Biochemical and biophysical characterization of the RAS family small GTPase protein DiRAS3.

DiRAS3, also called ARHI, is a RAS (sub)family small GTPase protein that shares 50-60% sequence identity with H-, K-, and N-RAS, with substitutions in key conserved G-box motifs and a unique 34 amino acid extension at its N-terminus. Unlike the RAS proto-oncogenes, DiRAS3 exhibits tumor suppressor properties. DiRAS3 function has been studied through genetics and cell biology, but there has been a lack of understanding of the biochemical and biophysical properties of the protein, likely due to its instability and poor solubility. To overcome this solubility issue, we engineered a DiRAS3 variant (C75S/C80S), which significantly improved soluble protein expression in E. coli. Recombinant DiRAS3 was purified by Ni-NTA and size exclusion chromatography (SEC). Concentration dependence of the SEC chromatogram indicated that DiRAS3 exists in monomer-dimer equilibrium. We then produced truncations of the N-terminal (ΔN) and both (ΔNC) extensions to the GTPase domain. Unlike full-length DiRAS3, the SEC profiles showed that ΔNC is monomeric while ΔN was monomeric with aggregation, suggesting that the N and/or C-terminal tail(s) contribute to dimerization and aggregation. The 1H-15N HSQC NMR spectrum of ΔNC construct displayed well-dispersed peaks similar to spectra of other GTPase domains, which enabled us to demonstrate that DiRAS3 has a GTPase domain that can bind GDP and GTP. Taken together, we conclude that, despite the substitutions in the G-box motifs, DiRAS3 can switch between nucleotide-bound states and that the N- and C-terminal extensions interact transiently with the GTPase domain in intra- and inter-molecular fashions, mediating weak multimerization of this unique small GTPase.

Wild-type sTREM2 blocks Aß aggregation and neurotoxicity, but the Alzheimer's R47H mutant increases Aß aggregation.

TREM2 is a pattern recognition receptor, expressed on microglia and myeloid cells, detecting lipids and Aß and inducing an innate immune response. Missense mutations (e.g., R47H) of TREM2 increase risk of Alzheimer's disease (AD). The soluble ectodomain of wild-type TREM2 (sTREM2) has been shown to protect against AD in vivo, but the underlying mechanisms are unclear. We show that Aß oligomers bind to cellular TREM2, inducing shedding of the sTREM2 domain. Wild-type sTREM2 bound to Aß oligomers (measured by single-molecule imaging, dot blots, and Bio-Layer Interferometry) inhibited Aß oligomerization and disaggregated preformed Aß oligomers and protofibrils (measured by transmission electron microscopy, dot blots, and size-exclusion chromatography). Wild-type sTREM2 also inhibited Aß fibrillization (measured by imaging and thioflavin T fluorescence) and blocked Aß-induced neurotoxicity (measured by permeabilization of artificial membranes and by loss of neurons in primary neuronal-glial cocultures). In contrast, the R47H AD-risk variant of sTREM2 is less able to bind and disaggregate oligomeric Aß but rather promotes Aß protofibril formation and neurotoxicity. Thus, in addition to inducing an immune response, wild-type TREM2 may protect against amyloid pathology by the Aß-induced release of sTREM2, which blocks Aß aggregation and neurotoxicity. In contrast, R47H sTREM2 promotes Aß aggregation into protofibril that may be toxic to neurons. These findings may explain how wild-type sTREM2 apparently protects against AD in vivo and why a single copy of the R47H variant gene is associated with increased AD risk.

Nanoluciferase complementation-based bioreporter reveals the importance of N-linked glycosylation of SARS-CoV-2 S for viral entry.

