Investigation of neurodegenerative diseases via the CHIP:HSP70 complex 

Heat Shock Protein 70 (Hsp70) is a chaperone protein that works to maintain cellular homeostasis through folding and refolding proteins. These proteins are vital in the cell’s response to stress via their ability to refold misfolded proteins. Hsp70 also has a key role in the protein degradation pathway, working with cochaperone proteins, such as CHIP, to ubiquitinate proteins to send them to be degraded. CHIP and Hsp70 bind together, forming the CHIP:Hsp70 capable of performing these tasks. CHIP autoubiquitinates itself when it is no longer needed. This autoubiquitination leads to its own degradation. CHIP has 9 known Lysine residues capable of ubiquitination. This ubiquitination leads to the dissociation of the CHIP/Hsp70 complex.

Protein quality control is a process vital to the maintenance of cellular homeostasis through ensuring proteins are correctly produced, folded, and degraded.1 If left unchecked, the accumulation of misfolded proteins can lead to the development of toxic aggregates capable of causing diseases such as Alzheimer’s, Parkinson, or cancer.2 One method cells use to prevent the buildup of misfolded proteins is by chaperone proteins, such as heat shock protein 70 (HSP70), which interact with their misfolded “client” protein and works to refold it back into its native state .1,3 If the misfolded protein is unable to be refolded, HSP70 can aid in the degradation of it instead.2 HSP70 works with its cochaperone, Carboxy-terminus of Hsp70 Interacting Protein (CHIP), to aid in the degradation pathway of client proteins, forming the CHIP:HSP70 complex capable of performing these tasks.2,3 Given the vital role HSP70 and its cochaperone, CHIP, play in the possible development of many chronic diseases, understanding their interactions can aid in the development of therapeutics for treating said diseases.2

CHIP is self-regulated through its ability to autoubiquitinate.3,4 This process is CHIP has the capability to ubiquitinate nine of its lysine residues, allowing it to dissociate from the CHIP:Hsp70 complex and to undergo degradation.5,6 The process of dissociation is not well understood, leading to our current research. Through designing CHIP mutants varying numbers of lysine residues mutated to arginine, we will be able to understand how the autoubiquitination process leads to the dissociation of the CHIP:Hsp70 complex and the eventual degradation of the CHIP cochaperone.4,5

Step 1: Purification of CHIPWT and CHIP9R

The plasmids of TOPO CHIPWT and TOPO CHIP9R were designed incorporating His6-tag and TEV recognition sites, and the plasmids were ordered from GenScript. The plasmids of proteins UbcH5b (E2), Hsp70, and ubiquitin will be designed incorporating His6-tag and TEV recognition sites (ENLYFQ) for effective protein purification. TOPO CHIPWT, TOPO CHIP9R, UbcH5b (E2), and ubiquitin were purified through Nickel affinity chromatography. These proteins were then cleaved with TEV protease enzyme, and the TEV-cleaved proteins were run through Nickel-frontal chromatography followed by Size exclusion chromatography (S75) to get pure protein. SDS-PAGE will be performed at each stage of purification to identify the protein-containing fractions and assess the purity of the proteins. The protein concentration was determined using the Pierce assay, then flash frozen in liquid nitrogen and kept at -80 °C.

Step 2: Sor-tagging of CHIPWT, CHIP9R, Ubiquitin

CHIPWT and CHIP9R were tagged with Cy3-LPETGG peptides (visible in 600 nM). 5 mM Cy3- LPETGG peptides, 50 μM of the respective protein (either CHIPWT and CHIP9R), sortase reaction buffer, and 50 μM of Sa sortase enzyme were mixed. Molecular grade water was added to reach a 500 μL reaction mixture. The sample incubated at room temperature for 5 hours in a shaker, followed by S75 chromatography to separate protein according to the size. Later, Nickel-frontal chromatography was used to separate the his-tagged sortase enzyme with sor-tagged CHIPWT and CHIP9R.

Step 3: Site-directed mutagenesis to generate CHIP9R mutants

Nine different mutants of CHIP9R are generated through site-directed mutagenesis using the Agilent mutagenesis kit. The mutation is confirmed through sequencing, being sent to Plasmidsaurus. Once the sequence is confirmed, the cell stock will be prepared, and respective protein will be purified as above. The purified mutated proteins will also be sor-tagged with Cy3-LPETGG peptides through sortase reaction, following the conditions as mentioned in step 2.

