Site-Specific Anchoring a Luminescent Tag in a Protein with Non-Emissive Iridium(III) Complex.

Site-specific modification of proteins with synthetic fluorescent tag effectively improves the resolution of imaging, and such a labeling method with negligible three-dimensional structural perturbations and minimal impact on the biological functions of proteins is of high interest to dissect the high-resolution activities of biomolecules in complex systems. To this end, several non-emissive iridium(III) complexes [Ir(C-N)2 (H2 O)2 ]+ OTF- (C-N denotes various cyclometalated ligands) were designed and synthesized. These complexes were tested for attaching a protein by coordinating to H/X (HisMet, HisHis, and HisCys) that are separated by i and i+4 in α-helix. Replacement of the two labile water ligands in the iridium(III) complex by a protein HisHis pair increases the luminescent intensity up to over 100 folds. This labeling approach has been demonstrated in a highly specific and efficient manner in a number of proteins, and it is also feasible for labeling target proteins in cell lysates.

Efficient Protein-Protein Couplings Mediated by Small Molecules under Mild Conditions.

Protein-protein coupling reactions under physiological conditions that do not impact the three-dimensional structures of the proteins are in high demand. Owing to the combination of phenylsulfonyl and aldehyde groups in 5-fluoro-4-(phenylsulfonyl)picolinaldehyde (FPPA), the fluorine substituent shows high reactivity toward free thiols. In FPPA, the fluorine is more reactive than phenylsulfonyl for free thiols. Thus the first quantitative nucleophilic substitution can be followed by selective substitution of phenylsulfonyl by an additional thiol or cyclization of aldehyde with a 1,2-aminothiol molecule. The FPPA mediated protein-protein coupling proceeds efficiently under mild conditions, resulting in stable protein conjugates. This coupling method has negligible 3D structural perturbations on the target proteins, and it produces overall intact, nearly traceless, and native structural folds of proteins. It is highly suitable for reconstruction of proteins that are difficult to make and segmental isotopic labeling of multidomain proteins.

Tailoring a Nanochaperone to Regulate α-Synuclein Assembly.

Protein misassembly leads to the formation of dysfunctional and toxic molecular species relating to neurodegeneration in Parkinson's disease and Alzheimer's disease. Here, we tailored a nanochaperone (αS-nChap) for α-synuclein to regulate its assembly. The αS-nChap is capable of i) specifically recognizing α-synuclein; ii) dynamically capturing and stabilizing monomeric α-synuclein and retarding oligomerization; iii) tightly capturing oligomeric α-synuclein to prevent fibrillization; and iv) transporting α-synuclein oligomers to the lysosomal degradation system. The regulation of α-synuclein assembly by αS-nChap was studied in vitro. Moreover, the role of αS-nChap preventing α-synuclein pathology in cells and protecting neurons from apoptosis was investigated. The strategy of tailoring a nanochaperone to regulate aberrant assembly of pathogenic proteins provides important insights into protein misfolding diseases. We foresee that αS-nChap has therapeutic value for Parkinson's disease.

Deficiency of IKKα in Macrophages Mitigates Fibrosis Progression in the Kidney after Renal Ischemia-Reperfusion Injury.

