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1.
Adv Sci (Weinh) ; 9(11): e2105170, 2022 04.
Artigo em Inglês | MEDLINE | ID: mdl-35166455

RESUMO

The cytokine interferon-gamma (IFN-γ) is a master regulator of innate and adaptive immunity involved in a broad array of human diseases that range from atherosclerosis to cancer. IFN-γ exerts it signaling action by binding to a specific cell surface receptor, the IFN-γ receptor (IFN-γR), whose activation critically depends on its partition into lipid nanodomains. However, little is known about the impact of specific lipids on IFN-γR signal transduction activity. Here, a new conserved cholesterol (chol) binding motif localized within its single transmembrane domain is identified. Through direct binding, chol drives the partition of IFN-γR2 chains into plasma membrane lipid nanodomains, orchestrating IFN-γR oligomerization and transmembrane signaling. Bioinformatics studies show that the signature sequence stands for a conserved chol-binding motif presented in many mammalian membrane proteins. The discovery of chol as the molecular switch governing IFN-γR transmembrane signaling represents a significant advance for understanding the mechanism of lipid selectivity by membrane proteins, but also for figuring out the role of lipids in modulating cell surface receptor function. Finally, this study suggests that inhibition of the chol-IFNγR2 interaction may represent a potential therapeutic strategy for various IFN-γ-dependent diseases.


Assuntos
Receptores de Interferon , Transdução de Sinais , Animais , Sítios de Ligação , Colesterol , Humanos , Interferon gama/metabolismo , Interferon gama/farmacologia , Lipídeos , Mamíferos/metabolismo , Receptores de Interferon/metabolismo , Receptor de Interferon gama
2.
Dev Cell ; 47(4): 479-493.e7, 2018 11 19.
Artigo em Inglês | MEDLINE | ID: mdl-30458139

RESUMO

While retrograde cargo selection in the Golgi is known to depend on specific signals, it is unknown whether anterograde cargo is sorted, and anterograde signals have not been identified. We suggest here that S-palmitoylation of anterograde cargo at the Golgi membrane interface is an anterograde signal and that it results in concentration in curved regions at the Golgi rims by simple physical chemistry. The rate of transport across the Golgi of two S-palmitoylated membrane proteins is controlled by S-palmitoylation. The bulk of S-palmitoylated proteins in the Golgi behave analogously, as revealed by click chemistry-based fluorescence and electron microscopy. These palmitoylated cargos concentrate in the most highly curved regions of the Golgi membranes, including the fenestrated perimeters of cisternae and associated vesicles. A palmitoylated transmembrane domain behaves similarly in model systems.


Assuntos
Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Lipoilação/fisiologia , Transporte Proteico/fisiologia , Transporte Biológico/fisiologia , Células Cultivadas , Humanos , Membranas Intracelulares/metabolismo
3.
FEBS Lett ; 591(1): 65-75, 2017 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-27928819

RESUMO

In this paper, we experimentally address the debate about why functional transfer of mitochondrial genes to the nucleus has been halted in some organismal groups and why cytosolic expression of mitochondrial proteins has proven remarkably difficult. By expressing all 13 human mitochondrial-encoded genes with strong mitochondrial-targeting sequences in the cytosol of human cells, we show that all proteins, except ATP8, are transported to the endoplasmic reticulum (ER). These results confirm and extend previous findings based on three mitochondrial genes lacking mitochondrial-targeting sequences that also were relocated to the ER during cytosolic expression. We conclude that subcellular protein targeting constitutes a major barrier to functional transfer of mitochondrial genes to the nuclear genome.


Assuntos
Genoma Mitocondrial , Mitocôndrias/genética , Retículo Endoplasmático/metabolismo , Genes Mitocondriais , Engenharia Genética , Células HeLa , Humanos , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Modelos Biológicos , Proteínas Recombinantes/metabolismo , Transfecção
4.
Nature ; 536(7615): 219-23, 2016 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-27487212