The ongoing COVID-19 pandemic has highlighted the immediate need for the development of antiviral therapeutics targeting different stages of the SARS-CoV-2 life cycle. We developed a bioluminescence-based bioreporter to interrogate the interaction between the SARS-CoV-2 viral spike (S) protein and its host entry receptor, angiotensin-converting enzyme 2 (ACE2). The bioreporter assay is based on a nanoluciferase complementation reporter, composed of two subunits, large BiT and small BiT, fused to the S receptor-binding domain (RBD) of the SARS-CoV-2 S protein and ACE2 ectodomain, respectively. Using this bioreporter, we uncovered critical host and viral determinants of the interaction, including a role for glycosylation of asparagine residues within the RBD in mediating successful viral entry. We also demonstrate the importance of N-linked glycosylation to the RBD's antigenicity and immunogenicity. Our study demonstrates the versatility of our bioreporter in mapping key residues mediating viral entry as well as screening inhibitors of the ACE2-RBD interaction. Our findings point toward targeting RBD glycosylation for therapeutic and vaccine strategies against SARS-CoV-2.

Differential interaction with TREM2 modulates microglial uptake of modified Aß species.

Rare coding variants of the microglial triggering receptor expressed on myeloid cells 2 (TREM2) confer an increased risk for Alzheimer's disease (AD) characterized by the progressive accumulation of aggregated forms of amyloid ß peptides (Aß). Aß peptides are generated by proteolytic processing of the amyloid precursor protein (APP). Heterogeneity in proteolytic cleavages and additional post-translational modifications result in the production of several distinct Aß variants that could differ in their aggregation behavior and toxic properties. Here, we sought to assess whether post-translational modifications of Aß affect the interaction with TREM2. Biophysical and biochemical methods revealed that TREM2 preferentially interacts with oligomeric Aß, and that phosphorylation of Aß increases this interaction. Phosphorylation of Aß also affected the TREM2 dependent interaction and phagocytosis by primary microglia and in APP transgenic mouse models. Thus, TREM2 function is important for sensing phosphorylated Aß variants in distinct aggregation states and reduces the accumulation and deposition of these toxic Aß species in preclinical models of Alzheimer's disease.

NMR in integrated biophysical drug discovery for RAS: past, present, and future.

Mutations in RAS oncogenes occur in ~ 30% of human cancers, with KRAS being the most frequently altered isoform. RAS proteins comprise a conserved GTPase domain and a C-terminal lipid-modified tail that is unique to each isoform. The GTPase domain is a 'switch' that regulates multiple signaling cascades that drive cell growth and proliferation when activated by binding GTP, and the signal is terminated by GTP hydrolysis. Oncogenic RAS mutations disrupt the GTPase cycle, leading to accumulation of the activated GTP-bound state and promoting proliferation. RAS is a key target in oncology, however it lacks classic druggable pockets and has been extremely challenging to target. RAS signaling has thus been targeted indirectly, by harnessing key downstream effectors as well as upstream regulators, or disrupting the proper membrane localization required for signaling, by inhibiting either lipid modification or 'carrier' proteins. As a small (20 kDa) protein with multiple conformers in dynamic equilibrium, RAS is an excellent candidate for NMR-driven characterization and screening for direct inhibitors. Several molecules have been discovered that bind RAS and stabilize shallow pockets through conformational selection, and recent compounds have achieved substantial improvements in affinity. NMR-derived insight into targeting the RAS-membrane interface has revealed a new strategy to enhance the potency of small molecules, while another approach has been development of peptidyl inhibitors that bind through large interfaces rather than deep pockets. Remarkable progress has been made with mutation-specific covalent inhibitors that target the thiol of a G12C mutant, and these are now in clinical trials. Here we review the history of RAS inhibitor development and highlight the utility of NMR and integrated biophysical approaches in RAS drug discovery.

Ultrasound-Guided Umbilical Venous Catheter Insertion With Alignment of the Umbilical Vein and Ductus Venosus.

Previous studies have highlighted the importance of confirming the position of an umbilical venous catheter (UVC) tip by an ultrasound (US) examination. However, methods for preventing insertion into the portal circulation under US guidance have not yet been established. We report 15 cases in which a UVC was successfully passed through the ductus venosus by compressing the upper abdomen near the portal sinus of the liver to align the umbilical vein and ductus venosus under US guidance. The UVC was inserted into the correct position in 14 of the 15 neonates (93%) without complications.

