1.  Below are the structures of three proteins that are synthesized on the rough endoplasmic reticulum. Illustrate how each would be oriented in the cell membrane if it is a membrane protein. If it is a soluble protein, indicate that by writing "soluble". (12 pts).

See the key in the glass case for these illustrations.

A. NH2__signal sequence-cleavage signal________________COOH
 
 
 

 a soluble protein
 
 
 

B. NH2____________+++signal sequence---________________COOH
 
 

single spanning membrane protein with its NH2 end in the cytoplasm and its COOH end outside the cell
 
 
 

C.  NH2__signal sequence-cleavage signal________stop transfer signal__________COOH

 single pass cell membrane protein with its NH2 end outside the cell and its COOH in the cytoplasm

2. Describe the steps by which a ribosome that has begun translating a protein in the cytoplasm is taken to the endoplasmic reticulum to continue synthesizing the protein there. (8 pts)

After the signal sequence has been translated in the cytoplasm, the signal recognition particle binds to the signal sequence and also to the A site of the ribosome, halting translation. This entire complex moves to the endoplasmic reticulum. A signal recognition particle receptor in the membrane binds to the signal recognition particle, anchoring the entire complex to the membrane. Then a different signal sequence binding protein in the ER membrane binds to the signal sequence. A trans-membrane protein channel forms next to the now anchored signal sequence. The signal recognition particle detaches from the A site of the ribosome and the ribosome attaches to the ER membrane protein and continues translation. The signal recognition particle leaves.
 
 

3.  What is Bip, and how does it function? (6 pts)

 Bip is a protein in the endoplasmic reticulum lumen that binds to proteins being synthesized there as they enter the lumen in an unfolded state. Specifically, it binds to bulky hydrophobic amino acid side chains, preventing them from misfolding and aggregating, thus clogging the transmembrane channel. Bip then hydrolyzes ATP so that it can detach from the protein to allow it now to fold correctly. If the folding is still incorrect, Bip will bind to it again, allowing the protein to have another chance to fold correctly. It will hydrolyze ATP and release the protein again. This cycle repeats until the protein is properly folded. Also, if the protein is to become part of a larger, oligomeric structure, Bip will bind as above until the other subunits are present and ready to interact with the protein. Then it will hydrolyze ATP, release, and allow the oligomeric structure to form. Bip is also called a chaperone protein.
 
 

4.  What roles are played by Rab, syntaxin-like protein, and vamp-like protein in the docking that occurs between a membrane-bound transport vesicle and one of the stacks of the Golgi complex? (9 pts)

 Rab, in its GTP-bound form, is anchored to the cytoplasmic leaflet of the transport vesicle. It lies next to the vamp-like protein, also in the vesicle membrane. As the vesicle approaches the target membrane, the vamp-like protein interacts with the syntaxin-like protein which is in the membrane of the golgi stack (the target). Rab monitors this interaction. If it is correct, meaning that the two membranes are supposed to fuse, Rab hydrolyzes GTP to GDP and changes its shape. Now, it cannot remain anchored to the vesicle membrane and it is released into the cytoplasm. This allows the two membranes to begin the stable fusion process. Rab will be returned to its GTP-bound, membrane-anchored form after binding to a protein that causes it to release GDP so that GTP can replace it.
 

5.  How does a cell increase the levels of calcium in its cytoplasm in response to a signalling molecule? Describe all steps. (8 pts).
 
 The signalling molecule binds to the receptor in the cell membrane. The receptor changes its shape when bound. It now can bind to the Gp protein in the membrane. The Gp protein has two subunits, Gbg and Ga. When the receptor binds, the two subunits separate. The Ga subunit is bound to GTP. It moves over and binds to phospholipase C, activating it. Phospholipase C cleaves PIP2 into InP3 and DAG. DAG remains in the membrane and activates protein kinase C which phosphorylates its substrates. The InP3 moves to the membrane of the endoplasmic reticulum and binds to a calcium channel, opening it. Calcium moves into the cytoplasm, increasing its concentration there. Some cells will also modify InP3 to InP4 which then moves to the cell membrane, binding to calcium channels there and opening them. Calcium then enters the cell and this also contributes to its increased concentration.
 
 

6. A disease has been identified that is caused by excessive amounts of glucose inside a cell. Research has ruled out any problems with cellular respiration in this cell. Therefore, other enzymes that regulate glucose levels inside cells must be the cause. Choose three possible candidate enzymes and explain why mutation within them could possibly cause this increase in glucose. (12 pts)

Possible choices: A.  glycogen synthase is needed to synthesize glycogen. If mutated, glucose not polymerized and its levels stay high. B.  Glycogen phosphorylase is needed to break down glycogen into glucose. It is activated by phosphorylation. If a mutation caused it to become active even if not phosphorylated or to be unable to be dephosphorylated by phosphatase to stop its activity, glycogen would be abnormally broken down and glucose levels would be high. C.  glycogen phosphorylase kinase activtes glycogen phosphorylase when it is itself phosphorylated. If mutated, it could be overactive, whether phosphorylated or not. This would abnormally activate glycogen phosphorylase and therefore abnormally break down glycogen, raising glucose levels. It is also dephosphorylated by phosphatase and therefore, if the mutation prevented this, it could similarly increase glucose level. D.  phosphatase dephosphorylates glycogen synthase, glycogen phosphorylase and glycogen phosphorylase kinase. If it is mutated and could not do this, these three enzymes, as explained above, would cause the glucose levels to remain high. E.  Phosphatase inhibitor binds phosphatase and inactivates it when it is phosphorylated. If it is mutated and binds even when not phosphorylated, it would inhibit phosphatase activity and allow the glucose level to remain high as described above.

