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1.
J Cell Biol ; 223(5)2024 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-38558237

RESUMO

The p24 family of proteins have been regarded as cargo receptors for endoplasmic reticulum (ER) to Golgi transport; however, their precise functions have yet to be revealed. In this issue, Pastor-Pareja and colleagues (https://doi.org/10.1083/jcb.202309045) show that the interaction of these proteins with Tango1 is critical for their localization at the ER exit site (ERES) and efficient transport of secretory proteins in Drosophila.


Assuntos
Translocador Nuclear Receptor Aril Hidrocarboneto , Drosophila , Retículo Endoplasmático , Animais , Translocador Nuclear Receptor Aril Hidrocarboneto/metabolismo , Vesículas Revestidas pelo Complexo de Proteína do Envoltório/metabolismo , Drosophila/citologia , Drosophila/metabolismo , Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Transporte Proteico/fisiologia , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo
2.
J Cell Biol ; 223(5)2024 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-38558238

RESUMO

Plants often adapt to adverse or stress conditions via differential growth. The trans-Golgi network (TGN) has been implicated in stress responses, but it is not clear in what capacity it mediates adaptive growth decisions. In this study, we assess the role of the TGN in stress responses by exploring the previously identified interactome of the Transport Protein Particle II (TRAPPII) complex required for TGN structure and function. We identified physical and genetic interactions between AtTRAPPII and shaggy-like kinases (GSK3/AtSKs) and provided in vitro and in vivo evidence that the TRAPPII phosphostatus mediates adaptive responses to abiotic cues. AtSKs are multifunctional kinases that integrate a broad range of signals. Similarly, the AtTRAPPII interactome is vast and considerably enriched in signaling components. An AtSK-TRAPPII interaction would integrate all levels of cellular organization and instruct the TGN, a central and highly discriminate cellular hub, as to how to mobilize and allocate resources to optimize growth and survival under limiting or adverse conditions.


Assuntos
Proteínas de Arabidopsis , Arabidopsis , Proteínas de Transporte , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Quinase 3 da Glicogênio Sintase/metabolismo , Fosforilação , Transporte Proteico , Rede trans-Golgi/metabolismo , Proteínas de Transporte/metabolismo
3.
J Cell Biol ; 223(7)2024 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-38578286

RESUMO

The AP-1 adaptor complex is found in all eukaryotes, but it has been implicated in different pathways in different organisms. To look directly at AP-1 function, we generated stably transduced HeLa cells coexpressing tagged AP-1 and various tagged membrane proteins. Live cell imaging showed that AP-1 is recruited onto tubular carriers trafficking from the Golgi apparatus to the plasma membrane, as well as onto transferrin-containing early/recycling endosomes. Analysis of single AP-1 vesicles showed that they are a heterogeneous population, which starts to sequester cargo 30 min after exit from the ER. Vesicle capture showed that AP-1 vesicles contain transmembrane proteins found at the TGN and early/recycling endosomes, as well as lysosomal hydrolases, but very little of the anterograde adaptor GGA2. Together, our results support a model in which AP-1 retrieves proteins from post-Golgi compartments back to the TGN, analogous to COPI's role in the early secretory pathway. We propose that this is the function of AP-1 in all eukaryotes.


Assuntos
Complexo de Golgi , Proteínas de Membrana , Transporte Proteico , Fator de Transcrição AP-1 , Humanos , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Membrana Celular/metabolismo , Endossomos/genética , Endossomos/metabolismo , Complexo de Golgi/genética , Complexo de Golgi/metabolismo , Células HeLa , Proteínas de Membrana/metabolismo , Rede trans-Golgi/metabolismo , Fator de Transcrição AP-1/genética , Fator de Transcrição AP-1/metabolismo
4.
Curr Biol ; 34(7): R267-R268, 2024 Apr 08.
Artigo em Inglês | MEDLINE | ID: mdl-38593766

RESUMO

In this Quick guide, Palmer and Berks introduce the twin-arginine translocation (Tat) systems. Tats are found in a variety of microbes and microbe-derived organelles, and are known to translocate folded substrate proteins across biological membranes.


