Affinity Precipitation Research Articles

Thy-1-mediated cell–cell contact induces astrocyte migration through the engagement of αVβ3 integrin and syndecan-4

Cell adhesion to the extracellular matrix proteins occurs through interactions with integrins that bind to Arg-Gly-Asp (RGD) tripeptides, and syndecan-4, which recognizes the heparin-binding domain of other proteins. Both receptors trigger signaling pathways, including those that activate RhoGTPases such as RhoA and Rac1. This sequence of events modulates cell adhesion to the ECM and cell migration. Using a neuron–astrocyte model, we have reported that the neuronal protein Thy-1 engages αVβ3 integrin and syndecan-4 to induce RhoA activation and strong astrocyte adhesion to their underlying substrate. Thus, because cell–cell interactions and strong cell attachment to the matrix are considered antagonistic to cell migration, we hypothesized that Thy-1 stimulation of astrocytes should preclude cell migration. Here, we studied the effect of Thy-1 expressing neurons on astrocyte polarization and migration using a wound-healing assay and immunofluorescence analysis. Signaling molecules involved were studied by affinity precipitation, western blotting and the usage of specific antibodies. Intriguingly, Thy-1 interaction with its two receptors was found to increase astrocyte polarization and migration. The latter events required interactions of these receptors with both the RGD-like sequence and the heparin-binding domain of Thy-1. Additionally, prolonged Thy-1-receptor interactions inhibited RhoA activation while activating FAK, PI3K and Rac1. Therefore, sustained engagement of integrin and syndecan-4 with the neuronal surface protein Thy-1 induces astrocyte migration. Interestingly we identify here, a cell–cell interaction that despite initially inducing strong cell attachment, favors cell migration upon persistent stimulation by engaging the same signaling receptors and molecules as those utilized by the extracellular matrix proteins to stimulate cell movement.

ZBP-89 Regulates Expression of Tryptophan Hydroxylase I and Mucosal Defense Against Salmonella Typhimurium in Mice

ZBP-89 (also ZNF148 or Zfp148) is a butyrate-inducible zinc finger transcription factor that binds to GC-rich DNA elements. Deletion of the N-terminal domain is sufficient to increase mucosal susceptibility to chemical injury and inflammation. We investigated whether conditional deletion of ZBP-89 from the intestinal and colonic epithelium of mice increases their susceptibility to pathogens such as Salmonella typhimurium. We generated mice with a conditional null allele of Zfp148 (ZBP-89(FL/FL)) using homologous recombination to flank Zfp148 with LoxP sites (ZBP-89(FL/FL)), and then bred the resulting mice with those that express VillinCre. We used microarray analysis to compare gene expression patterns in colonic mucosa between ZBP-89(ΔInt) and C57BL/6 wild-type mice (controls). Mice were gavaged with 2 isogenic strains of S. typhimurium after administration of streptomycin. Microarray analysis revealed that the colonic mucosa of ZBP-89(ΔInt) mice had reduced levels of tryptophan hydroxylase 1 (Tph1) messenger RNA, encoding the rate-limiting enzyme in enterochromaffin cell serotonin (5-hydroxytryptamine [5HT]) biosynthesis. DNA affinity precipitation demonstrated direct binding of ZBP-89 to the mouse Tph1 promoter, which was required for its basal and butyrate-inducible expression. ZBP-89(ΔInt) mice did not increase mucosal levels of 5HT in response to S. typhimurium infection, and succumbed to the infection 2 days before control mice. The ΔhilA isogenic mutant of S. typhimurium lacks this butyrate-regulated locus and stimulated, rather than suppressed, expression of Tph1 approximately 50-fold in control, but not ZBP-89(ΔInt), mice, correlating with fecal levels of butyrate. ZBP-89 is required for butyrate-induced expression of the Tph1 gene and subsequent production of 5HT in response to bacterial infection in mice. Reductions in epithelial ZBP-89 increase susceptibility to colitis and sepsis after infection with S. typhimurium, partly because of reduced induction of 5HT production in response to butyrate and decreased secretion of antimicrobial peptides.

