RESUMEN
In living systems, a complex network of protein-protein interactions (PPIs) underlies most biochemical events. The human protein-protein interactome has been surveyed using yeast two-hybrid (Y2H)- and mass spectrometry (MS)-based approaches such as affinity purification coupled to MS (AP-MS). Despite decades of systematic investigations and collaborative multi-disciplinary efforts, there is no "gold standard" for documenting PPIs. A surprisingly large fraction of the human interactome remains uncharted, which we refer to as the "dark interactome." In this review, we highlight the complexity of the human interactome and discuss the current status of the human reference interactome maps. We discuss why a large proportion of the human interactome has remained refractory to traditional approaches. We propose an experimental model that can enable the identification of the dark interactome in a cell-type-specific manner. We also propose a framework to implement when embarking on studies designed to rigorously identify and characterize protein interactions.
Asunto(s)
Mapeo de Interacción de Proteínas , Proteínas , Humanos , Mapeo de Interacción de Proteínas/métodos , Proteínas/química , Cromatografía de Afinidad/métodos , Saccharomyces cerevisiae/metabolismoRESUMEN
Normal protein-protein interactions (normPPIs) occur with high fidelity to regulate almost every physiological process. In cancer, this highly organised and precisely regulated network is disrupted, hijacked or reprogrammed resulting in oncogenic protein-protein interactions (oncoPPIs). OncoPPIs, which can result from genomic alterations, are a hallmark of many types of cancers. Recent technological advances in the field of mass spectrometry (MS)-based interactomics, structural biology and drug discovery have prompted scientists to identify and characterise oncoPPIs. Disruption of oncoPPI interfaces has become a major focus of drug discovery programs and has resulted in the use of PPI-specific drugs clinically. However, due to several technical hurdles, studies to build a reference oncoPPI map for various cancer types have not been undertaken. Therefore, there is an urgent need for experimental workflows to overcome the existing challenges in studying oncoPPIs in various cancers and to build comprehensive reference maps. Here, we discuss the important hurdles for characterising oncoPPIs and propose a three-phase multidisciplinary workflow to identify and characterise oncoPPIs. Systematic identification of cancer-type-specific oncogenic interactions will spur new opportunities for PPI-focused drug discovery projects and precision medicine.
Asunto(s)
Neoplasias , Medicina de Precisión , Carcinogénesis/genética , Humanos , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Neoplasias/metabolismo , Oncogenes , Mapeo de Interacción de ProteínasRESUMEN
The combination of four proteins and their paralogues including MBD2/3, GATAD2A/B, CDK2AP1 and CHD3/4/5, which we refer to as the MGCC module, form the chromatin remodelling module of the nucleosome remodelling and deacetylase (NuRD) complex. To date, mechanisms by which the MGCC module acquires paralogue-specific function and specificity have not been addressed. Understanding the protein-protein interaction (PPI) network of the MGCC subunits is essential for defining underlying mechanisms of gene regulation. Therefore, using pulldown followed by mass spectrometry analysis (PD-MS), we report a proteome-wide interaction network of the MGCC module in a paralogue-specific manner. Our data also demonstrate that the disordered C-terminal region of CHD3/4/5 is a gateway to incorporate remodelling activity into both ChAHP (CHD4, ADNP, HP1γ) and NuRD complexes in a mutually exclusive manner. We define a short aggregation-prone region (APR) within the C-terminal segment of GATAD2B that is essential for the interaction of CHD4 and CDK2AP1 with the NuRD complex. Finally, we also report an association of CDK2AP1 with the nuclear receptor co-repressor (NCOR) complex. Overall, this study provides insight into the possible mechanisms through which the MGCC module can achieve specificity and diverse biological functions.
