Quantifying Small Molecule-Induced Changes in Cellular Protein Expression and Posttranslational Modifications Using Isobaric Mass Tags

2014 ◽  
pp. 431-443 ◽  
Author(s):  
Isabelle Becher ◽  
Maria Fälth Savitski ◽  
Marcus Bantscheff
Keyword(s):  
Protein Expression ◽  
Small Molecule ◽  
Posttranslational Modifications ◽  
Cellular Protein ◽  
Induced Changes

Overview of Recent Progress in Protein-Expression Technologies for Small-Molecule Screening

2014 ◽  
Vol 19 (7) ◽  
pp. 1000-1013 ◽  
Author(s):  
John W. Cuozzo ◽  
Holly H. Soutter
Keyword(s):  
Protein Expression ◽  
Small Molecule ◽  
Posttranslational Modifications ◽  
Expression Systems ◽  
Design Strategies ◽  
Protein Targets ◽  
Small Molecule Screening ◽  
Active Research ◽  
Small Molecule Modulators ◽  
And Function

Production of novel soluble and membrane-localized protein targets for functional and affinity-based screening has often been limited by the inability of traditional protein-expression systems to generate recombinant proteins that have properties similar to those of their endogenous counterparts. Such targets have often been labeled as challenging. Although biological validation of these challenging targets for specific disease areas may be strong, discovery of small-molecule modulators can be greatly delayed or completely halted due to target-expression issues. In this article, the limitations of traditional protein-expression systems will be discussed along with new systems designed to overcome these challenges. Recent work in this field has focused on two major areas for both soluble and membrane targets: construct-design strategies to improve expression levels and new hosts that can carry out the posttranslational modifications necessary for proper target folding and function. Another area of active research has been on the reconstitution of solubilized membrane targets for both structural analysis and screening. Finally, the potential impact of these new systems on the output of small-molecule screening campaigns will be discussed.

Daidzein affects steroidogenesis and oestrogen receptor expression in medium ovarian follicles of pigs

2013 ◽  
Vol 61 (1) ◽  
pp. 85-98 ◽  
Author(s):  
Anna Nynca ◽  
Dominika Słonina ◽  
Olga Jablońska ◽  
Barbara Kamińska ◽  
Renata Ciereszko
Keyword(s):  
Protein Expression ◽  
Granulosa Cells ◽  
Oestrogen Receptor ◽  
Receptor Expression ◽  
Progesterone Production ◽  
Progesterone Secretion ◽  
Mrna And Protein Expression ◽  
Induced Changes ◽  
Reproductive Processes

Daidzein, a phytoestrogen present in soybean products used in swine feed, has been demonstrated to affect both reproductive and endocrine functions. The aims of this study were to examine the in vitro effects of daidzein on (1) progesterone (P4) and oestradiol (E2) secretion by porcine luteinised granulosa cells harvested from medium follicles, and (2) the mRNA and protein expression of oestrogen receptors α and β (ERα and ERβ) in these cells. The influence of E2 on P4 secretion and ERα and ERβ expression in the granulosa cells of pigs was also investigated. It was found that daidzein inhibited progesterone secretion by luteinised granulosa cells isolated from medium follicles. In contrast, E2 did not affect progesterone production by these cells. Moreover, daidzein did not alter the granulosal secretion of E2. Both daidzein and E2 decreased mRNA expression of ERα in the cells examined. The expression of ERβ mRNA was not affected by daidzein but was inhibited by E2. ERα protein was not detected while ERβ protein was found in the nuclei of the cells. Daidzein and E2 upregulated the expression of ERβ protein in the cells. In summary, the phytoestrogen daidzein directly affected the porcine ovary by inhibiting progesterone production and increasing ERβ protein expression. Daidzein-induced changes in follicular steroidogenesis and granulosal sensitivity to oestrogens may disturb reproductive processes in pigs.

scholarly journals Differentiation and Maturation Effect of All-trans Retinoic Acid on Cultured Fetal RPE and Stem Cell-Derived RPE Cells for Cell-Based Therapy

