Section 1 - ROS part 2

Question 1

Erythropoietin (def.)

Answer 1

30 kDa glycoprotein/cytokine for erythrocyte (red blood cell) precursors in the bone marrow

Question 2

Erythropoietin regulates ____ and is used as medicine against _____. Its expression is induced by ____

Answer 2

-red blood cell production
-anemia
-hypoxia, e.g. in COPD patients

Question 3

Erythropoietin is famously used in _____ because it increases ____ (2) by increasing ____

Answer 3

-endurance sports
-the oxygen carrying capacity of the blood and the aerobic respiratory capacity of the muscles
-red blood cell production

Question 4

negative downside of Erythropoietin

Answer 4

heart attacks

Question 5

Why climb Mount Everest is hard?

Answer 5

-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

Question 6

Tibetans in Everest have an adaption to high altitude which involves ___

Answer 6

a reduced response to hypoxia

Question 7

HIF-1 (____) is involved in oxygen sensing in mammals

Answer 7

Hypoxia-inducible factor 1

Question 8

How can we determine if a protein binds to a DNA sequence?

Answer 8

-EMSA assay

Question 9

EMSA assay does what?

Answer 9

analyzes DNA probes on a gel after incubation with protein fractions vs without incubation to determine whether protein binds to DNA (increase in MW)

Question 10

EMSA assay is a variation of ____

Answer 10

northern blot but uses predefined DNA oligos instead of RNA

Question 11

hypoxia-response element (def.)/ HRE

Answer 11

gene enhancer that provides induction of genes (ex. erythropoietin gene) under hypoxic conditions

Question 12

identification of enhancer sequence in DNA method

Answer 12

-sequence of enhancer is placed in front of luciferase-coding DNA and light emission is measured

Question 13

EMSA assay to determine proteins that bind to HRE

Answer 13

-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

Question 14

method to isolate proteins that bind to any given interactor

Answer 14

chromatography using the oligo sequences

Question 15

HIF-1 affinity purification

Answer 15

-Nuclear extracts were first run over a DEAE-sepharose column (binds DNA)
-specifically purified using the HIF-1-binding W-18 DNA sequences

Question 16

Two distinct HIF proteins were found: ____

Answer 16

HIF-1a and HIF-1B

Question 17

HIF-1a and HIF-1B were identified by ___

Answer 17

mass spectrometry

Question 18

The induction of proteins under hypoxic conditions were measured with ____

Answer 18

northern blot (measure the total RNA)

Question 19

northern blot steps

Answer 19

-gel electrophoresis of total mRNA
-transfer to membrane by blotting
-hybridize RNA with radioactive cDNA (remove RNA with RNase)
-image with X-ray film

Question 20

advantage of northern blot over western blot (protein)

Answer 20

• northern blot: more precise determination of protein production by assessing mRNA production
• results from Western blots could be clouded by previously present protein

Question 21

The mRNA for HIF-1a is also under the control of _____. It contains____ in its 3’ untranslated sequences that mediate _____

Answer 21

-oxygen tension
-RNA instability elements
-its destruction, when oxygen is present

Question 22

in hypoxia conditions, HIF-1a does what?

Answer 22

-transcription & translation of HIF-1a increase
-HIF-1a & HIF-1B increase transcription of genes (ex.VEGF gene)

Question 23

How are HIF-1A protein levels controlled?

Answer 23

poly-ubiquitinylation targets it for degradation by proteasome in O2 dependent manner

Question 24

in normoxia conditions, HIP-1a is ____ + method

Answer 24

-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

Question 25

immunoprecipitation method

Answer 25

-antibody against protein of interest binds to protein
-add protein to antibody to make antibody-protein complex insolubule
-centrifugation & removal of supernatant (pellet = antibody- protein complex)

Question 26

proteasome inhibitors block HIF-1a ____

Answer 26

degradation

Question 27

pVHL is what?

Answer 27

-tumour supressor protein

Question 28

VHL proteins does what?

Answer 28

targets hypoxia-inducible factors (ex. HIF-1a) for oxygen-dependent proteolysis

Question 29

VHL protein stands for ___

Answer 29

von Hippel Lindau protein

Question 30

von Hippel Lindau syndrome (def.)

Answer 30

-hereditary predisposition for a variety of tumors, in particular hemangioblastomas (blood vessel tumors)
-patients have abnormally high levels of VEGF (Vascular Endothelial Growth Factor)

Question 31

VEGF gene is under the influence of ____

Answer 31

HIF-1a, meaning that it is induced in hypoxia conditions

Question 32

General tendency of tumors to attract blood vessels to increase oxygen import also utilizes _____. It is very important for tumor development, because ____

Answer 32

-VEGF
-tumor tissue is often technically hypoxic

Question 33

Renal carcinoma cell line RCC4 loses ______ upon re-expression of VHL

Answer 33

-VEGF overproduction

Question 34

Renal carcinoma cell line RCC4 restores _____ upon re-expression of VHL, also restores clean hypoxic response (_____)`

Answer 34

-normal HIF-1a expression pattern
-increasing VEGF

Question 35

VHL protein absence in RCC4 cells compromises _____, leading to ______

Answer 35

-the entire HIF-1a signaling pathway
-aberrant VEGF expression that leads to tumor vascularization

Question 36

VHL and HIF-1 only bind in vitro when ____

Answer 36

a mammalian cell extract is present

Question 37

to undergo O2 dependent VHL-mediated degradation, HIF-a is _____

Answer 37

hydroxylated at Pro 564 by prolyl-hydroxylase

Question 38

Modification of proline to hydroxyproline in a synthetic peptide abolishes ______ in the in vitro VHL- HIF-a binding assay. The extract contains ____

Answer 38

-extract requirement
-prolyl-hydroxylase

Question 39

prolyl-hydroxylase needs _____/_____ and _____ for reaction with HIF-a

Answer 39

-iron/cobalt (replace iron in enzymatic reaction centers)
-oxygen

Question 40

PET scanning uses ____ to ____

Answer 40

18F-deoxyglucose (radioactive glucose)
-see sugar-hungry organs (ex. brain) + cancer cells

Question 41

The Warburg hypothesis (def.)

Answer 41

tumor cells mainly generate ATP by non-oxidative breakdown of glucose (a process called glycolysis).

Question 42

cells at the center of the tumor are typically ___ while cells are the edge (far from blood vessel) are ____

Answer 42

-normoxic around a blood vessel
-hypoxia

Question 43

Warburg hypothesis + effect on tumor cell growth

Answer 43

-reduced mitochondrial function by just using glycolysis + hypoxia conditions induces HIF-1a action (conserves oxygen) and prevents anoxic conditions

Question 44

mild hypoxia + tumor cells

Answer 44

mild hypoxia supports growth of tumor cells (attracts nutrients to the area)

Question 45

HIF-1a + tumor cells

Answer 45

-stimulates VEGF + growth of blood vessels