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1.
BMC Bioinformatics ; 21(Suppl 8): 266, 2020 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-32938368

RESUMO

BACKGROUND: The prediction of protein subcellular localization is a key step of the big effort towards protein functional annotation. Many computational methods exist to identify high-level protein subcellular compartments such as nucleus, cytoplasm or organelles. However, many organelles, like mitochondria, have their own internal compartmentalization. Knowing the precise location of a protein inside mitochondria is crucial for its accurate functional characterization. We recently developed DeepMito, a new method based on a 1-Dimensional Convolutional Neural Network (1D-CNN) architecture outperforming other similar approaches available in literature. RESULTS: Here, we explore the adoption of DeepMito for the large-scale annotation of four sub-mitochondrial localizations on mitochondrial proteomes of five different species, including human, mouse, fly, yeast and Arabidopsis thaliana. A significant fraction of the proteins from these organisms lacked experimental information about sub-mitochondrial localization. We adopted DeepMito to fill the gap, providing complete characterization of protein localization at sub-mitochondrial level for each protein of the five proteomes. Moreover, we identified novel mitochondrial proteins fishing on the set of proteins lacking any subcellular localization annotation using available state-of-the-art subcellular localization predictors. We finally performed additional functional characterization of proteins predicted by DeepMito as localized into the four different sub-mitochondrial compartments using both available experimental and predicted GO terms. All data generated in this study were collected into a database called DeepMitoDB (available at http://busca.biocomp.unibo.it/deepmitodb ), providing complete functional characterization of 4307 mitochondrial proteins from the five species. CONCLUSIONS: DeepMitoDB offers a comprehensive view of mitochondrial proteins, including experimental and predicted fine-grain sub-cellular localization and annotated and predicted functional annotations. The database complements other similar resources providing characterization of new proteins. Furthermore, it is also unique in including localization information at the sub-mitochondrial level. For this reason, we believe that DeepMitoDB can be a valuable resource for mitochondrial research.


Assuntos
Biologia Computacional/métodos , Proteínas Mitocondriais/genética , Transporte Proteico/genética , Animais , Humanos
2.
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
3.
PLoS One ; 15(7): e0234086, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32658928

RESUMO

Based on microRNA (miR) microarray analysis, we previously found that miR22-5p expression is decreased in the mid-luteal endometrium of women with minimal/mild endometriosis. Bioinformatics analysis predicted that miR22-5p targets ten-eleven translocation (TET2) 3'-untranslated region. This study aimed to determine the regulation and roles of miR22-5p in the pathogenesis of minimal/mild endometriosis-associated infertility. MiR22-5p and TET2 expression in the mid-luteal endometrium from women with or without minimal/mild endometriosis was analyzed. After transfection with miR22-5p mimics or inhibitor, TET2 expression was analyzed by quantitative reverse transcription (RT-q) PCR, western blotting and immunohistochemistry. 5-Hydroxymethylcytosine was determined by immunofluorescence and dot blotting. Expression and promoter methylation of estrogen receptor 2 (ESR2) was measured by RT-qPCR and western blotting, and by bisulfite sequencing, respectively. We first established that miR22-5p expression decreased and TET2 expression increased in minimal/mild endometriosis during implantation window. TET2 was found to be a direct target of miR22-5p. MiR22-5p regulated the expression of ESR2, but did not directly affect methylation of its promoter region (-197/+359). Our results suggest that an imbalance in miR22-5p expression in the mid-luteal endometrium may be involved in minimal/mild endometriosis-associated infertility.


Assuntos
Proteínas de Ligação a DNA/biossíntese , Implantação do Embrião , Endometriose/complicações , Endométrio/metabolismo , Receptor beta de Estrogênio/biossíntese , Infertilidade Feminina/etiologia , Fase Luteal/fisiologia , MicroRNAs/genética , Proteínas Proto-Oncogênicas/biossíntese , Regiões 3' não Traduzidas/genética , 5-Metilcitosina/análogos & derivados , 5-Metilcitosina/análise , Adulto , Células Cultivadas , Metilação de DNA , Proteínas de Ligação a DNA/genética , Receptor beta de Estrogênio/genética , Feminino , Regulação da Expressão Gênica , Genes Reporter , Humanos , Infertilidade Feminina/genética , MicroRNAs/antagonistas & inibidores , MicroRNAs/biossíntese , Regiões Promotoras Genéticas/genética , Transporte Proteico/genética , Proteínas Proto-Oncogênicas/genética , Células Estromais/metabolismo , Adulto Jovem
4.
PLoS Genet ; 16(7): e1008610, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32716926

