Erythrocyte Cytoskeletal Proteins Research Articles

Plasmodium falciparum exploits CD44 as a coreceptor for erythrocyte invasion

AbstractThe malaria parasite Plasmodium falciparum invades and replicates asexually within human erythrocytes. CD44 expressed on erythrocytes was previously identified as an important host factor for P falciparum infection through a forward genetic screen, but little is known about its regulation or function in these cells, nor how it may be used by the parasite. We found that CD44 can be efficiently deleted from primary human hematopoietic stem cells using CRISPR/Cas9 genome editing, and that the efficiency of ex vivo erythropoiesis to enucleated cultured red blood cells (cRBCs) is not affected by lack of CD44. However, the rate of P falciparum invasion was reduced in CD44-null cRBCs relative to isogenic wild-type control cells, validating CD44 as an important host factor for this parasite. We identified 2 P falciparum invasion ligands as binding partners for CD44, erythrocyte binding antigen 175 (EBA-175) and EBA-140 and demonstrated that their ability to bind to human erythrocytes relies primarily on their canonical receptors, glycophorin A and glycophorin C, respectively. We further show that EBA-175 induces phosphorylation of erythrocyte cytoskeletal proteins in a CD44-dependent manner. Our findings support a model in which P falciparum exploits CD44 as a coreceptor during invasion of human erythrocytes, stimulating CD44-dependent phosphorylation of host cytoskeletal proteins that alter host cell deformability and facilitate parasite entry.

Identification of Exported Plasmodium falciparum Proteins That Bind to the Erythrocyte Cytoskeleton.

Plasmodium proteins are exported to the erythrocyte cytoplasm to create an environment that supports parasite replication. Although hundreds of proteins are predicted to be exported through Plasmodium export element (PEXEL)-dependent and -independent mechanisms, the functions of exported proteins are largely uncharacterized. In this study, we used a biochemical screening approach to identify putative exported P. falciparum proteins that bound to inside-out vesicles prepared from erythrocytes. Out of 69 P. falciparum PEXEL-motif proteins tested, 18 bound to inside-out vesicles (IOVs) in two or more independent assays. Using co-affinity purifications followed by mass spectrometry, pairwise co-purification experiments, and the split-luciferase assay, we identified 31 putative protein–protein interactions between erythrocyte cytoskeletal proteins and predicted exported P. falciparum proteins. We further showed that PF3D7_1401600 binds to the spectrin-binding domain of erythrocyte ankyrin via its MESA erythrocyte cytoskeleton binding (MEC) motif and to the N-terminal domains of ankyrin and 4.1R through a fragment that required an intact Plasmodium helical interspersed sub-telomeric (PHIST) domain. Introduction of PF3D7_1401600 into erythrocyte ghosts increased retention in the microsphiltration assay, consistent with previous data that reported a reduction of rigidity in red blood cells infected with PF3D7_1401600-deficient parasites.

Biochemical Properties of Two Plasmodium malariae Cysteine Proteases, Malapain-2 and Malapain-4.

Cysteine proteases belonging to the falcipain (FP) family play a pivotal role in the biology of malaria parasites and have been extensively investigated as potential antimalarial drug targets. Three paralogous FP-family cysteine proteases of Plasmodium malariae, termed malapains 2–4 (MP2–4), were identified in PlasmoDB. The three MPs share similar structural properties with the FP-2/FP-3 subfamily enzymes and exhibit a close phylogenetic lineage with vivapains (VXs) and knowpains (KPs), FP orthologues of P. vivax and P. knowlesi. Recombinant MP-2 and MP-4 were produced in a bacterial expression system, and their biochemical properties were characterized. Both recombinant MP-2 and MP-4 showed enzyme activity across a broad range of pH values with an optimum activity at pH 5.0 and relative stability at neutral pHs. Similar to the FP-2/FP-3 subfamily enzymes in other Plasmodium species, recombinant MP-2 and MP-4 effectively hydrolyzed hemoglobin at acidic pHs. They also degraded erythrocyte cytoskeletal proteins, such as spectrin and band 3, at a neutral pH. These results imply that MP-2 and MP-4 are redundant hemoglobinases of P. malariae and may also participate in merozoite egression by degrading erythrocyte cytoskeletal proteins. However, compared with other FP-2/FP-3 enzymes, MP-2 showed a strong preference for arginine at the P2 position. Meanwhile, MP-4 showed a primary preference for leucine at the P2 position but a partial preference for phenylalanine. These different substrate preferences of MPs underscore careful consideration in the design of optimized inhibitors targeting the FP-family cysteine proteases of human malaria parasites.

