Characterization of Post-Translational Modifications to Calsequestrins of Cardiac and Skeletal Muscle

Calsequestrin is glycosylated and phosphorylated during its transit to its final destination in the junctional sarcoplasmic reticulum. To determine the significance and universal profile of these post-translational modifications to mammalian calsequestrin, we characterized, via mass spectrometry, the glycosylation and phosphorylation of skeletal muscle calsequestrin from cattle (B. taurus), lab mice (M. musculus) and lab rats (R. norvegicus) and cardiac muscle calsequestrin from cattle, lab rats and humans. On average, glycosylation of skeletal calsequestrin consisted of two N-acetylglucosamines and one mannose (GlcNAc₂Man₁), while cardiac calsequestrin had five additional mannoses (GlcNAc₂Man₆). Skeletal calsequestrin was not phosphorylated, while the C-terminal tails of cardiac calsequestrin contained between zero to two phosphoryls, indicating that phosphorylation of cardiac calsequestrin may be heterogeneous in vivo. Static light scattering experiments showed that the Ca²⁺-dependent polymerization capabilities of native bovine skeletal calsequestrin are enhanced, relative to the non-glycosylated, recombinant isoform, which our crystallographic studies suggest may be due to glycosylation providing a dynamic “guiderail”-like scaffold for calsequestrin polymerization. Glycosylation likely increases a polymerization/depolymerization response to changing Ca²⁺ concentrations, and proper glycosylation, in turn, guarantees both effective Ca²⁺ storage/buffering of the sarcoplasmic reticulum and localization of calsequestrin (Casq) at its target site.     

Epigenetic Modifications of Major Depressive Disorder

Major depressive disorder (MDD) is a chronic disease whose neurological basis and pathophysiology remain poorly understood. Initially, it was proposed that genetic variations were responsible for the development of this disease. Nevertheless, several studies within the last decade have provided evidence suggesting that environmental factors play an important role in MDD pathophysiology. Alterations in epigenetics mechanism, such as DNA methylation, histone modification and microRNA expression could favor MDD advance in response to stressful experiences and environmental factors. The aim of this review is to describe genetic alterations, and particularly altered epigenetic mechanisms, that could be determinants for MDD progress, and how these alterations may arise as useful screening, diagnosis and treatment monitoring biomarkers of depressive disorders.     

Influence of serine O-glycosylation or O-phosphorylation close to the vJun nuclear localisation sequence on nuclear import

Nuclear import triggered by the nuclear-localisation sequence (NLS) of the viral Jun (vJun) protein is mediated by phosphorylation of a serine close to the NLS. Since phosphorylation and glycosylation of serine residues are often in a reciprocal "yin-yang" relationship, we investigated whether glycosylation of this serine with O-linked N-acetylglucosamine (O-GlcNAc) would also regulate nuclear import via the vJun NLS. Peptides containing the vJun NLS with an adjacent O-phosphorylated, O-GlcNAc-functionalised or unmodified serine, and equipped with an N-terminal biotin or a 7-nitrobenz-2-oxa-1,3-diazolyl (NBD) fluorescent label, were synthesised on the solid phase by means of an Fmoc/Boc strategy and a Pd0-sensitive HYCRON linker. Fluorescence-polarisation measurements on the NBD-labelled peptides indicated that modification with phosphate or O-GlcNAc leads to a decrease in affinity to the import-mediating adapter protein, importin alpha, of about one order of magnitude compared to the unmodified NLS. Microinjection of biotinylated NLS peptide conjugated with fluorescently labelled avidin into NIH/3T3 and MDCK cells, revealed that avidin-unmodified-NLS peptide was rapidly imported into the nucleus. However, either phosphate or O-GlcNAc next to the NLS caused almost complete exclusion of the protein conjugate from nuclear import. These findings indicate that nuclear import by the vJun NLS might not be regulated by a "yin-yang" modification of an adjacent serine with phosphate or O-GlcNAc. Rather, negative regulation of binding between the polybasic NLS and importin by a negatively charged or a bulky, uncharged residue appears likely.     