2021 ◽  
Author(s):  
Tingyu Yan ◽  
Na Yang ◽  
Wei Hu ◽  
Xinxin Zhang ◽  
Xuedong Li ◽  
...  
Keyword(s):  
Stem Cell ◽  
Retinoic Acid ◽  
Protein Expression ◽  
Rpe Cells ◽  
Plating Density ◽  
Cell Based Therapy ◽  
Trans Retinoic Acid ◽  
All Trans Retinoic Acid ◽  
Gene And Protein Expression ◽  
Induced Changes

Abstract Background: Phase I/II clinical trials using fetal retinal pigment epithelium (fRPE), human embryonic stem cell (hESC)-derived RPE, or human induced pluripotent stem cell (hiPSC)-derived RPE as potential sources of materials for cell-based therapy to treat degenerative retinal diseases have been carried out during the past decade. Challenges for successful translational cell-based therapy include cell manufacture, cell quality, cell storage, and cell behavior in vivo. In this study, we investigated the culture-induced changes in passaged fetal RPE, hESC-RPE and hiPSC-RPE cells in vitro and explored the differentiation and maturation effect of all-trans retinoic acid (ATRA) on those RPE cells. Methods: A total of 9 fetal RPE cell lines, hESC-RPE and hiPSC-RPE cell lines were set up using previously described methods. The culture-induced changes in subsequent passages caused by manipulating plating density, dissociation method and repeated passaging were studied by microscope, real-time quantitative PCR, western blot and immunofluorescent assays. Gene and protein expression and functional characteristics of fRPE, hESC-RPE and hiPSC-RPE incubated with ATRA at different concentration were also evaluated.Results: Compared with fRPE, hESC-RPE and hiPSC-RPE showed decreased gene and protein expression of RPE markers. Passage 3 RPE of all three types seeded at a density of 6×105 and 9x105 cells/mL in basal medium maintained pigmented polygonal, cobblestone-like morphology. RPE cells underwent mesenchymal changes showing increased expression of mesenchymal markers including a-SMA, N-cadherin, fibronectin and decreased expression of RPE markers including RPE65, E-cadherin and ZO-1, as a subsequence of low plating density, inappropriate dissociated method, and repeated passaging. fRPE, hESC-RPE and iPSC-RPE treated by ATRA at different concentrations showed increased expression of RPE markers such as RPE65, bestrophin (BEST) and CRALBP, and increased expression of negative complement regulatory proteins (CRP) including complement factor H (CFH), CD46, CD55 and CD59, and increased transepithelial resistance (TER) as well.Conclusion: Although hESC and hiPSC-derived RPE are morphologically similar to fRPE, and also have the tendency to undergo epithelial-to-mesenchymal transition (EMT) changes during the culturing and passaging process in vitro, differences in protein and gene expression among three RPE types exist. Moreover, ATRA can increase RPE markers expression, as well as to increase the expression levels of CRPs gene and protein in fRPE and stem cell-derived RPE.

scholarly journals Enhanced production of recombinant serratiopeptidase in Escherichia coli and its characterization as a potential biosimilar to native biotherapeutic counterpart

2019 ◽  
Vol 18 (1) ◽  
Author(s):  
Vishal Srivastava ◽  
Shivam Mishra ◽  
Tapan K. Chaudhuri
Keyword(s):  
Protein Expression ◽  
Process Parameters ◽  
Recombinant Expression ◽  
Cellular Protein ◽  
Biologically Active ◽  
Fixed Dose ◽  
Antibiofilm Activity ◽  
Protein Accumulation ◽  
E Coli ◽  
Enhanced Production

