RESUMO
Acute myeloid leukemia (AML) is the most common form of acute leukemia in adults and the second most common among children. AML is characterized by aberrant proliferation of myeloid blasts in the bone marrow and impaired normal hematopoiesis. Despite the introduction of new drugs and allogeneic bone marrow transplantation, patients have poor overall survival rate with relapse as the major challenge, driving the demand for new therapeutic strategies. AML patients with high expression of the very long/long chain fatty acid transporter CD36 have poorer survival and very long chain fatty acid metabolism is critical for AML cell survival. Here we show that fatty acids are transferred from human primary adipocytes to AML cells upon co-culturing. A drug-like small molecule (SMS121) was identified by receptor-based virtual screening and experimentally demonstrated to target the lipid uptake protein CD36. SMS121 reduced the uptake of fatty acid into AML cells that could be reversed by addition of free fatty acids and caused decreased cell viability. The data presented here serves as a framework for the development of CD36 inhibitors to be used as future therapeutics against AML.
Assuntos
Ácidos Graxos , Leucemia Mieloide Aguda , Adulto , Criança , Humanos , Ácidos Graxos/uso terapêutico , Leucemia Mieloide Aguda/metabolismo , Medula Óssea/metabolismo , Doença Aguda , Técnicas de CoculturaRESUMO
Cancer invasion and metastasis are known to be potentiated by the expression of aquaporins (AQPs). Likewise, the expression levels of AQPs have been shown to be prognostic for survival in patients and have a role in tumor growth, edema, angiogenesis, and tumor cell migration. Thus, AQPs are key players in cancer biology and potential targets for drug development. Here, we present the single-particle cryo-EM structure of human AQP7 at 3.2-Å resolution in complex with the specific inhibitor compound Z433927330. The structure in combination with MD simulations shows that the inhibitor binds to the endofacial side of AQP7. In addition, cancer cells treated with Z433927330 show reduced proliferation. The data presented here serve as a framework for the development of AQP inhibitors.
Assuntos
Aquaporinas , Neoplasias , Humanos , Aquaporinas/metabolismo , Aquaporina 1/metabolismoRESUMO
Glucose transporters (GLUTs) are responsible for transporting hexose molecules across cellular membranes. In adipocytes, insulin stimulates glucose uptake by redistributing GLUT4 to the plasma membrane. In unstimulated adipose-like mouse cell lines, GLUT4 is known to be retained intracellularly by binding to TUG protein, while upon insulin stimulation, GLUT4 dissociates from TUG. Here, we report that the TUG homolog in human, ASPL, exerts similar properties, i.e., forms a complex with GLUT4. We describe the structural details of complex formation by combining biochemical assays with cross-linking mass spectrometry and computational modeling. Combined, the data suggest that the intracellular domain of GLUT4 binds to the helical lariat of ASPL and contributes to the regulation of GLUT4 trafficking by cooperative binding.
Assuntos
Proteínas de Transporte , Glucose , Humanos , Camundongos , Animais , Proteínas de Transporte/metabolismo , Transporte Proteico , Glucose/metabolismo , Proteínas Facilitadoras de Transporte de Glucose/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Insulina/metabolismoRESUMO
Long-distance migratory animals such as birds and bats have evolved to withstand selection imposed by pathogens across the globe, and pathogen richness is known to be particularly high in tropical regions. Immune genes, so-called Major Histocompatibility Complex (MHC) genes, are highly duplicated in songbirds compared to other vertebrates, and this high MHC diversity has been hypothesised to result in a unique adaptive immunity. To understand the rationale behind the evolution of the high MHC genetic diversity in songbirds, we determined the structural properties of an MHC class I protein, Acar3, from a long-distance migratory songbird, the great reed warbler Acrocephalus arundinaceus (in short: Acar). The structure of Acar3 was studied in complex with pathogen-derived antigens and shows an overall antigen presentation similar to human MHC class I. However, the peptides bound to Acar3 display an unusual conformation: Whereas the N-terminal ends of the peptides display enhanced flexibility, the conformation of their C-terminal halves is rather static. This uncommon peptide-binding mode in Acar3 is facilitated by a central Arg residue within the peptide-binding groove that fixes the backbone of the peptide at its central position, and potentially permits successful interactions between MHC class I and innate immune receptors. Our study highlights the importance of investigating the immune system of wild animals, such as birds and bats, to uncover unique immune mechanisms which may neither exist in humans nor in model organisms.
