Week 4-7 Flashcards
What’re the regulatory molecules of the hypoxia stress response signalling pathway
1) Hypoxia-inducible transcription factors (HIFs)
2) sensors (VHL)
3) the enzymes (HIF hydroxylases)
Why is oxygen so important for our bodies
What is hypoxia and what does it affect
WE NEED OXYGEN HOMEOSTASIS
- mitochondria utilizes >95% of O2 to make ATP = energy production and cell ability to maintain proper functions
Hypoxia:
- a deficiency in O2 delivered to the cells/body tissue that induces signalling pathways mediated by HIFs (hypoxia-inducible transcriptional factors)
- linked to the pathology of many diseases (cancer, cardiovascular diseases, stroke, COVID-19
What are the O2 partial pressures in the atmosphere, in blood, and in our tissues. Why does this happen and why is it important?
Lower conc. in vivo then in atmosphere
21% in atmosphere = normoxia for breadth/lungs
13% in alveoli/arterial blood
<5% in tissues and cells (considered hypoxia for cell cultures - requiring specific isolated hypoxia chambers)
Due to en route O2 consumption
importance?
- research uses in vivo cell cultures that are exposed to atmospheric levels of O2 conc (20%) meaning they are in HYPEROXIC conditions.
- we currently keep them in CO2 incubators (to mimic CO2 buffering system)
How does Hypoxia induce erythropoiesis
1) Erythropoietin (EPO) hormone produced by the kidneys, mediates bone marrow’s production of erythrocytes (red blood cells that transfer O2 thru the body)
2) under hypoxic stress, HETERODIMER: HIF-1 (hypoxia-inducible factor 1) binds to the short seq (HRE - hypoxia response element) downstream of the EPO gene and activates transcription
examples of this in the human body:
- pts w/kidney disease have anemia (deficiency in red blood cells) and this can be treated with EPO treatement
How does Hypoxia induce glycolysis and Angiogenesis, what is angiogenesis?
Glycolysis:
1. Glucose turns to Pyruvate which can turn into:
a) Acetyl-CoA for oxidation in Kreb’s or TCA cycle
b) or Lactate as glycolytic metabolism end product
- in hypoxia conditions, expression of LDHA (lactate dehydrogenase A) and PDK1 (pyruvate dehydrogenase kinase 1) inhibits PDH (pyruvate hydrogenase), tipping balance from oxidative to glycolytic metabolism
Angiogenesis: induced growth of new blood vessels from pre-existing vasculature during hypoxia
- During hypoxia, the HRE in the promoter region of growth factor VEGF (vascular endothelial GF), binds to HIF-1 and is secreted from the tumour (hypoxia allows up regulation of VEGF in the tumour and is secreted)
- VEGF increases blood vessel growth and movement towards the tumour
- Plenty of blood vessels is attached to the tumour supplying it with nutrients
What is the domain structure of HIF-1a and HIF-1B, which one is more complex, and why does it matter?
