The endocytosis‐mediating performances of two types of peptide ligands, cell receptor binding peptide (CRBP) and cell membrane penetrating peptide (CMPP), were analyzed and compared using a common carrier of peptide ligands‐human ferritin heavy chain (hFTH) nanoparticle. Twenty‐four copies of a CMPP(human immunodeficiency virus‐derived TAT peptide) and/or a CRBP (peptide ligand with strong and specific affinity for either human integrin(αvβ3) or epidermal growth factor receptor I (EGFR) that is overexpressed on various cancer cells) were genetically presented on the surface of each hFTH nanopariticle. The quantitative level of endocytosis and intracellular localization of fluorescence dye‐labeled CRBP‐ and CMPP‐presenting nanoparticles were estimated in the in vitro cultures of integrin‐ and EGFR‐overexpressing cancer and human dermal fibroblast cells(control). From the cancer cell cultures treated with the CMPP‐ and CRBP‐presenting nanoparticles, it was notable that CRBPs resulted in quantitatively higher level of endocytosis than CMPP (TAT) and successfully transported the nanoparticles to the cytosol of cancer cells depending on concentration and treatment period of time, whereas TAT‐mediated endocytosis localized most of the nanoparticles within endosomal vesicles under the same conditions. These novel findings provide highly useful informations to many researchers both in academia and in industry who are interested in developing anticancer drug delivery systems/carriers. 相似文献
Rab GTPases and their effectors, activators and guanine nucleotide exchange factors (GEFs) are essential for vesicular transport. Rab8 and its GEF Rabin8 function in formation of the cilium organelle important for developmental signaling and sensory reception. Here, we show by size exclusion chromatography and analytical ultracentrifugation that Rabin8 exists in equilibrium between dimers and tetramers. The crystal structure of tetrameric Rabin8 GEF domain reveals an occluded Rab8 binding site suggesting that this oligomer is enzymatically inactive, a notion we verify experimentally using Rabin8/Rab8 GEF assays. We outline a procedure for the purification of active dimeric Rabin8 GEF‐domain for in vitro activity assays. 相似文献
As a ubiquitous, highly pleiotropic and constitutively active serine/threonine protein kinase, casein kinase 2 (CK2) is closely associated with tumorigenesis by its overexpression in cancer cells. Here we report several proteolysis targeting chimeras (PROTACs) via “click reaction” to connect a CK2 inhibitor (CX-4945) and pomalidomide for degradation of CK2 protein. Among them, compound 2 degraded CK2 in a dose and time-dependent manner, and kept CK2 at a low basal level by recruiting ubiquitin-proteasome system. The degradation of CK2 resulted in the reduced phosphorylation of Akt and the up-regulation of p53. As a CK2 protein degrader, 2 showed the analogous cytotoxicity to CX-4945 but with a quite different mechanism of action from the CK2 inhibitor, hinting that degradation of CK2 proteins by PROTACs is a potential way for cancer treatments. 相似文献
Cdc14 protein phosphatase is critical for late mitosis progression in budding yeast, although its orthologs in other organisms, including mammalian cells, function as stress-responsive phosphatases. We found herein unexpected roles of Cdc14 in autophagy induction after nutrient starvation and target of rapamycin complex 1 (TORC1) kinase inactivation. TORC1 kinase phosphorylates Atg13 to repress autophagy under nutrient-rich conditions, but if TORC1 becomes inactive upon nutrient starvation or rapamycin treatment, Atg13 is rapidly dephosphorylated and autophagy is induced. Cdc14 phosphatase was required for optimal Atg13 dephosphorylation, pre-autophagosomal structure formation, and autophagy induction after TORC1 inactivation. In addition, Cdc14 was required for sufficient induction of ATG8 and ATG13 expression. Moreover, Cdc14 activation provoked autophagy even under normal conditions. This study identified a novel role of Cdc14 as the stress-responsive phosphatase for autophagy induction in budding yeast. 相似文献
Malarial parasites propagate asexually inside the erythrocytes of their vertebrate host. Six hours after invasion, the permeability of the host cell membrane to anions and small nonelectrolytes starts to increase and reaches its peak as the parasite matures. This increased permeability differs from the native transport systems of the normal erythrocyte in its solute selectivity pattern, its enthalpy of activation and its susceptibility to inhibitors, suggesting the appearance of new transport pathways. A biophysical analysis of the permeability data indicates that the selectivity barrier discriminates between permeants according to their hydrogen bonding capacity and has solubilization properties compared to those ofiso-butanol. The new permeability pathways could result from structural defects caused in the host cell membrane by the insertion of parasite-derived polypeptides. It is suggested that the unique transport properties of the new pathways be used to target drugs into infected cells, to affect the parasite either directly or through the modulation of the intraerythrocytic environment. The feasibility of drug targeting is demonstrated inin vitro cultures of the human malarial parasitePlasmodium falciparum. 相似文献
The interactions of liposomes with cells have been extensively studied to determine their potential use as vehicles for the delivery of drugs in vivo. Since intravenously administered liposomes are, for the most part, cleared by cells of the reticuloendothelial system (RES), considerable effort has been made to take advantage of this phenomenon rather than view it as an obstacle. Indeed, cells of the RES, in particular macrophages, have been shown to play a vital role in homeostasis and in host defence mechanisms against infection and neoplasia.
