Funded Projects

SARS-CoV-2 caused the COVID-19 pandemic and millions of deaths worldwide. Although vaccines were developed in record time, the natural cycle of immunity is short and the rise of new variants complicates the development of herd immunity. New drugs have been proposed as antivirals however, it is known that viruses also develop drug resistance. Therefore, it is necessary to find new therapeutic targets to cope with SARS-CoV-2 and new zoonotic coronaviruses to prevent new pandemics and another global health crisis. In this regard, a proven therapeutic target that is understudied is the inhibition of viral capping, a process that modifies the 5’UTR of the viral RNA to mimic the mammalian RNA. Capping prevents the degradation of viral RNA, improves translation, and prevents the detection of the innate cell immune system. Viral replication and capping take place in confined double-membrane vesicles (DMV) formed by host membranes and viral non-structural proteins (nsps). These processes cause severe stress and imbalance in the metabolism and bioenergetics of the host cell since high amounts of ATP and S-adenosylmethionine are used. Many metabolic pathways improve their efficiency by forming protein complexes, which avoid product inhibition and move the equilibrium of the reaction to the product. The replication-transcriptional (RTC) complex of SARS-CoV-2 was previously described; however little is known about the capping enzymes (nsp14-nsp10, nsp16-nsp10). Since capping enzymes are methyl transferases (MTases), which are strongly inhibited by the product of the reaction S-adenosylhomocysteine (SAH), and this product can only be hydrolyzed by a host SAH-hydrolase (AHCY), the need for host metabolites such as ATP, GTP,SAM and SAH hydrolysis, indicates a possible viral-host hybrid metabolon which is unknown. The overall goal of this proposal is to determine the existence of a hybrid viral-capping-host metabolic pool within the DMVs and the impact of these changes on the bioenergetics of the host. To address these knowledge gaps, we will take an integrated strategy using computational, biochemical, structural, and cell biology approaches. The aims of the proposal are: 1. Determine the existence of a viral methyltransferases-SAH hydrolase metabolon. Using AlphaFold 2 multimer software as a computational approach to predict the interactions of the methyl transferases nsp14-nsp16-nsp10 and nsp14, nsp16, nsp10 with AHCY. In parallel, these interactions will be tested by pull-down assays, using purified proteins and structural biology. Aim 2. Establish the localization of the methyltransferases from coronaviruses and S-adenosylmethionine hydrolase within the DMVs. The co-localization of capping enzymes nsp14, nsp16, and AHCY hydrolase within viral vesicles will be assessed by a time-course of MHV infection using lung-rat epithelial cells (L2), followed by subcellular fractionation, Co-immunoprecipitation as well as confocal microscopy using immunofluorescence. Aim 3. Assess the changes in the glycolysis and oxidative phosphorylation of the host upon viral replication and capping. The rate of external acidification (glycolysis-lactate production) and the rate of oxygen consumption (mitochondrial activity) will be measured in L2-MHV-infected cells using a seahorse analyzer. And the interaction of the glycolytic enzymes and mitochondria with SAM-metabolic enzymes and viral proteins in the DMV, will be tested as in aim 2.

Lysosomal damage is a major threat to cellular homeostasis and survival. Lysosomal damage has been implicated in many human diseases as well as normal ageing. However, the characterizations, functions and underlying molecular mechanisms of cellular responses to lysosomal damage (referred to as “lysosomal damage responses” hereafter) remain elusive. Our new publication uncovers that lysosomal damage can act as a hitherto unappreciated initiator of stress granule (SG) formation. SGs, the aggregation of messenger ribonucleoprotein condensates, help the cell to adapt to lysosomal damage situation and maintain cellular homeostasis by suppressing bulk translation and promoting selective protein synthesis. However, much remains to be understood regarding how lysosomal damage initiates SG formation as well as how SGs connect to the network of lysosomal damage responses to organize cellular adjustment. SGs have been implicated in the etiology of several disorders, and manipulation of SGs is emerging as a promising therapeutic avenue for disease treatment. The lysosomal damage as a signal for SG formation will be of relevance for multiple disease states. Thus, it is very important to study the intersection between lysosomal damage and SGs, which is relevant both to normal cellular functions and to dysfunctional lysosomes and SGs found in a wide range of human diseases. We have reported a set of galectin-based lysosomal damage responses to recognize, repair, recycle and replace damaged lysosomes. This galectin-based detection and signal-transduction system safeguards lysosomal quality and sets off downstream catabolic and anabolic processes of core cell regulatory mTOR and AMPK signaling. The discovery of SG formation upon lysosomal damage brings the consideration of global translational reprogramming into the network of lysosomal damage responses. Therefore, our overall objective is to identify the signaling pathway of SG formation upon lysosomal damage and its’s connectivity to the network of lysosomal damage responses to recognize, repair, recycle, replace and reprogramme damaged lysosomes. Our long-term goal is to understand cellular responses to lysosomal damage. The proposed studies detail the regulatory pathway of the new response to lysosomal damage, SG formation and link with our knowledge of galectin-based lysosomal damage responses could fill the gap in lysosomal damage field, extend the network of SGs and bring a paradigm shift in the current knowledge of cell stress opening a new field of study Completion of the proposed studies will provide

