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1.
Cells ; 9(5)2020 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-32354068

RESUMO

In the endomembrane system of mammalian cells, membrane traffic processes require a high degree of regulation in order to ensure their specificity. The range of molecules that participate in trafficking events is truly vast, and much attention to date has been given to the Rab family of small GTPases. However, in recent years, a role in membrane traffic for members of the Rho GTPase family, in particular Cdc42, has emerged. This prompted us to develop and apply an image-based high-content screen, initially focussing on the Golgi complex, using RNA interference to systematically perturb each of the 21 Rho family members and assess their importance to the overall organisation of this organelle. Analysis of our data revealed previously unreported roles for two atypical Rho family members, RhoBTB1 and RhoBTB3, in membrane traffic events. We find that depletion of RhoBTB3 affects the morphology of the Golgi complex and causes changes in the trafficking speeds of carriers operating at the interface of the Golgi and endoplasmic reticulum. In addition, RhoBTB3 was found to be present on these carriers. Depletion of RhoBTB1 was also found to cause a disturbance to the Golgi architecture, however, this phenotype seems to be linked to endocytosis and retrograde traffic pathways. RhoBTB1 was found to be associated with early endosomal intermediates, and changes in the levels of RhoBTB1 not only caused profound changes to the organisation and distribution of endosomes and lysosomes, but also resulted in defects in the delivery of two different classes of cargo molecules to downstream compartments. Together, our data reveal new roles for these atypical Rho family members in the endomembrane system.

2.
Int J Mol Sci ; 21(6)2020 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-32244909

RESUMO

One remarkable characteristic of eukaryotic cells is the complexity of their membrane systems [...].

3.
Bioinformatics ; 2020 Mar 06.
Artigo em Inglês | MEDLINE | ID: mdl-32142105

RESUMO

MOTIVATION: The subcellular location of a protein can provide useful information for protein function prediction and drug design. Experimentally determining the subcellular location of a protein is an expensive and time-consuming task. Therefore, various computer-based tools have been developed, mostly using machine learning algorithms, to predict the subcellular location of proteins. RESULTS: Here, we present a neural network based algorithm for protein subcellular location prediction. We introduce SCLpred-EMS a subcellular localization predictor powered by an ensemble of Deep N-to-1 Convolutional Neural Networks. SCLpred-EMS predicts the subcellular location of a protein into two classes, the endomembrane system and secretory pathway versus all others, with an MCC of 0.75-0.86 outperforming the other state-of-the-art web servers we tested. AVAILABILITY: SCLpred-EMS is freely available for academic users at http://distilldeep.ucd.ie/SCLpred2/. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

4.
Mol Ther ; 28(4): 1190-1199, 2020 Apr 08.
Artigo em Inglês | MEDLINE | ID: mdl-32059764

RESUMO

MicroRNAs that are overexpressed in cystic fibrosis (CF) bronchial epithelial cells (BEC) negatively regulate CFTR and nullify the beneficial effects of CFTR modulators. We hypothesized that it is possible to reverse microRNA-mediated inhibition of CFTR using CFTR-specific target site blockers (TSBs) and to develop a drug-device combination inhalation therapy for CF. Lead microRNA expression was quantified in a series of human CF and non-CF samples and in vitro models. A panel of CFTR 3' untranslated region (UTR)-specific locked nucleic acid antisense oligonucleotide TSBs was assessed for their ability to increase CFTR expression. Their effects on CFTR activity alone or in combination with CFTR modulators were measured in CF BEC models. TSB encapsulation in poly-lactic-co-glycolic acid (PLGA) nanoparticles was assessed as a proof of principle of delivery into CF BECs. TSBs targeting the CFTR 3' UTR 298-305:miR-145-5p or 166-173:miR-223-3p sites increased CFTR expression and anion channel activity and enhanced the effects of ivacaftor/lumacaftor or ivacaftor/tezacaftor in CF BECs. Biocompatible PLGA-TSB nanoparticles promoted CFTR expression in primary BECs and retained desirable biophysical characteristics following nebulization. Alone or in combination with CFTR modulators, aerosolized CFTR-targeting TSBs encapsulated in PLGA nanoparticles could represent a promising drug-device combination therapy for the treatment for CFTR dysfunction in the lung.

