Clinical metabolomics identifies blood serum branched chain amino acids as potential predictive biomarkers for chronic graft vs. host disease

The allogeneic hematopoietic stem cell transplantation procedure-the only curative therapy for many types of hematological cancers-is increasing, and graft vs. host disease (GVHD) is the main cause of morbidity and mortality after transplantation. Currently, GVHD diagnosis is clinically performed. Whereas, biomarker panels have been developed for acute GVHD (aGVHD), there is a lack of information about the chronic form (cGVHD). Using nuclear magnetic resonance (NMR) and gas chromatography coupled to time-of-flight (GC-TOF) mass spectrometry, this study prospectively evaluated the serum metabolome of 18 Brazilian patients who had undergone allogeneic hematopoietic stem cell transplantation (HSCT). We identified and quantified 63 metabolites and performed the metabolomic profile on day -10, day 0, day +10 and day +100, in reference to day of transplantation. Patients did not present aGVHD or cGVHD clinical symptoms at sampling times. From 18 patients analyzed, 6 developed cGVHD. The branched-chain amino acids (BCAAs) leucine and isoleucine were reduced and the sulfur-containing metabolite (cystine) was increased at day +10 and day +100. The area under receiver operating characteristics (ROC) curves was higher than 0.79. BCAA findings were validated by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) in 49 North American patients at day +100; however, cystine findings were not statistically significant in this patient set. Our results highlight the importance of multi-temporal and multivariate biomarker panels for predicting and understanding cGVHD9FUNDAÇÃO DE AMPARO À PESQUISA DO ESTADO DE SÃO PAULO - FAPESP2011/06441-

GVHD-derived plasma as a priming strategy of mesenchymal stem cells

Background: Mesenchymal stem cell (MSC) therapy is an important alternative for GVHD treatment, but a third of patients fail to respond to such therapy. Therefore, strategies to enhance the immunosuppressive potential of MSCs constitute an active area of investigation. Here, we proposed an innovative priming strategy based on the plasma obtained from GVHD patients and tested whether this approach could enhance the immunosuppressive capacity of MSCs. Methods: We obtained the plasma from healthy as well as acute (aGVHD) and chronic (cGVHD) GVHD donors. Plasma samples were characterized according to the TNF-α, IFN-γ, IL-10, IL-1β, IL-12p40, and IL-15 cytokine levels. The MSCs primed with such plasmas were investigated according to surface markers, morphology, proliferation, mRNA expression, and the capacity to control T cell proliferation and Treg generation. Results: Interestingly, 57% of aGVHD and 33% of cGVHD plasmas significantly enhanced the immunosuppressive potential of MSCs. The most suppressive MSCs presented altered morphology, and those primed with cGHVD displayed a pronounced overexpression of ICAM-1 on their surface. Furthermore, we observed that the ratio of IFN-γ to IL-10 cytokine levels in the plasma used for MSC priming was significantly correlated with higher suppressive potential and Treg generation induction by primed MSCs, regardless of the clinical status of the donor. Conclusions: This work constitutes an important proof of concept which demonstrates that it is possible to prime MSCs with biological material and also that the cytokine levels in the plasma may affect the MSC immunosuppressive potential, serving as the basis for the development of new therapeutic approaches for the treatment of immune diseases

FEZ proteins family (Fasciculation and Elongation protein Zeta) as bivalent transport adaptors : functional, structural and evolutionary aspects.

