Differential in-gel electrophoresis (DIGE) analysis of CHO cells under hyperosmotic pressure: osmoprotective effect of glycine betaine addition

The use of glycine betaine combined with hyperosmolality is known to be an efficient means for achieving high protein production in recombinant Chinese hamster ovary (rCHO) cells. In order to understand the intracellular events and identify the key factors in rCHO cells cultivated with glycine betaine under hyperosmotic conditions, two-dimensional differential in-gel electrophoresis (2D-DIGE) followed by mass spectrometric analysis was applied. Differentially expressed 19 protein spots were selected and 16 different kinds of proteins were successfully identified. The identified proteins were associated with cellular metabolism (PEPCK, GAPDH, and PK), cellular architecture (β-tubulin and β-actin), protein folding (GRP78 and OSP94), mRNA processing (Rbm34, ACF, and IPMK), and protein secretion (γ-COP). 2D-Western blot analysis of β-tubulin, GAPDH, Peroxidoxin-1, and GRP78 confirmed the proteomic findings. The proteins identified from this study, which are related to cell growth and antibody production, can be applied to cell engineering for maximizing the efficacy of the use of glycine betaine combined with hyperosmolality in rCHO cells.     

A DIGE approach for the assessment of differential expression of the CHO proteome under sodium butyrate addition: Effect of Bcl‐xL overexpression

Bcl‐x_L, a member of the Bcl‐2 family, is known to inhibit apoptosis of recombinant Chinese hamster ovary (rCHO) cells induced by the addition of sodium butyrate (NaBu), which is used for the elevated expression of recombinant protein. In order to understand the intracellular effects of Bcl‐x_L overexpression on CHO cells treated with NaBu, changes to the proteome caused by controlled Bcl‐x_L expression in rCHO cells producing erythropoietin (EPO) in the presence of 3 mM NaBu were evaluated using two‐dimensional differential in‐gel electrophoresis (2D‐DIGE) and MS analysis. The consequences of Bcl‐x_L overexpression were not limited to the apoptotic signaling pathway. Out of eight proteins regulated significantly by Bcl‐x_L overexpression in 3 mM NaBu addition culture, four proteins were related to cell survival (Iq motif‐containing GTPase‐activating protein 1), cell proliferation (dihydrolipoamide‐S‐acetyltransferase, guanine nucleotide binding protein alpha interacting 2), and repair of DNA damage (BRCA and CDKN1A interacting protein). Taken together, a DIGE approach reveals that overexpression of Bcl‐x_L not only inhibits apoptosis in the presence of NaBu but also affects cell proliferation and survival in various aspects. Biotechnol. Bioeng. 2010; 105: 358–367. © 2009 Wiley Periodicals, Inc.     

灵芝细胞中磷脂酸互作蛋白鉴定

灵芝是名贵药用真菌,三萜是灵芝的关键药效成分。前期研究发现,磷脂酶D (Phospholipase D,PLD) 产生的磷脂酸 (Phosphatidic acid,PA) 可调控三萜合成,为进一步阐明PA调控灵芝三萜合成的分子机制,研究采用PA-beads富集结合LC-MS/MS技术,鉴定灵芝细胞中PA互作蛋白,结果共鉴定到了19个PA互作蛋白,主要包括细胞色素P450单加氧酶 (GL22084)、特异性蛋白激酶MAPK (GL23765)、过氧化氢酶和细胞表面疏水性蛋白等。通过基因克隆、原核表达载体构建、蛋白诱导表达和分离纯化,获得了融合GST标签的GL22084和GL23765蛋白,采用GST-pull down实验,验证了灵芝GL22084和GL23765蛋白与PA互作。研究结果揭示了灵芝细胞中PA互作蛋白,为后续解析PLD介导的PA信号分子调控灵芝三萜合成的分子机理奠定了基础;同时,鉴定到的PA互作蛋白也为其他物种的PLD/PA信号通路相关研究提供借鉴。     

Proteomic understanding of intracellular responses of recombinant chinese hamster ovary cells adapted to grow in serum‐free suspension culture

