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PubMed2006

A counterintuitive Mg2+-dependent and modification-assisted functional folding of mitochondrial tRNAs.

Mitochondrial tRNAs (mtRNAs) often lack domains and posttranscriptional modifications that are found in cytoplasmic tRNAs. These structural and chemical elements normally stabilize the folding of cytoplasmic tRNAs into canonical structures that are competent for aminoacylation and translation. For example, the dihydrouridine (D) stem and loop domain is involved in the tertiary structure of cytoplasmic tRNAs through hydrogen bonds and a Mg2+ bridge to the ribothymidine (T) stem and loop domain. These interactions are often absent in mtRNA because the D-domain is truncated or missing. Using gel mobility shift analyses, UV, circular dichroism and NMR spectroscopies and aminoacylation assays, we have investigated the functional folding interactions of chemically synthesized and site-specifically modified mitochondrial and cytoplasmic tRNAs. We found that Mg2+ is critical for folding of the truncated D-domain of bovine mtRNAMet with the tRNA's T-domain. Contrary to the expectation that Mg2+ stabilizes RNA folding, the mtRNAMet D-domain structure was unfolded and relaxed, rather than stabilized in the presence of Mg2+. Because the D-domain is transcribed prior to the T-domain, we conclude that Mg2+ prevents misfolding of the 5'-half of bovine mtRNAMet facilitating its correct interaction with the T-domain. The interaction of the mtRNAMet D-domain with the T-domain was enhanced by a pseudouridine located in either the D or T-domains compared to that of the unmodified RNAs (Kd=25.3, 24.6 and 44.4 microM, respectively). Mg2+ also affected the folding interaction of a yeast mtRNALeu1, but had minimal effect on the folding of an Escherichia coli cytoplasmic tRNALeu. The D-domain modification, dihydrouridine, facilitated mtRNALeu folding. These data indicate that conserved modifications assist and stabilize the formation of the functional mtRNA tertiary structure.

Aminoacylation↗
PubMed2006

Multiple glucocorticoid receptor isoforms and mechanisms of post-translational modification.

Glucocorticoids regulate diverse physiological effects in virtually every organ and tissue in the body. Glucocorticoid actions are mediated through the glucocorticoid receptor (GR), a ligand-dependent transcriptional factor that activates or represses gene transcription. Since, the cloning of the human GR in 1985, research efforts have been focused on describing the mechanism of action exerted by one of the GR isoforms, GRalpha. However, recent studies from our lab and others have suggested that multiple isoforms of hGR are generated from one single gene and one mRNA species by the mechanisms of alternative RNA splicing and alternative translation initiation. These isoforms display diverse cytoplasm-to-nucleus trafficking patterns and distinct transcription activities. In addition, this new information predicts that each hGR protein can be subjected to a variety of post-translational modifications, such as phosphorylation, sumoylation and ubiquitination. The nature and degree of post-translational modification, as well as subcellular localization, may differentially modulate stability and function among the GR isoforms in different tissues providing an additional important mechanism for regulation of GR action. We outline the recent advances made in identifying the processes that generate and modify multiple GR isoforms and the post-translational modifications that contribute to the increasing diversity in the glucocorticoid signaling pathway.

Alternative Splicing↗
PubMed2004

Modification patterns in germinating barley--malting II.

Modification refers to breaking down of cell walls and the conversion of starch-to-sugars in the endosperm of grains by the action of enzymes released from the aleurone layer and possibly the scutellum during germination. Experimentalists have observed two completely different modification patterns in germinating barley. Based on an enzyme reaction, strongly nonlinear diffusion model developed in Part I of this series of papers on malting we examine simple models which predict modifications patterns similar to both observed patterns. We show that one of the observed patterns represents a natural propagation mode that acts as an attractor for the system. The speed of approach to this mode is strongly effected by initial conditions, a consequence of the enzymic nature of the reaction and the dramatic change of diffusivity brought about by the reaction.

Biological Transport↗
PubMed2006

The role of WRN in DNA repair is affected by post-translational modifications.

Werner syndrome (WS) is an autosomal recessive progeroid disease characterized by genomic instability. WRN gene encodes one of the RecQ helicase family proteins, WRN, which has ATPase, helicase, exonuclease and single stranded DNA annealing activities. There is accumulating evidence suggesting that WRN contributes to the maintenance of genomic integrity through its involvement in DNA repair, replication and recombination. The role of WRN in these pathways can be modulated by its post-translational modifications in response to DNA damage. Here, we review the functional consequences of post-translational modifications on WRN as well as specific DNA repair pathways where WRN is involved and discuss how these modifications affect DNA repair pathways.

