NF-κB/STAT5/miR-155 network targets PU.1 in FLT3-ITD-driven acute myeloid leukemia.

Almost 30% of all acute myeloid leukemias (AML) are associated with an internal tandem duplication (ITD) in the juxtamembrane domain of FMS-like tyrosine kinase 3 receptor (FLT3). Patients with FLT3-ITD mutations tend to have a poor prognosis. MicroRNAs (miRNAs) have a pivotal role in myeloid differentiation and leukemia. MiRNA-155 (MiR-155) was found to be upregulated in FLT3-ITD-associated AMLs. In this study, we discovered that FLT3-ITD signaling induces the oncogenic miR-155. We show in vitro and in vivo that miR-155 expression is regulated by FLT3-ITD downstream targets nuclear factor-κB (p65) and signal transducer and activator of transcription 5 (STAT5). Further, we demonstrate that miR-155 targets the myeloid transcription factor PU.1. Knockdown of miR-155 or overexpression of PU.1 blocks proliferation and induces apoptosis of FLT3-ITD-associated leukemic cells. Our data demonstrate a novel network in which FLT3-ITD signaling induces oncogenic miR-155 by p65 and STAT5 in AML, thereby targeting transcription factor PU.1.

YETI: Yeast Exploration Tool Integrator.

UNLABELLED: Yeast Exploration Tool Integrator (YETI) is a novel bioinformatics tool for the integrated visualization and analysis of functional genomic data sets from the budding yeast Saccharomyces cerevisiae. AVAILABILITY: YETI is freely available for use over the WWW, or download under license, at http://www.bru.ed.ac.uk/~orton/yeti.html

Human INCENP colocalizes with the Aurora-B/AIRK2 kinase on chromosomes and is overexpressed in tumour cells.

The inner centromere protein (INCENP), which has previously been described in chicken, frog and mouse, is required for correct chromosome segregation and cytokinesis. We have identified the human INCENP gene by library screening and reverse transcription-polymerase chain reaction (RT-PCR) and localized it to chromosomal region 11q12. HsINCENP is a single-copy gene that consists of 17 exons and covers 25 kb of genomic DNA. The gene is expressed at highest levels in the colon, testis and prostate, consistent with its likely role in cell proliferation. HsINCENP encodes a highly basic protein of 915 amino acids that localizes to metaphase chromosomes and to the mitotic spindle and equatorial cortex at anaphase. Recently we showed that INCENP is stockpiled in a complex with the Aurora-B/XAIRK2 kinase in Xenopus eggs. Here we demonstrate that, consistent with such an interaction, the two proteins colocalize on human metaphase chromosomes. Levels of Aurora-B are increased in several human cancers, and we show here that HsINCENP protein levels are also significantly increased in several colorectal cancer cell lines.

INCENP binds the Aurora-related kinase AIRK2 and is required to target it to chromosomes, the central spindle and cleavage furrow.

Cytoskeletal rearrangements during mitosis must be co-ordinated with chromosome movements. The 'chromosomal passenger' proteins [1], which include the inner centromere protein (INCENP [2]), the Aurora-related serine-threonine protein kinase AIRK2 [3,4] and the unidentified human autoantigen TD-60 [5], have been suggested to integrate mitotic events. These proteins are chromosomal until metaphase but subsequently transfer to the midzone microtubule array and the equatorial cortex during anaphase. Disruption of INCENP function affects both chromosome segregation and completion of cytokinesis [6,7], whereas interference with AIRK2 function primarily affects cytokinesis [3,8]. Here, we report that INCENP is stockpiled in Xenopus eggs in a complex with Xenopus AIRK2 (XAIRK2), and that INCENP and AIRK2 kinase bind one another in vitro. This association was found to be evolutionarily conserved. Sli15p, the binding partner of yeast Aurora kinase Ipl1p, can be recognized as an INCENP family member because of the presence of a conserved carboxy-terminal sequence region, which we term the IN box. This interaction between INCENP and Aurora kinase was found to be biologically relevant. INCENP and AIRK2 colocalized exactly in human cells, and INCENP was required to target AIRK2 correctly to centromeres and the central spindle.

Evolutionary, mechanistic, and predictive analyses of the hydroxymethyldihydropterin pyrophosphokinase family of proteins.

A prediction has been prepared ab initio for the secondary structure of the hydroxymethyldihydropterin pyrophosphokinase (HPPK) family of proteins starting from a set of aligned homologous protein sequences. Attempts to identify a fold by threading failed, judging by the inability to find a threading "hit" that had a secondary structure that was plausibly congruent to the predicted secondary structure for the HPPK family. Therefore, a set of tertiary structure models was assembled ab initio, where alternative models were built and used to select between alternative secondary structure models. This prediction report illustrates the importance of non-computational approaches to structure prediction at its present frontier, which is to obtain medium resolution models of tertiary structure.

