In Vitro and in Vivo Phosphorylation of Stomatal Phosphoenolpyruvate Carboxylase from Vicia faba L.

The in vitro and in vivo phosphorylation of stomatal phosphoenolpyruvate carboxylase [EC 4.1. 1.31] from Vicia faba L. was demonstrated by feeding the concentrated enzyme after 0–70% ammonium sulfate precipitation of guard cell protoplasts with ³²P and subsequent analysis of autoradiograms and Western immunoblots, after SDS-PAGE, of protein samples. The in vitro and in vivo results provide evidence for a radioactive labeling of the two stomatal PEPCase bands (112 and 110 kDa).     

Kv1.3 钾离子通道在卵巢癌细胞株中的表达及活性

目的:研究Kv1.3钾离子通道在SKOV3卵巢癌细胞中的表达及其在细胞增殖和细胞周期中的作用。方法:应用RT-PCR和免疫细胞化学鉴别Kv1.3钾离子通道在SKOV3卵巢癌细胞中的表达。应用MTT和流式细胞技术观察KV1.3钾离子通道对SKOV3卵巢癌细胞增殖及细胞周期的影响。结果:4-氨基吡啶是Kv1.3钾离子通道特异性阻滞剂。不同浓度的4-氨基吡啶可以明显抑制SKOV3细胞的增殖,并且细胞周期也受到影响。G0/G1细胞比例增加,S期和G2/M期细胞比例下降。结论:Kv1.3钾离子通道在SKOV3卵巢癌细胞中表达,并且在细胞增殖及细胞周期变换中扮演着重要的角色。     

Identification,Quantification, and System Analysis of Protein N‐ε Lysine Methylation in Anucleate Blood Platelets

Protein posttranslational modifications critically regulate a range of physiological and disease processes. In addition to tyrosine, serine, and threonine phosphorylation, reversible N‐ε acylation and alkylation of protein lysine residues also modulate diverse aspects of cellular function. Studies of lysine acyl and alkyl modifications have focused on nuclear proteins in epigenetic regulation; however, lysine modifications are also prevalent on cytosolic proteins to serve increasingly apparent, although less understood roles in cell regulation. Here, the methyl‐lysine (meK) proteome of anucleate blood platelets is characterized. With high‐resolution, multiplex MS methods, 190 mono‐, di‐, and tri‐meK modifications are identified on 150 different platelet proteins—including 28 meK modifications quantified by tandem mass tag (TMT) labeling. In addition to identifying meK modifications on calmodulin (CaM), GRP78 (HSPA5, BiP), and EF1A1 that have been previously characterized in other cell types, more novel modifications are also uncovered on cofilin, drebin‐like protein (DBNL, Hip‐55), DOCK8, TRIM25, and numerous other cytoplasmic proteins. Together, the results and analyses support roles for lysine methylation in mediating cytoskeletal, translational, secretory, and other cellular processes. MS data for this study have been deposited into the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD012217.     

Discovery of (S,E)-3-(2-fluorophenyl)-N-(1-(3-(pyridin-3-yloxy)phenyl)ethyl)-acrylamide as a potent and efficacious KCNQ2 (Kv7.2) opener for the treatment of neuropathic pain

Acrylamide (S)-6, a potent and efficacious KCNQ2 (Kv7.2) opener, demonstrated significant activity in two models of neuropathic pain and in the formalin test, suggesting that KCNQ2 openers may be useful in the treatment of neuropathic pain including diabetic neuropathy.     

