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.