RESUMO
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 (ß4 spectrin) were reported to cause both neuropathy and myopathy. Although the role of ß4 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 ß4 spectrin conditional knockout mouse, we show that ß4 spectrin does not contribute to muscle function. In addition, we show ß4 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, ß1 and ß2 spectrins are found in skeletal muscle, with α2 and ß1 spectrins being enriched at the postsynaptic neuromuscular junction (NMJ). Surprisingly, using muscle specific conditional knockout mice, we show that loss of α2 and ß2 spectrins had no effect on muscle health, function or the enrichment of ß1 spectrin at the NMJ. Muscle specific deletion of ß1 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 ß1 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 ß4 spectrin) is neurogenic in origin. ß1 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 ß1 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.
Assuntos
Junção Neuromuscular , Canais de Sódio , Espectrina , Animais , Humanos , Camundongos , Citoesqueleto de Actina/metabolismo , Músculo Esquelético/metabolismo , Doenças Musculares , Junção Neuromuscular/metabolismo , Espectrina/genética , Espectrina/análise , Espectrina/metabolismo , Canais de Sódio/metabolismoRESUMO
The spectrin-based membrane skeleton is a ubiquitous membrane-associated two-dimensional cytoskeleton underneath the lipid membrane of metazoan cells. Mutations of skeleton proteins impair the mechanical strength and functions of the membrane, leading to several different types of human diseases. Here, we report the cryo-EM structures of the native spectrin-actin junctional complex (from porcine erythrocytes), which is a specialized short F-actin acting as the central organizational unit of the membrane skeleton. While an α-/ß-adducin hetero-tetramer binds to the barbed end of F-actin as a flexible cap, tropomodulin and SH3BGRL2 together create an absolute cap at the pointed end. The junctional complex is strengthened by ring-like structures of dematin in the middle actin layers and by patterned periodic interactions with tropomyosin over its entire length. This work serves as a structural framework for understanding the assembly and dynamics of membrane skeleton and offers insights into mechanisms of various ubiquitous F-actin-binding factors in other F-actin systems.
Assuntos
Citoesqueleto , Eritrócitos , Animais , Humanos , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Citoesqueleto/metabolismo , Eritrócitos/citologia , Eritrócitos/metabolismo , Espectrina/análise , Espectrina/metabolismo , SuínosRESUMO
Despite being a major health concern, little is known about the pathophysiological changes that underly concussion. Nonetheless, emerging evidence suggests that selective damage to white matter axons, or diffuse axonal injury (DAI), disrupts brain network connectivity and function. While voltage-gated sodium channels (NaChs) and their anchoring proteins at the nodes of Ranvier (NOR) on axons are key elements of the brain's network signaling machinery, changes in their integrity have not been studied in context with DAI. Here, we utilized a clinically relevant swine model of concussion that induces evolving axonal pathology, demonstrated by accumulation of amyloid precursor protein (APP) across the white matter. Over a two-week follow-up post-concussion with this model, we found widespread loss of NaCh isoform 1.6 (Nav1.6), progressive increases in NOR length, the appearance of void and heminodes and loss of ßIV-spectrin, ankyrin G, and neurofascin 186 or their collective diffusion into the paranode. Notably, these changes were in close proximity, yet distinct from APP-immunoreactive swollen axonal profiles, potentially representing a unique, newfound phenotype of axonal pathology in DAI. Since concussion in humans is non-fatal, the clinical relevance of these findings was determined through examination of post-mortem brain tissue from humans with higher levels of acute traumatic brain injury. Here, a similar loss of Nav1.6 and changes in NOR structures in brain white matter were observed as found in the swine model of concussion. Collectively, this widespread and progressive disruption of NaChs and NOR appears to be a form of sodium channelopathy, which may represent an important substrate underlying brain network dysfunction after concussion.