A Non-Canonical Calmodulin Target Motif Comprising a Polybasic Region and Lipidated Terminal Residue Regulates Localization.

Calmodulin (CaM) is a Ca2+-sensor that regulates a wide variety of target proteins, many of which interact through short basic helical motifs bearing two hydrophobic 'anchor' residues. CaM comprises two globular lobes, each containing a pair of EF-hand Ca2+-binding motifs that form a Ca2+-induced hydrophobic pocket that binds an anchor residue. A central flexible linker allows CaM to accommodate diverse targets. Several reported CaM interactors lack these anchors but contain Lys/Arg-rich polybasic sequences adjacent to a lipidated N- or C-terminus. Ca2+-CaM binds the myristoylated N-terminus of CAP23/NAP22 with intimate interactions between the lipid and a surface comprised of the hydrophobic pockets of both lobes, while the basic residues make electrostatic interactions with the negatively charged surface of CaM. Ca2+-CaM binds farnesylcysteine, derived from the farnesylated polybasic C-terminus of KRAS4b, with the lipid inserted into the C-terminal lobe hydrophobic pocket. CaM sequestration of the KRAS4b farnesyl moiety disrupts KRAS4b membrane association and downstream signaling. Phosphorylation of basic regions of N-/C-terminal lipidated CaM targets can reduce affinity for both CaM and the membrane. Since both N-terminal myristoylated and C-terminal prenylated proteins use a Singly Lipidated Polybasic Terminus (SLIPT) for CaM binding, we propose these polybasic lipopeptide elements comprise a non-canonical CaM-binding motif.

Two Distinct Structures of Membrane-Associated Homodimers of GTP- and GDP-Bound KRAS4B Revealed by Paramagnetic Relaxation Enhancement.

KRAS homo-dimerization has been implicated in the activation of RAF kinases, however, the mechanism and structural basis remain elusive. We developed a system to study KRAS dimerization on nanodiscs using paramagnetic relaxation enhancement (PRE) NMR spectroscopy, and determined distinct structures of membrane-anchored KRAS dimers in the active GTP- and inactive GDP-loaded states. Both dimerize through an α4-α5 interface, but the relative orientation of the protomers and their contacts differ substantially. Dimerization of KRAS-GTP, stabilized by electrostatic interactions between R135 and E168, favors an orientation on the membrane that promotes accessibility of the effector-binding site. Remarkably, "cross"-dimerization between GTP- and GDP-bound KRAS molecules is unfavorable. These models provide a platform to elucidate the structural basis of RAF activation by RAS and to develop inhibitors that can disrupt the KRAS dimerization. The methodology is applicable to many other farnesylated small GTPases.

From Stores to Sinks: Structural Mechanisms of Cytosolic Calcium Regulation.

All eukaryotic cells have adapted the use of the calcium ion (Ca2+) as a universal signaling element through the evolution of a toolkit of Ca2+ sensor, buffer and effector proteins. Among these toolkit components, integral and peripheral proteins decorate biomembranes and coordinate the movement of Ca2+ between compartments, sense these concentration changes and elicit physiological signals. These changes in compartmentalized Ca2+ levels are not mutually exclusive as signals propagate between compartments. For example, agonist induced surface receptor stimulation can lead to transient increases in cytosolic Ca2+ sourced from endoplasmic reticulum (ER) stores; the decrease in ER luminal Ca2+ can subsequently signal the opening surface channels which permit the movement of Ca2+ from the extracellular space to the cytosol. Remarkably, the minuscule compartments of mitochondria can function as significant cytosolic Ca2+ sinks by taking up Ca2+ in a coordinated manner. In non-excitable cells, inositol 1,4,5 trisphosphate receptors (IP3Rs) on the ER respond to surface receptor stimulation; stromal interaction molecules (STIMs) sense the ER luminal Ca2+ depletion and activate surface Orai1 channels; surface Orai1 channels selectively permit the movement of Ca2+ from the extracellular space to the cytosol; uptake of Ca2+ into the matrix through the mitochondrial Ca2+ uniporter (MCU) further shapes the cytosolic Ca2+ levels. Recent structural elucidations of these key Ca2+ toolkit components have improved our understanding of how they function to orchestrate precise cytosolic Ca2+ levels for specific physiological responses. This chapter reviews the atomic-resolution structures of IP3R, STIM1, Orai1 and MCU elucidated by X-ray crystallography, electron microscopy and NMR and discusses the mechanisms underlying their biological functions in their respective compartments within the cell.

