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
How are different domains of NOX enzymes localized in the plasma membrane (membrane topology)
NOX 1,2,3, and 4 all built the same
1) they have e- transfer centres that allow it to go from cytoplasmic NADPH -> O to form superoxide outside the cell
2) superoxide reacts with itself = hydrogen peroxide which is a substrate for peroxidase
NOX5 structure builds off of NOX2 but w/an amino-terminal calmodulin-like domain that has 4 binding sites for calcium
DUOX1/2 is like the NOX5 +1 transmembrane a-helix and a domain homologous to peroxides (MPO) that is localized outside the PM where it refuses the ROS pumped from the core for its own function
what are the functional domains of the PHOX proteins and and their functions, how is the enzyme complex activated
NOX22 is a flavocytochrome (enzymatic component)
RAc = G protein
p67= activator
p47 = organizer
p40 = secondary adapter
activated using protein-protein/lipid interactions (SH3, PB1, PRR, AIR, T, Rac-binding tetra repeat domains, AD; PX and RAC tail)
in resting conditions the trimer complex (40/47/67) stay localized cause of AIR in the p47/SH3 domain preventing it from binding to PRR in p22phox
RAC-GDP is also localizeed cause its lipid tail is masked by RhoGDP-dissociation inhibitor
how is Phagocyte NADPH oxidase activated
this is responsible for generation of superoxides by neutrophils and others etc.
1) In norm conditions p22phox and NOX2 are attached (Flavocytochrome complex) while the rest, 40,47,67, and RAC are localized in the cytoplasm (all needed to activate NOX2)
2) pathogens, inflammatory mediators, or compounds mimic bacterial wall oligopeptides create a coordinated signalling process of protein phosphorylation, lipid metabolism, and GTPase activation
3) active protein kinase phosphorylates 47, allowing the whole complex+ GTP-activated-RAC to bind to p22phox
4) lipid metabolism synthesizes phosphtidylinntositol-phosphates accumulated on the complex and helps stabilizes it
How is NOX2 activated
1) phosphorylation of 47(organizer) recruits the 37phox complex to membrane while RAC-GTP translocates to membrane using its lipid-tail
2) conformational change of 47 unmasks PX domains and SH3 domains required for binding of PRR of phox22
3) RAC-GTP (dissociated from RhoGDI) orients phox67 (activator) using protein-protein interaction on activation domain
4 PX domain of phox47/40(secondary adapters) helps further OPTIMIZE the orientation of 67
What is chronic granulomatous disease, how is it diagnosed
- working phagocyte NADPH oxidase is required for neutrophils to assist ROS-induced killing of pathogens
CGD: is mutations in all BUT RAC. NOX2 (67% cause of cases), 47 (20%), and remainder divided between 67/22 and one case of 40
- CGD pts have neutrophils that generate less superoxide and bactericidal properties are impaired (bacterial/ fungal infections and inflammation called granulomas that contain masses of immune cells)
diagnosis:
direct measure of superoxide production using whole blood sample treated with activator of the phagocyte oxidase in presence of ROS-sensitive dye
yellow nitrobluee-tetrazolium - turns purple when reduced and stains cytoplasmic granules showing production of radicals under microscopy
Dihydrorhodamine (DHR) dye allows detected signals from cells after treatment with activator and using flow cytometry machine: histograms with 2 peaks shows ROS production
what are peroxisomes in terms of oxidative stress, how are they visualized? what are the pros and cons of overproduction?
