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1.
Cell Rep ; 40(3): 111103, 2022 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-35858567

RESUMO

Red blood cells (RBCs) (erythrocytes) are the simplest primary human cells, lacking nuclei and major organelles and instead employing about a thousand proteins to dynamically control cellular function and morphology in response to physiological cues. In this study, we define a canonical RBC proteome and interactome using quantitative mass spectrometry and machine learning. Our data reveal an RBC interactome dominated by protein homeostasis, redox biology, cytoskeletal dynamics, and carbon metabolism. We validate protein complexes through electron microscopy and chemical crosslinking and, with these data, build 3D structural models of the ankyrin/Band 3/Band 4.2 complex that bridges the spectrin cytoskeleton to the RBC membrane. The model suggests spring-like compression of ankyrin may contribute to the characteristic RBC cell shape and flexibility. Taken together, our study provides an in-depth view of the global protein organization of human RBCs and serves as a comprehensive resource for future research.


Assuntos
Anquirinas , Eritrócitos , Anquirinas/metabolismo , Citoesqueleto/metabolismo , Eritrócitos/metabolismo , Humanos , Proteoma/metabolismo , Espectrina/metabolismo
2.
Hear Res ; 423: 108564, 2022 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-35864018

RESUMO

Reports have proposed a putative role for ßV spectrin in outer hair cells (OHCs) of the cochlea. In an ongoing investigation of the role of the cytoskeleton in electromotility, we tested mice with a targeted exon deletion of ßV spectrin (Spnb5), and unexpectedly find that Spnb5(-/-) animals' auditory thresholds are unaffected. Similarly, these mice have normal OHC electromechanical activity (otoacoustic emissions) and non-linear capacitance. In contrast, magnitudes of auditory brainstem response (ABR) wave 1-amplitudes are significantly reduced. Evidence of a synaptopathy was absent with normal hair cell CtBP2 counts. In Spnb5(-/-) mice, the number of afferent and efferent nerve fibers is decreased. Consistent with this data, Spnb5 mRNA is present in Type I and II spiral ganglion neurons, but undetectable in OHCs. Together, these data establish that ßV spectrin is important for hearing, affecting neuronal structure and function. Significantly, these data support that ßV spectrin as is not functionally important to OHCs as has been previously suggested.


Assuntos
Células Ciliadas Auditivas Externas , Espectrina , Animais , Cóclea/fisiologia , Potenciais Evocados Auditivos do Tronco Encefálico , Células Ciliadas Auditivas Externas/fisiologia , Camundongos , Camundongos Knockout , Emissões Otoacústicas Espontâneas , Espectrina/genética , Espectrina/metabolismo
3.
Cell Rep ; 39(12): 110980, 2022 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-35732124

RESUMO

Tumor-suppressive cell competition is an evolutionarily conserved process that selectively removes precancerous cells to maintain tissue homeostasis. Using the polarity-deficiency-induced cell competition model in Drosophila, we identify Toll-6, a Toll-like receptor family member, as a driver of tension-mediated cell competition through α-Spectrin (α-Spec)-Yorkie (Yki) cascade. Toll-6 aggregates along the boundary between wild-type and polarity-deficient clones, where Toll-6 physically interacts with the cytoskeleton network protein α-Spec to increase mechanical tension, resulting in actomyosin-dependent Hippo pathway activation and the elimination of scrib mutant cells. Furthermore, we show that Spz5 secreted from fat body, the key innate organ in fly, facilitates the elimination of scrib clones by binding to Toll-6. These findings uncover mechanisms by which fat bodies remotely regulate tumor-suppressive cell competition of polarity-deficient tumors through inter-organ crosstalk and identified the Toll-6-α-Spec axis as an essential guardian that prevents tumorigenesis via tension-mediated cell elimination.


Assuntos
Proteínas de Drosophila , Animais , Competição entre as Células , Drosophila/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Corpo Adiposo/metabolismo , Transdução de Sinais/fisiologia , Espectrina/metabolismo
4.
Elife ; 112022 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-35762204

RESUMO

Microtubules are dynamic polymers consisting of αß-tubulin heterodimers. The initial polymerization process, called microtubule nucleation, occurs spontaneously via αß-tubulin. Since a large energy barrier prevents microtubule nucleation in cells, the γ-tubulin ring complex is recruited to the centrosome to overcome the nucleation barrier. However, a considerable number of microtubules can polymerize independently of the centrosome in various cell types. Here, we present evidence that the minus-end-binding calmodulin-regulated spectrin-associated protein 2 (CAMSAP2) serves as a strong nucleator for microtubule formation by significantly reducing the nucleation barrier. CAMSAP2 co-condensates with αß-tubulin via a phase separation process, producing plenty of nucleation intermediates. Microtubules then radiate from the co-condensates, resulting in aster-like structure formation. CAMSAP2 localizes at the co-condensates and decorates the radiating microtubule lattices to some extent. Taken together, these in vitro findings suggest that CAMSAP2 supports microtubule nucleation and growth by organizing a nucleation centre as well as by stabilizing microtubule intermediates and growing microtubules.