 If you chose proteins earlier in the pathway as your answer, for example, cyclic AMP dependent protein kinase, adenylate cyclase, Gs, or the epinephrine receptor, etc., you will also get credit if you accurately described the pathway from them to glucose levels.

7.  The ras protein is a common intracellular mediator of signal transduction events. When mutated, it contributes to malignancy. Describe what you know about how ras protein normally functions and which step in its normal cycling has gone wrong when it gets mutated. (8 pts).

Ras is active when bound to GTP. It can interact then with raf and activates its kinase activity, transducing the signal and eventually activating DNA synthesis genes and causing the cell cycle to progress into S phase. When ras no longer should transduce this signal, it binds to a GAP which activates ras’ s GTPase activity, causing the GTP to be hydrolyzed to GDP. In this form ras is inactive. To reactivate it, it must bind to a GEF which displaces the GDP, allowing GTP to bind and activating ras again. When ras becomes oncogenic, it has sustained a mutation that causes it to be unable to hydrolyze the GTP to GDP. Therefore the signal is constantly transduced and the cell is deregulated and enters mitosis in an uncontrolled fashion.
 

8.  What are SH2 domains and SH3 domains? (8 pts.)
 

SH2 domains are regions of proteins that transduce signals from activated receptor tyrosine kinases. These domains directly bind to phosphorylated tyrosines in the cytoplasmic domains of the active, dimeric receptors. The same protein, or another protein which interacts with it, will contain an SH3 domain. This domain further transduces the signal by binding to and activating another protein. For example, sos is activated by GRB2 (contains both and SH2 and an SH3 domain). Sos is a GEF which can activate ras to its GTP-binding form. Ras then further transduces the signal.
 

9.  How does a receptor tyrosine kinase respond when a signaling molecule binds to it? (5 pts)

The monomeric receptor, when bound by the signalling molecule, dimerizes with its identical partner. This activates the tyrosine kinase activity in the COOH domains of these receptors. The receptors autophosphorylate by attaching phosphates to each others tyrosines in the cytoplasmic domains. These will be recognized by SH2 containing proteins and the signal will be transduced.
 
 

10.  Illustrate an antibody molecule. On this illustration, show the location of the following: all cdr regions; all variable region domains in both the heavy and light chains; and all constant region domains in both the heavy and light chains. (12 pts)
 
 
 See the diagram in the glass cabinet or refer to figure 27-15 in the text.
 
 

 
11. Describe all gene segment rearrangements in the correct sequence that occur in a developing B cell as it rearranges to become a cell that makes a complete antibody molecule. Remember that allelic exclusion is part of this process.  (12 pts)
 

The germ cell differentiates by first recombing a selected D gene segment of the heavy chain locus next to a selected J region. Then a selected V region is recombined next to the DJ. The first allele in the developing B cell that successfully does this and begins to make heavy chain proteins prevents the second allele from recombining. If the first allele is not successful, the second allele recombines. If neither is successful, the B cell no longer differentiates. If the recombination successfully makes a heavy chain protein, the cell is now a pre-B cell. This cell now recombines a V gene segment from the kappa light chain locus next to a selected J gene segment. Again, if this produces a functional light chain, the second allele does not recombine. If not, the second allele recombines. If this recombination is still unsuccessful, the lambda light chain locus recombines one of its alleles. If unsuccessful, the second lambda light chain locus recombines. If still not successful, no further differentiation of the B cell occurs. Once a light chain can be make, the entire antibody molecule can be made. The constant region for the IgM is selected and a membrane-bound form of the IgM is made. The cell is now a virgin B lymphocyte. Shortly after, some IgD will also be made and put into the membrane by having the delta constant region segment transcribed and the u segment removed as an intron. This will also be a membrane-bound form
 
 
 

                                                     Group Work

Define the following:

A.  apoptosis
 

This is programmed cell death, an orderly, genetically directed death of a cell that occurs for the overall good of the organism. For example, it occurs during normal organogenesis and morphogenesis during development, in development of the immune system cells, and as a defense against cells who have damaged DNA that cannot be repaired. The apoptosis in the latter case keeps the cell from going on to divide into two cells containing damaged DNA that can lead to the activation of oncogenes or loss of tumor suppressor proteins, thus leading the cell along the road to malignancy.

B.  oncogene

This is an abnormally regulated normal gene that codes for a protein involved in moving the cell into mitosis. These proteins can be signalling molecules, receptors, intacellular modulators of growth signal pathways, or nuclear transcription factors involved in activating DNA synthesis-related genes and progress through the cell cycle. They can also be positive regulators of cell-cycle progression itself (for example cyclins or cdks). They may be normally silent genes that played a role during development but that are no longer needed. Mutations, insertional mutagenesis, translocations, or gene amplifications are several ways that these proto-oncogenes can become oncogenes. Only one allele needs to be altered for the growth deregulatory effect to occur.

C.  Tumor-suppressor gene

These are genes for proteins that repress movement through the cell cycle or that repair damaged DNA, thus protecting the accumulation of additional mutations. P53, Rb, BRCA1 and BRCA2 are examples. Tumor suppressors are recessive in nature. This means that both alleles of the gene must be mutated or lost for the cell to become deregulated.
 
 

Graders:

Q 1 and 2:  Ya Chen

Q 3 and 4:  Wei Chen

Q 5 and 6:  Rania Al-Shami

Q 7 and 8:  Sheba Argawal

Q 9 and 10: Joy He

Q 11 and Group Work: Dr. Schmieg