RESUMO
Targeted nanoparticles offer potential to selectively deliver therapeutics to cells; however, their subcellular fate following endocytosis must be understood to properly design mechanisms of drug release. Here we describe a nanoparticle platform and associated cell-based assay to observe lysosome trafficking of targeted nanoparticles in live cells. The nanoparticle platform utilizes two fluorescent dyes loaded onto PEG-poly(glutamic acid) and PEG-poly(Lysine) block co-polymers that also comprise azide reactive handles on PEG termini to attach antibody-based targeting ligands. Fluorophores were selected to be pH-sensitive (pHrodo Red) or pH-insensitive (Alexafluor 488) to report when nanoparticles enter low pH lysosomes. Dye-labelled block co-polymers were further assembled into polyion complex micelle nanoparticles and crosslinked through amide bond formation to form stable nano-scaffolds for ligand attachment. Cell binding and lysosome trafficking was determined in live cells by fluorescence imaging in 96-well plates and quantification of red- and green-fluorescence signals over time. The platform and assay was validated for selection of optimal antibody-derived targeting ligands directed towards CD22 for nanoparticle delivery. Kinetic analysis of uptake and lysosome trafficking indicated differences between ligand types and the ligand with the highest lysosome trafficking efficiency translated into effective DNA delivery with nanoparticles bearing the optimal ligand.
The ability of this pH-sensitive reporter platform to rapidly screen ligands in nanoparticle format will enable identification and production of targeted NPs with desired lysosome trafficking properties.
RESUMO
NO (nitric oxide) is an important regulator of neutrophil functions and has a key role in diverse pathophysiological conditions. NO production by nitric oxide synthases (NOS) is under tight control at transcriptional, translational, and post-translational levels including interactions with heterologous proteins owing to its potent chemical reactivity and high diffusibility; this limits toxicity to other cellular components and promotes signaling specificity. The protein-protein interactions govern the activity and spatial distribution of NOS isoform to regulatory proteins and to their intended targets. In comparison with the vast literature available for endothelial, macrophages, and neuronal cells, demonstrating neuronal NOS (nNOS) interaction with other proteins through the PDZ domain, neutrophil nNOS, however, remains unexplored. Neutrophil's key role in both physiological and pathological conditions necessitates the need for further studies in delineating the NOS mediated NO modulations in signaling pathways operational in them. nNOS has been linked to depression, schizophrenia, and Parkinson's disease, suggesting the importance of exploring nNOS/NO-mediated neutrophil physiology in relation to such neuronal disorders. The review thus presents the scenario of neutrophil nNOS from the genetics to the functional level, including protein-protein interactions governing its intracellular sequestration in diverse cell types, besides speculating possible regulation in neutrophils and also addressing their clinical implications.
Assuntos
Neutrófilos , Óxido Nítrico Sintase Tipo I/metabolismo , Humanos , Neurônios , Óxido NítricoRESUMO
BACKGROUND: Human angiotensin-converting enzyme 2 (ACE2), a type I transmembrane receptor physiologically acting as a carboxypeptidase enzyme within the renin-angiotensin system (RAS), is a critical mediator of infection by several severe acute respiratory syndrome (SARS) corona viruses. For instance, it has been demonstrated that ACE2 is the primary receptor for the SARS-CoV-2 entry to many human cells through binding to the viral spike S protein. Consequently, genetic variability in ACE2 gene has been suggested to contribute to the variable clinical manifestations in COVID-19. Many of those genetic variations result in missense variants within the amino acid sequence of ACE2. The potential effects of those variations on binding to the spike protein have been speculated and, in some cases, demonstrated experimentally. However, their effects on ACE2 protein folding, trafficking and subcellular targeting have not been established. RESULTS: In this study we aimed to examine the potential effects of 28 missense variants (V801G, D785N, R768W, I753T, L731F, L731I, I727V, N720D, R710H, R708W, S692P, E668K, V658I, N638S, A627V, F592L, G575V, A501T, I468V, M383I, G173S, N159S, N149S, D38E, N33D, K26R, I21T, and S19P) distributed across the ACE2 receptor domains on its subcellular trafficking and targeting through combinatorial approach involving in silico analysis and experimental subcellular localization analysis. Our data show that none of the studied missense variants (including 3 variants predicted to be deleterious R768W, G575V, and G173S) has a significant effect on ACE2 intracellular trafficking and subcellular targeting to the plasma membrane. CONCLUSION: Although the selected missense variants display no significant change in ACE2 trafficking and subcellular localization, this does not rule out their effect on viral susceptibility and severity. Further studies are required to investigate the effect of ACE2 variants on its expression, binding, and internalization which might explain the variable clinical manifestations associated with the infection.