RESUMO

Two-component systems and phosphorelays play central roles in the ability of bacteria to rapidly respond to changing environments. In E. coli and related enterobacteria, the complex Rcs phosphorelay is a critical player in the bacterial response to antimicrobial peptides, beta-lactam antibiotics, and other disruptions at the cell surface. The Rcs system is unusual in that an inner membrane protein, IgaA, is essential due to its negative regulation of the RcsC/RcsD/RcsB phosphorelay. While it is known that IgaA transduces signals from the outer membrane lipoprotein RcsF, how it interacts with the phosphorelay has remained unknown. Here we performed in vivo interaction assays and genetic dissection of the critical proteins and found that IgaA interacts with the phosphorelay protein RcsD, and that this interaction is necessary for regulation. Interactions between IgaA and RcsD within their respective periplasmic domains of these two proteins anchor repression of signaling. However, the signaling response depends on a second interaction between cytoplasmic loop 1 of IgaA and a truncated Per-Arndt-Sim (PAS-like) domain in RcsD. A single point mutation in the PAS-like domain increased interactions between the two proteins and blocked induction of the phosphorelay. IgaA may regulate RcsC, the histidine kinase that initiates phosphotransfer through the phosphorelay, indirectly, via its contacts with RcsD. Unlike RcsD, and unlike many other histidine kinases, the periplasmic domain of RcsC is dispensable for the response to signals that induce the Rcs phosphorelay system. The multiple contacts between IgaA and RcsD constitute a poised sensing system, preventing potentially toxic over-activation of this phosphorelay while enabling it to rapidly and quantitatively respond to signals.


Assuntos
Proteínas de Escherichia coli/genética , Proteínas de Membrana/genética , Fosfoproteínas/genética , Fosfotransferases/genética , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica/genética , Complexos Multienzimáticos/genética , Fosforilação/genética , Transporte Proteico/genética , Salmonella typhimurium/genética , Transdução de Sinais/genética
5.
Nat Commun ; 11(1): 3516, 2020 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-32665580

RESUMO

It is unclear whether the establishment of apical-basal cell polarity during the generation of epithelial lumens requires molecules acting at the plasma membrane/actin interface. Here, we show that the I-BAR-containing IRSp53 protein controls lumen formation and the positioning of the polarity determinants aPKC and podocalyxin. Molecularly, IRSp53 acts by regulating the localization and activity of the small GTPase RAB35, and by interacting with the actin capping protein EPS8. Using correlative light and electron microscopy, we further show that IRSp53 ensures the shape and continuity of the opposing plasma membrane of two daughter cells, leading to the formation of a single apical lumen. Genetic removal of IRSp53 results in abnormal renal tubulogenesis, with altered tubular polarity and architectural organization. Thus, IRSp53 acts as a membrane curvature-sensing platform for the assembly of multi-protein complexes that control the trafficking of apical determinants and the integrity of the luminal plasma membrane.


Assuntos
Membrana Celular/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Actinas/metabolismo , Polaridade Celular/genética , Polaridade Celular/fisiologia , Células Epiteliais/metabolismo , Feminino , Humanos , Morfogênese/genética , Morfogênese/fisiologia , Proteínas do Tecido Nervoso/genética , Transporte Proteico/genética , Transporte Proteico/fisiologia , Sialoglicoproteínas/genética , Sialoglicoproteínas/metabolismo , Proteínas rab de Ligação ao GTP/genética
6.
Nat Commun ; 11(1): 3182, 2020 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-32576858

RESUMO

Most eukaryotic genes produce alternative polyadenylation (APA) isoforms. Here we report that, unlike previously characterized cell lineages, differentiation of syncytiotrophoblast (SCT), a cell type critical for hormone production and secretion during pregnancy, elicits widespread transcript shortening through APA in 3'UTRs and in introns. This global APA change is observed in multiple in vitro trophoblast differentiation models, and in single cells from placentas at different stages of pregnancy. Strikingly, the transcript shortening is unrelated to cell proliferation, a feature previously associated with APA control, but instead accompanies increased secretory functions. We show that 3'UTR shortening leads to transcripts with higher mRNA stability, which augments transcriptional activation, especially for genes involved in secretion. Moreover, this mechanism, named secretion-coupled APA (SCAP), is also executed in B cell differentiation to plasma cells. Together, our data indicate that SCAP tailors the transcriptome during formation of secretory cells, boosting their protein production and secretion capacity.


