Himanshu Malhotra

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.

IL-27 inhibits IFN-γ induced autophagy by concomitant induction of JAK/PI3 K/Akt/mTOR cascade and up-regulation of Mcl-1 in Mycobacterium tuberculosis H37Rv infected macrophages

Interleukin-27 (IL-27), a key immunoregulatory cytokine plays an important role in host response to mycobacterial infection as neutralization of IL-27 augments intracellular killing of mycobacteria. Autophagy has a pivotal role in host immunity and is regulated by various cytokines. Here, we report that IL-27 inhibits IFN-γ and starvation induced autophagy and as a result blocks phagosome maturation and promotes intracellular survival of Mycobacterium tuberculosis H37Rv. Addition of exogenous IL-27 induces the activation of mTOR through JAK/PI3 K pathway and inhibits IFN-γ stimulated autophagy. Furthermore, blockade of JAKs obstructs the inhibitory effect of IL-27 on IFN-γ induced autophagy. Besides this, IL-27 also up-regulates Mcl-1 through PI3 K pathway. We further show that in mTOR or Mcl-1 silenced THP-1 cells, IL-27 could no longer inhibit IFN-γ mediated autophagy in M. tuberculosis H37Rv infected cells. Altogether, our study demonstrates that IL-27 by concurrent activation of JAK/PI3 K/Akt/mTOR cascade as well as up-regulation of Mcl-1 inhibits IFN-γ induced autophagy and elimination of intracellular mycobacteria in macrophages.

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

ABSTRACT Iron (Fe2+, Fe3+) 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.

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.

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 in vivo 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. FASEB J. 31, 2638–2648 (2017). www.fasebj.org

Functional Characterization of Monomeric GTPase Rab1 in the Secretory Pathway of Leishmania

Leishmania secretes a large number of its effectors to the extracellular milieu. However, regulation of the secretory pathway in Leishmania is not well characterized. Here, we report the cloning, expression, and characterization of the Rab1 homologue from Leishmania. We have found that LdRab1 localizes in Golgi in Leishmania. To understand the role of LdRab1 in the secretory pathway of Leishmania, we have generated transgenic parasites overexpressing GFP-LdRab1:WT, GFP-LdRab1:Q67L (a GTPase-deficient dominant positive mutant of Rab1), and GFP-LdRab1:S22N (a GDP-locked dominant negative mutant of Rab1). Surprisingly, our results have shown that overexpression of GFP-LdRab1:Q67L or GFP-LdRab1:S22N does not disrupt the trafficking and localization of hemoglobin receptor in Leishmania. To determine whether the Rab1-dependent secretory pathway is conserved in parasites, we have analyzed the role of LdRab1 in the secretion of secretory acid phosphatase and Ldgp63 in Leishmania. Our results have shown that overexpression of GFP-LdRab1:Q67L or GFP-LdRab1:S22N significantly inhibits the secretion of secretory acid phosphatase by Leishmania. We have also found that overexpression of GFP-LdRab1:Q67L or GFP-LdRab1:S22N retains RFP-Ldgp63 in Golgi and blocks the secretion of Ldgp63, whereas the trafficking of RFP-Ldgp63 in GFP-LdRab1:WT-expressing cells is unaltered in comparison with control cells. Taken together, our results have shown that the Rab1-regulated secretory pathway is well conserved, and hemoglobin receptor trafficking follows an Rab1-independent secretory pathway in Leishmania.

Secreted multifunctional Glyceraldehyde-3-phosphate dehydrogenase sequesters lactoferrin and iron into cells via a non-canonical pathway

Lactoferrin is a crucial nutritionally important pleiotropic molecule and iron an essential trace metal for all life. The current paradigm is that living organisms have evolved specific membrane anchored receptors along with iron carrier molecules for regulated absorption, transport, storage and mobilization of these vital nutrients. We present evidence for the existence of non-canonical pathway whereby cells actively forage these vital resources from beyond their physical boundaries, by secreting the multifunctional housekeeping enzyme Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) into the extracellular milieu. This effect’s an autocrine/paracrine acquisition of target ligand into the cell. Internalization by this route is extensively favoured even by cells that express surface receptors for lactoferrin and involves urokinase plasminogen activator receptor (uPAR). We also demonstrate the operation of this phenomenon during inflammation, as an arm of the innate immune response where lactoferrin denies iron to invading microorganisms by chelating it and then itself being sequestered into surrounding host cells by GAPDH.

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

BackgroundObtaining sufficient quantities of recombinant M.tb proteins using traditional approaches is often unsuccessful. Several enzymes of the glycolytic cycle are known to be multifunctional, however relatively few enzymes from M.tb H37Rv have been characterized in the context of their enzymatic and pleiotropic roles. One of the primary reasons is the difficulty in obtaining sufficient amounts of functionally active protein.ResultsIn the current study, using M.tb glyceraldehyde-3-phosphate dehydrogenase (GAPDH) we demonstrate that expression in E. coli or M. smegmatis results in insolubility and improper subcellular localization. In addition, expression of such conserved multisubunit proteins poses the problem of heteromerization with host homologues. Importantly the expression host dramatically affected the yield and functionality of GAPDH in terms of both enzymatic activity and moonlighting function (transferrin binding). The applicability of this system was further confirmed using two additional enzymes i.e. M.tb Pyruvate kinase and Enolase.ConclusionsOur studies establish that the attenuated strain M.tb H37Ra is a suitable host for the expression of highly hydrophobic, conserved, multimeric proteins of M.tb H37Rv. Significantly, this expression host overcomes the limitations of E. coli and M. smegmatis expression and yields recombinant protein that is qualitatively superior to that obtained by traditional methods. The current study highlights the fact that protein functionality (which is an an essential requirement for all in vitro assays and drug development) may be altered by the choice of expression host.

Reverse overshot water-wheel retroendocytosis of apotransferrin extrudes cellular iron

ABSTRACT 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, 55Fe-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. Summary: We present evidence that retroendocytosis of apotransferrin mediated by multifunctional GAPDH extrudes excess cellular Fe in a mechanism reminiscent of pumping out of water by a reverse overshot water-wheel.

Mycobacterium tuberculosis Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) Functions as a Receptor for Human Lactoferrin

Iron is crucial for the survival of living cells, particularly the human pathogen Mycobacterium tuberculosis (M.tb) which uses multiple strategies to acquire and store iron. M.tb synthesizes high affinity iron chelators (siderophores), these extract iron from host iron carrier proteins such as transferrin (Tf) and lactoferrin (Lf). Recent studies have revealed that M.tb may also relocate several housekeeping proteins to the cell surface for capture and internalization of host iron carrier protein transferrin. One of the identified receptors is the glycolytic enzyme Glyceraldehyde-3-phosphate dehydrogenase (GAPDH). This conserved multifunctional protein has been identified as a virulence factor in several other bacterial species. Considering the close structural and functional homology between the two major human iron carrier proteins (Tf and Lf) and the fact that Lf is abundantly present in lung fluid (unlike Tf which is present in plasma), we evaluated whether GAPDH also functions as a dual receptor for Lf. The current study demonstrates that human Lf is sequestered at the bacterial surface by GAPDH. The affinity of Lf-GAPDH (31.7 ± 1.68 nM) is higher as compared to Tf-GAPDH (160 ± 24 nM). Two GAPDH mutants were analyzed for their enzymatic activity and interaction with Lf. Lastly, the present computational studies offer the first significant insights for the 3D structure of monomers and assembled tetramer with the associated co-factor NAD+. Sequence analysis and structural modeling identified the surface exposed, evolutionarily conserved and functional residues and predicted the effect of mutagenesis on GAPDH.