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1.
Int J Mol Sci ; 21(19)2020 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-33019591

RESUMO

Tom70 is a versatile adaptor protein of 70 kDa anchored in the outer membrane of mitochondria in metazoa, fungi and amoeba. The tertiary structure was resolved for the Tom70 of yeast, showing 26 α-helices, most of them participating in the formation of 11 tetratricopeptide repeat (TPR) motifs. Tom70 serves as a docking site for cytosolic chaperone proteins and co-chaperones and is thereby involved in the uptake of newly synthesized chaperone-bound proteins in mitochondrial biogenesis. In yeast, Tom70 additionally mediates ER-mitochondria contacts via binding to sterol transporter Lam6/Ltc1. In mammalian cells, TOM70 promotes endoplasmic reticulum (ER) to mitochondria Ca2+ transfer by association with the inositol-1,4,5-triphosphate receptor type 3 (IP3R3). TOM70 is specifically targeted by the Bcl-2-related protein MCL-1 that acts as an anti-apoptotic protein in macrophages infected by intracellular pathogens, but also in many cancer cells. By participating in the recruitment of PINK1 and the E3 ubiquitin ligase Parkin, TOM70 can be implicated in the development of Parkinson's disease. TOM70 acts as receptor of the mitochondrial antiviral-signaling protein (MAVS) and thereby participates in the corresponding system of innate immunity against viral infections. The protein encoded by Orf9b in the genome of SARS-CoV-2 binds to TOM70, probably compromising the synthesis of type I interferons.


Assuntos
Imunidade Inata , Proteínas de Transporte da Membrana Mitocondrial/química , Animais , Betacoronavirus/genética , Sítios de Ligação , Humanos , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Fases de Leitura Aberta , Ligação Proteica , Transporte Proteico
2.
Sci Adv ; 6(35): eaba7910, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32923629

RESUMO

Targeting a universal host protein exploited by most viruses would be a game-changing strategy that offers broad-spectrum solution and rapid pandemic control including the current COVID-19. Here, we found a common YxxØ-motif of multiple viruses that exploits host AP2M1 for intracellular trafficking. A library chemical, N-(p-amylcinnamoyl)anthranilic acid (ACA), was identified to interrupt AP2M1-virus interaction and exhibit potent antiviral efficacy against a number of viruses in vitro and in vivo, including the influenza A viruses (IAVs), Zika virus (ZIKV), human immunodeficiency virus, and coronaviruses including MERS-CoV and SARS-CoV-2. YxxØ mutation, AP2M1 depletion, or disruption by ACA causes incorrect localization of viral proteins, which is exemplified by the failure of nuclear import of IAV nucleoprotein and diminished endoplasmic reticulum localization of ZIKV-NS3 and enterovirus-A71-2C proteins, thereby suppressing viral replication. Our study reveals an evolutionarily conserved mechanism of protein-protein interaction between host and virus that can serve as a broad-spectrum antiviral target.


Assuntos
Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Antivirais/farmacologia , Cinamatos/farmacologia , Infecções por Coronavirus/tratamento farmacológico , Infecções por HIV/tratamento farmacológico , Influenza Humana/tratamento farmacológico , Pneumonia Viral/tratamento farmacológico , ortoaminobenzoatos/farmacologia , Células A549 , Animais , Betacoronavirus/efeitos dos fármacos , Sítios de Ligação/genética , Linhagem Celular Tumoral , Chlorocebus aethiops , Infecções por Coronavirus/patologia , Cães , Células HEK293 , Infecções por HIV/patologia , HIV-1/efeitos dos fármacos , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Humanos , Vírus da Influenza A/efeitos dos fármacos , Influenza Humana/patologia , Células Madin Darby de Rim Canino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Coronavírus da Síndrome Respiratória do Oriente Médio/efeitos dos fármacos , Pandemias , Pneumonia Viral/patologia , Ligação Proteica/genética , Transporte Proteico/efeitos dos fármacos , RNA Viral/genética , Receptor de Interferon alfa e beta/genética , Fator de Crescimento Transformador beta1/metabolismo , Células Vero , Replicação Viral/efeitos dos fármacos , Zika virus/efeitos dos fármacos , Infecção por Zika virus/patologia
3.
Mol Genet Genomics ; 295(6): 1489-1500, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32948893

