anguo Ji

Methylene blue-mediated photodynamic therapy induces mitochondria-dependent apoptosis in HeLa Cell

Methylene blue (MB), a widely studied reagent, is investigated in this work for its usage in photodynamic therapy (PDT). PDT has been proved to be highly effective in the treatment of different types of cancers. Previous studies showed MB has both high affinity for mitochondria and high photodynamic efficiency. To elucidate the effects of MB in PDT, we analyzed PDT‐induced apoptosis in HeLa cells by introducing different doses of MB into the culture media. Our data showed that MB‐mediated PDT triggered intense apoptotic cell death through a series of steps, beginning with photochemical generation of reactive oxygen species. The release of cytochrome c and activation of caspase‐3 indicated that MB‐PDT‐mediated apoptosis in HeLa cells was executed by the mitochondria‐dependent apoptotic pathway. Importantly, proteomic studies confirmed that expression levels of several mitochondrial proteins were altered in MB‐PDT‐induced apoptosis, including TRAP1, mitochondrial elongation factor Tu and peroxiredoxin 3 isoform b. Western blot data showed that phosphorylation of ERK1/2 and PKA were reduced in MB‐PDT treated cells, indicating several signal molecules participating in this apoptotic cascade. Moreover, MB‐PDT induced an increase in the strength of interaction between Bcl‐xL and dephosphorylated Bad. This led to loss of the pro‐survival function of Bcl‐xL and resulted in mitochondria‐mediated apoptosis. This study provides solid evidence of a strong induction by MB‐PDT of a mitochondria‐dependent apoptosis cascade in HeLa cells. J. Cell. Biochem. 105: 1451–1460, 2008.

A comprehensive study of optimal conditions for naked plasmid DNA transfer into skeletal muscle by electroporation

Efficient gene transfer is a key factor in gene therapy. Reducing the damage caused by gene transfer to muscle by electroporation is very important for its clinical application. Extensive investigation of optimal conditions for gene transfer by electroporation is required. The parameters used for electroporation, including plasmid concentration; injection volume; the plasmid dose of the injection; the concentration of saline media; the size of plasmid DNA; the age of the mice; the lag time between plasmid injection and electroporation; and the effect of repeated gene transfer by electroporation, were systematically investigated in the present study. The efficiencies of gene transfer by electroporation in normal and rodent models of diabetes were also evaluated. We found that electroporation used for non‐viral gene transfer could be repeated in the same place in the muscle, but the expression efficiency was closely related to the muscle damage. Increasing pulse times could enhance the efficiency of gene transfer with a lower strength of electric field. It was better to use a higher plasmid concentration than to use a larger dose of plasmid and repeated injection to achieve a high level of transgene expression. Optimal conditions varied in different animal models, being milder for diabetic mice than for normal mice, and it was also shown that the conditions that worked well on these small rodents were not necessarily suitable for larger animals. Our results provide a comprehensive view of the factors that affect the efficiency of gene transfer into skeletal muscle by electroporation. Copyright

Deacetylation of TFEB promotes fibrillar Aβ degradation by upregulating lysosomal biogenesis in microglia

Microglia play a pivotal role in clearance of Aβ by degrading them in lysosomes, countering amyloid plaque pathogenesis in Alzheimer’s disease (AD). Recent evidence suggests that lysosomal dysfunction leads to insufficient elimination of toxic protein aggregates. We tested whether enhancing lysosomal function with transcription factor EB (TFEB), an essential regulator modulating lysosomal pathways, would promote Aβ clearance in microglia. Here we show that microglial expression of TFEB facilitates fibrillar Aβ (fAβ) degradation and reduces deposited amyloid plaques, which are further enhanced by deacetylation of TFEB. Using mass spectrometry analysis, we firstly confirmed acetylation as a previously unreported modification of TFEB and found that SIRT1 directly interacted with and deacetylated TFEB at lysine residue 116. Subsequently, SIRT1 overexpression enhanced lysosomal function and fAβ degradation by upregulating transcriptional levels of TFEB downstream targets, which could be inhibited when TFEB was knocked down. Furthermore, overexpression of deacetylated TFEB at K116R mutant in microglia accelerated intracellular fAβ degradation by stimulating lysosomal biogenesis and greatly reduced the deposited amyloid plaques in the brain slices of APP/PS1 transgenic mice. Our findings reveal that deacetylation of TFEB could regulate lysosomal biogenesis and fAβ degradation, making microglial activation of TFEB a possible strategy for attenuating amyloid plaque deposition in AD.

