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MicroScale Thermophoresis (MST) follows the movement of fluorescent-labelled biomolecules with different sizes along a temperature gradient.

A 1.5 Å resolution crystal structure of bovine trypsin at room temperature has been determined using 150 fs X-ray diffraction snapshots at the FemtoMAX beamline of the MAX IV facility., Protein dynamics contribute to protein function on different time scales. Ultrafast X-ray diffraction snapshots can visualize the location and amplitude of atom displacements after perturbation. Since amplitudes of ultrafast motions are small, high-quality X-ray diffraction data is necessary for detection. Diffraction from bovine trypsin crystals using single femtosecond X-ray pulses was recorded at FemtoMAX, which is a versatile beamline of the MAX IV synchrotron. The time-over-threshold detection made it possible that single photons are distinguishable even under short-pulse low-repetition-rate conditions. The diffraction data quality from FemtoMAX beamline enables atomic resolution investigation of protein structures. This evaluation is based on the shape of the Wilson plot, cumulative intensity distribution compared with theoretical distribution, I/σ, R merge/R meas and CC1/2 statistics versus resolution. The FemtoMAX beamline provides an interesting alternative to X-ray free-electron lasers when studying reversible processes in protein crystals.

Scientific reports 9(1), 19281 (1-14) (2019). doi:10.1038/s41598-019-55777-5, Low-frequency vibrations are crucial for protein structure and function, but only a few experimental techniques can shine light on them. The main challenge when addressing protein dynamics in the terahertz domain is the ubiquitous water that exhibit strong absorption. In this paper, we observe the protein atoms directly using X-ray crystallography in bovine trypsin at 100 K while irradiating the crystals with 0.5 THz radiation alternating on and off states. We observed that the anisotropy of atomic displacements increased upon terahertz irradiation. Atomic displacement similarities developed between chemically related atoms and between atoms of the catalytic machinery. This pattern likely arises from delocalized polar vibrational modes rather than delocalized elastic deformations or rigid-body displacements. The displacement correlation between these atoms were detected by a hierarchical clustering method, which can assist the analysis of other ultra-high resolution crystal structures. These experimental and analytical tools provide a detailed description of protein dynamics to complement the structural information from static diffraction experiments., Published by Macmillan Publishers Limited, part of Springer Nature, [London]

Interferon-gamma (IFNγ) producing T cells develop metabolic adaptation required for their effector functions in tumour biology, autoimmunity and antiviral defence.Using sorted CD4+cells we demonstrated that glycolytic switch and high glucose uptake in IFNγ-producing cells was associated with survivin expression. Inhibition of survivin restored glycolysis by upregulating the transcription of phosphofructokinase PFKFB3 and reducing glucose uptake. Integration of the whole-genome sequencing of the chromatin immunoprecipitated with survivin with transcription changes in CD4+cells after survivin inhibition revealed co-localization of survivin, IRF1 and SMAD3 in the regulatory elements paired to the differentially expressed genes. Western blot demonstrated direct binding of survivin to IRF1 and SMAD3. Functionally, inhibition of survivin repressed IFNγ signalling and activated SMAD3-dependent protein remodelling, which resulted in the effector-to-memory transition of CD4+cells. These findings demonstrate the key role of survivin in IFNγ-dependent metabolic adaptation and identify survivin inhibition as an attractive strategy to counteract these effects.

Mucus forms an important protective barrier that minimizes bacterial contact with the colonic epithelium. Intestinal mucus is organized in a complex network with several specific proteins, including the mucin-2 (MUC2) and the abundant IgGFc-binding protein, FCGBP. FCGBP is expressed in all intestinal goblet cells and is secreted into the mucus. It is comprised of repeated von Willebrand D (vWD) domain assemblies, most of which have a GDPH amino acid sequence that can be autocatalytically cleaved, as previously observed in the mucins MUC2 and mucin-5AC. However, the functions of FCGBP in the mucus are not understood. We show that all vWD domains of FCGBP with a GDPH sequence are cleaved and that these cleavages occur early during biosynthesis in the endoplasmic reticulum. All cleaved fragments, however, remain connected via a disulfide bond within each vWD domain. This cleavage generates a C-terminal-reactive Asp-anhydride that could react with other molecules, such as MUC2, but this was not observed. Quantitative analyses by MS showed that FCGBP was mainly soluble in chaotropic solutions, whereas MUC2 was insoluble, and most of the secreted FCGBP was not covalently bound to MUC2. Although FCGBP has been suggested to bind immunoglobulin G, we were unable to reproduce this binding in vitro using purified proteins. In conclusion, while the function of FCGBP is still unknown, our results suggest that it does not contribute to covalent crosslinking in the mucus, nor incorporate immunoglobulin G into mucus, instead the single disulfide bond linking each fragment could mediate controlled dissociation.

