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American Journal of Medical Genetics.... Jul 2024Pathogenic variants in TRIO, encoding the guanine nucleotide exchange factor, are associated with two distinct neurodevelopmental delay phenotypes: gain-of-function...
Pathogenic variants in TRIO, encoding the guanine nucleotide exchange factor, are associated with two distinct neurodevelopmental delay phenotypes: gain-of-function missense mutations within the spectrin repeats are causative for a severe developmental delay with macrocephaly (MIM: 618825), whereas loss-of-function missense variants in the GEF1 domain and truncating variants throughout the gene lead to a milder developmental delay and microcephaly (MIM: 617061). In three affected family members with mild intellectual disability/NDD and microcephaly, we detected a novel heterozygous TRIO variant at the last coding base of exon 31 (NM_007118.4:c.4716G>A). RNA analysis from patient-derived lymphoblastoid cells confirmed aberrant splicing resulting in the skipping of exon 31 (r.4615_4716del), leading to an in-frame deletion in the first Pleckstrin homology subdomain of the GEF1 domain: p.(Thr1539_Lys1572del). To test for a distinct gestalt, facial characteristics of the family members and 41 previously published TRIO cases were systematically evaluated via GestaltMatcher. Computational analysis of the facial gestalt suggests a distinguishable facial TRIO-phenotype not outlined in the existing literature.
Topics: Humans; Guanine Nucleotide Exchange Factors; Phenotype; Male; Female; RNA Splice Sites; Pedigree; Intellectual Disability; Microcephaly; Child; Child, Preschool; Developmental Disabilities; Exons; RNA Splicing; Facies; Protein Serine-Threonine Kinases
PubMed: 38517182
DOI: 10.1002/ajmg.a.63599 -
Spectrochimica Acta. Part A, Molecular... May 2024In this work, we investigated the oxidative stress-related biochemical alterations in red blood cells (RBCs) and their membranes with the use of spectroscopic...
In this work, we investigated the oxidative stress-related biochemical alterations in red blood cells (RBCs) and their membranes with the use of spectroscopic techniques. We aimed to show their great advantage for the in situ detection of lipid classes and secondary structures of proteins without the need for their extraction in the cellular environment. The exposition of the cells to peroxides, t-butyl hydroperoxide (tBOOH) or hydrogen peroxide (HO) led to different degradation processes encompassing the changes in the composition of membranes and structural modifications of hemoglobin (Hb). Our results indicated that tBOOH is generally a stronger oxidizing agent than HO and this observation was congruent with the activity of superoxide and glutathione peroxidase. ATR-FTIR and Raman spectroscopies of membranes revealed that tBOOH caused primarily the partial loss and peroxidation of the lipids resulting in loss of the integrity of membranes. In turn, both peroxides induced several kinds of damage in the protein layer, including the partial decrease of their content and irreversible aggregation of spectrin, ankyrin, and membrane-bound globin. These changes were especially pronounced on the membrane surface where stress conditions induced the formation of β-sheets and intramolecular aggregates, particularly for tBOOH. Interestingly, nano-FTIR spectroscopy revealed the lipid peroxidative damage on the membrane surface in both cases. As far as hemoglobin was concerned, tBOOH and HO caused the increase of the oxyhemoglobin species and conformational alterations of its polypeptide chain into β-sheets. Our findings confirm that applied spectroscopies effectively track the oxidative changes occurring in the structural components of red blood cells and the simplicity of conducting measurements and sample preparation can be readily applied to pharmacological and clinical studies.
Topics: Humans; Hydrogen Peroxide; Erythrocytes; Hemoglobins; Peroxides; Spectroscopy, Fourier Transform Infrared; Lipids; Oxidative Stress
PubMed: 38493515
DOI: 10.1016/j.saa.2024.124142 -
MBio Apr 2024Remodeling the erythrocyte membrane and skeleton by the malarial parasite is closely associated with intraerythrocytic development. However, the mechanisms underlying...
