Tau-Like Proteins in the Nervous System of Goldfish

This report describes the presence of a group of tau-like proteins (TLPs) in goldfish central nervous system. The TLPs were immunoreactive with antibodies that recognized the carboxy-terminal domain of mammalian tau, but not with antibodies that recognized the amino-terminus. The TLPs of goldfish exhibited the basic properties of tau proteins including neuronal specificity, structural heterogeneity, heat stability and the ability to co-assemble with tubulin. We propose that TLPs may represent a precursor of tau, that share the microtubule binding domain and the carboxy-terminal domain with mammalian tau proteins. In contrast the amino-terminus of the TLPs is much shorter and may represent a more variable domain of tau proteins.     

新型细胞骨架分隔丝的结构和功能研究进展

细胞骨架是细胞内的蛋白纤维网状结构,包括人们熟知的微管、微丝和中间纤维.目前研究表明分隔丝(septin filaments)是一类在真核生物中广泛分布的蛋白纤维,逐渐被认为是一种新型细胞骨架结构.分隔丝由可结合GTP的分隔丝蛋白单体(Septin)聚合形成异源复合体,进一步组装成纤维丝.分隔丝可形成纤维束,环状或笼状等结构,并与细胞膜或其他细胞骨架成分发生相互作用.在细胞内,分隔丝参与胞质分裂、细胞迁移、神经元发育和免疫等重要生理及病理过程.分隔丝结构或功能的异常与多种人类疾病如肿瘤等密切相关.本文将从分隔丝的结构、组装调控、功能及与人类疾病的关系等方面综述近年的研究进展.     

Role of Matricellular Proteins in Disorders of the Central Nervous System

Matricellular proteins (MCPs) are actively expressed non-structural proteins present in the extracellular matrix, which rapidly turnover and possess regulatory roles, as well as mediate cell–cell interactions. MCPs characteristically contain binding sites for other extracellular proteins, cell surface receptors, growth factors, cytokines and proteases, that provide structural support for surrounding cells. MCPs are present in most organs, including brain, and play a major role in cell–cell interactions and tissue repair. Among the MCPs found in brain include thrombospondin-1/2, secreted protein acidic and rich in cysteine family (SPARC), including Hevin/SC1, Tenascin C and CYR61/Connective Tissue Growth Factor/Nov family of proteins, glypicans, galectins, plasminogen activator inhibitor (PAI-1), autotaxin, fibulin and perisostin. This review summarizes the potential role of MCPs in the pathogenesis of major neurological disorders, including Alzheimer’s disease, amyotrophic lateral sclerosis, ischemia, trauma, hepatic encephalopathy, Down’s syndrome, autism, multiple sclerosis, brain neoplasms, Parkinson’s disease and epilepsy. Potential therapeutic opportunities of MCP’s for these disorders are also considered in this review.     

Age-Related Changes of Myelin Proteins in the Rat Peripheral Nervous System

Age-related changes in amounts of myelin proteins from rat sciatic nerve or spinal root were analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE). In the aged peripheral nerve myelin, the relative amounts of band 105K and proteins X and Y increased, whereas those of proteins P0 and P1 and band 190K decreased. Band 105K purified by preparative SDS-PAGE exhibited three bands of 105K, 28K, and 21K at the second electrophoresis. A repeated SDS-PAGE did not improve the purity of bank 105K, but increased the ratio of 21K to 28K. Compared with P0 protein, band 105K has a very similar peptide map pattern and amino acid composition, as well as the identical NH2 terminal residue, isoleucine. These findings suggest that band 105K is an aggregate form of P0 protein and its fragment, 21K. The 21K protein is a distinct entity from X protein. The quantitative and qualitative alterations in myelin proteins, as we report here, may reflect continuing demyelination and remyelination in aged peripheral nerves.     