RESUMO

Glucose, fructose and mannose are the preferred carbon/energy sources for the yeast Saccharomyces cerevisiae. Absence of preferred energy sources activates glucose derepression, which is regulated by the kinase Snf1. Snf1 phosphorylates the transcriptional repressor Mig1, which results in its exit from the nucleus and subsequent derepression of genes. In contrast, Snf1 is inactive when preferred carbon sources are available, which leads to dephosphorylation of Mig1 and its translocation to the nucleus where Mig1 acts as a transcription repressor. Here we revisit the role of the three hexose kinases, Hxk1, Hxk2 and Glk1, in glucose de/repression. We demonstrate that all three sugar kinases initially affect Mig1 nuclear localization upon addition of glucose, fructose and mannose. This initial import of Mig1 into the nucleus was temporary; for continuous nucleocytoplasmic shuttling of Mig1, Hxk2 is required in the presence of glucose and mannose and in the presence of fructose Hxk2 or Hxk1 is required. Our data suggest that Mig1 import following exposure to preferred energy sources is controlled via two different pathways, where (1) the initial import is regulated by signals derived from metabolism and (2) continuous shuttling is regulated by the Hxk2 and Hxk1 proteins. Mig1 nucleocytoplasmic shuttling appears to be important for the maintenance of the repressed state in which Hxk1/2 seems to play an essential role.


Assuntos
Núcleo Celular/metabolismo , Frutose/metabolismo , Glucose/metabolismo , Hexoquinase/metabolismo , Manose/metabolismo , Proteínas Repressoras/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Transporte Ativo do Núcleo Celular , Regulação Fúngica da Expressão Gênica , Hexoquinase/genética , Fosforilação , Transporte Proteico , Proteínas Repressoras/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genética
4.
Nat Commun ; 11(1): 4577, 2020 09 11.
Artigo em Inglês | MEDLINE | ID: mdl-32917881

RESUMO

Nuclear pore complexes (NPCs) are important for cellular functions beyond nucleocytoplasmic trafficking, including genome organization and gene expression. This multi-faceted nature and the slow turnover of NPC components complicates investigations of how individual nucleoporins act in these diverse processes. To address this question, we apply an Auxin-Induced Degron (AID) system to distinguish roles of basket nucleoporins NUP153, NUP50 and TPR. Acute depletion of TPR causes rapid and pronounced changes in transcriptomic profiles. These changes are dissimilar to shifts observed after loss of NUP153 or NUP50, but closely related to changes caused by depletion of mRNA export receptor NXF1 or the GANP subunit of the TRanscription-EXport-2 (TREX-2) mRNA export complex. Moreover, TPR depletion disrupts association of TREX-2 subunits (GANP, PCID2, ENY2) to NPCs and results in abnormal RNA transcription and export. Our findings demonstrate a unique and pivotal role of TPR in gene expression through TREX-2- and/or NXF1-dependent mRNA turnover.


Assuntos
Exodesoxirribonucleases/metabolismo , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Poro Nuclear/metabolismo , Fosfoproteínas/metabolismo , RNA Mensageiro/metabolismo , Linhagem Celular , Regulação da Expressão Gênica , Humanos , Ácidos Indolacéticos/metabolismo , Proteínas Nucleares , Proteínas de Transporte Nucleocitoplasmático , Transporte Proteico , Proteínas de Ligação a RNA , Transcriptoma , Dedos de Zinco
5.
PLoS Biol ; 18(8): e3000820, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32866173

RESUMO

Mutations in the gene encoding the microtubule-severing protein spastin (spastic paraplegia 4 [SPG4]) cause hereditary spastic paraplegia (HSP), associated with neurodegeneration, spasticity, and motor impairment. Complicated forms (complicated HSP [cHSP]) further include cognitive deficits and dementia; however, the etiology and dysfunctional mechanisms of cHSP have remained unknown. Here, we report specific working and associative memory deficits upon spastin depletion in mice. Loss of spastin-mediated severing leads to reduced synapse numbers, accompanied by lower miniature excitatory postsynaptic current (mEPSC) frequencies. At the subcellular level, mutant neurons are characterized by longer microtubules with increased tubulin polyglutamylation levels. Notably, these conditions reduce kinesin-microtubule binding, impair the processivity of kinesin family protein (KIF) 5, and reduce the delivery of presynaptic vesicles and postsynaptic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors. Rescue experiments confirm the specificity of these results by showing that wild-type spastin, but not the severing-deficient and disease-associated K388R mutant, normalizes the effects at the synaptic, microtubule, and transport levels. In addition, short hairpin RNA (shRNA)-mediated reduction of tubulin polyglutamylation on spastin knockout background normalizes KIF5 transport deficits and attenuates the loss of excitatory synapses. Our data provide a mechanism that connects spastin dysfunction with the regulation of kinesin-mediated cargo transport, synapse integrity, and cognition.