Asunto(s)
Ensamble y Desensamble de Cromatina/genética , Complejo Desacetilasa y Remodelación del Nucleosoma Mi-2/genética , Mapas de Interacción de Proteínas/genética , Proteoma/genética , ADN Helicasas/genética , Proteínas de Unión al ADN/genética , Regulación de la Expresión Génica , Humanos , Complejo Desacetilasa y Remodelación del Nucleosoma Mi-2/ultraestructura , Complejos Multiproteicos/genética , Nucleosomas/genética , Nucleosomas/ultraestructura , Proteínas Represoras/genética , Proteínas Supresoras de Tumor/genéticaRESUMEN
CCCTC-binding factor (CTCF) plays fundamental roles in transcriptional regulation and chromatin architecture maintenance. CTCF is also a tumour suppressor frequently mutated in cancer, however, the structural and functional impact of mutations have not been examined. We performed molecular and structural characterisation of five cancer-specific CTCF missense zinc finger (ZF) mutations occurring within key intra- and inter-ZF residues. Functional characterisation of CTCF ZF mutations revealed a complete (L309P, R339W, R377H) or intermediate (R339Q) abrogation as well as an enhancement (G420D) of the anti-proliferative effects of CTCF. DNA binding at select sites was disrupted and transcriptional regulatory activities abrogated. Molecular docking and molecular dynamics confirmed that mutations in residues specifically contacting DNA bases or backbone exhibited loss of DNA binding. However, R339Q and G420D were stabilised by the formation of new primary DNA bonds, contributing to gain-of-function. Our data confirm that a spectrum of loss-, change- and gain-of-function impacts on CTCF zinc fingers are observed in cell growth regulation and gene regulatory activities. Hence, diverse cellular phenotypes of mutant CTCF are clearly explained by examining structure-function relationships.
Asunto(s)
Factor de Unión a CCCTC/química , Factor de Unión a CCCTC/metabolismo , Regulación Neoplásica de la Expresión Génica , Mutación , Neoplasias/patología , Fenotipo , Dedos de Zinc , Apoptosis , Factor de Unión a CCCTC/genética , Proliferación Celular , Humanos , Neoplasias/genética , Neoplasias/metabolismo , Regiones Promotoras Genéticas , Relación Estructura-Actividad , Células Tumorales CultivadasRESUMEN
Separation of X and Y chromosome-bearing sperm is an appropriate method for the selection of desired sex of offspring to increase the profit in livestock industries. The purpose of this study was the production of a monoclonal antibody against recombinant bovine sex-determining region Y protein for separation Y sperm. The hybridoma cells from splenocytes of immunized female's balb/C mice and Sp2/0 cells were made. The binding affinity of our monoclonal antibody (mAbSRY2) was compared with mouse monoclonal SRY-15. The Western blot method indicated that mAbSRY2 successfully detected the rbSRY protein. The specificity and sensitivity of mAbSRY2 is comparable to SRY-15 commercially ones. The SRY gene in 100% of bull semen contains the Y chromosome that had the strongest binding affinity to mAbSRY2 was synthesized. In other words, the binding affinity of semen contains the X sperms near the negative control. In general, this immunological method can help to separate X from Y sperms. However, the mAbSRY2 is bind to Y-bearing sexed sperm, but in the future; the sexed sperms need to apply in farms.
Asunto(s)
Genes sry/inmunología , Preselección del Sexo/veterinaria , Espermatozoides/inmunología , Animales , Anticuerpos Monoclonales/farmacología , Bovinos , Femenino , Hibridomas , Masculino , Ratones Endogámicos BALB C , Preselección del Sexo/métodos , Bazo , Cromosoma Y/inmunologíaRESUMEN
The nucleosome remodeling and deacetylase (NuRD) complex is essential for metazoan development but has been refractory to biochemical analysis. We present an integrated analysis of the native mammalian NuRD complex, combining quantitative mass spectrometry, cross-linking, protein biochemistry, and electron microscopy to define the architecture of the complex. NuRD is built from a 2:2:4 (MTA, HDAC, and RBBP) deacetylase module and a 1:1:1 (MBD, GATAD2, and Chromodomain-Helicase-DNA-binding [CHD]) remodeling module, and the complex displays considerable structural dynamics. The enigmatic GATAD2 controls the asymmetry of the complex and directly recruits the CHD remodeler. The MTA-MBD interaction acts as a point of functional switching, with the transcriptional regulator PWWP2A competing with MBD for binding to the MTA-HDAC-RBBP subcomplex. Overall, our data address the long-running controversy over NuRD stoichiometry, provide imaging of the mammalian NuRD complex, and establish the biochemical mechanism by which PWWP2A can regulate NuRD composition.
