Functional expression of voltage-gated calcium channels in human melanoma

The expression of voltage-gated calcium channels (VGCCs) has not been reported previously in melanoma cells in spite of increasing evidence of a role of VGCCs in tumorigenesis and tumour progression. To address this issue we have performed an extensive RT-PCR analysis of VGCC expression in human melanocytes and a range of melanoma cell lines and biopsies. In addition, we have tested the functional expression of these channels using Ca(2+) imaging techniques and examined their relevance for the viability and proliferation of the melanoma cells. Our results show that control melanocytes and melanoma cells express channel isoforms belonging to the Ca(v) 1 and Ca(v) 2 gene families. Importantly, the expression of low voltage-activated Ca(v) 3 (T-type) channels is restricted to melanoma. We have confirmed the function of T-type channels as mediators of constitutive Ca(2+) influx in melanoma cells. Finally, pharmacological and gene silencing approaches demonstrate a role for T-type channels in melanoma viability and proliferation. These results encourage the analysis of T-type VGCCs as targets for therapeutic intervention in melanoma tumorigenesis and/or tumour progression.     

Up-regulation of BMP-2 antagonizes TGF-β1/ROCK-enhanced cardiac fibrotic signalling through activation of Smurf1/Smad6 complex

Rho‐associated kinase (ROCK) plays a critical role in pressure overload‐induced left ventricular remodelling. However, the underlying mechanism remains unclear. Here, we reported that TGF‐β1‐induced ROCK elevation suppressed BMP‐2 level and strengthened fibrotic response. Exogenous BMP‐2 supply effectively attenuated TGF‐β1 signalling pathway through Smad6‐Smurf‐1 complex activation. In vitro cultured cardiomyocytes, mechanical stretch up‐regulated cardiac TGF‐β1, TGF‐β1‐dependent ROCK and down‐regulated BMP‐2, but BMP‐2 level could be reversed through blocking TGF‐β1 receptor by SB‐431542 or inhibition of ROCK by Y‐27632. TGF‐β1 could also activate ROCK and suppress endogenous BMP‐2 level in a dose‐dependent manner. Knock‐down BMP‐2 enhanced TGF‐β1‐mediated PKC‐δ and Smad3 signalling cascades. In contrast, treatment with Y‐27632 or SB‐431542, respectively suppressed ROCK‐dependent PKC‐δ and Smad3 activation, but BMP‐2 was only up‐regulated by Y‐27632. In addition, BMP‐2 silencing abolished the effect of Y‐27632, but not SB‐431542 on suppression of TGF‐β1 pathway. Further experiments showed that Smad6 Smurf1 interaction were required for BMP‐2‐evoked antagonizing effects. Smad6 overexpression attenuated TGF‐β1‐induced activation of PKC‐δ and Smad3, promoted TGF‐β RI degradation in BMP‐2 knock‐down cardiomyocytes, and could be abolished after knocking‐down Smurf‐1, in which Smad6/Smurf1 complex formation was critically involved. In vivo data showed that pressure overload‐induced collagen deposition was attenuated, cardiac function was improved and TGF‐β1‐dependent activation of PKC‐δ and Smad3 was reduced after 2 weeks treatment with rhBMP‐2(0.5 mg/kg) or Y‐27632 (10 mg/kg) in mice that underwent surgical transverse aortic constriction. In conclusion, we propose that BMP‐2, as a novel fibrosis antagonizing cytokine, may have potential beneficial effect in attenuating pressure overload‐induced cardiac fibrosis.     

