In vitro incorporation of a cell-binding protein to a lentiviral vector using an engineered split intein enables targeted delivery of genetic cargo

A study was carried out to determine the in vitro interaction between ochratoxin A (OTA) and wine lactic acid bacteria (LAB). Fifteen strains belonging to five relevant oenological LAB species were grown in liquid synthetic culture medium containing OTA. The portion of OTA removed during the bacterial growth was 8 to 28%. The OTA removed from the supernatants was partially recovered (31 to 57%) from the bacterial pellet. Cell-free extracts of three representative strains were produced by disrupting cells in a French pressure cell. The ability of crude cell-free extracts to degrade OTA was studied. OTA was not degraded by cell-free extracts of wine LAB strains, and no degradation products of OTA were detected in the high-performance liquid chromatograms of the methanol extract of the bacterial pellet. On the basis of these results, we conclude that OTA removal by wine LAB is a cell-binding phenomenon. The chemistry and the molecular basis of OTA binding to wine LAB remains unknown.

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A cell-specific factor represses stimulation of transcription in vitro by transcriptional enhancer factor 1

Molecular and Cellular Biology ◽

10.1128/mcb.14.8.5290-5299.1994 ◽

1994 ◽

Vol 14 (8) ◽

pp. 5290-5299

Author(s):

S Chaudhary ◽

C Brou ◽

M E Valentin ◽

N Burton ◽

L Tora ◽

...

Keyword(s):

Hela Cell ◽

Binding Protein ◽

Activation Function ◽

Tata Binding Protein ◽

Lymphoid Cells ◽

Specific Factor ◽

Transcriptional Enhancer ◽

Cell Extracts ◽

A Cell

Transcription in HeLa cell extracts in vitro was stimulated 8- to 10-fold by a recombinant chimera, GAL-TEF-1, consisting of the DNA-binding domain of GAL4 and the activation function of the HeLa cell activator TEF-1. In contrast, only a 2- to 3-fold stimulation was obtained with GAL-TEF-1 in extracts from BJA-B lymphoid cells. Stimulation by GAL-TEF-1 in BJA-B extracts was dramatically increased by the addition of immunopurified HeLa cell TFIID, suggesting that BJA-B TFIID lacks or contains lower quantities of a TATA-binding-protein-associated factor(s) required for the activity of the TEF-1 activation function. However, chromatography, immunopurification, and transcriptional reconstitution experiments indicated that BJA-B extracts did not lack the previously identified TATA-binding-protein-associated factors required for TEF-1 activity but rather contained a negatively acting factor(s) which inhibited transactivation by GAL-TEF-1. These results indicate that the relative lack of activity of the TEF-1 activation function in vitro in BJA-B cell extracts does not result from the absence of positively acting factors from the presence of a cell-specific negatively acting factor(s).

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gp96 Is a Human Colonocyte Plasma Membrane Binding Protein for Clostridium difficile Toxin A

Infection and Immunity ◽

10.1128/iai.00326-08 ◽

2008 ◽

Vol 76 (7) ◽

pp. 2862-2871 ◽

Cited By ~ 62

Author(s):

Xi Na ◽

Ho Kim ◽

Mary P. Moyer ◽

Charalabos Pothoulakis ◽

J. Thomas LaMont

Keyword(s):

Plasma Membrane ◽

Clostridium Difficile ◽

Binding Protein ◽

Membrane Binding ◽

Cell Surface Receptor ◽

Toxin A ◽

Surface Receptor ◽

A Cell ◽

Plasma Membrane Binding

ABSTRACT Clostridium difficile toxin A (TxA), a key mediator of antibiotic-associated colitis, requires binding to a cell surface receptor prior to internalization. Our aim was to identify novel plasma membrane TxA binding proteins on human colonocytes. TxA was coupled with biotin and cross-linked to the surface of HT29 human colonic epithelial cells. The main colonocyte binding protein for TxA was identified as glycoprotein 96 (gp96) by coimmunoprecipitation and mass spectrum analysis. gp96 is a member of the heat shock protein family, which is expressed on human colonocyte apical membranes as well as in the cytoplasm. TxA binding to gp96 was confirmed by fluorescence immunostaining and in vitro coimmunoprecipitation. Following TxA binding, the TxA-gp96 complex was translocated from the cell membrane to the cytoplasm. Pretreatment with gp96 antibody decreased TxA binding to colonocytes and inhibited TxA-induced cell rounding. Small interfering RNA directed against gp96 reduced gp96 expression and cytotoxicity in colonocytes. TxA-induced inflammatory signaling via p38 and apoptosis as measured by activation of BAK (Bcl-2 homologous antagonist/killer) and DNA fragmentation were decreased in gp96-deficient B cells. We conclude that human colonocyte gp96 serves as a plasma membrane binding protein that enhances cellular entry of TxA, participates in cellular signaling events in the inflammatory cascade, and facilitates cytotoxicity.