Assuntos
Enzima de Conversão de Angiotensina 2/genética , COVID-19 , Enzima de Conversão de Angiotensina 2/metabolismo , COVID-19/genética , Humanos , Peptidil Dipeptidase A/genética , Ligação Proteica , SARS-CoV-2/genética , Glicoproteína da Espícula de Coronavírus/genéticaRESUMO
The vesicle-associated membrane protein 7 (VAMP7) is a SNARE protein of the longin family involved in a wide range of subcellular trafficking events, including neurite sprouting and elongation. The expression of the human gene SYBL1, encoding VAMP7, is finely regulated by alternative splicing. Among the minor isoforms identified so far, VAMP7j is the one most expressed and modulated in the human brain. Therefore, we focused on gaining functional evidence on VAMP7j, which lacks a functional SNARE motif but retains both the longin and transmembrane domains. In human SH-SY5Y cells, we found VAMP7j to modulate neuritogenesis by mediating transport of L1CAM toward the plasma membrane, in a fashion regulated by phosphorylation of the longin domain. VAMP7-mediated regulation of L1CAM trafficking seems at least to differentiate humans from rats, with VAMP7j CNS expression being restricted to primates, including humans. Since L1CAM is a central player in neuritogenesis and axon guidance, these findings suggest the species-specific splicing of SYBL1 is among the fine tuners of human neurodevelopmental complexity.
Assuntos
Molécula L1 de Adesão de Célula Nervosa , Neuroblastoma , Animais , Humanos , Ratos , Membrana Celular/metabolismo , Molécula L1 de Adesão de Célula Nervosa/genética , Molécula L1 de Adesão de Célula Nervosa/metabolismo , Neuroblastoma/metabolismo , Crescimento Neuronal , Proteínas R-SNARE/genética , Proteínas R-SNARE/metabolismo , Proteínas SNARE/metabolismoRESUMO
Much of plant development depends on cell-to-cell redistribution of the plant hormone auxin, which is facilitated by the plasma membrane (PM) localized PIN FORMED (PIN) proteins. Auxin export activity, developmental roles, subcellular trafficking, and polarity of PINs have been well studied, but their structure remains elusive besides a rough outline that they contain two groups of 5 alpha-helices connected by a large hydrophilic loop (HL). Here, we focus on the PIN1 HL as we could produce it in sufficient quantities for biochemical investigations to provide insights into its secondary structure. Circular dichroism (CD) studies revealed its nature as an intrinsically disordered protein (IDP), manifested by the increase of structure content upon thermal melting. Consistent with IDPs serving as interaction platforms, PIN1 loops homodimerize. PIN1 HL cytoplasmic overexpression in Arabidopsis disrupts early endocytic trafficking of PIN1 and PIN2 and causes defects in the cotyledon vasculature formation. In summary, we demonstrate that PIN1 HL has an intrinsically disordered nature, which must be considered to gain further structural insights. Some secondary structures may form transiently during pairing with known and yet-to-be-discovered interactors.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Proteínas Intrinsicamente Desordenadas , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Transporte Biológico , Ácidos Indolacéticos/metabolismo , Proteínas Intrinsicamente Desordenadas/genética , Proteínas Intrinsicamente Desordenadas/metabolismo , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Raízes de Plantas/metabolismoRESUMO
Enzymes, once considered static molecular machines acting in defined spatial patterns and sites of action, move to different intra- and extracellular locations, changing their function. This topological regulation revealed a close cross-talk between proteases and signaling events involving post-translational modifications, membrane tyrosine kinase receptors and G-protein coupled receptors, motor proteins shuttling cargos in intracellular vesicles, and small-molecule messengers. Here, we highlight recent advances in our knowledge of regulation and function of A Disintegrin And Metalloproteinase (ADAM) endopeptidases at specific subcellular sites, or in multimolecular complexes, with a special focus on ADAM10, and tumor necrosis factor-α convertase (TACE/ADAM17), since these two enzymes belong to the same family, share selected substrates and bioactivity. We will discuss some examples of ADAM10 activity modulated by changing partners and subcellular compartmentalization, with the underlying hypothesis that restraining protease activity by spatial segregation is a complex and powerful regulatory tool.
