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1.
Genome Biol ; 20(1): 133, 2019 07 08.
Artigo em Inglês | MEDLINE | ID: mdl-31287004

RESUMO

BACKGROUND: Genome-wide association studies (GWAS) have identified hundreds of loci associated with coronary artery disease (CAD) and blood pressure (BP) or hypertension. Many of these loci are not linked to traditional risk factors, nor do they include obvious candidate genes, complicating their functional characterization. We hypothesize that many GWAS loci associated with vascular diseases modulate endothelial functions. Endothelial cells play critical roles in regulating vascular homeostasis, such as roles in forming a selective barrier, inflammation, hemostasis, and vascular tone, and endothelial dysfunction is a hallmark of atherosclerosis and hypertension. To test this hypothesis, we generate an integrated map of gene expression, open chromatin region, and 3D interactions in resting and TNFα-treated human endothelial cells. RESULTS: We show that genetic variants associated with CAD and BP are enriched in open chromatin regions identified in endothelial cells. We identify physical loops by Hi-C and link open chromatin peaks that include CAD or BP SNPs with the promoters of genes expressed in endothelial cells. This analysis highlights 991 combinations of open chromatin regions and gene promoters that map to 38 CAD and 92 BP GWAS loci. We validate one CAD locus, by engineering a deletion of the TNFα-sensitive regulatory element using CRISPR/Cas9 and measure the effect on the expression of the novel CAD candidate gene AIDA. CONCLUSIONS: Our data support an important role played by genetic variants acting in the vascular endothelium to modulate inter-individual risk in CAD and hypertension.


Assuntos
Doença da Artéria Coronariana/genética , Proteínas de Transferência de Fosfolipídeos/genética , Sistemas CRISPR-Cas , Células Endoteliais/metabolismo , Epigenômica , Estudo de Associação Genômica Ampla , Humanos , Elementos Reguladores de Transcrição , Transcriptoma
2.
Nat Commun ; 10(1): 1606, 2019 04 08.
Artigo em Inglês | MEDLINE | ID: mdl-30962435

RESUMO

Vascular endothelial growth factor (VEGF) regulates vasculogenesis by using its tyrosine kinase receptors. However, little is known about whether Sec14-like phosphatidylinositol transfer proteins (PTP) are involved in this process. Here, we show that zebrafish sec14l3, one of the family members, specifically participates in artery and vein formation via regulating angioblasts and subsequent venous progenitors' migration during vasculogenesis. Vascular defects caused by sec14l3 depletion are partially rescued by restoration of VEGFR2 signaling at the receptor or downstream effector level. Biochemical analyses show that Sec14l3/SEC14L2 physically bind to VEGFR2 and prevent it from dephosphorylation specifically at the Y1175 site by peri-membrane tyrosine phosphatase PTP1B, therefore potentiating VEGFR2 signaling activation. Meanwhile, Sec14l3 and SEC14L2 interact with RAB5A/4A and facilitate the formation of their GTP-bound states, which might be critical for VEGFR2 endocytic trafficking. Thus, we conclude that Sec14l3 controls vasculogenesis in zebrafish via the regulation of VEGFR2 activation.


Assuntos
Neovascularização Fisiológica/fisiologia , Proteínas de Transferência de Fosfolipídeos/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/fisiologia , Animais , Animais Geneticamente Modificados , Proteínas de Transporte/metabolismo , Embrião não Mamífero , Desenvolvimento Embrionário/fisiologia , Técnicas de Silenciamento de Genes , Células HEK293 , Células Endoteliais da Veia Umbilical Humana , Humanos , Lipoproteínas/metabolismo , Proteínas de Transferência de Fosfolipídeos/genética , Transdução de Sinais/fisiologia , Transativadores/metabolismo , Proteínas rab5 de Ligação ao GTP/metabolismo
3.
Nat Commun ; 10(1): 1846, 2019 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-31015464

RESUMO

Transmembrane protein 16F (TMEM16F) is an enigmatic Ca2+-activated phospholipid scramblase (CaPLSase) that passively transports phospholipids down their chemical gradients and mediates blood coagulation, bone development and viral infection. Despite recent advances in the structure and function understanding of TMEM16 proteins, how mammalian TMEM16 CaPLSases open and close, or gate their phospholipid permeation pathways remains unclear. Here we identify an inner activation gate, which is established by three hydrophobic residues, F518, Y563 and I612, in the middle of the phospholipid permeation pathway of TMEM16F-CaPLSase. Disrupting the inner gate profoundly alters TMEM16F phospholipid permeation. Lysine substitutions of F518 and Y563 even lead to constitutively active CaPLSases that bypass Ca2+-dependent activation. Strikingly, an analogous lysine mutation to TMEM16F-F518 in TMEM16A (L543K) is sufficient to confer CaPLSase activity to the Ca2+-activated Cl- channel (CaCC). The identification of an inner activation gate can help elucidate the gating and permeation mechanism of TMEM16 CaPLSases and channels.


