The Role of Insulin-like Growth Factor-2 MRNA Binding Protein 1 (IMP1) in Intestinal Epithelial Homeostasis

The intestinal epithelium spans proliferating crypts at its base to differentiated villi at the luminal surface and renews itself every 3-5 days. It maintains a dynamic equilibrium between proliferation, differentiation and apoptosis. The regulation of homeostasis, response to injury, regeneration and transformation is a complex set of dynamic processes. mRNA binding proteins (RBPs) are newly recognized regulators of intestinal homeostasis. The RBP:mRNA complexes act as rheostats of key signaling processes by regulating expression of already transcribed RNAs. Their functional effects are tissue and context dependent. The manner in which RBPs operate and their interactions with other pivotal pathways in colorectal cancer provide a framework for new insights and potential therapeutic applications. This thesis focuses upon the interplay between LIN28B and IGF2 mRNA binding protein (IMP1) in the regulation of intestinal epithelial regeneration and malignant transformation, unraveling a new perspective on these processes. LIN28B, an mRNA binding protein, plays a critical role in regulating growth and proliferation in the intestinal epithelium. Previous work in our lab revealed that LIN28B promotes growth and tumorigenesis of the intestinal epithelium via suppression of mature let-7 miRNAs. LIN28B suppression of let-7 promotes upregulation of let-7 targets, including IMP1 (Insulin-like growth factor II mRNA-binding protein 1). Our lab has shown that transgenic mice expressing LIN28B from the mouse Vil1 promoter (Vil-Lin28b mice) have increased proliferation and tumor formation in the small intestine. IMP1 protein levels are upregulated in these mice epithelia and tumors but specific role of IMP1 in Lin28b-mediated tumorigenesis remains unknown. The current study tested the hypothesis that IMP1 may be required for LIN28B-mediated tumorigenesis and that LIN28B and IMP1 may cooperatively promote a tumor-initiating phenotype. Additionally, IMP1 hypomorphic mice exhibit severe intestinal growth defects, yet it’s role in adult epithelium is unclear. We investigated the mechanistic contribution of epithelial IMP1 to intestinal homeostasis and repair. We evaluated IMP1 expression in Crohn’s disease patients followed by unbiased ribosome profiling in IMP1 knockout cells. We used irradiation and dextran sodium sulphate (DSS) induced colitis as injury models to evaluate regeneration in intestinal epithelium lacking IMP1. These studies show that in the context of LIN28B overexpression, IMP1 loss led to increased tumor initiation and progression. Ribosome profiling and RNA sequencing revealed a potential role for IMP1 in negatively regulating the Wnt pathway, stem cell signature and other pathways associated with proliferation. This pro-proliferative effect with the loss of IMP1 has been previously observed in breast cancer and intestinal stroma. Additionally, IMP1 acts as a post-transcriptional regulator of gut epithelial repair post-colitis and irradiation, in part through modulation of autophagy. This study provides a new perspective on post-transcriptional regulation of autophagy as a contributing factor to the pathogenesis of inflammatory bowel disease. In total, these studies provide new insights into the role of IMP1 in regulating homeostasis, response to injury, and tumorigenesis in the intestine. It provides evidence that IMP1 regulates the expression of its targets at both the transcriptional and translational levels

Macromolecular recognition at the air-water interface: application of Langmuir-Blodgett technique

The proximate aim of this review is to investigate the specific interaction between two macromolecules, either two complementary strands of DNA or the binding of DNA with a protein. Although a lot of experiments have been done to address these issues, our aim here is different. We either create a dense brush of DNA chains at the air–water interface or orient a large protein, like RNA polymerase, such that they are amenable for specific interaction at the surface. The advantage of our system is that the macromolecules are stretched, oriented parallel to each other, and their concentrations can be made similar to these encountered in real nuclei. In this way we plan to construct an ‘artificial nucleus’. Other methods adopted so far can check for the possibility of collective behaviour and the effect of chain elongation or compaction. We have used Langmuir– Blodgett technique for the same and extensively performed FTIR and AFM experiments to monitor the L–B surface. Each macromolecule has been attached by one of its extremities to a hydrophobic buoy to keep it at the interface. Detailed thermodynamic analysis results in some interesting conclusions

Macromolecular recognition at the air-water interface: application of Langmuir-Blodgett technique

