2007 — 2011 |
Rohatgi, Rajat |
K99Activity Code Description: To support the initial phase of a Career/Research Transition award program that provides 1-2 years of mentored support for highly motivated, advanced postdoctoral research scientists. R00Activity Code Description: To support the second phase of a Career/Research Transition award program that provides 1 -3 years of independent research support (R00) contingent on securing an independent research position. Award recipients will be expected to compete successfully for independent R01 support from the NIH during the R00 research transition award period. |
Biochemical Mechanisms of Hedgehog Signaling
[unreadable] DESCRIPTION (provided by applicant): Aberrant activation of the Hedgehog signaling pathway has been implicated in the genesis and maintenance of human cancers that develop in multiple organ systems, including the skin, brain, lung, prostate, and pancreas. It is hypothesized that the pathway drives tumorigenesis by causing the activation and expansion of tissue stem cells. There are significant gaps in our understanding of how the mammalian Hedgehog signal is received and transduced. Deciphering the detailed biochemical mechanism of Hedgehog signaling will allow the development of novel therapeutics and preventative strategies for these lethal cancers. During my PhD training with Dr. Marc Kirschner, I used a combination of protein biochemistry and microscopy to dissect a pathway that links cell surface signals to the actin cytoskeleton. To apply a similar biochemical and cell-biological approach to Hedgehog signaling, I have developed novel antibodies and confocal microscopy- based assays to study the dynamics and interactions of proteins in the pathway. Based on this work, I have constructed a new model for Hedgehog signaling that highlights the importance of the primary cilium, a tiny projection found on the surface of most cells that has been recently implicated in the Hedgehog pathway and in human disease. In the independent phase of this proposal, I plan to use the above tools to understand how localization of the receptor, Patched 1, to the primary cilium affects its ability to sense the Sonic Hedgehog signal and to activate downstream signaling in mouse embryonic fibroblasts and human tumor cells. I will also use an unbiased immunoaffinity purification approach to discover novel interacting proteins and post-translational modifications that link Hedgehog pathway components to the primary cilium. In the K99 phase of this proposal, Dr. Matthew Scott, a pioneer in the analysis of Hedgehog signaling in human and mouse cancer, will serve as mentor. My time in his laboratory will provide a critical opportunity to gain experience in techniques for the analysis of Hedgehog signal transduction in tissue culture fibroblasts, tumor cells and mice and to develop optical and biochemical probes for the proposed imaging and protein- interaction analysis. Most importantly, it will put me in an ideal position for a tenure-track position in an oncology department, where I plan to spend ~80-90% of my time in research and teaching and 10-20% of my time in the care of cancer patients. [unreadable] [unreadable] [unreadable] [unreadable]
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2011 |
Rohatgi, Rajat |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
High-Throughput Imaging of Hedgehog Pathway Components At the Primary Cilium
DESCRIPTION (provided by applicant): The Sonic hedgehog (Shh) pathway is involved in various forms of cancer, including brain, skin, pancreatic, ovarian, and gastrointestinal neoplasms. Drugs targeting this pathway hold great promise for oncology, but the current generation of Shh pathway inhibitors cause emergence of drug resistance. Most of the known compounds that inhibit the Shh pathway were discovered by high-throughput screening based on the expression of an artificial target luciferase construct. This method, however, has some major drawbacks and yields a large number of false hits. Here, we propose to develop a novel high-throughput screening method based on subcellular localization of Shh pathway components to an organelle called the primary cilium. Our method will not only be more robust than the currently used screening methods, but the high-content data generated in the course of screening can be used to probe molecular mechanisms of Shh signaling and ciliary function. We will use this newly developed high-throughput screening assay to discover new Shh inhibitors that will prevent cancer cells from evading treatment. PUBLIC HEALTH RELEVANCE: The discovery of new pharmaceuticals is accomplished by screening vast collections of chemical compounds for their ability to block specific biochemical events found only in disease states, such as cancer. Certain forms of cancer, such as a childhood brain tumor medulloblastoma, skin cancer, and tumors of the pancreas, colon, and ovaries, are characterized by increased movement of proteins to a tiny structure in the cell called the primary cilium. We will develop a method of automatic monitoring protein movement to the primary cilium, which will enable us to test hundreds of thousands of chemical molecules for their ability to block this transport phenomenon in hopes of discovering a potential new generation of anti-cancer drugs.