Cells are able to hold their shape thanks to tube-like structures called microtubules that are made of hundreds of tubulin proteins. Microtubules are responsible for maintaining the uneven distribution of molecules throughout the cell, a phenomenon known as polarity that allows cells to differentiate into different types with various roles. A protein complex called the γ-tubulin ring complex (γ-TuRC) is necessary for microtubules to form. This protein helps bind the tubulin proteins together and stabilises microtubules. However, recent research has found that in highly polarized cells such as neurons, which have highly specialised regions, microtubules can form without γ-TuRC. Searching for the proteins that could be filling in for γ-TuRC in these cells some evidence has suggested that a group known as CAMSAPs may be involved, but it is not known how. To characterize the role of CAMSAPs, Imasaki, Kikkawa et al. studied how one of these proteins, CAMSAP2, interacts with tubulins. To do this, they reconstituted both CAMSAP2 and tubulins using recombinant biotechnology and mixed them in solution. These experiments showed that CAMSAP2 can help form microtubules by bringing together their constituent proteins so that they can bind to each other more easily. Once microtubules start to form, CAMSAP2 continues to bind to them, stabilizing them and enabling them to grow to full size. These results shed light on how polarity is established in cells such as neurons, muscle cells, and epithelial cells. Additionally, the ability to observe intermediate structures during microtubule formation can provide insights into the processes that these structures are involved in.


Assuntos
Espectrina , Tubulina (Proteína) , Proteínas Associadas aos Microtúbulos/metabolismo , Centro Organizador dos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Espectrina/metabolismo , Tubulina (Proteína)/metabolismo
5.
Nat Commun ; 13(1): 3196, 2022 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-35680881

RESUMO

Actin, spectrin, and associated molecules form a membrane-associated periodic skeleton (MPS) in neurons. The molecular composition and functions of the MPS remain incompletely understood. Here, using co-immunoprecipitation and mass spectrometry, we identified hundreds of potential candidate MPS-interacting proteins that span diverse functional categories. We examined representative proteins in several of these categories using super-resolution imaging, including previously unknown MPS structural components, as well as motor proteins, cell adhesion molecules, ion channels, and signaling proteins, and observed periodic distributions characteristic of the MPS along the neurites for ~20 proteins. Genetic perturbations of the MPS and its interacting proteins further suggested functional roles of the MPS in axon-axon and axon-dendrite interactions and in axon diameter regulation, and implicated the involvement of MPS interactions with cell adhesion molecules and non-muscle myosin in these roles. These results provide insights into the interactome of the MPS and suggest previously unknown functions of the MPS in neurons.


Assuntos
Proteômica , Espectrina , Actinas/metabolismo , Axônios/metabolismo , Moléculas de Adesão Celular/metabolismo , Membrana Celular/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Neurônios/metabolismo , Espectrina/metabolismo
6.
Biochem Biophys Res Commun ; 613: 127-132, 2022 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-35550199

RESUMO

Activation of calpain1 (CPN1) contributes to mitochondrial dysfunction during cardiac ischemia (ISC) - reperfusion (REP). Blockade of electron transport using amobarbital (AMO) protects mitochondria during ISC-REP, indicating that the electron transport chain (ETC) is a key source of mitochondrial injury. We asked if AMO treatment can decrease CPN1 activation as a potential mechanism of mitochondrial protection during ISC-REP. Buffer-perfused adult rat hearts underwent 25 min global ISC and 30 min REP. AMO (2.5 mM) or vehicle was administered for 1 min before ISC to block electron flow in the ETC. Hearts in the time control group were untreated and buffer perfused without ISC. Hearts were collected at the end of perfusion and used for mitochondrial isolation. ISC-REP increased both the cleavage of spectrin (indicating cytosolic CPN1 activation) in cytosol and the truncation of AIF (apoptosis inducing factor, indicating mitochondrial CPN1 activation) in subsarcolemmal mitochondria compared to time control. Thus, ISC-REP activated both cytosolic and mitochondrial CPN1. AMO treatment prevented the cleavage of spectrin and AIF during ISC-REP, suggesting that the transient blockade of electron transport during ISC decreases CPN1 activation. AMO treatment decreased the activation of PARP [poly(ADP-ribose) polymerase] downstream of AIF that triggers caspase-independent apoptosis. AMO treatment also decreased the release of cytochrome c from mitochondria during ISC-REP that prevented caspase 3 activation. These results support that the damaged ETC activates CPN1 in cytosol and mitochondria during ISC-REP, likely via calcium overload and oxidative stress. Thus, AMO treatment to mitigate mitochondrial-driven cardiac injury can decrease both caspase-dependent and caspase-independent programmed cell death during ISC-REP.