Aims. Acute kidney injury (AKI) can lead to chronic kidney disease (CKD), and macrophages play a key role in this process. The aim of this study was to discover the role of IκB kinase α (IKKα) in macrophages in the process of AKI-to-CKD transition. Main Methods. We crossed lyz2-Cre mice with IKKα-floxed mice to generate mice with IKKα ablation in macrophages (Mac IKKα-/-). A mouse renal ischemia/reperfusion injury (IRI) model was induced by clamping the renal artery for 45 minutes. Treated mice were evaluated for blood biochemistry, tissue histopathology, and fibrosis markers. Macrophages were isolated from the peritoneal cavity for coculturing with tubular epithelial cells (TECs) and flow cytometry analysis. Key Findings. We found that fibrosis and kidney function loss after IRI were significantly alleviated in Mac IKKα-/- mice compared with wild-type (WT) mice. The expression of fibrosis markers and the infiltration of M2 macrophages were decreased in the kidneys of Mac IKKα-/- mice after IRI. The in vitro experiment showed that the IRI TECs cocultured with IKKα-/- macrophages (KO MΦs) downregulated the fibrosis markers accompanied by a downregulation of Wnt/ß-catenin signaling. Significance. These data support the hypothesis that IKKα is involved in mediating macrophage polarization and increasing the expression of fibrosis-promoting inflammatory factors in macrophages. Therefore, knockdown of IKKα in macrophages may be a potential method that can be used to alleviate the AKI-to-CKD transition after IRI.

In-Cell Trityl-Trityl Distance Measurements on Proteins.

Double-electron electron resonance (DEER) can be used to track the structural dynamics of proteins in their native environment, the cell. This method provides the distance distribution between two spin labels attached at specific, well-defined positions in a protein. For the method to be viable under in-cell conditions, the spin label and its attachment to the protein should exhibit high chemical stability in the cell. Here we present low-temperature, trityl-trityl DEER distance measurements on two model proteins, PpiB (prolyl cis-trans isomerase from E. coli) and GB1 (immunoglobulin G-binding protein), doubly labeled with the trityl spin label, CT02MA. Both proteins gave in-cell distance distributions similar to those observed in vitro, with maxima at 4.5-5 nm, and the data were further compared with in-cell Gd(III)-Gd(III) DEER obtained for PpiB labeled with BrPSPy-DO3A-Gd(III) at the same positions. These results highlight the challenges of designing trityl tags suitable for in-cell distance determination at ambient temperatures on live cells.

Third BIR domain of XIAP binds to both Cu(II) and Cu(I) in multiple sites and with diverse affinities characterized at atomic resolution.

The X-chromosome linked inhibitor of apoptosis, XIAP, is mainly known as the inhibitor of caspases by direct interaction with caspases with its baculoviral IAP repeat (BIR) domains. XIAP has three BIR domains and each BIR domain contains a zinc binding site, normally known as zinc finger motif. Recent studies showed that XIAP is involved in copper homeostasis in cells and the BIR domains bind copper ion. However, structural details of the second and third BIR domain, BIR2 and BIR3, in XIAP, with copper as well as the binding modes are not known. In the present work we characterize the structural properties of BIR3 in solution by high resolution NMR and other biophysical techniques. The interaction of BIR3 with copper both in vitro and in cell lysates was analyzed. Our results show that BIR3 is able to form stable complexes both with Cu(II) and Cu(I), whereas zinc binding site is not affected and zinc retains tightly bound in the zinc finger during these interactions. Surprisingly, BIR3 has multiple binding sites for Cu(II) and Cu(I) but with varied binding affinities. In addition, the solvent exposed Cys351 is readily oxidized by Cu(II) resulting an intermolecular disulfide bond either between two BIR3 molecules or a mixed disulfide bond with glutathione in cell lysates.

Coordination of Platinum to α-Synuclein Inhibits Filamentous Aggregation in Solution.

Accumulation of filamentous aggregates of α-synuclein (AS) in Lewy bodies and neurites is characteristic of neurodegenerative diseases such as Parkinson's disease. Inhibition of AS fibrillation is helpful for understanding of AS aggregate structure and for developing chemical therapies. Herein, we report that the PtII -containing antitumor drug cisplatin suppresses filamentous aggregation of AS in solution. PtII thus contrasts strongly with reported transition-metal ions such as MnII , FeIII , and CuII , which accelerate AS aggregation. Interaction between PtII and the side chains of methionine and histidine residues was essential for inhibition of AS fibrillation. Binding of PtII to AS did not change the protein's overall random coil structure, as indicated by solution-state two-dimensional NMR and circular dichroism spectroscopy; and a solution of the AS⋅PtII complex remained free of filamentous aggregates. Our results constitute interesting new information about the biological chemistry of metal ions in Parkinson's disease and might open new lines of research into the suppression of filamentous aggregation.

