Characterizing Interactions of MT3 with Rab3A and with 14-3-3 Zeta to Find Evidence of the Role of MT3 in Alzheimer - PowerPoint PPT Presentation

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Characterizing Interactions of MT3 with Rab3A and with 14-3-3 Zeta to Find Evidence of the Role of MT3 in Alzheimer

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Title: Characterizing Interactions of MT3 with Rab3A and with 14-3-3 Zeta to Find Evidence of the Role of MT3 in Alzheimer


1
Characterizing Interactions of MT3 with Rab3A and
with 14-3-3 Zeta to Find Evidence of the Role of
MT3 in Alzheimers Disease
  • Cyprien Sarteau
  • 2007-2008

2
Armitage Lab University of Minnesota
3
Background Alzheimers Disease
4
Metallothionein-3
  • Yu et al. (2001) showed MT3 levels are reduced
    10-fold in AD brains
  • Uchida et al. (1991) showed MT3 inhibits growth
    promoting effects of AD brain extracts
  • Irie et al. (2001) showed MT3 has specific
    activity with amyloid ?-peptides

5
Rab3A
  • El Ghazi et al. (2006) showed Rab3A is part of
    the a multi-protein complex with MT3
  • Knipp et al. (2005) showed that Rab3A binds with
    MT3

6
14-3-3 zeta
  • El Ghazi et al. (2006) showed that 14-3-3 zeta is
    in a multi-protein complex with MT3
  • Umahara et al. (2004) showed that 14-3-3 zeta is
    localized at neurofibrillary tangles

7
Goals
  • Preparation
  • MT3, Rab3A native forms
  • 14-3-3 zeta tagged version
  • Purification
  • MT3, Rab3A size-exclusion and ion-exchange
    chromatography
  • 14-3-3 zeta protein tagging
  • Isothermal Titration Calorimetry (ITC)

8
Methods
Preparation
Purification
Isothermal Titration Calorimetry (ITC)
9
Purification Size-exclusion Chromatography
Size-exclusion Column
Unpurified Protein
Fractions
10
Size-exclusion Column
Fractions
11
Size-exclusion Column
Fractions
12
Size-exclusion Column
Fractions
13
Size-exclusion Column
Fractions
14
Size-exclusion Column
Fractions
15
Size-exclusion Column
Fractions
16
Size-exclusion Column
Fractions
17
Size-exclusion Column
Fractions
18
Size-exclusion Column
Fractions
19
Size-exclusion Column
Fractions containing separated proteins
20
Purification Ion-exchange Chromatography
Selected Fractions
Ion-exchange Column
Fractions
21
Ion-exchange Column
Fractions
22
Ion-exchange Column
Fractions
23
Ion-exchange Column
Fractions
24
Ion-exchange Column
Fractions
25
Ion-exchange Column
Fractions
26
Ion-exchange Column
Fractions
27
Ion-exchange Column
Fractions
28
Ion-exchange Column
Fractions
29
Ion-exchange Column
Fractions
30
Ion-exchange Column
Fractions
31
Ion-exchange Column
Fractions
32
Ion-exchange Column
Fractions
33
Ion-exchange Column
Fractions
34
Ion-exchange Column
Fractions
35
Ion-exchange Column
Fractions
36
Ion-exchange Column
Fractions
37
Ion-exchange Column
Fractions
38
Ion-exchange Column
Fractions
39
Ion-exchange Column
Fractions
40
Ion-exchange Column
Fractions containing separated proteins
41
Protein Yields
Results
42
Purification of MT3, Cadmium Profile of Fractions
from Size-exclusion Chromatography
43
Purification of MT3, Cadmium Profile of Fractions
from Size-exclusion Chromatography
44
Purification of MT3, Cadmium Profile of Fractions
from Ion-exchange Chromatography
45
Purification of MT3, Cadmium Profile of Fractions
from Ion-exchange Chromatography
46
Purification of MT3, UV Spectra at 254 nm and 280
nm of Fractions from Size-exclusion
Chromatography after DTT Reduction
47
Purification of MT3, UV Spectra at 254 nm and 280
nm of Fractions from Size-exclusion
Chromatography after DTT Reduction
48
SDS-PAGE
Mixtures of proteins
49
SDS-PAGE
50
Purification of Rab3A, SDS-PAGE Gel Displaying
Ion-exchange Chromatography
Key 1 Size markers 2 Empty 3 Pool 1 4
Pool 2 5 Pool 3 6 Pool 4 7 Pool 5 8 Pool
6 9 Pool 7 10 Pool 8 11 Pool 9 12 Pool 10
51
Purification of Rab3A, SDS-PAGE Gel Displaying
Ion-exchange Chromatography
Key 1 Size markers 2 Empty 3 Pool 1 4
Pool 2 5 Pool 3 6 Pool 4 7 Pool 5 8 Pool
6 9 Pool 7 10 Pool 8 11 Pool 9 12 Pool 10
52
Purification of 14-3-3 zeta, SDS-PAGE Gel of GST
Purification Procedures
Key 1 Size markers 2 Supernatant 3 Wash
1 4 Wash 2 5 Wash 3 6 Wash 4 7 Wash 5 8
Wash 6 9 Wash 7 10 Wash 8 11 Elutant 1 12
Elutant 2 13 Elutant 3 14 Elutant 4 15
Elutant 5
53
Purification of 14-3-3 zeta, SDS-PAGE Gel of GST
Purification Procedures
Key 1 Size markers 2 Supernatant 3 Wash
1 4 Wash 2 5 Wash 3 6 Wash 4 7 Wash 5 8
Wash 6 9 Wash 7 10 Wash 8 11 Elutant 1 12
Elutant 2 13 Elutant 3 14 Elutant 4 15
Elutant 5
54
Conclusions
Preparation
  • Successfully prepared MT3 and 14-3-3 zeta
  • Unable to determine success of Rab3A

55
Purification
  • Successfully purified MT3 and 14-3-3 zeta
  • Unsuccessful in the purification of Rab3A

56
Isothermal Titration Calorimetry
  • Unable to attempt ITC with Rab3A
  • Aggregation of 14-3-3 zeta made ITC of MT3 and
    14-3-3 zeta uninterruptible

57
Future Work
  • Purification of Rab3A
  • Specific assay
  • Protein tagging
  • Aggregation of 14-3-3 zeta
  • Adjust pH, temperature, or concentration
  • Surface Plasmon Resonance

58
Acknowledgements
  • Dr. Ian Armitage
  • Dr. Issam El Ghazi
  • Mrs. Lois Fruen
  • Breck Advanced Science Research Team

59
Characterizing Interactions of MT3 with Rab3A and
with 14-3-3 Zeta to Find Evidence of the Role of
MT3 in Alzheimers Disease
Cyprien Sarteau 2007-2008
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