Delineate the NF-kappaB interactome
Cellular processes including immune response are highly dependent on gene transcription. Although major advances have been made in the field of transcription for the past few decades, we are still far from complete understanding of this process. This is apparent from our inadequate knowledge about cancers and autoimmune diseases whose origin lies in our very own cells through deregulated gene expression. Transcription factor (TF) proteins are one of the basic components, which regulate transcription by binding to their cognate site/s on the promoter/enhancer regions of their target genes. TFs can be constitutive or inducible – the latter form requires a stimulating agent for its onset. To ensure proper regulation of target gene expression the TFs interact with various other components in the nucleus in addition to DNA. These components comprise of co-factors, co-activator proteins, nucleic acids (like non-coding RNAs) and other transcription factors. The primary focus of my research is to understand how TF interacts with multiple components in the nucleus to regulate and control gene transcription. My lab focuses on the NF-kappaB family of transcription factor and study its interaction with various other proteins that control the gene expression of its target genes. The outcome of this research will enrich our knowledge about the mechanisms of NF-kB regulated gene expression and have direct impact on the design of therapeutic agents for various diseases including cancers that are caused by deregulated activity of NF-kB in the diseased cells.
Research Project I: Molecular Mechanism of transcription activation of genes repressed by NF-kappaB
(a) Statement of problem: NF-B is majorly known for its transcription activation potential, while reports have also emerged highlighting NF-B’s activities as transcription repressor. p50 subunit of NF-B in its homodimeric form is known to repress a set of IRF3 target genes by masking the IRF3 cognate site through DNA binding. How such NF-B-repressed genes are activated remains poorly understood due to inadequate knowledge of the various molecular processes involved, such as DNA binding, NF-B dimer association and NF-B-coactivator protein interactions. Subtle changes in the biophysical parameters of these processes could regulate the transcriptional outcome of various genes.
(b) My lab contribution: We are working towards understanding the molecular mechanism of activation of genes repressed by NF-B using NMR spectroscopy and molecular biology techniques. In this regard our goal is ‘to study the mechanism of NF-B dimer removal from IRF binding site using NMR spectroscopy and molecular biology techniques’. NMR spectroscopy provides atomic level details of biomolecular interaction and is also used to study dynamics of these interactions. We have initiated the study of an ~85 kDa NF-B-DNA complex using NMR which is technically challenging. We have obtained the backbone assignments of this huge complex and progressing towards unraveling the mechanism of this process at atomic level details.
(c) Related Publications: 1). Raza T, Dhaka N, Joseph D, Dadhwal P, Kakita VMR, Atreya HS and Mukherjee SP* Insights into the NF-κB-DNA Interaction through NMR Spectroscopy. (2021) ACS Omega. 6:12877-12886.
Research Project II: Role of NF-kappaB dimer dynamics in NF-kappaB driven transcription
(a) Statement of problem: The NF-κΒ family is comprised of five members, namely, p50, p52, RelA,RelB and c-rel. The family members form 15 dimers in various combinations amongst themselves with RelA:p50 heterodimer being the most abundant followed by p50 homodimer; the other dimers either are reported to exist at very low concentrations physiologically or are not observed experimentally.However, many of these rare specific dimers play critical role in specific gene transcription. How such NF-κΒ dimers are formed and their role in NF-κΒ pathway remains to be investigated.
(b) My lab contribution: My lab is currently studying the role of preferential dimer composition on the NF-kB target gene regulation using a multidisciplinary approach using NMR spectroscopy and molecular biology techniques. Our experiments show RelA-p50 heterodimer to be the strongest dimer followed by p50 and then RelA homodimers. We show that the strength of the H-bonding network in the dimerization domain of an individual NF-B subunit undergo major change depending upon its partner NF-kB subunit.
(c) Related Publications:
Kumar M, Dhaka N, Raza T, Dadhwal P, Atreya HS and Mukherjee SP* Domain Stability Regulated through the Dimer Interface Controls the Formation Kinetics of a Specific NF-κB Dimer. (2021) Biochemistry. 60:513-523.
Kumar M Dadhwal P, Atreya HS and Mukherjee SP* Backbone Resonance Assignments of the Dimeric Domain of the p50 NF-kappaB Subunit. (2020) Biomol NMR Assign 14:9-11.
Research Project II: Understanding Gene Regulation and Targeting protein-protein interactions in a proto-oncogene system.
(a) Statement of problem: Bcl3 (B cell lymphoma 3) protein has an emerging role in a number of autoimmune pathologies and different cancers. It is identified as a proto-oncogene with a central role in regulating NF-kappaB signaling. Belonging to the IkappaB family of proteins, which is known for its inhibitory role of NF-B pathway, Bcl3 plays a dual role of transcription activation as well as repression of NF-B target genes. It accomplishes its role as a transcription regulator by interacting with p50 and p52 subunits of NF-kB in their homodimer form. Though the interaction of Bcl3 with p50 and p52 homodimers is well-established the details of the interaction active site of the proteins required for designing any potential drug target remains elusive. This is despite the fact that 3-dimensional crystal structures of the individual components of the Bcl3-p50/p52 complex, namely, Bcl3, p50 homodimer and p52 homodimer is now available for over a decade.
(b) My lab contribution: We hypothesize that the dynamic nature of the Bcl3-p50/p52 complex might be instrumental in formation of poorly or non-diffracting crystals or no crystal formation altogether of the complex. Given such a scenario we propose to use solution NMR spectroscopy to decipher the interaction of Bcl3 with p50 and p52 homodimers at the residue level along with various molecular biology techniques. Apart from the knowledge of Bcl3-p50/p52 interface, it is also intriguing that how the same Bcl3-p50/p52 complex plays a dual role of a transactivator as well as a transrepressor. To address this a detailed understanding of the interaction mechanism of the complex in presence of kappaB DNA is imperative. Thus, we aim to study Bcl3-p50/p52 complex in the presence and absence of kappaB.