Structure prediction in a post-genomic environment: a secondary and tertiary structural model for the initiation factor 5A family.

Two predictions have been prepared for the fold of initiation factor 5A (IF5A) starting from a set of homologous sequences. In the first, a secondary structural model was predicted for the protein in 1994, when only eleven homologs (and no eubacterial homologs) had been sequenced. The second was made recently, after genome projects had generated a total of 33 sequences for the protein family from species of all three kingdoms of life. With the second set of sequences, but not with the first, it was possible to predict that the N-terminal domain of the protein folds in a possibly open beta-barrel/sandwich core structure, with a short helix capping one side of the barrel. We place the pair of predictions in the public domain before an experimental structure is known. This example illustrates the impact of genome sequencing projects on structure prediction from sequence alignments.

A predicted consensus structure for the N-terminal fragment of the heat shock protein HSP90 family.

A secondary structure has been predicted for the heat shock protein HSP90 family from an aligned set of homologous protein sequences by using a transparent method in both manual and automated implementation that extracts conformational information from patterns of variation and conservation within the family. No statistically significant sequence similarity relates this family to any protein with known crystal structure. However, the secondary structure prediction, together with the assignment of active site positions and possible biochemical properties, suggest that the fold is similar to that seen in N-terminal domain of DNA gyrase B (the ATPase fragment).

A predicted consensus structure for the C terminus of the beta and gamma chains of fibrinogen.

A secondary structure has been predicted for the C termini of the fibrinogen beta and gamma chains from an aligned set of homologous protein sequences using a transparent method that extracts conformational information from patterns of variation and conservation, parsing strings, and patterns of amphiphilicity. The structure is modeled to form two domains, the first having a core parallel sheet flanked on one side by at least two helices and on the other by an antiparallel amphiphilic sheet, with an additional helix connecting the two sheets. The second domain is built entirely from beta strands.

Meeting review: the Second meeting on the Critical Assessment of Techniques for Protein Structure Prediction (CASP2), Asilomar, California, December 13-16, 1996.

In most fields of scientific endeavor, the outcomes of important experiments are not always known before the experiments are performed. But in protein structure prediction, algorithms are usually developed and tested in situations where the answers are known. In December 1996, the Second Meeting on the Critical Assessment of Techniques for Protein Structure Prediction (CASP2) was held in Asilomar, California to rectify this situation: protein sequences were provided in advance for which the experimental structure had not yet been published. Over 70 research groups provided bona fide predictions on 42 targets in four categories: comparative or 'homology' modeling, fold recognition or 'threading', ab initio structure predictions, and docking predictions. Since the previous CASP meeting in 1994, the role of fold recognition in structure prediction has increased enormously with the largest number of groups participating in this category. In this review, we highlight some of the important developments and give at least a qualitative sense of what kind of methods produced some of the better predictions.

Secondary structure prediction and unrefined tertiary structure prediction for cyclin A, B, and D.

We present heuristic-based predictions of the secondary and tertiary structures of cyclins A, B, and D, representatives of the cyclin superfamily. The list of suggested constraints for tertiary structure assembly was left unrefined in order to submit this report before an announced crystal structure for cyclin A becomes available. To predict these constraints, a master sequence alignment over 270 positions of cyclin types A, B, and D was adjusted based on individual secondary structure predictions for each type. We used new heuristics for predicting aromatic residues at protein-protein interfaces and to identify sequentially distinct regions in the protein chain that cluster in the folded structure. The boundaries of two conjectured domains in the cyclin fold were predicted based on experimental data in the literature. The domain that is important for interaction of the cyclins with cyclin-dependent kinases (CDKs) is predicted to contain six helices; the second domain in the consensus model contains both helices and a beta-sheet that is formed by sequentially distant regions in the protein chain. A plausible phosphorylation site is identified. This work represents a blinded test of the method for prediction of secondary and, to a lesser extent, tertiary structure from a set of homologous protein sequences. Evaluation of our predictions will become possible with the publication of the announced crystal structure.

The phospho-beta-galactosidase and synaptotagmin predictions.

Two bona fide consensus predictions of secondary and tertiary structure in a protein family, made and announced before experimental structures were known, are evaluated in light of the subsequently determined experimental structures. The first, for phospho-beta-galactosidase, identified the core strands of an 8-fold alpha-beta barrel, and identified the 8-fold alpha-beta barrel itself, which was found in the subsequently determined experimental structure to be the core folding domain. The second, for synaptotagmin, identified seven out of eight beta-strands in the structure correctly, missing only a noncore strand. Three preferred "topologies" were selected from several hundred thousand possible topologies of these seven predicted strands using a rule-based analysis. The subsequently determined experimental structure showed that these seven strands in synaptotagmin adopt one of the three preferred topologies. We were unable, however, to identify the correct topology from among these three topologies.

A predicted consensus structure for the protein kinase C2 homology (C2H) domain, the repeating unit of synaptotagmin.