Assuntos
Concussão Encefálica , Lesões Encefálicas , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Anquirinas/análise , Anquirinas/metabolismo , Axônios/patologia , Concussão Encefálica/patologia , Lesões Encefálicas/patologia , Humanos , Isoformas de Proteínas/metabolismo , Nós Neurofibrosos/química , Nós Neurofibrosos/metabolismo , Nós Neurofibrosos/patologia , Sódio/metabolismo , Canais de Sódio/análise , Canais de Sódio/metabolismo , Espectrina/análise , Espectrina/metabolismo , SuínosRESUMO
Recent work has established that axons have a periodic skeleton structure comprising of azimuthal actin rings connected via longitudinal spectrin tetramer filaments. This structure endows the axon with structural integrity and mechanical stability. Additionally, voltage-gated sodium channels follow the periodicity of the active-spectrin arrangement, spaced â¼190 nm segments apart. The impact of this periodic arrangement of sodium channels on the generation and propagation of action potentials is unknown. To address this question, we simulated an action potential using the Hodgkin-Huxley formalism in a cylindrical compartment, but instead of using a homogeneous distribution of voltage-gated sodium channels in the membrane, we applied the experimentally determined periodic arrangement. We found that the periodic distribution of voltage-gated sodium channels does not significantly affect the generation or propagation of action potentials but instead leads to large, localized sodium action currents caused by high-density sodium nanodomains. Additionally, our simulations show that the distance between periodic sodium channel strips could control axonal excitability, suggesting a previously underappreciated mechanism to regulate neuronal firing properties. Together, this work provides a critical new insight into the role of the periodic arrangement of sodium channels in axons, providing a foundation for future experimental studies.
Assuntos
Espectrina , Canais de Sódio Disparados por Voltagem , Actinas/química , Potenciais de Ação/fisiologia , Axônios/fisiologia , Sódio , Espectrina/análise , Espectrina/químicaRESUMO
Protein torsion angles define the backbone secondary structure of proteins. Magic-angle spinning (MAS) NMR methods using carbon detection have been developed to measure torsion angles by determining the relative orientation between two anisotropic interactionsâdipolar coupling or chemical shift anisotropy. Here we report a new proton-detection based method to determine the backbone torsion angle by recoupling NH and CH dipolar couplings within the HCANH pulse sequence, for protonated or partly deuterated samples. We demonstrate the efficiency and precision of the method with microcrystalline chicken α spectrin SH3 protein and the influenza A matrix 2 (M2) membrane protein, using 55 or 90 kHz MAS. For M2, pseudo-4D data detect a turn between transmembrane and amphipathic helices.
Assuntos
Ressonância Magnética Nuclear Biomolecular , Prótons , Espectrina/análise , Proteínas da Matriz Viral/análise , Proteínas Viroporinas/análise , Animais , Galinhas , Modelos Moleculares , Domínios de Homologia de srcRESUMO
Dynamic assembly of actin filaments is essential for many cellular processes. The rates of assembly and disassembly of actin filaments are intricately controlled by regulatory proteins that interact with the ends and the sides of filaments and with actin monomers. TIRF-based single-filament imaging techniques have proven instrumental in uncovering mechanisms of actin regulation. In this unit, novel single-filament approaches using microfluidics-assisted TIRF imaging are described. These methods can be used to grow anchored actin filaments aligned in a flow, thus making the analysis much easier as compared to open flow cell approaches. The microfluidic nature of the system also enables rapid change of biochemical conditions and allows simultaneous imaging of a large number of actin filaments. Support protocols for preparing microfluidic chambers and purifying spectrin-actin seeds used for nucleating anchored filaments are also described. © 2017 by John Wiley & Sons, Inc.
