Marked difference in membrane-protein-binding properties of the two isoforms of protein 4.1R expressed at early and late stages of erythroid differentiation

2008 ◽  
Vol 417 (1) ◽  
pp. 141-148 ◽  
Author(s):  
Wataru Nunomura ◽  
Marilyn Parra ◽  
Miwa Hebiguchi ◽  
Ken-Ichi Sawada ◽  
Narla Mohandas ◽  
...  
Keyword(s):  
Principal Component ◽  
Erythroid Differentiation ◽  
Band 3 ◽  
Binding Activity ◽  
Binding Affinities ◽  
Order Of Magnitude ◽  
Protein 4.1R ◽  
Terminal Erythroid Differentiation ◽  
Late Stages

Two major isoforms of protein 4.1R, a 135 kDa isoform (4.1R135) and an 80 kDa isoform (4.1R80), are expressed at distinct stages of terminal erythroid differentiation. The 4.1R135 isoform is exclusively expressed in early erythroblasts and is not present in mature erythrocytes, whereas the 4.1R80 isoform is expressed at late stages of erythroid differentiation and is the principal component of mature erythrocytes. These two isoforms differ in that the 4.1R135 isoform includes an additional 209 amino acids designated as the HP (head-piece) at the N-terminus of 4.1R80. In the present study, we performed detailed characterization of the interactions of the two 4.1R isoforms with various membrane-binding partners and identified several isoform-specific differences. Although both 4.1R135 and 4.1R80 bound to cytoplasmic domains of GPC (glycophorin C) and band 3, there is an order of magnitude difference in the binding affinities. Furthermore, although both isoforms bound CaM (calmodulin), the binding of 4.1R80 was Ca2+-independent, whereas the binding of 4.1R135 was strongly Ca2+-dependent. The HP of 4.1R135 mediates this Ca2+-dependent binding. Ca2+-saturated CaM completely inhibited the binding of 4.1R135 to GPC, whereas it strongly reduced the affinity of its binding to band 3. Interestingly, in spite of the absence of spectrin-binding activity, the 4.1R135 isoform was able to assemble on to the membrane of early erythroblasts suggesting that its ability to bind to membrane proteins is sufficient for its membrane localization. These findings enable us to offer potential new insights into the differential contribution of 4.1R isoforms to membrane assembly during terminal erythroid differentiation.

RUNX1 Directly Regulates Band 3 Transcription.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1735-1735
Author(s):  
Jingping Xie ◽  
Scott W. Hiebert ◽  
Mark J. Koury ◽  
Stephen J. Brandt
Keyword(s):  
Histone Acetylation ◽  
Gene Knockout ◽  
Regulatory Region ◽  
Erythroid Differentiation ◽  
Band 3 ◽  
Upstream Region ◽  
Pcr Analysis ◽  
Upstream Regulatory Region ◽  
Terminal Erythroid Differentiation ◽  
Chip Analysis

Abstract RUNX1 (AML1 or CBFA2) regulates the expression of a number of genes important to hematopoiesis. Gene knockout studies demonstrated that a heterodimeric complex of RUNX1 and its DNA binding partner, core binding factor-beta (CBFbeta), is essential for definitive hematopoiesis. Here, we report that RUNX1 directly represses expression of the Band 3 gene prior to terminal erythroid differentiation. Band 3 is one of four major components of the erythrocyte membrane skeleton and is important for maintenance of cytoskeletal architecture and electroneutral Cl-/HCO3− exchange across the red cell membrane. Band 3 expression, like that of beta-globin, increases dramatically with terminal erythroid differentiation. In a previous study, we identified an upstream region in the mouse Band 3 gene designated as B3URE (for Band 3 upstream regulatory region) bound by multiple transcription factors, including TAL1 (also known as SCL), RUNX1, Ldb1, and GATA1, that acts as an orientation- and position-independent and tissue-specific repressive element. Chromatin immunoprecipitation (ChIP) analysis showed that RUNX1 was associated with the B3URE in intact MEL cells and electrophoretic mobility shift analysis confirmed specific RUNX1 interaction with RUNX1 binding sites in the B3URE. Together with CBFbeta, RUNX1 inhibited reporter activity from a construct linking the B3URE with 1 kb of Band 3 promoter in transiently transfected MEL but not COS cells. DNA affinity precipitation analysis with wild-type and mutant oligos established that RUNX1 and CBFbeta in MEL cell nuclear extracts could interact with the B3URE in vitro and suggested that RUNX1 recruits TAL1 and Ldb1 to DNA. Northern blot and quantitative real-time PCR analysis demonstrated that enforced expression of RUNX1 dramatically inhibited dimethylsulfoxide (DMSO)-induced Band 3 gene expression. Quantitative ChIP analysis showed that histone acetylation in the B3URE increased more than 4-fold, while histone methylation decreased ~50% after 3 days of DMSO-induced differentiation. Over the same time frame, the promoter region underwent significantly less acetylation but more extensive demethylation. Finally, changes in B3URE acetylation and methylation were attenuated and inhibited, respectively, in RUNX1-transfected MEL cells relative to vector controls. In sum, these results demonstrate that the Band 3 gene is a direct target of RUNX1 in erythroid cells and indicate that the B3URE contributes to the tightly regulated expression of this gene in differentiating erythroid progenitors. One mechanism by which RUNX1 regulates Band 3 transcription may be by influencing histone acetylation/methylation in this upstream regulatory region.

