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1.
J Cell Sci ; 135(5)2022 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-34028531

RESUMO

Lipid droplets (LDs) are globular subcellular structures that store neutral lipids. LDs are closely associated with the endoplasmic reticulum (ER) and are limited by a phospholipid monolayer harboring a specific set of proteins. Most of these proteins associate with LDs through either an amphipathic helix or a membrane-embedded hairpin motif. Here, we address the question of whether integral membrane proteins can localize to the surface of LDs. To test this, we fused perilipin 3 (PLIN3), a mammalian LD-targeted protein, to ER-resident proteins. The resulting fusion proteins localized to the periphery of LDs in both yeast and mammalian cells. This peripheral LD localization of the fusion proteins, however, was due to a redistribution of the ER around LDs, as revealed by bimolecular fluorescence complementation between ER- and LD-localized partners. A LD-tethering function of PLIN3-containing membrane proteins was confirmed by fusing PLIN3 to the cytoplasmic domain of an outer mitochondrial membrane protein, OM14. Expression of OM14-PLIN3 induced a close apposition between LDs and mitochondria. These data indicate that the ER-LD junction constitutes a barrier for ER-resident integral membrane proteins.

2.
Proc Natl Acad Sci U S A ; 118(10)2021 Mar 09.
Artigo em Inglês | MEDLINE | ID: mdl-33674387

RESUMO

Lipid droplets (LDs) are intracellular organelles responsible for lipid storage, and they emerge from the endoplasmic reticulum (ER) upon the accumulation of neutral lipids, mostly triglycerides (TG), between the two leaflets of the ER membrane. LD biogenesis takes place at ER sites that are marked by the protein seipin, which subsequently recruits additional proteins to catalyze LD formation. Deletion of seipin, however, does not abolish LD biogenesis, and its precise role in controlling LD assembly remains unclear. Here, we use molecular dynamics simulations to investigate the molecular mechanism through which seipin promotes LD formation. We find that seipin clusters TG, as well as its precursor diacylglycerol, inside its unconventional ring-like oligomeric structure and that both its luminal and transmembrane regions contribute to this process. This mechanism is abolished upon mutations of polar residues involved in protein-TG interactions into hydrophobic residues. Our results suggest that seipin remodels the membrane of specific ER sites to prime them for LD biogenesis.

3.
Elife ; 102021 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-33522484

RESUMO

Cells store energy in the form of neutral lipids (NLs) packaged into micrometer-sized organelles named lipid droplets (LDs). These structures emerge from the endoplasmic reticulum (ER) at sites marked by the protein seipin, but the mechanisms regulating their biogenesis remain poorly understood. Using a combination of molecular simulations, yeast genetics, and fluorescence microscopy, we show that interactions between lipids' acyl-chains modulate the propensity of NLs to be stored in LDs, in turn preventing or promoting their accumulation in the ER membrane. Our data suggest that diacylglycerol, which is enriched at sites of LD formation, promotes the packaging of NLs into LDs, together with ER-abundant lipids, such as phosphatidylethanolamine. On the opposite end, short and saturated acyl-chains antagonize fat storage in LDs and promote accumulation of NLs in the ER. Our results provide a new conceptual understanding of LD biogenesis in the context of ER homeostasis and function.

4.
Microb Cell ; 7(8): 218-221, 2020 Jun 16.
Artigo em Inglês | MEDLINE | ID: mdl-32743002

RESUMO

Lipid droplets (LDs) are cellular compartments dedicated to the storage of metabolic energy in the form of neutral lipids, commonly known as "fat". The biogenesis of LDs takes place in the endoplasmic reticulum (ER), but its spatial and temporal organization is poorly understood. How exactly sites of LD formation are selected and the succession of proteins and lipids needed to mediate this process remains to be defined. In our current study we show that the yeast triacylglycerol (TAG)-synthases, Lro1 and Dga1 get recruited to discrete ER subdomains where they initiate TAG synthesis and hence LD formation (Choudhary et al. (2020), J Cell Biol). These ER subdomains are defined by yeast seipin, Fld1, and a regulator of diacylglycerol (DAG) production, Nem1. Both Fld1 and Nem1 are ER proteins which localize at contact sites between the ER and LDs. Interestingly, even in cells lacking LDs, Fld1 and Nem1 show punctate localization at ER subdomains independently of each other, but they are required together to recruit the TAG-synthases and hence create functional sites of LD biogenesis. Fld1/Nem1-containing ER subdomains recruit additional LD biogenesis factors, such as Yft2, Pex30, Pet10 and Erg6, and these membrane domains become enriched in DAG. In conclusion, Fld1 and Nem1 play a crucial role in defining ER subdomains for the recruitment of proteins and lipids needed to initiate LD biogenesis.

