Targeting CDK9 Reactivates Epigenetically Silenced Genes in Cancer.

Cyclin-dependent kinase 9 (CDK9) promotes transcriptional elongation through RNAPII pause release. We now report that CDK9 is also essential for maintaining gene silencing at heterochromatic loci. Through a live cell drug screen with genetic confirmation, we discovered that CDK9 inhibition reactivates epigenetically silenced genes in cancer, leading to restored tumor suppressor gene expression, cell differentiation, and activation of endogenous retrovirus genes. CDK9 inhibition dephosphorylates the SWI/SNF protein BRG1, which contributes to gene reactivation. By optimization through gene expression, we developed a highly selective CDK9 inhibitor (MC180295, IC50 = 5 nM) that has broad anti-cancer activity in vitro and is effective in in vivo cancer models. Additionally, CDK9 inhibition sensitizes to the immune checkpoint inhibitor α-PD-1 in vivo, making it an excellent target for epigenetic therapy of cancer.

Pharmacological stimulation of nuclear factor (erythroid-derived 2)-like 2 translation activates antioxidant responses.

Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is the master regulator of the antioxidant response, and its function is tightly regulated at the transcriptional, translational, and post-translational levels. It is well-known that Nrf2 is regulated at the protein level by proteasomal degradation via Kelch-like ECH-associated protein 1 (Keap1), but how Nrf2 is regulated at the translational level is less clear. Here, we show that pharmacological stimulation increases Nrf2 levels by overcoming basal translational repression. We developed a novel reporter assay that enabled identification of natural compounds that induce Nrf2 translation by a mechanism independent of Keap1-mediated degradation. Apigenin, resveratrol, and piceatannol all induced Nrf2 translation. More importantly, the pharmacologically induced Nrf2 overcomes Keap1 regulation, translocates to the nucleus, and activates the antioxidant response. We conclude that translational regulation controls physiological levels of Nrf2, and this can be modulated by apigenin, resveratrol, and piceatannol. Also, targeting this mechanism with novel compounds could provide new insights into prevention and treatment of multiple diseases in which oxidative stress plays a significant role.

Design, synthesis and SAR of new-di-substituted pyridopyrimidines as ATP-competitive dual PI3Kα/mTOR inhibitors.

PI3Kα/mTOR ATP-competitive inhibitors are considered as one of the promising molecularly targeted cancer therapeutics. Based on lead compound A from the literature, two similar series of 2-substituted-4-morpholino-pyrido[3,2-d]pyrimidine and pyrido[2,3-d]pyrimidine analogs were designed and synthesized as PI3Kα/mTOR dual inhibitors. Interestingly, most of the series gave excellent inhibition for both enzymes with IC50 values ranging from single to double digit nM. Unlike many PI3Kα/mTOR dual inhibitors, our compounds displayed selectivity for PI3Kα. Based on its potent enzyme inhibitory activity, selectivity for PI3Kα and good therapeutic index in 2D cell culture viability assays, compound 4h was chosen to be evaluated in 3D culture for its IC50 against MCF7 breast cancer cells as well as for docking studies with both enzymes.

Modulation of polyamine metabolic flux in adipose tissue alters the accumulation of body fat by affecting glucose homeostasis.

The continued rise in obesity despite public education, awareness and policies indicates the need for mechanism-based therapeutic approaches to help control the disease. Our data, in conjunction with other studies, suggest an unexpected role for the polyamine catabolic enzyme spermidine/spermine-N1-acetyltransferase (SSAT) in fat homeostasis. Our previous studies showed that deletion of SSAT greatly exaggerates weight gain and that the transgenic overexpression suppresses weight gain in mice on a high-fat diet. This discovery is substantial but the underlying molecular linkages are only vaguely understood. Here, we used a comprehensive systems biology approach, on white adipose tissue (WAT), to discover that the partition of acetyl-CoA towards polyamine catabolism alters glucose homeostasis and hence, fat accumulation. Comparative proteomics and antibody-based expression studies of WAT in SSAT knockout, wild type and transgenic mice identified nine proteins with an increasing gradient across the genotypes, all of which correlate with acetyl-CoA consumption in polyamine acetylation. Adipose-specific SSAT knockout mice and global SSAT knockout mice on a high-fat diet exhibited similar growth curves and proteomic patterns in their WAT, confirming that attenuated consumption of acetyl-CoA in acetylation of polyamines in adipose tissue drives the obese phenotype of these mice. Analysis of protein expression indicated that the identified changes in the levels of proteins regulating acetyl-CoA consumption occur via the AMP-activated protein kinase pathway. Together, our data suggest that differential expression of SSAT markedly alters acetyl-CoA levels, which in turn trigger a global shift in glucose metabolism in adipose tissue, thus affecting the accumulation of body fat.

