Focus on phosphohistidine

Summary. Phosphohistidine has been identified as an enzymic intermediate in numerous biochemical reactions and plays a functional role in many regulatory pathways. Unlike the phosphoester bond of its cousins (phosphoserine, phosphothreonine and phosphotyrosine), the phosphoramidate (P–N) bond of phosphohistidine has a high ΔG° of hydrolysis and is unstable under acidic conditions. This acid-lability has meant that the study of protein histidine phosphorylation and the associated protein kinases has been slower to progress than other protein phosphorylation studies. Histidine phosphorylation is a crucial component of cell signalling in prokaryotes and lower eukaryotes. It is also now becoming widely reported in mammalian signalling pathways and implicated in certain human disease states. This review covers the chemistry of phosphohistidine in terms of its isomeric forms and chemical derivatives, how they can be synthesized, purified, identified and the relative stabilities of each of these forms. Furthermore, we highlight how this chemistry relates to the role of phosphohistidine in its various biological functions.     

Role of osmoregulation in the actions of taurine

Summary. Taurine regulates an unusual number of biological phenomena, including heart rhythm, contractile function, blood pressure, platelet aggregation, neuronal excitability, body temperature, learning, motor behavior, food consumption, eye sight, sperm motility, cell proliferation and viability, energy metabolism and bile acid synthesis. Many of these actions are associated with alterations in either ion transport or protein phosphorylation. Although the effects on ion transport have been attributed to changes in membrane structure, they could be equally affected by a change in the activity of the affected transporters. Three common ways of altering transporter activity is enhanced expression, changes in the phosphorylation status of the protein and cytoskeletal changes. Interestingly, all three events are altered by osmotic stress. Since taurine is a key organic osmolyte in most cells, the possibility that the effects of taurine on ion transport could be related to its osmoregulatory activity was considered. This was accomplished by comparing the effects of taurine, cell swelling and cell shrinkage on the activities of key ion channels and ion transporters. The review also compares the phosphorylation cascades initiated by osmotic stress with some of the phosphorylation events triggered by taurine depletion or treatment. The data reveal that certain actions of taurine are probably caused by the activation of osmotic-linked signaling pathways. Nonetheless, some of the actions of taurine are unique and appear to be correlated with its membrane modulating and phosphorylation regulating activities. Received January 25, 2000/Accepted January 31, 2000     

Detection of histidine kinases via a filter-based assay and reverse-phase thin-layer chromatographic phosphoamino acid analysis

The methods that detect histidine phosphorylation have largely been either laborious or difficult to apply quantitatively. The major difficulty in assessing for its presence is its alkali-stable, acid-labile nature. While an assay that detects alkali-stable phosphorylation has been developed, it does not distinguish phosphohistidine from other alkali-stable phosphoamino acids. Using this established method, we extend the assay to facilitate the specific detection of phosphohistidine. We use the acid-lability of phosphohistidine as a defining feature in our approach for its detection. In addition, reverse-phase thin-layer chromatography was utilized to conclusively demonstrate the viability of the conditions that we implement in the assay for the selective detection of phosphohistidine. In summary, this report describes a rapid filter-based kinase assay that quantitatively measures histidine kinase activity, even in the presence of tyrosine kinase activity.     

Phosphorus-31 nuclear magnetic resonance study of the active site phosphohistidine and regulatory phosphoserine residues of rat liver ATP-citrate lyase

