Actin-binding proteins in a postsynaptic preparation: Lasp-1 is a component of central nervous system synapses and dendritic spines

CNS synapses are complex sites of cell-cell communication. Identification and characterization of the protein components of synapses will lead to a better understanding of the mechanisms of neurotransmission and plasticity. We applied multidimensional protein identification technology (MudPIT) to purified, guanidine-solubilized postsynaptic fractions to identify novel synaptically localized molecules. We identified several actin-associated proteins known to regulate actin polymerization and control cell motility in nonneural cells that have not previously been associated with CNS synaptic function. One of these is lasp-1, an actin-associated LIM and SH3 domain-containing protein. We show that lasp-1 is strongly expressed by CNS neurons and is concentrated at synaptic sites. Overall, the preponderance of actin-associated proteins in postsynaptic density fractions, and specifically those involved in actin reorganization, suggests that there are many modes by which the state of synaptic F-actin polymerization and, hence, synaptic physiology are affected.     

Support for a proposed retinoid-processing protein complex in apical retinal pigment epithelium

The interaction of cellular retinaldehyde-binding protein (CRALBP) with ERM (ezrin, radixin, moesin)-binding phosphoprotein 50 (EBP50) in retinal pigment epithelium (RPE) microsomes has led to the hypothesis that a retinoid-processing protein complex exists in apical RPE. Mouse RPE apical processes were isolated on wheat germ agglutinin-coated agarose beads. Proteomic analyses of the isolated apical RPE demonstrated the presence of CRALBP, EBP50, 11-cis-retinol dehydrogenase, cellular retinol-binding protein 1, and interphotoreceptor retinoid-binding protein. The results support the hypothesis that a visual cycle protein complex may serve in the localization and release of 11-cis-retinoid in the apical RPE.     

The future of regulatory toxicology: impact of the biotechnology revolution.

The molecular biology revolution and the advent of genomic and proteomic technologies are facilitating rapid advances in our understanding of the molecular details of cell and tissue function. These advances have the potential to transform toxicological and clinical practice, and are likely to lead to the supplementation or replacement of traditional biomarkers of cellular integrity, cell and tissue homeostasis, and morphological alterations that result from cell damage or death. New technologies that permit simultaneous monitoring of many hundreds, or thousands, of macro- and small molecules ("-omics" technologies) promise to allow functional monitoring of multiple (or perhaps all) key cellular pathways simultaneously. Elucidation of cellular responses to molecular damage, including evolutionarily conserved inducible molecular defense systems, suggests the possibility of new biomarkers based on molecular responses to functional perturbations and cellular damage. Our improved understanding of the molecular basis of various pathologies suggests that monitoring specific molecular responses may provide improved prediction of human outcomes. Responses that can be monitored directly in the human should provide "bridging biomarkers" that may eliminate much of the current uncertainty in extrapolating from laboratory models to human outcome. Another aspect of genomics is our enhanced ability to associate DNA sequence variations with biological outcomes and individual sensitivity. The human genome sequence has revealed that sequence variations are very common, and may be an important determinant of variation in biological outcomes. The impending availability of a complete human haplotype map linked to standard genetic markers greatly facilitates identification of genetic variations that convey sensitivity or resistance to chemical exposures. Genetic approaches have already linked a large number of genetic variants (polymorphisms) with human diseases and adverse reactions from exposure to drugs or toxicants, suggesting an important role in sensitivity to drugs and environmental agents, disease susceptibilities, and therapeutic responses. As these opportunities are transformed into reality, regulatory toxicological practice is likely to be shaped in the future by the combination of conventional pathology, toxicology, molecular genetics, biochemistry, cell biology, and computational bio-informatics-resulting in the broad application of molecular approaches to monitoring functional disturbances.     

The growing impact of click chemistry on drug discovery

Click chemistry is a modular approach that uses only the most practical and reliable chemical transformations. Its applications are increasingly found in all aspects of drug discovery, ranging from lead finding through combinatorial chemistry and target-templated in situ chemistry, to proteomics and DNA research, using bioconjugation reactions. The copper-(I)-catalyzed 1,2,3-triazole formation from azides and terminal acetylenes is a particularly powerful linking reaction, due to its high degree of dependability, complete specificity, and the bio-compatibility of the reactants. The triazole products are more than just passive linkers; they readily associate with biological targets, through hydrogen bonding and dipole interactions.     

Expression profiling of breast cancer cells by differential peptide display

Expression profiling of RNAs or proteins has become a promising means to investigate the heterogeneity of histopathologically defined classes of cancer. Peptides, representing degradation as well as processing products of proteins offer an even closer insight into cell physiology. Peptides are related to the turnover of cellular proteins and are capable to reflect disease-related changes in homoeostasis of the human body. Furthermore, peptides derived from tumor cells are potentially useful markers in the early detection of cancer.In this study, we introduced a method called differential peptide display (DPD) for separating, detecting, and identifying native peptides derived from whole cell extracts. This method is a highly standardized procedure, combining the power of reversed-phase chromatography with mass spectrometry. This technology is suitable to analyze cell lines, various tissue types and human body fluids. Peptide-based profiling of normal human mammary epithelial cells (HMEC) and the breast cancer cell line MCF-7 revealed complex peptide patterns comprising of up to 2300 peptides. Most of these peptides were common to both cell lines whereas about 8% differed in their abundance. Several of the differentially expressed peptides were identified as fragments of known proteins such as intermediate filament proteins, thymosins or Cathepsin D. Comparing cell lines with native tumors, overlapping peptide patterns were found between HMEC and a phylloides tumor (CP) on the one hand and MCF-7 cells and tissue from a invasive ductal carcinoma (DC) on the other hand.     

