Week 5 Flashcards
Distinguish the unique features of the different classes of ATPases.
P class:
Located primarily on Plasma membranes
Are autoPhosphorylated during catalysis
Examples: Na-K ATPase, Ca ATPases
V class:
Located in secretory Vesicles like synaptosomes
Transport H+ into vesicle, multimeric
F class:
Located in mitochondria (and chloroplasts)
Physiologically ATP is Formed (synthesized), multimeric
- Distinguish the unique features of the sodium-potassium ATPase
o Na+ concentration higher outside cell o K+ concentration higher inside cell
o ATP ADP
carrier protein undergoes conformational change
three Na+ are released on the extracellular side of membrane two K+ bind
dephosphorylation causes another conformational change
K+ ions are released to the intracellular side of membrane
o ions against concentration gradient o 3Na+,2K+,1ATP
- Describe the mechanism of ionophores.
Ionophores use a hydrophilic inside and a hydrophobic outside to form channels in cells membranes. Can be used as an antibiotic to kill bacteria. An example is Gramicidin!
- Explain why ion channels are considered allosteric proteins.
Because many can be bound by ligands away from the site of action and have their activity changed
- Postulate the mechanism for opening voltage-sensitive channels.
There are positively charged alpha helices that move toward the negatively charged side of the membrane when the membrane become depolarized. As they move up, they also move outward a little causing the channel to open
- Know examples of transporters that cause a specific human disease when mutation occurs.
ABC transport defect in ABCA-1 – Tangier’s Disease - Results in problems with cholesterol and heart attacks early in life
ABC(C7) – Cystic Fibrosis
Define an ABC transporter (features) and the role of specific ABC transporters in drug resistance.
- ABC transporters – transmembrane transporters that utilize the energy of ATP hydrolysis to carry out certain biological processes, including:
o translocation of various substrates across membranes
o non-transport-related processes such as translation of RNA and DNA repair - involved in
o tumor resistance
o cystic fibrosis - patients can develop resistance not only to the drug they’re taking but to several different types
of drugs
o increased excretion of the drug from the cell by ABC transporters
o i.e. ABCB1 protein – functions in pumping tumor suppression drugs out of the cell o confer resistance to most of Topoisomerase I or II inhibitors
LETS LOOK AT GLUTS
Glut1 – everywhere – glucose/galactose
Glut 2 – Liver, intestine, kidney - used for uptake and efflux – Gluocse/Gal/Fruc
Glut3 – Neurons – gluc/galac
Glut 4 – fat and muscle – regulated by insulin – ONLY glucose
Glut5 – intestine and sperm – fructose metabolizing tissues – fructose!
What proteins are responsible for driving the cell cycle?
Cyclin dependent Kinases (CDK) and cyclins basically run the whole show.
CDK’s must be bound to cyclin and CAK (cdk activating kinase) must phosphorylate CDK’s activating site for CDK be active.
Wee1 can inhibit by phosphorylating an inactivation site, but this can be reversed by Cdc25 phosphatase to activate the Cdk-cyclin complex once more! Hoorah!
Sorry, Cki (Cyclin-dependent kinase inhibitor) can stop the party AGAIN by binding and inhibiting the whole complex. Usually its for a good cause because they have to repair some DNA damage or something
How are positive feedback loops used in cell cycle regulation?
Positive feedback loops are used in the mitogen receptor – RAS – MAP kinase – E2F pathway!
Basically what happens is that after g1-CDK is activated its going to activate E2F by phophorylating Rb.
E2F is then going to go on to help transcribe G1/S cyclins and S cyclins. These activated cyclins and CDKs are actually going to positively enhance the activation of E2F again and again! BAM! Positive feedback!
- How is S-phase triggered? What are ORCs and pre-RCs? What features of this system control the block to re-replication? Why is overall Cdk activity very low in early G1?