Histone 3.3 participates in a self-sustaining cascade of apoptosis that contributes to the progression of chronic obstructive pulmonary disease.

RATIONALE: Shifts in the gene expression of nuclear protein in chronic obstructive pulmonary disease (COPD), a progressive disease that is characterized by extensive lung inflammation and apoptosis, are common; however, the extent of the elevation of the core histones, which are the major components of nuclear proteins and their consequences in COPD, has not been characterized, which is important because extracellular histones are cytotoxic to endothelial and airway epithelial cells. OBJECTIVES: To investigate the role of extracellular histones in COPD disease progression. METHODS: We analyzed the nuclear lung proteomes of ex-smokers with and without the disease. Further studies on the consequences of H3.3 were also performed. MEASUREMENTS AND MAIN RESULTS: A striking finding was a COPD-specific eightfold increase of hyperacetylated histone H3.3. The hyperacetylation renders H3.3 resistant to proteasomal degradation despite ubiquitination; when combined with the reduction in proteasome activity that is known for COPD, this resistance helps account for the increased levels of H3.3. Using anti-H3 antibodies, we found H3.3 in the airway lumen, alveolar fluid, and plasma of COPD samples. H3.3 was cytotoxic to lung structural cells via a mechanism that involves the perturbation of Ca(2+) homeostasis and mitochondrial toxicity. We used the primary human airway epithelial cells and found that the antibodies to either the C or N terminus of H3 could partially reverse H3.3 toxicity. CONCLUSIONS: Our data indicate that there is an uncontrolled positive feedback loop in which the damaged cells release acetylated H3.3, which causes more damage, adds H3.3 release, and contributes toward the disease progression.

Translational control of Nrf2 within the open reading frame.

Nuclear Factor Erythroid 2-Related Factor 2 (Nrf2) is a transcription factor that is essential for the regulation of an effective antioxidant and detoxifying response. The regulation of its activity can occur at transcription, translation and post-translational levels. Evidence suggests that under environmental stress conditions, new synthesis of Nrf2 is required - a process that is regulated by translational control and is not fully understood. Here we described the identification of a novel molecular process that under basal conditions strongly represses the translation of Nrf2 within the open reading frame (ORF). This mechanism is dependent on the mRNA sequence within the 3' portion of the ORF of Nrf2 but not in the encoded amino acid sequence. The Nrf2 translational repression can be reversed with the use of synonymous codon substitutions. This discovery suggests an additional layer of control to explain the reason for the low Nrf2 concentration under quiescent state.

Identification of Inhibitors of Tubulin Polymerization Using a CRISPR-Edited Cell Line with Endogenous Fluorescent Tagging of ß-Tubulin and Histone H1.

Tubulin is a protein that plays a critical role in maintaining cellular structure and facilitating cell division. Inhibiting tubulin polymerization has been shown to be an effective strategy for inhibiting the proliferation of cancer cells. In the past, identifying compounds that could inhibit tubulin polymerization has required the use of in vitro assays utilizing purified tubulin or immunofluorescence of fixed cells. This study presents a novel approach for identifying tubulin polymerization inhibitors using a CRISPR-edited cell line that expresses fluorescently tagged ß-tubulin and a nuclear protein, enabling the visualization of tubulin polymerization dynamics via high-content imaging analysis (HCI). The cells were treated with known tubulin polymerization inhibitors, colchicine, and vincristine, and the resulting phenotypic changes indicative of tubulin polymerization inhibition were confirmed using HCI. Furthermore, a library of 429 kinase inhibitors was screened, resulting in the identification of three compounds (ON-01910, HMN-214, and KX2-391) that inhibit tubulin polymerization. Live cell tracking analysis confirmed that compound treatment leads to rapid tubulin depolymerization. These findings suggest that CRISPR-edited cells with fluorescently tagged endogenous ß-tubulin can be utilized to screen large compound libraries containing diverse chemical families for the identification of novel tubulin polymerization inhibitors.

CRISPR/Cas9-Based Screening of FDA-Approved Drugs for NRF2 Activation: A Novel Approach to Discover Therapeutics for Non-Alcoholic Fatty Liver Disease.