Prevention of osteoporosis in ovariectomized mice by electroporational gene transfer of parathyroid hormone.

Non-viral vector, pCMV-Pth, with full-length human parathyroid hormone (prepro-hPTH) cDNA was constructed and delivered into the ovariectomized mouse quadriceps to explore the effects of electroporational gene transfer of PTH on the bone. The expression of hPTH in the transfected mice was detected by RT-PCR and radioimmunoassay. Mechanical testing and the analysis of bone mineral content demonstrated the improvement of bone properties. These results suggest that the electroporational gene taransfer of parathyroid hormone might be a promising method to prevent the bone loss in the postmenopausal women.

Evaluation of the accuracy of protein quantification using isotope TMPP‐labeled peptides

N‐succinimidyloxycarbonylmethyl tris(2,4,6‐trimethoxyphenyl) phosphonium bromide (TMPP‐Ac‐OSu) reacts rapidly, mildly, and specifically with the N‐terminals of proteins and peptides. Thus, it can be developed as an ideal isotope‐coded tag to be used in quantitative proteomics. Here, we present a strategy for light and heavy TMPP‐based quantitative proteomic analysis, in which peptides in a mixture can be quantified using an on‐tip TMPP derivatization approach. To demonstrate the accuracy of this strategy, light and heavy TMPP‐labeled peptides were combined at different ratios and subsequently analyzed by LC‐MS/MS. The MS spectra and scatter plots show that peptide and protein ratios were both consistent with the mixed ratios. We observed a linear correlation between protein ratios and the predicted ratios. In comparison with SILAC method, the TMPP labeling method produced similarly accurate quantitative results with low CVs. In conclusion, our results suggest that this isotope‐coded TMPP method achieved accurate quantification and compatibility with IEF‐based separation. With the inherent advantages of TMPP derivatization, we believe that it holds great promise for future applications in quantitative proteomics analysis.

Bone loss induced by ovariectomy in rats is prevented by gene transfer of parathyroid hormone or an Arg-Gly-Asp-containing peptide.

Osteoporosis is a major and growing healthcare concern as the population ages. The genes of both a Arg-Gly-Asp (RGD)-containing peptide and parathyroid hormone (PTH) were used to reduce bone loss induced by ovariectomy (OVX) in rats. Plasmids with either RGD or PTH gene were delivered into the quadriceps of OVX rats. The expression of the genes was detected by RT-PCR and radioimmunoassay. Analysis of bone mineral density, bone mechanical testing and bone mineral content indicated an improvement in bone properties in both RGD-transferred and PTH-transferred rats compared to OVX rats. Gene transfer of either RGD or PTH is therefore a possible approach to prevent bone loss in OVX rats thus providing a potential method to prevent osteoporosis in clinical situations.

A rapid and easy protein N-terminal profiling strategy using (N-Succinimidyloxycarbonylmethyl)tris(2,4,6-trimethoxyphenyl)phosphonium bromide (TMPP) labeling and StageTip

Protein N‐terminal profiling is crucial when characterizing biological functions and provides proteomic evidences for genome reannotations. However, most of the current N‐terminal enrichment approaches involve multiple chemical derivatizations and chromatographic separation processes which are time consuming and can contribute to N‐terminal peptide losses. In this study, a fast, one‐step approach utilizing (N‐Succinimidyloxycarbonylmethyl)tris(2,4,6‐trimethoxyphenyl)phosphonium bromide (TMPP) derivatization and StageTip separation was developed to enhance N‐terminal peptide enrichment and analysis. Based on the characteristics of TMPP‐derivatized samples, such as a higher hydrophobicity and increased likelihood to produce a and b ions in collision‐induced dissociation or HCD fragmentation modes, first the SDS‐PAGE was optimized to increase protein loading and gel entry and to remove unbound TMPP. Then, this process was combined with a simplified StageTip separation and a new scoring criterion (considering a, b and y ions) to identify more TMPP‐modified N‐terminal spectra. When utilizing a low amount of starting material (∼20 μg protein), a total of 581 yeast N‐terminal peptides were identified, with 485 of them being TMPP modified, in only about one third of the general experimental time. It is hoped that the workflow constructed herein will provide a fast and practical strategy for N‐terminomic studies.

