Signal transduction & biochemical defence

Question 1

Give three examples of stimuli that would trigger a response.

Answer 1

• Light
• mechanical touch
• pathogens
• Tastants
• Neurotransmitters
• Nutrients
• Odorants

Question 2

What are the eight concepts/features of signal transducing systems? Explain them in short.

Answer 2

a) Specificity: That the receptor and signaling molecule fits well together (many small non-covalent interactions) while other signaling molecules don’t.

b) Sensitivity: That the signaling molecule have a high affinity to the receptor, favoring binding.

c) Amplification: That the activation of one enzyme can activate more to make the response bigger, fast, in an enzymatic cascade.

d) Modularity: That the receptors can be assembled differently to convey different signals/responses. Modifications like phosphorylation provides reversibility of assembly.

e) Desensitization/adaptation: Receptor activation triggers feedback inhibition that turns off the receptor activity or removes it, to limit response and make the cell sensitive to size of stimuli.

f) Integration: Multiple signals are summed so that the resulting response comes from the integrated input from both signals.

g) Divergence: One signal can convey more than one response, eg to activate one process and inhibit another.

h) Localized response: When the receptor is clustered with the enzyme that shuts of the signal, the signal is confined locally and doesn’t spread.

Question 3

Name the four receptor types present in multicellular organisms.

Answer 3

• Gated Ion channels: Opens or closes in response to concentration of small ligand or membrane potential change.
• GPCRs (G-protein coupled receptors): External ligand binding activates an internal G-protein that in turn activates an enzyme generates a second messenger which conveys signal.
• Receptor tyrosine kinases: external ligand binding activates internal tyrosine kinase activity by autophosphorylation.
• Nuclear receptors. Intracellular receptor that act in the nucleus as transcription factors to regulate gene expression upon binding of hormones.

Question 4

How does gated ion channels work in short?

Answer 4

• Gated ion channels are found in excitable cells, like nerve cells, muscle cells and hormone releasing cells.
• They provide a regulated flow path for ions in response to stimuli, most common; Na+, K+, Ca2+ and Cl-.
• Those regulated by changed membrane potential are maintained by pumps that keep the membrane potential up, so that a signal can be relayed.

Question 5

Explain how gated ion channels work with a real life example.

Answer 5

A good example of gated ion channels are those in nerve cells, both in axon and synaps.

The action potential needed to relay the signal to the next neuron is dependent on voltage gated Na+ channels. They are closed when membrane potential is at resting potential (maintained by ion pumps) and opened when the membrane is depolarized. With the opening of a Na+ channel, the membrane in it’s close vicinity is opened, and the next is open, this moves the action potential through the axon to the synapse.

• In the synapse, there are voltage gated Ca2+ channels, and when the membrane is depolarized at the synapse, these open and results in a big influx of Ca2+.
• The high concentrations of Ca2+ triggers the fusing of NT filled vesicles to the synaptic membrane, releasing the NTs (ACh in this example) in the synaptic cleft. There, the ACh bind to ligand gated Na+ channels which open and an influx of Na+ cause a new action potential in the next neuron and signal is relayed.

Ca2+ usually have this role in other vesicle systems too!

Question 6

How does the binding of ACh affect the ligand gated ion channel?

Answer 6

When Ach binds, the alpha helices that form the channel change conformation, so that other residues (hydrophilic) are pointed inwards, which can let through specific ions.

Question 7

Explain how GPCRs work with a real life example.

Answer 7

A classic example of a GPCR signal relay system is beta-adrenergic receptors (epinephrine binding). Epinephrine (adrenaline) binds to the GCPR, and the binding causes a conformational change in the ligand binding part causes a change in the G-protein, that lowers its affinity to GDP and increases its affinity to GTP. When GTP binds, the alpha subunit of the G-protein disassociates and activates adenylyl cyclase, which in turn catalyzes the formation of cAMP (2nd messenger) that activates PKA that phosphorylates enzymes (amplification) that for example mobilize energy metabolism (FA synthesis and glycolysis for example) and shuts down energy requiring processes, basically causes the whole response to adrenaline.

