A computational framework for the design of optimal protein synthesis

Despite the establishment of design principles to optimize codon choice for heterologous expression vector design, the relationship between codon sequence and final protein yield remains poorly understood. In this work, we present a computational framework for the identification of a set of mutant codon sequences for optimized heterologous protein production, which uses a codon-sequence mechanistic model of protein synthesis. Through a sensitivity analysis on the optimal steady state configuration of protein synthesis we are able to identify the set of codons, that are the most rate limiting with respect to steady state protein synthesis rate, and we replace them with synonymous codons recognized by charged tRNAs more efficient for translation, so that the resulting codon-elongation rate is higher. Repeating this procedure, we iteratively optimize the codon sequence for higher protein synthesis rate taking into account multiple constraints of various types. We determine a small set of optimized synonymous codon sequences that are very close to each other in sequence space, but they have an impact on properties such as ribosomal utilization or secondary structure. This limited number of sequences can then be offered for further experimental study. Overall, the proposed method is very valuable in understanding the effects of the different properties of mRNA sequences on the final protein yield in heterologous protein production and it can find applications in synthetic biology and biotechnology.     

Differential expression of a WD protein during squamous differentiation of tracheal epithelial cells

The lining of the trachea consists of a pseudostratified, mucociliary epithelium that under a variety of conditions, such as vitamin A deficiency, toxic and mechanical injury, becomes a stratified squamous epithelium. Several in vitro cell culture models have been established to study the process of differentiation of airway epithelium. Such studies have indicated that mucosecretory differentiation of tracheal epithelial cells can be modulated by substratum. This study was undertaken to understand molecular mechanisms of squamous differentiation in tracheal epithelia. Primary cultured tracheal cells grown on uncoated filters were differentiated to single layer of squamous cells, whereas cells were grown as stratified columnar cells on collagen-I coated filters. The responses to secretagogues were altered according to culture conditions. DD-PCR revealed that FAK and a WD protein expression was increased in squamous tracheal epithelia. Expression of a WD protein was changed by the treatment of retinoic acid in various epithelial cells. These results indicated that squamous differentiation of tracheal cells changes the expression of a variety of genes, and that the experimental model for this study can be employed to study molecular mechanisms of squamous differentiation in airway epithelial cells.     

The rate of fluid shear stress is a potent regulator for the differentiation of mesenchymal stem cells

We have previously demonstrated that the rate of fluid shear stress (ΔSS) can manipulate the fate of mesenchymal stem cells (MSCs) to osteogenic or chondrogenic cells. However, whether ΔSS is comparable to other two means of induction medium and substrate stiffness that have been proven to be potent in differentiation control is unknown. In this study, we subjected MSCs to 1–7 days of osteogenic or chondrogenic chemical induction, or 1–4 days of 37 or 86 kPa of substrate stiffness induction, followed by 20 min of Fast ΔSS (0–0′) or Slow ΔSS (0–2′), which is a laminar FSS that linearly increased from 0 to 10 dyn/cm ² in 0 (Fast) or 2 min (Slow) and maintained at 10 dyn/cm ² for a total of 20 min. We found that 20 min of ΔSS could compete with 5 days' chemical and 2 days' substrate stiffness inductions. Our study confirmed that ΔSS is a powerful tool to control the differentiation of MSCs, which stressed the possible application in MSCs linage specification.     

Dlx5 is a positive regulator of chondrocyte differentiation during endochondral ossification

The process of endochondral ossification in which the bones of the limb are formed after generation of cartilage models is dependent on a precisely regulated program of chondrocyte maturation. Here, we show that the homeobox-containing gene Dlx5 is expressed at the onset of chondrocyte maturation during the conversion of immature proliferating chondrocytes into postmitotic hypertrophying chondrocytes, a critical step in the maturation process. Moreover, retroviral misexpression of Dlx5 during differentiation of the skeletal elements of the chick limb in vivo results in the formation of severely shortened skeletal elements that contain excessive numbers of hypertrophying chondrocytes which extend into ectopic regions, including sites normally occupied by immature chondrocytes. The expansion in the extent of hypertrophic maturation detectable histologically is accompanied by expanded and upregulated domains of expression of molecular markers of chondrocyte maturation, particularly type X collagen and osteopontin, and by expansion of mineralized cartilage matrix, which is characteristic of terminal hypertrophic differentiation. Furthermore, Dlx5 misexpression markedly reduces chondrocyte proliferation concomitant with promoting hypertrophic maturation. Taken together, these results indicate that Dlx5 is a positive regulator of chondrocyte maturation and suggest that it regulates the process at least in part by promoting conversion of immature proliferating chondrocytes into hypertrophying chondrocytes. Retroviral misexpression of Dlx5 also enhances formation of periosteal bone, which is derived from the Dlx5-expressing perichondrium that surrounds the diaphyses of the cartilage models. This suggests that Dlx5 may be involved in regulating osteoblast differentiation, as well as chondrocyte maturation, during endochondral ossification.     