To understand the intracellular responses in recombinant Chinese hamster ovary (rCHO) cells adapted to grow in serum‐free suspension culture, a proteomic approach was employed. After rCHO cells producing erythropoietin were adapted to grow in suspension culture with the two different serum‐free media (SFM4CHO? and SF‐L1), proteome analyses were carried out using 2‐D PAGE and based on spot intensities, 58 high‐intensity protein spots were selected. Of the 58 protein spots, which represented 34 different kinds of proteins, 55 were identified by MALDI‐TOF‐MS, and MS/MS. Compared with the results in serum‐containing medium, six proteins, four de novo synthesis of nucleotides‐related proteins (dihydrolipoamide S‐acetyltransferase, transaldolase, inosine‐5′‐monophosphate dehydrogenase 2, and lymphoid‐restricted membrane protein) and two molecular chaperones (heat shock protein 70 kDa and 60 kDa [HSC70, HSP60]) were significantly increased in SFM4CHO?. From the results of proteomic analysis, HSP60 and HSC70, which were increased in both SFM, were selected as candidate proteins for engineering and rCHO cell lines overexpressing these genes were constructed. Cells overexpressing HSP60 and/or HSC70 showed 10–15% enhanced cell concentration during serum‐free adaptation and 15–33% reduction in adaptation time. Taken together, identification of differentially expressed proteins in rCHO cells by a proteomic study can provide insights into understanding the intracellular events and clues to find candidate genes for cell engineering for improved performance of rCHO cells during adaptation to serum‐free suspension culture. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011     

Glucose‐regulated protein 78: A new partner of p53 in trophoblast

Although wild‐type p53 protein is overexpressed in first trimester trophoblast, it is inactive towards its target genes Metalloproteinase 2 and 9. This seems to be due to a complex mechanism of inactivation and stabilization of p53 relying on the formation of protein complexes involving the N‐terminus of p53. To detect the proteins associated with this sequence, we incubated biotinylated p53 N‐terminal peptide in cytotrophoblastic cell medium 24 h before lysis of cells. We purified the proteins retained on biotinylated peptide using a neutravidin affinity column. Proteins were then identified by peptide mass finger printing followed or not by peptide fragmentation sequencing. Among these proteins, we identified glucose‐regulated protein 78 (GRP78) and verified its interaction with p53 in trophoblastic cells by immunoprecipitation and Western blot analysis. Moreover, the decreased expression of GRP78 induced by GRP78siRNA or versipelostatin decreased the formation of high molecular weight p53 complexes and p53 monomer and increased trophoblastic invasion. These results suggest that GRP78 is involved in inactivation and stabilization of p53 and in the regulation of trophoblastic invasion.     

Bcl‐xL overexpression does not enhance specific erythropoietin productivity of recombinant CHO cells grown at 33°C and 37°C

Overexpression of bcl‐xL in recombinant Chinese hamster ovary (rCHO) cells has been known to suppress apoptotic cell death and thereby extend culture longevity during batch culture. However, its effect on specific productivity (q) of rCHO cells is controversial. This study attempts to investigate the effect of bcl‐xL overexpression on q of rCHO cells producing erythropoietin (EPO). To regulate the bcl‐xL expression level, the Tet‐off system was introduced in rCHO cells producing EPO (EPO‐off‐bcl‐xL). The bcl‐xL expression level was tightly controlled by doxycycline concentration. To evaluate the effect of bcl‐xL overexpression on specific EPO productivity (q_EPO) at different levels, EPO‐off‐bcl‐xL cells were cultivated at the two different culture temperatures, 33°C and 37°C. The q_EPO at 33°C and 37°C in the presence of 100 ng/mL doxycycline (without bcl‐xL overexpression) were 4.89 ± 0.21 and 3.18 ± 0.06 μg/10⁶cells/day, respectively. In the absence of doxycycline, bcl‐xL overexpression did not affect q_EPO significantly, regardless of the culture temperature, though it extended the culture longevity. Taken together, bcl‐xL overexpression showed no significant effect on the q_EPO of rCHO cells grown at 33°C and 37°C. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

