Genetic isolation of transport signals directing cell surface expression

Membrane proteins represent approximately 30% of the proteome in both prokaryotes and eukaryotes. The spatial localization of membrane-bound proteins is often determined by specific sequence motifs that may be regulated in response to physiological changes, such as protein interactions and receptor signalling. Identification of signalling motifs is therefore important for understanding membrane protein expression, function and transport mechanisms. We report a genetic isolation of novel motifs that confer surface expression. Further characterization showed that SWTY, one class of these isolated motifs with homology to previously reported forward transport motifs, has the ability to both override the RKR endoplasmic reticulum localization signal and potentiate steady-state surface expression. The genetically isolated SWTY motif is functionally interchangeable with a known motif in cardiac potassium channels and an identified motif in an HIV coreceptor, and operates by recruiting 14-3-3 proteins. This study expands the repertoire of and enables a screening method for membrane trafficking signals.     

H2AX phosphorylation regulated by p38 is involved in Bim expression and apoptosis in chronic myelogenous leukemia cells induced by imatinib

Increasing evidence suggests that histone H2AX plays a critical role in regulation of tumor cell apoptosis and acts as a novel human tumor suppressor protein. However, the action of H2AX in chronic myelogenous leukemia (CML) cells is unknown. The detailed mechanism and epigenetic regulation by H2AX remain elusive in cancer cells. Here, we report that H2AX was involved in apoptosis of CML cells. Overexpression of H2AX increased apoptotic sensitivity of CML cells (K562) induced by imatinib. However, overexpression of Ser139-mutated H2AX (blocking phosphorylation) decreased sensitivity of K562 cells to apoptosis. Similarly, knockdown of H2AX made K562 cells resistant to apoptotic induction. These results revealed that the function of H2AX involved in apoptosis is strictly related to its phosphorylation (Ser139). Our data further indicated that imatinib may stimulate mitogen-activated protein kinase (MAPK) family member p38, and H2AX phosphorylation followed a similar time course, suggesting a parallel response. H2AX phosphorylation can be blocked by p38 siRNA or its inhibitor. These data demonstrated that H2AX phosphorylation was regulated by p38 MAPK pathway in K562 cells. However, the p38 MAPK downstream, mitogen- and stress-activated protein kinase-1 and -2, which phosphorylated histone H3, were not required for H2AX phosphorylation during apoptosis. Finally, we provided epigenetic evidence that H2AX phosphorylation regulated apoptosis-related gene Bim expression. Blocking of H2AX phosphorylation inhibited Bim gene expression. Taken together, these data demonstrated that H2AX phosphorylation regulated by p38 is involved in Bim expression and apoptosis in CML cells induced by imatinib.     

The Presence of Thyroid-Stimulation Blocking Antibody Prevents High Bone Turnover in Untreated Premenopausal Patients with Graves’ Disease

Osteoporosis-related fractures are one of the complications of Graves’ disease. This study hypothesized that the different actions of thyroid-stimulating hormone receptor (TSHR) antibodies, both stimulating and blocking activities in Graves’ disease patients might oppositely impact bone turnover. Newly diagnosed premenopausal Graves’ disease patients were enrolled (n = 93) and divided into two groups: patients with TSHR antibodies with thyroid-stimulating activity (stimulating activity group, n = 83) and patients with TSHR antibodies with thyroid-stimulating activity combined with blocking activity (blocking activity group, n = 10). From the stimulating activity group, patients who had matched values for free T4 and TSH binding inhibitor immunoglobulin (TBII) to the blocking activity group were further classified as stimulating activity-matched control (n = 11). Bone turnover markers BS-ALP, Osteocalcin, and C-telopeptide were significantly lower in the blocking activity group than in the stimulating activity or stimulating activity-matched control groups. The TBII level showed positive correlations with BS-ALP and osteocalcin levels in the stimulating activity group, while it had a negative correlation with the osteocalcin level in the blocking activity group. In conclusion, the activation of TSHR antibody-activated TSH signaling contributes to high bone turnover, independent of the actions of thyroid hormone, and thyroid-stimulation blocking antibody has protective effects against bone metabolism in Graves’ disease.     