Genetic conflicts with Plasmodium parasites and functional constraints shape the evolution of erythrocyte cytoskeletal proteins

Plasmodium parasites exerted a strong selective pressure on primate genomes and mutations in genes encoding erythrocyte cytoskeleton proteins (ECP) determine protective effects against Plasmodium infection/pathogenesis. We thus hypothesized that ECP-encoding genes have evolved in response to Plasmodium-driven selection. We analyzed the evolutionary history of 15 ECP-encoding genes in primates, as well as of their Plasmodium-encoded ligands (KAHRP, MESA and EMP3). Results indicated that EPB42, SLC4A1, and SPTA1 evolved under pervasive positive selection and that episodes of positive selection tended to occur more frequently in primate species that host a larger number of Plasmodium parasites. Conversely, several genes, including ANK1 and SPTB, displayed extensive signatures of purifying selection in primate phylogenies, Homininae lineages, and human populations, suggesting strong functional constraints. Analysis of Plasmodium genes indicated adaptive evolution in MESA and KAHRP; in the latter, different positively selected sites were located in the spectrin-binding domains. Because most of the positively selected sites in alpha-spectrin localized to the domains involved in the interaction with KAHRP, we suggest that the two proteins are engaged in an arms-race scenario. This observation is relevant because KAHRP is essential for the formation of “knobs”, which represent a major virulence determinant for P. falciparum.

Plasmodium falciparum ligand binding to erythrocytes induce alterations in deformability essential for invasion.

The most lethal form of malaria in humans is caused by Plasmodium falciparum. These parasites invade erythrocytes, a complex process involving multiple ligand-receptor interactions. The parasite makes initial contact with the erythrocyte followed by dramatic deformations linked to the function of the Erythrocyte binding antigen family and P. falciparum reticulocyte binding-like families. We show EBA-175 mediates substantial changes in the deformability of erythrocytes by binding to glycophorin A and activating a phosphorylation cascade that includes erythrocyte cytoskeletal proteins resulting in changes in the viscoelastic properties of the host cell. TRPM7 kinase inhibitors FTY720 and waixenicin A block the changes in the deformability of erythrocytes and inhibit merozoite invasion by directly inhibiting the phosphorylation cascade. Therefore, binding of P. falciparum parasites to the erythrocyte directly activate a signaling pathway through a phosphorylation cascade and this alters the viscoelastic properties of the host membrane conditioning it for successful invasion.

Famine From Feast: Low Red Cell Vitamin C Levels in Diabetes

Famine From Feast: Low Red Cell Vitamin C Levels in Diabetes

Acute Kernicterus in a Neonate With O/B Blood Group Incompatibility and a Mutation in SLC4A1

We cared for a term female newborn, who at 108 hours of age, with a total serum bilirubin of 15.4 mg/dL, was discharged from the hospital on home phototherapy. At a return appointment 44 hours later, her total serum bilirubin was 41.7 mg/dL and signs of acute kernicterus were present. Maternal/fetal blood group O/B incompatibility was identified, with a negative direct antiglobulin test, which was positive on retesting. She had abundant spherocytes on blood smear, and these persisted at follow-up, but neither parent had spherocytes identified. A heterozygous SLC4A1(E508K) mutation (gene encoding erythrocyte membrane protein band 3) was found, and in silico predicted to result in damaged erythrocyte cytoskeletal protein function. No mutations were identified in other red cell cytoskeleton genes (ANK1, SPTA1, SPTB, EPB41, EPB42) and the UGT1A1 promoter region was normal. Neurologic follow-up at 2 and 4 months showed developmental delays consistent with mild kernicterus.

Fullerenol C60(OH)36 could associate to band 3 protein of human erythrocyte membranes

The present study was aimed at investigating the effect of fullerenol C60(OH)36 on chosen parameters of the human erythrocyte membrane and the preliminary estimation of the properties of fullerenol as a potential linking agent transferring the compounds (e.g., anticancer drugs) into the membrane of erythrocytes. The results obtained in this study confirm the impact of fullerenol on erythrocyte cytoskeletal transmembrane proteins, particularly on the band 3 protein. The presence of fullerenol in each of the concentrations used prevented degradation of the band 3 protein. The results show that changes in the morphology of red blood cells caused by high concentrations of fullerenol (up to 150mg/L) did not lead to increased red blood cell hemolysis or the leakage of potassium. Moreover, fullerenol slightly prevented hemolysis and potassium efflux. The protective effect of fullerenol at the concentration of 150mg/L was 20.3%, and similar results were obtained for the efflux of potassium. The study shows that fullerenol slightly changed the morphology of the cells and, therefore, altered the intracellular organization of erythrocytes through the association with cytoskeletal proteins.

