EFR3A is a conserved peripheral membrane protein required for the plasma membrane localization of the phosphatidylinositol-4 kinase (PI4KIIIα/PI4KA) complex and for regulating the responsiveness of G-protein-coupled receptors. Additionally, it was implicated in several other potentially unrelated physiological functions. In metazoan organisms, EFR3A is ubiquitously co-expressed with its paralog EFR3B which shares similar biological roles. This brief review summarizes the current knowledge regarding the potential roles of EFR3A in human disease states, including neurological and cardiovascular disorders, as well as various neoplasia-based diseases.

Background Hereditary spherocytosis (HS) is an erythrocytic membranopathy that belongs to a group of rare genetic disorders. Mutations in five genes, including ANK1, cause clinical manifestations of the disease. Identified variations in individual families provide a better understanding of the molecular basis of the disease. Methods In this study, we used two sequencing methods, whole exome sequencing (WES) and Sanger sequencing, analyzing gDNA and cDNA as templates, to detect and verify the variants putatively responsible for the clinical symptoms observed in a Polish family diagnosed with HS. Results We detected two variants that occur in cis in the ANK1 gene, a known missense mutation (NP_000028.3:p.V463I) and a novel frameshift mutation (NP_000028.3: p.V1626fs*64) that appears to be crucial for the probands. As shown by transcriptome studies, the mutant allele is not present at a detectable level. Conclusions We conclude that the molecular basis of this case is related to an unstable transcript of the mutant allele and that the direct cause of the HS is a deficiency of erythrocyte ankyrin leading to a disruption of the AE1-erythrocyte ankyrin-spectrin complex in the erythrocyte membrane.

Throughout the last 5 years, extensive research has been carried out towards the development of effective treatments for coronavirus disease 2019 (COVID-19). Regardless of the worldwide efforts, only a few drugs have passed clinical trials, and there is still a need to develop therapies, especially for those who are particularly vulnerable to a severe disease course. Maleimide-functionalized liposomes are proposed to serve as a platform for the immobilization, stabilization, and delivery of a short peptide sequence with high affinity towards severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, extensive optimizations should be performed in order to achieve features required for a reliable drug candidate, such as homogeneity of physical parameters and their long-term stability. Here, we present a step-by-step development process for maleimide-functionalized liposomes, which—once decorated with the SARS-CoV-2-binding peptide—could inhibit the infection progress of COVID-19. The main emphasis is placed on defining optimal lipid composition and formation conditions of PEGylated liposomes. We propose that the developed nanocarrier technology can be used as a universal platform for the construction of multiple antiviral agents.

Flotillin-binding protein networks serve as scaffolds, organizing lipid rafts and facilitating the recruitment of other raft-associated proteins such as receptors and downstream signaling molecules to regulate various intracellular pathways, including those involved in cell proliferation, migration, and endocytosis.Flotillins belong to the SPFH (stomatin/prohibitin/flotillin/HflK/C) domain-containing protein family, also known as the prohibitin homology (PHB) domain, which enables membrane association via acylation and hydrophobic hairpin motifs that anchor them to the inner leaflet of the plasma membrane. The functional diversity of flotillin proteins within membrane microdomains primarily stems from their interactions with other proteins.Data presented in this article characterize the proximal interactome of flotillin-2 within detergent-resistant membranes (DRMs) using BioID, a proximity-dependent biotinylation technique. Flotillin-2 was fused with the biotin ligase BirA* at either the N- or C-terminus and expressed in HeLa cells. DRMs were isolated through sucrose density gradient ultracentrifugation, and biotinylated proteins were purified using biotin–avidin affinity followed by label-free quantitative (LFQ) mass spectrometry.This approach identified a set of proteins significantly enriched in DRM fractions from cells expressing flotillin-2–BirA* fusions compared to control cells expressing BirA*–mEGFP. The two analyses allowed for relative quantification of ∼433 and ∼926 unique proteins across the N-terminal and C-terminal BirA* fusions, respectively. 28 (N-terminal) and 88 (C-terminal) proteins were observed as significantly enriched in flotillin-2 samples (28 and 43 with fold change ≥ 2). These enriched proteins are candidate interactors of flotillin-2 within membrane raft domains. Notably, the N-terminal fusion-associated proteins were significantly linked to specific biological processes such as dendritic transport and regulation of signal transduction, whereas the C-terminal fusion group showed enrichment in membrane biogenesis-related proteins. Among here presented DRM partners of flotillin-2, both previously known and earlier unreported interactors of this protein were found. Overall, our BioID-based analysis provides valuable insight into the flotillin-2 interactome in DRM fractions and lays the groundwork for future studies exploring the regulation of membrane lateral heterogeneity and the role of flotillin-mediated domains in signaling pathways, particularly those dysregulated in diseases such as cancer.

