The Interaction of Integrin αIIbβ3 with Fibrin Occurs through Multiple Binding Sites in the αIIb β-Propeller Domain

The currently available antithrombotic agents target the interaction of platelet integrin α_IIbβ₃ (GPIIb-IIIa) with fibrinogen during platelet aggregation. Platelets also bind fibrin formed early during thrombus growth. It was proposed that inhibition of platelet-fibrin interactions may be a necessary and important property of α_IIbβ₃ antagonists; however, the mechanisms by which α_IIbβ₃ binds fibrin are uncertain. We have previously identified the γ370–381 sequence (P3) in the γC domain of fibrinogen as the fibrin-specific binding site for α_IIbβ₃ involved in platelet adhesion and platelet-mediated fibrin clot retraction. In the present study, we have demonstrated that P3 can bind to several discontinuous segments within the α_IIb β-propeller domain of α_IIbβ₃ enriched with negatively charged and aromatic residues. By screening peptide libraries spanning the sequence of the α_IIb β-propeller, several sequences were identified as candidate contact sites for P3. Synthetic peptides duplicating these segments inhibited platelet adhesion and clot retraction but not platelet aggregation, supporting the role of these regions in fibrin recognition. Mutant α_IIbβ₃ receptors in which residues identified as critical for P3 binding were substituted for homologous residues in the I-less integrin α_Mβ₂ exhibited reduced cell adhesion and clot retraction. These residues are different from those that are involved in the coordination of the fibrinogen γ404–411 sequence and from auxiliary sites implicated in binding of soluble fibrinogen. These results map the binding of fibrin to multiple sites in the α_IIb β-propeller and further indicate that recognition specificity of α_IIbβ₃ for fibrin differs from that for soluble fibrinogen.     

Expression patterns and protein structure of a lipid transfer protein END1 from Arabidopsis

Arabidopsis END1-LIKE (AtEND1) was identified as a homolog of the barley endosperm-specific gene END1 and provides a model for the study of this class of genes and their products. The END1 is expressed in the endosperm transfer cells (ETC) of grasses. The ETC are responsible for transfer of nutrients from maternal tissues to the developing endosperm. Identification of several ETC-specific genes encoding lipid transfer proteins (LTP), including the END1, provided excellent markers for identification of ETC during seed development. To understand how AtEND1 forms complexes with lipid molecules, a three-dimensional (3D) molecular model was generated and reconciled with AtEND1 function. The spatial and temporal expression patterns of AtEND1 were examined in transgenic Arabidopsis plants transformed with an AtEND1 promoter-GUS fusion construct. The AtEND1 promoter was found to be seed and pollen specific. In contrast to ETC-specific expression of homologous genes in wheat and barley, expression of AtEND1 is less specific. It was observed in ovules and a few gametophytic tissues. A series of AtEND1 promoter deletions fused to coding sequence (CDS) of the uidA were transformed in Arabidopsis and the promoter region responsible for AtEND1 expression was identified. A 163 bp fragment of the promoter was found to be sufficient for both spatial and temporal patterns of expression reflecting that of AtEND1. Our data suggest that AtEND1 could be used as a marker gene for gametophytic tissues and developing endosperm. The role of the gene is unclear but it may be involved in fertilization and/or endosperm cellularization.     

FoldUnfold: web server for the prediction of disordered regions in protein chain

Identification of disordered regions in polypeptide chains is very important because such regions are essential for protein function. A new parameter, namely mean packing density of residues has been introduced to detect disordered regions in a protein sequence. We have demonstrated that regions with weak expected packing density would be responsible for the appearance of disordered regions. Our method (FoldUnfold) has been tested on datasets of globular proteins (559 proteins) and long disordered protein segments (129 proteins) and showed improved performance over some other widely used methods, such as DISOPRED, PONDR VL3H, IUPred and GlobPlot. AVAILABILITY: The FoldUnfold server is available for users at http://skuld.protres.ru/~mlobanov/ogu/ogu.cgi. There is a link to our server through the web site of DisProt (http://www.disprot.org/predictors.php).     