Assuntos
Quirópteros , Aves Canoras , Animais , Humanos , Aves Canoras/genética , Aves Canoras/metabolismo , Antígenos de Histocompatibilidade Classe I , Peptídeos/metabolismo , Apresentação de Antígeno , Antígenos HLARESUMO
Aquaglyceroporin 7 (AQP7) facilitates glycerol flux across the plasma membrane with a critical physiological role linked to metabolism, obesity, and associated diseases. Here, we present the single-particle cryo-EM structure of AQP7 determined at 2.55 Å resolution adopting two adhering tetramers, stabilized by extracellularly exposed loops, in a configuration like that of the well-characterized interaction of AQP0 tetramers. The central pore, in-between the four monomers, displays well-defined densities restricted by two leucine filters. Gas chromatography mass spectrometry (GC/MS) results show that the AQP7 sample contains glycerol 3-phosphate (Gro3P), which is compatible with the identified features in the central pore. AQP7 is shown to be highly expressed in human pancreatic α- and ß- cells suggesting that the identified AQP7 octamer assembly, in addition to its function as glycerol channel, may serve as junction proteins within the endocrine pancreas.
Assuntos
Aquagliceroporinas , Aquaporinas , Ilhotas Pancreáticas , Humanos , Aquaporinas/metabolismo , Glicerol/metabolismo , Microscopia Crioeletrônica , Ilhotas Pancreáticas/metabolismoRESUMO
Transition metals such as copper and zinc are essential elements required for the survival of most organisms, from bacteria to humans. Yet, elevated levels of these elements are highly toxic. The Copper TRansporter protein family (CTRs) represents the only identified copper uptake proteins in eukaryotes and hence serves as key components for the maintenance of appropriate levels of the metal. Moreover, CTRs have been proposed to serve as an entry point into cells of certain cancer drugs and to constitute attractive drug-targets for novel antifungals. Nevertheless, the structure, function, and regulation of the CTRs remain elusive, limiting valuable information also for applied sciences. To this end, here we report procedures to isolate a range of CTR members using Saccharomyces cerevisiae as a production host, focusing on three homologs, human CTR1, human CTR2, and Candida albicans CTR. Using forms C-terminally-linked to a protease cleavage sequence, Green Fluorescent Protein (GFP), and a His-tag, assessment of the localization, quantification and purification was facilitated. Cellular accumulation of the proteins was investigated via live-cell imaging. Detergents compatible with acceptable solubilization yields were identified and fluorescence-detection size-exclusion-chromatography (F-SEC) revealed preferred membrane extraction conditions for the targets. For purification purposes, the solubilized CTR members were subjected to affinity chromatography and SEC, reaching near homogeneity. The quality and quantity of the CTRs studied will permit downstream efforts to uncover imperative biophysical aspects of these proteins, paving the way for subsequent drug-discovery studies.
Assuntos
Cobre , Saccharomyces cerevisiae , Humanos , Cobre/metabolismo , Transporte Biológico , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Transportador de Cobre 1/metabolismo , Proteínas de Fluorescência Verde/metabolismoRESUMO
Measurement of protein-facilitated copper flux across biological membranes is a considerable challenge. Here, we demonstrate a straightforward microfluidic-derived approach for visualization and measurement of membranous Cu flux. Giant unilamellar vesicles, reconstituted with the membrane protein of interest, are prepared, surface-immobilized, and assessed using a novel quencher-sensor reporter system for detection of copper. With the aid of a syringe pump, the external buffer is exchanged, enabling consistent and precise exchange of solutes, without causing vesicle rupture or uneven local metal concentrations brought about by rapid mixing. This approach bypasses common issues encountered when studying heavy metal-ion flux, thereby providing a new platform for in vitro studies of metal homeostasis aspects that are critical for all cells, health, and disease.