Membrane-Associated Cytoskeletal Proteins in Squid Giant Axons

Abstract: Cytoskeletal proteins (e.g., tubulin, actin, and neurofilament proteins) in the squid giant axon are separable into KF-soluble and -insoluble forms. The KF-insoluble cytoskeletal components appear to constitute the major proteins in the subaxolemmal fibrous network on the inner surface of the axon. These cytoskeletal proteins and the subaxolemmal network are both highly soluble in KI solutions. Whereas giant axons tolerate prolonged perfusions in KF solutions with no loss of excitable properties, a relatively short perfusion with KI solution completely eliminates the excitability of the axon. The loss of this excitability correlates with the simultaneous dissolution of the subaxolemmal network of cytoskeletal proteins and the release of its proteins into the perfusate. These data support the hypothesis that cytoskeletal proteins associated with the inner surface of the axolemma are involved in the regulation of axonal excitability.     

Characterization and Biosynthesis of Cyclic-AMP-Binding Proteins in the Rat Central Nervous System

Cyclic-AMP-binding proteins in membrane and soluble fractions from rat forebrain were compared; membrane fractions included smooth and rough microsomes and a plasma membrane fraction enriched in synaptic membranes. Protein fractions were treated with 8-azido-[32P]cyclic AMP and ultraviolet irradiation to covalently tag cyclic-AMP-binding proteins. Labeled proteins were then analyzed by two-dimensional gel electrophoresis (2DGE) and fluorography. The soluble CNS proteins contained two major cyclic-AMP-binding species at 48K (48K 5.5 and 48K 5.45), differing slightly in their isoelectric points. Another protein was seen at 54K (54K 5.3) adjacent to the beta-tubulin subunits in the 2D electrophoretogram. The analysis of the smooth microsome and plasma membrane fractions differed from the soluble fraction in that there were two cyclic-AMP-binding proteins adjacent to the beta-tubulin region (54K 5.3 and 52K 5.3) differing slightly in apparent molecular weight. The membrane fractions also contained a cyclic-AMP-binding protein at 54K 5.8. The 52K 5.3 and 54K 5.8 species were unique to the membrane fractions. The rough microsomes did not contain detectable amounts of cyclic-AMP-binding proteins. Free polysomes were isolated from brain tissue, and translation products were analyzed by cyclic AMP affinity chromatography and immunopurification with antibodies to the brain specific type II regulatory subunit. The translation products that were found to bind cyclic AMP Sepharose are as follows: 48K 5.5, 48K 5.45, 52K 5.3, and 54K 5.8. These species comigrated with proteins that were photoaffinity-labeled in cytosol and membrane fractions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Myotubularin-Related (MTMR) Phospholipid Phosphatase Proteins in the Peripheral Nervous System

Myotubularin-related proteins (MTMRs) constitute a broad family of ubiquitously expressed phosphatases with 14 members in humans, of which eight are catalytically active phosphatases, while six are catalytically inactive. Active MTMRs possess 3-phosphatase activity toward both PtdIns3P and PtdIns(3, 5)P ₂ poliphosphoinositides (PPIn), suggesting an involvement in intracellular trafficking and membrane homeostasis. Among MTMRs, catalytically active MTMR2 and inactive MTMR13 have a nonredundant function in nerve. Loss of either MTMR2 or MTMR13 causes Charcot–Marie–Tooth type 4B1 and B2 neuropathy, respectively, characterized by demyelination and redundant loops of myelin known as myelin outfoldings. In Mtmr2-null mouse nerves, these aberrant foldings occur at 3–4 weeks after birth, a time when myelination is established, and Schwann cells are still elongating to reach the final internodal length. Moreover, Mtmr2-specific ablation in Schwann cells is both sufficient and necessary to provoke CMT4B1 with myelin outfoldings. MTMR2 phospholipid phosphatase might regulate intracellular trafficking events and membrane homeostasis in Schwann cells during postnatal nerve development. In this review, we will discuss recent findings on the MTMR family with a major focus on MTMR2 and MTMR13 and their putative role in Schwann cell biology.     