Assuntos
Proteínas de Escherichia coli , Sistema de Translocação de Argininas Geminadas , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Proteínas de Escherichia coli/metabolismo , Sistema de Translocação de Argininas Geminadas/metabolismo , Membrana Celular/metabolismo , Arginina/metabolismo , Transporte Proteico , Sinais Direcionadores de Proteínas , Proteínas de Bactérias/metabolismo
5.
Methods Mol Biol ; 2778: 83-99, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38478273

RESUMO

ß-barrel membrane proteins populate the outer membrane of Gram-negative bacteria, mitochondria, and chloroplasts, playing significant roles in multiple key cellular pathways. Characterizing the functions of these membrane proteins in vivo is often challenging due to the complex protein network in the periplasm of Gram-negative bacteria (or intermembrane space in mitochondria and chloroplasts) and the presence of other outer membrane proteins. In vitro reconstitution into lipid-bilayer-like environments such as nanodiscs or proteoliposomes provides an excellent method for examining the specific function and mechanism of these membrane proteins in an isolated system. Here, we describe the methodologies employed to investigate Slam, a 14-stranded ß-barrel membrane protein also known as the type XI secretion system that is responsible for translocating proteins across the outer membrane of many bacterial species.


Assuntos
Proteínas da Membrana Bacteriana Externa , Proteolipídeos , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteolipídeos/metabolismo , Mitocôndrias/metabolismo , Transporte Proteico , Bactérias Gram-Negativas/metabolismo
6.
Methods Mol Biol ; 2778: 201-220, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38478280

RESUMO

Mitochondrial ß-barrel proteins fulfill crucial roles in the biogenesis and function of the cell organelle. They mediate the import and membrane insertion of proteins and transport of small metabolites and ions. All ß-barrel proteins are made as precursors on cytosolic ribosomes and are imported into mitochondria. The ß-barrel proteins fold and assemble with partner proteins in the outer membrane. The in vitro import of radiolabelled proteins into isolated mitochondria is a powerful tool to investigate the import of ß-barrel proteins, the folding of the ß-barrel proteins, and their assembly into protein complexes. Altogether, the in vitro import assay is a versatile and crucial assay to analyze the mechanisms of the biogenesis of mitochondrial ß-barrel proteins.


Assuntos
Proteínas Mitocondriais , Proteínas de Saccharomyces cerevisiae , Proteínas Mitocondriais/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Mitocôndrias/metabolismo , Transporte Proteico , Proteínas de Transporte da Membrana Mitocondrial/metabolismo
7.
Methods Mol Biol ; 2778: 185-200, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38478279

RESUMO

All but a few mitochondrial proteins are translated into the cytosol and imported in via complicated and varied pathways. These processes occur over short time frames and, as such, are difficult to monitor with classical approaches such as Western blotting or autoradiography that require sample collection at discrete time points. The development of an assay based on a split version of the small luciferase-Mitoluc-has allowed us to monitor the import of proteins into mitochondria in high resolution and real time (Pereira et al., J Mol Biol 431:1689-1699, 2019). Luminescence measurements are acquired using a plate reader in the order of seconds. This allows scores of experiments to be conducted in parallel in a single multi-well plate and permits kinetic analysis yielding information about import mechanisms (Ford et al., Elife 11:e75426, 2022).