Abstract Background Serratia marcescens, a Gram-negative nosocomial pathogen secretes a 50 kDa multi-domain zinc metalloprotease called serratiopeptidase. Broad substrate specificity of serratiopeptidase makes it suitable for detergent and food processing industries The protein shows potent anti-inflammatory, anti-edemic, analgesic, antibiofilm activity and sold as an individual or fixed-dose enteric-coated tablets combined with other drugs. Although controversial, serratiopeptidase as drug is used in the treatment of chronic sinusitis, carpal tunnel syndrome, sprains, torn ligaments, and postoperative inflammation. Since the native producer of serratiopeptidase is a pathogenic microorganism, the current production methods need to be replaced by alternative approaches. Heterologous expression of serratiopeptidase in E. coli was tried before but not found suitable due to the limited yield, and other expression related issues due to its inherent proteolytic activity such as cytotoxicity, cell death, no expression, minimal expression, or inactive protein accumulation. Results Recombinant expression of mature form serratiopeptidase in E. coli seems toxic and resulted in the failure of transformation and other expression related issues. Although E. coli C43(DE3) cells, express protein correctly, the yield was compromised severely. Optimization of protein expression process parameters such as nutrient composition, induction point, inducer concentration, post-induction duration, etc., caused significant enhancement in serratiopeptidase production (57.9 ± 0.73% of total cellular protein). Expressed protein formed insoluble, enzymatically inactive inclusion bodies, and gave 40–45 mg/l homogenous (> 98% purity) biologically active and conformationally similar serratiopeptidase to the commercial counterpart upon refolding and purification. Conclusion Expression of mature serratiopeptidase in E. coli C43(DE3) cells eliminated the protein expression associated with toxicity issues. Further optimization of process parameters significantly enhanced the overexpression of protein resulting in the higher yield of pure and functionally active recombinant serratiopeptidase. The biological activity and conformational features of recombinant serratiopeptidase were very similar to the commercially available counterpart suggesting it-a potential biosimilar of therapeutic and industrial relevance.

REST/NRSF-induced changes of ChAT protein expression in the neocortex and hippocampus of the 3xTg-AD mouse model for Alzheimer's disease

Life Sciences ◽  
2014 ◽  
Vol 116 (2) ◽  
pp. 83-89 ◽  
Author(s):  
E. Orta-Salazar ◽  
A. Aguilar-Vázquez ◽  
H. Martínez-Coria ◽  
S. Luquín-De Anda ◽  
M. Rivera-Cervantes ◽  
...  
Keyword(s):  
Alzheimer’S Disease ◽  
Alzheimer's Disease ◽  
Mouse Model ◽  
Protein Expression ◽  
Induced Changes ◽  
Ad Mouse Model

Pregnancy-induced changes in ovine cerebral arteries

1992 ◽  
Vol 262 (1) ◽  
pp. R137-R143 ◽  
Author(s):  
A. D. Hull ◽  
D. M. Long ◽  
L. D. Longo ◽  
W. J. Pearce
Keyword(s):  
Cerebral Arteries ◽  
Calcium Ionophore ◽  
Cellular Protein ◽  
Calcium Ionophore A23187 ◽  
Ionophore A23187 ◽  
Cross Sectional ◽  
Pregnant Sheep ◽  
Relaxant Response ◽  
Cellular Protein Content ◽  
Induced Changes

We examined the effects of pregnancy on the ovine cerebral vasculature by comparing several characteristics of isolated endothelium-intact segments of three intracranial arteries including the middle cerebral (MCA), posterior communicating (PC), and basilar (BAS) arteries taken from pregnant sheep (138-143 days gestation, term approximately 145 days) and nonpregnant controls. For comparison, segments of the extracranial common carotid (COM) artery were also studied. With pregnancy, vessel water content increased (5.4-5.8%) in all arteries except the PC. Additionally, cellular protein content increased in all arteries (4.4-50.0%). Arterial stiffness, as determined by passive stress-strain determinations, was significantly decreased during pregnancy in the MCA but not in the larger arteries. Maximum contractile responses, when normalized to vessel wall cross-sectional area, were consistently greater in arteries from pregnant than in those from nonpregnant animals (10.1-49.7%). Relaxation to the endothelium-independent guanylate cyclase stimulator S-nitroso-N-acetyl penicillamine (SNAP) increased with pregnancy only in the distal MCA (approximately 17%). Endothelium-dependent relaxation to the calcium ionophore A23187 decreased only in the larger and more proximal COM (-39%). Thus pregnancy was associated with an increase in production of contractile force, a decrease in peripheral vascular stiffness, a decrease in the relaxant response to A23187 in the COM, and an increase in the relaxant response to SNAP in the MCA. Together, these findings indicate that pregnancy has widespread and important vessel specific cerebrovascular consequences that affect not only arterial composition, but also contractility and endothelial reactivity.

