Expression and Purification of Soluble, Active Heterodimeric Guanylyl Cyclase from Baculovirus

A method for expression and purification of active cytosolic heterodimeric histidine (His)-tagged guanylyl cyclase of the α1/β1 isoform has been developed using recombinant baculovirus-transfected insect cells. Confirmation of expression of active cyclase was obtained by both Western analysis and enzymatic activity. A His tag on the COOH-terminus of the α1 and β1 subunits allowed rapid purification of the heterodimeric form of guanylyl cyclase in a single affinity step using a nickel column. A second gel-filtration step was applied to reconstitute into the complex heme, a required cofactor. This was confirmed spectroscopically by absorbance in the Soret region. Like enzyme purified from tissue, the activity of recombinant guanylyl cyclase was increased by protoporphyrin IX and inhibited by both Zn- and Sn-protoporphyrin. The method described here should provide a general approach for the expression and purification of alternate forms of cytosolic guanylyl cyclase and facilitate mechanistic and structural studies of this important family of enzymes. Furthermore, the procedure demonstrates the utility of the His-tag system to purify multimeric proteins.     

Nucleotidyl Cyclase Activity of Particulate Guanylyl Cyclase A: Comparison with Particulate Guanylyl Cyclases E and F,Soluble Guanylyl Cyclase and Bacterial Adenylyl Cyclases Cyaa and Edema Factor

Guanylyl cyclases (GCs) regulate many physiological processes by catalyzing the synthesis of the second messenger cGMP. The GC family consists of seven particulate GCs (pGCs) and a nitric oxide-activated soluble GC (sGC). Rat sGC α₁β₁ possesses much broader substrate specificity than previously assumed. Moreover, the exotoxins CyaA from Bordetella pertussis and edema factor (EF) from Bacillus anthracis possess nucleotidyl cyclase (NC) activity. pGC-A is a natriuretic peptide-activated homodimer with two catalytic sites that act cooperatively. Here, we studied the NC activity of rat pGC-A in membranes of stably transfected HEK293 cells using a highly sensitive and specific HPLC-MS/MS technique. GTP and ITP were effective, and ATP and XTP were only poor, pGC-A substrates. In contrast to sGC, pGC-A did not use CTP and UTP as substrates. pGC-E and pGC-F expressed in bovine rod outer segment membranes used only GTP as substrate. In intact HEK293 cells, pGC-A generated only cGMP. In contrast to pGCs, EF and CyaA showed very broad substrate-specificity. In conclusion, NCs exhibit different substrate-specificities, arguing against substrate-leakiness of enzymes and pointing to distinct physiological functions of cyclic purine and pyrimidine nucleotides.     

瞬时受体电位M8离子通道功能及其翻译后修饰调节机制

瞬时受体电位M8(transient receptor potential melastatin 8, TRPM8)又称冷及薄荷醇感受器,位于细胞膜或细胞器膜上,是瞬时受体电位(transient receptor potential, TRP)通道超家族中的一员。TRPM8通道分布广泛,是一个非选择性阳离子通道,可作为冷热传感器和冷痛传感器进行信号传导,参与众多生物过程的调节,在维持细胞内外稳态、控制离子进出细胞方面具有重要作用。研究发现,蛋白质翻译后修饰(post-translational modification, PTM)通过调控TRPM8通道的功能,进而影响多种疾病的发生和发展。因此,探究TRPM8的翻译后修饰的过程,对深入了解TRPM8的功能及调控机制是十分必要的。目前,已报道的TRPM8翻译后修饰包括磷酸化、泛素化和糖基化等,它们能够调控蛋白质的相互作用和改变TRPM8离子通道的活性,从而调控细胞增殖、迁移和凋亡。值得注意的是,TRPM8的表达与前列腺癌、膀胱癌和乳腺癌等多种癌症密切相关。本文将从TRPM8离子通道的结构出发,系统地阐述TRPM8蛋白翻译后修饰和激动剂、...     

