Anti-proliferative effect of novel primary cetyl alcohol derived sophorolipids against human cervical cancer cells HeLa

<div><p>Sophorolipids (SLs) are glycolipid biosurfactants that have been shown to display anticancer activity. In the present study, we report anti-proliferative studies on purified forms of novel SLs synthesized using cetyl alcohol as the substrate (referred as SLCA) and their anticancer mechanism in human cervical cancer cells. Antiproliferative effect of column purified SLCA fractions (A, B, C, D, E and F) was examined in panel of human cancer cell lines as well as primary cells. Among these fractions, SLCA B and C significantly inhibited the survival of HeLa and HCT 116 cells without affecting the viability of normal human umbilical vein endothelial cells (HUVEC). The two fractions were identified as cetyl alcohol sophorolipids with non-hydroxylated tail differing in the degree of acetylation on sophorose head group. At an IC₅₀ concentration SLCA B (16.32 μg ml^-1) and SLCA C (14.14 μg ml^-1) blocked the cell cycle progression of HeLa cells at G1/S phase in time-dependent manner. Moreover, SLCA B and SLCA C induced apoptosis in HeLa cells through an increase in intracellular Ca²⁺ leading to depolarization of mitochondrial membrane potential and increase in the caspase-3, -8 and -9 activity. All these findings suggest that these SLCAs could be explored for their chemopreventive potential in cervical cancer.</p></div

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

Moonlighting glycolytic protein glyceraldehyde-3-phosphate dehydrogenase (GAPDH): an evolutionarily conserved plasminogen receptor on mammalian cells

Prokaryotic pathogens establish infection in mammals by capturing the proteolytic enzyme plasminogen (Plg) onto their surface to digest host extracellular matrix (ECM). One of the bacterial surface Plg receptors is the multifunctional glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). In a defensive response, the host mounts an inflammatory response, which involves infiltration of leukocytes to sites of inflammation. This requires macrophage exit from the blood and migration across basement membranes, a phenomenon dependent on proteolytic remodeling of the ECM utilizing Plg. The ability of Plg to facilitate inflammatory cell recruitment critically depends on receptors on the surface of phagocyte cells. Utilizing a combination of biochemical, cellular, knockdown, and approaches, we demonstrated that upon inflammation, macrophages recruit GAPDH onto their surface to carry out the same task of capturing Plg to digest ECM to aid rapid phagocyte migration and combat the invading pathogens. We propose that GAPDH is an ancient, evolutionarily conserved receptor that plays a key role in the Plg-dependent regulation of macrophage recruitment in the inflammatory response to microbial aggression, thus pitting prokaryotic GAPDH against mammalian GAPDH, with both involved in a conserved role of Plg activation on the surface of their respective cells, to conflicting ends.-Chauhan, A. S., Kumar, M., Chaudhary, S., Patidar, A., Dhiman, A., Sheokand, N., Malhotra, H., Raje, C. I., Raje, M. Moonlighting glycolytic protein glyceraldehyde-3-phosphate dehydrogenase (GAPDH): an evolutionarily conserved plasminogen receptor on mammalian cells

Moonlighting Protein Glyceraldehyde-3-Phosphate Dehydrogenase: A Cellular Rapid-Response Molecule for Maintenance of Iron Homeostasis in Hypoxia

Hypoxia triggers a rapid increase in iron demand to meet the requirements of enhanced erythropoiesis. The mobilization of iron stores from macrophage to plasma as holo-transferrin (Tf) from where it is accessible to erythroid precursor cells impacts iron homeostasis. Despite the immediate need for enhanced iron uptake by bone marrow cells, numerous studies have shown that transferrin receptor levels do not rise until more than 24 hours after the onset of hypoxia, suggesting the existence of heretofore unknown rapid response cellular machinery for iron acquisition in the early stages of cellular hypoxia. We performed flow cytometry to measure cell surface levels of TfR1, GAPDH, and Tf binding after hypoxia treatment. We utilized FRET analysis and co-immunoprecipitation methods to establish the interaction between Tf and GAPDH. In the current study, we demonstrated that hypoxia induces K562 cells to translocate the cytosolic moonlighting protein glyceraldehyde-3-phosphate dehydrogenase (GAPDH) onto cell surfaces and into the extracellular milieu to acquire transferrin-bound iron, even while levels of the classical transferrin receptor TfR1 (CD71) remain suppressed. GAPDH knockdown confirmed this protein's role in transferrin acquisition. Interestingly, macrophages did not show enhanced levels of extracellular GAPDH under hypoxia. Our results suggest the role of GAPDH-mediated Tf uptake as a rapid response mechanism by which cells acquire iron during the early stages of hypoxia. This is a tissue-specific phenomenon for the distinct requirements of cells that are consumers of iron versus cells that play a role in iron storage and recycling. This rapid deployment of an abundantly available multipurpose molecule allows hypoxic cells to internalize more Tf and maintain enhanced iron supplies in the early stages of hypoxia before specialized receptors can be synthesized and deployed to the cell membrane

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 (TB), has evolved mechanisms to evade host defenses and persist within host cells. Host directed therapies (HDTs) 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 P2X7, 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 pathogen. In infected macrophages wherein GAPDH had been knocked down, we observed an inhibition of LL-37 mediated autophagy which was rescued by GAPDH over expression. 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 suggests a moonlighting role of GAPDH in regulating cellular phenotypic responses of LL-37 resulting in reduction of M.tb burden