small ubiquitous cells that help generate hydrogen peroxide
- lipid biosynthesis, oxidation of fatty acids, catabolism of aa etc using different oxidases
- source for oxidative stress and sometimes producing superoxides/nitric oxides
- can be detected by transfecting w/GFP fused peroxisomal targeting peptides using fluorescent microscopy
Cons: bad for cells
Pros: SOD, catalase, peroxidase, and peroxiredoxins also helps inactivation/degrade ROS
- exposure of cultured peroxisomes to oxidative stress = peroxisome proliferation w/formation of tubular peroxisomes (higher surface that accomodates more antioxidants)
- NAC can be used to prevent this if wanted
what is the level of ROS generation in the Mitochondria
- large amounts of ROS in mito due to leakage from the ETC (complex I and III react with O2 to form superoxide)
- 0.5% O2 consumption by mito is reduced to sup oxidant = hydrogen peroxide
- superoxides produced att complex I = only in matrix
- produced at Complex III = matrix + inner mito space
- mito uses antioxidant enzymes to protect against ROS (converts superoxides to less toxic hydrogen peroxide or catalase, GPx, and Prx3 to inactivate/catabolize hydrogen peroxide)
- Inhibited ETC (rotenone inhibition of complex I) greatly increases ROS accumulation
What is the role of Heme in ROS production
- major source of production
- example is haemoglobin producing constant flux of superoxide (releases superoxides due to e- delocalization between heme-divalent Fe and O2 = Fe(III)
- to prevent superoxide side effects, erythrocytes have many antioxidant enzyms
- their discoid shaped + hydrogen peroxide = echinocytes (small wrinkled cell with thorns)
How is hydrogen peroxide produced
- spontaneous/non-spontaneous
Way 1: superoxide dismutation reactions (slow but relevant amts of it is produced) catalyzed by superoxide dismutass (SOD) - SOD converts high reactive superoxide anion radicals into less active hydrogen peroxide
SOD types: cytoplasm localized SOD1, mito SOD2, extracellular space SOD3 depending on Cu/Zn Manganese cofactors
Way 2) oxidases also catalyzes redox rxns using O2 as the e- acceptor
What is the enzyme Cytochrome P450 in the ER
- many e- transfer hemoproteins and membrane bound flavoproteins are localized in the ER which produces ROS but also contains this enzyme (in catalytic cycle of cytoP450 + heme ferrous state produces ROS), produced by leaky branches:
a) autodidact shunt:
- superoxide released by the autodidact of the oxy-ferrous complex
b) peroxide shunt:
- hydrogen peroxide produced vt protonation of the peroxycytochrome p450
- involved in metabolic conversion of lipophilic substrate by oxidizing them to become more polar/soluable = get secreted
- oxidizes substates using e- transfers from NADPH to NADPH-Cytochrome P450 reductase (purple) and cytochrome p450(blue)
what is myeloperoxidase (MPO)
neutrophil enzyme located in azurophil granules comprising of 2-5% of cellular proteins
- assists neutrophils in degrading pathogens by catalyzing production of HOCL in rxn (hydrogen peroxide + CL ions)
- moderate ROS (hydrogen peroxide) converts to more aggressive form when MPO uses it to form HOCL which is highly reactive and causes more pathogen destruction
explain nitric oxide synthase
nitric oxide + superoxide = peroxynitrite (another more aggressive ROS that can cause pathogen damage)
-nitric oxide (NO) is synthesized by NOS into forms endothelial, neuronal, and inducible
- NO is a function messenger for vasodilation, neurotransmission, and antimicrobial/anti-tumour activity
ROS toxicity vs ROS signalling
ROS is not just harmful but also essential physiological regulators of intracellular signalling pathways and cellular responses
harmful: O2 uses NOX2/DUOX2 -> superoxide uses:
1) Fe(III) to turn into hydroxyl radical,
2) SOD to turn into hydrogen peroxide + MPO to become HOCl,
3) or nitric oxide to be peroxynitrite (ONOO)
effects of ROX production by NOX enzymes:
a) moderate ROS = generation in neutrophils (phagocytes) important for microbicidal (killing of pathogens)
b) high ROS = tissue injury/hyperinflammation/cell damage
