RESUMO
"I don't know the question, but sex is definitely the answer!," was a Woody Allen quote cited by Fuller and Insel in an Editorial Comment in 2013 on the importance of cell sex in submissions to AJP-Cell Physiology, and in biomedical research in general. The notion that cell sex is important is axiomatic in studies on prostate cancer (LnCAP) or placental physiology (BeWo). Indeed, most researchers are aware that HeLa cells are female cervical derived, and CHO are female hamster ovary cells, yet beyond those well-known examples, it would be fair to assume that the sex of cells derived from kidney, lung, or liver, for example, is given cursory, if any thought. In the end, what possible impact could the presence or absence of a Y chromosome have on protein trafficking in a nonreproductive tissue, such as a pancreatic ß cell? However, this approach to cell, and indeed organismal physiology, seems to be in conflict with accumulating data, that show that far from being irrelevant, genes expressed off sex chromosomes have a broad-ranging impact on cells as diverse as neurons and renal cells. Moreover, it is also the policy of AJP-Cell Physiology that the source of all cells used (species, sex, etc.) should be clearly indicated when submitting an article for publication (https://journals.physiology.org/author-info.manuscript-composition). In 2013, we wrote a review examining how faithfully such requirements were adhered to in submissions to Cell Physiology. Nearly a decade later, it seems fitting to revisit the topic and ask if any improvements have been made in the description of cells and cell lines used in publications submitted to AJP-Cell Physiology.
Assuntos
Rim , Placenta , Gravidez , Masculino , Humanos , Feminino , Células HeLa , Pulmão , Fenômenos Fisiológicos Celulares/fisiologiaRESUMO
Why are my students not paying attention in class? Why are they not engaged? What am I doing wrong? These are questions that even seasoned faculty pose in moments of self-reflection. Although it is good practice for educators to continually evaluate their presentations for areas of improvement, it is unfortunately also true that questions of why students lack attention or engagement in class are often raised by school administrators as subtle implications of failure on the part of faculty. But why is it so hard for our students to pay attention? Why is it that students' focus can so easily ebb away during a class? Why are my students seemingly indifferent to the presented material? To address these issues, and to provide solutions that can be incorporated easily into class preparation and presentation, it is important to define precisely what we mean by attention and engagement. Are they the same thing, and if not, how do they differ? To what extent is the student responsible for acquiring and applying the material presented? Ultimately, of course, the student is accountable for learning the material and passing exams, but there are practices that we as educators often employ that erect hurdles and barriers to student learning, that can make the process significantly harder than it needs to be.NEW & NOTEWORTHY Although many articles and books exist describing various classroom strategies to increase student engagement, often advice to newer faculty is given as prescriptive ways to organize a class or to repeat what was given by the last person who presented the material. Yet irrespective of the structure of the class or learning environment, there are subtle hurdles that many faculty erect that hinder a student's progress. This Personal View discusses some of the potential barriers educators put up that deter effective student learning and, importantly, offers advice on how those barriers can be dismantled.
Assuntos
Aprendizagem , Estudantes , Humanos , DocentesRESUMO
Once thought to be exclusively an absorptive tissue, the intestine is now recognized as an important secretory tissue, playing a key role in body ion and fluid homeostasis. Given the intestine's role in fluid homeostasis, it is not surprising that important clinical pathologies arise from imbalances in fluid absorption and secretion. Perhaps the most important examples of this can be seen in enterotoxigenic secretory diarrheas with extreme fluid secretion, and Cystic Fibrosis with little or no fluid secretion. A mechanistic understanding of the cellular pathways regulating ion and fluid transport has been obtained from a variety of approaches and model systems. These have ranged from the intact intestine to a single intestinal epithelial cell type. Although for many years a reductionist approach has held sway for investigating intestinal transport, the growing realization that physiologic processes should really be examined within a physiological context has seen a marked increase in studies using models that are essentially mini-intestines in a dish. The aim of this chapter is to provide a historical context for our understanding of intestinal ion and fluid transport, and to highlight the model systems that have been used to acquire this knowledge.