Assuntos
Diferenciação Celular/fisiologia , Poliadenilação/fisiologia , Transporte Proteico/fisiologia , Transcriptoma , Regiões 3' não Traduzidas , Diferenciação Celular/genética , Linhagem da Célula , Proliferação de Células , Células-Tronco Embrionárias , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Isoformas de Proteínas , Transporte Proteico/genética , Estabilidade de RNA , RNA Mensageiro/metabolismo
8.
J Neurosci ; 40(25): 4954-4969, 2020 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-32354853

RESUMO

Glycine receptors (GlyRs) are the major mediators of fast synaptic inhibition in the adult human spinal cord and brainstem. Hereditary mutations to GlyRs can lead to the rare, but potentially fatal, neuromotor disorder hyperekplexia. Most mutations located in the large intracellular domain (TM3-4 loop) of the GlyRα1 impair surface expression levels of the receptors. The novel GLRA1 mutation P366L, located in the TM3-4 loop, showed normal surface expression but reduced chloride currents, and accelerated whole-cell desensitization observed in whole-cell recordings. At the single-channel level, we observed reduced unitary conductance accompanied by spontaneous opening events in the absence of extracellular glycine. Using peptide microarrays and tandem MS-based analysis methods, we show that the proline-rich stretch surrounding P366 mediates binding to syndapin I, an F-BAR domain protein involved in membrane remodeling. The disruption of the noncanonical Src homology 3 recognition motif by P366L reduces syndapin I binding. These data suggest that the GlyRα1 subunit interacts with intracellular binding partners and may therefore play a role in receptor trafficking or synaptic anchoring, a function thus far only ascribed to the GlyRß subunit. Hence, the P366L GlyRα1 variant exhibits a unique set of properties that cumulatively affect GlyR functionality and thus might explain the neuropathological mechanism underlying hyperekplexia in the mutant carriers. P366L is the first dominant GLRA1 mutation identified within the GlyRα1 TM3-4 loop that affects GlyR physiology without altering protein expression at the whole-cell and surface levels.SIGNIFICANCE STATEMENT We show that the intracellular domain of the inhibitory glycine receptor α1 subunit contributes to trafficking and synaptic anchoring. A proline-rich stretch in this receptor domain forms a noncanonical recognition motif important for the interaction with syndapin I (PACSIN1). The disruption of this motif, as present in a human patient with hyperekplexia led to impaired syndapin I binding. Functional analysis revealed that the altered proline-rich stretch determines several functional physiological parameters of the ion channel (e.g., faster whole-cell desensitization) reduced unitary conductance and spontaneous opening events. Thus, the proline-rich stretch from the glycine receptor α1 subunit represents a multifunctional intracellular protein motif.


Assuntos
Receptores da Glicina/genética , Receptores da Glicina/metabolismo , Rigidez Muscular Espasmódica/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Motivos de Aminoácidos , Animais , Humanos , Mutação , Ligação Proteica/genética , Estrutura Quaternária de Proteína , Transporte Proteico/genética , Receptores da Glicina/química
9.
Neuron ; 106(5): 759-768.e7, 2020 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-32243781

RESUMO

Autism spectrum disorder (ASD) is more prevalent in males; however, the etiology for this sex bias is not well understood. Many mutations on X-linked cell adhesion molecule NLGN4X result in ASD or intellectual disability. NLGN4X is part of an X-Y pair, with NLGN4Y sharing ∼97% sequence homology. Using biochemistry, electrophysiology, and imaging, we show that NLGN4Y displays severe deficits in maturation, surface expression, and synaptogenesis regulated by one amino acid difference with NLGN4X. Furthermore, we identify a cluster of ASD-associated mutations surrounding the critical amino acid in NLGN4X, and these mutations phenocopy NLGN4Y. We show that NLGN4Y cannot compensate for the functional deficits observed in ASD-associated NLGN4X mutations. Altogether, our data reveal a potential pathogenic mechanism for male bias in NLGN4X-associated ASD.


Assuntos
Moléculas de Adesão Celular Neuronais/genética , Cromossomos Humanos X/genética , Cromossomos Humanos Y/genética , Neurônios/metabolismo , Transtorno do Espectro Autista/genética , Moléculas de Adesão Celular Neuronais/metabolismo , Feminino , Predisposição Genética para Doença , Células HEK293 , Humanos , Células-Tronco Pluripotentes Induzidas , Deficiência Intelectual/genética , Masculino , Mutação , Transporte Proteico/genética
10.
Proc Natl Acad Sci U S A ; 117(18): 9884-9895, 2020 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-32321832