c) NOX1 activation in epithelial activates transducer signals via low ROS production = affects redox status, cell proliferation, and differentiation
what is redox mediated signalling
Redox is regulated using ROS by covalent modification of specific reactive cysteine residues in redox-sensitive target proteins
- oxidization of thiol group = reversible modification of enzymatic activity
1) formation of intramolecular and intermolecular disulphide bonds, the cross-link between regions in a polypeptide chain or between separate chains: (2 thiol groups oxidizes) OR (RSOH interacts with cysteines)
2) Oxidation to a sulfenic (RSOH) which is unstable and can further oxidize into Sulfinic (RSO2H - reversible), or Sulfonic (RSO3H) form which can be reduced’
3) Glutathionylation (RSSG) a reversible post translational protein modification where G-SH binds to protein via disulphide bond w/thiol
what’e the redox-sensitive signalling molecules and processes
ROS regulates the pathways through interactions w/molecules that have redox-reactive cysteine residues
uses transcription factor Nrf2 and enzymes that control thiol/disulfied exhange (Pre, Trx, Srx) - pre, teen, sr
What its the ROS-mediated Nrf2/Keap1-signalling
adaptive pathway:
- Keap1 is stress sensor w/cysteine rich protein and ability for conformation change when thiol groups are modified
Unstressed cells: nrF (TF) is bound to Keap1 and ubiquinates it using CUL3 sending it for proteosomal degradation
stressed: ROS-induced oxidative modification of cysteine residues inactive KEAP1 freeing the TF to go translocate to the nucleus, heterodimerizing with MAF, binding to ARE which induces expression of genes encoding antioxidant enzymes and cytoprotective genes
What’re the cytoprotective genes regulated by Nrf2
stress free NrF2 from sensor KEAP1 letting it up regulate synthesis of:
- superoxide dismutase, catalase, and peroxidase that are needed to inactivate ROS (hydrogen peroxide, superoxide)
- also upregulates enzymes that control thiol/disulfied redox exchange
What are Peroxiredoxins (Prxs)
- part of the peroxidases family that reduces and inactivates hydrogen peroxide and peroxides
- small, localized in all kingdoms, homodimers arranged head-to-tail orientation, and containing 2 cysteine residues:
a) Peroxidatic Cys (Cp): oxidizes hydrogen peroxide to cystein sulfenic acid
b) Resolving Cys (Cr): forms disulphide linkage with other cysteine (CP)
What is the reaction mechanisms of Prx enzymes
- peroxiredoxin has 6 families encoded by PRDX1-PRDX6 but is classified by three main subfamilies (based on location of Cr
- typical and atypica (I-V) need thioredoxin to regenerate, 1-cys needs (VI) needs glutathione
TYPICAL 2-cycteine peroxidredoxin(Prxs 1-IV):
1) contains both Cp/Cr. The Cp-SH is oxidized by H202 to form CP-SOH (sulfenic)
2) 2 molecules of H2O2 used to make disulphide bounds between CP-SOH and Cr-SH, antiparallel prx dimers are formed from 2 more disulphide bonds between prx molecules
3) Pre can be hyperoxidated using high conc. of H2O2 to convert Cp into CP-SO2H before it reacts with Cr-Sh. This leads to inactivation of Prxs peroxidase activity and result in protein aggregation
OR oxidation of Prx is reversible using Trx to reduce disulphide bonds
4) Srx can reduce hyperoxidized sulfinic group back to its active form (ATP-dependent) and regenerating PRX
ATYPICAL 2cys (Pre V):
functions same as typical 2-cys but with intramolecular disulphide linkage formed within each subunit
1-CYSTEINE peroxiredoxin (Prx VI):
only has Cp and during the catalytic cycle, Cys-SOH forms but doesn’t make disulphide bonds because there’s no Cys-SH, to be regenerated CP-SOH needs to be oxidized by glutathione (GSH) by glutathione S-transferase
explain the Thioredoxin system, what does it do, its structure?
thiol-disulfide reactions are crucial for redox homeostasis and function of peroxiredoxin and other redox enzymes is dependent on the thioredoxin system.
- it performs protein disulfied reductase (catalyzes disulfide bond reduction in many proteins by maintaining a pool of reduced thioredoxin Trx-SH2 through e-transfer)
- its a key antioxidant defence through disulphide reductase activity regulating the thiol-disulfide balance.