31P NMR has been used to investigate the nature of the two chemically distinct phosphorylation sites of ATP-citrate lyase from rat liver. The "regulatory" or "structural" phosphorylation site is acid stable and known to be phosphoserine. The "catalytic" site is very acid labile and has been suggested by different workers to contain either phosphohistidine or an acyl phosphate group. We have demonstrated the presence of both endogenous phosphoserine and phosphoserine introduced after treatment of the lyase with the catalytic subunit of cAMP-dependent protein kinase. This structural phosphate group could be titrated and was readily removed by alkaline phosphatase; these facts, together with the narrow line width of the 31P NMR signal, suggest that it is relatively mobile and located near the surface of the protein. 31P NMR spectra of ATP-citrate lyase that had previously been exposed to fairly high concentrations of potassium chloride (1.5 M), or that had been denatured in detergent and 2-mercaptoethanol, clearly identified phosphohistidine as the catalytic phosphate group. That phosphohistidine is indeed a catalytic intermediate was demonstrated by the disappearance of the resonance in the presence of the substrates citrate and coenzyme A. The line width of the phosphohistidine resonance indicated that the catalytic phosphohistidine residue has negligible residual mobility on the protein. These results are consistent with the pattern of earlier observations on the chemical environments of phospho groups that serve a regulatory or structural role as opposed to a catalytic function in proteins.     

Cellular thiol status-dependent inhibition of tumor cell growth via modulation of p27<Superscript>kip1</Superscript> translocation and retinoblastoma protein phosphorylation by 1′-acetoxychavicol acetate

Summary. 1′-Acetoxychavicol acetate (ACA) has been shown to inhibit tumor cell growth, but there is limited information on its effects on cell signaling and the cell cycle control pathway. In this study, we sought to determine how ACA alters cell cycle and its related control factors in its growth inhibitory effect in Ehrlich ascites tumor cells (EATC). ACA caused an accumulation of cells in the G1 phase and an inhibition of DNA synthesis, which were reversed by supplementation with N-acetylcysteine (NAC) or glutathione ethyl ester (GEE). Furthermore, ACA decreased hyperphosphorylated Rb levels and increased hypophosphorylated Rb levels. NAC and GEE also abolished the decease in Rb phosphorylation by ACA. As Rb phosphorylation is regulated by G1 cyclin dependent kinase and CDK inhibitor p27^kip1, which is an important regulator of the mammalian cell cycle, we estimated the amount of p27^kip1 levels by western blotting. Treatment with ACA had virtually no effect on the amount of p27^kip1 levels, but caused a decrease in phosphorylated p27^kip1 and an increase in unphosphorylated p27^kip1 as well as an increase in the nuclear localization of p27^kip1. These events were abolished in the presence of NAC or GEE. These results suggest that in EATC, cell growth inhibition elicited by ACA involves decreases in Rb and p27^kip1 phosphorylation and an increase in nuclear localization of p27^kip1, and these events are dependent on the cellular thiol status.     

Differential responses to anxiogenic drugs in a mouse model of panic disorder as revealed by Fos immunocytochemistry in specific areas of the fear circuitry

Summary. Sensitivity to pharmacological challenges has been reported in patients with panic disorder. We have previously validated transgenic mice overexpressing the neurotrophin-3 (NT-3) receptor, TrkC (TgNTRK3), as an engineered murine model of panic disorder. We could determine that TgNTRK3 mice presented increased cellularity in brain regions, such as the locus ceruleus, that are important neural substrates for the expression of anxiety in severe anxiety states. Here, we investigated the sensitivity to induce anxiety and panic-related symptoms by sodium lactate and the effects of various drugs (the α2-adrenoceptor antagonist, yohimbine and the adenosine antagonist, caffeine), in TgNTRK3 mice. We found enhanced panicogenic sensitivity to sodium lactate and an increased intensity and a differential pattern of Fos expression after the administration of yohimbine or caffeine in TgNTRK3. Our findings validate the relevance of the NT-3/TrkC system to pathological anxiety and raise the possibility that a specific set of fear-related pathways involved in the processing of anxiety-related information may be differentially activated in panic disorder.     