Haloferax mediterranei is an extremely halophilic archaeon, able to live in hypersaline environments with versatile nutritional requirements, whose study represents an excellent basis in the field of biotechnology. The transcriptional machinery in Archaea combines the eukaryotic basal apparatus and the bacterial regulation mechanisms. However, little is known about molecular mechanisms of gene expression regulation compared with Bacteria, particularly in Haloarchaea. The genome of Hfx. mediterranei contains a gene, lrp (HFX_RS01210), which encodes a transcriptional factor belonging to Lrp/AsnC family. It is located downstream of the glutamine synthetase gene (HFX_RS01205), an enzyme involved in ammonium assimilation and amino acid metabolism. To study this transcriptional factor more deeply, the lrp gene has been homologously overexpressed and purified under native conditions by two chromatographic steps, namely nickel affinity and gel filtration chromatography, showing that Lrp behaves asa tetrameric protein of approximately 67 kDa. Its promoter region has been characterized under different growth conditions using bgaH as a reporter gene. The amount of Lrp protein was also analyzed by Western blotting in different nitrogen sources and under various stress conditions. To sum up, regarding its involvement in the nitrogen cycle, it has been shown that its expression profile does not change in response to the nitrogen sources tested. Differences in its expression pattern have been observed under different stress conditions, such as in the presence of hydrogen peroxide or heavy metals. According to these results, the Lrp seems to be involved in a general response against stress factors, acting as a first-line transcriptional regulator. This research was funded by Universidad de Alicante, VIGROB-016.

A biomolecular ensemble exhibits different responses to a temperature gradient depending on its diffusion properties. MicroScale Thermophoresis technique exploits this effect and is becoming a popular technique for analyzing interactions of biomolecules in solution. When comparing affinities of related compounds, the reliability of the determined thermodynamic parameters often comes into question. The thermophoresis binding curves can be assessed by Bayesian inference, which provides a probability distribution for the dissociation constant of the interacting partners. By applying Bayesian machine learning principles, binding curves can be autonomously analyzed without manual intervention and without introducing subjective bias by outlier rejection. We demonstrate the Bayesian inference protocol on the known survivin:borealin interaction and on the putative protein-protein interactions between human survivin and two members of the human Shugoshin-like family (hSgol1 and hSgol2). These interactions were identified in a protein microarray binding assay against survivin and confirmed by MicroScale Thermophoresis.

Low-frequency vibrations are crucial for protein structure and function, but only a few experimental techniques can shine light on them. The main challenge when addressing protein dynamics in the terahertz domain is the ubiquitous water that exhibit strong absorption. In this paper, we observe the protein atoms directly using X-ray crystallography in bovine trypsin at 100 K while irradiating the crystals with 0.5 THz radiation alternating on and off states. We observed that the anisotropy of atomic displacements increases upon terahertz irradiation. Atomic displacement similarities develop between chemically related atoms and between atoms of the catalytic machinery. This pattern likely arise from delocalized polar vibrational modes rather than delocalized elastic deformations or rigid-body displacements. This method can ultimately reveal how the alignment of chemically related atoms and the underlying polar vibrational dynamics make a protein structure stable.

A Bayesian model which uses a Markov chain Monte Carlo algorithm has been developed to estimate structure-factor amplitude differences., In experimental research referencing two or more measurements to one another is a powerful tool to reduce the effect of systematic errors between different sets of measurements. The interesting quantity is usually derived from two measurements on the same sample under different conditions. While an elaborate experimental design is essential for improving the estimate, the data analysis should also maximally exploit the covariance between the measurements. In X-ray crystallography the difference between structure-factor amplitudes carries important information to solve experimental phasing problems or to determine time-dependent structural changes in pump–probe experiments. Here a multivariate Bayesian method was used to analyse intensity measurement pairs to determine their underlying structure-factor amplitudes and their differences. The posterior distribution of the model parameter was approximated with a Markov chain Monte Carlo algorithm. The described merging method is shown to be especially advantageous when systematic and random errors result in recording negative intensity measurements.