Assuntos
Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/ultraestrutura , Técnicas Analíticas Microfluídicas/instrumentação , Microscopia de Fluorescência/instrumentação , Imagem Óptica/instrumentação , Actinas/análise , Actinas/metabolismo , Actinas/ultraestrutura , Animais , Desenho de Equipamento , Humanos , Cinética , Técnicas Analíticas Microfluídicas/métodos , Microscopia de Fluorescência/métodos , Imagem Óptica/métodos , Espectrina/análise , Espectrina/metabolismo , Espectrina/ultraestruturaRESUMO
BACKGROUND: Old human red blood cells (RBCs) have a reduced surface area with respect to young RBCs. If this decrease occurred through the release of vesicles similar to the spectrin-free vesicles that are shed in vitro under different experimental conditions or during storage, there would be no decrease of membrane-skeleton, but only of lipid bilayer surface area, during RBC ageing in vivo. However, we observed a decrease in spectrin and other membrane-skeletal proteins in old RBCs. Because RBCs contain components of the ubiquitin-proteasome system and other hydrolytic systems for protein degradation, we asked whether increased membrane-skeleton fragments could be detected in older RBCs. METHODS: Four different anti-spectrin antibodies and an antibody anti-ubiquitin conjugates were used to analyse, by Western blotting, fragments of spectrin and other proteins in RBCs of different age separated in density gradients and characterized for their protein 4.1a/4.1b ratio as a cell age parameter. RESULTS: spectrin fragments do exist in RBCs of all ages, they represent a minute fraction of all spectrin, are membrane-bound and not cytoplasmic and do not increase with cell age. Besides spectrin, other membrane-skeletal components decrease with cell age. CONCLUSION: Observed results challenge the commonly accepted view that decrease in cell membrane throughout RBC life in vivo occurs via the release of spectrin-free vesicles.
Assuntos
Senescência Celular , Eritrócitos/citologia , Espectrina/análise , Citoesqueleto/metabolismo , Membrana Eritrocítica/metabolismo , Eritrócitos/metabolismo , Exossomos/metabolismo , Humanos , Proteínas de Membrana/metabolismo , Multimerização Proteica , Espectrina/metabolismo , UbiquitinaçãoRESUMO
BACKGROUND/AIMS: A high surface-to-volume ratio and a spectrin membrane-skeleton (MS) confer to the mammalian red blood cells (RBCs) their characteristic deformability, mechanical strength and structural stability. During their 120 days of circulatory life in humans, RBCs decrease in size, while remaining biconcave disks, owing to a coordinated decrease in membrane surface area and cell water. It is generally believed that part of the membrane is lost with the shedding of spectrin-free vesicles of the same type that can be obtained in vitro by different treatments. If this were true, an excess of MS would arise in old RBCs, with respect to the lipid bilayer. Aim of this paper was to investigate this aspect. METHODS: Quantification of spectrin by electrophoretic methods was carried out in RBCs of different age. RESULTS: Spectrin decreases, on a per cell basis, with RBC ageing. On the other hand, the membrane raft protein marker flotillin-2, while decreasing in the membrane of old cells, was found to be strongly depleted in the membrane of in vitro-induced vesicles. CONCLUSION: Part of the membrane-skeleton is probably lost together with part of the lipid bilayer in a balanced way. These findings point to a mechanism for the in vivo release of membrane that is different from that which is known to occur in vitro.
Assuntos
Senescência Celular , Membrana Eritrocítica/metabolismo , Eritrócitos/citologia , Micropartículas Derivadas de Células/metabolismo , Eritrócitos/metabolismo , Humanos , Proteínas de Membrana/análise , Proteínas de Membrana/metabolismo , Espectrina/análise , Espectrina/metabolismoRESUMO
The activities of calpain and caspase systems during ageing in Longissimus lumborum (LL) and Infraspinatus (IS) muscles of Italian Simmental young bulls (Bos taurus) were assessed. Samples from 10 animals were collected within 20 min of exsanguination (T0), after 48 h (T1) and 7 days (T2) post mortem. Calpain and caspase activity were evaluated based on the formation of αII spectrin cleavage products of 145 kDa (SBDP145) and 120 kDa (SBDP120), respectively. Caspase activity was also assessed by the presence of poly (adenosine diphosphate-ribose) polymerase-1 (PARP-1) cleavage product. At T0, LL showed higher levels of SBDP145 than IS (P < 0.01), while SBDP120 and PARP-1 degradation products were similar between muscles. At T1, no difference was found in the level of SBDP145 between muscles, while SBDP120 and PARP-1 cleavage products were not detected. At T2 neither αII spectrin nor PARP-1 cleavage products were found. LL and IS showed different proteolysis after slaughter that was influenced more by calpain than caspase activity, which was detectable only in the early post mortem period.