RESUMO

The factors and mechanisms involved in vacuolar transport in plants, and in particular those directing vesicles to their target endomembrane compartment, remain largely unknown. To identify components of the vacuolar trafficking machinery, we searched for Arabidopsis modified transport to the vacuole (mtv) mutants that abnormally secrete the synthetic vacuolar cargo VAC2. We report here on the identification of 17 mtv mutations, corresponding to mutant alleles of MTV2/VSR4, MTV3/PTEN2A MTV7/EREL1, MTV8/ARFC1, MTV9/PUF2, MTV10/VPS3, MTV11/VPS15, MTV12/GRV2, MTV14/GFS10, MTV15/BET11, MTV16/VPS51, MTV17/VPS54, and MTV18/VSR1 Eight of the MTV proteins localize at the interface between the trans-Golgi network (TGN) and the multivesicular bodies (MVBs), supporting that the trafficking step between these compartments is essential for segregating vacuolar proteins from those destined for secretion. Importantly, the GARP tethering complex subunits MTV16/VPS51 and MTV17/VPS54 were found at endoplasmic reticulum (ER)- and microtubule-associated compartments (EMACs). Moreover, MTV16/VPS51 interacts with the motor domain of kinesins, suggesting that, in addition to tethering vesicles, the GARP complex may regulate the motors that transport them. Our findings unveil a previously uncharacterized compartment of the plant vacuolar trafficking pathway and support a role for microtubules and kinesins in GARP-dependent transport of soluble vacuolar cargo in plants.


Assuntos
Proteínas de Arabidopsis/metabolismo , Transporte Proteico/genética , Vacúolos/metabolismo , Proteínas de Transporte Vesicular/genética , Alelos , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Vesículas Citoplasmáticas/genética , Vesículas Citoplasmáticas/metabolismo , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Complexo de Golgi/genética , Complexo de Golgi/metabolismo , Cinesina/genética , Cinesina/metabolismo , Microtúbulos/genética , Microtúbulos/metabolismo , Corpos Multivesiculares/genética , Corpos Multivesiculares/metabolismo , Mutação , Vacúolos/genética , Proteínas de Transporte Vesicular/metabolismo
11.
Infect Immun ; 88(5)2020 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-32122942

RESUMO

Legionella pneumophila, the etiological agent of Legionnaires' disease, employs an arsenal of hundreds of Dot/Icm-translocated effector proteins to facilitate replication within eukaryotic phagocytes. Several effectors, called metaeffectors, function to regulate the activity of other Dot/Icm-translocated effectors during infection. The metaeffector Lpg2505 is essential for L. pneumophila intracellular replication only when its cognate effector, SidI, is present. SidI is a cytotoxic effector that interacts with the host translation factor eEF1A and potently inhibits eukaryotic protein translation by an unknown mechanism. Here, we evaluated the impact of Lpg2505 on SidI-mediated phenotypes and investigated the mechanism of SidI function. We determined that Lpg2505 binds with nanomolar affinity to SidI and suppresses SidI-mediated inhibition of protein translation. SidI binding to eEF1A and Lpg2505 is not mutually exclusive, and the proteins bind distinct regions of SidI. We also discovered that SidI possesses GDP-dependent glycosyl hydrolase activity and that this activity is regulated by Lpg2505. We have therefore renamed Lpg2505 MesI (metaeffector of SidI). This work reveals novel enzymatic activity for SidI and provides insight into how intracellular replication of L. pneumophila is regulated by a metaeffector.


Assuntos
Proteínas de Bactérias/genética , Hidrolases/genética , Legionella pneumophila/genética , Biossíntese de Proteínas/genética , Animais , Células CHO , Linhagem Celular , Cricetulus , Células HEK293 , Interações Hospedeiro-Patógeno/genética , Humanos , Doença dos Legionários/microbiologia , Fator 1 de Elongação de Peptídeos/genética , Fagócitos/fisiologia , Transporte Proteico/genética
12.
Biochim Biophys Acta Mol Cell Res ; 1867(7): 118693, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32198023

RESUMO

Talin2 plays an important role in transduction of mechanical signals between extracellular matrix and actin cytoskeleton. Recent studies showed that talin2 is localized to invadopodia and regulates their maturation, subsequently cancer cell invasion and metastasis. However, the molecular mechanism whereby talin2 mediates invadopodium maturation is unknown. Here we show that ablation of talin2 in MDA-MB-231 cells inhibited the secretion of matrix metallopeptidase 9 (MMP9), a proteinase involved in extracellular matrix degradation in invadopodium maturation and metastasis. Furthermore, re-expression of talin2WT in talin2-KO cells rescued MMP9 secretion, but talin2S339C, a mutant with reduced ß-integrin binding, did not, indicating that the talin2-ß-integrin interaction is involved in the MMP9 secretion. Moreover, ablation of talin2 caused an accumulation of enlarged MMP9 vesicles. These vesicles co-localized with enlarged early, late endosomes and autophagosomes, suggesting talin2 controls MMP9 trafficking process. Therefore, these data suggest that talin2 regulates extracellular matrix degradation and invadopodium maturation by mediating MMP9 secretion.