5.
Sci Rep ; 9(1): 18216, 2019 Dec 03.
Artigo em Inglês | MEDLINE | ID: mdl-31796849

RESUMO

The World Health Organization has listed C. jejuni as one of 12 microorganisms on a global priority list for antibiotic resistance due to a rapid increase in strains resistant to fluoroquinolone antibiotics. This fluoroquinolone resistance is conferred through a single point mutation in the QRDR region within the gyrA gene known to be involved in DNA supercoiling. We have previously revealed that changes in DNA supercoilikng play a major role in the regulation of virulence in C. jejuni with relaxation of DNA supercoiling associated with increased attachment to and invasion of human epithelial cells. The aim of this study was to investigate whether fluoroquinolone resistant strains of C. jejuni displayed altered supercoiling associated phenotypes. A panel of fluoroquinolone resistant mutants were derived and shown to have a greater ability to form viable biofilms under aerobic conditions, invade epithelial cells and promote virulence in the Galleria mellonella model of infection. We thus report for the first time that fluoroquinolone resistance in C. jejuni is associated with an increase in virulence and the ability to form viable biofilms in oxygen rich environments. These altered phenotypes likely play a critical role in the continued increase in fluoroquinolone resistance observed for this important pathogen.

6.
Front Neurosci ; 13: 1051, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31680803

RESUMO

The hereditary spastic paraplegias (HSPs) are a group of inherited neurodegenerative conditions whose characteristic feature is degeneration of the longest axons within the corticospinal tract which leads to progressive spasticity and weakness of the lower limbs. Though highly genetically heterogeneous, the majority of HSP cases are caused by mutations in genes encoding proteins that are responsible for generating and organizing the tubular endoplasmic reticulum (ER). Despite this, the role of the ER within neurons, particularly the long axons affected in HSP, is not well understood. Throughout axons, ER tubules make extensive contacts with other organelles, the cytoskeleton and the plasma membrane. At these ER contacts, protein complexes work in concert to perform specialized functions including organelle shaping, calcium homeostasis and lipid biogenesis, all of which are vital for neuronal survival and may be disrupted by HSP-causing mutations. In this article we summarize the proteins which mediate ER contacts, review the functions these contacts are known to carry out within neurons, and discuss the potential contribution of disruption of ER contacts to axonopathy in HSP.

7.
Eur J Pharm Biopharm ; 144: 50-56, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31419585

RESUMO

Extracellular vesicles (EVs) are small lipid-enclosed particles that can carry various types of cargo, including proteins, nucleic acids and metabolites. They are known to be released by all cell types and can be taken up by other cells, leading to the transfer of the cargo they carry. As such, they represent an important type of intercellular signalling and a natural mechanism for transferring macromolecules between cells. This ability to transfer cargo could be harnessed to deliver therapeutic molecules. Indeed, a growing body of work has described the attempt by the field to utilise EVs to deliver a range of therapeutics including RNAi, CRISPR/Cas9 and chemotherapeutics, to a specific target tissue. However, there are numerous barriers associated with the use of EVs as therapeutic vehicles, including the challenge of efficiently loading therapeutics into EVs, avoiding clearance of the EVs from circulation, targeting the correct tissue type and the inefficiency of internalisation and functional delivery of the cargo. Despite these difficulties, EVs represent a tremendous therapeutic opportunity, both for the delivery of exogenous cargo, as well as the therapeutic benefit of targeting aberrant EV signalling or treating patients with natural EVs, such as those released by mesenchymal stem cells. This review describes current knowledge on the therapeutic potential of EVs and the challenges faced by the field. Many of these challenges are due to a lack of complete understanding of EV function, but further research in this area should continue to yield new solutions that will lead to the use of EVs in the clinic.


