Ubiquitin‐specific protease‐like 1 (USPL1) is a SUMO isopeptidase with essential,non‐catalytic functions

Isopeptidases are essential regulators of protein ubiquitination and sumoylation. However, only two families of SUMO isopeptidases are at present known. Here, we report an activity‐based search with the suicide inhibitor haemagglutinin (HA)‐SUMO‐vinylmethylester that led to the identification of a surprising new SUMO protease, ubiquitin‐specific protease‐like 1 (USPL1). Indeed, USPL1 neither binds nor cleaves ubiquitin, but is a potent SUMO isopeptidase both in vitro and in cells. C13orf22l—an essential but distant zebrafish homologue of USPL1—also acts on SUMO, indicating functional conservation. We have identified invariant USPL1 residues required for SUMO binding and cleavage. USPL1 is a low‐abundance protein that colocalizes with coilin in Cajal bodies. Its depletion does not affect global sumoylation, but causes striking coilin mislocalization and impairs cell proliferation, functions that are not dependent on USPL1 catalytic activity. Thus, USPL1 represents a third type of SUMO protease, with essential functions in Cajal body biology.     

Functional Effects of a Restrictive-Cardiomyopathy-Linked Cardiac Troponin I Mutation (R145W) in Transgenic Mice

The human cardiac troponin I (hcTnI) mutation R145W has been associated with restrictive cardiomyopathy. In this study, simultaneous measurements of ATPase activity and force in skinned papillary fibers from hcTnI R145W transgenic mice (Tg-R145W) were explored. Tg-R145W fibers showed an ∼ 13-16% increase in maximal Ca²⁺-activated force and ATPase activity compared to hcTnI wild-type transgenic mice. The force-generating cross-bridge turnover rate (g) and the energy cost (ATPase/force) were the same in all groups of fibers. Also, the Tg-R145W fibers showed a large increase in the Ca²⁺ sensitivity of both force development and ATPase. In intact fibers, the mutation caused prolonged force and intracellular [Ca²⁺] transients and increased time to peak force. Analysis of force and Ca²⁺ transients showed that there was a 40% increase in peak force in Tg-R145W muscles, which was likely due to the increased Ca²⁺ transient duration. The above cited results suggest that: (1) there would be an increase in resistance to ventricular filling during diastole resulting from the prolonged force and Ca²⁺ transients that would result in a decrease in ventricular filling (diastolic dysfunction); and (2) there would be a large (approximately 53%) increase in force during systole, which may help to partly compensate for diastolic dysfunction. These functional results help to explain the mechanisms by which these mutations give rise to a restrictive phenotype.     

Denaturation and unfolding of human anaphylatoxin C3a: an unusually low covalent stability of its native disulfide bonds

The complement C3a anaphylatoxin is a major molecular mediator of innate immunity. It is a potent activator of mast cells, basophils and eosinophils and causes smooth muscle contraction. Structurally, C3a is a relatively small protein (77 amino acids) comprising a N-terminal domain connected by 3 native disulfide bonds and a helical C-terminal segment. The structural stability of C3a has been investigated here using three different methods: Disulfide scrambling; Differential CD spectroscopy; and Reductive unfolding. Two uncommon features regarding the stability of C3a and the structure of denatured C3a have been observed in this study. (a) There is an unusual disconnection between the conformational stability of C3a and the covalent stability of its three native disulfide bonds that is not seen with other disulfide proteins. As measured by both methods of disulfide scrambling and differential CD spectroscopy, the native C3a exhibits a global conformational stability that is comparable to numerous proteins with similar size and disulfide content, all with mid-point denaturation of [GdmCl]_1/2 at 3.4-5 M. These proteins include hirudin, tick anticoagulant protein and leech carboxypeptidase inhibitor. However, the native disulfide bonds of C3a is 150-1000 fold less stable than those proteins as evaluated by the method of reductive unfolding. The 3 native disulfide bonds of C3a can be collectively and quantitatively reduced with as low as 1 mM of dithiothreitol within 5 min. The fragility of the native disulfide bonds of C3a has not yet been observed with other native disulfide proteins. (b) Using the method of disulfide scrambling, denatured C3a was shown to consist of diverse isomers adopting varied extent of unfolding. Among them, the most extensively unfolded isomer of denatured C3a is found to assume beads-form disulfide pattern, comprising Cys³⁶-Cys⁴⁹ and two disulfide bonds formed by two pair of consecutive cysteines, Cys²²-Cys²³ and Cys⁵⁶-Cys⁵⁷, a unique disulfide structure of polypeptide that has not been documented previously.     