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2012 |
Rohatgi, Rajat |
DP2Activity Code Description: To support highly innovative research projects by new investigators in all areas of biomedical and behavioral research. |
Reconstructing Primary Cilia
DESCRIPTION (Provided by the applicant) Abstract: The primary cilium is a micron-scale signaling machine that projects from most cells in our bodies and is required for the detection and processing of optical, chemical and mechanical signals. Birth defects, obesity, mental retardation, and cystic organs are just a subset of the disease phenotypes seen in patients with damaged cilia. While an extensive parts list for cilia consisting of hundreds of proteins has been compiled over the last decade, a major challenge going forward is to understand how these proteins work together to allow the self-assembly and function of this remarkable structure. This proposal is based on the idea that major advances in our understanding of primary cilia will result from a systematic effort to reconstitute the assembly and function of this organelle in vitro using cell-free extracts. This biochemical analysis will draw on quantitative genetic interaction analysis of cilia proteins implicated in human diseases. I present a blueprint for this approach, integrating traditional protein biochemistry, modern imaging, and combinatorial RNAi, to understanding how centrosomes, membranes, and soluble factors cooperate to form functional cilia. This reconstitution will allow a high-resolution analysis of proteins encoded by cilia disease genes and, ultimately, the development of biochemical strategies to repair and even re-engineer ciliary function. Public Health Relevance: Most cells in our bodies communicate with the environment through an antenna-like structure called the primary cilium. Damage to cilia can cause a variety of diseases in humans, including birth defects, obesity, mental retardation, blindness, and kidney abnormalities. By understanding how signals are processed in this important cellular communication center, we hope to understand cilia-related diseases and develop new strategies to repair cilia function.
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2014 — 2017 |
Covey, Douglas F (co-PI) [⬀] Rohatgi, Rajat |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Signal Transduction by Oxysterols
DESCRIPTION (provided by applicant): Oxysterols are a class of endogenous cellular lipids derived from cholesterol that have been implicated in the pathophysiology of atherosclerosis, inborn errors of metabolism, inflammation and cancer. In many cases, the proteins and molecular pathways through which these enigmatic molecules exert their powerful biological effects remain unknown. We established collaboration between a synthetic organic chemist and a cell biologist to understand how oxysterols activate the Hedgehog (Hh) signaling system, a pathway that plays important roles in development, regeneration and cancer. We discovered that a specific oxysterol, 20(S)-OHC, is an allosteric activator of the 7-pass transmembrane protein Smoothened (Smo), a human oncoprotein and key drug target in oncology. This finding significantly expands the regulatory scope of oxysterols as signaling molecules, demonstrating their capacity to function as direct agonists for both a human on co-protein and a signaling receptor. Based on preliminary work, we hypothesize that endogenous 20(S)-OHC functions as a second-messenger in Hh signaling. Using a combination of mutagenesis, photo affinity labeling, and mass spectrometry, we will map the region of Smo that interacts with 20(S)-OHC to provide a biochemical portrait of this novel class of receptor-ligand interaction (Aim 1). Using quantitative mass spectrometry and a click chemistry-based imaging assay, we will ask if Hedgehog signaling can alter cellular levels or distribution of 20(S)-OHC (Aim 2). Finally, we will develop and characterize novel Hh pathway inhibitors that are inspired by oxysterol scaffolds (Aim 3). We expect three major outcomes to emerge from the successful completion of this project: (1) An answer to the question of how Smo is regulated in cells, perhaps the longest-standing mystery in the Hh pathway, (2) a biochemical understanding of how oxysterols engage and regulate 7-pass signaling receptors, and (3) the development of an integrative toolkit that can be deployed to dissect any other oxysterol- regulated cellular process. To accomplish these goals, we have recruited a team of investigators with complementary expertise in cell biology, protein biochemistry, synthetic chemistry, and mass spectrometry.
