Protease inhibitors‐based therapy induces acquired spherocytic‐like anaemia and ineffective erythropoiesis in chronic hepatitis C virus patients

Lupo, Fabio; Russo, Roberta; Iolascon, Achille; Ieluzzi, Donatella; Siciliano, Angela; Toniutto, Pierluigi; Piovesan, Sara; Raffetti, Elena; Turrini, Francesco Michelangelo; Dissegna, D.; Donato, Francesco; Alberti, Alfredo; Zuliani, Valeria; Fattovich, Giovanna; Franceschi, Lucia De

doi:10.1111/liv.12900

Cited by 11 publications

(6 citation statements)

References 42 publications

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“…Proteins were separated by 1D-electrophoresis, transferred to membranes, and probed with the following specific antibodies: anti-Lyn antibody (2732, Cell Signaling, USA), anti-phospho-Lyn (P-Lyn; Cell Signaling, USA), anti-Syk (Cell Signaling; USA) and anti-phospho-Syk (P-Syk; Cell Signaling; USA); anti-SH-PTP1 (SantaCruz; USA); Phospho-Tyr 564 SH-PTP1 (anti-P-SH-PTP1 564; Cell Signaling, USA). Densitometric analysis of band intensities was carried out by Quantity One analysis software (Bio-Rad, Hercules, CA, USA) (De Franceschi et al, 1997 , 2011 ; Perrotta et al, 2005 ; Lupo et al, 2016 ).…”

Section: Methodsmentioning

confidence: 99%

Red Blood Cell Homeostasis: Pharmacological Interventions to Explore Biochemical, Morphological and Mechanical Properties

Cluitmans

Gevi

Siciliano

et al. 2016

Front. Mol. Biosci.

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During their passage through the circulation, red blood cells (RBCs) encounter severe physiological conditions consisting of mechanical stress, oxidative damage and fast changes in ionic and osmotic conditions. In order to survive for 120 days, RBCs adapt to their surroundings by subtle regulation of membrane organization and metabolism. RBC homeostasis depends on interactions between the integral membrane protein band 3 with other membrane and cytoskeletal proteins, and with key enzymes of various metabolic pathways. These interactions are regulated by the binding of deoxyhemoglobin to band 3, and by a signaling network revolving around Lyn kinase and Src family kinase-mediated phosphorylation of band 3. Here we show that manipulation of the interaction between the lipid bilayer and the cytoskeleton, using various pharmacological agents that interfere with protein-protein interactions and membrane lipid organization, has various effects on: (1) morphology, as shown by high resolution microscopy and quantitative image analysis; (2) organization of membrane proteins, as indicated by immunofluorescence confocal microscopy and quantitative as well as qualitative analysis of vesicle generation; (3) membrane lipid organization, as indicated by flow cytometric analysis of phosphatidylserine exposure; (4) deformability, as assessed in capillary-mimicking circumstances using a microfluidics system; (5) deformability as determined using a spleen-mimicking device; (6) metabolic activity as indicated by metabolomics. Our data show that there is a complex relationship between red cell morphology, membrane organization and deformability. Also, our data show that red blood cells have a relatively high resistance to disturbance of membrane organization in vitro, which may reflect their capacity to withstand mechanical, oxidative and osmotic stress in vivo.

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Section: Methodsmentioning

confidence: 99%

Red Blood Cell Homeostasis: Pharmacological Interventions to Explore Biochemical, Morphological and Mechanical Properties

Cluitmans

Gevi

Siciliano

et al. 2016

Front. Mol. Biosci.

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“…In these patients oxidative stress, induced by drugs, damages membrane-cytoskeletal stability, reducing a-and β-spectrins. 30 Alterations in the RBCs membrane are also present in neuroacanthocytosis, a heterogeneous group of diseases that include chorea-acanthocytosis, McLeod and Huntington's disease-like syndromes. These conditions are characterized by alterations of post-translational modifications, mostly phosphorylation, of erythrocyte membrane proteins and significant neurological symptoms.…”

