The study of protein-protein interactions is an essential process to understand the biological functions of proteins and the underlying mechanisms. Co-immunoprecipitation coupled with mass spectrometry (CoIP-MS) is one of the most extensively used high-throughput techniques to discover novel protein-protein interactions. However, the traditional CoIP process uses whole cell lysate, disrupts cellular organization, and leads to potential false positives by inducing artificial protein-protein interactions. Here, we have developed a strategy by combining subcellular fractionation with CoIP-MS to study the interacting proteins of the complement component 1, q subcomponent binding protein (C1QBP) in the mitochondria. Using this method, a novel C1QBP interacting protein, dihydrolipoyllysine-residue acetyltransferase component of pyruvate dehydrogenase complex, mitochondrial (DLAT) was identified and validated. Furthermore, the activity of the pyruvate dehydrogenase (PDH) was found to be affected by the expression level of C1QBP. These results provide novel insights regarding the mitochondrial function of C1QBP in the regulation of cellular energy metabolism. This method could also be used to analyze the subcellular protein-protein interactions for other proteins of interest.

The Y-box-binding protein 1 (YBX1) plays a critical role in tumorigenesis by promoting cell proliferation, overriding cell-cycle check points, and enhancing genomic instability. In this study, the interactome of YBX1 in renal cell carcinoma (RCC) was analyzed by coimmunoprecipitation and mass spectrometry to better understand its function and regulatory mechanism. A total of 129 proteins were identified as potential YBX1 binding partners. The interaction between the complement component 1, q subcomponent binding protein (C1QBP), and YBX1 was further confirmed by immunoprecipitation and Western blotting. Knockdown of C1QBP enhanced the phosphorylation of YBX1and its nuclear translocation, indicating that C1QBP negatively regulated YBX1 activation. The clinical significance of these two proteins was analyzed in the tissues from 52 RCC patients by immunohistochemistry. Expression of YBX1 was markedly elevated in the carcinoma tissues, and its nuclear expression was associated with histological T stage and metastasis. Meanwhile, the level of C1QBP in the carcinoma tissues was significantly lower than that in the adjacent healthy tissues, which was negatively correlated with the nuclear localization of YBX1 in the RCC tissues (P = 0.011). These data suggest that C1QBP is a novel regulator of YBX1, and the expression of C1QBP and the nuclear expression of YBX1 could both be used as independent prognostic makers for cancer progression in the RCC patients. The proteomics data have been deposited to the ProteomeXchange with identifier PXD001493.

Highly fluorescent quantum dot (QD) probes have attracted great attention in the field of cancer detection. But before they can be applied in clinical practice, further work is required. In this study, QDs were prepared by chemical method and the obtained QDs were characterized by ultraviolet irradiation; the bioconjugation effects between QDs and α-fetoprotein (AFP) monoclonal antibodies were analyzed by fluorescence (FL) imaging and Coomassie Blue assay; the diameter of the obtained QD probes were measured by transmission electronic microscopy (TEM); size distribution and zeta potential were studied by a laser diffraction particle size analyzer; the optical properties of the QD probes were analyzed by UV-Vis absorption spectrometry and fluorescence spectrometry; the targeting capability of the obtained probes was evaluated by labeling Hepatocellular Carcinoma (HCC) cells. The results showed that QDs had a high FL property, AFP monoclonal antibodies could steadily couple with QDs; the diameter of QDs became slightly larger after bioconjugation, and the hydrodynamic size of QDs also increased from ca. 10 nm to about 95 nm after QD coupling with antibodies. The absorption properties of QDs did not remarkably change before and after combination with antibodies; a blue shift of about 4 nm to 5 nm of the FL peaks occurred after QD coupling with AFP antibodies, which is probably mainly attributed to the electronegativity increase of the QD surface due to amine groups in the PEG molecules; and the formed FL probes could specifically and accurately target the AFP antigen in HCCs. These FL probes could be designed and constructed according to the clinical practice needs.