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cAMP-Responsive Element Binding Protein: A Vital Link in Embryonic Hormonal Adaptation

Endocrinology ◽

10.1210/en.2012-2096 ◽

2013 ◽

Vol 154 (6) ◽

pp. 2208-2221 ◽

Cited By ~ 13

Author(s):

Maria Schindler ◽

Sünje Fischer ◽

René Thieme ◽

Bernd Fischer ◽

Anne Navarrete Santos

Keyword(s):

Transcription Factors ◽

Target Genes ◽

Binding Protein ◽

Cell Lineage ◽

Element Binding Protein ◽

A Cell ◽

Responsive Element ◽

Camp Responsive Element Binding

Abstract The transcription factor cAMP responsive element-binding protein (CREB) and activating transcription factors (ATFs) are downstream components of the insulin/IGF cascade, playing crucial roles in maintaining cell viability and embryo survival. One of the CREB target genes is adiponectin, which acts synergistically with insulin. We have studied the CREB-ATF-adiponectin network in rabbit preimplantation development in vivo and in vitro. From the blastocyst stage onwards, CREB and ATF1, ATF3, and ATF4 are present with increasing expression for CREB, ATF1, and ATF3 during gastrulation and with a dominant expression in the embryoblast (EB). In vitro stimulation with insulin and IGF-I reduced CREB and ATF1 transcripts by approximately 50%, whereas CREB phosphorylation was increased. Activation of CREB was accompanied by subsequent reduction in adiponectin and adiponectin receptor (adipoR)1 expression. Under in vivo conditions of diabetes type 1, maternal adiponectin levels were up-regulated in serum and endometrium. Embryonic CREB expression was altered in a cell lineage-specific pattern. Although in EB cells CREB localization did not change, it was translocated from the nucleus into the cytosol in trophoblast (TB) cells. In TB, adiponectin expression was increased (diabetic 427.8 ± 59.3 pg/mL vs normoinsulinaemic 143.9 ± 26.5 pg/mL), whereas it was no longer measureable in the EB. Analysis of embryonic adipoRs showed an increased expression of adipoR1 and no changes in adipoR2 transcription. We conclude that the transcription factors CREB and ATFs vitally participate in embryo-maternal cross talk before implantation in a cell lineage-specific manner. Embryonic CREB/ATFs act as insulin/IGF sensors. Lack of insulin is compensated by a CREB-mediated adiponectin expression, which may maintain glucose uptake in blastocysts grown in diabetic mothers.

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Hyaluronan and a cell-associated hyaluronan binding protein regulate the locomotion of ras-transformed cells.

The Journal of Cell Biology ◽

10.1083/jcb.112.5.1041 ◽

1991 ◽

Vol 112 (5) ◽

pp. 1041-1047 ◽

Cited By ~ 127

Author(s):

E A Turley ◽

L Austen ◽

K Vandeligt ◽

C Clary

Keyword(s):

Binding Sites ◽

Binding Protein ◽

Experimental Period ◽

Cell Binding ◽

Ras Gene ◽

Hybrid Gene ◽

Cell Locomotion ◽

A Cell ◽

Dose Dependent ◽

Hyaluronan Binding

Hyaluronan (HA) and one of its cell binding sites, fibroblast hyaluronan binding protein (HABP), is shown to contribute to the regulation of 10T1/2 cell locomotion that contain an EJ-ras-metallothionein (MT-1) hybrid gene. Promotion of the ras-hybrid gene with zinc sulfate acutely stimulates, by 6-10-fold, cell locomotion. After 10 h, locomotion drops to two- to threefold above that of uninduced cells. Several observations indicate increased locomotion is partly regulated by HA. These include the ability of a peptide that specifically binds HA (HABR) to reduce locomotion, the ability of HA (0.001-0.1 micrograms/ml), added at 10-30 h after induction to stimulate locomotion back to the original, acute rate, and the ability of an mAb specific to a 56-kD fibroblast HABP to block locomotion. Further, both HA and HABP products are regulated by induction of the ras gene. The effect of exogenous HA is blocked by HABR, is dose-dependent and specific in that chondroitin sulfate or heparan have no significant effect. Stimulatory activity is retained by purified HA and lost upon digestion with Streptomyces hyaluronidase indicating that the activity of HA resides in its glycosaminoglycan chain. Uninduced cells are not affected by HA, HABR, or mAb and production of HA or HABP is not altered during the experimental period. These results suggest that ras-transformation activates an HA/HABP locomotory mechanism that forms part of an autocrine motility mechanism. Reliance of induced cells on HA/HABP for locomotion is transient and specific to the induced state.

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A cell-specific factor represses stimulation of transcription in vitro by transcriptional enhancer factor 1.

