RESUMEN
The objective of this study was to develop a thermotriggered, polymer-based liposomal drug carrier with an activatable magnetic resonance imaging (MRI) contrast property for monitoring the release of substances and for localized tumor therapy. The multimodal thermoactivatable polymer-grafted liposomes (MTPLs) were tested to investigate whether the accumulation of MTPLs in colon-26 grafted tumors could be visualized in vivo using MRI and optical imaging, whether MTPLs induce signal enhancement, reflecting the release of their contents, after triggering by short-term heating (42.5°C for 10 minutes) 9 hours after MTPL administration (late-phase triggering), and whether MTPLs can provide a sufficient antitumor effect. The imaging and therapeutic properties of MTPLs were tested both in vitro and in vivo (BALB/c nude mice: heated group with MTPLs (n = 5), nonheated group with MTPLs (n = 5), heated group with doxorubicin-free MTPLs (n = 5), nonheated group with manganese-free MTPLs (n = 5), and kinetics observation group (n = 3); N = 23). Through in vivo MRI and fluorescent imaging, the MTPLs were shown to have significantly accumulated in the grafted colon-26 tumors 8 hours after administration. Delayed thermotriggering (9 hours after administration) caused MR signal enhancement, reflecting the release of their contents, after a short exposure to tolerable heat. In addition, significant antitumor effects were observed after treatment. The proposed polymer-based activatable MTPLs with a "delayed thermotrigger" provide a promising technology for cancer theranostics that allows minimal adverse effects and rapid interactive therapy.
Asunto(s)
Antineoplásicos/administración & dosificación , Liposomas , Neoplasias/tratamiento farmacológico , Polímeros/química , Humanos , Imagen por Resonancia Magnética , Neoplasias/patologíaRESUMEN
ZFAT is a transcriptional regulator, containing eighteen C2H2-type zinc-fingers and one AT-hook, involved in autoimmune thyroid disease, apoptosis, and immune-related cell survival. We determined the solution structures of the thirteen individual ZFAT zinc-fingers (ZF) and the tandemly arrayed zinc-fingers in the regions from ZF2 to ZF5, by NMR spectroscopy. ZFAT has eight uncommon bulged-out helix-containing zinc-fingers, and six of their structures (ZF4, ZF5, ZF6, ZF10, ZF11, and ZF13) were determined. The distribution patterns of the putative DNA-binding surface residues are different among the ZFAT zinc-fingers, suggesting the distinct DNA sequence preferences of the N-terminal and C-terminal zinc-fingers. Since ZFAT has three to five consecutive tandem zinc-fingers, which may cooperatively function as a unit, we also determined two tandemly arrayed zinc-finger structures, between ZF2 to ZF4 and ZF3 to ZF5. Our NMR spectroscopic analysis detected the interaction between ZF4 and ZF5, which are connected by an uncommon linker sequence, KKIK. The ZF4-ZF5 linker restrained the relative structural space between the two zinc-fingers in solution, unlike the other linker regions with determined structures, suggesting the involvement of the ZF4-ZF5 interfinger linker in the regulation of ZFAT function.
Asunto(s)
Proteínas de Unión al ADN/química , Conformación Proteica , Factores de Transcripción/química , Dedos de Zinc/genética , Secuencia de Aminoácidos/genética , Animales , Proteínas de Unión al ADN/genética , Regulación de la Expresión Génica , Humanos , Ratones , Resonancia Magnética Nuclear Biomolecular , Estructura Terciaria de Proteína , Relación Estructura-Actividad , Tiroiditis Autoinmune/genética , Factores de Transcripción/metabolismo , Transcripción GenéticaRESUMEN
Spliceostatin A (SSA) is a methylated derivative of an antitumor natural product FR901464, which specifically binds and inhibits the SF3b spliceosome sub-complex. To investigate the selective antitumor activity of SSA, we focused on the regulation of vascular endothelial growth factor (VEGF) mRNA, since VEGF is a key regulatory component in tumor angiogenesis and known for the intricate regulation of mRNA processing, such as alternative splicing. We found that in HeLa cells SSA reduced the amount of both mRNA and protein of VEGF. Spliceostatin A not only inhibited the splicing reaction of VEGF pre-mRNA but also reduced the total amount of VEGF's transcripts, while SSA affected GAPDH mRNA to a lesser extent. Given a significant reduction in VEGF gene expression, SSA was expected to possess anti-angiogenic activity in vivo. Indeed, SSA inhibited cancer cell-derived angiogenesis in vivo in a chicken chorioallantoic membrane (CAM) assay. The inhibition of angiogenesis with SSA was abolished by addition of exogenous VEGF. We also performed global gene expression analyses of HeLa cells and found that the expression levels of 38% of total genes including VEGF decreased to <50% of the basal levels following 16 h of SSA treatment. These results suggest that the global interference of gene expression including VEGF in tumor cells is at least one of the mechanisms by which SSA (or FR901464) exhibits its strong antitumor activity.