RESUMO

Signal recognition particle (SRP) is a universally conserved protein-RNA complex that mediates co-translational protein translocation and membrane insertion by targeting translating ribosomes to membrane translocons. The existence of parallel co- and post-translational transport pathways, however, raises the question of the cellular substrate pool of SRP and the molecular basis of substrate selection. Here we determine the binding sites of bacterial SRP within the nascent proteome of Escherichia coli at amino acid resolution, by sequencing messenger RNA footprints of ribosome-nascent-chain complexes associated with SRP. SRP, on the basis of its strong preference for hydrophobic transmembrane domains (TMDs), constitutes a compartment-specific targeting factor for nascent inner membrane proteins (IMPs) that efficiently excludes signal-sequence-containing precursors of periplasmic and outer membrane proteins. SRP associates with hydrophobic TMDs enriched in consecutive stretches of hydrophobic and bulky aromatic amino acids immediately on their emergence from the ribosomal exit tunnel. By contrast with current models, N-terminal TMDs are frequently skipped and TMDs internal to the polypeptide sequence are selectively recognized. Furthermore, SRP binds several TMDs in many multi-spanning membrane proteins, suggesting cycles of SRP-mediated membrane targeting. SRP-mediated targeting is not accompanied by a transient slowdown of translation and is not influenced by the ribosome-associated chaperone trigger factor (TF), which has a distinct substrate pool and acts at different stages during translation. Overall, our proteome-wide data set of SRP-binding sites reveals the underlying principles of pathway decisions for nascent chains in bacteria, with SRP acting as the dominant triaging factor, sufficient to separate IMPs from substrates of the SecA-SecB post-translational translocation and TF-assisted cytosolic protein folding pathways.


Assuntos
Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Proteínas de Membrana/metabolismo , Peptídeos/metabolismo , Biossíntese de Proteínas , Proteoma/metabolismo , Partícula de Reconhecimento de Sinal/metabolismo , Sítios de Ligação , Escherichia coli/genética , Proteínas de Escherichia coli/biossíntese , Interações Hidrofóbicas e Hidrofílicas , Proteínas de Membrana/biossíntese , Peptidilprolil Isomerase/metabolismo , Periplasma/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Transporte Proteico , Proteoma/biossíntese , Proteômica , RNA Bacteriano/metabolismo , RNA Mensageiro/metabolismo , Ribossomos/metabolismo , Especificidade por Substrato
5.
Proc Natl Acad Sci U S A ; 112(33): 10154-61, 2015 Aug 18.
Artigo em Inglês | MEDLINE | ID: mdl-26195779

RESUMO

Mitochondria are energy-producing organelles in eukaryotic cells considered to be of bacterial origin. The mitochondrial genome has evolved under selection for minimization of gene content, yet it is not known why not all mitochondrial genes have been transferred to the nuclear genome. Here, we predict that hydrophobic membrane proteins encoded by the mitochondrial genomes would be recognized by the signal recognition particle and targeted to the endoplasmic reticulum if they were nuclear-encoded and translated in the cytoplasm. Expression of the mitochondrially encoded proteins Cytochrome oxidase subunit 1, Apocytochrome b, and ATP synthase subunit 6 in the cytoplasm of HeLa cells confirms export to the endoplasmic reticulum. To examine the extent to which the mitochondrial proteome is driven by selective constraints within the eukaryotic cell, we investigated the occurrence of mitochondrial protein domains in bacteria and eukaryotes. The accessory protein domains of the oxidative phosphorylation system are unique to mitochondria, indicating the evolution of new protein folds. Most of the identified domains in the accessory proteins of the ribosome are also found in eukaryotic proteins of other functions and locations. Overall, one-third of the protein domains identified in mitochondrial proteins are only rarely found in bacteria. We conclude that the mitochondrial genome has been maintained to ensure the correct localization of highly hydrophobic membrane proteins. Taken together, the results suggest that selective constraints on the eukaryotic cell have played a major role in modulating the evolution of the mitochondrial genome and proteome.


Assuntos
Genoma Mitocondrial/genética , Proteínas Mitocondriais/metabolismo , Proteínas de Bactérias/metabolismo , Núcleo Celular/genética , Proteínas de Cloroplastos/metabolismo , Biologia Computacional , Citocromos b/metabolismo , Citosol/metabolismo , Retículo Endoplasmático/metabolismo , Células HeLa/metabolismo , Humanos , Interações Hidrofóbicas e Hidrofílicas , Proteínas de Membrana/genética , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Fosforilação Oxidativa , Filogenia , Dobramento de Proteína , Estrutura Terciária de Proteína , Partícula de Reconhecimento de Sinal/metabolismo , Termodinâmica
6.
Nat Commun ; 6: 7688, 2015 Jul 09.
Artigo em Inglês | MEDLINE | ID: mdl-26158910

RESUMO

The high pathogenicity of the Ebola virus reflects multiple concurrent processes on infection. Among other important determinants, Ebola fusogenic glycoprotein (GP) has been associated with the detachment of infected cells and eventually leads to vascular leakage and haemorrhagic fever. Here we report that the membrane-anchored GP is sufficient to induce the detachment of adherent cells. The results show that the detachment induced through either full-length GP1,2 or the subunit GP2 depends on cholesterol and the structure of the transmembrane domain. These data reveal a novel molecular mechanism in which GP regulates Ebola virus assembly and suggest that cholesterol-reducing agents could be useful as therapeutics to counteract GP-mediated cell detachment.