Chemical biology techniques unlock the secrets of casein kinase 2 regulation by phosphorylation and glycosylation

The key to understanding: The application of expressed protein ligation and protein microarrays enabled an unparalleled insight into the complex interaction of phosphorylation and glycosylation on casein kinase 2 and its biological outcome.     

Post-Translational Modification and Secretion of Azelaic Acid Induced 1 (AZI1), a Hybrid Proline-Rich Protein from Arabidopsis

Arabidopsis EARLI-type hybrid proline-rich proteins (HyPRPs) consist of a putative N-terminal secretion signal, a proline-rich domain (PRD), and a characteristic eight-cysteine-motif (8-CM). They have been implicated in biotic and abiotic stress responses. AZI1 is required for systemic acquired resistance and it has recently been identified as a target of the stress-induced mitogen-activated protein kinase MPK3. AZI1 gel migration properties strongly indicate AZI1 to undergo major post-translational modifications. These occur in a stress-independent manner and are unrelated to phosphorylation by MAPKs. As revealed by transient expression of AZI1 in Nicotiana benthamiana and Tropaeolum majus, the Arabidopsis protein is similarly modified in heterologous plant species. Proline-rich regions, resembling arabinogalactan proteins point to a possible proline hydroxylation and subsequent O-glycosylation of AZI1. Consistently, inhibition of prolyl hydroxylase reduces its apparent protein size. AZI1 secretion was examined using Arabidopsis protoplasts and seedling exudates. Employing Agrobacterium-mediated leaf infiltration of N. benthamiana, we attempted to assess long-distance movement of AZI1. In summary, the data point to AZI1 being a partially secreted protein and a likely new member of the group of hydroxyproline-rich glycoproteins. Its dual location suggests AZI1 to exert both intra- and extracellular functions.     

Highly Efficient Cell‐Penetrating Probes of Protein Arginine Deiminases for Functional Proteomics

Protein arginine deiminases (PADs) have recently emerged at the forefront of drug‐discovery programs for several human disorders. Despite this, a precise understanding of their functional roles in human biology remains to be fully elucidated. This report highlights a recent development of next‐generation activity‐based PAD probes that are highly efficient, cell active, and metabolically stable. This discovery should rapidly expedite functional assignments of PAD biology in both normal and diseased cells, thereby leading to the development of PAD‐targeted therapeutics in the near future.     

Cell‐Free Protein Synthesis: Pros and Cons of Prokaryotic and Eukaryotic Systems

From its start as a small‐scale in vitro system to study fundamental translation processes, cell‐free protein synthesis quickly rose to become a potent platform for the high‐yield production of proteins. In contrast to classical in vivo protein expression, cell‐free systems do not need time‐consuming cloning steps, and the open nature provides easy manipulation of reaction conditions as well as high‐throughput potential. Especially for the synthesis of difficult to express proteins, such as toxic and transmembrane proteins, cell‐free systems are of enormous interest. The modification of the genetic code to incorporate non‐canonical amino acids into the target protein in particular provides enormous potential in biotechnology and pharmaceutical research and is in the focus of many cell‐free projects. Many sophisticated cell‐free systems for manifold applications have been established. This review describes the recent advances in cell‐free protein synthesis and details the expanding applications in this field.     

Synergistic Effect of Abrasive and Sonication for Release of Carbohydrate and Protein from Algae

While algae have demonstrated significant potential as a raw material due to the high intracellular concentration of carbohydrates and proteins, a primary limitation as a biofuel feedstock is due to the fact that an economically feasible method of extraction has yet to be offered. Algae samples, acquired from a local waste treatment facility, were combined with abrasive materials and subjected to ultrasonication for specific time intervals. It was found that the synergistic effect of sonication in the presence of abrasive material, such as silicon beads or sand, could extract adequate protein and carbohydrate to be utilized in fermentation processes.     