Assuntos
Anoctaminas/metabolismo , Membrana Celular/metabolismo , Ativação do Canal Iônico/fisiologia , Proteínas de Transferência de Fosfolipídeos/metabolismo , Fosfolipídeos/metabolismo , Anoctamina-1/genética , Anoctamina-1/metabolismo , Anoctaminas/genética , Técnicas de Inativação de Genes , Células HEK293 , Humanos , Interações Hidrofóbicas e Hidrofílicas , Ativação do Canal Iônico/genética , Isoleucina/metabolismo , Lisina/genética , Lisina/metabolismo , Mutagênese , Fenilalanina/genética , Fenilalanina/metabolismo , Proteínas de Transferência de Fosfolipídeos/genética , Tirosina/metabolismo
4.
J Biol Chem ; 294(15): 5970-5979, 2019 04 12.
Artigo em Inglês | MEDLINE | ID: mdl-30760526

RESUMO

The P-type ATPase protein family includes, in addition to ion pumps such as Ca2+-ATPase and Na+,K+-ATPase, also phospholipid flippases that transfer phospholipids between membrane leaflets. P-type ATPase ion pumps translocate their substrates occluded between helices in the center of the transmembrane part of the protein. The large size of the lipid substrate has stimulated speculation that flippases use a different transport mechanism. Information on the functional importance of the most centrally located helices M5 and M6 in the transmembrane domain of flippases has, however, been sparse. Using mutagenesis, we examined the entire M5-M6 region of the mammalian flippase ATP8A2 to elucidate its possible function in the lipid transport mechanism. This mutational screen yielded an informative map assigning important roles in the interaction with the lipid substrate to only a few M5-M6 residues. The M6 asparagine Asn-905 stood out as being essential for the lipid substrate-induced dephosphorylation. The mutants N905A/D/E/H/L/Q/R all displayed very low activities and a dramatic insensitivity to the lipid substrate. Strikingly, Asn-905 aligns with key ion-binding residues of P-type ATPase ion pumps, and N905D was recently identified as one of the mutations causing the neurological disorder cerebellar ataxia, mental retardation, and disequilibrium (CAMRQ) syndrome. Moreover, the effects of substitutions to the adjacent residue Val-906 (i.e. V906A/E/F/L/Q/S) suggest that the lipid substrate approaches Val-906 during the translocation. These results favor a flippase mechanism with strong resemblance to the ion pumps, despite a location of the translocation pathway in the periphery of the transmembrane part of the flippase protein.


Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Transferência de Fosfolipídeos/metabolismo , Adenosina Trifosfatases/química , Adenosina Trifosfatases/genética , Substituição de Aminoácidos , Animais , Asparagina/química , Asparagina/genética , Asparagina/metabolismo , Bovinos , Células HEK293 , Humanos , Mutagênese Sítio-Dirigida , Mutação de Sentido Incorreto , Proteínas de Transferência de Fosfolipídeos/química , Proteínas de Transferência de Fosfolipídeos/genética , Fosforilação
5.
Fish Shellfish Immunol ; 87: 32-42, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30593902

RESUMO

Phospholipid scramblases (PLSCRs) are a family of transmembrane proteins known to be responsible for Ca2+-mediated bidirectional phospholipid translocation in the plasma membrane. Apart from the scrambling activity of PLSCRs, recent studies revealed their diverse other roles, including antiviral defense, tumorigenesis, protein-DNA interactions, apoptosis regulation, and cell activation. Nonetheless, the biological and transcriptional functions of PLSCRs in fish have not been discovered to date. Therefore, in this study, two new members related to the PLSCR1 family were identified in the red lip mullet (Liza haematocheila) as MuPLSCR1like-a and MuPLSCR1like-b, and their characteristics were studied at molecular and transcriptional levels. Sequence analysis revealed that MuPLSCR1like-a and MuPLSCR1like-b are composed of 245 and 228 amino acid residues (aa) with the predicted molecular weights of 27.82 and 25.74 kDa, respectively. A constructed phylogenetic tree showed that MuPLSCR1like-a and MuPLSCR1like-b are clustered together with other known PLSCR1 and -2 orthologues, thus pointing to the relatedness to both PLSCR1 and PLSCR2 families. Two-dimensional (2D) and 3D graphical representations illustrated the well-known 12-stranded ß-barrel structure of MuPLSCR1like-a and MuPLSCR1like-b with transmembrane orientation toward the phospholipid bilayer. In analysis of tissue-specific expression, the highest expression of MuPLSCR1like-a was observed in the intestine, whereas MuPLSCR1like-b was highly expressed in the brain, indicating isoform specificity. Of note, we found that the transcription of MuPLSCR1like-a and MuPLSCR1like-b was significantly upregulated when the fish were stimulated with poly(I:C), suggesting that such immune responses target viral infections. Overall, this study provides the first experimental insight into the characteristics and immune-system relevance of PLSCR1-related genes in red lip mullets.