Unstructural Biology of TRP Ion Channels: The Role of Intrinsically Disordered Regions in Channel Function and Regulation

The first genuine high-resolution single particle cryo-electron microscopy structure of a membrane protein determined was a transient receptor potential (TRP) ion channel, TRPV1, in 2013. This methodical breakthrough opened up a whole new world for structural biology and ion channel aficionados alike. TRP channels capture the imagination due to the sheer endless number of tasks they carry out in all aspects of animal physiology. To date, structures of at least one representative member of each of the six mammalian TRP channel subfamilies as well as of a few non-mammalian families have been determined. These structures were instrumental for a better understanding of TRP channel function and regulation. However, all of the TRP channel structures solved so far are incomplete since they miss important information about highly flexible regions found mostly in the channel N- and C-termini. These intrinsically disordered regions (IDRs) can represent between a quarter to almost half of the entire protein sequence and act as important recruitment hubs for lipids and regulatory proteins. Here, we analyze the currently available TRP channel structures with regard to the extent of these “missing” regions and compare these findings to disorder predictions. We discuss select examples of intra- and intermolecular crosstalk of TRP channel IDRs with proteins and lipids as well as the effect of splicing and post-translational modifications, to illuminate their importance for channel function and to complement the prevalently discussed structural biology of these versatile and fascinating proteins with their equally relevant ’unstructural’ biology.     

Glucagon-Like Peptide-2 (GLP-2) Modulates the cGMP Signalling Pathway by Regulating the Expression of the Soluble Guanylyl Cyclase Receptor Subunits in Cultured Rat Astrocytes

The aim of this work was to study the effect of glucagon-like peptide-2 (GLP-2) on the cyclic guanosine monophosphate (cGMP) signalling pathway and whether insulin or epidermal growth factor (EGF) might modulate the effects of GLP-2. GLP-2 produced a dose-dependent decrease in intracellular sodium nitroprusside-induced cGMP production. However, insulin induced an increase in the levels of cGMP that was dose-dependently decreased by the addition of GLP-2. By contrast, EGF induced a decrease in cGMP production, which was further reduced by the addition of GLP-2. To assess whether variations in cGMP production might be related with changes in some component of soluble guanylyl cyclase (sGC), the expression of the ??1, ??2, and ??1 subunits were determined by Western blot analysis. At 1?h, GLP-2 produced a decrease in the expression of both ??1 and ??1 in the cytosolic fraction, but at 24?h only ??1was reduced. As expected, insulin induced an increase in the expression of both subunits after 1?h of incubation; this was decreased by the addition of GLP-2. Likewise, incubation with EGF for 24?h produced a decrease in the expression of both subunits that was maximal when GLP-2 was added. In addition, incubation with insulin for 1?h produced an increase in the expression of the ??2 subunit, which was reduced by the addition of GLP-2. These results suggest that GLP-2 inhibits cGMP production by decreasing the cellular content of at least one subunit of the heterodimeric active form of the sGC, independently of the presence of insulin or EFG. This may open new insights into the actions of this neuropeptide.     

Determinants of Voltage-Gated Potassium Channel Surface Expression and Localization in Mammalian Neurons

Neurons strictly regulate expression of a wide variety of voltage-dependent ion channels in their surface membranes to achieve precise yet dynamic control of intrinsic membrane excitability. Neurons also exhibit extreme morphological complexity that underlies diverse aspects of their function. Most ion channels are preferentially targeted to either the axonal or somatodendritic compartments, where they become further localized to discrete membrane subdomains. This restricted accumulation of ion channels enables local control of membrane signaling events in specific microdomains of a given compartment. Voltage-dependent K⁺ (Kv) channels act as potent modulators of diverse excitatory events such as action potentials, excitatory synaptic potentials, and Ca²⁺ influx. Kv channels exhibit diverse patterns of cellular expression, and distinct subtype-specific localization, in mammalian central neurons. Here we review the mechanisms regulating the abundance and distribution of Kv channels in mammalian neurons and discuss how dynamic regulation of these events impacts neuronal signaling.     