Assuntos
Mitocôndrias Cardíacas , Traumatismo por Reperfusão Miocárdica , Animais , Calpaína/metabolismo , Caspases/metabolismo , Transporte de Elétrons , Isquemia/metabolismo , Mitocôndrias Cardíacas/metabolismo , Traumatismo por Reperfusão Miocárdica/metabolismo , Ratos , Reperfusão , Espectrina/metabolismo
7.
Sci Rep ; 12(1): 5854, 2022 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-35393465

RESUMO

Beta-4 (ß4)-spectrin, encoded by the gene Sptbn4, is a cytoskeleton protein found at nodes and the axon initial segments (AIS). Sptbn4 mutations are associated with myopathy, neuropathy, and auditory deficits in humans. Related to auditory dysfunction, however, the expression and roles of ß4-spectrin at axon segments along the myelinated axon in the developing auditory brain are not well explored. We found during postnatal development, ß4-spectrin is critical for voltage-gated sodium channel (Nav) clustering at the heminode along the nerve terminal, but not for the formation of nodal and AIS structures in the auditory brainstem. Presynaptic terminal recordings in Sptbn4geo mice, ß4-spectrin null mice, showed an elevated threshold of action potential and increased failures during action potential train at high-frequency. Sptbn4geo mice exhibited a slower central conduction and showed no startle responses, but had normal cochlear function. Taken together, the lack of ß4-spectrin impairs Nav clustering at the heminode along the nerve terminal and the temporal fidelity and reliability of presynaptic spikes, leading to central auditory processing deficits during postnatal development.


Assuntos
Axônios , Espectrina , Animais , Axônios/metabolismo , Encéfalo/metabolismo , Análise por Conglomerados , Camundongos , Reprodutibilidade dos Testes , Espectrina/genética , Espectrina/metabolismo
8.
Nat Commun ; 13(1): 1326, 2022 03 14.
Artigo em Inglês | MEDLINE | ID: mdl-35288568

RESUMO

Defective angiogenesis underlies over 50 malignant, ischemic and inflammatory disorders yet long-term therapeutic applications inevitably fail, thus highlighting the need for greater understanding of the vast crosstalk and compensatory mechanisms. Based on proteomic profiling of angiogenic endothelial components, here we report ßIV-spectrin, a non-erythrocytic cytoskeletal protein, as a critical regulator of sprouting angiogenesis. Early loss of endothelial-specific ßIV-spectrin promotes embryonic lethality in mice due to hypervascularization and hemorrhagic defects whereas neonatal depletion yields higher vascular density and tip cell populations in developing retina. During sprouting, ßIV-spectrin expresses in stalk cells to inhibit their tip cell potential by enhancing VEGFR2 turnover in a manner independent of most cell-fate determining mechanisms. Rather, ßIV-spectrin recruits CaMKII to the plasma membrane to directly phosphorylate VEGFR2 at Ser984, a previously undefined phosphoregulatory site that strongly induces VEGFR2 internalization and degradation. These findings support a distinct spectrin-based mechanism of tip-stalk cell specification during vascular development.


