USP15 promotes the apoptosis of degenerative nucleus pulposus cells by suppressing the PI3K/AKT signalling pathway

ObjectivesDegenerative disc disease is characterized by an enhanced breakdown of its existing nucleus pulposus (NP) matrix due to the dysregulation of matrix enzymes and factors. Ubiquitin‐specific protease 15 (USP15) is reported to be abnormal in certain human diseases. However, its role in NP degeneration remains unclear. Therefore, we aimed to explore the function of USP15 in degenerative NP cell specimens.MethodsWe induced gene silencing and overexpression of USP15 in degenerative NP cells using RNA interference (RNAi) and a lentiviral vector, respectively. qRT‐PCR and Western blotting were used to determine gene and protein expression levels. Cell apoptosis was analysed via flow cytometry. Protein interaction was examined by performing a co‐immunoprecipitation assay. Furthermore, the PI3K inhibitor LY294002 and agonist IGF‐1 were used to investigate the link between USP15 and AKT in NP degeneration.ResultsWe found that USP15 was up‐regulated in degenerative NP cells and that its overexpression accelerated the process of apoptosis. Moreover, USP15 expression levels negatively correlated with AKT phosphorylation in degenerative NP cells. Furthermore, targeting and silencing USP15 with miR‐338‐3p and studying its interaction with FK506‐binding protein 5 (FKBP5) revealed enhancement of FKBP5 ubiquitination, indicating that USP15 is a component of the FKBP5/AKT signalling pathway in degenerative NP cells.ConclusionsOur results show that USP15 exacerbates NP degradation by deubiquitinating and stabilizing FKBP5. This in turn results in the suppression of AKT phosphorylation in degenerative NP cells. Therefore, our study provides insights into the understanding of USP15 function as a potential molecule in the network of NP degeneration.     

Membrane dynamics of γ‐secretase with the anterior pharynx‐defective 1B subunit

The four‐subunit protease complex γ‐secretase cleaves many single‐pass transmembrane (TM) substrates, including Notch and β‐amyloid precursor protein to generate amyloid‐β (Aβ), central to Alzheimer's disease. Two of the subunits anterior pharynx‐defective 1 (APH‐1) and presenilin (PS) exist in two homologous forms APH1‐A and APH1‐B, and PS1 and PS2. The consequences of these variations are poorly understood and could affect Aβ production and γ‐secretase medicine. Here, we developed the first complete structural model of the APH‐1B subunit using the published cryo‐electron microscopy (cryo‐EM) structures of APH1‐A (Protein Data Bank: 5FN2, 5A63, and 6IYC). We then performed all‐atom molecular dynamics simulations at 303 K in a realistic bilayer system to understand both APH‐1B alone and in γ‐secretase without and with substrate C83‐bound. We show that APH‐1B adopts a 7TM topology with a water channel topology similar to APH‐1A. We demonstrate direct transport of water through this channel, mainly via Glu84, Arg87, His170, and His196. The apo and holo states closely resemble the experimental cryo‐EM structures with APH‐1A, however with subtle differences: The substrate‐bound APH‐1B γ‐secretase was quite stable, but some TM helices of PS1 and APH‐1B rearranged in the membrane consistent with the disorder seen in the cryo‐EM data. This produces different accessibility of water molecules for the catalytic aspartates of PS1, critical for Aβ production. In particular, we find that the typical distance between the catalytic aspartates of PS1 and the C83 cleavage sites are shorter in APH‐1B, that is, it represents a more closed state, due to interactions with the C‐terminal fragment of PS1. Our structural‐dynamic model of APH‐1B alone and in γ‐secretase suggests generally similar topology but some notable differences in water accessibility which may be relevant to the protein's existence in two forms and their specific function and location.     