Assuntos
Proteína ADAM10/metabolismo , Animais , Humanos , Modelos Biológicos , Processamento de Proteína Pós-Traducional , Transporte Proteico , Transdução de Sinais , Especificidade por SubstratoRESUMO
Abnormalities of the peripheral myelin protein 22 (PMP22) gene, including duplication, deletion and point mutations are a major culprit in Type 1 Charcot-Marie-Tooth (CMT) diseases. The complete absence of PMP22 alters cholesterol metabolism in Schwann cells, which likely contributes to myelination deficits. Here, we examined the subcellular trafficking of cholesterol in distinct models of PMP22-linked neuropathies. In Schwann cells from homozygous Trembler J (TrJ) mice carrying a Leu16Pro mutation, cholesterol was retained with TrJ-PMP22 in the Golgi, alongside a corresponding reduction in its plasma membrane level. PMP22 overexpression, which models CMT1A caused by gene duplication, triggered cholesterol sequestration to lysosomes, and reduced ATP-binding cassette transporter-dependent cholesterol efflux. Conversely, lysosomal targeting of cholesterol by U18666A treatment increased wild type (WT)-PMP22 levels in lysosomes. Mutagenesis of a cholesterol recognition motif, or CRAC domain, in human PMP22 lead to increased levels of PMP22 in the ER and Golgi compartments, along with higher cytosolic, and lower membrane-associated cholesterol. Importantly, cholesterol trafficking defects observed in PMP22-deficient Schwann cells were rescued by WT but not CRAC-mutant-PMP22. We also observed that myelination deficits in dorsal root ganglia explants from heterozygous PMP22-deficient mice were improved by cholesterol supplementation. Collectively, these findings indicate that PMP22 is critical in cholesterol metabolism, and this mechanism is likely a contributing factor in PMP22-linked hereditary neuropathies. Our results provide a basis for understanding how altered expression of PMP22 impacts cholesterol metabolism.
Assuntos
Mutação , Animais , Doença de Charcot-Marie-Tooth/genética , Colesterol , Camundongos , Mutação/genética , Proteínas da Mielina , Células de SchwannRESUMO
Neurotrophin signaling via receptor tyrosine kinases is essential for the development and function of the nervous system in vertebrates. TrkB activation and signaling show substantial differences to other receptor tyrosine kinases of the Trk family that mediate the responses to nerve growth factor and neurotrophin-3. Growing evidence suggests that TrkB cell surface expression is highly regulated and determines the sensitivity of neurons to brain-derived neurotrophic factor (BDNF). This translocation of TrkB depends on co-factors and modulators of cAMP levels, N-glycosylation, and receptor transactivation. This process can occur in very short time periods and the resulting rapid modulation of target cell sensitivity to BDNF could represent a mechanism for fine-tuning of synaptic plasticity and communication in complex neuronal networks. This review focuses on those modulatory mechanisms in neurons that regulate responsiveness to BDNF via control of TrkB surface expression.
Assuntos
Fator Neurotrófico Derivado do Encéfalo/metabolismo , Glicoproteínas de Membrana/metabolismo , Plasticidade Neuronal/imunologia , Neurônios/metabolismo , Receptor trkB/metabolismo , Humanos , Transdução de SinaisRESUMO
Recent findings have revealed the role of membrane traffic in the signaling of transforming growth factor-ß (TGF-ß). These findings originate from the pivotal function of TGF-ß in development, cell proliferation, tumor metastasis, and many other processes essential in malignancy. Actin and unconventional myosin have crucial roles in subcellular trafficking of receptors; research has also revealed a growing number of unconventional myosins that have crucial roles in TGF-ß signaling. Unconventional myosins modulate the spatial organization of endocytic trafficking and tether membranes or transport them along the actin cytoskeletons. Current models do not fully explain how membrane traffic forms a bridge between TGF-ß and the downstream effectors that produce its functional responsiveness, such as cell migration. In this review, we present a brief overview of the current knowledge of the TGF-ß signaling pathway and the molecular components that comprise the core pathway as follows: ligands, receptors, and Smad mediators. Second, we highlight key role(s) of myosin motor-mediated protein trafficking and membrane domain segregation in the modulation of the TGF-ß signaling pathway. Finally, we review future challenges and provide future prospects in this field.
