Protein histidine phosphorylation: increased stability of thiophosphohistidine.

Posttranslational phosphorylation of proteins is an important event in many cellular processes. Whereas phosphoesters of serine, threonine and tyrosine have been extensively studied, only limited information is available for other amino acids modified by a phosphate group. The formation of phosphohistidine residues in proteins has been discovered in prokaryotic organisms as well as in eukaryotic cells. The ability to biochemically analyze phosphohistidine residues in proteins, however, is severely hampered by its extreme lability under acidic conditions. In our studies we have found that by replacing the phosphate linked to the histidine residue with a thiophosphate, a phosphohistidine derivative with increased stability is formed. This allows the analysis of phosphohistidine-containing proteins by established biochemical techniques and will greatly aid in the investigation of the role of this posttranslational modification in cellular processes.     

CONSTANS and the CCAAT box binding complex share a functionally important domain and interact to regulate flowering of Arabidopsis

The CCT (for CONSTANS, CONSTANS-LIKE, TOC1) domain is found in 45 Arabidopsis thaliana proteins involved in processes such as photoperiodic flowering, light signaling, and regulation of circadian rhythms. We show that this domain exhibits similarities to yeast HEME ACTIVATOR PROTEIN2 (HAP2), which is a subunit of the HAP2/HAP3/HAP5 trimeric complex that binds to CCAAT boxes in eukaryotic promoters. Moreover, we demonstrate that CONSTANS (CO), which promotes Arabidopsis flowering, interacts with At HAP3 and At HAP5 in yeast, in vitro, and in planta. Mutations in CO that delay flowering affect residues highly conserved between CCT and the DNA binding domain of HAP2. Taken together, these data suggest that CO might replace At HAP2 in the HAP complex to form a trimeric CO/At HAP3/At HAP5 complex. Flowering was delayed by overexpression of At HAP2 or At HAP3 throughout the plant or in phloem companion cells, where CO is expressed. This phenotype was correlated with reduced abundance of FLOWERING LOCUS T (FT) mRNA and no change in CO mRNA levels. At HAP2 or At HAP3 overexpression may therefore impair formation of a CO/At HAP3/At HAP5 complex leading to reduced expression of FT. During plant evolution, the number of genes encoding HAP proteins was greatly amplified, and these proteins may have acquired novel functions, such as mediating the effect of CCT domain proteins on gene expression.     

Effect of laminin-1 on intestinal cell differentiation involves inhibition of nuclear nucleolin

Intestinal epithelial cells are characterized by continuous renewal and differentiation events, which may be influenced by the basement membrane, and in particular laminins, which are major components of this specialized extracellular matrix. The function and signaling pathways of laminins in these processes are still poorly documented. In this study, we investigated the possible role and the subcellular localization of nucleolin, a nuclear shuttling protein, in relation to differentiation of human intestinal epithelial Caco2/TC7 cells triggered by exogenous laminin-1. Immunofluorescence and Western blot analysis indicated that laminin-1 induced early differentiation of the cells concomitantly to a decrease in nuclear nucleolin and its a cell surface location. We also showed that both effects of laminin-1 on Caco2/TC7 cells--induction of the differentiation marker sucrase-isomaltase and redistribution of nucleolin--could be mediated by a beta1-integrin dependent cascade that implicated activation of the p38 MAPK pathway. In addition, knock-down of nucleolin expression by the small interfering RNA strategy mimicked the effect of laminin-1 as it resulted in the induction of cell polarization and differentiation. Thus, our study suggests that changes in the subcellular distribution and expression level of nucleolin play an important role in intestinal cell differentiation and relay the signaling pathway induced by laminin-1.     

Identification of brain cell death associated proteins in human post-mortem cerebrospinal fluid

Following any form of brain insult, proteins are released from damaged tissues into the cerebrospinal fluid (CSF). This body fluid is therefore an ideal sample to use in the search for biomarkers of neurodegenerative disorders and brain damage. In this study, we used human post-mortem CSF as a model of massive brain injury and cell death for the identification of such protein markers. Pooled post-mortem CSF samples were analyzed using a protocol that combined immunoaffinity depletion of abundant CSF proteins, off-gel electrophoresis, SDS-PAGE and protein identification by LC-MS/MS. A total of 299 proteins were identified, of which 172 proteins were not previously described to be present in CSF. Of these 172 proteins, more than 75% have been described as intracellular proteins suggesting that they were released from damaged cells. Immunoblots of a number of proteins were performed on individual post-mortem CSF samples and confirmed elevated concentrations in post-mortem CSF compared to ante-mortem CSF. Interestingly, among the proteins specifically identified in the post-mortem CSF, several have been previously described as biochemical markers of brain damage.     

Neopterin Is a Cerebrospinal Fluid Marker for Treatment Outcome Evaluation in Patients Affected by Trypanosoma brucei gambiense Sleeping Sickness

Background

Post-therapeutic follow-up is essential to confirm cure and to detect early treatment failures in patients affected by sleeping sickness (HAT). Current methods, based on finding of parasites in blood and cerebrospinal fluid (CSF) and counting of white blood cells (WBC) in CSF, are imperfect. New markers for treatment outcome evaluation are needed. We hypothesized that alternative CSF markers, able to diagnose the meningo-encephalitic stage of the disease, could also be useful for the evaluation of treatment outcome.