A member of the Plasmodium falciparum PHIST family binds to the erythrocyte cytoskeleton component band 4.1

BackgroundPlasmodium falciparum parasites export more than 400 proteins into the cytosol of their host erythrocytes. These exported proteins catalyse the formation of knobs on the erythrocyte plasma membrane and an overall increase in erythrocyte rigidity, presumably by modulating the endogenous erythrocyte cytoskeleton. In uninfected erythrocytes, Band 4.1 (4.1R) plays a key role in regulating erythrocyte shape by interacting with multiple proteins through the three lobes of its cloverleaf-shaped N-terminal domain. In P. falciparum-infected erythrocytes, the C-lobe of 4.1R interacts with the P. falciparum protein mature parasite-infected erythrocyte surface antigen (MESA), but it is not currently known whether other P. falciparum proteins bind to other lobes of the 4.1R N-terminal domain.MethodsIn order to identify novel 4.1R interacting proteins, a yeast two-hybrid screen was performed with a fragment of 4.1R containing both the N- and α-lobes. Positive interactions were confirmed and investigated using site-directed mutagenesis, and antibodies were raised against the interacting partner to characterise it’s expression and distribution in P. falciparum infected erythrocytes.ResultsYeast two-hybrid screening identified a positive interaction between the 4.1R N- and α-lobes and PF3D7_0402000. PF3D7_0402000 is a member of a large family of exported proteins that share a domain of unknown function, the PHIST domain. Domain mapping and site-directed mutagenesis established that it is the PHIST domain of PF3D7_0402000 that interacts with 4.1R. Native PF3D7_0402000 is localized at the parasitophorous vacuole membrane (PVM), and colocalizes with a subpopulation of 4.1R.DiscussionThe function of the majority of P. falciparum exported proteins, including most members of the PHIST family, is unknown, and in only a handful of cases has a direct interaction between P. falciparum-exported proteins and components of the erythrocyte cytoskeleton been established. The interaction between 4.1R and PF3D7_0402000, and localization of PF3D7_0402000 with a sub-population of 4.1R at the PVM could indicate a role in modulating PVM structure. Further investigation into the mechanisms for 4.1R recruitment is needed.ConclusionPF3D7_0402000 was identified as a new binding partner for the major erythrocyte cytoskeletal protein, 4.1R. This interaction is consistent with a growing body of literature that suggests the PHIST family members function by interacting directly with erythrocyte proteins.

Malaria Parasite Signal Peptide Peptidase is an ER‐Resident Protease Required for Growth but not for Invasion

The establishment of parasite infection within the human erythrocyte is an essential stage in the development of malaria disease. As such, significant interest has focused on the mechanics that underpin invasion and on characterization of parasite molecules involved. Previous evidence has implicated a presenilin-like signal peptide peptidase (SPP) from the most virulent human malaria parasite, Plasmodium falciparum, in the process of invasion where it has been proposed to function in the cleavage of the erythrocyte cytoskeletal protein Band 3. The role of a traditionally endoplasmic reticulum (ER) protease in the process of red blood cell invasion is unexpected. Here, using a combination of molecular, cellular and chemical approaches we provide evidence that PfSPP is, instead, a bona fide ER-resident peptidase that remains intracellular throughout the invasion process. Furthermore, SPP-specific drug inhibition has no effect on erythrocyte invasion whilst having low micromolar potency against intra-erythrocytic development. Contrary to previous reports, these results show that PfSPP plays no role in erythrocyte invasion. Nonetheless, PfSPP clearly represents a potential chemotherapeutic target to block parasite growth, supporting ongoing efforts to develop antimalarial-targeting protein maturation and trafficking during intra-erythrocytic development.

Single-cell electrical lysis of erythrocytes detects deficiencies in the cytoskeletal protein network

The network of erythrocyte cytoskeletal proteins significantly influences erythrocyte physical and biological properties. Here we show that the kinetics of erythrocyte lysis during exposure to an electric field is sensitively correlated with defects in the cytoskeletal network. Histograms compiled from single-cell electrical lysis data show characteristics of erythrocyte populations that are deficient in a specific cytoskeletal protein, revealing the presence of cell subpopulations.

