Influence of N-terminal acetylation and C-terminal proteolysis on the analgesic activity of beta-endorphin.

Removal of one, two and four amino-acid residues from the C-terminus of beta-endorphin ('lipotropin C-Fragment', lipotropin residues 61--91) led to the formation of peptides with progressively decreased analgesic potency; there was no change in the persistence of the analgesic effects. The four C-terminal residues are thus important for the activity of beta-endorphin, but not for the duration of action. Removal of eight amino-acid residues from the N-terminus provided a peptide that had no specific affinity for brain opiate receptors in vitro and was devoid of analgesic properties. The N-terminal sequence of beta-endorphin is therefore necessary for the production of analgesia, whereas the C-terminal residues confer potency. The N alpha-acetyl form of beta-endorphin had no specific affinity for brain opiate receptors in vitro and possessed no significant analgesic properties. Since lipotropin C'-Fragment (lipotropin residues 61--87) and the N alpha-acetyl derivative of beta-endorphin occur naturally in brain and pituitary and are only weakly active or inactive as opiates, it is suggested that proteolysis at the C-terminus and acetylation of the N-terminus of beta-endorphin may constitute physiological mechanisms for inactivation of this potent analgesic peptide.     

Autophosphorylation of JAK2 on tyrosines 221 and 570 regulates its activity

The tyrosine kinase JAK2 is a key signaling protein for at least 20 receptors in the cytokine/hematopoietin receptor superfamily and is a component of signaling by insulin receptor and several G-protein-coupled receptors. However, there is only limited knowledge of the physical structure of JAK2 or which of the 49 tyrosines in JAK2 are autophosphorylated. In this study, mass spectrometry and two-dimensional peptide mapping were used to determine that tyrosines 221, 570, and 1007 in JAK2 are autophosphorylated. Phosphorylation of tyrosine 570 is particularly robust. In response to growth hormone, JAK2 was rapidly and transiently phosphorylated at tyrosines 221 and 570, returning to basal levels by 60 min. Analysis of the sequences surrounding tyrosines 221 and 570 in JAK2 and tyrosines in other proteins that are phosphorylated in response to ligands that activate JAK2 suggests that the YXX[L/I/V] motif is one of the motifs recognized by JAK2. Experiments using JAK2 with tyrosines 221 and 570 mutated to phenylalanine suggest that tyrosines 221 and 570 in JAK2 may serve as regulatory sites in JAK2, with phosphorylation of tyrosine 221 increasing kinase activity and phosphorylation of tyrosine 570 decreasing kinase activity and thereby contributing to rapid termination of ligand activation of JAK2.     

Reversible acetylation on Lys501 regulates the activity of RNase II

RNase II, a 3′ to 5′ processive exoribonuclease, is the major hydrolytic enzyme in Escherichia coli accounting for ∼90% of the total activity. Despite its importance, little is actually known about regulation of this enzyme. We show here that one residue, Lys501, is acetylated in RNase II. This modification, reversibly controlled by the acetyltransferase Pka, and the deacetylase CobB, affects binding of the substrate and thus decreases the catalytic activity of RNase II. As a consequence, the steady-state level of target RNAs of RNase II may be altered in the cells. We also find that under conditions of slowed growth, the acetylation level of RNase II is elevated and the activity of RNase II decreases, emphasizing the importance of this regulatory process. These findings indicate that acetylation can regulate the activity of a bacterial ribonuclease.     

Essential role of two tyrosines and two tryptophans on the photoprotection activity of the Orange Carotenoid Protein

Photosynthetic organisms have developed photoprotective mechanisms to protect themselves from lethal high light intensities. One of these mechanisms involves the dissipation of excess absorbed light energy into heat. In cyanobacteria, light activation of a soluble carotenoid protein, the Orange Carotenoid Protein (OCP), binding a keto carotenoid, is the key inducer of this mechanism. Blue-green light absorption triggers structural changes within the carotenoid and the protein, leading to the conversion of a dark orange form into a red active form. Here we report the role in photoconversion and photoprotection of individual conserved tyrosines and tryptophans surrounding the rings of the carotenoid. Our results demonstrate that the interaction between the keto group of the carotenoid and Tyr201 and Trp288 is essential for OCP photoactivity. In addition, these amino acids are responsible for carotenoid affinity and specificity. We have already demonstrated that the aromatic character of Tyr44 and Trp110 interacting with the hydroxyl ring is critical. Here we show that the replacement of Tyr44 by Ser affects the stability of the red form avoiding its accumulation at any temperature, while Trp110Ser is affected in the energy necessary to the orange to red conversion and in the interaction with the antenna. Collectively our data support the idea that the red form is essential for photoprotection but not sufficient. Specific conformational changes occurring in the protein seem to be critical to the events leading to energy dissipation.     

Demethylase activity is directed by histone acetylation

Mammalian genomes are compartmentalized into dense inactive chromatin that is hypermethylated and active open chromatin that is hypomethylated. It is generally accepted that this bimodal pattern of methylation is established during development and is then faithfully inherited through subsequent cell divisions by a maintenance DNA methyltransferase (DNMT1). The pattern of methylation is believed to direct local histone acetylation states. In contrast to this well accepted consensus, we show here using a transient transfection model that an active demethylase is involved in shaping patterns of methylation in somatic cells. Demethylase activity is directed by the state of histone acetylation, and therefore, the resulting methylation pattern is determined by local histone acetylation states contrary to the accepted model. Our data support a new model suggesting that the pattern of methylation is maintained by a dynamic balance of methylation and demethylation activities and the local state of histone acetylation. This provides a simple mechanism for explaining why active genes are not methylated.     