Membrane-bound plasma platelet activating factor acetylhydrolase acts on substrate in the aqueous phase.

Human plasma platelet activating factor acetylhydrolase (pPAF-AH) is a phospholipase A(2) that specifically hydrolyzes the sn-2 ester of platelet activating factor (PAF) and of phospholipids with oxidatively truncated sn-2 fatty acyl chains. pPAF-AH is bound to lipoproteins in vivo, and it binds essentially irreversibly to anionic and zwitterionic phospholipid vesicles in vitro and hydrolyzes PAF and PAF analogues. Substrate hydrolysis also occurs in the absence of vesicles, with a maximum rate reached at the critical micelle concentration. A novel pre-steady-state kinetic analysis with enzyme tightly bound to vesicles and with a substrate that undergoes slow intervesicle exchange establishes that pPAF-AH accesses its substrate from the aqueous phase and thus is not an interfacial enzyme. Such a mechanism readily explains why this enzyme displays dramatic specificity for phospholipids with short sn-2 chains or with medium-length, oxidatively truncated sn-2 chains since a common feature of these lipids is their relatively high water solubility. It also explains why the enzymatic rate drops as the length of the sn-1 chain is increased. pPAF-AH shows broad specificity toward phospholipids with different polar headgroups. Additional results are that PAF undergoes intervesicle exchange on the subminute time scale and it does not undergo transbilayer movement over tens of minutes.     

Crystal Structures and Biochemical Analyses Suggest a Unique Mechanism and Role for Human Glycyl-tRNA Synthetase in Ap4A Homeostasis

