The human ABCB6 protein is the functional homologue of HMT-1 proteins mediating cadmium detoxification.

ABCB6 belongs to the family of ATP-binding cassette (ABC) transporters, which transport various molecules across extra- and intra-cellular membranes, bearing significant impact on human disease and pharmacology. Although mutations in the ABCB6 gene have been linked to a variety of pathophysiological conditions ranging from transfusion incompatibility to pigmentation defects, its precise cellular localization and function is not understood. In particular, the intracellular localization of ABCB6 has been a matter of debate, with conflicting reports suggesting mitochondrial or endolysosomal expression. ABCB6 shows significant sequence identity to HMT-1 (heavy metal tolerance factor 1) proteins, whose evolutionarily conserved role is to confer tolerance to heavy metals through the intracellular sequestration of metal complexes. Here, we show that the cadmium-sensitive phenotype of Schizosaccharomyces pombe and Caenorhabditis elegans strains defective for HMT-1 is rescued by the human ABCB6 protein. Overexpression of ABCB6 conferred tolerance to cadmium and As(III) (As2O3), but not to As(V) (Na2HAsO4), Sb(V), Hg(II), or Zn(II). Inactivating mutations of ABCB6 abolished vacuolar sequestration of cadmium, effectively suppressing the cadmium tolerance phenotype. Modulation of ABCB6 expression levels in human glioblastoma cells resulted in a concomitant change in cadmium sensitivity. Our findings reveal ABCB6 as a functional homologue of the HMT-1 proteins, linking endolysosomal ABCB6 to the highly conserved mechanism of intracellular cadmium detoxification.

Subcellular Duplex DNA and G-Quadruplex Interaction Profiling of a Hexagonal PtII Metallacycle.

Metal-driven self-assembly afforded a multitude of fascinating supramolecular coordination complexes (SCCs) with applications as catalysts, host-guest, and stimuli-responsive systems. However, the interest in the biological applications of SCCs is only starting to emerge and thorough characterization of their behavior in biological milieus is still lacking. Herein, we report on the synthesis and detailed in-cell tracking of a Pt2 L2 metallacycle. We show that our hexagonal supramolecule accumulates in cancer cell nuclei, exerting a distinctive blue fluorescence staining of chromatin resistant to UV photobleaching selectively in nucleolar G4-rich regions. SCC co-localizes with epitopes of the quadruplex-specific antibody BG4 and replaces other well-known G4 stabilizers. Moreover, the photophysical changes accompanying the metallacycle binding to G4s in solution (fluorescence quenching, absorption enhancement) also take place intracellularly, allowing its subcellular interaction tracking.

p53 Loss Mediates Hypersensitivity to ETS Transcription Factor Inhibition Based on PARylation-Mediated Cell Death Induction.

The small-molecule E26 transformation-specific (ETS) factor inhibitor YK-4-279 was developed for therapy of ETS/EWS fusion-driven Ewing's sarcoma. Here we aimed to identify molecular factors underlying YK-4-279 responsiveness in ETS fusion-negative cancers. Cell viability screenings that deletion of P53 induced hypersensitization against YK-4-279 especially in the BRAFV600E-mutated colon cancer model RKO. This effect was comparably minor in the BRAF wild-type HCT116 colon cancer model. Out of all ETS transcription factor family members, especially ETS1 overexpression at mRNA and protein level was induced by deletion of P53 specifically under BRAF-mutated conditions. Exposure to YK-4-279 reverted ETS1 upregulation induced by P53 knock-out in RKO cells. Despite upregulation of p53 by YK-4-279 itself in RKOp53 wild-type cells, YK-4-279-mediated hyperphosphorylation of histone histone H2A.x was distinctly more pronounced in the P53 knock-out background. YK-4-279-induced cell death in RKOp53-knock-out cells involved hyperPARylation of PARP1, translocation of the apoptosis-inducible factor AIF into nuclei, and induction of mitochondrial membrane depolarization, all hallmarks of parthanatos. Accordingly, pharmacological PARP as well as BRAFV600E inhibition showed antagonistic activity with YK-4-279 especially in the P53 knock-out background. Taken together, we identified ETS factor inhibition as a promising strategy for the treatment of notoriously therapy-resistant p53-null solid tumours with activating MAPK mutations.

Spatial signal repression as an additional role of Sprouty2 protein variants.