Assuntos
Ácido Glutâmico/metabolismo , Cinesina/metabolismo , Transtornos da Memória/metabolismo , Transtornos da Memória/fisiopatologia , Memória de Curto Prazo , Neurônios/metabolismo , Espastina/deficiência , Tubulina (Proteína)/metabolismo , Potenciais de Ação , Animais , Membrana Celular/metabolismo , Espinhas Dendríticas/metabolismo , Espinhas Dendríticas/ultraestrutura , Potenciais Pós-Sinápticos Excitadores , Hipocampo/patologia , Hipocampo/fisiopatologia , Camundongos Knockout , Microtúbulos/metabolismo , Microtúbulos/ultraestrutura , Atividade Motora , Neurônios/patologia , Neurônios/ultraestrutura , Transporte Proteico , Espastina/metabolismo , Sinapses/metabolismo , Sinapses/ultraestrutura , Vesículas Sinápticas/metabolismo
6.
Nat Commun ; 11(1): 3825, 2020 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-32732874

RESUMO

The malaria parasite interfaces with its host erythrocyte (RBC) using a unique organelle, the parasitophorous vacuole (PV). The mechanism(s) are obscure by which its limiting membrane, the parasitophorous vacuolar membrane (PVM), collaborates with the parasite plasma membrane (PPM) to support the transport of proteins, lipids, nutrients, and metabolites between the cytoplasm of the parasite and the cytoplasm of the RBC. Here, we demonstrate that the PV has structure characterized by micrometer-sized regions of especially close apposition between the PVM and the PPM. To determine if these contact sites are involved in any sort of transport, we localize the PVM nutrient-permeable and protein export channel EXP2, as well as the PPM lipid transporter PfNCR1. We find that EXP2 is excluded from, but PfNCR1 is included within these regions of close apposition. We conclude that the host-parasite interface is structured to segregate those transporters of hydrophilic and hydrophobic substrates.


Assuntos
Lipídeos , Malária Falciparum/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Plasmodium falciparum/metabolismo , Proteínas de Protozoários/metabolismo , Transporte Biológico , Membrana Celular/metabolismo , Citoplasma/metabolismo , Citoplasma/parasitologia , Eritrócitos/metabolismo , Eritrócitos/parasitologia , Interações Hospedeiro-Parasita , Humanos , Membranas Intracelulares/metabolismo , Membranas Intracelulares/parasitologia , Malária Falciparum/parasitologia , Plasmodium falciparum/fisiologia , Transporte Proteico , Vacúolos/metabolismo , Vacúolos/parasitologia
7.
Nat Commun ; 11(1): 3802, 2020 07 30.
Artigo em Inglês | MEDLINE | ID: mdl-32732903

RESUMO

The Sec translocon moves proteins across lipid bilayers in all cells. The Sec channel enables passage of unfolded proteins through the bacterial plasma membrane, driven by the cytosolic ATPase SecA. Whether SecA generates mechanical force to overcome barriers to translocation posed by structured substrate proteins is unknown. Here, we kinetically dissect Sec-dependent translocation by monitoring translocation of a folded substrate protein with tunable stability at high time resolution. We find that substrate unfolding constitutes the rate-limiting step during translocation. Using single-molecule force spectroscopy, we also define the response of the protein to mechanical force. Relating the kinetic and force measurements reveals that SecA generates at least 10 piconewtons of mechanical force to actively unfold translocating proteins, comparable to cellular unfoldases. Combining biochemical and single-molecule measurements thus allows us to define how the SecA motor ensures efficient and robust export of proteins that contain stable structure.


Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Desdobramento de Proteína , Canais de Translocação SEC/metabolismo , Proteínas SecA/metabolismo , Estresse Mecânico , Membrana Celular/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Bicamadas Lipídicas/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Metotrexato/metabolismo , NADP/metabolismo , Transporte Proteico , Proteínas SecA/genética , Tetra-Hidrofolato Desidrogenase/metabolismo
8.
PLoS Genet ; 16(8): e1008820, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32750048

RESUMO

The core planar polarity proteins are essential mediators of tissue morphogenesis, controlling both the polarised production of cellular structures and polarised tissue movements. During development the core proteins promote planar polarisation by becoming asymmetrically localised to opposite cell edges within epithelial tissues, forming intercellular protein complexes that coordinate polarity between adjacent cells. Here we describe a novel protein complex that regulates the asymmetric localisation of the core proteins in the Drosophila pupal wing. DAnkrd49 (an ankyrin repeat protein) and Bride of Doubletime (Bdbt, a non-canonical FK506 binding protein family member) physically interact, and regulate each other's levels in vivo. Loss of either protein results in a reduction in core protein asymmetry and disruption of the placement of trichomes at the distal edge of pupal wing cells. Post-translational modifications are thought to be important for the regulation of core protein behaviour and their sorting to opposite cell edges. Consistent with this, we find that loss of DAnkrd49 or Bdbt leads to reduced phosphorylation of the core protein Dishevelled and to decreased Dishevelled levels both at cell junctions and in the cytoplasm. Bdbt has previously been shown to regulate activity of the kinase Discs Overgrown (Dco, also known as Doubletime or Casein Kinase Iε), and Dco itself has been implicated in regulating planar polarity by phosphorylating Dsh as well as the core protein Strabismus. We demonstrate that DAnkrd49 and Bdbt act as dominant suppressors of Dco activity. These findings support a model whereby Bdbt and DAnkrd49 act together to modulate the activity of Dco during planar polarity establishment.


Assuntos
Caseína Quinase Iépsilon/metabolismo , Polaridade Celular , Proteínas de Drosophila/metabolismo , Morfogênese , Proteínas de Ligação a Tacrolimo/metabolismo , Animais , Caseína Quinase Iépsilon/genética , Proteínas Desgrenhadas/genética , Proteínas Desgrenhadas/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster , Mutação com Perda de Função , Ligação Proteica , Transporte Proteico , Proteínas de Ligação a Tacrolimo/genética , Asas de Animais/citologia , Asas de Animais/crescimento & desenvolvimento
9.
PLoS Genet ; 16(8): e1008941, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32760060

RESUMO

Apolipoprotein B-containing lipoproteins (B-lps) are essential for the transport of hydrophobic dietary and endogenous lipids through the circulation in vertebrates. Zebrafish embryos produce large numbers of B-lps in the yolk syncytial layer (YSL) to move lipids from yolk to growing tissues. Disruptions in B-lp production perturb yolk morphology, readily allowing for visual identification of mutants with altered B-lp metabolism. Here we report the discovery of a missense mutation in microsomal triglyceride transfer protein (Mtp), a protein that is essential for B-lp production. This mutation of a conserved glycine residue to valine (zebrafish G863V, human G865V) reduces B-lp production and results in yolk opacity due to aberrant accumulation of cytoplasmic lipid droplets in the YSL. However, this phenotype is milder than that of the previously reported L475P stalactite (stl) mutation. MTP transfers lipids, including triglycerides and phospholipids, to apolipoprotein B in the ER for B-lp assembly. In vitro lipid transfer assays reveal that while both MTP mutations eliminate triglyceride transfer activity, the G863V mutant protein unexpectedly retains ~80% of phospholipid transfer activity. This residual phospholipid transfer activity of the G863V mttp mutant protein is sufficient to support the secretion of small B-lps, which prevents intestinal fat malabsorption and growth defects observed in the mttpstl/stl mutant zebrafish. Modeling based on the recent crystal structure of the heterodimeric human MTP complex suggests the G865V mutation may block triglyceride entry into the lipid-binding cavity. Together, these data argue that selective inhibition of MTP triglyceride transfer activity may be a feasible therapeutic approach to treat dyslipidemia and provide structural insight for drug design. These data also highlight the power of yolk transport studies to identify proteins critical for B-lp biology.


Assuntos
Proteínas de Transporte/genética , Lipídeos/genética , Lipoproteínas/genética , Triglicerídeos/genética , Animais , Fígado Gorduroso/genética , Fígado Gorduroso/patologia , Trato Gastrointestinal/metabolismo , Humanos , Imunoprecipitação , Gotículas Lipídicas/metabolismo , Lipoproteínas/metabolismo , Mutação de Sentido Incorreto/genética , Mutação Puntual/genética , Transporte Proteico/genética , Triglicerídeos/metabolismo , Peixe-Zebra/genética
10.
Yakugaku Zasshi ; 140(8): 1007-1012, 2020.
Artigo em Japonês | MEDLINE | ID: mdl-32741858

