Selective inhibition of a regulatory subunit of protein phosphatase 1 restores proteostasis

Many biological processes are regulated through the selective dephosphorylation of proteins. Protein serine-threonine phosphatases are assembled from catalytic subunits bound to diverse regulatory subunits that provide substrate specificity and subcellular localization. We describe a small molecule, guanabenz, that bound to a regulatory subunit of protein phosphatase 1, PPP1R15A/GADD34, selectively disrupting the stress-induced dephosphorylation of the α subunit of translation initiation factor 2 (eIF2α). Without affecting the related PPP1R15B-phosphatase complex and constitutive protein synthesis, guanabenz prolonged eIF2α phosphorylation in human stressed cells, adjusting the protein production rates to levels manageable by available chaperones. This favored protein folding and thereby rescued cells from protein misfolding stress. Thus, regulatory subunits of phosphatases are drug targets, a property used here to restore proteostasis in stressed cells.     

植物PP2C蛋白磷酸酶负调控ABA信号转导途径研究进展

PP2C(PP2C-type protein phosphatases)蛋白磷酸酶是一类丝氨酸/苏氨酸残基蛋白磷酸酶,植物体内目前已经发现了4种PP2C蛋白磷酸酶:ABI,AtP2C-HABl,AtPP2CA以及MP2C.大量的研究表明植物PP2C蛋白磷酸酶参与了ABA信号转导途径的负调控功能.就高等植物PP2C的分类及其对ABA信号转导途径的负调控功能的研究进展进行了综述.     

植物2C类蛋白磷酸酶及其在逆境信号转导中的作用

2C类蛋白磷酸酶(PP2C)是一类丝氨酸/苏氨酸残基蛋白磷酸酶,以单体酶的形式广泛存在于生物体中,参与多种信号途径。大量研究表明,植物PP2C负调控ABA信号转导途径及多种逆境胁迫转导途径。本文对高等植物PP2C的分类及其对多种逆境信号转导途径的调控功能研究进行了综述与展望。     

Autophagy as a regulated pathway of cellular degradation

Macroautophagy is a dynamic process involving the rearrangement of subcellular membranes to sequester cytoplasm and organelles for delivery to the lysosome or vacuole where the sequestered cargo is degraded and recycled. This process takes place in all eukaryotic cells. It is highly regulated through the action of various kinases, phosphatases, and guanosine triphosphatases (GTPases). The core protein machinery that is necessary to drive formation and consumption of intermediates in the macroautophagy pathway includes a ubiquitin-like protein conjugation system and a protein complex that directs membrane docking and fusion at the lysosome or vacuole. Macroautophagy plays an important role in developmental processes, human disease, and cellular response to nutrient deprivation.     

OSBP is a cholesterol-regulated scaffolding protein in control of ERK 1/2 activation

Oxysterol-binding protein (OSBP) is the founding member of a family of sterol-binding proteins implicated in vesicle transport, lipid metabolism, and signal transduction. Here, OSBP was found to function as a cholesterol-binding scaffolding protein coordinating the activity of two phosphatases to control the extracellular signal-regulated kinase (ERK) signaling pathway. Cytosolic OSBP formed a approximately 440-kilodalton oligomer with a member of the PTPPBS family of tyrosine phosphatases, the serine/threonine phosphatase PP2A, and cholesterol. This oligomer had dual specific phosphatase activity for phosphorylated ERK (pERK). When cell cholesterol was lowered, the oligomer disassembled and the level of pERK rose. The oligomer also disassembled when exposed to oxysterols. Increasing the amount of OSBP oligomer rendered cells resistant to the effects of cholesterol depletion and decreased the basal level of pERK. Thus, cholesterol functions through its interaction with OSBP outside of membranes to regulate the assembly of an oligomeric phosphatase that controls a key signaling pathway in the cell.     

Differential phosphorylation of c-Abl in cell cycle determined by cdc2 kinase and phosphatase activity

The product of the c-abl proto-oncogene (c-Abl) is phosphorylated on three sites during interphase and seven additional sites during mitosis. Two interphase and all mitotic c-Abl sites are phosphorylated by cdc2 kinase isolated from either interphase or mitotic cells, with the mitotic cdc2 having an 11-fold higher activity. Inhibition of phosphatases with okadaic acid in interphase cells leads to the phosphorylation of c-Abl mitotic sites, indicating that those sites are preferentially dephosphorylated during interphase. The differential phosphorylation of c-Abl in the cell cycle is therefore determined by an equilibrium between cdc2 kinase and protein phosphatase activities. Treatment of interphase cells with okadaic acid leads to a rounded morphology similar to that observed during mitosis.     

Protein kinase activity closely associated with a reconstituted calcium-activated potassium channel.