Assuntos
Proteínas de Transferência de Fosfolipídeos/metabolismo , Smegmamorpha/genética , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Membrana Celular/metabolismo , Proteínas de Transferência de Fosfolipídeos/química , Proteínas de Transferência de Fosfolipídeos/genética , Filogenia , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Smegmamorpha/imunologia , Smegmamorpha/metabolismo
6.
Biochim Biophys Acta Biomembr ; 1861(3): 619-630, 2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30543784

RESUMO

Fluorescence resonance energy transfer (FRET) assays and membrane binding determinations were performed using three phosphatidylinositol transfer proteins, including the yeast Sec14 and two mammalian proteins PITPα and PITPß. These proteins were able to specifically bind the fluorescent phosphatidylcholine analogue NBD-PC ((2-(12-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)dodecanoyl-1-hexadecanoyl-sn-glycero-3-phosphocholine)) and to transfer it to small unilamellar vesicles (SUVs). Rate constants for transfer to vesicles comprising 100% PC were slower for all proteins than when increasing percentages of phosphatidylinositol were incorporated into the same SUVs. The rates of ligand transfer by Sec14 were insensitive to the inclusion of equimolar amounts of another anionic phospholipid phosphatidylserine (PS), but the rates of ligand transfer by both mammalian PITPs were strikingly enhanced by the inclusion of phosphatidic acid (PA) in the receptor SUV. Binding of Sec14 to immobilized bilayers was substantial, while that of PITPα and PITPß was 3-7 times weaker than Sec14 depending on phospholipid composition. When small proportions of the phosphoinositide PI(4)P were included in receptor SUVs (either with PI or not), Sec14 showed substantially increased rates of NBD-PC pick-up, whereas the PITPs were unaffected. The data are supportive of a role for PITPß as functional PI transfer protein in vivo, but that Sec14 likely has a more elaborate function.


Assuntos
Transferência Ressonante de Energia de Fluorescência/métodos , Técnicas In Vitro/métodos , Metabolismo dos Lipídeos , Fosfatidilinositóis/metabolismo , Proteínas de Transferência de Fosfolipídeos/metabolismo , Animais , Azóis/química , Azóis/metabolismo , Transporte Biológico , Proteínas de Transporte/metabolismo , Bovinos , Humanos , Ligantes , Nitrobenzenos/química , Nitrobenzenos/metabolismo , Fosfatidilcolinas/química , Fosfatidilcolinas/metabolismo , Proteínas de Transferência de Fosfolipídeos/química , Proteínas de Transferência de Fosfolipídeos/genética , Fosfolipídeos/química , Fosfolipídeos/metabolismo , Ligação Proteica , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
7.
Proc Natl Acad Sci U S A ; 115(26): 6709-6714, 2018 06 26.
Artigo em Inglês | MEDLINE | ID: mdl-29891673

RESUMO

The peptidoglycan cell wall provides an essential protective barrier in almost all bacteria, defining cellular morphology and conferring resistance to osmotic stress and other environmental hazards. The precursor to peptidoglycan, lipid II, is assembled on the inner leaflet of the plasma membrane. However, peptidoglycan polymerization occurs on the outer face of the plasma membrane, and lipid II must be flipped across the membrane by the MurJ protein before its use in peptidoglycan synthesis. Due to its central role in cell wall assembly, MurJ is of fundamental importance in microbial cell biology and is a prime target for novel antibiotic development. However, relatively little is known regarding the mechanisms of MurJ function, and structural data for MurJ are available only from the extremophile Thermosipho africanus Here, we report the crystal structure of substrate-free MurJ from the gram-negative model organism Escherichia coli, revealing an inward-open conformation. Taking advantage of the genetic tractability of E. coli, we performed high-throughput mutagenesis and next-generation sequencing to assess mutational tolerance at every amino acid in the protein, providing a detailed functional and structural map for the enzyme and identifying sites for inhibitor development. Lastly, through the use of sequence coevolution analysis, we identify functionally important interactions in the outward-open state of the protein, supporting a rocker-switch model for lipid II transport.