Assuntos
Metaloproteinase 9 da Matriz/genética , Podossomos/genética , Talina/genética , Matriz Extracelular/genética , Regulação da Expressão Gênica/genética , Humanos , Metaloproteinase 9 da Matriz/biossíntese , Podossomos/fisiologia , Ligação Proteica/genética , Transporte Proteico/genética
13.
Biochim Biophys Acta Mol Basis Dis ; 1866(6): 165746, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32105825

RESUMO

In the mitochondria of healthy cells, Apoptosis-Inducing factor (AIF) is required for the optimal functioning of the respiratory chain machinery, mitochondrial integrity, cell survival, and proliferation. In all analysed species, it was revealed that the downregulation or depletion of AIF provokes mainly the post-transcriptional loss of respiratory chain Complex I protein subunits. Recent progress in the field has revealed that AIF fulfils its mitochondrial pro-survival function by interacting physically and functionally with CHCHD4, the evolutionarily-conserved human homolog of yeast Mia40. The redox-regulated CHCHD4/Mia40-dependent import machinery operates in the intermembrane space of the mitochondrion and controls the import of a set of nuclear-encoded cysteine-motif carrying protein substrates. In addition to their participation in the biogenesis of specific respiratory chain protein subunits, CHCHD4/Mia40 substrates are also implicated in the control of redox regulation, antioxidant response, translation, lipid homeostasis and mitochondrial ultrastructure and dynamics. Here, we discuss recent insights on the AIF/CHCHD4-dependent protein import pathway and review current data concerning the CHCHD4/Mia40 protein substrates in metazoan. Recent findings and the identification of disease-associated mutations in AIF or in specific CHCHD4/Mia40 substrates have highlighted these proteins as potential therapeutic targets in a variety of human disorders.


Assuntos
Fator de Indução de Apoptose/genética , Complexo I de Transporte de Elétrons/genética , Mitocôndrias/genética , Proteínas de Transporte da Membrana Mitocondrial/genética , Fator de Indução de Apoptose/metabolismo , Cisteína/genética , Cisteína/metabolismo , Dissulfetos/metabolismo , Complexo I de Transporte de Elétrons/metabolismo , Regulação da Expressão Gênica , Humanos , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética , Mutação/genética , Transporte Proteico/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo
14.
Biochim Biophys Acta Mol Basis Dis ; 1866(6): 165726, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32061767

RESUMO

Mitochondrial diseases are considered rare genetic disorders characterized by defects in oxidative phosphorylation (OXPHOS). They can be provoked by mutations in nuclear DNA (nDNA) or mitochondrial DNA (mtDNA). MERRF (Myoclonic Epilepsy with Ragged-Red Fibers) syndrome is one of the most frequent mitochondrial diseases, principally caused by the m.8344A>G mutation in mtDNA, which affects the translation of all mtDNA-encoded proteins and therefore impairs mitochondrial function. In the present work, we evaluated autophagy and mitophagy flux in transmitochondrial cybrids and fibroblasts derived from a MERRF patient, reporting that Parkin-mediated mitophagy is increased in MERRF cell cultures. Our results suggest that supplementation with coenzyme Q10 (CoQ), a component of the electron transport chain (ETC) and lipid antioxidant, prevents Parkin translocation to the mitochondria. In addition, CoQ acts as an enhancer of autophagy and mitophagy flux, which partially improves cell pathophysiology. The significance of Parkin-mediated mitophagy in cell survival was evaluated by silencing the expression of Parkin in MERRF cybrids. Our results show that mitophagy acts as a cell survival mechanism in mutant cells. To confirm these results in one of the main affected cell types in MERRF syndrome, mutant induced neurons (iNs) were generated by direct reprogramming of patients-derived skin fibroblasts. The treatment of MERRF iNs with Guttaquinon CoQ10 (GuttaQ), a water-soluble derivative of CoQ, revealed a significant improvement in cell bioenergetics. These results indicate that iNs, along with fibroblasts and cybrids, can be utilized as reliable cellular models to shed light on disease pathomechanisms as well as for drug screening.