Consists of
1) Thioredoxin (Trx) that reduces disulphide bonds into two S-H groups
2) and thioredoxin reductase (TrxR) a selenoenzyme reduced by NADPH that reduces disulphide bonds in oxidized thioredoxin (Trx-S2)
electron transfer process (NADPH->TrxR - > Trx) maintains pool of reduced thioredoxins = catalyzes disulphide bond reduction in proteins
What is reduce state and redox potential, how do you calculate redox and glutathione potential
- transfer of e- sometimes in redox pairs (one acts as a donor, the other acceptor eg NAD+/NADH)
- glutathione and GSH/GSSG redox pair its key in redox regulation
ratio between the two is high and varies (intracellular = 3-10mM, extracellular blood plasma= 2-10 µm)
redox potential is calculated using Nernsts eqt: standard reduction - [2.3(8.3)(temp) / #e-(9.6 x 104)] - oxidant ratio
glutathione: depends on ratio of GSSG/GHS and GHS^2 conc.
What is the accumulation of H2O2 around lipid rafts
when we want local accumulation of ROS (eg. when we want H2O2 for signalling growth factors like PDGF or EGF for wound healing/tissue regeneration
- these growth factors activate PTKs (supports phosphorylation of effector proteins and the stead-state lvl of protein tyrosine phosphorylation in cells required for proliferation
- however cells also express PTPs (removed P’s) which need to be inhibited in order for cell activation/induced growth factors
flow chart PTK, NOX, and SRC are all in the lipid rafts
- PTK phosphorylates growth factors, NOX production of H2O2, and SRC production of Prx I
- PTK is inhibited by PTP, but H2O2 from NOX can can stop PTP, but we need PRx from src in order for the H2O2 to not be degraded first before it does its job
what is the redox compartmentalization and redox changes in cell
- overal redox potential across different compartments is negative
- conc. of reduced molecules in live cells is higher than oxidized molecules)
- redox potential is still different among intracellular compartments and organelles (most reduced is mito followed by nucleus, cyto, and extracellular space)
- cellular/extracelllullar redox microenviron is oxidized in association with cell proliferation to differentiation, arrest, and apoptosis
what is the plasma redox potential and aging
- reduce potential of tissues largely depends on the redox pairs (GSH/GSSG and Cys/CysSSCys) and can be calculated using blood plasma
- results of the study showed linear correlation between increase of cys/CysSSCys and aging until capacity of GSSH antioxidant system at 45-50 where it declines rapidly
what is the relationship between oxidative stress/redox state and health
- redox pair states in blood plasma could be used as quantitative measures of oxidative stress/ predictive health markers (-62 mV as initial values)
- more oxidized values are for ppl aging, not taking antioxidants, smokers, drinkers, and diseased
- redox regulation is more complex having to also consider other redox pairs
- redox regulation implies factors supporting reduced microenvironment and inhibiting oxidative stress has positive side effects on health/aging (fruits are high in antioxidants)
What is the role of miRNAs and biogenesis and action
Central DOGMA = DNA->RNA->Protein
but some genes don’t encode proteins, but instead miRNA (18-26 nucleotides) that control amt of proteins made by other protein-coding genes (effects depend on type of mRNAs present)
1)miRNAs are transcribed by RNA polymerase II as pri-miRNA hairpins
2) endonucleus enzyme Drosha makes initial cleavage in it creating 70 nucleotide pre-miRNA
3) nuclear transporter exportin5 moves it out of the nucleus to cytoplasm by diffusion (inner channel of pore complex)
4) RNAse dicer processes it into double stranded miRNA (22 nucleotides) that binds to Argonaute (a core protein in RISC)
- aka it cuts hairpin so its double stranded and not connected
5) miRNA-RISC complex associates with mRNAs at 3’UTR and either blocks protein translation or induce mRNA degradation depending on compatibility of miRNA/mRNA
What’re examples of pre-miRNAs
- first discovered in C/elegans as RNAs encoded by Lin-4 (worms) and Lin-7 (Elgan) and kick-started miRNA field
- those genes regulated embryogenesis and larval development specific to worms
- all are hairpin loop structures with 70 nucleotide imperfect base pairing that suggest each strand can produce different mature miRNAs
How do you find out more about an miRNA
all current info is on the microRNA database and has not changed since 2018
- each entry represents a predicted hairpin portion of the transcription (mir) w/its location and seq of the mature seq (miR)
- miRNA nomenclature includes 3-letter prefix, word miR, and the humber
numbered sequentially by discovery date
- 3p and 5p specify origin
what’re the targets of miRNA-mRNA interactions, how are they identified, and how is it interacted with
- > 60% of genes
- 1 miRNA can recognize multiple targets
- critical spot for targeting is the “seed seq” of miRNA (first 2-8 nucleotides starting from 5’)
- different bindings based on Watson-crick match AU and C
- perfect complimentary = degradation of mRNA
- imperfect = inhibition of protein synthesis
main types:
6mer = perfect match between miRNA seed/mRNA for 6 nucleotides
7mer-m8 = 2-8 of the seed
8mer = 2-8+ A across from nucleotide 1
7mer-A1: 2-7 + A across from nucleotide 1
How is stress and miRNAs connected, steps?