Assuntos
Cobre , Microfluídica , Lipídeos , Membranas , Proteínas , Lipossomas UnilamelaresRESUMO
In eukaryotes, iron-sulfur clusters are essential cofactors for numerous physiological processes, but these clusters are primarily biosynthesized in mitochondria. Previous studies suggest mitochondrial ABCB7-type exporters are involved in maturation of cytosolic iron-sulfur proteins. However, the molecular mechanism for how the ABCB7-type exporters participate in this process remains elusive. Here, we report a series of cryo-electron microscopy structures of a eukaryotic homolog of human ABCB7, CtAtm1, determined at average resolutions ranging from 2.8 to 3.2 Å, complemented by functional characterization and molecular docking in silico. We propose that CtAtm1 accepts delivery from glutathione-complexed iron-sulfur clusters. A partially occluded state links cargo-binding to residues at the mitochondrial matrix interface that line a positively charged cavity, while the binding region becomes internalized and is partially divided in an early occluded state. Collectively, our findings substantially increase the understanding of the transport mechanism of eukaryotic ABCB7-type proteins.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Proteínas Ferro-Enxofre , Proteínas Mitocondriais , Microscopia Crioeletrônica , Humanos , Ferro/metabolismo , Proteínas Ferro-Enxofre/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Simulação de Acoplamento Molecular , Enxofre/metabolismoRESUMO
Copper (Cu) is one of the most abundant trace metals in all organisms, involved in a plethora of cellular processes. Yet elevated concentrations of the element are harmful, and interestingly prokaryotes are more sensitive for environmental Cu stress than humans. Various transport systems are present to maintain intracellular Cu homeostasis, including the prokaryotic plasmid-encoded multiprotein pco operon, which is generally assigned as a defense mechanism against elevated Cu concentrations. Here we structurally and functionally characterize the outer membrane component of the Pco system, PcoB, recovering a 2.0 Å structure, revealing a classical ß-barrel architecture. Unexpectedly, we identify a large opening on the extracellular side, linked to a considerably electronegative funnel that becomes narrower towards the periplasm, defining an ion-conducting pathway as also supported by metal binding quantification via inductively coupled plasma mass spectrometry and molecular dynamics (MD) simulations. However, the structure is partially obstructed towards the periplasmic side, and yet flux is permitted in the presence of a Cu gradient as shown by functional characterization in vitro. Complementary in vivo experiments demonstrate that isolated PcoB confers increased sensitivity towards Cu. Aggregated, our findings indicate that PcoB serves to permit Cu import. Thus, it is possible the Pco system physiologically accumulates Cu in the periplasm as a part of an unorthodox defense mechanism against metal stress. These results point to a previously unrecognized principle of maintaining Cu homeostasis and may as such also assist in the understanding and in efforts towards combatting bacterial infections of Pco-harboring pathogens.
Assuntos
Cobre , Proteínas de Membrana , Transporte Biológico , Cobre/metabolismo , Homeostase , Humanos , Proteínas de Membrana/metabolismo , Periplasma/metabolismoRESUMO
Superantigens (SAgs) are bacterial enterotoxins produced by Staphylococcus aureus. Staphylococcal enterotoxin type A (SEA), a staphylococcal superantigen, has been shown to bind to the cytokine signalling receptor glycoprotein 130 (gp130). The structural details, as well as the exact physiological role of this interaction, remain unclear. Here, we describe the structural details of the SEA-gp130 complex by combining crosslinking mass spectrometry and computational modelling. Interestingly, SEA is not able to bind gp130-homologues from rat and mouse. Our data suggest that SEA may interact with human gp130 in a different manner than other known gp130-ligands. Moreover, the fact that SEA does not bind mouse or rat gp130 suggests that SAgs have additional mechanisms of action in humans.