5.
Biol Open ; 9(6)2020 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-32554483

RESUMO

Members of the CAP/SCP/TAPS superfamily have been implicated in many different physiological processes, including pathogen defense, sperm maturation and fertilization. The mode of action of this class of proteins, however, remains poorly understood. The genome of Saccharomyces cerevisiae encodes three CAP superfamily members, Pry1-3. We have previously shown that Pry1 function is required for the secretion of sterols and fatty acids. Here, we analyze the function of Pry3, a GPI-anchored cell wall protein. Overexpression of Pry3 results in strong reduction of mating efficiency, providing for a cell-based readout for CAP protein function. Mating inhibition is a conserved function of the CAP domain and depends on highly conserved surface exposed residues that form part of a putative catalytic metal-ion binding site. Pry3 displays polarized cell surface localization adjacent to bud scars, but is absent from mating projections. When overexpressed, however, the protein leaks onto mating projections, suggesting that mating inhibition is due to mislocalization of the protein. Trapping of the CAP domain within the cell wall through a GPI-anchored nanobody results in a dose-dependent inhibition of mating, suggesting that a membrane proximal CAP domain inhibits a key step in the mating reaction, which is possibly related to the function of CAP domain proteins in mammalian fertilization.This article has an associated First Person interview with the first author of the paper.

6.
Sci Rep ; 10(1): 8268, 2020 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-32427974

RESUMO

Sphingosine-1-phosphate is a signaling molecule involved in the control of cell migration, differentiation, survival and other physiological processes. This sphingolipid metabolite can be degraded by the action of sphingosine-1-phosphate lyase (SPL) to form hexadecenal and ethanolamine phosphate. The importance of SPL-mediated ethanolamine phosphate formation has been characterized in only few cell types. We show that in the protozoan parasite Trypanosoma brucei, expression of TbSpl is essential for cell survival. Ablation of TbSpl expression increased sphingosine-1-phosphate levels and reduced de novo formation and steady-state levels of the glycerophospholipid phosphatidylethanolamine (PE). Growth of TbSpl-depleted parasites could be in part rescued by ethanolamine supplementation to the growth medium, indicating that the main function of TbSpl is to provide ethanolamine phosphate for PE synthesis. In contrast to most cell types analyzed, where SPL localizes to the endoplasmic reticulum, we found by high-resolution microscopy that TbSpl is a mitochondrial protein. In spite of its mitochondrial localization, TbSpl depletion had no apparent effect on mitochondrial morphology but resulted in aggregation of acidocalcisomes. Our results link mitochondria to sphingolipid metabolism and suggest possible roles for PE in acidocalcisome function.

7.
J Cell Biol ; 219(7)2020 Jul 06.
Artigo em Inglês | MEDLINE | ID: mdl-32349126

RESUMO

Lipid droplets (LDs) are fat storage organelles that originate from the endoplasmic reticulum (ER). Relatively little is known about how sites of LD formation are selected and which proteins/lipids are necessary for the process. Here, we show that LDs induced by the yeast triacylglycerol (TAG)-synthases Lro1 and Dga1 are formed at discrete ER subdomains defined by seipin (Fld1), and a regulator of diacylglycerol (DAG) production, Nem1. Fld1 and Nem1 colocalize to ER-LD contact sites. We find that Fld1 and Nem1 localize to ER subdomains independently of each other and of LDs, but both are required for the subdomains to recruit the TAG-synthases and additional LD biogenesis factors: Yft2, Pex30, Pet10, and Erg6. These subdomains become enriched in DAG. We conclude that Fld1 and Nem1 are both necessary to recruit proteins to ER subdomains where LD biogenesis occurs.