Assuntos
Espectrina , Fator A de Crescimento do Endotélio Vascular , Animais , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Camundongos , Neovascularização Fisiológica , Proteômica , Transdução de Sinais , Espectrina/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo
9.
Sci Rep ; 12(1): 1726, 2022 02 02.
Artigo em Inglês | MEDLINE | ID: mdl-35110634

RESUMO

Recent structural studies of ß-III-spectrin and related cytoskeletal proteins revealed N-terminal sequences that directly bind actin. These sequences are variable in structure, and immediately precede a conserved actin-binding domain composed of tandem calponin homology domains (CH1 and CH2). Here we investigated in Drosophila the significance of the ß-spectrin N-terminus, and explored its functional interaction with a CH2-localized L253P mutation that underlies the neurodegenerative disease spinocerebellar ataxia type 5 (SCA5). We report that pan-neuronal expression of an N-terminally truncated ß-spectrin fails to rescue lethality resulting from a ß-spectrin loss-of-function allele, indicating that the N-terminus is essential to ß-spectrin function in vivo. Significantly, N-terminal truncation rescues neurotoxicity and defects in dendritic arborization caused by L253P. In vitro studies show that N-terminal truncation eliminates L253P-induced high-affinity actin binding, providing a mechanistic basis for rescue. These data suggest that N-terminal sequences may be useful therapeutic targets for small molecule modulation of the aberrant actin binding associated with SCA5 ß-spectrin and spectrin-related disease proteins.


Assuntos
Actinas/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Neurônios/metabolismo , Espectrina/metabolismo , Ataxias Espinocerebelares/metabolismo , Animais , Animais Geneticamente Modificados , Sítios de Ligação , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Feminino , Masculino , Mutação , Plasticidade Neuronal , Neurônios/patologia , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Espectrina/genética , Ataxias Espinocerebelares/genética , Ataxias Espinocerebelares/patologia
10.
Bioengineered ; 13(3): 5756-5768, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-35188072

RESUMO

Circular RNAs (circRNAs) are closely linked with human cancer development such as non-small-cell lung cancer (NSCLC). However, the characteristics and specific functions of most circRNAs in NSCLC remained unknown. Previous studies have suggested that circRNA SOD2 (CircSOD2) expression was upregulated in a number of cancers. This study aimed to explore the functions of circSOD2 in NSCLC advancement with epithelial-mesenchymal transition (EMT). Expression profile analysis of circSOD2, miR-2355-5p, and calmodulin-regulated spectrin-associated protein 2 (CAMSAP2) was detected by real-time quantitative PCR (RT-qPCR). Transwell assay, cell migration assay, CCK8, ELISA, RIP assay, RNA pull-down assay, and Western blot analysis were performed to evaluate the functions of circSOD2, miR-2355-5p, and CAMSAP2. We found elevated expression of circSOD2 and CAMSAP2 while reduced expression of miR-2355-5p in NSCLC tumor tissues. Silencing or overexpression of CircSOD2 resulted in increased or decreased expression of miR-2355-5p, respectively. Mechanically, we showed that silencing of CircSOD2 and overexpression of miR-2355-5p resulted in the reduced rate of NSCLC cell proliferation. Inhibition of miR-2355-5p reversed the changes induced via silencing of CircSOD2. MiR-2355-5p binds to the CircSOD2 promoter and triggered its stimulation, which further activated circSOD2 expression. CircSOD2 suppression impaired lung cancer cell growth, cell migration, prohibited cell cycle progression, and in vivo tumor growth by targeting miR-2355-5p expression in NSCLC tissues. Meanwhile, increased expression of CAMSAP2 reversed the changes stimulated by the elevated level of miR-2355-5p in NSCLC progression. This innovative signaling axis CircSOD2/miR-2355-5p/CAMSAP2 illustrated the new horizon to investigate NSCLC tumorigenesis and provided new prognosis and treatment of NSCLC.


Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , MicroRNAs , Calmodulina/genética , Calmodulina/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Movimento Celular/genética , Proliferação de Células/genética , Transição Epitelial-Mesenquimal/genética , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Pulmão/patologia , Neoplasias Pulmonares/patologia , MicroRNAs/genética , MicroRNAs/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , RNA Circular/genética , Espectrina/genética , Espectrina/metabolismo
11.
IUBMB Life ; 74(5): 474-487, 2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-35184374