scholarly journals Mitochondria Clearance Mechanisms Via Interaction with Macrophages in Erythropoiesis

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2320-2320
Author(s):  
Chong Yang ◽  
Toshio Suda ◽  
Xiaoxuan Lin ◽  
Mitsuhiro Endo
Keyword(s):  
Large Scale ◽  
Conflicts Of Interest ◽  
Terminal Differentiation ◽  
Erythroid Differentiation ◽  
Transcriptional Pattern ◽  
Mitochondria Transfer ◽  
Erythroid Maturation ◽  
Terminal Erythroid Differentiation ◽  
Late Stages

Abstract Adult erythropoiesis involves a series of well-coordinated events resulting in the production of mature red blood cells. One of such events is the mitochondria clearance, which is known to occur cell-autonomously via autophagy-dependent mechanisms. Interestingly, we identified a sequential changes in the transcriptional pattern during terminal erythroid differentiation based on the expression of several macroautophagy (e.g. Atg3, Atg5, Atg7 and Atg10) and non-canonical mitophagy (e.g. Pink1, Park2, Bnip3l/Nix, P62 and Ulk1) genes. Hence we hypothesize that the progressive reduction in mitochondria during terminal erythroid differentiation is directed by distinct transcriptionally-regulated programs. Notably, we revealed a gradual reduction of the expression of lysosome related genes (e.g. Lamp1, CD63, and Atp6v) and lysosomal activities from early to late stages of terminal differentiation. On the other hand, P62-Pink1-Parkin mediated ubiquitin proteasome degradation of mitochondria proteins seems to be more prominent during late stage erythropoiesis. Hence our data suggest that mitochondria clearance is predominantly mediated by canonical autophagy during early stages of terminal differentiation, whereas non-canonical mitophagy pathway seem to play a more predominant role to regulate late stages erythroid maturation. Next, we discovered mitochondria transfer activities from erythroblasts to their niche. In the context of erythropoiesis, macrophages are known to interact closely with erythroblasts to provide a specialized niche for erythroid precursors to proliferate, differentiate and enucleate. We showed defective erythropoiesis after macrophage depletion in the bone marrow. Subsequently, we identified a tendency for early erythroblasts to associate with macrophages and isolated those erythroblasts from mito-dendra2 mice with trackable mitochondria to establish a murine primary cell co-culture system. Then we report mitochondria transfer activities in the erythroid niche via different modes including direct uptake, micro-vesicle transfer and tunnelling nanotubes (TNT). Interestingly, interchangeable structures between micro-vesicles and TNTs have been observed, suggesting an interplay between cytoskeleton and membrane lipid molecules in the mitochondria transfer mechanisms. Furthermore, mitochondria transfer activities have also been observed in the co-culture of mito-dendra2 erythroid cells with a macrophage cell line, RAW cells, and are significantly enhanced by the activation of the RAW cells via Tfe3 activation. In summary, our findings may provide insight into the mitochondria clearance machineries that mediates erythroid maturation to fulfil the clinical demand for large scale transfusable blood cell production in vitro. Disclosures No relevant conflicts of interest to declare.

