蛋白质组学在干细胞研究中的应用

蛋白质组学技术通过整合多项技术来分析生物体的全部蛋白质成分,通过考察不同状态下细胞或组织蛋白质组的变化情况来了解细胞活动的分子机理。干细胞分化过程中受外界条件的影响其蛋白表达模式也表现出一定的差异,对干细胞分化过程中进行蛋白质组学研究将有利于从蛋白质分子水平上阐明干细胞的分化机理。本文对蛋白质组学及其在干细胞研究中的应用加以评述。     

Neurogenesis and neuronal regeneration in the adult fish brain

Fish are distinctive in their enormous potential to continuously produce new neurons in the adult brain, whereas in mammals adult neurogenesis is restricted to the olfactory bulb and the hippocampus. In fish new neurons are not only generated in structures homologous to those two regions, but also in dozens of other brain areas. In some regions of the fish brain, such as the optic tectum, the new cells remain near the proliferation zones in the course of their further development. In others, as in most subdivisions of the cerebellum, they migrate, often guided by radial glial fibers, to specific target areas. Approximately 50% of the young cells undergo apoptotic cell death, whereas the others survive for the rest of the fish’s life. A large number of the surviving cells differentiate into neurons. Two key factors enabling highly efficient brain repair in fish after injuries involve the elimination of damaged cells by apoptosis (instead of necrosis, the dominant type of cell death in mammals) and the replacement of cells lost to injury by newly generated ones. Proteome analysis has suggested well over 100 proteins, including two dozen identified ones, to be involved in the individual steps of this phenomenon of neuronal regeneration.     

Proteomic identification of processes and pathways characteristic of osmoregulatory tissues in spiny dogfish shark (Squalus acanthias)

We used dogfish shark (Squalus acanthias) as a model for proteome analysis of six different tissues to evaluate tissue-specific protein expression on a global scale and to deduce specific functions and the relatedness of multiple tissues from their proteomes. Proteomes of heart, brain, kidney, intestine, gill, and rectal gland were separated by two-dimensional gel electrophoresis (2DGE), gel images were matched using Delta 2D software and then evaluated for tissue-specific proteins. Sixty-one proteins (4%) were found to be in only a single type of tissue and 535 proteins (36%) were equally abundant in all six tissues. Relatedness between tissues was assessed based on tissue-specific expression patterns of all 1465 consistently resolved protein spots. This analysis revealed that tissues with osmoregulatory function (kidney, intestine, gill, rectal gland) were more similar in their overall proteomes than non-osmoregulatory tissues (heart, brain). Sixty-one proteins were identified by MALDI-TOF/TOF mass spectrometry and biological functions characteristic of osmoregulatory tissues were derived from gene ontology and molecular pathway analysis. Our data demonstrate that the molecular machinery for energy and urea metabolism and the Rho-GTPase/cytoskeleton pathway are enriched in osmoregulatory tissues of sharks. Our work provides a strong rationale for further study of the contribution of these mechanisms to the osmoregulation of marine sharks.     

Schistosoma japonicum translationally controlled tumour protein,which is associated with the development of female worms,as a target for control of schistosomiasis

Schistosomiasis is a globally important helminthic disease of both humans and animals, and is the second most common parasitic disease after malaria. Although praziquantel is extensively used for treatment of parasitic diseases, drug resistance has been reported. Therefore, new drugs and effective vaccines are needed for continuous control of schistosomiasis. Eggs produced by schistosomes are responsible for the occurrence and spread of schistosomiasis. Revealing the reproductive mechanism of schistosomes will help to control this disease. In this study, the proteomic profiles of single-sex infected female worms and bisexual infected mature female worms of Schistosoma japonicum at 18, 21, 23 and 25 days p.i. were identified with isobaric tags for relative quantitation-coupled liquid chromatography–tandem mass spectrometry. Differentially expressed proteins were subsequently used for bioinformatic analysis. Six highly expressed differentially expressed proteins in mature female worms were selected and long-term interference with small interfering RNA (siRNA) was conducted to determine biological functions. SiRNA against S. japonicum translationally controlled tumour protein (SjTCTP) resulted in the most significant effect on the growth and development of MF worms. Sjtctp mRNA expression gradually increased over time with a high level of expression maintained at 25–42 days p.i., while levels were significantly higher in mature female worms than male and SF worms. The subsequent animal immune protection experiments showed that recombinant SjTCTP (rSjTCTP) reduced the number of adults by 44.7% (P < 0.01), average egg burden per gram of liver by 57.94% (P < 0.01), egg hatching rate by 47.57% (P < 0.01), and oviposition of individual females by 43.16%. rSjTCTP induced higher levels of serum IgG, IL-2, and IL-10 in mice. Collectively, these results show that SjTCTP is vital to reproduction of female worms and, thus, is a candidate antigen for immune protection.     