The role of endoplasmic reticulum‐related BiP/GRP78 in interferon gamma‐induced persistent Chlamydia pneumoniae infection

Direct interaction of Chlamydiae with the endoplasmic reticulum (ER) is essential in intracellular productive infection. However, little is known about the interplay between Chlamydiae and the ER under cellular stress conditions that are observed in interferon gamma (IFN‐γ) induced chlamydial persistent infection. ER stress responses are centrally regulated by the unfolded protein response (UPR) under the control of the ER chaperone BiP/GRP78 to maintain cellular homeostasis. In this study, we could show that the ER directly contacted with productive and IFN‐γ‐induced persistent inclusions of Chlamydia pneumoniae (Cpn). BiP/GRP78 induction was observed in the early phase but not in the late phase of IFN‐γ‐induced persistent infection. Enhanced BiP/GRP78 expression in the early phase of IFN‐γ‐induced persistent Cpn infection was accompanied by phosphorylation of the eukaryotic initiation factor‐2α (eIF2α) and down‐regulation of the vesicle‐associated membrane protein‐associated protein B. Loss of BiP/GRP78 function resulted in enhanced phosphorylation of eIF2α and increased host cell apoptosis. In contrast, enhanced BiP/GRP78 expression in IFN‐γ‐induced persistent Cpn infection attenuated phosphorylation of eIF2α upon an exogenous ER stress inducer. In conclusion, ER‐related BiP/GRP78 plays a key role to restore cells from stress conditions that are observed in the early phase of IFN‐γ‐induced persistent infection.     

Effect of Bcl‐xL overexpression on apoptosis and autophagy in recombinant Chinese hamster ovary cells under nutrient‐deprived condition

Upon nutrient deprivation during culture, recombinant Chinese hamster ovary (rCHO) cells are subjected to two types of programmed cell death (PCD), apoptosis and autophagy. To investigate the effect of Bcl‐x_L overexpression on apoptosis and autophagy in rCHO cells, an erythropoietin (EPO)‐producing rCHO cell line with regulated Bcl‐x_L overexpression (EPO‐off‐Bcl‐x_L) was established using the Tet‐off system. The expression level of Bcl‐x_L in EPO‐off‐Bcl‐x_L cells was tightly regulated by doxycycline in a dose‐dependent manner. Bcl‐x_L overexpression enhanced cell viability and extended culture longevity in batch culture. Upon nutrient depletion in the later stage of batch culture, Bcl‐x_L overexpression suppressed apoptosis by inhibiting the activation of caspase‐3 and ‐7. Simultaneously, Bcl‐x_L overexpression also delayed autophagy, characterized by LC3‐II accumulation. Immunoprecipitation analysis with a Flag‐tagged Bcl‐x_L revealed that Bcl‐x_L interacts with Bax and Bak, essential mediators of caspase‐dependent apoptosis, as well as with Beclin‐1, an essential mediator of autophagy, and may inhibit their pro‐cell death function. Taken together, it was found that Bcl‐x_L overexpression inhibits both apoptosis and autophagy in rCHO cell culture. Biotechnol. Bioeng. 2009;103: 757–766. © 2009 Wiley Periodicals, Inc.     

Apoptosis induced by staurosporine alters chaperone and endoplasmic reticulum proteins: Identification by quantitative proteomics

Apoptosis contributes to cell death after cerebral ischaemia. A quantitative proteomics approach has been employed to define alterations in protein levels in apoptosis induced with staurosporine (STS). Human neuroblastoma derived SH-SY5Y cells were treated with STS (500 nM for 6 h) to induce apoptosis. Quantitative 2-DE was used to determine the changing protein levels with MALDI-TOF MS identification of proteins. Of the 154 proteins analysed, 13 proteins were significantly altered as a result of the apoptotic stimulus; ten of the proteins showed an increase in level with STS and were identified as heat shock cognate 71 (Hsc71), two isoforms of heat shock protein 70 (Hsp70), glucose regulated protein 78 (GRP78), F-actin capping protein, stress-induced phosphoprotein 1, chromatin assembly factor 1 (CAF-1), protein disulphide isomerase A3 (PDI A3) precursor, transitional ER ATPase and actin interacting protein 1 (AIP 1). Three proteins which displayed significant decrease in levels with STS were identified as tubulin, vimentin and glucose regulated protein 94 (GRP94). The functional roles and subcellular locations of these proteins collectively indicate that STS-induced apoptosis provokes induces an unfolded protein response involving molecular chaperones, cochaperones and structural proteins indicative of ER stress.     

