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.     

Mammalian histidine kinases

Protein phosphorylation is one of the most ubiquitous and important types of post-translational modification for the regulation of cell function. The importance of two-component histidine kinases in bacteria, fungi and plants has long been recognised. In mammals, the regulatory roles of serine/threonine and tyrosine kinases have attracted most attention. However, the existence of histidine kinases in mammalian cells has been known for many years, although little is still understood about their biological roles by comparison with the hydroxyamino acid kinases. In addition, with the exception of NDP kinase, other mammalian histidine kinases remain to be identified and characterised. NDP kinase is a multifunctional enzyme that appears to act as a protein histidine kinase and as such, to regulate the activation of some G-proteins. Histone H4 histidine kinase activity has been shown to correlate with cellular proliferation and there is evidence that it is an oncodevelopmental marker in liver. This review mainly concentrates on describing recent research on these two types of histidine kinase. Developments in methods for the detection and assay of histidine kinases, including mass spectrometric methods for the detection of phosphohistidines in proteins and in-gel kinase assays for histone H4 histidine kinases, are described. Little is known about inhibitors of mammalian histidine kinases, although there is much interest in two-component histidine kinase inhibitors as potential antibiotics. The inhibition of a histone H4 histidine kinase by genistein is described and that of two-component histidine kinase inhibitors of structurally-related mammalian protein kinases. In addition, recent findings concerning mammalian protein histidine phosphatases are briefly described.     

Production of neosolaniol monoacetate by an undescribedFusarium species resemblingF camptoceras

Cultures of 12 South African isolates of an undescribedFusarium species resembling but distinct fromF camptoceras were analysed for the presence of diacetoxyscirpenol (DAS), neosolaniol monoacetate (NMA), and T-2 toxin, by capillary gas chromatography utilizing electron capture detection. No DAS or T-2 toxin could be detected in any of the cultures of the isolates. NMA was, however, detected in 10 of the 12 isolates at levels ranging from 310 to 2060 ng/g. The method used, was primarily developed for the determination of DAS and T-2 toxin in fungal cultures and grain samples but was found to be suitable for the coextraction of NMA at an average recovery of 80.8%, with a detection limit in the order of 100 ng/g. Supportive evidence for the presence of the NMA was obtained by capillary gas chromatography / mass spectrometry. Regarded as a relatively rare trichothecene, NMA has never been reported to occur naturally and has previously been shown to be produced by only a fewFusarium strains.     

The sensitivity of Afromontane tarns in the Maloti-Drakensberg region of South Africa and Lesotho to acidic deposition

Despite their remoteness from sources of atmospheric pollutant emissions, the Afromontane tarns in the Maloti-Drakensberg region are perfect candidates to study the negative effects of acidifying atmospheric pollution, because mountain lakes are widely recognised as sentinel ecosystems, unimpacted by direct human disturbance within their catchments. Thirty-four tarns were sampled in the Maloti-Drakensberg region and most were found to be extremely sensitive to acidic deposition, as indicated by their low acid neutralising capacity. There are very few studies of freshwater critical loads for any region within South Africa. The steady-state water chemistry model (SSWC) was adapted and used to determine critical loads, whereas exceedance was estimated relative to modelled regional deposition data, in order to understand the risk of harmful effects to aquatic ecosystems. Seventy-six percent of sampled sites across the Maloti-Drakensberg would exceed critical loads even at the lowest modelled deposition levels, but there are no current measured deposition data for the region. The sensitivity of the Maloti-Drakensberg tarns needs to be considered in future policy formulation regarding acceptable levels of acidifying atmospheric pollution from South Africa’s energy sector and indicates the need for assessing aquatic ecosystem impacts in other regions of South Africa.     

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.     

Sampling Rhyparida nitida Clark (Coleoptera: Chrysomelidae) and symptoms of their damage on sugarcane

Sampling statistics were determined for larvae, pupae and adults of the chrysomelid Rhyparida nitida associated with sugarcane in Australia and for symptoms of their damage. Iwao's patchiness regression was inappropriate for modelling the mean–variance relationships of the insect counts. Taylor's power law was used to model these data and relationships were developed for counts of small, medium and large larvae, all larvae combined, pupae and adults. The mean–variance relationships of counts of live shoots and shoots killed by larvae of R. nitida were modelled using Iwao's patchiness regression; Taylor's power law was not appropriate to either data set. Relationships to determine sample sizes for fixed levels of precision and fixed-precision-level stop lines for sequential sampling of the different stages and live and dead shoots were also developed. Neither the ln(x + 1) transformation nor the Healy and Taylor transformation consistently standardised the mean–variance relationships of insect counts and the appropriate transformation should be selected on a case-by-case basis. Counts of both live and dead shoots were adequately transformed by the Iwao and Kuno transformation.     

Stable triazolylphosphonate analogues of phosphohistidine

Histidine-phosphorylated proteins and the corresponding kinases are important components of bacterial and eukaryotic cell-signalling pathways, and are therefore potential drug targets. The study of these biomolecules has been hampered by the lability of the phosphoramidate functional group in the phosphohistidines and the lack of generic antibodies. Herein, the design and concise synthesis of stable triazolylphosphonate analogues of N1- and N3-phosphohistidine, and derivatives suitable for bioconjugation, are described.     

Asymmetric introgression between sympatric molestus and pipiens forms of Culex pipiens (Diptera: Culicidae) in the Comporta region, Portugal

Background  

Culex pipiens L. is the most widespread mosquito vector in temperate regions. This species consists of two forms, denoted molestus and pipiens, that exhibit important behavioural and physiological differences. The evolutionary relationships and taxonomic status of these forms remain unclear. In northern European latitudes molestus and pipiens populations occupy different habitats (underground vs. aboveground), a separation that most likely promotes genetic isolation between forms. However, the same does not hold in southern Europe where both forms occur aboveground in sympatry. In these southern habitats, the extent of hybridisation and its impact on the extent of genetic divergence between forms under sympatric conditions has not been clarified. For this purpose, we have used phenotypic and genetic data to characterise Cx. pipiens collected aboveground in Portugal. Our aims were to determine levels of genetic differentiation and the degree of hybridisation between forms occurring in sympatry, and to relate these with both evolutionary and epidemiological tenets of this biological group.     

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.