Assuntos
Enterotoxinas , Receptores de Citocinas , Animais , Receptor gp130 de Citocina , Enterotoxinas/metabolismo , Glicoproteínas , Humanos , Camundongos , Ratos , SuperantígenosRESUMO
Aquaporins play a crucial role in water homeostasis in the human body, and recently the physiological importance of aquaporins as glycerol channels have been demonstrated. The aquaglyceroporins (AQP3, AQP7, AQP9 and AQP10) represent key glycerol channels, enabling glycerol flux across the membranes of cells. Adipocytes are the major source of glycerol and during lipolysis, glycerol is released to be metabolized by other tissues through a well-orchestrated process. Here we show that both AQP3 and AQP7 bind to the lipid droplet protein perilipin 1 (PLIN1), suggesting that PLIN1 is involved in the coordination of the subcellular translocation of aquaglyceroporins in human adipocytes. Moreover, in addition to aquaglyceroporins, we discovered by transcriptome sequencing that AQP1 is expressed in human primary adipocytes. AQP1 is mainly a water channel and thus is thought to be involved in the response to hyper-osmotic stress by efflux of water during hyperglycemia. Thus, this data suggests a contribution of both orthodox aquaporin and aquaglyceroporin in human adipocytes to maintain the homeostasis of glycerol and water during fasting and feeding.
Assuntos
Aquaporina 1/genética , Aquaporina 3/genética , Aquaporinas/genética , Hiperglicemia/genética , Perilipina-1/genética , Adipócitos/metabolismo , Aquagliceroporinas/genética , Aquagliceroporinas/metabolismo , Aquaporina 3/metabolismo , Aquaporinas/metabolismo , Regulação da Expressão Gênica/genética , Glicerol/metabolismo , Homeostase/genética , Humanos , Hiperglicemia/metabolismo , Hiperglicemia/patologia , Transcriptoma/genética , Água/metabolismoRESUMO
Staphylococcal enterotoxins (SE) pose a great threat to human health due to their ability to bypass antigen presentation and activate large amounts of conventional T cells resulting in a cytokine storm potentially leading to toxic shock syndrome. Unconventional T- and NK cells are also activated by SE but the mechanisms remain poorly understood. In this study, the authors aimed to explore the underlying mechanism behind SE-mediated activation of MAIT-, γδ T-, and NK cells in vitro. CBMC or PBMC were stimulated with the toxins SEA, SEH, and TSST-1, and cytokine and cytotoxic responses were analyzed with ELISA and flow cytometry. All toxins induced a broad range of cytokines, perforin and granzyme B, although SEH was not as potent as SEA and TSST-1. SE-induced IFN-γ expression in MAIT-, γδ T-, and NK cells was clearly reduced by neutralization of IL-12, while cytotoxic compounds were not affected at all. Kinetic assays showed that unconventional T cell and NK cell-responses are secondary to the response in conventional T cells. Furthermore, co-cultures of isolated cell populations revealed that the ability of SEA to activate γδ T- and NK cells was fully dependent on the presence of both monocytes and αß T cells. Lastly, it was found that SE provoked a reduced and delayed cytokine response in infants, particularly within the unconventional T and NK cell populations. This study provides novel insights regarding the activation of unconventional T- and NK cells by SE, which contribute to understanding the vulnerability of young children towards Staphylococcus aureus infections.
Assuntos
Monócitos , Linfócitos T , Criança , Pré-Escolar , Citocinas , Enterotoxinas/farmacologia , Humanos , Células Matadoras Naturais , Leucócitos Mononucleares , Staphylococcus aureus , Superantígenos/farmacologiaRESUMO
It has been suggested that intake of polar lipids may beneficially modulate various metabolic variables. The purpose of this study was to evaluate the effect of oat polar lipids on postprandial and second meal glycemic regulation, blood lipids, gastrointestinal hormones, and subjective appetite-related variables in healthy humans. In a randomized design, twenty healthy subjects ingested four liquid cereal-based test beverages (42 g of available carbohydrates) containing: i. 30 g of oat oil with a low concentration (4%) of polar lipids (PLL), ii. 30 g of oat oil containing a high concentration (40%) of polar lipids (PLH), iii. 30 g of rapeseed oil (RSO), and iv. no added lipids (NL). The products were served as breakfast meals followed by a standardized lunch. Test variables were measured at fasting and during 3 h after breakfast and two additional hours following a standardized lunch. PLH reduced glucose and insulin responses after breakfast (0-120 min) compared to RSO, and after lunch (210-330 min) compared to RSO and PLL (p < 0.05). Compared to RSO, PLH resulted in increased concentrations of the gut hormones GLP-1 and PYY after the standardized lunch (p < 0.05). The results suggest that oat polar lipids have potential nutraceutical properties by modulating acute and second meal postprandial metabolic responses.