Asunto(s)
Regulación de la Expresión Génica/genética , Histona Desacetilasas/metabolismo , Nucleosomas/metabolismo , Humanos , Modelos MolecularesRESUMEN
The nucleosome remodelling and deacetylase complex (NuRD) is a widely conserved regulator of gene expression. The determination of the subunit composition of the complex and identification of its binding partners are important steps towards understanding its architecture and function. The question of how these properties of the complex vary across different cell types has not been addressed in detail to date. Here, we set up a two-step purification protocol coupled to liquid chromatography-tandem mass spectrometry to assess NuRD composition and interaction partners in three different cancer cell lines, using label-free intensity-based absolute quantification (iBAQ). Our data indicate that the stoichiometry of the NuRD complex is preserved across our three different cancer cell lines. In addition, our interactome data suggest ZNF219 and SLC25A5 as possible interaction partners of the complex. To corroborate this latter finding, in vitro and cell-based pull-down experiments were carried out. These experiments indicated that ZNF219 can interact with RBBP4, GATAD2A/B and chromodomain helicase DNA binding 4, whereas SLC25A5 might interact with MTA2 and GATAD2A.
Asunto(s)
Línea Celular Tumoral/química , Complejo Desacetilasa y Remodelación del Nucleosoma Mi-2/metabolismo , Proteínas de Neoplasias/metabolismo , Mapas de Interacción de Proteínas , Translocador 2 del Nucleótido Adenina/metabolismo , Centrifugación por Gradiente de Densidad , Cromatografía de Afinidad , Cromatografía Liquida , Proteínas de Unión al ADN/metabolismo , Perfilación de la Expresión Génica , Humanos , Complejo Desacetilasa y Remodelación del Nucleosoma Mi-2/química , Complejo Desacetilasa y Remodelación del Nucleosoma Mi-2/genética , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patología , Mapeo de Interacción de Proteínas/métodos , Subunidades de Proteína , ARN Mensajero/biosíntesis , ARN Neoplásico/biosíntesis , Proteínas Recombinantes/metabolismo , Espectrometría de Masas en Tándem , Dedos de ZincRESUMEN
The nucleosome remodelling and deacetylase (NuRD) complex is essential for the development of complex animals. NuRD has roles in regulating gene expression and repairing damaged DNA. The complex comprises at least six proteins with two or more paralogues of each protein routinely identified when the complex is purified from cell extracts. To understand the structure and function of NuRD, a map of direct subunit interactions is needed. Dozens of published studies have attempted to define direct inter-subunit connectivities. We propose that conclusions reported in many such studies are in fact ambiguous for one of several reasons. First, the expression of many NuRD subunits in bacteria is unlikely to lead to folded, active protein. Second, interaction studies carried out in cells that contain endogenous NuRD complex can lead to false positives through bridging of target proteins by endogenous components. Combining existing information on NuRD structure with a protocol designed to minimize false positives, we report a conservative and robust interaction map for the NuRD complex. We also suggest a 3D model of the complex that brings together the existing data on the complex. The issues and strategies discussed herein are also applicable to the analysis of a wide range of multi-subunit complexes. ENZYMES: Micrococcal nuclease (MNase), EC 3.1.31.1; histone deacetylase (HDAC), EC 3.5.1.98.
Asunto(s)
Complejo Desacetilasa y Remodelación del Nucleosoma Mi-2/química , Nucleosomas/química , Mapeo de Interacción de Proteínas/métodos , Animales , Artefactos , Western Blotting , Escherichia coli , Células HEK293 , Células HeLa , Histona Desacetilasa 1/química , Humanos , Ratones , Modelos Moleculares , Conformación Proteica , Pliegue de Proteína , Subunidades de Proteína , Conejos , Proteínas Recombinantes de Fusión/química , ReticulocitosRESUMEN
Producing meiosis-competent germ cells (GCs) from embryonic stem cells (ESCs) is essential for developing advanced therapies for infertility. Here, a novel approach is presented for generation of GCs from ESCs. In this regard, microparticles (MPs) have been developed from alginate sulfate loaded with bone morphogenetic protein 4 (BMP4). The results here show that BMP4 release from alginate sulfate MPs is significantly retarded by the sulfated groups compared to neat alginate. Then, BMP4-laden MPs are incorporated within the aggregates during differentiation of GCs from ESCs. It is observed that BMP4-laden MPs increase GC differentiation from ESCs at least twofold compared to the conventional soluble delivery method. Interestingly, following meiosis induction, Dazl, an intrinsic factor that enables GCs to enter meiosis, and two essential meiosis genes (Stra8 and Smc1b) are upregulated significantly in MP-induced aggregates compared to aggregates, which are formed by the conventional method. Together, these data show that controlled delivery of BMP4 during ESC differentiation into GC establish meiosis-competent GCs which can serve as an attractive GC source for reproductive medicine.
