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BMC Systems Biology Sep 2012A hub protein is one that interacts with many functional partners. The annotation of hub proteins, or more generally the protein-protein interaction "degree" of each...
BACKGROUND
A hub protein is one that interacts with many functional partners. The annotation of hub proteins, or more generally the protein-protein interaction "degree" of each gene, requires quality genome-wide data. Data obtained using yeast two-hybrid methods contain many false positive interactions between proteins that rarely encounter each other in living cells, and such data have fallen out of favor.
RESULTS
We find that protein "stickiness", measured as network degree in ostensibly low quality yeast two-hybrid data, is a more predictive genomic metric than the number of functional protein-protein interactions, as assessed by supposedly higher quality high throughput affinity capture mass spectrometry data. In the yeast Saccharomyces cerevisiae, a protein's high stickiness, but not its high number of functional interactions, predicts low stochastic noise in gene expression, low plasticity of gene expression across different environments, and high probability of forming a homo-oligomer. Our results are robust to a multiple regression analysis correcting for other known predictors including protein abundance, presence of a TATA box and whether a gene is essential. Once the higher stickiness of homo-oligomers is controlled for, we find that homo-oligomers have noisier and more plastic gene expression than other proteins, consistent with a role for homo-oligomerization in mediating robustness.
CONCLUSIONS
Our work validates use of the number of yeast two-hybrid interactions as a metric for protein stickiness. Sticky proteins exhibit low stochastic noise in gene expression, and low plasticity in expression across different environments.
Topics: False Positive Reactions; Gene Expression; Mass Spectrometry; Molecular Sequence Annotation; Protein Binding; Protein Multimerization; Protein Structure, Quaternary; Proteins; Regression Analysis; Two-Hybrid System Techniques
PubMed: 23017156
DOI: 10.1186/1752-0509-6-128 -
Experimental Eye Research Nov 2019Cataract-associated gene discovery in human and animal models have informed on key aspects of human lens development, homeostasis and pathology. Additionally, in vitro...
Cataract-associated gene discovery in human and animal models have informed on key aspects of human lens development, homeostasis and pathology. Additionally, in vitro models such as the culture of permanent human lens epithelium-derived cell lines (LECs) have also been utilized to understand the molecular biology of lens cells. However, these resources remain uncharacterized, specifically regarding their global gene expression and suitability to model lens cell biology. Therefore, we sought to molecularly characterize gene expression in the human LEC, SRA01/04, which is commonly used in lens studies. We first performed short tandem repeat (STR) analysis and validated SRA01/04 LEC for its human origin, as recommended by the eye research community. Next, we used Illumina HumanHT-12 v3.0 Expression BeadChip arrays to gain insights into the global gene expression profile of SRA01/04. Comparative analysis of SRA01/04 microarray data was performed using other resources such as the lens expression database iSyTE (integrated Systems Tool for Eye gene discovery), the cataract gene database Cat-Map and the published lens literature. This analysis showed that SRA01/04 significantly expresses >40% of the top iSyTE lens-enriched genes (313 out of 749) across different developmental stages. Further, SRA01/04 also significantly expresses ~53% (168 out of 318) of cataract-associated genes in Cat-Map. We also performed comparative gene expression analysis between SRA01/04 cells and the previously validated mouse LEC 21EM15. To gain insight into whether SRA01/04 reflects epithelial or fiber cell characteristics, we compared its gene expression profile to previously reported differentially expressed genes in isolated mouse lens epithelial and fiber cells. This analysis suggests that SRA01/04 has reduced expression of several fiber cell-enriched genes. In agreement with these findings, cell culture analysis demonstrates that SRA01/04 has reduced potential to initiate spontaneous lentoid body formation compared to 21EM15 cells. Next, to independently validate SRA01/04 microarray gene expression, we subjected several candidate genes to RT-PCR and RT-qPCR assays. This analysis demonstrates that SRA01/04 supports expression of many key genes associated with lens development and cataract, including CRYAB, CRYBB2, CRYGS, DKK3, EPHA2, ETV5, GJA1, HSPB1, INPPL1, ITGB1, PAX6, PVRL3, SFRP1, SPARC, TDRD7, and VIM, among others, and therefore can be relevant for understanding the mechanistic basis of these factors. At the same time, SRA01/04 cells do not exhibit robust expression of several genes known to be important to lens biology and cataract such as ALDH1A1, COL4A6, CP, CRYBA4, FOXE3, HMX1, HSF4, MAF, MEIS1, PITX3, PRX, SIX3, and TRPM3, among many others. Therefore, the present study offers a rich transcript-level resource for case-by-case evaluation of the potential advantages and limitations of SRA01/04 cells prior to their use in downstream investigations. In sum, these data show that the human LEC, SRA01/04, exhibits lens epithelial cell-like character reflected in the expression of several lens-enriched and cataract-associated genes, and therefore can be considered as a useful in vitro resource when combined with in vivo studies to gain insight into specific aspects of human lens epithelial cells.