Cellular mechanical properties, collectively referred to as mechanotype, play a role in cell physiology and pathology, including cell proliferation, survival, metabolism, stem cell differentiation, immune cell migration, and cancer metastasis. Cell deformability and contractility are two key characteristics that determine the mechanotype of a cell. We have focused on understanding how cellular mechanotype is regulated by microenvironmental inputs that have been implicated in cell invasion, such as glucose levels. Hyperglycemia (HG) is prevalent in obesity and diabetes, which in turn are factors facilitating cancer progression.
The effects of HG on cellular mechanotype are the focus of this project. The mentored principal investigator (mPI) has developed a novel cell mechanotyping tool to probe cell deformability, called parallel microfiltration (PMF). In this project, using PMF and related technologies, we will define effects of glucose on cell mechanotype in two distinct model systems: breast cancer cells and macrophages. Our hypothesis is that the HG effects on cellular mechanotype have critical consequences on cell migration, invasiveness, and anoikis. Our long-term objective is to identify pathways that regulate cell mechanotype, migration, and survival under HG conditions, which is of translational relevance and health significance in the context of cancer and immune responses. The specific aims are:
1. Determine how glucose regulates the mechanotype of cancer and immune cells. In this aim, we will define the mechanistic basis of glucose-mediated mechanotype regulation that results in alterations in cell migration. We will use two models: (i) breast cancer cells; and (ii) macrophages. We will employ a novel mechanotyping technique invented by the mPI and collaborators.

2. Delineate how glucose-mediated mechanotype alterations affect cell survival. In this aim, we will determine how regulators and mediators of mechanotype dynamics influence anoikis of cancer cells.

Pilot Projects

In this project, we hypothesized that stromal/immune cells in the TM promote GBM cell invasion. Our hypothesis is based on our preliminary data generated using our unique multiple-sampling scheme and single-cell/single-nucleus analysis performed on matched TM-, tumor core- and SVZ-dissociated tissues of 3 genetically diverse GBM patients. Specifically, we found that the Activated Leukocyte Cell Adhesion Molecule (ALCAM) is upregulated in the TM compared to matched tumor core and SVZ. Interestingly, ALCAM expression has been correlated with poor outcome in several cancer models, including GBM. However, in contrast to other cancers, such as melanoma, in GBM the soluble form of this adhesion molecule promotes cell invasion and tumor growth. We will test our hypothesis by pursuing the following two specific aims:
#1: Define the cell types expressing ALCAM in the TM by single-cell/single-nucleus transcriptomics and functional phenotyping analysis; #2: Identify mechanisms contributing to glioblastoma invasion by functional phenotyping analysis of ALCAM-expressing cells isolated from the TM and treated in vitro with ionizing radiation and TMZ. 
Our expected outcome is to identify the mechanisms by which ALCAM-expressing cells in the TM contribute to the emergence of the recurrent tumor. 
Of note, this research represents a completely novel investigation in the laboratory of the PI and there is no overlap between this project and other funded projects.

