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1.
Cell ; 173(2): 443-455.e12, 2018 04 05.
Artigo em Inglês | MEDLINE | ID: mdl-29576450

RESUMO

Hereditary xerocytosis is thought to be a rare genetic condition characterized by red blood cell (RBC) dehydration with mild hemolysis. RBC dehydration is linked to reduced Plasmodium infection in vitro; however, the role of RBC dehydration in protection against malaria in vivo is unknown. Most cases of hereditary xerocytosis are associated with gain-of-function mutations in PIEZO1, a mechanically activated ion channel. We engineered a mouse model of hereditary xerocytosis and show that Plasmodium infection fails to cause experimental cerebral malaria in these mice due to the action of Piezo1 in RBCs and in T cells. Remarkably, we identified a novel human gain-of-function PIEZO1 allele, E756del, present in a third of the African population. RBCs from individuals carrying this allele are dehydrated and display reduced Plasmodium infection in vitro. The existence of a gain-of-function PIEZO1 at such high frequencies is surprising and suggests an association with malaria resistance.


Assuntos
Anemia Hemolítica Congênita/patologia , População Negra/genética , Hidropisia Fetal/patologia , Canais Iônicos/genética , Malária/patologia , Alelos , Anemia Hemolítica Congênita/genética , Animais , Desidratação , Modelos Animais de Doenças , Eritrócitos/citologia , Eritrócitos/metabolismo , Deleção de Genes , Genótipo , Humanos , Hidropisia Fetal/genética , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/deficiência , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/genética , Canais Iônicos/química , Malária/genética , Malária/parasitologia , Malária/prevenção & controle , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenótipo , Plasmodium berghei/crescimento & desenvolvimento , Plasmodium berghei/patogenicidade , Linfócitos T/citologia , Linfócitos T/metabolismo
2.
Chemistry ; 29(20): e202203958, 2023 Apr 06.
Artigo em Inglês | MEDLINE | ID: mdl-36617500

RESUMO

Here, we present remarkable epoxyketone-based proteasome inhibitors with low nanomolar in vitro potency for blood-stage Plasmodium falciparum and low cytotoxicity for human cells. Our best compound has more than 2,000-fold greater selectivity for erythrocytic-stage P. falciparum over HepG2 and H460 cells, which is largely driven by the accommodation of the parasite proteasome for a D-amino acid in the P3 position and the preference for a difluorobenzyl group in the P1 position. We isolated the proteasome from P. falciparum cell extracts and determined that the best compound is 171-fold more potent at inhibiting the ß5 subunit of P. falciparum proteasome when compared to the same subunit of the human constitutive proteasome. These compounds also significantly reduce parasitemia in a P. berghei mouse infection model and prolong survival of animals by an average of 6 days. The current epoxyketone inhibitors are ideal starting compounds for orally bioavailable anti-malarial drugs.


Assuntos
Antimaláricos , Plasmodium , Camundongos , Animais , Humanos , Inibidores de Proteassoma/química , Complexo de Endopeptidases do Proteassoma/química , Plasmodium falciparum , Antimaláricos/farmacologia
3.
Molecules ; 25(7)2020 Mar 31.
Artigo em Inglês | MEDLINE | ID: mdl-32244512

RESUMO

Marine Cyanobacteria (blue-green algae) have been shown to possess an enormous potential to produce structurally diverse natural products that exhibit a broad spectrum of potent biological activities, including cytotoxic, antifungal, antiparasitic, antiviral, and antibacterial activities. Here, we report the isolation and structure determination of palstimolide A, a complex polyhydroxy macrolide with a 40-membered ring that was isolated from a tropical marine cyanobacterium collected at Palmyra Atoll. NMR-guided fractionation in combination with MS2-based molecular networking and isolation via HPLC yielded 0.7 mg of the pure compound. The small quantity isolated along with the presence of significant signal degeneracy in both the 1H and 13C-NMR spectra complicated the structure elucidation of palstimolide A. Various NMR experiments and solvent systems were employed, including the LRHSQMBC experiment that allows the detection of long-range 1H-13C correlation data across 4-, 5-, and even 6-bonds. This expanded NMR data set enabled the elucidation of the palstimolide's planar structure, which is characterized by several 1,5-disposed hydroxy groups as well as a tert-butyl group. The compound showed potent antimalarial activity with an IC50 of 223 nM as well as interesting anti-leishmanial activity with an IC50 of 4.67 µM.