A secondary structure has been predicted for the protein kinase C2 regulatory domain found in homologous form in synaptotagmin, some phospholipases, and some GTP activated proteins. The proposed structure is built from seven consecutive beta strands followed by a terminal alpha helix. Considerations of overall surface exposure of individual secondary structural elements suggest that these are packed into a 2-sheet beta sandwich structure, with one of only three of the many possible folds being preferred.

A consensus prediction of the secondary structure for the 6-phospho-beta-D-galactosidase superfamily.

Two separate unrefined models for the secondary structure of two subfamilies of the 6-phospho-beta-D-galactosidase superfamily were independently constructed by examining patterns of variation and conservation within homologous protein sequences, assigning surface, interior, parsing, and active site residues to positions in the alignment, and identifying periodicities in these. A consensus model for the secondary structure of the entire superfamily was then built. The prediction tests the limits of an unrefined prediction made using this approach in a large protein with substantial functional and sequence divergence within the family. The protein belongs to the (alpha-beta class), with the core beta strands aligned parallel. The supersecondary structural elements that are readily identified in this model is a parallel beta sheet built by strands C, D, and E, with helices 2 and 3 connecting strands (C+D) and (D+E), respectively, and an analogous beta-alpha unit (strand G and helix 7) toward the end of the sequence. The resemblance of the supersecondary model to the tertiary structure formed by 8-fold alpha-beta barrel proteins is almost certainly not coincidental.

Predicted secondary and supersecondary structure for the serine-threonine-specific protein phosphatase family.

A bona fide consensus prediction for the secondary and supersecondary structure of the serine-threonine specific protein phosphatases is presented. The prediction includes assignments of active site segments, an internal helix, and a region of possible 3(10) helical structure. An experimental structure for a member of this family of proteins should appear shortly, allowing this prediction to be evaluated.

Bona fide prediction of aspects of protein conformation. Assigning interior and surface residues from patterns of variation and conservation in homologous protein sequences.

Heuristics have been developed for analyzing patterns of conservation and variation within a set of aligned homologous protein sequences for the purpose of assigning amino acids whose side-chains lie on the surface and inside the folded structure of a protein. These were used in several recent bona fide predictions of the secondary structure of proteins from sequence data, made and published before crystallographic information became available. Heuristics based on concurrent hydrophilic variation identify positions that lie on the surface. Heuristics based on concurrent hydrophobic conservation and variation identify positions lying in the interior. These heuristics are described here in detail and their performance evaluated when applied to seven protein families with known three-dimensional structures. The performance of individual heuristics is shown to depend on the nature of the multiple alignment within the protein family, and a strategy is presented for obtaining surface and interior assignments useful for predicting secondary structure.

A secondary structure prediction of the hemorrhagic metalloprotease family.

A secondary structure has been predicted for the hemorrhagic metalloproteases using a method developed in Zurich that extracts structural information from patterns of conservation and variation in homologous protein sequences. This prediction tests the limits of the method when applied to a small number of homologous sequences that have undergone only modest evolutionary divergence. Predictions were also obtained using a neural network developed by Sander and coworkers, to date the best fully automated method for predicting secondary structure, and using the classical Chou-Fasman and GOR heuristics. The predictions are different. No crystal structure is known within this protein family, but one is expected shortly. Therefore, this prediction should contribute significantly to the evaluation of the relative merits of these prediction methods.

Predicting the conformation of proteins. Man versus machine.

Two types of approaches for predicting the conformation of proteins from sequence data have lately received attention: 'black box' tools that generate fully automated predictions of secondary structure from a set of homologous protein sequences, and methods involving the expertise of a human biochemist who is assisted, but not replaced, by computer tools. A friendly controversy has emerged as to which approach offers a brighter future. In fact, both are necessary. Nevertheless, a snapshot of the controversy at this instant offers much insight into the structure prediction problem itself.

The nitrogenase MoFe protein. A secondary structure prediction.

Surface residues, interior residues, and parsing residues, together with a secondary structure derived from these, are predicted for the MoFe nitrogenase protein in advance of a crystal structure of the protein, scheduled shortly to appear in Nature. By publishing this prediction, we test our method for predicting the conformation of proteins from patterns in the divergent evolution of homologous protein sequences in a way that places the method 'at risk'.

Predicted secondary structure for the Src homology 3 domain.

A de novo secondary structure prediction has been prepared for Src homology domain 3, in advance of any crystallographic information concerning any member of this interesting protein family. The prediction can be compared with a crystal structure that will be published in Nature on October 29, 1992. The prediction is based on analysis of a multiple alignment of homologous proteins. The patterns of variation and conservation of amino acids across the alignment allow the determination of surface and internal positions, which then allow the assignment of secondary structure. The prediction is quite different both in method and, in this case, result from predictions based on propensities (e.g. Garnier-Osgurthorpe-Robson) of particular amino acids to appear in particular types of secondary structure.