Section 1 - ROS part 2 Flashcards
(45 cards)
Erythropoietin (def.)
30 kDa glycoprotein/cytokine for erythrocyte (red blood cell) precursors in the bone marrow
Erythropoietin regulates ____ and is used as medicine against _____. Its expression is induced by ____
-red blood cell production
-anemia
-hypoxia, e.g. in COPD patients
Erythropoietin is famously used in _____ because it increases ____ (2) by increasing ____
-endurance sports
-the oxygen carrying capacity of the blood and the aerobic respiratory capacity of the muscles
-red blood cell production
negative downside of Erythropoietin
heart attacks
Why climb Mount Everest is hard?
-air on top as less oxygen
-as you climb, you exhale more vigorously to try to get more oxygen and reduce co2
-exhale too much co2, increasing blood ph
-high blood ph = nauseated and dizzy
Tibetans in Everest have an adaption to high altitude which involves ___
a reduced response to hypoxia
HIF-1 (____) is involved in oxygen sensing in mammals
Hypoxia-inducible factor 1
How can we determine if a protein binds to a DNA sequence?
-EMSA assay
EMSA assay does what?
analyzes DNA probes on a gel after incubation with protein fractions vs without incubation to determine whether protein binds to DNA (increase in MW)
EMSA assay is a variation of ____
northern blot but uses predefined DNA oligos instead of RNA
hypoxia-response element (def.)/ HRE
gene enhancer that provides induction of genes (ex. erythropoietin gene) under hypoxic conditions
identification of enhancer sequence in DNA method
-sequence of enhancer is placed in front of luciferase-coding DNA and light emission is measured
EMSA assay to determine proteins that bind to HRE
-identify enhancer sequence
-synthesis radioactive ds oligos that have those sequence
-incubate with normal and hypoxic (from cells undergoing hypoxia) protein lysates
-analyze in polyacrylamide gel (to separate high MV of proteins) to distinguish between free/bound ds oligos
method to isolate proteins that bind to any given interactor
chromatography using the oligo sequences
HIF-1 affinity purification
-Nuclear extracts were first run over a DEAE-sepharose column (binds DNA)
-specifically purified using the HIF-1-binding W-18 DNA sequences
Two distinct HIF proteins were found: ____
HIF-1a and HIF-1B
HIF-1a and HIF-1B were identified by ___
mass spectrometry
The induction of proteins under hypoxic conditions were measured with ____
northern blot (measure the total RNA)
northern blot steps
-gel electrophoresis of total mRNA
-transfer to membrane by blotting
-hybridize RNA with radioactive cDNA (remove RNA with RNase)
-image with X-ray film
advantage of northern blot over western blot (protein)
- northern blot: more precise determination of protein production by assessing mRNA production
- results from Western blots could be clouded by previously present protein
The mRNA for HIF-1a is also under the control of _____. It contains____ in its 3’ untranslated sequences that mediate _____
-oxygen tension
-RNA instability elements
-its destruction, when oxygen is present
in hypoxia conditions, HIF-1a does what?
-transcription & translation of HIF-1a increase
-HIF-1a & HIF-1B increase transcription of genes (ex.VEGF gene)
How are HIF-1A protein levels controlled?
poly-ubiquitinylation targets it for degradation by proteasome in O2 dependent manner
in normoxia conditions, HIP-1a is ____ + method
-degraded
-HIF-1a is oxidized by proline hyroxylase
-OH residues on protein is recognized by pVHL and other proteins
-those proteins promote poly-ubiquitylation = degradation in proteasome