Assuntos
Metabolismo Energético/genética , Síndrome MERRF/genética , Ubiquinona/análogos & derivados , Ubiquitina-Proteína Ligases/genética , Autofagia/genética , Células Cultivadas , DNA Mitocondrial/genética , Fibroblastos/efeitos dos fármacos , Humanos , Peroxidação de Lipídeos/efeitos dos fármacos , Síndrome MERRF/tratamento farmacológico , Síndrome MERRF/metabolismo , Síndrome MERRF/patologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias/genética , Mitocôndrias/patologia , Mitofagia/genética , Fosforilação Oxidativa/efeitos dos fármacos , Transporte Proteico/genética , Ubiquinona/metabolismo , Ubiquinona/farmacologia
15.
J Virol ; 94(9)2020 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-32075937

RESUMO

Dendritic cells (DCs) are one of the earliest targets of HIV-1 infection acting as a "Trojan horse," concealing the virus from the innate immune system and delivering it to T cells via virological synapses (VS). To explicate how the virus is trafficked through the cell to the VS and evades degradation, a high-throughput small interfering RNA screen targeting membrane trafficking proteins was performed in monocyte-derived DCs. We identified several proteins including BIN-1 and RAB7L1 that share common roles in transport from endosomal compartments. Depletion of target proteins resulted in an accumulation of virus in intracellular compartments and significantly reduced viral trans-infection via the VS. By targeting endocytic trafficking and retromer recycling to the plasma membrane, we were able to reduce the virus's ability to accumulate at budding microdomains and the VS. Thus, we identify key genes involved in a pathway within DCs that is exploited by HIV-1 to traffic to the VS.IMPORTANCE The lentivirus human immunodeficiency virus (HIV) targets and destroys CD4+ T cells, leaving the host vulnerable to life-threatening opportunistic infections associated with AIDS. Dendritic cells (DCs) form a virological synapse (VS) with CD4+ T cells, enabling the efficient transfer of virus between the two cells. We have identified cellular factors that are critical in the induction of the VS. We show that ADP-ribosylation factor 1 (ARF1), bridging integrator 1 (BIN1), and Rab GTPases RAB7L1 and RAB8A are important regulators of HIV-1 trafficking to the VS and therefore the infection of CD4+ T cells. We found these cellular factors were essential for endosomal protein trafficking and formation of the VS and that depletion of target proteins prevented virus trafficking to the plasma membrane by retaining virus in intracellular vesicles. Identification of key regulators in HIV-1 trans-infection between DC and CD4+ T cells has the potential for the development of targeted therapy to reduce trans-infection of HIV-1 in vivo.


Assuntos
Células Dendríticas/imunologia , Infecções por HIV/genética , HIV-1/imunologia , Sinapses Imunológicas/metabolismo , Fator 1 de Ribosilação do ADP/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Linfócitos T CD4-Positivos/imunologia , Células Dendríticas/virologia , Infecções por HIV/virologia , HIV-1/patogenicidade , Ensaios de Triagem em Larga Escala/métodos , Humanos , Monócitos/metabolismo , Proteínas Nucleares/metabolismo , Cultura Primária de Células , Transporte Proteico/genética , Proteínas Supressoras de Tumor/metabolismo , Vírion/metabolismo , Replicação Viral , Produtos do Gene gag do Vírus da Imunodeficiência Humana/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo
16.
Development ; 147(3)2020 02 03.
Artigo em Inglês | MEDLINE | ID: mdl-31964775

RESUMO

Both the establishment of neuronal polarity and axonal growth are crucial steps in the development of the nervous system. The local translation of mRNAs in the axon provides precise regulation of protein expression, and is now known to participate in axon development, pathfinding and synaptic formation and function. We have investigated the role of miR-26a in early stage mouse primary cortical neuron development. We show that micro-RNA-26a-5p (miR-26a) is highly expressed in neuronal cultures, and regulates both neuronal polarity and axon growth. Using compartmentalised microfluidic neuronal cultures, we identified a local role for miR-26a in the axon, where the repression of local synthesis of GSK3ß controls axon development and growth. Removal of this repression in the axon triggers local translation of GSK3ß protein and subsequent transport to the soma, where it can impact axonal growth. These results demonstrate how the axonal miR-26a can regulate local protein translation in the axon to facilitate retrograde communication to the soma and amplify neuronal responses, in a mechanism that influences axon development.