A new strategy for the synthesis of dinucleotides loaded with glycosylated amino acids--investigations on in vitro non-natural amino acid mutagenesis for glycoprotein synthesis

The in vitro non-natural amino acid mutagenesis method provides the opportunity to introduce non-natural amino acids site-specifically into proteins. To this end, a chemically synthesised aminoacylated dinucleotide is enzymatically ligated to a truncated suppressor transfer RNA. The loaded suppressor tRNA is then used in translation reactions to read an internal stop codon. Here we report an advanced and general strategy for the synthesis of the aminoacyl dinucleotide. The protecting group pattern developed for the dinucleotide facilitates highly efficient aminoacylation, followed by one-step global deprotection. The strategy was applied to the synthesis of dinucleotides loaded with 2-acetamido-2-deoxy-glycosylated amino acids, including N- and O-beta-glycosides and O- and C-alpha-glycosides of amino acids, thus enabling the extension of in vitro non-natural amino acid mutagenesis towards the synthesis of natural glycoproteins of high biological interest. We demonstrate the incorporation of the glycosylamino acids--although with low suppression efficiency--into the human interleukin granulocyte-colony stimulating factor (hG-CSF), as verified by the ELISA technique.     

藻类水华防治技术研究

随着全球水体富营养化的加剧,有害藻类的暴发日趋频繁,给环境、生态和经济造成了巨大的损失。有效控制水华的爆发是环境科学领域的一个难题,笔者对国内外水华防治措施进行了综述,并提出了藻类水华防治方法的发展方向。溶藻细菌一般分离自暴发水华的水体,这种土著细菌在工程应用中具有安全、特异、高效等特点。作为一种新的生物控藻手段显示出了广泛的应用前景。     

湛江海域6种经济海藻的营养成分分析

分析采自湛江海域的6种经济海藻的营养成分。除海蒿子外,碳水化合物是这些海藻的主要营养成分,平均为39.49%;粗蛋白含量4.08%~23.70%;粗脂肪含量最低,其中的4种皆不足藻体干重的0.5%;矿物质含量丰富,异枝麒麟菜的Fe,Mn,Zn的含量都较高,分别为261.20 mg/100g,62.27 mg/100g,206.10 mg/100g;氨基酸含量较高,均符合1973年联合国粮农组织/世界卫生组织（FAO/WHO）推荐的理想蛋白质模式;不少种类中镉、铅、砷等含量超出我国食品卫生标准。海藻体内的营养成分含量因海藻种类和生长海域的不同而有差异。     

表面活性剂对粉体的有机化改性

本文研究了用表面活性剂对无机粉体碳酸钙,钛白粉,白炭黑,凹凸棒土进行有机化改性的方法,制备了改性样品,测定了样品的比表面,接触角在液体石蜡中的粘度等物化性能,还考察了带有反应性基团的新型阳离子表面活性剂（ＣＤＭＡＡＢ）所改性样品等天然橡胶力学性能的影响,结果表明上述有机化改性是可行的。     

Tau Protein Modifications and Interactions: Their Role in Function and Dysfunction

Tau protein is abundant in the central nervous system and involved in microtubule assembly and stabilization. It is predominantly associated with axonal microtubules and present at lower level in dendrites where it is engaged in signaling functions. Post-translational modifications of tau and its interaction with several proteins play an important regulatory role in the physiology of tau. As a consequence of abnormal modifications and expression, tau is redistributed from neuronal processes to the soma and forms toxic oligomers or aggregated deposits. The accumulation of tau protein is increasingly recognized as the neuropathological hallmark of a number of dementia disorders known as tauopathies. Dysfunction of tau protein may contribute to collapse of cytoskeleton, thereby causing improper anterograde and retrograde movement of motor proteins and their cargos on microtubules. These disturbances in intraneuronal signaling may compromise synaptic transmission as well as trophic support mechanisms in neurons.     

太湖蓝藻的成因与启示

随着经济的快速发展、人类活动的加强,湖泊及其湖滨带的退化日益严重。介绍了太湖发生蓝藻的根源,从生态学原理上分析了太湖蓝藻的成因,概括了湖滨带的退化机理及现状。太湖水质变坏值得其他城市引以为戒,如果等到暴发水华,就需要付出更多的人力物力来解决富营养化问题。     

Effects of Bofu-Tsusho-San on Diabetes and Hyperlipidemia Associated with AMP-Activated Protein Kinase and Glucose Transporter 4 in High-Fat-Fed Mice