Assuntos
Antimaláricos/química , Antimaláricos/farmacologia , Macrolídeos/química , Macrolídeos/farmacologia , Organismos Aquáticos/química , Cianobactérias/química , Espectroscopia de Ressonância Magnética , Estrutura Molecular , Testes de Sensibilidade Parasitária , Relação Estrutura-Atividade
4.
Nat Commun ; 11(1): 1780, 2020 04 14.
Artigo em Inglês | MEDLINE | ID: mdl-32286267

RESUMO

A promising new compound class for treating human malaria is the imidazolopiperazines (IZP) class. IZP compounds KAF156 (Ganaplacide) and GNF179 are effective against Plasmodium symptomatic asexual blood-stage infections, and are able to prevent transmission and block infection in animal models. But despite the identification of resistance mechanisms in P. falciparum, the mode of action of IZPs remains unknown. To investigate, we here combine in vitro evolution and genome analysis in Saccharomyces cerevisiae with molecular, metabolomic, and chemogenomic methods in P. falciparum. Our findings reveal that IZP-resistant S. cerevisiae clones carry mutations in genes involved in Endoplasmic Reticulum (ER)-based lipid homeostasis and autophagy. In Plasmodium, IZPs inhibit protein trafficking, block the establishment of new permeation pathways, and cause ER expansion. Our data highlight a mechanism for blocking parasite development that is distinct from those of standard compounds used to treat malaria, and demonstrate the potential of IZPs for studying ER-dependent protein processing.


Assuntos
Antimaláricos/farmacologia , Plasmodium falciparum/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Concentração Inibidora 50 , Espectrometria de Massas , Proteínas de Protozoários/metabolismo , Pirazóis/farmacologia , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/metabolismo , Via Secretória/efeitos dos fármacos
5.
Circulation ; 117(9): 1207-15, 2008 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-18285563

RESUMO

BACKGROUND: Peripheral arterial disease (PAD) caused by occlusive atherosclerosis of the lower extremity has 2 major clinical manifestations. Critical limb ischemia is characterized by rest pain and/or tissue loss and has a > or = 40% risk of death and major amputation. Intermittent claudication causes pain on walking, has no tissue loss, and has amputation plus mortality rates of 2% to 4% per year. Progression from claudication to limb ischemia is infrequent. Risk factors in most PAD patients overlap. Thus, we hypothesized that genetic variations may be linked to presence or absence of tissue loss in PAD. METHODS AND RESULTS: Hindlimb ischemia (murine model of PAD) was induced in C57BL/6, BALB/c, C57BL/6 x BALB/c (F1), F1 x BALB/c (N2), A/J, and C57BL/6J-Chr7(A/J)/NaJ chromosome substitution strains. Mice were monitored for perfusion recovery and tissue necrosis. Genome-wide scanning with polymorphic markers across the 19 murine autosomes was performed on the N2 mice. Greater tissue loss and poorer perfusion recovery occurred in BALB/c than in the C57BL/6 strain. Analysis of 105 N2 progeny identified a single quantitative trait locus on chromosome 7 that exhibited significant linkage to both tissue necrosis and extent of perfusion recovery. Using the appropriate chromosome substitution strain, we demonstrate that C57BL/6-derived chromosome 7 is required for tissue preservation. CONCLUSIONS: We have identified a quantitative trait locus on murine chromosome 7 (LSq-1) that is associated with the absence of tissue loss in a preclinical model of PAD and may be useful in identifying gene(s) that influence PAD in humans.


Assuntos
Cromossomos de Mamíferos/genética , Membro Posterior/irrigação sanguínea , Membro Posterior/cirurgia , Isquemia/genética , Isquemia/cirurgia , Locos de Características Quantitativas/genética , Animais , Feminino , Membro Posterior/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Especificidade da Espécie
6.
Nat Commun ; 10(1): 488, 2019 01 30.
Artigo em Inglês | MEDLINE | ID: mdl-30700707

RESUMO

The exoerythrocytic stage of Plasmodium infection is a critical window for prophylactic intervention. Using genome-wide dual RNA sequencing of flow-sorted infected and uninfected hepatoma cells we show that the human mucosal immunity gene, mucin-13 (MUC13), is strongly upregulated during Plasmodium exoerythrocytic hepatic-stage infection. We confirm MUC13 transcript increases in hepatoma cell lines and primary hepatocytes. In immunofluorescence assays, host MUC13 protein expression distinguishes infected cells from adjacent uninfected cells and shows similar colocalization with parasite biomarkers such as UIS4 and HSP70. We further show that localization patterns are species independent, marking both P. berghei and P. vivax infected cells, and that MUC13 can be used to identify compounds that inhibit parasite replication in hepatocytes. This data provides insights into host-parasite interactions in Plasmodium infection, and demonstrates that a component of host mucosal immunity is reprogrammed during the progression of infection.