Transcription factor HIF1 is composed of 2 subunits that assemble into a heterodimer in the nucleus:
* both have DNA-binding domain bHLH and dimerization domain PAS
HIF-1a:
- O2-labile cytoplasmic protein w/half-life of <5min in normoxia due to proteasomal degradation
- also has transactivation domain at c-term split into a sandwich of TAD-N and TAD-C with a inhibitory ID in-between
- TAD-N has 2 pro and 1 Asp in TAD-C that can be hydroxylated by two different types of HIF hydroxylases
- hydroxylation occurs at normoxia conditions using O2 as a substrate
HIF-1B: stable nuclear protein
HIF hydroxylases:
1) PHDs
2) FIH1
in normal conditions, O2 is used by PHD and FIH1 to hydroxylate TAD-N/TAD-C in HIF-1a inhibiting the whole molecule from working = no hypoxia signalling pathway
What’re the consequences of HIF-1a hydroxylation
1) hypoxia stress sensor VHL binds to hydroxylated prolines TAD-N, recruits E3 ligase to ubiquinate it and send for proteosomal degradation (degrades whole protein)
2) hydroxylation of asparagine prevents binding of the co-activator protein p300 to HIF-1a (required for activation of the whole HIF-1) (can’t activate)
as a result, under normoxia, HIF-1a not available to activate the overall gene for hypoxia signalling pathway
How does Hypoxia stabilize the HIF-1a protein
1) In hypoxia, no O2 present to be used for hydroxylation (VHL can’t bind and no ubiqutination/proteasomal deviation) and P300 can bind = activation
2) stabilized HIF-1a can travel to nucleus and dimerizes with HIF-1B
3) DNA binding is now mediated by the bHLH and PAS domains and the whole heterodimer is free to bind to the HRE promoter region of other genes to induce their expression (eg. EPO PDK1, VEGF)
What are VHLs, what do they cause, what are common characteristics of it’s byproducts
- a sensor for hypoxia stress response
- a tumour suppressor gene (LOF causes VHL syndrome: tumours in eyes, kidneys, tissues)
- VHL uses (ELC, ELB, CUL2, RBX1) to ubiquinate HIF-1a. But inactive/missing VHL leads to accumulation of HIF1 = cell proliferation, neovascularization of tumours
All VHL disease tumours:
a) are angiogenic (elevated VEGF = excessive blood vessel growths)
b) high hematocrit (elevated EPO = high red blood cell count
What is the chemical reaction scheme of PHDs
1) PHDs uses O2, co-substrate 2-oxoglutarate and co-factor Fe(II) to hydroxylate HIF-1a prolines and sends it for proteasomal degradation
2) if any is missing, HIF-1a avoids degradation and induces hypoxia related genes instead this happens when conditions are (hypoxia, iron chelation, and 2-oxoglutarate analogues aka lack of these things)
How was hypoxia studied in cell cultures
Option A: animals cells were grown in hypoxia chambers that maintained O2 at 1-5%
Option B: Used chemical inhibitors of HIF-1a hydroxylation was used to mimic hypoxia signalling
either way, hypoxia stress signalling pathway was induced and western blots for HIF-1a was used to justify the hypoxia response in cells (results should show HIF-1a increase under hypoxia)
hypoxia response could also be tested in cells transfected with a reporter gene (luciferase)
Related to hypoxia, what is the definition for high altitudes, and what do we know about human adaptation to it
- 2500m above sea level is high altitude
- populations like the Ethiopians have lived in chronic hypoxia state
- genomic has studied several genes that underlie high-altitude adaptive phenotypes developed by these people (related to major components of HIF pathway)
What is Oxidative Stress, what is oxidants, name all the ones that are important oxidants and antioxidants for this course
STRESS: imbalance between excessive ROS and loss of antioxidants (AOX) defences
OXIDANTS occur internally/externally (radiation etc) and oxidizes other molecules (most common is ROS - oxygen radicals/non radicals with unpaired electrons)
non-radicals:
- hydrogen peroxide - H2O2 *
- hypochlorous acid - HOCL
- peroxynitrite - ONOO
(ONOO HOCL 22)
radicals:
Hydroxyl radical - OH *
superoxide radical - O2 *
lipid peroxyl radical
nitric oxide radical - NO
SHH is the 3 important ones
Antioxidants
The Cat Pressed Paused on the GpS
- Thioredoxin
- catalase
- peroxidase
- peroxiredoxin
- glutathione peroxidase
- superoxide dismutases
what is ROS, what’re the major types, and how are they formed
O2 has 2 unpaired electrons with parallel spins (BIRADICAL) and will not react with stable molecules (because they’re paired and spinning antiparallel
- an exception is when e- are transferred to O2 one at a time producing ROS (monoradical/nnonradical) this happens enzymatically/non-enzymatically
Types:
1) superoxide anion radical (O2 - 1e)
2) hydrogen peroxide (O2 + 2e)
3) hydroxyl radical (O2 + 3e)
can further form peroxynitrite and hypochlorous acid from this finishing the trio (ONOO HOCl H202)
What do we need to know about hydroxyl radicals and how are they formed (2 ways)
- only ROS produced non-enzymatically in vitro (externally) by radiation-induced homolytic fission of H20 or H2O2 (radiolysis/photolytic cleavage)
- specifically UV irradiation (H2O absorbs wavelengths <350nm)
- or in vivo (internal)
1) Fenton reaction between H2O2 and FE(II) to form FERRIC iron (III)
2) reacts with superoxide anion radical to reform ferrous ions
combination of these 2 (harder-weiss rxn) = hydroxyl radicals
what are the common properties among ROS
- high reduction potential (V) = they can oxidize molecules with lower/neg reduction potentials by taking from them (LEO the lion says GER)
- out of SHH, hydrogen peroxide is lowest reactivity and highest stability/ intracellular conc.