Physiological girdling of pine trees via phloem chilling: proof of concept

Quantifying below-ground carbon (C) allocation is particularly difficult as methods usually disturb the root-mycorrhizal-soil continuum. We reduced C allocation below ground of loblolly pine trees by: (1) physically girdling trees and (2) physiologically girdling pine trees by chilling the phloem. Chilling reduced cambium temperatures by approximately 18 degrees C. Both methods rapidly reduced soil CO2 efflux, and after approximately 10 days decreased net photosynthesis (P(n)), the latter indicating feedback inhibition. Chilling decreased soil-soluble C, indicating that decreased soil CO2 efflux may have been mediated by a decrease in root C exudation that was rapidly respired by microbes. These effects were only observed in late summer/early autumn when above-ground growth was minimal, and not in the spring when above-ground growth was rapid. All of the effects were rapidly reversed when chilling was ceased. In fertilized plots, both chilling and physical girdling methods reduced soil CO2 efflux by approximately 8%. Physical girdling reduced soil CO2 efflux by 26% in non-fertilized plots. This work demonstrates that phloem chilling provides a non-destructive alternative to reducing the movement of recent photosynthate below the point of chilling to estimate C allocation below ground on large trees.     

蝉花虫草活性成分的抗惊厥作用

本研究采用活性追踪的方法,逐步从人工培养蝉花虫草分离获得A（50%乙醇回流提取）、B（膜分离）、C（大孔树脂洗脱）和D（Sephadex LH20柱纯化）等4种样品,单一化合物D纯度为98.62%,鉴定为N6‐(2‐羟乙基)腺苷[N6‐(2‐hydroxyethyl)‐adenosine,HEA]。研究各样品对戊四唑（pentylenetetrazol,PTZ）诱导的小鼠惊厥模型的影响,以及选择性腺苷A1受体（AA1R）拮抗剂DPCPX或选择性腺苷A2A受体（AA2AR）拮抗剂ZM241385对HEA作用的影响,并采用苏木精-伊红（hematoxylin-eosin,HE）染色、免疫组化（immunohistochemical,IHC）染色和蛋白质免疫印迹（Western blot,WB）等技术进一步探究HEA抗惊厥作用的机制。结果表明,各样品（i.p.）均有抗惊厥活性,HEA（40–60mg/kg,i.p.）能显著延长惊厥小鼠的存活时间和降低死亡率,DPCPX（2mg/kg,i.p.）能够拮抗HEA的抗惊厥作用,而ZM241385无此作用;HE、IHC和WB的结果进一步揭示DPCPX显著降低HEA的作用。综上所述,蝉花虫草的HEA具有抗惊厥作用,并且可能通过激活腺苷A1受体而起作用。     

Tight junction biogenesis during early development

The tight junction (TJ) is an essential component of the differentiated epithelial cell required for polarised transport and intercellular integrity and signalling. Whilst much can be learnt about how the TJ is constructed and maintained and how it functions using a wide range of cellular systems, the mechanisms of TJ biogenesis within developmental models must be studied to gain insight into this process as an integral part of epithelial differentiation. Here, we review TJ biogenesis in the early mammalian embryo, mainly considering the mouse but also including the human and other species, and, briefly, within the amphibian embryo. We relate TJ biogenesis to inherent mechanisms of cell differentiation and biosynthesis occurring during cleavage of the egg and the formation of the first epithelium. We also evaluate a wide range of exogenous cues, including cell-cell interactions, protein kinase C signalling, gap junctional communication, Na⁺/K⁺-ATPase and cellular energy status, that may contribute to TJ biogenesis in the embryo and how these may shape the pattern of early morphogenesis.     