Polymer Nanoparticle–Protein Interface. Evaluation of the Contribution of Positively Charged Functional Groups to Protein Affinity

Cationic-functionalized polymer nanoparticles (NPs) show strikingly distinct affinities to proteins depending on the nature of the cationic functional group. N-Isopropylacrylamide (NIPAm) polymer NPs incorporating three types of positively charged functional groups (guanidinium, primary amino, and quaternary ammonium groups) were prepared by precipitation polymerization. The affinities to fibrinogen, a protein with an isoelectric point (pI) of 5.5, were compared using UV-vis spectrometry and a quartz crystal microbalance (QCM). Guanidinium-containing NPs showed the highest affinity to fibrinogen. The observation is attributed to strong, specific interactions with carboxylate groups on the protein surface. The affinity of the positively charged NPs to proteins with a range of pIs revealed that protein-NP affinity is due to a combination of ionic, hydrogen bonding, and hydrophobic interactions. Protein affinity can be modulated by varying the composition of these functional monomers in the acrylamide NPs. Engineered NPs containing the guanidinium group with hydrophobic and hydrogen bonding functional groups were used in an affinity precipitation for the selective separation of fibrinogen from a plasma protein mixture. Circular dichroism (CD) revealed that the protein was not denatured in the process of binding or release.

Y-39983 downregulates RhoA/Rho-associated kinase expression during its promotion of axonal regeneration

Y-39983, a selective Rho-associated kinase (ROCK) inhibitor, promotes axonal regeneration of damaged retinal ganglion cells (RGCs). The present study investigated the effects of Y-39983 on RhoA/ROCK expression during promotion of axonal regeneration using a rat optic nerve crush (ONC) model. Herein, we demonstrated that Y-39983 significantly enhanced the survival and axonal regeneration of RGCs after ONC. Using a pull‑down assay and affinity precipitation to examine the activity of RhoA, we detected the decreased expression of active-RhoA after using Y-39983. The expression of ROCK1 and ROCK2 was significantly decreased as demonstrated by RT-PCR, immunohistochemistry and western blot analysis. The downregulation of active-RhoA, ROCK1 and ROCK2 expression by Y-39983 coincided with the appearance of larger numbers of regenerating axons. In conclusion, Y-39983 downregulated the expression of active-RhoA, ROCK1 and ROCK2 during its promotion of axonal regeneration.

Direct Interaction between a Precursor Mature Domain and Transport Component Tha4 during Twin Arginine Transport of Chloroplasts

Proteins destined for the thylakoid lumen of chloroplasts must cross three membranes en route. The chloroplast twin arginine translocation (cpTat) system facilitates the transport of about one-half of all proteins that cross the thylakoid membrane in chloroplasts. Known mechanistic features of the cpTat system are drastically different from other known translocation systems, notably in its formation of a transient complex to transport fully folded proteins utilizing only the protonmotive force generated during photosynthesis for energy. However, key details, such as the structure and composition of the translocation pore, are still unknown. One of the three transmembrane cpTat components, Tha4, is thought to function as the pore by forming an oligomer. Yet, little is known about the topology of Tha4 in thylakoid, and little work has been done to detect precursor-Tha4 interactions, which are expected if Tha4 is the pore. Here, we present evidence of the interaction of the precursor with Tha4 under conditions leading to transport, using cysteine substitutions on the precursor and Tha4 and disulfide bond formation in pea (Pisum sativum). The mature domain of a transport-competent precursor interacts with the amphipathic helix and amino terminus of functional Tha4 under conditions leading to transport. Detergent solubilization of thylakoids post cross linking and blue-native polyacrylamide gel electrophoresis analysis shows that Tha4 is found in a complex containing precursor and Hcf106 (i.e. the cpTat translocase). Affinity precipitation of the cross-linked complex via Tha4 clearly demonstrates that the interaction is with full-length precursor. How these data suggest a role for Tha4 in cpTat transport is discussed.