Lysosomal Targeting of the CLN7 Membrane Glycoprotein and Transport Via the Plasma Membrane Require a Dileucine Motif

CLN7 is a polytopic lysosomal membrane protein deficient in variant late infantile neuronal ceroid lipofuscinosis, a neurodegenerative lysosomal storage disorder. In this study fluorescence protease protection assays and mutational analyses revealed the N‐ and C‐terminal tails of CLN7 in the cytosol and two N‐glycosylation sites at N371 and N376. Both partially and non‐glycosylated CLN7 were correctly transported to lysosomes. To identify lysosomal targeting motifs, we generated CD4‐chimera fused to the N‐ and C‐terminal domains of CLN7. Lysosomal localization of the chimeric proteins requires a consensus acidic dileucine‐based motif in the N‐terminus and two tandem tyrosine‐based signals in the C‐terminus. Mutation of these sorting motifs resulted in cell surface redistribution of CD4 chimeras. However, the dileucine‐based motif is of critical importance for lysosomal localization of the full‐length CLN7 in different cell lines. Cell surface biotinylation revealed that at equilibrium 22% of total CLN7 is localized at the plasma membrane. Mutation of the dileucine motif or the co‐expression of dominant‐negative mutant dynamin K44A led to a further increase of CLN7 at the plasma membrane. Our data demonstrate that CLN7 contains several cytoplasmic lysosomal targeting signals of which the N‐terminal dileucine‐based motif is required for the predominant lysosomal targeting along the indirect pathway and clathrin‐mediated endocytosis of CLN7.     

Optimization of tetracycline-responsive recombinant protein production and effect on cell growth and ER stress in mammalian cells

The inducible T-REx system and other inducible expression systems have been developed in order to control the expression levels of recombinant protein in mammalian cells. In order to study the effects of heterologous protein expression on mammalian host behavior, the gene for recombinant Human transferrin (hTf) was integrated into HEK-293 cells and expressed under the control of the T-REx inducible technology (293-TetR-Hyg-hTf) or using a constitutive promoter (293-CMV-hTf). A number of inducible clones with variable expression levels were identified for the T-REx system with levels of hTf for the high expressing clones nearly double those obtained using the constitutive cytomegalovirus (CMV) promoter. The level of transferrin produced was found to increase proportionately with tetracycline concentration between 0 and 1 mug/mL with no significant increases in transferrin production above 1 mug/mL. As a result, the optimal induction time and tetracycline concentrations were determined to be the day of plating and 1 mug/mL, respectively. Interestingly, the cells induced to express transferrin, 293-TetR-Hyg-hTf, exhibited lower viable cell densities and percent viabilities than the uninduced cultures for multiple clonal isolates. In addition, the induction of transferrin expression was found to cause an increase in the expression of the ER-stress gene, BiP, that was not observed in the uninduced cells. However, both uninduced and induced cell lines containing the hTf gene exhibited longer survival in culture than the control cells, possibly as a result of the positive effects of hTf on cell survival. Taken together, these results suggest that the high level expression of complex proteins in mammalian cells can limit the viable cell densities of cells in culture as a result of cellular stresses caused by generating proteins that may be difficult to fold or are otherwise toxic to cells. The application of inducible systems such as the T-REx technology will allow us to optimize protein production while limiting the negative effects that result from these cellular stresses.     

Genome-wide survey of prokaryotic O-protein phosphatases

Complex and diverse signal transduction circuits are responsible for the efficient functioning of cellular network. Protein kinases and O-protein phosphatases are primarily responsible for propagating such stimuli within a eukaryotic cell. However, there is limited understanding of O-protein phosphatases in the prokaryotic genomes. The availability of complete genome sequence information for several prokaryotes permits a genome-wide survey of O-protein phosphatases. The distribution of the various protein phosphatase families has been observed to be mosaic, with the exception of the members of the phospho protein family P (PPP), which is consistent with previous studies. The PPP family is ubiquitous in the prokaryotic world and undergoes the highest sequence divergence within a genome amongst phosphatases studied. The co-occurrence of low molecular mass tyrosine phosphatase (LMWPc) and PPP domain in a single polypeptide suggests that the protein present in Archaeoglobus fulgidus might represent the progenitor for all protein phosphatases. The curation of data on prokaryotic protein phosphatases provides a convenient framework for the analysis of domain architectures and for characterising structural and functional properties of this important family of signalling proteins.     