Orientador: Jörg KobargTese (doutorado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: As proteínas humanas FEZ1 e FEZ2 (fasciculation and elongation protein zeta) são ortólogas da proteína UNC-76 de C. elegans e estão envolvidas no crescimento e na fasciculação dos axônios através de interações que envolvem kinesinas, mitocôndrias e vesículas sinápticas. Além disso, algumas evidências sugerem a participação de FEZ1 na etiologia da esquizofrenia, no ciclo viral, além da resistência à quimioterápicos. Sua estrutura intrinsecamente desordenada, com coiled-coil ao longo da sequência, pode contribuir para sua função. Nós exploramos a evolução molecular da família de proteínas FEZ com ênfase no ramo dos vertebrados. Através do perfil do interactoma comparado entre FEZ1 e FEZ2 de Homo sapiens e UNC-76 de C. elegans foi observado um padrão de conservação das interações proteínaproteína entre FEZ1 e UNC-76, que explicam a capacidade de FEZ1 resgatar os defeitos causados por mutações em unc-76 em nematoides, de acordo com o descrito por Bloom e colaboradores em 1997. Além disso, caracterizamos a interação entre FEZ1 e SCOCO (short coil-coiled) por SAXS (Small Angle X-ray Scattering). Essa interação já foi descrita previamente entre os seus ortólogos UNC-76 e UNC-69, que cooperam no crescimento axonal. Um estado de heterotetramérico foi observado, consistindo de duas moléculas GST-SCOCO interagindo com duas moléculas de 6xHis-FEZ1 dimerizadas. Por PAGE (Polyacrylamide Gel Electrophoresis, eletroforese em gel de poli-acrilamida), SAXS, Espectrometria de Massas e Ressonância Magnética Nuclear, constatamos que FEZ1 dimeriza envolvendo a formação de ponte dissulfeto. In vivo, este estado dimérico de forma covalente pode ser importante para o transporte mediado por kinesinas de proteínas ao longo dos microtúbulos. Assim, FEZ1 pode ser classificada como uma proteína adaptadora do transporte, dimérica e bivalente, essencial para o crescimento axonal e organização pré-sináptica normal e transporte de cargas. A agregação de novos parceiros de interação encontrada para a proteína FEZ2 poderia ser interpretada como aquisição de novas funções moleculares e pode ter ocorrido nos primeiros estágios da evolução dos cordadosAbstract: The human proteins FEZ1 and FEZ2 (fasciculation and elongation protein zeta 1) are orthologs of the protein UNC-76 from C. elegans, involved in growth and fasciculation of axons, through interactions that involve kinesins, mitochondria and synaptic vesicles. Moreover, some evidence suggests involvement of FEZ1 in the etiology of schizophrenia, in addition to the viral cycle and resistance to chemotherapy. Its structure intrinsically disordered, with coiled-coil along the sequence, can contribute to its function. We have explored the molecular evolution of the FEZ protein family with emphasis on the vertebrata branch. Analyzing the interactome profile of the FEZ1 and FEZ2 from Homo sapiens and UNC-76 from C. elegans we observed a conserved pattern of protein-protein interactions among FEZ1 and UNC-76 that explain the ability of FEZ1 to rescue the defects caused by unc-76 mutations in nematodes, according to Bloom and co-workers in 1997. Furthermore, we characterized the interaction between FEZ1 and SCOCO (short coiled-coil protein) by SAXS (Small Angle X-ray Scattering). This interaction has been previously reported between their orthologs UNC-76 and UNC-69 that cooperate in axonal outgrowth. A heterotetrameric state was observed, which consists of two GST-SCOCO molecules attached to two FEZ1 molecules. By PAGE (Polyacrylamide Gel Electrophoresis), SAXS, Mass Spectrometry and Nuclear Magnetic Resonance we defined that FEZ1 dimerizes involving formation of disulfide bond. In vivo this covalent mediated dimeric state could be important for kinesin mediated protein transport along the microtubule. Thereby, FEZ1 may be classified as a dimeric and bivalent transport adaptor, essential to axon outgrowth and normal pre-synaptic organization and transport of cargoes. The aggregation of new interaction partners found for the FEZ2 protein could be interpreted as the acquisition of new molecular functions and may have occurred in the early stages of chordate evolutionDoutoradoBioquimicaDoutor em Biologia Funcional e Molecula

Calcium in the Backstage of Malaria Parasite Biology

International audienceThe calcium ion (Ca 2+ ) is a ubiquitous second messenger involved in key biological processes in prokaryotes and eukaryotes. In Plasmodium species, Ca 2+ signaling plays a central role in the parasite life cycle. It has been associated with parasite development, fertilization, locomotion, and host cell infection. Despite the lack of a canonical inositol-1,4,5-triphosphate receptor gene in the Plasmodium genome, pharmacological evidence indicates that inositol-1,4,5-triphosphate triggers Ca 2+ mobilization from the endoplasmic reticulum. Other structures such as acidocalcisomes, food vacuole and mitochondria are proposed to act as supplementary intracellular Ca 2+ reservoirs. Several Ca 2+ -binding proteins (CaBPs) trigger downstream signaling. Other proteins with no EF-hand motifs, but apparently involved with CaBPs, are depicted as playing an important role in the erythrocyte invasion and egress. It is also proposed that a cross-talk among kinases, which are not members of the family of Ca 2+ -dependent protein kinases, such as protein kinases G, A and B, play additional roles mediated indirectly by Ca 2+ regulation. This statement may be extended for proteins directly related to invasion or egress, such as SUB1, ERC, IMC1I, IMC1g, GAP45 and EBA175. In this review, we update our understanding of aspects of Ca 2+ -mediated signaling correlated to the developmental stages of the malaria parasite life cycle

Structural Analysis of Intermolecular Interactions in the Kinesin Adaptor Complex Fasciculation and Elongation Protein Zeta 1/ Short Coiled-Coil Protein (FEZ1/SCOCO)