Assuntos
Proteínas de Escherichia coli/química , Proteínas de Transferência de Fosfolipídeos/química , Cristalografia por Raios X , Proteínas de Escherichia coli/genética , Evolução Molecular , Biblioteca Gênica , Bacilos Gram-Negativos Anaeróbios Retos, Helicoidais e Curvos/enzimologia , Sequenciamento de Nucleotídeos em Larga Escala , Modelos Moleculares , Mutação , Proteínas de Transferência de Fosfolipídeos/genética , Conformação Proteica , Proteínas Recombinantes de Fusão/química , Relação Estrutura-Atividade
8.
Proc Natl Acad Sci U S A ; 115(30): E7033-E7042, 2018 07 24.
Artigo em Inglês | MEDLINE | ID: mdl-29925604

RESUMO

Phospholipid scramblases externalize phosphatidylserine to facilitate numerous physiological processes. Several members of the structurally unrelated TMEM16 and G protein-coupled receptor (GPCR) protein families mediate phospholipid scrambling. The structure of a TMEM16 scramblase shows a membrane-exposed hydrophilic cavity, suggesting that scrambling occurs via the ?credit-card" mechanism where lipid headgroups permeate through the cavity while their tails remain associated with the membrane core. Here we show that afTMEM16 and opsin, representatives of the TMEM16 and GCPR scramblase families, transport phospholipids with polyethylene glycol headgroups whose globular dimensions are much larger than the width of the cavity. This suggests that transport of these large headgroups occurs outside rather than within the cavity. These large lipids are scrambled at rates comparable to those of normal phospholipids and their presence in the reconstituted vesicles promotes scrambling of normal phospholipids. This suggests that both large and small phospholipids can move outside the cavity. We propose that the conformational rearrangements underlying TMEM16- and GPCR-mediated credit-card scrambling locally deform the membrane to allow transbilayer lipid translocation outside the cavity and that both mechanisms underlie transport of normal phospholipids.


Assuntos
Anoctaminas/metabolismo , Metabolismo dos Lipídeos/fisiologia , Proteínas de Transferência de Fosfolipídeos/metabolismo , Receptores Acoplados a Proteínas-G/metabolismo , Anoctaminas/genética , Transporte Biológico Ativo/fisiologia , Células HEK293 , Humanos , Proteínas de Transferência de Fosfolipídeos/genética , Receptores Acoplados a Proteínas-G/genética , Saccharomyces cerevisiae
9.
Int J Mol Sci ; 19(6)2018 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-29912162

RESUMO

The Ca2+-activated phospholipid scramblase and ion channel TMEM16F is expressed in podocytes of renal glomeruli. Podocytes are specialized cells that form interdigitating foot processes as an essential component of the glomerular filter. These cells, which participate in generation of the primary urine, are often affected during primary glomerular diseases, such as glomerulonephritis and secondary hypertensive or diabetic nephropathy, which always leads to proteinuria. Because the function of podocytes is known to be controlled by intracellular Ca2+ signaling, it is important to know about the role of Ca2+-activated TMEM16F in these cells. To that end, we generated an inducible TMEM16F knockdown in the podocyte cell line AB8, and produced a conditional mouse model with knockout of TMEM16F in podocytes and renal epithelial cells of the nephron. We found that knockdown of TMEM16F did not produce proteinuria or any obvious phenotypic changes. Knockdown of TMEM16F affected cell death of tubular epithelial cells but not of glomerular podocytes when analyzed in TUNEL assays. Surprisingly, and in contrast to other cell types, TMEM16F did not control intracellular Ca2+ signaling and was not responsible for Ca2+-activated whole cell currents in podocytes. TMEM16F levels in podocytes were enhanced after inhibition of the endolysosomal pathway and after treatment with angiotensin II. Renal knockout of TMEM16F did not compromise renal morphology and serum electrolytes. Taken together, in contrast to other cell types, such as platelets, bone cells, and immune cells, TMEM16F shows little effect on basal properties of podocytes and does not appear to be essential for renal function.


Assuntos
Anoctaminas/genética , Proteínas de Transferência de Fosfolipídeos/genética , Podócitos/metabolismo , Potenciais de Ação , Animais , Anoctaminas/metabolismo , Apoptose , Sinalização do Cálcio , Células HEK293 , Humanos , Camundongos , Fenótipo , Proteínas de Transferência de Fosfolipídeos/metabolismo , Podócitos/fisiologia
10.
Proc Natl Acad Sci U S A ; 115(12): 3066-3071, 2018 03 20.
Artigo em Inglês | MEDLINE | ID: mdl-29507235