Assuntos
Imunidade nas Mucosas/fisiologia , Malária/imunologia , Malária/metabolismo , Mucinas/metabolismo , Carcinoma Hepatocelular/imunologia , Carcinoma Hepatocelular/parasitologia , Linhagem Celular , Células Cultivadas , Proteínas de Choque Térmico HSP70/metabolismo , Hepatócitos/parasitologia , Hepatócitos/patologia , Interações Hospedeiro-Parasita , Humanos , Imunidade nas Mucosas/genética , Neoplasias Hepáticas/imunologia , Plasmodium berghei/patogenicidade
7.
ACS Infect Dis ; 4(4): 531-540, 2018 04 13.
Artigo em Inglês | MEDLINE | ID: mdl-29542317

RESUMO

To develop new drugs and vaccines for malaria elimination, it will be necessary to discover biological interventions, including small molecules that act against Plasmodium vivax exoerythrocytic forms. However, a robust in vitro culture system for P. vivax is still lacking. Thus, to study exoerythrocytic forms, researchers must have simultaneous access to fresh, temperature-controlled patient blood samples, as well as an anopheline mosquito colony. In addition, researchers must rely on native mosquito species to avoid introducing a potentially dangerous invasive species into a malaria-endemic region. Here, we report an in vitro culture system carried out on site in a malaria-endemic region for liver stage parasites of P. vivax sporozoites obtained from An. darlingi, the main malaria vector in the Americas. P. vivax sporozoites were obtained by dissection of salivary glands from infected An. darlingi mosquitoes and purified by Accudenz density gradient centrifugation. HC04 liver cells were exposed to P. vivax sporozoites and cultured up to 9 days. To overcome low P. vivax patient parasitemias, potentially lower mosquito vectorial capacity, and humid, nonsterile environmental conditions, a new antibiotic cocktail was included in tissue culture to prevent contamination. Culturing conditions supported exoerythrocytic (EEF) P. vivax liver stage growth up to 9 days and allowed for maturation into intrahepatocyte merosomes. Some of the identified small forms were resistant to atovaquone (1 µM) but sensitive to the phosphatidylinositol 4-kinase inhibitor, KDU691 (1 µM). This study reports a field-accessible EEF production process for drug discovery in a malaria-endemic site in which viable P. vivax sporozoites are used for drug studies using hepatocyte infection. Our data demonstrate that the development of meaningful, field-based resources for P. vivax liver stage drug screening and liver stage human malaria experimentation in the Amazon region is feasible.


Assuntos
Técnicas de Cultura de Células/métodos , Hepatócitos/parasitologia , Parasitologia/métodos , Plasmodium vivax/crescimento & desenvolvimento , Animais , Anopheles/parasitologia , Linhagem Celular , Humanos , Peru , Plasmodium vivax/isolamento & purificação , Glândulas Salivares/parasitologia
8.
Science ; 359(6372): 191-199, 2018 01 12.
Artigo em Inglês | MEDLINE | ID: mdl-29326268

RESUMO

Chemogenetic characterization through in vitro evolution combined with whole-genome analysis can identify antimalarial drug targets and drug-resistance genes. We performed a genome analysis of 262 Plasmodium falciparum parasites resistant to 37 diverse compounds. We found 159 gene amplifications and 148 nonsynonymous changes in 83 genes associated with drug-resistance acquisition, where gene amplifications contributed to one-third of resistance acquisition events. Beyond confirming previously identified multidrug-resistance mechanisms, we discovered hitherto unrecognized drug target-inhibitor pairs, including thymidylate synthase and a benzoquinazolinone, farnesyltransferase and a pyrimidinedione, and a dipeptidylpeptidase and an arylurea. This exploration of the P. falciparum resistome and druggable genome will likely guide drug discovery and structural biology efforts, while also advancing our understanding of resistance mechanisms available to the malaria parasite.