Ca2+-Binding S100 Proteins in the Central Nervous System

A number of neurodegenerative disorders have been attributed to abberrations of intracellular Ca²⁺ homeostasis regulated by Ca²⁺-binding proteins. This chapter will focus on the S100B and S100A6 proteins, which are highly expressed in the central nervous system. Their protein structures, localizations, and association with brain pathology as well as their potential use in clinical diagnostics will be discussed.     

原生动物的细胞骨架蛋白及其功能组件

目前在原生动物中发现了许多新的细胞骨架蛋白,如中心元蛋白、副鞭毛杆蛋白等。深入研究发现,原生动物的细胞骨架在细胞的模式形成,细胞核的遗传中也具有重要作用。从功能组件角度着眼研究细胞骨架的功能,将有助于了解细胞骨架的进化机制。     

Process Formation in Astrocytes: Modulation of Cytoskeletal Proteins

Studies on primary astrocytes cultured in vitro have shown that process formation involves changes in cytoskeletal proteins and release of tension on the substratum. Actin filament reorganization has previously been found to be the major cytoskeletal change occurring during process formation. These changes are relatively rapid with breakdown of the actin web and release of contacts occur within 15 min. of cyclic AMP treatment. The former is regulated by myosin light chain (MLC) and actin depolymerizing factor (ADF), with MLC involved in the initial release of contractile tension and ADF in both initial and longer term actin breakdown. Our results show that the dephosphorylation of MLC is due to the phosphorylation and inactivation of myosin light chain kinase (MLCK) in response to cyclic AMP. To further study the mechanisms underlying the process formation in astrocytes we used endothelin-1 (ET-1), a vasopeptide which has been shown to inhibit process formation in astrocytes and sodium fluoride which is a general phosphatase inhibitor. We observe an increase in phosphorylation of MLC on inhibition of process formation. To study the role of adhesion in process formation we used suspension cultures of astrocytes. Our results with the astrocytes in suspension suggest that the process formation in astrocytes is adhesion dependent and the changes in ADF and MLC occur only when there is process formation.     

RBC-NOS-Dependent S-Nitrosylation of Cytoskeletal Proteins Improves RBC Deformability

Background

Red blood cells (RBC) possess a nitric oxide synthase (RBC-NOS) whose activation depends on the PI3-kinase/Akt kinase pathway. RBC-NOS-produced NO exhibits important biological functions like maintaining RBC deformability. Until now, the cellular target structure for NO, to exert its influence on RBC deformability, remains unknown. In the present study we analyzed the modification of RBC-NOS activity by pharmacological treatments, the resulting influence on RBC deformability and provide first evidence for possible target proteins of RBC-NOS-produced NO in the RBC cytoskeletal scaffold.

Methods/Findings

Blood from fifteen male subjects was incubated with the NOS substrate L-arginine to directly stimulate enzyme activity. Direct inhibition of enzyme activity was induced by L-N5-(1-Iminoethyl)-ornithin (L-NIO). Indirect stimulation and inhibition of RBC-NOS were achieved by applying insulin and wortmannin, respectively, substances known to affect PI3-kinase/Akt kinase pathway. The NO donor sodium nitroprusside (SNP) and the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) were additionally applied as NO positive and negative controls, respectively. Immunohistochemical staining was used to determine phosphorylation and thus activation of RBC-NOS. As a marker for NO synthesis nitrite was measured in plasma and RBCs using chemiluminescence detection. S-nitrosylation of erythrocyte proteins was determined by biotin switch assay and modified proteins were identified using LC-MS. RBC deformability was determined by ektacytometry. The data reveal that activated RBC-NOS leads to increased NO production, S-nitrosylation of RBC proteins and RBC deformability, whereas RBC-NOS inhibition resulted in contrary effects.

Conclusion/Significance

This study first-time provides strong evidence that RBC-NOS-produced NO modifies RBC deformability through direct S-nitrosylation of cytoskeleton proteins, most likely α- and β-spectrins. Our data, therefore, gain novel insights into biological functions of RBC-NOS by connecting impaired RBC deformability abilities to specific posttranslational modifications of RBC proteins. By identifying likely NO-target proteins in RBC, our results will stimulate new therapeutic approaches for patients with microvascular disorders.     