Effects of Y361‐auto‐phosphorylation on structural plasticity of the HIPK2 kinase domain

The dual‐specificity activity of the homeodomain interacting protein kinase 2 (HIPK2) is regulated by cis‐auto‐phosphorylation of tyrosine 361 (Y361) on the activation loop. Inhibition of this process or substitution of Y361 with nonphosphorylatable amino acid residues result in aberrant HIPK2 forms that show altered functionalities, pathological‐like cellular relocalization, and accumulation into cytoplasmic aggresomes. Here, we report an in vitro characterization of wild type HIPK2 kinase domain and of two mutants, one at the regulating Y361 (Y361F, mimicking a form of HIPK2 lacking Y361 phosphorylation) and another at the catalytic lysine 228 (K228A, inactivating the enzyme). Gel filtration and thermal denaturation analyzes along with equilibrium binding experiments and kinase assays performed in the presence or absence of ATP‐competitors were performed. The effects induced by mutations on overall stability, oligomerization and activity support the existence of different conformations of the kinase domain linked to Y361 phosphorylation. In addition, our in vitro data are consistent with both the cross‐talk between the catalytic site and the activation loop of HIPK2 and the aberrant activities and accumulation previously reported for the Y361 nonphosphorylated HIPK2 in mammalian cells.     

Phosphorylation and Fucosylation of Myelin Protein In Vitro by Sciatic Nerve from Developing Rats

Abstract: Proteins of the paniculate fraction of sciatic nerve of rats ranging from 1 to 55 days of age were analyzed by polyacrylamide gel electrophoresis. The major myelin protein, P₀, could not be detected at 1 day of age, but by 10 days it comprised from 15 to 20% of the particulate protein, the same proportion as in adult rats. Growth of nerve continued throughout the period studied. Rat sciatic nerves were incubated with [³²P]orthophosphate or [³H]fucose. Particulate matter proteins from sciatic nerve (and in certain cases proteins of myelin purified from sciatic nerve) were separated by polyacrylamide disc gel electrophoresis and the distribution of protein and of radioactivity along the gels was determined. [³²P]Phosphate appeared to label all myelin proteins. Labeling with fucose was more specific; myelin basic proteins were not fucosylated. A developmental study showed that sciatic nerves from 2-day-old rats could incorporate radioactive fucose and [³²P]-phosphate into several proteins at the P₀ region of polyacrylamide gels. Specific radioactivity of [³H]fucose in P₀ protein was highest in preparations from 5-day-old rats and declined by 80% over the next 5 days as it was diluted by accumulating myelin. The specific radioactivity of incorporated [³²P] phosphate was high at the early age points and declined as a result of the accumulation of compact myelin. The results indicate an association of fucosylation and/or phosphorylation with some step in the formation of myelin.     

Effects of Calcium, Strontium, and Barium Ions on Phosphorylation of Hippocampal Proteins In Vitro

Abstract: Calcium ion alone or in the presence of added calmodulin stimulated in vitro transfer of ³²P from [γ³²P]ATP into several proteins of mitochondrial and synaptosomal particulate fractions from rat brain. Strontium ion was capable of substituting for calcium ion in this stimulation, but barium ion lacked this capacity. These results bring into question the hypothesis that calciumdependent protein phosphorylation of synaptic proteins is intrinsic to neurotransmitter release during neurotransmission, but they do not rule out that possibility.     

In Vitro and In Vivo Proteomic Comparison of Human Neural Progenitor Cell‐Induced Photoreceptor Survival

Retinal degenerative diseases lead to blindness with few treatments. Various cell‐based therapies are aimed to slow the progression of vision loss by preserving light‐sensing photoreceptor cells. A subretinal injection of human neural progenitor cells (hNPCs) into the Royal College of Surgeons (RCS) rat model of retinal degeneration has aided in photoreceptor survival, though the mechanisms are mainly unknown. Identifying the retinal proteomic changes that occur following hNPC treatment leads to better understanding of neuroprotection. To mimic the retinal environment following hNPC injection, a co‐culture system of retinas and hNPCs is developed. Less cell death occurs in RCS retinal tissue co‐cultured with hNPCs than in retinas cultured alone, suggesting that hNPCs provide retinal protection in vitro. Comparison of ex vivo and in vivo retinas identifies nuclear factor (erythroid‐derived 2)‐like 2 (NRF2) mediated oxidative response signaling as an hNPC‐induced pathway. This is the first study to compare proteomic changes following treatment with hNPCs in both an ex vivo and in vivo environment, further allowing the use of ex vivo modeling for mechanisms of retinal preservation. Elucidation of the protein changes in the retina following hNPC treatment may lead to the discovery of mechanisms of photoreceptor survival and its therapeutic for clinical applications.     

