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The antimalarial agents artemisinins inhibit cytomegalovirus (CMV) in vitro and in vivo, but their target(s) has been elusive. Using a biotin-labeled artemisinin, we identified the intermediate filament protein vimentin as an artemisinin target, validated by detailed biochemical and biological assays. We provide insights into the dynamic and unique modulation of vimentin, depending on the stage of human CMV (HCMV) replication. In vitro, HCMV entry and viral progeny are reduced in vimentin-deficient fibroblasts, compared with control cells. Similarly, mouse CMV (MCMV) replication in vimentin knockout mice is significantly reduced compared with controls in vivo, confirming the requirement of vimentin for establishment of infection. Early after HCMV infection of human foreskin fibroblasts vimentin level is stable, but as infection proceeds, vimentin is destabilized, concurrent with its phosphorylation and virus-induced calpain activity. Intriguingly, in vimentin-overexpressing cells, HCMV infection is reduced compared with control cells. Binding of artesunate, an artemisinin monomer, to vimentin prevents virus-induced vimentin degradation, decreasing vimentin phosphorylation at Ser-55 and Ser-83 and resisting calpain digestion. In vimentin-deficient fibroblasts, the anti-HCMV activity of artesunate is reduced compared with controls. In summary, an intact and stable vimentin network is important for the initiation of HCMV replication but hinders its completion. Artesunate binding to vimentin early during infection stabilizes it and antagonizes subsequent HCMV-mediated vimentin destabilization, thus suppressing HCMV replication. Our target discovery should enable the identification of vimentin-binding sites and compound moieties for binding.

Manganese (Mn) is an essential micronutrient required for the normal development of many organs, including the brain. Although its roles as a cofactor in several enzymes and in maintaining optimal physiology are well-known, the overall biological functions of Mn are rather poorly understood. Alterations in body Mn status are associated with altered neuronal physiology and cognition in humans, and either overexposure or (more rarely) insufficiency can cause neurological dysfunction. The resultant balancing act can be viewed as a hormetic U-shaped relationship for biological Mn status and optimal brain health, with changes in the brain leading to physiological effects throughout the body and vice versa. This review discusses Mn homeostasis, biomarkers, molecular mechanisms of cellular transport, and neuropathological changes associated with disruptions of Mn homeostasis, especially in its excess, and identifies gaps in our understanding of the molecular and biochemical mechanisms underlying Mn homeostasis and neurotoxicity.

Mammalian target of rapamycin complex 1 (mTORC1) promotes cell growth and proliferation in response to nutrients and growth factors. Amino acids induce lysosomal translocation of mTORC1 via the Rag GTPases. Growth factors activate Ras homolog enriched in brain (Rheb), which in turn activates mTORC1 at the lysosome. Amino acids and growth factors also induce the phospholipase D (PLD)–phosphatidic acid (PA) pathway, required for mTORC1 signaling through mechanisms that are not fully understood. Here, using human and murine cell lines, along with immunofluorescence, confocal microscopy, endocytosis, PLD activity, and cell viability assays, we show that exogenously supplied PA vesicles deliver mTORC1 to the lysosome in the absence of amino acids, Rag GTPases, growth factors, and Rheb. Of note, pharmacological or genetic inhibition of endogenous PLD prevented mTORC1 lysosomal translocation. We observed that precancerous cells with constitutive Rheb activation through loss of tuberous sclerosis complex subunit 2 (TSC2) exploit the PLD–PA pathway and thereby sustain mTORC1 activation at the lysosome in the absence of amino acids. Our findings indicate that sequential inputs from amino acids and growth factors trigger PA production required for mTORC1 translocation and activation at the lysosome.