Assuntos
Calpaína/metabolismo , Caspases/metabolismo , Carne/análise , Músculo Esquelético/metabolismo , Poli(ADP-Ribose) Polimerase-1/metabolismo , Espectrina/metabolismo , Animais , Calpaína/análise , Caspases/análise , Bovinos , Masculino , Poli(ADP-Ribose) Polimerase-1/análise , Mudanças Depois da Morte , Proteólise , Espectrina/análise , Fatores de TempoRESUMO
Superresolution fluorescence microscopy recently revealed a ~190 nm periodic cytoskeleton lattice consisting of actin, spectrin, and other proteins underneath the membrane of cultured hippocampal neurons. Whether the periodic cytoskeleton lattice is a structural feature of all neurons and how it is modified when axons are ensheathed by myelin forming glial cells is not known. Here, STED nanoscopy is used to demonstrate that this structure is a commonplace of virtually all neuron types in vitro. To check how the subcortical meshwork is modified during myelination, we studied sciatic nerve fibers from adult mice. Periodicity of both actin and spectrin was uncovered at the internodes, indicating no substantial differences between unmyelinated and myelinated axons. Remarkably, the actin/spectrin pattern was also detected in glial cells such as cultured oligodendrocyte precursor cells. Altogether our work shows that the periodic subcortical cytoskeletal meshwork is a fundamental characteristic of cells in the nervous system and is not a distinctive feature of neurons, as previously thought.
Assuntos
Actinas/análise , Axônios/química , Sistema Nervoso/química , Espectrina/análise , Animais , Feminino , Masculino , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência , Neuroglia/química , Ratos WistarRESUMO
The kidney displays specialized regions devoted to filtration, selective reabsorption, and electrolyte and metabolite trafficking. The polarized membrane pumps, channels, and transporters responsible for these functions have been exhaustively studied. Less examined are the contributions of spectrin and its adapter ankyrin to this exquisite functional topography, despite their established contributions in other tissues to cellular organization. We have examined in the rodent kidney the expression and distribution of all spectrins and ankyrins by qPCR, Western blotting, immunofluorescent and immuno electron microscopy. Four of the seven spectrins (αΙΙ, ßΙ, ßΙΙ, and ßΙΙΙ) are expressed in the kidney, as are two of the three ankyrins (G and B). The levels and distribution of these proteins vary widely over the nephron. αΙΙ/ßΙΙ is the most abundant spectrin, found in glomerular endothelial cells; on the basolateral membrane and cytoplasmic vesicles in proximal tubule cells and in the thick ascending loop of Henle; and less so in the distal nephron. ßΙΙΙ spectrin largely replaces ßΙΙ spectrin in podocytes, Bowman's capsule, and throughout the distal tubule and collecting ducts. ßΙ spectrin is only marginally expressed; its low abundance hinders a reliable determination of its distribution. Ankyrin G is the most abundant ankyrin, found in capillary endothelial cells and all tubular segments. Ankyrin B populates Bowman's capsule, podocytes, the ascending thick loop of Henle, and the distal convoluted tubule. Comparison to the distribution of renal protein 4.1 isoforms and various membrane proteins indicates a complex relationship between the spectrin scaffold, its adapters, and various membrane proteins. While some proteins (e.g. ankyrin B, ßΙΙΙ spectrin, and aquaporin 2) tend to share a similar distribution, there is no simple mapping of different spectrins or ankyrins to most membrane proteins. The implications of this data are discussed.