Sprouty2 (Spry2) is a prominent member of a protein family with crucial functions in the modulation of signal transduction. One of its main actions is the repression of mitogen-activated protein kinase (MAPK) pathway in response to growth factor-induced signalling. A common single nucleotide polymorphism within the Spry2 gene creates two protein variants where a proline adjacent to the serine rich domain is converted to an additional serine. Both protein variants perform similar functions although their efficiency in fulfilling these tasks varies. In this report, we used biochemical fractionation methods as well as confocal microscopy to analyse quantitative and qualitative differences in the distribution of Spry2 variants. We found that Spry2 proteins localize not solely to the plasma membrane, but also to other membrane engulfed compartments like for example the Golgi apparatus. In these less dense organelles, predominantly slower migrating forms reside indicating that posttranslational modification contributes to the distribution profile of Spry2. However there is no significant difference in the distribution of the two variants. Additionally, we found that Spry2 could be found exclusively in membrane fractions irrespective of the mitogen availability and the phosphorylation status. Considering the interference of extracellular signal-regulated kinase (ERK) activation in the cytoplasm, both Spry2 variants inhibited the levels of phosphorylated ERK (pERK) significantly to a similar extent. In contrast, the induction profiles of pERK levels were completely different in the nuclei. Again, both Spry2 variants diminished the levels of pERK. While the proline variant lowered the activation throughout the observation period, the serine variant failed to interfere with immediate accumulation of nuclear pERK levels, but the signal duration was shortened. Since the extent of the pERK inhibition in the nuclei was drastically more pronounced than in the cytoplasm, we conclude that Spry2 - in addition to its known functions as a repressor of general ERK phosphorylation - functions as a spatial repressor of nucleic ERK activation. Accordingly, a dominant negative version of Spry2 was only able to enhance the pERK levels of serum-deprived cells in the cytosol, while in the nucleus the intensity of the pERK signal in response to serum addition was significantly increased.

ABCB6 Resides in Melanosomes and Regulates Early Steps of Melanogenesis Required for PMEL Amyloid Matrix Formation.

Genetically inheritable pigmentation defects provide a unique opportunity to reveal the function of proteins contributing to melanogenesis. Dyschromatosis universalis hereditaria (DUH) is a rare pigmentary genodermatosis associated with mutations in the ABCB6 gene. Here we use optical and electron microscopy imaging combined with biochemical tools to investigate the localization and function of ABCB6 in pigment cells. We show that ABCB6 localizes to the membrane of early melanosomes and lysosomes of the human melanocytic cell line MNT-1. Depletion of ABCB6 by siRNA impaired PMEL amyloidogenesis in early melanosomes and induced aberrant accumulation of multilamellar aggregates in pigmented melanosomes. PMEL fibril formation and normal maturation of pigmented melanosomes could be restored by the overexpression of wild-type ABCB6 but not by variants containing an inactivating catalytic mutation (K629M) or the G579E DUH mutation. In line with the impairment of PMEL matrix formation in the absence of ABCB6, morphological analysis of the retinal pigment epithelium of ABCB6 knockout mice revealed a significant decrease of melanosome numbers. Our study extends the localization of ABCB6 to melanosomes, suggesting a potential link between the function of ABCB6 and the etiology of DUH to amyloid formation in pigment cells.

Photounbinding of calmodulin from a family of CaM binding peptides.

BACKGROUND: Recent studies have shown that fluorescently labeled antibodies can be dissociated from their antigen by illumination with laser light. The mechanism responsible for the photounbinding effect, however, remains elusive. Here, we give important insights into the mechanism of photounbinding and show that the effect is not restricted to antibody/antigen binding. METHODOLOGY/PRINCIPAL FINDINGS: We present studies of the photounbinding of labeled calmodulin (CaM) from a set of CaM-binding peptides with different affinities to CaM after one- and two-photon excitation. We found that the photounbinding effect becomes stronger with increasing binding affinity. Our observation that photounbinding can be influenced by using free radical scavengers, that it does not occur with either unlabeled protein or non-fluorescent quencher dyes, and that it becomes evident shortly after or with photobleaching suggest that photounbinding and photobleaching are closely linked. CONCLUSIONS/SIGNIFICANCE: The experimental results exclude surface effects, or heating by laser irradiation as potential causes of photounbinding. Our data suggest that free radicals formed through photobleaching may cause a conformational change of the CaM which lowers their binding affinity with the peptide or its respective binding partner.