RESUMO
We previously showed that calcineurin B homologous protein 1 (CHP1) interacts with nuclear apoptosis-inducing protein kinase DRAK2, and that overexpression of DRAK2 induces the nuclear accumulation of CHP1, although CHP1 usually resides in the cytoplasm [Matsumoto et al. (2001) J. Biochem. 130, 217-225]. Here we show that CHP1 has two functional nuclear export signal (NES) sequences in its carboxyl-terminal region. Treatment of several cell lines with leptomycin B, a specific inhibitor of CRM1-dependent nuclear export, induces the nuclear accumulation of CHP1. Moreover, CHP1-GFP fusion proteins with deletions or point mutations affecting the two putative NES sequences accumulate in the nucleus to a greater extent than wild-type CHP1-GFP. Tagging glutathione S-transferase-GFP fusion protein with each NES sequence caused a shift in their intracellular localization from all over the cells to the cytoplasm. These results suggest that after CHP1 has entered the nucleus, it is exported to the cytoplasm in an NES-dependent manner.
Assuntos
Calcineurina/metabolismo , Proteínas de Ligação ao Cálcio/biossíntese , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Transporte Ativo do Núcleo Celular/genética , Sequência de Aminoácidos , Animais , Células CHO , Células COS , Calcineurina/química , Calcineurina/genética , Proteínas de Ligação ao Cálcio/análise , Proteínas de Ligação ao Cálcio/genética , Núcleo Celular/química , Núcleo Celular/genética , Chlorocebus aethiops , Cricetinae , Citoplasma/química , Citoplasma/genética , Proteínas de Fluorescência Verde , Humanos , Líquido Intracelular/química , Líquido Intracelular/metabolismo , Proteínas Luminescentes/genética , Camundongos , Dados de Sequência Molecular , Células NIH 3T3 , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , RatosRESUMO
Bionanocapsules (BNCs) are hollow nanoparticles that are composed of L protein (the hepatitis B virus surface antigen) and show specific affinity for human hepatocytes. The pre-S1 peptide displayed on the surface of BNCs is the specific ligand for binding to the receptor on human hepatocytes. Therefore, BNCs are not delivered to other tissues, such as the brain. The aim of the present study was to develop a novel drug delivery system (DDS) targeting brain tumors using BNCs that selectively targeted brain tumors. Epidermal growth factor receptor (EGFR), especially a constitutively active genomic sequence deletion variant of EGFR (EGFRvIII), is overexpressed in human glioblastoma. In the present study, we replaced the pre-S1 peptide with the antibody affinity motif of protein A and made hybrid BNCs conjugated with anti-human EGFR antibody recognizing EGFRvIII. The hybrid BNCs were efficiently delivered to glioma cells but not normal glial cells. Moreover, we confirmed the specific delivery of the hybrid BNCs to brain tumors in an in vivo brain tumor model. These results suggest that this new approach using BNCs is a promising system for brain tumor-targeted drug delivery.
Assuntos
Anticorpos Monoclonais/metabolismo , Neoplasias Encefálicas/metabolismo , Portadores de Fármacos , Receptores ErbB/metabolismo , Glioma/metabolismo , Nanocápsulas , Proteína Estafilocócica A/metabolismo , Animais , Animais Recém-Nascidos , Anticorpos Monoclonais/química , Afinidade de Anticorpos , Especificidade de Anticorpos , Astrócitos/metabolismo , Transporte Biológico , Linhagem Celular Tumoral , Receptores ErbB/imunologia , Corantes Fluorescentes/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Ratos , Ratos Wistar , Rodaminas/metabolismo , Proteína Estafilocócica A/químicaRESUMO
The Na+/H+ exchangers (NHEs) comprise a family of membrane proteins that catalyze the electroneutral exchange of Na+ and H+. Calcineurin homologous protein (CHP) acts as a crucial cofactor for NHE activity through direct interaction with the carboxyl-terminal tail region of NHEs. We have cloned a new rat CHP isoform (rCHP2) and characterized the binding property to NHEs and the tissue distribution. rCHP2 binds to the juxtamembrane region of plasma membrane-type NHE isoforms (NHE1-5) in vivo and in vitro as well as rCHP1 (original rat CHP). Interestingly, CHP2 is predominantly expressed in the small and large intestine although rCHP1 shows relatively ubiquitous expression at both the mRNA and protein levels. In situ hybridization experiments demonstrated the abundant expression of CHP2 in the epithelial cell layer of villi of the small intestine in contrast with the expression of CHP1 in both the epithelial layer and connective tissues. These results suggest that CHP2 functions in the absorptive epithelium for the intestine with NHE(s).