RESUMO

Transmembrane protein 16F (TMEM16F) is a Ca2+-dependent phospholipid scramblase that translocates phospholipids bidirectionally between the leaflets of the plasma membrane. Phospholipid scrambling of TMEM16F causes exposure of phosphatidylserine in activated platelets to induce blood clotting and in differentiated osteoblasts to promote bone mineralization. Despite the importance of TMEM16F-mediated phospholipid scrambling in various biological reactions, the fundamental features of the scrambling reaction remain elusive due to technical difficulties in the preparation of a platform for assaying scramblase activity in vitro. Here, we established a method to express and purify mouse TMEM16F as a dimeric molecule by constructing a stable cell line and developed a microarray containing membrane bilayers with asymmetrically distributed phospholipids as a platform for single-molecule scramblase assays. The purified TMEM16F was integrated into the microarray, and monitoring of phospholipid translocation showed that a single TMEM16F molecule transported phospholipids nonspecifically between the membrane bilayers in a Ca2+-dependent manner. Thermodynamic analysis of the reaction indicated that TMEM16F transported 4.5 × 104 lipids per second at 25 °C, with an activation free energy of 47 kJ/mol. These biophysical features were similar to those observed with channels, which transport substrates by facilitating diffusion, and supported the stepping-stone model for the TMEM16F phospholipid scramblase.


Assuntos
Anoctaminas/metabolismo , Proteínas de Transferência de Fosfolipídeos/metabolismo , Fosfolipídeos/química , Fosfolipídeos/metabolismo , Animais , Anoctaminas/genética , Linhagem Celular , Cinética , Membranas Artificiais , Camundongos , Proteínas de Transferência de Fosfolipídeos/genética , Análise Serial de Proteínas
11.
Proc Natl Acad Sci U S A ; 115(7): E1667-E1674, 2018 02 13.
Artigo em Inglês | MEDLINE | ID: mdl-29382763

RESUMO

TMEM16F, which is activated by elevation of intracellular calcium to trigger phospholipid scrambling and the collapse of lipid bilayer asymmetry to mediate important cellular functions such as blood coagulation, also generates a small-conductance calcium-activated cation current. How TMEM16F activation may be regulated is an open question. By recording TMEM16F Ca2+-activated current, we found that the TMEM16F Ca2+-response is desensitized by a brief exposure to high intracellular Ca2+, which is associated with depletion of phosphatidylinositol-(4, 5)-bisphosphate (PIP2) from the inner leaflet of the membrane. Application of artificial or natural PIP2 restores TMEM16F channel activity. PIP2 modulation of TMEM16F requires the presence of several positively charged amino acids in its cytoplasmic N-terminal domain. TMEM16F interaction with PIP2 works synergistically with membrane depolarization to facilitate Ca2+-gating of TMEM16F. Our study reveals the dependence of TMEM16F activity on phosphoinositides and provides one mechanism for TMEM16F activation to be strictly regulated in the cell membrane.


Assuntos
Anoctaminas/metabolismo , Cálcio/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Proteínas de Transferência de Fosfolipídeos/metabolismo , Animais , Anoctaminas/química , Anoctaminas/genética , Linhagem Celular , Membrana Celular/genética , Membrana Celular/metabolismo , Humanos , Camundongos , Proteínas de Transferência de Fosfolipídeos/química , Proteínas de Transferência de Fosfolipídeos/genética , Domínios Proteicos
12.
PLoS Pathog ; 14(1): e1006766, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29293671

RESUMO

Herpes simplex virus (HSV) entry is associated with Akt translocation to the outer leaflet of the plasma membrane to promote a complex signaling cascade. We hypothesized that phospholipid scramblase-1 (PLSCR1), a calcium responsive enzyme that flips phosphatidylserines between membrane leaflets, might redistribute Akt to the outside during entry. Confocal imaging, biotinylation of membrane proteins and flow cytometric analysis demonstrated that HSV activates PLSCR1 and flips phosphatidylserines and Akt to the outside shortly following HSV-1 or HSV-2 exposure. Translocation was blocked by addition of a cell permeable calcium chelator, pharmacological scramblase antagonist, or transfection with small interfering RNA targeting PLSCR1. Co-immunoprecipitation and proximity ligation studies demonstrated that PLSCR1 associated with glycoprotein L at the outer leaflet and studies with gL deletion viruses indicate that this interaction facilitates subsequent restoration of the plasma membrane architecture. Ionomycin, a calcium ionophore, also induced PLSCR1 activation resulting in Akt externalization, suggesting a previously unrecognized biological phenomenon.