Emerging chemistry and biology in protein glutathionylation

Protein S-glutathionylation serves a regulatory role in proteins and modulates distinct biological processes implicated in health and diseases. Despite challenges in analyzing the dynamic and reversible nature of S-glutathionylation, recent chemical and biological methods have significantly advanced the field of S-glutathionylation, culminating in selective identification and detection, structural motif analysis, and functional studies of S-glutathionylation. This review will highlight emerging studies of protein glutathionylation, beginning by introducing biochemical tools that enable mass spectrometric identification and live-cell imaging of S-glutathionylation. Next, it will spotlight recent examples of S-glutathionylation regulating physiology and inflammation. Lastly, we will feature two emerging lines of glutathionylation research in cryptic cysteine glutathionylation and protein C-glutathionylation.     

Recent advances in induced proximity modalities

Challenging disease targets necessitate new approaches for therapeutic intervention. Rewiring protein-biomolecule interactions with proximity-inducing agents extends intervention opportunities beyond target agonism or inhibition. Spanning varied molecular phenotypes and diverse target classes, proximity-inducing agents demonstrate immense potential across target degradation, cleavage, and post-translational editing. Here, we review a selection of exciting developments in the concepts and mechanisms of induced proximity-driven strategies from the last two years. Key technological advances that enable these discoveries and expand the scope of targets and machinery for induced-proximity modalities are highlighted.     

Extracellular vesicles from thyroid cancer harbor a functional machinery involved in extracellular matrix remodeling

Extracellular vesicles (EVs) participate in cell-stroma crosstalk within the tumor microenvironment and fibroblasts (Fb) contribute to tumor promotion in thyroid cancer. However, the role of tumor-stroma derived EVs still needs to be deciphered. We hypothesized that the interaction of thyroid tumor cells with Fb would liberate EVs with a specific proteomic profile, which would have an impact on EV-functionality in thyroid tumor progression-related events. Tumor (TPC-1, 8505c) and non-tumor (NThyOri) thyroid cells were co-cultured with human Fb. EVs, obtained by ultracentrifugation of conditioned media, were characterized by nanoparticle tracking analysis and western blotting. EV-proteomic analysis was performed by mass-spectrometry, and metalloproteinases (MMPs) were studied by zymography. EV-exchange was evaluated using immunofluorescence, confocal microscopy and FACS. EVs expressed classical exosome markers, with EVs from thyroid tumor cell-Fb co-cultures showing a proteomic profile related to extracellular matrix (ECM) remodeling. Bidirectional crosstalk between Fb and TPC-1 cells produced significantly more EVs than their isolated cells, and potentiated EV-functionality. In line with this, Fb-TPC-1 derived EVs induced MMP2 activation in NThyOri supernatants, and MMP2 activity could be evidenced in Fb and TPC-1 contact-independent co-cultures. Besides, MMP2 interactors allowed us to discriminate between EVs from thyroid tumoral and non-tumoral milieus. Interestingly, Fb internalized more EVs from TPC-1 than from NThyOri producing cells. Fb and thyroid tumor cell crosstalk produces specialized EVs with an ECM remodeling proteomic profile, enabling activation of MMP2 and possibly facilitating ECM-degradation, which is potentially linked with thyroid tumor progression.     

Preparing to Work with Big Data in Proteomics – A Report on the HUPO‐PSI Spring Workshop

The Human Proteome Organisation Proteomics Standards Initiative (HUPO‐PSI) was established in 2002 with the aim of defining community standards for data representation in proteomics and facilitating data comparison, exchange and verification. The 2013 annual spring workshop was hosted by the University of Liverpool, UK and concentrated on updating and refining the existing standards in the light of new methodologies and technologies. To control the inflation of file sizes, strategies for file compression, particularly for mzML files, were explored. Best practices for encoding information such as protein grouping and PTM localisation were refined and documented. Additional example files for the mzQuantML format were designed to provide support for selected reaction monitoring techniques. Enhancements to the PSI Common Query Interface (PSICQUIC) and PSI‐MI XML were discussed. Finally, the group engaged in discussion on how the existing work of the HUPO‐PSI can be leveraged by the Metabolomics Standards Initiative to improve the capture of metabolite data.