PubMed · 4571801
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H J Weiss. 1973. Bleeding disorders due to abnormal platelet function.. https://doi.org/10.1016/s0025-7125(16)32295-7
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PURPOSE: Diphthamide is a post-translationally modified histidine essential for messenger RNA translation and ribosomal protein synthesis. We present evidence for DPH5 as a novel cause of embryonic lethality and profound neurodevelopmental delays (NDDs). METHODS: Molecular testing was performed using exome or genome sequencing. A targeted Dph5 knockin mouse (C57BL/6Ncrl-Dph5em1Mbp/Mmucd) was created for a DPH5 p.His260Arg homozygous variant identified in 1 family. Adenosine diphosphate-ribosylation assays in DPH5-knockout human and yeast cells and in silico modeling were performed for the identified DPH5 potential pathogenic variants. RESULTS: DPH5 variants p.His260Arg (homozygous), p.Asn110Ser and p.Arg207Ter (heterozygous), and p.Asn174LysfsTer10 (homozygous) were identified in 3 unrelated families with distinct overlapping craniofacial features, profound NDDs, multisystem abnormalities, and miscarriages. Dph5 p.His260Arg homozygous knockin was embryonically lethal with only 1 subviable mouse exhibiting impaired growth, craniofacial dysmorphology, and multisystem dysfunction recapitulating the human phenotype. Adenosine diphosphate-ribosylation assays showed absent to decreased function in DPH5-knockout human and yeast cells. In silico modeling of the variants showed altered DPH5 structure and disruption of its interaction with eEF2. CONCLUSION: We provide strong clinical, biochemical, and functional evidence for DPH5 as a novel cause of embryonic lethality or profound NDDs with multisystem involvement and expand diphthamide-deficiency syndromes and ribosomopathies.
Myosin subfragment 1 hydrophobicity was found to be sensitive to the occupancy and nature of bound nucleotide at its active site, as shown by changes in elution behavior of unmodified and chemically modified S1 during phenyl hydrophobic chromatography. The elution properties of S1 were unaltered by alkylation of SH1 (Cys-707) with N-ethylmaleimide or by covalent bridging between SH1 and SH2 (Cys-697) with p-phenylenedimaleimide with trapping of MgADP. Although addition of MgADP or MgATP to the elution buffers had minimal effect on the elution properties of these modified S1 species, the presence of these nucleotides was found to produce differential effects with unmodified S1. With MgADP, where S1 is in the S1** MgADP state, the elution times were decreased slightly, whereas with MgATP, where S1 is primarily in the S1** MgADP.Pi state, the elution times were significantly lowered, indicating reduced accessibility for the immobilized phenyl ligand. Stable S1 ternary complexes, formed with MgADP and various Pi analogues, showed elution times similar to that for S1 in the buffers containing MgATP. Thus, two main classes of nucleotide-induced S1 conformations can be defined according to their interaction with immobilized phenyl. These nucleotide-induced changes in S1 hydrophobicity correlate well with reported changes in radius of gyration of S1 associated with different states of the bound nucleotide [Wakabayashi, K., Tokunga, M., Kohno, I., Sugimoto, Y.; Hamanaka, T., Takezawa, Y., Wakabayashi, T., & Amemiya, Y. (1992) Science 258, 443-447], suggesting that the observed hydrophobicity interaction may be measuring accessibility of the immobilized phenyl ligand into a hydrophobic crevice, and that this crevice is closed or tightened when S1 is in the S1** MgADP.Pi state.
Several bacterial-expressed recombinant fragments encompassing the extracellular part of the beta 3 subunit of the integrin alpha IIb beta 3 were shown to recognize and bind soluble and immobilized forms of fibrinogen. Two of them, designated as rIII-11 (beta 3 274-368) and rIII-13 (beta 3 274-403), did not contain the established RGD-ligand binding sequence. In fact, they interacted, in a Ca(2+)-independent manner, with the C-terminal part of the fibrinogen gamma chain. Both beta 3 fragments blocked the participation of fibrinogen in the induction of platelet aggregation induced by adenosine diphosphate. Fragment rIII-13 was recognized by the anti-beta 3 monoclonal antibody B2A. This antibody, which possesses an epitope exposed on both resting and activated platelets, inhibited fibrinogen binding as well as platelet adhesion and aggregation. In conclusion, the results demonstrated that the 274-368 sequence of the beta 3 subunit of integrin alpha IIb beta 3 constitutes a fibrinogen ligand binding domain, distinct from the RGD-binding site, that is required for both platelet adhesion and aggregation.