oxidation & chlorination important
• Highly reactive with sulphur: oxidizes methionine residues in bacterial
  cytosol and inner membrane
• Bacterial proteins oxidized include those involved in synthesis

Question 20

Chronic granulomatous disease (CGD)

Answer 20

• 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

Question 21

Diseases linked to ROS

Answer 21

Acute respiratory distress syndrome, ageing, Alzheimer, atherosclerosis, cancer, cardiovascular disease, diabetes, inflammation, inflammatory joint disease, obesity, Parkinson, pulmonary fibrosis, rheumatoid arthritis, vascular disease

Question 22

Cancers linked to ROS

Answer 22

Bladder, brain tumour, breast, cervical, gastric, liver, lung, melanoma, multiple myeloma, leukaemia, lymphoma, oral, ovarian, pancreatic, prostate, sarcoma

Question 23

Is NADPH oxidase just for phagocytes?

Answer 23

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

Question 24

Isoforms of nitric oxide synthase

Answer 24

Nitric oxide synthase: 3 isoforms
- neuronal (ncNOS) (cytosol)
- endothelial (ecNOS) (membrane)
- macrophage (macNOS or iNOS – inducible NOS) (cytosol)

Question 25

Role of NO

Answer 25

- diffuses across the plasma membrane - reacts with ROS - acts as a signalling molecule

Question 26

Bactericidal effects of MPO, iNOs and NADPH oxidase

Answer 26

MPO - oxidation, chlorination, tyrosine nitration iNOS - S-nitrosylation, disruption of Fe-S- or ZnS clusters NADPH oxidase - oxidation

Question 27

Examples of RNS in disease (excess RNS)

Answer 27

- atherosclerosis - asthma - systemic lupus erythematosus (SLE)

Question 28

Atherosclerosis

Answer 28

-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

Question 29

Asthma

Answer 29

NO: exhaled levels correlate with clinical symptoms

Question 30

Systemic lupus erythematosus (SLE):

Answer 30

iNOS activity increases with disease progression Risk of excess peroxynitrite formation by combination of NO and ROI Peroxynitrite: modifies self antigens, lipid peroxidation

Question 31

What are ROS are responsible for?

Answer 31

ROS are responsible for O2-dependent killing of bacteria by phagocytes through the activity of NADPH oxidase and myeloperoxidase (MPO)

Question 32

What is known about NADPH oxidase?

Answer 32

NADPH oxidase now known to comprise several homologs and is referred to as NOX, with NOX2 being the phagocyte oxidase

Question 33

What does ROS include?

Answer 33

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

Question 34

What does RNS do?

Answer 34

RNS include nitric oxide and nitrogen dioxide and act as signalling molecules as well as combining with ROS to form compounds such as peroxynitrite

Question 35

What do deficiencies result in?

Answer 35

Deficiencies results in increased risk of infections, excess associated with numerous diseases and cancers