Vascular endothelial growth factor A (VEGF) signals primarily through its cognate receptor VEGF receptor-2 (VEGFR-2) to control vasculogenesis and angiogenesis, key physiological processes in cardiovascular disease and cancer. In human umbilical vein endothelial cells (HUVECs), knockdown of protein kinase D-1 (PKD1) or PKD2 down-regulates VEGFR-2 expression and inhibits VEGF-induced cell proliferation and migration. However, how PKD regulates VEGF signaling is unclear. Previous bioinformatics analyses have identified binding sites for the transcription factor activating enhancer-binding protein 2 (AP2) in the VEGFR-2 promoter. Using ChIP analyses, here we found that PKD knockdown in HUVECs increases binding of AP2β to the VEGFR-2 promoter. Luciferase reporter assays with serial deletions of AP2-binding sites within the VEGFR-2 promoter revealed that its transcriptional activity negatively correlates with the number of these sites. Next we demonstrated that AP2β up-regulation decreases VEGFR-2 expression and that loss of AP2β enhances VEGFR-2 expression in HUVECs. In vivo experiments confirmed increased VEGFR-2 immunostaining in the spinal cord of AP2β knockout mouse embryos. Mechanistically, we observed that PKD phosphorylates AP2β at Ser258 and Ser277 and suppresses its nuclear accumulation. Inhibition of PKD activity with a pan-PKD inhibitor increased AP2β nuclear localization, and overexpression of both WT and constitutively active PKD1 or PKD2 reduced AP2β nuclear localization through a Ser258- and Ser277-dependent mechanism. Furthermore, substitution of Ser277 in AP2β increased its binding to the VEGFR-2 promoter. Our findings uncover evidence of a molecular pathway that regulates VEGFR-2 expression, insights that may shed light on the etiology of diseases associated with aberrant VEGF/VEGFR signaling.

The essential metal manganese becomes neurotoxic at elevated levels. Yet, the mechanisms by which brain manganese homeostasis is regulated are unclear. Loss-of-function mutations in SLC30A10, a cell surface–localized manganese efflux transporter in the brain and liver, induce familial manganese neurotoxicity. To elucidate the role of SLC30A10 in regulating brain manganese, we compared the phenotypes of whole-body and tissue-specific Slc30a10 knockout mice. Surprisingly, unlike whole-body knockouts, brain manganese levels were unaltered in pan-neuronal/glial Slc30a10 knockouts under basal physiological conditions. Further, although transport into bile is a major route of manganese excretion, manganese levels in the brain, blood, and liver of liver-specific Slc30a10 knockouts were only minimally elevated, suggesting that another organ compensated for loss-of-function in the liver. Additional assays revealed that SLC30A10 was also expressed in the gastrointestinal tract. In differentiated enterocytes, SLC30A10 localized to the apical/luminal domain and transported intracellular manganese to the lumen. Importantly, endoderm-specific knockouts, lacking SLC30A10 in the liver and gastrointestinal tract, had markedly elevated manganese levels in the brain, blood, and liver. Thus, under basal physiological conditions, brain manganese is regulated by activity of SLC30A10 in the liver and gastrointestinal tract, and not the brain or just the liver. Notably, however, brain manganese levels of endoderm-specific knockouts were lower than whole-body knockouts, and only whole-body knockouts exhibited manganese-induced neurobehavioral defects. Moreover, after elevated exposure, pan-neuronal/glial knockouts had higher manganese levels in the basal ganglia and thalamus than controls. Therefore, when manganese levels increase, activity of SLC30A10 in the brain protects against neurotoxicity.

Progesterone receptor membrane component 1 (PGRMC1) is a heme-binding protein implicated in a wide range of cellular functions. We previously showed that PGRMC1 binds to cytochromes P450 in yeast and mammalian cells and supports their activity. Recently, the paralog PGRMC2 was shown to function as a heme chaperone. The extent of PGRMC1 function in cytochrome P450 biology and whether PGRMC1 is also a heme chaperone are unknown. Here, we examined the function of Pgrmc1 in mouse liver using a knockout model and found that Pgrmc1 binds and stabilizes a broad range of cytochromes P450 in a heme-independent manner. Proteomic and transcriptomic studies demonstrated that Pgrmc1 binds more than 13 cytochromes P450 and supports maintenance of cytochrome P450 protein levels posttranscriptionally. In vitro assays confirmed that Pgrmc1 KO livers exhibit reduced cytochrome P450 activity consistent with reduced enzyme levels. Mechanistic studies in cultured cells demonstrated that PGRMC1 stabilizes cytochromes P450 and that binding and stabilization do not require PGRMC1 binding to heme. Importantly, Pgrmc1-dependent stabilization of cytochromes P450 is physiologically relevant, as Pgrmc1 deletion protected mice from acetaminophen-induced liver injury. Finally, evaluation of Y113F mutant Pgrmc1, which lacks the axial heme iron-coordinating hydroxyl group, revealed that proper iron coordination is not required for heme binding, but is required for binding to ferrochelatase, the final enzyme in heme biosynthesis. PGRMC1 was recently identified as the causative mutation in X-linked isolated pediatric cataract formation. Together, these results demonstrate a heme-independent function for PGRMC1 in cytochrome P450 stability that may underlie clinical phenotypes.

