Human Cytomegalovirus Immediate-Early 1 Protein Rewires Upstream STAT3 to Downstream STAT1 Signaling Switching an IL6-Type to an IFNγ-Like Response.
... results also reveal that IE1, a protein considered to be a key activator of the hCMV productive cycle, has an unanticipated role in tempering viral replication. Author Summary Our previous work has shown that the human cytomegalovirus (hCMV) major immediate-early 1 protein (IE1) modulates host cell signaling pathways involving proteins of the signal transducer ... to STAT1 Signaling tested genes (S1 Fig). Finally, we tested the effects of a mutant STAT3 protein (STAT3α_Y705F), which is expressed to similar levels as the wild-type protein and resistant to Y705 but not S727 phosphorylation (Fig 2E, left panel). This mutant protein is known to act in a trans-dominant negative fashion on expression of STAT3-responsive ... of the indicated HA-tagged wild-type or mutant IE1 proteins were treated with dox for 72 h. Whole cell protein extracts were prepared and analyzed by immunoblotting for IE1 (HA tag) and GAPDH. (C) TetR cells without (w/o) or with inducible expression of the indicated HA-tagged wild-type or mutant IE1 proteins were treated with dox for 72 h. Whole cell
Interaction between plate make and protein in protein crystallisation screening.
... different proteins. From our analysis of protein crystallisation, we found a significant interaction between plate make and the specific protein being crystallised. Conclusions/Significance: Protein crystal structure determination is the principal method for determining protein structure but is limited by the need to produce crystals of the protein under ... of Introduction Protein crystallography is the major structural biology technique, accounting for more than 80% of solved protein structures, and commercially available 96-well plates are essential components of modern high-throughput protein crystallisation condition screening . Vapour diffusion is the most frequently used technique for protein crystallisation ... crystallisation condition or reservoir solution and the protein drop, within a sealed plate well. The protein drop contains the protein mixed with the crystallisation condition. The hanging and sitting drop vapour diffusion formats utilise 96-well plates in different ways. For the hanging drop method, the protein drop is suspended above the well solution
Characterization of SLCO5A1/OATP5A1, a solute carrier transport protein with non-classical function.
... The proteins mAno1His (lanes 5–7) and SLC26A3 (lanes 8, 9) served as dissociation control for the oligomeric protein structure. (2, homodimer of the high-mannoseglycosylated SLCO5A1 protein; 1, monomer of the high-mannose-glycosylated SLCO5A1 protein; #, monomer of the complex-glycosylated SLCO5A1 protein) . doi:10.1371/journal.pone.0083257.g002 protein ... mutant SLCO5A1 proteins do not transport known OATP substrates Human WT and mutant SLCO5A1 N-glycosylated proteins are expressed in intracellular membranes and on the plasma membrane of HeLa cells The SLCO5A1 protein was found to be expressed on the plasma membrane of X. laevis oocytes (Fig. 2A). To study whether the human SLCO5A1 protein shares transport ... e83257 Biochemical Properties and Function of OATP5A1 tagged WT and mutant (L33F) SLCO5A1 protein. Protein expression was induced with tetracycline for 48 h. The proteins were deglycosylated with either endoglycosidase H (EndoH) (E) or PNGase F (P). Tubulin served as loading control. C) Protein expression of the YFP-tagged WT SLCO5A1 or its L33F mutant after induction
Analysis of human protein replacement stable cell lines established using snoMEN-PR vector.