Targeting inhibition of GluR1 Ser845 phosphorylation with an RNA aptamer that blocks AMPA receptor trafficking

Phosphorylation at glutamate receptor subunit 1(GluR1) Ser845 residue has been widely accepted to involve in GluR1-containing α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor trafficking, but the in vivo evidence has not yet been established. One of the main obstacles is the lack of effective methodologies to selectively target phosphorylation at single amino acid residue. In this study, the Escherichia  coli -expressed glutathione- S -transferase-tagged intracellular carboxyl-terminal domain of GluR1 (cGluR1) was phosphorylated by protein kinase A for in vitro selection. We have successfully selected aptamers which effectively bind to phospho-Ser845 cGluR1 protein, but without binding to phospho-Ser831 cGluR1 protein. Moreover, pre-binding of the unphospho-cGluR1 protein with these aptamers inhibits protein kinase A-mediated phosphorylation at Ser845 residue. In contrast, the pre-binding of aptamer A2 has no effect on protein kinase C-mediated phosphorylation at Ser831 residue. Importantly, the representative aptamer A2 can effectively bind the mammalian GluR1 that inhibited GluR1/GluR1-containing AMPA receptor trafficking to the cell surface and abrogated forskolin-stimulated phosphorylation at GluR1 Ser845 in both green fluorescent protein–GluR1-transfected human embryonic kidney cells and cultured rat cortical neurons. The strategy to use aptamer to modify single-residue phosphorylation is expected to facilitate evaluation of the potential role of AMPA receptors in various forms of synaptic plasticity including that underlying psychostimulant abuse.     

2-D DIGE analysis of UV-C radiation-responsive proteins in globe artichoke leaves

Plants respond to ultraviolet stress inducing a self-defence through the regulation of specific gene family members. The UV acclimation is the result of biochemical and physiological processes, such as enhancement of the antioxidant enzymatic system and accumulation of UV-absorbing phenolic compounds (e.g. flavonoids). Globe artichoke is an attractive species for studying the protein network involved in UV stress response, being characterized by remarkable levels of inducible antioxidants. Proteomic tools can assist the evaluation of the expression patterns of UV-responsive proteins and we applied the difference in-gel electrophoresis (DIGE) technology for monitoring the globe artichoke proteome variation at four time points following an acute UV-C exposure. A total of 145 UV-C-modulated proteins were observed and 119 were identified by LC-MS/MS using a ～144,000 customized Compositae protein database, which included about 19,000 globe artichoke unigenes. Proteins were Gene Ontology (GO) categorized, visualized on their pathways and their behaviour was discussed. A predicted protein interaction network was produced and highly connected hub-like proteins were highlighted. Most of the proteins differentially modulated were chloroplast located, involved in photosynthesis, sugar metabolisms, protein folding and abiotic stress. The identification of UV-C-responsive proteins may contribute to shed light on the molecular mechanisms underlying plant responses to UV stress.     

Toll样受体3抑制人类巨噬细胞感染HIV的机制研究

Toll样受体（Toll like receptor,TLR）是固有免疫系统中的病原模式识别受体,在巨噬细胞抗感染免疫中发挥重要作用。TLR3特异性识别双链RNA,诱导细胞内多重信号传导,引发巨噬细胞产生抗病毒活性。本研究以TLR3激活剂多聚次黄苷酸-胞苷酸（polyinosinie：polycytidylic acid,Polyl：C）刺激人类巨噬细胞,发现能显著抑制胞内HIV病毒感染和复制。Poly I：C刺激后,巨噬细胞I型干扰素（interferon,IFN）和抗HIV胞嘧啶脱氨酶（APOBEC3G,A3G）表达水平显著上调;且具有抗HIV作用的MicroRNA（miRNA-28,125b,150,223,and382）的表达也显著上调。本研究初步揭示了TLR3激活后抗HIV感染的机制。     

Predicting RNA secondary structures with pseudoknots by MCMC sampling

The most probable secondary structure of an RNA molecule, given the nucleotide sequence, can be computed efficiently if a stochastic context-free grammar (SCFG) is used as the prior distribution of the secondary structure. The structures of some RNA molecules contain so-called pseudoknots. Allowing all possible configurations of pseudoknots is not compatible with context-free grammar models and makes the search for an optimal secondary structure NP-complete. We suggest a probabilistic model for RNA secondary structures with pseudoknots and present a Markov-chain Monte-Carlo Method for sampling RNA structures according to their posterior distribution for a given sequence. We favor Bayesian sampling over optimization methods in this context, because it makes the uncertainty of RNA structure predictions assessable. We demonstrate the benefit of our method in examples with tmRNA and also with simulated data. McQFold, an implementation of our method, is freely available from http://www.cs.uni-frankfurt.de/~metzler/McQFold.