Transcutaneous bilirubin measurement during phototherapy in term neonates.

BACKGROUND: We tested whether direct transcutaneous bilirubin (TcB) measurement from an area unexposed to phototherapy is reliable for estimation of total serum bilirubin (TSB) in neonates during phototherapy and whether it contributes to reduction in TSB blood sampling in phototherapy decision making. METHODS: This was a retrospective observational study of term neonates who received phototherapy in the mother's room. TSB and TcB from the neonate's sternum were measured before and during phototherapy and compared using linear regression analysis and Bland-Altman plot, respectively. Various cut-offs of TcB for estimating TSB during phototherapy at >72 h after birth were analyzed. RESULTS: There were moderate correlations between TSB and TcB before (r = 0.56) and during (r = 0.47) phototherapy in 125 neonates. The mean difference (TSB-TcB) before and during phototherapy was 1.2 ± 1.7 mg/dL and 1.0 ± 1.7 mg/dL, respectively. The 95% limits of agreement for the difference before and during phototherapy ranged from -2.1 to 4.5 and from -2.3 to 4.3 mg/dL, respectively. For TSB ≤18 mg/dL during phototherapy, a TcB cut-off of 14 mg/dL had a specificity of 1.0; with this method, 43% of the TSB measurements could have been avoided. CONCLUSIONS: Direct measurement of TcB during phototherapy using a bed-type device is a reliable method to estimate TSB in term neonates and would contribute to a reduction in blood sampling. It cannot, however, be used as a substitute for TSB measurement.

Acquired palatal groove and delayed oral feeding in preterm infants.

BACKGROUND: Acquired palatal groove has been reported in the 1970s and 1980s, but its current incidence in Japanese newborns is unclear. The aims of this study were to determine the incidence of palatal groove in preterm infants and to evaluate whether this condition affects oral feeding ability. METHODS: We conducted a prospective observational study among very low-birthweight infants born at Takatsuki General Hospital, Osaka, between March and October in 2010. The shape of the hard palate was classified into three types: normal, narrow high-arched palate, and palatal groove. RESULTS: Among the 37 enrolled infants, 14 (38%) had palatal groove. In particular, among the 29 infants with birthweight <1000 g, palatal groove was observed in 48% of these patients, and only 10% were normal. Infants with palatal groove were ventilated for considerably more days with oral endotracheal tube than those without palate groove, even after adjustment for gestational age, birthweight, and duration of oral duodenal tube placement (OR, 1.11). Establishment of oral feeding and disappearance of choking on milk were considerably delayed in infants with palatal groove. Transient oral feeding difficulty requiring thickened-feed intervention was observed only in infants with palatal groove; on multi-regression analysis this difficulty seemed to be induced by the palatal groove. CONCLUSIONS: Palatal groove formation induced by oral endotracheal intubation occurs with a high frequency in preterm infants, and this is likely to affect oral feeding ability.

Effect of Volume Guarantee in Preterm Infants on High-Frequency Oscillatory Ventilation: A Pilot Study.