In this article, we present an overview of liposome-cell interactions, with particular emphasis on the techniques used to monitor the interaction of liposomes with macrophages. Specifically, we discuss methodologies which can be used to differentiate between liposome-cell fusion, adsorption and endocytosis in vitro. In addition, we outline the various strategies that have been employed for both actively and passively targeting liposomes to macrophages in vivo. We also review the rationale and various techniques for designing liposomes for enhanced macrophage uptake, which, in certain cases, results in the selective release of liposome-entrapped compounds in situ. 相似文献
We have successfully disrupted thecftr (cystic fibrosis transmembrane conductance regulator) gene at its endogenous locus in embryonic stem cells by gene targeting.
We are using a double replacement strategy to introduce subtle mutations into exon 10. We report here the first step of creating
a null mutation by insertion of a functionalhprt (hypoxanthine phosphoribosyl transferase) mini-gene into exon 10 of thecftr gene. Targeted embryonic stem cell clones were identified by PCR screening and confirmed by Southern blot analysis. One of
thecftr targeted clones has been injected into recipient blastocysts and shown to contribute to chimaeras. The targeted clones will
now be used as the starting point for a second gene targeting step to remove thehprt gene in exon 10 with the concomitant introduction of the ΔF508 mutation or other mutations. 相似文献
We wish to construct a mouse model for the human inherited disease cystic fibrosis. We describe here the successful targeting
in embryonal stem cells of the murine homologue (Cftr) of the cystic fibrosis transmembrane conductance regulator gene, as the first critical step towards this end. The targeting
event precisely disrupts exon 10, the site of the major mutation in patients with cystic fibrosis. The targeted cells are
pluripotent and competent to form chimaeras. 相似文献
We used a heterologous system (transgenic Nicotiana tabacum L.) to investigate the processing, assembly and targeting of phytohemagglutinin (PHA), the lectin of the common bean, Phaseolus vulgaris L. In the bean, this glycoprotein accumulates in the protein bodies of the storage parenchyma cells in the cotyledons, and each polypeptide has a high-mannose glycan attached to Asn12 and a complex glycan on Asn60. The gene for PHA-L, dlec2, with 1200 basepairs (bp) 5 upstream and 1600 bp 3 downstream from the coding sequence was introduced into tobacco using Agrobacterium-mediated transformation (T. Voelker et al., 1987, EMBO J. 6, 3571–3577). Examination of thin sections of tobacco seeds by immunocytochemistry with antibodies against PHA showed that PHA-L accumulated in the amorphous matrix of the protein bodies in the embryo and endosperm. This localization was confirmed using a non-aqueous method to isolate the protein bodies from mature tobacco seeds. The biochemical analysis of tobacco PHA indicated that the signal peptide had been correctly removed, and that the polypeptides formed 6.4 S oligomers; tobacco PHA had a high-mannose glycan at Asn12 and a complex glycan at Asn60. The presence of the complex glycan shows that transport to the protein bodies was mediated by the Golgi complex. At seed maturity, a substantial portion of the PHA-L remained associated with the endoplasmic reticulum and the Golgi complex, as indicated by fractionation experiments using aqueous media and the presence of two high-mannose glycans on some of the polypeptides. Taken together, these data show that insertion of the nascent PHA into the endoplasmic reticulum, signal peptide processing, glycosylation, assembly into oligomers, glycan modification in the Golgi, and targeting of the protein occur faithfully in this heterologous system, although transport may not be as efficient as in bean cotyledons.Abbreviations Asn
asparagine
- Endo H
endoglycosidase H
- HPLC
high-performance liquid chromatography
- IgG
immunoglobulin G
- Mr
relative molecular mass
- PAGE
polyacrylamide gel electrophoresis
- PHA
phytohemagglutinin
- SDS
sodium dodecylsulfate
- TFMS
trifluoromethanesulfonic acid 相似文献