RESUMO

Hemoglobin oxidation due to oxidative stress and disease conditions leads to the generation of ROS (reactive oxygen species) and membrane attachment of hemoglobin in-vivo, where its redox activity leads to peroxidative damage of membrane lipids and proteins. Spectrin, the major component of the red blood cell (RBC) membrane skeleton, is known to interact with hemoglobin and, here this interaction is shown to increase hemoglobin peroxidase activity in the presence of reducing substrate ABTS (2', 2'-Azino-Bis-3-Ethylbenzothiazoline-6-Sulfonic Acid). It is also shown that in the absence of reducing substrate, spectrin forms covalently cross-linked aggregates with hemoglobin which display no peroxidase activity. This may have implications in the clearance of ROS and limiting peroxidative damage. Spectrin is found to modulate the peroxidase activity of different hemoglobin variants like A, E, and S, and of isolated globin chains from each of these variants. This may be of importance in disease states like sickle cell disease and HbE-ß-thalassemia, where increased oxidative damage and free globin subunits are present due to the defects inherent in the hemoglobin variants associated with these diseases. This hypothesis is corroborated by lipid peroxidation experiments. The modulatory role of spectrin is shown to extend to other heme proteins, namely catalase and cytochrome-c. Experiments with free heme and Raman spectroscopy of heme proteins in the presence of spectrin show that structural alterations occur in the heme moiety of the heme proteins on spectrin binding, which may be the structural basis of increased enzyme activity.


Assuntos
Hemeproteínas , Antioxidantes , Catalase/genética , Heme , Hemoglobinas/genética , Hemoglobinas/metabolismo , Peroxidase/genética , Peroxidases/genética , Espécies Reativas de Oxigênio , Espectrina/química , Espectrina/genética , Espectrina/metabolismo
12.
FASEB J ; 36(3): e22220, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-35195928

RESUMO

Erythroid differentiation (ED) is a complex cellular process entailing morphologically distinct maturation stages of erythroblasts during terminal differentiation. Studies of actin filament (F-actin) assembly and organization during terminal ED have revealed essential roles for the F-actin pointed-end capping proteins, tropomodulins (Tmod1 and Tmod3). Tmods bind tropomyosins (Tpms), which enhance Tmod capping and F-actin stabilization. Tmods can also nucleate F-actin assembly, independent of Tpms. Tmod1 is present in the red blood cell (RBC) membrane skeleton, and deletion of Tmod1 in mice leads to a mild compensated anemia due to mis-regulated F-actin lengths and membrane instability. Tmod3 is not present in RBCs, and global deletion of Tmod3 leads to embryonic lethality in mice with impaired ED. To further decipher Tmod3's function during ED, we generated a Tmod3 knockout in a mouse erythroleukemia cell line (Mel ds19). Tmod3 knockout cells appeared normal prior to ED, but showed defects during progression of ED, characterized by a marked failure to reduce cell and nuclear size, reduced viability, and increased apoptosis. Tmod3 does not assemble with Tmod1 and Tpms into the Triton X-100 insoluble membrane skeleton during ED, and loss of Tmod3 had no effect on α1,ß1-spectrin and protein 4.1R assembly into the membrane skeleton. However, F-actin, Tmod1 and Tpms failed to assemble into the membrane skeleton during ED in absence of Tmod3. We propose that Tmod3 nucleation of F-actin assembly promotes incorporation of Tmod1 and Tpms into membrane skeleton F-actin, and that this is integral to morphological maturation and cell survival during erythroid terminal differentiation.


Assuntos
Citoesqueleto de Actina/metabolismo , Eritroblastos/citologia , Eritropoese , Leucemia Eritroblástica Aguda/metabolismo , Tropomodulina/metabolismo , Animais , Linhagem Celular Tumoral , Eritroblastos/metabolismo , Leucemia Eritroblástica Aguda/sangue , Camundongos , Multimerização Proteica , Espectrina/metabolismo , Tropomodulina/genética
13.
Transl Res ; 243: 78-88, 2022 05.
Artigo em Inglês | MEDLINE | ID: mdl-34979321

RESUMO

Spectrin, as one of the major components of a plasma membrane-associated cytoskeleton, is a cytoskeletal protein composed of the modular structure of α and ß subunits. The spectrin-based skeleton is essential for preserving the integrity and mechanical characteristics of the cell membrane. Moreover, spectrin regulates a variety of cell processes including cell apoptosis, cell adhesion, cell spreading, and cell cycle. Dysfunction of spectrins is implicated in various human diseases including hemolytic anemia, neurodegenerative diseases, ataxia, heart diseases, and cancers. Here, we briefly discuss spectrins function as well as the clinical manifestations and currently known molecular mechanisms of human diseases related to spectrins, highlighting that strategies for targeting regulation of spectrins function may provide new avenues for therapeutic intervention for these diseases.