The CMP-sialic acid transporter SLC35A1 and UDP-galactose transporter SLC35A2 are two well-characterized nucleotide sugar transporters with distinctive substrate specificities. Mutations in either induce congenital disorders of glycosylation. Despite the biomedical relevance, mechanisms of substrate specificity are unclear. To address this critical issue, we utilized a structure-guided mutagenesis strategy and assayed a series of SLC35A2 and SLC35A1 mutants using a rescue approach. Our results suggest that three pockets in the central cavity of each transporter provide substrate specificity. The pockets comprise (1) nucleobase (residues E52, K55, and Y214 of SLC35A1; E75, K78, N235, and G239 of SLC35A2); (2) middle (residues Q101, N102, and T260 of SLC35A1; Q125, N126, Q129, Y130, and Q278 of SLC35A2); and (3) sugar (residues K124, T128, S188, and K272 of SLC35A1; K148, T152, S213, and K297 of SLC35A2) pockets. Within these pockets, two components appear to be especially critical for substrate specificity. Y214 (for SLC35A1) and G239 (for SLC35A2) in the nucleobase pocket appear to discriminate cytosine from uracil. Furthermore, Q129 and Q278 of SLC35A2 in the middle pocket appear to interact specifically with the β-phosphate of UDP while the corresponding A105 and A253 residues in SLC35A1 do not interact with CMP, which lacks a β-phosphate. Overall, our findings contribute to a molecular understanding of substrate specificity and coordination in SLC35A1 and SLC35A2 and have important implications for the understanding and treatment of diseases associated with mutations or dysregulations of these two transporters.

Beclin 1 (BECN1) is a key regulator of autophagy, a critical catabolic homeostasis pathway that involves sequestration of selected cytoplasmic components by multilayered vesicles called autophagosomes, followed by lysosomal fusion and degradation. BECN1 is a core component of class III phosphatidylinositol-3-kinase complexes responsible for autophagosome nucleation. Without heterologous binding partners, BECN1 forms an antiparallel homodimer via its coiled-coil domain (CCD). However, the last 16 CCD residues, composing an "overlap helix" (OH), have been crystallized in two mutually exclusive states: either as part of the CCD or packed against the C-terminal β-α repeated, autophagy-specific domain (BARAD). Here, using CD spectroscopy, isothermal titration calorimetry, and small-angle X-ray scattering, we show that in the homodimeric state, the OH transitions between these two different packing states, with the predominant state comprising the OH packed against the BARAD, contrary to expectations based on known BECN1 interactions with heterologous partners. We confirmed this observation by comparing the impact of mutating four residues that mediate packing of the OH against both the CCD and BARAD on structure and stability of the CCD, the OH+BARAD, and the two-domain CCD–BARAD. Last, we used cellular assays to demonstrate that mutation of these OH-interface residues abrogates starvation-induced up-regulation of autophagy but does not affect basal autophagy. In summary, we have identified a BECN1 helical region that transitions between packing as part of either one of two conserved domains (i.e. the CCD or the BARAD). Our findings have important implications for the relative stability of autophagy-inactive and autophagy-active BECN1 complexes.

