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51.
Evolutionarily conserved SR proteins (serine/arginine-rich proteins) are important factors for alternative splicing and their activity is modulated by SRPKs (SR protein-specific kinases). We previously identified Dsk1p (dis1-suppressing protein kinase) as the orthologue of human SRPK1 in fission yeast. In addition to its similarity of gene structure to higher eukaryotes, fission yeast Schizosaccharomyces pombe is a unicellular eukaryotic organism in which alternative splicing takes place. In the present study, we have revealed for the first time that SR proteins, Srp1p and Srp2p, are the in vivo substrates of Dsk1p in S. pombe. Moreover, the cellular localization of the SR proteins and Prp2p splicing factor is dependent on dsk1(+): Dsk1p is required for the efficient nuclear localization of Srp2p and Prp2p, while it promotes the cytoplasmic distribution of Srp1p, thereby differentially influencing the destinations of these proteins in the cell. The present study offers the first biochemical and genetic evidence for the in vivo targets of the SRPK1 orthologue, Dsk1p, in S. pombe and the significant correlation between Dsk1p-mediated phosphorylation and the cellular localization of the SR proteins, providing information about the physiological functions of Dsk1p. Furthermore, the results demonstrate that the regulatory function of SRPKs in the nuclear targeting of SR proteins is conserved from fission yeast to human, indicating a general mechanism of reversible phosphorylation to control the activities of SR proteins in RNA metabolism through cellular partitioning.  相似文献   
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Background

Protein transduction is safer than viral vector-mediated transduction for the delivery of a therapeutic protein into a cell. Fusion proteins with an arginine-rich cell-penetrating peptide have been produced in E. coli, but the low solubility of the fusion protein expressed in E. coli impedes the large-scale production of fusion proteins from E. coli.

Results

Expressed protein ligation is a semisynthetic method to ligate a bacterially expressed protein with a chemically synthesized peptide. In this study, we developed expressed protein ligation-based techniques to conjugate synthetic polyarginine peptides to Cre recombinase. The conjugation efficiency of this technique was higher than 80%. Using this method, we prepared semisynthetic Cre with poly-L-arginine (ssCre-R9), poly-D-arginine (ssCre-dR9) and biotin (ssCre-dR9-biotin). We found that ssCre-R9 was delivered to the cell to a comparable level or more efficiently compared with Cre-R11 and TAT-Cre expressed as recombinant fusion proteins in E. coli. We also found that the poly-D-arginine cell-penetrating peptide was more effective than the poly-L-arginine cell-penetrating peptide for the delivery of Cre into cell. We visualized the cell transduced with ssCre-dR9-biotin using avidin-FITC.

Conclusions

Collectively, the results demonstrate that expressed protein ligation is an excellent technique for the production of cell-permeable Cre recombinase with polyarginine cell-penetrating peptides. In addition, this approach will extend the use of cell-permeable proteins to more sophisticated applications, such as cell imaging.