8.
PLoS Pathog ; 14(10): e1007300, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30335852

RESUMO

Despite causing considerable damage to host tissue at the onset of parasitism, invasive helminths establish remarkably persistent infections in both animals and plants. Secretions released by these obligate parasites during host invasion are thought to be crucial for their persistence in infection. Helminth secretions are complex mixtures of molecules, most of which have unknown molecular targets and functions in host cells or tissues. Although the habitats of animal- and plant-parasitic helminths are very distinct, their secretions share the presence of a structurally conserved group of proteins called venom allergen-like proteins (VALs). Helminths abundantly secrete VALs during several stages of parasitism while inflicting extensive damage to host tissue. The tight association between the secretion of VALs and the onset of parasitism has triggered a particular interest in this group of proteins, as improved knowledge on their biological functions may assist in designing novel protection strategies against parasites in humans, livestock, and important food crops.


Assuntos
Alérgenos/imunologia , Produtos Agrícolas/imunologia , Proteínas de Helminto/imunologia , Helmintos/imunologia , Interações Hospedeiro-Parasita/imunologia , Infecções por Nematoides/parasitologia , Peçonhas/imunologia , Animais , Infecções por Nematoides/imunologia
9.
PLoS One ; 13(8): e0201932, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30080909

RESUMO

Members of the Cysteine-rich secretory protein, Antigen 5 and Pathogenesis-related 1 (CAP) protein superfamily are important virulence factors in fungi but remain poorly characterized on molecular level. Here, we investigate the cellular localization and molecular function of Rbe1p and Rbt4p, two CAP family members from the human pathogen Candida albicans. We unexpectedly found that Rbe1p localizes to budding sites of yeast cells in a disulfide bond-dependent manner. Furthermore, we show that Rbe1p and Rbt4p bind free cholesterol in vitro and export cholesteryl acetate in vivo. These findings suggest a previously undescribed role for Rbe1p in cell wall-associated processes and a possible connection between the virulence attributes of fungal CAP proteins and sterol binding.


Assuntos
Candida albicans/fisiologia , Candidíase/microbiologia , Proteínas Fúngicas/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Transporte Biológico , Colesterol/química , Colesterol/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Humanos , Modelos Moleculares , Conformação Molecular , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Transporte Proteico , Esteróis/química , Esteróis/metabolismo , Relação Estrutura-Atividade , Virulência
10.
Int J Parasitol ; 48(5): 359-369, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29505764

RESUMO

Heligmosomoides polygyrus bakeri is a model parasitic hookworm used to study animal and human helminth diseases. During infection, the parasite releases excretory/secretory products that modulate the immune system of the host. The most abundant protein family in excretory/secretory products comprises the venom allergen-like proteins (VALs), which are members of the SCP/TAPS (sperm-coating protein/Tpx/antigen 5/pathogenesis related-1/Sc7) superfamily. There are >30 secreted Heligmosomoides polygyrus VAL proteins (HpVALs) and these proteins are characterised by having either one or two 15 kDa CAP (cysteine-rich secretory protein (CRISP)/antigen 5/pathogenesis related-1) domains. The first known HpVAL structure, HpVAL-4, refined to 1.9 Šis reported. HpVAL-4 was produced as a homogeneously glycosylated protein in leaves of Nicotiana benthamiana infiltrated with recombinant plasmids, making this plant expression platform amenable for the production of biological products. The overall topology of HpVAL-4 is a three layered αßα sandwich between a short N-terminal loop and a C-terminal cysteine rich extension. The C-terminal cysteine rich extension has two strands stabilized by two disulfide bonds and superposes well with the previously reported extension from the human hookworm Necator americanus Ancylostoma secreted protein-2 (Na-ASP-2). The N-terminal loop is connected to alpha helix 2 via a disulfide bond previously observed in Na-ASP-2. HpVAL-4 has a central cavity that is more similar to the N-terminal CAP domain of the two CAP Na-ASP-1 from Necator americanus. Unlike Na-ASP-2, mammalian CRISP, and the C-terminal CAP domain of Na-ASP-1, the large central cavity of HpVAL-4 lacks the two histidines required to coordinate divalent cations. HpVAL-4 has both palmitate-binding and sterol-binding cavities and is able to complement the in vivo sterol export phenotype of yeast mutants lacking their endogenous CAP proteins. More studies are required to determine endogenous binding partners of HpVAL-4 and unravel the possible impact of sterol binding on immune-modulatory functions.