Assuntos
Anticolesterolemiantes/farmacologia , Adesão Celular/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Colesterol/metabolismo , Citocalasina B/farmacologia , Ebolavirus/efeitos dos fármacos , Sinvastatina/farmacologia , Proteínas Virais de Fusão/efeitos dos fármacos , beta-Ciclodextrinas/farmacologia , Animais , Células COS , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Chlorocebus aethiops , Ebolavirus/metabolismo , Ebolavirus/patogenicidade , Citometria de Fluxo , Células HEK293 , Células HeLa , Células Endoteliais da Veia Umbilical Humana , Humanos , Microscopia Confocal , Estrutura Terciária de Proteína , Proteínas Virais de Fusão/metabolismo , Fatores de Virulência/metabolismo
7.
Biochim Biophys Acta ; 1838(8): 2066-70, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24796501

RESUMO

Specific interactions between transmembrane proteins and sphingolipids is a poorly understood phenomenon, and only a couple of instances have been identified. The best characterized example is the sphingolipid-binding motif VXXTLXXIY found in the transmembrane helix of the vesicular transport protein p24. Here, we have used a simple motif-probability algorithm (MOPRO) to identify proteins that contain putative sphingolipid-binding motifs in a dataset comprising proteomes from mammalian organisms. From these motif-containing candidate proteins, four with different numbers of transmembrane helices were selected for experimental study: i) major histocompatibility complex II Q alpha chain subtype (DQA1), ii) GPI-attachment protein 1 (GAA1), iii) tetraspanin-7 TSN7, and iv), metabotropic glutamate receptor 2 (GRM2). These candidates were subjected to photo-affinity labeling using radiolabeled sphingolipids, confirming all four candidate proteins as sphingolipid-binding proteins. The sphingolipid-binding motifs are enriched in the 7TM family of G-protein coupled receptors, predominantly in transmembrane helix 6. The ability of the motif-containing candidate proteins to bind sphingolipids with high specificity opens new perspectives on their respective regulation and function.


Assuntos
Motivos de Aminoácidos , Membrana Celular/metabolismo , Proteínas de Membrana/metabolismo , Fragmentos de Peptídeos/metabolismo , Esfingolipídeos/metabolismo , Receptor de Asialoglicoproteína/metabolismo , Western Blotting , Células HeLa , Humanos , Glicoproteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Tetraspaninas/metabolismo
8.
FEBS Lett ; 587(21): 3480-6, 2013 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-24055247

RESUMO

Sdh3/Shh3, a subunit of mitochondrial succinate dehydrogenase, contains transmembrane domains with a hydrophobicity comparable to that of endoplasmic reticulum (ER) proteins. Here, we show that a C-terminal reporter fusion to Sdh3/Shh3 results in partial mis-targeting of the protein to the ER. This mis-targeting is mediated by the signal recognition particle (SRP) and depends on the length of the C-terminal tail. These results imply that if nuclear-encoded mitochondrial proteins contain strongly hydrophobic transmembrane domains and a long C-terminal tail, they have the potential to be recognized by SRP and mis-targeted to the ER.


Assuntos
Retículo Endoplasmático/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Membranas Mitocondriais/metabolismo , Succinato Desidrogenase/metabolismo , Sequência de Aminoácidos , Interações Hidrofóbicas e Hidrofílicas , Proteínas de Fusão de Membrana/química , Proteínas de Fusão de Membrana/metabolismo , Proteínas de Transporte da Membrana Mitocondrial/química , Dados de Sequência Molecular , Transporte Proteico , Saccharomyces cerevisiae/metabolismo , Partícula de Reconhecimento de Sinal/metabolismo , Succinato Desidrogenase/química
9.
J Biol Chem ; 287(20): 16399-409, 2012 May 11.
Artigo em Inglês | MEDLINE | ID: mdl-22431723