ORC’s are origins of replication in eukaryotic DNA. Proteins are there to mark the spot. Pre-replication complexes (pre-RC’s) form at the ORC’s at the beginning of G1 because there is very low CDK activity. S-CDK arrives and triggers S-phase as it phosphorylates in order to degrade and inactivate Cdc6 and Cdt1. These proteins will stay phosphorylated throughout the rest of the cell-cycle, keeping S-phase from reoccurring because they can’t form the pre-RC whole phosphorylation keeps them inactive.
- How does growth factor signaling and the MAP kinase pathway function in the regulation of cell division? What are early and late response genes (and what are some examples of each)? What is the mechanism of pRb inhibition of the E2F family of transcription factors, and what releases this inhibition? How is G1-Cdk activity shut off?
So basically growth factor comes in and binds to the receptor which activates Ras. Ras activates MAP kinase, which does a phosphorylation thing and then activates Myc, a gene regulatory protein which helps cyclin D to be expressed. This then activates the CDK
CDK goes on to phosphorylate and remove Rb from E2F. E2F will go on to help transcribe a bunch of G1/S and S cyclins that will continue to positively feedback more cyclins and finally enter into S-phase with DNA synthesis.
- What are cell cycle checkpoints?
G1 Checkpoint
- Is environment favorable?
- Enter S phase!
G2 Checkpoint
- Is all DNA replicated?
- Is environment favorable?
- Enter Mitosis!
Metaphase Checkpoint
- Are all chromosomes attached to the spindle?
- Exit Mitosis!
What are the main molecules involved in sensing and transducing DNA damage into cell cycle arrest
DNA damage aactivates ATM kinase, which activates Chk1/Chk2.
Chk1/Chk2 activate p53 by phosphorylation.
P53 then binds to the regulatory region of the p21 gene and transcribes/translates p21
P21 is a CDK inhibitor, so this inactivates the CDK and arrests cell cycle until the damage is taken care of.
- Use your knowledge of electrochemical gradients and equilibrium potentials to determine a cell’s resting membrane potential, and be able to determine the direction of flow for Na+, K+ and Cl- for any value of Vm.
K+ is -88 mV (-83 is hyperkalemia)
Na+ is +60 mV
Cl- is -61 mV
- Apply Ohm’s law to cells
V=IR or I=VC
- List normal values for intra- and extra-cellular concentrations of the primary electrolytes
Intra/extra Na: 10/140 K: 150/4 Ca: .0001/1 Cl: 20/100
Calculate equilibrium potentials by using the Nernst equation
Nernst Equation = (61/z) x ([]out/[]in)
- What are the major pathways that regulate and execute apoptosis?
Two types of signals are commonly associated with induction of apoptosis: specific signaling events at the cell surface, and cytoplasmic/nuclear damage. Signaling events trigger the extrinsic apoptotic pathway, and cell damage triggers the intrinsic apoptotic pathway.
A number of different triggers and conditions can lead cells to initiate apoptosis. Typically, the net result of these signals is the activation of a set of proteases that are responsible for the destruction of the cell. These proteases are called caspases, so named due to their function as cysteine-aspartic acid proteases.
Compare extrinsic and intrinsic pathways of apoptosis
Extrinsic:
Activated by signaling events
-Ligands – binds and activate members of TNF (tumor necrosis factor) family,
-Receptors – integral proteins of TNF family, Form trimmers, create a DISC (death-inducing signaling complex) scaffold when bound to recruit caspases
-Capsases – activate after associating with DISCs, effector caspases then destroy cytoskeletal, nuclear, and regulatory proteins
Intrinsic:
This pathway is set off by DNA damage or cell stress. (heat shock, UV, starvation, ROS, ER stress, cytosketetal perturbation
Important thing that happens is mitochondrial dysfunction, causing cytochrome C to be realeased and cause the formation fo the apoptosome
The apoptosome is formed wen released cytochrome-C associated with Apaf1. This structure forms a scaffold similar to the DISC in the extrinsic pathway. You still get the initator and then effector caspases bidning to it doing their thing.
How is cell stress transduced into apoptosis
Cell stresses act through p53. P53 has the ability to send the cell into cycle arrest or apoptosis once it is activated. Arf removes mdm2 to activate p53.