Molecular and Cellular Biology ◽

10.1128/mcb.14.8.5290 ◽

1994 ◽

Vol 14 (8) ◽

pp. 5290-5299 ◽

Cited By ~ 15

Author(s):

S Chaudhary ◽

C Brou ◽

M E Valentin ◽

N Burton ◽

L Tora ◽

...

Keyword(s):

Hela Cell ◽

Binding Protein ◽

Activation Function ◽

Tata Binding Protein ◽

Lymphoid Cells ◽

Specific Factor ◽

Transcriptional Enhancer ◽

Cell Extracts ◽

A Cell

Transcription in HeLa cell extracts in vitro was stimulated 8- to 10-fold by a recombinant chimera, GAL-TEF-1, consisting of the DNA-binding domain of GAL4 and the activation function of the HeLa cell activator TEF-1. In contrast, only a 2- to 3-fold stimulation was obtained with GAL-TEF-1 in extracts from BJA-B lymphoid cells. Stimulation by GAL-TEF-1 in BJA-B extracts was dramatically increased by the addition of immunopurified HeLa cell TFIID, suggesting that BJA-B TFIID lacks or contains lower quantities of a TATA-binding-protein-associated factor(s) required for the activity of the TEF-1 activation function. However, chromatography, immunopurification, and transcriptional reconstitution experiments indicated that BJA-B extracts did not lack the previously identified TATA-binding-protein-associated factors required for TEF-1 activity but rather contained a negatively acting factor(s) which inhibited transactivation by GAL-TEF-1. These results indicate that the relative lack of activity of the TEF-1 activation function in vitro in BJA-B cell extracts does not result from the absence of positively acting factors from the presence of a cell-specific negatively acting factor(s).

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Possible Involvement of Heparin-Binding Protein in Antibody-Mediated Non-Hemolytic Transfusion Reaction.

Blood ◽

10.1182/blood.v110.11.4014.4014 ◽

2007 ◽

Vol 110 (11) ◽

pp. 4014-4014

Author(s):

Rika A. Furuta ◽

Kazuta Yasui ◽

Nobuki Matsuyama ◽

Yasuo Fukumori ◽

Takafumi Kimura ◽

...

Keyword(s):

Cell Activation ◽

Binding Protein ◽

Heparin Binding ◽

Human Antibodies ◽

Heparin Binding Protein ◽

Hemolytic Transfusion Reaction ◽

A Cell ◽

Cell Culture Assay ◽

Transfusion Reactions

Abstract In antibody-mediated non-hemolytic transfusion reactions, Transfusion-Related Acute Lung Injury (TRALI) tends to occur immediately after a blood transfusion. Leukocyte antibodies or immune complexes have been frequently shown to be associated with the syndrome, although the mechanisms by which they induce TRALI are poorly understood. To explore the mechanism of TRALI, an in vitro whole blood cell culture assay was established in which cells were stimulated by human antibodies and the activation of neutrophils was monitored by a cell surface marker (Mac-1) with FCM. To identify soluble mediators that were released instantly following immune stimulation, we examined the release of soluble factors, including, Perforin, IL-6, TNF-a, and Heparin binding protein (HBP) using ELISAs. In addition, the involvement of two neutrophil FcγRs (FcγRIIIb and FcγRIIa, also known as CD16 and CD32, respectively) in release of HBP was examined during antibody-induced cell activation using anti-FcγR blocking antibodies. Substantial amounts of HBP were released within 30 min upon stimulation by human antibodies, although other soluble mediators were not released within such an eary period. Furthermore, the release of HBP was mediated via signals though both FcγRIIIb and FcγRIIa. HBP that have been known as a multifunctional molecule in early inflammation appears to be a one of the primary effector molecules of antibody-mediated non-hemolytic transfusion reactions including TRALI.

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Evaluation of Elastin-Like Polypeptides for Tumor Targeted Delivery of Doxorubicin to Glioblastoma

Molecules ◽

10.3390/molecules24183242 ◽

2019 ◽

Vol 24 (18) ◽

pp. 3242 ◽

Cited By ~ 4

Author(s):

Sonja Dragojevic ◽

Rebecca Mackey ◽

Drazen Raucher

Keyword(s):