Assuntos
Antimaláricos/farmacologia , Resistência a Medicamentos/genética , Genoma de Protozoário , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/genética , Ativação Metabólica , Alelos , Variações do Número de Cópias de DNA , Evolução Molecular Direcionada , Resistência a Múltiplos Medicamentos/genética , Genes de Protozoários , Metabolômica , Mutação , Plasmodium falciparum/crescimento & desenvolvimento , Seleção Genética , Fatores de Transcrição/química , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
9.
J Med Chem ; 60(15): 6721-6732, 2017 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-28696697

RESUMO

Naturally derived chemical compounds are the foundation of much of our pharmacopeia, especially in antiproliferative and anti-infective drug classes. Here, we report that a naturally derived molecule called carmaphycin B is a potent inhibitor against both the asexual and sexual blood stages of malaria infection. Using a combination of in silico molecular docking and in vitro directed evolution in a well-characterized drug-sensitive yeast model, we determined that these compounds target the ß5 subunit of the proteasome. These studies were validated using in vitro inhibition assays with proteasomes isolated from Plasmodium falciparum. As carmaphycin B is toxic to mammalian cells, we synthesized a series of chemical analogs that reduce host cell toxicity while maintaining blood-stage and gametocytocidal antimalarial activity and proteasome inhibition. This study describes a promising new class of antimalarial compound based on the carmaphycin B scaffold, as well as several chemical structural features that serve to enhance antimalarial specificity.


Assuntos
Antimaláricos/farmacologia , Dipeptídeos/farmacologia , Oligopeptídeos/farmacologia , Plasmodium falciparum/efeitos dos fármacos , Inibidores de Proteassoma/farmacologia , Antimaláricos/síntese química , Artemisininas/farmacologia , Dipeptídeos/síntese química , Desenho de Fármacos , Ensaios Enzimáticos , Células Hep G2 , Humanos , Simulação de Acoplamento Molecular , Oligopeptídeos/síntese química , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma/síntese química , Saccharomyces cerevisiae/efeitos dos fármacos
10.
mBio ; 7(4)2016 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-27381290

RESUMO

UNLABELLED: Mutations in the Plasmodium falciparum cyclic amine resistance locus (PfCARL) are associated with parasite resistance to the imidazolopiperazines, a potent class of novel antimalarial compounds that display both prophylactic and transmission-blocking activity, in addition to activity against blood-stage parasites. Here, we show that pfcarl encodes a protein, with a predicted molecular weight of 153 kDa, that localizes to the cis-Golgi apparatus of the parasite in both asexual and sexual blood stages. Utilizing clustered regularly interspaced short palindromic repeat (CRISPR)-mediated gene introduction of 5 variants (L830V, S1076N/I, V1103L, and I1139K), we demonstrate that mutations in pfcarl are sufficient to generate resistance against the imidazolopiperazines in both asexual and sexual blood-stage parasites. We further determined that the mutant PfCARL protein confers resistance to several structurally unrelated compounds. These data suggest that PfCARL modulates the levels of small-molecule inhibitors that affect Golgi-related processes, such as protein sorting or membrane trafficking, and is therefore an important mechanism of resistance in malaria parasites. IMPORTANCE: Several previous in vitro evolution studies have implicated the Plasmodium falciparum cyclic amine resistance locus (PfCARL) as a potential target of imidazolopiperazines, potent antimalarial compounds with broad activity against different parasite life cycle stages. Given that the imidazolopiperazines are currently being tested in clinical trials, understanding their mechanism of resistance and the cellular processes involved will allow more effective clinical usage.


Assuntos
Antimaláricos/farmacologia , Resistência a Múltiplos Medicamentos , Loci Gênicos , Mutação , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/genética , Proteínas de Protozoários/genética , Recombinação Genética
11.
Sci Rep ; 6: 27806, 2016 06 13.
Artigo em Inglês | MEDLINE | ID: mdl-27291296

RESUMO

The spiroindolones, a new class of antimalarial medicines discovered in a cellular screen, are rendered less active by mutations in a parasite P-type ATPase, PfATP4. We show here that S. cerevisiae also acquires mutations in a gene encoding a P-type ATPase (ScPMA1) after exposure to spiroindolones and that these mutations are sufficient for resistance. KAE609 resistance mutations in ScPMA1 do not confer resistance to unrelated antimicrobials, but do confer cross sensitivity to the alkyl-lysophospholipid edelfosine, which is known to displace ScPma1p from the plasma membrane. Using an in vitro cell-free assay, we demonstrate that KAE609 directly inhibits ScPma1p ATPase activity. KAE609 also increases cytoplasmic hydrogen ion concentrations in yeast cells. Computer docking into a ScPma1p homology model identifies a binding mode that supports genetic resistance determinants and in vitro experimental structure-activity relationships in both P. falciparum and S. cerevisiae. This model also suggests a shared binding site with the dihydroisoquinolones antimalarials. Our data support a model in which KAE609 exerts its antimalarial activity by directly interfering with P-type ATPase activity.