Assuntos
Axônios/metabolismo , Córtex Cerebral/citologia , Glicogênio Sintase Quinase 3 beta/metabolismo , MicroRNAs/metabolismo , Neurônios/metabolismo , Animais , Linhagem Celular Tumoral , Polaridade Celular/genética , Glicogênio Sintase Quinase 3 beta/genética , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/genética , Neurogênese/genética , Biossíntese de Proteínas , Transporte Proteico/genética , Transfecção
17.
J Biol Chem ; 295(9): 2544-2554, 2020 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-31974161

RESUMO

Mammalian mitochondria assemble four complexes of the respiratory chain (RCI, RCIII, RCIV, and RCV) by combining 13 polypeptides synthesized within mitochondria on mitochondrial ribosomes (mitoribosomes) with over 70 polypeptides encoded in nuclear DNA, translated on cytoplasmic ribosomes, and imported into mitochondria. We have previously observed that mitoribosome assembly is inefficient because some mitoribosomal proteins are produced in excess, but whether this is the case for other mitochondrial assemblies such as the RCs is unclear. We report here that pulse-chase stable isotope labeling with amino acids in cell culture (SILAC) is a valuable technique to study RC assembly because it can reveal considerable differences in the assembly rates and efficiencies of the different complexes. The SILAC analyses of HeLa cells indicated that assembly of RCV, comprising F1/Fo-ATPase, is rapid with little excess subunit synthesis, but that assembly of RCI (i.e. NADH dehydrogenase) is far less efficient, with dramatic oversynthesis of numerous proteins, particularly in the matrix-exposed N and Q domains. Unassembled subunits were generally degraded within 3 h. We also observed differential assembly kinetics for individual complexes that were immunoprecipitated with complex-specific antibodies. Immunoprecipitation with an antibody that recognizes the ND1 subunit of RCI co-precipitated a number of proteins implicated in FeS cluster assembly and newly synthesized ubiquinol-cytochrome c reductase Rieske iron-sulfur polypeptide 1 (UQCRFS1), the Rieske FeS protein in RCIII, reflecting some coordination between RCI and RCIII assemblies. We propose that pulse-chase SILAC labeling is a useful tool for studying rates of protein complex assembly and degradation.


Assuntos
Complexo I de Transporte de Elétrons/genética , Proteínas com Ferro-Enxofre/genética , Mitocôndrias/genética , NADH Desidrogenase/genética , ATPases Translocadoras de Prótons/genética , Técnicas de Cultura de Células/métodos , Núcleo Celular/genética , DNA/genética , Transporte de Elétrons/genética , Complexo I de Transporte de Elétrons/química , Células HeLa , Humanos , Marcação por Isótopo/métodos , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/genética , Ribossomos Mitocondriais/metabolismo , NADH Desidrogenase/química , Peptídeos/genética , Transporte Proteico/genética , ATPases Translocadoras de Prótons/química
18.
J Biol Chem ; 295(7): 2068-2083, 2020 02 14.
Artigo em Inglês | MEDLINE | ID: mdl-31915245

RESUMO

Many secretory proteins are activated by cleavage at specific sites. The proprotein convertases (PCs) form a family of nine secretory subtilisin-like serine proteases, seven of which cleave at specific basic residues within the trans-Golgi network, granules, or at the cell surface/endosomes. The seventh member, PC7, is a type-I transmembrane (TM) protein with a 97-residue-long cytosolic tail (CT). PC7 sheds human transferrin receptor 1 (hTfR1) into soluble shTfR1 in endosomes. To better understand the physiological roles of PC7, here we focused on the relationship between the CT-regulated trafficking of PC7 and its ability to shed hTfR1. Deletion of the TMCT resulted in soluble PC7 and loss of its hTfR1 shedding activity. Extensive CT deletions and mutagenesis analyses helped us zoom in on three residues in the CT, namely Glu-719, Glu-721, and Leu-725, that are part of a novel motif, EXEXXXL725, critical for PC7 activity on hTfR1. NMR studies of two 14-mer peptides mimicking this region of the CT and its Ala variants revealed that the three exposed residues are on the same side of the molecule. This led to the identification of adaptor protein 2 (AP-2) as a protein that recognizes the EXEXXXL725 motif, thus representing a potentially new regulator of PC7 trafficking and cleavage activity. Immunocytochemistry of the subcellular localization of PC7 and its Ala variants of Leu-725 and Glu-719 and Glu-721 revealed that Leu-725 enhances PC7 localization to early endosomes and that, together with Glu-719 and Glu-721, it increases the endosomal activity of PC7 on hTfR1.