Assuntos
Avena/química , Desjejum/fisiologia , Índice Glicêmico/efeitos dos fármacos , Lipídeos/administração & dosagem , Almoço/fisiologia , Período Pós-Prandial/efeitos dos fármacos , Adulto , Apetite/efeitos dos fármacos , Biomarcadores/sangue , Fatores de Risco Cardiometabólico , Estudos Cross-Over , Feminino , Hormônios Gastrointestinais/sangue , Humanos , Lipídeos/sangue , Lipídeos/química , Masculino , Método Simples-CegoRESUMO
The human microbiome can produce metabolites that modulate insulin signaling. Type 2 diabetes patients have increased circulating concentrations of the microbially produced histidine metabolite, imidazole propionate (ImP) and administration of ImP in mice resulted in impaired glucose tolerance. Interestingly, the fecal microbiota of the patients had increased capacity to produce ImP, which is mediated by the bacterial enzyme urocanate reductase (UrdA). Here, we describe the X-ray structures of the ligand-binding domains of UrdA in four different states, representing the structural transitions along the catalytic reaction pathway of this unexplored enzyme linked to disease in humans. The structures in combination with functional data provide key insights into the mechanism of action of UrdA that open new possibilities for drug development strategies targeting type 2 diabetes.
Assuntos
Imidazóis/metabolismo , Oxirredutases/metabolismo , Shewanella/enzimologia , Ácido Urocânico/metabolismo , Arginina/metabolismo , Domínio Catalítico , Flavina-Adenina Dinucleotídeo/metabolismo , Imidazóis/química , Cinética , Ligantes , Modelos Moleculares , Oxirredutases/química , Conformação Proteica , Domínios Proteicos , Especificidade por Substrato , Termodinâmica , Ácido Urocânico/químicaAssuntos
Citarabina , Leucemia Mieloide Aguda , Antimetabólitos Antineoplásicos/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Citarabina/uso terapêutico , Transportador de Glucose Tipo 1/genética , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genéticaRESUMO
A challenging question in evolutionary theory is the origin of cell division and plausible molecular mechanisms involved. Here, we made the surprising observation that complexes formed by short alpha-helical peptides and oleic acid can create multiple membrane-enclosed spaces from a single lipid vesicle. The findings suggest that such complexes may contain the molecular information necessary to initiate and sustain this process. Based on these observations, we propose a new molecular model to understand protocell division.
Assuntos
Células Artificiais/química , Divisão Celular , Lactalbumina/química , Membranas/química , Ácido Oleico/química , Vesículas Citoplasmáticas/química , Peptídeos/químicaRESUMO
The aquaglyceroporin 7 (AQP7) facilitates permeation of glycerol through cell membranes and is crucial for lipid metabolism in humans. Glycerol efflux in human adipocytes is controlled by translocation of AQP7 to the plasma membrane upon hormone stimulation. Here we present two X-ray structures of human AQP7 at 1.9 and 2.2 Å resolution. The structures combined with molecular dynamics simulations suggest that AQP7 is a channel selective for glycerol and that glycerol may hamper water permeation through the channel. Moreover, the high resolution of the structures facilitated a detailed analysis of the orientation of glycerol in the pore, disclosing unusual positions of the hydroxyl groups. The data suggest that glycerol is conducted by a partly rotating movement through the channel. These observations provide a framework for understanding the basis of glycerol efflux and selectivity in aquaglyceroporins and pave the way for future design of AQP7 inhibitors.