Assuntos
Antimaláricos/metabolismo , Indóis/metabolismo , ATPases do Tipo-P/metabolismo , Compostos de Espiro/metabolismo , Sequência de Aminoácidos , Antimaláricos/química , Antimaláricos/farmacologia , Sítios de Ligação , Sistemas CRISPR-Cas/genética , Citosol/química , Citosol/efeitos dos fármacos , Farmacorresistência Fúngica , Indóis/química , Indóis/farmacologia , Concentração Inibidora 50 , Simulação de Acoplamento Molecular , ATPases do Tipo-P/antagonistas & inibidores , ATPases do Tipo-P/genética , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/enzimologia , Estrutura Terciária de Proteína , ATPases Translocadoras de Prótons/antagonistas & inibidores , ATPases Translocadoras de Prótons/genética , ATPases Translocadoras de Prótons/metabolismo , Proteínas de Protozoários/antagonistas & inibidores , Proteínas de Protozoários/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/enzimologia , Proteínas de Saccharomyces cerevisiae/antagonistas & inibidores , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Alinhamento de Sequência , Análise de Sequência de DNA , Compostos de Espiro/química , Compostos de Espiro/farmacologia , Relação Estrutura-Atividade , Sequenciamento Completo do Genoma
12.
Nat Microbiol ; 1: 16166, 2016 Sep 19.
Artigo em Inglês | MEDLINE | ID: mdl-27642791

RESUMO

A molecular understanding of drug resistance mechanisms enables surveillance of the effectiveness of new antimicrobial therapies during development and deployment in the field. We used conventional drug resistance selection as well as a regime of limiting dilution at early stages of drug treatment to probe two antimalarial imidazolopiperazines, KAF156 and GNF179. The latter approach permits the isolation of low-fitness mutants that might otherwise be out-competed during selection. Whole-genome sequencing of 24 independently derived resistant Plasmodium falciparum clones revealed four parasites with mutations in the known cyclic amine resistance locus (pfcarl) and a further 20 with mutations in two previously unreported P. falciparum drug resistance genes, an acetyl-CoA transporter (pfact) and a UDP-galactose transporter (pfugt). Mutations were validated both in vitro by CRISPR editing in P. falciparum and in vivo by evolution of resistant Plasmodium berghei mutants. Both PfACT and PfUGT were localized to the endoplasmic reticulum by fluorescence microscopy. As mutations in pfact and pfugt conveyed resistance against additional unrelated chemical scaffolds, these genes are probably involved in broad mechanisms of antimalarial drug resistance.

13.
J Vis Exp ; (106): e53214, 2015 Dec 04.
Artigo em Inglês | MEDLINE | ID: mdl-26709459

RESUMO

The genetic variation responsible for the sickle cell allele (HbS) enables erythrocytes to resist infection by the malaria parasite, P. falciparum. The molecular basis of this resistance, which is known to be multifactorial, remains incompletely understood. Recent studies found that the differential expression of erythrocyte microRNAs, once translocated into malaria parasites, affect both gene regulation and parasite growth. These miRNAs were later shown to inhibit mRNA translation by forming a chimeric RNA transcript via 5' RNA fusion with discreet subsets of parasite mRNAs. Here, the techniques that were used to study the functional role and putative mechanism underlying erythrocyte microRNAs on the gene regulation and translational potential of P. falciparum, including the transfection of modified synthetic microRNAs into host erythrocytes, will be detailed.  Finally, a polysome gradient method is used to determine the extent of translation of these transcripts. Together, these techniques allowed us to demonstrate that the dysregulated levels of erythrocyte microRNAs contribute to cell-intrinsic malaria resistance of sickle erythrocytes.