Assuntos
Sistemas de Liberação de Medicamentos/métodos , Vesículas Extracelulares/metabolismo , Animais , Sistemas CRISPR-Cas/efeitos dos fármacos , Humanos , Células-Tronco Mesenquimais/efeitos dos fármacos , Ácidos Nucleicos/metabolismo , Proteínas/metabolismo , Interferência de RNA/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos
8.
Int J Mol Sci ; 20(16)2019 Aug 12.
Artigo em Inglês | MEDLINE | ID: mdl-31408960

RESUMO

Autophagy (particularly macroautophagy) is a bulk degradation process used by eukaryotic cells in order to maintain adequate energy levels and cellular homeostasis through the delivery of long-lived proteins and organelles to the lysosome, resulting in their degradation. It is becoming increasingly clear that many of the molecular requirements to fulfil autophagy intersect with those of conventional and unconventional membrane trafficking pathways. Of particular interest is the dependence of these processes on multiple members of the Rab family of small GTP binding proteins. Rab33b is a protein that localises to the Golgi apparatus and has suggested functions in both membrane trafficking and autophagic processes. Interestingly, mutations in the RAB33B gene have been reported to cause the severe skeletal disorder, Smith-McCort Dysplasia; however, the molecular basis for Rab33b in this disorder remains to be determined. In this review, we focus on the current knowledge of the participation of Rab33b and its interacting partners in membrane trafficking and macroautophagy, and speculate on how its function, and dysfunction, may contribute to human disease.


Assuntos
Autofagia , Mapas de Interação de Proteínas , Proteínas rab de Ligação ao GTP/metabolismo , Animais , Transporte Biológico , Retículo Endoplasmático/metabolismo , Endossomos/metabolismo , Complexo de Golgi/metabolismo , Humanos , Lisossomos/metabolismo , Osteocondrodisplasias/metabolismo
9.
EMBO Rep ; 20(10): e47625, 2019 10 04.
Artigo em Inglês | MEDLINE | ID: mdl-31432619

RESUMO

Rab and Arl guanine nucleotide-binding (G) proteins regulate trafficking pathways essential for the formation, function and composition of primary cilia, which are sensory devices associated with Sonic hedgehog (Shh) signalling and ciliopathies. Here, using mammalian cells and zebrafish, we uncover ciliary functions for Rab35, a multitasking G protein with endocytic recycling, actin remodelling and cytokinesis roles. Rab35 loss via siRNAs, morpholinos or knockout reduces cilium length in mammalian cells and the zebrafish left-right organiser (Kupffer's vesicle) and causes motile cilia-associated left-right asymmetry defects. Consistent with these observations, GFP-Rab35 localises to cilia, as do GEF (DENND1B) and GAP (TBC1D10A) Rab35 regulators, which also regulate ciliary length and Rab35 ciliary localisation. Mammalian Rab35 also controls the ciliary membrane levels of Shh signalling regulators, promoting ciliary targeting of Smoothened, limiting ciliary accumulation of Arl13b and the inositol polyphosphate 5-phosphatase (INPP5E). Rab35 additionally regulates ciliary PI(4,5)P2 levels and interacts with Arl13b. Together, our findings demonstrate roles for Rab35 in regulating cilium length, function and membrane composition and implicate Rab35 in pathways controlling the ciliary levels of Shh signal regulators.

10.
Small ; 15(37): e1902033, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31334922

RESUMO

There is a high demand for advanced, image-based, automated high-content screening (HCS) approaches to facilitate phenotypic screening in 3D cell culture models. A major challenge lies in retaining the resolution of fine cellular detail but at the same time imaging multicellular structures at a large scale. In this study, a confocal microscopy-based HCS platform in optical multiwell plates that enables the quantitative morphological profiling of populations of nonuniform spheroids obtained from HT-29 human colorectal cancer cells is described. This platform is then utilized to demonstrate a quantitative dissection of the penetration of synthetic nanoparticles (NP) in multicellular 3D spheroids at multiple levels of scale. A pilot RNA interference-based screening validates this methodology and identifies a subset of RAB GTPases that regulate NP trafficking in these spheroids. This technology is suitable for high-content phenotyping in 3D cell-based screening, providing a framework for nanomedicine drug development as applied to translational oncology.