Targeted Delivery ◽

Drug Carrier ◽

In Vitro Cytotoxicity ◽

Free Drug ◽

Nuclear Accumulation ◽

Therapeutic Approaches ◽

Normal Tissues ◽

C Terminus ◽

A Cell

To increase treatment efficiency for glioblastoma, we have developed a system to selectively deliver chemotherapeutic doxorubicin (Dox) to Glioblastoma (GBM) tumors. This carrier is based on elastin-like polypeptide (ELP), which is soluble at physiological temperatures but undergoes a phase transition and accumulates at tumor sites with externally applied, mild (40–41 °C) hyperthermia. The CPP-ELP-Dox conjugate consists of a cell penetrating peptide (CPP), which facilitates transcytosis through the blood brain barrier and cell entry, and a 6-maleimidocaproyl hydrazone derivative of doxorubicin at the C-terminus of ELP. The acid-sensitive hydrazone linker ensures release of Dox in the lysosomes/endosomes after cellular uptake of the drug conjugate. We have shown that CPP-ELP-Dox effectively inhibits cell proliferation in three GBM cell lines. Both the free drug and CPP-ELP-Dox conjugate exhibited similar in vitro cytotoxicity, although their subcellular localization was considerably different. The Dox conjugate was mainly dispersed in the cytoplasm, while free drug had partial nuclear accumulation in addition to cytoplasmic distribution. The intracellular Dox concentration was increased in the CPP-ELP-Dox cells compared to that in the cells treated with free Dox, which positively correlates with cytotoxic activity. In summary, our findings demonstrate that CPP-ELP-Dox effectively kills GBM cells. Development of such a drug carrier has the potential to greatly improve current therapeutic approaches for GBM by increasing the specificity and efficacy of treatment and reducing cytotoxicity in normal tissues.

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Structural Analysis of the Role of Pseudomonas aeruginosa Penicillin-Binding Protein 5 in β-Lactam Resistance

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00505-13 ◽

2013 ◽

Vol 57 (7) ◽

pp. 3137-3146 ◽

Cited By ~ 25

Author(s):

Jeffrey D. Smith ◽

Malika Kumarasiri ◽

Weilie Zhang ◽

Dusan Hesek ◽

Mijoon Lee ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Active Site ◽

Binding Protein ◽

Main Function ◽

Penicillin Binding Protein ◽

Active Site Residues ◽

Content Type ◽

A Cell ◽

Dynamics Simulations

ABSTRACTPenicillin-binding protein 5 (PBP5) is one of the most abundant PBPs inPseudomonas aeruginosa. Although its main function is that of a cell walldd-carboxypeptidase, it possesses sufficient β-lactamase activity to contribute to the ability ofP. aeruginosato resist the antibiotic activity of the β-lactams. The study of these dual activities is important for understanding the mechanisms of antibiotic resistance byP. aeruginosa, an important human pathogen, and to the understanding of the evolution of β-lactamase activity from the PBP enzymes. We purified a soluble version ofP. aeruginosaPBP5 (designated Pa sPBP5) by deletion of its C-terminal membrane anchor. Underin vitroconditions, Pa sPBP5 demonstrates bothdd-carboxypeptidase and expanded-spectrum β-lactamase activities. Its crystal structure at a 2.05-Å resolution shows features closely resembling those of the class A β-lactamases, including a shortened loop spanning residues 74 to 78 near the active site and with respect to the conformations adopted by two active-site residues, Ser101 and Lys203. These features are absent in the related PBP5 ofEscherichia coli. A comparison of the two Pa sPBP5 monomers in the asymmetric unit, together with molecular dynamics simulations, revealed an active-site flexibility that may explain its carbapenemase activity, a function that is absent in theE. coliPBP5 enzyme. Our functional and structural characterizations underscore the versatility of this PBP5 in contributing to the β-lactam resistance ofP. aeruginosawhile highlighting how broader β-lactamase activity may be encoded in the structural folds shared by the PBP and serine β-lactamase classes.

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The RNA m6A binding protein YTHDF2 promotes the B cell to plasma cell transition.

10.1101/2021.07.21.453193 ◽

2021 ◽

Author(s):

David Turner ◽

Alexander Saveliev ◽

Fiamma Salerno ◽

Louise S Matheson ◽

Michael Screen ◽

...

Keyword(s):

B Cells ◽

B Cell ◽

Rna Binding ◽

Plasma Cells ◽

Rna Binding Proteins ◽

Binding Protein ◽

Chimeric Mouse ◽

B Cell Differentiation ◽

A Cell

To identify roles of RNA binding proteins (RBPs) in the differentiation of B cells to antibody-secreting plasma cells we performed a CRISPR/Cas9 knockout screen of 1213 mouse RBPs for their ability to affect proliferation and/or survival, and the emergence of differentiated CD138+ cells in vitro. We identified RBPs that promoted the appearance of CD138+ cells including CSDE1 and STRAP, as well as RBPs that inhibited CD138+ cell appearance such as EIF3 subunits EIF3K and EIF3L. Furthermore, we identified RBPs that share the property of recruiting the CCR4-NOT complex to their target transcripts have the potential to mediate opposing outcomes on B cell differentiation. One such RBP, the m6A binding protein YTHDF2 promotes the appearance of CD138+ cells in vitro. In chimeric mouse models YTHDF2-deficient B cells formed germinal centers in a cell-autonomous manner, however plasma cells failed to accumulate.

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