Culture in embryonic kidney serum and xeno-free media as renal cell carcinoma and renal cell carcinoma cancer stem cells research model

The use of fetal bovine serum hinders obtaining reproducible experimental results and should also be removed in hormone and growth factor studies. In particular hormones found in FBS act globally on cancer cell physiology and influence transcriptome and metabolome. The aim of our study was to develop a renal carcinoma serum free culture model optimized for (embryonal) renal cells in order to select the best study model for downstream auto-, para- or endocrine research. Secondary aim was to verify renal carcinoma stem cell culture for this application. In the study, we have cultured renal cell carcinoma primary tumour cell line (786-0) as well as human kidney cancer stem cells in standard 2D monolayer cultures in Roswell Park Memorial Institute Medium or Dulbecco’s Modified Eagle’s Medium and Complete Human Kidney Cancer Stem Cell Medium, respectively. Serum-free, animal-component free Human Embryonic Kidney 293 media were tested. Our results revealed that xeno-free embryonal renal cells optimized culture media provide a useful tool in RCC cancer biology research and at the same time enable effective growth of RCC. We propose bio-mimic RCC cell culture model with specific serum-free and xeno-free medium that promote RCC cell viability

An artificial CO-releasing metalloprotein built by histidine-selective metallation.

We report the design and synthesis of an aquacarbonyl Ru(II) dication cis-[Ru(CO)2(H2O)4](2+) reagent for histidine (His)-selective metallation of interleukin (IL)-8 at site 33. The artificial, non-toxic interleukin (IL)-8-Ru(II)(CO)2 metalloprotein retained IL-8-dependent neutrophil chemotactic activity and was shown to spontaneously release CO in live cells.We thank the European Commission (Marie Curie CIG to G.J.L.B., Marie Curie IEF to O.B.), FCT Portugal (FCT Investigator to G.J.L.B.) and the EPSRC for generous funding.This is the final published version. It first appeared at http://pubs.rsc.org/en/Content/ArticleLanding/2015/CC/c4cc10204e#!divAbstract

Effect of DMSO on Protein Structure and Interactions Assessed by Collision-Induced Dissociation and Unfolding

Given the frequent use of DMSO in biochemical and biophysical assays, it is desirable to understand the influence of DMSO concentration on the dissociation or unfolding behavior of proteins. In this study, the effects of DMSO on the structure and interactions of avidin and Mycobacterium tuberculosis (Mtb) CYP142A1 were assessed through collision-induced dissociation (CID) and collision-induced unfolding (CIU) as monitored by nanoelectrospray ionization–ion mobility–mass spectrometry (nESI-IM-MS). DMSO concentrations higher than 4% (v/v) destabilize the avidin tetramer toward dissociation and unfolding, via both its effects on charge state distribution (CSD) as well as at the level of individual charge states. In contrast, DMSO both protects against heme loss and increases the stability of CYP142A1 toward unfolding even up to 40% DMSO. Tandem MS/MS experiments showed that DMSO could modify the dissociation pathway of CYP142A1, while CIU revealed the protective effect of the heme group on the structure of CYP142A1.D.S.-H.C. acknowledges the Croucher Foundation and the Cambridge Commonwealth, European and International Trust for receipt of a Croucher Cambridge International Scholarship. M.E.K. was supported by a Commonwealth (University of Cambridge) Scholarship awarded in conjunc-tion with the Cambridge Commonwealth Trust and Cam-bridge Overseas Trust. K.J.M. and A.G.C. were supported by grants from the UK BBSRC (Biotechnology and Biological Sciences Research Council (BB/I019669/1 and BB/I019227/1)

Solvent content of protein crystals from diffraction intensities by Independent Component Analysis

An analysis of the protein content of several crystal forms of proteins has been performed. We apply a new numerical technique, the Independent Component Analysis (ICA), to determine the volume fraction of the asymmetric unit occupied by the protein. This technique requires only the crystallographic data of structure factors as input.Comment: 9 pages, 2 figures, 1 tabl

Insight into Protein Conformation and Subcharging by DMSO from Native Ion Mobility Mass Spectrometry

Electrospray ionization-mass spectrometry (ESI-MS) interfaced with ion-mobility (IM) spectrometry has enabled the study of protein structure and interactions under native-like conditions. In biological assays, dimethyl sulfoxide (DMSO) is often included as a co-solvent to dissolve organic molecules. While low levels of DMSO are known to reduce the charge of protein ions generated by ESI, the exact mechanism by which this occurs has been debated. In this study, we describe the first application of IM–MS to study the effect of DMSO subcharging on native protein conformation. We find that at low concentrations, DMSO induces modest (1-2 %), but repeatable, reductions in protein collision-cross sections (CCSs) of four different protein complexes, avidin, concanavalin A, alcohol dehydrogenase, and pyruvate kinase, as measured by traveling-wave (TW) IM–MS. Individual protein charge states also experienced compaction in size, suggesting that this effect could not be attributed to the shift of charge state distribution by DMSO alone.D.S.-H. Chan acknowledges the support of the Croucher Foundation and the Cambridge Commonwealth, European and International Trust for receipt of a Croucher Cambridge International Scholarship

Mining 2:2 Complexes from 1:1 Stoichiometry: Formation of Cucurbit[8]uril−Diarylviologen Quaternary Complexes Favored by Electron-Donating Substituents

