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Proteasome

Proteasome. WHAT WOULD YOU SEE IF YOU RAN THE THE WHOLE PROTEIN EXTRACT FROM A CELL?. WHAT INFORMATION DOES THIS GIVE YOU?. MUTATIONS

Michelle
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Proteasome

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  1. Proteasome

  2. WHAT WOULD YOU SEE IF YOU RAN THE THE WHOLE PROTEIN EXTRACT FROM A CELL?

  3. WHAT INFORMATION DOES THIS GIVE YOU?

  4. MUTATIONS • Given a eukaryotic gene with one intron. You isolate mutations that change the sequence of the transcript in introns or exons. For each of these mutations, indicate what you expect to see in terms of the levels and sizes of the corresponding mRNAs and the levels and size of the encoded proteins. • - Missense mutation that results in an amino acid change in the protein without affecting the structure of the protein. • Missense mutation that results in an amino acid change in the protein that destabilizes the protein (conformational change). • - Mutation that inserts an earlier stop codon. • - Mutation that eliminates stop codon. • - Mutation that eliminates splicing of intron (there are many outcomes here).

  5. Consequences of protein misfloding-disease -Alzheimer's -Huntigton's -Prion Diseases

  6. Aggregate Fiber PrPc PrPSc

  7. RNA molecules stimulate prion protein conversionNATHAN R. DELEAULT, RALF W. LUCASSEN & SURACHAI SUPATTAPONENature 2003 Oct16 2003 Vol. 425: 717-720 • Prions ProteinaceousInfectious particle • An infectious agent composed of protein and devoid of nucleic acid • Suspected cause of fatal human and animal diseases • Prions are chemically identical to cellular protein (PrPc), but conformationaly different • Different solubility, structure,and stability • Prions are able to convert PrPc to the prion form PrPSc (scrapie)

  8. PrPc = The cellular form of the prion protein PrPSc = The prion (or Scrapie) form of the prion protein + = Pure PrPSc can convert PrPc but it requires 50 fold molar excess PrPSc

  9. PrPc = The cellular form of the prion protein PrPSc = The prion (or Scrapie) form of the prion protein + = + = Cellular cofactors + What is the specific cellular cofactor??

  10. Tissue from PrPSc hamster infected brain (0.1%w/v) + Tissue from uninfected hamster brain (5% w/v) incubate treat with protease (should remove all PrPc) Probe for PrP via western blot starting PrPSc PrPres (protease-resistant PrPSc-like protein) PrPres amplification assay

  11. DRAW EXPECTED BANDS ON THE GEL Tissue from PrPSc hamster infected brain- With Protease Tissue from uninfected hamster brain -With Protease Tissue from PrPSc hamster infected brain - No Protease Tissue from uninfected hamster brain - No Protease

  12. PrPres amplification assay Tissue from PrPSc hamster infected brain (0.1%w/v) + Tissue from uninfected hamster brain (5% w/v) incubate treat with protease (should remove all PrPc) Probe for PrP via western blot starting PrPSc PrPres (protease-resistant PrPSc-like protein) DESCRIBE ALL THE STEPS OF AN EXPERIMENT THAT SHOWS THAT THE COFACTOR IS RNA.

  13. Cellular co-factor appears to be RNA IS RNA NECESSARY OR SUFFICIENT?

  14. It looks like it is ssRNA, not dsRNA, RNA:DNA, DNA, ATP or HSPG

  15. ASSUMING THAT THE CO-FACTOR IS NOT RNA,WHAT WOULD THE AUTHORS SEE IF THEY ADD Rnase THAT HAS SOME PROTEASE IN IT (BAD BATCH OF ENZYME) versus GOOD Rnase (GOOD BATCH)? MAP OUT THE EXPERIMENT IN DETAIL AND SHOW THE GEL WITH BOTH RESULTS. M SC RNase RNase (BAD) HOW CAN YOU MAKE SURE YOUR RNase IS NOT CONTAMINATED WITH PROTEASE? DETAIL THE EXPERIMENT.

  16. Effects are not due to protease contamination in enzyme mixtures

  17. Enzyme activity is required +EDTA +EDTA +DEPC

  18. THE END PRODUCTS OF RNA DIGESTION BY RNase ARE CYCLIC 2', 3'-NUCLEOTIDES. DO YOU THINK THERE IS ANOTHER INTERPRETATION WHY THE RNase IS INHIBITING PrPres FORMATION? DETAIL THE EXPERIMENT. HINT: YOU CAN BUY SOLUTIONS OF CYCLIC 2', 3'-NUCLEOTIDES.

  19. End products of RNA digestion not inhibiting PrPres amplification in RNase treated samples

  20. 1) Do the Results so far show that RNA is necessary or sufficient For the amplification? How can you prove the other (assume you can isolate purified molecules in a test tube.

  21. Total RNA from hamsters can reconstruct amplification in nuclease treated extracts

  22. Cellular cofactor for PrPres amplification: • is not sensitive to RNase V1 (dsRNA) • is not sensitive to RNase H (DNA:RNA) • is not sensitive to DNase • is not sensitive to apyrase (ATP) • is not sensitive to inactive RNase • is not sensitive to byproducts of RNA digestion • is not sensitive to heparinase III • is sensitive to RNase • can be reconstituted in RNase treated samples by the addition of purified total RNA Looks like it might be the RNA

  23. What size RNA is required for this reaction? T: Total RNA R: Retentate RNA (>300 nt) Size Filtration Column F: Filtrate RNA (<300 nt)

  24. Active RNA is >300 nucleotides in length activity remains in retentate

  25. Will just any RNA do? Specific RNA molecules are need for PrPres amplification

  26. To sum up • This does not change the protein only hypothesis • Potentially useful in making new and better diagnostic tools • Suggest mechanism for strain diversity and species barriers

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