Acetylation regulates monopolar attachment at multiple levels during meiosis I in fission yeast

In fission yeast, meiotic mono-orientation of sister kinetochores is established by cohesion at the core centromere, which is established by a meiotic cohesin complex and the kinetochore protein Moa1. The cohesin subunit Psm3 is acetylated by Eso1 and deacetylated by Clr6. We show that in meiosis, Eso1 is required for establishing core centromere cohesion during S phase, whereas Moa1 is required for maintaining this cohesion after S phase. The clr6-1 mutation suppresses the mono-orientation defect of moa1Δ cells, although the Clr6 target for this suppression is not Psm3. Thus, several acetylations are crucial for establishing and maintaining core centromere cohesion.     

Calmodulin binding to cellular FLICE-like inhibitory protein modulates Fas-induced signalling

We and others have demonstrated that Fas-mediated apoptosis is a potential therapeutic target for cholangiocarcinoma. Previously, we reported that CaM (calmodulin) antagonists induced apoptosis in cholangiocarcinoma cells through Fas-related mechanisms. Further, we identified a direct interaction between CaM and Fas with recruitment of CaM into the Fas-mediated DISC (death-inducing signalling complex), suggesting a novel role for CaM in Fas signalling. Therefore we characterized the interaction of CaM with proteins recruited into the Fas-mediated DISC, including FADD (Fas-associated death domain)-containing protein, caspase 8 and c-FLIP {cellular FLICE [FADD (Fas-associated death domain)-like interleukin 1beta-converting enzyme]-like inhibitory protein}. A Ca(2+)-dependent direct interaction between CaM and FLIP(L), but not FADD or caspase 8, was demonstrated. Furthermore, a 37.3+/-5.7% increase (n=6, P=0.001) in CaM-FLIP binding was observed at 30 min after Fas stimulation, which returned to the baseline after 60 min and correlated with a Fas-induced increase in intracellular Ca(2+) that reached a peak at 30 min and decreased gradually over 60 min in cholangiocarcinoma cells. A CaM antagonist, TFP (trifluoperazine), inhibited the Fas-induced increase in CaM-FLIP binding concurrent with inhibition of ERK (extracellular-signal-regulated kinase) phosphorylation, a downstream signal of FLIP. Direct binding between CaM and FLIP(L) was demonstrated using recombinant proteins, and a CaM-binding region was identified in amino acids 197-213 of FLIP(L). Compared with overexpression of wild-type FLIP(L) that resulted in decreased spontaneous as well as Fas-induced apoptosis, mutant FLIP(L) with deletion of the CaM-binding region resulted in increased spontaneous and Fas-induced apoptosis in cholangiocarcinoma cells. Understanding the biology of CaM-FLIP binding may provide new therapeutic targets for cholangiocarcinoma and possibly other cancers.     

Clustering analysis of proteins from microbial genomes at multiple levels of resolution

Background

Microbial genomes at the National Center for Biotechnology Information (NCBI) represent a large collection of more than 35,000 assemblies. There are several complexities associated with the data: a great variation in sampling density since human pathogens are densely sampled while other bacteria are less represented; different protein families occur in annotations with different frequencies; and the quality of genome annotation varies greatly. In order to extract useful information from these sophisticated data, the analysis needs to be performed at multiple levels of phylogenomic resolution and protein similarity, with an adequate sampling strategy.

Results

Protein clustering is used to construct meaningful and stable groups of similar proteins to be used for analysis and functional annotation. Our approach is to create protein clusters at three levels. First, tight clusters in groups of closely-related genomes (species-level clades) are constructed using a combined approach that takes into account both sequence similarity and genome context. Second, clustroids of conservative in-clade clusters are organized into seed global clusters. Finally, global protein clusters are built around the the seed clusters. We propose filtering strategies that allow limiting the protein set included in global clustering.The in-clade clustering procedure, subsequent selection of clustroids and organization into seed global clusters provides a robust representation and high rate of compression. Seed protein clusters are further extended by adding related proteins. Extended seed clusters include a significant part of the data and represent all major known cell machinery. The remaining part, coming from either non-conservative (unique) or rapidly evolving proteins, from rare genomes, or resulting from low-quality annotation, does not group together well. Processing these proteins requires significant computational resources and results in a large number of questionable clusters.

Conclusion

The developed filtering strategies allow to identify and exclude such peripheral proteins limiting the protein dataset in global clustering. Overall, the proposed methodology allows the relevant data at different levels of details to be obtained and data redundancy eliminated while keeping biologically interesting variations.