Asunto(s)
Proteína Morfogenética Ósea 4/química , Diferenciación Celular/efectos de los fármacos , Células Madre Embrionarias/efectos de los fármacos , Células Madre Mesenquimatosas/efectos de los fármacos , Alginatos/administración & dosificación , Alginatos/química , Animales , Proteína Morfogenética Ósea 4/administración & dosificación , Micropartículas Derivadas de Células/química , Células Madre Embrionarias/citología , Células Germinativas/efectos de los fármacos , Células Germinativas/crecimiento & desarrollo , Ácido Glucurónico/administración & dosificación , Ácido Glucurónico/química , Ácidos Hexurónicos/administración & dosificación , Ácidos Hexurónicos/química , Meiosis/genética , Células Madre Mesenquimatosas/citología , RatonesRESUMEN
Sex determining region Y gene (SRY) is located on Y chromosome and encodes a protein with 229 amino acids. In this study, ORF region of SRY with a length of 690 bp was synthesized using PCR and ligated to pET28a (+), then transformed in E.coli DH5α. E.coli BL21 (DE3) strain was chosen to express recombinant bovine SRY protein. A set of optimization steps was taken including different concentrations of IPTG, glucose, and temperatures at differed incubation times after the induction. Results showed that temperature points and different concentrations of IPTG and glucose had a significant effect (p < 0.01) on total protein and recombinant bovine SRY. After purification, various temperatures and concentrations of IPTG showed meaningful effects (p < 0.01) on the solubility of expressed recombinant SRY. Highest soluble rSRY protein amount was achieved where 0.5 mM IPTG and 0.5% glucose was used at 20°C during induction. In the absence of glucose, the highest amount of soluble recombinant SRY levels were achieved at the concentrations of 0.8 mM of IPTG at 28°C, 20°C, and 1.5 mM IPTG at 37°C during induction for 16, 24, and 8 hours, respectively. Regarding the results obtained in this study, it could be stated that by decreasing temperature and inducer concentration, soluble bovine SRY protein expression increases.
Asunto(s)
Bovinos/genética , Genes sry/genética , Proteína de la Región Y Determinante del Sexo/genética , Cromosoma Y/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Masculino , Sistemas de Lectura Abierta/genética , Dominios Proteicos , Proteínas Recombinantes , Análisis de Secuencia de ADN/veterinaria , Proteína de la Región Y Determinante del Sexo/aislamiento & purificación , Proteína de la Región Y Determinante del Sexo/metabolismo , Solubilidad , TemperaturaRESUMEN
The nucleosome remodeling and deacetylase (NuRD) complex remodels the genome in the context of both gene transcription and DNA damage repair. It is essential for normal development and is distributed across multiple tissues in organisms ranging from mammals to nematode worms. In common with other chromatin-remodeling complexes, however, its molecular mechanism of action is not well understood and only limited structural information is available to show how the complex is assembled. As a step towards understanding the structure of the NuRD complex, we have characterized the interaction between two subunits: the metastasis associated protein MTA1 and the histone-binding protein RBBP4. We show that MTA1 can bind to two molecules of RBBP4 and present negative stain electron microscopy and chemical crosslinking data that allow us to build a low-resolution model of an MTA1-(RBBP4)2 subcomplex. These data build on our understanding of NuRD complex structure and move us closer towards an understanding of the biochemical basis for the activity of this complex.
