A yeast Chaperone overexpression screen identifies modifiers of Huntington Toxicity Ruta Chitale1, Megha Desai2, Amitabha Majumdar2, Tania Bose1 1
2
Institute of Bioinformatics and Biotechnology, Pune University
Abstract
National Centre for Cell Sciences, Pune, India.
Yeast Chaperone Overexpression screen
Huntington’s Disease (HD) is a neurodegenerative disorder associated with memory defects. It is caused due to expansion in the Poly Q repeat region of the Huntingtin protein (Htt) that is responsible for its aggregation resulting in formation of intracellular inclusions. Moreover, imbalance between protein folding and increasing load of Htt aggregates disrupts proteostasis and eventually leads to neuronal death that triggers the onset of HD. However, Molecular chaperones are known to mitigate the misfolding of these proteins by remodeling/refolding. Thus, we hypothesize that certain Hsp40/DnaJ domain chaperones could interfere with the aggregation process thereby, giving rise to non-toxic aggregation states. Using a yeast based overexpression approach, DnaJ domain chaperones were overexpressed in Htt background and modified phenotypes were analyzed using spot assays, growth curves, SDD-AGE (for protein aggregates) and fluorescence microscopy-based screening of the aggregate morphologies. The screen resulted in a set of chaperone enhancers (increase the toxicity) and suppressors (decrease the toxicity). Thus, our genetic modifier screen has identified chaperone proteins that modulate Htt aggregation and could help in development of therapeutic approaches.
A.
Characterization of Htt aggregate Morphologies A.
B.
Morphology
CG-8476
CG-9828
C. Misfolded protein
Genetic Mutations
Low
P58ipK, Mrj, CG-5001
Diffuse cytoplasm with aggregates
Low
CG-11035, JJJ2
CG-4164
Single aggregate
Low
CG-4164, CG-30156
Double aggregate
Low
JdpC, Hsp70-Aa
Multiple aggregates
Medium
Sec63
Ring like aggregates
Medium
Droj2
High
CG-14650, CG-8476, CG-9828
Very high
Csp
Luciferase Mrj Droj2 CG-14650 Luciferase Csp Hsp70-Aa
Large aggregates C.
JJJ2
Fibre like aggregate
Luciferase Cellular Stress
Genetic Mutations
Cellular Stress
P58ipK
B.
Luciferase
C. Enhancer
CG-5001
B.
Luciferase N1-17
Poly Q
Proline rich
Folded protein
< Q30
Aggregates
JdpC Suppressor
Chaperones
Glucose (Plasmid “Off”)
> Q45 Fig.1. A. HD is a result of genetic as well as environmental factors that lead to formation of protein aggregates inside the neurons. B. Toxic and aggregating Htt protein is formed due to expansion in the PolyQ (CAG) repeat region and the toxicity is directly proportional to the number of repeats. C. Chaperones are known to refold the misfolded as well as aggregated proteins and bring them back to their original functional state, thereby reducing the toxicity.
A Yeast model for HD A.
B. Q25
Q103
Glucose (Plasmid “Off”)
Galactose (Plasmid “On”)
Galactose (Plasmid “On”)
Fig.3. A. Spot assay of mutant Htt in presence of various DnaJ domain chaperones; Luciferase acts as a control for all chaperone experiments. B. Growth curves of mutant Htt in presence of DnaJ domain chaperones. C. Semi denaturing – AGE showing smear like banding indicative of aggregates, where as mutant Htt overexpressed with chaperones show lighter smear indicative of reduced aggregates.
Aggregation states of mutant Htt in presence of chaperones Luciferase
Q25
CG-8476
Csp
P58ipk
CG-30156
CG-5001
CG-9828
JdpC
Sec63
JJJ2
CG-4164
CG-14650
Hsp70-Aa
Droj2
E. Aggregate
D. Chaperones
CG-11035 Diffuse Suppressors
Mrj
Amyloid Enhancers
Fig.2. A. Confocal microscopy of yeast cells expressing Wildtype (Q25) and Mutant(Q103) Htt after 16hrs of induction via a galactose inducible promoter. B. Spot assay to monitor growth deficit of the mutant Htt. C. Growth curve experiment highlighting difference in growth curves after 16 hrs of induction, proving that aggregation results in growth defect. D. Semi denaturing – AGE shows a crisp band in the wildtype, where as a smear in the mutant indicative of aggregation. E. Yeast chaperone screen to identify enhancers and suppressors of Htt toxicity.
Fig.5. A. Characterization of aggregation states of mutant Htt, diffuse cytoplasm being the least toxic where as fibre/amyloids being the most toxic. B. Fold change Log2 graph with weighted scores for each chaperone, negative score indicates more toxicity, positive score indicates less toxicity. C. Fluorescence Recovery after Photobleaching (FRAP) data indicates non-dynamic nature of the aggregates.
Conclusion
Q103
C.
Diffuse cytoplasm
CG-30156
Sec63
Background
Chaperone
Luciferase
Luciferase
A.
Toxicity
Luciferase CG-11035
Fig.4. Confocal microscopy based screening of mutant Htt in presence of DnaJ domain chaperones. In presence of certain chaperones Htt was seen diffused throughout the cytoplasm, whereas in presence of the others various cytoplasmic foci were seen. .
Our study shows that mutant Htt protein forms intracellular inclusions that are non dynamic in nature and severely affect growth of the cells. The Hsp40/DnaJ domain chaperone screen reveals certain modifiers of Htt toxicity when screened using multiple approaches. Our screen has revealed a set of enhancers of the Htt toxicity: Csp and Droj2 that might act by converting the aggregates into more stable amyloid conformation. These amyloid structures are known to damage the membranes and also trap important cellular constituents thus, contributing to toxicity. On the other hand, we have also found out a set of suppressors which decrease Htt toxicity: CG-11035, CG-8476, CG-4164, Mrj, CG-14650, JJJ2, P58ipK, and CG-5001. These might act by refolding, degradation or to fly and human systems by looking for orthologs of these chaperones and into by breaking larger aggregates smaller ones. This listThus, of modifiers of carrying out similar assays in order to look for increased survival. our toxicity could be extrapolatedand to fly preliminary screen could act as a baseline to studyHttthese genetic interactions aggregate human systems by HD. looking for mechanisms as well as spur new therapeutic and approaches to combat orthologs of these chaperones and Remodelling to Stable carrying out similar assays in order to Native state amyloid forms look for increased survival. Thus, our preliminary screen could act as a Breaking into baseline to study these genetic Enhancer smaller interactions and mechanisms as well Degradation aggregates as spur new therapeutic approaches to combat HD. Suppressor Fig. 6. Probable mechanism of action of chaperones.