Assuntos
Miosinas/metabolismo , Transdução de Sinais , Fator de Crescimento Transformador beta/metabolismo , Animais , Vesículas Revestidas por Clatrina/metabolismo , Endocitose , Humanos , Microdomínios da Membrana/metabolismo , Transporte ProteicoRESUMO
Pentabromopseudilin (PBrP) is a marine antibiotic isolated from the marine bacteria Pseudomonas bromoutilis and Alteromonas luteoviolaceus. PBrP exhibits antimicrobial, anti-tumour, and phytotoxic activities. In mammalian cells, PBrP is known to act as a reversible and allosteric inhibitor of myosin Va (MyoVa). In this study, we report that PBrP is a potent inhibitor of transforming growth factor-ß (TGF-ß) activity. PBrP inhibits TGF-ß-stimulated Smad2/3 phosphorylation, plasminogen activator inhibitor-1 (PAI-1) protein production and blocks TGF-ß-induced epithelial-mesenchymal transition in epithelial cells. PBrP inhibits TGF-ß signalling by reducing the cell-surface expression of type II TGF-ß receptor (TßRII) and promotes receptor degradation. Gene silencing approaches suggest that MyoVa plays a crucial role in PBrP-induced TßRII turnover and the subsequent reduction of TGF-ß signalling. Because, TGF-ß signalling is crucial in the regulation of diverse pathophysiological processes such as tissue fibrosis and cancer development, PBrP should be further explored for its therapeutic role in treating fibrotic diseases and cancer.
Assuntos
Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Miosina Tipo V/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Pirróis/farmacologia , Receptores de Fatores de Crescimento Transformadores beta/antagonistas & inibidores , Fator de Crescimento Transformador beta/antagonistas & inibidores , Alteromonas/química , Animais , Linhagem Celular , Relação Dose-Resposta a Droga , Células HEK293 , Células Hep G2 , Humanos , Vison , Estrutura Molecular , Miosina Tipo V/metabolismo , Proteínas Serina-Treonina Quinases/biossíntese , Pseudomonas/química , Pirróis/química , Pirróis/isolamento & purificação , Receptor do Fator de Crescimento Transformador beta Tipo II , Receptores de Fatores de Crescimento Transformadores beta/biossíntese , Relação Estrutura-Atividade , Fator de Crescimento Transformador beta/metabolismoRESUMO
Endomembrane trafficking is one of the most prominent cytological features of eukaryotes. Given their widespread distribution and specialization, coiled-coil domains, coatomer domains, small GTPases and Longin domains are considered primordial 'building blocks' of the membrane trafficking machineries. Longin domains are conserved across eukaryotes and were likely to be present in the Last Eukaryotic Common Ancestor. The Longin fold is based on the α-ß-α sandwich architecture and a unique topology, possibly accounting for the special adaptation to the eukaryotic trafficking machinery. The ancient Per ARNT Sim (PAS) and cGMP-specific phosphodiesterases, Adenylyl cyclases and FhlA (GAF) family domains show a similar architecture, and the identification of prokaryotic counterparts of GAF domains involved in trafficking provides an additional connection for the endomembrane system back into the pre-eukaryotic world. Proteome-wide, comparative bioinformatic analyses of the domains reveal three binding regions (A, B and C) mediating either specific or conserved protein-protein interactions. While the A region mediates intra- and inter-molecular interactions, the B region is involved in binding small GTPases, thus providing an evolutionary connection among major building blocks in the endomembrane system. Finally, we propose that the peculiar interaction surface of the C region of the Longin domain allowed it to extensively integrate into the endomembrane trafficking machinery in the earliest stages of building the eukaryotic cell.