Step 4: Ubiquitination reaction to generate monoubiquitinated CHIP and CHIP mutants

The monoubiquitinated CHIP will be generated through ubiquitination reaction using Ko ubiquitin. The additional proteins that are required for the ubiquitination assay are His6-E1 (UbcE1) and ATP/MgCl2, which will be purchased from R&D. First, 0.5 μM E1, 10 μM E2, 250 μM Ko ubiquitin, and 15 mM ATP/MgCl2 will be mixed in a tube and incubated on ice for 30 minutes to generate the E2~Ub conjugate. After that, 4 μM CHIP will be mixed with E2~Ub conjugate mixture at 0, 5, 15, and 30-minute time points and incubated in a ProFlex heat cycler at 37 °C. The reaction will be quenched by adding 4X LDS buffer with 2 mM DTT, followed by heating at 95 °C for 10 minutes. The formation of monoubiquitinated CHIP will be confirmed visualizing it with the LI-COR Odyssey Fc Imager.

Step 5: Interaction study using BLI

The BLItz instrument (ForteBio) will be used for the BLI assays, and ubiquitination assays will be performed with a setting of each step at a specific time. Biosensor tips will be immersed in 50 mM HEPES and 50 mM NaCl, pH 7.0, for at least 10 min before each run. All the assays were performed at 37 °C by housing the BLItz in a Peltier incubator. Anti-GST biosensors will be used for the assay. The ubiquitination assay started with an initial baseline step (step 1) to wash the sensor with buffer (50 mM HEPES, 50 mM NaCl, pH 7.0). In step 2, GST Hsp70 will be loaded onto the sensor, followed by another baseline step (step 3). In the association step (step 4), the sensor tip was dipped in a reaction mixture containing monoubiquitinated CHIP. Finally, the dissociation step (step 5) was carried out in buffer (50 mM HEPES, 50 mM NaCl, pH 7.0) to remove any unbound molecules from the biosensor. A similar interaction study will be done on various CHIP mutants with Hsp70.

See poster above:

Figure 4: Ni-frontal run of TEV-cleaved WT-CHIP. Lane 1 is a ladder & lanes 2 – 12 exhibit the presence of TEV-cleaved CHIP-WT ~35 kDa. 

Figure 5: Ni-frontal run of TEV-cleaved CHIP-9R. Lane 1 is a ladder & lanes 6 – 10 exhibit the presence of TEV-cleaved CHIP-9R ~35 kDa. 

Figure 6: S75 run of Ac-K Cy3-Sortase labeled CHIP-WT.  Lane 1 is a ladder & lanes 3 – 5 show the presence of Ac-K Cy3-sortase labeled CHIP-WT at ~35 kDa. 

Figure 7: Ni-frontal run of Ac-K Cy3-Sortase labeled CHIP-WT.  Lane 1 is a ladder & lanes 2 – 10 show the presence of Ac-K Cy3-sortase labeled CHIP-WT at ~35 kDa. 

Figure 8: CHIP-WT showed polyubiquitination within 15 minutes of the reaction.  

Figure 9: CHIP-9R showed no ubiquitination after an hour of the reaction.  

As research continues, more of the CHIP variants will be generated via site-directed mutagenesis. Along with the mutagenesis, kinetic assays of the CHIP variants, in association with Hsp70, will be performed.

National Institutes of Health through grant R35GM128595 to R. Page

Special thanks to Dr. Richard C. Page, Page lab members, and Miami University

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2.   Edkins, A. L. (2015). CHIP: A co-chaperone for degradation by the proteasome. Subcellular Biochemistry, 78, 219–242. https://doi.org/10.1007/978-3-319-11731-7_11

3.   Mcdonough, H., & Patterson, C. (2003). CHIP: a link between the chaperone and proteasome systems. In Cell Stress & Chaperones (Vol. 8, Issue 4). Cell Stress Society International.

4.   Zhang, M., Windheim, M., Roe, S. M., Peggie, M., Cohen, P., Prodromou, C., & Pearl, L. H. (2005). Chaperoned ubiquitylation - Crystal structures of the CHIP U box E3 ubiquitin ligase and a CHIP-Ubc13-Uev1a complex. Molecular Cell, 20(4), 525–538. https://doi.org/10.1016/j.molcel.2005.09.023

5.   Graf, C., Stankiewicz, M., Nikolay, R., & Mayer, M. P. (2010). Insights into the conformational dynamics of the E3 ubiquitin ligase CHIP in complex with chaperones and E2 enzymes. Biochemistry, 49(10), 2121–2129. https://doi.org/10.1021/bi901829f

6.   VanPelt, J., & Page, R. C. (2017). Unraveling the CHIP:Hsp70 complex as an information processor for protein quality control. In Biochimica et Biophysica Acta - Proteins and Proteomics (Vol. 1865, Issue 2, pp. 133–141). Elsevier B.V. https://doi.org/10.1016/j.bbapap.2016.11.005