An Intergrated Approach for Matching Metals and Metallodrugs to Proteins

Keynote Lecture (Abstract)The effect of metals in biology effects is double-edged. Metal ions operate, on one hand, as cofactors for around 40% enzymes, on the other hand, they also exhibit toxic effects. Some metal ions, although being not essential, have been widely used in human healthcare as either therapeutic agents or diagnosis agents. To understand the molecular mechanism of a metallodrug, it is crucial to match metals to proteins at a proteome-wide scale [1,2]. We used an integrated approach consisting of gel electrophoresis and inductively coupled plasma mass spectrometry, LA-ICP-MS, IMAC and bioinformatic approach to identify metal-associated proteins using bismuth antiulcer drug as an example [3,4]. Using continuous-flow gel electrophoresis in combination with ICP-MS, we developed a comprehensive and robust strategy to readily identify metal-associated proteins as well as to quantify the metals for fast metallome/proteome-wide profiling of metal-binding proteins. At the same time, we have developed a tunable fluorescent method to visualize metalbinding proteins and histidine-rich proteins directly in cells. To match metals to proteins, we also established a bioinformatic method which allows potential metal-binding proteins both sequentially and spaciously to be searched [5-7]. Surprisingly, histidine-rich proteins and motifs(HRMs) are commonly found in proteins. We systematically analyzed the proteomes of 675 prokaryotes including 50 archaea and 625 bacteria for HRMs, and show that HRMs are extensively distributed in prokaryotic proteomes, with the majority (62%) of histidine-rich proteins (HRPs) being involved in metal homeostasis. Importantly, the occurrence of histidine-rich proteins (motifs) in the proteomes of prokaryotes is related to their habitats.published_or_final_versio

Seeing is believing; tracking metalloproteins by fluorescent probe in vivo and in vitro

Abstract no. EuAsC2S-12/S1-OP22Extensive genome research has shown that around 1/4 to 1/3 proteins are metalloproteins (or metal-binding proteins) with various metal ions incorporated with proteins for either structural or functional purposes. Thus, metalloproteomics/metallomics are developed to investigate the molecular mechanism of metal-related biological processes and the entirety of metal/metalloid species within a cell or tissue type[1]. Fluorescence labeling is probably the best method in view of its capability in providing rapid and sensitive identification in living biological systems. In spite of the development of fluorescent proteins, synthetic small-molecule fluorescence agents have been utilized to identify specific targets in cells, while metal-chelation methodology has been extensively applied to the study of metal-oriented biological process[2]. Although different types of metal-responsive sensors have been developed to label cellular metals[3], tracking of metal-binding proteins in living cells by fluorescence is still highly anticipated. In this work, novel fluorescent probe was designed to label metalloproteins both in vivo and in vitro. The protein partners of several metal ions such as Ni2+ (Histidine-rich proteins in particular), Bi3+, Cr3+ have been identified by the agent. The fluorescent agent exhibited “turnon” response to the targets in SDS-PAGE, and its excellent permeability enabled “lighting up” of targeted proteins in living cells, providing valuable information on metalloprotein spatial distribution in biology.postprintThe 12th EuroAsia Conference on Chemical Sciences (EuAsC2S-12), Corfu, Greece, 16-21 April 2012

Fasting plasma zeaxanthin response to Fructus barbarum L. (wolfberry; Kei Tze) in a food-based human supplementation trial

Antioxidant Research Group, Faculty of Health & Social Sciences2004-2005 > Academic research: refereed > Publication in refereed journalVersion of RecordPublishe

Selective binding of Hpnl towards Ni(II) and Bi(III)

Poster-5Histidine-rich protein Hpn and histidine- and glutamine-rich protein Hpn-like (Hpnl) in Helicobacter pylori have been corroborated to be crucial to nickel homeostasis.[1-3] Nickel supply to hydrogenases and ureases might be disrupted owing to the interaction of metallodrugs, such as bismuth antiulcer drugs, with Hpnl, which may subsequently disturb the functions of the essential …postprin

The Diurnal Variation on Cardiovascular Endurance Performance of Secondary School Athlete Student

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Selective interaction of Hpn-like protein with nickel, zinc and bismuth in vitro and in cells by FRET

Hpn-like (Hpnl) is a unique histidine- and glutamine-rich protein found only in Helicobacter pylori and plays a role on nickel homeostasis.Weconstructed the fluorescent sensor proteins CYHpnl and CYHpnl_1-48 (C-terminal glutamine-rich region truncated) using enhanced cyan and yellow fluorescent proteins (eCFP and eYFP) as the donor–acceptor pair to monitor the interactions of Hpnl with metal ions and to elucidate the role of conserved Glu-rich sequence in Hpnl by fluorescence resonance energy transfer (FRET). CYHpnl and CYHpnl_1-48 exhibited largest responses towards Ni(II) and Zn(II) over other metals studied and the binding of Bi(III) to CYHpnl was observed in the presence of an excess amount of Bi(III) ions (Kd =115±4.8 μM). Moreover, both CYHpnl and CYHpnl_1-48 showed positive FRET responses towards the binding to Ni(II) and Zn(II) in Escherichia coli cells overexpressing CYHpnl and CYHpnl_1-48, whereas a decrease in FRET upon Bi(III)-binding in E. coli cells overexpressing the latter. Our study provides clear evidence on Hpnl binding to nickel in cells, and intracellular interaction of Hpnl with Bi(III) could disrupt the protein function, thus probably contributing to the efficacy of Bi(III) drugs against H. pylori.postprin