Chemical phosphorylation of histidine-containing peptides based on the sequence of histone H4 and their dephosphorylation by protein histidine phosphatase

Using peptides based on the amino acid sequences surrounding the two histidine residues in histone H4, we have investigated the kinetics of the phosphorylation and dephosphorylation reactions of their histidine residues, when reacted with potassium phosphoramidate, by ¹H NMR. We have been able to estimate rate constants for the reactions and have shown that there are differences in the kinetics between the two peptides. The kinetics of hydrolysis of phosphoramidate was measured by ³¹P NMR and protein histidine phosphatase (PHP) was shown to catalyse the reaction. We have shown that the dephosphorylation of the phosphohistidine of the phosphopeptides is catalysed by PHP. In terms of substrate specificity, there is a small preference for 1-phosphohistidine compared to 3-phosphohistidine, although the rate accelerations for hydrolysis induced by the enzyme were 1100- and 33,333-fold, respectively. The kinetics of both the phosphorylation and dephosphorylation reactions depend on the amino acid sequence surrounding the histidine. PHP shows greater substrate specificity for the peptide whose sequence is similar to that around histidine 18 of histone H4. PHP was unable to catalyse the dephosphorylation of histone H4 that had been phosphorylated with a histone H4 histidine kinase.     

Influence of ascorbate and the Mehler peroxidase reaction on non-photochemical quenching of chlorophyll fluorescence in maize mesophyll chloroplasts

 Non-photochemical quenching of chlorophyll fluorescence (NPQ) and quantum yield of photosystem II (PSII) were studied with intact mesophyll chloroplasts of maize (Zea mays L.) during the initial minutes of illumination using the pulse-modulated chlorophyll fluorescence technique. Non-photochemical quenching was rapidly reversible in the dark at any point during illumination, which is indicative of energy-dependent dissipation of energy (mediated via thylakoid ΔpH changes and ascorbate-dependent synthesis of zeaxanthin). In chloroplasts suspensions including 15 mM ascorbate in the medium, with addition of oxaloacetate and pyruvate, the PSII yield, rate of reduction of oxaloacetate and phosphorylation of pyruvate reached a maximum after approximately 2 min of illumination. Under these conditions, which promote phosphorylation and a decreased ΔpH across the thylakoid membrane, NPQ rose to a maximum after 2–3 min of illumination, dropped to a minimum after about 6 min, and then increased to a steady-state level. A rather similar pattern was observed when leaves were illuminated following a 30-min dark period. Providing chloroplasts with higher levels of ascorbate (60 mM), prevented the transient drop in NPQ. Anaerobic conditions or addition of potassium cyanide caused a decrease in PSII yield, providing evidence for operation of the ascorbate-dependent Mehler-peroxidase reaction. These conditions also strongly suppressed the transient drop in NPQ. Dithiothreitol, an inhibitor of violaxanthin de-epoxidase, caused a large drop in NPQ even in the presence of high levels of ascorbate. The results suggest that the decline of NPQ occurs in response to an increase in lumen pH after initiation of phosphorylation, that this decline can be suppressed by conditions where ascorbate is not limiting for violaxanthin de-epoxidase, and that the increase of NPQ after such a decline is the result of development of energy dissipation in PSII reaction centers. Received: 13 August 1999 / Accepted: 17 September 1999     

Mutational study of human phosphohistidine phosphatase: effect on enzymatic activity

Although protein histidine phosphorylation is estimated to account for about 6% of total protein phosphorylation in eukaryotes, knowledge on histidine phosphorylation and dephosphorylation is still limited. Recently, a few reports have appeared on a mammalian 14-kDa phosphohistidine phosphatase, also named protein histidine phosphatase. Molecular cloning of the protein has opened possibilities for exploring its properties and physiological role. In the present work, we have searched for potential active site residues in the human phosphohistidine phosphatase by point mutations of conserved histidine and arginine residues to alanine. When assayed by the phosphohistidine-containing peptide succinyl-Ala-His(P)-Pro-Phe-p-nitroanilide, mutants H53A and H102A showed no detectable activity. Compared to the wild-type recombinant enzyme, the specific activity of mutant R45A was decreased by one order of magnitude, that of mutant R78A was decreased by about 30%, while that of mutant H81A was essentially unchanged. These results will facilitate future studies of the reaction mechanism, substrate binding, and molecular structure of the phosphohistidine phosphatase.     