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2015 — 2016 |
Rohatgi, Rajat |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Molecular Dissection of Signal Transduction At Primary Cilia
DESCRIPTION (provided by applicant): Primary cilia are antenna-like organelles that project from the surfaces of most cells in our bodies and serve as important signaling centers in vertebrate development. Ciliary defects cause a number of inherited human diseases, the ciliopathies, manifested by phenotypes across organ systems. The molecular mechanisms by which signals are transmitted from the cilium to the nucleus remain poorly understood. The Hedgehog (Hh) pathway, implicated in development, cancer and regeneration, is orchestrated at cilia in vertebrates. We have used proteomic methods to identify a ciliary protein complex (the EvC complex) that positively regulates Hh signaling at primary cilia. Two of the proteins in this complex, Efcab7 and Iqce, are novel positive regulators of Hh signaling, and the other two, Evc and Evc2, are mutated in Ellis van Creveld syndrome and Weyers acrodental dysostosis, human ciliopathies characterized by defective Hh signaling in skeletal, cardiac and orofacial tissues. In the three aims of this proposal, we will test the model that this complex mediates signaling between the membrane protein Smoothened (Smo) and the Gli transcription factors at a unique signaling microdomain (the EvC Zone) at the base of cilia. The mechanism by which Smo regulates the Gli proteins remains one of the long-standing mysteries in vertebrate Hh signaling, despite the fact that this step is the target for Hh drugs in oncology. In Aim 1, we wil identify the specific step in signaling regulated by the EvC complex by testing for interactions with known Hh components and by detailed in vitro epistasis analysis enabled by CRISPR/Cas9-mediated single and double gene knockouts in cultured cells. Based on preliminary data, particular emphasis will be placed on the regulation of Protein Kinase A and Sufu, two universal negative regulators that function between Smo and the Gli proteins. Guided by deep phylogenetic analysis, we have identified conserved domains and sequence elements in EvC complex proteins and used a battery of binding assays to map the contact points between the four proteins. In Aim 2, we use a gene replacement strategy to test the cellular functions of these domains in mediating complex assembly, EvC zone localization, and Hh signaling. Finally, in Aim 3 we will expand our successful proteomic pipeline to identify proteins that bind to Efcab7, Iqce and Evc using tandem affinity purification and proximity biotinylation to complement the more directed investigations outlined in Aims 1 and 2. This work promises to reveal the molecular mechanism of a mysterious and therapeutically relevant step in Hh signaling and to illuminate the pathophysiology of a cilia-related congenital malformation syndrome.