Assuntos
Evolução Molecular , Proteínas de Transporte Vesicular/química , Adaptação Fisiológica/genética , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Dados de Sequência Molecular , Plantas , Estrutura Terciária de Proteína , Transporte Proteico , Homologia de Sequência de Aminoácidos , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismoRESUMO
The RNA-binding protein HuR is crucial for normal intestinal mucosal regeneration by modulating the stability and translation of target mRNAs, but the exact mechanism underlying HuR trafficking between the cytoplasm and nucleus remains largely unknown. Here we report a novel function of transcription factor JunD in the regulation of HuR subcellular localization through the control of importin-α1 expression in intestinal epithelial cells (IECs). Ectopically expressed JunD specifically inhibited importin-α1 at the transcription level, and this repression is mediated via interaction with CREB-binding site that was located at the proximal region of importin-α1 promoter. Reduction in the levels of importin-α1 by JunD increased cytoplasmic levels of HuR, although it failed to alter whole cell HuR levels. Increased levels of endogenous JunD by depleting cellular polyamines also inhibited importin-α1 expression and increased cytoplasmic HuR levels, whereas JunD silencing rescued importin-α1 expression and enhanced HuR nuclear translocation in polyamine-deficient cells. Moreover, importin-α1 silencing protected IECs against apoptosis, which was prevented by HuR silencing. These results indicate that JunD regulates HuR subcellular distribution by downregulating importin-α1, thus contributing to the maintenance of gut epithelium homeostasis.
Assuntos
Proteína Semelhante a ELAV 1/genética , Células Epiteliais/metabolismo , Mucosa Intestinal/metabolismo , Proteínas Proto-Oncogênicas c-jun/genética , Proteínas de Ligação a RNA/genética , Transcrição Gênica/genética , alfa Carioferinas/genética , Animais , Apoptose/genética , Sítios de Ligação/genética , Células CACO-2 , Linhagem Celular , Linhagem Celular Tumoral , Citoplasma/genética , Citoplasma/metabolismo , Epitélio/metabolismo , Regulação da Expressão Gênica/genética , Humanos , Poliaminas/metabolismo , Regiões Promotoras Genéticas/genética , Estabilidade de RNA/genética , RNA Mensageiro/genética , RatosRESUMO
ATG9A is the only polytopic protein of the mammalian autophagy-related protein family whose members regulate autophagosome formation during macroautophagy. At steady state, ATG9A localizes to several intracellular sites, including the Golgi apparatus, endosomes and the plasma membrane, and it redistributes towards autophagosomes upon autophagy induction. Interestingly, the transport of yeast Atg9 to the pre-autophagosomal structure depends on its self-association, which is mediated by a short amino acid motif located in the C-terminal region of the protein. Here, we investigated whether the residues that align with this motif in human ATG9A (V515-C519) are also required for its trafficking in mammalian cells. Interestingly, our findings support that human ATG9A self-interacts as well, and that this process promotes transport of ATG9A molecules through the Golgi apparatus. Furthermore, our data reveal that the transport of ATG9A out of the ER is severely impacted after mutation of the conserved V515-C519 motif. Nevertheless, the mutated ATG9A molecules could still interact with each other, indicating that the molecular mechanism of self-interaction differs in mammalian cells compared to yeast. Using sequential amino acid substitutions of glycine 516 and cysteine 519, we found that the stability of ATG9A relies on both of these residues, but that only the former is required for efficient transport of human ATG9A from the endoplasmic reticulum to the Golgi apparatus.
Assuntos
Proteínas Relacionadas à Autofagia/metabolismo , Glicina/química , Proteínas de Membrana/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Alanina/química , Motivos de Aminoácidos , Proteínas Relacionadas à Autofagia/genética , Membrana Celular/metabolismo , Cisteína/química , Retículo Endoplasmático/metabolismo , Deleção de Genes , Complexo de Golgi/metabolismo , Células HeLa , Humanos , Proteínas de Membrana/genética , Microscopia de Fluorescência , Domínios Proteicos , Transporte Proteico , Proteínas de Transporte Vesicular/genéticaRESUMO
The first layer of plant immunity is activated by cell surface receptor-like kinases (RLKs) and proteins (RLPs) that detect infectious pathogens. Constitutive interaction with the SUPPRESSOR OF BIR1 (SOBIR1) RLK contributes to RLP stability and kinase activity. As RLK activation requires transphosphorylation with a second associated RLK, it remains elusive how RLPs initiate downstream signaling. We employed live-cell imaging, gene silencing and coimmunoprecipitation to investigate the requirement of associated kinases for functioning and ligand-induced subcellular trafficking of Cf RLPs that mediate immunity of tomato against Cladosporium fulvum. Our research shows that after elicitation with matching effector ligands Avr4 and Avr9, BRI1-ASSOCIATED KINASE 1/SOMATIC EMBRYOGENESIS RECEPTOR KINASE 3 (BAK1/SERK3) associates with Cf-4 and Cf-9. BAK1/SERK3 is required for the effector-triggered hypersensitive response and resistance of tomato against C. fulvum. Furthermore, Cf-4 interacts with SOBIR1 at the plasma membrane and is recruited to late endosomes upon Avr4 trigger, also depending on BAK1/SERK3. These observations indicate that RLP-mediated resistance and endocytosis require ligand-induced recruitment of BAK1/SERK3, reminiscent of BAK1/SERK3 interaction and subcellular fate of the FLAGELLIN SENSING 2 (FLS2) RLK. This reveals that diverse classes of cell surface immune receptors share common requirements for initiation of resistance and endocytosis.