Lysine catabolism, an effective versatile regulator of lysine level in plants

Lysine is a nutritionally important essential amino acid, whose synthesis in plants is strongly regulated by the rate of its synthesis. Yet, lysine level in plants is also finely controlled by a super-regulated catabolic pathway that catabolizes lysine into glutamate and acetyl Co-A. The first two enzymes of lysine catabolism are synthesized from a single LKR/SDH gene. Expression of this gene is subject to compound developmental, hormonal and stress-associated regulation. Moreover, the LKR/SDH gene of different plant species encodes up to three distinct polypeptides: (i) a bifunctional enzyme containing the linked lysine-ketoglutarate (LKR) and saccharopine dehydrogenase (SDH) whose LKR activity is regulated by its linked SDH enzyme; (ii) a monofunctional SDH encoded by an internal promoter, which is a part of the coding DNA region of the LKR/SDH gene; and (iii) a monofunctional, highly potent LKR that is formed by polyadenylation within an intron. LKR activity in the bifunctional LKR/SDH polypeptide is also post-translationally regulated by phosphorylation by casein kinase-2 (CK2), but the consequence of this regulation is still unknown. Why is lysine metabolism super-regulated by synthesis and catabolism? A hypothesis addressing this important question is presented, suggesting that lysine may serve as a regulator of plant growth and interaction with the environment.     

Protein histidine phosphorylation: increased stability of thiophosphohistidine.

Posttranslational phosphorylation of proteins is an important event in many cellular processes. Whereas phosphoesters of serine, threonine and tyrosine have been extensively studied, only limited information is available for other amino acids modified by a phosphate group. The formation of phosphohistidine residues in proteins has been discovered in prokaryotic organisms as well as in eukaryotic cells. The ability to biochemically analyze phosphohistidine residues in proteins, however, is severely hampered by its extreme lability under acidic conditions. In our studies we have found that by replacing the phosphate linked to the histidine residue with a thiophosphate, a phosphohistidine derivative with increased stability is formed. This allows the analysis of phosphohistidine-containing proteins by established biochemical techniques and will greatly aid in the investigation of the role of this posttranslational modification in cellular processes.     

Identification of phosphopeptides by MALDI Q-TOF MS in positive and negative ion modes after methyl esterification

We have developed an efficient, sensitive, and specific method for the detection of phosphopeptides present in peptide mixtures by MALDI Q-TOF mass spectrometry. Use of the MALDI Q-TOF enables selection of phosphopeptides and characterization by CID of the phosphopeptides performed on the same sample spot. However, this type of experiment has been limited by low ionization efficiency of phosphopeptides in positive ion mode while selecting precursor ions of phosphopeptides. Our method entails neutralizing negative charges on acidic groups of nonphosphorylated peptides by methyl esterification before mass spectrometry in positive and negative ion modes. Methyl esterification significantly increases the relative signal intensity generated by phosphopeptides in negative ion mode compared with positive ion mode and greatly increases selectivity for phosphopeptides by suppressing the signal intensity generated by acidic peptides in negative ion mode. We used the method to identify 12 phosphopeptides containing 22 phosphorylation sites from low femtomolar amounts of a tryptic digest of beta-casein and alpha-s-casein. We also identified 10 phosphopeptides containing five phosphorylation sites from an in-gel tryptic digest of 100 fmol of an in vitro autophosphorylated fibroblast growth factor receptor kinase domain and an additional phosphopeptide containing another phosphorylation site when 500 fmol of the digest was examined. The results demonstrate that the method is a fast, robust, and sensitive means of characterizing phosphopeptides present in low abundance mixtures of phosphorylated and nonphosphorylated peptides.     

