PubMed · 6935988
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R E Smith, G A Saab. 1981. Platelet dysfunction in acute lymphatic leukemia.. https://pubmed.ncbi.nlm.nih.gov/6935988/
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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.
The ars operon of plasmid R773 encodes an ATP-dependent extrusion pump for arsenite and antimonite in Escherichia coli. The ArsA ATPase is the catalytic subunit of the pump protein, with two nucleotide binding consensus sequences, one in the NH2-terminal half and one in the COOH-terminal half of the protein. A 12-residue consensus sequence (DTAPTGHTIRLL) has been identified in ArsA homologs from eubacteria, archebacteria, fungi, plants, and animals. ArsA enzymes were constructed containing single tryptophan residues at either end of this conserved sequence. The emission spectrum of the fluorescence of the tryptophan on the COOH-terminal end (Trp-159) indicated a relatively hydrophilic environment for this residue. An increase in intrinsic tryptophan fluorescence and a blue shift of the maximum emission wavelength were observed upon addition of MgATP, indicating movement of Trp-159 into a relatively less polar environment. No fluorescence response was observed with MgADP, with nonhydrolyzable ATP analogs, or with MgATP by catalytically inactive enyzmes. This suggests that the location Trp-159 is shifted only during hydrolysis of ATP. In contrast, the emission spectrum of Trp-141, located on the NH2-terminal side of the consensus sequence, indicated a relatively nonpolar environment. The maximum emission wavelength red shifted upon addition of MgADP. MgATP slowly produced a response that correlated with product formation, suggesting that the environment of Trp-141 is sensitive only to MgADP binding. Thus, during ATP hydrolysis the COOH-terminal end of the conserved domain moves into a less polar environment, whereas the NH2-terminal end moves into a more hydrophilic environment as product is formed. A hypothesis is presented in which the conserved domain of ArsA and homologs is an energy transduction domain involved in transmission of the energy of ATP hydrolysis to biological functions such as transport.
Increased energy metabolism in the circulating blood platelet plays an essential role in platelet plug formation and clot retraction. This increased energy consumption is mainly due to enhanced anaerobic consumption of glucose via the glycolytic pathway. The aim of the present study was to determine the role of glucose transport as a potential rate-limiting step for human platelet glucose metabolism. We measured in isolated platelet preparations the effect of thrombin and ADP activation, on glucose transport (2-deoxyglucose uptake), and the cellular distribution of the platelet glucose transporter (GLUT), GLUT-3. Thrombin (0.5 U/ml) caused a pronounced shape change and secretion of most alpha-granules within 10 min. During that time glucose transport increased approximately threefold, concomitant with a similar increase in expression of GLUT-3 on the plasma membrane as observed by immunocytochemistry. A major shift in GLUT-3 labeling was observed from the alpha-granule membranes in resting platelets to the plasma membrane after thrombin treatment. ADP induced shape change but no significant alpha-granule secretion. Accordingly, ADP-treated platelets showed no increased glucose transport and no increased GLUT-3 labeling on the plasma membrane. These studies suggest that, in human blood platelets, increased energy metabolism may be precisely coupled to the platelet activation response by means of the translocation of GLUT-3 by regulated secretion of alpha-granules. Observations in megakaryocytes and platelets freshly fixed from blood confirmed the predominant GLUT-3 localization in alpha-granules in the isolated cells, except that even less GLUT-3 is present at the plasma membrane in the circulating cells (approximately 15%), indicating that glucose uptake may be upregulated five to six times during in vivo activation of platelets.