Metalloprotease gp63 (Leishmania donovani gp63 (Ldgp63)) is a critical virulence factor secreted by Leishmania. However, how newly synthesized Ldgp63 exits the endoplasmic reticulum (ER) and is secreted by this parasite is unknown. Here, we cloned, expressed, and characterized the GTPase LdSar1 and other COPII components like LdSec23, LdSec24, LdSec13, and LdSec31 from Leishmania to understand their role in ER exit of Ldgp63. Using dominant-positive (LdSar1:H74L) and dominant-negative (LdSar1:T34N) mutants of LdSar1, we found that GTP-bound LdSar1 specifically binds to LdSec23, which binds, in turn, with LdSec24(1–702) to form a prebudding complex. Moreover, LdSec13 specifically interacted with His6–LdSec31(1–603), and LdSec31 bound the prebudding complex via LdSec23. Interestingly, dileucine 594/595 and valine 597 residues present in the Ldgp63 C-terminal domain were critical for binding with LdSec24(703–966), and GFP–Ldgp63L594A/L595A or GFP–Ldgp63V597S mutants failed to exit from the ER. Moreover, Ldgp63-containing COPII vesicle budding from the ER was inhibited by LdSar1:T34N in an in vitro budding assay, indicating that GTP-bound LdSar1 is required for budding of Ldgp63-containing COPII vesicles. To directly demonstrate the function of LdSar1 in Ldgp63 trafficking, we coexpressed RFP–Ldgp63 along with LdSar1:WT–GFP or LdSar1:T34N–GFP and found that LdSar1:T34N overexpression blocks Ldgp63 trafficking and secretion in Leishmania. Finally, we noted significantly compromised survival of LdSar1:T34N–GFP–overexpressing transgenic parasites in macrophages. Taken together, these results indicated that Ldgp63 interacts with the COPII complex via LdSec24 for Ldgp63 ER exit and subsequent secretion.

Manganese is an essential metal that becomes toxic at elevated levels. Loss-of-function mutations in SLC30A10, a cell-surface-localized manganese efflux transporter, cause a heritable manganese metabolism disorder resulting in elevated manganese levels and parkinsonian-like movement deficits. The underlying disease mechanisms are unclear; therefore, treatment is challenging. To understand the consequences of loss of SLC30A10 function at the organism level, we generated Slc30a10 knock-out mice. During early development, knock-outs were indistinguishable from controls. Surprisingly, however, after weaning and compared with controls, knock-out mice failed to gain weight, were smaller, and died prematurely (by ∼6–8 weeks of age). At 6 weeks, manganese levels in the brain, blood, and liver of the knock-outs were ∼20–60-fold higher than controls. Unexpectedly, histological analyses revealed that the brain and liver of the knock-outs were largely unaffected, but their thyroid exhibited extensive alterations. Because hypothyroidism leads to growth defects and premature death in mice, we assayed for changes in thyroid and pituitary hormones. At 6 weeks and compared with controls, the knock-outs had markedly reduced thyroxine levels (∼50–80%) and profoundly increased thyroid-stimulating hormone levels (∼800–1000-fold), indicating that Slc30a10 knock-out mice develop hypothyroidism. Importantly, a low-manganese diet produced lower tissue manganese levels in the knock-outs and rescued the phenotype, suggesting that manganese toxicity was the underlying cause. Our unanticipated discovery highlights the importance of determining the role of thyroid dysfunction in the onset and progression of manganese-induced disease and identifies Slc30a10 knock-out mice as a new model for studying thyroid biology.

δ, a small protein found in most Gram-positive bacteria was, for a long time, thought to be a subunit of RNA polymerase (RNAP) and was shown to be involved in recycling of RNAP at the end of each round of transcription. However, how δ participates in both up-regulation and down-regulation of genes in vivo remains unclear. We have recently shown, in addition to the recycling of RNAP, δ functions as a transcriptional activator by binding to an A-rich sequence located immediately upstream of the -35 element, consequently facilitating the open complex formation. The result had explained the mechanism of up-regulation of the genes by δ. Here, we show that Bacillus subtilis δ could also function as a transcriptional repressor. Our results demonstrate that δ binds to an A-rich sequence located near the -35 element of the spo0B promoter, the gene involved in the regulatory cascade of bacterial sporulation and inhibits the open complex formation due to steric clash with σ region 4.2. We observed a significant increase in the mRNA level of the spo0B gene in a δ-knock-out strain of B. subtilis compared with the wild-type. Thus, the results report a novel function of δ, and suggest the mechanism of down-regulation of genes in vivo by the protein.