Altered phosphorylation of cytoskeleton proteins in sickle red blood cells: The role of protein kinase C, Rac GTPases, and reactive oxygen species

The small Rho GTPases Rac1 and Rac2 regulate actin structures and mediate reactive oxygen species (ROS) production via NADPH oxidase in a variety of cells. We have demonstrated that deficiency of Rac1 and Rac2 GTPases in mice disrupts the normal hexagonal organization of the RBC cytoskeleton and reduces erythrocyte deformability. This is associated with increased phosphorylation of adducin at Ser-724, (corresponding to Ser-726 in human erythrocytes), a domain target of protein kinase C (PKC). PKC phosphorylates adducin and leads to decreased F-actin capping and dissociation of spectrin from actin, implicating a significant role of such phosphorylation in cytoskeletal remodeling. We evaluated adducin phosphorylation in erythrocytes from patients with sickle cell disease and found it consistently increased at Ser-726. In addition, ROS concentration is elevated in sickle erythrocytes by 150–250% compared to erythrocytes from normal control individuals. Here, we review previous studies demonstrating that altered phosphorylation of erythrocyte cytoskeletal proteins and increased ROS production result in disruption of cytoskeleton stability in healthy and sickle cell erythrocytes. We discuss in particular the known and potential roles of protein kinase C and the Rac GTPases in these two processes.

Solution structure and glycophorin C binding studies of the protein 4.1R FERM α‐lobe domain

Red blood cell protein 4.1 (4.1R, an 80 kDa isoform),1 one of the major erythrocyte cytoskeletal proteins, forms two major ternary complexes in red blood cells, 4.1Ractin-spectrin and 4.1R-p55-glycophorin C (GPC), and plays important roles in maintaining cell morphology and membrane mechanical properties.1–7 This protein consists of four functional domains: the FERM (4.1 and ezrin/radixin/moesin)8 domain (also termed the N-terminal 30 kDa membrane-binding domain), the 16 kDa regulatory domain, the spectrin-actin-binding (SAB) domain, and the C-terminal 22/24 kDa domain. The 4.1R FERM domain is a multifunctional domain that forms a cloverleaf-like fold containing three structural and functional domains (N-lobe, a-lobe, and C-lobe domains).9 In the 4.1R-p55-GPC ternary complex, the 4.1R FERM a-lobe domain interacts with the N-terminal cytoplasmic domain of GPC, the 4.1R FERM C-lobe10 and/or N-lobe domain11 interacts with the p55 HOOK domain, and the p55 PDZ domain interacts with the C-terminal cytoplasmic domain of GPC.10,12–14 We previously examined the specific binary interaction between the p55 PDZ domain and the C-terminal cytoplasmic domain of GPC by NMR, as the first step to obtain structural insights into the 4.1R-p55-GPC ternary complex.15,16 Our NMR studies clearly revealed how the p55 PDZ domain interacts with two key C-terminal residues, Tyr126 and Ile128, of GPC at the atomic level.16 We have now focused on the binary interaction between the 4.1R FERM a-lobe domain and the N-terminal cytoplasmic domain of GPC, as the second step toward our goal. In the binary interaction between 4.1R and GPC, the 4.1R-binding site on GPC reportedly corresponds to the Nterminal 12 amino acid residues of its cytoplasmic domain (residues 82–93), and the RHK (residues Arg86-His87Lys88) motif is probably crucial for binding the 4.1R FERM a-lobe domain.13 In contrast, the GPC-binding site on the 4.1R FERM domain reportedly corresponds to the sequence encoded by exon 8 (residues Tyr94-Arg166) in the 4.1R alobe domain, and the ELEE motif (residues Glu153Glu156) is considered to be a good candidate for the GPC-binding site.9,10 However, there is no experimental evidence that the ELEE motif contributes to the interaction with the N-terminal cytoplasmic domain of GPC. To better understand this interaction mode, we prepared the 4.1R FERM a-lobe domain (residues Asp83-Tyr187) and performed an NMR analysis of this domain with the N-terminal cytoplasmic domain of GPC. Here, we report the first NMR-derived structure of the 4.1R FERM a-lobe domain and propose a new GPC-binding site on the 4.1R FERM a-lobe domain, based on our NMR experiments.