Assuntos
Antígenos CD/genética , Citosol/metabolismo , Transporte Proteico/genética , Receptores da Transferrina/genética , Subtilisinas/genética , Fator de Transcrição AP-2/genética , Motivos de Aminoácidos/genética , Sequência de Aminoácidos/genética , Antígenos CD/química , Membrana Celular/genética , Movimento Celular/genética , Citosol/química , Endossomos/genética , Células HEK293 , Humanos , Receptores da Transferrina/química , Subtilisinas/química , Rede trans-Golgi/genética
19.
Hum Mol Genet ; 29(2): 320-334, 2020 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-31915823

RESUMO

Deficiency of the adaptor protein complex 4 (AP-4) leads to childhood-onset hereditary spastic paraplegia (AP-4-HSP): SPG47 (AP4B1), SPG50 (AP4M1), SPG51 (AP4E1) and SPG52 (AP4S1). This study aims to evaluate the impact of loss-of-function variants in AP-4 subunits on intracellular protein trafficking using patient-derived cells. We investigated 15 patient-derived fibroblast lines and generated six lines of induced pluripotent stem cell (iPSC)-derived neurons covering a wide range of AP-4 variants. All patient-derived fibroblasts showed reduced levels of the AP4E1 subunit, a surrogate for levels of the AP-4 complex. The autophagy protein ATG9A accumulated in the trans-Golgi network and was depleted from peripheral compartments. Western blot analysis demonstrated a 3-5-fold increase in ATG9A expression in patient lines. ATG9A was redistributed upon re-expression of AP4B1 arguing that mistrafficking of ATG9A is AP-4-dependent. Examining the downstream effects of ATG9A mislocalization, we found that autophagic flux was intact in patient-derived fibroblasts both under nutrient-rich conditions and when autophagy is stimulated. Mitochondrial metabolism and intracellular iron content remained unchanged. In iPSC-derived cortical neurons from patients with AP4B1-associated SPG47, AP-4 subunit levels were reduced while ATG9A accumulated in the trans-Golgi network. Levels of the autophagy marker LC3-II were reduced, suggesting a neuron-specific alteration in autophagosome turnover. Neurite outgrowth and branching were reduced in AP-4-HSP neurons pointing to a role of AP-4-mediated protein trafficking in neuronal development. Collectively, our results establish ATG9A mislocalization as a key marker of AP-4 deficiency in patient-derived cells, including the first human neuron model of AP-4-HSP, which will aid diagnostic and therapeutic studies.


Assuntos
Complexo 4 de Proteínas Adaptadoras/genética , Complexo 4 de Proteínas Adaptadoras/metabolismo , Proteínas Relacionadas à Autofagia/metabolismo , Proteínas de Membrana/metabolismo , Transporte Proteico/genética , Paraplegia Espástica Hereditária/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Rede trans-Golgi/metabolismo , Complexo 4 de Proteínas Adaptadoras/deficiência , Subunidades beta do Complexo de Proteínas Adaptadoras/metabolismo , Adolescente , Autofagossomos/metabolismo , Autofagia/genética , Linhagem Celular , Criança , Pré-Escolar , Feminino , Fibroblastos/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Ferro/metabolismo , Mutação com Perda de Função , Masculino , Proteínas Associadas aos Microtúbulos/metabolismo , Mitocôndrias/metabolismo , Neurogênese/genética , Neurônios/metabolismo , Paraplegia Espástica Hereditária/genética , Rede trans-Golgi/genética
20.
Adv Exp Med Biol ; 1232: 271-276, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31893420

RESUMO

Adaptation to hypoxia is essential for regulating the survival and functions of hypoxic cells; it is mainly mediated by the hypoxia-inducible factor 1 (HIF1). The alpha subunit of HIF1 (HIF1α) is a well-known regulatory component of HIF1, which is tightly controlled by various types of HIF1α-regulating processes. Previous research has shown that microtubule-regulated HIF1α nuclear translocation is a key factor for HIF1 activation under hypoxia. In this review, we summarize experimental reports on the role of microtubule-associated factors, such as microtubule, dynein, and dynein adaptor protein, in nuclear translocation of HIF1α. Based upon scientific evidence, we propose a bicaudal D homolog (BICD) as a novel HIF1α translocation regulating factor. A deeper understanding of the mechanism of the action of regulatory factors in controlling HIF1α nuclear translocation will provide novel insights into cell biology under hypoxia.


Assuntos
Transporte Ativo do Núcleo Celular , Subunidade alfa do Fator 1 Induzível por Hipóxia , Transporte Ativo do Núcleo Celular/genética , Hipóxia Celular/fisiologia , Núcleo Celular/metabolismo , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Microtúbulos/metabolismo , Transporte Proteico/genética
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