Topics: Biomarkers; Cell Line; Ephrin-A2; Epithelial Cells; Eye Proteins; Gene Expression; Humans; Lens, Crystalline; Molecular Biology; Oligonucleotide Array Sequence Analysis; Osteonectin; PAX6 Transcription Factor; Real-Time Polymerase Chain Reaction; Receptor, EphA2; Ribonucleoproteins; alpha-Crystallin B Chain; beta-Crystallin B Chain
PubMed: 31479653
DOI: 10.1016/j.exer.2019.107787 -
International Journal of Hyperthermia :... Aug 2005Although the majority of studies on the protective effect of individual hsps have concentrated on the major inducible heat shock protein Hsp70, a variety of evidence... (Review)
Review
Although the majority of studies on the protective effect of individual hsps have concentrated on the major inducible heat shock protein Hsp70, a variety of evidence suggests that the small heat shock protein Hsp27 may have a more potent protective effect in the nervous system. Thus, for example, in cultured neurones over-expression of Hsp70 can protect against subsequent exposure to thermal or ischaemic stress but not against exposure to some other stressful stimuli, whereas over-expression of Hsp27 protects against a variety of stresses. Similarly, although transgenic animals over-expressing Hsp70 are protected against cardiac ischaemia, more equivocal results have been obtained in terms of their protection against cerebral ischaemia and other stresses to the nervous system. In contrast, transgenic animals over-expressing Hsp27 have recently been shown to show neuroprotection as well as being protected against cardiac ischaemia. Recent findings have also implicated Hsp27 and related proteins in human disease. Thus, it has been demonstrated that mutation of either Hsp27 or the related protein hsp22 can be observed in specific families with hereditary motor neuropathy caused by premature axonal loss, possibly due to neuronal death and subsequent degeneration. Moreover, the mutations are associated with a reduced ability to promote neuronal survival compared to the wild type protein. Hence, Hsp27 appears to be a potent protective factor for neuronal cells whose mutation results in neuronal cell death and disease, whilst enhanced expression of the wild type protein may be a therapeutic option for human diseases involving excessive neuronal cell death.
Topics: Animals; Cell Survival; Gene Expression; Genetic Therapy; HSP27 Heat-Shock Proteins; Heat-Shock Proteins; Humans; Mice; Molecular Chaperones; Mutation; Neoplasm Proteins; Nervous System Diseases; Neurons; Rats
PubMed: 16048837
DOI: 10.1080/02656730400023664 -
The Journal of Biological Chemistry Jan 2007PepT1 is an intestinal epithelial apical membrane transporter that is expressed in the small intestine, with little or no expression in the normal colon. However, we...
