A structure filter for the Eukaryotic Linear Motif Resource

<p>Abstract</p> <p>Background</p> <p>Many proteins are highly modular, being assembled from globular domains and segments of natively disordered polypeptides. Linear motifs, short sequence modules functioning independently of protein tertiary structure, are most abundant in natively disordered polypeptides but are also found in accessible parts of globular domains, such as exposed loops. The prediction of novel occurrences of known linear motifs attempts the difficult task of distinguishing functional matches from stochastically occurring non-functional matches. Although functionality can only be confirmed experimentally, confidence in a putative motif is increased if a motif exhibits attributes associated with functional instances such as occurrence in the correct taxonomic range, cellular compartment, conservation in homologues and accessibility to interacting partners. Several tools now use these attributes to classify putative motifs based on confidence of functionality.</p> <p>Results</p> <p>Current methods assessing motif accessibility do not consider much of the information available, either predicting accessibility from primary sequence or regarding any motif occurring in a globular region as low confidence. We present a method considering accessibility and secondary structural context derived from experimentally solved protein structures to rectify this situation. Putatively functional motif occurrences are mapped onto a representative domain, given that a high quality reference SCOP domain structure is available for the protein itself or a close relative. Candidate motifs can then be scored for solvent-accessibility and secondary structure context. The scores are calibrated on a benchmark set of experimentally verified motif instances compared with a set of random matches. A combined score yields 3-fold enrichment for functional motifs assigned to high confidence classifications and 2.5-fold enrichment for random motifs assigned to low confidence classifications. The structure filter is implemented as a pipeline with both a graphical interface via the ELM resource <url>http://elm.eu.org/</url> and through a Web Service protocol.</p> <p>Conclusion</p> <p>New occurrences of known linear motifs require experimental validation as the bioinformatics tools currently have limited reliability. The ELM structure filter will aid users assessing candidate motifs presenting in globular structural regions. Most importantly, it will help users to decide whether to expend their valuable time and resources on experimental testing of interesting motif candidates.</p

A nonsynonymous SNP in the ITGB3 gene disrupts the conserved membrane-proximal cytoplasmic salt bridge in the αIIbβ3 integrin and cosegregates dominantly with abnormal proplatelet formation and macrothrombocytopenia

We report a 3-generation pedigree with 5 individuals affected with a dominantly inherited macrothrombocytopenia. All 5 carry 2 nonsynonymous mutations resulting in a D723H mutation in the beta3 integrin and a P53L mutation in glycoprotein (GP) Ibalpha. We show that GPIbalpha-L53 is phenotypically silent, being also present in 3 unaffected pedigree members and in 7 of 1639 healthy controls. The beta3-H723 causes constitutive, albeit partial, activation of the alphaIIbbeta3 complex by disruption of the highly conserved cytoplasmic salt bridge with arginine 995 in the alphaIIb integrin as evidenced by increased PAC-1 but not fibrinogen binding to the patients' resting platelets. This was confirmed in CHO alphaIIbbeta3-H723 transfectants, which also exhibited increased PAC-1 binding, increased adhesion to von Willebrand factor (VWF) in static conditions and to fibrinogen under shear stress. Crucially, we show that in the presence of fibrinogen, alphaIIbbeta3-H723, but not wild-type alphaIIbbeta3, generates a signal that leads to the formation of proplatelet-like protrusions in transfected CHO cells. Abnormal proplatelet formation was confirmed in the propositus's CD34+ stem cell-derived megakaryocytes. We conclude that the constitutive activation of the alphaIIbbeta3-H723 receptor causes abnormal proplatelet formation, leading to incorrect sizing of platelets and the thrombocytopenia observed in the pedigree

Molecular dissection of the mechanism by which EWS/FLI expression compromises actin cytoskeletal integrity and cell adhesion in Ewing sarcoma

Ewing sarcoma is the second-most-common bone cancer in children. Driven by an oncogenic chromosomal translocation that results in the expression of an aberrant transcription factor, EWS/FLI, the disease is typically aggressive and micrometastatic upon presentation. Silencing of EWS/FLI in patient-derived tumor cells results in the altered expression of hundreds to thousands of genes and is accompanied by dramatic morphological changes in cytoarchitecture and adhesion. Genes encoding focal adhesion, extracellular matrix, and actin regulatory proteins are dominant targets of EWS/FLI-mediated transcriptional repression. Reexpression of genes encoding just two of these proteins, zyxin and α5 integrin, is sufficient to restore cell adhesion and actin cytoskeletal integrity comparable to what is observed when the EWS/FLI oncogene expression is compromised. Using an orthotopic xenograft model, we show that EWS/FLI-induced repression of α5 integrin and zyxin expression promotes tumor progression by supporting anchorage-independent cell growth. This selective advantage is paired with a tradeoff in which metastatic lung colonization is compromised