基于Petri网的基因调控网络建模与分析

如何有效描述与分析复杂的基因调控网络(GRN)是生物学家研究基因表达调控机制的关键步骤.现有大部分方法在建模过程中忽略了生物中广泛存在的协同作用,模型预测结果与实际生物行为之间存在误差.基于混合函数Petri网(HFPN)理论提出了一种对基因调控网络进行定量分析的新方法.首先简要介绍GRN与HFPN的基础理论,然后为HFPN引入两类新元素:逻辑库所与逻辑变迁,描述基因调控网络的逻辑规则以及转录因子间的协同作用,最后构建海胆endo16基因调控网络的Petri网模型,并预测模型在不同位点发生突变时的基因表达水平变化.分析结果与文献实验数据相一致,验证了方法的正确性.     

The role of STIM1 and SOCE in smooth muscle contractility

Contraction is a central feature for skeletal, cardiac and smooth muscle; this unique feature is largely dependent on calcium (Ca²⁺) signaling and therefore maintenance of internal Ca²⁺ stores. Stromal interaction molecule 1 (STIM1) is a single-pass transmembrane protein that functions as a Ca²⁺ sensor for the activation store-operated calcium channels (SOCCs) on the plasma membrane in response to depleted internal sarco(endo)plasmic (S/ER) reticulum Ca²⁺ stores. STIM1 was initially characterized in non-excitable cells; however, evidence from both animal models and human mutations suggests a role for STIM1 in modulating Ca²⁺ homeostasis in excitable tissues as well. STIM1-dependent SOCE is particularly important in tissues undergoing sustained contraction, leading us to believe STIM1 may play a role in smooth muscle contraction. To date, the role of STIM1 in smooth muscle is unknown. In this review, we provide a brief overview of the role of STIM1-dependent SOCE in striated muscle and build off that knowledge to investigate whether STIM1 contributes to smooth muscle contractility. We conclude by discussing the translational implications of targeting STIM1 in the treatment of smooth muscle disorders.     

Refolding the unfoldable: A systematic approach for renaturation of Bacillus circulans xylanase

Xylanases are important polysaccharide‐cleaving catalysts for the pulp and paper, animal feeds and biofuels industries. They have also proved to be valuable model systems for understanding enzymatic catalysis, with one of the best studied being the GH11 xylanase from Bacillus circulans (Bcx). However, proteins from this class are very recalcitrant to refolding in vitro. This both limits their high level expression in heterologous hosts, and prevents experimental approaches, such as peptide ligation or chemical modifications, to probe and engineer their stability and function. To solve this problem, a systematic screening approach was employed to identify suitable buffer conditions for renaturing Bcx in vitro. The fractional factorial screen employed identified starting conditions for refolding, which were then refined and developed into a generic protocol for renaturing preparative amounts of active Bcx in a 50–60% yield from inclusion bodies. The method is robust and proved equally proficient at refolding circularly permuted versions that carry cysteine mutations. This general approach should be applicable to related GH11 xylanases, as well as proteins adopting a similar β‐jellyroll fold, that are otherwise recalcitrant to refolding in vitro.     

Head-to-tail oligomerization of calsequestrin: a novel mechanism for heterogeneous distribution of endoplasmic reticulum luminal proteins

Many proteins retained within the endo/sarcoplasmic reticulum (ER/SR) lumen express the COOH-terminal tetrapeptide KDEL, by which they continuously recycle from the Golgi complex; however, others do not express the KDEL retrieval signal. Among the latter is calsequestrin (CSQ), the major Ca2+-binding protein condensed within both the terminal cisternae of striated muscle SR and the ER vacuolar domains of some neurons and smooth muscles. To reveal the mechanisms of condensation and establish whether it also accounts for ER/SR retention of CSQ, we generated a variety of constructs: chimeras with another similar protein, calreticulin (CRT); mutants truncated of COOH- or NH2-terminal domains; and other mutants deleted or point mutated at strategic sites. By transfection in L6 myoblasts and HeLa cells we show here that CSQ condensation in ER-derived vacuoles requires two amino acid sequences, one at the NH2 terminus, the other near the COOH terminus. Experiments with a green fluorescent protein GFP/CSQ chimera demonstrate that the CSQ-rich vacuoles are long-lived organelles, unaffected by Ca2+ depletion, whose almost complete lack of movement may depend on a direct interaction with the ER. CSQ retention within the ER can be dissociated from condensation, the first identified process by which ER luminal proteins assume a heterogeneous distribution. A model is proposed to explain this new process, that might also be valid for other luminal proteins.     