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2016 — 2020 |
Rohatgi, Rajat |
R35Activity Code Description: To provide long term support to an experienced investigator with an outstanding record of research productivity. This support is intended to encourage investigators to embark on long-term projects of unusual potential. |
Biochemical and Cell Biological Mechanisms of Signal Transduction Through the Hedgehog Pathway
? DESCRIPTION (provided by applicant): The Hedgehog (Hh) pathway is a cell-cell communication system that plays important roles in development, regeneration and cancer. Hh signaling is orchestrated in vertebrates at primary cilia, antenna-like organelles that project fro the surfaces of most cells in our bodies and serve as signaling centers in development. Mutations in cilia genes cause a number of inherited human diseases called ciliopathies, many of which are characterized by birth defects and other phenotypes attributable to aberrant Hh signaling. Despite its importance as a target in cancer and regenerative medicine, many of the steps in Hh signaling remain poorly understood at the biochemical and cell biological level. My research program is focused on the major unsolved mechanistic questions in the vertebrate Hh pathway, with a particular emphasis on ciliary mechanisms that mediate signal propagation and transcriptional activation. The Hh signal is transmitted across the membrane by the 7-pass transmembrane protein Smo, the target for all anti-Hh drugs in clinical use. However, we do not understand the mechanism by which Smo is activated in response to Hh ligands, nor do we know how activated Smo in turn signals to the Glioblastoma (Gli) family of transcription factors. Primary cilia play an important role in both steps- Hh ligands promote the accumulation of Smo in the ciliary membrane, a critical step in signaling that eventually leads to the activation of Gl proteins as they traffic through the ciliary compartment. Delineating this mechanism will provide a valuable paradigm for how TM receptors relay signals from the ciliary membrane to the nucleus. Three major questions under investigation are (1) how Smo is activated by the main Hh receptor Patched 1 through endogenous small molecule ligands, (2) how activated Smo in the ciliary membrane transmits signals to the Gli proteins and (3) how Gli proteins are converted into transcriptional activators. We have made progress in each of these areas. Using new chemical tools to probe the interaction between Smo and oxysterols, endogenous lipids that can activate Hh signaling, we identified and structurally characterized a previously unknown ligand-binding site with regulatory potential in Smo. Using comparative proteomics, we identified a ciliary membrane protein complex that engages Smo at cilia in response to Hh signals and is required for Smo signaling. Finally, we have characterized both dynamic phosphorylation and protein association events that play critical role in Gli activation. Our work is supported by productive collaborations with investigators who have expertise in synthetic chemistry, structural biology, mass spectrometry and embryology. The successful completion of this project will provide (1) an answer to the question of Smo regulation, perhaps the longest-standing mystery in the Hh pathway, (2) an understanding of how ciliary protein trafficking drives signaling and how these processes are corrupted in ciliopathies, and (3) new strategies to monitor and modulate the pathway in Hh-related diseases.
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2019 |
Rohatgi, Rajat |
R35Activity Code Description: To provide long term support to an experienced investigator with an outstanding record of research productivity. This support is intended to encourage investigators to embark on long-term projects of unusual potential. |
Supplement Application For An Olympus Automated Microscope
? DESCRIPTION (provided by applicant): The Hedgehog (Hh) pathway is a cell-cell communication system that plays important roles in development, regeneration and cancer. Hh signaling is orchestrated in vertebrates at primary cilia, antenna-like organelles that project fro the surfaces of most cells in our bodies and serve as signaling centers in development. Mutations in cilia genes cause a number of inherited human diseases called ciliopathies, many of which are characterized by birth defects and other phenotypes attributable to aberrant Hh signaling. Despite its importance as a target in cancer and regenerative medicine, many of the steps in Hh signaling remain poorly understood at the biochemical and cell biological level. My research program is focused on the major unsolved mechanistic questions in the vertebrate Hh pathway, with a particular emphasis on ciliary mechanisms that mediate signal propagation and transcriptional activation. The Hh signal is transmitted across the membrane by the 7-pass transmembrane protein Smo, the target for all anti-Hh drugs in clinical use. However, we do not understand the mechanism by which Smo is activated in response to Hh ligands, nor do we know how activated Smo in turn signals to the Glioblastoma (Gli) family of transcription factors. Primary cilia play an important role in both steps- Hh ligands promote the accumulation of Smo in the ciliary membrane, a critical step in signaling that eventually leads to the activation of Gl proteins as they traffic through the ciliary compartment. Delineating this mechanism will provide a valuable paradigm for how TM receptors relay signals from the ciliary membrane to the nucleus. Three major questions under investigation are (1) how Smo is activated by the main Hh receptor Patched 1 through endogenous small molecule ligands, (2) how activated Smo in the ciliary membrane transmits signals to the Gli proteins and (3) how Gli proteins are converted into transcriptional activators. We have made progress in each of these areas. Using new chemical tools to probe the interaction between Smo and oxysterols, endogenous lipids that can activate Hh signaling, we identified and structurally characterized a previously unknown ligand-binding site with regulatory potential in Smo. Using comparative proteomics, we identified a ciliary membrane protein complex that engages Smo at cilia in response to Hh signals and is required for Smo signaling. Finally, we have characterized both dynamic phosphorylation and protein association events that play critical role in Gli activation. Our work is supported by productive collaborations with investigators who have expertise in synthetic chemistry, structural biology, mass spectrometry and embryology. The successful completion of this project will provide (1) an answer to the question of Smo regulation, perhaps the longest-standing mystery in the Hh pathway, (2) an understanding of how ciliary protein trafficking drives signaling and how these processes are corrupted in ciliopathies, and (3) new strategies to monitor and modulate the pathway in Hh-related diseases.
