Proteome alterations in erythrocytes with PIEZO1 gain-of-function mutations

Abstract

Gain-of-function (GoF) mutations in PIEZO1 cause dehydrated hereditary stomatocytosis (DHS) or hereditary xerocytosis, an autosomal dominant hemolytic anemia characterized by high reticulocyte count, a tendency to macrocytosis, and mild jaundice, as well as by other variably penetrant clinical features, such as perinatal edema, severe thromboembolic complications after splenectomy, and hepatic iron overload. PIEZO1 mutations in DHS lead to slowing inactivation kinetics of the ion channel and/or facilitating channel opening in response to physiological stimuli. To characterize the alterations of red blood cell proteome in PIEZO1 mutated patients, we used a differential approach to compare the proteome of DHS patients (16 patients from 13 unrelated pedigrees) versus healthy subjects. We identified new actors in the regulation of the complex landscape of ion and volume balance of erythrocytes mediated by PIEZO1. Particularly, the main impaired processes in DHS patients were: ion homeostasis, transmembrane transport, regulation of vesicle-mediated transport, and the proteasomal catabolic process. Functional assays demonstrated a co-expression of PIEZO1 and Band3 when PIEZO1 was activated. Moreover, the alteration of the vesicle-mediated transport was functionally demonstrated by the increased vesiculation rate in DHS patients compared to healthy controls. This finding provides also an explanation of the pathogenetic mechanism underlying the increased thrombotic rate observed in these patients. Finally, the newly identified proteins, involved in the intracellular signaling pathways altered by PIEZO1 mutations, could be used in the future as potential druggable targets in DHS.

Proteomic signature profiling in the cortex of dairy cattle unravels the physiology of brain aging

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Preliminary evaluation of the proteomic profiling in the hippocampus of aged grazing cattle

“…Hippocampus samples from 4 adult and 5 aged dairy cattle were treated as reported in Cozzolino et al (2021) to carry out a label free shotgun differential proteomics analysis ( Andolfo et al, 2023 ). Peptide mixtures were dissolved in 100 μL of formic acid 0.2% and 2 μL were analyzed on an LTQ Orbitrap XL (Thermo Fisher Scientific) coupled to the nanoACQUITY UPLC system (Waters; Bertini et al, 2021 ).…”

“…The Perseus Software (1.6.15.0) was used for statistical analysis of differentially expressed proteins, by analyzing LFQ Intensity values, according to normalization and imputation criteria reported in ( Andolfo et al, 2023 ). Proteins with more than 70% of non-valid values (zeros) were removed, and the data were normalized by applying a logarithmic scale [log 2 (LFQ)].…”

One gene, two opposite phenotypes: a case report of hereditary anemia due to a loss-of-function variant in the <i>EPAS1</i> gene

“…To evaluate the effect of the mutation on the expression of EPAS1 protein, we measured the amount of wildtype protein in patients and compared it to the amount in healthy controls using a multiple reaction monitoring approach. 14 Briefly, 50 μg of protein extracts were obtained from three patients and three controls. For each protein, at least three double- A B C D charged prototypic peptides were selected with at least three transitions (Figure 2A), in silico predicted by using Skyline.…”