Assuntos
Proteínas de Arabidopsis/metabolismo , Endocitose , Proteínas Fúngicas/metabolismo , Imunidade Vegetal , Proteínas Serina-Treonina Quinases/metabolismo , Receptores de Superfície Celular/metabolismo , Membrana Celular/metabolismo , Cladosporium , Endossomos/metabolismo , Ligantes , Solanum lycopersicum/imunologia , Solanum lycopersicum/microbiologia , Modelos Biológicos , Plantas Geneticamente Modificadas , Ligação Proteica , Nicotiana/genéticaRESUMO
The ubiquitously expressed glucocorticoid receptor (GR) is a major drug target for inflammatory disease, but issues of specificity and target tissue sensitivity remain. We now identify high potency, non-steroidal GR ligands, GSK47867A and GSK47869A, which induce a novel conformation of the GR ligand-binding domain (LBD) and augment the efficacy of cellular action. Despite their high potency, GSK47867A and GSK47869A both induce surprisingly slow GR nuclear translocation, followed by prolonged nuclear GR retention, and transcriptional activity following washout. We reveal that GSK47867A and GSK47869A specifically alter the GR LBD structure at the HSP90-binding site. The alteration in the HSP90-binding site was accompanied by resistance to HSP90 antagonism, with persisting transactivation seen after geldanamycin treatment. Taken together, our studies reveal a new mechanism governing GR intracellular trafficking regulated by ligand binding that relies on a specific surface charge patch within the LBD. This conformational change permits extended GR action, probably because of altered GR-HSP90 interaction. This chemical series may offer anti-inflammatory drugs with prolonged duration of action due to altered pharmacodynamics rather than altered pharmacokinetics.
Assuntos
Anti-Inflamatórios/farmacologia , Benzamidas/farmacologia , Proteínas de Choque Térmico HSP90/metabolismo , Indazóis/farmacologia , Receptores de Glucocorticoides/metabolismo , Androstadienos/química , Androstadienos/farmacologia , Anti-Inflamatórios/química , Benzamidas/química , Benzoquinonas/farmacologia , Dexametasona/química , Dexametasona/farmacologia , Fluticasona , Células HeLa , Humanos , Doenças do Sistema Imunitário , Indazóis/química , Lactamas Macrocíclicas/farmacologia , Ligantes , Terapia de Alvo Molecular , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas/genética , Transporte Proteico , Receptores de Glucocorticoides/agonistas , Ativação Transcricional/efeitos dos fármacosRESUMO
The osteoblastic expression of RANKL, which is essential for the communication between osteoblastic cells and osteoclastogenic cells, is stimulated by locally acting or circulating osteotropic cytokines and hormones such as PTH and 1,25-(OH)2-D3 during the bone remodeling process. However, mechanisms those control subcellular trafficking events, membrane expression and extracellular secretion of the newly synthesized RANKL are still not well understood. In our previous study, we have found that the deficiency of osteoblastic Arl6ip5 (ADP-ribosylation-like factor 6 interacting protein 5), an endoplasmic reticulum (ER)-localized protein belonging to the prenylated rab-acceptor-family, enhanced osteoclastogenesis by increasing RANKL transcription in an ER stress dependent signaling. Here we found that over-expression of hemagglutinin (HA)-tagged Arl6ip5 in UAMS32 stromal/osteoblastic cells inhibited osteoclastogenesis, decreased the amount of soluble RANKL in culture supernatant and increased RANKL retention in ER. Moreover, Arl6ip5 bound with RANKL and disturbed the RANKL-OPG complex in UAMS-32 cells. Finally, 1 to 36 amino acid deletion on the NH2 lumen terminus of Arl6ip5 impaired the interaction between Arl6ip5 and RANKL, restored the level of soluble RANKL and the osteoclastogenic ability. These findings indicated that Arl6ip5 was an anti-catabolic factor by binding with RANKL and disturbing its subcellular trafficking in osteoblast.