Assuntos
Luminescência , Mitocôndrias , Cinética , Mitocôndrias/metabolismo , Transporte Proteico , Medições Luminescentes
8.
Commun Biol ; 7(1): 273, 2024 Mar 12.
Artigo em Inglês | MEDLINE | ID: mdl-38472392

RESUMO

Membrane-enclosed organelles are defining features of eukaryotes in distinguishing these organisms from prokaryotes. Specification of distinct membranes is critical to assemble and maintain discrete compartments. Small GTPases and their regulators are the signaling molecules that drive membrane-modifying machineries to the desired location. These signaling molecules include Rab and Rag GTPases, roadblock and longin domain proteins, and TRAPPC3-like proteins. Here, we take a structural approach to assess the relatedness of these eukaryotic-like proteins in Asgard archaea, the closest known prokaryotic relatives to eukaryotes. We find that the Asgard archaea GTPase core domains closely resemble eukaryotic Rabs and Rags. Asgard archaea roadblock, longin and TRAPPC3 domain-containing proteins form dimers similar to those found in the eukaryotic TRAPP and Ragulator complexes. We conclude that the emergence of these protein architectures predated eukaryogenesis, however further adaptations occurred in proto-eukaryotes to allow these proteins to regulate distinct internal membranes.


Assuntos
Proteínas Monoméricas de Ligação ao GTP , Proteínas Monoméricas de Ligação ao GTP/química , Archaea/metabolismo , Transporte Proteico
9.
FASEB J ; 38(5): e23519, 2024 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-38457249

RESUMO

ARL3 is essential for cilia development, and mutations in ARL3 are closely associated with ciliopathies. In a previous study, we observed distinct phenotypes of retinal dystrophy in patients with heterozygous ARL3T31A and compound heterozygous ARL3T31A/C118F mutations, indicating that different mutation types may exert diverse effects on their functions. Here, we generated transformed immortal fibroblast cells from patients carrying heterozygous ARL3T31A and compound heterozygous ARL3T31A/C118F mutations, and systematically evaluated their cilia morphology and function, which were further validated in ARPE-19 cells. Results showed that both ARL3T31A and ARL3T31A/C118F mutations led to a decrease in cilium formation. The ARL3T31A/C118F mutations caused significantly elongated cilia and impaired retrograde transport, whereas the ARL3T31A mutation did not induce significant changes in fibroblasts. RNA-sequencing results indicated that compared to ARL3T31A , ARL3T31A/C118F fibroblasts exhibited a higher enrichment of biological processes related to neuron projection development, tissue morphogenesis, and extracellular matrix (ECM) organization, with noticeable alterations in pathways such as ECM-receptor interaction, focal adhesion, and TGF-ß signaling. Similar changes were observed in the proteomic results in ARPE-19 cells. Core regulated genes including IQUB, UNC13D, RAB3IP, and GRIP1 were specifically downregulated in the ARL3T31A/C118F group, and expressions of IQUB, NPM2, and SLC38A4 were further validated. Additionally, IQUB showed a rescuing effect on the overlong cilia observed in ARL3T31A/C118F fibroblasts. Our results not only enhance our understanding of ARL3-related diseases but also provide new insights into the analysis of heterozygous and compound heterozygous mutations in genetics.


Assuntos
Cílios , Proteômica , Humanos , Cílios/genética , Cílios/metabolismo , Transporte Proteico , Fatores de Ribosilação do ADP/genética , Fatores de Ribosilação do ADP/metabolismo , Mutação , Fibroblastos/metabolismo , Proteínas de Membrana/metabolismo
10.
Elife ; 122024 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-38466628

RESUMO

Secretory proteins are sorted at the trans-Golgi network (TGN) for export into specific transport carriers. However, the molecular players involved in this fundamental process remain largely elusive. Here, we identified the human transmembrane protein TGN46 as a receptor for the export of secretory cargo protein PAUF in CARTS - a class of protein kinase D-dependent TGN-to-plasma membrane carriers. We show that TGN46 is necessary for cargo sorting and loading into nascent carriers at the TGN. By combining quantitative fluorescence microscopy and mutagenesis approaches, we further discovered that the lumenal domain of TGN46 encodes for its cargo sorting function. In summary, our results define a cellular function of TGN46 in sorting secretory proteins for export from the TGN.