This study was undertaken to examine the effect and mechanism of Bofu-tsusho-san formula (BO) on hyperglycemia and hyperlipidemia and in mice fed with a high-fat (HF) diet. The C57BL/6J mice were received control/HF diet for 12 weeks, and oral administration of BO (at three doses) or rosiglitazone (Rosi) or vehicle for the last 4 weeks. Blood, skeletal muscle and tissues were examined by means of measuring glycaemia and dyslipidaemia-associated events. BO treatment effectively prevented HF diet-induced increases in the levels of triglyceride (TG), free fatty acid (FFA) and leptin (p < 0.01, p < 0.01, p < 0.01, respectively). BO treatment exhibited reduced both visceral fat mass and hepatic triacylglycerol content; moreover, BO treatment displayed significantly decreased both the average area of the cut of adipocytes and ballooning of hepatocytes. BO treatment exerted increased the protein contents of glucose transporter 4 (GLUT4) in skeletal muscle, and caused lowered blood glucose levels. BO treatment displayed increased levels of phosphorylated AMP-activated protein kinase (AMPK) in both skeletal muscle and liver tissue. Furthermore, BO reduced the hepatic expression of glucose-6-phosphatase (G6Pase) and phosphenolpyruvate carboxykinase (PEPCK) and glucose production. Therefore, it is possible that the activation of AMPK by BO leads to diminished gluconeogenesis in liver tissue. BO increased hepatic expressions of peroxisome proliferator-activated receptor α (PPARα), whereas down-regulating decreasing expressions of fatty acid synthesis, including sterol regulatory element binding protein 1c (SREBP1c) and fatty acid synthase (FAS), resulting in a decrease in circulating triglycerides. This study originally provides the evidence that amelioration of dyslipidemic and diabetic state by BO in HF-fed mice occurred by regulation of GLUT4, SREBP1c, FAS, PPARα, adiponectin and AMPK phosphorylation.     

Photocontrol of Protein Activity in Cultured Cells and Zebrafish with One‐ and Two‐Photon Illumination

We have implemented a noninvasive optical method for the fast control of protein activity in a live zebrafish embryo. It relies on releasing a protein fused to a modified estrogen receptor ligand binding domain from its complex with cytoplasmic chaperones, upon the local photoactivation of a nonendogenous caged inducer. Molecular dynamics simulations were used to design cyclofen‐OH, a photochemically stable inducer of the receptor specific for 4‐hydroxy‐tamoxifen (ER^T2). Cyclofen‐OH was easily synthesized in two steps with good yields. At submicromolar concentrations, it activates proteins fused to the ER^T2 receptor. This was shown in cultured cells and in zebrafish embryos through emission properties and subcellular localization of properly engineered fluorescent proteins. Cyclofen‐OH was successfully caged with various photolabile protecting groups. One particular caged compound was efficient in photoinducing the nuclear translocation of fluorescent proteins either globally (with 365 nm UV illumination) or locally (with a focused UV laser or with two‐photon illumination at 750 nm). The present method for photocontrol of protein activity could be used more generally to investigate important physiological processes (e.g., in embryogenesis, organ regeneration and carcinogenesis) with high spatiotemporal resolution.     

The Structure and Dynamics of BmR1 Protein from Brugia malayi: In Silico Approaches

Brugia malayi is a filarial nematode, which causes lymphatic filariasis in humans. In 1995, the disease has been identified by the World Health Organization (WHO) as one of the second leading causes of permanent and long-term disability and thus it is targeted for elimination by year 2020. Therefore, accurate filariasis diagnosis is important for management and elimination programs. A recombinant antigen (BmR1) from the Bm17DIII gene product was used for antibody-based filariasis diagnosis in “Brugia Rapid”. However, the structure and dynamics of BmR1 protein is yet to be elucidated. Here we study the three dimensional structure and dynamics of BmR1 protein using comparative modeling, threading and ab initio protein structure prediction. The best predicted structure obtained via an ab initio method (Rosetta) was further refined and minimized. A total of 5 ns molecular dynamics simulation were performed to investigate the packing of the protein. Here we also identified three epitopes as potential antibody binding sites from the molecular dynamics average structure. The structure and epitopes obtained from this study can be used to design a binder specific against BmR1, thus aiding future development of antigen-based filariasis diagnostics to complement the current diagnostics.