Assuntos
Membrana Celular/metabolismo , Fosfatidilserinas/metabolismo , Proteínas de Transferência de Fosfolipídeos/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Simplexvirus/fisiologia , Regulação para Cima , Internalização do Vírus , Animais , Transporte Biológico/efeitos dos fármacos , Ionóforos de Cálcio/farmacologia , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Membrana Celular/virologia , Cercopithecus aethiops , Deleção de Genes , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Proteínas de Transferência de Fosfolipídeos/agonistas , Proteínas de Transferência de Fosfolipídeos/antagonistas & inibidores , Proteínas de Transferência de Fosfolipídeos/genética , Transporte Proteico/efeitos dos fármacos , Interferência de RNA , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Simplexvirus/efeitos dos fármacos , Propriedades de Superfície/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos , Proteínas do Envelope Viral/genética , Proteínas do Envelope Viral/metabolismo , Internalização do Vírus/efeitos dos fármacos
13.
Cell Mol Life Sci ; 75(9): 1657-1670, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29124309

RESUMO

Mutations in the human TMEM16E (ANO5) gene are associated both with the bone disease gnathodiaphyseal dysplasia (GDD; OMIM: 166260) and muscle dystrophies (OMIM: 611307, 613319). However, the physiological function of TMEM16E has remained unclear. We show here that human TMEM16E, when overexpressed in mammalian cell lines, displayed partial plasma membrane localization and gave rise to phospholipid scrambling (PLS) as well as non-selective ionic currents with slow time-dependent activation at highly depolarized membrane potentials. While the activity of wild-type TMEM16E depended on elevated cytosolic Ca2+ levels, a mutant form carrying the GDD-causing T513I substitution showed PLS and large time-dependent ion currents even at low cytosolic Ca2+ concentrations. Contrarily, mutation of the homologous position in the Ca2+-activated Cl- channel TMEM16B paralog hardly affected its function. In summary, these data provide the first direct demonstration of Ca2+-dependent PLS activity for TMEM16E and suggest a gain-of-function phenotype related to a GDD mutation.


Assuntos
Anoctaminas/genética , Mutação com Ganho de Função , Osteogênese Imperfeita/genética , Fosfolipídeos/metabolismo , Animais , Anoctaminas/metabolismo , Células CHO , Cricetinae , Cricetulus , Ativação Enzimática/genética , Células HEK293 , Humanos , Osteogênese Imperfeita/metabolismo , Proteínas de Transferência de Fosfolipídeos/genética , Proteínas de Transferência de Fosfolipídeos/metabolismo , Células Tumorais Cultivadas
14.
Cancer Lett ; 414: 136-146, 2018 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-29104146

RESUMO

Necroptosis has been reported to be involved in cisplatin-induced cell death, but the mechanisms underlying the occurrence of necroptosis are not fully elucidated. In this study, we show that apart from apoptosis, cisplatin induces necroptosis in A549 cells. The alleviation of cell death by two necroptosis inhibitors-necrostatin-1 (Nec-1) and necrosulfonamide (NSA), and the phosphorylation of mixed lineage kinase domain-like protein (MLKL) at serine 358, suggest the involvement of receptor-interacting protein kinase 1 (RIPK1)-RIPK3-MLKL signaling in cisplatin-treated A549 cells. Additionally, the initiation of cisplatin-induced necroptosis relies on autocrine tumor necrosis factor alpha (TNF-α). Furthermore, we present the first evidence that phosphatidylinositol transfer protein alpha (PITPα) is involved in MLKL-mediated necroptosis by interacting with the N terminal MLKL on its sixth helix and the preceding loop, which facilitates MLKL oligomerization and plasma membrane translocation in necroptosis. Silencing of PITPα expression interferes with MLKL function and reduces cell death. Our data elucidate that cisplatin-treated lung cancer cells undergo a new type of programmed cell death called necroptosis and shed new light on how MLKL translocates to the plasma membrane.


Assuntos
Apoptose/efeitos dos fármacos , Cisplatino/farmacologia , Proteínas de Transferência de Fosfolipídeos/metabolismo , Proteínas Quinases/metabolismo , Transdução de Sinais , Células A549 , Antineoplásicos/administração & dosagem , Antineoplásicos/farmacologia , Morte Celular/efeitos dos fármacos , Cisplatino/administração & dosagem , Células HEK293 , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Necrose , Proteínas de Transferência de Fosfolipídeos/genética , Fosforilação , Ligação Proteica , Interferência de RNA
15.
J Cell Biol ; 217(1): 269-282, 2018 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-29187527

RESUMO

Functional heterogeneity within the lipid droplet (LD) pool of a single cell has been observed, yet the underlying mechanisms remain enigmatic. Here, we report on identification of a specialized LD subpopulation characterized by a unique proteome and a defined geographical location at the nucleus-vacuole junction contact site. In search for factors determining identity of these LDs, we screened ∼6,000 yeast mutants for loss of targeting of the subpopulation marker Pdr16 and identified Ldo45 (LD organization protein of 45 kD) as a crucial targeting determinant. Ldo45 is the product of a splicing event connecting two adjacent genes (YMR147W and YMR148W/OSW5/LDO16). We show that Ldo proteins cooperate with the LD biogenesis component seipin and establish LD identity by defining positioning and surface-protein composition. Our studies suggest a mechanism to establish functional differentiation of organelles, opening the door to better understanding of metabolic decisions in cells.