RESUMO

We previously showed that increased permeability of the blood-brain barrier (BBB) after ischemic stroke enables extravasation of nano-sized liposomes and accumulation in the ischemic region, and that delivery of neuroprotective agents using liposomal drug delivery systems (DDS) is applicable for treating cerebral ischemia/reperfusion (I/R) injury. However, entry of liposomes into the brain parenchyma was limited in the early stages after I/R possibly due to microvascular dysfunction induced by pathological progression. As such, new approaches to overcome the BBB are needed. Leukocytes can pass through inflamed BBB in I/R region due to membrane proteins displayed on their surface. We thus hypothesized that incorporation of leukocyte membrane proteins onto liposomal membranes would impart leukocyte-mimicking functions to liposomes and that leukocyte-mimetic liposomes (LM-Lipo) may pass through inflamed endothelial cells and BBB, similar to leukocytes. LM-Lipo prepared using intermembrane protein transfer from human leukemia cells showed significantly increased association to inflamed human umbilical vein endothelial cells relative to plain liposomes. Moreover, LM-Lipo passed through inflamed endothelial cell layer by regulating intercellular junctions. These results suggest that imparting leukocyte-like properties to liposomes via intermembrane protein transfer would be an effective strategy to overcome inflamed endothelial barriers. In this review, we describe our findings on ischemic stroke treatment using liposomal DDS and the potential of LM-Lipo to overcome inflamed endothelial barriers.


Assuntos
Barreira Hematoencefálica/metabolismo , Sistemas de Liberação de Medicamentos , Desenvolvimento de Medicamentos , Leucócitos , Lipossomos/metabolismo , Proteínas de Membrana/metabolismo , Fármacos Neuroprotetores/administração & dosagem , Acidente Vascular Cerebral/tratamento farmacológico , Acidente Vascular Cerebral/metabolismo , Humanos , Permeabilidade , Transporte Proteico , Traumatismo por Reperfusão/tratamento farmacológico , Traumatismo por Reperfusão/metabolismo
11.
Nat Commun ; 11(1): 3922, 2020 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-32764664

RESUMO

The Plasmodium falciparum chloroquine resistance transporter (PfCRT) is a key contributor to multidrug resistance and is also essential for the survival of the malaria parasite, yet its natural function remains unresolved. We identify host-derived peptides of 4-11 residues, varying in both charge and composition, as the substrates of PfCRT in vitro and in situ, and show that PfCRT does not mediate the non-specific transport of other metabolites and/or ions. We find that drug-resistance-conferring mutations reduce both the peptide transport capacity and substrate range of PfCRT, explaining the impaired fitness of drug-resistant parasites. Our results indicate that PfCRT transports peptides from the lumen of the parasite's digestive vacuole to the cytosol, thereby providing a source of amino acids for parasite metabolism and preventing osmotic stress of this organelle. The resolution of PfCRT's native substrates will aid the development of drugs that target PfCRT and/or restore the efficacy of existing antimalarials.


Assuntos
Antimaláricos/farmacologia , Cloroquina/farmacologia , Proteínas de Membrana Transportadoras/metabolismo , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/metabolismo , Proteínas de Protozoários/metabolismo , Animais , Transporte Biológico Ativo , Resistência a Medicamentos/genética , Feminino , Interações Hospedeiro-Parasita/genética , Interações Hospedeiro-Parasita/fisiologia , Humanos , Malária Falciparum/tratamento farmacológico , Malária Falciparum/metabolismo , Malária Falciparum/parasitologia , Proteínas de Membrana Transportadoras/genética , Modelos Biológicos , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Oligopeptídeos/metabolismo , Oócitos/metabolismo , Plasmodium falciparum/genética , Transporte Proteico , Proteínas de Protozoários/genética , Xenopus laevis
12.
Nat Commun ; 11(1): 3944, 2020 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-32769983

RESUMO

Triacylglycerols (TG) are synthesized at the endoplasmic reticulum (ER) bilayer and packaged into organelles called lipid droplets (LDs). LDs are covered by a single phospholipid monolayer contiguous with the ER bilayer. This connection is used by several monotopic integral membrane proteins, with hydrophobic membrane association domains (HDs), to diffuse between the organelles. However, how proteins partition between ER and LDs is not understood. Here, we employed synthetic model systems and found that HD-containing proteins strongly prefer monolayers and returning to the bilayer is unfavorable. This preference for monolayers is due to a higher affinity of HDs for TG over membrane phospholipids. Protein distribution is regulated by PC/PE ratio via alterations in monolayer packing and HD-TG interaction. Thus, HD-containing proteins appear to non-specifically accumulate to the LD surface. In cells, protein editing mechanisms at the ER membrane would be necessary to prevent unspecific relocation of HD-containing proteins to LDs.