... and characterised two such partial protein replacement human cell lines (snoMENPR). Quantitative mass spectrometry was used to analyse the specificity of knock-down and replacement at the protein level and also showed an increased pull-down efficiency of protein complexes containing exogenous, tagged proteins in the protein replacement cell lines, as ... enrichment of GFP– SMN1-associated proteins (protein replacement cell line IP versus the control IP: heavy/light ratio) and the y axis represents enrichment of GFP–SMN1-associated proteins (over-expression cell line IP versus the control IP: medium/light ratio) (Figure S5). Contaminant proteins are clustered around the origin, while proteins specifically co-purifying ... motor neurons protein (SMN) are examples of proteins where toxic effects of overexpression have been reported. UBF belongs to the sequence-nonspecific class of high mobility group (HMG) proteins and functions in RNA polymerase I transcription [9,10]. UBF1 depletion using siRNA leads to inhibition of rRNA transcription Introduction Methods for studying protein
Exploiting the yeast L-A viral capsid for the in vivo assembly of chimeric VLPs as platform in vaccine development and foreign protein expression.
... heterologous model protein (GFP), (ii) a per se toxic protein (K28 a-subunit), and (iii) a particle-associated and fully recyclable biotechnologically relevant enzyme (esterase A). Thus, yeast viral Gag represents a unique platform for the in vivo assembly of chimeric VLPs, equally attractive and useful in vaccine development and recombinant protein production. Citation: ... copies of a viral capsid and/or envelope protein capable to self-assemble into VLPs of defined spherical symmetry in vivo [1–3]. Currently, VLPs composed of a structural protein are often used as particulate antigen in the design of prototype vaccines as they possess several advantages over conventional monomeric protein immunogens . Firstly, most VLPs ... version of the immunodominant phosphoprotein pp65 from human cytomegalovirus (HCMV) to modify the inner surface of the capsid. The truncated protein (Dpp65) comprised the Cterminal amino acids 358-561 of pp65 flanked by the CD8+ T-cell epitopes AE44 and AE45  at its N- and C-terminus, respectively. The resulting Gag/Dpp65 protein fusion (101 kDa) Academic
Examination of the ligand-binding and enzymatic properties of a bilin-binding protein from the poisonous caterpillar Lonomia obliqua.
... modeling, that lopap exists as a tetrameric protein having one catalytic site per monomer. This remarkable finding would bestow a new functional activity on the lipocalin family of proteins which normally bind small molecule ligands or other proteins. Based on sequence similarity, lopap belongs to the biliverdinbinding protein (BBP) family. Members of this ... Federal do Rio Grande do Sul, Porto Alegre RS, Brazil Abstract The bilin-binding proteins (BBP) from lepidopteran insects are members of the lipocalin family of proteins and play a special role in pigmentation through the binding of biliverdin IXc. Lopap, a BBP-like protein from the venom of the toxic caterpillar Lonomia obliqua has been reported to ... BBPLo cDNA places it in the lipocalin protein family which is characterized by an eight-stranded antiparallel b-barrel structure with a hydrophobic central binding cavity (Fig. 2). BBPLo is 50% identical to the biliverdin-binding protein I (BvBPI) from Samia cynthia ricini and 35% identical to the bilin-binding protein (BBP) from Pieris brassicae, and
CELF family RNA-binding protein UNC-75 regulates two sets of mutually exclusive exons of the unc-32 gene in neuron-specific manners in Caenorhabditis elegans.
... fluorescent protein cDNA cassettes and by introducing artificial termination codons into two of the three mutually exclusive exons in each construct (Figure 1C). From these minigenes, we expect expression of Venus-fusion protein (E4a-Venus), monomeric red fluorescent protein (mRFP)-fusion protein (E4b-mRFP) and enhanced cyan fluorescent protein (ECFP)-fusion protein ... upstream of either of two fluorescent protein cDNA cassettes and by introducing an artificial termination codon into one of the two mutually exclusive exons in each construct (Figure 1D). From these minigenes, we expect expression of enhanced green fluorescent protein (EGFP)-fusion protein (E7a-EGFP) and mRFP-fusion protein (E7b-mRFP) when exon 7a and ... recombinant proteins for each of the three RRMs and performed an EMSA (Figure 6B). The RRM3 protein (Figure 6B, right, lanes 12–16) as well as full-length UNC-75 (lanes 17,18) shifted the mobility of Probe 2-1-1, while the RRM1 or RRM2 protein did not (lanes 1-11), indicating that only RRM3 can bind to Probe 21-1 by itself. So we used only RRM3 protein
Fluorescent-protein stabilization and high-resolution imaging of cleared, intact mouse brains.