Objectives Although adding volume guarantee (VG) to conventional ventilation has been a well-established respiratory management for preterm infants, the evidence of VG combined with high-frequency oscillatory ventilation (HFOV) has not been studied well. The aim of this study was to investigate the effect of VG added to HFOV on respiratory and other physiological parameters. Methods We conducted a pilot study in extremely low-birth-weight infants ventilated with HFOV + VG with stable pulmonary status after 28 days of age. VG was applied for 6 hours and removed for the following 6 hours, and data were collected during these 12 hours. Results Six neonates were included in this study (gestational age: 22w5d-23w6d, birthweight: 424-584 g). High-frequency expired tidal volume per weight and amplitude were similar between periods with and without VG. Fluctuation of SpO2, but not heart rate, was significantly smaller when babies were ventilated with VG than without VG. Fluctuation of minute volume and carbon dioxide diffusion coefficient significantly increased after VG removal. The proportion of time with SpO2 < 80% was decreased by VG overall, especially in three cases. Conclusion This pilot study suggests VG combined with HFOV attenuates fluctuation of SpO2 and CO2 clearance, which may prevent hypoxemia and hypocapnia.

Structural insights into endoplasmic reticulum stored calcium regulation by inositol 1,4,5-trisphosphate and ryanodine receptors.

The two major calcium (Ca²âº) release channels on the sarco/endoplasmic reticulum (SR/ER) are inositol 1,4,5-trisphosphate and ryanodine receptors (IP3Rs and RyRs). They play versatile roles in essential cell signaling processes, and abnormalities of these channels are associated with a variety of diseases. Structural information on IP3Rs and RyRs determined using multiple techniques including X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy and cryo-electron microscopy (EM), has significantly advanced our understanding of the mechanisms by which these Ca²âº release channels function under normal and pathophysiological circumstances. In this review, structural advances on the understanding of the mechanisms of IP3R and RyR function and dysfunction are summarized. This article is part of a Special Issue entitled: 13th European Symposium on Calcium.

Reliability of Total Bilirubin Measurements in Whole Blood from Preterm Neonates Using a Blood Gas Analyzer.

BACKGROUND: Blood gas analyses are usually required more frequently in preterm neonates than in term neonates. If total bilirubin (TB) levels in whole blood measured using a blood gas analyzer are reliable, blood sampling for total serum bilirubin (TSB) levels alone can be reduced in preterm neonates. We investigated the reliability of measuring TB levels in whole blood from preterm neonates using the latest generation blood gas analyzer. METHODS: TB measured on an ABL90 FLEX blood gas analyzer and TSB analyzed in the hospital laboratory were simultaneously analyzed. TB and TSB levels (300 data sets in 85 preterm neonates) were compared using linear regression and Bland-Altman difference plots. RESULTS: Concordance correlation coefficient analysis showed a strong relationship between TB and TSB levels (a CCC value of 0.94) with a Pearson's coefficient of 0.97 and a bias correction of 0.97. Bland-Altman difference p lots demonstrated that, on average, TB tended to underestimate the TSB, with a mean (95% confidence interval) bias of -0.7 (-0.6 to -0.8) mg/dL. CONCLUSIONS: Whole blood TB levels measured using an ABL90 FLEX are reliable and can lead to a reduction in blood sampling for TSB in preterm neonates.

CaBP1, a neuronal Ca2+ sensor protein, inhibits inositol trisphosphate receptors by clamping intersubunit interactions.