With the rising prevalence of obesity, non-alcoholic fatty liver disease (NAFLD) now affects 20-25% of the global population. NAFLD, a progressive condition associated with oxidative stress, can result in cirrhosis and liver cancer in 10% and 3% of patients suffering NAFLD, respectively. Therapeutic options are currently limited, emphasizing the need for novel treatments. In this study, we examined the potential of activating the transcription factor NRF2, a crucial player in combating oxidative stress, as an innovative approach to treating NAFLD. Utilizing a CRISPR/Cas9-engineered human HEK293T cell line, we were able to monitor the expression of heme oxygenase-1 (HMOX1), an NRF2 target, using a Nanoluc luciferase tag. Our model was validated using a known NRF2 activator, after which we screened 1200 FDA-approved drugs, unearthing six compounds (Disulfiram, Thiostrepton, Auranofin, Thimerosal, Halofantrine, and Vorinostat) that enhanced NRF2 activity and antioxidant response. These compounds demonstrated protective effects against oxidative stress induced by hydrogen peroxide and lipid droplets accumulation in vitro with hepatoma HUH-7 cells. Our study underscores the utility of CRISPR/Cas9 tagging with Nanoluc luciferase in identifying potential NRF2 activators, paving the way for potential NAFLD therapeutics.

Secreted folate receptor γ drives fibrogenesis in metabolic dysfunction-associated steatohepatitis by amplifying TGFß signaling in hepatic stellate cells.

Hepatic fibrosis is the primary determinant of mortality in patients with metabolic dysfunction-associated steatohepatitis (MASH). Transforming growth factor-ß (TGFß), a master profibrogenic cytokine, is a promising therapeutic target that has not yet been translated into an effective therapy in part because of liabilities associated with systemic TGFß antagonism. We have identified that soluble folate receptor γ (FOLR3), which is expressed in humans but not in rodents, is a secreted protein that is elevated in the livers of patients with MASH but not in those with metabolic dysfunction-associated steatotic liver disease, those with type II diabetes, or healthy individuals. Global proteomics showed that FOLR3 was the most highly significant MASH-specific protein and was positively correlated with increasing fibrosis stage, consistent with stimulation of activated hepatic stellate cells (HSCs), which are the key fibrogenic cells in the liver. Exposure of HSCs to exogenous FOLR3 led to elevated extracellular matrix (ECM) protein production, an effect synergistically potentiated by TGFß1. We found that FOLR3 interacts with the serine protease HTRA1, a known regulator of TGFBR, and activates TGFß signaling. Administration of human FOLR3 to mice induced severe bridging fibrosis and an ECM pattern resembling human MASH. Our study thus uncovers a role of FOLR3 in enhancing fibrosis.

Dosing strategies for de novo once-daily extended release tacrolimus in kidney transplant recipients based on CYP3A5 genotype.

BACKGROUND: Tacrolimus extended-release tablets have been Food and Drug Administration-approved for use in the de novo kidney transplant population. Dosing requi rements often vary for tacrolimus based on several factors including variation in metabolism based on CYP3A5 expression. Patients who express CYP3A5 often require higher dosing of immediate-release tacrolimus, but this has not been established for tacrolimus extended-release tablets in the de novo setting. AIM: To obtain target trough concentrations of extended-release tacrolimus in de novo kidney transplant recipients according to CYP3A5 genotype. METHODS: Single-arm, prospective, single-center, open-label, observational study (ClinicalTrials.gov: NCT037 13645). Life cycle pharma tacrolimus (LCPT) orally once daily at a starting dose of 0.13 mg/kg/day based on actual body weight. If weight is more than 120% of ideal body weight, an adjusted body weight was used. LCPT dose was adjusted to maintain tacrolimus trough concentrations of 8-10 ng/mL. Pharmacogenetic analysis of CYP3A5 genotype was performed at study conclusion. RESULTS: Mean time to therapeutic tacrolimus trough concentration was longer in CYP3A5 intermediate and extensive metabolizers vs CYP3A5 non-expressers (6 d vs 13.5 d vs 4.5 d; P = 0.025). Mean tacrolimus doses and weight-based doses to achieve therapeutic concentration were higher in CYP3A5 intermediate and extensive metabolizers vs CYP3A5 non-expressers (16 mg vs 16 mg vs 12 mg; P = 0.010) (0.20 mg/kg vs 0.19 mg/kg vs 0.13 mg/kg; P = 0.018). CYP3A5 extensive metabolizers experienced lower mean tacrolimus trough concentrations throughout the study period compared to CYP3A5 intermediate metabolizers and non-expressers (7.98 ng/mL vs 9.18 ng/mL vs 10.78 ng/mL; P = 0 0.008). No differences were identified with regards to kidney graft function at 30-d post-transplant. Serious adverse events were reported for 13 (36%) patients. CONCLUSION: Expression of CYP3A5 leads to higher starting doses and incremental dosage titration of extended-release tacro limus to achieve target trough concentrations. We suggest a higher starting dose of 0.2 mg/kg/d for CYP3A5 expressers.