RESUMO

Many plasma membrane proteins are anchored to the membrane via a C-terminal glycosylphosphatidylinositol (GPI) moiety. The GPI anchor is attached to the protein in the endoplasmic reticulum by transamidation, a reaction in which a C-terminal GPI-attachment signal is cleaved off concomitantly with addition of the GPI moiety. GPI-attachment signals are poorly conserved on the sequence level but are all composed of a polar segment that includes the GPI-attachment site followed by a hydrophobic segment located at the very C terminus of the protein. Here, we show that efficient GPI modification requires that the hydrophobicity of the C-terminal segment is "marginal": less hydrophobic than type II transmembrane anchors and more hydrophobic than the most hydrophobic segments found in secreted proteins. We further show that the GPI-attachment signal can be modified by the transamidase irrespective of whether it is first released into the lumen of the endoplasmic reticulum or is retained in the endoplasmic reticulum membrane.


Assuntos
Aminoaciltransferases/metabolismo , Retículo Endoplasmático/metabolismo , Glicosilfosfatidilinositóis/metabolismo , Proteínas de Membrana/metabolismo , Processamento de Proteína Pós-Traducional/fisiologia , Sinais Direcionadores de Proteínas/fisiologia , Aminoaciltransferases/genética , Retículo Endoplasmático/genética , Glicosilfosfatidilinositóis/genética , Humanos , Interações Hidrofóbicas e Hidrofílicas , Proteínas de Membrana/genética
10.
Nature ; 481(7382): 525-9, 2012 Jan 09.
Artigo em Inglês | MEDLINE | ID: mdl-22230960

RESUMO

Functioning and processing of membrane proteins critically depend on the way their transmembrane segments are embedded in the membrane. Sphingolipids are structural components of membranes and can also act as intracellular second messengers. Not much is known of sphingolipids binding to transmembrane domains (TMDs) of proteins within the hydrophobic bilayer, and how this could affect protein function. Here we show a direct and highly specific interaction of exclusively one sphingomyelin species, SM 18, with the TMD of the COPI machinery protein p24 (ref. 2). Strikingly, the interaction depends on both the headgroup and the backbone of the sphingolipid, and on a signature sequence (VXXTLXXIY) within the TMD. Molecular dynamics simulations show a close interaction of SM 18 with the TMD. We suggest a role of SM 18 in regulating the equilibrium between an inactive monomeric and an active oligomeric state of the p24 protein, which in turn regulates COPI-dependent transport. Bioinformatic analyses predict that the signature sequence represents a conserved sphingolipid-binding cavity in a variety of mammalian membrane proteins. Thus, in addition to a function as second messengers, sphingolipids can act as cofactors to regulate the function of transmembrane proteins. Our discovery of an unprecedented specificity of interaction of a TMD with an individual sphingolipid species adds to our understanding of why biological membranes are assembled from such a large variety of different lipids.


Assuntos
Membrana Celular/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Esfingolipídeos/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Sítios de Ligação , Células CHO , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/metabolismo , Biologia Computacional , Sequência Conservada , Cricetinae , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Multimerização Proteica , Estrutura Terciária de Proteína , Transporte Proteico , Sistemas do Segundo Mensageiro/fisiologia , Esfingomielinas/metabolismo , Especificidade por Substrato
11.
EMBO J ; 30(6): 1003-11, 2011 Mar 16.
Artigo em Inglês | MEDLINE | ID: mdl-21326212

RESUMO

While overall hydrophobicity is generally recognized as the main characteristic of transmembrane (TM) α-helices, the only membrane system for which there are detailed quantitative data on how different amino acids contribute to the overall efficiency of membrane insertion is the endoplasmic reticulum (ER) of eukaryotic cells. Here, we provide comparable data for TIM23-mediated membrane protein insertion into the inner mitochondrial membrane of yeast cells. We find that hydrophobicity and the location of polar and aromatic residues are strong determinants of membrane insertion. These results parallel what has been found previously for the ER. However, we see striking differences between the effects elicited by charged residues flanking the TM segments when comparing the mitochondrial inner membrane and the ER, pointing to an unanticipated difference between the two insertion systems.