Assuntos
Proteínas de Transporte/metabolismo , Osteoblastos/metabolismo , Ligante RANK/metabolismo , Frações Subcelulares/metabolismo , Animais , Linhagem Celular , Regulação da Expressão Gênica/fisiologia , Proteínas de Choque Térmico , Proteínas de Membrana Transportadoras , Camundongos , Osteoblastos/ultraestrutura , Regulação para Cima/fisiologiaRESUMO
Eradication of Mycobacterium tuberculosis (MTB) infection requires daily administration of combinations of rifampin (RIF), isoniazid [isonicotinylhydrazine (INH)], pyrazinamide, and ethambutol, among other drug therapies. To facilitate and optimize MTB therapeutic selections, a mononuclear phagocyte (MP; monocyte, macrophage, and dendritic cell)-targeted drug delivery strategy was developed. Long-acting nanoformulations of RIF and an INH derivative, pentenyl-INH (INHP), were prepared, and their physicochemical properties were evaluated. This included the evaluation of MP particle uptake and retention, cell viability, and antimicrobial efficacy. Drug levels reached 6 µg/10(6) cells in human monocyte-derived macrophages (MDMs) for nanoparticle treatments compared with 0.1 µg/10(6) cells for native drugs. High RIF and INHP levels were retained in MDM for >15 d following nanoparticle loading. Rapid loss of native drugs was observed in cells and culture fluids within 24 h. Antimicrobial activities were determined against Mycobacterium smegmatis (M. smegmatis). Coadministration of nanoformulated RIF and INHP provided a 6-fold increase in therapeutic efficacy compared with equivalent concentrations of native drugs. Notably, nanoformulated RIF and INHP were found to be localized in recycling and late MDM endosomal compartments. These were the same compartments that contained the pathogen. Our results demonstrate the potential of antimicrobial nanomedicines to simplify MTB drug regimens.
Assuntos
Antituberculosos/farmacologia , Endossomos/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Nanopartículas , Células Cultivadas , Endossomos/metabolismo , Humanos , Macrófagos/metabolismo , Frações Subcelulares/metabolismoRESUMO
Human immunodeficiency virus-1 (HIV-1) enters the brain early during infection and leads to severe neuronal damage and central nervous system impairment. HIV-1 envelope glycoprotein 120 (gp120), a neurotoxin, undergoes intracellular trafficking and transport across neurons; however mechanisms of gp120 trafficking in neurons are unclear. Our results show that mannose binding lectin (MBL) that binds to the N-linked mannose residues on gp120, participates in intravesicular packaging of gp120 in neuronal subcellular organelles and also in subcellular trafficking of these vesicles in neuronal cells. Perinuclear MBL:gp120 vesicular complexes were observed and MBL facilitated the subcellular trafficking of gp120 via the endoplasmic reticulum (ER) and Golgi vesicles. The functional carbohydrate recognition domain of MBL was required for perinuclear organization, distribution and subcellular trafficking of MBL:gp120 vesicular complexes. Nocodazole, an agent that depolymerizes the microtubule network, abolished the trafficking of MBL:gp120 vesicles, suggesting that these vesicular complexes were transported along the microtubule network. Live cell imaging confirmed the association of the MBL:gp120 complexes with dynamic subcellular vesicles that underwent trafficking in neuronal soma and along the neurites. Thus, our findings suggest that intracellular MBL mediates subcellular trafficking and transport of viral glycoproteins in a microtubule-dependent mechanism in the neurons.