A recent report suggested that the thioredoxin-dependent metabolic regulation, which is widespread in all domains of life, existed in methanogenic archaea about 3.5 billion years ago. We now show that the respective electron delivery enzyme (thioredoxin reductase, TrxR), although structurally similar to flavin-containing NADPH-dependent TrxRs (NTR), lacked an NADPH-binding site and was dependent on reduced coenzyme F420 (F420H2), a stronger reductant with a mid-point redox potential (E'0) of -360 mV; E'0 of NAD(P)H is -320 mV. Because F420 is a deazaflavin, this enzyme was named deazaflavin-dependent flavin-containing thioredoxin reductase (DFTR). It transferred electrons from F420H2 to thioredoxin via protein-bound flavin; Km values for thioredoxin and F420H2 were 6.3 and 28.6 μm, respectively. The E'0 of DFTR-bound flavin was approximately -389 mV, making electron transfer from NAD(P)H or F420H2 to flavin endergonic. However, under high partial pressures of hydrogen prevailing on early Earth and present day deep-sea volcanoes, the potential for the F420/F420H2 pair could be as low as -425 mV, making DFTR efficient. The presence of DFTR exclusively in ancient methanogens and mostly in the early Earth environment of deep-sea volcanoes and DFTR's characteristics suggest that the enzyme developed on early Earth and gave rise to NTR. A phylogenetic analysis revealed six more novel-type TrxR groups and suggested that the broader flavin-containing disulfide oxidoreductase family is more diverse than previously considered. The unprecedented structural similarities between an F420-dependent enzyme (DFTR) and an NADPH-dependent enzyme (NTR) brought new thoughts to investigations on F420 systems involved in microbial pathogenesis and antibiotic production.

Background: The mechanism of FGF16-mediated invasions in ovarian cancer cells was not reported.

Differential functions of Rab5 isoforms in endocytosis are not well characterized. Here, we cloned, expressed, and characterized Rab5a and Rab5b from Leishmania and found that both of them are localized in the early endosome. To understand the role of LdRab5 isoforms in different modes of endocytosis in Leishmania, we generated transgenic parasites overexpressing LdRab5a, LdRab5b, or their dominant-positive (LdRab5a:Q93L and LdRab5b:Q80L) or dominant-negative mutants (LdRab5a:N146I and LdRab5b:N133I). Using LdRab5a or its mutants overexpressing parasites, we found that LdRab5a specifically regulates the fluid-phase endocytosis of horseradish peroxidase and also specifically induced the transport of dextran-Texas Red to the lysosomes. In contrast, cells overexpressing LdRab5b or its mutants showed that LdRab5b explicitly controls receptor-mediated endocytosis of hemoglobin, and overexpression of LdRab5b:WT enhanced the transport of internalized Hb to the lysosomes in comparison with control cells. To unequivocally demonstrate the role of Rab5 isoforms in endocytosis in Leishmania, we tried to generate null-mutants of LdRab5a and LdRab5b parasites, but both were lethal indicating their essential functions in parasites. Therefore, we used heterozygous LdRab5a(+/-) and LdRab5b(+/-) cells. LdRab5a(+/-) Leishmania showed 50% inhibition of HRP uptake, but hemoglobin endocytosis was uninterrupted. In contrast, about 50% inhibition of Hb endocytosis was observed in LdRab5b(+/-) cells without any significant effect on HRP uptake. Finally, we tried to identify putative LdRab5a and LdRab5b effectors. We found that LdRab5b interacts with clathrin heavy chain and hemoglobin receptor. However, LdRab5a failed to interact with the clathrin heavy chain, and interaction with hemoglobin receptor was significantly less. Thus, our results showed that LdRab5a and LdRab5b differentially regulate fluid phase and receptor-mediated endocytosis in Leishmania.