Question 8

How is the activated G-protein inactivated?

Answer 8

The activated G-protein is inactivated by intrinsic GTPase activity, which reforms GDP and cause it to reassociate with the GPCR.

Question 9

Explain how receptor tyrosine kinases work with a real life example.

Answer 9

An example of receptor tyrosine kinases is the insulin receptor. When insulin binds to the external portion, the internal part of the receptor dimerizes and undergo autophosphorylation on its c-terminal tyr residues, forming a complex with kinase activity. The kinase phosphorylates IRS-1 (intracellular protein) which forms a complex that move on to bind to Ras. The binding to Ras causes a conformational change so that it has higher affinity to GTP than GDP, and is activated. Activated Ras binds and activated Raf-1, which phosphorylated MAK, which in turn phosphorylates ERK that moves into the nucleus and phosphorylates TFs that stimulate transcription of cell division genes.

Question 10

How exactly does the autophosphorylation of the tyr residues on the receptor tyrosine kinase activate it?

Answer 10

The conformational changes brought on by the phosphorylation of the three tyrosine’s in each part moves an Asp that blocks the active site away, unblocking it so the the target substrate can bind.

Question 11

Explain how nuclear receptors work in detail.

Answer 11

1. A hormone bound to a serum binding protein is released in the target tissue, and the small and non-polar hormone diffuses through the outer membrane into the nucleus.
2. In the nucleus, it binds to a nuclear receptor, which changes the conformation of the receptor so that it forms dimers with other hormone-receptor complexes which binds to HREs (Hormone responsive elements) in the DNA adjacent to specific genes.
3. The receptor-hormone complexes attracts coactivator/corepressor proteins and with them, regulates transcription of the adjacent gene(s), increasing or decreasing the rate of mRNA formation.
4. The altered levels of the gene produce a cellular response to the hormone. (a lot slower process than the membrane bound receptors).

Question 12

Recognition – vs binding affinity. Are they related, interdependent? What happens if signals…
- …are interpreted wrongly?
- …trigger unjustified response?

Answer 12

It is important for receptors to have high specificity, but if the binding affinity is too high, that would lead to the triggering of bigger responses than needed. So a balance is needed!

Question 13

What are hormones?

Answer 13

Hormones are chemical messengers that activate cellular responses in other tissues than where they’re synthesized. They are a diverse group of molecules including peptides, amines, eicosanoids etc. Signaling can be either via nuclear receptors (small non polar hormones) or membrane bound receptors + 2nd messenger.

Question 14

Give one example of a steroid hormone and one peptide hormone.

Answer 14

Sterioid: Estradiol, testosterone
Peptide: insulin, glukagon, some NTs.

Question 15

Peptide hormones often go through post-translational modifications to get to their active form. Name and explain the process of one.

Answer 15

Insulin is modified post translationally. It is translated as preproinsulin, a signal sequence is cleaved away to form proinsulin and then the C-peptide is removed to form the tertiary structure of insulin.

Question 16

Hormones are excreted from endocrine organs, name three and what hormone they excrete.

Answer 16

• The pancreas release glucagon and insulin.
• the testis and ovaries release testosterone and estrogen.
• The adrenal glands release epinephrine.

Question 16

Hormones have a hierarchy, what does this mean?

Answer 16

Hormonal signaling is dependent and regulated by other hormonal signaling. So the stimuli that the hormonal signaling is the response to comes into the central nervous system, which in turn triggers the release of hormones from the hypothalamus –> pituitary and from there the hormones stimulates a range of hormones depending on the stimuli.

Question 17

Some metabolism is tissue specific, name three tissues that are central in metabolism of proteins, lipids and carbohydrates.