11.
PLoS One ; 14(1): e0210337, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30689642

RESUMO

Therapeutic modulation of protein interactions is challenging, but short linear motifs (SLiMs) represent potential targets. Focal adhesions play a central role in adhesion by linking cells to the extracellular matrix. Integrins are central to this process, and many other intracellular proteins are components of the integrin adhesome. We applied a peptide network targeting approach to explore the intracellular modulation of integrin function in platelets. Firstly, we computed a platelet-relevant integrin adhesome, inferred via homology of known platelet proteins to adhesome components. We then computationally selected peptides from the set of platelet integrin adhesome cytoplasmic and membrane adjacent protein-protein interfaces. Motifs of interest in the intracellular component of the platelet integrin adhesome were identified using a predictor of SLiMs based on analysis of protein primary amino acid sequences (SLiMPred), a predictor of strongly conserved motifs within disordered protein regions (SLiMPrints), and information from the literature regarding protein interactions in the complex. We then synthesized peptides incorporating these motifs combined with cell penetrating factors (tat peptide and palmitylation for cytoplasmic and membrane proteins respectively). We tested for the platelet activating effects of the peptides, as well as their abilities to inhibit activation. Bioactivity testing revealed a number of peptides that modulated platelet function, including those derived from α-actinin (ACTN1) and syndecan (SDC4), binding to vinculin and syntenin respectively. Both chimeric peptide experiments and peptide combination experiments failed to identify strong effects, perhaps characterizing the adhesome as relatively robust against within-adhesome synergistic perturbation. We investigated in more detail peptides targeting vinculin. Combined experimental and computational evidence suggested a model in which the positively charged tat-derived cell penetrating part of the peptide contributes to bioactivity via stabilizing charge interactions with a region of the ACTN1 negatively charged surface. We conclude that some interactions in the integrin adhesome appear to be capable of modulation by short peptides, and may aid in the identification and characterization of target sites within the complex that may be useful for therapeutic modulation.


Assuntos
Adesões Focais/química , Adesões Focais/fisiologia , Integrinas/química , Integrinas/fisiologia , Peptídeos/química , Peptídeos/farmacologia , Motivos de Aminoácidos , Sequência de Aminoácidos , Plaquetas/efeitos dos fármacos , Plaquetas/fisiologia , Adesões Focais/efeitos dos fármacos , Células HeLa , Humanos , Integrinas/genética , Modelos Moleculares , Peptídeos/genética , Ativação Plaquetária/efeitos dos fármacos , Ativação Plaquetária/fisiologia , Adesividade Plaquetária/efeitos dos fármacos , Adesividade Plaquetária/fisiologia , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Mapas de Interação de Proteínas , Vinculina/metabolismo
12.
Small ; 15(2): e1803758, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30565853

RESUMO

Nanoparticles (NPs) are now used in numerous technologies and serve as carriers for several new classes of therapeutics. Studies of the distribution of NPs in vivo demonstrate that they can be transported through biological barriers and are concentrated in specific tissues. Here, transport behavior, and final destination of polystyrene NPs are reported in primary mouse cortical neurons and SH-SY5Y cells, cultured in two-compartmental microfluidic devices. In both cell types, negative polystyrene NPs (PS(-)) smaller than 100 nm are taken up by the axons, undergo axonal retrograde transport, and accumulate in the somata. Examination of NP transport reveals different transport mechanisms depending on the cell type, particle charge, and particle internalization by the lysosomes. In cortical neurons, PS(-) inside lysosomes and 40 nm positive polystyrene NPs undergo slow axonal transport, whereas PS(-) outside lysosomes undergo fast axonal transport. Inhibition of dynein in cortical neurons decreases the transport velocity and cause a dose-dependent reduction in the number of accumulated PS(-), suggesting that the fast axonal transport is dynein mediated. These results show that the axonal retrograde transport of NPs depends on the endosomal pathway taken and establishes a means for screening nanoparticle-based therapeutics for diseases that involve neurons.