Methodology/Principal findings

Cerebrospinal fluid from patients affected by Trypanosoma brucei gambiense HAT and followed for two years after treatment was investigated. The population comprised stage 2 (S2) patients either cured or experiencing treatment failure during the follow-up. IgM, neopterin, B2MG, MMP-9, ICAM-1, VCAM-1, CXCL10 and CXCL13 were first screened on a small number of HAT patients (n = 97). Neopterin and CXCL13 showed the highest accuracy in discriminating between S2 cured and S2 relapsed patients (AUC 99% and 94%, respectively). When verified on a larger cohort (n = 242), neopterin resulted to be the most efficient predictor of outcome. High levels of this molecule before treatment were already associated with an increased risk of treatment failure. At six months after treatment, neopterin discriminated between cured and relapsed S2 patients with 87% specificity and 92% sensitivity, showing a higher accuracy than white blood cell numbers.

Conclusions/Significance

In the present study, neopterin was highlighted as a useful marker for the evaluation of the post-therapeutic outcome in patients suffering from sleeping sickness. Detectable levels of this marker in the CSF have the potential to shorten the follow-up for HAT patients to six months after the end of the treatment.     

NATURAL VARIATION IN EPIGENETIC GENE REGULATION AND ITS EFFECTS ON PLANT DEVELOPMENTAL TRAITS

In plants, epigenetic variation contributes to phenotypic differences in developmental traits. At the mechanistic level, this variation is conferred by DNA methylation and histone modifications. We describe several examples in which changes in gene expression caused by variation in DNA methylation lead to alterations in plant development. In these examples, the presence of repeated sequences or transposons within the promoters of the affected genes are associated with DNA methylation and gene inactivation. Small interfering RNAs expressed from these sequences recruit DNA methylation to the gene. Some of these methylated alleles are unstable giving rise to revertant sectors during mitosis and to progeny in which the methylated state is lost. However, others are stable for many generations and persist through speciation. These examples indicate that although DNA methylation influences gene expression, this is frequently dependent on classical changes to DNA sequence such as transposon insertions. By contrast, forms of histone methylation cause repression of gene expression that is stably inherited through mitosis but that can also be erased over time or during meiosis. A striking example involves the induction of flowering by exposure to low winter temperatures in Arabidopsis thaliana and its relatives. Histone methylation participates in repression of expression of an inhibitor of flowering during cold. In annual, semelparous species such as A. thaliana, this histone methylation is stably inherited through mitosis after return from cold to warm temperatures allowing the plant to flower continuously during spring and summer until it senesces. However, in perennial, iteroparous relatives the histone modification rapidly disappears when temperatures rise, allowing expression of the floral inhibitor to increase and limiting flowering to a short interval. In this case, epigenetic histone modifications control a key adaptive trait, and their pattern changes rapidly during evolution associated with life‐history strategy. We discuss these examples of epigenetic developmental traits with emphasis on the underlying mechanisms, their stability, and adaptive value.     

ABRF-PRG04: differentiation of protein isoforms.

Accurate protein identification sometimes requires careful discrimination between closely related protein isoforms that may differ by as little as a single amino acid substitution or post-translational modification. The ABRF Proteomics Research Group sent a mixture of three picomoles each of three closely related proteins to laboratories who requested it in the form of intact proteins, and participating laboratories were asked to identify the proteins and report their results. The primary goal of the ABRF-PRG04 Study was to give participating laboratories a chance to evaluate their capabilities and practices with regards to sample fractionation (1D- or 2D-PAGE, HPLC, or none), protein digestion methods (in-solution, in-gel, enzyme choice), and approaches to protein identification (instrumentation, use of software, and/or manual techniques to facilitate interpretation), as well as determination of amino acid or post-translational modifications. Of the 42 laboratories that responded, 8 (19%) correctly identified all three isoforms and N-terminal acetylation of each, 16 (38%) labs correctly identified two isoforms, 9 (21%) correctly identified two isoforms but also made at least one incorrect identification, and 9 (21%) made no correct protein identifications. All but one lab used mass spectrometry, and data submitted enabled a comparison of strategies and methods used.     

T cell and antibody reactivity with the Borrelia burgdorferi 60-kDa heat shock protein in Lyme arthritis.

The reactivity of cloned T cells and serum antibodies, obtained from patients with chronic Lyme arthritis, with expressed recombinant B. burgdorferi 60-kDa heat shock protein homologue (HSP60) was analyzed. The expressed recombinant Borrelia burgdorferi HSP60 was bound by antibodies in the sera of patients with Lyme arthritis, but not by control sera. A T cell clone (CR253), isolated from one of four patients examined, exhibited an HLA-DR2 restricted proliferative response to the expressed recombinant B. burgdorferi HSP60. This T cell clone specifically recognized the HSP60 of B. burgdorferi and did not proliferate in response to the human, mycobacterial, or Escherichia coli HSP60 homologues. The epitope recognized by this cloned T cell, located between amino acids 260 and 274, is in a region of the spirochetal HSP60 that is not conserved between bacteria and eukaryotes.