Glyceraldehyde-3-Phosphate Dehydrogenase Binds with Spike Protein and Inhibits the Entry of SARS-CoV-2 into Host Cells.

INTRODUCTION: Coronavirus disease 2019 caused by coronavirus-2 (SARS-CoV-2) has emerged as an aggressive viral pandemic. Health care providers confront a challenging task for rapid development of effective strategies to combat this and its long-term after effects. Virus entry into host cells involves interaction between receptor-binding domain (RBD) of spike (S) protein S1 subunit with angiotensin converting enzyme present on host cells. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a moonlighting enzyme involved in cellular glycolytic energy metabolism and micronutrient homeostasis. It is deployed in various cellular compartments and the extra cellular milieu. Though it is known to moonlight as a component of mammalian innate immune defense machinery, till date its role in viral restriction remains unknown. METHOD: Recombinant S protein, the RBD, and human GAPDH protein were used for solid phase binding assays and biolayer interferometry. Pseudovirus particles expressing four different strain variants of S protein all harboring ZsGreen gene as marker of infection were used for flow cytometry-based infectivity assays. RESULTS: Pseudovirus entry into target cells in culture was significantly inhibited by addition of human GAPDH into the extracellular medium. Binding assays demonstrated that human GAPDH binds to S protein and RBD of SARS-CoV-2 with nanomolar affinity. CONCLUSIONS: Our investigations suggest that this interaction of GAPDH interferes in the viral docking with hACE2 receptors, thereby affecting viral ingress into mammalian cells.

Chronic hyperglycemia impairs anti-microbial function of macrophages in response to Mycobacterium tuberculosis infection.

Diabetes mellitus (DM) is a major risk factor for tuberculosis (TB), though the underlying mechanisms linking DM and TB remain ambiguous. Macrophages are a key player in the innate immune response and their phagocytic ability is enhanced in response to microbial infections. Upon infection or inflammation, they also repel invading pathogens by generating; reactive oxygen species (ROS), reactive nitrogen species (RNS), pro-inflammatory cytokines (IL-1ß and IL-6), and anti-inflammatory cytokines (IL-10). However, the robustness of these innate defensive capabilities of macrophages when exposed to hyperglycemia remains unclear. In our current work, we explored the production of these host defense molecules in response to challenge with Mycobacterium tuberculosis (Mtb) infection and lipopolysaccharide (LPS) stimulation. Utilizing peritoneal macrophages from high-fat diet + streptozotocin induced diabetic mice and hyperglycemic THP-1-derived macrophages as model systems; we found that LPS stimulation and Mtb infection were ineffective in stimulating the production of ROS, RNS, and pro-inflammatory cytokines in cells exposed to hyperglycemia. On the contrary, an increase in production of anti-inflammatory cytokines was observed. To confirm the mechanism of decreased anti-bacterial activity of the diabetic macrophage, we explored activation status of these compromised macrophages and found decreased surface expression of activation (TLR-4) and differentiation markers (CD11b and CD11c). We postulate that this could be the cause for higher susceptibility for Mtb infection among diabetic individuals.

Excess iron aggravates the severity of COVID-19 infection.

Coronavirus disease-19 (COVID-19) can induce severe inflammation of the lungs and respiratory system. Severe COVID-19 is frequently associated with hyper inflammation and hyper-ferritinemia. High iron levels are known to trigger pro-inflammatory effects. Cumulative iron loading negatively impacts on a patients innate immune effector functions and increases the risk for infection related complications. Prognosis of severe acute respiratory SARS-CoV-2 patients may be impacted by iron excess. Iron is an essential co-factor for numerous essential cellular enzymes and vital cellular operations. Viruses hijack cells in order to replicate, and efficient replication requires an iron-replete host. Utilizing iron loaded cells in culture we evaluated their susceptibility to infection by pseudovirus expressing the SARS-CoV-2 spike protein and resultant cellular inflammatory response. We observed that, high levels of iron enhanced host cell ACE2 receptor expression contributing to higher infectivity of pseudovirus. In vitro Cellular iron overload also synergistically enhanced the levels of; reactive oxygen species, reactive nitrogen species, pro-inflammatory cytokines (IL-1ß, IL-6, IL-8 & TNF-α) and chemokine (CXCL-1&CCL-4) production in response to inflammatory stimulation of cells with spike protein. These results were confirmed using an in vivo mouse model. In future, limiting iron levels may be a promising adjuvant strategy in treating viral infection.