Asunto(s)
Inhibidores de la Angiogénesis/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Neovascularización Patológica/genética , Piranos/farmacología , Compuestos de Espiro/farmacología , Factor A de Crecimiento Endotelial Vascular/genética , Inhibidores de la Angiogénesis/química , Animales , Western Blotting , Embrión de Pollo , Membrana Corioalantoides/irrigación sanguínea , Membrana Corioalantoides/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Células HeLa , Humanos , Estructura Molecular , Neovascularización Patológica/metabolismo , Neovascularización Patológica/prevención & control , Análisis de Secuencia por Matrices de Oligonucleótidos , Piranos/química , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Compuestos de Espiro/química , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
SWIRM is a conserved domain found in several chromatin-associated proteins. Based on their sequences, the SWIRM family members can be classified into three subfamilies, which are represented by Swi3, LSD1, and Ada2. Here we report the SWIRM structure of human MYb-like, Swirm and Mpn domain-containing protein-1 (MYSM1). The MYSM1 SWIRM structure forms a compact HTH-related fold comprising five alpha-helices, which best resembles the Swi3 SWIRM structure, among the known SWIRM structures. The MYSM1 and Swi3 SWIRM structures are more similar to the LSD1 structure than the Ada2alpha structure. The SWIRM domains of MYSM1 and LSD1 lacked DNA binding activity, while those of Ada2alpha and the human Swi3 counterpart, SMARCC2, bound DNA. The dissimilarity in the DNA-binding ability of the MYSM1 and SMARCC2 SWIRM domains might be due to a couple of amino acid differences in the last helix. These results indicate that the SWIRM family has indeed diverged into three structural subfamilies (Swi3/MYSM1, LSD1, and Ada2 types), and that the Swi3/MYSM1-type subfamily has further diverged into two functionally distinct groups. We also solved the structure of the SANT domain of MYSM1, and demonstrated that it bound DNA with a similar mode to that of the c-Myb DNA-binding domain.
Asunto(s)
Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Factores de Transcripción/química , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , ADN/metabolismo , Humanos , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Datos de Secuencia Molecular , Filogenia , Pliegue de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Alineación de Secuencia , Soluciones , Relación Estructura-Actividad , Transactivadores , Proteasas Ubiquitina-EspecíficasRESUMEN
GCN2 is the alpha-subunit of the only translation initiation factor (eIF2alpha) kinase that appears in all eukaryotes. Its function requires an interaction with GCN1 via the domain at its N-terminus, which is termed the RWD domain after three major RWD-containing proteins: RING finger-containing proteins, WD-repeat-containing proteins, and yeast DEAD (DEXD)-like helicases. In this study, we determined the solution structure of the mouse GCN2 RWD domain using NMR spectroscopy. The structure forms an alpha + beta sandwich fold consisting of two layers: a four-stranded antiparallel beta-sheet, and three side-by-side alpha-helices, with an alphabetabetabetabetaalphaalpha topology. A characteristic YPXXXP motif, which always occurs in RWD domains, forms a stable loop including three consecutive beta-turns that overlap with each other by two residues (triple beta-turn). As putative binding sites with GCN1, a structure-based alignment allowed the identification of several surface residues in alpha-helix 3 that are characteristic of the GCN2 RWD domains. Despite the apparent absence of sequence similarity, the RWD structure significantly resembles that of ubiquitin-conjugating enzymes (E2s), with most of the structural differences in the region connecting beta-strand 4 and alpha-helix 3. The structural architecture, including the triple beta-turn, is fundamentally common among various RWD domains and E2s, but most of the surface residues on the structure vary. Thus, it appears that the RWD domain is a novel structural domain for protein-binding that plays specific roles in individual RWD-containing proteins.