... bleaching . An apparent disadvantage is the apparent trade-off between the degree of clearing and the preservation of fluorescence from proteinaceous fluorophores (XFPs), such as EGFP (Enhanced Green Fluorescent Protein) : most protocols that clear tissue effectively also result in the loss of XFP fluorescence, either immediately [4,7] or within hours ... [22,23]. The modified rabies virus (RABVΔG-EGFP(EnvA)) was envelope A (EnvA)-pseudotyped, glycoprotein-deficient and expressed EGFP. The two recombinant adeno-associated viruses expressed the TVA receptor (rAAV-mRFP1-IRES-TVA virus) and the rabies virus glycoprotein (RG) (rAAV-RG virus), respectively. After stereotaxic injection of the virus cocktail, ... of the brains of adult and PLOS ONE | DOI:10.1371/journal.pone.0124650 May 20, 2015 9 / 26 Fluorescent Protein Imaging in Cleared Mouse Brain PLOS ONE | DOI:10.1371/journal.pone.0124650 May 20, 2015 10 / 26 Fluorescent Protein Imaging in Cleared Mouse Brain Fig 5. Visualization of neurons monosynaptically connected to RABVΔG-EGFP(EnvA)
The regulation of skeletal muscle protein turnover during the progression of cancer cachexia in the Apc(Min/+) mouse.
... autophagy-related proteins. We did not observe any differences in autophagy-related protein expression between wild-type and weight stable ApcMin/+ mice (Figure S4A and B). In support of our protein degradation measurements, which show non-ATP dependent protein degradation increased after the onset of cachexia, no autophagy-related proteins were induced ... Muscle Protein Turnover during Cachexia Figure 4. Autophagy is increased during late stage cachexia in the ApcMin/+ mouse. A) Upper representative western blot of Beclin-1 protein. Lower Quantification of Beclin-1 protein normalized to weight stable ApcMin/+ mice, B) Upper representative western blot of Atg7 protein. Lower Quantification of Atg7 protein. ... | e24650 Skeletal Muscle Protein Turnover during Cachexia PLoS ONE | www.plosone.org 5 September 2011 | Volume 6 | Issue 9 | e24650 Skeletal Muscle Protein Turnover during Cachexia Figure 1. Muscle protein synthesis and IGF-1/mTOR signaling are reduced during the progression of cachexia in ApcMin/+ mice. Protein synthesis and IGF-1 expression
Lack of the matricellular protein SPARC (secreted protein, acidic and rich in cysteine) attenuates liver fibrogenesis in mice.
... Lack of the Matricellular Protein SPARC (Secreted Protein, Acidic and Rich in Cysteine) Attenuates Liver Fibrogenesis in Mice Catalina Atorrasagasti1, Estanislao Peixoto1., Jorge B. Aquino1,2., Ne´ stor Kippes1, Mariana ... Navarra, Pamplona, Espan˜ a, 5 Gene Therapy Laboratory, Fundacio´ n Instituto Leloir, Buenos Aires, Argentina Abstract Introduction: Secreted Protein, Acidic and Rich in Cysteine (SPARC) is a matricellular protein involved in many biological processes and found over-expressed in cirrhotic livers. By mean of a genetic approach we herein provide evidence ... promising approaches for liver fibrosis treatment. Citation: Atorrasagasti C, Peixoto E, Aquino JB, Kippes N, Malvicini M, et al. (2013) Lack of the Matricellular Protein SPARC (Secreted Protein, Acidic and Rich in Cysteine) Attenuates Liver Fibrogenesis in Mice. PLoS ONE 8(2): e54962. doi:10.1371/journal.pone.0054962 Editor: Wing-Kin Syn, Institute
Induction of cell stress in neurons from transgenic mice expressing yellow fluorescent protein: implications for neurodegeneration research.