Epigenetic Down-Regulation of NFkB-Mediated FAS Gene Transcription During Progression of Myelodysplastic Syndromes

AbstractAbstract 3503 BackgroundCD95/Fas, a member of the death receptor family, can induce cell death upon binding of its ligand, FasL Suppression of Fas signaling leads to autoimmune diseases while overexpression of Fas and FasL accounts for inappropriate apoptosis of erythroid cells in low grade myelodysplastic syndromes (MDS). The progression of MDS into secondary acute myeloid leukaemia (sAML) is associated with inhibition of hematopoietic progenitor cell death which involves the methylation of FAS gene promoter. We have previously demonstrated that FAS gene expression was increased in MDS and down-regulated when disease progresses to sAML. We further investigated the molecular mechanisms of FAS gene extinction during MDS progression. The transcription factor NFκB is functionnal as homo or heterodimer and is composed of proteins p65 or p50. The constitutive activation of NFkB pathway identified in solid tumors and in diverse hematological malignancies was suggested to promote tumor cell survival.The aim of the present work was to further investigate the interplay between FAS gene and NFkB during the progression of MDS to sAML. Methods85 consecutive patients with a diagnosis of MDS and 15 age-matched controls with normal bone marrow (BM) were included between 2005 and 2011. Using cell lines and primary normal and leukemic bone marrow cells, we explored the p65/RelA NFkB subunit localisation by immunofluorescence and immunoblotting, inhibited NFkB pathway with the IKKa inhibitor BAY11–7082, quantified FAS transcript and protein by RT-qPCR and flow cytometry respectively, and explored p65/RelA binding to DNA by pull-down and chromatin immunoprecipitation (ChIP). ResultsIn order to explore the role of NFkB in FAS gene expression, we used the HL-60 human leukemic cells which spontaneously express Fas at their surface and the SW-480 colon carcinoma cells which do not. We show that TNFα increased FAS gene and Fas receptor expression only in HL-60 cells indicating a cell type-dependent regulation of FAS gene expression. We conclude that NFkB is responsible for FAS gene expression in HL60 cell line.The FAS gene promoter contains putative NFκB sites in the 5’ UTR region spreading between −899 and the transcription start site. By using in vitro DNA affinity precipitation experiments, we show that P65/RelA bound to each of the 3 putative NFκB binding sites on FAS promoter upon TNFa treatment. We conclude that p65/RelA binds to the chromatin at FAS promoter in a cell type-dependent manner to regulate FAS gene expression in cells.Then, we hypothesized that DNA methylation in FAS gene 5'UTR regulatory regions may prevent the docking of NFkB. ChIP Experiments identified one active mark H3K9/14Ac and none of the two repressive marks H3K27me3 and H3K9me2 in HL60 cell line while the repressive mark H3K9me2 was expressed in SW480 cell line These results suggested that the chromatin conformation at FAS promoter was open in Fas-positive HL-60 cells, and repressed in Fas-negative SW-480 cells. By treatment with azacitidine, which stimulated the expression of Fas in SW-480 but not in HL-60 cells and by experiments using TNFa, we show that NFkB-dependent FAS gene expression is epigenetically regulated.We used ChIP to explore the binding of p65/RelA to the chromatin at FAS promoter in 4 Fas-expressing and 6 Fas-negative MDS samples. The expression of Fas was proportional to the recruitment of p65/RelA at the gene promoter. Among 16 MDS samples where FAS protein expression was positive, BAY11–7082 was able to decrease Fas expression in 12 of them. In addition, we show that azacitidine rescues NFκB-induced Fas expression in primary MDS/sAML cells. Altogether, NFκB controls the transcription of FAS gene in primary BM cells from MDS or sAML and azacitidine can induce FAS promoter demethylation, thus facilitates p65/RelA binding and FAS gene transcription in myeloid cells. ConclusionThe present study demonstrates that NF-kB directly promotes FAS gene expression in normal and myelodysplastic hematopoietic progenitor cells and that, when MDS progress into sAML. The inhibition of FAS gene expression observed in sAML is due to the methylation of NFkB binding site in the gene promoter, which can be reversed by the demethylating agent 5-azacitidine. The increased expression of FAS gene induced by 5-azacitidine now deserves to be tested as a biomarker of the drug activity. Disclosures:No relevant conflicts of interest to declare.