Evaluation of ubiquitinated proteins by proteomics reveals the role of the ubiquitin proteasome system in the regulation of Grp75 and Grp78 chaperone proteins during intestinal inflammation

The ubiquitin proteasome system (UPS) is the major pathway of intracellular protein degradation and may be involved in the pathophysiology of inflammatory bowel diseases or irritable bowel syndrome. UPS specifically degrades proteins tagged with an ubiquitin chain. We aimed to identify polyubiquitinated proteins during inflammatory response in intestinal epithelial HCT‐8 cells by a proteomic approach. HCT‐8 cells were incubated with interleukin 1β, tumor necrosis factor‐α, and interferon‐γ for 2 h. Total cellular protein extracts were separated by 2D gel electrophoresis and analyzed by an immunodetection using antiubiquitin antibody. Differential ubiquitinated proteins were then identified by LC‐ESI MS/MS. Seven proteins were differentially ubiquitinated between control and inflammatory conditions. Three of them were chaperones: Grp75 and Hsc70 were more ubiquitinated (p < 0.05) and Grp78 was less ubiquitinated (p < 0.05) under inflammatory conditions. The results for Grp75 and Grp78 were then confirmed in HCT‐8 cells and in 2‐4‐6‐trinitrobenzen sulfonic acid induced colitis in rats mimicking inflammatory bowel disease by immunoprecipitation. No difference was observed in irritable bowel syndrome like model. In conclusion, we showed that a proteomic approach is suitable to identify ubiquitinated proteins and that UPS‐regulated expression of Grp75 and Grp78 may be involved in inflammatory response. Further studies should lead to the identification of ubiquitin ligases responsible for Grp75 and Grp78 ubiquitination.     

BAG3 sensitizes cancer cells exposed to DNA damaging agents via direct interaction with GRP78

Bcl-2 associated athanogene 3 (BAG3) has a modular structure that contains a BAG domain, a WW domain, a proline-rich (PxxP) domain to mediate potential interactions with chaperons and other proteins that participate in more than one signal transduction. In search for novel interacting partners, the current study identified that 78 kDa glucose-regulated protein (GRP78) was a novel partner interacting with BAG3. Interaction between GRP78 and BAG3 was confirmed by coimmunoprecipitation and glutathione S-transferase (GST) pulldown. We also identified that the ATPase domain of GRP78 and BAG domain of BAG3 mediated their interaction. Counterintuitive for a prosurvival protein, BAG3 was found to promote the cytotoxicity of breast cancer MCF7, thyroid cancer FRO and glioma U87 cells subjected to genotoxic stress. In addition, the current study demonstrated that BAG3 interfered with the formation of the antiapoptotic GRP78-procaspase-7 complex, which resulted in an increased genotoxic stress-induced cytotoxicity in cancer cells. Furthermore, overexpression of GRP78 significantly blocked the enhancing effects of BAG3 on activation of caspase-7 and induction of apoptosis by genotoxic stress. Overall, these results suggested that through direct interaction BAG3 could prevent the antiapoptotic effect of GRP78 upon genotoxic stress.     