Membrane palmitoylated protein 1 (MPP1), a protein found to directly interact with flotillins, has been shown to play a crucial role as a raft-capturing molecule, modulating dynamics of flotillin-nanodomains and affects plasma membrane (PM) organisation in native erythroid cells. This study aims to reconstitute the flotillin-MPP1 complexes in a minimal membrane-based system to check its ability to govern domain formation and modulate fluidity and phase separation of membranes comprising simple ternary lipid mixtures. Using recombinant flotillins reconstituted into giant unilamellar vesicles (GUVs) and fluorescence lifetime imaging (FLIM), we have shown that MPP1 promotes membrane remodelling and triggers the coexistence of liquid-ordered (Lo) and liquid-disordered (Ld) domains. Additionally, we examined whether palmitoylation of MPP1 affects lipid bilayers and demonstrated that it exerts a certain influence on membrane organisation. Our data highlights that flotillin-MPP1 assemblies are sufficient and necessary to modulate the lateral organisation of lipid bilayers, pointing to their crucial role in PM organisation. Additionally, we propose a new toolset for successful flotillin reconstitution in GUVs, which is a viable platform compatible with a wide spectrum of flotillin-based studies on model membrane systems.

The EFR3 (Eighty-Five Requiring 3) protein and its homologs are rather poorly understood eukaryotic plasma membrane peripheral proteins. They belong to the armadillo-like family of superhelical proteins. In higher vertebrates two paralog genes, A and B were found, each expressing at least 2–3 protein isoforms. EFR3s are involved in several physiological functions, mostly including phosphatidyl inositide phosphates, e.g., phototransduction (insects), GPCRs, and insulin receptors regulated processes (mammals). Mutations in the EFR3A were linked to several types of human disorders, i.e., neurological, cardiovascular, and several tumors. Structural data on the atomic level indicate the extended superhelical rod-like structure of the first two-thirds of the molecule with a typical armadillo repeat motif (ARM) in the N-terminal part and a triple helical motif in its C-terminal part. EFR3s’ best-known molecular function is anchoring the giant phosphatidylinositol 4-kinase A complex to the plasma membrane crucial for cell signaling, also linked directly to the KRAS mutant oncogenic function. Another function connected to the newly uncovered interaction of EFR3A with flotillin-2 may be the participation of the former in the organization and regulation of the membrane raft domain. This review presents EFR3A as an intriguing subject of future studies.

Transdifferentiation, also known as direct reprogramming, is the transformation of one terminally differentiated cell type into another mature cell type, while bypassing the stage of pluripotency. In leukemia, this phenomenon has a dual significance: on the one hand, it is an adaptive mechanism driving tumor survival and resistance to treatment, and on the other, it offers a potential opportunity for innovative therapies. Of particular interest is the directional transdifferentiation (mostly partial) toward dendritic cell-like phenotypes, which increases the immunogenicity of cancer cells. Mastering this process could define a new generation of immunotherapies that leverage the inherent plasticity of leukemic cells to achieve therapeutic benefits. In this brief review, we attempt to gather information concerning the molecular mechanism of this process and point to the role of dendritic cells as a crucial element of anticancer, particularly anti-leukemia innate and acquired, immunity. Thus, in vitro and in vivo techniques of inducing transformation of the leukemia cells into cancer antigen-presenting cells and the application of these technologies in current and future therapies are discussed.