scholarly journals Gene and Protein Expression Pilot Profiling and Biomarkers in an Experimental Mouse Model of Hypertensive Glaucoma

2009 ◽  
Vol 234 (8) ◽  
pp. 918-930 ◽  
Author(s):  
Molly M. Walsh ◽  
Haiqing Yi ◽  
Julie Friedman ◽  
Kyoung-in Cho ◽  
Nomingerel Tserentsoodol ◽  
...  
Keyword(s):  
Protein Expression ◽  
Serum Amyloid ◽  
Cell Integrity ◽  
Molecular Processes ◽  
Experimental Glaucoma ◽  
Molecular Events ◽  
Gene And Protein Expression ◽  
Inflammatory Processes ◽  
Ganglion Neurons ◽  
Induced Changes

Glaucoma is a group of genetically heterogeneous neurodegenerative disorders causing the degeneration of the ganglion neurons of the retina. Increased intraocular pressure (IOP) is a hallmark risk factor promoting the death of ganglion neurons of the retina in glaucoma. Yet, the molecular processes underlying the degeneration of these neurons by increased IOP are not understood. To gain insight into the early molecular events and discover biomarkers induced by IOP, we performed gene and protein expression profiling to compare retinas of eyes with and without high IOP in a rodent model of experimental glaucoma. This pilot study found that the IOP-mediated changes in the transcription levels of a restricted set of genes implicated in peroxisomal and mitochondrial function, modulation of neuron survival and inflammatory processes, were also accompanied by changes in the levels of proteins encoded by the same genes. With the exception of the inflammatory markers, serum amyloid-A1 (SAA1) and serum amyloid-A2 (SAA2), the IOP-induced changes in protein expression were restricted to ganglion neurons of the retina and they were detected also in the vitreous, thus suggesting an early IOP-mediated loss of ganglion cell integrity. Interestingly, SAA1 and SAA2 were induced in retinal microglia cells, whereas they were reduced in sera of IOP-responsive mice. Hence, this study defines novel IOP-induced molecular processes, biomarkers and sources thereof, and it further validates the extension of the analyses herein reported to other genes modulated by IOP.

scholarly journals Influence of Acute High Glucose on Protein Abundance Changes in Murine Glomerular Mesangial Cells

2016 ◽  
Vol 2016 ◽  
pp. 1-11 ◽  
Author(s):  
Michelle T. Barati ◽  
James C. Gould ◽  
Sarah A. Salyer ◽  
Susan Isaacs ◽  
Daniel W. Wilkey ◽  
...  
Keyword(s):  
Protein Expression ◽  
Posttranslational Modifications ◽  
High Glucose ◽  
Mesangial Cells ◽  
Expression Patterns ◽  
Growth Conditions ◽  
Glomerular Mesangial Cells ◽  
Two Dimensional Gel Electrophoresis ◽  
Glucose Levels ◽  
Flight Mass Spectrometry

The effects of acute exposure to high glucose levels as experienced by glomerular mesangial cells in postprandial conditions and states such as in prediabetes were investigated using proteomic methods. Two-dimensional gel electrophoresis and matrix assisted laser desorption ionization time of flight mass spectrometry methods were used to identify protein expression patterns in immortalized rat mesangial cells altered by 2 h high glucose (HG) growth conditions as compared to isoosmotic/normal glucose control (NG⁎) conditions. Unique protein expression changes at 2 h HG treatment were measured for 51 protein spots. These proteins could be broadly grouped into two categories: (1) proteins involved in cell survival/cell signaling and (2) proteins involved in stress response. Immunoblot experiments for a protein belonging to both categories, prohibitin (PHB), supported a trend for increased total expression as well as significant increases in an acidic PHB isoform. Additional studies confirmed the regulation of proteasomal subunit alpha-type 2 and the endoplasmic reticulum chaperone and oxidoreductase PDI (protein disulfide isomerase), suggesting altered ER protein folding capacity and proteasomal function in response to acute HG. We conclude that short term high glucose induces subtle changes in protein abundances suggesting posttranslational modifications and regulation of pathways involved in proteostasis.

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