Altered Axonal Transport of Cytoskeletal Proteins in the Mutant Diabetic Mouse

Polypeptides in the motor axons of the sciatic nerve in 120-day-old normal and diabetic mice C57BL/Ks (db/db) were labeled by injection of [35S]methionine into the ventral horn of the spinal cord. At 8, 15, and 25 days after the injection, the distribution of radiolabeled polypeptides along the sciatic nerve was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Four major radiolabeled polypeptides, tentatively identified as actin, tubulin, and the two lightest subunits of the neurofilament triplet, were studied in both diabetic and control mice. In the diabetic animals, the two polypeptides identified as actin and tubulin showed a reduction of average velocity of migration along the sciatic nerve, resulting in a higher fraction of radioactivity in the proximal part of the sciatic nerve, whereas the front of radioactivity (advancing at maximal velocity) moved at a normal rate. In contrast, both the average and maximal velocities of the two neurofilament subunits were slower in the diabetic mice than in the control mice. These results indicate that the axonal transport of the cytoskeletal proteins is differentially affected in the course of diabetic neuropathy, and may suggest that the impairment concerns mainly the proteins carried by the slowest component of axonal transport.     

Distribution of Cytoskeletal Proteins in Neomycin-Induced Protrusions of Human Fibroblasts

The organization of actin, tubulin, and vimentin was studied in protruding lamellae of human fibroblasts induced by the aminoglycoside antibiotic neomycin, an inhibitor of the phosphatidylinositol cycle. Neomycin stimulates the simultaneous protrusion of lamellae in all treated cells, and the lamellae remain extended for about 15–20 min, before gradually withdrawing. The pattern and distribution of actin, tubulin, and vimentin during neomycin stimulation were analyzed by fluorescence and electron microscopy. F-actin in the newly formed lamellae is localized in a marginal band at the leading edge. Tubulin is colocalized with F-actin in the marginal band, but the newly formed lamellae are initially devoid of microtubules. Over a period of 10 to 20 min after the addition of neomycin, microtubules grow into the lamellae from the adjacent cytoplasm, while the intensity of tubulin staining of the marginal band decreases. Distribution of vimentin remains unchanged in neomycin-treated cells and vimentin filaments do not enter the new protrusions. Treatment of cells with colchicine and Taxol do not inhibit neomycin-induced protrusion but protrusions are no longer localized at the ends of cell processes and occur all around the cell periphery. We conclude that actin filaments are the major component of the cytoskeleton involved in generating protrusions. Microtubules and, possibly, intermediate filaments control the pattern of protrusions by their interaction with actin filaments.     

The Changes of Cytoskeletal Proteins in Plasma of Acrylamide-Induced Rats

Acrylamide (ACR) is a known industrial neurotoxic chemical that can induce neurodegeneration. Cytoskeletal protein aggregation is a pathological hallmark of neurodegenerative disorders. This study was an initial exploration on cytoskeletal proteins in plasma as potential biomarkers of ACR neurotoxicity. Low and high ACR groups received 20 mg/kg and 40 mg/kg ACR by intraperitoneal injection in adult Wistar rats and control group received physiological saline. Rats were all killed after 8 weeks to evaluate the levels of neurofilament(NF)-L, NF-M, NF-H, β-actin, α-tubulin, β-tubulin, tau, MAP₂ proteins in plasma using both SDS-PAGE and western blotting. Compared with the control, the levels of NF-L, NF-M, NF-H, β-actin, tau, MAP₂ proteins decreased and the level of α-tubulin increased in high ACR group, the levels of α-tubulin, β-tubulin and MAP₂ increased in low ACR group. The results suggested that the changes of these proteins might be relevant to the neurotoxicity of ACR. Some of the cytoskeletal proteins in plasma might be used as marker of biological effect in ACR induced neuropathy.