The role of substance P in stress and anxiety responses

Summary. Substance P (SP) is one of the most abundant peptides in the central nervous system and has been implicated in a variety of physiological and pathophysiological processes including stress regulation, as well as affective and anxiety-related behaviour. Consistent with these functions, SP and its preferred neurokinin 1 (NK1) receptor has been found within brain areas known to be involved in the regulation of stress and anxiety responses. Aversive and stressful stimuli have been shown repeatedly to change SP brain tissue content, as well as NK1 receptor binding. More recently it has been demonstrated that emotional stressors increase SP efflux in specific limbic structures such as amygdala and septum and that the magnitude of this effect depends on the severity of the stressor. Depending on the brain area, an increase in intracerebral SP concentration (mimicked by SP microinjection) produces mainly anxiogenic-like responses in various behavioural tasks. Based on findings that SP transmission is stimulated under stressful or anxiety-provoking situations it was hypothesised that blockade of NK1 receptors may attenuate stress responses and exert anxiolytic-like effects. Preclinical and clinical studies have found evidence in favour of such an assumption. The status of this research is reviewed here.     

磷酸化组氨酸研究进展

蛋白质磷酸化是生物体内一种广泛存在的蛋白质翻译后修饰形式,这种氨基酸与磷酸基团共价连接的修饰模式对蛋白质结构和功能起到了重要调节作用.目前天然蛋白质中发现的可磷酸化位点主要有9种氨基酸残基,其中包括以磷酰胺连接的磷酸化组氨酸.虽然该磷酸化形式在原核生物与真核生物中都起到了重要的调节作用,但对于其生物学功能的研究长期存在技术困难.由于磷酸化组氨酸本身不同于其他磷酸化氨基酸的化学性质,如存在异构体、化学不稳定等,其在传统的研究方法中容易发生水解去磷酸化.随着现代生物化学与分子生物学技术的不断进步,人们针对含有磷酸化组氨酸的蛋白质构建了新的制备、分离与表征策略,本领域也因此开始迅速发展.本文从磷酸化组氨酸的化学结构入手,分析其两种异构体的主要理化性质与化学反应特性,并概述了基于此发展的新型化学生物学研究手段以及对于磷酸化组氨酸生物功能的研究进展.     

Preparation of β-nitroalanine using the Easton three-component coupling method

Summary. A simple one-step preparation of β-nitroalanine has been developed using the Easton three-component coupling method. To date one limitation of this method has been that use of nitromethane as the nitroalkane component does not yield β-nitroalanine. We report that use of the dipotassium salt of nitroacetic acid in the Easton three-component coupling gives β-nitroalanine in high yield, presumably via facile decarboxylation of a β-nitroaspartate intermediate.     

Detection and analysis of protein histidine phosphorylation

Protein histidine phosphorylation is well established as an important part of signalling systems in bacteria, fungi and plants and there is growing evidence of its role in mammalian cell biology. Compared to phosphoserine, phosphothreonine and phosphotyrosine, phosphohistidine is relatively labile, especially under the acidic conditions that were developed to analyse protein phosphorylation. In recent years, there has been an increasing impetus to develop specific methods for the analysis of histidine phosphorylation and assay of histidine kinase activity. Most recently attention has focussed on the application of mass spectrometry to this end. This review provides an overview of methods available for the detection and analysis of phosphohistidine in phosphoproteins, with particular emphasis on the application of mass spectrometric techniques.     

The necessity of functional proteomics: protein species and molecular function elucidation exemplified by in vivo alpha A crystallin N-terminal truncation

Summary. Ten years after the establishment of the term proteome, the science surrounding it has yet to fulfill its potential. While a host of technologies have generated lists of protein names, there are only a few reported studies that have examined the individual proteins at the covalent chemical level defined as protein species in 1997 and their function. In the current study, we demonstrate that this is possible with two-dimensional gel electrophoresis (2-DE) and mass spectrometry by presenting clear evidence of in vivo N-terminal alpha A crystallin truncation and relating this newly detected protein species to alpha crystallin activity regulation by protease cleavage in the healthy young murine lens. We assess the present state of technology and suggest a shift in resources and paradigm for the routine attainment of the protein species level in proteomics.