     

Immunochemical study of chromatin non-histone proteins

Summary Rabbit antibodies against rat thymus and liver chromatin are obtained. Antithymus IgG are found to interact only with homologous chromatin, while antiliver IgG interact with both liver and thymus chromatin. After preincubation of antiliver IgG with thymus chromatin the antibodies interact with homologous chromatin only. Thus chromatin of both organs contains tissue-specific proteins. Antiliver and antithymus IgG are used to investigate a distribution of tissue-specific and tissue-non-specific immunogenic proteins in thymocyte nuclei. It is shown that these proteins are practically lacking in nuclear membranes, matrix, nuclear sap, nucleous chromatin and do not participate in attaching DNA to the nuclear matrix.Abbreviations NHP non-histone chromatin proteins - IgG immunoglobulins G - PMSF PhMeSO₃     

Comparative study of lymphocyte non-histone proteins

The non-histone chromosomal proteins of bovine lymphocytes were investigated by the two dimensional gel electrophoresis of O'Farrell. The 0.35 M NaCl extractable proteins from lymphocyte nuclei, the high mobility group proteins (HMG) and some proteins released from nuclei by DNase I were compared on the basis of their electrophoretic patterns.     

Presence of non-histone proteins in nucleosomes

It has been established that nucleosomes are made of histones and DNA fragments. The purpose of this work to establish whether some non-histone proteins are also present in these chromatin subunits. We have found that nucleosome preparations contain phosphorylated non-histone proteins and protein kinases by sucrose gradient analysis. In order to establish whether these proteins are actually bound to nucleosomes or if they represent unbound or aggregated proteins, the following experiments were performed. (a) Free non-histone proteins and proteins released from chromatin by DNase overdigestion were analyzed by sucrose gradient centrifugation. No phosphoproteins but some phosvitin kinase activity was found in the part of the gradient which contained the nucleosomes. It could be assumed that part of the phosphoproteins are bound to nucleosomes. (b) A digestion of nucleosomes with DNase I suppressed the phosvitin kinase activity in the 11-S region of the gradient. (c) High ionic strength, which extracted non-histone proteins, suppressed the phosvitin kinase activity in the nucleosome region. Part of phosvitin kinase and of nuclear phosphoproteins are therefore bound to nucleosomes and are released by nuclease digestion and by high ionic strength.     

Lectin activity of chromatin non-histone proteins

Non-histone proteins from chromatin of sea urchin embryos were found to possess the ability to agglutinate erythrocytes.     

Role of non-histone proteins in chromatin solubility

Treatment of chromatin gel with low ionic strength solution of tRNA has produced the dioxyribonucleoprotein (dnptRNA) in which only part of non-histone proteins was removed without loss of any major histone fraction. The solubility of DNP in the presence of 0.15 M NaCl and 1 to 5 mM MgCl2 was considerably higher than that of initial untreated chromatin. It has been assumed that the solubility of chromatin depended primarily on some non-histone proteins and not on H1 histone.     

Immune signalling: SHP-2 docks at multiple ports

The protein tyrosine phosphatase SHP-2 functions in many diverse signalling pathways. The recent identification of a SHP-2-binding protein as a homologue of the Grb2-associated adaptor protein Gab1 sheds light on the role of SHP-2 in immune signalling.     

Antibacterial peptides and proteins with multiple cellular targets.

Native antimicrobial peptides and proteins represent bridges between innate and adaptive immunity in mammals. On the one hand they possess direct bacterial killing properties, partly by disintegrating bacterial membranes, and some also by inhibiting functions of intracellular biopolymers. On the other, native antimicrobial peptides and proteins upregulate the host defense as chemoattractants or by various additional immunostimulatory effects. Structure-activity relationship studies indicate that residues responsible for the activities on bacterial membranes or for the secondary functions do not perfectly overlap. In reality, in spite of the relatively short size (18-20 amino acid residues) of some of these molecules, the functional domains can frequently be separated, with the cell-penetrating fragments located at the C-termini and the protein binding domains found upstream. As a cumulative effect, multifunctional and target-specific (agonist or antagonist) antimicrobial peptides and proteins interfere with more than one bacterial function at low concentrations, eliminating toxicity concerns of the earlier generations of antibacterial peptides observed in the clinical setting.     

Histone and non-histone proteins from rabbit mammary gland at late pregnancy and early lactation

The content of non-histone protein in epithelial cell chromatin from rabbit mammary gland increased during late pregnancy and early lactation up to 30%, while the histone content remained unchanged. No qualitative changes, as judged by polyacrylamide-gel electrophoresis, were found during accumulation of non-histone proteins at the onset of lactation. The chromatin isolated at lactation, assayed either by polylysine or Toluidine Blue binding, contained slightly more available DNA-phosphate than the chromatin isolated at pregnancy.