Discovery of a novel periplasmic protein that forms a complex with a trimeric autotransporter adhesin and peptidoglycan

Trimeric autotransporter adhesins (TAAs), fibrous proteins on the cell surface of Gram‐negative bacteria, have attracted attention as virulence factors. However, little is known about the mechanism of their biogenesis. AtaA, a TAA of Acinetobacter sp. Tol 5, confers nonspecific, high adhesiveness to bacterial cells. We identified a new gene, tpgA, which forms a single operon with ataA and encodes a protein comprising two conserved protein domains identified by Pfam: an N‐terminal SmpA/OmlA domain and a C‐terminal OmpA_C‐like domain with a peptidoglycan (PGN)‐binding motif. Cell fractionation and a pull‐down assay showed that TpgA forms a complex with AtaA, anchoring it to the outer membrane (OM). Isolation of total PGN‐associated proteins showed TpgA binding to PGN. Disruption of tpgA significantly decreased the adhesiveness of Tol 5 because of a decrease in surface‐displayed AtaA, suggesting TpgA involvement in AtaA secretion. This is reminiscent of SadB, which functions as a specific chaperone for SadA, a TAA in Salmonella species; however, SadB anchors to the inner membrane, whereas TpgA anchors to the OM through AtaA. The genetic organization encoding the TAA–TpgA‐like protein cassette can be found in diverse Gram‐negative bacteria, suggesting a common contribution of TpgA homologues to TAA biogenesis.     

The HSP70 heat shock response in the Antarctic fish <Emphasis Type="Italic">Harpagifer antarcticus</Emphasis>

Members of the HSP70 gene family comprising the constitutive (HSC70) and inducible (HSP70) genes, plus GRP78 (Glucose-regulated protein 78 kDa) were surveyed for expression levels via Q-PCR after both an acute 2-h heat shock experiment and a time course assay in the Antarctic plunderfish Harpagifer antarcticus. In general, down regulation of all genes was observed during the course of the heat shock experiments. This thermally induced down regulation was particularly acute for the GRP78 gene, which at one time point was more than 100-fold down regulated. These results demonstrate the loss of the heat shock response in H. antarcticus, a basal member of the Notothenioidei. This finding is discussed with reference to the survival of Notothenioids during observed ocean warming and also the reorganisation of cellular protein mechanisms of species living in extreme environments.     

Bovine adenovirus‐3 protein VIII associates with eukaryotic initiation factor‐6 during infection

Adenovirus protein VIII appears to connect core with the inner surface of the adenovirus capsid. Because protein–protein interactions are central to virus replication, identification of proteins interacting with protein VIII may help in understanding their role in adenovirus infection. Our yeast 2‐hybrid assay indicated that protein VIII interacts with eukaryotic initiation factor 6 (eIF6). These findings were confirmed by Glutathione S‐transferase‐pull down assay, bimolecular fluorescent complementation assay, and coimmunoprecipitation assay in plasmid DNA transfected and bovine adenovirus‐3 (BAdV‐3) infected cells. The C‐terminus amino acids 147 to 174 of protein VIII and N‐terminus amino acids 44 to 97 of eIF6 are involved in these interactions. Polysome analysis demonstrated increased level of 60S ribosomal subunit and decreased level of 80S complex in protein VIII expressing cells or BAdV‐3 infected cells. Our results suggest that formation of functional 80S ribosome appears impaired in the presence of protein VIII at late times post infection. We speculate that this impaired ribosome assembly may be responsible for the inhibition of cellular mRNA translation observed late in adenovirus infected cells. Moreover, analysis of recombinant BAdV‐3 expressing mutant protein VIII (deletion of eIF6 interacting domain) suggests that interaction of protein VIII and eIF6 may help in preferential translation of adenovirus genes during late phase of adenovirus infection.     

