Proteins as an infectious agent

1
(No Transcript)
2
(No Transcript)
3
(No Transcript)
4
(No Transcript)
5
(No Transcript)
6
(No Transcript)
7
(No Transcript)
8
WHAT WOULD YOU SEE IF YOU RAN THE THE WHOLE
PROTEIN EXTRACT FROM A CELL?
9
WHAT INFORMATION DOES THIS GIVE YOU?
10

• 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).

11
(No Transcript)
12
(No Transcript)
13
(No Transcript)
14
RNA molecules stimulate prion protein
conversionNATHAN R. DELEAULT, RALF W. LUCASSEN
SURACHAI SUPATTAPONENature 2003 Oct16 2003 Vol.
425 717-720

• Prions Proteinaceous Infectious 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)
  

15
PrPc The cellular form of the prion protein
PrPSc The prion (or Scrapie) form of the prio
n protein



Pure PrPSc can convert PrPc but it requires 50
fold molar excess PrPSc
16
PrPc The cellular form of the prion protein
PrPSc The prion (or Scrapie) form of the prio
n protein





Cellular cofactors

What is the specific cellular cofactor??
17
PrPres amplification assay
18
(No Transcript)
19
DESCRIBE ALL THE STEPS OF AN EXPERIMENT THAT SHO
WS THAT THE COFACTOR
IS RNA.
20
IS RNA NECESSARY OR SUFFICIENT?
21
It looks like it is ssRNA, not dsRNA, RNADNA, D
NA, ATP or HSPG
22
HOW CAN YOU MAKE SURE YOUR RNase
IS NOT CONTAMINATED WITH PROTEASE?
DETAIL THE EXPERIMENT.
23
Effects are not due to protease contamination
in enzyme mixtures
24
Enzyme activity is required
EDTA
EDTA
DEPC
25
THE END PRODUCTS OF RNA DIGESTION BY RNase ARE
CYCLIC 2', 3'-NUCLEOTIDES. DO YOU THINK THERE
IS ANOTHER INTERPRETATION WHY THE RNase IS INHIBI
TING PrPres FORMATION? DETAIL THE EXPERIMENT. H
INT YOU CAN BUY SOLUTIONS OF CYCLIC 2',
3'-NUCLEOTIDES.
26
End products of RNA digestion not inhibiting
PrPres amplification in RNase treated samples
27

• 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.

28
Total RNA from hamsters can reconstruct
amplification
in nuclease treated extracts
29
Cellular cofactor for PrPres amplification

• is not sensitive to RNase V1 (dsRNA)
• is not sensitive to RNase H (DNARNA)
• 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
30
(No Transcript)
31
(No Transcript)
32
Will just any RNA do?
Specific RNA molecules are need for PrPres
amplification
33
(No Transcript)
34
(No Transcript)