PepT1 is an intestinal epithelial apical membrane transporter that is expressed in the small intestine, with little or no expression in the normal colon. However, we previously demonstrated that colonic PepT1 may be expressed during chronic inflammation. To begin elucidating inflammatory hPepT1 signaling, we herein investigated the long term leptin treatments, on PepT1 expression and activity in Caco2-BBE cells, and began to reveal the involved signaling pathways. We successfully cloned the 723-bp hPepT1 promoter region and identified the human transcription initiation site 86 bp upstream from the translation start site. Leptin treatment dose- and time-dependently increased hPepT1 promoter and transport activities in Caco2-BBE cells, with maximal activity observed in cells treated with 100 nM leptin for 8 h. Under these conditions, we observed 2-fold increases in hPepT1 mRNA and protein expression, as well as increased transport activity. Our molecular analyses of possible signal-transduction pathways revealed that leptin treatment enhanced the intracellular levels of cAMP and phosphorylated cAMP-response element-binding protein (CREB) protein in Caco2-BBE cells, whereas our deletion, mutation, and CDX2 overexpression analyses demonstrated that interaction of the Cdx2 and phosphorylated CREB transcription factors was essential for leptin-induced hPepT1 transcription in Caco2-BBE cells. Our results indicate that leptin, which is increased in inflamed colonic mucosa, triggers colonic expression of hPepT1 via the CREB and Cdx2 transcription factors. These findings provide important new insights into the mechanisms of intestinal inflammation and may suggest new therapeutic modalities in the future.
Topics: 5' Flanking Region; Amino Acid Sequence; Base Sequence; CDX2 Transcription Factor; CREB-Binding Protein; Caco-2 Cells; Cloning, Molecular; Cyclic AMP; Cyclic AMP Response Element-Binding Protein; Enteritis; Gene Expression; Homeodomain Proteins; Humans; Intestinal Mucosa; Leptin; Molecular Sequence Data; Peptide Transporter 1; Promoter Regions, Genetic; Symporters; Transcription, Genetic
PubMed: 16963449
DOI: 10.1074/jbc.M604267200 -
Microbial Cell Factories Dec 2018The secretion of recombinant disulfide-bond containing proteins into the periplasm of Gram-negative bacterial hosts, such as E. coli, has many advantages that can...
BACKGROUND
The secretion of recombinant disulfide-bond containing proteins into the periplasm of Gram-negative bacterial hosts, such as E. coli, has many advantages that can facilitate product isolation, quality and activity. However, the secretion machinery of E. coli has a limited capacity and can become overloaded, leading to cytoplasmic retention of product; which can negatively impact cell viability and biomass accumulation. Fine control over recombinant gene expression offers the potential to avoid this overload by matching expression levels to the host secretion capacity.
RESULTS
Here we report the application of the RiboTite gene expression control system to achieve this by finely controlling cellular expression levels. The level of control afforded by this system allows cell viability to be maintained, permitting production of high-quality, active product with enhanced volumetric titres.
CONCLUSIONS
The methods and systems reported expand the tools available for the production of disulfide-bond containing proteins, including antibody fragments, in bacterial hosts.
Topics: Gene Expression; Protein Transport; Recombinant Proteins
PubMed: 30577801
DOI: 10.1186/s12934-018-1047-z -
Scientific Reports Mar 2020Protein overexpression sometimes causes cellular defects, although the underlying mechanism is still unknown. A protein's expression limit, which triggers cellular...
Protein overexpression sometimes causes cellular defects, although the underlying mechanism is still unknown. A protein's expression limit, which triggers cellular defects, is a useful indication of the underlying mechanism. In this study, we developed an experimental method of estimating the expression limits of target proteins in the human embryonic kidney cell line HEK293 by measuring the proteins' expression levels in cells that survived after the high-copy introduction of plasmid DNA by which the proteins were expressed under a strong cytomegalovirus promoter. The expression limits of nonfluorescent target proteins were indirectly estimated by measuring the levels of green fluorescent protein (GFP) connected to the target proteins with the self-cleaving sequence P2A. The expression limit of a model GFP was ~5.0% of the total protein, and sustained GFP overexpression caused cell death. The expression limits of GFPs with mitochondria-targeting signals and endoplasmic reticulum localization signals were 1.6% and 0.38%, respectively. The expression limits of four proteins involved in vesicular trafficking were far lower compared to a red fluorescent protein. The protein expression limit estimation method developed will be valuable for defining toxic proteins and consequences of protein overexpression.