Assuntos
Eletrólitos , Animais , Transporte Biológico/fisiologia , Fibrose Cística/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Homeostase/fisiologia , Humanos , IntestinosRESUMO
The eukaryotic cell is organized into membrane-delineated compartments that are characterized by specific cadres of proteins sustaining biochemically distinct cellular processes. The appropriate subcellular localization of proteins is key to proper organelle function and provides a physiological context for cellular processes. Disruption of normal trafficking pathways for proteins is seen in several genetic diseases, where a protein's absence for a specific subcellular compartment leads to organelle disruption, and in the context of an individual, a disruption of normal physiology. Importantly, several drug therapies can also alter protein trafficking, causing unwanted side effects. Thus, a deeper understanding of trafficking pathways needs to be appreciated as novel therapeutic modalities are proposed. Despite the promising efficacy of novel therapeutic agents, the intracellular bioavailability of these compounds has proved to be a potential barrier, leading to failures in treatments for various diseases and disorders. While endocytosis of drug moieties provides an efficient means of getting material into cells, the subsequent release and endosomal escape of materials into the cytosol where they need to act has been a barrier. An understanding of cellular protein/lipid trafficking pathways has opened up strategies for increasing drug bioavailability. Approaches to enhance endosomal exit have greatly increased the cytosolic bioavailability of drugs and will provide a means of investigating previous drugs that may have been shelved due to their low cytosolic concentration.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Fibrose Cística/tratamento farmacológico , Animais , Endossomos/metabolismo , Humanos , Transporte Proteico/efeitos dos fármacosRESUMO
BACKGROUND: Resveratrol, a natural phenolic compound, has been reported to rescue mutant ΔF508 CFTR in expression systems and primary epithelial cells. Although this implies a therapeutic benefit to patients with CF, investigations were performed using resveratrol concentrations greatly in excess of those achievable in plasma. We evaluated the efficacy of resveratrol as a CFTR corrector in relevant primary airway cells, using physiologically achievable resveratrol concentrations. METHODS: Cells expressing wt or ΔF508 CFTR were exposed to chronic or acute resveratrol. CFTR mRNA and protein expression were monitored. The effects of resveratrol on primary ΔF508 human airway cells were evaluated by equivalent current analysis using modified Ussing chambers. RESULTS: Consistent with previously published data in heterologous expression systems, high doses of resveratrol increased CFTR expression; however physiologically relevant concentrations were without effect. In contrast to heterologous expression systems, resveratrol was unable to increase mutant CFTR channel activity in primary airway cells. Elevated amiloride-sensitive currents, indicative of sodium transport and characteristically elevated in CF airway cells, were also unaffected by resveratrol. CONCLUSIONS: High concentrations of resveratrol can increase CFTR mRNA and protein in some cell types. In addition, acute resveratrol exposure can stimulate CFTR mediated chloride secretion, probably by increasing cellular cAMP levels. Resveratrol at physiologically achievable levels yielded no benefit in primary ΔF508 airway cells, either in terms of amiloride-sensitive currents of CFTR currents. GENERAL SIGNIFICANCE: Taken together, our results do not support the use of resveratrol supplements as a therapy for patients with cystic fibrosis. It is possible that further modifications of the resveratrol backbone would yield a more efficacious compound.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/genética , Fibrose Cística/tratamento farmacológico , Mutação , Estilbenos/uso terapêutico , AMP Cíclico/análise , Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/fisiologia , Células HEK293 , Humanos , ResveratrolRESUMO
In the current climate of curriculum reform, the traditional lecture has come under fire for its perceived lack of effectiveness. Indeed, several institutions have reduced their lectures to 15 min in length based upon the "common knowledge" and "consensus" that there is a decline in students' attention 10-15 min into lectures. A review of the literature on this topic reveals many discussions referring to prior studies but scant few primary investigations. Alarmingly, the most often cited source for a rapid decline in student attention during a lecture barely discusses student attention at all. Of the studies that do attempt to measure attention, many suffer from methodological flaws and subjectivity in data collection. Thus, the available primary data do not support the concept of a 10- to 15-min attention limit. Interestingly, the most consistent finding from a literature review is that the greatest variability in student attention arises from differences between teachers and not from the teaching format itself. Certainly, even the most interesting material can be presented in a dull and dry fashion, and it is the job of the instructor to enhance their teaching skills to provide not only rich content but also a satisfying lecture experience for the students.