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2020 |
Fordyce, Polly Morrell (co-PI) [⬀] Rohatgi, Rajat Salzman, Julia [⬀] |
R56Activity Code Description: To provide limited interim research support based on the merit of a pending R01 application while applicant gathers additional data to revise a new or competing renewal application. This grant will underwrite highly meritorious applications that if given the opportunity to revise their application could meet IC recommended standards and would be missed opportunities if not funded. Interim funded ends when the applicant succeeds in obtaining an R01 or other competing award built on the R56 grant. These awards are not renewable. |
Orthocoding For Spatial Sequencing
Project Summary The 3D spatial context of a cell determines which genes and RNA isoforms it expresses, enabling specialized cell functions fundamental to multicellular life. In typical single-cell RNA-seq (scRNA-seq), the first step of cell dissociation erases the spatial context of the cell. This flaw creates an urgent need for a technology that has the same throughput of scRNA-seq but also encodes the cells? spatial context. Although a new wave of spatial transcriptomic technologies based on sequencing has emerged recently, all suffer from severe limitations: low efficiency (~1-2% of the Drop-Seq efficiency), providing 2D resolution only, failure to discriminate cell boundaries and requiring specialized or expensive equipment. These limitations are intrinsic and result from their shared reliance on cDNA synthesis in situ by from a solid support. Imaging-based technologies have higher spatial resolution but require more equipment, time for protocol execution, have limited gene measurement throughput, and cannot profile RNA isoforms or other sequence variants. To overcome these limitations in state-of-the-art spatial transcriptomic methods, we propose to develop Orthocode, an innovative paradigm for statistically-driven spatial transcriptomics, grounded in proof-of-principle molecular experiments, and cutting-edge statistical theory. Orthocode achieves > 50x or higher sensitivity compared to current approaches by encoding and recovering spatial information from simple, inexpensive and efficient molecular biology protocols. The experimental Orthocode protocol has two steps: 1) a pool of two types of ?location-encoding oligos? (a) barcoded emitter oligos produce copies of themselves that diffuse locally and (b) ?receptors? record the barcodes of nearby emitters are coupled to cells; 2) cells coupled to location- encoding oligos that have together record the spatial position of the cell, are isolated and input into scRNA-seq workflows, eg. Drop-seq and sequenced. Orthocode then employs a rigorous statistical analysis of the barcode profiles of location encoding oligos to triangulate the location of each sequenced cell. This rigorously reasoned experimental design and prototype development builds Orthocode from the simplest test systems to prototypes that will allow unprecedented spatial transcriptomic resolution in tissues to address a critical unmet need in biomedicine. The Orthocode paradigm can be generalized beyond RNA profiling to spatial measurements of proteins, DNA and epigenetic modifications and is a potential breakthrough innovation in deep-sequencing based spatial ?omics.