Assuntos
Alérgenos/química , Proteínas de Helminto/química , Nematospiroides dubius/fisiologia , Peçonhas/química , Sequência de Aminoácidos , Animais , Modelos Moleculares , Conformação Proteica
11.
Curr Biol ; 28(6): 915-926.e9, 2018 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-29526591

RESUMO

Lipid droplets (LDs) store fats and play critical roles in lipid and energy homeostasis. They form between the leaflets of the endoplasmic reticulum (ER) membrane and consist of a neutral lipid core wrapped in a phospholipid monolayer with proteins. Two types of ER-LD architecture are thought to exist and be essential for LD functioning. Maturing LDs either emerge from the ER into the cytoplasm, remaining attached to the ER by a narrow membrane neck, or stay embedded in the ER and are surrounded by ER membrane. Here, we identify a lipid-based mechanism that controls which of these two architectures is favored. Theoretical modeling indicated that the intrinsic molecular curvatures of ER phospholipids can determine whether LDs remain embedded in or emerge from the ER; lipids with negative intrinsic curvature such as diacylglycerol (DAG) and phosphatidylethanolamine favor LD embedding, while those with positive intrinsic curvature, like lysolipids, support LD emergence. This prediction was verified by altering the lipid composition of the ER in S. cerevisiae using mutants and the addition of exogenous lipids. We found that fat-storage-inducing transmembrane protein 2 (FIT2) homologs become enriched at sites of LD generation when biogenesis is induced. DAG accumulates at sites of LD biogenesis, and FIT2 proteins may promote LD emergence from the ER by reducing DAG levels at these sites. Altogether, our findings suggest that cells regulate LD integration in the ER by modulating ER lipid composition, particularly at sites of LD biogenesis and that FIT2 proteins may play a central role in this process.


Assuntos
Proteínas de Transporte de Cátions/metabolismo , Glicoproteínas/metabolismo , Gotículas Lipídicas/metabolismo , Gotículas Lipídicas/fisiologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Transporte de Cátions/fisiologia , Simulação por Computador , Diglicerídeos/metabolismo , Diglicerídeos/fisiologia , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/fisiologia , Glicoproteínas/fisiologia , Proteínas Associadas a Gotículas Lipídicas/metabolismo , Proteínas Associadas a Gotículas Lipídicas/fisiologia , Metabolismo dos Lipídeos/fisiologia , Proteínas de Membrana/metabolismo , Fosfatidiletanolaminas/metabolismo , Fosfolipídeos/fisiologia , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiologia
12.
Int J Parasitol ; 48(5): 371-378, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29501266

RESUMO

Brugia malayi is a causative agent of lymphatic filariasis, a major tropical disease. The infective L3 parasite stage releases immunomodulatory proteins including the venom allergen-like proteins (VALs), which are members of the SCP/TAPS (Sperm-coating protein/Tpx/antigen 5/pathogenesis related-1/Sc7) superfamily. BmVAL-1 is a major target of host immunity with >90% of infected B. malayi microfilaraemic cases being seropositive for antibodies to BmVAL-1. This study is part of ongoing efforts to characterize the structures and functions of important B. malayi proteins. Recombinant BmVAL-1 was produced using a plant expression system, crystallized and the structure was solved by molecular replacement and refined to 2.1 Å, revealing the characteristic alpha/beta/alpha sandwich topology of eukaryotic SCP/TAPS proteins. The protein has more than 45% loop regions and these flexible loops connect the helices and strands, which are longer than predicted based on other parasite SCP/TAPS protein structures. The large central cavity of BmVAL-1 is a prototypical CRISP cavity with two histidines required to bind divalent cations. The caveolin-binding motif (CBM) that mediates sterol binding in SCP/TAPS proteins is large and open in BmVAL-1 and is N-glycosylated. N-glycosylation of the CBM does not affect the ability of BmVAL-1 to bind sterol in vitro. BmVAL-1 complements the in vivo sterol export phenotype of yeast mutants lacking their endogenous SCP/TAPS proteins. The in vitro sterol-binding affinity of BmVAL-1 is comparable with Pry1, a yeast sterol transporting SCP/TAPS protein. Sterol binding of BmVAL-1 is dependent on divalent cations. BmVAL-1 also has a large open palmitate-binding cavity, which binds palmitate comparably to tablysin-15, a lipid-binding SCP/TAPS protein. The central cavity, CBM and palmitate-binding cavity of BmVAL-1 are interconnected within the monomer with channels that can serve as pathways for water molecules, cations and small molecules.