The Extracytoplasmic Function Sigma Factor EcfO Protects Bacteroides fragilis against Oxidative Stress

The anaerobe Bacteroides fragilis is a highly aerotolerant, opportunistic pathogen that is an important component of the human intestinal microbiota. Aerotolerance has been linked to a robust oxidative stress response, which in turn is necessary for maximal virulence in a mouse intra-abdominal abscess model. During oxidative stress, there is a dynamic change in gene expression that encompasses a third of the genome, but there is a paucity of information on factors that control this response. A large number of transcription regulators, including about 14 extracytoplasmic function (ECF) sigma factors, are affected by oxidative stress, and one of these, EcfO, was used as a model of ECF sigma factor activity during stress. Genetic and biochemical experiments showed that EcfO was located in an operon with a structurally unique anti-sigma factor, Reo. Cells deleted for EcfO were impaired during exposure to oxygen or other forms of oxidative stress, whereas reo mutants were more resistant to stress. Protein-protein interaction experiments demonstrated that Reo directly interacts with and regulates the activity of EcfO. Expression microarray and chromatin affinity precipitation assays were used to identify target genes regulated by EcfO, and an EcfO recognition sequence was identified. The results revealed that EcfO controls a regulon of novel lipoproteins whose distribution in nature is restricted to members of the Bacteroidetes phylum.

ELP-z and ELP-zz capturing scaffolds for the purification of immunoglobulins by affinity precipitation

The increasing demand of monoclonal antibodies for therapeutic applications along with the high manufacturing cost have made it necessary to evaluate better process options and technologies for their purification. Affinity precipitation is an attractive alternative to traditional chromatographic methods by affording effective purification using a simple environmental trigger. The feature of elastin-like-protein (ELP) fused with antibody binding domains has already been explored for the purification of antibodies. However, ELP when fused with the bulkier domains such as Protein A, resulted in lower protein production. In this study, ELP was fused to smaller synthetic IgG binding domains such as the z or zz domain, resulting in up to 10-fold higher level of production. Both ELP-z and ELP-zz bind tightly to human immunoglobulin (HIgG) with a dissociation constant of 768±142nM and 68±23nM, respectively. Owing to the higher binding affinity, the use of ELP-zz resulted in more than 99% recovery of HIgG in four repeated binding and elution cycles with no observable decrease in the purification performance. The same binding and elution cycle was successfully implemented for the purification of monoclonal antibodies from hybridoma culture supernatant with close to 100% recovery.

Affinity precipitation of cellulase using pH-response polymer with Cibacron Blue F3GA

A pH-response polymer PMMDN was synthesized and then ligand Cibacron Blue F3GA was immobilized on the polymer for affinity precipitation of cellulase. The isoelectric point of the affinity polymer was 5.0 and the recovery was 99.8% in presence of 0.8mol/l Na2SO4. Optimal adsorptions could be achieved at pH 7.2 and 1.0mol/l NaCl. FTIR confirmed that ligand Cibacron Blue F3GA was immobilized on the polymer and cellulase was adsorbed on PMMDN–Cibacron Blue F3GA. The optimal eluant was 0.1mol/lTris–HCl (pH 7.1) with 20.0% glycol. The elution recovery of total cellulase activity and endo-glucanase activity was almost 84.4% and 99.8%, respectively. The SDS–PAGE showed the main bands with a molecular weight corresponding to that of the native cellulase.

Role of smart polymers in protein purification and refolding

Affinity precipitation is a non-chromatographic method which is useful for purification and refolding of proteins. Quite often, a stimuli-sensitive polymer can be identified which selectively binds to the desired protein. For separation, the protein can be recovered from the precipitate of the protein-smart polymer complex. In case of a refolding experiment, binding of the solubilized protein (in its denatured form) with the polymer leads to the refolding of the protein.