A putative spermidine synthase interacts with flagellar switch protein FliM and regulates motility in Helicobacter pylori

The flagellar motor is an important virulence factor in infection by many bacterial pathogens. Motor function can be modulated by chemotactic proteins and recently appreciated proteins that are not part of the flagellar or chemotaxis systems. How these latter proteins affect flagellar activity is not fully understood. Here, we identified spermidine synthase SpeE as an interacting partner of switch protein FliM in Helicobacter pylori using pull‐down assay and mass spectrometry. To understand how SpeE contributes to flagellar motility, a speE‐null mutant was generated and its motility behavior was evaluated. We found that deletion of SpeE did not affect flagellar formation, but induced clockwise rotation bias. We further determined the crystal structure of the FliM‐SpeE complex at 2.7 Å resolution. SpeE dimer binds to FliM with micromolar binding affinity, and their interaction is mediated through the β1' and β2' region of FliM middle domain. The FliM‐SpeE binding interface partially overlaps with the FliM surface that interacts with FliG and is essential for proper flagellar rotational switching. By a combination of protein sequence conservation analysis and pull‐down assays using FliM and SpeE orthologues in E. coli, our data suggest that FliM‐SpeE association is unique to Helicobacter species.     

Identification of in vitro phosphorylation sites in the Arabidopsis thaliana somatic embryogenesis receptor‐like kinases

The Arabidopsis thaliana somatic embryogenesis receptor‐like kinase (SERK) family consists of five leucine‐rich repeat receptor‐like kinases (LRR‐RLKs) with diverse functions such as brassinosteroid insensitive 1 (BRI1)‐mediated brassinosteroid perception, development and innate immunity. The autophosphorylation activity of the kinase domains of the five SERK proteins was compared and the phosphorylated residues were identified by LC‐MS/MS. Differences in autophosphorylation that ranged from high activity of SERK1, intermediate activities for SERK2 and SERK3 to low activity for SERK5 were noted. In the SERK1 kinase the C‐terminally located residue Ser‐562 controls full autophosphorylation activity. Activation loop phosphorylation, including that of residue Thr‐462 previously shown to be required for SERK1 kinase activity, was not affected. In vivo SERK1 phosphorylation was induced by brassinosteroids. Immunoprecipitation of CFP‐tagged SERK1 from plant extracts followed by MS/MS identified Ser‐303, Thr‐337, Thr‐459, Thr‐462, Thr‐463, Thr‐468, and Ser‐612 or Thr‐613 or Tyr‐614 as in vivo phosphorylation sites of SERK1. Transphosphorylation of SERK1 by the kinase domain of the main brassinosteroid receptor BRI1 occurred only on Ser‐299 and Thr‐462. This suggests both intra‐ and intermolecular control of SERK1 kinase activity. Conversely, BRI1 was transphosphorylated by the kinase domain of SERK1 on Ser‐887. BRI1 kinase activity was not required for interaction with the SERK1 receptor in a pull down assay.     

HepG2细胞中与戊型肝炎病毒衣壳蛋白相互作用蛋白的初步研究

利用重组戊型肝炎病毒(HEV)衣壳蛋白片段p239(368~606 aa)形成的类病毒颗粒作为亲和层析诱饵蛋白,HepG2为细胞模型,筛选与p239类病毒颗粒特异性相互作用蛋白。经过二维电泳分离,MALDI-TOF-MS分析鉴定得到GRP78/Bip,HSP90,alpha-tubulin及P43四个与p239有相互作用的候选蛋白。GRP78/Bip为热休克蛋白家族成员,体外免疫共沉淀实验结果证实,其与p239有特异性的结合。此研究结果为深入研究HEV的感染过程如吸附、入胞,以及HEV的致病机理提供了有益线索。     

Combining affinity enrichment,cross‐linking with photo amino acids,and mass spectrometry for probing protein kinase D2 interactions

We present a novel approach that relies on the affinity capture of protein interaction partners from a complex mixture, followed by their covalent fixation via UV‐induced activation of incorporated diazirine photoreactive amino acids (photo‐methionine and photo‐leucine). The captured protein complexes are enzymatically digested and interacting proteins are identified and quantified by label‐free LC/MS analysis. Using HeLa cell lysates with photo‐methionine and photo‐leucine‐labeled proteins, we were able to capture and preserve protein interactions that are otherwise elusive in conventional pull‐down experiments. Our approach is exemplified for mapping the protein interaction network of protein kinase D2, but has the potential to be applied to any protein system. Data are available via ProteomeXchange with identifiers PXD005346 (photo amino acid incorporation) and PXD005349 (enrichment experiments).