Genetic engineering of proteins with cell membrane permeability

Rojas, Mauricio; Donahue, John P.; Tan, Zhongjia; Lin, Yao‐Zhong

doi:10.1038/nbt0498-370

Cited by 152 publications

(94 citation statements)

References 26 publications

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“…22 Therefore, we analyzed the effect of Pp-YLKTK-mts, a potent inhibitor of STAT3 activation. 23 Inhibition of activated STAT3 resulted in 2.3-fold decreased Ngn3 transcript levels (Po0.05; n ¼ 3) (Figure 1b) and in decreased numbers of Ngn3-immunopositive cells (0.1270.02% compared to 4.7870.28% in the absence of inhibitor; n ¼ 3; Po0.001) (Figure 4a). After 72 h of STAT3-inhibitor treatment, the number of insulin-expressing cells is also strongly reduced (0.370.07% in the presence of inhibitor compared to 9.271.5% in the absence of inhibitor; n ¼ 3; Po0.01) (Figure 4b).…”

Section: Role Of Jak/stat-signalling In Ngn3 Expressionmentioning

confidence: 98%

Ngn3 expression during postnatal in vitro beta cell neogenesis induced by the JAK/STAT pathway

Baeyens¹,

Bonné²,

German³

et al. 2006

Cell Death Differ

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The basic helix-loop-helix protein Neurogenin3 specifies precursor cells of the endocrine pancreas during embryonic development, and is thought to be absent postnatally. We have studied Ngn3 expression during in vitro generation of beta-cells from adult rat exocrine pancreas tissue treated with epidermal growth factor and leukaemia inhibitory factor. This treatment induced a transient expression of both Ngn3 and its upstream activator hepatocyte nuclear factor 6. Inhibition of EGF and LIF signalling by pharmacological antagonists of the JAK2/STAT3 pathway, or knockdown of Ngn3 by RNA interference prevented the generation of new insulin-positive cells. This study demonstrates that in vitro growth factor stimulation can induce recapitulation of an embryonic endocrine differentiation pathway in adult dedifferentiated exocrine cells. This could prove to be important for understanding the mechanism of beta-cell regeneration and for therapeutic ex vivo neogenesis of beta cells. Keywords: neurogenin3; transdifferentiation; beta cell; pancreas; diabetes Abbreviations: CK20, cytokeratin 20; EGF, epidermal growth factor; eGFP, enhanced green fluorescent protein; Hnf6, hepatocyte nuclear factor 6; JAK2, Janus kinase 2; LIF, leukaemia inhibitory factor; Ngn3, neurogenin 3; Pdx1, pancreatic and duodenal homeobox 1; STAT3, signal transducer and activator of transcription 3 IntroductionThere is currently a lot of interest in the development of a treatment for diabetes that is based on beta-cell replacement or regeneration. Beta-cells could be (re-)generated by stimulation of beta-cell replication and/or (trans-)differentiation of stem/progenitor cells. It has been demonstrated that, during adult life, beta-cell maintenance and compensatory growth in mice results primarily from proliferation of the pre-existing beta cells. 1 However, numerous reports of alternative sources of beta-cell 'neogenesis' in response to severe tissue injury have been described. [2][3][4][5][6] The pancreas is composed of exocrine tissue, comprising acinar and duct cells, and of endocrine tissue that contains beta-, alpha-, delta-and pancreatic polypeptide cells. A possible source of progenitor cells is the acinar cell population that is the most abundant in adult pancreas and is known to retain a remarkable plasticity both in vivo and in vitro. 7-9 Isolated acinar cells can undergo dedifferentiation in vitro whereby they lose their zymogen granules and gain a duct-like phenotype including expression of the duct cell marker cytokeratin20. 8,10 External stimuli can induce a phenotypical switch in these cells towards a hepatocyte-like phenotype. 7 We have recently developed a model for in vitro beta-cell neogenesis based upon growth factor stimulation of dedifferentiated acinar pancreatic cells. 11 The molecular regulation of this transdifferentiation from acinar to beta cells is unknown at present. Although it is known that during pancreatic regeneration the exocrine population can dedifferentiate towards an embryonic-like state in vivo, 12 in v...