Expression and Regulation of Soluble Epoxide Hydrolase in Adipose Tissue

Obesity is an increasingly important public health issue reaching epidemic proportions. Visceral obesity has been defined as an important element of the metabolic syndrome and expansion of the visceral fat mass has been shown to contribute to the development of insulin resistance and cardiovascular disease. To identify novel contributors to cardiovascular and metabolic abnormalities in obesity, we analyzed the adipose proteome and identified soluble epoxide hydrolase (sEH) in the epididymal fat pad from C57BL/6J mice that received either a regular diet or a “western diet.” sEH was synthesized in adipocytes and expression levels increased upon differentiation of 3T3‐L1 preadipocytes. Although normalized sEH mRNA and protein levels did not differ in the fat pads from mice receiving a regular or a “western diet,” total adipose sEH activity was higher in the obese mice, even after normalization for body weight. Furthermore, peroxisome proliferator–activated receptor γ (PPARγ) agonists increased the expression of sEH in mature 3T3‐L1 adipocytes in vitro and in adipose tissue in vivo. Considering the established role for sEH in inflammation, cardiovascular diseases, and lipid metabolism, and the suggested involvement of sEH in the development of type 2 diabetes, our study has identified adipose sEH as a potential novel therapeutic target that might affect the development of metabolic and cardiovascular abnormalities in obesity.     

Bimodal Opioid Regulation of Cyclic AMP Formation: Implications for Positive and Negative Coupling of Opiate Receptors to Adenylyl Cyclase

Abstract: A μ-selective opiate receptor agonist, sufentanil, can either increase or decrease the stimulated formation of cyclic AMP (cAMP) in the myenteric plexus. The direction of the opioid modulation of this second messenger depends on the concentration of opioid used. Low doses of opioid enhance, whereas higher concentrations inhibit, the magnitude of cAMP that is formed in response to electrical stimulation. Opioids exert this dual regulation on only stimulated cAMP formation. Basal levels are not affected. Opioid facilitation and inhibition of stimulated cAMP formation are blocked by naloxone, indicating mediation by opiate receptors. Because all experiments were conducted in the presence of a phosphodiesterase inhibitor, it is highly unlikely that opioid regulation of stimulated cAMP formation is due to changes in the rate of its degradation. Positive and negative coupling of μ-opiate receptors to adenylyl cyclase is the most plausible explanation for the bimodal opioid effects on cAMP content. The marked parallel between the current observations and the previously reported bimodal opioid regulation of evoked enkephalin release is consistent with the hypothesis that adenylyl cyclase is one biochemical substrate for the bimodal opiate receptor-coupled regulatory mechanism governing the stimulated release of this opioid peptide.     

神经营养素脑内功能及其表达调节

神经营养素（neurotrophin,NT）是与神经生长因子同源的一类神经营养因子,它们在神经系统的分化和发育过程中起着重要作用,并具有治疗某些脑疾病的潜在应用价值．文章较全面地阐述了NT在脑内既相互交叉又有各自特性的生理功能,并系统地介绍了脑内NT表达调节方面的研究进展．     

Ion Channel Gene Expression in Lung Adenocarcinoma: Potential Role in Prognosis and Diagnosis

Ion channels are known to regulate cancer processes at all stages. The roles of ion channels in cancer pathology are extremely diverse. We systematically analyzed the expression patterns of ion channel genes in lung adenocarcinoma. First, we compared the expression of ion channel genes between normal and tumor tissues in patients with lung adenocarcinoma. Thirty-seven ion channel genes were identified as being differentially expressed between the two groups. Next, we investigated the prognostic power of ion channel genes in lung adenocarcinoma. We assigned a risk score to each lung adenocarcinoma patient based on the expression of the differentially expressed ion channel genes. We demonstrated that the risk score effectively predicted overall survival and recurrence-free survival in lung adenocarcinoma. We also found that the risk scores for ever-smokers were higher than those for never-smokers. Multivariate analysis indicated that the risk score was a significant prognostic factor for survival, which is independent of patient age, gender, stage, smoking history, Myc level, and EGFR/KRAS/ALK gene mutation status. Finally, we investigated the difference in ion channel gene expression between the two major subtypes of non-small cell lung cancer: adenocarcinoma and squamous-cell carcinoma. Thirty ion channel genes were identified as being differentially expressed between the two groups. We suggest that ion channel gene expression can be used to improve the subtype classification in non-small cell lung cancer at the molecular level. The findings in this study have been validated in several independent lung cancer cohorts.     

Thrombin Regulation of Cell Function through Protease-Activated Receptors: Implications for Therapeutic Intervention

The serine protease thrombin is well recognized as being pivotal to the maintenance of hemostasis under both normal and pathological conditions. Its cellular actions are mediated through a unique family of protease-activated receptors (PARs). These receptors represent a novel family of G protein-coupled receptors that undergo proteolytic cleavage of their amino terminus and subsequent autoactivation by a tethered peptide ligand. This paper reviews the consequences of PAR activation in thrombosis, vascular injury, inflammation, tissue injury, and within the tumor microenvironment.     