Assuntos
Atenção/fisiologia , Avaliação Educacional , Estudantes , Ensino/tendências , Avaliação Educacional/métodos , Humanos , Aprendizagem/fisiologia , Estudantes/psicologia , Ensino/psicologia , Fatores de TempoRESUMO
BACKGROUND: The membrane anchored kinase, LMTK2, is a serine/threonine kinase predominantly localized to endosomal compartments. LMTK2 has been shown to be involved in the trafficking of the CFTR ion channel, the androgen receptor, as well as modulating neurodegeneration. As a membrane anchored protein, LMTK2 must be exported from the ER, yet the mechanisms whereby LMTK2 is sequestered within the ER for efficient export are unknown. METHODS: Sequence analysis of the carboxyl tail of LMTK2 revealed a putative di-acidic ER export motif. Site-directed mutagenesis was utilized to ablate this potential motif. Subcellular fractionation, immunofluorescence microscopy, and transferrin recycling assays were used to determine the consequence of mutating LMTK2's export motif. RESULTS: Mutation of the di-acidic export motif led to ER retention of LMTK2, and an increase in protein half-life and a concomitant loss of LMTK2 from its appropriate terminal destination. Loss of LMTK2 from endosomal compartments by preventing its release from the ER is linked to a reduction in transferrin recycling. CONCLUSIONS: We have identified a di-acidic ER export motif within the carboxyl tail of the membrane anchored kinase LMTK2. This sequence is used by LMTK2 for its efficient export from the ER.
Assuntos
Retículo Endoplasmático/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Endossomos/metabolismo , Humanos , Proteínas de Membrana/genética , Dados de Sequência Molecular , Proteínas Serina-Treonina Quinases/genética , Estabilidade Proteica , Transporte Proteico , Alinhamento de Sequência , Transferrina/metabolismoRESUMO
Do you know the sex of your cells? Not a question that is frequently heard around the lab bench, yet thanks to recent research is probably one that should be asked. It is self-evident that cervical epithelial cells would be derived from female tissue and prostate cells from a male subject (exemplified by HeLa and LnCaP, respectively), yet beyond these obvious examples, it would be true to say that the sex of cell lines derived from non-reproductive tissue, such as lung, intestine, kidney, for example, is given minimal if any thought. After all, what possible impact could the presence of a Y chromosome have on the biochemistry and cell biology of tissues such as the exocrine pancreatic acini? Intriguingly, recent evidence has suggested that far from being irrelevant, genes expressed on the sex chromosomes can have a marked impact on the biology of such diverse tissues as neurons and renal cells. It is also policy of AJP-Cell Physiology that the source of all cells utilized (species, sex, etc.) should be clearly indicated when submitting an article for publication, an instruction that is rarely followed (http://www.the-aps.org/mm/Publications/Info-For-Authors/Composition). In this review we discuss recent data arguing that the sex of cells being used in experiments can impact the cell's biology, and we provide a table outlining the sex of cell lines that have appeared in AJP-Cell Physiology over the past decade.