Nitrated Alpha-Synuclein and Microglial Neuroregulatory Activities

Microglial neuroinflammatory responses affect the onset and progression of Parkinson’s disease (PD). We posit that such neuroinflammatory responses are, in part, mediated by microglial interactions with nitrated and aggregated α-synuclein (α-syn) released from Lewy bodies as a consequence of dopaminergic neuronal degeneration. As disease progresses, secretions from α-syn-activated microglia can engage neighboring glial cells in a cycle of autocrine and paracrine amplification of neurotoxic immune products. Such pathogenic processes affect the balance between a microglial neurotrophic and neurotoxic signature. We now report that microglia secrete both neurotoxic and neuroprotective factors after exposure to nitrated α-syn (N-α-syn). Proteomic (surface enhanced laser desorption–time of flight, 1D sodium dodecyl sulfate electrophoresis, and liquid chromatography-tandem mass spectrometry) and limited metabolomic profiling demonstrated that N-α-syn-activated microglia secrete inflammatory, regulatory, redox-active, enzymatic, and cytoskeletal proteins. Increased extracellular glutamate and cysteine and diminished intracellular glutathione and secreted exosomal proteins were also demonstrated. Increased redox-active proteins suggest regulatory microglial responses to N-α-syn. These were linked to discontinuous cystatin expression, cathepsin activity, and nuclear factor-kappa B activation. Inhibition of cathepsin B attenuated, in part, N-α-syn microglial neurotoxicity. These data support multifaceted microglia functions in PD-associated neurodegeneration.     

蛋白质组学——一门正在崛起的蛋白质分子生物学

人类基因组计划(HGP)全部基因测定的完成,标志着分子生物学已跨入后基因时代。由于基因组学不能阐明蛋白质表达的水平与时间、翻译后修饰、蛋白质与蛋白质相互作用等众多问题,人们于是提出了蛋白质组学概念,并以此来探讨细胞的结构与功能以及生命活动的本质和规律。本文以蛋白质组学的形成背景、分类、蛋白质的分离和鉴定方法、空间结构的预测及蛋白质组学在疾病的诊治、药物开发及对其它生物科学的贡献作一综述。     

生物医学新兴学科与中药现代化——现代组合成分药物的研究

中药的特色是通过多成分、多靶点的整体调控作用来系统纠正疾病造成的机体失衡。中药在治疗多基因、多因素引起的某些复杂疾病方面相对西药(通常是单一化学成分药物)有独到优势。但以天然动植物为来源的中药在质量控制和疗效科学依据方面的不足严重影响了中药在现代社会的应用。目前,组合治疗现代医学和系统生物学的发展为中药现代化提供了新的机会。提出应用现代组合成分药物和组合治疗新概念,科学阐明中药药效的学术观点;并提出综合应用蛋白质组学和化学生物学等生物医学新兴科技从中药中开发现代组合成分新药的技术。     

Proteomic biomarkers of endometrial cancer risk in women with polycystic ovary syndrome: a systematic review and biomarker database integration

Abstract

There is a need for research studies into the molecular mechanisms underpinning the link between polycystic ovary syndrome (PCOS) and endometrial cancer (EC) to facilitate screening and to encourage the development of novel strategies to prevent disease progression. The objective of this review was to identify proteomic biomarkers of EC risk in women with PCOS. All eligible published studies on proteomic biomarkers for EC identified through the literature were evaluated. Proteomic biomarkers for EC were then integrated with an updated previously published database of all proteomic biomarkers identified so far in PCOS women. Nine protein biomarkers were similarly either under or over expressed in women with EC and PCOS in various tissues. These include transgelin, pyruvate kinase M1/M2, gelsolin-like capping protein (macrophage capping protein), glutathione S-transferase P, leucine aminopeptidase (cytosol aminopeptidase), peptidyl-prolyl cis-transisomerase, cyclophilin A, complement component C4A and manganese-superoxide dismutase. If validated, these biomarkers may provide a useful framework on which the knowledge base in this area could be developed and will facilitate future mathematical modelling to enhance screening and prevention of EC in women with PCOS who have been shown to be at increased risk.     

Cell biology,chemogenomics and chemoproteomics – application to drug discovery

Cell biology has added immensely to the understanding of basic biologic concepts. However, scientists need to use cell biology more in the proteomic–genomic revolution. The authors have developed two novel techniques: transitional structural chemogenomics (TSCg) and transitional structural chemoproteomics (TSCp). TSCg is used to regulate gene expression by using ultrasensitive small-molecule drugs that target nucleic acids. By using chemicals to target transitional changes in the helical conformations of single-stranded (ss) and double-stranded (ds) DNA (e.g., B- to Z-DNA) and RNA (e.g., A- to Z-RNA), gene expression can be regulated (i.e., turning genes ‘on/off’ and variably controlling them). Alternative types of ds- and ssDNA and RNA (e.g., cruciform DNA) and other multistranded nucleic acids (e.g., triplex-DNA) are also targeted by this method. The authors’ second technique, TSCp, targets a protein before, during or after post-translational modifications, which alters the protein’s structure and function. These novel methods represent the next step in the evolution of chemical genomics and chemical proteomics. In addition, a novel multi-stranded (alternative) DNA, RNA and plasmid microarray has been developed that allows for the immobilization of intact, non-denatured dsDNA, alternative (i.e., exotic) and other multiple-stranded nucleic acids. This represents the next generation of nucleic acid microarrays, which will aid in the characterization of nucleic acids, studying the ageing process and improving the drug discovery process. The authors discuss how cell biology can be used to enhance genomics and proteomics. Cell biology will play a greater role during the postgenomic age and will help to enhance the omics/omes and drug discovery. It is the authors’ hope that these novel approaches can be used together with cellular biologic techniques to make major contributions towards understanding and manipulating different genomes.