Asunto(s)
Histona Desacetilasas/química , Nucleosomas/química , Subunidades de Proteína/química , Proteínas Represoras/química , Proteína 4 de Unión a Retinoblastoma/química , Proteína 7 de Unión a Retinoblastoma/química , Secuencia de Aminoácidos , Animales , Clonación Molecular , Secuencia Conservada , Reactivos de Enlaces Cruzados/química , Expresión Génica , Células HEK293 , Histona Desacetilasas/genética , Histona Desacetilasas/metabolismo , Humanos , Cinética , Modelos Moleculares , Mutación , Nucleosomas/metabolismo , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Estructura Secundaria de Proteína , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Proteína 4 de Unión a Retinoblastoma/genética , Proteína 4 de Unión a Retinoblastoma/metabolismo , Proteína 7 de Unión a Retinoblastoma/genética , Proteína 7 de Unión a Retinoblastoma/metabolismo , Alineación de Secuencia , Termodinámica , Transactivadores , Transcripción GenéticaRESUMEN
OBJECTIVE: Genetic modification of human embryonic stem cells (hESCs) is critical for their extensive use as a fundamental tool for cell therapy and basic research. Despite the fact that various methods such as lipofection and electroporation have been applied to transfer the gene of interest (GOI) into the target cell line, however, there are few re- ports that compare all parameters, which influence transfection efficiency. In this study, we examine all parameters that affect the efficiency of electroporation and lipofection for transient and long-term gene expression in three different cell lines to introduce the best method and determinant factor. MATERIALS AND METHODS: In this experimental study, both electroporation and lipofection approaches were employed for genetic modification. pCAG-EGFP was applied for tran- sient expression of green fluorescent protein in two genetically different hESC lines, Roy- an H5 (XX) and Royan H6 (XY), as well as human foreskin fibroblasts (hFF). For long-term EGFP expression VASA and OLIG2 promoters (germ cell and motoneuron specific genes, respectively), were isolated and subsequently cloned into a pBluMAR5 plasmid backbone to drive EGFP expression. Flow cytometry analysis was performed two days after trans- fection to determine transient expression efficiency. Differentiation of drug resistant hESC colonies toward primordial germ cells (PGCs) was conducted to confirm stable integration of the transgene. RESULTS: Transient and stable expression suggested a variable potential for different cell lines against transfection. Analysis of parameters that influenced gene transformation ef- ficiency revealed that the vector concentrations from 20-60 µg and the density of the sub- jected cells (5×10(5)and 1×10(6)cells) were not as effective as the genetic background and voltage rate. The present data indicated that in contrast to the circular form, the linearized vector generated more distinctive drug resistant colonies. CONCLUSION: Electroporation was an efficient tool for genetic engineering of hESCs compared to the chemical method. The genetic background of the subjected cell line for transfection seemed to be a fundamental factor in each gene delivery method. For each cell line, optimum voltage rate should be calculated as it has been shown to play a crucial role in cell death and rate of gene delivery.
RESUMEN
The human Y chromosome has an inevitable role in male fertility because it contains many genes critical for spermatogenesis and the development of the male gonads. Any genetic variation or epigenetic modification affecting the expression pattern of Y chromosome genes may thus lead to male infertility. In this study, we performed isoform-level gene expression profiling of Y chromosome genes within the azoospermia factor (AZF) regions, their X chromosome counterparts, and few autosomal paralogues in testicular biopsies of 12 men with preserved spermatogenesis and 68 men with nonobstructive azoospermia (NOA) (40 Sertoli-cell-only syndrome (SCOS) and 28 premiotic maturation arrest (MA)). This was undertaken using quantitative real-time PCR (qPCR) at the transcript level and Western blotting (WB) and immunohistochemistry (IHC) at the protein level. We profiled the expression of 41 alternative transcripts encoded by 14 AZFa, AZFb, and AZFc region genes (USP9Y, DDX3Y, XKRY, HSFY1, CYORF15A, CYORF15B, KDM5D, EIF1AY, RPS4Y2, RBMY1A1, PRY, BPY2, DAZ1, and CDY1) as well as their X chromosome homologue transcripts and a few autosomal homologues. Of the 41 transcripts, 18 were significantly down-regulated in men with NOA when compared with those of men with complete spermatogenesis. In contrast, the expression of five transcripts increased significantly in NOA patients. Furthermore, to confirm the qPCR results at the protein level, we performed immunoblotting and IHC experiments (based on 24 commercial and homemade antibodies) that detected 10 AZF-encoded proteins. In addition, their localization in testis cell types and organelles was determined. Interestingly, the two missing proteins, XKRY and CYORF15A, were detected for the first time. Finally, we focused on the expression patterns of the significantly altered genes in 12 MA patients with successful sperm retrieval compared to those of 12 MA patients with failed sperm retrieval to predict the success of sperm retrieval in azoospermic men. We showed that HSFY1-1, HSFY1-3, BPY2-1, KDM5C2, RBMX2, and DAZL1 transcripts could be used as potential molecular markers to predict the presence of spermatozoa in MA patients. In this study, we have identified isoform level signature that can be used to discriminate effectively between MA, SCOS, and normal testicular tissues and suggests the possibility of diagnosing the presence of mature sperm cell in azoospermic men to prevent additional testicular sperm extraction (TESE) surgery.