... in corresponding changes at the protein level, expression levels for 2 proteins selected from the PCR array (caspase 1 and CCL3) were validated in YFP-16 mouse spinal cord using quantitative fluorescent western blot (Figure 1B,C). Expression levels of another cell stress protein not included on the array stress inducible protein 1 (STI1) – remained unchanged ... stress proteins in the spinal cord of wild-type and YFP-16 mice. Both caspase 1 (Casp1) and CCL3 had increased expression in YFP-expressing tissue, whereas STI1 (a stress protein not on the array) remained at the same levels found in wild-type mice and YFP (FP) was only present in YFP-16 tissue. C - Bar chart (mean 6 s.e.m.) showing quantification of protein ... autoxidation of green fluorescent protein. Proc Natl Acad Sci USA 91: 12501–12504. 10. Liu HS, Jan MS, Chou CK, Chen PH, Ke NJ (1999) Is green fluorescent protein toxic to the living cells? Biochem Biophys Res Comm 260: 712–717. 11. Huang WY, Aramburu J, Douglas PS, Izumo S (2000) Transgenic expression of green fluorescence protein can cause dilated cardiomyopathy.
The fission yeast RNA binding protein Mmi1 regulates meiotic genes by controlling intron specific splicing and polyadenylation coupled RNA turnover.
... cells. Mmi1 works with Rrp6, a nuclear 3’ to 5’ exonuclease component of the exosome, to target transcripts for degradation [3,5–7]. Other proteins probably involved include Red1 , and the poly A binding protein Pab2 [6,8] (which forms a complex with Rrp6 . Hyperadenylation of transcripts targeted by Mmi1 has been observed in rrp6 mutants [6,7], ... promotes hyperadenylation. This hyperadenylation also depends on the polyA polymerase Pla1  and probably Pab2. Hyperadenylated transcripts are rapidly degraded by Rrp6. Protein- protein interactions are shown by dotted lines based on the following evidence: Mmi1-Pfs2: genetic interactions (data not shown); Mmi1-Rna15: yeast two-hybrid and co-IP ; ... promote extremely long polyA tails of up to a kilobase. The hyperadenylated transcripts are then targeted for degradation by the nuclear exonuclease Rrp6. The nuclear polyA binding protein Pab2 assists this hyperadenylation-mediated RNA decay. Rrp6 also targets other hyperadenylated transcripts, which become hyperadenylated in an unknown, but Mmi1-independent
Identification of human microRNA-like sequences embedded within the protein-encoding genes of the human immunodeficiency virus.
... with Dysregulated Proteins in HIV-Infected Cells We used the program ‘‘GeneSet2miRNA’’ to identify potential microRNAs that could impact the activities of the proteins whose expression had been shown to be dysregulated in HIV-infected RH9 T-cells [25,26]. For example, the microtubule-actin crosslinking factor 1 (MACF1)  is one of the proteins which ... a strong binding affinity to this protein. MACF1 has an actin-regulated ATPase activity necessary for cross-linking actin to other cytoskeletal proteins. In addition, MACF isoforms act as positive regulators of Wnt receptor signaling pathway and are essential for controlling focal adhesions assembly. Actin-related proteins also play critical roles during ... replication enhances production of free fatty acids, low density lipoproteins and many key proteins involved in lipid metabolism: a proteomics study. PLoS One 3: e3003. 26. Rasheed S, Yan JS, Hussain A, Lai B (2009) Proteomic characterization of HIVmodulated membrane receptors, kinases and signaling proteins involved in novel angiogenic pathways. J Transl Med
Novel method for isolation of murine clara cell secretory protein-expressing cells with traces of stemness.