Single Cell Transfection through Precise Microinjection with Quantitatively Controlled Injection Volumes

Cell transfection is a technique wherein foreign genetic molecules are delivered into cells. To elucidate distinct responses during cell genetic modification, methods to achieve transfection at the single-cell level are of great value. Herein, we developed an automated micropipette-based quantitative microinjection technology that can deliver precise amounts of materials into cells. The developed microinjection system achieved precise single-cell microinjection by pre-patterning cells in an array and controlling the amount of substance delivered based on injection pressure and time. The precision of the proposed injection technique was examined by comparing the fluorescence intensities of fluorescent dye droplets with a standard concentration and water droplets with a known injection amount of the dye in oil. Injection of synthetic modified mRNA (modRNA) encoding green fluorescence proteins or a cocktail of plasmids encoding green and red fluorescence proteins into human foreskin fibroblast cells demonstrated that the resulting green fluorescence intensity or green/red fluorescence intensity ratio were well correlated with the amount of genetic material injected into the cells. Single-cell transfection via the developed microinjection technique will be of particular use in cases where cell transfection is challenging and genetically modified of selected cells are desiredpublished_or_final_versio

Phosphorylation of nucleophosmin at threonine 234/237 is associated with HCC metastasis

Hepatocellular carcinoma (HCC) is frequently complicated by the occurrence of intrahepatic and extrahepatic metastases, leading to poor prognosis. To improve the prognosis for HCC patients, there is an urgent need to understand the molecular mechanisms of metastasis in HCC. Since protein Serine/Threonine phosphorylation emerges to be an important posttranslational modification critical in signaling process associated with cell proliferation, survival and metastasis, we employed a pair of primary tumor-derived and corresponding lung-metastatic counterparts (PLC/PRF/5-PT and PLC/PRF/5-LM) and aimed to identify these changes using CelluSpot Serine/Threonine kinase peptide array. Upon analysis, we found phosphorylated level of nucleophosmin (NPM) at Threonine 234/237 (p-NPM-Thr234/237) had remarkably high level in metastatic HCC cells (PLC-LM) than the corresponding primary HCC cell line (PLC-PT). Similar observation was observed in another match primary and their metastatic counterparts (MHCC-97L and MHCC-97H). By immunohistochemical staining, p-NPM-Thr234/237 was consistently found to be preferentially expressed in metastatic HCCs when compared with primary HCC in 28 HCC cases (p < 0.0001). By overexpressing Flag-tagged NPM and its phosphorylation site mutant (Thr234/237A) into low p-NPM-Thr234/237 expressing cells (Hep3B and Huh7) using a lentiviral based approach, we demonstrated that p-NPM-Thr234/237 is critical in invasion and migration of HCC cells, and this effect was mediated by cyclin-dependent kinase 1 (CDK1). Wild-type NPM was found to physically interact with a metastatic gene, ROCK2, and defective in Thr234/237 phosphorylation decreased its binding affinity, resulting in decrease in ROCK2 mediated signaling pathway. Identification of CDK1/p-NPM/ROCK2 signaling pathway provides a novel target for molecular therapy against HCC metastasis.published_or_final_versio

Scallop swimming kinematics and muscle performance: modelling the effects of "within-animal" variation in temperature sensitivity

Escape behaviour was investigated in Queen scallops (Aequipecten opercularis) acclimated to 5, 10 or 15 degrees C and tested at their acclimation temperature. Scallops are active molluscs, able to escape from predators by jet-propelled swimming using a striated muscle working in opposition to an elastic hinge ligament. The first cycle of the escape response was recorded using high-speed video ( 250 Hz) and whole-animal velocity and acceleration determined. Muscle shortening velocity, force and power output were calculated using measurements of valve movement and jet area, and a simple biomechanical model. The average shortening speed of the adductor muscle had a Q(10) of 2.04, significantly reducing the duration of the jetting phase of the cycle with increased temperature. Muscle lengthening velocity and the overall duration of the clap cycle were changed little over the range 5 - 15 degrees C, as these parameters were controlled by the relatively temperature-insensitive, hinge ligament. Improvements in the average power output of the adductor muscle over the first clap cycle ( 222 vs. 139 W kg(-1) wet mass at 15 and 5 degrees C respectively) were not translated into proportional increases in overall swimming velocity, which was only 32% higher at 15 degrees C ( 0.37m s(-1)) than 5 degrees C (0.28 m s(-1))

Evidence for solar cycles in a late Holocene speleothem record from Dongge Cave, China

The association between solar activity and Asian monsoon (AM) remains unclear. Here we evaluate the possible connection between them based on a precisely-dated, high-resolution speleothem oxygen isotope record from Dongge Cave, southwest China during the past 4.2 thousand years (ka). Without being adjusted chronologically to the solar signal, our record shows a distinct peak-to-peak correlation with cosmogenic nuclide 14C, total solar irradiance (TSI) and sunspot number (SN) at multi-decadal to centennial timescales. Further cross-wavelet analyses between our calcite δ18O and atmospheric 14C show statistically strong coherence at three typical periodicities of ~80, 200 and 340 years, suggesting important roles of solar activities in modulating AM changes at those timescales. Our result has further indicated a better correlation between our calcite δ18O record and atmospheric 14C than between our record and TSI. This better correlation may imply that the Sun–monsoon connection is dominated most likely by cosmic rays and oceanic circulation (both associated to atmospheric 14C), instead of the direct solar heating (TSI)