SESSION 11 Flashcards
Outline the mechanisms involved in targeting proteins to nuclear targeting
The nucleus contains many proteins but none of them are actually synthesised there; they are made on ribosomes in the cytoplasm and must be transported into the nucleus
Proteins destined for the nucleus can pass through nuclear pores, protein channels that span the nuclear membrane
Proteins that need to enter the nucleus contain a nuclear localisation sequence (NLS). This is usually a run of 4-8 basic amino acids which is contained somewhere within the primary sequence of the protein
Import of nuclear proteins involves a number of proteins that can shuttle between the cytosol and nucleus, with the aid of the protein Importin:
1) a fully folded protein with a NLS is bound by Importin a and B in the cytosol
2) the resulting complex binds to the nuclear pore and translocates into the nucleus in an energy dependent mechanism
3) once inside the nucleus the nuclear protein is released and the Importins bind to a small GTPase protein known as Ran
4) Importins are exported from the nucleus and can be recycled to transport more nuclear proteins
5) Ran is transported back to the nucleus following hydrolysis of GTP
Outline the mechanisms involved in targeting proteins to mitochondria targeting (matrix proteins)
Mitochondrial proteins synthesised in the cytosol are transported into mitochondria in an unfolded state
Proteins destined for the mitochondrial matrix contain an amphipathethic N- terminal signal sequence of 10-80 amino acids
These unfolded proteins are stabilised in the cytosol by interaction with molecular chaperones such as Mitochondrial Import Stimulation Factor (MSF)
The signal sequence is recognised by proteins in the mitochondrial outer membrane that forms protein import channel; these are now as TOM proteins (Translocase of the Outer Membrane)
Proteins destined for the matrix are transported across the inner mitochondrial membrane by TIM proteins (Translocase of the Inner Membrane). This process requires energy in the form of ATP and a membrane potential (DY)
Proteins of the inner mitochondrial membrane also contain additional signals that stop them entering the matrix
Once across the inner mitochondrial membrane the N- terminal signal sequence is removed by mitochondrial processing peptidase (MPP). The protein folds into its naive conformation in an ATP- dependent process helped by the chaperones such as mitochondrial Hsp70.
Outline the mechanisms involved in targeting proteins to lysosomal targeting
Lysosomal hydrolysed are targeted to lysosomes by the addition of a mannose- 6- phosphate (M6P) signal to N-linked oligosaccharides on the protein
This reaction occurs in the golgi and involves 2 enzymes:
- N- actylglucosamine phosphotransferase
- N- acetylglucosmine phosphoglycosidase
Proteins destined for lysosomes are targeted for the addition of M6P groups by the presence of a signal patch, a sequence of several amino acids from different parts of the amino acid sequence
M6P groups on the targeted proteins are recognised by M6P receptors at the trans Golgi and vesicles are pinched ff for transport to the lysosomes.
Once at the lysosome, he acid pH causes dissociation of the protein and receptor. The receptor recycles back to the Golgi for further use. The phosphate group is removed from the M6P group on the protein to ensure that the protein does not return to the Golgi with the receptor
Describe I- cell disease
Genetic defects in the N- acetylglucosamine phosphotransferase enzyme result in a lack of M6P addition to lysosomal targeted proteins
In I- cell disease this results in lysosomal hydrolysed being mistargeted for secretion and results in large amounts of these proteins present in the blood and urine
Outline the mechanisms involved in targeting proteins to retention of proteins within the ER
Proteins such as protein disulphide isomerase and signal peptidase that are resident within the lumen of the ER can sometimes be lost when vesicles are pinched off and transported to the Golgi. There is a selective method that allows ER- resident proteins that have escaped to the Golgi to be retrieved
ER resident proteins have the sequence Lys- Asp- Glu- Leu (KDEL) near the C- terminus
If these proteins are transported to the Golgi they will interact with KDEL receptors in the Golgi, a process that is enhanced by the low pH there
ER proteins bound to the receptor are returned to the ER in transport vesicles. ER resident proteins dissociate from the receptor in the neutral condition within the ER and the KDEL receptor is transported back to the Golgi.
Describe the structure and mode of action of selected antibiotics and growth inhibitors
Target: Bacterial cell wall
Drug: penicillin
Mode of action: inhibits transpeptidase that forms cross- links in cell wall. Osmotic pressure causes cell lysis
Target: Bacterial transcription
Drug: Rifampicin
Mode of action: binds to bacterial RNA polymerase preventing transcription
Target: Bacterial protein synthesis
Drug: tetracycline
Mode of action: competes with tRNA at A site of bacterial ribosome
Target: Anti- Folates in cancer therapy
Drug: methotrexate
Mode of action: impairs the synthesis of tetrahydrofuran evidence, which is essential for DNA synthesis, from folic acid. Competitively inhibits dihydrofolate reductive (DHFR)
Provide an overview of the general mechanisms by which cells can become resistance to an antibiotic or drug
High rate of division
High rate of division in both bacteria and cancer cells means that there is a higher rate of mutation
Positive mutations, such as drug resistance will be positively selected for and breed a population of drug resistant cells
Decreased influx
For drugs, which require to be taken up by their target cells to take effect (Rifampicin, Tetracycline, Methotrexate), influx can be reduced
Cells achieve this by expressing a reduced amount or altered version that reduces affinity of the carrier protein that allows the drug tough the cell membrane
Increased efflux
P- Glycoprotein, our multi- drug resistance protein 1 (MDR1) is a protein similar in structure to CFTR that is responsible for the efflux of Toxic products from a cell
In many cancers, expression of P- Glycoprotein is up- regulated. This allows the cells to increase the efflux of chemotherapy drugs, e.g. Methotrexate
Increased transcription of target
If the drug targets a specific product of transcription, e.g. A ribosome or enzyme, the cell can acquire resistance by increasing the transcription of the target to overwhelm the drug
Altered target
Specific target of drug acquired a mutation, lowering the affinity of the drug for it