UNLABELLED
Remodeling the erythrocyte membrane and skeleton by the malarial parasite is closely associated with intraerythrocytic development. However, the mechanisms underlying this association remain unclear. In this study, we present evidence that erythrocytic α-spectrin, but not β-spectrin, was dynamically ubiquitinated and progressively degraded during the intraerythrocytic development of from the ring to the schizont stage. We further observed an upregulated expression of phosphatidylinositol 3-kinase (PfPI3K) in the infected red blood cells during the intraerythrocytic development of the parasite. The data indicated that PfPI3K phosphorylated and activated erythrocytic ubiquitin-protein ligase, leading to increased α-spectrin ubiquitination and degradation during development. We further revealed that inhibition of the activity of PfPI3K impaired development and infectivity in mice. These findings collectively unveil an important mechanism of PfPI3K-ubiquitin-mediated degradation of α-spectrin during the intraerythrocytic development of species. Proteins in the PfPI3K regulatory pathway are novel targets for effective treatment of severe malaria.
IMPORTANCE
is the causative agent of severe malaria that causes millions of deaths globally. The parasite invades human red blood cells and induces a cascade of alterations in erythrocytes for development and proliferation. Remodeling the host erythrocytic cytoskeleton is a necessary process during parasitization, but its regulatory mechanisms remain to be elucidated. In this study, we observed that erythrocytic α-spectrin is selectively degraded after invasion, while β-spectrin remained intact. We found that the α-spectrin chain was profoundly ubiquitinated by E3 ubiquitin ligase and degraded by the 26S proteasome. E3 ubiquitin ligase activity was regulated by phosphatidylinositol 3-kinase (PfPI3K) signaling. Additionally, blocking the PfPI3K-ubiquitin-proteasome pathway in -infected red blood cells reduced parasite proliferation and infectivity. This study deepens our understanding of the regulatory mechanisms of host and malarial parasite interactions and paves the way for the exploration of novel antimalarial drugs.
Topics: Humans; Animals; Mice; Plasmodium falciparum; Spectrin; Erythrocytes; Malaria, Falciparum; Ubiquitin; Phosphatidylinositol 3-Kinase; Ubiquitin-Protein Ligases
PubMed: 38470053
DOI: 10.1128/mbio.03510-23 -
Oncology Mar 2024Nuclear envelope spectrin repeat protein (Nesprin) 1 encoded by SYNE1, crucially regulates the morphology and functions of the cell. Mutations in the SYNE1 gene are...
INTRODUCTION
Nuclear envelope spectrin repeat protein (Nesprin) 1 encoded by SYNE1, crucially regulates the morphology and functions of the cell. Mutations in the SYNE1 gene are associated with various diseases; however, their significance in renal cell carcinoma (RCC) remains unknown. In this study, we have investigated the association of SYNE1/Nesprin1 with the progression and prognosis of clear cell RCC (ccRCC).
METHODS
In silico analyses of publicly available datasets of patients with RCC were performed. Based on the cohort data, Nesprin1 expression in nephrectomized tissue samples acquired from patients with ccRCC was analyzed using immunohistochemical staining. The invasion, migration, and proliferation of the SYNE1-knockdown human RCC cell lines were analyzed in vitro; moreover, RNA sequencing and Gene Set Enrichment Analysis were conducted to study the molecular mechanism underlying the association of SYNE1/Nesprin1 with prognosis of RCC.
RESULTS
Patients with RCC-associated SYNE1 gene mutations exhibited significantly worse overall and progression-free survivals. Patients with Nesprin1-negative ccRCC tumors exhibit significantly poorer overall, cancer-specific, and recurrence-free survival rates than those recorded in the Nesprin1-positive group. SYNE1 knockdown enhanced the invasion and migration of RCC cells, however, it did not influence the proliferation of cells. RNA sequencing and Gene Set Enrichment Analysis revealed that SYNE1 knockdown significantly altered the expression of genes associated with oxidative phosphorylation. Consistently, patients with RCC exhibiting low SYNE1 expression, who were treated with the vascular endothelial growth factor receptor inhibitor sunitinib, had worse progression-free survival.
CONCLUSIONS
The results indicate that the expression of SYNE1/Nesprin1 and SYNE1 mutations in patients with RCC are closely linked to their prognosis and responsiveness to sunitinib treatment.