Modulation of the activity of potassium and other ion channels is an essential feature of nervous system function. The open probability of a large conductance Ca(2+)-activated K+ channel from rat brain, incorporated into planar lipid bilayers, is increased by the addition of adenosine triphosphate (ATP) to the cytoplasmic side of the channel. This modulation takes place without the addition of protein kinase, requires Mg2+, and is mimicked by an ATP analog that serves as a substrate for protein kinases but not by a nonhydrolyzable ATP analog. Addition of protein phosphatase 1 reverses the modulation by MgATP. Thus, there may be an endogenous protein kinase activity firmly associated with this K+ channel. Some ion channels may exist in a complex that contains regulatory protein kinases and phosphatases.     

A selective inhibitor of eIF2alpha dephosphorylation protects cells from ER stress

Most protein phosphatases have little intrinsic substrate specificity, making selective pharmacological inhibition of specific dephosphorylation reactions a challenging problem. In a screen for small molecules that protect cells from endoplasmic reticulum (ER) stress, we identified salubrinal, a selective inhibitor of cellular complexes that dephosphorylate eukaryotic translation initiation factor 2 subunit alpha (eIF2alpha). Salubrinal also blocks eIF2alpha dephosphorylation mediated by a herpes simplex virus protein and inhibits viral replication. These results suggest that selective chemical inhibitors of eIF2alpha dephosphorylation may be useful in diseases involving ER stress or viral infection. More broadly, salubrinal demonstrates the feasibility of selective pharmacological targeting of cellular dephosphorylation events.     

PirB restricts ocular-dominance plasticity in visual cortex

Experience can alter synaptic connectivity throughout life, but the degree of plasticity present at each age is regulated by mechanisms that remain largely unknown. Here, we demonstrate that Paired-immunoglobulin-like receptor B (PirB), a major histocompatibility complex class I (MHCI) receptor, is expressed in subsets of neurons throughout the brain. Neuronal PirB protein is associated with synapses and forms complexes with the phosphatases Shp-1 and Shp-2. Soluble PirB fusion protein binds to cortical neurons in an MHCI-dependent manner. In mutant mice lacking functional PirB, cortical ocular-dominance plasticity is more robust at all ages. Thus, an MHCI receptor is expressed in central nervous system neurons and functions to limit the extent of experience-dependent plasticity in the visual cortex throughout life. PirB is also expressed in many other regions of the central nervous system, suggesting that it may function broadly to stabilize neural circuits.     

Identification of Hedgehog pathway components by RNAi in Drosophila cultured cells

Classical genetic screens can be limited by the selectivity of mutational targeting, the complexities of anatomically based phenotypic analysis, or difficulties in subsequent gene identification. Focusing on signaling response to the secreted morphogen Hedgehog (Hh), we used RNA interference (RNAi) and a quantitative cultured cell assay to systematically screen functional roles of all kinases and phosphatases, and subsequently 43% of predicted Drosophila genes. Two gene products reported to function in Wingless (Wg) signaling were identified as Hh pathway components: a cell surface protein (Dally-like protein) required for Hh signal reception, and casein kinase 1alpha, a candidate tumor suppressor that regulates basal activities of both Hh and Wg pathways. This type of cultured cell-based functional genomics approach may be useful in the systematic analysis of other biological processes.     

Greatwall phosphorylates an inhibitor of protein phosphatase 2A that is essential for mitosis

Entry into mitosis in eukaryotes requires the activity of cyclin-dependent kinase 1 (Cdk1). Cdk1 is opposed by protein phosphatases in two ways: They inhibit activation of Cdk1 by dephosphorylating the protein kinases Wee1 and Myt1 and the protein phosphatase Cdc25 (key regulators of Cdk1), and they also antagonize Cdk1's own phosphorylation of downstream targets. A particular form of protein phosphatase 2A (PP2A) containing a B55δ subunit (PP2A- B55δ) is the major protein phosphatase that acts on model CDK substrates in Xenopus egg extracts and has antimitotic activity. The activity of PP2A-B55δ is high in interphase and low in mitosis, exactly opposite that of Cdk1. We report that inhibition of PP2A-B55δ results from a small protein, known as α-endosulfine (Ensa), that is phosphorylated in mitosis by the protein kinase Greatwall (Gwl). This converts Ensa into a potent and specific inhibitor of PP2A-B55δ. This pathway represents a previously unknown element in the control of mitosis.     