The proximate aim of this review is to investigate the specific interaction between two macromolecules, either two complementary strands of DNA or the binding of DNA with a protein. Although a lot of experiments have been done to address these issues, our aim here is different. We either create a dense brush of DNA chains at the air-water interface or orient a large protein, like RNA polymerase, such that they are amenable for specific interaction at the surface. The advantage of our system is that the macromolecules are stretched, oriented parallel to each other, and their concentrations can be made similar to these encountered in real nuclei. In this way we plan to construct an 'artificial nucleus'. Other methods adopted so far can check for the possibility of collective behaviour and the effect of chain elongation or compaction. We have used Langmuir Blodgett technique for the same and extensively performed FTIR and AFM experiments to monitor the L-B surface. Each macromolecule has been attached by one of its extremities to a hydrophobic buoy to keep it at the interface. Detailed thermodynamic analysis results in some interesting conclusions

Thermodynamic and spectroscopic studies on the nickel arachidate-RNA polymerase Langmuir-Blodgett monolayer

The Langmuir-Blodgett (LB) monolayers offer a unique system to study molecular interaction at the air-water interface with reduced dimensionality. In order to develop this further to follow macromolecular interactions at equilibrium, we first characterized the Ni (II)-arachidate (NiA) monolayer at varying conditions. Subsequently, the interaction between NiA and histidine-tagged RNA polymerase (HisRNAP) were also studied. LB films of arachidic acid-NiA and NiA-RNAP with different mole fractions were fabricated systematically. Surface pressure versus area per molecule (P-A) isotherms were registered, and the excess Gibbs energy of mixing was calculated. The LB films were then deposited on solid supports for Fourier transform infrared (FTIR) spectroscopic measurements. The FTIR spectra revealed the change in the amount of incorporated Ni (II) ions into the arachidic acid monolayer with the change in pH and the increasing mole fraction of RNAP in the NiA monolayer with its increasing concentration in the subphase. The system developed here seems to be robust and can be utilized to follow macromolecular interactions

Thermodynamic and Spectroscopic Studies on the Nickel Arachidate-RNA Polymerase Langmuir-Blodgett Monolayer

The Langmuir-Blodgett (LB) monolayers offer a unique system to study molecular interaction at the air-water interface with reduced dimensionality. In order to develop this further to follow macromolecular interactions at equilibrium, we first characterized the Ni (II)-arachidate (NiA) monolayer at varying conditions. Subsequently, the interaction between NiA and histidine-tagged RNA polymerase (HisRNAP) were also studied. LB films of arachidic acid-NiA and NiA-RNAP with different mole fractions were fabricated systematically. Surface pressure versus area per molecule (P-A) isotherms were registered, and the excess Gibbs energy of mixing was calculated. The LB films were then deposited on solid supports for Fourier transform infrared (FTIR) spectroscopic measurements. The FTIR spectra revealed the change in the amount of incorporated Ni (II) ions into the arachidic acid monolayer with the change in pH and the increasing mole fraction of RNAP in the NiA monolayer with its increasing concentration in the subphase. The system developed here seems to be robust and can be utilized to follow macromolecular interactions

Sequence Specific Interaction between Promoter DNA and Escherichia coli RNA Polymerase: Comparative Thermodynamic Analysis with One Immobilized Partner

Sequence specific interaction between DNA and protein molecules has been a subject of active investigation for decades now. Here, we have chosen single promoter containing bacteriophage Delta D-III T7 DNA and Escherichia coli RNA polymerase and followed their recognition at the air-water interface by using the surface plasmon resonance (SPR) technique, where the movement of one of the reacting species is restricted by way of arraying them on an immobilized support. For the Langmuir monolayer studies, we used a RNA polymerase with a histidine tag attached to one of its subunits, thus making it an xcellent substrate for Ni(II) ions, while the SPR Studies were done using biotin-labeled DNA immobilized on a streptavidin-coated chip. Detailed analysis of the thermodynamic parameters as a function of concentration and temperature revealed that the interaction of RNA polymerase with T7 DNA is largely entropy driven (83 (+/- 12) kcal mol(-1)) with a positive enthalpy of 13.6 (+/- 3.6) kcal mol(-1), The free energy of reaction determined by SPR and Langmuir-Blodgett technique was -11 (+/- 2) and -15.6 kcal mol(-1), respectively. The ability of these methods to retain the specificity of the recognition process was also established

