Puthalakath Laboratory
Department of Biochemistry
Research - Apoptosis regulation
Regulation of Bcl-2 family proteins during apoptosis
Apoptosis (programmed cell death) plays a prominent role in the maintenance of self-tolerance in the immune system. A young adult mouse with 100-200 million thymocytes generates between 20-40 million new thymocytes per day. However, the number of T cells that leave the thymus and enter the peripheral T cell pool is only about 2-3% of the number initially generated indicating that massive death must occur. Immature thymocytes are subjected to stringent selection. Thymocytes that fail to receive a signal via their T cell receptor (TCR) will undergo apoptosis (death by neglect). Only those thymocytes expressing a T cell receptor capable of interacting with peptides presented by MHC molecules with moderate affinity are positively selected and migrate to the periphery. In contrast, thymocytes with receptors that bind to self-peptide-MHC complexes with high affinity (autoreactive thymocytes) are negatively selected and undergo apoptosis. Hence negative selection ensures tolerance to normal tissues and prevents autoimmunity.
BIM, an apoptosis inducing Bcl-2 family protein, is crucial for the induction of thymic negative selection. In mouse where this gene is deleted, thymic negative selection fails to occur. Also, my recent results show that this protein is induced by Endoplasmic reticulum stress (ER stress). In response to ER stress in thymocytes, this gene is transcriptionally induced and contributes to cell death. Thymocytes subjected to negative selection undergo ER stress (see the figure) and therefore the question we are asking is both positive and negative selections are the extension of the Unfolded Protein Response (UPR) i.e. moderate affinity MHC-peptide interaction results in limited UPR and therefore positive selection and high affinity (self antigen) MHC-peptide interaction results in sustained UPR (ER stress) which leads to apoptosis. A substantial part of this project would involve generation of transgenic mice expressing misfolded proteins in the thymic compartment and making crosses with relevant TCR transgenics and analysing negative selection in vivo as well as in vitro using Foetal Thymic Organ Culture (FTOC). This will be done in collaboration with Dr. Wu Li from the Walter and Eliza Hall Institute for Medical Research. 
BIM is also known to be a tumor suppressor. It is deleted in Mantle cell lymphoma. Also, in mouse models, it has been shown to be involved in the suppression of c-Myc induced lymphoma. Thus the involvement of BH3-only pro apoptotic proteins such as BIM and PUMA in tumor suppression has lead to a new generation of cancer chemotherapeutics called “The BH3 mimetics”. BH3 mimetic compounds activate the adaptor molecules such as BAX and BAK leading to apoptosis. However, in many cancers, the adaptor molecule BAX is deleted or down regulated and thus it may render these cancers resistant to this new generation drugs. Thus, understanding the molecular mechanism of BAX regulation may help in understanding chemo resistance in these cancers and may lead to better therapeutics. This work is being carried out with Dr. Geoff Lindeman from the Victorian Breast Cancer Research Consortium.
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