How do cell survival pathways function?
Apparently to survive, most cells need to receive continuous signaling in order to suppress apoptosis. Regulation of signals or competition for survivial factors is used to remove tissue in embryonic development
Survival factors allow cell to keep producing anti-apoptotic protein Bcl-2
Viral proteins can use survival factors to their advantage. Ike inhibiting the apoptotic abilities of BAX – whoa! Keeps virus from dying before it wants to!
What is the significance of how Bcl-2 functions as an oncogene?
Bcl-2 inhibits autophagosome creation. Bcl-2 also prevents apoptosis. The ratio of Bcl-2 to BAX helps determine this. The more Bcl-2 there is compared to BAX, the less likely it will apoptose.
Tumor cells interact with neighboring cells. They send signals to fibroblasts telling them to autophage. Lactate and pyruvate are released by the autophaging cells and used for growth and proliferation by the cancer cells! Because so many mitochondria are lost from fibroblast in this process, they have to use glycolysis a lot for energy, which is where the reverse Warburg effect comes from.
What is the “Reverse Warburg Effect?”
Warburg effect is when tumors use glucose preferentially, but still don’t get much energy from oxidative phosphorylation. Reverse Warburg effect is when cells surrounding the tumor are actually using more glucose and making less oxidative phosphorylation energy as well!
Trace the biosynthesis of NO
- Acetylcholine released by nerve terminals in the blood vessel wall activates NO synthase in endothelial cells lining the blood vessel
- causes the endothelial cells to produce NO
- NO diffuses out of the endothelial cells and into the underlying smooth muscle cells
- binds to and activates guanylyl cyclase – produces cyclic GMP
- cGMP triggers a response that causes the smooth muscle cells to relax
- enhances blood flow through the blood vessel
Identify common neurotransmitters, hormones, growth factors, and gaseous signal molecules.
NTs – histamine, ACh GABA
Hormones – cortisol, estradiol, glucagon, insulin, testosterone
GF’s – EGF, PDGF, NGF
Gaseous – NO, CO, H2S
Identify the three main classes of cell-surface receptors.
Ion channel receptors
Receptors that are kinases, or bind kinases
Heptahelical or G-protein-coupled receptors
Describe G proteins and their function in membrane events.
Basically their membrane surface protein is bound by a signal.
This binding will activate the alpha/beta/epsilon subunits by exchanging a GDP for GTP.
These subunits can then go on to activate a host of different signal cascades
(adenylyl cyclase, cAMP, directly opening channels, IP3 and DAG creation, Ca release)
Describe the source of cAMP and other second messengers.
- Adenylyl Cyclase – converts ATPcAMP
o AC – second messenger – released when g protein binds receptor - diacylglycerol, phosphatidyl inositol, Ca2+
o PLC – breaks down phospholipid (phosphatidyl inositol bisphosphate)
DAG and IP3 – second messengers o IP3 – migrates to ER (high calcium content)
causes release of Ca
activates PKC
o DAG – can further bind/activate PKC
Trace the intracellular events leading to activation of nuclear transcription via protein kinase A.
Signal binds GPCR receptor. G subunit activated by GTP G subunit activates phospholipase C PLC cleaves PIP2 to form DAG and IP3 IP3 acts on ER channel to release Ca++ Ca++ and DAG together activate Protein Kinase C Wow.
Know the function of the ryanodine receptor and its association with malignant hyperthermia.
Ryanodine receptors mediate the release of calcium ions from the sarcoplasmic reticulum, an essential step in muscle contraction.
Ryanodine receptors are very close to mitochondria and calcium release from RyR has been shown to regulate ATP production in heart and pancreas cells.
Use Ca++ or IP3 to stimulate Ca release! Woo! Positive feedback!
If it gets too crazy without stopping then you can get malignant hyperthermia, which is bad. This comes about de to a mutation in the recptor along with anesthesia use. Too much ATP is being hydrolyzed and creates heat.