Assuntos
Fenômenos Fisiológicos Celulares/fisiologia , Caracteres Sexuais , Cromossomo X/fisiologia , Cromossomo Y/fisiologia , Animais , Linhagem Celular , Feminino , Humanos , MasculinoRESUMO
The lemur family of protein kinases has gained much interest in recent years as they are involved in a variety of cellular processes including regulation of axonal transport and endosomal trafficking, modulation of synaptic functions, memory and learning, and they are centrally placed in several intracellular signalling pathways. Numerous studies have also implicated role of the lemur kinases in the development and progression of a wide range of cancers, cystic fibrosis, and neurodegenerative diseases. However, parallel discoveries and inaccurate prediction of their kinase activity have resulted in a confusing and misleading nomenclature of these proteins. Herein, a group of international scientists with expertise in lemur family of protein kinases set forth a novel nomenclature to rectify this problem and ultimately help the scientific community by providing consistent information about these molecules.
Assuntos
Fibrose Cística , Lemur , Animais , Proteínas Quinases , Fosforilação , Transporte AxonalRESUMO
Lemur tyrosine kinase 2 (LMTK2) is a novel membrane-anchored kinase reported to be involved in several normal and pathophysiological conditions, including endosomal membrane recycling, prostate cancer, and neurodegeneration. In this study, we have investigated the topology and orientation of LMTK2 within cellular membranes utilizing fluorescence protease protection. Appending the green fluorescent protein to either the amino or carboxyl terminus of LMTK2, we were able to determine which side of intracellular membrane these regions were located. Our results indicate that LMTK2 is an integral membrane protein in which both the amino and carboxyl termini are exposed to the cytoplasm. Moreover, this topology places the kinase active site within the cytoplasm.
Assuntos
Proteínas de Membrana/química , Proteínas Serina-Treonina Quinases/química , Domínio Catalítico/fisiologia , Citoplasma/enzimologia , Proteínas de Fluorescência Verde/química , Células HEK293 , Humanos , Rim/citologia , Rim/enzimologiaRESUMO
Dynasore, a small molecule inhibitor of dynamin, was used to probe the role of dynamin in the endocytosis of wild-type and mutant CFTR (cystic fibrosis transmembrane conductance regulator). Internalization of both wild-type and 'temperature-corrected' DeltaF508 CFTR was markedly inhibited by a short exposure to dynasore, implicating dynamin as a key element in the endocytic internalization of both wild-type and mutant CFTR. The inhibitory effect of dynasore was readily reversible upon washout of dynasore from the growth media. Corr-4 ({2-(5-chloro-2-methoxy-phenylamino)-4'-methyl-[4,5']-bithiazolyl-2'-yl}-phenyl-methanonone), a pharmacological corrector of DeltaF508 CFTR biosynthesis, caused a marked increase in the cell surface expression of mutant CFTR. Co-incubation of DeltaF508 CFTR expressing cells with Corr-4 and dynasore caused a significantly greater level of cell surface CFTR than that observed in the presence of Corr-4 alone. These results argue that inhibiting the endocytic internalization of mutant CFTR provides a novel therapeutic target for augmenting the benefits of small molecule correctors of mutant CFTR biosynthesis.
Assuntos
Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Fibrose Cística/metabolismo , Regulação para Baixo , Hidrazonas/farmacologia , Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Endocitose/efeitos dos fármacos , Células HeLa , Humanos , Transporte Proteico/efeitos dos fármacosRESUMO
The octameric exocyst complex is associated with the junctional complex and recycling endosomes and is proposed to selectively tether cargo vesicles directed toward the basolateral surface of polarized Madin-Darby canine kidney (MDCK) cells. We observed that the exocyst subunits Sec6, Sec8, and Exo70 were localized to early endosomes, transferrin-positive common recycling endosomes, and Rab11a-positive apical recycling endosomes of polarized MDCK cells. Consistent with its localization to multiple populations of endosomes, addition of function-blocking Sec8 antibodies to streptolysin-O-permeabilized cells revealed exocyst requirements for several endocytic pathways including basolateral recycling, apical recycling, and basolateral-to-apical transcytosis. The latter was selectively dependent on interactions between the small GTPase Rab11a and Sec15A and was inhibited by expression of the C-terminus of Sec15A or down-regulation of Sec15A expression using shRNA. These results indicate that the exocyst complex may be a multipurpose regulator of endocytic traffic directed toward both poles of polarized epithelial cells and that transcytotic traffic is likely to require Rab11a-dependent recruitment and modulation of exocyst function, likely through interactions with Sec15A.