Assuntos
Gotículas Lipídicas/metabolismo , Proteínas de Membrana/genética , Proteínas de Transferência de Fosfolipídeos/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Subunidades gama da Proteína de Ligação ao GTP/genética , Subunidades gama da Proteína de Ligação ao GTP/metabolismo , Gotículas Lipídicas/classificação , Metabolismo dos Lipídeos/fisiologia , Proteínas de Membrana/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteoma , Saccharomyces cerevisiae/metabolismo
16.
FEBS Lett ; 591(24): 4056-4066, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-29171872

RESUMO

Scramblases redistribute phospholipids in biological membranes. Phospholipid scramblase 3 (PLSCR3), which is located in mitochondria, has been reported to be involved in cardiolipin distribution from the inner to the outer membrane, thus regulating cellular processes such as apoptosis or mitophagy. However, the localization and topology of this protein has not been convincingly addressed to support a role in intermembrane phospholipid transfer. Here, we studied PLSCR3 topology within mitochondria. We show that PLSCR3 inserts in the inner membrane (IM) via its C-terminal transmembrane helix, whereas its N-terminal portion is oriented toward the intermembrane space where it is activated by calcium. Our results suggest that PLSCR3, via its C-terminal transmembrane domain, participates in the bidirectional movement of phospholipids within the IM.


Assuntos
Mitocôndrias/enzimologia , Proteínas de Transferência de Fosfolipídeos/química , Animais , Cardiolipinas/química , Cardiolipinas/metabolismo , Cristalografia por Raios X , Membranas Intracelulares/metabolismo , Masculino , Modelos Moleculares , Proteínas de Transferência de Fosfolipídeos/genética , Proteínas de Transferência de Fosfolipídeos/metabolismo , Filogenia , Ratos , Ratos Sprague-Dawley , Saccharomyces cerevisiae
17.
Nat Commun ; 8(1): 1246, 2017 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-29093443

RESUMO

Yes-associated protein (YAP) is a recently discovered growth-promoting transcription coactivator that has been shown to regulate the malignancy of various cancers. How YAP is regulated is not fully understood. Here, we show that one of the factors regulating YAP is phosphatidylserine (PS) in recycling endosomes (REs). We use proximity biotinylation to find proteins proximal to PS. Among these proteins are YAP and multiple proteins related to YAP signalling. Knockdown of ATP8A1 (an RE PS-flippase) or evectin-2 (an RE-resident protein) and masking of PS in the cytoplasmic leaflet of membranes, all suppress nuclear localization of YAP and YAP-dependent transcription. ATP8A1 knockdown increases the phosphorylated (activated) form of Lats1 that phosphorylates and inactivates YAP, whereas evectin-2 knockdown reduces the ubiquitination and increased the level of Lats1. The proliferation of YAP-dependent metastatic cancer cells is suppressed by knockdown of ATP8A1 or evectin-2. These results suggest a link between a membrane phospholipid and cell proliferation.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Adenosina Trifosfatases/genética , Proteínas de Membrana/genética , Fosfatidilserinas/metabolismo , Proteínas de Transferência de Fosfolipídeos/genética , Fosfoproteínas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Animais , Biotinilação , Células COS , Núcleo Celular/metabolismo , Proliferação de Células , Cercopithecus aethiops , Endossomos/metabolismo , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Fosforilação , Transporte Proteico/genética , Transdução de Sinais , Ubiquitinação
18.
Clin Chim Acta ; 475: 157-163, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-29074220

RESUMO

BACKGROUND: Colorectal cancer (CRC) is one of the most common malignancies worldwide, and early diagnosis is vital to improving prognoses. We explored the diagnostic potential of a multiplex autoantibody panel as a biomarker for the detection of CRC by ELISA. METHODS: In total, 192 serum samples (92 CRC and 100 matched controls) were tested against a panel of 12 tumor-associated antigens (TAAs): RPH3AL, RPL36, SLP2, p53, survivin, ANAXA4, SEC61B, CCCAP, NYCO16, NMDAR, PLSCR1, and HDAC5. Individual and combined autoantibody signatures were examined. RESULTS: Compared to individual autoantibody markers, the combinations of TAAs provided better discrimination between tumorous and normal sera. The overall sensitivity of a selected panel of four antibodies (anti-SLP2, -p53, -SEC61B, and -PLSCR1) was 64.1%, with a specificity of 80% that increased to 83.7% when carcinoembryonic antigen (CEA) measurement was added. Furthermore, the sensitivity of the panel of four antibodies for early and advanced stages of CRC was 66.7% and 62%, increasing to 88.3% and 84%, respectively, when CEA was added. CONCLUSIONS: We identified a panel of four antibodies as a promising diagnostic biomarker for the detection of CRC.


