Biology subjects

Perfetto, M.

Publications and source records attributed to Perfetto, M..

4 recordsLinked to original sources

bioRxiv2023

FAM210B is dispensable for erythroid differentiation in adult mice

Iron is required for redox homeostasis but poses toxicity risks due to its redox activity. Erythropoiesis hence requires tight regulation of iron utilization for hemoglobin synthesis. The requirement for iron in erythropoiesis has necessitated the evolution of mechanisms to handle the iron required for hemoglobinization. FAM210B was identified as a regulator of mitochondrial iron import and heme synthesis in erythroid cell culture and zebrafish models. Here, we demonstrate that while FAM210B is required for erythroid differentiation and heme synthesis under standard cell culture conditions, holotransferrin supplementation was sufficient to chemically complement the iron-deficient phenotype. To investigate the role of FAM210B in erythropoiesis, we used knockout mice. While Fam210b-/- mice were viable and did not exhibit overt erythropoietic defects in the bone marrow, the male mice exhibited an increase in serum transferrin suggesting sex-specific alterations in systemic iron sensing. Upon phlebotomy-induced stress erythropoiesis, Fam210b-/- mice exhibited differences in serum transferrin levels, and more starkly, had markedly smaller spleens indicating defects in stress response. Fam210b-/- males had defects in neutrophil and monocyte numbers, as well as decreased erythroid progenitor numbers during erythropoietic stress. Together, our findings show that Fam210b plays a key role in splenic response to erythropoietic stress Our findings reveal a critical role for FAM210B in mediating splenic stress erythropoiesis and suggest it may act as a sex-specific regulator potentially linked to androgen signaling.

developmental biology↗

bioRxiv2020

CLPX regulates erythroid heme synthesis by control of mitochondrial heme synthesis enzymes and iron utilization

Heme is a prosthetic group that plays a critical role in catalyzing life-essential redox reactions in all cells, including critical metabolic processes. Heme synthesis must be tightly co-regulated with cellular requirements in order to maximize utilization and minimize toxicity. Terminally differentiating erythroid cells have an extremely high demand for heme for hemoglobin synthesis. While the enzymatic reactions of heme synthesis are extremely well studied, the mechanisms by which the mitochondrial homeostatic machinery interacts with and regulates heme synthesis are poorly understood. Knowledge of these regulatory mechanisms are key to understanding how red cells couple heme production with heme demand. Heme synthesis is tightly regulated by the mitochondrial AAA+ unfoldase CLPX, which has been reported to promote heme synthesis by activation of yeast {delta}-aminolevulinate synthase (ALAS/Hem1). CLPX was also reported to mediate heme-induced turnover of ALAS1 in human cells. However, a mutation in the ATP binding domain of CLPX that abrogated ATP binding caused an increase in ALAS activity, contrary to previous predictions that CLPX activated ALAS. Using loss-of-function assays in murine cells and zebrafish, we interrogated the mechanisms by which CLPX regulates erythroid heme synthesis. We found that consistent with previous studies, CLPX is required for erythroid heme synthesis. We show that ALAS2 stability and activity were both increased in the absence of CLPX, suggesting that CLPX primarily regulates ALAS2 by control of its turnover. However, we also showed that CLPX is required for PPOX activity and maintenance of FECH levels, likely accounting for the heme deficiency in the absence of CLPX. Lastly, CLPX is required for iron metabolism during erythroid terminal differentiation. Our results show that the role of CLPX in heme synthesis is not conserved across eukaryotes. Our studies reveal a potential mechanism for the role of CLPX in anemia and porphyria, and reveal multiple nodes at which heme synthesis is regulated by the mitochondrial housekeeping machinery.

biochemistry↗

bioRxiv2019

The RNA helicase DDX3 induces neural crest by promoting AKT activity

Mutations in the RNA helicase DDX3 have emerged as a frequent cause of intellectual disability in humans. Because many patients carrying DDX3 mutations have additional defects in craniofacial structures and other tissues containing neural crest (NC)-derived cells, we hypothesized that DDX3 is also important for NC development. Using Xenopus tropicalis as a model, we show that DDX3 is required for normal NC induction and craniofacial morphogenesis by regulating AKT kinase activity. Depletion of DDX3 decreases AKT activity and AKT-dependent inhibitory phosphorylation of GSK3{beta}, leading to reduced levels of {beta}-catenin and Snai1, two GSK3{beta} substrates that are critical for NC induction. DDX3 function in regulating these downstream signaling events during NC induction is likely mediated by RAC1, a small GTPase whose translation depends on the RNA helicase activity of DDX3. These results suggest an evolutionarily conserved role of DDX3 in NC development by promoting AKT activity, and provide a potential mechanism for the NC-related birth defects displayed by patients harboring mutations in DDX3 and its downstream effectors in this signaling cascade.

developmental biology↗

bioRxiv2019

A new transgenic reporter line reveals Wnt-dependent Snail2 re-expression and cranial neural crest differentiation in Xenopus

During vertebrate embryogenesis, the cranial neural crest (CNC) forms at the neural plate border and subsequently migrates and differentiates into many types of cells. The transcription factor Snail2, which is induced by canonical Wnt signaling to be expressed in the early CNC, is pivotal for CNC induction and migration in Xenopus. However, snail2 expression is silenced during CNC migration, and its roles at later developmental stages remain unclear. We generated a transgenic X. tropicalis line that expresses enhanced green fluorescent protein (eGFP) driven by the snail2 promoter/enhancer, and observed eGFP expression not only in the pre-migratory and migrating CNC, but also the differentiating CNC. This transgenic line can be used directly to detect deficiencies in CNC development at various stages, including subtle perturbation of CNC differentiation. In situ hybridization and immunohistochemistry confirm that Snail2 is reexpressed in the differentiating CNC. Using a separate transgenic Wnt reporter line, we show that canonical Wnt signaling is also active in the differentiating CNC. Blocking Wnt signaling shortly after CNC migration causes reduced snail2 expression and impaired differentiation of CNC-derived head cartilage structures. These results suggest that Wnt signaling drives the reexpression of snail2 in the post-migratory CNC and regulates CNC differentiation.

developmental biology↗