Characterization of Multiple Isoforms of Protein 4.1R Expressed During Erythroid Terminal Differentiation

Blood ◽  
1998 ◽  
Vol 92 (11) ◽  
pp. 4404-4414 ◽  
Author(s):  
P. Gascard ◽  
G. Lee ◽  
L. Coulombel ◽  
I. Auffray ◽  
M. Lum ◽  
...  
Keyword(s):  
Primary Structure ◽  
Splice Variants ◽  
Cell Lineage ◽  
Erythroid Differentiation ◽  
Membrane Properties ◽  
Exon 2 ◽  
Erythroid Terminal Differentiation ◽  
And Function ◽  
Protein 4.1R

In erythrocytes, 80-kD protein 4.1R regulates critical membrane properties of deformability and mechanical strength. However, previously obtained data suggest that multiple isoforms of protein 4.1, generated by alternative pre-mRNA splicing, are expressed during erythroid differentiation. Erythroid precursors use two splice acceptor sites at the 5′ end of exon 2, thereby generating two populations of 4.1 RNA: one that includes an upstream AUG-1 in exon 2′ and encodes high molecular weight isoforms, and another that skips AUG-1 in exon 2′ and encodes 4.1 by initiation at a downstream AUG-2 in exon 4. To begin an analysis of the complex picture of protein 4.1R expression and function during erythropoiesis, we determined the number and primary structure of 4.1R isoforms expressed in erythroblasts. We used reverse-transcription polymerase chain reaction to amplify and clone full-length coding domains from the population of 4.1R cDNA containing AUG-1 and the population excluding AUG-1. We observed an impressive repertoire of 4.1R isoforms that included 7 major and 11 minor splice variants, thus providing the first definitive characterization of 4.1R primary structures in a single-cell lineage. 4.1R isoforms, transfected into COS-7 cells, distributed to the nucleus, cytoplasm, plasma membrane, and apparent centrosome. We confirmed previous studies showing that inclusion of exon 16 was essential for efficient nuclear localization. Unexpectedly, immunochemical analysis of COS-7 cells transfected with an isoform lacking both AUG-1 and AUG-2 documented that a previously unidentified downstream translation initiation codon located in exon 8 can regulate expression of 4.1R. We speculate that the repertoire of primary structure of 4.1R dictates its distinct binding partners and functions during erythropoiesis.

scholarly journals Preparation and properties of human red-cell ankyrin

1989 ◽  
Vol 264 (2) ◽  
pp. 423-428 ◽  
Author(s):  
J C Pinder ◽  
K S Smith ◽  
A Pekrun ◽  
W B Gratzer
Keyword(s):  
Gel Filtration ◽  
Membrane Vesicles ◽  
Band 3 ◽  
Binding Activity ◽  
Anion Exchange Column ◽  
Intact Cells ◽  
Calmodulin Binding ◽  
Intact Membrane ◽  
Order Of Magnitude ◽  
Triton X

We describe a procedure for the preparation of ankyrin from human red cells with a yield of 2-3 mg of protein from 30 ml of packed cells. This represents an improvement of an order of magnitude over the usual earlier procedure. Moreover, the product is, in our hands, much more stable against adsorption and proteolysis, and can in general be stored for at least 2 months at 4 degrees C without significant decrease in concentration and binding activity. The preparation depends on the release of the ankyrin-band-3 complex from the membrane cytoskeleton when intact cells are lysed in a medium containing concentrated Triton X-100. The complex is dissociated at high ionic strength, and the final purification is achieved by gel filtration in a medium containing 2 M-Tris or 0.6 M-NaBr. The ankyrin contains all the progression of components present in the intact membrane. All react with affinity-purified polyclonal anti-ankyrin antibodies, and all give widely similar patterns of peptides in partial proteolytic digests. The ankyrin is fully active, as judged by its capacity to bind to band-3-containing membrane vesicles and to Sepharose-coupled spectrin. All components bind to the membrane vesicles. Purified components 2.1 and 2.2, as well as the calmodulin-binding cytoskeletal constituent adducin, can be isolated in pure form by a single anion-exchange column step.