Assuntos
Proteína gp120 do Envelope de HIV/metabolismo , Lectina de Ligação a Manose/metabolismo , Neurônios/metabolismo , Organelas/metabolismo , Western Blotting , Linhagem Celular Tumoral , Células Cultivadas , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Complexo de Golgi/efeitos dos fármacos , Complexo de Golgi/metabolismo , Proteína gp120 do Envelope de HIV/genética , HIV-1 , Humanos , Imunoprecipitação , Lectina de Ligação a Manose/genética , Microscopia Confocal , Microtúbulos/efeitos dos fármacos , Microtúbulos/metabolismo , Neuritos/efeitos dos fármacos , Neuritos/metabolismo , Neurônios/efeitos dos fármacos , Nocodazol/farmacologia , Organelas/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Vesículas Transportadoras/efeitos dos fármacos , Vesículas Transportadoras/metabolismo , Moduladores de Tubulina/farmacologiaRESUMO
Classically associated with gap junction-mediated intercellular communication, connexin43 (Cx43) is increasingly recognized to possess non-canonical biological functions, including gene expression regulation. However, the mechanisms governing the localization and role played by Cx43 in the nucleus, namely in transcription modulation, remain unknown. Using comprehensive and complementary approaches encompassing biochemical assays, super-resolution and immunogold transmission electron microscopy, we demonstrate that Cx43 localizes to the nuclear envelope of different cell types and in cardiac tissue. We show that translocation of Cx43 to the nucleus relies on Importin-ß, and that Cx43 significantly impacts the cellular transcriptome, likely by interacting with transcriptional regulators. In vitro patch-clamp recordings from HEK293 and adult primary cardiomyocytes demonstrate that Cx43 forms active channels at the nuclear envelope, providing evidence that Cx43 can participate in nucleocytoplasmic shuttling of small molecules. The accumulation of nuclear Cx43 during myogenic differentiation of cardiomyoblasts is suggested to modulate expression of genes implicated in this process. Altogether, our study provides new evidence for further defining the biological roles of nuclear Cx43, namely in cardiac pathophysiology.
Assuntos
Conexina 43 , Membrana Nuclear , Humanos , Comunicação Celular , Conexina 43/genética , Conexina 43/metabolismo , Expressão Gênica , Células HEK293 , Miócitos Cardíacos/metabolismo , Membrana Nuclear/metabolismoRESUMO
Hepatitis B virus (HBV) capsid assembly is traditionally thought to occur predominantly in the cytoplasm, where the virus gains access to the virion egress pathway. To better define sites of HBV capsid assembly, we carried out single cell imaging of HBV Core protein (Cp) subcellular trafficking over time under conditions supporting genome packaging and reverse transcription in Huh7 hepatocellular carcinoma cells. Time-course analyses including live cell imaging of fluorescently tagged Cp derivatives showed Cp to accumulate in the nucleus at early time points (~24 h), followed by a marked re-distribution to the cytoplasm at 48 to 72 h. Nucleus-associated Cp was confirmed to be capsid and/or high-order assemblages using a novel dual label immunofluorescence strategy. Nuclear-to-cytoplasmic re-localization of Cp occurred predominantly during nuclear envelope breakdown in conjunction with cell division, followed by strong cytoplasmic retention of Cp. Blocking cell division resulted in strong nuclear entrapment of high-order assemblages. A Cp mutant, Cp-V124W, predicted to exhibit enhanced assembly kinetics, also first trafficked to the nucleus to accumulate at nucleoli, consistent with the hypothesis that Cp's transit to the nucleus is a strong and constitutive process. Taken together, these results provide support for the nucleus as an early-stage site of HBV capsid assembly, and provide the first dynamic evidence of cytoplasmic retention after cell division as a mechanism underpinning capsid nucleus-to-cytoplasm relocalization. IMPORTANCE Hepatitis B virus (HBV) is an enveloped, reverse-transcribing DNA virus that is a major cause of liver disease and hepatocellular carcinoma. Subcellular trafficking events underpinning HBV capsid assembly and virion egress remain poorly characterized. Here, we developed a combination of fixed and long-term (>24 h) live cell imaging technologies to study the single cell trafficking dynamics of the HBV Core Protein (Cp). We demonstrate that Cp first accumulates in the nucleus, and forms high-order structures consistent with capsids, with the predominant route of nuclear egress being relocalization to the cytoplasm during cell division in conjunction with nuclear membrane breakdown. Single cell video microscopy demonstrated unequivocally that Cp's localization to the nucleus is constitutive. This study represents a pioneering application of live cell imaging to study HBV subcellular transport, and demonstrates links between HBV Cp and the cell cycle.