Assuntos
Eritrócitos/fisiologia , MicroRNAs/administração & dosagem , MicroRNAs/genética , Plasmodium falciparum/genética , Polirribossomos/genética , Ribossomos/genética , Transfecção/métodos , Eritrócitos/parasitologia , Humanos , Malária Falciparum/genética , Malária Falciparum/parasitologia , MicroRNAs/sangue , Plasmodium falciparum/metabolismo , Polirribossomos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ribossomos/metabolismo
14.
Cancer Metab ; 1(1): 23, 2013 Dec 23.
Artigo em Inglês | MEDLINE | ID: mdl-24359630

RESUMO

BACKGROUND: A variety of oncogenic and environmental factors alter tumor metabolism to serve the distinct cellular biosynthetic and bioenergetic needs present during oncogenesis. Extracellular acidosis is a common microenvironmental stress in solid tumors, but little is known about its metabolic influence, particularly when present in the absence of hypoxia. In order to characterize the extent of tumor cell metabolic adaptations to acidosis, we employed stable isotope tracers to examine how acidosis impacts glucose, glutamine, and palmitate metabolism in breast cancer cells exposed to extracellular acidosis. RESULTS: Acidosis increased both glutaminolysis and fatty acid ß-oxidation, which contribute metabolic intermediates to drive the tricarboxylic acid cycle (TCA cycle) and ATP generation. Acidosis also led to a decoupling of glutaminolysis and novel glutathione (GSH) synthesis by repressing GCLC/GCLM expression. We further found that acidosis redirects glucose away from lactate production and towards the oxidative branch of the pentose phosphate pathway (PPP). These changes all serve to increase nicotinamide adenine dinucleotide phosphate (NADPH) production and counter the increase in reactive oxygen species (ROS) present under acidosis. The reduced novel GSH synthesis under acidosis may explain the increased demand for NADPH to recycle existing pools of GSH. Interestingly, acidosis also disconnected novel ribose synthesis from the oxidative PPP, seemingly to reroute PPP metabolites to the TCA cycle. Finally, we found that acidosis activates p53, which contributes to both the enhanced PPP and increased glutaminolysis, at least in part, through the induction of G6PD and GLS2 genes. CONCLUSIONS: Acidosis alters the cellular metabolism of several major metabolites, which induces a significant degree of metabolic inflexibility. Cells exposed to acidosis largely rely upon mitochondrial metabolism for energy generation to the extent that metabolic intermediates are redirected away from several other critical metabolic processes, including ribose and glutathione synthesis. These alterations lead to both a decrease in cellular proliferation and increased sensitivity to ROS. Collectively, these data reveal a role for p53 in cellular metabolic reprogramming under acidosis, in order to permit increased bioenergetic capacity and ROS neutralization. Understanding the metabolic adaptations that cancer cells make under acidosis may present opportunities to generate anti-tumor therapeutic agents that are more tumor-specific.

15.
Cancer Res ; 72(2): 491-502, 2012 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-22135092

RESUMO

Within solid tumor microenvironments, lactic acidosis, and hypoxia each have powerful effects on cancer pathophysiology. However, the influence that these processes exert on each other is unknown. Here, we report that a significant portion of the transcriptional response to hypoxia elicited in cancer cells is abolished by simultaneous exposure to lactic acidosis. In particular, lactic acidosis abolished stabilization of HIF-1α protein which occurs normally under hypoxic conditions. In contrast, lactic acidosis strongly synergized with hypoxia to activate the unfolded protein response (UPR) and an inflammatory response, displaying a strong similarity to ATF4-driven amino acid deprivation responses (AAR). In certain breast tumors and breast tumor cells examined, an integrative analysis of gene expression and array CGH data revealed DNA copy number alterations at the ATF4 locus, an important activator of the UPR/AAR pathway. In this setting, varying ATF4 levels influenced the survival of cells after exposure to hypoxia and lactic acidosis. Our findings reveal that the condition of lactic acidosis present in solid tumors inhibits canonical hypoxia responses and activates UPR and inflammation responses. Furthermore, these data suggest that ATF4 status may be a critical determinant of the ability of cancer cells to adapt to oxygen and acidity fluctuations in the tumor microenvironment, perhaps linking short-term transcriptional responses to long-term selection for copy number alterations in cancer cells.