Homozygous mutations in SLC30A10 lead to the development of familial manganese-induced parkinsonism. We previously demonstrated that SLC30A10 is a cell surface-localized manganese efflux transporter, and parkinsonism-causing mutations block its trafficking and efflux activity. Interestingly, other transporters in the SLC30 family mediate zinc efflux. Determining the mechanisms that allow SLC30A10 to transport manganese, which are unclear, is essential to understand its role in parkinsonism. Here, we generated a predicted structure of SLC30A10, based on the structure of the bacterial zinc transporter YiiP, and performed functional studies. In YiiP, side chains of residues Asp-45 and Asp-49 in the second and His-153 and Asp-157 in the fifth transmembrane segments coordinate zinc and are required for transport. In SLC30A10, the corresponding residues are Asn-43 and Asp-47 in the second and His-244 and Asp-248 in the fifth transmembrane segments. Surprisingly, although alanine substitution of Asp-248 abolished manganese efflux, that of Asn-43 and Asp-47 did not. Instead, side chains of charged or polar residues adjacent to Asp-248 in the first (Glu-25) or fourth (Asn-127) transmembrane segments were required. Further analyses revealed that residues His-333 and His-350 in the cytoplasmic C-terminal domain were required for full activity. However, the C-terminal domain failed to transfer manganese transport capability to a related zinc transporter. Overall, our results indicate that residues in the transmembrane and C-terminal domains together confer optimal manganese transport capability to SLC30A10 and suggest that the mechanism of ion coordination in the transmembrane domain of SLC30A10 may be substantially different from that in YiiP/other SLC30 proteins.

Most bacterial RNA polymerases (RNAP) contain five conserved subunits, viz. 2α, β, β′, and ω. However, in many Gram-positive bacteria, especially in fermicutes, RNAP is associated with an additional factor, called δ. For over three decades since its identification, it had been thought that δ functioned as a subunit of RNAP to enhance the level of transcripts by recycling RNAP. In support of the previous observations, we also find that δ is involved in recycling of RNAP by releasing the RNA from the ternary complex. We further show that δ binds to RNA and is able to recycle RNAP when the length of the nascent RNA reaches a critical length. However, in this work we decipher a new function of δ. Performing biochemical and mutational analysis, we show that Bacillus subtilis δ binds to DNA immediately upstream of the promoter element at A-rich sequences on the abrB and rrnB1 promoters and facilitates open complex formation. As a result, δ facilitates RNAP to initiate transcription in the second scale, compared with minute scale in the absence of δ. Using transcription assay, we show that δ-mediated recycling of RNAP cannot be the sole reason for the enhancement of transcript yield. Our observation that δ does not bind to RNAP holo enzyme but is required to bind to DNA upstream of the −35 promoter element for transcription activation suggests that δ functions as a transcriptional regulator.

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.

Exosomes are cell-derived extracellular vesicles thought to promote intercellular communication by delivering specific content to target cells. The aim of this study was to determine whether endothelial cell (EC)-derived exosomes could regulate the phenotype of hepatic stellate cells (HSCs). Initial microarray studies showed that fibroblast growth factor 2 induced a 2.4-fold increase in mRNA levels of sphingosine kinase 1 (SK1). Exosomes derived from an SK1-overexpressing EC line increased HSC migration 3.2-fold. Migration was not conferred by the dominant negative SK1 exosome. Incubation of HSCs with exosomes was also associated with an 8.3-fold increase in phosphorylation of AKT and 2.5-fold increase in migration. Exosomes were found to express the matrix protein and integrin ligand fibronectin (FN) by Western blot analysis and transmission electron microscopy. Blockade of the FN-integrin interaction with a CD29 neutralizing antibody or the RGD peptide attenuated exosome-induced HSC AKT phosphorylation and migration. Inhibition of endocytosis with transfection of dynamin siRNA, the dominant negative dynamin GTPase construct Dyn2K44A, or the pharmacological inhibitor Dynasore significantly attenuated exosome-induced AKT phosphorylation. SK1 levels were increased in serum exosomes derived from mice with experimental liver fibrosis, and SK1 mRNA levels were up-regulated 2.5-fold in human liver cirrhosis patient samples. Finally, S1PR2 inhibition protected mice from CCl4-induced liver fibrosis. Therefore, EC-derived SK1-containing exosomes regulate HSC signaling and migration through FN-integrin-dependent exosome adherence and dynamin-dependent exosome internalization. These findings advance our understanding of EC/HSC cross-talk and identify exosomes as a potential target to attenuate pathobiology signals.

