Abstract:Endoplasmic reticulum associated degradation (ERAD) is a mechanism, which recognizes misfolded and unfolded proteins of the ER and retrotranslocates them into the cytoplasm for degradation by the ubiquitin-protesome machinery. Since it was discovered, many researches have conducted to contribute to understanding the mechanism of this conserved pathway. Recently, with the significant progresses in identification of new components revolved in substrate recognition, retrotranslocation and ubiquitylation, as well as the development of new techniques in this area, the specific molecular mechanism of ERAD becomes more clear. The recent progress in the ER stress response and the roles of the components related to ERAD process are summarized with focuses on their molecular mechanisms to provide an overview of this field. Also, the model ERAD substrates and the novel strategies recently developed are included.

Abstract:Identification of post-translational modifications is one of the most challenging tasks in proteomics, and the analysis of glycosylation is a very important yet difficult one among all post-translational modifications, which has attracted more and more attention in recent years. Mass spectrometry provides an effective way for the high-throughput analysis of glycosylation. Comparing with most of the other post-translational modifications, glycans are large and hetorogeneous, and glycans themselves could be fragmented in tandem mass spectrometry, in particular, the fragmentation patterns of glycans are quite different from those of peptides, resulting in difficulties in simultaneously identifying glycans and peptides of intact glycopeptides using proteomic analytical methods and software tools. The identification of intact N-glycopeptides is a hot spot in glycosylation research, for which various mass spectrometry-based analytical methods have been developed in recent years, including deglycosylation for the identification of N-glycosylated sites, electron transfer dissociation for the identification of peptide backbones, the combination of higher energy collisional dissociation and electron transfer dissociation or the combination of collision-induced dissociation and MS3 for complete identification of intact N-glycopeptides. In this article, we reviewed these analytical methods, and briefly pointed out the deficiencies of existing software tools, and suggested some future work.

Abstract:Preeclampsia (PE) is a severe pregnancy syndrome with multi-organ vascular dysfunction and classical symptoms, such as, maternal hypertension, proteinuria, glomerular endotheliosis and insufficient trophoblastic invasion. Although quite bit of animal models have been established for PE study, different animal models have both advantages and disadvantages. To date, there is still no one ideal animal model for PE study. This is also the reason that the etiology of PE is still unknown, and no effective treatments are available for PE. In the light of providing references for selecting the right animal model for investigations and establishing ideal animal models, we summarized the characteristic of different animal models for PE in this review.

Abstract:P-loop NTPases (GTPase and ATPase) are widely employed in both prokaryotes and eukaryotes to regulate various cellular processes. YchF and YihA are two highly conserved NTPases in bacteria, but their cellular roles remain elusive. Previous data revealed that the ribosome or ribosomal subunits are binding partners of these two NTPases. Here, we examined the binding preferences of Escherichia coli YchF and YihA to the 30S, 50S and 70S ribosomes in the presence of different nucleotides, and assayed whether these binding preferences were associated with the stimulation of their NTPase activities. Our data show that YchF and YihA display a strong preference for the 70S and 50S, respectively. While the 70S ribosome, but not the 50S or 30S, promotes both the ATPase and GTPase activities of YchF, YihA responds to both the 50S and 70S, with the moderate GTPase stimulation (～8.8 fold) seen in the presence of the 70S ribosome.

Abstract:Oligomers, rather than polymers and fibrils, of protein aggregates are thought to be cytotoxic, which is a milestone in the study of protein misfolding and aggregation. Abnormally high level of uric ribose in type 2 diabetic patients and ribosylated animal models indicate that diabetes is not only correlated with metabolic dysfunction in glucose but also ribose. Here, using ribosylation of bovine serum albumin (BSA), we show that ribosylated BSA aggregates and proceeds from a monomer and onto an oligomer and polymer, observed with fluorescence spectrophotometer, atomic force microscopy, transmission electron microscopy and size exclusion chromatography. Ribosylated monomer showed severely cytotoxic to SH-SY5Y cells (a human neuroblastoma cell line) under the observations by assays of CCK-8, LDH activity, TUNEL staining, caspase-3 activity and flow-cytometry, whereas ribosylated oligomer and polymer did not. The cytotoxic effect of the ribosylated monomer likely occurs by inducing neuronal apoptosis through activation of the receptor of AGEs (RAGE) associated with mitogen-activated protein kinases (MAPK) pathways.

Abstract:Chronic granulomatous disease (CGD) is an inherited disorder of the immune system due to mutations in gp91phox gene which coding for NOX2 protein. Bacterial and fungal infections are most common infections in CGD patients. β-glucan (curdlan) is the main component of the cell wall of Candida albicans. At present, the molecular mechanism of dectin-1 in macrophage which involved in immune inflammation induced by curdlan is not very clear. In order to study the molecular mechanism, we estimated the level of ROS production by luminal and Ampled Red; estimated levels of IL-1β by ELISA; and estimated NF-κB signaling pathway by immunofluorescence assay. Our results showed that curdlan induced ROS production in macrophage in a dose-dependent way. Dectin-1 deficiency can partially reduce ROS generation and partially block the activation of NF-κB signaling pathway. NOX2 deficient mice displayed high level of IL-1β after curdlan injection and IL-1β level decreased in NOX2 KO mice when dectin-1 was knocked out. IL-1β level in Dectin-1 KO mice was as same as in WT mice while significantly different from Dectin-1/NOX2 KO mice. In summary, curdlan can activate NOX2 to generate ROS through recognition of foreign pathogens by receptor dectin-1 which existed in macrophage cell surface. Besides that, immune response induced by curdlan activated NF-κB signaling pathway and increased IL-1β level through dectin-1. Dectin-1 was involved in this process, but it was not the only pattern recognition receptor.