Regulation of Macrophage Cell Surface GAPDH Alters LL-37 Internalization and Downstream Effects in the Cell.

Mycobacterium tuberculosis (M.tb), the major causative agent of tuberculosis, has evolved mechanisms to evade host defenses and persist within host cells. Host-directed therapies against infected cells are emerging as an effective option. Cationic host defense peptide LL-37 is known to internalize into cells and induce autophagy resulting in intracellular killing of M.tb. This peptide also regulates the immune system and interacts with the multifunctional protein glyceraldehyde-3-phosphate dehydrogenase (GAPDH) inside macrophages. Our investigations revealed that GAPDH moonlights as a mononuclear cell surface receptor that internalizes LL-37. We confirmed that the surface levels of purinergic receptor 7, the receptor previously reported for this peptide, remained unaltered on M.tb infected macrophages. Upon infection or cellular activation with IFNγ, surface recruited GAPDH bound to and internalized LL-37 into endocytic compartments via a lipid raft-dependent process. We also discovered a role for GAPDH in LL-37-mediated autophagy induction and clearance of intracellular pathogens. In infected macrophages wherein GAPDH had been knocked down, we observed an inhibition of LL-37-mediated autophagy which was rescued by GAPDH overexpression. This process was dependent on intracellular calcium and p38 MAPK pathways. Our findings reveal a previously unknown process by which macrophages internalize an antimicrobial peptide via cell surface GAPDH and suggest a moonlighting role of GAPDH in regulating cellular phenotypic responses of LL-37 resulting in reduction of M.tb burden.

Host glyceraldehyde-3-phosphate dehydrogenase-mediated iron acquisition is hijacked by intraphagosomal Mycobacterium tuberculosis.

Availability of iron is a key factor in the survival and multiplication of Mycobacterium tuberculosis (M.tb) within host macrophage phagosomes. Despite host cell iron regulatory machineries attempts to deny supply of this essential micronutrient, intraphagosomal M.tb continues to access extracellular iron. In the current study, we report that intracellular M.tb exploits mammalian secreted Glyceraldehyde 3-phosphate dehydrogenase (sGAPDH) for the delivery of host iron carrier proteins lactoferrin (Lf) and transferrin (Tf). Studying the trafficking of iron carriers in infected cells we observed that sGAPDH along with the iron carrier proteins are preferentially internalized into infected cells and trafficked to M.tb containing phagosomes where they are internalized by resident mycobacteria resulting in iron delivery. Collectively our findings provide a new mechanism of iron acquisition by M.tb involving the hijack of host sGAPDH. This may contribute to its successful pathogenesis and provide an option for targeted therapeutic intervention.

Exposure of a specific pleioform of multifunctional glyceraldehyde 3-phosphate dehydrogenase initiates CD14-dependent clearance of apoptotic cells.

Rapid clearance of apoptotic cells by phagocytes is crucial for organogenesis, tissue homeostasis, and resolution of inflammation. This process is initiated by surface exposure of various 'eat me' ligands. Though phosphatidylserine (PS) is the best recognized general recognition ligand till date, recent studies have shown that PS by itself is not sufficient for clearance of apoptotic cells. In this study, we have identified a specific pleioform of GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) that functions as an 'eat me' signal on apoptotic cell surface. This specific form of GAPDH which is exposed on surface of apoptotic cells was found to interact with CD14 present on plasma membrane of phagocytes leading to their engulfment. This is the first study demonstrating the novel interaction between multifunctional GAPDH and the phagocytic receptor CD14 resulting in apoptotic cell clearance (efferocytosis).