Assuntos
Anquirinas/análise , Rim/química , Espectrina/análise , Animais , Anquirinas/genética , Western Blotting , Citoesqueleto/ultraestrutura , Éxons/genética , Rim/fisiologia , Rim/ultraestrutura , Proteínas de Membrana/análise , Camundongos , Camundongos Endogâmicos C57BL , Microscopia de Fluorescência , Microscopia Imunoeletrônica , Reação em Cadeia da Polimerase , Isoformas de Proteínas/análise , Isoformas de Proteínas/genética , RNA Mensageiro/análise , RNA Mensageiro/genética , Organismos Livres de Patógenos Específicos , Espectrina/genéticaRESUMO
BACKGROUND: The cardiac cytoskeleton plays key roles in maintaining myocyte structural integrity in health and disease. In fact, human mutations in cardiac cytoskeletal elements are tightly linked to cardiac pathologies, including myopathies, aortopathies, and dystrophies. Conversely, the link between cytoskeletal protein dysfunction and cardiac electric activity is not well understood and often overlooked in the cardiac arrhythmia field. METHODS AND RESULTS: Here, we uncover a new mechanism for the regulation of cardiac membrane excitability. We report that ßII spectrin, an actin-associated molecule, is essential for the posttranslational targeting and localization of critical membrane proteins in heart. ßII spectrin recruits ankyrin-B to the cardiac dyad, and a novel human mutation in the ankyrin-B gene disrupts the ankyrin-B/ßII spectrin interaction, leading to severe human arrhythmia phenotypes. Mice lacking cardiac ßII spectrin display lethal arrhythmias, aberrant electric and calcium handling phenotypes, and abnormal expression/localization of cardiac membrane proteins. Mechanistically, ßII spectrin regulates the localization of cytoskeletal and plasma membrane/sarcoplasmic reticulum protein complexes, including the Na/Ca exchanger, ryanodine receptor 2, ankyrin-B, actin, and αII spectrin. Finally, we observe accelerated heart failure phenotypes in ßII spectrin-deficient mice. CONCLUSIONS: Our findings identify ßII spectrin as critical for normal myocyte electric activity, link this molecule to human disease, and provide new insight into the mechanisms underlying cardiac myocyte biology.
Assuntos
Arritmias Cardíacas/patologia , Arritmias Cardíacas/fisiopatologia , Citoesqueleto/fisiologia , Miócitos Cardíacos/patologia , Miócitos Cardíacos/fisiologia , Espectrina/fisiologia , Sequência de Aminoácidos , Animais , Anquirinas/genética , Anquirinas/fisiologia , Arritmias Cardíacas/genética , Proteínas de Transporte/genética , Proteínas de Transporte/fisiologia , Modelos Animais de Doenças , Insuficiência Cardíaca/genética , Insuficiência Cardíaca/patologia , Insuficiência Cardíaca/fisiopatologia , Humanos , Proteínas de Membrana/fisiologia , Camundongos , Camundongos Knockout , Proteínas dos Microfilamentos/deficiência , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/fisiologia , Microtúbulos/fisiologia , Dados de Sequência Molecular , Mutação/genética , Fenótipo , Espectrina/análise , Espectrina/químicaRESUMO
Biomarkers for diffuse axonal injury could have utilities for the acute diagnosis and clinical care of concussion, including those related to sports. The calpain-derived αII-spectrin N-terminal fragment (SNTF) accumulates in axons after traumatic injury and increases in human blood after mild traumatic brain injury (mTBI) in relation to white matter abnormalities and persistent cognitive dysfunction. However, SNTF has never been evaluated as a biomarker for sports-related concussion. Here, we conducted longitudinal analysis of serum SNTF in professional ice hockey players, 28 of whom had a concussion, along with 45 players evaluated during the preseason, 17 of whom were also tested after a concussion-free training game. Compared with preseason levels, serum SNTF increased at 1 h after concussion and remained significantly elevated from 12 h to 6 days, before declining to preseason baseline. In contrast, serum SNTF levels were unchanged after training. In 8 players, postconcussion symptoms resolved within a few days, and in these cases serum SNTF levels were at baseline. On the other hand, for the 20 players withheld from play for 6 days or longer, serum SNTF levels rose from 1 h to 6 days postconcussion, and at 12-36 h differed significantly from the less-severe concussions (p=0.004). Serum SNTF exhibited diagnostic accuracy for concussion, especially so with delayed return to play (area under the curve=0.87). Multi-variate analyses of serum SNTF and tau improved the diagnostic accuracy, the relationship with the delay in return to play, and the temporal window beyond tau alone. These results provide evidence that blood SNTF, a biomarker for axonal injury after mTBI, may be useful for diagnosis and prognosis of sports-related concussion, as well as for guiding neurobiologically informed decisions on return to play.