Bacterial infection, environmental and chemical toxicants, and inflammation are common sources of injury that
damage the intestinal epithelial barrier and directly affect the proliferation and differentiation of epithelial cells
arising from intestinal crypts. Chronic infection or toxicant exposure often lead to systemic diseases.
Understanding the mechanistic underpinning that drive injury-induced epithelial changes is essential to
facilitate barrier regeneration and prevent long term dysfunction and disease. Enteroendocrine cells (EECs)
are derived from secretory progenitors and function as sentinels, communicating changes in the intestinal
milieu to the rest of the body. These specialized cells undergo hyperplasia in the early stages of various
intestinal metabolic and inflammatory diseases. It is now appreciated that EEC hyperplasia is likely a negative
response that contributes to intestinal and systemic dysfunction by abnormal hormone and peptide secretion.
Autophagy is an essential process in the function of intestinal secretory lineage cells, goblet and Paneth cells, and in the epithelial response to injury and regeneration. However, its role in EECs, also a secretory lineage cell, remains underdeveloped. Thus, our goals are to define autophagy’s role in EEC function and hormone secretion in the early stages of intestinal injury and recovery. We have previously developed human intestinal organoids (HIOs) in homeostasis and pathogen infected conditions, to characterize HIOs as an intestinal pathophysiological model. Our preliminary data of HIOs injured by environmental toxicants or bacterial toxins founds that there is a rapid increase in EEC differentiation, suggesting EEC hyperplasia is a ubiquitous response to intestinal injury. We found that autophagic genes and WNT pathway genes are upregulated in EECs and secretory progenitors, respectively. Thus, our central hypothesis is that WNT-driven transcription factors promote differentiation of secretory progenitors to EECs and the autophagy process is needed to proper function and hormone/peptide secretion. The following Aims will explore our hypothesis in a mechanistic manner. The first aim will determine the mechanism by which autophagy regulates EEC function and hormone/peptide secretion. The second aim will define the role of increasing WNT signaling in EEC differentiation. Successful completion of this proposal will help us understand the mechanistic underpinnings of intestinal injury that may promote disease progression from enteroendocrine cells using a relevant, yet biologically diverse model.

Graduated Projects

Increasing evidence is showing cells use organelle contact sites to transport lipids, which is an essential process to maintain cellular lipid homeostasis for human health. Indeed, the genes mediating formation of organelle contact sites have been linked to type II diabetes, neurodegenerative diseases, and cancer. Lysosomes play a critical role in processing and transporting lipids derived from different processes, such as autophagy. Autophagy maintains the basal level of lipid metabolites in response to environmental nutrient status, in which lysosomes not only process and deliver lipids but also regulate autophagy by controlling mTORC1, the master of cell growth and metabolism. However, the molecules constructing lysosome-organelle contact sites and the commanding signals regulating the contact dynamics are still largely unknown. Cancer cells have abnormal nutrient environments, exhibit different lysosome dynamics, and hijack autophagy to reprogram lipid metabolism, so it is very important to understand the role of lysosome dynamics in signaling and autophagy and identify the key molecules, which could serve as potential targets for anticancer purpose. The main objectives of this application are to study how lysosome change their dynamics (motility, positioning, and contacts) to function in signaling and lipid trafficking in response to excess environmental lipids in the context of obesity-related cancer. The central hypothesis is that during cancer development, altered nutrient environment changes lysosome dynamics and consequently alters lipid metabolism through mTORC1 and autophagy, which serves as an adaptive way to provide lipids and energy for cancer cells. Our recent discovery of the protein complex BORC and other components of the lysosome-dynamics machinery provides unprecedented opportunities to examine their roles in lipid metabolism. My preliminary results indicate deletion of BORC suppressed tumor growth and at cell level caused cholesterol accumulation in lysosomes and reduced lipid droplets. Further analysis revealed BORC-regulated HOPS complex interacted with ER complex NRZ, which is probably responsible for the formation of lysosome-ER contacts to transport cholesterol from lysosomes to ER. I will continue these studies to uncover how the regulation of lipid transport and signaling by lysosome dynamics promotes cancer cell malignancy and look for the potential anticancer targets for drug development.