Phosphorylation of Myelin-Associated Glycoprotein In Vivo and In Vitro Occurs Only in the Cytoplasmic Domain of the Large Isoform

Myelin-associated glycoprotein (MAG) was radioactively labelled with 32P both in intact brain and in myelin membrane preparations. Chemical deglycosylation of the phosphorylated products revealed that only one of the MAG isoforms (L-MAG) is labelled in vitro. Furthermore, the phosphorylation events in vivo and in vitro are confined to the cytoplasmic portion of the L-MAG isoform. Tryptic mapping of L-MAG labelled both in vivo and in vitro revealed that the majority of the sites phosphorylated in intact brain are also phosphorylated in myelin membrane preparations; however, the extent of phosphorylation at individual sites is variable. The results demonstrate that partially purified myelin membrane preparations can be used to study the enzymes responsible for MAG phosphorylation and dephosphorylation events in vivo.     

Effect of Oral Administration of Tri-o-cresyl Phosphate on In Vitro Phosphorylation of Membrane and Cytosolic Proteins from Chicken Brain

Abstract: The effects of a single oral dose of 750 mg/kg tri- o -cresyl phosphate (TOCP) on the endogenous phosphorylation of specific brain proteins were assessed in male adult chickens following the development of delayed neurotoxicity. Phosphorylation of crude synaptosomal (P₂) membrane and synaptosomal cytosolic proteins was assayed in vitro by using [γ-³²P]ATP as phosphate donor. Following resolution of brain proteins by sodium dodecyl sulfate polyacrylamide gel electrophoresis, specific protein phosphorylation was detected by autoradiography and quantified by microdensitometry. TOCP administration enhanced the phosphorylation of both cytosolic (M_r 65,000 and 55,000) and membrane (20,000) proteins by as much as 146% and 200%, respectively.     

Modulation of Native TREK-1 and Kv1.4 K<Superscript>+</Superscript> Channels by Polyunsaturated Fatty Acids and Lysophospholipids

The modulation of TREK-1 leak and Kv1.4 voltage-gated K⁺ channels by fatty acids and lysophospholipids was studied in bovine adrenal zona fasciculata (AZF) cells. In whole-cell patch-clamp recordings, arachidonic acid (AA) (1–20 µM) dramatically and reversibly increased the activity of bTREK-1, while inhibiting bKv1.4 current by mechanisms that occurred with distinctly different kinetics. bTREK-1 was also activated by the polyunsaturated cis fatty acid linoleic acid but not by the trans polyunsaturated fatty acid linolelaidic acid or saturated fatty acids. Eicosatetraynoic acid (ETYA), which blocks formation of active AA metabolites, failed to inhibit AA activation of bTREK-1, indicating that AA acts directly. Compared to activation of bTREK-1, inhibition of bKv1.4 by AA was rapid and accompanied by a pronounced acceleration of inactivation kinetics. Cis polyunsaturated fatty acids were much more effective than trans or saturated fatty acids at inhibiting bKv1.4. ETYA also effectively inhibited bKv1.4, but less potently than AA. bTREK-1 current was markedly increased by lysophospholipids including lysophosphatidyl choline (LPC) and lysophosphatidyl inositol (LPI). At concentrations from 1–5 µM, LPC produced a rapid, transient increase in bTREK-1 that peaked within one minute and then rapidly desensitized. The transient lysophospholipid-induced increases in bTREK-1 did not require the presence of ATP or GTP in the pipette solution. These results indicate that the activity of native leak and voltage-gated K⁺ channels are directly modulated in reciprocal fashion by AA and other cis unsaturated fatty acids. They also show that lysophospholipids enhance bTREK-1, but with a strikingly different temporal pattern. The modulation of native K⁺ channels by these agents differs from their effects on the same channels expressed in heterologous cells, highlighting the critical importance of auxiliary subunits and signaling. Finally, these results reveal that AZF cells express thousands of bTREK-1 K⁺ channels that lie dormant until activated by metabolites including phospholipase A₂ (PLA₂)-generated fatty acids and lysophospholipids. These metabolites may alter the electrical and secretory properties of AZF cells by modulating bTREK-1 and bKv1.4 K⁺ channels.     