Extracellular Acetylated Histone 3.3 Induces Inflammation and Lung Tissue Damage.

Extracellular histones, part of the protein group known as damage-associated molecular patterns (DAMPs), are released from damaged or dying cells and can instigate cellular toxicity. Within the context of chronic obstructive pulmonary disease (COPD), there is an observed abundance of extracellular histone H3.3, indicating potential pathogenic implications. Notably, histone H3.3 is often found hyperacetylated (AcH3.3) in the lungs of COPD patients. Despite these observations, the specific role of these acetylated histones in inducing pulmonary tissue damage in COPD remains unclear. To investigate AcH3.3's impact on lung tissue, we administered recombinant histones (rH2A, rH3.3, and rAcH3.3) or vehicle solution to mice via intratracheal instillation. After 48 h, we evaluated the lung toxicity damage and found that the rAcH3.3 treated animals exhibited more severe lung tissue damage compared to those treated with non-acetylated H3.3 and controls. The rAcH3.3 instillation resulted in significant histological changes, including alveolar wall rupture, epithelial cell damage, and immune cell infiltration. Micro-CT analysis confirmed macroscopic structural changes. The rAcH3.3 instillation also increased apoptotic activity (cleavage of caspase 3 and 9) and triggered acute systemic inflammatory marker activation (TNF-α, IL-6, MCP-3, or CXCL-1) in plasma, accompanied by leukocytosis and lymphocytosis. Confocal imaging analysis confirmed lymphocytic and monocytic/macrophage lung infiltration in response to H3.3 and AcH3.3 administration. Taken together, our findings implicate extracellular AcH3.3 in inducing cytotoxicity and acute inflammatory responses, suggesting its potential role in promoting COPD-related lung damage progression.

Regulation of polyamine metabolism by translational control.

Polyamines are low molecular weight, positively charged compounds that are ubiquitous in all living cells. They play a crucial role in many biochemical processes including regulation of transcription and translation, modulation of enzyme activities, regulation of ion channels and apoptosis. A strict balance between synthesis, catabolism and excretion tightly controls the cellular concentration of polyamines. The concentrations of rate-limiting enzymes in the polyamine synthesis and degradation pathways are regulated at different levels, including transcription, translation and degradation. Polyamines can modulate the translation of most of the enzymes required for their synthesis and catabolism through feedback mechanisms that are unique for each enzyme. Translational control is associated with cis-acting and trans-acting factors that can be influenced by the concentration of polyamines through mechanisms that are not completely understood. In this review, we present an overview of the translational control mechanisms of the proteins in the polyamine pathway, including ornithine decarboxylase (ODC), ODC antizyme, S-adenosylmethionine decarboxylase and spermidine/spermine N(1) acetyltransferase, highlighting the areas where more research is needed. A better understanding of the translational control of these enzymes would offer the possibility of a novel pharmacological intervention against cancer and other diseases.

Pneumocystis S-adenosylmethionine transport: a potential drug target.

Pneumocystis pneumonia (PCP) is a life-threatening condition in immunosuppressed patients. Current treatments are inadequate, and new drug leads are needed. This fungus depends on its host for S-adenosylmethionine (AdoMet), a critical metabolic intermediate ordinarily synthesized by individual cells as needed. Pneumocystis contains a gene coding for the AdoMet-synthesizing enzyme methionine ATP transferase (MAT), and the protein is expressed. However, the fungus lacks MAT activity, and infection causes the depletion of host plasma AdoMet. The uptake of Pneumocystis AdoMet was shown to be exquisitely specific, which suggests the transport of AdoMet as a potential drug target. Here we report on the discovery of PcPET8, a Pneumocystis gene with homology to mitochondrial AdoMet transporters. When expressed by Saccharomyces cerevisiae, it locates properly to the mitochondrion and complements a strain of S. cerevisiae lacking its native mitochondrial AdoMet transporter. The importance of AdoMet transport is demonstrated by the ability of the AdoMet analogue sinefungin to block the uptake of Pneumocystis AdoMet and inhibit growth in culture. Because PcPET8 is likely critical for Pneumocystis, the yeast construct has potential as a surrogate for testing compounds against Pneumocystis.

