A naturally occurring mutation in the SLC21A6 gene causing impaired membrane localization of the hepatocyte uptake transporter

The organic anion transporter SLC21A6 (also known as OATP2, OATP-C, or LST-1) is involved in the hepatocellular uptake of a variety of endogenous and xenobiotic substances and drugs. We analyzed 81 human liver samples by immunoblotting and found one with a strongly reduced amount of SLC21A6 protein suggesting mutations in the SLC21A6 gene. The SLC21A6 cDNA from this sample contained five base pair changes in one allele; three of the mutations resulted in amino acid substitutions designated SLC21A6-N130D, SLC21A6-P155T, and SLC21A6-L193R. The former two were polymorphisms (SLC21A6*1b and SLC21A6*4), whereas SLC21A6-L193R represents the first naturally occurring mutation identified in one allele of the SLC21A6 gene, which affects protein maturation and organic anion transport. We introduced each of the mutations into the SLC21A6 cDNA and established stably transfected MDCKII cells expressing the respective mutant SLC21A6 protein. Immunofluorescence microscopy and uptake measurements were used to study localization and transport properties of the mutated proteins. Both proteins carrying the polymorphisms were sorted to the lateral membrane like wild-type SLC21A6, but their transport properties for the substrates cholyltaurine and 17beta-glucuronosyl estradiol were altered. Importantly, most of the mutant protein SLC21A6-L193R was retained intracellularly, and this single amino acid exchange abolished transport function.     

Site-directed perturbation of protein kinase C- integrin interaction blocks carcinoma cell chemotaxis

Polarized cell movement is an essential requisite for cancer metastasis; thus, interference with the tumor cell motility machinery would significantly modify its metastatic behavior. Protein kinase C alpha (PKC alpha) has been implicated in the promotion of a migratory cell phenotype. We report that the phorbol ester-induced cell polarization and directional motility in breast carcinoma cells is determined by a 12-amino-acid motif (amino acids 313 to 325) within the PKC alpha V3 hinge domain. This motif is also required for a direct association between PKC alpha and beta 1 integrin. Efficient binding of beta 1 integrin to PKC alpha requires the presence of both NPXY motifs (Cyto-2 and Cyto-3) in the integrin distal cytoplasmic domains. A cell-permeant inhibitor based on the PKC-binding sequence of beta 1 integrin was shown to block both PKC alpha-driven and epidermal growth factor (EGF)-induced chemotaxis. When introduced as a minigene by retroviral transduction into human breast carcinoma cells, this inhibitor caused a striking reduction in chemotaxis towards an EGF gradient. Taken together, these findings identify a direct link between PKC alpha and beta 1 integrin that is critical for directed tumor cell migration. Importantly, our findings outline a new concept as to how carcinoma cell chemotaxis is enhanced and provide a conceptual basis for interfering with tumor cell dissemination.     

Stabilisation of TG- and AG-containing antiparallel DNA triplexes by triplex-binding ligands

We have used DNase I footprinting to examine the interaction of several triplex-binding ligands with antiparallel TG- and AG-containing triplexes. We find that although a 17mer TG-containing oligonucleotide on its own fails to produce a footprint at concentrations as high as 30 µM, this interaction can be stabilised by several ligands. Within a series of disubstituted amidoanthraquinones we find that the 2,7- regioisomer affords the best stabilisation of this TG triplex, though the 1,8- isomer also stabilises this interaction to some extent. By contrast the 1,5- and 2,6- regioisomers show no interaction with TG triplexes. Similar studies with a 13mer AG-containing oligonucleotide show the opposite pattern of stabilisation: the 2,6- and 1,5- isomers stabilise this triplex, but the 2,7- and 1,8-compounds do not. The polycyclic compound BePI strongly stabilises TG- but not AG-containing triplexes, while a substituted naphthylquinoline interacts with both antiparallel triplex motifs.     