Brown and Beige (or brite) adipose tissues burn lipid by converting chemical energy into heat, and have been considered as a new therapeutic target to counteract obesity. The regulation of thermogenic function in adipose tissue at the transcriptional level has been extensively studied in the past several years. However, the upstream signaling pathways that control the transcriptional machinery of thermogenic genes and effector mechanisms in brown or beige adipocytes remain largely unknown. Our recent study demonstrated that overactivation of mTOR Complex 1 (mTORC1) signaling is associated with impaired thermogenic function in vivo (Liu et al., 2014, Cell Metabolism). Consistent with this, our preliminary data showed that inactivation of mTORC1 by adipose-specific ablation of raptor, a key component of mTORC1, up-regulated the expression of thermogenic genes in brown adipose tissue (BAT) and inguinal white adipose tissue (iWAT or beige fat). In addition, we also found that the autophagy, a pathway downstream of mTORC1 is activated by cold stress and a β3-adrenoceptor agonist in vivo and in cells, and inhibition of autophagy diminishes β3-adrenoceptor agonist- induced UCP1 expression in primary adipocytes. In support of this, inhibition of autophagy by adipose-specific deletion of autophagy protein 7 (ATG7) suppresses basal and cold-induced energy expenditure, lipolysis and UCP1 expression in iWAT in vivo. Based on these findings, we hypothesize that autophagy plays a critical role in regulating the browning of white adipose tissue and mediates the beneficial effect of mTORC1 inhibition on thermogenesis in human brown adipocytes. We will first determine whether and how inhibiting mTORC1 and autophagy alters beige adipocyte differentiation and thermogenesis via cell-autonomous mechanisms. We will then investigate whether autophagy is an essential effector downstream of mTORC1 in regulating thermogenesis in WAT using adipose-specific autophagy-related protein 7 (ATG7)/raptor double KO mice. Lastly, we will delineate the role of mTORC1 and autophagy in regulating thermogenesis in primary human brown adipocytes. This study will lead to the identification of the mTORC1/autophagy pathway as a critical regulator of beige adipocyte differentiation and recruitment in response to various environmental factors. In addition, elucidation of the underlying signaling mechanisms involved in the browning of white fat may reveal promising new anti-obesity drug targets and lead to novel therapeutic approaches for obesity-associated metabolic diseases.

Brown and brown‐like (beige or brite) adipocytes in adipose tissue have strong anti‐obesity and anti‐ diabetic benefits. However, the mechanisms underlying the browning of white adipose tissue remains largely unknown. This proposed study investigates how mTORC1 and autophagy pathways cooperate to regulate the recruitment and activation of beige adipocytes in white adipose tissue. The results from this study may lead to the identification of potential therapeutic targets for the treatment of obesity and its associated metabolic diseases.

The apically located inter-cellular tight junctions (TJ) within the intestinal epithelium act as a paracellular barrier and prevent permeation of noxious luminal antigens. Loss of intestinal TJ barrier function is a key pathogenic factor in intestinal disorders and inflammatory bowel disease (IBD). Emerging evidence shows that defects in autophagy play an important role in the susceptibility, etiology, and progression of IBD. Although clinical data and animal studies show a direct link between defective intestinal TJ barrier and intestinal inflammation in IBD patients and animal models of IBD, the role of autophagy in the regulation of intestinal epithelial TJ barrier remains unknown. Our preliminary studies indicated that autophagy plays a key role in the enhancement of intestinal TJ barrier. Specifically, autophagy reduces paracellular TJ permeability by degradation of the pore forming tight junction protein claudin-2 and increasing protein levels of barrier protective transmembrane TJ protein occludin. Induction of autophagy causes a selective increase in lysosomal targeting of claudin-2 from the membrane and causes an increase in membrane retention of occludin. Thus, our central hypothesis is that autophagy selectively modulates TJ membrane protein composition to induce an enhancement of the intestinal TJ barrier. The overall specific aims of this application are: Specific Aim 1. To delineate the intracellular vesicular trafficking mechanisms in autophagy-induced enhancement of intestinal epithelial TJ barrier. Specific Aim 2. To elucidate the role of intracellular signaling in autophagy regulation of intestinal TJ barrier. Specific Aim 3. To delineate the role of autophagy in intestinal TJ barrier function and inflammation in animal models of IBD. This proposal will provide novel insights into the crucial role that autophagy plays in the homeostasis of intestinal barrier and bridge the gap in scientific knowledge that will be important for therapeutic efforts against IBD.

