SUMMER WEBINAR SERIES 2022

Inositol pyrophosphates (PP-IPs), such as diphosphoinositol pentakisphosphate (IP7) and bisdiphosphoinositol tetrakisphosphate (IP8), are a class of ubiquitous, energy rich metabolites, whose synthesis is catalysed by two groups of enzymes, IP6 kinases and PP-IP5 kinases. Since the discovery of PP-IPs in the early 1990s, significant progress has been made in uncovering pleiotropic roles for these small molecules in cellular physiology. PP-IPs exert their effect on proteins in two ways – allosteric regulation by direct binding, or posttranslational regulation by serine pyrophosphorylation, a modification unique to PP-IPs. Serine pyrophosphorylation is achieved by Mg 2+ -dependent, but enzyme independent transfer of a β-phosphate moiety from PP-IPs to a prephosphorylated serine residue located in an intrinsically disordered region, amidst acidic amino acid residues. We have demonstrated that serine pyrophosphorylation by PP-IPs regulates diverse cellular processes, including rRNA synthesis, dynein-driven vesicle transport, and protein stability. However, our understanding of the molecular details of this phosphotransfer process from pyrophospho-inositol to generate pyrophospho-serine, is still nascent. Our current knowledge of the importance of protein pyrophosphorylation, and recent advances in understanding the mechanism of this important yet under-appreciated posttranslational modification will be discussed.

A large fraction of the human proteome consists of RBPs. These proteins perform diverse functions in cells. Many of these RBPs like FUS, EWSR1, TAF15, TDP43 and hnRNAP1, which are present at high concentrations in the nucleus of the mammalian cells, contain low complexity domains making them prone to undergo liquid-liquid phase separation, a physicochemical process to make micron scale condensates in both the cellular environment and in vitro. In cells, these RBPs are present at concentrations where they can readily phase separate into condensates. We are currently studying how do cells control the formation of RBP condensates at specific times and locations? Additionally, these phase separation prone proteins tend to aggregate and form solid-like assemblies in the cytoplasm in neurodegenerative diseases- Amyotrophic Lateral Sclerosis and Frontotemporal Lobar Degeneration, the underlying mechanism for which is unclear. In this talk, Dr. Shovamayee Maharana will present evidence to elucidate the mechanisms which inhibit the spontaneous formation of RBP condensates and solid-like transitions in cells.

Many double-stranded RNA-binding domains (dsRBDs) interact with topologically distinct dsRNAs in biological pathways pivotal to viral replication, cancer causation, neurodegeneration, etc. In this study, we hypothesized that the adaptability of dsRBDs is essential to target distinct dsRNA substrates. We employed a model dsRBD and a few toplogocally distinct dsRNAs to test the systematic shape-dependence of RNA on the dsRBD-binding using NMR spectroscopy and molecular modeling. We used NMR line-broadening, microsecond timescale dynamics measurements using relaxation experiments, and atomistic molecular simulations to show a distinct binding pattern for the dsRBD with the topologically distinct dsRNAs, and a role of intrinsic dynamical basis for the substrate promiscuity for dsRBD proteins.

Diffuse large B-cell lymphoma (DLBCL) is one of the most aggressive forms of lymphoma with poor response to standard-of-care (R-CHOP) therapy. This poor response and acquired drug resistance to R-CHOP chemotherapy is partially explained by its remarkable heterogeneity. Genomics based studies in the past had been identified recurrently mutated oncogenes in DLBCL e.g., BCL6, KMT2D, CREBBP, EP300 etc. Despite the discovery of such recurrent genomic alterations, there are very limited options available for DLBCL patients’ treatment, which indicate towards the urgency of identifying novel mechanisms of DLBCL tumorigenesis and drug targets. In our recent study, we looked at super-enhancers (SEs) regions and their involvement in DLBCL tumorigenesis and in this talk, Dr. Jeetender Chugh will talk about this study.

In nature, hosts can face multiple pathogens simultaneously. While this might warrant activation of different immune mechanisms to counter mixed infections, an expansion of diverse immune arms together can collectively increase the costs of immunity, reducing the rate of adaptation against pathogens. However, a comparative experimental framework is missing. Here, we used several Tribolium beetle lines evolving against either a single or a combination of pathogens with divergent within-host growth dynamics— Fast-growing Bacillus thuringiensis (Bt); Slow-growing Pseudomonas entomophila (Pe); A combination of both (M). Although we began by imposing an equivalent selection pressure (~60% mortality in all lines), resistance could evolve most rapidly against Pe by overexpressing antimicrobial peptides and lysozyme (within 12-generations), whereas resistance against fast-growing Bt did not evolve yet, possibly because beetles could not invest more in fast-acting immunity relevant to Bt-clearance such as cytotoxic phenoloxidase. Expectedly, resistance evolution against M was delayed (~17-generations), perhaps due to increased costs of an extended immune repertoire. Finally, we found a novel cost of immune responses in ancestral lines where they compromised the germline DNA-repair and increased the mutational load in offspring, but evolution with pathogens rapidly reversed this effect by improving germline maintenance to reduce the deleterious mutation transmission.

HER2+ breast cancer patients presenting with either synchronous (S-BM) or metachronous (MBM) brain metastases have poor survival outcomes. Although relatively rare, HER2+ breast cancer patients with synchronous brain metastases have a median overall survival of around 6 months. In contrast, metachronous brain metastases or relapse are observed in approximately fifty percent of HER2+ breast cancer patients considered disease free after a variable length of time post primary diagnosis and treatment. It is believed that disseminated latent residual cells (Lat) that survive current therapies are responsible for the metastatic relapses or metachronous metastasis. How disseminated tumor cells survive as latent/dormant entities for extended period of time before initiating metachronous metastasis is poorly understood. Moreover, the basis for disparate metastatic fitness among disseminated tumor cells of similar oncotype within a distal organ is unknown and vital for better clinical management. Through phenotypic screen in mice, we have isolated isogenic HER2+ S-BM, Lat and M-BM brain metastatic cells. Employing those isogenic HER2+ breast cancer brain metastasis models, we show metabolic diversity and plasticity within brain-tropic cells determines metastatic fitness. Lactate secreted by aggressive metastatic cells (S-BM and M-BM) or lactate supplementation to mice bearing latent residual disease limits innate immune surveillance and triggers overt metastasis. Attenuating lactate metabolism in S-BM impedes metastasis, while M-BM adapt and survive as latent residual disease. In contrast to S-BM, Lat and M-BM survive in equilibrium with innate immune surveillance, oxidize glutamine and maintain cellular redox homeostasis through the anionic amino acid transporter xCT. Likewise, xCT expression was significantly higher in matched metachronous brain metastatic samples compared to primary tumors from HER2+ breast cancer patients. Genetic or pharmacological blockade of xCT eradicates residual disease and brain metastatic relapse in these preclinical models. In sum, by investigating phenotypically distinct brain-tropic S-BM, Lat and M-BM cells, we uncovered the impact of metabolic diversity and adaptations on metastatic fitness and identified targetable metabolic vulnerabilities.