Asunto(s)
Azoospermia/genética , Cromosomas Humanos X , Cromosomas Humanos Y , Perfilación de la Expresión Génica , Testículo/patología , Adulto , Azoospermia/patología , Humanos , Masculino , Persona de Mediana EdadRESUMEN
Although it is apparent that chromosome complement mediates sexually dimorphic expression patterns of some proteins that lead to functional differences, there has been insufficient evidence following the manipulation of the male-specific region of the Y chromosome (MSY) gene expression during neural development. In this study, we profiled the expression of 23 MSY genes and 15 of their X-linked homologues during neural cell differentiation of NTERA-2 human embryonal carcinoma cell line (NT2) cells in three different developmental stages using qRT-PCR, Western blotting, and immunofluorescence. The expression level of 12 Y-linked genes significantly increased over neural differentiation, including RBMY1, EIF1AY, DDX3Y, HSFY1, BPY2, PCDH11Y, UTY, RPS4Y1, USP9Y, SRY, PRY, and ZFY. We showed that siRNA-mediated knockdown of DDX3Y, a DEAD box RNA helicase enzyme, in neural progenitor cells impaired cell cycle progression and increased apoptosis, consequently interrupting differentiation. Label-free quantitative shotgun proteomics based on a spectral counting approach was then used to characterize the proteomic profile of the cells after DDX3Y knockdown. Among 917 reproducibly identified proteins detected, 71 proteins were differentially expressed following DDX3Y siRNA treatment compared with mock treated cells. Functional grouping indicated that these proteins were involved in cell cycle, RNA splicing, and apoptosis, among other biological functions. Our results suggest that MSY genes may play an important role in neural differentiation and demonstrate that DDX3Y could play a multifunctional role in neural cell development, probably in a sexually dimorphic manner.
Asunto(s)
Diferenciación Celular/genética , Cromosomas Humanos Y , ARN Helicasas DEAD-box/genética , Neuronas/citología , Apoptosis/genética , Puntos de Control del Ciclo Celular/genética , Células Cultivadas , Eliminación de Gen , Humanos , Masculino , Antígenos de Histocompatibilidad Menor , Neurogénesis , ProteómicaRESUMEN
BACKGROUND: Human granulocyte colony-stimulating factor (hG-CSF) can serve as valuable biopharmaceutical for research and treatment of the human blood cancer. Transplastomic plants have been emerged as a new and high potential candidate for production of recombinant biopharmaceutical proteins in comparison with transgenic plants due to extremely high level expression, biosafety and many other advantages. METHODS: hG-CSF gene was cloned into pCL vector between prrn16S promoter and TpsbA terminator. The recombinant vector was coated on nanogold particles and transformed to lettuce chloroplasts through biolistic method. Callogenesis and regeneration of cotyledonary explants were obtained by Murashige and Skoog media containing 6-benzylaminopurine and 1-naphthaleneacetic acid hormones. The presence of hG-CSF gene in plastome was studied with four specific PCR primers and expression by Western immunoblotting. RESULTS: hG-CSF gene cloning was confirmed by digestion and sequencing. Transplastomic lettuce lines were regenerated and subjected to molecular analysis. The presence of hG-CSF in plastome was confirmed by PCR using specific primers designed from the plastid genome. Western immunoblotting of extracted protein from transplastomic plants showed a 20-kDa band, which verified the expression of recombinant protein in lettuce chloroplasts. CONCLUSIONS: This study is the first report that successfully express hG-CSF gene in lettuce chloroplast. The lettuce plastome can provide a cheap and safe expression platform for producing valuable biopharmaceuticals for research and treatment.