Assuntos
Membrana Celular/metabolismo , Bicamadas Lipídicas/metabolismo , Gotículas Lipídicas/metabolismo , Proteínas de Membrana/metabolismo , Triglicerídeos/metabolismo , Dicroísmo Circular , Retículo Endoplasmático/metabolismo , Proteínas de Membrana/química , Simulação de Dinâmica Molecular , Domínios Proteicos , Transporte Proteico , Triglicerídeos/química
13.
PLoS One ; 15(8): e0237982, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32817700

RESUMO

Some organisms, like Trichomonas vaginalis, contain mitochondria-related hydrogen-producing organelles, called hydrogenosomes. The protein targeting into these organelles is proposed to be similar to the well-studied mitochondria import. Indeed, S. cerevisiae mitochondria and T. vaginalis hydrogenosomes share some components of protein import complexes. However, it is still unknown whether targeting signals directing substrate proteins to hydrogenosomes can support in other eukaryotes specific mitochondrial localization. To address this issue, we investigated the intracellular localization of three hydrogenosomal tail-anchored proteins expressed in yeast cells. We observed that these proteins were targeted to both mitochondria and ER with a variable dependency on the mitochondrial MIM complex. Our results suggest that the targeting signal of TA proteins are only partially conserved between hydrogenosomes and yeast mitochondria.


Assuntos
Retículo Endoplasmático/metabolismo , Regulação Fúngica da Expressão Gênica , Mitocôndrias/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/citologia , Trichomonas vaginalis/citologia , Transporte Proteico , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Trichomonas vaginalis/genética , Trichomonas vaginalis/metabolismo
14.
PLoS One ; 15(8): e0237514, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32790781

RESUMO

Golgi phosphoprotein 3 (GOLPH3) is a peripheral membrane protein localized at the trans-Golgi network that is also distributed in a large cytosolic pool. GOLPH3 has been involved in several post-Golgi protein trafficking events, but its precise function at the molecular level is not well understood. GOLPH3 is also considered the first oncoprotein of the Golgi apparatus, with important roles in several types of cancer. Yet, it is unknown how GOLPH3 is regulated to achieve its contribution in the mechanisms that lead to tumorigenesis. Binding of GOLPH3 to Golgi membranes depends on its interaction to phosphatidylinositol-4-phosphate. However, an early finding showed that GTP promotes the binding of GOLPH3 to Golgi membranes and vesicles. Nevertheless, it remains largely unknown whether this response is consequence of the function of GTP-dependent regulatory factors, such as proteins of the RAB family of small GTPases. Interestingly, in Drosophila melanogaster the ortholog of GOLPH3 interacts with- and behaves as effector of the ortholog of RAB1. However, there is no experimental evidence implicating GOLPH3 as a possible RAB1 effector in mammalian cells. Here, we show that human GOLPH3 interacted directly with either RAB1A or RAB1B, the two isoforms of RAB1 in humans. The interaction was nucleotide dependent and it was favored with GTP-locked active state variants of these GTPases, indicating that human GOLPH3 is a bona fide effector of RAB1A and RAB1B. Moreover, the expression in cultured cells of the GTP-locked variants resulted in less distribution of GOLPH3 in the Golgi apparatus, suggesting an intriguing model of GOLPH3 regulation.


Assuntos
Complexo de Golgi/metabolismo , Proteínas de Membrana/metabolismo , Proteínas rab1 de Ligação ao GTP/metabolismo , Células HeLa , Humanos , Proteínas de Membrana/genética , Transporte Proteico , Proteínas rab1 de Ligação ao GTP/genética , Rede trans-Golgi
15.
Virology ; 548: 31-38, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32838944

RESUMO

Entering the nucleus is important for Porcine circovirus type 2 (PCV2) replication. Karyopherins (KPNs) mediate the nuclear import of many cytoplasmic proteins. Our previous study showed that KPNA3 is involved in interferon production during PCV2 infection induced by Poly I:C and ISD (Interferon stimulatory DNA). However, it remains unclear whether PCV2 replication is associated with KPNA3. In the present study, knockdown of KPNA3 promoted the replication of PCV2, whereas overexpression of KPNA3 inhibited PCV2 replication in PK-15 cells. Furthermore, KPNA3 knockdown inhibited IRF3 and reduced the expression of antiviral genes including IFN-ß, ISG54, Mx1 and ISG56, while the opposite results were obtained after KPNA3 overexpression. KPNA3 knockdown also promoted p65 nuclear translocation and increased the mRNA expression of IL-10 and IL-1ß. These results suggested that KPNA3 facilitates IRF3 entry into the nucleus and the production of an antiviral response, resulting in PCV2 replication inhibition and blockage of NF-κB signal activation.