Assuntos
Polaridade Celular , Endocitose , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Proteínas de Transporte Vesicular/metabolismo , Animais , Permeabilidade da Membrana Celular , Cães , Regulação para Baixo/genética , Endossomos/metabolismo , Imunoglobulina A/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Ligação Proteica , Subunidades Proteicas/metabolismo , Transporte Proteico , Coelhos , Ratos , Proteínas Recombinantes de Fusão/metabolismo , Transferrina/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Rede trans-Golgi/metabolismoRESUMO
The initial entry of papillomaviruses into their target cells has been shown to occur by clathrin-mediated endocytosis and caveolae-mediated endocytosis. These mechanisms entail the formation of nascent-coated vesicles at the plasma membrane. Such coated vesicles, clathrin or caveolin, form and pinch-off in a controlled mechanism that involves several proteins including dynamin. Dynamin is a GTPase that forms a dynamin ring at the stem connecting the nascent vesicle to the plasma membrane. In a still not fully characterized mechanism, dynamin's contraction and twisting results in the scission of the vesicle. In an effort to better characterize the role and molecular mechanisms of dynamin's function, researchers have identified dynasore, a dynamin GTPase inhibitor that prevents the scission of dynamin-dependent endocytic vesicles. Here, we have tested if infection by pseudovirus corresponding to the oncogenic human papillomavirus type 16 and bovine papillomavirus type 1 can be blocked by dynasore. We present data demonstrating that dynasore can block infection of human papillomavirus type 16 and bovine papillomavirus type 1 pseudovirions in a dose- and time-dependent manner with equal efficiency. Presently, there is no available therapy that can block infection by a wide range of papillomavirus regardless of species or genotypes. Targeting dynamin may lead to the rational design of drug able to prevent infection by papillomaviruses, and by other infectious agents dependent on this protein for initial internalization into target cells. Whether such an approach will prove successful needs further investigation.
Assuntos
Papillomavirus Bovino 1/efeitos dos fármacos , Dinaminas/antagonistas & inibidores , Papillomavirus Humano 16/efeitos dos fármacos , Hidrazonas/farmacologia , Antivirais/administração & dosagem , Antivirais/farmacologia , Papillomavirus Bovino 1/patogenicidade , Linhagem Celular , Relação Dose-Resposta a Droga , Sistemas de Liberação de Medicamentos , GTP Fosfo-Hidrolases/antagonistas & inibidores , Papillomavirus Humano 16/patogenicidade , Humanos , Hidrazonas/administração & dosagem , Fatores de TempoRESUMO
The cystic fibrosis transmembrane conductance regulator (CFTR) is a Cl-selective anion channel expressed in epithelial tissues. Mutations in CFTR lead to the genetic disease cystic fibrosis (CF). Within each epithelial cell, CFTR interacts with a large number of transient macromolecular complexes, many of which are involved in the trafficking and targeting of CFTR. Understanding how these complexes regulate the trafficking and fate of CFTR, provides a singular insight not only into the patho-physiology of cystic fibrosis, but also provides potential drug targets to help cure this debilitating disease.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/fisiologia , Fibrose Cística/genética , Fibrose Cística/fisiopatologia , Endocitose/fisiologia , Animais , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Humanos , Alinhamento de Sequência , Relação Estrutura-Atividade , Vesículas Transportadoras/fisiologiaRESUMO
Regulatory elements present in the promoter of a gene drive the expression of the gene in response to various stimuli. Lemur Tyrosine Kinase 2 (LMTK2) is a membrane-anchored Serine/Threonine kinase involved in endosomal protein trafficking and androgen signaling amongst other processes. Previous studies have shown this protein to be of therapeutic importance in cystic fibrosis and prostate cancer. However, nothing is known about the endogenous expression of this protein and its regulation. In this study, we analyzed the gene encoding human LMTK2, to look for possible regulatory elements that could affect its expression. Interestingly, the human lmtk2 gene contains a consensus TPA (12- O-Tetradecanoylphorbol-13-acetate)-responsive element (TRE) in the region preceding its start codon. The element with the sequence TGAGTCA modulates LMTK2 expression in response to treatment with TPA, a synthetic Protein Kinase C (PKC) activator. It serves as the binding site for c-Fos, a member of the Activator Protein -1 (AP-1) transcription factor complex, which is transactivated by PKC. We observed that TPA, at low concentrations, increases the promoter activity of LMTK2, which leads to a subsequent increase in the mRNA transcript and protein levels. This modulation occurs through binding of the AP-1 transcription factor complex to the lmtk2 promoter. Thus, our current study has established LMTK2 as a TPA-responsive element-containing gene, which is upregulated downstream of PKC activation. Considering the involvement of LMTK2 in intracellular processes as well as pathological conditions, our findings demonstrate a way to modulate intracellular LMTK2 levels pharmacologically for potentially therapeutic purposes.