Glyceraldehyde-3-Phosphate Dehydrogenase Facilitates Macroautophagic Degradation of Mutant Huntingtin Protein Aggregates.

Protein aggregate accumulation is a pathological hallmark of several neurodegenerative disorders. Autophagy is critical for clearance of aggregate-prone proteins. In this study, we identify a novel role of the multifunctional glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in clearance of intracellular protein aggregates. Previously, it has been reported that though clearance of wild-type huntingtin protein is mediated by chaperone-mediated autophagy (CMA), however, degradation of mutant huntingtin (mHtt with numerous poly Q repeats) remains impaired by this route as mutant Htt binds with high affinity to Hsc70 and LAMP-2A. This delays delivery of misfolded protein to lysosomes and results in accumulation of intracellular aggregates which are degraded only by macroautophagy. Earlier investigations also suggest that mHtt causes inactivation of mTOR signaling, causing upregulation of autophagy. GAPDH had earlier been reported to interact with mHtt resulting in cellular toxicity. Utilizing a cell culture model of mHtt aggregates coupled with modulation of GAPDH expression, we analyzed the formation of intracellular aggregates and correlated this with autophagy induction. We observed that GAPDH knockdown cells transfected with N-terminal mutant huntingtin (103 poly Q residues) aggregate-prone protein exhibit diminished autophagy. GAPDH was found to regulate autophagy via the mTOR pathway. Significantly more and larger-sized huntingtin protein aggregates were observed in GAPDH knockdown cells compared to empty vector-transfected control cells. This correlated with the observed decrease in autophagy. Overexpression of GAPDH had a protective effect on cells resulting in a decreased load of aggregates. Our results demonstrate that GAPDH assists in the clearance of protein aggregates by autophagy induction. These findings provide a new insight in understanding the mechanism of mutant huntingtin aggregate clearance. By studying the molecular mechanism of protein aggregate clearance via GAPDH, we hope to provide a new approach in targeting and understanding several neurodegenerative disorders.

Moonlighting glyceraldehyde-3-phosphate dehydrogenase (GAPDH) modulates protein aggregation.

Onset of protein aggregation reflects failure of the cellular folding machinery to keep aggregation-prone protein from misfolding and accumulating into a non-degradable state. FRET based analysis and biochemical data reveal that cytosolic prion (cyPrP) and httQ-103 interact with the multifunctional protein glyceraldehyde-3-phosphate dehydrogenase (GAPDH) leading to few detectable aggregates in GAPDH-over expressing cells.The preventive effect of GAPDH suggests that this abundant and long-lived cytoplasmic protein has an active role in the shielding and maintenance, in soluble form of proteins as heterogeneous as huntingtin and cyPrP.

Mycobacterium tuberculosis glyceraldehyde-3-phosphate dehydrogenase plays a dual role-As an adhesin and as a receptor for plasmin(ogen).

The spread of infection is directly determined by the ability of a pathogen to invade and infect host tissues. The process involves adherence due to host-pathogen interactions and traversal into deeper tissues. Mycobacterium tuberculosis (Mtb) primarily infects the lung but is unique in its ability to infect almost any other organ of the human host including immune privileged sites such as the central nervous system (CNS). The extreme invasiveness of this bacterium is not fully understood. In the current study, we report that cell surface Mtb glyceraldehyde-3-phosphate dehydrogenase (GAPDH) functions as a virulence factor by multiple mechanisms. Firstly, it serves as a dual receptor for both plasminogen (Plg) and plasmin (Plm). CRISPRi-mediated silencing of this essential enzyme confirmed its role in the recruitment of Plg/Plm. Our studies further demonstrate that soluble GAPDH can re-associate on Mtb bacilli to promote plasmin(ogen) recruitment. The direct association of plasmin(ogen) via cell surface GAPDH or by the re-association of soluble GAPDH enhanced bacterial adherence to and traversal across lung epithelial cells. Furthermore, the association of GAPDH with host extracellular matrix (ECM) proteins coupled with its ability to recruit plasmin(ogen) may endow cells with the ability of directed proteolytic activity vital for tissue invasion.