PubMed: 38442705
DOI: 10.1159/000536539 -
The Journal of Physiology Mar 2024Spectrins function together with actin as obligatory subunits of the submembranous cytoskeleton. Spectrins maintain cell shape, resist mechanical forces, and stabilize...
Spectrins function together with actin as obligatory subunits of the submembranous cytoskeleton. Spectrins maintain cell shape, resist mechanical forces, and stabilize ion channel and transporter protein complexes through binding to scaffolding proteins. Recently, pathogenic variants of SPTBN4 (β4 spectrin) were reported to cause both neuropathy and myopathy. Although the role of β4 spectrin in neurons is mostly understood, its function in skeletal muscle, another excitable tissue subject to large forces, is unknown. Here, using a muscle specific β4 spectrin conditional knockout mouse, we show that β4 spectrin does not contribute to muscle function. In addition, we show β4 spectrin is not present in muscle, indicating the previously reported myopathy associated with pathogenic SPTBN4 variants is neurogenic in origin. More broadly, we show that α2, β1 and β2 spectrins are found in skeletal muscle, with α2 and β1 spectrins being enriched at the postsynaptic neuromuscular junction (NMJ). Surprisingly, using muscle specific conditional knockout mice, we show that loss of α2 and β2 spectrins had no effect on muscle health, function or the enrichment of β1 spectrin at the NMJ. Muscle specific deletion of β1 spectrin also had no effect on muscle health, but, with increasing age, resulted in the loss of clustered NMJ Na channels. Together, our results suggest that muscle β1 spectrin functions independently of an associated α spectrin to maintain Na channel clustering at the postsynaptic NMJ. Furthermore, despite repeated exposure to strong forces and in contrast to neurons, muscles do not require spectrin cytoskeletons to maintain cell shape or integrity. KEY POINTS: The myopathy found in pathogenic human SPTBN4 variants (where SPTBN4 is the gene encoding β4 spectrin) is neurogenic in origin. β1 spectrin plays essential roles in maintaining the density of neuromuscular junction Nav1.4 Na channels. By contrast to the canonical view of spectrin organization and function, we show that β1 spectrin can function independently of an associated α spectrin. Despite the large mechanical forces experienced by muscle, we show that spectrins are not required for muscle cell integrity. This is in stark contrast to red blood cells and the axons of neurons.
Topics: Mice; Animals; Humans; Spectrin; Actin Cytoskeleton; Neuromuscular Junction; Muscle, Skeletal; Muscular Diseases
PubMed: 38441922
DOI: 10.1113/JP285894 -
Frontiers in Cellular Neuroscience 2024Neuronal connectivity is regulated during normal brain development with the arrangement of spines and synapses being dependent on the morphology of dendrites. Further,...
Neuronal connectivity is regulated during normal brain development with the arrangement of spines and synapses being dependent on the morphology of dendrites. Further, in multiple neurodevelopmental and aging disorders, disruptions of dendrite formation or shaping is associated with atypical neuronal connectivity. We showed previously that Pdlim5 binds delta-catenin and promotes dendrite branching. We report here that Pdlim5 interacts with PalmD, a protein previously suggested by others to interact with the cytoskeleton (e.g., adducin/spectrin) and to regulate membrane shaping. Functionally, the knockdown of PalmD or Pdlim5 in rat primary hippocampal neurons dramatically reduces branching and conversely, PalmD exogenous expression promotes dendrite branching as does Pdlim5. Further, we show that each proteins' effects are dependent on the presence of the other. In summary, using primary rat hippocampal neurons we reveal the contributions of a novel Pdlim5:PalmD protein complex, composed of functionally inter-dependent components responsible for shaping neuronal dendrites.