Nuclear forward scattering of synchrotron radiation by deoxymyoglobin

Nuclear forward scattering of synchrotron radiation is used to determine the quadrupole splitting and the mean square displacement of the iron atom in deoxymyoglobin in the temperature range between 50 K and 243 K. Above 200 K an abnormally fast decay of the forward scattered intensity at short times after the synchrotron flash is observed, which is caused by protein-specific motions. The results strongly support the picture that protein dynamics seen at the position of the iron can be understood by harmonic motions in the low temperature regime while in the physiological regime diffusive motions in limited space are present. The shape of the resonance broadening function is investigated. An inhomogeneous broadening with a Lorentzian distribution indicating dipole interactions results in a better agreement with the experimental data than the common Gaussian distribution. Received: 30 August 1999 / Revised version: 22 October 1999 / Accepted: 6 December 1999     

Increased expression of mature cathepsin B in aging rat liver

Senescence has been proposed as an important safeguard against neoplasia. One of the hallmarks of cellular senescence in vitro as well as human aging in vivo is a reduced intracellular protein catabolism. The pathways affected and the mechanisms responsible for the decrease in overall protein turnover in aging cells are not well understood. Our aim was to determine whether or not expression of one of the major hepatic lysosomal cysteine peptidases, cathepsin B, changes during aging of Sprague-Dawley rats. Cathepsin B activity was assessed in whole rat liver homogenates, and was found to be increased fourfold (P< or =0.001) in aged livers compared with younger counterparts. This was paralleled by an at least a twofold increase in mature cathepsin B protein. Nonetheless, Northern blot analysis of total liver RNA revealed no change in steady-state levels of cathepsin B mRNAs. These findings seem to contradict the present dogma according to which aging tissues have a reduced intracellular capacity to catabolise proteins. We propose that our earlier observation of the accumulation of T-kininogen, a potent but reversible cysteine peptidase inhibitor, in aging rat liver may provide a plausible explanation for this discrepancy.     

Bis‐ and Tetrakis(carboxylato)platinum(IV) Complexes with Mixed Axial Ligands – Synthesis,Characterization, and Cytotoxicity

A series of twelve novel diamminetetrakis(carboxylato)platinum(IV) and 18 novel bis(carboxylato)dichlorido(ethane‐1,2‐diamine)platinum(IV) complexes with mixed axial carboxylato ligands was synthesized and characterized by multinuclear ¹H‐, ¹³C‐, ¹⁵N‐, and ¹⁹⁵Pt‐NMR spectroscopy. Their cytotoxic potential was evaluated (by MTT assay) against three human cancer cell lines derived from ovarian teratocarcinoma (CH1/PA‐1), lung (A549), and colon carcinoma (SW480). In the cisplatin‐sensitive CH1/PA‐1 cancer cell line, diamminetetrakis(carboxylato)platinum(IV) complexes showed IC₅₀ values in the low micromolar range, whereas, for the most lipophilic compounds of the bis(carboxylato)dichlorido(ethane‐1,2‐diamine)platinum(IV) series, IC₅₀ values in the nanomolar range were found.     

Ruthenium versus platinum: interactions of anticancer metallodrugs with duplex oligonucleotides characterised by electrospray ionisation mass spectrometry

The binding of the ruthenium-based anticancer drug candidates KP1019, NAMI-A and RAPTA-T towards different double-stranded oligonucleotides was probed by electrospray ionisation mass spectrometry and compared with that of the widely used platinum-based chemotherapeutics cisplatin, carboplatin and oxaliplatin. It was found that the extent of adduct formation decreased in the following order: cisplatin > oxaliplatin > NAMI-A > RAPTA-T > carboplatin > KP1019. In addition to the characterisation of the adducts formed with the DNA models, the binding sites of the metallodrugs on the oligonucleotides were elucidated employing top-down tandem mass spectrometry and were found to be similar for all the metallodrugs studied, irrespective of the sequence of the oligonucleotide. A strong preference for guanine residues was established.     