Assuntos
Espectrina , Adesão Celular , Ciclo Celular , Membrana Celular/metabolismo , Humanos , Espectrina/química , Espectrina/metabolismo
14.
Mol Biol Cell ; 33(3): ar28, 2022 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-35020457

RESUMO

Red blood cell (RBC) shape and deformability are supported by a planar network of short actin filament (F-actin) nodes (∼37 nm length, 15-18 subunits) interconnected by long spectrin strands at the inner surface of the plasma membrane. Spectrin-F-actin network structure underlies quantitative modeling of forces controlling RBC shape, membrane curvature, and deformation, yet the nanoscale organization and dynamics of the F-actin nodes in situ are not well understood. We examined F-actin distribution and dynamics in RBCs using fluorescent-phalloidin labeling of F-actin imaged by multiple microscopy modalities. Total internal reflection fluorescence and Zeiss Airyscan confocal microscopy demonstrate that F-actin is concentrated in multiple brightly stained F-actin foci ∼200-300 nm apart interspersed with dimmer F-actin staining regions. Single molecule stochastic optical reconstruction microscopy imaging of Alexa 647-phalloidin-labeled F-actin and computational analysis also indicates an irregular, nonrandom distribution of F-actin nodes. Treatment of RBCs with latrunculin A and cytochalasin D indicates that F-actin foci distribution depends on actin polymerization, while live cell imaging reveals dynamic local motions of F-actin foci, with lateral movements, appearance and disappearance. Regulation of F-actin node distribution and dynamics via actin assembly/disassembly pathways and/or via local extension and retraction of spectrin strands may provide a new mechanism to control spectrin-F-actin network connectivity, RBC shape, and membrane deformability.


Assuntos
Citoesqueleto de Actina , Actinas , Membrana Eritrocítica , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Membrana Eritrocítica/metabolismo , Eritrócitos/metabolismo , Espectrina/metabolismo
15.
J Neurosci ; 42(1): 2-15, 2022 01 05.
Artigo em Inglês | MEDLINE | ID: mdl-34785580

RESUMO

Ankyrin scaffolding proteins are critical for membrane domain organization and protein stabilization in many different cell types including neurons. In the cerebellum, Ankyrin-R (AnkR) is highly enriched in Purkinje neurons, granule cells, and in the cerebellar nuclei (CN). Using male and female mice with a floxed allele for Ank1 in combination with Nestin-Cre and Pcp2-Cre mice, we found that ablation of AnkR from Purkinje neurons caused ataxia, regional and progressive neurodegeneration, and altered cerebellar output. We show that AnkR interacts with the cytoskeletal protein ß3 spectrin and the potassium channel Kv3.3. Loss of AnkR reduced somatic membrane levels of ß3 spectrin and Kv3.3 in Purkinje neurons. Thus, AnkR links Kv3.3 channels to the ß3 spectrin-based cytoskeleton. Our results may help explain why mutations in ß3 spectrin and Kv3.3 both cause spinocerebellar ataxia.SIGNIFICANCE STATEMENT Ankyrin scaffolding proteins localize and stabilize ion channels in the membrane by linking them to the spectrin-based cytoskeleton. Here, we show that Ankyrin-R (AnkR) links Kv3.3 K+ channels to the ß3 spectrin-based cytoskeleton in Purkinje neurons. Loss of AnkR causes Purkinje neuron degeneration, altered cerebellar physiology, and ataxia, which is consistent with mutations in Kv3.3 and ß3 spectrin causing spinocerebellar ataxia.


Assuntos
Anquirinas/metabolismo , Citoesqueleto/metabolismo , Células de Purkinje/metabolismo , Canais de Potássio Shaw/metabolismo , Espectrina/metabolismo , Animais , Sobrevivência Celular/fisiologia , Feminino , Masculino , Camundongos , Ataxias Espinocerebelares/genética
16.
Cardiovasc Res ; 118(4): 1046-1060, 2022 03 16.
Artigo em Inglês | MEDLINE | ID: mdl-33576403