Assuntos
Proteínas de Membrana , Rede trans-Golgi , Humanos , Proteínas de Membrana/metabolismo , Transporte Proteico/fisiologia , Rede trans-Golgi/metabolismo
11.
Brief Bioinform ; 25(2)2024 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-38426320

RESUMO

Protein subcellular localization (PSL) is very important in order to understand its functions, and its movement between subcellular niches within cells plays fundamental roles in biological process regulation. Mass spectrometry-based spatio-temporal proteomics technologies can help provide new insights of protein translocation, but bring the challenge in identifying reliable protein translocation events due to the noise interference and insufficient data mining. We propose a semi-supervised graph convolution network (GCN)-based framework termed TransGCN that infers protein translocation events from spatio-temporal proteomics. Based on expanded multiple distance features and joint graph representations of proteins, TransGCN utilizes the semi-supervised GCN to enable effective knowledge transfer from proteins with known PSLs for predicting protein localization and translocation. Our results demonstrate that TransGCN outperforms current state-of-the-art methods in identifying protein translocations, especially in coping with batch effects. It also exhibited excellent predictive accuracy in PSL prediction. TransGCN is freely available on GitHub at https://github.com/XuejiangGuo/TransGCN.


Assuntos
Proteômica , Mineração de Dados , Espectrometria de Massas , Transporte Proteico
12.
Mol Genet Genomics ; 299(1): 39, 2024 Mar 22.
Artigo em Inglês | MEDLINE | ID: mdl-38519717

RESUMO

Chorea-acanthocytosis (ChAc) is a rare autosomal recessive neurodegenerative disorder characterized by a variety of involuntary movements, predominantly chorea, and the presence of acanthocytosis in peripheral blood smears. ChAc is caused by mutations in the vacuolar protein sorting-associated protein 13A (VPS13A) gene. The aim of the present study was to conduct a clinical and genetic analysis of five patients with suspected ChAc in Iran. This study included five patients who were referred to the genetic department of the Endocrinology and Metabolism Research Institute between 2020 and 2022, with a suspicion of ChAc. Clinical features and the presence of characteristic MRI findings were evaluated in the patients. Whole-exome sequencing (WES) followed by Sanger sequencing was employed to identify the disease-causing variants. The functional effects of novel mutations were analyzed by specific bioinformatics prediction tools. WES and data analysis revealed the presence of five distinct VPS13A mutations in the patients, four of which were novel. These included one nonsense mutation (p.L984X), and three splice site mutations (c.755-1G>A, c.144+1 G>C, c.2512+1G>A). All mutations were validated by Sanger sequencing, and in silico analysis predicted that all mutations were pathogenic. This study provides the first molecular genetic characteristics of Iranian patients with ChAc, identifying four novel mutations in the VPS13A gene. These findings expand the VPS13A variants spectrum and confirm the clinical variability in ChAc patients.


Assuntos
Neuroacantocitose , Humanos , Neuroacantocitose/genética , Neuroacantocitose/patologia , Irã (Geográfico) , Proteínas de Transporte Vesicular/genética , Transporte Proteico , Mutação
13.
Med Sci (Paris) ; 40(3): 267-274, 2024 Mar.
Artigo em Francês | MEDLINE | ID: mdl-38520102

RESUMO

The characterization of the structural and functional organization of eukaryotic cells has revealed the membrane compartments and machinery required for vesicular protein transport. Most proteins essential for intercellular communication contain an N-terminal signal sequence enabling them to be incorporated into the biosynthetic or conventional secretory pathway, in which proteins are sequentially transported through the endoplasmic reticulum (ER) and the Golgi apparatus. However, major research studies have shown the existence of alternative secretory routes that are independent of the ER-Golgi and designated as unconventional secretory pathways. These pathways involve a large number of players that may divert specific compartments from their primary function in favor of secretory roles. The comprehensive description of these processes is therefore of utmost importance to unveil how proteins secreted through these alternative pathways control cell homeostasis or contribute to disease development.


