Starved Dictyostelium cells aggregate into groups of nearly 105 cells. AMPK is a highly conserved serine/threonine protein kinase consisting of a catalytic and two regulatory subunits. As multi-cellular development in Dictyostelium is initiated upon starvation, we explored the role of the energy sensor, AMPK, which shows significant similarity to human AMPK and is expressed throughout development. Deletion of the ampkα gene results in the formation of numerous small-sized aggregates that develop asynchronously to form few fruiting bodies with small sori and long stalks. On the other hand, ampkαOE cells form fruiting bodies with small stalks and large sori when compared with wild-type, Ax2. A minimum of 5% ampkα− cells in a chimaera with Ax2 cells was sufficient to reduce the aggregate size. Also, the conditioned media collected from ampkα− cells triggered Ax2 cells to form smaller aggregates. The starved ampkα− cells showed low glucose levels and formed large aggregates when glucose was supplied exogenously. Interestingly, ampkα− cells exhibit abnormal cell-type patterning with increased prestalk region and a concomitant reduction of prespore region. In addition, there was a loss of distinct prestalk/prespore boundary in the slugs.

Autophagy is a degradation process, wherein long-lived proteins, damaged organelles, and protein aggregates are degraded to maintain cellular homeostasis. Upon starvation, 5′-AMP-activated protein kinase (AMPK) initiates autophagy. We show that ampkα − cells exhibit 50% reduction in pinocytosis and display defective phagocytosis. Re-expression of AMPKα in ampkα − cells co-localizes with red fluorescence protein-tagged bacteria. The ampkα − cells show reduced cell survival and autophagic flux under basal and starvation conditions. Co-immunoprecipitation studies show conservation of the AMPK-ATG1 axis in basal autophagy. Computational analyses suggest that the N-terminal region of DdATG1 is amenable for interaction with AMPK. Furthermore, β-actin was found to be a novel interacting partner of AMPK, attributed to the alteration in macropinocytosis and phagocytosis in the absence of AMPK. Additionally, ampkα − cells exhibit enhanced polyubiquitinated protein levels and allied large ubiquitin-positive protein aggregates.Our findings suggest that AMPK provides links among pinocytosis, phagocytosis, autophagy, and is a requisite for basal autophagy in Dictyostelium.

The translationally controlled tumor protein (TCTP) is a highly conserved multifunctional protein, preferentially expressed in mitotically active tissues and is a potential biomarker and a therapeutic target for lung cancers. An understanding of the biology of this molecule and model systems for the screening of drugs is still awaited. In the absence of complete crystal structure, NMR structures as templates were used for homology modeling and MD optimization of both Dictyostelium discoideum and human TCTPs, which was followed by pocket-site prediction, ligand screening and docking. Rescoring of TCTP-ligand complexes was done using MD and MM-PBSA approaches. D. discoideum TCTP was expressed under a constitutive promoter and the endogenous RNA in multicellular structures formed was localized by in situ hybridization. Based on the interactions and binding energy scores, two novel compounds were identified as the best potential inhibitors that could be further used for the development of drug candidates. Inhibition of cell proliferation was observed in the strain overexpressing Dictyostelium TCTP and in situ hybridization results show them to be localized in the prestalk (dying cell population) cells. D. discoideum and human TCTPs share similar dynamic behaviors; overexpression of Dictyostelium TCTP inhibits cell proliferation. D. discoideum could be used as a model system for understanding the biology of this molecule and also for drug screening.

Prostate cancer (PC) is one of the leading cancers in men, raising a serious health issue worldwide. Due to lack of suitable biomarker, their inhibitors and the platform for testing those inhibitors result in poor prognosis of PC. AMP-activated protein kinase (AMPK) is a highly conserved protein kinase found in eukaryotes that is involved in growth and development, and also acts as a therapeutic target for PC. The aim of the present study is to identify novel potent inhibitors of AMPK and propose a simple cellular model system for understanding its biology. Structural modelling and MD simulations were performed to construct and refine the 3D models of Dictyostelium and human AMPK. Binding mechanisms of different drug compounds were studied by performing molecular docking, molecular dynamics and MM-PBSA methods. Two novel drugs were isolated having higher binding affinity over the known drugs and hydrophobic forces that played a key role during protein-ligand interactions. The study also explored the simple cellular model system for drug screening and understanding the biology of a therapeutic target by performing in vitro experiments.

Translationally controlled tumor protein (TCTP) is a multifunctional protein implicated in various types of cellular processes involving growth and development of an organism. Here, we identified tctp gene in Dictyostelium discoideum and unraveled its function. The sequence analysis of D. discoideum TCTP (DdTCTP) showed its conservation among eukaryotes. Transcript of DdTCTP was highly expressed at the initial time points of development and protein is localized both in the cytoplasm and nucleus. Disruption of tctp was achieved by BSR cassette using double homologous recombination method. Abrogation of tctp resulted in reduced cell proliferation but increased cell size. Additionally, development was delayed by 4 h wherein small-sized aggregates and fruiting bodies were produced by tctp − cells while larger aggregates and fruiting bodies were produced by tctp OE cells concordant with the fact that TCTP regulates prestalk/prespore ratio and cell-type differentiation. tctp − cells produced round spores with reduced viability and stalk cells are arranged in septate pattern as compared to polyhedral manner of wild type. Abrogation of tctp resulted in aberrant localization of cell type specific markers and show low proclivity toward prespore/spore region, in presence of wild type cells.