Electronic supplementary material

The online version of this article (doi:10.1186/s12896-015-0126-z) contains supplementary material, which is available to authorized users.  相似文献   
55.
Mycobacterial arabinogalactan (AG) is an essential cell wall component of mycobacteria and a frequent structural and bio‐synthetical target for anti‐tuberculosis (TB) drug development. Here, we report that mycobacterial AG is recognized by galectin‐9 and exacerbates mycobacterial infection. Administration of AG‐specific aptamers inhibits cellular infiltration caused by Mycobacterium tuberculosis (Mtb) or Mycobacterium bovis BCG, and moderately increases survival of Mtb‐infected mice or Mycobacterium marinum‐infected zebrafish. AG interacts with carbohydrate recognition domain (CRD) 2 of galectin‐9 with high affinity, and galectin‐9 associates with transforming growth factor β‐activated kinase 1 (TAK1) via CRD2 to trigger subsequent activation of extracellular signal‐regulated kinase (ERK) as well as induction of the expression of matrix metalloproteinases (MMPs). Moreover, deletion of galectin‐9 or inhibition of MMPs blocks AG‐induced pathological impairments in the lung, and the AG‐galectin‐9 axis aggravates the process of Mtb infection in mice. These results demonstrate that AG is an important virulence factor of mycobacteria and galectin‐9 is a novel receptor for Mtb and other mycobacteria, paving the way for the development of novel effective TB immune modulators.  相似文献   
56.
GABAergic neurons and oligodendrocytes originate from progenitors within the ventral telencephalon. However, the molecular mechanisms that control neuron-glial cell-fate segregation, especially how extrinsic factors regulate cell-fate changes, are poorly understood. We have discovered that the Wnt receptor Ryk promotes GABAergic neuron production while repressing oligodendrocyte formation in the ventral telencephalon. We demonstrate that Ryk controls the cell-fate switch by negatively regulating expression of the intrinsic oligodendrogenic factor Olig2 while inducing expression of the interneuron fate determinant Dlx2. In addition, we demonstrate that Ryk is required for GABAergic neuron induction and oligodendrogenesis inhibition caused by Wnt3a stimulation. Furthermore, we showed that the cleaved intracellular domain of Ryk is sufficient to regulate the cell-fate switch by regulating the expression of intrinsic cell-fate determinants. These results identify Ryk as a multi-functional receptor that is able to transduce extrinsic cues into progenitor cells, promote GABAergic neuron formation, and inhibit oligodendrogenesis during ventral embryonic brain development.  相似文献   
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In the search for new efficacious antibiotics, biosynthetic engineering offers attractive opportunities to introduce minor alterations to antibiotic structures that may overcome resistance. Dbv29, a flavin-containing oxidase, catalyzes the four-electron oxidation of a vancomycin-like glycopeptide to yield A40926. Structural and biochemical examination of Dbv29 now provides insights into residues that govern flavinylation and activity, protein conformation and reaction mechanism. In particular, the serendipitous discovery of a reaction intermediate in the crystal structure led us to identify an unexpected opportunity to intercept the normal enzyme mechanism at two different points to create new teicoplanin analogs. Using this method, we synthesized families of antibiotic analogs with amidated and aminated lipid chains, some of which showed marked potency and efficacy against multidrug resistant pathogens. This method offers a new strategy for the development of chemical diversity to combat antibacterial resistance.  相似文献   
59.
Lyu  Chuqiao  Wang  Lei  Zhang  Juhua 《BMC genomics》2018,19(10):905-165

Background

The DNase I hypersensitive sites (DHSs) are associated with the cis-regulatory DNA elements. An efficient method of identifying DHSs can enhance the understanding on the accessibility of chromatin. Despite a multitude of resources available on line including experimental datasets and computational tools, the complex language of DHSs remains incompletely understood.

Methods

Here, we address this challenge using an approach based on a state-of-the-art machine learning method. We present a novel convolutional neural network (CNN) which combined Inception like networks with a gating mechanism for the response of multiple patterns and longterm association in DNA sequences to predict multi-scale DHSs in Arabidopsis, rice and Homo sapiens.

Results

Our method obtains 0.961 area under curve (AUC) on Arabidopsis, 0.969 AUC on rice and 0.918 AUC on Homo sapiens.

Conclusions

Our method provides an efficient and accurate way to identify multi-scale DHSs sequences by deep learning.
  相似文献   
60.
Photodynamic therapy (PDT), a photochemotherapeutic regimen used to treat several diseases, including cancer, exerts its effects mainly through induction of cell death. Using human epidermoid carcinoma A431 cells as a model, we previously showed that distinct cell death types could be triggered by protocols that selectively delivered Photofrin (a clinically approved photosensitizer) to different subcellular sites (Hsieh et al. [2003] J Cell Physiol 194: 363–375]. Here, the responses elicited by PDT in A431 cells containing intracellular organelle‐localized Photofrin were further characterized. Two prominent cell phenotypes were observed under these conditions: one characterized by perinuclear vacuole (PV) formation 2–8 h after PDT followed by cell recovery or shrinkage within 48 h, and a second characterized by typical apoptotic features appearing within 4 h after PDT. DCFDA‐sensitive reactive oxygen species formed proximal to PVs during the response to PDT, covering areas in which both endoplasmic reticulum (ER) and the Golgi complex were located. Biochemical analyses showed that Photofrin‐PDT also induced JNK activation and altered the protein secretion profile. A more detailed examination of PV formation revealed that PVs were derived from the ER. The alteration of ER structure induced by PDT was similar to that triggered by thapsigargin, an ER Ca2+‐ATPase inhibitor that perturbs Ca2+ homeostasis, suggesting a role for Ca2+ in the formation of PVs. Microtubule dynamics did not significantly affect PV formation. This study demonstrates that cells in which intracellular organelles are selectively loaded with Photofrin mount a novel response to ER stress induced by PDT. J. Cell. Biochem. 111: 821–833, 2010. © 2010 Wiley‐Liss, Inc.  相似文献   
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