The transition from the formation of the RNA polymerase (RNAP)-promoter open complex step to the productive elongation complex step involves "promoter escape" of RNAP. From the structure of RNAP, a promoter escape model has been proposed that suggests that the interactions between σR4 and RNAP and σR4 and DNA are destabilized upon transition to elongation. This accounts for the reduced affinity of σ to RNAP and stochastic release of σ. However, as the loss of interaction of σR4 with RNAP results in the release of intact σ, assessing this interaction remains challenging to be experimentally verified. Here we study the promoter escape model using a two-component σ factor YvrI and YvrHa from Bacillus subtilis that independently contributes to the functions of σR4 and σR2 in a RNAP-promoter complex. Our results show that YvrI, which mimics σR4, is released gradually as transcription elongation proceeds, whereas YvrHa, which mimics σR2 is retained throughout the elongation complexes. Thus our result validates the proposed model for promoter escape and also suggests that promoter escape involves little or no change in the interaction of σR2 with RNAP.

Adequate availability of iron is important for cellular energy metabolism. Catecholamines such as epinephrine and norepinephrine promote energy expenditure to adapt to conditions that arose due to stress. To restore the energy balance, epinephrine/norepinephrine-exposed cells may face higher iron demand. So far, no direct role of epinephrine/norepinephrine in cellular iron homeostasis has been reported. Here we show that epinephrine/norepinephrine regulates iron homeostasis components such as transferrin receptor-1 and ferritin-H in hepatic and skeletal muscle cells by promoting the binding of iron regulatory proteins to iron-responsive elements present in the UTRs of transferrin receptor-1 and ferritin-H transcripts. Increased transferrin receptor-1, decreased ferritin-H, and increased iron-responsive element-iron regulatory protein interaction are also observed in liver and muscle tissues of epinephrine/norepinephrine-injected mice. We demonstrate the role of epinephrine/norepinephrine-induced generation of reactive oxygen species in converting cytosolic aconitase (ACO1) into iron regulatory protein-1 to bind iron-responsive elements present in UTRs of transferrin receptor-1 and ferritin-H. Our study further reveals that mitochondrial iron content and mitochondrial aconitase (ACO2) activity are elevated by epinephrine/norepinephrine that are blocked by the antioxidant N-acetyl cysteine and iron regulatory protein-1 siRNA, suggesting involvement of reactive oxygen species and iron regulatory protein-1 in this mechanism. This study reveals epinephrine and norepinephrine as novel regulators of cellular iron homeostasis.