Assuntos
Acidose Láctica/genética , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Acidose Láctica/metabolismo , Fator 4 Ativador da Transcrição/genética , Fator 4 Ativador da Transcrição/metabolismo , Hipóxia Celular/genética , Linhagem Celular Tumoral , Feminino , Amplificação de Genes , Regulação Neoplásica da Expressão Gênica , Genômica , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Subunidade alfa do Fator 1 Induzível por Hipóxia/biossíntese , Transdução de Sinais , Transfecção , Microambiente Tumoral
16.
Cell Host Microbe ; 12(2): 187-99, 2012 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-22901539

RESUMO

Erythrocytes carrying a variant hemoglobin allele (HbS), which causes sickle cell disease and resists infection by the malaria parasite Plasmodium falciparum. The molecular basis of this resistance, which has long been recognized as multifactorial, remains incompletely understood. Here we show that the dysregulated microRNA (miRNA) composition, of either heterozygous HbAS or homozygous HbSS erythrocytes, contributes to resistance against P. falciparum. During the intraerythrocytic life cycle of P. falciparum, a subset of erythrocyte miRNAs translocate into the parasite. Two miRNAs, miR-451 and let-7i, were highly enriched in HbAS and HbSS erythrocytes, and these miRNAs, along with miR-223, negatively regulated parasite growth. Surprisingly, we found that miR-451 and let-7i integrated into essential parasite messenger RNAs and, via impaired ribosomal loading, resulted in translational inhibition. Hence, sickle cell erythrocytes exhibit cell-intrinsic resistance to malaria in part through an atypical miRNA activity, which may represent a unique host defense strategy against complex eukaryotic pathogens.


Assuntos
Eritrócitos/parasitologia , Hemoglobina Falciforme/genética , Malária Falciparum/genética , MicroRNAs/metabolismo , Plasmodium falciparum/genética , Biossíntese de Proteínas , Transporte Biológico , Células Cultivadas , Regulação para Baixo , Eritrócitos/metabolismo , Hemoglobina Falciforme/metabolismo , Humanos , Malária Falciparum/metabolismo , Malária Falciparum/parasitologia , MicroRNAs/genética , Plasmodium falciparum/crescimento & desenvolvimento , Plasmodium falciparum/metabolismo
17.
Mol Biochem Parasitol ; 179(1): 42-6, 2011 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-21605599

RESUMO

In the malaria parasite Plasmodium falciparum, global studies of translational regulation have been hampered by the inability to isolate malaria polysomes. We describe here a novel method for polysome profiling in P. falciparum, a powerful approach which allows both a global view of translation and the measurement of ribosomal loading and density for specific mRNAs. Simultaneous lysis of infected erythrocytes and parasites releases stable, intact malaria polysomes, which are then purified by centrifugation through a sucrose cushion. The polysomes are resuspended, separated by velocity sedimentation and then fractionated, yielding a characteristic polysome profile reflecting the global level of translational activity in the parasite. RNA isolated from specific fractions can be used to determine the density of ribosomes loaded onto a particular transcript of interest, and is free of host ribosome contamination. Thus, our approach opens translational regulation in malaria to genome-wide analysis.


Assuntos
Parasitologia/métodos , Plasmodium falciparum/metabolismo , Polirribossomos/metabolismo , Biossíntese de Proteínas , RNA Mensageiro/metabolismo
18.
Methods Mol Biol ; 667: 193-203, 2010.
Artigo em Inglês | MEDLINE | ID: mdl-20827535

RESUMO

Human mature erythrocytes are terminally differentiated cells that have lost their nuclei and organelles during development. Even though mature erythrocytes lack ribosomal and other large-sized RNAs, they still retain small-sized RNAs. We have recently shown that there are abundant and diverse species of microRNAs in mature erythrocytes through the use of several different techniques, including northern blot, miRNA microarray, and real-time PCR. Furthermore, fractionation and genomic analysis has revealed that erythrocyte microRNA expression is different from that of reticulocytes or leukocytes and that mature erythrocytes contribute the majority of microRNA expression in whole blood. Therefore, global analysis of microRNA expression in circulating erythrocytes has the potential to provide mechanistic insights into erythrocyte biology and erythrocyte-related disorders. Here, we have provided the detailed methods for isolating and characterizing the microRNAs from human mature erythrocytes to enable such researches into human diseases involving erythrocytes.


Assuntos
Eritrócitos/fisiologia , MicroRNAs , Biomarcadores/metabolismo , Eritrócitos/citologia , Perfilação da Expressão Gênica/instrumentação , Perfilação da Expressão Gênica/métodos , Humanos , MicroRNAs/genética , MicroRNAs/isolamento & purificação , MicroRNAs/metabolismo , Análise em Microsséries/instrumentação , Análise em Microsséries/métodos
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