Answer 17

• The liver is the most important metabolically active tissue. The portal vein is a direct route from the digestive organs to the liver, and the liver therefore has first access to ingested nutrients. The liver is responsible for urea excretion and recycling of amino acids AND glucose and lipid synthesis. All these processes produce base materials for synthesis of nucleotides and other important compounds too. The liver has amazing metabolic plasticity, with an enzyme turnover rate 5-10 times faster than other tissues.
• Adipose tissue stores, mobilizes and synthesizes lipids.
• The pancreas release glucagon and insulin which is central in glucose homeostasis.

Question 18

Explain the Cori cycle in short.

Answer 18

The Cori cycle is the pathway for extra energy supply in muscle. At rapid contractions in muscle, the muscle start to break down glycogen to produce ATP anaerobically, forming lactate in the process. The lactate is then transported via the blood as blood lactate to the liver, where the lactate is converted to glucose via gluconeogenesis. The glucose is then transported to the muscles again to reform the glycogen used up for rapid contractions.

Question 19

Explain the metabolic pathway for glucose from ingestion, through liver out in the body.

Answer 19

When carbohydrates are broken down in the GI system, the monosaccharides are taken up in the epithelial cells of the intestine and transported via the portal vein to the liver. In the liver, the glucose is quickly phosphorylated by hexokinase IV into Glucose-6-phosphate. From there, depending on the energy state:
1. If glucose is scarce, dephosphorylation happens to yield free glucose that can diffuse out into the blood.
2. Glucose 6-phosphate not immediately needed to form blood glucose is converted to liver glycogen, or has one of several
other fates:
3. Go through glycolysis and PDHc to form Acetyl-CoA that go into the CA cycle + ETC to provide energy to the hepatocytes. (Normally, however, fatty acids are the preferred fuel for ATP production in hepatocytes, as other tissues have high glucose needs)
4. Acetyl-CoA can also serve as the precursor of fatty acids, which are incorporated into TAGs and phospholipids, and of cholesterol. Much of the lipid synthesized in the liver is
transported to other tissues by blood lipoproteins.
5. Go through the PPP (pentose-phosphate pathway) and form Ribose-5-phosphate used in nucleotide synthesis.

Question 20

What is the normal range of blood glucose concentration (in mg/100mL)?

Answer 20

90-60 mg/mL is considered normal, below that we get subtle neurological signs of energy depletion like hunger, trembling, sweating and this is where glucagon, epinephrine and cortisol are released. Below 40 is critical, and lead to lethargy/coma and prolonged time below can lead to brain damage or death.

The rather small differences between these states is the reason for very tight regulation to maintain glucose homeostasis, mainly by the hormones insulin and glucagon.

Question 21

In what organ and what cells is glucagon and insulin synthesized?

Answer 21

Both insulin and glucagon are produced in the pancreas:
- β cells produce insulin
- α cells produce glucagon
Both of these cell types exits as islets - small “colonies” of the same cell type in a cluster.

Question 22

How is insulin secreted?

Answer 22

When the blood glucose levels are high, a lot of glucose is taken up by the pancreatic β cells, in there, its phosphorylated to glucose-6-phosphate which goes through glycolysis + Ca + oxidative phosphorylation which higher the ATP levels in the cell. The high levels of ATP inhibits ATP gated K+ channels in the outer membrane, which causes depolarization of the membrane. The depolarization causes voltage gated Ca2+ channels to open and the influx triggers fusion of ER vesicles filled with insulin to fuse with the outer membrane and release the insulin out in the blood to different targets, promoting synthesis and storage of energy rich molecules and downregulation of their breakdown.

Glucagon basically have the reverse effect.

Question 23

What happens in the liver at a well fed state?

Answer 23

During a well fed state, the metabolism is focused on energy storage. Insulin release stimulates several metabolic processes in the liver:
- Glucose uptake
- Glycogen synthesis
- Glycolysis -> Acetyl-CoA -> Fatty acid synthesis (a little for fuel)
- TAG (triacylglycerol) synthesis and storage in adipose tissue.

All of this contributes to lowering the blood glucose levels which eventually stops insulin release (so kind of feedback regulation)

Question 24

What happens in the liver at a low blood glucose state?