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2020 — 2021 |
Rohatgi, Rajat |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Unconventional Signaling by the R-Spondin Family of Wnt Regulators
Project Summary Unconventional signaling by the R-spondin family of WNT regulators The four R-spondins (RSPOs1-4) are vertebrate-specific secreted proteins that dramatically amplify signaling through the WNT/?-catenin pathway, a cell-cell communication system that regulates tissue patterning during development and regenerative responses in adults. Mutations that damage the RSPO signaling circuit cause structural birth defects, exemplified by limb truncation, lung hypoplasia and craniofacial malformations seen in patients with mutations in RSPO2. In adults, RSPOs function as niche-derived signals required for the renewal of epithelial stem cells in multiple tissues, including the intestine, skin, and bone. RSPOs amplify WNT signals by simultaneously binding to two receptors: Leucine-rich repeat-containing G-protein coupled receptors (LGRs) and the ZNRF3/RNF43 transmembrane E3 ubiquitin ligases. RSPOs increase WNT receptor levels only in specific cell types, such as intestinal stem cells, that express LGR receptors and thus allow the strength of the WNT signal (a potentially oncogenic signal) to be tightly controlled in time and space. While LGRs had been considered obligate high-affinity receptors for RSPOs, we made the unexpected discovery (along with other groups) that RSPOs can amplify WNT signals in the absence of all LGRs (?LGR-independent signaling?) or in the absence of ZNRF3/RNF43 (?ZNRF3/RNF43-independent signaling?). Distinct from the intensively studied LGR-mediated signaling in intestinal stem cells, LGR-independent signaling is particularly relevant for RSPO- related birth defects: it is the dominant mode of signaling during development of the limbs, lungs and cardiovascular system. Our preliminary results show that alternate, undiscovered receptors must exist that mediate some cellular responses to RSPOs. To identify these receptors and associated signaling components, we propose an innovative strategy that combines ligand engineering with genome-wide loss-of-function screens in cultured cells. In Aim 1, we use chimeric and mutant RSPO ligands in cell and organoid culture systems to test the hypothesis that Heparan Sulfate Proteoglycans (HSPGs) can function as RSPO co-receptors to transduce LGR-independent signals. In Aim 2, we use engineered RSPO ligands constrained to signal through either LGR-independent or ZNRF3/RNF43-independent modes to search for the required genes using CRISPR- based screens and haploid genetic screens. Successful completion of this project will identify the signaling machinery that mediates cellular responses to RSPOs in developmental and regenerative contexts, thereby improving our understanding of WNT-related structural birth defects and providing new strategies to enhance WNT-regulated regenerative responses in a tissue-selective manner.
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2021 |
Gunn, Teresa M (co-PI) [⬀] Lo, Cecilia W. Rohatgi, Rajat |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
A Protein Traffic Control System That Regulates Left-Right Patterning and Heart Development
Project Summary A protein traffic control system that regulates left-right patterning and heart development Structural birth defects represent the leading cause of infant deaths. Congenital Heart Defects (CHDs) are the most common structural birth defects, affecting ~40,000 babies each year. Amongst CHDs, a disproportionate burden of mortality and morbidity is due to ?severe? CHDs, defined as those that require surgery or a procedure before the first year of life. The molecular mechanisms that drive severe CHDs are incompletely understood, hampering preventative, diagnostic and therapeutic advances. Data from mouse studies and human birth registries have revealed a striking association between severe CHDs and heterotaxy, defects in left-right patterning of visceral organs. By integrating the expertise of three investigators in signal transduction, mouse development, human genetics and CHDs, we have identified a novel cell-surface ubiquitination pathway (the ?MMM pathway?) that plays widespread roles in the patterning of tissues during development. Disruption of this pathway leads to a characteristic syndrome of heterotaxy with severe CHDs in embryonic mice, along with defects in other tissues such as the limb, skeleton and face. Three dimensional reconstructions of the intracardiac anatomy of MMM mutant embryos reveal the presence of severe CHDs also often seen in human patients, including double outlet right ventricle and transposition of the great arteries. The MMM pathway is anchored at the cell surface by a receptor-like ubiquitin ligase complex composed of MEGF8, a single-pass transmembrane protein, and MGRN1, a RING superfamily E3 ligase. This unique membrane-tethered ubiquitination machine attenuates signaling through the iconic Hedgehog (Hh) pathway. Mechanistically, the MMM components decrease the abundance of the Hh transducer Smoothened (SMO) by direct ubiquitination, thereby reducing the sensitivity of target cells to Hh ligands. We propose to test the hypothesis that the MMM pathway functions as a traffic control system for signaling receptors that regulate left-right patterning and cardiac development. Our first aim is focused on understanding the biochemical function and developmental roles of MOSMO, an uncharacterized tetraspan membrane protein that we identified as a third component of the MMM pathway. In the second aim, we test whether the heterotaxy and CHDs seen in MMM mutant embryos are caused by elevated Hh signaling strength at critical periods in development and also search for other signaling receptors regulated by the MMM pathway. Finally, we leverage our comprehensive biochemical and developmental assays for MMM proteins to test the functionality of rare coding variants in MMM genes seen in human patients with severe CHDs. Successful completion of this project will uncover trafficking and signaling mechanisms that underlie the long-observed link between left-right patterning and heart development and consequently advance our understanding of the molecular pathophysiology of severe CHDs.