Assuntos
Aquaporinas/química , Aquaporinas/metabolismo , Glicerol/metabolismo , Transporte Biológico , Cristalografia por Raios X , Humanos , Modelos Moleculares , Simulação de Dinâmica Molecular , Estrutura Secundária de Proteína , Água/metabolismoRESUMO
Presented here is an engineered protein domain, based on Protein A, that displays a calcium-dependent binding to antibodies. This protein, ZCa, is shown to efficiently function as an affinity ligand for mild purification of antibodies through elution with ethylenediaminetetraacetic acid. Antibodies are commonly used tools in the area of biological sciences and as therapeutics, and the most commonly used approach for antibody purification is based on Protein A using acidic elution. Although this affinity-based method is robust and efficient, the requirement for low pH elution can be detrimental to the protein being purified. By introducing a calcium-binding loop in the Protein A-derived Z domain, it has been re-engineered to provide efficient antibody purification under mild conditions. Through comprehensive analyses of the domain as well as the ZCa-Fc complex, the features of this domain are well understood. This novel protein domain provides a very valuable tool for effective and gentle antibody and Fc-fusion protein purification.
Assuntos
Anticorpos Monoclonais/química , Anticorpos Monoclonais/isolamento & purificação , Cromatografia de Afinidade , Engenharia de Proteínas , Anticorpos Monoclonais/imunologia , Afinidade de Anticorpos , Cálcio/química , Ensaio de Imunoadsorção Enzimática , Ligantes , Modelos Moleculares , Conformação Molecular , Engenharia de Proteínas/métodosRESUMO
Activating signaling mutations are common in acute leukemia with KMT2A (previously MLL) rearrangements (KMT2A-R). These mutations are often subclonal and their biological impact remains unclear. Using a retroviral acute myeloid mouse leukemia model, we demonstrate that FLT3 ITD , FLT3 N676K , and NRAS G12D accelerate KMT2A-MLLT3 leukemia onset. Further, also subclonal FLT3 N676K mutations accelerate disease, possibly by providing stimulatory factors. Herein, we show that one such factor, MIF, promotes survival of mouse KMT2A-MLLT3 leukemia initiating cells. We identify acquired de novo mutations in Braf, Cbl, Kras, and Ptpn11 in KMT2A-MLLT3 leukemia cells that favored clonal expansion. During clonal evolution, we observe serial genetic changes at the Kras G12D locus, consistent with a strong selective advantage of additional Kras G12D . KMT2A-MLLT3 leukemias with signaling mutations enforce Myc and Myb transcriptional modules. Our results provide new insight into the biology of KMT2A-R leukemia with subclonal signaling mutations and highlight the importance of activated signaling as a contributing driver.
Assuntos
Evolução Clonal , Rearranjo Gênico , Histona-Lisina N-Metiltransferase/genética , Leucemia Mieloide/genética , Mutação , Proteína de Leucina Linfoide-Mieloide/genética , Doença Aguda , Animais , Linhagem Celular Tumoral , Células Cultivadas , Regulação Leucêmica da Expressão Gênica , Leucemia Mieloide/patologia , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas de Fusão Oncogênica/genética , Proteínas Proto-Oncogênicas p21(ras)/genéticaRESUMO
Large amounts of pure and homogenous protein are a prerequisite for several biochemical and biophysical analyses, and in particular if aiming at resolving the three-dimensional protein structure. Here we describe the production of the rat glucose transporter 1 (GLUT1), a membrane protein facilitating the transport of glucose in cells. The protein is recombinantly expressed in the yeast Pichia pastoris. It is easily maintained and large-scale protein production in shaker flasks, as commonly performed in academic research laboratories, results in relatively high yields of membrane protein. The purification protocol describes all steps needed to obtain a pure and homogenous GLUT1 protein solution, including cell growth, membrane isolation, and chromatographic purification methods.