13.
Mol Pharm ; 15(5): 1878-1891, 2018 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-29590755

RESUMO

The field of tissue engineering is increasingly recognizing that gene therapy can be employed for modulating in vivo cellular response thereby guiding tissue regeneration. However, the field lacks a versatile and biocompatible gene delivery platform capable of efficiently delivering transgenes to mesenchymal stem cells (MSCs), a cell type often refractory to transfection. Herein, we describe the extensive and systematic exploration of three architectural variations of star-shaped poly(l-lysine) polypeptide (star-PLL) with varying number and length of poly(l-lysine) arms as potential nonviral gene delivery vectors for MSCs. We demonstrate that star-PLL vectors are capable of self-assembling with pDNA to form stable, cationic nanomedicines. Utilizing high content screening, live cell imaging, and mechanistic uptake studies we confirm the intracellular delivery of pDNA by star-PLLs to MSCs is a rapid process, which likely proceeds via a clathrin-independent mechanism. We identify a star-PLL composition with 64 poly(l-lysine) arms and five l-lysine subunits per arm as a particularly efficient vector that is capable of delivering both reporter genes and the therapeutic transgenes bone morphogenetic protein-2 and vascular endothelial growth factor to MSCs. This composition facilitated a 1000-fold increase in transgene expression in MSCs compared to its linear analogue, linear poly(l-lysine). Furthermore, it demonstrated comparable transgene expression to the widely used vector polyethylenimine using a lower pDNA dose with significantly less cytotoxicity. Overall, this study illustrates the ability of the star-PLL vectors to facilitate efficient, nontoxic nucleic acid delivery to MSCs thereby functioning as an innovative nanomedicine platform for tissue engineering applications.


Assuntos
DNA/administração & dosagem , DNA/química , Portadores de Fármacos/química , Células-Tronco Mesenquimais/efeitos dos fármacos , Nanopartículas/química , Peptídeos/química , Polilisina/química , Animais , Proteína Morfogenética Óssea 2/genética , Células Cultivadas , Clatrina/genética , Sistemas de Liberação de Medicamentos/métodos , Técnicas de Transferência de Genes , Genes Reporter/genética , Terapia Genética/métodos , Vetores Genéticos/genética , Ácidos Nucleicos/genética , Polietilenoimina/química , Polímeros/química , Ratos , Engenharia Tecidual/métodos , Transfecção/métodos , Transgenes/genética , Fator A de Crescimento do Endotélio Vascular/genética
14.
Handb Exp Pharmacol ; 245: 191-225, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29071510

RESUMO

Rare bone disorders are a heterogeneous group of diseases, initially associated with mutations in type I procollagen (PC) genes. Recent developments from dissection at the molecular and cellular level have expanded the list of disease-causing proteins, revealing that disruption of the machinery that handles protein secretion can lead to failure in PC secretion and in several cases result in skeletal dysplasia. In parallel, cell-based in vitro studies of PC trafficking pathways offer clues to the identification of new disease candidate genes. Together, this raises the prospect of heritable bone disorders as a paradigm for biosynthetic protein traffic-related diseases, and an avenue through which therapeutic strategies can be explored.Here, we focus on human syndromes linked to defects in type I PC secretion with respect to the landscape of biosynthetic and protein transport steps within the early secretory pathway. We provide a perspective on possible therapeutic interventions for associated heritable craniofacial and skeletal disorders, considering different orders of complexity, from the cellular level by manipulation of proteostasis pathways to higher levels involving cell-based therapies for bone repair and regeneration.


Assuntos
Colágeno Tipo I/genética , Diástase Óssea/genética , Retículo Endoplasmático/metabolismo , Animais , Regeneração Óssea , Colágeno Tipo I/metabolismo , Diástase Óssea/tratamento farmacológico , Diástase Óssea/etiologia , Humanos , Transporte Proteico , Proteostase
15.
Nanoscale ; 9(41): 15911-15922, 2017 Oct 26.
Artigo em Inglês | MEDLINE | ID: mdl-29019498