Topics: Cell Death; Cytomegalovirus; DNA; Endoplasmic Reticulum; Gene Expression; Green Fluorescent Proteins; HEK293 Cells; Humans; Luminescent Proteins; Methotrexate; Plasmids; Promoter Regions, Genetic; Protein Biosynthesis; Protein Transport; Tetrahydrofolate Dehydrogenase; Red Fluorescent Protein
PubMed: 32179769
DOI: 10.1038/s41598-020-61646-3 -
Microbial Cell Factories Dec 2013Membrane protein research is frequently hampered by the low natural abundance of these proteins in cells and typically relies on recombinant gene expression. Different...
BACKGROUND
Membrane protein research is frequently hampered by the low natural abundance of these proteins in cells and typically relies on recombinant gene expression. Different expression systems, like mammalian cells, insect cells, bacteria and yeast are being used, but very few research efforts have been directed towards specific host cell customization for enhanced expression of membrane proteins. Here we show that by increasing the intracellular membrane production by interfering with a key enzymatic step of lipid synthesis, enhanced expression of membrane proteins in yeast is achieved.
RESULTS
We engineered the oleotrophic yeast, Yarrowia lipolytica, by deleting the phosphatidic acid phosphatase, PAH1, which led to massive proliferation of endoplasmic reticulum (ER) membranes. For all eight tested representatives of different integral membrane protein families, we obtained enhanced protein accumulation levels and in some cases enhanced proteolytic integrity in the ∆pah1 strain. We analysed the adenosine A2AR G-protein coupled receptor case in more detail and found that concomitant induction of the unfolded protein response in the ∆pah1 strain enhanced the specific ligand binding activity of the receptor. These data indicate an improved quality control mechanism for membrane proteins accumulating in yeast cells with proliferated ER.
CONCLUSIONS
We conclude that redirecting the metabolic flux of fatty acids away from triacylglycerol- and sterylester-storage towards membrane phospholipid synthesis by PAH1 gene inactivation, provides a valuable approach to enhance eukaryotic membrane protein production. Complementary to this improvement in membrane protein quantity, UPR co-induction further enhances the quality of the membrane protein in terms of its proper folding and biological activity. Importantly, since these pathways are conserved in all eukaryotes, it will be of interest to investigate similar engineering approaches in other cell types of biotechnological interest, such as insect cells and mammalian cells.
Topics: Gene Expression; Membrane Proteins; Phenotype; Protein Engineering
PubMed: 24321035
DOI: 10.1186/1475-2859-12-122 -
Plant Physiology Oct 1995The phytotoxin coronatine and the plant growth regulator methyl jasmonate (MeJA) inhibit the growth of Arabidopsis seedlings. Coronatine and MeJA induced the...
The phytotoxin coronatine and the plant growth regulator methyl jasmonate (MeJA) inhibit the growth of Arabidopsis seedlings. Coronatine and MeJA induced the accumulation of an approximately 29-kD protein in wild-type seedlings but not in seedlings of the coi1 mutant, which is insensitive to both compounds. The approximately 29-kD protein was recognized not only by antibodies raised against the partially purified polypeptide, but also by antibodies raised against vegetative storage proteins (VSPs) from soybean (29 kD) and poplar (32 kD). In the absence of added MeJA/coronatine, the VSP-like protein was highly expressed in flowers and siliques but not in seeds, seedlings, or mature leaves of wild-type Arabidopsis. By contrast, this protein could not be detected in coi1 seedlings treated with coronatine or MeJA, and it was found in very low levels in the male sterile flowers of coi1. A transcript corresponding to the gene of the Arabidopsis 27-kD VSP precursor shows the same pattern of expression as the VSP-like protein. Significantly, the VSP-like protein was not detected in green siliques or seeds obtained from coi1 flowers fertilized with wild-type pollen. We conclude that the VSP-like protein is normally expressed in maternal tissues, where it is regulated by COI1, but is not essential for the development of siliques.
Topics: Acetates; Amino Acids; Arabidopsis; Base Sequence; Cyclopentanes; DNA Primers; Gene Expression; Genes, Plant; Indenes; Molecular Sequence Data; Oxylipins; Plant Proteins; Polymerase Chain Reaction; RNA, Messenger; RNA, Plant; Seeds; Transcription, Genetic
PubMed: 7480347
DOI: 10.1104/pp.109.2.567 -
Journal of Nippon Medical School =... Aug 2003Cisplatin is a widely used drug in cancer chemotherapy and resistance to cisplatin is a major limitation for its successful application. Intracellular inactivation of...
