Inositol Polyphosphate Phosphatidylinositol 5-Phosphatase9 （At5PTase9） Controls Plant Salt Tolerance by Regulating Endocytosis

Phosphatidylinositol 5-phosphatases （5PTases） components of membrane trafficking system. Recently, we that hydrolyze the 5＇ position of the inositol ring are key reported that mutation in AtSPTase7 gene reduced produc- tion of reactive oxygen species （ROS） and decreased expression of stress-responsive genes, resulting in increased salt sensitivity. Here, we describe an even more salt-sensitive 5ptase mutant, At5ptase9, which also hydrolyzes the 5＇ phos- phate groups specifically from membrane-bound phosphatidylinositides. Interestingly, the mutants were more tolerant to osmotic stress. We analyzed the main cellular processes that may be affected by the mutation, such as production of ROS, influx of calcium, and induction of salt-response genes. The At5ptase9 mutants showed reduced ROS produc- tion and Ca2~ influx, as well as decreased fluid-phase endocytosis. Inhibition of endocytosis by phenylarsine oxide or Tyrphostin A23 in wild-type plants blocked these responses. Induction of salt-responsive genes in wild-type plants was also suppressed by the endocytosis inhibitors. Thus, inhibition of endocytosis in wild-type plants mimicked the salt stress responses, observed in the AtSptase9 mutants. In summary, our results show a key non-redundant role of At5PTase7 and 9 isozymes, and underscore the localization of membrane-bound Ptdlns in regulating plant salt tolerance by coordinating the endocytosis, ROS production, Ca2＋ influx, and induction of stress-responsive genes.     

HPLC法分析乳清蛋白对糖尿病模型小鼠血浆氨基酸组分的影响

为寻求有效的氨基酸分离方法并探讨乳清蛋白对2型糖尿病防治的作用机制,采用HPLC法分析乳清蛋白中氨基酸组分及含量;分别以0％、10％、20％和40％的乳清蛋白(WP)灌胃1型、2型糖尿病模型组、正常组小鼠,4周后观察各组血浆氨基酸的变化.乳清蛋白中亮氨酸、异亮氨酸、缬氨酸分别占氨基酸总量的14.40％、5.93％和5....     

Immunohistochemical localization of Bis protein in the rat central nervous system

We studied the distribution of Bis (Bcl-2 interacting death suppressor) protein in the adult rat brain and spinal cord using immunohistochemistry. Immunoreactivity was observed in specific neuronal populations in distinct nuclei. The most intensely labeled cells were associated with the motor system, including most cranial nerve motor nuclei, Purkinje cells of the cerebellum, the red nucleus, and the ventral motor neurons of the spinal cord. Bis protein was also expressed in several structures associated with the ventricular system, including the subventricular zone of the lateral ventricle and its rostral extension, in the subcommissural organ, and in tanycytes, radial glial cells in the hypothalamus. Using double-labeling techniques, Bis-immunoreactive cells in the rostral migratory stream, coexpressing Bcl-2, were confirmed as glial fibrillary acidic protein-positive astrocytes comprising the glial tubes. The widespread distribution of Bis suggests that this protein has broader functions in the adult rat central nervous system than previously thought, and that it could be associated with a particular role in the rostral migratory system.J.-H. Lee and M.-Y. Lee contributed equally to this study. This work was supported by the KOSEF through the Cell Death Disease Research Center of MRC at the Catholic University of Korea (R13-2002-005-01001-0) and the Catholic Medical Center Research Foundation grant made in the program year of 2002     

ATP synthases: insights into their motor functions from sequence and structural analyses

ATP synthases are motor complexes comprised of F₀ and F₁ parts that couple the proton gradient across the membrane to the synthesis of ATP by rotary catalysis. Although a great deal of information has been accumulated regarding the structure and function of ATP synthases, their motor functions are not fully understood. For this reason, we performed the alignments and analyses of the protein sequences comprising the core of the ATP synthase motor complex, and examined carefully the locations of the conserved residues in the subunit structures of ATP synthases. A summary of the findings from this bioinformatic study is as follows. First, we found that four conserved regions in the sequence of subunit are clustered into three patches in its structure. The interactions of these conserved patches with the and subunits are likely to be critical for energy coupling and catalytic activity of the ATP synthase. Second, we located a four-residue cluster at the N-terminal domain of mitochondrial OSCP or bacterial (or chloroplast) subunit which may be critical for the binding of these subunits to F₁. Third, from the localizations of conserved residues in the subunits comprising the rotors of ATP synthases, we suggest that the conserved interaction site at the interface of subunit c and (mitochondria) or (bacteria and chloroplasts) may be important for connecting the rotor of F₁ to the rotor of F₀. Finally, we found the sequence of mitochondrial subunit b to be highly conserved, significantly longer than bacterial subunit b, and to contain a shorter dimerization domain than that of the bacterial protein. It is suggested that the different properties of mitochondrial subunit b may be necessary for interaction with other proteins, e.g., the supernumerary subunits.     

Near-Complete Structure and Model of Tel1ATM from Chaetomium thermophilum Reveals a Robust Autoinhibited ATP State

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ProDCoNN: Protein design using a convolutional neural network

Designing protein sequences that fold to a given three-dimensional (3D) structure has long been a challenging problem in computational structural biology with significant theoretical and practical implications. In this study, we first formulated this problem as predicting the residue type given the 3D structural environment around the C _α atom of a residue, which is repeated for each residue of a protein. We designed a nine-layer 3D deep convolutional neural network (CNN) that takes as input a gridded box with the atomic coordinates and types around a residue. Several CNN layers were designed to capture structure information at different scales, such as bond lengths, bond angles, torsion angles, and secondary structures. Trained on a very large number of protein structures, the method, called ProDCoNN (protein design with CNN), achieved state-of-the-art performance when tested on large numbers of test proteins and benchmark datasets.     

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.