Previously, we reported a new missense mutation in the ANK1 gene that correlated with the hereditary spherocytosis phenotype. This mutation, resulting in L1340P substitution (HGMD CM149731), likely leads to the changes in the conformation of the ankyrin ZZUD domain important for ankyrin binding to spectrin. Here, we report the molecular and physiological effects of this mutation. First, we assessed the binding activity of human β-spectrin to the mutated ZZUDL1340P domain of ankyrin using two different experimental approaches—the study of association and dissociation responses of the spectrin–ankyrin binding domain and a sedimentation assay. In addition, we documented the changes in morphology caused by the overexpressed ankyrin ZZUD domain in human cell models. Our results prove the key role of the L1340 aa residue for the correct alignment of the ZZUD domain of ankyrin, which results in binding the latter with spectrin within the erythrocyte membrane. Replacing L1340 with a proline residue disrupts the spectrin-binding activity of ankyrin.

Background Membrane rafts play a crucial role in the regulation of many important biological processes. Our previous data suggest that specific interactions of flotillins with MPP1 are responsible for membrane raft domain organization and regulation in erythroid cells. Interaction of the flotillin-based protein network with specific membrane components underlies the mechanism of raft domain formation and regulation, including in cells with low expression of MPP1. Methods We sought to identify other flotillin partners via the immobilized recombinant flotillin-2-based affinity approach and mass spectrometry technique. The results were further confirmed via immunoblotting and via co-immunoprecipitation. In order to study the effect of the candidate protein on the physicochemical properties of the plasma membrane, the gene was knocked down via siRNA, and fluorescence lifetime imaging microscopy and spot-variation fluorescence correlation spectroscopy was employed. Results EFR3A was identified as a candidate protein that interacts with flotillin-2. Moreover, this newly discovered interaction was demonstrated via overlay assay using recombinant EFR3A and flotillin-2. EFR3A is a stable component of the detergent-resistant membrane fraction of HeLa cells, and its presence was sensitive to the removal of cholesterol. While silencing the EFR3A gene, we observed decreased order of the plasma membrane of living cells or giant plasma membrane vesicles derived from knocked down cells and altered mobility of the raft probe, as indicated via fluorescence lifetime imaging microscopy and spot-variation fluorescence correlation spectroscopy. Moreover, silencing of EFR3A expression was found to disturb epidermal growth factor receptor and phospholipase C gamma phosphorylation and affect epidermal growth factor-dependent cytosolic Ca 2+ concentration. Conclusions Altogether, our results suggest hitherto unreported flotillin-2-EFR3A interaction, which might be responsible for membrane raft organization and regulation. This implies participation of this interaction in the regulation of multiple cellular processes, including those connected with cell signaling which points to the possible role in human health, in particular human cancer biology.

Hereditary spherocytosis (HS) refers to the group of the most frequently occurring non-immune hereditary hemolytic anemia in people of Caucasian central or northern European ancestry. HS is mainly associated with pathogenic variants of genes encoding defects in five membrane proteins, including anion exchanger 1 encoded by the SLC4A1 gene. In this study, in a family affected with HS, we identified a hitherto unreported AE1 defect, variant p.G720W. The result of it is most likely the HS phenotype. Molecular dynamics simulation study of the AE1 transmembrane domain may indicate reasonable changes in AE1 domain structure, i.e., significant displacement of the tryptophan residue towards the membrane surface connected with possible changes in AE1 function. The WES analysis verified by classical sequencing in conjunction with biochemical analysis and molecular simulation studies shed light on the molecular mechanism underlying this case of hereditary spherocytosis, for which the newly discovered AE1 variant p.G720W seems crucial.