RESUMO

AIMS: The transcription factor Tbx5 controls cardiogenesis and drives Scn5a expression in mice. We have identified two variants in TBX5 encoding p. D111Y and p. F206L Tbx5, respectively, in two unrelated patients with structurally normal hearts diagnosed with long QT (LQTS) and Brugada (BrS) syndrome. Here, we characterized the consequences of each variant to unravel the underlying disease mechanisms. METHODS AND RESULTS: We combined clinical analysis with in vivo and in vitro electrophysiological and molecular techniques in human-induced pluripotent stem-cell-derived cardiomyocytes (hiPSC-CMs), HL-1 cells, and cardiomyocytes from mice trans-expressing human wild-type (WT) or mutant proteins. Tbx5 increased transcription of SCN5A encoding cardiac Nav1.5 channels, while repressing CAMK2D and SPTBN4 genes encoding Ca/calmodulin kinase IIδ (CaMKIIδ) and ßIV-spectrin, respectively. These effects significantly increased Na current (INa) in hiPSC-CMs and in cardiomyocytes from mice trans-expressing Tbx5. Consequently, action potential (AP) amplitudes increased and QRS interval narrowed in the mouse electrocardiogram. p. F206L Tbx5 bound to the SCN5A promoter failed to transactivate it, thus precluding the pro-transcriptional effect of WT Tbx5. Therefore, p. F206L markedly decreased INa in hiPSC-CM, HL-1 cells and mouse cardiomyocytes. The INa decrease in p. F206L trans-expressing mice translated into QRS widening and increased flecainide sensitivity. p. D111Y Tbx5 increased SCN5A expression but failed to repress CAMK2D and SPTBN4. The increased CaMKIIδ and ßIV-spectrin significantly augmented the late component of INa (INaL) which, in turn, significantly prolonged AP duration in both hiPSC-CMs and mouse cardiomyocytes. Ranolazine, a selective INaL inhibitor, eliminated the QT and QTc intervals prolongation seen in p. D111Y trans-expressing mice. CONCLUSIONS: In addition to peak INa, Tbx5 critically regulates INaL and the duration of repolarization in human cardiomyocytes. Our original results suggest that TBX5 variants associate with and modulate the intensity of the electrical phenotype in LQTS and BrS patients.


Assuntos
Síndrome de Brugada , Células-Tronco Pluripotentes Induzidas , Síndrome do QT Longo , Potenciais de Ação/fisiologia , Animais , Síndrome de Brugada/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Síndrome do QT Longo/metabolismo , Camundongos , Miócitos Cardíacos/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.5/genética , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo , Técnicas de Patch-Clamp , Espectrina/metabolismo , Espectrina/farmacologia
17.
Sci Transl Med ; 13(624): eabk2267, 2021 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-34910547

RESUMO

The prevalence of nonalcoholic steatohepatitis (NASH) and liver cancer is increasing. De novo lipogenesis and fibrosis contribute to disease progression and cancerous transformation. Here, we found that ß2-spectrin (SPTBN1) promotes sterol regulatory element (SRE)­binding protein (SREBP)­stimulated lipogenesis and development of liver cancer in mice fed a high-fat diet (HFD) or a western diet (WD). Either hepatocyte-specific knockout of SPTBN1 or siRNA-mediated therapy protected mice from HFD/WD-induced obesity and fibrosis, lipid accumulation, and tissue damage in the liver. Biochemical analysis suggested that HFD/WD induces SPTBN1 and SREBP1 cleavage by CASPASE-3 and that the cleaved products interact to promote expression of genes with sterol response elements. Analysis of human NASH tissue revealed increased SPTBN1 and CASPASE-3 expression. Thus, our data indicate that SPTBN1 represents a potential target for therapeutic intervention in NASH and liver cancer.


Assuntos
Neoplasias , Hepatopatia Gordurosa não Alcoólica , Animais , Dieta Hiperlipídica/efeitos adversos , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Neoplasias/metabolismo , Hepatopatia Gordurosa não Alcoólica/genética , Espectrina/metabolismo
18.
Nat Commun ; 12(1): 6357, 2021 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-34737284

RESUMO

In the central nervous system (CNS), functional tasks are often allocated to distinct compartments. This is also evident in the Drosophila CNS where synapses and dendrites are clustered in distinct neuropil regions. The neuropil is separated from neuronal cell bodies by ensheathing glia, which as we show using dye injection experiments, contribute to the formation of an internal diffusion barrier. We find that ensheathing glia are polarized with a basolateral plasma membrane rich in phosphatidylinositol-(3,4,5)-triphosphate (PIP3) and the Na+/K+-ATPase Nervana2 (Nrv2) that abuts an extracellular matrix formed at neuropil-cortex interface. The apical plasma membrane is facing the neuropil and is rich in phosphatidylinositol-(4,5)-bisphosphate (PIP2) that is supported by a sub-membranous ßHeavy-Spectrin cytoskeleton. ßHeavy-spectrin mutant larvae affect ensheathing glial cell polarity with delocalized PIP2 and Nrv2 and exhibit an abnormal locomotion which is similarly shown by ensheathing glia ablated larvae. Thus, polarized glia compartmentalizes the brain and is essential for proper nervous system function.