This study designs a novel nanoparticle system with core–shell structure based on pullulan and poly(β-amino) ester (PBAE) for the hepatoma-targeted codelivery of gene and chemotherapy agent. Plasmid DNA expressing green fluorescent protein (pEGFP), as a model gene, was fully condensed with cationic PBAE to form the inner core of PBAE/pEGFP polycomplex. Methotrexate (MTX), as a model chemotherapy agent, was conjugated to pullulan by ester bond to synthesize polymeric prodrug of MTX-PL. MTX-PL was then adsorbed on the surface of PBAE/pEGFP polycomplex to form MTX-PL/PBAE/pEGFP nanoparticles with a classic core–shell structure. MTX-PL was also used as a hepatoma targeting moiety, because of its specific binding affinity for asialoglycoprotein receptor (ASGPR) overexpressed by human hepatoma HepG2 cells. MTX-PL/PBAE/pEGFP nanoparticles realized the efficient transfection of pEGFP in HepG2 cells and exhibited significant inhibitory effect on the cell proliferation. In HepG2 tumor-bearing nude mice, MTX-PL/PBAE/pEGFP nanoparticles were mainly distributed in the tumor after 24 h postintravenous injection. Altogether, this novel codelivery system with a strong hepatoma-targeting property achieved simultaneous delivery of gene and chemotherapy agent into tumor at both cellular and animal levels.Keywords: chemotherapy agent; gene; hepatoma; poly(β-amino) ester; pullulan

This study reports the design of a novel pH-sensitive nanoparticle carrier based on pullulan for realizing intracellular drug delivery. A series of pullulan derivatives (UCPA) were synthesized by conjugation of both urocanic acid (a pH-sensitive grafted moiety) and cholesterol succinate (a hydrophobically modified moiety) to pullulan. UCPAs exhibited amphiphilic and pH-sensitive properties, and their responding pH value was around 6.5. UCPA nanoparticles prepared by the precipitation method had roughly spherical shapes, and sizes ranging from 150 to 300 nm. Doxorubicin (DOX), as a model antitumor drug, was physically loaded into the UCPA nanoparticles and its in vitro release at different pH values was studied using a dialysis method. UCPA-1 nanoparticles, with the degree of substitution (DS) of urocanyl and cholesterol moieties of 6.8% and 3.5%, respectively, exhibited relatively high drug-loading capability and strong in vitro pH-induced drug release. The results of MTT assays, flow cytometric analyses and confocal microscopy observations confirmed that the UCPA-1 nanoparticles can realize the intracellular delivery of DOX after internalization and enhanced cytotoxicity of DOX against MCF-7 cells.

Microfluidic devices have emerged as revolutionary, novel platforms for in vitro drug evaluation. In this work, we developed a facile method for evaluating antihypertensive drugs using a microfluidic chip. This microfluidic chip was generated using the elastic material poly(dimethylsiloxane) (PDMS) and a microchannel structure that simulated a blood vessel as fabricated on the chip. We then cultured human umbilical vein endothelial cells (HUVECs) inside the channel. Different pressures and shear stresses could be applied on the cells. The generated vessel mimics can be used for evaluating the safety and effects of antihypertensive drugs. Here, we used hydralazine hydrochloride as a model drug. The results indicated that hydralazine hydrochloride effectively decreased the pressure-induced dysfunction of endothelial cells. This work demonstrates that our microfluidic system provides a convenient and cost-effective platform for studying cellular responses to drugs under mechanical pressure.Keywords: antihypertensive drug evaluation; endothelial cells; mechanical pressure; microfluidic chip;

Chemotaxis plays an important role in metastasis. In our previous studies, we reported that protein kinase C ζ (PKCζ) mediated cancer cell chemotaxis by regulating cytoskeleton rearrangement and cell adhesion. To further study the molecular mechanism of chemotaxis, mass spectrometry-based approaches were employed to investigate the interactome of PKCζ and its changes upon stimulation by epidermal growth factor (EGF). As a result, 233 proteins were identified as potential PKCζ binding partners. Label free quantification was applied to examine the quantitative changes of these interactions involved in the EGF induced chemotaxis. Fifteen identified proteins were enriched and 9 proteins were reduced in the presence of EGF (≥1.5 folds, p ≤ 0.05). The interaction between cofilin-1 (CFL1) and PKCζ was evidenced and this interaction was enhanced in the EGF induced chemotaxis signaling transduction. In addition, novel PKCζ interacting proteins potentially related with chemotaxis were characterized, such as isoform 1 of nucleophosmin (NPM1). Furthermore, Western blotting and chemotaxis assays were also applied to validate the proteomics result and explore its biological implications. Collectively, the combination of quantitative proteomics and biological assays provides a powerful strategy for elucidating the signaling pathway of tumor cell chemotaxis.