Role of ornithine decarboxylase in regulation of estrogen receptor alpha expression and growth in human breast cancer cells

Our previous studies demonstrated that specific polyamine analogues, oligoamines, down-regulated the activity of a key polyamine biosynthesis enzyme, ornithine decarboxylase (ODC), and suppressed expression of estrogen receptor alpha (ERα) in human breast cancer cells. However, the mechanism underlying the potential regulation of ERα expression by polyamine metabolism has not been explored. Here, we demonstrated that RNAi-mediated knockdown of ODC (ODC KD) down-regulated the polyamine pool, and hindered growth in ERα-positive MCF7 and T47D and ERα-negative MDA-MB-231 breast cancer cells. ODC KD significantly induced the expression and activity of the key polyamine catabolism enzymes, spermine oxidase (SMO) and spermidine/spermine N (1)-acetyltransferase (SSAT). However, ODC KD-induced growth inhibition could not be reversed by exogenous spermidine or overexpression of antizyme inhibitor (AZI), suggesting that regulation of ODC on cell proliferation may involve the signaling pathways independent of polyamine metabolism. In MCF7 and T47D cells, ODC KD, but not DFMO treatment, diminished the mRNA and protein expression of ERα. Overexpression of antizyme (AZ), an ODC inhibitory protein, suppressed ERα expression, suggesting that ODC plays an important role in regulation of ERα expression. Decrease of ERα expression by ODC siRNA altered the mRNA expression of a subset of ERα response genes. Our previous analysis showed that oligoamines disrupt the binding of Sp1 family members to an ERα minimal promoter element containing GC/CA-rich boxes. By using DNA affinity precipitation and mass spectrometry analysis, we identified ZBTB7A, MeCP2, PARP-1, AP2, and MAZ as co-factors of Sp1 family members that are associated with the ERα minimal promoter element. Taken together, these data provide insight into a novel antiestrogenic mechanism for polyamine biosynthesis enzymes in breast cancer.

Regulation of nicotinic acetylcholine receptor turnover by MuRF1 connects muscle activity to endo/lysosomal and atrophy pathways

Muscle atrophy is a process of muscle wasting induced under a series of catabolic stress conditions, such as denervation, disuse, cancer cachexia, heart and renal failure, AIDS, and aging. Neuromuscular junctions (NMJs), the synapses between motor neurons and muscle fibers undergo major changes in atrophying muscles, ranging from mild morphological alterations to complete disintegration. In this study, we hypothesized that remodeling of NMJs and muscle atrophy could be linked together. To test this, we examined if a major atrophy-promoting E3 ubiquitin ligase, MuRF1, is involved in the maintenance of NMJs. Immunofluorescence revealed that MuRF1 is highly enriched close to the NMJ. Affinity precipitation and in vivo imaging showed that MuRF1 interacts in endocytic structures with both, acetylcholine receptor, the primary postsynaptic protein of the NMJ, as well as with Bif-1, an autophagy- and endocytosis-regulating factor. In vivo imaging, radio labeling, and weighing approaches demonstrated that metabolic destabilization of acetylcholine receptors and muscle atrophy induced by denervation were significantly rescued in MuRF1-KO animals. Notably, interaction with Bif-1, and the rescue of AChR lifetime and muscle atrophy were specific to MuRF1 but not MuRF2. Our data demonstrate an involvement of MuRF1 in membrane protein-turnover, including the degradation of AChRs at the NMJ under atrophying conditions where MuRF1 also interacts and associates with Bif-1.