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Expresión Génica , Factor Estimulante de Colonias de Granulocitos/genética , Lactuca/genética , Plastidios/genética , Cloroplastos/metabolismo , Clonación Molecular , Vectores Genéticos , Genoma de Planta/genética , Glucuronidasa/metabolismo , Humanos , Immunoblotting , Plantas Modificadas Genéticamente , Reacción en Cadena de la Polimerasa , RegeneraciónRESUMEN
BACKGROUND: RNA-binding motif gene on Y chromosome (RBMY), a germ cell-specific nuclear protein, is known as a key factor in spermatogenesis and disorders associated with this protein have been recognized to be related to male infertility. Although it was suggested that this protein could have different functions during germ cell development, no studies have been conducted to uncover the mechanism of this potential function yet. Here, we analyzed the expression pattern of RBMY protein isoforms in testis compared to NT2, a testicular germ cell cancer-derived cell line, to test probability of differential expression of RBMY protein isoforms at different spermatogenesis stages. METHODS: Full length and a segment of RBMY gene were cloned and expressed in E. coli. Anti-human RBMY antibody was produced in rabbit using the recombinant proteins as antigen. Western-blot and immunofluorescence were conducted for detection and comparison of RBMY protein isoforms. RESULTS: Selected segment of RBMY protein resulted in producing a mono-specific antibody. As results shows, only the longest isoform of RBMY was expressed at protein level in NT2 cell line, while three isoforms of this protein were detected in the whole testis lysate. CONCLUSION: The results imply that different alternative splicing may happen in testis cells and probably difference of RBMY function during spermatogenesis is due to the differential expression of RBMY protein isoforms. These results and further experiments on RBMY isoforms can help to obtain a better understanding of the function of this protein, which may increase our knowledge about spermatogenesis and causes of male infertility.
Asunto(s)
Cromosomas Humanos Y/metabolismo , Neoplasias de Células Germinales y Embrionarias/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Unión al ARN/metabolismo , Neoplasias Testiculares/metabolismo , Testículo/metabolismo , Anticuerpos/metabolismo , Western Blotting , Línea Celular Tumoral , Clonación Molecular , Codón/genética , Técnica del Anticuerpo Fluorescente , Humanos , Masculino , Neoplasias de Células Germinales y Embrionarias/patología , Proteínas Nucleares/inmunología , Isoformas de Proteínas/metabolismo , Proteínas de Unión al ARN/inmunología , Proteínas Recombinantes/biosíntesis , Reproducibilidad de los Resultados , Neoplasias Testiculares/patología , Testículo/patologíaRESUMEN
The Chromosome-centric Human Proteome Project (C-HPP) aims to systematically map the entire human proteome with the intent to enhance our understanding of human biology at the cellular level. This project attempts simultaneously to establish a sound basis for the development of diagnostic, prognostic, therapeutic, and preventive medical applications. In Iran, current efforts focus on mapping the proteome of the human Y chromosome. The male-specific region of the Y chromosome (MSY) is unique in many aspects and comprises 95% of the chromosome's length. The MSY continually retains its haploid state and is full of repeated sequences. It is responsible for important biological roles such as sex determination and male fertility. Here, we present the most recent update of MSY protein-encoding genes and their association with various traits and diseases including sex determination and reversal, spermatogenesis and male infertility, cancers such as prostate cancers, sex-specific effects on the brain and behavior, and graft-versus-host disease. We also present information available from RNA sequencing, protein-protein interaction, post-translational modification of MSY protein-coding genes and their implications in biological systems. An overview of Human Y chromosome Proteome Project is presented and a systematic approach is suggested to ensure that at least one of each predicted protein-coding gene's major representative proteins will be characterized in the context of its major anatomical sites of expression, its abundance, and its functional relevance in a biological and/or medical context. There are many technical and biological issues that will need to be overcome in order to accomplish the full scale mapping.