Title: Sécrétion non conventionnelle - Nouvelles perspectives dans le trafic des protéines. Abstract: L'étude de l'organisation structurale et fonctionnelle des cellules eucaryotes a révélé les compartiments membranaires ainsi que la machinerie nécessaires au trafic vésiculaire des protéines. La plupart des protéines essentielles à la communication intercellulaire contiennent une séquence signal leur permettant d'être incorporées dans la voie de sécrétion conventionnelle, par laquelle les protéines sont transportées séquentiellement dans le réticulum endoplasmique (RE) puis l'appareil de Golgi. Cependant, les cellules eucaryotes sont également dotées de voies de sécrétion alternatives ou voies de sécrétion non conventionnelles, qui mettent en jeu de nombreux acteurs susceptibles de détourner certains compartiments de leurs fonctions principales au profit de fonctions sécrétoires.


Assuntos
Células Eucarióticas , Proteínas , Humanos , Transporte Proteico , Proteínas/metabolismo , Células Eucarióticas/metabolismo , Retículo Endoplasmático/metabolismo , Complexo de Golgi , Via Secretória
16.
Commun Biol ; 7(1): 366, 2024 Mar 26.
Artigo em Inglês | MEDLINE | ID: mdl-38531947

RESUMO

The flagellar type III secretion system (fT3SS) switches substrate specificity from rod-hook-type to filament-type upon hook completion, terminating hook assembly and initiating filament assembly. The C-terminal cytoplasmic domain of FlhA (FlhAC) forms a homo-nonameric ring and is directly involved in substrate recognition, allowing the fT3SS to coordinate flagellar protein export with assembly. The highly conserved GYXLI motif (residues 368-372) of FlhAC induces dynamic domain motions of FlhAC required for efficient and robust flagellar protein export by the fT3SS, but it remains unknown whether this motif is also important for ordered protein export by the fT3SS. Here we analyzed two GYXLI mutants, flhA(GAAAA) and flhA(GGGGG), and provide evidence suggesting that the GYXLI motif in FlhAC requires the flagellar ATPase complex not only to efficiently remodel the FlhAC ring structure for the substrate specificity switching but also to correct substrate recognition errors that occur during flagellar assembly.


Assuntos
Proteínas de Bactérias , Proteínas de Membrana , Proteínas de Bactérias/metabolismo , Proteínas de Membrana/metabolismo , Transporte Proteico , Salmonella , ATPases Translocadoras de Prótons/metabolismo
17.
PLoS Biol ; 22(3): e3002567, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38470934

RESUMO

PEX5, the peroxisomal protein shuttling receptor, binds newly synthesized proteins in the cytosol and transports them to the organelle. During its stay at the peroxisomal protein translocon, PEX5 is monoubiquitinated at its cysteine 11 residue, a mandatory modification for its subsequent ATP-dependent extraction back into the cytosol. The reason why a cysteine and not a lysine residue is the ubiquitin acceptor is unknown. Using an established rat liver-based cell-free in vitro system, we found that, in contrast to wild-type PEX5, a PEX5 protein possessing a lysine at position 11 is polyubiquitinated at the peroxisomal membrane, a modification that negatively interferes with the extraction process. Wild-type PEX5 cannot retain a polyubiquitin chain because ubiquitination at cysteine 11 is a reversible reaction, with the E2-mediated deubiquitination step presenting faster kinetics than PEX5 polyubiquitination. We propose that the reversible nonconventional ubiquitination of PEX5 ensures that neither the peroxisomal protein translocon becomes obstructed with polyubiquitinated PEX5 nor is PEX5 targeted for proteasomal degradation.