1679P - Heat Shock Protein 90 Inhibition Downregulates C-Kit Expression in Gist Cells Through Diminishing Transcription and Enhancing Protein Degradation via both Autophagy and Proteasome Pathways

ABSTRACT Background Gastrointestinal stromal tumor (GIST) is a mutant oncoprotein c-Kit droved cancer. Patients suffered drug resistance after prolonged standard treatment of tyrosine kinase inhibitor (TKI), imatinib mesylate (IM). Inhibition of heat-shock protein 90 (Hsp90), a chaperone responsible for protein maturation and stability, causing its client proteins degradation, as c-Kit, is a developing therapy for cancer. However, the mechanisms of Hsp90 inhibition-induced c-Kit downregulation in post-translational and transcriptional levels have rarely been investigated. Methods and results Our data showed that NVP-AUY922 (AUY922), a new class of Hsp90 inhibitor, inhibited the growth of GIST882 and GIST48 cells with mutant c-Kit protein expression that was accompanied with reduction of both total and phosphor-c-Kit protein and induction of apoptosis. Pharmacological inhibition on either autophagy or proteasome degradation pathway could partially reverse endogenous c-Kit turnover and AUY922-induced c-Kit reduction. These findings were further confirmed by the co-localization of c-Kit with either LC-3B or acridine orange-labeled autophagosome in confocal microscopy, and delay of c-Kit degradation by silencing autophagosome essential Beclin-1 protein. In addition, AUY922 could also reduce c-Kit mRNA without affecting its degradation. Our preliminary data showed that activities and nuclear levels of several transcription factors in GIST cells were diminished after AUY922 treatment. DNA affinity precipitation assay and chromatin immunoprecipitation (ChIP) will be performed to validate the binding of the candidate transcript factors on the c-Kit promoter. Conclusions This is the first report showing the involvement of autophagy in endogenous as well as Hsp90 inhibitor-induced c-Kit degradation. Moreover, AUY922 treatment resulted in downregulation of c-Kit through protein degradation and transcriptional mRNA regulation in GIST cells. These findings address the mechanisms of AUY922 on c-Kit protein downregulation and highlight the potential use of AUY922 in TKI-refractory, c-Kit-expressing GIST. Disclosure All authors have declared no conflicts of interest.

P53 Acts as a Co-Repressor to Regulate Keratin 14 Expression during Epidermal Cell Differentiation

During epidermal cell differentiation, keratin 14 (K14) expression is down-regulated, p53 expression varies, and the expression of the p53 target genes, p21 and 14-3-3σ, increases. These trends suggest that the relative transcriptional activity of p53 is increased during epidermal cell differentiation. To determine the relationship between K14 and p53, we constructed K14 promoters of various sizes and found that wild-type p53 could repress the promoter activity of all of the K14 promoter constructs in H1299 cells. K14-p160 contains an SP1 binding site mutation that prevents p53 from repressing K14 expression. Using a DNA affinity precipitation assay, we confirmed that p53 forms a complex with SP1 at the SP1 binding site between nucleotides -48 and -43 on the K14 promoter. Thus, our data indicate that p53 acts as a co-repressor to down-regulate K14 expression by binding to SP1. Next, we used a 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced epidermal cell differentiation model to examine the inhibition of K14 expression caused by increased p53 activity. Human ovarian teratocarcinoma C9 cells were treated with TPA to induce differentiation. Over-expression of the dominant negative p53 mutant ΔTAp53, which inhibits p53 activity, prevented the TPA-induced K14 down-regulation in C9 cells. Furthermore, treatment of normal primary human foreskin keratinocytes (PHFK) with the p53 inhibitor pifithrin-α (PFT-α) showed that the inhibition of p53 activity relieves K14 repression during epidermal cell differentiation. Finally, we found that TPA induces the phosphorylation of p53 at residue 378, which enhances the affinity of p53 to bind to Sp1 and repress K14 expression.