Answer 24

During a low glucose state, glucagon release counteracts the low glucose levels by promoting glucose (or other energy compound) formation and inhibits insulin release. Glycogen binds to GPCR –> cAMP –> PKA
- dephosphorylation of glucose-6-phosphate to get free glucose out in the blood
- Upregulation of Gluconeogenesis, both from glycerol, OAA and amino acids.
- Fatty acid degradation to Acetyl-CoA –> ketone bodies.
- Breakdown of liver glucagon to G6P to free glucose.

Question 25

What happens in the liver at a fasting/starvation blood glucose state?

Answer 25

During a fasting/starvation state all available resources go primarily to the brain, glucose and ketone bodies to complement.
- The depletion of OAA causes Acetyl-CoA to accumulate, which stimulates ketone body formation and inhibits PDHc.
- amino acid breakdown to synthesize CA cycle components.
- During prolonged fasting protein is broken down to form pyruvate to use in gluconeogenesis. (not sustainable of course, as you need muscle to eat/fight or flight.

Question 26

Epinephrine release regulates insulin and glucagon, how?

Answer 26

Epinephrine release upregulates glucagon and inhibits insulin, to mobilize as much energy as possible to avoid the danger.

The effects of epinephrine is very similar to that of glucagon, but specialized for fight or flight, like upregulating glycolysis in muscle.

Question 27

The immune system consists of two molecular defense systems, which and which one produces antibodies?

Answer 27

The immune system is comprised of the:
- Humoral defense system: B-lymphocytes which produce antibodies, and the
- Cellular defense system: Macrophages and T-lymphocytes; T-killer cells that patrol the system and T-helper cells that release cytokines.

Question 28

What are antigens and how are they recognized by antibodies?

Answer 28

Antibodies (Ab) bind to antigens (Ag), which are anything “non-self”. Large antigens display several recognition sites for antibodies, called epitopes. Epitopes can be a range of things, like amino acids or carbohydrates (most common) or other molecules. Even small antigens have many epitopes.

Antibodies are bivalent, meaning they have two identical binding sites for the epitope, which allows for chain formation/aggregation of the antigens that is good targets for macrophages.

Question 29

The immune system remember an an antigen, which is the basis for vaccines, how?

Answer 29

Each B-lymphocyte encodes for a unique antigen. When a B-lymphocyte encounters an antigen, a clonal expansion occurs. The bulk of the clones differentiate into plasma cells that mass produce the antibody and release it into the circulation to tackle the antigen, but some differentiate into memory cells that persist even after the antigen has disappeared. The next time the same antigen is encountered, these memory cells are activated and the response is much faster!

Question 30

Explain the structure of an antibody.

Answer 30

Antibodies have a constant region (the bulk of the Y structure) which is the same for all antibodies and a variable region, which is different in all antibodies and bind to epitopes (The tips of the Y), with two light chains and two heavy chains - tetramer connected by disulfide bridges. The bottom of the Y structure is also called the FC region, ant this part interacts with surface receptors of immune cells, like macrophages.

Question 31

There are five major classes of antibodies, which?

Answer 31

• IgM: The first contact Ab, produced in early stages of exposure to Ag. Pentamer of the basic Ig unit with 10 binding sites. It’s very large so it can’t penetrate blood vessels.
• IgG: The most common/abundant Ab and the mainly produced one in response to repeated exposure. Monomer with two binding sites. Can be membrane anchored to for example memory cells or free in the blood.
• IgA: First line of defense, monomer or dimer, found in body secretions like saliva and breast milk.
• IgD: The mysterious one, monomer and surface receptor on B-cells.
• IgE: The problematic one, monomer and involved in allergic reactions. The constant region binds to mast cells which produce histamine.

Question 32

IgG can’t be expressed in E. Coli, still there’s a way to use E. coli as model in antibody studies, how?

Answer 32

Abs can be digested, cleaving the disulfide bond between the heavy and light chain. This produces a Fab fragment that can be expressed in E. Coli.

Question 33

Antibodies are a good example of induced fit, what is that?

