R1 Retrotransposons in Drosophila melanogaster are Transcribed by RNA Polymerase I Upon Heat Shock

The ribosomal RNA genes of Drosophila melanogaster reside within centromere-proximal nucleolar organizers on both the X and Y chromosomes. Each locus contains between 200-300 tandem repeat rDNA units that encode 18S, 5.8S, and 28S ribosomal RNAs (rRNAs) for ribosome biogenesis. In arthropods like Drosophila, about 60% of rDNA genes are inserted with R1 and/or R2 retrotransposons at specific sites within the 28S regions; these units likely fail to produce functional 28S rRNA. We showed previously that R2 expression increases upon nucleolar stress caused by the loss of a ribosome assembly factor, the Nucleolar Phosphoprotein of 140 kDa (Nopp140). Here we show that R1 expression is selectively induced by heat shock. Actinomycin D, but not α-amanitin, blocked R1 expression in S2 cells upon heat shock, indicating that R1 is transcribed by Pol I. RT-PCR analysis confirmed read-through transcription by Pol I from the 28S gene region into R1. Using a genome wide precision run-on sequencing (PRO-seq) data set available at NCBI-GEO, we showed that Pol I activity on R1 elements is negligible under the normal non-heat shock condition but increases dramatically upon heat shock. We propose that prior to heat shock, Pol I pauses within ~350 bp of the 5’ end of R1 wherein we find ‘pause button’ like sequence motifs, and that heat shock releases Pol I for read-through transcription into R1

Mycobacterium tuberculosis acquires iron by cell-surface sequestration and internalization of human holo-transferrin

Mycobacterium tuberculosis (M.tb), which requires iron for survival, acquires this element by synthesizing iron-binding molecules known as siderophores and by recruiting a host iron-transport protein, transferrin, to the phagosome. The siderophores extract iron from transferrin and transport it into the bacterium. Here we describe an additional mechanism for iron acquisition, consisting of an M.tb protein that drives transport of human holo-transferrin into M.tb cells. The pathogenic strain M.tb H37Rv expresses several proteins that can bind human holo-transferrin. One of these proteins is the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH, Rv1436), which is present on the surface of M.tb and its relative Mycobacterium smegmatis. Overexpression of GAPDH results in increased transferrin binding to M.tb cells and iron uptake. Human transferrin is internalized across the mycobacterial cell wall in a GAPDH-dependent manner within infected macrophages

Moonlighting cell-surface GAPDH recruits apotransferrin to effect iron egress from mammalian cells

Iron (Fe, Fe) homeostasis is a tightly regulated process, involving precise control of iron influx and egress from cells. Although the mechanisms of its import into cells by iron carrier molecules are well characterized, iron export remains poorly understood. The current paradigm envisages unique functions associated with specialized macromolecules for its cellular import (transferrin receptors) or export (ferroportin, also known as SLC40A1). Previous studies have revealed that iron-depleted cells recruit glyceraldehyde-3- phosphate dehydrogenase (GAPDH), a multitasking, 'moonlighting' protein, to their surface for internalization of the iron carrier holotransferrin. Here, we report that under the converse condition of intracellular iron excess, cells switch the isoform of GAPDH on their surface to one that now recruits iron-free apotransferrin in close association with ferroportin to facilitate the efflux of iron. Increased expression of surface GAPDH correlated with increased apotransferrin binding and enhanced iron export from cells, a capability lost in GAPDH-knockdown cells. These findings were confirmed in vivo utilizing a rodent model of iron overload. Besides identifying for the first time an apotransferrin receptor, our work uncovers the two-way switching of multifunctional molecules to manage cellular micronutrient requirements

Mycobacterium tuberculosis H37Ra: a surrogate for the expression of conserved, multimeric proteins of M.tb H37Rv

Additional file 3. Details of primers used, experimental and theoretical molecular weights, pI values and details of post-translational modifications in GAPDH

Prevalence of Metabolic Syndrome in Urban India

Background. Metabolic syndrome (MS) is characterised by a constellation of individual risk factors of cardiovascular disease. Materials and Methods. The current study was a population-based survey of cohort of subjects in the metropolitan city of Mumbai. A total of 548 subjects, who attended the CARDIAC evaluation camp, were recruited in the study. Participants with complete fasting lipid profiles, blood glucose, and known cardiac risk markers were evaluated. Results. On applying modified NCEP ATP III, we found out that nearly 95% of the subjects had at least one abnormal parameter. We found the prevalence of MS in our study population to be 19.52%. The prevalence of MS in males was almost double than females (P = .008). The overall prevalence of BMI (>23 kg/m2) was 79.01%. Increased hypertriglyceridemia and decreased levels of HDL-C were found to be more in males (P < .0001). Conclusion. The low percentage of subjects with normal and controlled parameters suggests that there is a need for awareness programs and lifestyle interventions for the prevention and control of MS

Secreted glyceraldehye-3-phosphate dehydrogenase is a multifunctional autocrine transferrin receptor for cellular iron acquisition

