ROS Signaling

Question 1

NOX/DUOX Family

Answer 1

The NOX/DUOX family in humans consists of NADPH Oxidase 1-5 and Dual Oxidase 1-2.

Question 2

Phox/NOX2/CYBB/gp91^phox

Answer 2

Named “phagocyte oxidase” as it is the dominant isoform present on phagocytes and is used to kill phagocytosed microbes.

Forms the core of the catalytic complex and associates with two heme prosthetic groups. One heme binds NADPH, the other binds FAD as a cosubstrate.

Constitutively bound to p22^phox/CYBA. Together these proteins form flavocytochrome b₅₅₈.

Question 3

Flavocytochrome b₅₅₈ Localization

Answer 3

Neutrophils:

PM, Secondary Granules, Tertiary Granules

BMMΦ:

PM, Rab11⁺ Recycling Endosomes, Rab5⁺ Early Endosomes

Dendritic Cells:

Phagosomes, Lysosomes

Question 4

Activation of NOX2

Answer 4

Multiple stimuli (including FcγRI, CR, IL-8, Leukotriene-B₄, etc…) may induce PKC, IRAK4, or IκBKγ activation.

These kinases proceed to phosphorylate p40^phox, p47^phox, and p67^phox. Upon phosphorylation, these components self-assemble into the p40-p47-p67^phox complex and translocate to flavocytochrome b₅₅₈. At this point either Rac1 or Rac2 small Rho GTPase are recruited to catalyze GTP activity which helps drive the production of O^2-.

Question 5

LyGDI

Answer 5

GDP-dissociation inhibitor that binds to Rac1 and Rac2, preventing association with flavocytochrome b₅₅₈. Effectively inhibits ROS production.

Upon phagocytosis in MΦ, CARD9 is activated to bind LyGDI and prevent it from inhibiting Rac1/2 activity, thus enabling ROS production.

Question 6

Direct toxicity as a mechanism of antimicrobial ROS activity

Answer 6

Oxidation, peroxidation, hydroxylation, or chlorination of proteins, lipids, nucleic acids, iron-sulfur clusters, or heme prosthetic groups.

Chemical modification of these groups impairs their function and eventually the accumulated destruction of molecules performing housekeeping functions destroys the cell.

Question 7

Granule serine protease activity as a mechanism of antimicrobial ROS activity

Answer 7

When O^2- is pumped into a phagosome, it quickly reacts to form hydrogen peroxide, thus sequestering two hydrogen molecules and raising the pH. The charge differential that the pumped-in electrons creates also must be balanced, and this is done by diffusion of K⁺ ions.

Phagosomes contain many cationic proteases that are initially sequestered. However, the rising pH deprotonates them, reducing their positive charge, and K⁺ displaces them from their sequestering anionic proteoglycan granule matrix. This activates the proteases, enabeling digestion of the contents of the phagosome.

Notable proteases with such activity include elastase and cathepsin G.

Question 8

NETosis as a mechanism of antimicrobial ROS activity

Answer 8

H₂O₂ signaling triggers the initiation of NETosis in neutrophils. NETosis both traps and kills microbes, as wel as enabeling the recruitment of more inflammatory cells via the release of chemokines upon cell death.

Question 9

ROS Signaling as a mechanism of antimicrobial ROS activity

Answer 9

ROS signaling may be picked up by immune cells, epithelial cells, and endothelial cells. This primes immune cells for antimicrobial functions, induces CAM epression in endothelial cells, and triggers innate immune signaling in epithelial cells.

Question 10

Isoforms of Nitric Oxide Synthase

Answer 10

NOS1 / neuronal NOS (nNOS)

NOS2 / inducible NOS (iNOS)

NOS3 / endothelial NOS (eNOS)

All catalyze the NADPH-dependent oxidation of L-arginine into N^ω​-hydroxy-L-arginine, then the NADPH-dependent oxidation of N^ω​-hydroxy-L-arginine into L-citrulline + ^·NO.

Question 11

NOS1 / nNOS

Answer 11

Neuronal nitric oxide synthase

Constitutively expressed, Ca²⁺-dependent enzyme which serves a homeostatic function.

Question 12

NOS2 / iNOS

Answer 12

Inducible nitric oxide synthase

Induced in response to PAMPs, DAMPs, and pro-inflammatory signaling molecules.

Question 13

NOS3 / eNOS

Answer 13

Endothelial nitric oxide synthase

Constitutively expressed, Ca2+-dependent enzyme which serves a homeostatic function.

Question 14

Negative regulators of iNOS in MΦ

Answer 14

TGF-β, IL-4, IL-13, glucocorticoids

(anti-inflammatory and Th2-type cytokines)

These exert control on mRNA and protein stability, as well as levels of arginase, an enyme that catalyzes the destruction of the NOS substrate L-arginine.

Question 15

^{<strong>·</strong>}NO in Dendritic Cells

Answer 15

Produced by DC iNOS in response to cytokines and TLR ligands. Promotes presentation via MHC Class II and expression of B7.1 and B7.2.

Question 16

p67^phox

Answer 16

An accessory protein whose exact function is unclear.

Required for oxidase activity and inhibited by NADPH dialdehyde. May be involved with the final transfer of electrons to O₂.

Question 17

p47^phox

Answer 17

The component responsible for carrying the p40-p47-p67^phox complex to the membrane proteins in order to assemble the active oxidase. Activated by serine phosphorylation by PKC or Akt.

Demonstrably nonessential, as at high enough [p67^phox], superoxide production takes place in the absence of p47. However, this component is essential for the neutrophil. Patients whose neutrophils lack p47^phox suffer chronic granulomatous disease, as do patients whose neutrophils lack p67, gp91, and p22.

Question 18

p22^phox

Answer 18

Stabilizing binding partner of gp91^phox/NOX2, and most of the other NADPH oxidases. Contains the binding site for p47^phox.

Question 19

p40^phox

Answer 19

Seems to serve a regulatory role, but the exact nature of that role is unclear. Generally nonessential for Phox activity.

Question 20

Chronic Granulomatous Disease

Answer 20

Caused by a deficiency in NOX2 activity in neutrophils. Results in the inability to produce superoxide and characterized by severe infections, often beginning early in life. Patients are particularly prone to Aspergillus infection resulting in intractable pneumonia. Also prone to develop chronic inflammatory diseases.

Current treatments are limited to antibiotics, antifungal agents, and IFN-γ. Males are at increased risk as NOX2 is coded for on the X chromosome (80% of diagnosed patients are male).

Question 21

ROS and NFkB

Answer 21

• ROS inhibits the phosphorylation of I𝜅Bα
  
  • Typical I𝜅Bα inactivation: Activation of some serine kinase ⇒ S phosphorylation of serines 32 and 36 ⇒ ubiquitination and proteasomal degradation
  • ROS-mediated: Activation of NFkB inducing kinase (NIK) or SYK ( ⇒ Y phosphorylation of tyrosine 42 ⇒ dissociation (?ubiquitination)