<div><p>Cytoskeleton and protein trafficking processes, including vesicle transport to synapses, are key processes in neuronal differentiation and axon outgrowth. The human protein FEZ1 (fasciculation and elongation protein zeta 1 / UNC-76, in <i>C. elegans</i>), SCOCO (short coiled-coil protein / UNC-69) and kinesins (e.g. kinesin heavy chain / UNC116) are involved in these processes. Exploiting the feature of FEZ1 protein as a bivalent adapter of transport mediated by kinesins and FEZ1 protein interaction with SCOCO (proteins involved in the same path of axonal growth), we investigated the structural aspects of intermolecular interactions involved in this complex formation by NMR (Nuclear Magnetic Resonance), cross-linking coupled with mass spectrometry (MS), SAXS (Small Angle X-ray Scattering) and molecular modelling. The topology of homodimerization was accessed through NMR (Nuclear Magnetic Resonance) studies of the region involved in this process, corresponding to FEZ1 (92-194). Through studies involving the protein in its monomeric configuration (reduced) and dimeric state, we propose that homodimerization occurs with FEZ1 chains oriented in an anti-parallel topology. We demonstrate that the interaction interface of FEZ1 and SCOCO defined by MS and computational modelling is in accordance with that previously demonstrated for UNC-76 and UNC-69. SAXS and literature data support a heterotetrameric complex model. These data provide details about the interaction interfaces probably involved in the transport machinery assembly and open perspectives to understand and interfere in this assembly and its involvement in neuronal differentiation and axon outgrowth.</p> </div

Interaction between FEZ1 and SCOCO.

<p>A) Purified recombinant proteins FEZ1 and SCOCO were incubated, chemically cross-linked, digested with trypsin, and analyzed by MS. MS/MS spectra were manually validated for b and y ion series of the α (peptide of FEZ1) and β (peptide of SCOCO) chains. B) General scheme of FEZ1 and SCOCO proteins cross-linked. Coiled-coils: box, alpha-helix prediction: gray. Amino acids 261-279 in FEZ1 correspond to the mininal interaction region of UNC-69/SCOCO in UNC-76/FEZ1.C) Best conformation based on both cross-link distance and energy value of the in silico modeled complex. FEZ1 is colored in green, and SCOCO is depicted in deep blue. The peptides identified in the MS analysis are shown in orange and the lysine residue in red. DSS is represented in yellow.</p

Purification of the FEZ1-SCOCO complex and GST-SCOCO and SAXS experimental

<p>data A) SDS-PAGE 10% of the 6His-FEZ1 (1-392) and GST-SCOCO (2-82) protein complex. The complex was analyzed by SAXS at 1.10 mg/mL in PBS buffer solution. The complex poly-dispersity was 28,0% according to DLS assay. B) SAXS (Small Angle X-ray Scattering) experiments of 6His-FEZ1 (1-392) interacting with GST-SCOCO (2-82). C) SDS-PAGE 10% of the GST-SCOCO (2-82) protein. D) The protein was analyzed by SAXS at 0.84 mg/mL in PBS buffer solution. The figure shows the experimental intensity points (empty symbols) and the theoretical fit (continuous line) obtained with the GNOM program package for both samples. Insets in both panels (B) and (C) show the linear behavior of the data in the Guinier region. R_g and D_max values obtained from the GNOM fitting for GST-SCOCO and FEZ1-SCOCO samples were (28.7 ±1, approx. 95 Ǻ) and (107±1, approx. 340 Ǻ), respectively. SAXS data of FEZ1 protein were previously published [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076602#B17" target="_blank">17</a>].</p

FEZ1 is an anti-parallel dimer.

<p>15N-edited NOESY-HSQC of dimers and monomers of FEZ1 (92-194) shows contacts between side chains of amino acids that occur in the homodimer, preferably in an anti-parallel topology. These contacts occur between residues 123 and 143, 127 and 139, 130 and 137, and are indicated in the sequence shown at the bottom of the figure.</p

FEZ1 homodimerization involves few amino acids residues.

<p>A) Amino acid sequence of human FEZ1 protein (92-194). The numbers indicate the position of each amino acid residue within the full-length sequence. The first three residues, unnumbered, are generated by the recombinant protein’s cleavage with TEV protease. B) 15N-HSQC FEZ1 (92-194) Nuclear Magnetic Resonance (NMR) spectra. HSQC shows chemical shifts in reduced monomeric protein (black) and non-reduced dimeric protein (red). The spectrum was obtained in spectrometer 600 MHz. For the series of experiments, isotope 15N was introduced in minimal medium for growth of bacteria and induction of protein expression. C) steady-state heteronuclear NOE experiments with dimers and monomers. Heteronuclear NOEs intensities of the monomer were subtracted from those of the dimer, resulting in the differential pattern of relaxation corresponding to amino acids probably present in the region of homodimerization.</p