Background The long held view is that mammalian cells obtain transferrin (Tf) bound iron utilizing specialized membrane anchored receptors. Here we report that, during increased iron demand, cells secrete the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) which enhances cellular uptake of Tf and iron. Methods These observations could be mimicked by utilizing purified GAPDH injected into mice as well as when supplemented in culture medium of model cell lines and primary cell types that play a key role in iron metabolism. Transferrin and iron delivery was evaluated by biochemical, biophysical and imaging based assays. Results This mode of iron uptake is a saturable, energy dependent pathway, utilizing raft as well as non-raft domains of the cell membrane and also involves the membrane protein CD87 (uPAR). Tf internalized by this mode is also catabolized. Conclusions Our research demonstrates that, even in cell types that express the known surface receptor based mechanism for transferrin uptake, more transferrin is delivered by this route which represents a hidden dimension of iron homeostasis. General significance Iron is an essential trace metal for practically all living organisms however its acquisition presents major challenges. The current paradigm is that living organisms have developed well orchestrated and evolved mechanisms involving iron carrier molecules and their specific receptors to regulate its absorption, transport, storage and mobilization. Our research uncovers a hidden and primitive pathway of bulk iron trafficking involving a secreted receptor that is a multifunctional glycolytic enzyme that has implications in pathological conditions such as infectious diseases and cancer

Comparison between the Biofilm Desorption Abilities of T4 and MS2 Coliphages

Biofilms are a collection of microorganisms that adhere to a surface where they continue to grow. Firmly established biofilms can be hazardous to human health. Chemical and biological as well as combination methods are being tested to control biofilms. The elucidation of the biofilm disruption capabilities of individual bacteriophages has received limited attention. Although the treatment of biofilms with a combination of bacteriophages is effective, the extent to which DNA and RNA coliphages individually desorb biofilms is not well understood. Here, we show that both T4 and MS2 coliphages desorbed natural biofilms. Individual incubations of the equivalent viral load of T4 and MS2 coliphages with natural biofilms resulted in similar desorption of these biofilms. We also note that the nutrient deprivation significantly reduced biofilm growth. However, the biofilm desorption upon nutrient deprivation was similar to that observed with both T4 and MS2 phages

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

Reverse overshot water-wheel retroendocytosis of apotransferrin extrudes cellular iron

Iron (Fe), a vital micronutrient for all organisms, must be managed judiciously because both deficiency or excess can trigger severe pathology. Although cellular Fe import is well understood, its export is thought to be limited to transmembrane extrusion through ferroportin (also known as Slc40a1), the only known mammalian Fe exporter. Utilizing primary cells and cell lines (including those with no discernible expression of ferroportin on their surface), we demonstrate that upon Fe loading, the multifunctional enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which is recruited to the cell surface, 'treadmills' apotransferrin in and out of the cell. Kinetic analysis utilizing labeled ligand, GAPDH-knockdown cells, (55)Fe-labeled cells and pharmacological inhibitors of endocytosis confirmed GAPDH-dependent apotransferrin internalization as a prerequisite for cellular Fe export. These studies define an unusual rapid recycling process of retroendocytosis for cellular Fe extrusion, a process mirroring receptor mediated internalization that has never before been considered for maintenance of cellular cationic homeostasis. Modulation of this unusual pathway could provide insights for management of Fe overload disorders

Exosomes: Tunable Nano Vehicles for Macromolecular Delivery of Transferrin and Lactoferrin to Specific Intracellular Compartment

Due to their abundant ubiquitous presence, rapid uptake and increased requirement in neoplastic tissue, the delivery of the iron carrier macromolecules transferrin (Tf) and lactoferrin (Lf) into mammalian cells is the subject of intense interest for delivery of drugs and other target molecules into cells. Utilizing exosomes obtained from cells of diverse origin we confirmed the presence of the multifunctional protein glyceraldehyde-3-phosphate dehydrogenase (GAPDH) which has recently been characterized as a Tf and Lf receptor. Using a combination of biochemical, biophysical and imaging based methodologies, we demonstrate that GAPDH present in exosomes captures Tf and Lf and subsequently effectively delivers these proteins into mammalian cells. Exosome vesicles prepared had a size of 51.2 ± 23.7 nm. They were found to be stable in suspension with a zeta potential (ζ-potential) of -28.16 ± 1.15 mV. Loading of Tf/Lf did not significantly affect ζ-potential of the exosomes. The carrier protein loaded exosomes were able to enhance the delivery of Tf/Lf by 2 to 3 fold in a diverse panel of cell types. Ninety percent of the internalized cargo via this route was found to be specifically delivered into late endosome and lysosomes. We also found exosomes to be tunable nano vehicles for cargo delivery by varying the amount of GAPDH associated with exosome. The current study opens a new avenue of research for efficient delivery of these vital iron carriers into cells employing exosomes as a nano delivery vehicle