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Section: Role Of Jak/stat-signalling In Ngn3 Expressionmentioning

confidence: 98%

Ngn3 expression during postnatal in vitro beta cell neogenesis induced by the JAK/STAT pathway

Baeyens¹,

Bonné²,

German³

et al. 2006

Cell Death Differ

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“…We are currently investigating the role of the MTP, including a modified 12-amino acid residue membrane-translocating sequence from the signal sequence of Kaposi fibroblast growth factor 25 and the homeodomain of the transcription factor Antennapedia from Drosophila, 26 in combination with various toxins (diphtheria toxin A-chain, Pseudomonas exotoxin). Our study shows that the use of a cleavable adapter in the construction of ITs has several advantages: (i) the cytotoxic activity is maintained while reducing the half-life, thus possibly limiting side effects such as VLS and (ii) the permutation between toxic moiety and ligand is greatly increased.…”

Section: The Adapter Reduces Nonspecific Toxicitymentioning

confidence: 99%

A cleavable adapter to reduce nonspecific cytotoxicity of recombinant immunotoxins

Heisler

Keller

Tauber

et al. 2002

Intl Journal of Cancer

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One of the problems associated with the administration of immunotoxins is hypersensitivity reaction such as vascular leak syndrome. This may be prevented by decreasing the plasma half-life. To improve immunotoxins with respect to reduced side effects, we have previously described the development of a cleavable adapter. This adapter links the toxic moiety and ligand that are usually directly coupled. In our study, the cytotoxicity of saporin linked either directly or via the adapter to epidermal growth factor (EGF) was evaluated in vitro. The immunotoxins exhibited similar cytotoxic activity towards A-431 and HER14 cells (IC 50 < 10 nM). The supernatant from 6 hr cultures of HER14 cells incubated in the presence of the adapter-containing immunotoxin exhibited a significantly reduced cytotoxicity as compared to the directly coupled immunotoxin. Western blotting revealed that the adapter was cleaved, thus supporting our proposal that cleavable adapters may reduce nonspecific effects. A similar reduced half-life was detected in platelet-poor plasma. In contrast MCF-7 cells remain unaffected by the immunotoxins. This was shown to be due to the absence of detectable EGF-receptor in comparison to A-431 and HER14 cells as determined by Western blotting. Furthermore, we could show that the adapter does not exert an effect on the N-glycosidase activity of saporin. These results suggest that the use of cleavable adapters may be a useful tool in immunotoxins for reducing the killing of surrounding noncancerous cells due to nonspecific binding.

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“…Linkage of so-called cell-penetrating peptides (CPPs) 1 to other molecules mediates the non-invasive import of these cargo molecules into cells ex vivo as well as in whole animals (4,5). Cargos have been peptides (6,7), proteins as large as 120 kDa (5,8), oligonucleotides (9), plasmids (10), peptide nucleic acids (11), and even nanoparticles (12).…”

mentioning

confidence: 99%

A Stepwise Dissection of the Intracellular Fate of Cationic Cell-penetrating Peptides

Fischer

Köhler

Fotin‐Mleczek

et al. 2004

Journal of Biological Chemistry

270

289

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The role of endosomal acidification and retrograde transport for the uptake of the highly basic cell-penetrating peptides penetratin, Tat, and oligoarginine was investigated. The effect of a panel of drugs that interfere with discrete steps of endocytosis or Golgi-mediated transport on uptake and cellular distribution of fluorescein-labeled peptide analogues was probed by confocal microscopy, flow cytometry, and fluorescence spectroscopy of whole cell lysates. The analyses were carried out in MC57 fibrosarcoma cells and in HeLa cells. While MC57 fibrosarcoma cells showed some vesicular fluorescence and a pronounced cytoplasmic fluorescence, in HeLa cells little cytoplasmic fluorescence was observed. In MC57 cells the inhibitors of endosomal acidification chloroquine and bafilomycin A1 abolished the release of the peptides into the cytoplasm. Release into the cytosol preserved endosomal integrity. In addition, cellular uptake of the peptides was inhibited by brefeldin A, a compound interfering with trafficking in the transGolgi network. In contrast, nordihydroguaiaretic acid, a drug that stimulates the rapid retrograde movement of both Golgi stacks and trans-Golgi network to the endoplasmic reticulum, promoted a cytoplasmic localization of Tat peptides in peptide-pulsed HeLa cells. The effects of these drugs on trafficking shared characteristics with those reported for the trafficking of plant and bacterial toxins, such as cholera toxin, which reach the cytoplasm by means of retrograde transport. A sequence comparison revealed a common stretch of 8 -10 amino acids with high sequence homology to the Tat peptide. The structural and functional data therefore strongly suggest a common mechanism of import for cationic cell-penetrating peptides and the toxins.

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Genetic engineering of proteins with cell membrane permeability

Cited by 152 publications

References 26 publications

Ngn3 expression during postnatal in vitro beta cell neogenesis induced by the JAK/STAT pathway

Ngn3 expression during postnatal in vitro beta cell neogenesis induced by the JAK/STAT pathway

A cleavable adapter to reduce nonspecific cytotoxicity of recombinant immunotoxins

A Stepwise Dissection of the Intracellular Fate of Cationic Cell-penetrating Peptides

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