Aminoacyl-tRNA synthetases catalyze the attachment of amino acids to their cognate tRNAs for protein synthesis. However, the aminoacylation reaction can be diverted to produce diadenosine tetraphosphate (Ap4A), a universal pleiotropic signaling molecule needed for cell regulation pathways. The only known mechanism for Ap4A production by a tRNA synthetase is through the aminoacylation reaction intermediate aminoacyl-AMP, thus making Ap4A synthesis amino acid-dependent. Here, we demonstrate a new mechanism for Ap4A synthesis. Crystal structures and biochemical analyses show that human glycyl-tRNA synthetase (GlyRS) produces Ap4A by direct condensation of two ATPs, independent of glycine concentration. Interestingly, whereas the first ATP-binding pocket is conserved for all class II tRNA synthetases, the second ATP pocket is formed by an insertion domain that is unique to GlyRS, suggesting that GlyRS is the only tRNA synthetase catalyzing direct Ap4A synthesis. A special role for GlyRS in Ap4A homeostasis is proposed.Aminoacyl-tRNA synthetases (AARSs)⁴ are considered to be among the earliest proteins to have emerged during evolution. As a family of typically 20 members (one for each amino acid), AARSs catalyze the first step of protein synthesis by linking each amino acid onto the 3′-end of its cognate tRNA harboring the trinucleotide anticodon. Through evolution, the role of AARSs has also been broadened with expanded functions (reviewed in Refs. 1 and 2). These expanded functions often involve direct interaction partners. For example, human tyrosyl-tRNA synthetase interacts with chemokine receptor CXCR1 to induce cell migration (3); human glutaminyl-tRNA synthetase interacts with ASK1 to regulate apoptosis (4); human tryptophanyl-tRNA synthetase interacts with VE-cadherin to inhibit angiogenesis (5); human lysyl-tRNA synthetase interacts with the Gag protein of human immunodeficiency virus to facilitate viral assembly (6); and human glutamyl-prolyl-tRNA synthetase interacts with L13a and glyceraldehyde-3-phosphate dehydrogenase to form the GAIT complex for translational silencing to regulate inflammation (7). However, functional expansion also can be achieved indirectly via reaction products of AARSs. As examples, Lys-tRNA^Lys and Ala-tRNA^Ala are used to aminoacylate cytoplasmic membrane phosphatidylglycerol of Staphylococcus aureus and Pseudomonas aeruginosa, respectively, to enhance drug resistance in these microorganisms (8).In addition to tRNA aminoacylation, the majority of AARSs have the capacity to catalyze a side reaction to form diadenosine oligophosphates (ApnA) in the absence of cognate tRNA (9). These reactions of AARS are the most well known sources of ApnA in vivo (10). ApnA are made up of two adenosine moieties linked at the 5′-end of the ribose by a chain of two to six phosphates. In the 4 decades following the discovery of these molecules by Zamecnik et al. (10), ApnA have been linked to highly diverse physiological effects in prokaryotic and eukaryotic cells, including various types of mammalian cells and tissues, and to assorted functions associated with the nucleus, membrane receptors, and activities in the cytoplasm (reviewed in Refs. 11 and 12). The concentrations of ApnA molecules in vivo respond to numerous factors, including cell proliferation status, glucose level, heat shock, oxidative stress, and interferon stimulation. They have emerged as extracellular and intracellular signaling molecules (as pleiotropically acting “alarmones” (13) and second messengers (14)) implicated in the maintenance and regulation of vital cellular functions.The aminoacylation reaction proceeds in two steps. First, the amino acid is activated by condensation with ATP to form aminoacyl-AMP, the enzyme-bound intermediate. The aminoacyl moiety is then transferred to the 3′-end of the cognate tRNA. When tRNA is absent, the enzyme-bound aminoacyl-AMP can be attacked by the γ-phosphate of a second ATP molecule to form diadenosine tetraphosphate (Ap4A), the most common diadenosine oligophosphate produced by a tRNA synthetase (see Fig. 1A). The presence of tRNA in most cases inhibits Ap4A synthesis (11). Therefore, a subgroup of tRNA synthetases that requires tRNA as cofactor for synthesis of aminoacyl-AMP is not capable of producing Ap4A. This group includes tRNA synthetases that are specific for arginine, glutamine, and glutamic acid and an unusual class I lysyl-tRNA synthetase (LysRS).Open in a separate windowFIGURE 1.Amino acid-independent synthesis of Ap4A by human GlyRS. A, conventional mechanism for synthesis of Ap4A by a tRNA synthetase. The first step of the reaction involves the generation of an enzyme-bound aminoacyl-AMP (aa-AMP), which is then attacked either by cognate tRNA to form aminoacyl-tRNA or by ATP to form Ap4A. B, mechanism used by human GlyRS to produce Ap4A by direct condensation of two ATPs. C–E, synthesis of Ap4A by GlyRS, LysRS, and TyrRS, respectively, in the presence (●) and absence (○) of cognate amino acid. Amounts of Ap4A produced were quantitated from the TLC sheets and are plotted on the right. GlyRS activity was measured in triplicate, and the plotted values reflect the mean ± S.E.Although the amino acid recycles, the above mechanism requires the presence of the amino acid for the production of Ap4A via the aminoacyl-AMP intermediate. Using biochemical analyses and determinations of co-crystal structures, we demonstrate in this work that human glycyl-tRNA synthetase (GlyRS) produces Ap4A by direct condensation of two ATPs in the absence of glycine. Thus, the mechanism for GlyRS to synthesize Ap4A is decoupled from aminoacylation. Furthermore, GlyRS is likely to be the only synthetase that produces Ap4A by this mechanism. Our results raise the possibility that GlyRS plays a special role in Ap4A homeostasis.     

Overexpression of uncoupling protein 4 promotes proliferation and inhibits apoptosis and differentiation of preadipocytes

Uncoupling proteins are a family of mitochondrial proteins involved in energy metabolism. We previously showed that uncoupling protein 4 (UCP4) is differentially expressed in omental adipose tissue in diet-induced obese and normal rats. However, the effect of UCP4 on adipocytes is unclear. In this work, we established a stable preadipocyte cell line overexpressing UCP4 to observe the direct effect of UCP4 on adipocytes. Cells overexpressing UCP4 showed significantly attenuated differentiation of preadipocytes into adipocytes. During differentiation, expression of adipogenesis-associated markers such as fatty acid synthetase, peroxisome proliferator-activated receptor gamma, CCAAT enhancer binding protein alpha, adipocyte lipid binding protein and lipoprotein lipase were downregulated. Preadipoctes expressing UCP4 grew faster and more of them stayed in S phase compared to control cells. In addition, UCP4 overexpression protected preadipocytes from apoptosis induced by serum deprivation. Our results demonstrate that overexpression of UCP4 can promote proliferation and inhibit apoptosis and differentiation of preadipocytes.     