RESUMO

Candida albicans is the lead fungal pathogen of nosocomial bloodstream infections worldwide and has mortality rates of 43%. Nanoparticles have been identified as a means to improve medical outcomes for Candida infections, enabling sample concentration, serving as contrast agents for in vivo imaging, and delivering therapeutics. However, little is known about how nanoparticles interact with the fungal cell wall. In this report we used laser scanning confocal microscopy to examine the interaction of fluorescent polystyrene nanoparticles of specific surface chemistry and diameter with C. albicans and mutant strains deficient in various C. albicans surface proteins. Carboxylate-functionalized nanoparticles adsorbed mainly to the hyphae of wild-type C. albicans. The dissociative binding constant of the nanoparticles was ∼150, ∼30 and ∼2.5 pM for 40, 100 nm and 200 nm diameter particles, respectively. A significant reduction in particle binding was observed with a Δals3 strain compared to wild-type strains, identifying the Als3 adhesin as the main mediator of this nanoparticle adhesion. In the absence of Als3, nanoparticles bound to germ tubes and yeast cells in a pattern resembling the localization of Als1, indicating Als1 also plays a role. Nanoparticle surface charge was shown to influence binding - positively charged amine-functionalized nanoparticles failed to bind to the hyphal cell wall. Binding of carboxylate-functionalized nanoparticles was observed in the presence of serum, though interactions were reduced. These observations show that Als3 and Als1 are important targets for nanoparticle-mediated diagnostics and therapeutics, and provide direction for optimal diameter and surface characteristics of nanoparticles that bind to the fungal cell wall.


Assuntos
Candida albicans/efeitos dos fármacos , Ácidos Carboxílicos/farmacologia , Parede Celular/efeitos dos fármacos , Nanopartículas , Hifas
16.
J Genet Genomics ; 44(10): 493-501, 2017 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-29037990

RESUMO

The VCP-Ufd1-Npl4 complex regulates proteasomal processing within cells by delivering ubiquitinated proteins to the proteasome for degradation. Mutations in VCP are associated with two neurodegenerative diseases, amyotrophic lateral sclerosis (ALS) and inclusion body myopathy with Paget's disease of the bone and frontotemporal dementia (IBMPFD), and extensive study has revealed crucial functions of VCP within neurons. By contrast, little is known about the functions of Npl4 or Ufd1 in vivo. Using neuronal-specific knockdown of Npl4 or Ufd1 in Drosophila melanogaster, we infer that Npl4 contributes to microtubule organization within developing motor neurons. Moreover, Npl4 RNAi flies present with neurodegenerative phenotypes including progressive locomotor deficits, reduced lifespan and increased accumulation of TAR DNA-binding protein-43 homolog (TBPH). Knockdown, but not overexpression, of TBPH also exacerbates Npl4 RNAi-associated adult-onset neurodegenerative phenotypes. In contrast, we find that neuronal knockdown of Ufd1 has little effect on neuromuscular junction (NMJ) organization, TBPH accumulation or adult behaviour. These findings suggest the differing neuronal functions of Npl4 and Ufd1 in vivo.


Assuntos
Proteínas de Transporte/fisiologia , Proteínas de Drosophila/fisiologia , Drosophila melanogaster/fisiologia , Neurônios/fisiologia , Proteína com Valosina/metabolismo , Animais , Proteínas de Transporte/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/citologia , Técnicas de Silenciamento de Genes , Neurônios/metabolismo
17.
Tissue Cell ; 49(2 Pt A): 163-169, 2017 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-27720426

RESUMO

The Golgi complex is the central unit of the secretory pathway, modifying, processing and sorting proteins and lipids to their correct cellular localisation. Changes to proteins at the Golgi complex can have deleterious effects on the function of this organelle, impeding trafficking routes through it, potentially resulting in disease. It is emerging that several Rho GTPase proteins, namely Cdc42, RhoBTB3, RhoA and RhoD are at least in part localised to the Golgi complex, and a number of studies have shown that dysregulation of their levels or activity can be associated with cellular changes which ultimately drive cancer progression. In this mini-review we highlight some of the recent work that explores links between form and function of the Golgi complex, Rho GTPases and cancer.