the most reactive/dangerous is hydroxyl radical, and superoxide is in the middle
- all ROS can oxidize and damage all molecules w/DUAL functions (causes apoptosis or cell proliferation due to
what is the oxidative damage to nucleic acids
most common cause of DNA lesions and mods to nucleotide bases is ROS-induced-oxidation of Guanine to 8-oxoguanine which results in CG to AT substitution during DNA replication = mutations
also induces fragmentation of deoxyribose/ribose rings of nucleic acid
results in: point mutations, changes in gene expression, single/double stranded dna breaks
What is the oxidative damage to lipids
called LIPID PEROXIDATION:
- unsaturated (double bonded+) lipids are susceptible to ROS (especially hydroxyl)
1) initiation
- (ROS rxn w/allylic Hydrogen and allylic Carbon = unstable radicals)
2) propagation
- (lipid radicals + O2 = lipid peroxyl radical which then reacts with unsaturated free lipid = new lipid radical/peroxide = another round lipid oxidation = continual free radical chain reaction
3) termination
antioxidants inactivate radicals. if AOX is impaired, accumulated lipid peroxides change membrane integrity, fluidity, permeability, enzyme activity. (eg. butter when warmed)
what is the oxidative damage to proteins
caused by ROS-induced modification of individual amino acid and their functional group (specifically thiol or SH groups of cysteines which results in SOH, SO2H, SO3H)
- changes to protein structure/function, turnover, and loss/occasional gains of activity
- diseases/aging
- fragmentation of peptide chains (aggregate)
- enzyme activity, ion transport, proteolysis, autoimmune responses
what are Glycans and what is the oxidative damage to them
carbohydrates that form a glycocalyx coat on surface of cells
- modifications to monosaccharides and glycan cleavage, fragmentation, and degradation
- accumulation of AGE
- impairment of intracellular contacts, induction of inflammation, and modification to functions of extracellular matrix
what is the clinical relevance of biomarkers of oxidative stress
can be detected used to detect oxidative stress (eg. direct and indirect measurements of ROS, antioxidants, and products)
the ones related to specific conditions are called specialized biomarkers
What’re the common sources of ROS in cells, what is an extra function of ROS
- plasma membrane, mitochondria, peroxisomes, endoplasmic reticulum, and lysosomal granules
- ROS are products of these enzymes but can also function a paracrine signals as hydrogen peroxide can cross from one cell to another through aquapores
What is the NOX family of NADPH oxidases
a major source of superoxide assembled in the plasma membrane
members:
- NOX2 (important part of phagocyte NADPH oxidase in neutrophils, a phagocytic cell in blood that generates ros to kill pathogens)
Breakdown the domain structure of NOX/DUOX and what does it do
all have 6 highly conserved transmembrane domain
- TM III and V each have 2 histidines that bind to asymmetrical hemes (functionality for carrying O, reduction, and transfer)
- Cytoplasmic C-term domain has NADPH and FAD binding domains
Function:
single e- transporter from NADPH ->FAD -> 1st Heme -> 2nd Heme -> O2 so it can reduce it into superoxide anion radical
- NOX5, DUOX1/DUOX2 also have Ca2+ binding domains
- DUOX1/2 also have peroxidase-like domains