Thermodynamic and electrostatic properties of ternary Oxytricha nova TEBP-DNA complex

Telomeres constitute the nucleoprotein ends of eukaryotic chromosomes which are essential for their proper function. Telomere end binding protein (TEBP) from Oxytricha nova was among the first telomeric proteins, which were well characterized biologically. TEBP consists of two protein subunits (alpha, beta) and forms a ternary complex with single stranded telomeric DNA containing tandem repeats TTTTGGGG. This work presents the characterization of the thermodynamic and electrostatic properties of this complex by computational chemistry methods (continuum Poisson-Boltzmann and solvent accessible surface calculations). Our calculations give a new insight into molecular properties of studied system. Based on the thermodynamic analysis we provide a rationale for the experimental observation that alpha and ssDNA forms a binary complex and the beta subunit joins alpha:ssDNA complex only after the latter is formed. Calculations of distribution of the molecular electrostatic potential for protein subunits alone and for all possible binary complexes revealed the important role of the "guiding funnel" potential generated by alpha:ssDNA complex. This potential may help the beta subunit to dock to the already formed alpha:DNA intermediate in highly steric and electrostatic favorable manner. Our pK(a) calculations of TEBP are able to explain the experimental mobility shifts of the complex in electrophoretic non-denaturating gels.     

NMDAR/PKC通路介导基底外侧杏仁核的长时程增强

本文在大鼠杏仁核脑片上刺激外囊（external capsule,EC）,在基底外侧杏仁核（basolateral amygdala,BLA）记录场电位,观察能否在杏仁核诱导长时程增强（long—term potentiation,LTP）,并探讨杏仁核LTP形成的可能机制。应用两串间隔10min的0频率波刺激EC,可见BLA的场电位明显增强,持续时间在30min以上。EC—BLA的LTP表现为通路特异性,可被N-甲基-D-天冬氨酸受体（N-methyl—D—aspartate receptor,NMDAR）阻断剂D,L-2-氨基-5磷酸基戊酸（APV）阻断。在灌流液中加入蛋白激酶C（protein kinaseC,PKC）抑制剂chelerythrine chloride,对BLA的基础场电位和配对脉冲比率（pairedpulse ratio,PPR）没有影响;但在chelerythrine chloride存在的情况下,两串θ频率波刺激不能诱导出LTP;若于两串高频刺激10min后,在灌流液中加入chelerythrine chloride不能阻断BLA的LTP。上述结果表明,EC—BLA的LTP为NMDAR依赖性,PKC参与BLA的LTP诱导和早期维持。     

Isolation of legumin-like protein from Phaseolus aureus and Phaseolus vulgaris

An 11S seed globulin has been isolated from Phaseolus aureus and P. vulgaris by zonal isoelectric precipitation and the MWs of the constituent subunits determined. The protein of P. vulgaris occurs in the protein body fraction and its chemical composition, including the N-terminal amino acids and amino acid composition has been determined. The similarity between the 11S globulin of the two Phaseolus spp. and legumin from other leguines is discussed.     

Light-Induced CREB Phosphorylation and Gene Expression in Rat Retinal Cells

Abstract: The signal pathway for light-induced expression of c- fos and the neuropeptide somatostatin (SS) in rat retinal cells was investigated. Flashing light induced c- fos and SS mRNA in the inner nuclear layer and the ganglion cell layer. As both c- fos and SS genes have a cyclic AMP response element (CRE) in their promoters, CRE-binding protein (CREB) phosphorylation in retinal cells was examined with a phospho-CREB-specific antibody. Both flashing light and administration of the L-type Ca²⁺ channel activator Bay K 8644 induced phosphorylation of CREB in the nuclei of the amacrine cells and the ganglion cells where c- fos /SS mRNAs were expressed. These cells could be double-stained with anti-calmodulin kinase II (anti-CaM kinase II) monoclonal antibody and phospho-CREB-specific polyclonal antiserum after Bay K 8644 administration, indicating the colocalization of phosphorylated CREB at Ser¹³³ and CaM kinase II in the neural retina.