Assuntos
Complexo de Golgi/genética , Neoplasias/genética , Proteínas rho de Ligação ao GTP/genética , Complexo de Golgi/metabolismo , Complexo de Golgi/patologia , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Neoplasias/metabolismo , Neoplasias/patologia , Transporte Proteico/genética , Proteína cdc42 de Ligação ao GTP/genética , Proteínas rho de Ligação ao GTP/metabolismo , Proteína rhoA de Ligação ao GTP/genética
18.
Proteomics ; 17(1-2)2017 01.
Artigo em Inglês | MEDLINE | ID: mdl-27957805

RESUMO

Phylogeny is often used to compare entire families of genes/proteins. We previously showed that classification of Caenorhabditis elegans Rho GTPases on the basis of their enzymatic properties was significantly different from sequence alignments. To further develop this concept, we have developed an integrated approach to classify C. elegans small GTPases based on functional data comprising affinity for GTP, sub-cellular localization, tissue distribution and silencing impact. This analysis led to establish a novel functional classification for small GTPases. To test the relevance of this classification in mammals, we focused our attention on the human orthologs of small GTPases from a specific group comprising arf-1.2, evl-20, arl-1, Y54E10BR.2, unc-108 and rab-7. We then tested their involvement in protein secretion and membrane traffic in mammalian systems. Using this approach we identify a novel network containing 18 GTPases, and 23 functionally interacting proteins, conserved between C. elegans and mammals, which is involved in membrane traffic and protein secretion.


Assuntos
Membrana Celular/metabolismo , Transporte Proteico/fisiologia , Proteínas ras/metabolismo , Animais , Caenorhabditis elegans/metabolismo , Humanos , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Transporte Proteico/genética , Proteômica/métodos
19.
Histochem Cell Biol ; 147(4): 439-451, 2017 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-27999994

RESUMO

In this study, we carry out a systematic characterisation of the YIPF family of proteins with respect to their subcellular localisation profile, membrane topology and functional effects on the endomembrane system. YIPF proteins primarily localise to the Golgi complex and can be grouped into trans-Golgi-localising YIPFs (YIPF1 and YIPF2) and cis-Golgi-localising YIPFs (YIPF3, YIPF4 and YIPF5), with YIPF6 and YIPF7 showing a broader profile being distributed throughout the Golgi stack. YIPF proteins have a long soluble N-terminal region, which is orientated towards the cytosol, followed by 5 closely stacked transmembrane domains, and a C terminus, orientated towards the lumen of the Golgi. The significance of YIPF proteins for the maintenance of the morphology of the Golgi was tested by RNA interference, revealing a number of specific morphological changes to this organelle on their depletion. We propose a role for this family of proteins in regulating membrane dynamics in the endomembrane system.


Assuntos
Proteínas de Membrana/metabolismo , Complexo de Golgi/metabolismo , Células HeLa , Humanos , Células Tumorais Cultivadas
20.
Methods Mol Biol ; 1470: 39-48, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27581283

RESUMO

RNA interference (RNAi) has become an essential tool for molecular and cellular biologists to dissect cell function. In recent years its application has been extended to genome-wide studies, enabling the systematic identification of new cell regulation mechanisms and drug targets. In this chapter, a protocol for a genome-wide RNAi screen coupled to high-content microscopy is presented. Specifically we describe key features of assay design, plate layout, and a protocol for forward transfection of small interfering RNAs (siRNAs) in a 384-well plate format. As an example of its application in identifying modulators of membrane trafficking, we also provide a protocol to measure the efficacy of intracellular delivery of the B subunit of Shiga-like toxin to the Golgi complex. Finally we show an automated image analysis routine that can be used to extract single cell data from the screen, thereby providing a quantitative ranking of how a large panel of siRNAs affects this biological process.


Assuntos
Membrana Celular/metabolismo , Biologia Molecular/métodos , RNA Interferente Pequeno , Transfecção/métodos , Animais , Membrana Celular/genética , Células Cultivadas , Imunofluorescência , Genoma , Processamento de Imagem Assistida por Computador , Mamíferos , Microscopia de Fluorescência/instrumentação , Microscopia de Fluorescência/métodos , Biologia Molecular/instrumentação , Interferência de RNA , RNA Interferente Pequeno/genética , Toxina Shiga II/metabolismo
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