B-Raf is critical for MAPK activation during mitosis and is regulated in an M phase-dependent manner in Xenopus egg extracts

Activation of the MAPK cascade during mitosis is critical for spindle assembly and normal mitotic progression. The underlying regulatory mechanisms that control activation of the MEK/MAPK cascade during mitosis are poorly understood. Here we purified and characterized the MEK kinase activity present in Xenopus M phase-arrested egg extracts. Our results show that B-Raf was the critical MEK kinase required for M phase activation of the MAPK pathway. Consistent with this, B-Raf was activated and underwent hyperphosphorylation in an M phase-dependent manner. Interestingly B-Raf hyperphosphorylation at mitosis occurred, at least in part, as a consequence of a feedback loop involving MAPK-mediated phosphorylation within a conserved C-terminal SPKTP motif. The kinase activity of a B-Raf mutant defective at both phosphorylation sites was substantially greater than its wild type counterpart when incubated in Xenopus M phase egg extracts. Furthermore suppression of MAPK feedback at mitosis enhanced B-Raf activity, whereas constitutive activation of MAPK at mitosis strongly suppressed B-Raf activity. These results suggest that feedback phosphorylation by MAPK negatively regulates B-Raf activity at mitosis. Collectively our data demonstrate for the first time a role for B-Raf at mitosis and provide new insight into understanding the regulation and function of B-Raf during cell proliferation.     

Millimeter‐wave emissivity as a metric for the non‐contact diagnosis of human skin conditions

A half‐space electromagnetic model of human skin over the band 30–300 GHz was constructed and used to model radiometric emissivity. The model showed that the radiometric emissivity rose from 0.4 to 0.8 over this band, with emission being localized to a layer approximately one millimeter deep in the skin. Simulations of skin with differing water contents associated with psoriasis, eczema, malignancy, and thermal burn wounds indicated radiometry could be used as a non‐contact technique to detect and monitor these conditions. The skin emissivity of a sample of 30 healthy volunteers, measured using a 95 GHz radiometer, was found to range from 0.2 to 0.7, and the experimental measurement uncertainty was ±0.002. Men on average were found to have an emissivity 0.046 higher than those of women, a measurement consistent with men having thicker skin than women. The regions of outer wrist and dorsal forearm, where skin is thicker, had emissivities 0.06–0.08 higher than the inner wrist and volar forearms where skin is generally thinner. Recommendations are made to develop a more sophisticated model of the skin and to collect larger data sets to obtain a deeper understanding of the signatures of human skin in the millimeter wave band. Bioelectromagnetics. 38:559–569, 2017. © 2017 The Authors. Bioelectromagnetics published by Wiley Periodicals, Inc.     

The N Terminus of the Retinoblastoma Protein Inhibits DNA Replication via a Bipartite Mechanism Disrupted in Partially Penetrant Retinoblastomas

The N-terminal domain of the retinoblastoma (Rb) tumor suppressor protein (RbN) harbors in-frame exon deletions in partially penetrant hereditary retinoblastomas and is known to impair cell growth and tumorigenesis. However, how such RbN deletions contribute to Rb tumor- and growth-suppressive functions is unknown. Here we establish that RbN directly inhibits DNA replication initiation and elongation using a bipartite mechanism involving N-terminal exons lost in cancer. Specifically, Rb exon 7 is necessary and sufficient to target and inhibit the replicative CMG helicase, resulting in the accumulation of inactive CMGs on chromatin. An independent N-terminal loop domain, which forms a projection, specifically blocks DNA polymerase α (Pol-α) and Ctf4 recruitment without affecting DNA polymerases ε and δ or the CMG helicase. Individual disruption of exon 7 or the projection in RbN or Rb, as occurs in inherited cancers, partially impairs the ability of Rb/RbN to inhibit DNA replication and block G₁-to-S cell cycle transit. However, their combined loss abolishes these functions of Rb. Thus, Rb growth-suppressive functions include its ability to block replicative complexes via bipartite, independent, and additive N-terminal domains. The partial loss of replication, CMG, or Pol-α control provides a potential molecular explanation for how N-terminal Rb loss-of-function deletions contribute to the etiology of partially penetrant retinoblastomas.