Pilocarpine-induced status epilepticus causes N-methyl-D-aspartate receptor-dependent inhibition of microsomal Mg(2+)/Ca(2+) ATPase-mediated Ca(2+) uptake

Status epilepticus is associated with sustained and elevated levels of cytosolic Ca(2+). To elucidate the mechanisms associated with changes of cytosolic Ca(2+) after status epilepticus, this study was initiated to evaluate the effect of pilocarpine-induced status epilepticus on Mg(2+)/Ca(2+) ATPase-mediated Ca(2+) uptake in microsomes isolated from rat cortex, because the Ca(2+) uptake mechanism plays a major role in regulating intracellular Ca(2+) levels. The data demonstrated that the initial rate and overall Ca(2+) uptake in microsomes from pilocarpine treated animals were significantly inhibited compared with those in microsomes from saline-treated control animals. It was also shown that the inhibition of Ca(2+) uptake caused by status epilepticus was not an artifact of increased Ca(2+) release from microsomes, selective isolation of damaged microsomes from the homogenate, or decreased Mg(2+)/Ca(2+) ATPase protein in the microsomes. Pretreatment with the NMDA antagonist dizocilpine maleate blocked status epilepticus-induced inhibition of the initial rate and overall Ca(2+) uptake. The data suggest that inhibition of microsomal Mg(2+)/Ca(2+) ATPase Ca(2+) uptake is involved in NMDA-dependent deregulation of cytosolic Ca(2+) homeostasis associated with status epilepticus.     

Inhibition of Ubiquitin Proteasome System Rescues the Defective Sarco(endo)plasmic Reticulum Ca2+-ATPase (SERCA1) Protein Causing Chianina Cattle Pseudomyotonia

A missense mutation in ATP2A1 gene, encoding sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA1) protein, causes Chianina cattle congenital pseudomyotonia, an exercise-induced impairment of muscle relaxation. Skeletal muscles of affected cattle are characterized by a selective reduction of SERCA1 in sarcoplasmic reticulum membranes. In this study, we provide evidence that the ubiquitin proteasome system is involved in the reduced density of mutated SERCA1. The treatment with MG132, an inhibitor of ubiquitin proteasome system, rescues the expression level and membrane localization of the SERCA1 mutant in a heterologous cellular model. Cells co-transfected with the Ca²⁺-sensitive probe aequorin show that the rescued SERCA1 mutant exhibits the same ability of wild type to maintain Ca²⁺ homeostasis within cells. These data have been confirmed by those obtained ex vivo on adult skeletal muscle fibers from a biopsy from a pseudomyotonia-affected subject. Our data show that the mutation generates a protein most likely corrupted in proper folding but not in catalytic activity. Rescue of mutated SERCA1 to sarcoplasmic reticulum membrane can re-establish resting cytosolic Ca²⁺ concentration and prevent the appearance of pathological signs of cattle pseudomyotonia.     

Intracellular- and extracellular-derived Ca influence phospholipase A2-mediated fatty acid release from brain phospholipids

Docosahexaenoic acid (DHA) and arachidonic acid (AA) are found in high concentrations in brain cell membranes and are important for brain function and structure. Studies suggest that AA and DHA are hydrolyzed selectively from the sn-2 position of synaptic membrane phospholipids by Ca²⁺-dependent cytosolic phospholipase A₂ (cPLA₂) and Ca²⁺-independent phospholipase A₂ (iPLA₂), respectively, resulting in increased levels of the unesterified fatty acids and lysophospholipids. Cell studies also suggest that AA and DHA release depend on increased concentrations of Ca²⁺, even though iPLA₂ has been thought to be Ca²⁺-independent. The source of Ca²⁺ for activation of cPLA₂ is largely extracellular, whereas Ca²⁺ released from the endoplasmic reticulum can activate iPLA₂ by a number of mechanisms. This review focuses on the role of Ca²⁺ in modulating cPLA₂ and iPLA₂ activities in different conditions. Furthermore, a model is suggested in which neurotransmitters regulate the activity of these enzymes and thus the balanced and localized release of AA and DHA from phospholipid in the brain, depending on the primary source of the Ca²⁺ signal.