evidence for weakened activity of mycobacterial chaperonins
DESCRIPTION
Evidence for Weakened Activity of Mycobacterial Chaperonins. C. M. Santosh Kumar, Garima Khare , C. V. Srikanth , Anil K Tyagi , Abhijit A. Sardesai and Shekhar C. Mande. Activity Restoration of a Naturally Inactive Chaperonin via Facilitated Oligomerization. - PowerPoint PPT PresentationTRANSCRIPT
![Page 1: Evidence for Weakened Activity of Mycobacterial Chaperonins](https://reader035.vdocuments.us/reader035/viewer/2022062501/568168f2550346895ddff8af/html5/thumbnails/1.jpg)
Evidence for Weakened Activity of Mycobacterial Chaperonins
Activity Restoration of a Naturally Inactive Chaperonin via Facilitated Oligomerization
C. M. Santosh Kumar, Garima Khare, C. V. Srikanth, Anil K Tyagi, Abhijit A. Sardesai and Shekhar C. Mande
Centre for DNA Fingerprinting and Diagnostics, Bldg. 7, Gruhakalpa, 5-4-399 / B,Nampally, Hyderabad - 500 001
![Page 2: Evidence for Weakened Activity of Mycobacterial Chaperonins](https://reader035.vdocuments.us/reader035/viewer/2022062501/568168f2550346895ddff8af/html5/thumbnails/2.jpg)
GroEL-GroES Chaperonin Complex• Best studied molecular chaperones• Two proteins – GroEL & GroESGroEL - Tetradecamer of 58 kDa subunits• Two isologus heptameric rings• Hydrophobic cavities bind protein substrates• ATP dependent GroES - Heptamer of 10 kDa, acts as a lid GroES
GroEL
~ 10% of newly synthesized proteins
![Page 3: Evidence for Weakened Activity of Mycobacterial Chaperonins](https://reader035.vdocuments.us/reader035/viewer/2022062501/568168f2550346895ddff8af/html5/thumbnails/3.jpg)
• Two copies cpn60s: Mtb groEL1 (cpn60.1) & Mtb groEL2 (cpn60.2)
• Share 70% homology with E. coli counterpart, GroEL• Dimeric form & ATP independent• Cannot help refolding but prevent aggregation
GroEL of M. tuberculosis
Qamra and Mande., J. Bact. (2004)Qamra, Srinivas and Mande., JMB (2004)
Unusual GroELs of M. tuberculosis
GroEL
Cpn60.2
![Page 4: Evidence for Weakened Activity of Mycobacterial Chaperonins](https://reader035.vdocuments.us/reader035/viewer/2022062501/568168f2550346895ddff8af/html5/thumbnails/4.jpg)
Substrate Recognition or Oligomerization ?
![Page 5: Evidence for Weakened Activity of Mycobacterial Chaperonins](https://reader035.vdocuments.us/reader035/viewer/2022062501/568168f2550346895ddff8af/html5/thumbnails/5.jpg)
Mtb groEL1 & Mtb groEL2 cloned along with cohort Mtb groES
E. coli SV2 (groEL Ts mutant) and LG6 (GroES/L depletion strain)
Mtb groELs are expressed but not able to complement
In vivo Complementation Studies
I. Mtb GroES + GroEL1
II. Mtb GroES + GroEL2
III. E. coli GroES + GroEL
IV. Vector
PgroERBS
E. coli groES E. coli groEL
PlacRBS
E. coli groES
MG1655 LG6
E. coli groEL
![Page 6: Evidence for Weakened Activity of Mycobacterial Chaperonins](https://reader035.vdocuments.us/reader035/viewer/2022062501/568168f2550346895ddff8af/html5/thumbnails/6.jpg)
Gene Shuffling
E. coli SV2
0.2 % L-arabinose
Incubated at 30 ℃ and 42 ℃
![Page 7: Evidence for Weakened Activity of Mycobacterial Chaperonins](https://reader035.vdocuments.us/reader035/viewer/2022062501/568168f2550346895ddff8af/html5/thumbnails/7.jpg)
Sequence Analysis of Gene Shuffled Mutants
Apical domain is plastic & equatorial domain is conserved
![Page 8: Evidence for Weakened Activity of Mycobacterial Chaperonins](https://reader035.vdocuments.us/reader035/viewer/2022062501/568168f2550346895ddff8af/html5/thumbnails/8.jpg)
Apical Domain
Intermediate Domain
Equatorial Domain
Equatorial Domains Exchanged
Impaired Oligomerization
– Activity Determinant
![Page 9: Evidence for Weakened Activity of Mycobacterial Chaperonins](https://reader035.vdocuments.us/reader035/viewer/2022062501/568168f2550346895ddff8af/html5/thumbnails/9.jpg)
Complementation Bacteriophage Plaquing
Bacteriophage
SV2 Lawn
0.2 % L-arabinose
Incubated at 30 ℃
![Page 10: Evidence for Weakened Activity of Mycobacterial Chaperonins](https://reader035.vdocuments.us/reader035/viewer/2022062501/568168f2550346895ddff8af/html5/thumbnails/10.jpg)
Resident GroEL Contamination?