Bioinformatics Analysis of Genes and Pathways of CD11b+/Ly6Cintermediate Macrophages after Renal Ischemia-Reperfusion Injury.

Renal ischemia-reperfusion injury (IRI) is a major cause of acute kidney injury (AKI), which could induce the poor prognosis. The purpose of this study was to characterize the molecular mechanism of the functional changes of CDllb+/Ly6Cintermediate macrophages after renal IRI. The gene expression profiles of CDllb+/Ly6Cintermcdiate macrophages of the sham surgery mice, and the mice 4 h, 24 h and 9 days after renal IRI were downloaded from the Gene Expression Omnibus database. Analysis of mRNA expression profiles was conducted to identify differentially expressed genes (DEGs), biological processes and pathways by the series test of cluster. Protein-protein interaction network was constructed and analysed to discover the key genes. A total of 6738 DEGs were identified and assigned to 20 model profiles. DEGs in profile 13 were one of the predominant expression profiles, which are involved in immune cell chemotaxis and proliferation. Signet analysis showed that Atp5al, Atp5o, Cox4i, Cdc42, Rac2 and Nhp2 were the key genes involved in oxidation-reduction, apoptosis, migration, M1-M2 differentiation, and proliferation of macrophages. RPS18 may be an appreciate reference gene as it was stable in macrophages. The identified DEGs and their enriched pathways investigate factors that may participate in the functional changes of CD 1lb+Ly6Cintermediate macrophages after renal IRI. Moreover, the vital gene Nhp2 may involve the polarization of macrophages, which may be a new target to affect the process of AKI.

Stable and rigid DTPA-like paramagnetic tags suitable for in vitro and in situ protein NMR analysis.

Organic synthesis of a ligand with high binding affinities for paramagnetic lanthanide ions is an effective way of generating paramagnetic effects on proteins. These paramagnetic effects manifested in high-resolution NMR spectroscopy are valuable dynamic and structural restraints of proteins and protein-ligand complexes. A paramagnetic tag generally contains a metal chelating moiety and a reactive group for protein modification. Herein we report two new DTPA-like tags, 4PS-PyDTTA and 4PS-6M-PyDTTA that can be site-specifically attached to a protein with a stable thioether bond. Both protein-tag adducts form stable lanthanide complexes, of which the binding affinities and paramagnetic tensors are tunable with respect to the 6-methyl group in pyridine. Paramagnetic relaxation enhancement (PRE) effects of Gd(III) complex on protein-tag adducts were evaluated in comparison with pseudocontact shift (PCS), and the results indicated that both 4PS-PyDTTA and 4PS-6M-PyDTTA tags are rigid and present high-quality PREs that are crucially important in elucidation of the dynamics and interactions of proteins and protein-ligand complexes. We also show that these two tags are suitable for in-situ protein NMR analysis.

Single-armed phenylsulfonated pyridine derivative of DOTA is rigid and stable paramagnetic tag in protein analysis.

Single-armed DOTA-like phenylsulfonated pyridine derivatives are rigid and stable paramagnetic tags for site-specific labeling of proteins. Their reactions with a solvent-exposed protein thiol group generate a stable C-S bond and produce one single paramagnetic species in solution NMR. The generated large paramagnetic effects yield valuable long-range structural restraints for proteins.

3D structure determination of a protein in living cells using paramagnetic NMR spectroscopy.

Determining the three-dimensional structure of a protein in living cells remains particularly challenging. We demonstrated that the integration of site-specific tagging proteins and GPS-Rosetta calculations provides a fast and effective way of determining the structures of proteins in living cells, and in principle the interactions and dynamics of protein-ligand complexes.