The tight junctions (TJ) present between intestinal epithelial cells act as a paracellular barrier and serve as a first line of defense against permeation of noxious antigens present in the intestinal lumen. Defective intestinal tight junction (TJ) barrier allows penetration of harmful luminal antigens in the gut which in turn leads to intestinal inflammation. Autophagy is a normal process that helps cell survival by recycling the nutrients and energy via degradation and turnover of the misfolded or unnecessary proteins. Recent studies have shown that mutations in autophagy related genes and defects in autophagy process are risk factors for inflammatory bowel disease (IBD) including Crohn disease (CD). The purpose of this grant application is to elucidate the mechanisms involved in autophagy-mediated enhancement of the intestinal epithelial tight junction barrier. Clinically, maintenance of mucosal barrier in the intestine is critical for the prevention of intestinal mucosal damage and therapeutic success in IBD cases. This study will provide novel insights into the crucial role of autophagy in enhancement of intestinal barrier and prevention of intestinal inflammation.

Although ignored until recently for not complying with the central dogma of molecular biology, non-coding RNAs (ncRNAs) are emerging as important novel regulators of diverge cellular processes. The capacity of ncRNAs to engage in “molecular multitasking” positions them to link multiple genetic risk factors for polygenic human genetic disorders, such as psychiatric diseases, into functional networks. Circular RNAs (circRNAs) are a novel category of non-coding RNAs that are derived from the back-splicing and covalent joining of exons and introns of protein-coding genes, yet lack the capacity to become translated into protein. Recent studies have suggested that circRNAs are enriched in the brain, are developmentally regulated, and are abundant in dendrites and synapses. Despite this, nothing is known about the function of circRNAs in the mammalian brain and their potential involvement in autophagy and neuropsychiatric disease. Autophagy is a coordinated lysosomal process for the degradation and recycling of cellular components, organelles, and protein aggregates that has been heavily implicated in the pathophysiology of neurodegenerative disorders. However, recent data suggest that numerous autophagy-associated genes display reduced expression in postmortem brains of subjects with psychiatric disorders and that neurons utilize autophagy during normal neuronal development and function. Despite the above, the importance of autophagy in psychiatric disease pathogenesis and pathophysiology has not been fully elucidated. Our proposed research aims in examining the role of altered in psychiatric disorders circRNAs that are either derived from autophagy-related genes or are upstream regulators of autophagy gene expression in neuronal autophagy and development and function. I specifically propose the following 3 aims. Aim 1: Test the hypothesis that alterations in psychiatric disease-associated circRNAs dysregulate linear gene expression related to autophagy and neuronal development and function. Aim 2: Test the hypothesis that manipulation of psychiatric disease-related circRNAs can alter autophagy and neuronal development and function. Aim 3: Test the hypothesis that treatment with autophagy-inducing drugs can rescue circRNA-mediated alterations in neuronal development and function. Taken together our proposed research will elucidate the mechanisms that underlie the effects of autophagy-associated circRNAs on neuronal autophagy and function, while examining in parallel their relevance for psychiatric disease therapeutics.

Received an independent R01; Completed April 2019

The relative ability of cell autonomous HIV-1 restriction factors to interfere with the viral life cycle contributes to a host’s level of susceptibility to infection. Pharmacological enhancement of restriction factor efficacy would be a novel approach to treating HIV infection. However, the mechanistic basis for HIV blockage by restriction factors is not completely understood hampering efforts to employ restriction factor-based host directed therapies. The tripartite motif (TRIM) family of proteins consists of more than 70 members in humans, several of which have been identified as antiviral restriction factors. In this role, TRIMs can diminish viral replication directly by interfering with the viral life cycle or indirectly by fine tuning cellular innate immune responses. TRIM family member TRIM5α accomplishes both of these: first, it prevents retroviral infection of cells by a hitherto unexplained mechanism. Second, TRIM5α also acts as a pattern recognition receptor, promoting the establishment of an antiviral cellular state via the activation of inflammatory signaling pathways upon retroviral recognition. Although TRIMs appear to employ multiple approaches in antiretroviral defense, one strikingly common feature among the TRIM family is that many if not all TRIMs are involved in the regulation and execution of autophagy. In addition to its role as a known defense mechanism against intracellular pathogens (including HIV-1), autophagy is also increasingly recognized as a means of reducing or fine tuning inflammation. Here, we propose to test the hypothesis that autophagy underlies TRIM action in protecting cells against HIV-1 infection and in modulating the TRIM-dependent inflammatory signaling. The studies proposed here have several overarching goals. First, they seek to improve our understanding of the molecular mechanism whereby rhesus TRIM5α both regulates autophagy and directs the autophagic degradation of incoming HIV-1 capsids (Aim 1). Second, they will determine if modulations of the autophagy pathway affect TRIM5α-dependent activation of pro-inflammatory signaling upon lentiviral infection. Finally, they will address whether human TRIMs other than TRIM5α that restrict HIV also employ autophagy in their antiviral actions (Aim 2). We have assembled a team of autophagy and HIV experts to address these questions. Our studies have the potential to uncover the mode of action of several known antiretroviral proteins and lay the groundwork for our understanding of how TRIMs as a family can both positively and negatively affect inflammation. We expect these studies to show that autophagy is a unifying aspect of diverse TRIM actions in HIV defense. Since autophagy can be pharmacologically manipulated, our findings may indicate that modulations of autophagy could be a therapeutic approach to dealing with TRIM-related diseases including HIV/AIDS. Our expertise in TRIMs and autophagy, along with the financial and institutional support to be provided should the COBRE application be funded will ensure successful completion of these aims.

