Under hostile conditions, bacteria elicit stress response. Such stress response is regulated by a secondary messenger called (p)ppGpp. (p)ppGpp is involved in wide range of functions such as GTP homeostasis, biofilm formation and cell growth. Its regulation and mode of action is not well understood. This work has been initiated with an aim to gain insights into the molecular basis of stress response. (p)ppGpp was discovered on the chromatogram of cell extract from starved E. coli cells. (p)ppGpp is synthesized and hydrolyzed by Rel/SpoT in Gram negative bacteria (such as E. coli), and by bifunctional enzyme called Rel in Gram positive bacteria (such as Mycobacteria). The obvious question that comes in our mind is how bifunctional Rel enzyme decides on synthesis or hydrolysis in Gram positive bacteria such as Mycobacterium? In our laboratory, it has been shown that N-terminal domain of Rel shows unregulated (p)ppGpp synthesis implying regulatory role of C-terminal domain. Also, concurrent increase in anisotropy of Rel C-terminal domain with the increase in concentration of pppGpp has been observed indicating the binding of pppGpp to the C-terminal domain. We performed Isothermal Calorimetry experiment to confirm that pppGpp binds with C-terminal domain of Rel enzyme. For identification of the binding region, small molecule analogue 8-azido-pppGpp has been synthesized. This analogue is UV-crosslinked with C-terminal domain of Rel and specificity of the interaction has been determined by gel based crosslinking experiments. Crosslinked protein has been subjected to the ingel¬trypsin digestion and analyzed by mass spectrometry. We identified two crosslinked peptides in the mass spectra of trypsin digest in case of the crosslinked protein where identity of the parent peptide is confirmed by MS-MS analysis. Site directed mutagenesis has been carried out based on the conservation of residues in the crosslinked peptides. Isothermal Calorimetry analysis has been done where Rel C-terminal domain mutants are titrated with pppGpp in order to detect any defect in binding due to the mutations. Mutations leading to the reduced binding affinity of pppGpp to Rel C-terminal domain have been introduced in the full length Rel protein and activity assays are carried out so as to evaluate the effects of mutations on synthesis and hydrolysis activity. In mutants, synthesis activity is found to be increased with the concomitant reduction in hydrolysis activity. This indicates the feedback loop where pppGpp binds to Rel C-terminal domain to regulate it own synthesis and hydrolysis. In E. coli, pppGpp binds to RNA polymerase and modulates the transcription. The region where it binds is controversial. In addition, whether ppGpp and pppGpp have different binding site on RNA polymerase is not known. The latter question becomes important in the light of evidence where differential regulation of transcription by ppGpp and pppGpp have been indicated. We found that ppGpp and pppGpp have an overlapping binding site on RNA polymerase. The 8-azido-ppGpp has been mapped on β and β’ subunits whereas binding site of 8-azido-pppGpp has been located on the β’ subunit. We observed that the 8-azido¬pppGpp labels RNA polymerase more efficiently than ppGpp. pppGpp can compete out ppGpp as illustrated by DRaCALA assay and gel based crosslinking experiment. However, the RNAP from B. subtilis does not bind to (p)ppGpp. (p)ppGpp is ubiquitous in bacteria but absent in mammals. Thus, blocking (p)ppGpp synthesis would impede the survival of bacteria without having any effect on humans. Recently, Relacin compound has been synthesized by another group in order to inhibit (p)ppGpp synthesis. The limitations of this compound are the requirement of high concentration (5mM) for inhibition and low permeability across the membrane. Taking hints from the latter compound, we acetylated the nd 2’, 3’ and 5’ position of ribose ring and benzoylated the 2position of guanine moiety in guanosine molecule. We observed significant inhibition of in vitro pppGpp synthesis and biofilm formation. More studies will be conducted in near future to test these compounds for their plausible functions. In collaboration with Prof. Jayaraman (Organic Chemistry, IISc), many artificial glycolipids are synthesized and tested for biological function. We observed that synthetic glycolipids exhibit a profound effect as inhibitors of the key mycobacterial functions. These analogs impede biofilm formation and can plausibly affect long term survival. Glycolipid analogs can compete with natural glycolipids, thus may help in understanding their functions. Our past and recent studies have showed that the synthetic glycolipids act as inhibitors of mycobacterial growth, sliding motility and biofilm formation. The major lacuna of these glycolipid inhibitors is the requirement of high concentration. Their inhibitions at nanomolar concentrations remain to be achieved. Issues surrounding the thick, waxy mycobacterial cell wall structures will continue to be the focus in manifold approaches to mitigate detrimental effects of mycobacterial pathogens. In chapter 1, introduction to the research work has been written and role of (p)ppGpp and its functions have been discussed. In chapter 2, novel binding site of pppGpp on Rel C-terminal domain and its regulatory role have been discussed. In chapter 3, differential binding of ppGpp and pppGpp to RNA polymerase has been discussed. In chapter 4, studies on natural and synthetic analogues of pppGpp have been presented. In chapter 5, synthetic glycolipids studies have been described. Chapter 6 summarizes all the chapters.

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Indiana University-Purdue University Indianapolis (IUPUI) / Mammary ductal epithelial cell growth is controlled by microenvironmental signals in serum under both normal physiological settings and during breast cancer progression. Importantly, the effects of several of these microenvironmental signals are mediated by the activities of the tumor suppressor protein kinases of the Hippo pathway. Canonically, Hippo protein kinases inhibit cellular growth through the phosphorylation and inactivation of the oncogenic transcriptional co-activator Yes-Associated Protein (YAP). This study defines an alternative mechanism whereby Hippo protein kinases induce growth arrest via the phosphorylation of the long isoform of Angiomotin (Amot130). Specifically, serum starvation is found to activate the Hippo protein kinase, Large Tumor Suppressor (LATS), which phosphorylates the adapter protein Amot130 at serine-175. Importantly, wild-type Amot130 potently inhibits mammary epithelial cell growth, unlike the Amot130 serine-175 to alanine mutant, which cannot be phosphorylated at this residue. The growth-arrested phenotype of Amot130 is likely a result of its mechanistic response to LATS signaling. Specifically, LATS activity promotes the association of Amot130 with the ubiquitin ligase Atrophin-1 Interacting Protein 4 (AIP4). As a consequence, the Amot130-AIP4 complex amplifies LATS tumor suppressive signaling by stabilizing LATS protein steady state levels via preventing AIP4-targeted degradation of LATS. Additionally, AIP4 binding to Amot130 leads to the ubiquitination and stabilization of Amot130. In turn, the Amot130-AIP4 complex signals the ubiquitination and degradation of YAP. This inhibition of YAP activity by Amot130 requires both AIP4 and the ability of Amot130 to be phosphorylated by LATS. Together, these findings significantly modify the current view that the phosphorylation of YAP by Hippo protein kinases is sufficient for YAP inhibition and cellular growth arrest. Based upon these results, the inhibition of cellular growth in the absence of serum more accurately involves the stabilization of Amot130 and LATS, which together inhibit YAP activity and mammary epithelial cell growth.