PubMed · 4372871
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R B Rucker. 1974. Calcium binding to elastin.. https://doi.org/10.1007/978-1-4684-0943-7_10
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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.
Hop is a 60-kDa protein characterized by its ability to bind the two chaperones, hsp70 and hsp90. We have tested the function of Hop using an assay for the refolding of denatured firefly luciferase. We show that Hop is involved in the process of refolding thermally denatured firefly luciferase in rabbit reticulocyte lysate. Hop also stimulates refolding by hsp70 and Ydj-1 in a purified refolding system. Hsp90 can also stimulate refolding, and optimal refolding is observed in the presence of both Hop and hsp90. Similar stimulation was observed when Hop was replaced by its yeast homolog Sti1. In assays of the binding of Hop to hsp70 and hsp90, Hop preferentially forms a complex with ADP-bound hsp70, and this process is unaffected by the presence of hsp90. Hop does not alter the ATPase activity or the rate of ADP dissociation of hsp70. Hop also appears to bind to the ADP-bound form of hsp90, blocking the ATP-dependent conversion of hsp90 to a form capable of interacting with p23. Conversely, once p23 is bound to hsp90, Hop binding is diminished. These results confirm that Hop provides a physical link between hsp70 and hsp90 and also indicate that Hop modulates the activities of both of these chaperone proteins.
Different methods for studying the concurrent effects of two linear inhibitors on a single enzyme have been published, including the fractional product of Webb, the Yonetani-Theorell plot or the method of Chou and Talalay. Recently the use of combination plots has also been advocated for this purpose. We have evaluated the applicability of these methods and found that most of them depend on assumptions about the mechanism of action of the inhibitors. If the mechanism of action is not completely understood, or if some assumptions about the mechanism are unfounded, the parameters obtained may be meaningless. Even if these assumptions are correct, the interaction can be advantageously measured using an alternative representation that does not require a knowledge of the inhibition constants and allows experimental data to be retrieved from the plot. In other cases it is the interpretation of the results rather than the validity of the method that is misleading. A common mistake is to take the exclusivity of the effects of two inhibitors as exclusivity of their binding. We show that this assumption is seldom justified. In any case, it is possible to decide whether the combination of two or more inhibitors is more effective than their individual use by means of isobolographic analysis, even when no information about their mechanism of action is available.