Assuntos
Alérgenos/química , Brugia Malayi/fisiologia , Filariose Linfática/prevenção & controle , Proteínas de Helminto/química , Vacinas/imunologia , Peçonhas/química , Animais , Proteínas de Helminto/fisiologia , Humanos , Lipídeos/química , Modelos Moleculares , Ligação Proteica , Conformação Proteica
13.
FEBS Lett ; 592(8): 1304-1311, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29125629

RESUMO

In their natural habitat, yeast cells are constantly challenged by changing environmental conditions and a fierce competition for limiting resources. To thrive under such conditions, cells need to adapt and divide quickly, and be able to neutralize the toxic compounds secreted by their neighbors. Proteins like the pathogen-related yeast, Pry proteins, which belong to the large CAP/SCP/TAPS superfamily, may have an important role in this function. CAP proteins are conserved from yeast to man and are characterized by a unique αßα sandwich fold. They are mostly secreted glycoproteins and have been implicated in many different physiological processes including pathogen defense, virulence, venom toxicity, and sperm maturation. Yeast members of this family bind and export sterols as well as fatty acids, and they render cells resistant to eugenol, an antimicrobial compound present in clove oil. CAP family members might thus exert their various physiological functions through binding, sequestration, and neutralization of such small hydrophobic compounds.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas do Citoesqueleto/metabolismo , Metabolismo dos Lipídeos/fisiologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Transporte Biológico Ativo/fisiologia , Proteínas do Citoesqueleto/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
14.
Sci Rep ; 7(1): 15310, 2017 11 10.
Artigo em Inglês | MEDLINE | ID: mdl-29127407

RESUMO

Tick-borne relapsing fever (RF) borreliosis is a neglected disease that is often misdiagnosed. RF species circulating in the United States include Borrelia turicatae, which is transmitted by argasid ticks. Environmental adaptation by RF Borrelia is poorly understood, however our previous studies indicated differential regulation of B. turicatae genes localized on the 150 kb linear megaplasmid during the tick-mammalian transmission cycle, including bta121. This gene is up-regulated by B. turicatae in the tick versus the mammal, and the encoded protein (BTA121) is predicted to be surface localized. The structure of BTA121 was solved by single-wavelength anomalous dispersion (SAD) using selenomethionine-derivative protein. The topology of BTA121 is unique with four helical domains organized into two helical bundles. Due to the sequence similarity of several genes on the megaplasmid, BTA121 can serve as a model for their tertiary  structures. BTA121 has large interconnected tunnels and cavities that can accommodate ligands, notably long parallel helices, which have a large hydrophobic central pocket. Preliminary in-vitro studies suggest that BTA121 binds lipids, notably palmitate with a similar order of binding affinity as tablysin-15, a known palmitate-binding protein. The reported data will guide mechanistic studies to determine the role of BTA121 in the tick-mammalian transmission cycle of B. turicatae.


Assuntos
Proteínas de Bactérias , Infecções por Borrelia/metabolismo , Borrelia , Ácido Palmítico/química , Doenças Transmitidas por Carrapatos/metabolismo , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Borrelia/química , Borrelia/metabolismo , Cristalografia por Raios X , Humanos , Ligação Proteica , Domínios Proteicos
15.
PLoS One ; 12(10): e0186840, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29073188

RESUMO

Here we made an attempt to obtain partial structural information on the topology of multispan integral membrane proteins of yeast by isolating organellar membranes, removing peripheral membrane proteins at pH 11.5 and introducing chemical crosslinks between vicinal amino acids either using homo- or hetero-bifunctional crosslinkers. Proteins were digested with specific proteases and the products analysed by mass spectrometry. Dedicated software tools were used together with filtering steps optimized to remove false positive crosslinks. In proteins of known structure, crosslinks were found only between loops residing on the same side of the membrane. As may be expected, crosslinks were mainly found in very abundant proteins. Our approach seems to hold to promise to yield low resolution topological information for naturally very abundant or strongly overexpressed proteins with relatively little effort. Here, we report novel XL-MS-based topology data for 17 integral membrane proteins (Akr1p, Fks1p, Gas1p, Ggc1p, Gpt2p, Ifa38p, Ist2p, Lag1p, Pet9p, Pma1p, Por1p, Sct1p, Sec61p, Slc1p, Spf1p, Vph1p, Ybt1p).