Members of the TRIM family of proteins have been identified as being capable of limiting the ability of HIV to infect and replicate within cells, but the mechanism(s) underlying their action have not been fully uncovered. In this proposal, we test the hypothesis that autophagy, a pathway that can be manipulated with existing drugs, underlies the action of TRIM proteins in antiviral defense. These studies will further our understanding of the cellular functions of TRIM proteins at a molecular level and may provide the groundwork for therapeutic approaches to the diverse diseases, including HIV/AIDS, in which TRIMs play a role.

The function of T helper (TH) cells, the central organizers of adaptive immunity, is specified by the effector cytokines they produce. Regulation of TH cell cytokine secretion is not well understood and represents an important gap in our knowledge. Our recent data indicate that membrane- associated nucleic acid binding protein (Mnab, encoded by Rc3h2) is required for TH cell effector cytokine secretion. Mnab shares with its paralog Roquin (encoded by Rc3h1) a highly conserved N-terminus but possesses a unique hydrophobic C-terminus. Roquin is important for control of follicular helper T (Tfh) cell development through repression of Icos mRNA. Recently, Mnab was shown to play a redundant role with Roquin in Tfh cell development via repression of Icos and Ox40 mRNAs. Whether Mnab also targets other mRNAs and regulates function of other TH lineages is unclear. Our preliminary studies demonstrated that a Mnab deficiency led to profound defects in effector cytokine production in TH1, TH2 and TH17 cells, which differs from its known function, suggesting an important role of the distinct C-terminus of Mnab. Based on our preliminary data, we formulate a novel hypothesis: Mnab targets mRNAs encoding proteins in the stress response-autophagy pathway, a common pathway that is critical in TH cell effector cytokine production. The overall specific aims of this project are: Aim 1. Determine the role of Mnab and UPR-autophagy in TH cell function. Aim 2. Delineate the molecular mechanism whereby Mnab controls mRNA stability. Aim 3. Determine the role of Mnab in TH cell function in vivo using disease models. By addressing our hypothesis, these studies will reveal a novel post-transcription control mechanism of TH cell effector function. Manipulation of the corresponding pathways may be of therapeutic benefit in human disease, such as autoimmune and inflammatory disorders.

The function of T helper (TH) cells, the central organizers of adaptive immunity, is specified by the effector cytokines they produce. Regulation of TH cell cytokine secretion is not well understood and represents an important gap in our knowledge. In our preliminary studies, we found that in vitro, deficiency of membrane-associated nucleic acid binding protein (Mnab) led to profound defects in TH cell effector cytokine production. How Mnab regulates TH cell effector function is entirely unclear. We hypothesize that Mnab modulates stability of mRNAs encoding proteins in a common pathway that is critical in TH cell cytokine production. Our current data support a novel hypothesis that Mnab stabilizes mRNAs in the stress response-autophagy pathway that is required for secretory cells. In the proposed study, we will further understand how Mnab controls TH cell cytokine secretion and whether Mnab regulates TH cell function in disease models. Our studies will reveal a novel post-transcriptional control mechanism of TH cell effector function. Manipulation of the corresponding pathways may be of therapeutic benefit in human disease, such as autoimmune and inflammatory disorders.