Investigation of Interaction between heat shock protein 70.14 (Hsp 70.14) and RBBP6 RING finger domain

Despite improvement in cancer treatment modalities, cancer remains one of the leading causes of death worldwide. Studies show that in 2012, about 14.1 million new cancer cases resulting in 8.2 million deaths were reported worldwide according to the WHO. Over the years, several unsuccessful attempts to completely eradicate cancer diseases from human life has led to variety application of therapies (such as immunotherapy, targeted therapy, antibody based therapy, chemotherapy, surgery) aimed at finding treatment and radical cure for this disease. Although, there have been major improvements in the application of these therapies, however, challenges of non-specificity, as well as several side effects, resulting from killing of normal cells by chemotherapeutic agents are still widespread. This has led to urgent and continuous need for alternative therapies aimed at finding alternative drugs, or improving the current available treatment modalities. In the same vein, researchers have exploited therapeutic potential of heat shock proteins (Hsps) as an alternative intervention against cancer because of their ever-presence in the pathology of different facets of cancer development. Studies have shown that tumour cells require Hsp chaperonin activities for survival and proliferation than normal cells, because most oncoproteins in invading cells are often unfolded, therefore require chaperonage protection of Hsps for survival. Hsp70.14, alternatively known as HSP70L1, is an Hsp70 variant that lacks the c-terminal domain but contains substrate binding domain and the ATPase domain. The postulated interaction of this protein with the RING finger domain of RBBP6, suggested their role in protein quality control system and chaperone-mediated ubiquitination. Therefore, this study aimed to recombinantly express, purify, characterize and investigate the interaction between Hsp70.14 and RING finger domain of RBBP6. In silico analysis using homology modelling and subsequent validation by Ramachandran plot revealed that modelled structures were of good quality. Molecular docking studies show a strong interaction between the two proteins with a high binding score (KD) of 16038. The recombinant proteins were successfully expressed in BL21 cells and subsequently purified iv using GSH-agarose and on cobalt-recharged Nikel affinity chromatography columns. Structural characterization of the proteins using far UV, CD spectroscopy, tryptophan fluorescence and SE-HPLC showed that proteins were properly folded, predominantly α-helices, absorbs light at 280 nm and contain no impurities or protein aggregates. ANS binding studies reveals more hydrophobic patches in Hsp70.14 than RING and complex. The binding results obtained from this study using molecular docking, size exclusion high pressure liquid chromatography and ANS binding assay revealed there was indeed an interaction between Hsp70.14 and RING finger domain of RBBP6, which holds a major therapeutic potential towards the development of alternative cancer drugs.

Grantor dc:publisher.institution

University of Zululand

Year dc:date.issued

2019

Author dc:creator

• Ikwegbue, Paul

Advisor dc:contributor.advisor

• Kappo, A.P.

• Heat shock protein
• Cancer diseases

Licence

• https://devspace.unizulu.ac.za/bitstreams/e261b6a4-3d51-44fa-95b6-99ccec70d41e/download