Section: Other Conditionsmentioning

confidence: 99%

New insights on hereditary erythrocyte membrane defects

et al. 2016

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© Ferrata Storti Foundationmary role in the removal of aged RBCs. In order to perform these journeys RBCs must possess and maintain a significant deformability. The main author of this property is certainly the membrane, that ensures both mechanical stability and deformability. After the first proposed model of the RBC membrane skeleton 36 years ago, 1 containing the core elements of the modern model, many additional proteins have been discovered during the intervening decades, and their structures and interactions have been defined. RBC membrane structure has been extensively covered by excellent reviews.2,3 Herein we summarize the main concepts. RBC membrane is composed by a fluid double layer of lipids in which approximately 20 major proteins and at least 850 minor ones are embedded. 4 The membrane is attached to an intracellular cytoskeleton by protein-protein and lipid-protein interactions that confer the erythrocyte shape, stability and deformability. The transmembrane proteins have mainly a transporter function. However, several of these also have a structural function, usually performed by an intracytoplasmic domain interacting with cytoskeletal proteins.The lipid bilayer acts as a barrier for the retention of cations and anions within the red cells, while it allows water molecules to pass through freely. Human erythrocytes have high intracellular K + and low intracellular Na + contents when compared with the corresponding ion concentrations in the plasma. The maintenance of this cation gradient between the cell and its environment involves a passive outward movement of K + , which is pumped back by the action of an ATP-dependent Na + /K + pump in exchange for Na+ ions. This protein belongs to a class of transmembrane proteins with a transport function (Figure 1). The third and more important component of the RBC membrane is the cytoskeleton, a protein network that laminates the inner surface of the membrane. Spectrin aand β-chains, proteins 4.1, or 4.1R, and actin are the main components of this skeleton, maintaining the biconcave shape of the RBC. These components are connected to each other in two protein complexes; ankyrin and protein 4.1 complex. The former is composed by band 3 tetramers, Rh, RhAG, CD47, glycophorin A and protein 4.2. Whereas the protein 4.1 complex is composed by band 3 dimers binding adducins a-and β-, glycophorin C, GLUT1 and stomatin (Figure 1). The ends of spectrin tetramers converge toward a protein 4.1 complex (junctional complex). Electron microscopy (EM) shows that this latter links the tail of six spectrin tetramers, forming a pseudo-hexagonal arrangement. 5Spectrin tetramers include anion transporters (band 3 or chloride/bicarbonate exchange). The capability of these transporters to form aggregates could define the half-life of RBCs, causing antibody binding and removal by the spleen. Defects that interrupt this vertical structure (spectrin-actin interaction) underlie the biochemical and molecular basis of hereditary spherocytosis (HS), whereas defects in horizo...

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“…22,23,25,28,30 Syk-phosphorylated membranes were used as substrates, as described in De Franceschi et al 14 …”

Section: Phosphorylation Of Erythrocyte Membranesmentioning

confidence: 99%

A new molecular link between defective autophagy and erythroid abnormalities in chorea-acanthocytosis

Lupo

Tibaldi

Mattè

et al. 2016

Blood

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Key Points• In chorea-acanthocytosis, spiculated red cells are characterized by heightened Lyn kinase activity and dysregulated autophagy.• Regulation of protein turnover by autophagy plays a key role in erythropoiesis and red cell integrity.Chorea-acanthocytosis is one of the hereditary neurodegenerative disorders known as the neuroacanthocytoses. Chorea-acanthocytosis is characterized by circulating acanthocytes deficient in chorein, a protein of unknown function. We report here for the first time that chorea-acanthocytosis red cells are characterized by impaired autophagy, with cytoplasmic accumulation of active Lyn and of autophagy-related proteins Ulk1 and Atg7. In chorea-acanthocytosis erythrocytes, active Lyn is sequestered by HSP90-70 to form high-molecular-weight complexes that stabilize and protect Lyn from its proteasomal degradation, contributing to toxic Lyn accumulation. An interplay between accumulation of active Lyn and autophagy was found in chorea-acanthocytosis based on Lyn coimmunoprecipitation with Ulk1 and Atg7 and on the presence of Ulk1 in Lyncontaining high-molecular-weight complexes. In addition, chorein associated with Atg7 in healthy but not in chorea-acanthocytosis erythrocytes. Electron microscopy detected multivesicular bodies and membrane remnants only in circulating chorea-acanthocytosis red cells. In addition, reticulocyte-enriched chorea-acanthocytosis red cell fractions exhibited delayed clearance of mitochondria and lysosomes, further supporting the impairment of authophagic flux. Because autophagy is also important in erythropoiesis, we studied in vitro CD341 -derived erythroid precursors. In chorea-acanthocytosis, we found (1) dyserythropoiesis; (2) increased active Lyn; (3) accumulation of a marker of autophagic flux and autolysososme degradation; (4) accumlation of Lamp1, a lysosmal membrane protein, and LAMP1-positive aggregates; and (5) reduced clearance of lysosomes and mitochondria. Our results uncover in choreaacanthocytosis erythroid cells an association between accumulation of active Lyn and impaired autophagy, suggesting a link between chorein and autophagic vesicle trafficking in erythroid maturation. (Blood. 2016;128(25):2976-2987

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Protease inhibitors‐based therapy induces acquired spherocytic‐like anaemia and ineffective erythropoiesis in chronic hepatitis C virus patients

Abstract: The bimodal pattern of anaemia in chronic HCV patients on triple therapy might be because of acquired spherocytic-like anaemia in the early phase, followed by hyporegenerative anaemia, most likely related to the combined effects of PR and TRV or BOC on erythropoiesis.

Cited by 11 publications

References 42 publications

Red Blood Cell Homeostasis: Pharmacological Interventions to Explore Biochemical, Morphological and Mechanical Properties

Red Blood Cell Homeostasis: Pharmacological Interventions to Explore Biochemical, Morphological and Mechanical Properties

New insights on hereditary erythrocyte membrane defects

A new molecular link between defective autophagy and erythroid abnormalities in chorea-acanthocytosis

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