PubMed: 38414752
DOI: 10.3389/fncel.2024.1315941 -
Biomolecules Feb 2024The protein 4.1R is an essential component of the erythrocyte membrane skeleton, serving as a key structural element and contributing to the regulation of the membrane's... (Review)
Review
The protein 4.1R is an essential component of the erythrocyte membrane skeleton, serving as a key structural element and contributing to the regulation of the membrane's physical properties, including mechanical stability and deformability, through its interaction with spectrin-actin. Recent research has uncovered additional roles of 4.1R beyond its function as a linker between the plasma membrane and the membrane skeleton. It has been found to play a crucial role in various biological processes, such as cell fate determination, cell cycle regulation, cell proliferation, and cell motility. Additionally, 4.1R has been implicated in cancer, with numerous studies demonstrating its potential as a diagnostic and prognostic biomarker for tumors. In this review, we provide an updated overview of the gene and protein structure of 4.1R, as well as its cellular functions in both physiological and pathological contexts.
Topics: Membrane Proteins; Cytoskeletal Proteins; Spectrin; Actins; Erythrocyte Membrane
PubMed: 38397451
DOI: 10.3390/biom14020214 -
Applied Magnetic Resonance Mar 2024Here we review applications of site-directed spin labeling (SDSL) with engineered cysteines in proteins, to study the structural dynamics of muscle and non-muscle...
Here we review applications of site-directed spin labeling (SDSL) with engineered cysteines in proteins, to study the structural dynamics of muscle and non-muscle proteins, using and developing the electron paramagnetic resonance (EPR) spectroscopic techniques of dipolar EPR, double electron electron resonance (DEER), saturation transfer EPR (STEPR), and orientation measured by EPR. The SDSL technology pioneered by Wayne Hubbell and collaborators has greatly expanded the use of EPR, including the measurement of distances between spin labels covalently attached to proteins and peptides. The Thomas lab and collaborators have applied these techniques to elucidate dynamic interactions in the myosin-actin complex, myosin-binding protein C, calmodulin, ryanodine receptor, phospholamban, utrophin, dystrophin, β-III-spectrin, and Aurora kinase. The ability to design and engineer cysteines in proteins for site-directed covalent labeling has enabled the use of these powerful EPR techniques to measure distances, while showing that they are complementary with optical spectroscopy measurements.
PubMed: 38371230
DOI: 10.1007/s00723-023-01623-x -
BioRxiv : the Preprint Server For... Feb 2024During development, planes of cells give rise to complex tissues and organs. The proper functioning of these tissues is critically dependent on proper inter- and...
BACKGROUND
During development, planes of cells give rise to complex tissues and organs. The proper functioning of these tissues is critically dependent on proper inter- and intra-cellular spatial orientation, a feature known as planar cell polarity (PCP). To study the genetic and environmental factors affecting planar cell polarity investigators must often manually measure cell orientations, which is a time-consuming endeavor.
METHODOLOGY
To automate cell counting and planar cell polarity data collection we developed a Fiji/ImageJ plug-in called PCP Auto Count (PCPA). PCPA analyzes binary images and identifies "chunks" of white pixels that contain "caves" of infiltrated black pixels. Inner ear sensory epithelia including cochleae (P4) and utricles (E17.5) from mice were immunostained for βII-spectrin and imaged on a confocal microscope. Images were preprocessed using existing Fiji functionality to enhance contrast, make binary, and reduce noise. An investigator rated PCPA cochlear angle measurements for accuracy using a 1-5 agreement scale. For utricle samples, we directly compared PCPA derived measurements against manually derived angle measurements using concordance correlation coefficients (CCC) and Bland-Altman limits of agreement. Finally, PCPA was tested against a variety of images copied from publications examining PCP in various tissues and across various species.
RESULTS
PCPA was able to recognize and count 99.81% of cochlear hair cells (n = 1,1541 hair cells) in a sample set, and was able to obtain ideally accurate planar cell polarity measurements for over 96% of hair cells. When allowing for a <10° deviation from "perfect" measurements, PCPA's accuracy increased to >98%. When manual angle measurements for E17.5 utricles were compared, PCPA's measurements fell within -9 to +10 degrees of manually obtained mean angle measures with a CCC of 0.999. Qualitative examination of example images of Drosophila ommatidia, mouse ependymal cells, and mouse radial progenitors revealed a high level of accuracy for PCPA across a variety of stains, tissue types, and species. Altogether, the data suggest that the PCPA plug-in suite is a robust and accurate tool for the automated collection of cell counts and PCP angle measurements.
PubMed: 38352473
DOI: 10.1101/2024.01.30.578047