The expression of functional human parathyroid hormone in a gene therapy model for osteoporosis

The pro-peptide sequence of human parathyroid hormone (hPTH) is essential for the efficient translocation of the nascent polypeptide and the precise cleavage at residue +1 from the cleavage site. If residue +1 is not a serine, the bioactivity of hPTH decreases dramatically. In order to express the functional hPTH properly in a gene therapy model, we constructed three sets of eukaryotic expression vectors: one combining hPTH cDNA with its pre-sequence or its prepro-sequence; one combining hPTH cDNA with the insulin pre-sequence or with the insulin pre-sequence and the hPTH pro-sequence; one combining pro-insulin cDNA with the hPTH pre-sequence or prepro-sequence. Radioimmunoassay and reverse transcription/polymerase chain reaction showed that transgenes were expressed in the animal model. The hPTH expressed by the vectors with the pro-sequence of PTH improved bone mechanical properties and bone mineral content, whereas the expressed insulin decreased blood glucose. Our results demonstrated that the pro-sequence of hPTH was required for skeletal muscle cells to secrete functional hPTH into the circulation of our gene therapy model. However, the function of expressed insulin was not affected by the fusion of the hPTH pro-sequence. We concluded that pre-sequence, pro-sequence and mature hPTH acted as an integrated unit in skeletal muscle to guarantee the proper processing and release of functional hPTH into the circulation.

Transfer of a gene containing the Arg-Gly-Asp peptide prolongs the bleeding time of mice

Peptides containing Arg-Gly-Asp (RGD) have been used to decrease thrombosis by competitive inhibition of the integrin glycoprotein, αIIb/β3a, in platelets. However, they have a short half-life in vivo. A naked plasmid, pCMV-RGD, was transferred into the skeletal muscle of mice and RGD gene expression was observed by RT-PCR. The bleeding time between control mice and RGD-transferred mice was prolonged from the 10th day to the 80th day after gene transfer while the blood glucose and serum insulin-like proteins remained at normal levels. These results provided a convenient and effective approach to relieve patients from thrombi in a single step over a relatively long period.

Mitosis-specific MRN complex promotes a mitotic signaling cascade to regulate spindle dynamics and chromosome segregation

Significance The Mre11–Rad50–Nbs1 (MRN) complex is well known for participating in DNA damage response pathways and mediating the ATM-dependent phosphorylation signaling cascade. Hypomorphic mutations in the human MRN complex have been identified in autosomal recessive genetic diseases, ataxia-telangiectasia–like disorder, and Nijmegen breakage syndrome. Here, we show that MRN forms a mitosis-specific complex with a protein, MMAP, which mediates a mitotic signaling cascade between PLK1 and KIF2A. We demonstrate that the assembly of this complex is crucial for normal spindle dynamics during mitosis. Thus, our study describes a signaling cascade in which PLK1-dependent phosphorylation promotes the assembly of the MRN–MMAP–PLK1–KIF2A complex, leading to mitotic spindle turnover and chromosome alignment. The MRE11–RAD50–NBS1 (MRN) complex is well known for participating in DNA damage response pathways in all phases of cell cycle. Here, we show that MRN constitutes a mitosis-specific complex, named mMRN, with a protein, MMAP. MMAP directly interacts with MRE11 and is required for optimal stability of the MRN complex during mitosis. MMAP colocalizes with MRN in mitotic spindles, and MMAP-deficient cells display abnormal spindle dynamics and chromosome segregation similar to MRN-deficient cells. Mechanistically, both MMAP and MRE11 are hyperphosphorylated by the mitotic kinase, PLK1; and the phosphorylation is required for assembly of the mMRN complex. The assembled mMRN complex enables PLK1 to interact with and activate the microtubule depolymerase, KIF2A, leading to spindle turnover and chromosome segregation. Our study identifies a mitosis-specific version of the MRN complex that acts in the PLK1–KIF2A signaling cascade to regulate spindle dynamics and chromosome distribution.