Calcium-binding protein 1 (CaBP1) is a neuron-specific member of the calmodulin superfamily that regulates several Ca(2+) channels, including inositol 1,4,5-trisphosphate receptors (InsP3Rs). CaBP1 alone does not affect InsP3R activity, but it inhibits InsP3-evoked Ca(2+) release by slowing the rate of InsP3R opening. The inhibition is enhanced by Ca(2+) binding to both the InsP3R and CaBP1. CaBP1 binds via its C lobe to the cytosolic N-terminal region (NT; residues 1-604) of InsP3R1. NMR paramagnetic relaxation enhancement analysis demonstrates that a cluster of hydrophobic residues (V101, L104, and V162) within the C lobe of CaBP1 that are exposed after Ca(2+) binding interact with a complementary cluster of hydrophobic residues (L302, I364, and L393) in the ß-domain of the InsP3-binding core. These residues are essential for CaBP1 binding to the NT and for inhibition of InsP3R activity by CaBP1. Docking analyses and paramagnetic relaxation enhancement structural restraints suggest that CaBP1 forms an extended tetrameric turret attached by the tetrameric NT to the cytosolic vestibule of the InsP3R pore. InsP3 activates InsP3Rs by initiating conformational changes that lead to disruption of an intersubunit interaction between a "hot-spot" loop in the suppressor domain (residues 1-223) and the InsP3-binding core ß-domain. Targeted cross-linking of residues that contribute to this interface show that InsP3 attenuates cross-linking, whereas CaBP1 promotes it. We conclude that CaBP1 inhibits InsP3R activity by restricting the intersubunit movements that initiate gating.

Simultaneous administration of 2-aminoethyl diphenylborinate and chloroquine reverses chloroquine resistance in malaria parasites.

A nearly complete reversal of chloroquine (CQ) resistance in the CQ-resistant Plasmodium falciparum K-1 strain, with a significant decrease in the mean ± standard deviation (SD) 50% inhibitory concentration (IC50) from 1,050 ± 95 nM to 14 ± 2 nM, was achieved in vitro by the simultaneous administration of 2-aminoethyl diphenylborinate (2-APB). The CQ resistance-reversing activity of 2-APB, which showed the same efficacy as verapamil, was also observed in an in vivo mouse infection model with the CQ-resistant Plasmodium chabaudi AS(30CQ) strain.

A dominant negative form of inositol 1,4,5-trisphosphate receptor induces metacyclogenesis and increases mitochondrial density in Trypanosoma cruzi.

Inositol 1,4,5-trisphosphate receptor (IP3R) is a key regulator of intracellular Ca(2+) concentration that release Ca(2+) from Ca(2+) stores in response to various external stimuli. IP3R also works as a signal hub which form a platform for interacting with various proteins involved in diverse cell signaling. Previously, we have identified an IP3R homolog in the parasitic protist, Trypanosoma cruzi (TcIP3R). Parasites expressing reduced or increased levels of TcIP3R displayed defects in growth, transformation, and infectivity. In the present study, we established parasitic strains expressing a dominant negative form of TcIP3R, named DN-TcIP3R, to further investigate the physiological role(s) of TcIP3R. We found that the growth of epimastigotes expressing DN-TcIP3R was significantly slower than that of parasites with TcIP3R expression levels that were approximately 65% of wild-type levels. The expression of DN-TcIP3R in epimastigotes induced metacyclogenesis even in the normal growth medium. Furthermore, these epimastigotes showed the presence of dense mitochondria under a transmission electron microscope. Our findings confirm that TcIP3R is crucial for epimastigote growth, as previously reported. They also suggest that a strong inhibition of the IP3R-mediated signaling induces metacyclogenesis and that mitochondrial integrity is closely associated with this signaling.

Newborn rat response to single vs. combined cGMP-dependent pulmonary vasodilators.