The appearance of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and its spread all over the world is the cause of the coronavirus disease 2019 (COVID-19) pandemic, which has recently resulted in almost 400 million confirmed cases and 6 million deaths, not to mention unknown long-term or persistent side effects in convalescent individuals. In this short review, we discuss approaches to treat COVID-19 that are based on current knowledge of the mechanisms of viral cell receptor recognition, virus-host membrane fusion, and inhibition of viral RNA and viral assembly. Despite enormous progress in antiviral therapy and prevention, new effective therapies are still in great demand.

MPP1 (membrane palmitoylated protein 1) belongs to the MAGUK (membrane-associated guanylate kinase homologs) scaffolding protein family. These proteins organize molecules into complexes, thereby maintaining the structural heterogeneity of the plasma membrane (PM). Our previous results indicated that direct, high-affinity interactions between MPP1 and flotillins (raft marker proteins) display dominant PM-modulating capacity in erythroid cells. In this study, with high-resolution structured illuminated imaging, we investigated how these complexes are organized within erythroid cells on the nanometer scale. Furthermore, using other spectroscopic techniques, namely fluorescence recovery after photobleaching (FRAP) and spot-variation fluorescence correlation spectroscopy (svFCS), we revealed that MPP1 acts as a key raft-capturing molecule, regulating temporal immobilization of flotillin-based nanoclusters, and controls local concentration and confinement of sphingomyelin and Thy-1 in raft nanodomains. Our data enabled us to uncover molecular principles governing the key involvement of MPP1-flotillin complexes in the dynamic nanoscale organization of PM of erythroid cells.

172 Background: Mechanisms of tumor invasion are not well defined. PTEN, a key tumor suppressor frequently inactivated in epithelial cancers, acts as a central node that controls tumor invasion. Despite PI-3 kinase-phospho-Akt pathway activation resulting in enhanced tumor growth, prostate tumors with PTEN loss undergo p53-mediated senescence that restricts tumor invasion. Methods: ABI1 downregulation is associated with epithelial-mesenchymal transition in highly invasive prostate tumors; these tumors frequently loose PTEN; therefore we set to examine genetic interaction of ABI1 and PTEN using novel mouse model of prostate cancer. We analyzed the correlation of ABI1 and PTEN expression in human PCa tumor tissue. Results: Here, using Abi1/Pten KO mouse model we identified a novel mechanism that guards tumor invasion. In Pten-null tumors upregulation of Abi1 leads to sequestration of activated Src kinase. In the absence of Abi1, this regulation is lost leading to activation of non-canonical WNT-SRC-STAT3 axis and enhanced invasion through activation of MMP2 activity. This molecular mechanism explains progression of tumors with Pten loss from PIN to invasive carcinoma upon concomitant Abi1 inactivation. In human tumors with low Abi1 and Pten are associated with aggressive phenotype, biochemical recurrence and metastasis. Conclusions: ABI1 acts as failsafe mechanism in PTEN null tumors by restricting SRC-mediated tumor invasion. ABI1 might have a predictive value in clinical setting in context of PTEN levels.

Pyrimidine 5′-nucleotidase deficiency is a rare erythrocyte enzymopathy. Here we report two cases of hemolytic anemia in brothers of Polish origin that are associated with a very rare mutation. Heterozygous deletion in the NT5C3A gene (c.444_446delGTT), inherited most likely from their asymptomatic mother, resulted in a single amino acid residue deletion (p.F149del) in cytosolic pyrimidine 5′-nucleotidase. However, only the mutated transcript was present in the reticulocyte transcriptome of both patients. Only residual activity of pyrimidine 5′-nucleotidase in the brothers’ erythrocytes could be observed when compared with the controls, including their asymptomatic father and sister. Western blot showed no sign of the presence of 5′-nucleotidase protein in the erythrocytes of both studied patients. The 2.5-fold reduction of the purine/pyrimidine ratio observed only in the brothers’ erythrocytes confirms the correlation of the results of molecular analysis, including whole-exome sequencing, with the phenotype of the pyrimidine 5′-nucleotidase deficiency. Altogether, our results may substantiate the hypothesis of the heterogeneity of the molecular basis of the defect involving both the mutation presented here and negative regulation of expression of the “normal” allele.