Protein expression profile in the differentiation of rat bone marrow stromal cells into Schwann cell-like cells

During the last decade, increasing evidence suggested that bone marrow stromal cells (MSCs) have the potential to differentiate into neural lineages. Many studies have reported that MSCs showed morphological changes and expressed a limited number of neural proteins under experimental conditions. However, no proteomic studies on MSCs differentiated into Schwann cell-like cells have been reported. In this study, we isolated MSCs from adult Sprague-Dawley rat femur and tibia bone marrows and induced the cells in vitro under specific conditions. By using two-dimensional gel electrophoresis (2-DE), we compared the protein profiles of MSCs before and after induced differentiation. We obtained 792 protein spots in the protein profile by 2-DE, and found that 74 spots changed significantly before and after the differentiation using PDQuest software, with 43 up-regulated and 31 down-regulated. We analyzed these 74 spots by a matrix assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS) and by database searching, and found that they could be grouped into various classes, including cytoskeleton and structure proteins, growth factors, metabolic proteins, chaperone proteins, receptor proteins, cell cycle proteins, calcium binding proteins, and other proteins. These proteins also include neural and glial proteins, such as BDNF, CNTF and GFAP. The results may provide valuable proteomic information about the differentiation of MSCs into Schwann cell-like cells. Supported by National High-Tech Research and Development Program of China (Grant No. 2006AA02A128) and National Natural Science Foundation of China (Grant No. 30670667).     

History of protein crystallography in China

China has a strong background in X-ray crystallography dating back to the 1920s. Protein crystallography research in China was first developed following the successful synthesis of insulin in China in 1966. The subsequent determination of the three-dimensional structure of porcine insulin made China one of the few countries which could determine macromolecular structures by X-ray diffraction methods in the late 1960s and early 1970s. After a slow period during the 1970s and 1980s, protein crystallography in China has reached a new climax with a number of outstanding accomplishments. Here, I review the history and progress of protein crystallography in China and detail some of the recent research highlights, including the crystal structures of two membrane proteins as well as the structural genomics initiative in China.     

A map of human protein interactions derived from co‐expression of human mRNAs and their orthologs

The human protein interaction network will offer global insights into the molecular organization of cells and provide a framework for modeling human disease, but the network's large scale demands new approaches. We report a set of 7000 physical associations among human proteins inferred from indirect evidence: the comparison of human mRNA co‐expression patterns with those of orthologous genes in five other eukaryotes, which we demonstrate identifies proteins in the same physical complexes. To evaluate the accuracy of the predicted physical associations, we apply quantitative mass spectrometry shotgun proteomics to measure elution profiles of 3013 human proteins during native biochemical fractionation, demonstrating systematically that putative interaction partners tend to co‐sediment. We further validate uncharacterized proteins implicated by the associations in ribosome biogenesis, including WBSCR20C, associated with Williams–Beuren syndrome. This meta‐analysis therefore exploits non‐protein‐based data, but successfully predicts associations, including 5589 novel human physical protein associations, with measured accuracies of 54±10%, comparable to direct large‐scale interaction assays. The new associations’ derivation from conserved in vivo phenomena argues strongly for their biological relevance.     

Protein kinases predominately expressed in human ES cell lines during differentiation

Capacity of human embryonic stem cells (ESC) for unlimited proliferation and differentiation make them an attractive object in fundamental science and medicine. Little is known about the mechanisms that direct cells to particular differentiation or sustain them in an undifferentiated state. Activation of these mechanisms is determined by gene expression mediated by cascades of signal transduction. Protein kinases are essential components of signal pathways. The study of protein kinases expression in ESC and embryoid bodies facilitates a better understanding of the processes underlying the differentiation stages. We isolated cDNA libraries with fragments of catalytic domains of protein kinases expressed in human ESC and embryoid bodies (EB) of hESM01 and hESM02 cell lines. Using Northern hybridization, we revealed a high level of protein kinases MAK-V in human ESC. Expressions of MAK-V, A-RAF-1, MAPK3, IGF1R, NEK3, and NEK7 in ESC and EB in hESM01 and hESM02 cell lines were compared by the semiquantitative method RT-PCR.     

基于人类蛋白质图集的蛋白质表达的总论

新的人类蛋白质图集4.0版本上已经含有了对应5000个人类基因的6000多种抗体。这个版本里已经拥有500多万张高分辨率的免疫组化和激光共聚焦图片。每张图片都是经过优秀的病理学家的注释,从而为功能研究提供知识储备,也可以进行正常和病理组织中蛋白质表达谱的查询和文献检索。一个新的结构实现了,它包括了所有预测的基因(大约20400个),并且带有可视化的所有编码蛋白质基因的特征。一个新的搜索工具也已经启动了,它可以执行高级检索功能,包括染色体定位、蛋白质分级和(或)组织特异性的检索。蛋白质图集作为一种搜索工具可以发现癌症诊断学的潜在生物标志物。     

Characterization of the EGFR interactome reveals associated protein complex networks and intracellular receptor dynamics