Assuntos
Encéfalo/metabolismo , Proteínas de Drosophila/metabolismo , Neuroglia/metabolismo , Neurônios/metabolismo , Neurópilo/metabolismo , Espectrina/metabolismo , Animais , Linhagem da Célula , Drosophila , Glicoproteínas/metabolismo , Larva , Proteínas do Tecido Nervoso/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo
19.
Biochem Biophys Res Commun ; 581: 68-73, 2021 12 03.
Artigo em Inglês | MEDLINE | ID: mdl-34656850

RESUMO

A spontaneous missense mutation in the alpha II spectrin (αII) gene, replacing a highly conserved arginine 1098 with the glutamine (R1098Q), causes progressive neurodegeneration in heterozygous mutant mice. The molecular mechanism underlying this phenotype is unknown but the accumulation of 150kD αII breakdown products in brains of homozygous mutant embryos suggests an imbalance in the substrate level control of αII cleavage by calpains. This is further supported by in silico simulation predicting unmasked calpain target site and increased spectrin scaffold bending and flexibility of R1098Q mutant peptide. Here, using spectroscopic and in situ enzymatic techniques, we aimed at obtaining direct experimental support for the impact of R1098Q mutation on the αII stability and its propensity for calpain-mediated degradation. Thermal circular dichroism analyses performed on recombinant wildtype and R1098Q mutant αII peptides, composed of spectrin repeat 9-10 revealed that although both had very similar secondary structure contents, thermal stability curve profiles varied and the observed midpoint of the unfolding transition (Tm) was 5.5 °C lower for the R1098Q peptide. Yet, the dynamic light scattering profiles of both peptides closely overlapped, implying the same thermal propensity to aggregate. Calpain digestion of plate-bound αII peptides with and without added calmodulin revealed an enhancement of the R1098Q peptide digestion rate relative to WT control. In summary, these results support the unstable scaffold structure of the R1098Q peptide as contributing to its enhanced intrinsic sensitivity to calpain and suggest physiologic relevance of a proper calpain/spectrin balance in preventing neurodegeneration.


Assuntos
Arginina/química , Calpaína/química , Glutamina/química , Mutação de Sentido Incorreto , Peptídeos/química , Espectrina/química , Substituição de Aminoácidos , Arginina/metabolismo , Calpaína/metabolismo , Ensaio de Imunoadsorção Enzimática , Expressão Gênica , Glutamina/metabolismo , Humanos , Peptídeos/genética , Peptídeos/metabolismo , Estabilidade Proteica , Proteólise , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Soluções , Espectrina/genética , Espectrina/metabolismo
20.
Molecules ; 26(20)2021 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-34684832

RESUMO

Androgens have been shown to have a beneficial effect on brain injury and lower reactive astrocyte expression after TBI. Androgen receptors (ARs) are known to mediate the neuroprotective effects of androgens. However, whether ARs play a crucial role in TBI remains unknown. In this study, we investigated the role of ARs in TBI pathophysiology, using AR knockout (ARKO) mice. We used the controlled cortical impact model to produce primary and mechanical brain injuries and assessed motor function and brain-lesion volume. In addition, the AR knockout effects on necrosis and autophagy were evaluated after TBI. AR knockout significantly increased TBI-induced expression of the necrosis marker alpha-II-spectrin breakdown product 150 and astrogliosis marker glial fibrillary acidic protein. In addition, the TBI-induced astrogliosis increase in ARKO mice lasted for three weeks after a TBI. The autophagy marker Beclin-1 was also enhanced in ARKO mice compared with wild-type mice after TBI. Our results also indicated that ARKO mice showed a more unsatisfactory performance than wild-type mice in a motor function test following TBI. Further, they were observed to have more severe lesions than wild-type mice after injury. These findings strongly suggest that ARs play a role in TBI.


Assuntos
Lesões Encefálicas Traumáticas/fisiopatologia , Receptores Androgênicos/deficiência , Animais , Autofagia , Proteína Beclina-1/metabolismo , Encéfalo/fisiologia , Encéfalo/fisiopatologia , Lesões Encefálicas Traumáticas/etiologia , Lesões Encefálicas Traumáticas/patologia , Modelos Animais de Doenças , Feminino , Proteína Glial Fibrilar Ácida/metabolismo , Humanos , Masculino , Camundongos , Camundongos Knockout , Transtornos Motores/patologia , Transtornos Motores/fisiopatologia , Receptores Androgênicos/genética , Receptores Androgênicos/fisiologia , Espectrina/metabolismo
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