Assuntos
Proteínas de Membrana/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Reagentes para Ligações Cruzadas/química , Espectrometria de Massas , Proteínas de Membrana/química , Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/química
16.
J Biol Chem ; 292(50): 20558-20569, 2017 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-29042440

RESUMO

Moniliophthora perniciosa is the causative agent of witches' broom disease, which devastates cacao cultures in South America. This pathogenic fungus infects meristematic tissues and derives nutrients from the plant apoplast during an unusually long-lasting biotrophic stage. To survive, the fungus produces proteins to suppress the plant immune response. Proteins of the PR-1 (pathogenesis-related 1)/CAP superfamily have been implicated in fungal virulence and immune suppression. The genome of M. perniciosa encodes 11 homologues of plant PR-1 proteins, designated MpPR-1 proteins, but their precise mode of action is poorly understood. In this study, we expressed MpPR-1 proteins in a yeast model lacking endogenous CAP proteins. We show that some members of the MpPR-1 family bind and promote secretion of sterols, whereas others bind and promote secretion of fatty acids. Lipid binding by purified MpPR-1 occurs with micromolar affinity and is saturable in vitro Sterol binding by MpPR-1 requires the presence of a flexible loop region containing aromatic amino acids, the caveolin-binding motif. Remarkably, MpPR-1 family members that do not bind sterols can be converted to sterol binders by a single point mutation in the caveolin-binding motif. We discuss the possible implications of the lipid-binding activity of MpPR-1 family members with regard to the mode of action of these proteins during M. perniciosa infections.


Assuntos
Agaricales/metabolismo , Ácidos Graxos não Esterificados/metabolismo , Proteínas Fúngicas/metabolismo , Esteróis/metabolismo , Agaricales/química , Agaricales/patogenicidade , Motivos de Aminoácidos , Sequência de Aminoácidos , Substituição de Aminoácidos , Ligação Competitiva , Cacau/microbiologia , Colesterol/química , Colesterol/metabolismo , Ácidos Graxos não Esterificados/química , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Deleção de Genes , Cinética , Ligantes , Mutagênese Sítio-Dirigida , Ácido Palmítico/química , Ácido Palmítico/metabolismo , Mutação Puntual , Conformação Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Esteróis/química
17.
Sci Rep ; 7(1): 7818, 2017 08 10.
Artigo em Inglês | MEDLINE | ID: mdl-28798297

RESUMO

The pathogenic fungi Moniliophthora perniciosa causes Witches' Broom Disease (WBD) of cacao. The structure of MpPR-1i, a protein expressed by M. perniciosa when it infects cacao, are presented. This is the first reported de novo structure determined by single-wavelength anomalous dispersion phasing upon soaking with selenourea. Each monomer has flexible loop regions linking the core alpha-beta-alpha sandwich topology that comprise ~50% of the structure, making it difficult to generate an accurate homology model of the protein. MpPR-1i is monomeric in solution but is packed as a high ~70% solvent content, crystallographic heptamer. The greatest conformational flexibility between monomers is found in loops exposed to the solvent channel that connect the two longest strands. MpPR-1i lacks the conserved CAP tetrad and is incapable of binding divalent cations. MpPR-1i has the ability to bind lipids, which may have roles in its infection of cacao. These lipids likely bind in the palmitate binding cavity as observed in tablysin-15, since MpPR-1i binds palmitate with comparable affinity as tablysin-15. Further studies are required to clarify the possible roles and underlying mechanisms of neutral lipid binding, as well as their effects on the pathogenesis of M. perniciosa so as to develop new interventions for WBD.


Assuntos
Agaricales/metabolismo , Cacau/microbiologia , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Agaricales/química , Sítios de Ligação , Cristalografia por Raios X , Modelos Moleculares , Palmitatos/metabolismo , Doenças das Plantas/microbiologia , Ligação Proteica , Conformação Proteica
18.
Cell Death Differ ; 24(12): 2044-2053, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-28800132