Erythropoietin promotes axonal regeneration after optic nerve crush in vivo by inhibition of RhoA/ROCK signaling pathway

We investigated whether the RhoA/ROCK pathway was involved in the effect of erythropoietin (EPO) to promote retinal ganglion cells (RGCs) axonal regeneration in a rat optic nerve crush (ONC) model. We demonstrated that both EPO and ROCK inhibitor Y-27632 significantly enhanced RGCs survival and axon regeneration in vivo, and the effects of these agents were additive. Expression of active-RhoA was decreased after EPO or Y-27632 per pull down assay and affinity precipitation. Administration of EPO and Y-27632 cocktail resulted in even more RhoA inactivation, decreased expression of ROCK-1 and ROCK-2, and increased expression of growth associated protein-43 (GAP-43) protein per immunohistochemistry and western blot analysis. Down-regulation of active-RhoA, ROCK-1, and ROCK-2 expression by EPO coincided with the appearance of larger numbers of regenerating axons. In conclusion, the RhoA/ROCK signaling pathway was involved in the EPO effect to promote RGCs axon regeneration after ONC.

α-Amylase immobilization on gelatin: Optimization of process variables

α-Amylase was extracted and purified from soybean seeds to apparent homogeneity by affinity precipitation. The homogeneous enzyme preparation was immobilized on gelatin matrix using glutaraldehyde as an organic hardener. Response surface methodology (RSM) and 3-level-3-factor Box–Behnken design was employed to evaluate the effects of immobilization parameters, such as gelatin concentration, glutaraldehyde concentration and hardening time on the activity of immobilized α-amylase. The results showed that 20% gelatin (w/v), 10% glutaraldehyde (v/v) and 1 h hardening time yielded an optimum immobilization of 82.5%.

EMERGING TRENDS IN DESIGNING SHORT AND EFFICIENT PROTEIN PURIFICATION PROTOCOLS

Protein purification is carried out in both academi c and industrial sectors. Both non-chromatographic approaches (precipitation, crystallization, partiti oning) and chromatographic methods are used. When recombinant proteins are over expressed in foreign hosts, inclusion bodies are obtained. Many purification methods can be used to refold proteins . Many strategies allow one to avoid formation of inclusion bodies. The concept of affinity based sep aration extends to non-chromatographic methods as well. Affinity precipitation and Macro-(affinity li gand) Facilitated Three Phase Partitioning (MLFTPP) are two examples of this. Produced as fusion protei ns, most of the proteins are purified via interacti on of their affinity tag coupled to a solid matrix. Na notechnology has made magnetic based methods even more powerful. Expanded bed chromatography, aqueous two phase systems and MLFTPP allow one to deal directly with feed containing particulate matt er. Membrane based separations are another powerful option. Finally crystallization at the ind ustrial level has further evolved to reoccupy an important place in protein purification. Overall, t he main trends are: integrate upstream and downstream phases and reduce the number of steps required for purifying a protein. This has become possible by adopting variety of non-chromatographic methods. More important, bringing affinity based steps earlier in the purification have helped achie ving this.

Affinity Precipitation of Active Rho-GEFs Using a GST-tagged Mutant Rho Protein (GST-RhoA(G17A)) from Epithelial Cell Lysates

Proteins of the Rho family of small GTPases are central regulators of the cytoskeleton, and control a large variety of cellular processes, including cell migration, gene expression, cell cycle progression and cell adhesion 1. Rho proteins are molecular switches that are active in GTP-bound and inactive in GDP-bound state. Their activation is mediated by a family of Guanine-nucleotide Exchange Factor (GEF) proteins. Rho-GEFs constitute a large family, with overlapping specificities 2. Although a lot of progress has been made in identifying the GEFs activated by specific signals, there are still many questions remaining regarding the pathway-specific regulation of these proteins. The number of Rho-GEFs exceeds 70, and each cell expresses more than one GEF protein. In addition, many of these proteins activate not only Rho, but other members of the family, contributing further to the complexity of the regulatory networks. Importantly, exploring how GEFs are regulated requires a method to follow the active pool of individual GEFs in cells activated by different stimuli. Here we provide a step-by-step protocol for a method used to assess and quantify the available active Rho-specific GEFs using an affinity precipitation assay. This assay was developed a few years ago in the Burridge lab 3,4 and we have used it in kidney tubular cell lines 5,6,7. The assay takes advantage of a "nucleotide free" mutant RhoA, with a high affinity for active GEFs. The mutation (G17A) renders the protein unable to bind GDP or GTP and this state mimics the intermediate state that is bound to the GEF. A GST-tagged version of this mutant protein is expressed and purified from E. coli, bound to glutathione sepharose beads and used to precipitate active GEFs from lysates of untreated and stimulated cells. As most GEFs are activated via posttranslational modifications or release from inhibitory bindings, their active state is preserved in cell lysates, and they can be detected by this assay8. Captured proteins can be probed for known GEFs by detection with specific antibodies using Western blotting, or analyzed by Mass Spectrometry to identify unknown GEFs activated by certain stimuli.