Identification and characterization of site‐specific N‐glycosylation in the potassium channel Kv3.1b

The potassium ion channel Kv3.1b is a member of a family of voltage‐gated ion channels that are glycosylated in their mature form. In the present study, we demonstrate the impact of N‐glycosylation at specific asparagine residues on the trafficking of the Kv3.1b protein. Large quantities of asparagine 229 (N229)‐glycosylated Kv3.1b reached the plasma membrane, whereas N220‐glycosylated and unglycosylated Kv3.1b were mainly retained in the endoplasmic reticulum (ER). These ER‐retained Kv3.1b proteins were susceptible to degradation, when co‐expressed with calnexin, whereas Kv3.1b pools located at the plasma membrane were resistant. Mass spectrometry analysis revealed a complex type Hex₃HexNAc₄Fuc₁ glycan as the major glycan component of the N229‐glycosylated Kv3.1b protein, as opposed to a high‐mannose type Man₈GlcNAc₂ glycan for N220‐glycosylated Kv3.1b. Taken together, these results suggest that trafficking‐dependent roles of the Kv3.1b potassium channel are dependent on N229 site‐specific glycosylation and N‐glycan structure, and operate through a mechanism whereby specific N‐glycan structures regulate cell surface expression.     

A Comparison of GFP‐Tagged Clathrin Light Chains with Fluorochromated Light Chains In Vivo and In Vitro

Clathrin triskelia consist of three heavy chains and three light chains (LCs). Green fluorescent protein (GFP)‐tagged LCs are widely utilized to follow the dynamics of clathrin in living cells, but whether they reflect faithfully the behavior of clathrin triskelia in cells has not been investigated yet thoroughly. As an alternative approach, we labeled purified LCs either with Alexa 488 or Cy3 dye and compared them with GFP‐tagged LC variants. Cy3‐labeled light chains (Cy3‐LCs) were microinjected into HeLa cells either directly or in association with heavy chains. Within 1–2 min the Cy3‐LC heavy chain complexes entered clathrin‐coated structures, whereas uncomplexed Cy3‐LC did not within 2 h. These findings show that no significant exchange of LCs occurs over the time–course of an endocytic cycle. To explore whether GFP‐tagged LCs behave functionally like endogenous LCs, we characterized them biochemically. Unlike wild‐type LCs, recombinant LCs with a GFP attached to either end did not efficiently inhibit clathrin assembly in vitro, whereas Cy3‐ and Alexa 488‐labeled LC behaved similar to wild‐type LCs in vitro and in vivo. Thus, fluorochromated LCs are a valuable tool for investigating the complex behavior of clathrin in living cells.     

Changes in In Vitro Brain and Spinal Cord Protein Phosphorylation After a Single Oral Administration of Tri-o-Cresyl Phosphate to Hens