Influence of gamma-chain amino-terminal acetylation on subunit assembly of human fetal hemoglobin

A human hemoglobin F subunit recombination study was performed to determine the relative efficiency of recombination of amino-terminally acetylated gamma-chains and non-acetylated chains with alpha-chains. The results of this work suggested that the acetylated gamma Ic-chains combined more readily with the alpha-chains than the non-acetylated gamma o-chains. An important factor in the function and assembly of multi-subunit macromolecules is the interaction of the unlike subunits. A model system for the study of such interactions has been the protein hemoglobin. With respect to the hemoglobin molecule, it has been noted that relative affinities of normal and mutant subunits for the unlike subunits can have a significant influence on hemoglobin synthesis at the post-translation level, i.e., subunit assembly [1,2]. A similar mechanism may control the formation of human Hb FIc, a hemoglobin with NH2-terminally acetylated gamma- (gamma Ic)-chains. In this instance acetylation may occur on the ribosomes before subunit assembly. A previous report from our laboratory showed a slight increase in the relative proportion of Hb FIc in cord blood samples with over 5% Hb Bart's [3]. The data were interpreted to be due to an influence of alpha-chain deficiency (alpha-thalassemia) on the formation of Hb FIc and Hb Fo tetramers by a preference of alpha-chains for gamma Ic-chains over gamma o- (non-acetylated gamma)-chains. The present study involves an examination of the relative affinity of the gamma Ic- and gamma o-chains for the normal alpha-chains in an in vitro recombination system.     

Influence of core histone acetylation on SV40 minichromosome replication in vitro

We have used the SV40 in vitro replication system to analyze the replication efficiencies of SV40 minichromosomes associated with normal or hyperacetylated histones. We found that elongation of replication occurs with higher efficiency in hyperacetylated minichromosomes in comparison with normal minichromosomes. Our results indicate that the movement of the replication machinery through nucleosomal DNA is facilitated by charge neutralization due to acetylation of the histone tails. Edited by: A. Wolffe     

Effect of acetylation and permethylation on the conformation and candidacidal activity of salivary histatin-5

Summary Salivary histatins provide the non-immune defense against oral pathogens such as Candida albicans. The structural requirements of histatin-5 for anticandida activity were examined with respect to its ability to adopt helical structures, its electrostatic interactions and the hydrogen-bonding potency of its basic residues. For this purpose, the lysine and/or histidine residues of histatin-5 were chemically modified by acetylation and permethylation. Acetylated histatin-5 retained its ability to adopt helical structures in 2,2,2-trifluoroethanol, but completely lost its ability to kill yeast cells. In contrast, permethylated histatin-5 shows very little tendency to adopt a helical structure, but retained significant anticandida activity. The results suggest that the candidacidal activity can arise even when the histatin does not have the ability to adopt helical structures. The candidacidal activity of the derivatives is discussed in terms of electrostatic interactions and hydrogen-bonding potency.     

Identification of the iodine-sensitive tyrosines in porcine pepsin

Porcine pepsin was iodinated at pH 6.0 and 37° with a 13-fold molar excess of [¹²⁵I]triiodide. Loss of proteolytic activity levelled off at 75 ± 5%, and 2.8 ± 0.1 gram atoms of iodine were incorporated per mole of enzyme. Pepsin, iodinated in this manner, was digested with chymotrypsin. The bulk of the label was located in two peptides with the sequences: Leu-Gly-Gly-Ile-Asp-Ser-Ser-diiodoTyr-Tyr and Ile-Gly-Asp-Glu-Pro-Leu-Asn-iodoTyr. The latter peptide is the N-terminal sequence of pepsin. It is postulated that one or both of these tyrosines may form part of the secondary binding site of pepsin.     

Regulatory role of acetylation on enzyme activity and fluxes of energy metabolism pathways

BackgroundMost of the enzymes involved in the central carbon metabolism are acetylated in Lys residues. It has been claimed that this covalent modification represents a novel regulatory mechanism by which both enzyme/transporter activities and pathway fluxes can be modulated.MethodsTo establish which enzymes are regulated by acetylation, a systematic experimental analysis of activities and acetylation profile for several energy metabolism enzymes and pathway fluxes was undertaken in cells and mitochondria.ResultsThe majority of the glycolytic and neighbor enzymes as well as mitochondrial enzymes indeed showed Lys-acetylation, with GLUT1, HPI, CS, ATP synthase displaying comparatively lower acetylation patterns. The incubation of cytosolic and mitochondrial fractions with recombinant Sirt-3 produced lower acetylation signals, whereas incubation with acetyl-CoA promoted protein acetylation. Significant changes in acetylation levels of MDH and IDH-2 from rat liver mitochondria revealed no change in their activities. Similar observations were attained for the cytosolic enzymes from AS-30D and HeLa cells. A minor but significant (23%) increase in the AAT-MDH complex activity induced by acetylation was observed. To examine this question further, AS-30D and HeLa cells were treated with nicotinamide and valproic acid. These compounds promoted changes in the acetylation patterns of glycolytic proteins, although their activities and the glycolytic flux (as well as the OxPhos flux) revealed no clear correlation with acetylation.ConclusionAcetylation seems to play no predominant role in the control of energy metabolism enzyme activities and pathway fluxes.General significanceThe physiological function of protein acetylation on energy metabolism pathways remains to be elucidated.