miRNA mutations exhibit phenotypic crisis under stressful conditions
- either restores or reprograms they gene expression patterns
does this by:
1) changing lvls of miRNA
2) changing lvls of mRNA targets
3) change in the mode of action of miRNA-protein complex
4) changes in the activity of the complex
How does miRNA mutations change its conc lvls in stressed vs nonstressed conditions?
- heat maps show that hypoxia changes miRNA expression in a time-dependent manner
example: an mRNA target has 3 miRNA binding sites (A,B,C)
untressed: target is repressed by miRNA A/B and has basal lvl expression
stressed:
- targets decrease if miRNA C increases (all 3 sites inhibited)
- increases if B decreases (only A is inhibited)
how does miRNA mutations change levels of mRNA targets
mechanism 1: Target mimicry
- individual miRNA interacts with diff target mRNAs (mimics of target)
- if mimic conc. increases due to stress, it competes for the same miRNA and provides relief in repression of targets = increasing their conc.
mechanism 2: create/deletion of miRNA binding sites
- when stress expresses different isoforms of the target where the binding sites could be created/deleted
miRNA sites created = expression of isoforms decrease
deleted = expression of isoforms stabilized
How does miRNA mutations changes the activity of the miRNA-protein complex
Argonaute which is a part of the RISC complex is an example.
still not much known but a change in the activity of miRNA-protein complex when stressed is the result of:
a) post-translational modification of components in the complex
b) direct association with other co-factors
c) activation/repression from nearby RNA-binding proteins
d) sequestration to subcellular locations (stress granules or P-bodies)
How does miRNA mutations change the mode of action of miRNA-protein complexes
- stress alters the balance between major modes
a) accelerating mRNA degradation
b) inhibiting translation
miRNA-RISC and bound mRNA can aggregate into granules:
i) P-bodies (foci where mRNAs are destroyed, contains degradation machinery like argonaute)
ii) Stress granules (cytoplasmic mRNP for translation initiation containing initiation factors = storage/stress signalling)
What are the mechanisms that control the expression of eukaryotic genes
2 mechanisms require TF and miRNAs
1) TF binds to diff regulatory seqs in the promotor region of genes and either activates/inhibit expression
2)TFs bound at distant enhancers interact with general TFs at the promoter (intervening DNA forms loops)
shows no diff between TF binding to DNA upstream of promotor vs distant enhancers
3) in the cyto, miRNA work post-transcriptionally to bind complimentary seqs in protein-coding mRNA at 3’UTR (blocks translation or induce degradation)
what is the timing patterns of mRNA target expression
stress-induced TFs can regulate expression of mRNA and miRNA resulting In different expression timing patterns
1) takes time for mature functional miRNA to accumulate after activation
2) miRNA is stable while changes to mRNA is immediate
3) stimuli implies at low basal while inhibition implies at high basal lvl for both
Pulse basal: low miRNA and mRNA
Lag basal: high low
Decrease: high high
Reinforced decrease: low high