Background:The oncogenic protein survivin is a marker of severe rheumatoid arthritis (RA). High serum levels of Survivin predict progressive joint damage1and poor treatment response2.Objectives:To study the role of survivin in the transcriptional regulation of phenotype in CD4+T cells.Methods:CD4+T cells of RA female patients were isolated from the perpheral blood. Activated CD4+cells were treated with survivin inhibitor YM155. Transcriptional analysis was done by RNAseq (Illumina) and conventional qPCR. Chromatin of CD4 cells was immunoprecipitated using polyclonal antibodies to survivin and subjected to deep sequencing (survivin ChIPseq, Hiseq2000, Illumina) and aligned to GRCh38. Statistical analysis of differentially expressed genes (DEG) was done in R-studio using Benjamini-Hochberg adjustment for multiple testing (Bioconductor, DESeq2 package).Results:Survivin ChIPseq of the activated CD4+T cells was enriched with the genes engaged in regulatory transcription factor specific DNA binding (GO:0000987, adj p=0.0005) and RNA polymerase II regulatory transcription (GO:0000978, adj p = 0.0004). Among survivin targets were the genes of HOX-B cluster and TALE family proteins MEIS, PKNOX and PBX1 controlling early leukopoesis and T cell maturation. Inhibition of survivin in PBMC resulted in significant upregulation of PBX1 (p=0.023), MEIS3 (p=0.0036), similar tendency was observed for HOXB6 and HOXC4 genes. RNAseq analysis CD4 cells of RA patients with different transcription of PBX1, identified 1636 genes (adj p+cells with low PBX1 (p=0.0005). Among the core transcription factors of T helper cell differentiation, we identifed NF-kB1 and NF-kB2, TBX21, IRF4, IRF8 and STAT3, BATF and BATF3. This followed by significantly higher TNF, IFNg and IL17A and IL17F in PBX1lo CD4 T cells. The pathway enrichment analysis of DEG identified strong over-representation of cytokine-specific genes (GO:005125, GO:0005126, GO:0048018, GO:0030545, FDR q-values 10-12-10-9). The genes of IL4, IL5, IL13, IL9, IL3 and CSF2 located within the chromosome 5 were common for all GO-lists, and were higher in PBX1lo, but none of those genes was identified by survivin-ChIPseq or PBX1-ChIPseq. Analysis of ChIPseq data identified the genes of STAT3, IRF4, IRF8 and BATF as common targets for PBX1 and survivin.Conclusion:This genome-wide analysis indicates that survivin regulates transcription of the TALE family protein PBX1 in CD4+ T cells, which has essential effect for differentiation and phenotype of Th subsets. Low PBX1 in RA patients is associated with terminally differentiated effector CD4+ T cells.References:[1]Svensson, B.et.al.Smoking in combination with antibodies to cyclic citrullinated peptides is associated with persistently high levels of survivin in early rheumatoid arthritis: a prospective cohort study.Arthritis Res Ther16, R12 (2014).[2]Levitsky, A.et.al.Serum survivin predicts responses to treatment in active rheumatoid arthritis: a post hoc analysis from the SWEFOT trial.BMC Med13, 247 (2015).Disclosure of Interests:None declared

Although virtual and augmented reality (VR and AR) techniques have been used extensively in specialized laboratories, only recently did they become affordable, reaching wider consumer markets. With increased availability, it is timely to examine the roles that VR and AR may play in teaching structural biology and in experiencing complex data sets such as macromolecular structures. This guide is suitable for those teachers of structural biology who do not have a deep knowledge of information technologies. This study focuses on three questions: 1) How can teachers of structural biology produce and disseminate VR/AR-ready educational material with established and user-friendly software tools?; 2) What are the positive and negative experiences reported by test participants when performing identical learning tasks in the VR and AR environments?; and 3) How do the test participants perceive prerecorded narration during VR/AR exploration? © 2018 International Union of Biochemistry and Molecular Biology, 47(1):16-24, 2018.

Survivin is a protein functionally important for cell division, apoptosis, and possibly, for micro-RNA biogenesis. It is an established marker of malignant cell transformation. In non-malignant conditions, the unique properties of survivin make it indispensable for homeostasis of the immune system. Indeed, it is required for the innate and adaptive immune responses, controlling differentiation and maintenance of CD4+ and CD8+ memory T-cells, and in B cell maturation. Recently, survivin has emerged as an important player in the pathogenesis of autoimmune diseases. Under the conditions of unreserved inflammation, survivin enhances antigen presentation, maintains persistence of autoreactive cells, and supports production of autoantibodies. In this context, survivin takes its place as a diagnostic and prognostic marker in rheumatoid arthritis, psoriasis, systemic sclerosis and pulmonary arterial hypertension, neuropathology and multiple sclerosis, inflammatory bowel diseases and oral lichen planus. In this review, we summarise the knowledge about non-malignant properties of survivin and focus on its engagement in cellular and molecular pathology of autoimmune diseases. The review highlights utility of survivin measures for clinical applications. It provides rational for the survivin inhibiting strategies and presents results of recent reports on survivin inhibition in modern therapies of cancers and autoimmune diseases.