GroELMEF active in vivo – GroES dependent
Ptac based vector
PgroERBS
E. coli groES E. coli groEL
PBADRBS
E. coli groES
MG1655
MGM100
E. coli groEL
![Page 11: Evidence for Weakened Activity of Mycobacterial Chaperonins](https://reader035.vdocuments.us/reader035/viewer/2022062501/568168f2550346895ddff8af/html5/thumbnails/11.jpg)
E. coli GroEL
GroELMEF
GroELMER
GroELSp24
GroELSp32
Expressed in E. coli BL21 (DE3)
Auto Induction System or L-arabinose
Ammonium Sulphate Extraction
Ion Exchange or Hydrophobic Interaction Chromatography
Gel Filtration
Protein Purification
GroELMEF exists as oligomer
![Page 12: Evidence for Weakened Activity of Mycobacterial Chaperonins](https://reader035.vdocuments.us/reader035/viewer/2022062501/568168f2550346895ddff8af/html5/thumbnails/12.jpg)
ATPase Activity
Prevention of Aggregation
Biochemical CharacterizationSubstrate Protein Refolding
![Page 13: Evidence for Weakened Activity of Mycobacterial Chaperonins](https://reader035.vdocuments.us/reader035/viewer/2022062501/568168f2550346895ddff8af/html5/thumbnails/13.jpg)
Summary• Mtb rGroEL - Inherently Inactive Chaperones
• GroELSp24 & GroELSp32
o Efficiency of Substrate Binding
• GroELMEF - Functional Chaperone
o Complementation
o Exists as Oligomer - Refolding & ATPase Activity
o Stability
• GroELMER - Inactive as Chaperone
o Prevents Substrate Protein Aggregation
Impaired Oligomerization of Mtb rGroELs – Reduced Activity
![Page 14: Evidence for Weakened Activity of Mycobacterial Chaperonins](https://reader035.vdocuments.us/reader035/viewer/2022062501/568168f2550346895ddff8af/html5/thumbnails/14.jpg)
Factor Mediated Oligomerization?
• Active ATPase• Slow Growing Mtb
– 24 hrs doubling time
• Residues mutated to Serine or Threonine• Eukaryotic like STPKs• Heat regulated oligomerization in chaperones
Can Mtb Tolerate Robust GroEL?
Heat or Phosphorylation?
![Page 15: Evidence for Weakened Activity of Mycobacterial Chaperonins](https://reader035.vdocuments.us/reader035/viewer/2022062501/568168f2550346895ddff8af/html5/thumbnails/15.jpg)
What Forms in Mtb?
Mtb lysates resolved by Gel filtration Superdex S200 16/60
Peaks corresponding to different Oligomeric forms of GroEL
Normallized fraction of each peak probed - α-GroEL1 antibody
Mtb lysates resolved on Native PAGE
Probed with α-GroEL1 antibody
37 ℃ 42 ℃
Native PAGE SDS PAGE
GroEL1 predominates in three forms
![Page 16: Evidence for Weakened Activity of Mycobacterial Chaperonins](https://reader035.vdocuments.us/reader035/viewer/2022062501/568168f2550346895ddff8af/html5/thumbnails/16.jpg)
Is GroEL Phosphorylated?Co-immuno Precipitation
Tetradecameric form is Phosphorylated on Serine residue
Which Form of GroEL?
Peaks corresponding to different Oligomeric forms of GroEL
α-Ser-P antibody
Western Blotting
![Page 17: Evidence for Weakened Activity of Mycobacterial Chaperonins](https://reader035.vdocuments.us/reader035/viewer/2022062501/568168f2550346895ddff8af/html5/thumbnails/17.jpg)
Acrylamide plugs(Oligomeric forms of GroEL)
Mass-Spec Analysis
MALDI-TOF
MS/MSPeptides with serine
ESVEDAVAAAK
P
S393 is Phosphorylated
Which Serine?
![Page 18: Evidence for Weakened Activity of Mycobacterial Chaperonins](https://reader035.vdocuments.us/reader035/viewer/2022062501/568168f2550346895ddff8af/html5/thumbnails/18.jpg)
Summary
• Mycobacterial GroEL1 Exists in Multiple Forms
o Heptameric Form - Ambient Conditions
o Tetra-decameric Form - Elevated Temperatures
• Phosphorylation
o S393 is Phosphorylated – No Threonine
o Switch Between Heptameric & Tetra-decameric Form
• GroEL2 is not phosphorylated!
Oligomerization (Activity) is Phosphorylation Mediated
![Page 19: Evidence for Weakened Activity of Mycobacterial Chaperonins](https://reader035.vdocuments.us/reader035/viewer/2022062501/568168f2550346895ddff8af/html5/thumbnails/19.jpg)
Mitochondrial & Choloroplast Hsp60 Levy et. al., Eur. J. Biochem. 2001Dickson et. al., J. Biol. Chem. 2000
The Model
![Page 20: Evidence for Weakened Activity of Mycobacterial Chaperonins](https://reader035.vdocuments.us/reader035/viewer/2022062501/568168f2550346895ddff8af/html5/thumbnails/20.jpg)
ACKNOWLEDGEMENTS
• CDFD, Hyderabad • Department of Biotechnology, India• Council of Scientific and Industrial Research, India• Wellcome Trust, UK