RESUMO
The recent FDA approval of two drugs to treat the basic defect in cystic fibrosis has given hope to patients and their families battling this devastating disease. Over many years, with heavy financial investment from Vertex Pharmaceuticals and the Cystic Fibrosis Foundation, pre-clinical evaluation of thousands of synthetic drugs resulted in the production of Kalydeco and Orkambi. Yet, despite the success of this endeavor, many other compounds have been proposed as therapeutic agents in the treatment of CF. Of note, several of these compounds are naturally occurring, and are present in spices from the grocery store and over the counter preparations in health food stores. In this short review, we look at three such compounds, genistein, curcumin, and resveratrol, and evaluate the scientific support for their use as therapeutic agents in the treatment of patients with CF.
RESUMO
Progression from early forms of prostate cancer to castration-resistant disease is associated with an increase in signal transduction activity. The majority of castration-resistance cancers persist in the expression of the androgen receptor (AR), as well as androgen-dependent genes. The AR is regulated not only by it associated steroid hormone, but also by manifold regulatory and signaling molecules, including several kinases. We undertook evaluation of the role of Lemur Tyrosine Kinase 2 (LMTK2) in modulating AR activity, as several Genome Wide Association Studies (GWAS) have shown a marked association of LMTK2 activity with the development of prostate cancer. We confirm that not only is LMTK2 mRNA reduced in prostate cancer tissue, but also LMTK2 protein levels are markedly diminished. Knockdown of LMTK2 protein in prostate cell lines greatly increased the transcription of androgen-responsive genes. In addition, LMTK2 knockdown led to an increase in prostate cancer stem cell populations in LNCaP cells, indicative of increased tumorogenicity. Using multiple approaches, we also demonstrate that LMTK2 interacts with the AR, thus putting LMTK2 as a component of a signaling complex modulating AR activity. Our finding that LMTK2 is a negative regulator of AR activity defines a novel cellular pathway for activation of AR-responsive genes in castrate resistant-prostate cancer. Moreover, pharmacologic manipulation of LMTK2 activity will provide a novel therapeutic target for more effective treatments for patients with castrate-resistant prostate cancer.