Presynaptic Localization and Possible Function of Calcium-Activated Chloride Channel Anoctamin 1 in the Mammalian Retina

Calcium (Ca²⁺)-activated chloride (Cl⁻) channels (CaCCs) play a role in the modulation of action potentials and synaptic responses in the somatodendritic regions of central neurons. In the vertebrate retina, large Ca²⁺-activated Cl⁻ currents (I_Cl(Ca)) regulate synaptic transmission at photoreceptor terminals; however, the molecular identity of CaCCs that mediate I_Cl(Ca) remains unclear. The transmembrane protein, TMEM16A, also called anoctamin 1 (ANO1), has been recently validated as a CaCC and is widely expressed in various secretory epithelia and nervous tissues. Despite the fact that tmem16a was first cloned in the retina, there is little information on its cellular localization and function in the mammalian retina. In this study, we found that ANO1 was abundantly expressed as puncta in 2 synaptic layers. More specifically, ANO1 immunoreactivity was observed in the presynaptic terminals of various retinal neurons, including photoreceptors. I_Cl(Ca) was first detected in dissociated rod bipolar cells expressing ANO1. I_Cl(Ca) was abolished by treatment with the Ca²⁺ channel blocker Co²⁺, the L-type Ca²⁺ channel blocker nifedipine, and the Cl⁻ channel blockers 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) and niflumic acid (NFA). More specifically, a recently discovered ANO1-selective inhibitor, T16A_inh-A01, and a neutralizing antibody against ANO1 inhibited I_Cl(Ca) in rod bipolar cells. Under a current-clamping mode, the suppression of I_Cl(Ca) by using NPPB and T16A_inh-A01 caused a prolonged Ca²⁺ spike-like depolarization evoked by current injection in dissociated rod bipolar cells. These results suggest that ANO1 confers I_Cl(Ca) in retinal neurons and acts as an intrinsic regulator of the presynaptic membrane potential during synaptic transmission.     

心脏瓣膜病患者围手术期血浆BNP水平的表达及与心功能的关系

目的:通过探讨心脏瓣膜病患者围手术期血浆脑钠肽(BNP)水平的表达与心功能的关系,为相关治疗提供参考。方法:选择2014年10月~2015年10月本院收治的心脏瓣膜病患者共50例,分别于术前1 d、术后6 h、24 h、48 h和术后7 d测量患者血浆BNP水平,并观测患者左心射血分数(LVEF)、心功能分级、术后呼吸机辅助时间以及住院期间并发症发生情况。结果:术前不同心功能分级患者血浆BNP水平的总体差异有统计学意义(F=45.767,P0.05),心功能分级越高,血浆BNP水平越高;心脏功能IV级患者血浆BNP水平高于Ⅲ级,Ⅲ级患者血浆BNP水平高于II级(P0.05)。术后LVEF50%和LVEF≥50%患者,术前1 d血浆BNP水平分别为(724.21±132.16)pg/m L和(428.64±149.31)pg/m L,差异有统计学意义(t=6.628,P0.001)。不同时间点患者血浆BNP水平的总体差异有统计学意义(F=29.003,P0.001)。与术前比较,术后24 h、术后48 h和术后7 d时血浆BNP水平均显著升高(P0.05);术后48h时BNP浓度最高,随后开始下降。术后呼吸机辅助使用时间24 h和≥24 h的患者,术前1 d血浆BNP水平的差异有统计学意义(t=2.378,P=0.021)。结论:心脏瓣膜病患者围手术期血浆BNP水平的变化能够准确的反映患者心功能情况,对患者术前病情评估和术后临床处理具有一定的指导意义。`     

Function and Regulation of the Mammalian Copper-transporting ATPases: Insights from Biochemical and Cell Biological Approaches

Copper is an essential trace element that plays a very important role in cell physiology. In humans, disruption of normal copper homeostasis leads to severe disorders, such as Menkes disease and Wilson's disease. Recent genetic, cell biological, and biochemical studies have begun to dissect the molecular mechanisms involved in transmembrane transport and intracellular distribution of copper in mammalian cells. In this review, we summarize the advances that have been made in understanding of structure, function, and regulation of the key human copper transporters, the Menkes disease and Wilson's disease proteins.