A global role for EKLF in definitive and primitive erythropoiesis

AbstractErythroid Kruppel-like factor (EKLF, KLF1) plays an important role in definitive erythropoiesis and β-globin gene regulation but failure to rectify lethal fetal anemia upon correction of globin chain imbalance suggested additional critical EKLF target genes. We employed expression profiling of EKLF-null fetal liver and EKLF-null erythroid cell lines containing an inducible EKLF-estrogen receptor (EKLF-ER) fusion construct to search for such targets. An overlapping list of EKLF-regulated genes from the 2 systems included α-hemoglobin stabilizing protein (AHSP), cytoskeletal proteins, hemesynthesis enzymes, transcription factors, and blood group antigens. One EKLF target gene, dematin, which encodes an erythrocyte cytoskeletal protein (band 4.9), contains several phylogenetically conserved consensus CACC motifs predicted to bind EKLF. Chromatin immunoprecipitation demonstrated in vivo EKLF occupancy at these sites and promoter reporter assays showed that EKLF activates gene transcription through these DNA elements. Furthermore, investigation of EKLF target genes in the yolk sac led to the discovery of unexpected additional defects in the embryonic red cell membrane and cytoskeleton. In short, EKLF regulates global erythroid gene expression that is critical for the development of primitive and definitive red cells.

Identification and biochemical characterization of vivapains, cysteine proteases of the malaria parasite Plasmodium vivax.

Cysteine proteases play important roles in the life cycles of malaria parasites. Cysteine protease inhibitors block haemoglobin hydrolysis and development in Plasmodium falciparum, suggesting that the cysteine proteases of this major human pathogen, termed falcipains, are appropriate therapeutic targets. To expand our understanding of plasmodial proteases to Plasmodium vivax, the other prevalent human malaria parasite, we identified and cloned genes encoding the P. vivax cysteine proteases, vivapain-2 and vivapain-3, and functionally expressed the proteases in Escherichia coli. The vivapain-2 and vivapain-3 genes predicted papain-family cysteine proteases, which shared a number of unusual features with falcipain-2 and falcipain-3, including large prodomains and short N-terminal extensions on the catalytic domain. Recombinant vivapain-2 and vivapain-3 shared properties with the falcipains, including acidic pH optima, requirements for reducing conditions for activity and hydrolysis of substrates with positively charged residues at P1 and Leu at P2. Both enzymes hydrolysed native haemoglobin at acidic pH and the erythrocyte cytoskeletal protein 4.1 at neutral pH, suggesting similar biological roles to the falcipains. Considering inhibitor profiles, the vivapains were inhibited by fluoromethylketone and vinyl sulphone inhibitors that also inhibited falcipains and have demonstrated potent antimalarial activity.

A biotin derivative blocks parasite induced novel permeation pathways in Plasmodium falciparum-infected erythrocytes

The malaria parasite Plasmodium falciparum infects human erythrocytes, and it induces an increased rate of uptake into the infected cell of a range of solutes, including essential nutrients required for parasite development. Several models have been proposed for the mechanism(s) underlying parasite-induced solute uptake, each differing with respect to the site of entry into infected cells. We show that a biotin derivative that is excluded from non-infected erythrocytes gains access to infected erythrocytes via a pathway that is inhibited by compounds shown previously to block the pathways responsible for the increased uptake of solutes. The derivative was found to bind erythrocyte cytoskeletal proteins and to hemoglobin, providing evidence that the novel pathways are in the erythrocyte membrane and allow direct access of solutes to the erythrocyte cytosol. The derivative inhibited its own uptake and blocked the parasite-induced transport of other solutes. In whole-cell patch-clamp analyses, biotinylation of infected erythrocytes caused significant decrease in a parasite-induced outward rectifying conductance. In vitro, biotinylation of trophozoite-stage parasitized erythrocytes delayed parasite development. Treatment of infected cells in the final developmental stage abrogated the parasite's ability to complete development. The data are consistent with the novel pathways playing an important role in parasite growth.

Cysteine proteases of malaria parasites: targets for chemotherapy.