重金属胁迫培养对微生物蛋白质含量的影响

[目的]研究重金属胁迫培养对微生物蛋白质含量的影响。[方法]选择4种典型微生物（大肠杆菌、枯草芽孢杆菌、啤酒酵母、链霉菌）为试验材料,采用传统微生物培养方法,在不同浓度的Hg2＋、Cd2＋、Cr6＋和Pb2＋重金属离子下进行胁迫培养,通过测定4种典型微生物的蛋白质含量,探讨重金属胁迫对4种微生物蛋白质合成的影响。[结果]低浓度的重金属对4种微生物蛋白质的合成有一定的促进作用,随着重金属浓度的增加,微生物蛋白质的合成均受到不同程度的抑制。枯草芽孢杆菌对这4种重金属的耐受性比其他3种微生物要强,4种重金属离子在比较大的质量浓度范围内（5～50 mg/L）对枯草芽孢杆菌蛋白质的合成有促进作用。低浓度Cr6＋对大肠杆菌蛋白质的合成有较强的促进作用;Pb2＋对大肠杆菌蛋白质合成有明显的抑制作用,对其他3种微生物蛋白质合成在一定浓度范围内有促进作用;低浓度的Cd2＋对4种微生物蛋白质的合成都有促进作用。[结论]该研究为探讨微生物对重金属胁迫的生理响应提供了理论依据。     

Involvement of RNA in the synthesis of proteins

We can now have considerable confidence that the broad features of protein synthesis are understood. The involvement of RNA is very much more complicated than was imagined in 1953. There is not one functional RNA. Instead, protein synthesis demands the ordered interaction of three classes of RNA-ribosomal, soluble, and messenger. Many important questions, however, remain unanswered. For instance, there is no theoretical framework for the ribosomal subunits, nor for that matter, do we understand the functional significance of ribosomal RNA. Most satisfying is the realization that all the steps in protein replication will be shown to involve well-understood chemical forces. As yet we do not know all the details. For example, are the DNA base pairs involved in messenger RNA selection of the corresponding amino-acyl-sRNA? With luck, this will soon be known. We can thus have every expectation that future progress in understanding selective protein synthesis (and its consequences for embryology) will have a similarly well-defined and, when understood, easy-to-comprehend chemical basis (62).     

Molecular mimicry regulates ABA signaling by SnRK2 kinases and PP2C phosphatases

Abscisic acid (ABA) is an essential hormone for plants to survive environmental stresses. At the center of the ABA signaling network is a subfamily of type 2C protein phosphatases (PP2Cs), which form exclusive interactions with ABA receptors and subfamily 2 Snfl-related kinase (SnRK2s). Here, we report a SnRK2-PP2C complex structure, which reveals marked similarity in PP2C recognition by SnRK2 and ABA receptors. In the complex, the kinase activation loop docks into the active site of PP2C, while the conserved ABA-sensing tryptophan of PP2C inserts into the kinase catalytic cleft, thus mimicking receptor-PP2C interactions. These structural results provide a simple mechanism that directly couples ABA binding to SnRK2 kinase activation and highlight a new paradigm of kinase-phosphatase regulation through mutual packing of their catalytic sites.     

Endosperm protein synthesis in maize mutants with increased lysine content

The endosperm proteins of the maize mutants, opaque-2, opaque-7, floury-2, brittle-2, and the double mutant of opaque-2 and brittle-2, were separated into five soluble fractions by the Landry-Moureaux method. As compared to their isogenic normal counterparts, the mutant endosperms had higher concentrations of albumins, globulins, and glutelin-3, and lower concentrations of prolamines. The combination of the opaque-2 and brittle-2 genes enhanced this difference. Although the four mutant genes are located on three different chromosomes, they exert a similar effect on endosperm protein composition. Five other starchmodifying mutants with high lysine content resemble the brittle-2 mutant in endosperm protein composition, when the gene is present either singly or combined with opaque-2. Therefore, the pattern of protein synthesis in all maize mutants with high lysine concentrations may be either identical or very similar. Because no synergistic effect on lysine concentration is obtained when floury-2 is combined with opaque-2, different pathways leading to reduced zein synthesis may exist in the floury and starch-modifying mutants with high lysine concentrations.     

苦瓜性别分化程序表达的研究（英文）

苦瓜的性别分化过程要先经过一个两性期,再分别向雄性或雌性的方向发育. 对两性期和雄,雌花早期发育的3个典型时期的花蕾进行毛细管电泳分析,结果表明,一种分子量为11 kD的蛋白质在雌花发育的3个时期都存在,并且含量变化很小,很可能是雌花程序表达中的一种"关键蛋白";类似地,一种分子量为30 kD的蛋白质很可能是雄花程序表达中的一种"关键蛋白".此外,蛋白质合成抑制剂亚胺环己酮抑制离体两性花的体外性别分化,而核酸合成抑制剂3-脱氧腺苷则不影响两性花的体外性别分化.     

ARID4B在牛磺酸调节成肌细胞C2C12蛋白质合成中的作用

肌肉细胞蛋白质合成能力与畜禽产肉量性状有关,试验旨在探究转录因子AT富集区4B(AT-rich interaction domain 4B,ARID4B)对牛磺酸(Taurine,Tau)调节成肌细胞C2C12蛋白质合成的影响.向体外培养C2C12细胞培养液中分别添加0、60、120、180和240μmol·L-1 T...