Abstract:Autophagy and apoptosis are considered as two main kinds of programmed cell death. The crosstalk between autophagy and apoptosis is crucial for illustration of anti-tumor drug’s effects. Triptolide, a diterpene compound extracted from Tripterygium wilfordii Hook F, attracts increasing attention from researchers worldwide for its broad anti-tumor spectrum. Triptolide could bind to XPB (a subunit of the transcription factor TFⅡH) and leads to RNA polymerase Ⅱ-mediated transcription inhibition. Also, triptolide induces autophagy and apoptosis processes in cancer cells. Through regulating different kinds of related proteins, tumor repressor p53, plays key roles in the crosstalk between autophagy and apoptosis. Thus, in this study, the authors focused on triptolide induced p53-dependent autophagy and apoptosis processes in HeLa cells, and to demonstrate the regulation of crosstalk through p53-dependent transcription inhibitor and autophagy inhibitors treatment. Using CCK-8 assay and clonogenic assay, triptolide shows significant inhibition effect on HeLa cells. The apoptosis analysis by Western blot has shown that triptolide induces the cleavage of caspase3 and nuclear poly (ADP-ribose) polymerase (PARP). Immuno-fluorescence and Western blot assay have shown that triptolide also leads to LC3-Ⅱ accumulation and p62 degradations. Collectively, triptolide induces autophagy and apoptosis in a time- and dose-dependent manner. Meanwhile, p53 is significantly up-regulated and the mammalian target of rapamycin (mTOR) is down-regulated with triptolide treatment, and ultimately results in activation of autophagy and apoptosis. Western blot assay have shown that triptolide-induced autophagy and apoptosis are partly inhibited by utilizing p53-dependent transcription inhibitor (Pifithrin-α). Furthermore, the combination treatment of autophagy inhibitors with triptolide enhances triptolide induced apoptosis, and significantly improves inhibition effect of triptolide in HeLa cells. Triptolide induces p53-dependent autophagy and apoptosis in HeLa cells. p53 functions as a key regulator in the triptolide induced autophagy and apoptosis crosstalk.

Abstract:Muscle satellite cells play an important role in the growth and development as well as postnatal injury and repair of skeletal muscles, however, it keeps unclear about muscle satellite cell plasticity, effect and mechanism in atrophied muscle. In the present study, we investigated the changes in the satellite cell number and potency of proliferation and differentiation during weightless muscle atrophy induced by mouse hindlimb suspension, and found that the number of quiet satellite cells significantly increased but that of activated cells significantly decreased, and the potential of cell myogenic differentiation also appeared a reduced tendency. Moreover, we verified the above changes of muscle satellite cell plasticity in the cultures of ex vivo single myofibers isolated from weightless atrophied soleus. Finally, Smad3 knockout mice and its wild-type littermates were subjected to hindlimb suspension induced muscle atrophy, and the variability analysis demonstrated that the key role of Smad3 in regulating the plasticity of muscle satellite cells weightless muscle atrophy.

Abstract:Loading cryoprotectants (CPAs) into oocytes is critical to restrain ice formation during cryopreservation. However, high concentration of CPAs may cause osmotic and toxic damage to oocytes. In order to minimize the osmotic damage, a microfluidic device for loading CPAs into oocytes was designed and fabricated in this study. 30% (v/v) Me₂SO was linear loaded into porcine MⅡ-stage oocyte with the microfluidic device, the intracellular CPA concentration, the cell volumetric changes and the effect on the survival rate and developmental rate of oocytes were investigated and compared with the traditional methods including the one-step method and the step-wise methods. The results showed that the microfluidic method can realize continuous CPA loading to oocytes, reduce the osmotic shock to cells and enhance the cell viability. The lowest penetration volume of oocyte reached 0.86V₀. The cell viability was 92.8% with the microfluidic method, ～33% higher than one-step method, ～16.3% higher than two-step method, and no significant difference with four-step method. After parthenogenetic activation and cultured in vitro, the cleavage rate and the blastocyst rate of oocytes were 75.8% and 27.4%, which were significantly higher than the one-step method and the step-wise methods (P < 0.05). In conclusion, linear loading CPAs with the microfluidic method can significantly alleviate the osmotic damage to oocytes, which may provide a new path for oocyte cryopreservation.

Abstract:Effects of Protein Corona on Intracellular Uptake and Cytotoxicity of PM₁

Abstract:Perceiving Beauteousness with Our Peripheral Vision