Multiplex Gene Tagging with CRISPR-Cas9 for Live-Cell Microscopy and Application to Study the Role of SARS-CoV-2 Proteins in Autophagy, Mitochondrial Dynamics, and Cell Growth.

The lack of efficient tools to label multiple endogenous targets in cell lines without staining or fixation has limited our ability to track physiological and pathological changes in cells over time via live-cell studies. Here, we outline the FAST-HDR vector system to be used in combination with CRISPR-Cas9 to allow visual live-cell studies of up to three endogenous proteins within the same cell line. Our approach utilizes a novel set of advanced donor plasmids for homology-directed repair and a streamlined workflow optimized for microscopy-based cell screening to create genetically modified cell lines that do not require staining or fixation to accommodate microscopy-based studies. We validated this new methodology by developing two advanced cell lines with three fluorescent-labeled endogenous proteins that support high-content imaging without using antibodies or exogenous staining. We applied this technology to study seven severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2/COVID-19) viral proteins to understand better their effects on autophagy, mitochondrial dynamics, and cell growth. Using these two cell lines, we were able to identify the protein ORF3a successfully as a potent inhibitor of autophagy, inducer of mitochondrial relocalization, and a growth inhibitor, which highlights the effectiveness of live-cell studies using this technology.

Discovery and SAR of Novel Disubstituted Quinazolines as Dual PI3Kalpha/mTOR Inhibitors Targeting Breast Cancer.

The dual PI3Kα/ m TOR inhibitors represent a promising molecularly targeted therapy for cancer. Here, we documented the discovery of new 2,4-disubstituted quinazoline analogs as potent dual PI3Kα/sm TOR inhibitors. Our structure based chemistry endeavor yielded six excellent compounds 9e, 9f, 9g, 9k, 9m, and 9o with single/double digit nanomolar IC50 values against both enzymes and acceptable aqueous solubility and stability to oxidative metabolism. One of those analogs, 9m, possessed a sulfonamide substituent, which has not been described for this chemical scaffold before. The short direct synthetic routes, structure-activity relationship, in vitro 2D cell culture viability assays against normal fibroblasts and 3 breast cancer cell lines, and in vitro 3D culture viability assay against MCF7 cells for this series are described.

Passive transfer of Plasmodium falciparum MSP-2 pseudopeptide-induced antibodies efficiently controlled parasitemia in Plasmodium berghei-infected mice.

We have developed monoclonal antibodies directed against the pseudopeptide psi-130, derived from the highly conserved malarial antigen Plasmodium falciparum merozoite surface protein 2 (MSP-2), for obtaining novel molecular tools with potential applications in the control of malaria. Following isotype switching, these antibodies were tested for their ability to suppress blood-stage parasitemia through passive immunization in malaria-infected mice. Some proved totally effective in suppressing a lethal blood-stage challenge infection and others reduced malarial parasitemia. Protection against P. berghei malaria following Ig passive immunization can be associated with specific immunoglobulins induced by a site-directed designed MSP-2 reduced amide pseudopeptide.

A novel assay platform for the detection of translation modulators of spermidine/spermine acetyltransferase.

Spermidine/spermine-N1-acetyltransferase (SSAT) is a mitochondrial-localized enzyme that is highly inducible and tightly controlled and is the rate-limiting enzyme in polyamine catabolism. It is known that SSAT is induced when polyamine level increases. Although multiple mechanisms have been implicated, translational control is thought to be paramount. Previous studies with transgenic and knockout mice suggested that for certain human conditions, the modulation of SSAT levels could offer therapeutic benefits. Besides polyamines and their analogs, certain stimuli can increase SSAT levels, suggesting that the development of reporters for high throughput screening can lead to the identification of novel pharmacophores that can modulate SSAT translation. Here we report the development and validation of a luciferase-based biosensor system for the identification of compounds that are able to either promote or prevent the translation of SSAT. The system uses HEK293T cells transfected with a construct composed of SSAT mRNA modified to lack upstream open reading frame (uORF) function, is mutated to reduce translational repression and is linked with luciferase. As a proof of principle of the utility of the SSAT translation sensor, we screened the Prestwick drug library (1,200 FDA Approved compounds). The library contained 15 compounds that activated SSAT translation by at least 40% more than the basal expression, but none exceeded the positive control N1, N11-diethylnorspermine. On the other hand, 38 compounds were found to strongly inhibit SSAT translation. We conclude that this biosensor can lead to the identification of novel pharmacophores that are able to modulate the translation of SSAT.