... secretory protein (CCSP), also known as CC10, CC16, Clara cell antigen, secretoglobin 1A1 (SCGB1A1) or uteroglobin, Clara cells also contribute surfactant apoproteins A, B and D, proteases, anti-microbial peptides, several cytokines and chemokines, and mucins in the extracellular fluid lining airspaces. CCSP is the most abundant secretory protein found ... cell secretory protein and the cells that express it. Cytotherapy 11: 676– 687. 4. Stripp BR, Lund J, Mango GW, Doyen KC, Johnston C, et al. (1996) Clara cell secretory protein: a determinant of PCB bioaccumulation in mammals. American Journal of Physiology 271: L656–664. 5. Hackett BP, Shimizu N, Gitlin JD (1992) Clara cell secretory protein gene expression ... Annals of the New York Academy of Sciences 923: 249–267. 9. Stripp BR, Reynolds SD, Boe IM, Lund J, Power JH, et al. (2002) Clara cell secretory protein deficiency alters clara cell secretory apparatus and the protein composition of airway lining fluid. American Journal of Respiratory Cell and Molecular Biology 27: 170–178. 10. Stripp BR, Reynolds SD,
A coarse-grained elastic network atom contact model and its use in the simulation of protein dynamics and the prediction of the effect of mutations.
... mutations with respect to protein stability and compare the ability of ENCoM and ENCoMns to a large number of existing methods specifically designed for the prediction of the effect of single point mutations on protein stability. Finally, we use ENCoM to predict the effect of mutations on protein function in the absence of any effects on protein stability. Results Correlation ... may not only affect protein stability but also protein function. While experimental data is less abundant, one protein in particular, dihydrofolate reductase (DHFR) from E. coli, has been widely used experimentally to understand this relationship [69,70]. Recently, Boher et al.  have analyzed the effect of the G121V mutation on protein dynamics in ... effect of mutations on protein dynamics or stability. It has been shown that different amino acids interact differently and that single mutations can have a high impact on protein function and stability [41–43]. Mutations on non-catalytic residues that participate into concerted (correlated) movements have been shown to disrupt protein function in NMR
Myocardial Ablation of G Protein-Coupled Receptor Kinase 2 (GRK2) Decreases Ischemia/Reperfusion Injury through an Anti-Intrinsic Apoptotic Pathway.
... FADD (Fas-associated death domain protein) , resulting in the activation of caspase-8 and downstream caspase-3 . The ‘‘intrinsic’’ pathway utilizes mitochondria to produce cell death through opening of the mitochondrial permeability transition pore (mPTP), triggering the sudden release of cytochrome C and other proteins from the intermembrane space ... were measured. For signaling studies, hearts were harvested at either 15 min post-I/R for preparation of cardiac protein lysates and subsequent analysis as described in the next section. For measurements of cytosolic cytochrome c and BcL-2 proteins (Bad, Bcl-2 and Bcl-xL), 30 min of reperfusion was utilized. Finally, at 3 hrs post-I/R another set of ... doi:10.1371/journal.pone.0066234.g005 experiments were performed. First, we measured Bcl-2 and Bcl-xL (anti-apoptotic) protein levels in hearts subjected to a Sham procedure or I/R injury (30 min of reperfusion). As shown in Fig. 6D–F, baseline levels of Bcl-2 (E) and Bcl-xL (F) protein were not changed between GRK2 KO and control mice. After I/R, the level of Bcl-2 and Bcl-xL
Escherichia coli Protein Expression System for Acetylcholine Binding Proteins (AChBPs).
... Ac-AChBP and the tagged protein can be used in studies not affected by presence of fusion partners. Fractions corresponding to the protein peak were found to be >90% pure when analyzed on SDS-PAGE gels, and therefore suitable for functional as well as structural studies requiring highly purified protein samples. The final yield of purified proteins was found ... homologous binding protein. Proceedings of the National Academy of Sciences. 2012; 109(23):9173–8. 46. Dyson MR, Shadbolt SP, Vincent KJ, Perera RL, McCafferty J. Production of soluble mammalian proteins in Escherichia coli: identification of protein features that correlate with successful expression. BMC biotechnology. 2004; 4(1):1. 47. Baker RT. Protein expression ... AChBP in E. coli Large scale protein production for Ls- and Ac-AChBPs were carried out in 2–6 L of 2x Yeast extract Tryptone (2xYT) media using the optimized expression conditions. Expression was performed in 2xYT media, which is capable of supporting higher biomass than LB and therefore improves protein expression levels. Protein expression was induced
Characterization of Danio rerio Mn2+-dependent ADP-ribose/CDP-alcohol diphosphatase, the structural prototype of the ADPRibase-Mn-like protein family.