A 1:1 binding stoichiometry of a host−guest complex need not consist of a single host and guest. Diarylviologens containing electron-donating substituents complexed with cucurbit[8]uril (CB[8]) in a 1:1 stoichiometry exhibit abnormally large binding enthalpies compared to typical enthalpy changes observed for 1:1 binary complexes. Here, several CB[8]-mediated host−guest complexes, which were previously reported as 1:1 binary complexes, are verified to be 2:2 quaternary complexes by a combination of isothermal titration calorimetry, $^1$H, NOESY, and ROESY NMR, and ion mobility mass spectrometry, clearly indicating a binding motif of two partially overlapping diarylviologens held in place with two CB[8] molecules. Formation of 2:2 quaternary complexes is favored by electron-donating substituents, while electron-withdrawing substituents typically result in 1:1 binary complexes. The stacking of two highly conjugated diarylviologens in one quaternary motif affords the complexes enhanced conductance when considered as a single-molecular conductor. Moreover, an additional conducting signal previously observed for this “supramolecular” conductor can be readily understood with our 2:2 complexation model, corresponding to a parallel conductance pathway. Therefore, a 2:2 quaternary complex model grants a greater understanding of such supramolecular complexes, enabling the design of engineered, hierarchical structures and functional materials.The authors thank the Leverhulme Trust (project: ‘Natural material innovation for sustainable living’), the Marie Curie FP7 SASSYPOL ITN (607602) programme, and EPSRC (EP/ L504920/1) for funding

Crystal structure of the N-terminal domain of human Timeless and its interaction with Tipin

Human Timeless is involved in replication fork stabilization, S-phase checkpoint activation and establishment of sister chromatid cohesion. In the cell, Timeless forms a constitutive heterodimeric complex with Tipin. Here we present the 1.85 Å crystal structure of a large N-terminal segment of human Timeless, spanning amino acids 1-463, and we show that this region of human Timeless harbours a partial binding site for Tipin. Furthermore, we identify minimal regions of the two proteins that are required for the formation of a stable Timeless-Tipin complex and provide evidence that the Timeless-Tipin interaction is based on a composite binding interface comprising different domains of Timeless.Wellcome Trust Investigator Award [104641/Z/14/Z to L.P.]; Boehringer-Ingelheim Fonds PhD Fellowship; Janggen-Pöhn-Stiftung Awards; Swiss National Science Foundation (to S.H.). Funding for open access charge: Wellcome Trust

Structural characterization of CYP144A1 - a cytochrome P450 enzyme expressed from alternative transcripts in Mycobacterium tuberculosis.

Mycobacterium tuberculosis (Mtb) causes the disease tuberculosis (TB). The virulent Mtb H37Rv strain encodes 20 cytochrome P450 (CYP) enzymes, many of which are implicated in Mtb survival and pathogenicity in the human host. Bioinformatics analysis revealed that CYP144A1 is retained exclusively within the Mycobacterium genus, particularly in species causing human and animal disease. Transcriptomic annotation revealed two possible CYP144A1 start codons, leading to expression of (i) a "full-length" 434 amino acid version (CYP144A1-FLV) and (ii) a "truncated" 404 amino acid version (CYP144A1-TRV). Computational analysis predicted that the extended N-terminal region of CYP144A1-FLV is largely unstructured. CYP144A1 FLV and TRV forms were purified in heme-bound states. Mass spectrometry confirmed production of intact, His6-tagged forms of CYP144A1-FLV and -TRV, with EPR demonstrating cysteine thiolate coordination of heme iron in both cases. Hydrodynamic analysis indicated that both CYP144A1 forms are monomeric. CYP144A1-TRV was crystallized and the first structure of a CYP144 family P450 protein determined. CYP144A1-TRV has an open structure primed for substrate binding, with a large active site cavity. Our data provide the first evidence that Mtb produces two different forms of CYP144A1 from alternative transcripts, with CYP144A1-TRV generated from a leaderless transcript lacking a 5'-untranslated region and Shine-Dalgarno ribosome binding site

Ctf4 Is a Hub in the Eukaryotic Replisome that Links Multiple CIP-Box Proteins to the CMG Helicase

Replisome assembly at eukaryotic replication forks connects the DNA helicase to DNA polymerases and many other factors. The helicase binds the leading-strand polymerase directly, but is connected to the Pol α lagging-strand polymerase by the trimeric adaptor Ctf4. Here, we identify new Ctf4 partners in addition to Pol α and helicase, all of which contain a "Ctf4-interacting-peptide" or CIP-box. Crystallographic analysis classifies CIP-boxes into two related groups that target different sites on Ctf4. Mutations in the CIP-box motifs of the Dna2 nuclease or the rDNA-associated protein Tof2 do not perturb DNA synthesis genome-wide, but instead lead to a dramatic shortening of chromosome 12 that contains the large array of rDNA repeats. Our data reveal unexpected complexity of Ctf4 function, as a hub that connects multiple accessory factors to the replisome. Most strikingly, Ctf4-dependent recruitment of CIP-box proteins couples other processes to DNA synthesis, including rDNA copy-number regulation.We gratefully acknowledge the support of the Medical Research Council (core grant MC_UU_12016/13), the Wellcome Trust (references 097945/B/11/Z for flow cytometry, 102943/Z/13/Z for award to K.L., and 104641/Z/14/Z for award to L.P.), and the Gates Cambridge PhD programme (A.C.S.) for funding our work