Assuntos
Neoplasias da Próstata/tratamento farmacológico , TYK2 Quinase/metabolismo , Animais , Linhagem Celular Tumoral , Proliferação de Células/fisiologia , Progressão da Doença , Células HEK293 , Humanos , Lemur , Masculino , Terapia de Alvo Molecular , Neoplasias da Próstata/enzimologia , Neoplasias da Próstata/genética , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Neoplasias de Próstata Resistentes à Castração/enzimologia , Neoplasias de Próstata Resistentes à Castração/genética , Receptores Androgênicos/metabolismo , Transdução de Sinais , TYK2 Quinase/biossíntese , TYK2 Quinase/genéticaRESUMO
Androgens and androgen receptors play essential roles in the development and progression of prostate cancer, a disease that claims roughly 28,000 lives annually. In addition to androgen biding, androgen receptor activity can be regulated via several post-translational modifications such as ubiquitination, acetylation, phosphorylation, methylation & SUMO-ylation. Off these modifications, phosphorylation has been the most extensively studied. Modification by phosphorylation can alter androgen receptor localization, protein stability and transcriptional activity, ultimately leading to changes in the biology of cancer cells and cancer progression. Understanding, role of phosphorylated androgen receptor species holds the key to identifying a potential therapeutic drug target for patients with prostate cancer and castrate resistant prostate cancer. Here, we present a brief review of recently discovered protein kinases phosphorylating AR, focusing on the functional role of phosphorylated androgen receptor species in prostate cancer and castrate resistant prostate cancer.
RESUMO
The correct topology and orientation of integral membrane proteins are essential for their proper function, yet such information has not been established for many membrane proteins. A simple technique called fluorescence protease protection (FPP) is presented, which permits the determination of membrane protein topology in living cells. This technique has numerous advantages over other methods for determining protein topology, in that it does not require the availability of multiple antibodies against various domains of the membrane protein, does not require large amounts of protein, and can be performed on living cells. The FPP method employs the spatially confined actions of proteases on the degradation of green fluorescent protein (GFP) tagged membrane proteins to determine their membrane topology and orientation. This simple approach is applicable to a wide variety of cell types, and can be used to determine membrane protein orientation in various subcellular organelles such as the mitochondria, Golgi, endoplasmic reticulum and components of the endosomal/recycling system. Membrane proteins, tagged on either the N-termini or C-termini with a GFP fusion, are expressed in a cell of interest, which is subject to selective permeabilization using the detergent digitonin. Digitonin has the ability to permeabilize the plasma membrane, while leaving intracellular organelles intact. GFP moieties exposed to the cytosol can be selectively degraded through the application of protease, whereas GFP moieties present in the lumen of organelles are protected from the protease and remain intact. The FPP assay is straightforward, and results can be obtained rapidly.
Assuntos
Proteínas de Fluorescência Verde/química , Proteínas de Membrana/química , Peptídeo Hidrolases/química , Membrana Celular/química , Membrana Celular/metabolismo , Citosol/química , Citosol/metabolismo , Digitonina/química , Retículo Endoplasmático/química , Retículo Endoplasmático/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Humanos , Proteínas de Membrana/metabolismo , Microscopia de Fluorescência/métodos , Mitocôndrias/química , Mitocôndrias/metabolismo , Peptídeo Hidrolases/metabolismo , Proteínas Recombinantes de Fusão/químicaRESUMO
The cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel composed of 1480 amino acids. The major mutation responsible for cystic fibrosis results in loss of amino acid residue, F508 (F508del). Loss of F508 in CFTR alters the folding pathway resulting in endoplasmic-reticulum-associated degradation. This study investigates the role of synonymous codon in the expression of CFTR and CFTR F508del in human HEK293 cells. DNA encoding the open reading frame (ORF) for CFTR containing synonymous codon replacements was expressed using a heterologous vector integrated into the genome. The results indicate that the codon usage greatly affects the expression of CFTR. While the promoter strength driving expression of the ORFs was largely unchanged and the mRNA half-lives were unchanged, the steady-state levels of the mRNA varied by as much as 30-fold. Experiments support that this apparent inconsistency is attributed to nonsense mediated decay independent of exon junction complex. The ratio of CFTR/mRNA indicates that mRNA containing native codons was more efficient in expressing mature CFTR as compared to mRNA containing synonymous high-expression codons. However, when F508del CFTR was expressed after codon optimization, a greater percentage of the protein escaped endoplasmic-reticulum-associated degradation resulting in considerable levels of mature F508del CFTR on the plasma membrane, which showed channel activity. These results indicate that codon usage has an effect on mRNA levels and protein expression, for CFTR, and likely on chaperone-assisted folding pathway, for F508del CFTR.