Assuntos
Cisteína , Lisina , Animais , Ratos , Proteínas de Transporte/metabolismo , Cisteína/metabolismo , Lisina/metabolismo , Receptor 1 de Sinal de Orientação para Peroxissomos/química , Receptor 1 de Sinal de Orientação para Peroxissomos/metabolismo , Transporte Proteico , Ubiquitinação
18.
Clin Nucl Med ; 49(5): e213-e214, 2024 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-38465981

RESUMO

ABSTRACT: The cancer specificity of fibroblast activation protein inhibitor (FAPI) uptake remains understudied topic, and several cases of false-positive FAPI PET/CT findings have been reported. We present 2 patients of differentiated thyroid cancer with thyroglobulin elevation and negative iodine scintigraphy syndrome who underwent 68 Ga-FAPI PET/CT and 18 F-FDG PET/CT for evaluation of any possible sites for metastatic disease. Although no focus of metastatic disease was found in these patients, remarkable findings were noticed instead. Nonmalignant FAPI uptake was evident in the gallbladder, uterus, and degenerative changes, whereas these uptake were discordant or partially concordant with those in FDG's, also CT images showed no underlying abnormality.


Assuntos
Fluordesoxiglucose F18 , Segunda Neoplasia Primária , Quinolinas , Feminino , Humanos , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada , Transporte Proteico , Transporte Biológico , Radioisótopos de Gálio
19.
Cell Rep ; 43(3): 113913, 2024 Mar 26.
Artigo em Inglês | MEDLINE | ID: mdl-38442016

RESUMO

The self-incompatibility system evolves in angiosperms to promote cross-pollination by rejecting self-pollination. Here, we show the involvement of Exo84c in the SI response of both Brassica napus and Arabidopsis. The expression of Exo84c is specifically elevated in stigma during the SI response. Knocking out Exo84c in B. napus and SI Arabidopsis partially breaks down the SI response. The SI response inhibits both the protein secretion in papillae and the recruitment of the exocyst complex to the pollen-pistil contact sites. Interestingly, these processes can be partially restored in exo84c SI Arabidopsis. After incompatible pollination, the turnover of the exocyst-labeled compartment is enhanced in papillae. However, this process is perturbed in exo84c SI Arabidopsis. Taken together, our results suggest that Exo84c regulates the exocyst complex vacuolar degradation during the SI response. This process is likely independent of the known SI pathway in Brassicaceae to secure the SI response.


Assuntos
Proteínas de Arabidopsis , Arabidopsis , Brassicaceae , Brassicaceae/genética , Brassicaceae/metabolismo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Pólen/metabolismo , Transporte Proteico , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo
20.
Brief Bioinform ; 25(2)2024 Jan 22.
Artigo em Inglês | MEDLINE | ID: mdl-38487847

RESUMO

Causal discovery is a powerful tool to disclose underlying structures by analyzing purely observational data. Genetic variants can provide useful complementary information for structure learning. Recently, Mendelian randomization (MR) studies have provided abundant marginal causal relationships of traits. Here, we propose a causal network pruning algorithm MRSL (MR-based structure learning algorithm) based on these marginal causal relationships. MRSL combines the graph theory with multivariable MR to learn the conditional causal structure using only genome-wide association analyses (GWAS) summary statistics. Specifically, MRSL utilizes topological sorting to improve the precision of structure learning. It proposes MR-separation instead of d-separation and three candidates of sufficient separating set for MR-separation. The results of simulations revealed that MRSL had up to 2-fold higher F1 score and 100 times faster computing time than other eight competitive methods. Furthermore, we applied MRSL to 26 biomarkers and 44 International Classification of Diseases 10 (ICD10)-defined diseases using GWAS summary data from UK Biobank. The results cover most of the expected causal links that have biological interpretations and several new links supported by clinical case reports or previous observational literatures.


Assuntos
Algoritmos , Estudo de Associação Genômica Ampla , Causalidade , Fenótipo , Transporte Proteico , Análise da Randomização Mendeliana , Polimorfismo de Nucleotídeo Único
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