Characterization of Multiple Isoforms of Protein 4.1R Expressed During Erythroid Terminal Differentiation

Blood ◽  
1998 ◽  
Vol 92 (11) ◽  
pp. 4404-4414 ◽  
Author(s):  
P. Gascard ◽  
G. Lee ◽  
L. Coulombel ◽  
I. Auffray ◽  
M. Lum ◽  
...  
Keyword(s):  
Primary Structure ◽  
Splice Variants ◽  
Cell Lineage ◽  
Erythroid Differentiation ◽  
Membrane Properties ◽  
Exon 2 ◽  
Erythroid Terminal Differentiation ◽  
And Function ◽  
Protein 4.1R

Abstract In erythrocytes, 80-kD protein 4.1R regulates critical membrane properties of deformability and mechanical strength. However, previously obtained data suggest that multiple isoforms of protein 4.1, generated by alternative pre-mRNA splicing, are expressed during erythroid differentiation. Erythroid precursors use two splice acceptor sites at the 5′ end of exon 2, thereby generating two populations of 4.1 RNA: one that includes an upstream AUG-1 in exon 2′ and encodes high molecular weight isoforms, and another that skips AUG-1 in exon 2′ and encodes 4.1 by initiation at a downstream AUG-2 in exon 4. To begin an analysis of the complex picture of protein 4.1R expression and function during erythropoiesis, we determined the number and primary structure of 4.1R isoforms expressed in erythroblasts. We used reverse-transcription polymerase chain reaction to amplify and clone full-length coding domains from the population of 4.1R cDNA containing AUG-1 and the population excluding AUG-1. We observed an impressive repertoire of 4.1R isoforms that included 7 major and 11 minor splice variants, thus providing the first definitive characterization of 4.1R primary structures in a single-cell lineage. 4.1R isoforms, transfected into COS-7 cells, distributed to the nucleus, cytoplasm, plasma membrane, and apparent centrosome. We confirmed previous studies showing that inclusion of exon 16 was essential for efficient nuclear localization. Unexpectedly, immunochemical analysis of COS-7 cells transfected with an isoform lacking both AUG-1 and AUG-2 documented that a previously unidentified downstream translation initiation codon located in exon 8 can regulate expression of 4.1R. We speculate that the repertoire of primary structure of 4.1R dictates its distinct binding partners and functions during erythropoiesis.

scholarly journals Constitutive expression of GATA-1 interferes with the cell-cycle regulation

Blood ◽  
1996 ◽  
Vol 87 (9) ◽  
pp. 3711-3721 ◽  
Author(s):  
A Dubart ◽  
PH Romeo ◽  
W Vainchenker ◽  
D Dumenil
Keyword(s):  
Cell Cycle ◽  
Target Genes ◽  
Constitutive Expression ◽  
Erythroid Differentiation ◽  
Serum Starvation ◽  
Nih3t3 Cells ◽  
Hematopoietic Cell Line ◽  
Cycle Regulation ◽  
Terminal Erythroid Differentiation ◽  
Late Stages

GATA-1, mainly expressed during erythroid differentiation, has been shown to regulate the genes specifically expressed in the late stages of erythropoiesis and to protect erythroid cells from apoptosis, suggesting that it might interfere with the cell cycle. By expressing the retrovirally transduced human GATA-1 cDNA in NIH3T3 fibroblasts, we have shown that GATA-1 alone was unable to transactivate its erythroid- specific target genes in these nonerythroid cells. However, GATA-1 expression had a dramatic effect on the proliferation of these fibroblasts. The cloning efficiency of the GATA-1-expressing fibroblasts was maintained but their S phase was greatly elongated and their G1 and G2/M phases were reduced, impairing substantially their proliferation. When cultured at low serum concentrations for 48 hours, GATA-1-expressing fibroblasts failed to accumulate in the G0/G1 phases but did not become serum independent. GATA-1-expressing fibroblasts expressed D1, A, and B1 cyclin mRNAs under conditions of serum starvation or at confluence, whereas these cyclin mRNAs were downregulated in the parental NIH3T3 cells cultured under the same conditions. Moreover, these effects of GATA-1 expression on proliferation were not limited to NIH3T3 cells, since different clones of hGATA-1 virus-infected FDCP-1 cells, a murine interleukin-3- dependent hematopoietic cell line, had a slower growth rate than control cells. Based on these data, we hypothesize that GATA-1 plays a role in the regulation of the cell cycle during terminal erythroid differentiation.