Assuntos
Traumatismos em Atletas/sangue , Concussão Encefálica/sangue , Hóquei , Doenças Profissionais/sangue , Espectrina/análise , Adulto , Traumatismos em Atletas/fisiopatologia , Biomarcadores/sangue , Concussão Encefálica/fisiopatologia , Lesão Axonal Difusa/sangue , Humanos , Masculino , Doenças Profissionais/fisiopatologia , Síndrome Pós-Concussão/sangue , Síndrome Pós-Concussão/fisiopatologia , Índice de Gravidade de DoençaRESUMO
The scaffolding protein ankyrin-G is required for Na(+) channel clustering at axon initial segments. It is also considered essential for Na(+) channel clustering at nodes of Ranvier to facilitate fast and efficient action potential propagation. However, notwithstanding these widely accepted roles, we show here that ankyrin-G is dispensable for nodal Na(+) channel clustering in vivo. Unexpectedly, in the absence of ankyrin-G, erythrocyte ankyrin (ankyrin-R) and its binding partner ßI spectrin substitute for and rescue nodal Na(+) channel clustering. In addition, channel clustering is also rescued after loss of nodal ßIV spectrin by ßI spectrin and ankyrin-R. In mice lacking both ankyrin-G and ankyrin-R, Na(+) channels fail to cluster at nodes. Thus, ankyrin R-ßI spectrin protein complexes function as secondary reserve Na(+) channel clustering machinery, and two independent ankyrin-spectrin protein complexes exist in myelinated axons to cluster Na(+) channels at nodes of Ranvier.
Assuntos
Anquirinas/análise , Nós Neurofibrosos/química , Canais de Sódio/análise , Espectrina/análise , Animais , Anquirinas/metabolismo , Feminino , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Nós Neurofibrosos/metabolismo , Ratos , Canais de Sódio/metabolismo , Espectrina/metabolismoRESUMO
Ankyrin-G and ßII-spectrin colocalize at sites of cell-cell contact in columnar epithelial cells and promote lateral membrane assembly. This study identifies two critical inputs from lipids that together provide a rationale for how ankyrin-G and ßII-spectrin selectively localize to Madin-Darby canine kidney (MDCK) cell lateral membranes. We identify aspartate-histidine-histidine-cysteine 5/8 (DHHC5/8) as ankyrin-G palmitoyltransferases required for ankyrin-G lateral membrane localization and for assembly of lateral membranes. We also find that ßII-spectrin functions as a coincidence detector that requires recognition of both ankyrin-G and phosphoinositide lipids for its lateral membrane localization. DHHC5/8 and ßII-spectrin colocalize with ankyrin-G in micrometer-scale subdomains within the lateral membrane that are likely sites for palmitoylation of ankyrin-G. Loss of either DHHC5/8 or ankyrin-G-ßII-spectrin interaction or ßII-spectrin-phosphoinositide recognition through its pleckstrin homology domain all result in failure to build the lateral membrane. In summary, we identify a functional network connecting palmitoyltransferases DHHC5/8 with ankyrin-G, ankyrin-G with ßII-spectrin, and ßII-spectrin with phosphoinositides that is required for the columnar morphology of MDCK epithelial cells.