Inhaled nitric oxide (NO) and other cGMP- or cAMP-dependent pulmonary vasodilators are often used in combination for the treatment of the persistent pulmonary hypertension of the newborn syndrome. There is in vitro evidence to indicate that NO downregulate the pulmonary vascular response to cGMP-dependent agonists raising concern as to whether a synergistic effect is observed when employing a combined strategy in newborns. Hypothesizing that a synergistic effect is absent, we evaluated newborn and juvenile rat pulmonary arteries to determine the individual and combined vasodilatory effect of cGMP- and cAMP-dependent agonists. In precontracted near-resistance pulmonary arteries, the addition of sildenafil reduced vasorelaxation response to NO donor S-nitroso-N-acetyl penicillamine (SNAP). A similar decrease in SNAP-induced vasodilation was observed in arteries pretreated with BAY 41-2272 (10(-9) M), a soluble guanylate cyclase stimulator cGMP, and its downstream protein kinase activator. cGMP also reduced the vasorelaxant response to the cAMP-dependent forskolin. Inhibition of endogenous vascular NO generation enhanced SNAP-induced relaxation. The present data suggest that the mechanism involved in the cGMP desensitization to other relaxant agonists involves downregulation of the small heat shock protein HSP20 and is evident in rat pulmonary and systemic vascular smooth muscle cells. In newborn rats with chronic hypoxia-induced pulmonary hypertension, the combination of sildenafil and inhaled NO resulted in a lesser reduction in pulmonary vascular resistance compared with their individual effect. These data suggest that clinical exposure to one cGMP-dependent pulmonary vasodilator may affect the response to other cGMP- or cAMP-mediated agonists.

Inositol 1,4,5-trisphosphate receptor regulates replication, differentiation, infectivity and virulence of the parasitic protist Trypanosoma cruzi.

In animals, inositol 1,4,5-trisphosphate receptors (IP3 Rs) are ion channels that play a pivotal role in many biological processes by mediating Ca(2+) release from the endoplasmic reticulum. Here, we report the identification and characterization of a novel IP3 R in the parasitic protist, Trypanosoma cruzi, the pathogen responsible for Chagas disease. DT40 cells lacking endogenous IP3 R genes expressing T. cruzi IP3 R (TcIP3 R) exhibited IP3 -mediated Ca(2+) release from the ER, and demonstrated receptor binding to IP3 . TcIP3 R was expressed throughout the parasite life cycle but the expression level was much lower in bloodstream trypomastigotes than in intracellular amastigotes or epimastigotes. Disruption of two of the three TcIP3 R gene loci led to the death of the parasite, suggesting that IP3 R is essential for T. cruzi. Parasites expressing reduced or increased levels of TcIP3 R displayed defects in growth, transformation and infectivity, indicating that TcIP3 R is an important regulator of the parasite's life cycle. Furthermore, mice infected with T. cruzi expressing reduced levels of TcIP3 R exhibited a reduction of disease symptoms, indicating that TcIP3 R is an important virulence factor. Combined with the fact that the primary structure of TcIP3 R has low similarity to that of mammalian IP3 Rs, TcIP3 R is a promising drug target for Chagas disease.

An interplay between 2 signaling pathways: melatonin-cAMP and IP3-Ca2+ signaling pathways control intraerythrocytic development of the malaria parasite Plasmodium falciparum.

Plasmodium falciparum spends most of its asexual life cycle within human erythrocytes, where proliferation and maturation occur. Development into the mature forms of P. falciparum causes severe symptoms due to its distinctive sequestration capability. However, the physiological roles and the molecular mechanisms of signaling pathways that govern development are poorly understood. Our previous study showed that P. falciparum exhibits stage-specific spontaneous Calcium (Ca(2+)) oscillations in ring and early trophozoites, and the latter was essential for parasite development. In this study, we show that luzindole (LZ), a selective melatonin receptor antagonist, inhibits parasite growth. Analyses of development and morphology of LZ-treated P. falciparum revealed that LZ severely disrupted intraerythrocytic maturation, resulting in parasite death. When LZ was added at ring stage, the parasite could not undergo further development, whereas LZ added at the trophozoite stage inhibited development from early into late schizonts. Live-cell Ca(2+) imaging showed that LZ treatment completely abolished Ca(2+) oscillation in the ring forms while having little effect on early trophozoites. Further, the melatonin-induced cAMP increase observed at ring and late trophozoite stage was attenuated by LZ treatment. These suggest that a complex interplay between IP3-Ca(2+) and cAMP signaling pathways is involved in intraerythrocytic development of P. falciparum.