Differences in binding behavior of (−)‐epigallocatechin gallate to β‐lactoglobulin heterodimers (AB) compared to homodimers (A) and (B)

The lipocalin β‐lactoglobulin (β‐LG) exists in different natural genetic variants—of which β‐LG A and B are predominant in bovine milk. At physiological conditions the protein dimerizes—building homodimers of β‐LG A and β‐LG B and heterodimers of β‐LG AB. Although β‐LG is one of the most intensely characterized lipocalins, the interaction behavior of ligands with hetero‐ and homodimers of β‐LG is largely unknown. The present findings revealed significant differences for hetero‐ and homodimers regarding ligand binding capacity as tested with a model ligand (i.e. surface binding (?)‐epigallocatechin gallate (EGCG)). These findings were confirmed using FT‐IR, where the addition of EGCG influenced the β‐sheet backbone of homodimer A and B with significantly higher intensity compared to heterodimer AB. Further, shape analysis by SAXS revealed oligomerization of both types of dimers upon addition of EGCG; however, homodimer A and B produced significantly larger aggregates compared to the heterodimer AB. In summary, the present study revealed that EGCG showed significantly different interaction reactivity (binding sites, aggregation size and conformational changes) to the hetero and homodimers of β‐LG in the order β‐LG A > B > AB. The results suggest that conformational differences between homodimers and heterodimers strongly influence the EGCG binding ability. This may also occur with other polyphenols and ligands of β‐LG and gives not only important information for β‐LG binding studies, but may also apply for polymorphisms of other self‐aggregating lipocalins. Copyright © 2015 John Wiley & Sons, Ltd.     

Spatially distinct binding of Cdc42 to PAK1 and N-WASP in breast carcinoma cells

While a significant amount is known about the biochemical signaling pathways of the Rho family GTPase Cdc42, a better understanding of how these signaling networks are coordinated in cells is required. In particular, the predominant subcellular sites where GTP-bound Cdc42 binds to its effectors, such as p21-activated kinase 1 (PAK1) and N-WASP, a homolog of the Wiskott-Aldritch syndrome protein, are still undetermined. Recent fluorescence resonance energy transfer (FRET) imaging experiments using activity biosensors show inconsistencies between the site of local activity of PAK1 or N-WASP and the formation of specific membrane protrusion structures in the cell periphery. The data presented here demonstrate the localization of interactions by using multiphoton time-domain fluorescence lifetime imaging microscopy (FLIM). Our data here establish that activated Cdc42 interacts with PAK1 in a nucleotide-dependent manner in the cell periphery, leading to Thr-423 phosphorylation of PAK1, particularly along the lengths of cell protrusion structures. In contrast, the majority of GFP-N-WASP undergoing FRET with Cy3-Cdc42 is localized within a transferrin receptor- and Rab11-positive endosomal compartment in breast carcinoma cells. These data reveal for the first time distinct spatial association patterns between Cdc42 and its key effector proteins controlling cytoskeletal remodeling.     

Rodent cells support key functions of the human immunodeficiency virus type 1 pathogenicity factor Nef

After infection with human immunodeficiency virus (HIV), progression toward immunodeficiency is governed by a complex interplay of viral and host determinants. The viral accessory protein Nef is a key factor for the development of AIDS. Strains of HIV and simian immunodeficiency virus that lack functional nef genes either do not induce AIDS or do so only after a significant delay. The validity of a transgenic-small-animal model for de novo infection by HIV will depend on its ability to recapitulate the actions of critical factors of viral pathogenicity, such as Nef. We assessed the ability of rat, mouse, and hamster cells to support key effector functions of Nef. In cell lines from rodents, the subcellular distribution of wild-type HIV type 1 strain SF2 Nef and mutants was comparable to that in human cells. Nef downregulated human CD4 from the cell surface, was associated with p21-activated kinase activity, and enhanced the infectivity of HIV-1 virions. Importantly, these Nef-induced effects, as well as the downregulation of rat CD4 and major histocompatibility complex class I molecules, could also be demonstrated in primary T lymphocytes and macrophages from human CD4-transgenic rats. Thus, HIV-1 Nef exerts key functions in rodent cells. In line with our ongoing efforts to establish a transgenic-rat model of HIV disease, these results indicate that important aspects of viral pathogenesis could be addressed in a transgenic-rodent model permissive for de novo infection and that such a model would be valuable for evaluating the function of Nef in vivo.