Assuntos
Anquirinas/metabolismo , Proteínas de Membrana/metabolismo , Fosfatidilinositóis/metabolismo , Espectrina/metabolismo , Animais , Anquirinas/análise , Anquirinas/fisiologia , Membrana Celular/metabolismo , Polaridade Celular , Cães , Células Epiteliais/metabolismo , Células Epiteliais/ultraestrutura , Técnicas de Silenciamento de Genes , Lipoilação , Proteínas de Membrana/análise , Proteínas de Membrana/fisiologia , Modelos Biológicos , Espectrina/análise , Espectrina/fisiologiaRESUMO
A pttg gene knockout affects the functional state of erythron in mice which could be associated with structural changes in the structure of erythrocyte membranes. The pttg gene knockout causes a significant modification of fatty acids composition of erythrocyte membrane lipids by reducing the content of palmitic acid and increasing of polyunsaturated fatty acids amount by 18%. Analyzing the erythrocyte surface architectonics of mice under pttg gene knockout, it was found that on the background of reduction of the functionally complete biconcave discs population one could observe an increase of the number of transformed cells at different degeneration stages. Researches have shown that in mice with a pttg gene knockout compared with a control group of animals cytoskeletal protein--beta-spectrin was reduced by 17.03%. However, there is a reduction of membrane protein band 3 by 33.04%, simultaneously the content of anion transport protein band 4.5 increases by 35.2% and protein band 4.2 by 32.1%. The lectin blot analysis has helped to reveal changes in the structure of the carbohydrate determinants of erythrocyte membrane glycoproteins under conditions of directed pttg gene inactivation, accompanied by changes in the type of communication, which joins the terminal residue in carbohydrate determinant of glycoproteins. Thus, a significant redistribution of protein and fatty acids contents in erythrocyte membranes that manifested in the increase of the deformed shape of red blood cells is observed underpttg gene knockout.
Assuntos
Citoesqueleto de Actina/química , Membrana Eritrocítica/química , Securina/deficiência , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/ultraestrutura , Animais , Proteína 1 de Troca de Ânion do Eritrócito/análise , Proteína 1 de Troca de Ânion do Eritrócito/metabolismo , Western Blotting , Proteínas do Citoesqueleto/análise , Proteínas do Citoesqueleto/metabolismo , Membrana Eritrocítica/metabolismo , Membrana Eritrocítica/ultraestrutura , Ácidos Graxos Insaturados/análise , Ácidos Graxos Insaturados/metabolismo , Lipídeos de Membrana/análise , Lipídeos de Membrana/metabolismo , Proteínas de Membrana/análise , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Microscopia , Proteínas de Transporte de Monossacarídeos/análise , Proteínas de Transporte de Monossacarídeos/metabolismo , Ácido Palmítico/análise , Ácido Palmítico/metabolismo , Lectinas de Plantas/química , Securina/genética , Espectrina/análise , Espectrina/metabolismoRESUMO
BACKGROUND: The two isoforms of carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), 1 with a long cytoplasmic domain (CEACAM1-L) and 1 with a short (CEACAM1-S), are involved in different signaling pathways. ß2-spectrin (ß2SP) is an adaptor protein that plays critical roles in the proper control of Smad access to activate receptors involved in regulation of TGF-ß signaling. In this study, we examined the association between CEACAM1 isoform balance and hepatocellular carcinoma (HCC) malignant potential and investigated the possibility of a molecular interaction between CEACAM1 and ß2SP. METHODS: Immunohistochemical analysis was carried out with CEACAM1-L or CEACAM1-S antibodies on 154 HCC tissues to correlate with the factors of malignancy. Invasion assay was performed for the effect of CEACAM1 expression on HCC cell lines. Moreover, immunohistochemical analysis and immunoprecipitation analysis were performed to investigate the association between CEACAM1 isoform balance and ß2SP. RESULTS: In immunohistochemical analysis, CEACAM1-L expression dominance was a risk factor for HCC recurrence (p = 0.04) and was significantly associated with a shorter survival compared with CEACAM1-S expression dominance. Invasion assay indicated that CEACAM1-4L-transfected HLF and PLC/PRF/5 cells showed significantly increased invasion (p < 0.0001) and CEACAM1-4S-transfected HLF cells showed significantly decreased invasion. Immunohistochemical analysis of ß2SP suggested that the HCCs with CEACAM1-L-dominant expression were more strongly stained with ß2SP than the HCCs with CEACAM1-S-dominant expression (p = 0.013), and coprecipitation assays indicated that CEACAM1-L could bind to ß2SP. CONCLUSIONS: CEACAM1-L may enhance the HCC invasiveness through an interaction with ß2SP and subsequent effects on TGF-ß signaling.