Answer 33

When the variable region binds to an epitope, the conformation of the antibody changes so that the binding gets stronger.

Question 34

How do you produce polyclonal antibodies?

Answer 34

Expose an animal to the antigen and it will produce antibodies for it.

Question 35

How do you produce monoclonal antibodies?

Answer 35

After exposure to an antigen you have many different antibodies (polyclonal response) but for analytical purposes you might want a specific one in large amounts, and here’s how:

1) Immunize mouse with antigen

2) Repeat immunization (‘boosting’) => primarily IgG is produced + somatic mutations (‘mature’ antibodies)

3) Sacrifice mouse → extract spleen → isolate spleen cells (rich in B-lymphocytes)

4) Fuse B-ly with mouse myeloma cells => hybrid cell (‘hybridoma’)
- Characteristics: Produces Ab (from spleen cell genes), immortalized (from myeloma cell genes)

5) Screen hybridoma cells for excretion of desired antibodies

6) Grow to large(r) quantities

7) Purify Ab and boom you got it.

Question 36

How is the huge diversity of the variable region of antibodies acheived?

Answer 36

• Variability in the LC: The Ig-gene consists of genes for the variable region, the J segments (“joining”) and the C segment. There are about 300 V segments, 4 J segments and one C segments. These sites are very prone to recombination in B-lymphosites, so during maturation, recombination in these genes result in deletion of DNA between random V and J segment creates the mature light chain gene, and after transcription, any remaining J segments between the light chain gene and the C gene is spliced away so that the processed mRNA only have one of each, that is then translated into the protein. This results in extremely high diversity (~1200 diff combinations) just by the different combinations in each b-lymphocyte.
• Variability in the HC: The rearrangement of the heavy chain similar, but with a D fragment instead, which can produce a large number of different LC. The combination of any of these together with the already diverse light chain results in even higher diversity.
• Somatic mutations: Somatic mutations can occur after differentiation, which further increases diversity! in total about 10^7-10^12 different combinations in humans.

Question 37

The detoxification systems in the body consists of phase I and II enzymes, what kind of reactions do they catalyze?

Answer 37

Phase I enzymes catalyze insertion of oxygen into non-polar compounds, which facilitates excretion by making the compounds become (more) water soluble.
- Oxidation (eg by cytochrome P450)
- Hydrolysis (eg by epoxide hydrolase)

Phase II enzymes: catalyze conjugation reactions, addition of (polar) group to compounds to facilitate export and excretion, or make the products less reactive.

Question 38

Explain the term “bioactivation”.

Answer 38

Bioactivation is “the other side of the coin” of well meant phase I reactions. Basically bioactivation causes an unreactive compound to get toxic/reactive. Some compounds are harmless in their natural form, like aflatoxin, but when Cyt P450 adds an oxygen to the C=C bond it is converted to an epoxide that is higly reactive towards DNA. This is the main cause of liver cancer.

These detoxification reaction must always be taken into account when designing drugs, as the drugs can be converted into other (potentially harmful) products otherwise.

Question 39

Name one Phase II reaction.

Answer 39

Conjugation reactions catalyzed by transferases:

• Glutathione conjugation: Catalyzed by glutathione transferases, major players, they make up ~10% of all liver proteins and functions as protection against ROS (radical quencher). Once the conjugate is done, it acts as a flag and is actively transported out of the cell to be excreted.
• Sulfation: addition of sulforyl (SO3) catalyzed by sulfotransferases.
• Acetylation
• Glucuronidation

Question 40

Give an example on a phase I reaction that can lead to cancer.

Answer 40

When smoking cigarettes, one of the inhaled substances, benzo(a)pyrene is included. This is an unreactive molecule, but when cytochrome P450 enzymes encounter it, they insert oxygen through oxidation of carbon double bonds. This leads to the formation of an epoxide, that is reduced by epoxide hydrolases, which are then further oxidized by Cyt P450 to an epoxide. Some of the resulting products from these reactions can be highly reactive, and can disrupt/break DNA which can cause mutations, that can potentially lead to cancer.