Assuntos
Proteínas de Membrana/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Proteínas de Membrana/química , Proteínas do Complexo de Importação de Proteína Precursora Mitocondrial , Proteínas Mitocondriais/química , Modelos Biológicos , Dados de Sequência Molecular , Estrutura Secundária de Proteína
12.
J Proteome Res ; 10(4): 1848-59, 2011 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-21210718

RESUMO

The cell envelope of Escherichia coli is an essential structure that modulates exchanges between the cell and the extra-cellular milieu. Previous proteomic analyses have suggested that it contains a significant number of proteins with no annotated function. To gain insight into these proteins and the general organization of the cell envelope proteome, we have carried out a systematic analysis of native membrane protein complexes. We have identified 30 membrane protein complexes (6 of which are novel) and present reference maps that can be used for cell envelope profiling. In one instance, we identified a protein with no annotated function (YfgM) in a complex with a well-characterized periplasmic chaperone (PpiD). Using the guilt by association principle, we suggest that YfgM is also part of the periplasmic chaperone network. The approach we present circumvents the need for engineering of tags and protein overexpression. It is applicable for the analysis of membrane protein complexes in any organism and will be particularly useful for less-characterized organisms where conventional strategies that require protein engineering (i.e., 2-hybrid based approaches and TAP-tagging) are not feasible.


Assuntos
Proteínas de Escherichia coli/análise , Escherichia coli/química , Proteínas de Membrana/análise , Chaperonas Moleculares/análise , Complexos Multiproteicos/química , Cromatografia por Troca Iônica/métodos , Eletroforese em Gel Bidimensional/métodos , Proteínas de Escherichia coli/classificação , Proteínas de Escherichia coli/isolamento & purificação , Espectrometria de Massas/métodos , Proteínas de Membrana/classificação , Proteínas de Membrana/isolamento & purificação , Chaperonas Moleculares/classificação , Chaperonas Moleculares/isolamento & purificação , Peso Molecular , Complexos Multiproteicos/isolamento & purificação , Filogenia , Proteoma/análise , Proteômica/métodos
13.
Bioinformatics ; 25(22): 3020-5, 2009 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-19720675

RESUMO

MOTIVATION: Certain protein domains are known to preferentially interact with other domains. Several approaches have been proposed to predict domain-domain interactions, and over nine datasets are available. Our aim is to analyse the coverage and quality of the existing resources, as well as the extent of their overlap. With this knowledge, we have the opportunity to merge individual domain interaction networks to construct a comprehensive and reliable database. RESULTS: In this article we introduce a new approach towards comparing domain-domain interaction networks. This approach is used to compare nine predicted domain and protein interaction networks. The networks were used to generate a database of unified domain interactions, UniDomInt. Each interaction in the dataset is scored according to the benchmarked reliability of the sources. The performance of UniDomInt is an improvement compared to the underlying source networks and to another composite resource, Domine. AVAILABILITY: http://sonnhammer.sbc.su.se/download/UniDomInt/


Assuntos
Biologia Computacional/métodos , Proteínas/química , Sítios de Ligação , Bases de Dados de Proteínas , Mapeamento de Interação de Proteínas , Estrutura Terciária de Proteína
14.
Bioinformatics ; 25(10): 1264-70, 2009 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-19289446

RESUMO

MOTIVATION: Correct prediction of residue-residue contacts in proteins that lack good templates with known structure would take ab initio protein structure prediction a large step forward. The lack of correct contacts, and in particular long-range contacts, is considered the main reason why these methods often fail. RESULTS: We propose a novel hidden Markov model (HMM)-based method for predicting residue-residue contacts from protein sequences using as training data homologous sequences, predicted secondary structure and a library of local neighborhoods (local descriptors of protein structure). The library consists of recurring structural entities incorporating short-, medium- and long-range interactions and is general enough to reassemble the cores of nearly all proteins in the PDB. The method is tested on an external test set of 606 domains with no significant sequence similarity to the training set as well as 151 domains with SCOP folds not present in the training set. Considering the top 0.2 x L predictions (L = sequence length), our HMMs obtained an accuracy of 22.8% for long-range interactions in new fold targets, and an average accuracy of 28.6% for long-, medium- and short-range contacts. This is a significant performance increase over currently available methods when comparing against results published in the literature. AVAILABILITY: http://predictioncenter.org/Services/FragHMMent/.


Assuntos
Biologia Computacional/métodos , Cadeias de Markov , Proteínas/química , Bases de Dados de Proteínas , Modelos Moleculares , Dobramento de Proteína , Estrutura Secundária de Proteína
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