Assuntos
Antígenos CD/análise , Carcinoma Hepatocelular/química , Carcinoma Hepatocelular/patologia , Moléculas de Adesão Celular/análise , Neoplasias Hepáticas/química , Neoplasias Hepáticas/patologia , Recidiva Local de Neoplasia/química , Espectrina/análise , Idoso , Antígenos CD/genética , Antígenos CD/metabolismo , Apoptose , Carcinoma Hepatocelular/genética , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Humanos , Neoplasias Hepáticas/genética , Masculino , Pessoa de Meia-Idade , Invasividade Neoplásica , Isoformas de Proteínas/análise , Estudos Retrospectivos , Transdução de Sinais , Proteína Smad3/metabolismo , Espectrina/metabolismo , Taxa de Sobrevida , Transfecção , Fator de Crescimento Transformador beta/metabolismoRESUMO
The goal of this project was to determine whether biochemical markers of brain damage can be used to diagnose and assess the severity of injury in a rat model of penetrating ballistic-like brain injury (PBBI). To determine the relationship between injury magnitude and biomarker levels, rats underwent three discrete PBBI severity levels defined by the magnitude of the ballistic component of the injury, calibrated to equal 5%, 10%, or 12.5% of total rat brain volume. Cortex, cerebrospinal fluid (CSF), and blood were collected at multiple time points. Levels of three biomarkers (αII-spectrin breakdown product [SBDP150], glial fibrillary acidic protein [GFAP], and ubiquitin C-terminal hydrolase-L1 [UCH-L1]), were measured using quantitative immunoblotting and/or enzyme-linked immunosorbent assays. In injured cortex, SBDP150 and GFAP levels were increased significantly over controls. Cortical SBDP150 was elevated at 1 day but not 7 days, and GFAP at 7 days but not 1 day. At their respective time points, mean levels of SBDP150 and GFAP biomarkers in the cortex rose stepwise as injury magnitude increased. In the CSF, increasing severity of PBBI was associated with increasing concentrations of both neuronal and glial biomarkers acutely at 1 day after injury, but no trends were observed at 7 days. In plasma, SBDP150 was elevated at 5 min after 10% PBBI and at 6 h after 12.5% PBBI. UCH-L1 levels in plasma were elevated acutely at 5 min post-injury reflecting injury severity and rapidly decreased within 2 h. Overall, our results support the conclusion that biomarkers are effective indicators of brain damage after PBBI and may also aid in the assessment of injury magnitude.
Assuntos
Biomarcadores/análise , Proteína Glial Fibrilar Ácida/análise , Traumatismos Cranianos Penetrantes/metabolismo , Espectrina/análise , Ubiquitina Tiolesterase/análise , Animais , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Immunoblotting , Masculino , Ratos , Ratos Sprague-DawleyAssuntos
Proteína 1 de Troca de Ânion do Eritrócito/genética , Mutação , Esferocitose Hereditária/genética , Alelos , Proteína 1 de Troca de Ânion do Eritrócito/química , Proteína 1 de Troca de Ânion do Eritrócito/metabolismo , Estudos de Coortes , Análise Mutacional de DNA , Éxons , Estudos de Associação Genética , Heterozigoto , Humanos , Íntrons , RNA Mensageiro/metabolismo , Espectrina/análise , Esferocitose Hereditária/sangue , Esferocitose Hereditária/metabolismoRESUMO
Factors which may influence on erythrocytes in peripheral blood while exacerbation of herpes infection are studied. The data showed that herpes infection changed quantitative spectrum of proteins in erythrocytes. In cellular membranes of erythrocytes lipid peroxidation increases that results in increase of microviscosity of membranes. Disconnection of hemoglobin with 2, 3-DPhG was defined. This results in decreased hemoglobin oxygenation and change of erythrocytes form. The data showed that herpes virus in exacerbation leads to tissue hypoxia in pregnant women.