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2021 |
Rohatgi, Rajat |
R35Activity Code Description: To provide long term support to an experienced investigator with an outstanding record of research productivity. This support is intended to encourage investigators to embark on long-term projects of unusual potential. |
Signal Transduction in Development and Disease
Project Summary Signal transduction in development and disease (PI: Rohatgi) The goals of my research program are to uncover new regulatory mechanisms in cell-cell communication pathways, to understand how these mechanisms are damaged in disease states, and to devise new strategies to repair their function. Over the last 4.5 years, funding from the NIGMS has supported 23 publications across four different research areas in my laboratory: Hedgehog (Hh) signaling, WNT signaling, drug resistance mechanisms and intrinsically disordered proteins. Trainees involved in MIRA-supported research have won competitive fellowships (including a K99/R00 award from the NIGMS) and obtained independent group leader positions in both academia and industry. The next project period will tackle major unsolved problems in the vertebrate Hh and WNT signaling systems, two iconic cell-cell communication pathways that coordinate the construction of tissues during development and their subsequent maintenance throughout adult life. Despite the importance of these pathways in human diseases ranging from birth defects to cancer and degenerative conditions, many steps in Hh and WNT signaling remain poorly understood at the biochemical and cell biological level. In the Hh pathway, our focus is on understanding how a signal is detected at the cell surface and transmitted across the plasma membrane to transcriptional effectors in the cytoplasm. These signaling steps in the vertebrate Hh pathway depend on primary cilia, antenna-like organelles that project from the surfaces of most cells and are implicated in human birth defect syndromes called ?ciliopathies.? Major questions under investigation include (1) how Patched 1 (PTCH1), the receptor for Hh ligands, regulates the function of Smoothened (SMO), the protein that transmits the signal across the membrane, (2) how SMO is activated at primary cilia and (3) how SMO signals to the Glioblastoma (GLI) family of transcription factors. Our MIRA- supported work has led to a new paradigm in transmembrane signaling: the use of cholesterol accessibility in the ciliary membrane as a second messenger to communicate the signal between PTCH1 and SMO. Our focus in the WNT pathway is on the multi-protein ?-catenin destruction complex that suppresses WNT signaling by promoting the degradation of ?-catenin. Defects in this complex drive the vast majority of colorectal cancer, a disease with an increasing burden (especially amongst people <50 years of age) predicted to cause over 1 million deaths yearly by 2030. Our emphasis is on uncovering differences in the genetic and biochemical requirements for oncogenic (mutation-driven) and physiological (ligand-driven) WNT signaling, since any successful anti-WNT drug will have to distinguish between the two to achieve an acceptable therapeutic index. Our work is supported by long-term collaborations and embraces a broad range of techniques that span structural biology, lipid biochemistry, CRISPR/Cas9-based genetic screens and microscopy. The successful completion of this project will provide a deep mechanistic understanding of these fundamental cell-cell communication systems and new strategies to monitor and modulate these pathways in human diseases.
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