RESUMO

Sphingolipids are structural components of cell membranes that have signaling roles to regulate many activities, including mitochondrial function and cell death. Sphingolipid metabolism is integrated with numerous metabolic networks, and dysregulated sphingolipid metabolism is associated with disease. Here, we describe a monogenic yeast model for sphingolipid accumulation. A csg2Δ mutant cannot readily metabolize and accumulates the complex sphingolipid inositol phosphorylceramide (IPC). In these cells, aberrant activation of Ras GTPase is IPC-dependent, and accompanied by increased mitochondrial reactive oxygen species (ROS) and reduced mitochondrial mass. Survival or death of csg2Δ cells depends on nutritional status. Abnormal Ras activation in csg2Δ cells is associated with impaired Snf1/AMPK protein kinase, a key regulator of energy homeostasis. csg2Δ cells are rescued from ROS production and death by overexpression of mitochondrial catalase Cta1, abrogation of Ras hyperactivity or genetic activation of Snf1/AMPK. These results suggest that sphingolipid dysregulation compromises metabolic integrity via Ras and Snf1/AMPK pathways.


Assuntos
Mitocôndrias/metabolismo , Esfingolipídeos/metabolismo , Morte Celular , Humanos , Espécies Reativas de Oxigênio , Transdução de Sinais
19.
Methods Mol Biol ; 1645: 361-368, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28710641

RESUMO

Sterols are major constituents of the plasma membrane of eukaryotic cells and serve as a precursor for several classes of signaling molecules, including steroids and hydroxy sterols. They maintain the functionality and permeability barrier of the plasma membrane through lipid-lipid and lipid-protein interactions. The S. cerevisiae pathogen-related yeast proteins 1, 2, and 3 (Pry) belong to a large protein superfamily known as CAP/SCP/TAPS. Members of this superfamily have been implicated in a wide variety of processes, including immune defense in mammals and plants, pathogen virulence, sperm maturation and fertilization, venom toxicity, and prostate and brain cancer. Pry proteins bind and export sterols in vivo and the purified Pry1 protein binds sterols and related small hydrophobic compounds in vitro. Here we describe a method to determine lipid binding of a purified protein in vitro.


Assuntos
Membrana Celular/química , Lipídeos/química , Esteróis/química , Membrana Celular/genética , Células Eucarióticas/química , Proteínas dos Microfilamentos/química , Proteínas dos Microfilamentos/genética , Ligação Proteica , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética
20.
J Biol Chem ; 292(20): 8304-8314, 2017 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-28365570

RESUMO

Members of the CAP superfamily (cysteine-rich secretory proteins, antigen 5, and pathogenesis-related 1 proteins), also known as SCP superfamily (sperm-coating proteins), have been implicated in many physiological processes, including immune defenses, venom toxicity, and sperm maturation. Their mode of action, however, remains poorly understood. Three proteins of the CAP superfamily, Pry1, -2, and -3 (pathogen related in yeast), are encoded in the Saccharomyces cerevisiae genome. We have shown previously that Pry1 binds cholesterol in vitro and that Pry function is required for sterol secretion in yeast cells, indicating that members of this superfamily may generally bind sterols or related small hydrophobic compounds. On the other hand, tablysin-15, a CAP protein from the horsefly Tabanus yao, has been shown to bind leukotrienes and free fatty acids in vitro Therefore, here we assessed whether the yeast Pry1 protein binds fatty acids. Computational modeling and site-directed mutagenesis indicated that the mode of fatty acid binding is conserved between tablysin-15 and Pry1. Pry1 bound fatty acids with micromolar affinity in vitro, and its function was essential for fatty acid export in cells lacking the acyl-CoA synthetases Faa1 and Faa4. Fatty acid binding of Pry1 is independent of its capacity to bind sterols, and the two sterol- and fatty acid-binding sites are nonoverlapping. These results indicate that some CAP family members, such as Pry1, can bind different lipids, particularly sterols and fatty acids, at distinct binding sites, suggesting that the CAP domain may serve as a stable, secreted protein domain that can accommodate multiple ligand-binding sites.


Assuntos
Proteínas de Ligação a Ácido Graxo/metabolismo , Proteínas dos Microfilamentos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Acil Coenzima A/química , Acil Coenzima A/genética , Acil Coenzima A/metabolismo , Coenzima A Ligases/química , Coenzima A Ligases/genética , Coenzima A Ligases/metabolismo , Simulação por Computador , Proteínas de Ligação a Ácido Graxo/química , Proteínas de Ligação a Ácido Graxo/genética , Proteínas dos Microfilamentos/química , Proteínas dos Microfilamentos/genética , Mutagênese Sítio-Dirigida , Domínios Proteicos , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética
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