Gefitinib Synergizes with Irinotecan to Suppress Hepatocellular Carcinoma via Antagonizing Rad51-Mediated DNA-Repair

Chemotherapy is the only choice for most of the advanced hepatocellular carcinoma (HCC) patients, while few agents were available, making it an urgent need to develop new chemotherapy strategies. A phase II clinical trial suggested that the efficacy of irinotecan in HCC was limited due to dose-dependent toxicities. Here, we found that gefitinib exhibited synergistic activity in combination with SN-38, an active metabolite of irinotecan, in HCC cell lines. And the enhanced apoptosis induced by gefitinib plus SN-38 was a result from caspase pathway activation. Mechanistically, gefitinib dramatically promoted the ubiquitin–proteasome-dependent degradation of Rad51 protein, suppressed the DNA repair, gave rise to more DNA damages, and ultimately resulted in the synergism of these two agents. In addition, the increased antitumor efficacy of gefitinib combined with irinotecan was further validated in a HepG2 xenograft mice model. Taken together, our data demonstrated for the first time that the combination of irinotecan and gefitinib showed potential benefit in HCC, which suggests that Rad51 is a promising target and provides a rationale for clinical trials investigating the efficacy of the combination of topoisomerase I inhibitors and gefitinib in HCC.     

The molecular weight and concentration of dextran sulfate affect cell growth and antibody production in CHO cell cultures

To investigate the effect of dextran sulfate (DS), a widely used anti‐aggregation agent, on cell growth and monoclonal antibody (mAb) production including the quality attributes, DS with the three different MWs (4,000 Da, 15,000 Da, and 40,000 Da) at various concentrations (up to 1 g/L) was added to suspension cultures of two different recombinant CHO (rCHO) cell lines producing mAb, SM‐0.025 and CS13‐1.00. For both cell lines, the addition of DS, regardless of the MW and concentration of DS used, improved cell growth and viability in the decline phase of growth. However, it increased mAb production only in the CS13‐1.00 cells. Among the three different MWs, 40,000 Da DS was most effective in attenuating cell aggregation during the cultures of CS13‐1.00 cells, and showed the highest maximum mAb concentration. For SM‐0.025 cells, it significantly decreased specific mAb productivity, particularly at a high concentration of DS. Overall, DS addition did not negatively affect the quality attributes of mAbs (aggregation, charge variation, and glycosylation), though its efficacy on mAb quality depended on the MW and concentration of DS and cell lines. For both cell lines, the addition of DS did not affect N‐glycosylation of mAbs and decreased basic charge variants in mAbs. For CS13‐1.00 cells, the mAb monomer increased with the addition of 40,000 Da DS at 0.3–1.0 g/L. Taken together, to maximize the beneficial effect of DS addition on mAb production, the optimal MW and concentration of DS should be determined for each specific rCHO cell line. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1113–1122, 2016     

High Ca2+-phosphate transfection efficiency in low-density neuronal cultures

This protocol describes a high-efficiency Ca2+-phosphate transfection method with low cell toxicity. The Ca2+-phosphate transfection method is widely used in transfecting neurons because of its low cell toxicity and simplicity in use, but the efficiency is typically low (approximately 1-5%). To solve this problem we have developed a new Ca2+-phosphate transfection protocol that increases the efficiency by 10-fold (< or = 60%), while maintaining low cell toxicity. First, it is critical to have gentle mixing of the DNA-Ca2+ solution with phosphate buffer to form a homogeneous snowlike precipitate (particle size 1-3 microm). Second, the precipitate should be dissolved using a slightly acidic culture medium to reduce cell toxicity. The high efficiency of this new protocol makes it possible to transfect single autaptic neurons as well as mature neurons (15-82 days in vitro) for gene functional analysis. The total time required for the protocol is 2-4 h (including 45 min-3 h incubation time).