The effect of a single oral 750 mg/kg dose of tri-o-cresyl phosphate (TOCP) on the endogenous phosphorylation of brain and spinal cord proteins was assessed in hens during the development of and recovery from delayed neurotoxicity. Crude membrane and cytosolic fractions were prepared from the brains and spinal cords of control and TOCP-treated hens at 1, 7, 14, 21, 35, and 55 days after treatment. Brain and spinal cord protein phosphorylation with [gamma-32P]ATP was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), autoradiography, and microdensitometry. TOCP administration conferred calcium and calmodulin dependence on the phosphorylation of a few brain cytosolic proteins and caused an increase in the phosphorylation of a number of other cytosolic and membrane proteins. This effect of TOCP was large in magnitude, and its time course reflected the onset of and recovery from the signs of ataxia and paralysis associated with delayed neurotoxicity in the hen. The molecular weights (Mr) and maximal phosphorylation (percent of control) for the most prominently affected bands were as follows: brain cytosol--50K (183%), 55K (575%), 60K (529%), 65K (273%), and 70K (548%); brain membranes--50K (622%) and 60K (697%); and spinal cord cytosol--20K (182%). The role of endogenous phosphorylation reactions in and their potential usefulness as biochemical indicators of delayed neurotoxicity are being explored further.     

Simultaneous Quantification of the Acetylome and Succinylome by ‘One‐Pot’ Affinity Enrichment

Protein posttranslational modifications (PTMs) are of increasing interest in biomedical research, yet studies rarely examine more than one PTM. One barrier to multi‐PTM studies is the time cost for both sample preparation and data acquisition, which scale linearly with the number of modifications. The most prohibitive requirement is often the need for large amounts of sample, which must be increased proportionally with the number of PTM enrichment steps. Here, a streamlined, quantitative label‐free proteomic workflow—“one‐pot” PTM enrichment—that enables comprehensive identification and quantification of peptides containing acetylated and succinylated lysine residues from a single sample containing as little as 1 mg mitochondria protein is described. Coupled with a label‐free, data‐independent acquisition (DIA), 2235 acetylated and 2173 succinylated peptides with the one‐pot method are identified and quantified and peak areas are shown to be highly correlated between the one‐pot and traditional single‐PTM enrichments. The ‘one‐pot’ method makes possible detection of multiple PTMs occurring on the same peptide, and it is shown that it can be used to make unique biological insights into PTM crosstalk. Compared to single‐PTM enrichments, the one‐pot workflow has equivalent reproducibility and enables direct assessment of PTM crosstalk from biological samples in less time from less tissue.     

KCNE1 and KCNE3 β-Subunits Regulate Membrane Surface Expression of Kv12.2 K+ Channels In Vitro and Form a Tripartite Complex In Vivo

Voltage-gated potassium channels that activate near the neuronal resting membrane potential are important regulators of excitation in the nervous system, but their functional diversity is still not well understood. For instance, Kv12.2 (ELK2, KCNH3) channels are highly expressed in the cerebral cortex and hippocampus, and although they are most likely to contribute to resting potassium conductance, surprisingly little is known about their function or regulation. Here we demonstrate that the auxiliary MinK (KCNE1) and MiRP2 (KCNE3) proteins are important regulators of Kv12.2 channel function. Reduction of endogenous KCNE1 or KCNE3 expression by siRNA silencing, significantly increased macroscopic Kv12.2 currents in Xenopus oocytes by around 4-fold. Interestingly, an almost 9-fold increase in Kv12.2 currents was observed with the dual injection of KCNE1 and KCNE3 siRNA, suggesting an additive effect. Consistent with these findings, over-expression of KCNE1 and/or KCNE3 suppressed Kv12.2 currents. Membrane surface biotinylation assays showed that surface expression of Kv12.2 was significantly increased by KCNE1 and KCNE3 siRNA, whereas total protein expression of Kv12.2 was not affected. KCNE1 and KCNE3 siRNA shifted the voltages for half-maximal activation to more hyperpolarized voltages, indicating that KCNE1 and KCNE3 may also inhibit activation gating of Kv12.2. Native co-immunoprecipitation assays from mouse brain membranes imply that KCNE1 and KCNE3 interact with Kv12.2 simultaneously in vivo, suggesting the existence of novel KCNE1-KCNE3-Kv12.2 channel tripartite complexes. Together these data indicate that KCNE1 and KCNE3 interact directly with Kv12.2 channels to regulate channel membrane trafficking.