Improving ex vivo skin permeation of non-steroidal anti-inflammatory drugs: enhancing extemporaneous transformation of liposomes into planar lipid bilayers.

Transdermal delivery of active principles is a versatile method widely used in medicine. The main drawback for the transdermal route, however, is the low efficiency achieved in the absorption of many drugs, mostly due to the complexity of the skin barrier. To improve drug delivery through the skin, we prepared and characterized liposomes loaded with ibuprofen and designed pharmaceutical formulations based on the extemporaneous addition of penetration enhancer (PE) surfactants. Afterwards, permeation and release studies were carried out. According to the permeation studies, the ibuprofen liposomal formulation supplemented with PEs exhibited similar therapeutic effects, but at lower doses (20%) comparing with a commercial formulation used as a reference. Atomic force microscopy (AFM) was used to investigate the effect caused by PEs on the adsorption mechanism of liposomal formulations onto the skin. Non-fused liposomes, bilayers and multilayered lipid structures were observed. The transformation of vesicles into planar structures is proposed as a possible rationale for explaining the lower doses required when a liposome formulation is supplemented with surfactant PEs.

Xenopus HJURP and condensin II are required for CENP-A assembly.

Centromeric protein A (CENP-A) is the epigenetic mark of centromeres. CENP-A replenishment is necessary in each cell cycle to compensate for the dilution associated to DNA replication, but how this is achieved mechanistically is largely unknown. We have developed an assay using Xenopus egg extracts that can recapitulate the spatial and temporal specificity of CENP-A deposition observed in human cells, providing us with a robust in vitro system amenable to molecular dissection. Here we show that this deposition depends on Xenopus Holliday junction-recognizing protein (xHJURP), a member of the HJURP/Scm3 family recently identified in yeast and human cells, further supporting the essential role of these chaperones in CENP-A loading. Despite little sequence homology, human HJURP can substitute for xHJURP. We also report that condensin II, but not condensin I, is required for CENP-A assembly and contributes to retention of centromeric CENP-A nucleosomes both in mitosis and interphase. We propose that the chromatin structure imposed by condensin II at centromeres enables CENP-A incorporation initiated by xHJURP.

Epigenetic specification of centromeres by CENP-A.

Centromeres are the chromosomal loci that direct the formation of the kinetochores. These macromolecular assemblies mediate the interaction between chromosomes and spindle microtubules and thereby power chromosome movement during cell division. They are also the sites of extensive regulation of the chromosome segregation process. Except in the case of budding yeast, centromere identity does not rely on DNA sequence but on the presence of a special nucleosome that contains a histone H3 variant known as CenH3 or CENP-A (Centromere Protein A). It has been therefore proposed that CENP-A is the epigenetic mark of the centromere. Upon DNA replication the mark is diluted two-fold and must be replenished to maintain centromere identity. What distinguishes CENP-A nucleosomes from those containing histone H3, how CENP-A nucleosomes are incorporated specifically into centromeric chromatin, and how this incorporation is coordinated with other cell cycle events are key issues that have been the focus of intensive research over the last decade. Here we review some of the highlights of this research.

Kinase activation and transformation by NUP214-ABL1 is dependent on the context of the nuclear pore.

Genetic alterations causing constitutive tyrosine kinase activation are observed in a broad spectrum of cancers. Thus far, these mutant kinases have been localized to the plasma membrane or cytoplasm, where they engage proliferation and survival pathways. We report that the NUP214-ABL1 fusion is unique among these because of its requisite localization to the nuclear pore complex for its transforming potential. We show that NUP214-ABL1 displays attenuated transforming capacity as compared to BCR-ABL1 and that NUP214-ABL1 preferentially transforms T cells, which is in agreement with its unique occurrence in T cell acute lymphoblastic leukemia. Furthermore, NUP214-ABL1 differs from BCR-ABL1 in subcellular localization, initiation of kinase activity, and signaling and lacks phosphorylation on its activation loop. In addition to delineating an unusual mechanism for kinase activation, this study provides new insights into the spectrum of chromosomal translocations involving nucleoporins by indicating that the nuclear pore context itself may play a central role in transformation.

Nup214-Nup88 nucleoporin subcomplex is required for CRM1-mediated 60 S preribosomal nuclear export.

The nuclear pore complex (NPC) conducts macromolecular transport to and from the nucleus and provides a kinetic/hydrophobic barrier composed of phenylalanine-glycine (FG) repeats. Nuclear transport is achieved through permeation of this barrier by transport receptors. The transport receptor CRM1 facilitates export of a large variety of cargoes. Export of the preribosomal 60 S subunit follows this pathway through the adaptor protein NMD3. Using RNA interference, we depleted two FG-containing cytoplasmically oriented NPC complexes, Nup214-Nup88 and Nup358, and investigated CRM1-mediated export. A dramatic defect in NMD3-mediated export of preribosomes was found in Nup214-Nup88-depleted cells, whereas only minor export defects were evident in other CRM1 cargoes or upon depletion of Nup358. We show that the large C-terminal FG domain of Nup214 is not accessible to freely diffusing molecules from the nucleus, indicating that it does not conduct 60 S preribosomes through the NPC. Consistently, derivatives of Nup214 lacking the FG-repeat domain rescued the 60 S export defect. We show that the coiled-coil region of Nup214 is sufficient for 60 S nuclear export, coinciding with recruitment of Nup88 to the NPC. Our data indicate that Nup214 plays independent roles in NPC function by participating in the kinetic/hydrophobic barrier through its FG-rich domain and by enabling NPC gating through association with Nup88.

Supraphysiological nuclear export signals bind CRM1 independently of RanGTP and arrest at Nup358.

Leucine-rich nuclear export signals (NESs) mediate rapid nuclear export of proteins via interaction with CRM1. This interaction is stimulated by RanGTP but remains of a relatively low affinity. In order to identify strong signals, we screened a 15-mer random peptide library for CRM1 binding, both in the presence and absence of RanGTP. Under each condition, strikingly similar signals were enriched, conforming to the NES consensus sequence. A derivative of an NES selected in the absence of RanGTP exhibits very high affinity for CRM1 in vitro and stably binds without the requirement of RanGTP. Localisation studies and RNA interference demonstrate inefficient CRM1-mediated export and accumulation of CRM1 complexed with the high-affinity NES at nucleoporin Nup358. These results provide in vivo evidence for a nuclear export reaction intermediate. They suggest that NESs have evolved to maintain low affinity for CRM1 to allow efficient export complex disassembly and release from Nup358.

Nup358/RanBP2 attaches to the nuclear pore complex via association with Nup88 and Nup214/CAN and plays a supporting role in CRM1-mediated nuclear protein export.

Nuclear pore complexes (NPCs) traverse the nuclear envelope (NE), providing a channel through which nucleocytoplasmic transport occurs. Nup358/RanBP2, Nup214/CAN, and Nup88 are components of the cytoplasmic face of the NPC. Here we show that Nup88 localizes midway between Nup358 and Nup214 and physically interacts with them. RNA interference of either Nup88 or Nup214 in human cells caused a strong reduction of Nup358 at the NE. Nup88 and Nup214 showed an interdependence at the NPC and were not affected by the absence of Nup358. These data indicate that Nup88 and Nup214 mediate the attachment of Nup358 to the NPC. We show that localization of the export receptor CRM1 at the cytoplasmic face of the NE is Nup358 dependent and represents its empty state. Also, removal of Nup358 causes a distinct reduction in nuclear export signal-dependent nuclear export. We propose that Nup358 provides both a platform for rapid disassembly of CRM1 export complexes and a binding site for empty CRM1 recycling into the nucleus.