Growth factor receptor mediated signaling is meanwhile recognized as a complex signaling network, which is initiated by recruiting specific patterns of adaptor proteins to the intracellular domain of epidermal growth factor receptor (EGFR). Approaches to globally identify EGFR‐binding proteins are required to elucidate this network. We affinity‐purified EGFR with its interacting proteins by coprecipitation from lysates of A431 cells. A total of 183 proteins were repeatedly detected in high‐resolution MS measurements. For 15 of these, direct interactions with EGFR were listed in the iRefIndex interaction database, including Grb2, shc‐1, SOS1 and 2, STAT 1 and 3, AP2, UBS3B, and ERRFI. The newly developed Cytoscape plugin ModuleGraph allowed retrieving and visualizing 93 well‐described protein complexes that contained at least one of the proteins found to interact with EGFR in our experiments. Abundances of 14 proteins were modulated more than twofold upon EGFR activation whereof clathrin‐associated adaptor complex AP‐2 showed 4.6‐fold enrichment. These proteins were further annotated with different cellular compartments. Finally, interactions of AP‐2 proteins and the newly discovered interaction of CIP2A could be verified. In conclusion, a powerful technique is presented that allowed identification and quantitative assessment of the EGFR interactome to provide further insight into EGFR signaling.     

Autoimmune regulator, Aire, is a novel regulator of chondrocyte differentiation

Chondrocyte differentiation is controlled by various regulators, such as Sox9 and Runx2, but the process is complex. To further understand the precise underlying molecular mechanisms of chondrocyte differentiation, we aimed to identify a novel regulatory factor of chondrocyte differentiation using gene expression profiles of micromass-cultured chondrocytes at different differentiation stages. From the results of microarray analysis, the autoimmune regulator, Aire, was identified as a novel regulator. Aire stable knockdown cells, and primary cultured chondrocytes obtained from Aire^−/− mice, showed reduced mRNA expression levels of chondrocyte-related genes. Over-expression of Aire induced the early stages of chondrocyte differentiation by facilitating expression of Bmp2. A ChIP assay revealed that Aire was recruited on an Airebinding site (T box) in the Bmp2 promoter region in the early stages of chondrocyte differentiation and histone methylation was modified. These results suggest that Aire can facilitate early chondrocyte differentiation by expression of Bmp2 through altering the histone modification status of the promoter region of Bmp2.     

The effect of the dw gene on the muscle protein turnover rate in chickens

Fractional rates (%/day) of muscle protein synthesis and degradation of the genotypes Dw/Dw and dw/dw of male White Plymouth Rock chickens were determined by measuring the output of N-methylhistidine (N-MH) in the excreta at 2, 4, and 8 weeks of age. The fractional growth rate of dw/dw was significantly lower (P<0.05) than that of Dw/Dw at 2 weeks of age but not at 4 and 8 weeks of age. No significant differences in the degradation rate (K _d; %/day) were found at any age. A significant difference (P<0.05) between genotypes in the rate of synthesis (K _s; %/day) was found at 2 weeks of age (Dw/Dw=11.8, dw/dw=9.9) but not at 4 and 8 weeks of age. These results suggest that the dw gene has a depressing effect on the synthesis rate of muscle protein, and the difference between genotypes in the growth rate at the early stage is a reflection of this effect.     

Proteome dynamics during contractile and metabolic differentiation of bovine foetal muscle

Contractile and metabolic properties of bovine muscles play an important role in meat sensorial quality, particularly tenderness. Earlier studies based on Myosin heavy chain isoforms analyses and measurements of glycolytic and oxidative enzyme activities have demonstrated that the third trimester of foetal life in bovine is characterized by contractile and metabolic differentiation. In order to complete this data and to obtain a precise view of this phase and its regulation, we performed a proteomic analysis of Semitendinosus muscle from Charolais foetuses analysed at three stages of the third trimester of gestation (180, 210 and 260 days). The results complete the knowledge of important changes in the profiles of proteins from metabolic and contractile pathways. They provide new insights about proteins such as Aldehyde dehydrogenase family, Enolase, Dihydrolipoyl dehydrogenase, Troponin T or Myosin light chains isoforms. These data have agronomical applications not only for the management of beef sensorial quality but also in medical context, as bovine myogenesis appears very similar to human one.     

Enzymology and significance of protein histidine methylation

Cells synthesize proteins using 20 standard amino acids and expand their biochemical repertoire through intricate enzyme-mediated post-translational modifications (PTMs). PTMs can either be static and represent protein editing events or be dynamically regulated as a part of a cellular response to specific stimuli. Protein histidine methylation (Hme) was an elusive PTM for over 5 decades and has only recently attracted considerable attention through discoveries concerning its enzymology, extent, and function. Here, we review the status of the Hme field and discuss the implications of Hme in physiological and cellular processes. We also review the experimental toolbox for analysis of Hme and discuss the strengths and weaknesses of different experimental approaches. The findings discussed in this review demonstrate that Hme is widespread across cells and tissues and functionally regulates key cellular processes such as cytoskeletal dynamics and protein translation. Collectively, the findings discussed here showcase Hme as a regulator of key cellular functions and highlight the regulation of this modification as an emerging field of biological research.