Alcoholism can result in fatty liver that can progress to steatohepatitis, cirrhosis, and liver cancer. Mice fed alcohol develop fatty liver through endocannabinoid activation of hepatic CB(1) cannabinoid receptors (CB(1)R), which increases lipogenesis and decreases fatty acid oxidation. Chronic alcohol feeding also up-regulates CB(1)R in hepatocytes in vivo, which could be replicated in vitro by co-culturing control hepatocytes with hepatic stellate cells (HSC) isolated from ethanol-fed mice, implicating HSC-derived mediator(s) in the regulation of hepatic CB(1)R (Jeong, W. I., Osei-Hyiaman, D., Park, O., Liu, J., Bátkai, S., Mukhopadhyay, P., Horiguchi, N., Harvey-White, J., Marsicano, G., Lutz, B., Gao, B., and Kunos, G. (2008) Cell Metab. 7, 227-235). HSC being a rich source of retinoic acid (RA), we tested whether RA and its receptors may regulate CB(1)R expression in cultured mouse hepatocytes. Incubation of hepatocytes with RA or RA receptor (RAR) agonists increased CB(1)R mRNA and protein, the most efficacious being the RARgamma agonist CD437 and the pan-RAR agonist TTNPB. The endocannabinoid 2-arachidonoylglycerol (2-AG) also increased hepatic CB(1)R expression, which was mediated indirectly via RA, because it was absent in hepatocytes from mice lacking retinaldehyde dehydrogenase 1, the enzyme catalyzing the generation of RA from retinaldehyde. The binding of RARgamma to the CB(1)R gene 5' upstream domain in hepatocytes treated with RAR agonists or 2-AG was confirmed by chromatin immunoprecipitation and electrophoretic mobility shift and antibody supershift assays. Finally, TTNPB-induced CB(1)R expression was attenuated by small interfering RNA knockdown of RARgamma in hepatocytes. We conclude that RARgamma regulates CB(1)R expression and is thus involved in the control of hepatic fat metabolism by endocannabinoids.

Ceruloplasmin (Cp), a copper-containing protein, plays a significant role in body iron homeostasis as aceruloplasminemia patients and Cp knock-out mice exhibit iron overload in several tissues including liver and brain. Several other functions as oxidant, as antioxidant, and in nitric oxide metabolism are also attributed to Cp. Despite its role in iron oxidation and other biological oxidation reactions the regulation of Cp by reactive oxygen species (ROS) remains unexplored. Cp is synthesized in liver as a secretory protein and predominantly as a glycosylphosphatidylinositol-anchored membrane-bound form in astroglia. In this study we demonstrated that Cp expression is decreased by an mRNA decay mechanism in response to extracellular (H2O2) or intracellular oxidative stress (by mitochondrial chain blockers rotenone or antimycin A) in both hepatic and astroglial cells. The promotion of Cp mRNA decay is conferred by its 3′-untranslated region (UTR). When chloramphenicol acetyltransferase (CAT) gene was transfected as a chimera with Cp 3′-UTR in hepatic or astroglial cells, in response to either H2O2, rotenone, or antimycin A, the expression of CAT transcript was decreased, whereas expression of a 3′-UTR-less CAT transcript remained unaffected. RNA gel shift assay showed significant reduction in 3′-UTR-binding protein complex by ROS in both cell types that was reversed by the antioxidant N-acetylcysteine suggesting that ROS affects RNA-protein complex formation to promote Cp mRNA decay. Our finding is not only the first demonstration of regulation of Cp by ROS by a novel post-transcriptional mechanism but also provides a mechanism of iron deposition in neurodegenerative diseases.

Macrophage infiltration into adipose tissue during obesity and their phenotypic conversion from anti-inflammatory M2 to proinflammatory M1 subtype significantly contributes to develop a link between inflammation and insulin resistance; signaling molecule(s) for these events, however, remains poorly understood. We demonstrate here that excess lipid in the adipose tissue environment may trigger one such signal. Adipose tissue from obese diabetic db/db mice, high fat diet-fed mice, and obese diabetic patients showed significantly elevated fetuin-A (FetA) levels in respect to their controls; partially hepatectomized high fat diet mice did not show noticeable alteration, indicating adipose tissue to be the source of this alteration. In adipocytes, fatty acid induces FetA gene and protein expressions, resulting in its copious release. We found that FetA could act as a chemoattractant for macrophages. To simulate lipid-induced inflammatory conditions when proinflammatory adipose tissue and macrophages create a niche of an altered microenvironment, we set up a transculture system of macrophages and adipocytes; the addition of fatty acid to adipocytes released FetA into the medium, which polarized M2 macrophages to M1. This was further confirmed by direct FetA addition to macrophages. Taken together, lipid-induced FetA from adipocytes is an efficient chemokine for macrophage migration and polarization. These findings open a new dimension for understanding obesity-induced inflammation.