Assuntos
Núcleo Celular/metabolismo , Infecções por Circoviridae/veterinária , Circovirus/fisiologia , Doenças dos Suínos/metabolismo , alfa Carioferinas/metabolismo , Animais , Núcleo Celular/genética , Infecções por Circoviridae/genética , Infecções por Circoviridae/metabolismo , Infecções por Circoviridae/virologia , Circovirus/genética , Interações Hospedeiro-Patógeno , Interleucina-10/genética , Interleucina-10/metabolismo , NF-kappa B/genética , NF-kappa B/metabolismo , Transporte Proteico , Transdução de Sinais , Suínos , Doenças dos Suínos/genética , Doenças dos Suínos/virologia , Replicação Viral , alfa Carioferinas/genética
16.
Nat Commun ; 11(1): 4187, 2020 08 21.
Artigo em Inglês | MEDLINE | ID: mdl-32826901

RESUMO

EHBP1 is an adaptor protein that regulates vesicular trafficking by recruiting Rab8 family members and Eps15-homology domain-containing proteins 1/2 (EHD1/2). It also links endosomes to the actin cytoskeleton. However, the underlying molecular mechanism of activation of EHBP1 actin-binding activity is unclear. Here, we show that both termini of EHBP1 have membrane targeting potential. EHBP1 associates with PI(3)P, PI(5)P, and phosphatidylserine via its N-terminal C2 domain. We show that in the absence of Rab8 family members, the C-terminal bivalent Mical/EHBP Rab binding (bMERB) domain forms an intramolecular complex with its central calponin homology (CH) domain and auto-inhibits actin binding. Rab8 binding to the bMERB domain relieves this inhibition. We have analyzed the CH:bMERB auto-inhibited complex and the active bMERB:Rab8 complex biochemically and structurally. Together with structure-based mutational studies, this explains how binding of Rab8 frees the CH domain and allows it to interact with the actin cytoskeleton, leading to membrane tubulation.


Assuntos
Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Proteínas dos Microfilamentos/química , Proteínas dos Microfilamentos/metabolismo , Proteínas rab de Ligação ao GTP/química , Proteínas rab de Ligação ao GTP/metabolismo , Citoesqueleto de Actina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Transporte/genética , Proteínas dos Microfilamentos/genética , Modelos Moleculares , Fosfatos de Fosfatidilinositol/metabolismo , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Domínios e Motivos de Interação entre Proteínas , Transporte Proteico/fisiologia , Alinhamento de Sequência , Proteínas de Transporte Vesicular , Proteínas rab de Ligação ao GTP/genética
17.
Life Sci ; 258: 118195, 2020 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-32781073

RESUMO

AIMS: The estrogen-ERα axis participates in osteoblast maturation. This study was designed to further evaluated the roles of the estrogen-ERα axis in bone healing and the possible mechanisms. MAIN METHODS: Female ICR mice were created a metaphyseal bone defect in the left femurs and administered with methylpiperidinopyrazole (MPP), an inhibitor of ERα. Bone healing was evaluated using micro-computed tomography. Colocalization of ERα with alkaline phosphatase (ALP) and ERα translocation to mitochondria were determined. Levels of ERα, ERß, PECAM-1, VEGF, and ß-actin were immunodetected. Expression of chromosomal Runx2, ALP, and osteocalcin mRNAs and mitochondrial cytochrome c oxidase (COX) I and COXII mRNAs were quantified. Angiogenesis was measured with immunohistochemistry. KEY FINDINGS: Following surgery, the bone mass was time-dependently augmented in the bone-defect area. Simultaneously, levels of ERα were specifically upregulated and positively correlated with bone healing. Administration of MPP to mice consistently decreased levels of ERα and bone healing. As to the mechanisms, osteogenesis was enhanced in bone healing, but MPP attenuated osteoblast maturation. In parallel, expressions of osteogenesis-related ALP, Runx2, and osteocalcin mRNAs were induced in the injured zone. Treatment with MPP led to significant inhibition of the alp, runx2, and osteocalcin gene expressions. Remarkably, administration of MPP lessened translocation of ERα to mitochondria and expressions of mitochondrial energy production-related coxI and coxII genes. Furthermore, exposure to MPP decreased levels of PECAM-1 and VEGF in the bone-defect area. SIGNIFICANCE: The present study showed the contributions of the estrogen-ERα axis to bone healing through stimulation of energy production, osteoblast maturation, and angiogenesis.