New drugs to treat malaria are urgently needed. Cysteine proteases of malaria parasites offer potential new chemotherapeutic targets. Cysteine protease inhibitors block parasite hemoglobin hydrolysis and development, indicating that cysteine proteases play a key role in hemoglobin degradation, a necessary function of erythrocytic trophozoites. These inhibitors also block the rupture of erythrocytes by mature parasites, suggesting an additional role for cysteine proteases in the hydrolysis of erythrocyte cytoskeletal proteins. Recent studies have shown that the repertoire of cysteine proteases of malaria parasites is larger than was previously realized. Plasmodium falciparum, the most virulent human malaria parasite, expresses three papain-family cysteine proteases, known as falcipains. All three proteases are expressed by trophozoites and hydrolyze hemoglobin at acidic pH, suggesting roles in this process. Falcipain-2 also hydrolyzes ankyrin at neutral pH, suggesting additional activity against erythrocyte cytoskeletal targets. Multiple orthologs of the falcipains have been identified in other plasmodial species. Analysis of orthologs from animal model rodent parasites identified similar features, but some noteworthy biochemical differences between the cysteine proteases. These differences must be taken into account in interpreting in vivo experiments. A number of small molecule cysteine protease inhibitors blocked parasite hemoglobin hydrolysis and development, and inhibitory effects against parasites generally correlated with inhibition of falcipain-2. Some compounds also cured mice infected with otherwise lethal malaria infections. Current research priorities are to better characterize the biological roles and biochemical features of the falcipains. In addition, efforts to identify optimal falcipain inhibitors as antimalarials are underway.

Hydrolysis of erythrocyte proteins by proteases of malaria parasites.

During the intraerythrocytic phase of the life cycle, malaria parasites hydrolyze host proteins. Hemoglobin is processed into individual amino acids, which are used for parasite protein synthesis. Erythrocyte cytoskeletal proteins are cleaved during erythrocyte invasion and rupture. A number of plasmodial proteases that appear to be responsible for key cleavages of host proteins have recently been characterized. Hemoglobin hydrolysis appears to be mediated by acid cysteine, aspartic, and metalloproteases, and then a neutral aminopeptidase. Cysteine and aspartic proteases that hydrolyze hemoglobin can also cleave host cytoskeletal proteins, and these and additional proteases likely cleave the cytoskeleton to mediate erythrocyte rupture and invasion. Various protease inhibitors block parasite development, suggesting that key proteases may be appropriate chemotherapeutic targets. Recent advances in the characterization of plasmodial proteases should facilitate the analysis of the specific roles of these enzymes and expedite the progress of drug discovery efforts directed against them.

Erythrocyte membrane and cytoskeletal protein glycation and oxidation in short-term diabetic rabbits.

The objective of this study was to elucidate the glycation and oxidation processes in plasma and erythrocyte membrane proteins as well as the major erythrocyte cytoskeletal protein, spectrin, using a short-term experimental rabbit diabetes model. Diabetes was induced with a single-dose alloxan injection. Spectrin was purified from erythrocyte ghosts with selective solubilization followed by gel filtration chromatography techniques, and tested for purity using sodium dodecyl sulfate-poly-acrylamide gel electrophoresis. Glycation in plasma proteins was measured as fructosamine using the nitroblue tetrazolium method, and in erythrocyte membrane and purified spectrin, as ketoamine equivalents, by the hydrazine/phenylhydrazine method. Protein oxidation in plasma, erythrocyte membrane proteins, and purified spectrin was evaluated in terms of sulfhydryl oxidation, based on cis-dichlorodiammine platinum (II) binding. Carbonyl formation was also measured in plasma and membrane proteins. Sulfhydryl oxidation, carbonyl groups and glycated protein levels showed statistically significant differences between the diabetic and control groups for both the plasma and the erythrocyte membrane proteins. The cis-dichlorodiammine platinum (II) binding was significantly different in diabetic rabbit erythrocyte spectrin, while glycation was not significantly different for this protein. Our data clearly demonstrate that both protein glycation and oxidation are biochemical alterations occurring in diabetes, even of short duration.

New insights into functions of erythroid proteins in nonerythroid cells.

This article presents new insights into potential roles that three erythrocyte cytoskeletal proteins, protein 4.1, ankyrin, and spectrin, may play in nonerythroid nucleated cells. Each of these proteins is encoded by several closely related genes characterized by complex alternative splicing of their pre-mRNA, thus resulting in the cellular expression of a broad repertoire of isoforms that can adopt tissue- and cell-specific distribution. This could account for the presence of skeletal networks in intracellular organelles such as lysosomes, the Golgi apparatus, or the nucleus. In addition to providing structural support to cell membranes, these cytoskeletal proteins regulate the functions of various transmembrane proteins they interact with, in particular ion channels, as well as the activity of membrane-bound enzymes. Thus, they appear to be key players in major unsuspected cell functions such as protein sorting, dynamics of nuclear architecture during mitosis, or regulation of signal transduction pathways.