Membrane dynamics of γ‐secretase with the anterior pharynx‐defective 1B subunit

The four‐subunit protease complex γ‐secretase cleaves many single‐pass transmembrane (TM) substrates, including Notch and β‐amyloid precursor protein to generate amyloid‐β (Aβ), central to Alzheimer's disease. Two of the subunits anterior pharynx‐defective 1 (APH‐1) and presenilin (PS) exist in two homologous forms APH1‐A and APH1‐B, and PS1 and PS2. The consequences of these variations are poorly understood and could affect Aβ production and γ‐secretase medicine. Here, we developed the first complete structural model of the APH‐1B subunit using the published cryo‐electron microscopy (cryo‐EM) structures of APH1‐A (Protein Data Bank: 5FN2, 5A63, and 6IYC). We then performed all‐atom molecular dynamics simulations at 303 K in a realistic bilayer system to understand both APH‐1B alone and in γ‐secretase without and with substrate C83‐bound. We show that APH‐1B adopts a 7TM topology with a water channel topology similar to APH‐1A. We demonstrate direct transport of water through this channel, mainly via Glu84, Arg87, His170, and His196. The apo and holo states closely resemble the experimental cryo‐EM structures with APH‐1A, however with subtle differences: The substrate‐bound APH‐1B γ‐secretase was quite stable, but some TM helices of PS1 and APH‐1B rearranged in the membrane consistent with the disorder seen in the cryo‐EM data. This produces different accessibility of water molecules for the catalytic aspartates of PS1, critical for Aβ production. In particular, we find that the typical distance between the catalytic aspartates of PS1 and the C83 cleavage sites are shorter in APH‐1B, that is, it represents a more closed state, due to interactions with the C‐terminal fragment of PS1. Our structural‐dynamic model of APH‐1B alone and in γ‐secretase suggests generally similar topology but some notable differences in water accessibility which may be relevant to the protein's existence in two forms and their specific function and location.     

Staphylococcus aureus Lpl protein triggers human host cell invasion via activation of Hsp90 receptor

Staphylococcus aureus is a facultative intracellular pathogen. Recently, it has been shown that the protein part of the lipoprotein‐like lipoproteins (Lpls), encoded by the lpl cluster comprising of 10 lpls paralogue genes, increases pathogenicity, delays the G2/M phase transition, and also triggers host cell invasion. Here, we show that a recombinant Lpl1 protein without the lipid moiety binds directly to the isoforms of the human heat shock proteins Hsp90α and Hsp90ß. Synthetic peptides covering the Lpl1 sequence caused a twofold to fivefold increase of S. aureus invasion in HaCaT cells. Antibodies against Hsp90 decrease S. aureus invasion in HaCaT cells and in primary human keratinocytes. Additionally, inhibition of ATPase function of Hsp90 or silencing Hsp90α expression by siRNA also decreased the S. aureus invasion in HaCaT cells. Although the Hsp90ß is constitutively expressed, the Hsp90α isoform is heat‐inducible and appears to play a major role in Lpl1 interaction. Pre‐incubation of HaCaT cells at 39°C increased both the Hsp90α expression and S. aureus invasion. Lpl1‐Hsp90 interaction induces F‐actin formation, thus, triggering an endocytosis‐like internalisation. Here, we uncovered a new host cell invasion principle on the basis of Lpl‐Hsp90 interaction.     

Revealing interaction between sulfobutylether‐β‐cyclodextrin and reserpine by chemiluminescence and site‐directed molecular docking

The host–guest interaction between sulfobutylether‐β‐cyclodextrin (SBE‐β‐CD) and reserpine (RSP) is described using flow injection‐chemiluminescence (FI‐CL) and site‐directed molecular docking methods. It was found that RSP could inhibit the CL intensity produced by a luminol/SBE‐β‐CD system. The decrease in CL intensity was logarithmic over an RSP concentration range of 0.03 to 700.0 nM, giving a regression equation of ?I = 107.1lgC_RES + 186.1 with a detection limit of 10 pM (3σ). The CL assay was successfully applied in the determination of RSP in injection, saliva and urine samples with recoveries in the range 93.5–106.1%. Using the proposed CL model, the binding constant (K_CD‐R) and the stoichiometric ratio of SBE‐β‐CD/RSP were calculated to be 7.4 × 10⁶ M^‐1 and 1 : 1, respectively. Using molecular docking, it was confirmed that luminol binds to the small cavity of SBE‐β‐CD with a nonpolar interaction, while RSP targeted the larger cavity of SBE‐β‐CD and formed a 1 : 1 complex with hydrogen bonds. The proposed new CL method has the potential to become a powerful tool for revealing the host–guest interaction between CDs and drugs, as well as monitoring drugs with high sensitivity. Copyright © 2013 John Wiley & Sons, Ltd.