Generic monitoring tools for water characteristics assessment

2003 ◽  
Vol 3 (1-2) ◽  
pp. 351-357
Author(s):  
S. Le Bonté ◽  
M.-N. Pons ◽  
O. Potier ◽  
S. Chanel ◽  
M. Baklouti
Keyword(s):  
Municipal Wastewater ◽  
Principal Component ◽  
Toxic Substances ◽  
Capacity Curves ◽  
Water Characteristics ◽  
Monitoring Tools ◽  
Visible Spectra ◽  
On Line ◽  
Mining Procedure

An adaptive principal component analysis applied to sets of data provided by global analytical methods (UV-visible spectra, buffer capacity curves, respirometric tests) is proposed as a generic procedure for on-line and fast characterization of wastewater. The data-mining procedure is able to deal with a large amount of information, takes into account the normal variations of wastewater composition related to human activity, and enables a rapid detection of abnormal situations such as the presence of toxic substances by comparison of the actual wastewater state with a continuously updated reference. The procedure has been validated on municipal wastewater.

scholarly journals A Methodology Based on FT-IR Data Combined with Random Forest Model to Generate Spectralprints for the Characterization of High-Quality Vinegars

Foods ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 1411
Author(s):  
José Luis P. Calle ◽  
Marta Ferreiro-González ◽  
Ana Ruiz-Rodríguez ◽  
Gerardo F. Barbero ◽  
José Á. Álvarez ◽  
...  
Keyword(s):  
Random Forest ◽  
Raw Materials ◽  
Principal Component ◽  
Hierarchical Cluster ◽  
Raw Material ◽  
Support Vector ◽  
Protected Designation Of Origin ◽  
Ft Ir

Sherry wine vinegar is a Spanish gourmet product under Protected Designation of Origin (PDO). Before a vinegar can be labeled as Sherry vinegar, the product must meet certain requirements as established by its PDO, which, in this case, means that it has been produced following the traditional solera and criadera ageing system. The quality of the vinegar is determined by many factors such as the raw material, the acetification process or the aging system. For this reason, mainly producers, but also consumers, would benefit from the employment of effective analytical tools that allow precisely determining the origin and quality of vinegar. In the present study, a total of 48 Sherry vinegar samples manufactured from three different starting wines (Palomino Fino, Moscatel, and Pedro Ximénez wine) were analyzed by Fourier-transform infrared (FT-IR) spectroscopy. The spectroscopic data were combined with unsupervised exploratory techniques such as hierarchical cluster analysis (HCA) and principal component analysis (PCA), as well as other nonparametric supervised techniques, namely, support vector machine (SVM) and random forest (RF), for the characterization of the samples. The HCA and PCA results present a clear grouping trend of the vinegar samples according to their raw materials. SVM in combination with leave-one-out cross-validation (LOOCV) successfully classified 100% of the samples, according to the type of wine used for their production. The RF method allowed selecting the most important variables to develop the characteristic fingerprint (“spectralprint”) of the vinegar samples according to their starting wine. Furthermore, the RF model reached 100% accuracy for both LOOCV and out-of-bag (OOB) sets.

scholarly journals Semi-automated regional classification of the style of activity of slow rock-slope deformations using PS InSAR and SqueeSAR velocity data

Landslides ◽  
2021 ◽  
Author(s):  
Chiara Crippa ◽  
Elena Valbuzzi ◽  
Paolo Frattini ◽  
Giovanni B. Crosta ◽  
Margherita C. Spreafico ◽  
...  
Keyword(s):  
Rock Slope ◽  
Progressive Failure ◽  
Principal Component ◽  
Cost Effective ◽  
Displacement Rate ◽  
Machine Learning Classification ◽  
Management Perspective ◽  
Alpine Environments

AbstractLarge slow rock-slope deformations, including deep-seated gravitational slope deformations and large landslides, are widespread in alpine environments. They develop over thousands of years by progressive failure, resulting in slow movements that impact infrastructures and can eventually evolve into catastrophic rockslides. A robust characterization of their style of activity is thus required in a risk management perspective. We combine an original inventory of slow rock-slope deformations with different PS-InSAR and SqueeSAR datasets to develop a novel, semi-automated approach to characterize and classify 208 slow rock-slope deformations in Lombardia (Italian Central Alps) based on their displacement rate, kinematics, heterogeneity and morphometric expression. Through a peak analysis of displacement rate distributions, we characterize the segmentation of mapped landslides and highlight the occurrence of nested sectors with differential activity and displacement rates. Combining 2D decomposition of InSAR velocity vectors and machine learning classification, we develop an automatic approach to characterize the kinematics of each landslide. Then, we sequentially combine principal component and K-medoids cluster analyses to identify groups of slow rock-slope deformations with consistent styles of activity. Our methodology is readily applicable to different landslide datasets and provides an objective and cost-effective support to land planning and the prioritization of local-scale studies aimed at granting safety and infrastructure integrity.

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