... ADPRibaseMn-like proteins form a unique SCOP family . The structural prototype of this family is a zebrafish protein, encoded by Danio rerio gene Zgc:64213. It was chosen a few years ago for crystallographic structure determination [7,8] as a hypothetical protein then lacking structurally- and biochemically-studied close homologues. The structure of this protein, ... and structural studies of proteins from diverse origins, among other things, are needed. Also, to obtain firm correlation between structure and function, it is necessary to have both aspects studied by experiments performed on the same protein. So far, the ADPRibase-Mn prototypes of structure and function correspond to different proteins, one from zebrafish, ... instance, the Mn2+-dependent YfcE protein from E. coli contains two Zn2+ ions when crystallyzed, but they probably bound the protein during purification and reflect a di-Mn2+ center . Similarly, zebrafish ADPRibase-Mn has been modeled with two Zn2+ ions in the dinuclear center , while metal analysis of the recombinant protein before crystallization
Dengue Virus Non-structural Protein 1 Modulates Infectious Particle Production via Interaction with the Structural Proteins.
... polarity, encoding for a polyprotein that is co- and post-translationally processed into three structural proteins (capsid, prM, and envelope) and seven nonstructural proteins (NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5) . After viral entry and release of the genomic RNA into the cytoplasm of infected cells, newly synthesized viral proteins induce massive remodeling ... possibly also subviral particles. In spite of comparable protein amounts in the cell lysates, no specific signal was detected in case of the non-tagged NS1WT or for the NS5 protein, confirming specificity of the NS1HA-immunoprecipitation. To corroborate the interaction between NS1 and the structural proteins, we performed analogous pull-down experiments ... the corresponding pre-immune (PIS) antiserum and protein A-Sepharose beads. After extensive washing, eluted protein complexes were analyzed by western-blotting using polyclonal anti-NS1 and anti-prM or mouse monoclonal anti-E or anti-C specific antibodies as specified on the right of each panel. DENV proteins are indicated with arrowheads, asterisks
Relation of Absolute or Relative Adiposity to Insulin Resistance, Retinol Binding Protein-4, Leptin, and Adiponectin in Type 2 Diabetes
... 0.24 2.62 6.37 21.70 28.41 0.59 5.11 10.18 117.66 67.99 0.11 6.25 12.96 127.14 80.48 0.37 SD, standard deviation; ApoA, apolipoprotein A; hsCRP, high sensitivity C-reactive protein; RBP4, retinol binding protein- 4. Table 2. Correlation between absolute regional adiposity, insulin sensitivity, and adipokines in type 2 diabetes (absolute ... total fat mass; PFM, peripheral fat mass; CFM, central fat mass; KITT, rate constant for plasma glucose disappearance; RBP4, retinol binding protein- 4; ApoA, apolipoprotein A; hsCRP, high sensitivity C-reactive protein. http://e-dmj.org Diabetes Metab J 2012;36:415-421 417 Kim YL, et al. Association between insulin sensitivity and relative adiposity ... central fat mass/total fat mass; PFM/CFM ratio, peripheral ratio; KITT, rate constant for plasma glucose disappearance; RBP4, retinol binding protein- 4; ApoA, apolipoprotein A; hsCRP, high sensitivity C-reactive protein. 0.75 0.70 Trunk/total body fat mass ratio Free fatty acid 1,250 Trunk/total body fat ratio Free fatty acid 0.65 1,000 0.60