Membrane fusion, transport, Receptor Tyrosine Kinase

Question 1

Understand the importance of sub-cellular protein targeting.

Answer 1

A cell has a lot of compartments through which it needs to move big proteins without compromising the membranes’ integrity - thus transport vesicles made from the membrane of the organelle of origin are necessary.This (vesicular transport) is the basic principle of transporting substances from one intracellular compartment to another and specificity of these vehicles to their targets is critical for normal function.Incorrect transport frequently results in disease.

Question 2

What are the basic principles of membrane and viral fusion?

Answer 2

Membranes don’t automatically merge when they get close together.An example with two membranes: initially they are coated with water molecules. First you have to remove the water before you can fuse the lipids; then you need to overcome charge repulsion between lipids in order to allow a greater efficiency of merging. Also need to make sure these specific lipids should be merging in the first place (specificity of membrane fusion).

Question 3

What are the three types of SNARE proteins? Their shapes?

Answer 3

VAMPs: transmembrane domain at one end, with a helical domain in it → located on transport vesicle.Syntaxin: transmembrane domain, still a helical domain ➝ located on target membrane.SNAP25: no transmembrane domain, two helical domains, fatty acid binding region that more or less acts as a membrane-binding domain ➝ located on target membrane.Note that there are different flavors of each protein, each of which can only bind to certain flavors of their counterparts.

Question 4

How do SNARE proteins work?

Answer 4

The helical domains of all three proteins serve to bind with each other; they form an aligned bundle or tetramer.All vesicles contain VAMPs. All target membranes contain syntaxin and SNAP25.When the vesicle nears the target membrane, the helical domains on the vesicle bind to those on the target membrane. This action effectively presses the two lipid layers together, squeezes out water and overcomes charge repulsion. It also promotes lipid fusion.To do this, the helical binding needs to be extremely strong, and so it is.

Question 5

How is SNARE based fusion regulated?

Answer 5

Enzyme that regulates the dissociation of SNARE proteins: NSF protein. Forms a kind of turning barrel around the SNARE complex and twists it apart with the help of a lot of ATP.After being unwound, syntaxin becomes unfolded; need another protein, Sec1, to refold it properly.SNARE cycle: VAMP on vesicle, syntaxin and SNAP25 on target membrane, helical binding and lipid fusion, NSF-mediated unwinding, refolding of syntaxin with Sec1.

Question 6

What is the mechanism of viral fusion and how is it regulated?

Answer 6

The class of so called enveloped viruses (includes HIV, ebola, and influenza) also needs to go through membrane fusion in order to infect cells.Viruses have developed a fusion machinery that is remarkably similar to SNARE fusion. This machinery also achieves both objectives of fusion, ensures that it is efficient and specific.Virus fusion is generally achieved with the help of a specialized viral fusion protein. This protein contains transmembrane domain at the one end (inserted into viral membrane) and highly hydrophobic fusion domain. Normally, fusion domain is folded and hidden within viral protein. Upon receiving specific signal (e.g. influenza = change in pH), the fusion domain is exposed and inserted into target cell membrane. That is immediately followed by refolding of fusion protein that causes the fusion of viral and cellular membranes.

Question 7

Know the difference between primary and secondary active transport.

Answer 7

Primary active transporters use ATP to do work.Secondary active transporters use Na+ leak into the cell to do work (this includes cotransporters and antiport/exchange ports).The main primary active transporters are Na+/K, Ca++, and H+ on cell membranes.There are also some within the organelles in cells.Secondary active transport is much more prevalent.

Question 8

Define cotransport and exchange transport.

Answer 8

There are two basic types of secondary active transporters, those that move different solute species in the same direction (cotransport), and those that move solute in opposite directions (antiport, or exchange).

Question 9

Be able to determine which direction a non-electrogenic secondary active transporter will operate, given the equilibrium potentials of the participating ions and the membrane potential.

Answer 9

Electrogenic: one cycle produces a net charge transfer across the membrane (e.g. H+).Thus non-electrogenic are transporters that don’t change the net charge across the membrane. Therefore, they tend to equalize ion concentration (e.g. glucose).

Question 10

How can electrogenic secondary active transporters change based on membrane potential?

Answer 10

They can reverse direction!

Question 11

Understand the non-existence, but conceptually useful idea, of the H+/K+ exchanger.

Answer 11

There are several clinical situations that suggest the presence of a system that will exchange K+ for H+, and vice versa. While it is conceptually simple (and useful) to think in terms of an H+/K+ exchanger, the reality is that such a transporter probably does not exist.Rather, the process evidently involves different transporters, perhaps working in pairs in parallel, the upshot being hydrogen/potassium exchange.

Question 12

Know how cells can concentrate glucose inside, even though the glucose transporter cannot pump glucose against its concentration gradient

Answer 12

Inside cell, glucose gets phosphorylated, which makes it unable to fit into the glucose transporter - thus glucose flow is more or less unidirectional into the cell.Note that glucose transporters are normally sequestered within vesicles in the cell until insulin signals the cell to make those vesicles merge with the cell surface and transport glucose into the cell.

Question 13

Concerning hyperkalemia: know two mechanisms by which cells can be encouraged to take up potassium from the ECF.

Answer 13

One can encourage cells to take up the potassium from the ECF by giving glucose and insulin.Second way is administering bicarbonate.

Question 14

Describe mechanism of receptor tyrosine kinase activation.

Answer 14

Ligand binding drives dimerization, which activates catalytic activity of the kinase resulting in Tyrosine autophosphorylation at specific sites.

Question 15

Explain molecular mechanism of stimulation of ras GTPase by receptor tyrosine kinases.

Answer 15

Tyrosine Phosphorylation of receptor causes SH2 domain (on Grb2) to bind the kinase. The SH3 domain (also on Grb2) binds the Sos protein. Sos is now local to Ras which allows easy activation.

Question 16

Describe mechanism of action of two main classes of RTK-targeted anti-cancer agents (antibodies and TKI’s)

Answer 16

Primary role of antibodies is to block ligand binding to the receptor (Cetuximab). This prevents ligand dependent activation of the receptor. This occurs extracellularly.TKIs inhibit catalytic activity (usually) by binding in substrate-binding site of the kinase (Gefitinib). These are small molecules that can get into cells.

Question 17

List tumor cell characteristics that predict clinical response to EGFR-targeted therapeutics (Gefinitib).

Answer 17

Positive response to EGFR TKI correlated with receptor mutations that may “activate” the receptor, EGFR amplification or overexpression as determined by FISH or immunohistochemistry.

Question 18

Describe mechanism of resistance to TKI’s.

Answer 18

Acquired resistance- second site mutations in EGFR arising or selected in patients who initially benefit from therapy but then acquire resistance and disease progression. These mutations block inhibitor binding to the kinase active site. A perfect example of Darwinian natural selection. May be able to design new inhibitors to avoid this problem. Activation of other receptors like Met or ErbB2. Combine inhibitors or make dual specificity inhibitors? Primary resistance- if the tumor has a Ras mutation inhibiting the receptor further up the pathway will not be effective.