Assuntos
Regeneração Óssea , Diferenciação Celular , Metabolismo Energético , Receptor alfa de Estrogênio/metabolismo , Neovascularização Fisiológica , Osteoblastos/citologia , Transdução de Sinais , Fosfatase Alcalina/metabolismo , Animais , Peso Corporal/efeitos dos fármacos , Regeneração Óssea/efeitos dos fármacos , Calo Ósseo/efeitos dos fármacos , Calo Ósseo/patologia , Diferenciação Celular/efeitos dos fármacos , Cromossomos de Mamíferos/metabolismo , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/genética , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Metabolismo Energético/efeitos dos fármacos , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Camundongos Endogâmicos ICR , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Neovascularização Fisiológica/efeitos dos fármacos , Tamanho do Órgão/efeitos dos fármacos , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Osteocalcina/metabolismo , Osteogênese/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Pirazóis/administração & dosagem , Pirazóis/farmacologia , Regulação para Cima/efeitos dos fármacos , Cicatrização/efeitos dos fármacos
18.
PLoS One ; 15(8): e0237448, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32790800

RESUMO

We established that Endosidin2 (ES2) affected the trafficking routes of both newly synthesized and endocytic pools of PIN-FORMED2 (PIN2) in Arabidopsis root epidermal cells. PIN2 populations accumulated in separated patches, which gradually merged into large and compact ES2 aggregates (ES2As). FM4-64 endocytic tracer labeled ES2As as well. Both PIN2 pools also appeared in vacuoles. Accelerated endocytosis of PIN2, its aggregation in the cytoplasm, and redirection of PIN2 flows to vacuoles led to a substantial reduction of the abundance of this protein in the plasma membrane. Whereas PIN-FORMED3 and PIN-FORMED4 also aggregated in the cytoplasm, SYT1 was not sensitive to ES2 treatment and did not appear either in the cytoplasmic aggregates or vacuoles. Ultrastructural analysis revealed that ES2 affects the Golgi apparatus so that stacks acquired cup-shape and even circular shape surrounded by several vesicles. Abnormally shaped Golgi stacks, stack remnants, multi-lamellar structures, separated Golgi cisterna rings, tubular structures, and vesicles formed discrete clusters.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Endocitose/efeitos dos fármacos , Limoninas/farmacologia , Proteínas de Membrana Transportadoras/metabolismo , Parede Celular/metabolismo , Citoplasma/metabolismo , Complexo de Golgi/metabolismo , Raízes de Plantas/citologia , Raízes de Plantas/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Transporte Proteico/efeitos dos fármacos , Sinaptotagmina I/metabolismo
19.
Nat Commun ; 11(1): 4044, 2020 08 13.
Artigo em Inglês | MEDLINE | ID: mdl-32792536

RESUMO

A growing number of optogenetic tools have been developed to reversibly control binding between two engineered protein domains. In contrast, relatively few tools confer light-switchable binding to a generic target protein of interest. Such a capability would offer substantial advantages, enabling photoswitchable binding to endogenous target proteins in cells or light-based protein purification in vitro. Here, we report the development of opto-nanobodies (OptoNBs), a versatile class of chimeric photoswitchable proteins whose binding to proteins of interest can be enhanced or inhibited upon blue light illumination. We find that OptoNBs are suitable for a range of applications including reversibly binding to endogenous intracellular targets, modulating signaling pathway activity, and controlling binding to purified protein targets in vitro. This work represents a step towards programmable photoswitchable regulation of a wide variety of target proteins.


Assuntos
Optogenética/métodos , Biologia Sintética/métodos , Animais , Células HEK293 , Humanos , Camundongos , Células NIH 3T3 , Ligação Proteica , Transporte Proteico/fisiologia , Transdução de Sinais/fisiologia
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