Xuesong Sun

Application of immobilized metal affinity chromatography in proteomics

It has been proved that the progress of proteomics is mostly determined by the development of advanced and sensitive protein separation technologies. Immobilized metal affinity chromatography (IMAC) is a powerful protein fractionation method used to enrich metal-associated proteins and peptides. In proteomics, IMAC has been widely employed as a prefractionation method to increase the resolution in protein separation. The combination of IMAC with other protein analytical technologies has been successfully utilized to characterize metalloproteome and post-translational modifications. In the near future, newly developed IMAC integrated with other proteomic methods will greatly contribute to the revolution of expression, cell-mapping and structural proteomics.

Identification and characterization of metallodrug binding proteins by (metallo)proteomics

The success of cisplatin in clinic has stimulated great interest in the development and application of metal-based drugs for therapeutic and diagnostic purposes. However, the treatment efficiency of metallodrugs suffers from side-effects and drug resistance. To overcome these challenges, targets of these metal-based drugs should be identified in order to understand the molecular mechanisms of actions of these compounds and to the intrinsic or acquired drug resistance by cancer cells and infectious microbes. This review summaries some of the recent developments in the identification of binding proteins and their target sites of platinum-, ruthenium-, gold-, arsenic- and bismuth-containing agents by proteomics and metalloproteomics, which may provide a rational basis for the design of new metal-based drugs.

Crystal structure and metal binding properties of the lipoprotein MtsA, responsible for iron transport in Streptococcus pyogenes.

An ability to acquire iron is essential for the viability and growth of almost all organisms and in pathogenic bacteria is strongly correlated with virulence. The cell surface lipoprotein MtsA, a component of the MtsABC transporter of Streptococcus pyogenes, acts as the primary receptor for inorganic iron by this significant human pathogen. Iron is bound as Fe(2+), with the participation of bicarbonate. The crystal structure of MtsA has been determined and refined at 1.8 A resolution (R = 0.167, and R(free) = 0.194). MtsA has the classic bacterial metal binding receptor (MBR) fold, with the Fe(2+) ion bound to the side chains of His68, His140, Glu206, and Asp281, at a totally enclosed site between the two domains of the protein. The absence of bicarbonate from the binding site suggests that it is displaced during the final stages of metal binding. Both the fold and metal binding site are most similar to those of the manganese receptors PsaA and MntC, consistent with the similar coordination requirements of Fe(2+) and Mn(2+). Binding studies confirm a 10-fold preference for Fe(2+) over Mn(2+), although both may be carried in vivo. Mutational analysis of the binding site shows that His140 is critical for a fully functional binding site but that Glu206 is dispensable. The crystal structure explains the distinct roles of these ligands and also reveals potential secondary binding sites that may explain the binding behavior of MtsA for metal ions other than Fe(2+).

Identification of Proteins Related to Nickel Homeostasis in Helicobater pylori by Immobilized Metal Affinity Chromatography and Two-Dimensional Gel Electrophoresis

Helicobacter pylori (H. pylori) is a widespread human pathogen causing peptic ulcers and chronic gastritis. Maintaining nickel homeostasis is crucial for the establishment of H. pylori infection in humans. We used immobilized-nickel affinity chromatography to isolate Ni-related proteins from H. pylori cell extracts. Two-dimensional gel electrophoresis and mass spectrometry were employed to separate and identify twenty two Ni-interacting proteins in H. pylori. These Ni-interacting proteins can be classified into several general functional categories, including cellular processes (HspA, HspB, TsaA, and NapA), enzymes (Urease, Fumarase, GuaB, Cad, PPase, and DmpI), membrane-associated proteins (OM jhp1427 and HpaA), iron storage protein (Pfr), and hypothetical proteins (HP0271, HP jhp0216, HP jhp0301, HP0721, HP0614, and HP jhp0118). The implication of these proteins in nickel homeostasis is discussed.

Lipoprotein MtsA of MtsABC in Streptococcus pyogenes primarily binds ferrous ion with bicarbonate as a synergistic anion

Lipoprotein MtsA is a critical component of MtsABC responsible for iron binding and transport in the Gram‐positive bacterium Streptococcus pyogenes. The present collective experimental data establish that Fe2+ is the primary binding ion for MtsA under optimal physiologically relevant conditions. The binding affinities of MtsA to metal ions are Fe2+ > Fe3+ >Cu2+ > Mn2+ > Zn2+. We report for the first time that bicarbonate is required as a synergistic anion for stable ferrous binding to MtsA, similar to the iron binding in human transferrin. This work provides valuable information, which helps to understand iron metabolism in bacteria, and creates a basis for developing strategies to suppress bacterial infection.

Iron depletion decreases proliferation and induces apoptosis in a human colonic adenocarcinoma cell line, Caco2.

Iron is essential for maintaining cellular metabolism of most organisms. Iron chelators such as desferrioxamine have been used clinically in the treatment of iron overload diseases. In the present study, we used human colon adenocarcinoma cells as a proliferating cell model to validate that desferrioxamine inhibits cell proliferation and induces apoptosis. Proteomic analysis revealed that proteins involved in cell proliferation, signal transduction, metabolism and protein synthesis were significantly regulated by the availability of iron, rendering a close correlation between cell apoptosis and the disturbance of mitochondrial, signaling and metabolic pathways. These results provide new insights into the mechanisms of cell proliferation inhibition attributed to iron depletion.

The low pKa value of iron-binding ligand Tyr188 and its implication in iron release and anion binding of human transferrin.

2D NMR–pH titrations were used to determine pK a values for four conserved tyrosine residues, Tyr45, Tyr85, Tyr96 and Tyr188, in human transferrin. The low pK a of Tyr188 is due to the fact that the iron‐binding ligand interacts with Lys206 in open‐form and with Lys296 in the closed‐form of the protein. Our current results also confirm the anion binding of sulfate and arsenate to transferrin and further suggest that Tyr188 is the actual binding site for the anions in solution. These data indicate that Tyr188 is a critical residue not only for iron binding but also for chelator binding and iron release in transferrin.

Fractionation of Proteins by Immobilized Metal Affinity Chromatography

It is widely known that the progress of proteomics mostly depends on the development of more advanced and sensitive protein separation technologies. Immobilized metal affinity chromatography (IMAC) is a useful protein fractionation method used to enrich metal associated proteins. IMAC represents an affinity separation approach based on the interaction between proteins and metal ions immobilized on a solid support. By changing various metal ions and other experimental conditions such as pH and elution composition, IMAC can selectively isolate metal-binding protein fractions for further specific proteomic analysis. The combination of IMAC with other protein analytical technologies has been successfully applied in characterizing metalloproteome and post-translational modifications. In the future, newly developed IMAC techniques integrated with other proteomic methods will significantly contribute to the further development of proteomics.

Purification, crystallization and preliminary X-ray analysis of Mycobacterium tuberculosis folylpolyglutamate synthase (MtbFPGS)

The gene encoding Mycobacterium tuberculosis FPGS (MtbFPGS; Rv2447c) has been cloned and the protein (51 kDa) expressed in Escherichia coli. The purified protein was crystallized either by the batch method in the presence of adenosine diphosphate (ADP) and CoCl2 or by vapour diffusion in the presence of ADP, dihydrofolate and CaCl2. X-ray diffraction data to approximately 2.0 and 2.6 A resolution were collected at the Stanford Synchrotron Radiation Laboratory (SSRL) for crystals grown under the respective conditions. Both crystals belong to the cubic space group P2(1)3, with a unit-cell parameter of 112.6 and 111.8 A, respectively. Structure determination is proceeding.