Tianfan Cheng

Identification of metal-associated proteins in cells by using continuous-flow gel electrophoresis and inductively coupled plasma mass spectrometry.

Metals and metalloids are crucial for life and indispensable for a series of biological processes. It is estimated that a quarter to one third of all proteins require metals to carry out their functions, and roughly half of the known enzymes uses a particular metal as a cofactor. In spite of the prevalence and importance of metalloproteins, they are generally poorly characterized in many organisms. A recent study demonstrated that the microbial metalloproteome is much more extensive and diverse than we presently know. Currently, it is impossible to predict, genome-wide, the numbers and types of metals used by organisms and to define any metalloproteome until the proteins are fully characterized owing to diverse and poorly recognized metal coordination sites. Moreover, metals/metalloids have long been used for therapeutic purposes, for example, arsenic trioxide for the treatment of acute promyelocytic leukemia. The detailed molecular mechanisms, however, are still not fully understood owing to the complex functions of metals in biological systems. A robust and convenient approach, by which metals/metalloids can be mapped to their associated proteins proteome-wide is urgently needed. Such a methodology will improve our understanding of the molecular mechanisms of metal-dependent biological processes and profoundly promote metallomics research, an integrated biometal science complementary to genomics and proteomics. Gel electrophoresis has been one of the commonly used methods for separation and analysis of proteins based on their molecular mass and charge; however, it fails to provide information on metal identity and content for metalloproteins. The lack of convenient subsequent methods for specific metal detection confines its application on providing metalrelated information of corresponding proteins. Although laser ablation inductively coupled plasma mass spectrometry (LAICP-MS) and synchrotron X-ray fluorescence spectrometry (SXFS) have been used for the identification of metalbinding proteins on gels and in tissues/organs, either compromised sensitivity originating from the sample introduction system or limited accessibility to the synchrotron facility prevents their routine applications. Other strategies such as metal isotope radioautography, which employs unique metal isotopes to label metalloproteins, are also very inconvenient for daily usage. Herein, a new strategy based on column-type gel electrophoresis coupled with a metal-specific detection system, that is ICP-MS, was developed (Figure 1a), allowing both metals and their associated proteins to be examined comprehensively. Since the strategy can be used to analyze and at the same time to separate and isolate proteins, it can readily be applied to not only detect metalloproteins and/or metalbound proteins with a sensitivity at the femtomole level, but also conveniently integrate current proteomics with metallomics. We further showed the bismuth profile in cell lysates of Helicobacter pylori upon treatment with colloidal bismuth subcitrate (CBS) and further characterized metal-binding features of H. pylori SlyD (HpSlyD) inside cells. The column-type gel adopted the traditional slab gel preparation. Both native and denaturing conditions could be applied, and the gel compositions varied with the protein targets of interest. To validate the feasibility of the column gel system, three metal-binding proteins, Cu-bound bovine serum albumin (Cu-BSA), Cu,Zn superoxide dismutase (Cu,ZnSOD), and diferric transferrin (Fe2-Tf), were mixed and subjected to separation. Three bands, corresponding to Fe2Tf, Cu,Zn-SOD, and BSA, were visualized on a CoomassieBlue-stained slab gel (Figure 1b). The proteins separated by column-type gel electrophoresis gave rise to migration profiles similar to those observed in classical slab gel under comparable conditions. The elutes from the column gel system were split into two parts by using a T connection, with one for online metal measurement by ICP-MS and the other for protein identification through biological mass spectrometry analysis of the collected fractions (Figure 1a). It is noted that one peak was observed in either the Zn or Fe profile corresponding to SOD and transferrin, respectively, indicative of association of Zn ions with SOD and binding of Fe ions to Tf; whereas there are two peaks in the Cu profile, with each corresponding to a distinct molecular mass, thus suggesting that copper binds to both SOD and BSA. The amounts of metals were quantifiable (Figure S2 in [*] Dr. L. Hu, Dr. T. Cheng, Dr. B. He, Y. Wang, Y.-T. Lai, Prof. H. Sun Department of Chemistry, The University of Hong Kong Pokfulam, Hong Kong (P. R. China) E-mail: hsun@hku.hk

Interaction of SlyD with HypB of Helicobacter pylori facilitates nickel trafficking

SlyD from Helicobacter pylori interacts with the [NiFe] hydrogenase accessory protein HypB through its IF domain. HpSlyD delivers Ni(2+) to HpHypB, leading to the enhancement of GTPase activity of HpHypB and implying the facilitation of Ni(2+) delivery from HpHypB to [NiFe] hydrogenase.

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. We constructed 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.

The role of citrate, lactate and transferrin in determining titanium release from surgical devices into human serum.

The presence of ionic titanium in the serum of patients with titanium implants is currently unexplained. This is presumed due to corrosion, and yet the serum titanium concentration measured in patients is far greater than that predicted by its solubility. The binding of titanium ion as Ti(IV) to human transferrin (hTF) in serum indicates that Ti(IV) ions interact with human physiology. This is an intriguing finding since there is currently no known role for titanium ions in human physiology. Thus, understanding the factors that determine in vivo titanium ion release is relevant to further understanding this metal’s interactions with human biochemistry. The present study sought to determine the extent of titanium ion release of into human serum in vitro, and the role of citrate, lactate and hTF in this process. It was found that, when surgical devices of commercially pure titanium were placed into human serum, citrate and lactate concentrations were the prime determinants of titanium release. Crystallography revealed Ti(IV) bound to hTF in the presence of citrate alone, signalling that citrate can act as an independent ligand for Ti(IV) binding to hTF. Based on these findings, a two-stage process of titanium ion release into human serum that is dependent upon both citrate and hTF is proposed to explain the ongoing presence of titanium ion in human subjects with implanted titanium devices.