Polyamine-regulated translation of spermidine/spermine-N1-acetyltransferase.

Rapid synthesis of the polyamine catabolic enzyme spermidine/spermine-N(1)-acetyltransferase (SSAT) in response to increased polyamines is an important polyamine homeostatic mechanism. Indirect evidence has suggested that there is an important control mechanism involving the release of a translational repressor protein that allows the immediate initiation of SSAT protein synthesis without RNA transcription, maturation, or translocation. To identify a repressor protein, we used a mass spectroscopy-based RNA-protein interaction system and found six proteins that bind to the coding region of SSAT mRNA. Individual small interfering RNA (siRNA) experiments showed that nucleolin knockdown enhances SSAT translation. Nucleolin exists in several isoforms, and we report that the isoform that binds to SSAT mRNA undergoes autocatalysis in the presence of polyamines, a result suggesting that there is a negative feedback system that helps control the cellular content of polyamines. Preliminary molecular interaction data show that a nucleolin isoform binds to a 5' stem-loop of the coding region of SSAT mRNA. The glycine/arginine-rich C terminus of nucleolin is required for binding, and the four RNA recognition motif domains are included in the isoform that blocks SSAT translation. Understanding SSAT translational control mechanisms has the potential for the development of therapeutic strategies against cancer and obesity.

Mechanism and tissue specificity of nicotine-mediated lung S-adenosylmethionine reduction.

We previously reported that chronic nicotine infusion blocks development of Pneumocystis pneumonia. This discovery developed from our work demonstrating the inability of this fungal pathogen to synthesize the critical metabolic intermediate S-adenosylmethionine and work by others showing nicotine to cause lung-specific reduction of S-adenosylmethionine in guinea pigs. We had found nicotine infusion to cause increased lung ornithine decarboxylase activity (rate-controlling enzyme of polyamine synthesis) and hypothesized that S-adenosylmethionine reduction is driven by up-regulated polyamine biosynthesis. Here we report a critical test of our hypothesis; inhibition of ornithine decarboxylase blocks the effect of nicotine on lung S-adenosylmethionine. Further support is provided by metabolite analyses showing nicotine to cause a strong diversion of S-adenosylmethionine toward polyamine synthesis and away from methylation reactions; these shifts are reversed by inhibition of ornithine decarboxylase. Because the nicotine effect on Pneumocystis is so striking, we considered the possibility of tissue specificity. Using laser capture microdissection, we collected samples of lung alveolar regions (site of infection) and respiratory epithelium for controls. We found nicotine to cause increased ornithine decarboxylase protein in alveolar regions but not airway epithelium; we conclude that tissue specificity likely contributes to the effect of nicotine on Pneumocystis pneumonia. Earlier we reported that the full effect of nicotine requires 3 weeks of treatment, and here we show recovery is symmetrical, also requiring 3 weeks after treatment cessation. Because this time frame is similar to pneumocyte turnover time, the shift in polyamine metabolism may occur as new pneumocytes are produced.

Identifying and characterising the Plasmodium falciparum RhopH3 Plasmodium vivax homologue.

Four Plasmodium species cause malaria in humans, Plasmodium falciparum being the most widely studied to date. All Plasmodium species have paired club-shaped organelles towards their apical extreme named rhoptries that contain many lipids and proteins which are released during target cell invasion. P. falciparum RhopH3 is a rhoptry protein triggering important immune responses in patients from endemic regions. It has also been shown that anti-RhopH3 antibodies inhibit in vitro invasion of erythrocytes. Recent immunisation studies in mice with the Plasmodium yoelii and Plasmodium berghei RhopH3 P. falciparum homologue proteins found that they are able to induce protection in murine models. This study described identifying and characterising RhopH3 protein in Plasmodium vivax; it is encoded by a seven exon gene and expressed during the parasite's asexual stage. PvRhopH3 has similar processing to its homologue in P. falciparum and presents a cellular immunolocalisation pattern characteristic of rhoptry proteins.