Assuntos
Antígenos de Neoplasias/genética , Autoanticorpos/sangue , Biomarcadores Tumorais/sangue , Neoplasias Colorretais/diagnóstico , Imunoensaio , Adulto , Idoso , Idoso de 80 Anos ou mais , Antígenos de Neoplasias/imunologia , Proteínas Sanguíneas/genética , Proteínas Sanguíneas/imunologia , Antígeno Carcinoembrionário/genética , Antígeno Carcinoembrionário/imunologia , Estudos de Casos e Controles , Neoplasias Colorretais/sangue , Neoplasias Colorretais/genética , Neoplasias Colorretais/imunologia , Detecção Precoce de Câncer/métodos , Feminino , Humanos , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Proteínas de Transferência de Fosfolipídeos/genética , Proteínas de Transferência de Fosfolipídeos/imunologia , Prognóstico , Canais de Translocação SEC/genética , Canais de Translocação SEC/imunologia , Sensibilidade e Especificidade , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/imunologia
19.
Curr Opin Plant Biol ; 40: 158-168, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-29017091

RESUMO

SEC14 lipid transfer proteins are important regulators of phospholipid metabolism. Structural, genetic and cell biological studies in yeast suggest that they help phosphatidylinositol (PtdIns)/phosphoinositide (PIP) kinases to overcome their intrinsic inefficiency to recognize membrane-embedded substrate, thereby playing a key role in PIP homeostasis. Genomes of higher plants encode a high number and diversity of SEC14 proteins, often in combination with other domains. The Arabidopsis SEC14-Nlj16 protein AtSFH1, an important regulator of root hair development, plays an important role in the establishment of PIP microdomains. Key to this mechanism is a highly specific interaction of the Nlj16 domain with PtdIns(4,5)P2 and an interaction-triggered oligomerization of the protein. Nlj16/PtdIns(4,5)P2 interaction depends on a polybasic motif similar to those identified in other regulatory proteins.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/fisiologia , Polaridade Celular , Metabolismo dos Lipídeos , Proteínas de Transferência de Fosfolipídeos/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/fisiologia , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Transferência de Fosfolipídeos/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
20.
Br J Cancer ; 117(12): 1798-1809, 2017 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-29024940

RESUMO

BACKGROUND: Anoctamin (ANO)/transmembrane member 16 (TMEM16) proteins mediate diverse physiological and pathophysiological functions including cancer cell proliferation. The present study aimed to identify the role of ANOs in pancreatic cancer. METHODS: In an initial screen of ANOs, ANO9/TMEM16J was overexpressed in pancreatic cancer cells, and its role in the pathogenesis of pancreatic cancer was evaluated using an integrated in vitro and in vivo approach. To determine clinical relevance of the experimental findings, the prognostic value of ANO9 was evaluated in patients with pancreatic cancer. RESULTS: The ANO9 mRNA and protein levels were increased in pancreatic cancer-derived cells. Exogenous expression of ANO9 in PANC-1 cells significantly increased cell proliferation in cell cultures and in mice. In contrast, knockdown of ANO9 in AsPC-1, BxPC-3, and Capan-2 cells strongly inhibited cell proliferation. Mechanistic analysis suggested that physical association of ANO9 with epidermal growth factor receptor (EGFR) underlies ANO9-induced cell proliferation. Knockdown of ANO9 augmented the effects of the EGFR inhibitor and the cytotoxic agent on pancreatic cancer cell proliferation. In addition, high ANO9 expression is a poor prognostic factor in patients with pancreatic cancer. CONCLUSIONS: The ANO9/TMEM16J appears to be a clinically useful prognostic marker for pancreatic cancer and a potential therapeutic target.


Assuntos
Anoctaminas/genética , Anoctaminas/metabolismo , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Receptores ErbB/metabolismo , Proteínas de Membrana/metabolismo , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Proteínas de Transferência de Fosfolipídeos/genética , Proteínas de Transferência de Fosfolipídeos/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Antibacterianos/uso terapêutico , Antineoplásicos/farmacologia , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Carcinogênese , Carcinoma Ductal Pancreático/tratamento farmacológico , Linhagem Celular Tumoral , Proliferação de Células/genética , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacologia , Intervalo Livre de Doença , Doxiciclina/uso terapêutico , Receptores ErbB/antagonistas & inibidores , Cloridrato de Erlotinib/farmacologia , Feminino , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Masculino , Proteínas de Membrana/genética , Camundongos , Pessoa de Meia-Idade , Transplante de Neoplasias , Neoplasias Pancreáticas/tratamento farmacológico , Prognóstico , RNA Mensageiro/metabolismo , Taxa de Sobrevida , Ensaio Tumoral de Célula-Tronco
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