Langmuir Monolayer as a Tool toward Visualization of a Specific DNA-Protein Complex

Immobilization and imaging of protein molecules and protein-DNAcomplexes on a Langmuir-Blodgett (LB) substrate have been explored here. We have prepared a nickel-arachidate (NiA) monolayer and characterized it through pressure-area isotherm on a LB trough. Recombinant RNA polymerase from Escherichia coli, where the largest subunit was replaced with the same gene having a series of histidine amino acids at the C-terminus end of the protein, was immobilized over the Ni-arachidate monolayer through a Ni(II)-histidine interaction. A single molecule of RNA polymerase could be seen through intermittent-contact atomic force microscopy (AFM). Under the condition of the formation of the LB monolayer, the enzyme molecules were arrayed and transcriptionally active. Interestingly, they could pick up sequence specificDNAmolecules from the subphase in an oriented fashion.Onthe other hand, preformed RNA polymerase Ni(II)-arachidate monolayers bound DNA haphazardly when no surface pressure was employed

Nonspecific Interaction between DNA and Protein allows for Cooperativity: A Case Study with Mycobacterium DNA Binding Protein

Different DNA-binding proteins have different interaction modes with DNA. Sequence-specific DNA protein interaction has been mostly associated with regulatory processes inside a cell, and as such extensive studies have been made. Adequate data is also available on nonspecific DNA protein interaction, as an intermediate to protein's search for its cognate partner. Multidomain nonspecific DNA protein interaction involving physical sequestering of DNA has often been implicated to regulate gene expression indirectly. However, data available on this type of interaction is limited. One such interaction is the binding of DNA with mycobacterium DNA binding proteins. We have used the Langmuir-Blodgett technique to evaluate for the first time the kinetics and thermodynamics of Mycobacterium smegmatis Dps 1 binding to DNA. By immobilizing one of the interacting partners, we have shown that, when a kinetic bottleneck is applied, the binding mechanism showed cooperative binding (n = 2.72) at lower temperatures, but the degree of cooperativity gradually reduces (n = 1.38) as the temperature was increased We have also compared the kinetics and thermodynamics of sequence-specific and nonspecific DNA protein interactions under the same set of conditions

Langmuir Monolayer as a Tool toward Visualization of a Specific DNA-Protein Complex

Immobilization and imaging of protein molecules and protein-DNAcomplexes on a Langmuir-Blodgett (LB) substrate have been explored here. We have prepared a nickel-arachidate (NiA) monolayer and characterized it through pressure-area isotherm on a LB trough. Recombinant RNA polymerase from Escherichia coli, where the largest subunit was replaced with the same gene having a series of histidine amino acids at the C-terminus end of the protein, was immobilized over the Ni-arachidate monolayer through a Ni(II)-histidine interaction. A single molecule of RNA polymerase could be seen through intermittent-contact atomic force microscopy (AFM). Under the condition of the formation of the LB monolayer, the enzyme molecules were arrayed and transcriptionally active. Interestingly, they could pick up sequence specificDNAmolecules from the subphase in an oriented fashion.Onthe other hand, preformed RNA polymerase Ni(II)-arachidate monolayers bound DNA haphazardly when no surface pressure was employed

Langmuir monolayer as a tool toward visualization of a specific DNA-protein complex

Immobilization and imaging of protein molecules and protein-DNA complexes on a Langmuir-Blodgett (LB) substrate have been explored here. We have prepared a nickel-arachidate (NiA) monolayer and characterized it through pressure-area isotherm on a LB trough. Recombinant RNA polymerase from Escherichia coli, where the largest subunit was replaced with the same gene having a series of histidine amino acids at the C-terminus end of the protein, was immobilized over the Ni-arachidate monolayer through a Ni(II)-histidine interaction. A single molecule of RNA polymerase could be seen through intermittent-contact atomic force microscopy (AFM). Under the condition of the formation of the LB monolayer, the enzyme molecules were arrayed and transcriptionally active. Interestingly, they could pick up sequence specific DNA molecules from the subphase in an oriented fashion. On the other hand, preformed RNA polymerase Ni(II)-arachidate monolayers bound DNA haphazardly when no surface pressure was employed