Additional file 2: Detailed data on the analyzed variant NM_001166118.3: c.444_446delGTT detected WES and analyzed using Ingenuity Variant Analysis plugin (QIAGEN, CA, USA).

Hereditary spherocytosis (HS), the most commonly inherited hemolytic anemia in northern Europeans, comprises a group of diseases whose heterogeneous genetic basis results in a variable clinical presentation. High-throughput genome sequencing methods have made a leading contribution to the recent progress in research on and diagnostics of inherited diseases and inspired us to apply whole exome sequencing (WES) to identify potential mutations in HS. The data presented here reveal a novel mutation probably responsible for HS in a single Polish family. Patients with clinical evidence of HS (clinical symptoms, hematological data, and EMA test) were enrolled in the study. The examination of the resulting WES data showed a number of polymorphisms in 71 genes associated with known erythrocyte pathologies (including membranopathies, enzymopathies, and hemoglobinopathies). Only a single SPTB gene variant indicated the possible molecular mechanism of the disease in the studied family. The new missense mutation p.C183Y was identified using WES in the SPTB gene, which is most likely the cause of clinical symptoms typical of hereditary spherocytosis (membranopathy) due to structural and functional impairments of human β-spectrin. This mutation allows for a better understanding of the molecular mechanism(s) of one of the membranopathies, hereditary spherocytosis.

Extensive studies showed the crucial role of ATP binding cassette (ABC) transporter ABCA1 in organizing the lipid microenvironment at the plasma membrane (PM) of living cells. However, the exact role of this protein in terms of lipid redistribution and lateral reorganization of the PM is still being discussed. Here, we took advantage of the spot variation fluorescence correlation spectroscopy (svFCS) to investigate the molecular dynamics of the ABCA1 expressed at the PM of Chinese hamster ovary cells (CHO-K1). We confirmed that this protein is strongly confined into the raft nanodomains. Next, in agreement with our previous observations, we showed that amphotericin B does not affect the diffusion properties of an active ABCA1 in contrary to inactive mutant ABCA1MM. We also evidenced that ApoA1 influences the molecular diffusion properties of ABCA1. Finally, we showed that the molecular confinement of ABCA1 depends on the cholesterol content in the PM, but presumably, this is not the only factor responsible for that. We concluded that the molecular dynamics of ABCA1 strongly depends on its activity and the PM composition. We hypothesize that other factors than lipids (i.e., proteins) are responsible for the strong confinement of ABCA1 in PM nanodomains which possibility has to be elucidated.

Flotillins are the major structural proteins in erythroid raft domains. We have shown previously that the dynamic nanoscale organization of raft domains in erythroid cells may depend on flotillin-MPP1 interactions. Here, by using molecular dynamic simulations and a surface plasmon resonance-based approach we determined that high-affinity complexes of MPP1 and flotillins are formed via a so far unidentified region within the D5 domain of MPP1. Significantly, this particular "flotillin binding motif" is of key physiological importance, as overexpression of peptides containing this motif inhibited endogenous MPP1-flotillin interaction in erythroid precursor cells, thereby causing lateral disorganization of raft domains. This was reflected by both reduction in the plasma membrane order and markedly decreased activation of signal transduction via the raft-dependent insulin receptor pathway. Our data highlight new molecular details concerning the mechanism whereby MPP1 functionally links flotillins to exert their physiological role in raft domain formation.