Cisplatin is a widely used drug in cancer chemotherapy and resistance to cisplatin is a major limitation for its successful application. Intracellular inactivation of cisplatin and detoxification of reactive oxygen species (ROS) by glutathione (a crucial cellular antioxidant) is a mechanism for cisplatin resistance. During cDNA microarray analyses of differential gene expression between a cisplatin-resistant A2780CP70 human ovarian carcinoma cell line and its parental A2780 cell line, we discovered that frataxin gene expression was frequently overexpressed in the cisplatin-resistant variant. Decreased expression of frataxin protein is associated with Friedreich's ataxia (FRDA) which is a neurodegenerative disease involving ROS-mediated cellular damage. Recent evidence suggests that frataxin might detoxify ROS via activation of glutathione peroxidase and elevation of thiols. To exploit potential involvement of frataxin gene in the development of resistance to cisplatin, we compared the levels of frataxin gene and protein in the cisplatin-resistant A2780CP70 ovarian carcinoma cell line and its parental A2780 cell line. We found that frataxin mRNA and protein expressions were elevated in the cisplatin-resistant cells. Our results suggest a potential role for cisplatin as an inducer of frataxin expression and implies that this gene may be a potential target for modulating the response to cisplatin. This is the first report showing an association between frataxin expression and cisplatin resistance.
Topics: Cisplatin; Friedreich Ataxia; Gene Expression; Humans; Iron-Binding Proteins; Tumor Cells, Cultured; Frataxin
PubMed: 12928720
DOI: 10.1272/jnms.70.367 -
PloS One 2019Magnetic resonance imaging (MRI) is a non-invasive imaging modality used in longitudinal cell tracking. Previous studies suggest that MagA, a putative iron transport...
Magnetic resonance imaging (MRI) is a non-invasive imaging modality used in longitudinal cell tracking. Previous studies suggest that MagA, a putative iron transport protein from magnetotactic bacteria, is a useful gene-based magnetic resonance contrast agent. Hemagglutinin-tagged MagA was stably expressed in undifferentiated embryonic mouse teratocarcinoma, multipotent P19 cells to provide a suitable model for tracking these cells during differentiation. Western blot and immunocytochemistry confirmed the expression and membrane localization of MagA in P19 cells. Surprisingly, elemental iron analysis using inductively-coupled plasma mass spectrometry revealed significant iron uptake in both parental and MagA-expressing P19 cells, cultured in the presence of iron-supplemented medium. Withdrawal of this extracellular iron supplement revealed unexpected iron export activity in P19 cells, which MagA expression attenuated. The influence of iron supplementation on parental and MagA-expressing cells was not reflected by longitudinal relaxation rates. Measurement of transverse relaxation rates (R2* and R2) reflected changes in total cellular iron content but did not clearly distinguish MagA-expressing cells from the parental cell type, despite significant differences in the uptake and retention of total cellular iron. Unlike other cell types, the reversible component R2' (R2* ‒ R2) provided only a moderately strong correlation to amount of cellular iron, normalized to amount of protein. This is the first report to characterize MagA expression in a previously unrecognized iron exporting cell type. The interplay between contrast gene expression and systemic iron metabolism substantiates the potential for diverting cellular iron toward the formation of a novel iron compartment, however rudimentary when using a single magnetotactic bacterial gene expression system like magA. Since relatively few mammalian cells export iron, the P19 cell line provides a tractable model of ferroportin activity, suitable for magnetic resonance analysis of key iron-handling activities and their influence on gene-based MRI contrast.
Topics: Animals; Cation Transport Proteins; Cell Line, Tumor; Cell Tracking; Contrast Media; Gene Expression; Genes, Reporter; Iron; Magnetic Resonance Imaging; Mice; Multipotent Stem Cells
PubMed: 31170273
DOI: 10.1371/journal.pone.0217842