Affinity Precipitation of Active Rho-GEFs Using a GST-tagged Mutant Rho Protein (GST-RhoA(G17A)) from Epithelial Cell Lysates

Proteins of the Rho family of small GTPases are central regulators of the cytoskeleton, and control a large variety of cellular processes, including cell migration, gene expression, cell cycle progression and cell adhesion 1. Rho proteins are molecular switches that are active in GTP-bound and inactive in GDP-bound state. Their activation is mediated by a family of Guanine-nucleotide Exchange Factor (GEF) proteins. Rho-GEFs constitute a large family, with overlapping specificities 2. Although a lot of progress has been made in identifying the GEFs activated by specific signals, there are still many questions remaining regarding the pathway-specific regulation of these proteins. The number of Rho-GEFs exceeds 70, and each cell expresses more than one GEF protein. In addition, many of these proteins activate not only Rho, but other members of the family, contributing further to the complexity of the regulatory networks. Importantly, exploring how GEFs are regulated requires a method to follow the active pool of individual GEFs in cells activated by different stimuli. Here we provide a step-by-step protocol for a method used to assess and quantify the available active Rho-specific GEFs using an affinity precipitation assay. This assay was developed a few years ago in the Burridge lab 3,4 and we have used it in kidney tubular cell lines 5,6,7. The assay takes advantage of a "nucleotide free" mutant RhoA, with a high affinity for active GEFs. The mutation (G17A) renders the protein unable to bind GDP or GTP and this state mimics the intermediate state that is bound to the GEF. A GST-tagged version of this mutant protein is expressed and purified from E. coli, bound to glutathione sepharose beads and used to precipitate active GEFs from lysates of untreated and stimulated cells. As most GEFs are activated via posttranslational modifications or release from inhibitory bindings, their active state is preserved in cell lysates, and they can be detected by this assay8. Captured proteins can be probed for known GEFs by detection with specific antibodies using Western blotting, or analyzed by Mass Spectrometry to identify unknown GEFs activated by certain stimuli.

Regulation of Transcription Factor Twist Expression by the DNA Architectural Protein High Mobility Group A2 during Epithelial-to-Mesenchymal Transition

Deciphering molecular mechanisms that control epithelial-to-mesenchymal transition (EMT) contributes to our understanding of how tumor cells become invasive and competent for intravasation. We have established that transforming growth factor β activates Smad proteins, which induce expression of the embryonic factor high mobility group A2 (HMGA2), which causes mesenchymal transition. HMGA2 associates with Smad complexes and induces expression of an established regulator of EMT, the zinc finger transcription factor Snail. We now show that HMGA2 can also induce expression of a second regulator of EMT, the basic helix-loop-helix transcription factor Twist. Silencing of endogenous Twist demonstrated that this protein acts in a partially redundant manner together with Snail. Double silencing of Snail and Twist reverts mesenchymal HMGA2-expressing cells to a more epithelial phenotype when compared with single silencing of Snail or Twist. Furthermore, HMGA2 can directly associate with A:T-rich sequences and promote transcription from the Twist promoter. The new evidence proposes a model whereby HMGA2 directly induces multiple transcriptional regulators of the EMT program and, thus, is a potential biomarker for carcinomas displaying EMT during progression to more advanced stages of malignancy.