Animals↗
PubMed2005

Epigenetic control of ovarian function: the emerging role of histone modifications.

The dynamic nature of the ovarian follicle makes it an ideal model to study the coordinated activation and inactivation of genes related to cell growth and differentiation. Much progress has been made in identifying transcription factors that promote the transcription of ovarian genes mediating gonadotropin action and steroidogenesis, but how these factors promote transcription in the context of chromatin is not well understood. Over the past 5 years, epigenetic regulation of ovarian genes through histone modifications has been the focus of an increasing number of studies. Several coactivators and corepressors associated with transcription factors are in fact histone acetyltransferases and histone deacetylases mediating the hyperacetylation and hypoacetylation of histones, respectively. Hyperacetylation of lysine residues in the core histone tails promotes chromatin alterations that favor transcription, whereas hypoacetylation of histones promotes gene silencing or repression. Not only does the acetylation status of the core histones determine whether chromatin remodeling occurs, but histone phosphorylation and methylation may serve equally important roles. For example, the combination of histone H3 phosphorylation and acetylation concertedly favors transcription. In addition, specific lysine methylations (e.g., K9 of histone H3) repress gene expression whereas other methylations promote gene expression. It is most likely the combination of histone modification events that regulate the initiation of transcription. Understanding how ovarian hormones control specific histone modifications will help us understand how follicular cells can switch from active gene pools governing cell proliferation to those gene groups controlling terminal differentiation. Progress in elucidating the ovarian specific regulation of histone modifying enzymes as well as identification of their target gene pools at different stages of the follicular cycle is expected in the next few years.

Acetylation↗
PubMed2006

Essential hypertension seems to result from melatonin-induced epigenetic modifications in area postrema.

Essential hypertension is a complex multifactorial disorder with epigenetic and environmental factors contributing to its prevalence. Epigenetic system is a genetic regulatory mechanism that allows humans to maintain extraordinarily stable patterns of gene expression over many generations. Sympathetic nervous system plays a major role in the maintenance of hypertension and the rostral ventrolateral medulla is the main source of this sympathetic activation. A possible mechanism to explain the sympathetic hyperactivity in the rostral ventrolateral medulla is an action of the area postrema. Area postrema seems to be the region where a shift of the set-point to a higher operating pressure occurs resulting in hypertension. But, how can a shift occur in the area postrema. We propose that melatonin-induced epigenetic modifications in the neurons of area postrema plays a role in this shift. Area postrema is reported to contain high levels of melatonin receptors that play a role in the epigenetic modifications in certain cells. Environmental stressors cause epigenetic modifications in the neurons of area postrema via the pineal hormone melatonin and these changes lead to a shift in the set-point to a higher operating pressure. This signal is then sent via efferent projections to key medullary sympathetic nuclei in rostral ventrolateral medulla resulting in increases in sympathetic nerve activity. This model may explain the long-term alterations in sympathetic activity in essential hypertension.

Area Postrema↗
PubMed2006

Post-translational modifications of Trypanosoma cruzi histone H4.

Histone tails provide sites for a variety of post-translational modifications implicated in the control of gene expression and chromatin assembly. As both histones and control of gene expression in trypanosomes are highly divergent compared to most eukaryotes, post-translational modifications of Trypanosoma cruzi histones were investigated. After in vivo incubation of live parasites with radiolabeled precursors, histone H4 mainly incorporates [(3)H]-acetyl, and to a lesser extent [(3)H]-methyl residues. In contrast, histone H3 preferentially incorporates [(3)H]-methyl residues. The modifications of histone H4 were further characterized by mass spectrometry. MALDI-TOF-TOF-MS analysis revealed that peptides from histone H4 amino-terminus, obtained by either endoproteinase Glu-C or endoproteinase Arg-C digestion, contain isoforms with 14 and 42Da additions, suggesting the presence of simultaneous acetylations and/or methylations. Tandem mass spectrometry analysis demonstrated that the N-terminal alanine is methylated, and lysine residues at positions 4, 10, 14 and 57 are acetylated; lysine at position 18 is mono-methylated, while arginine at position 53 is dimethylated. Immunoblotting analyses using specific antibodies raised against synthetic and acetylated peptides of T. cruzi histone H4 indicate that lysine 4 is acetylated in the majority of histone H4, while other acetylations at the N-terminus portion of histone H4 are less abundant.

Acetylation↗
PubMed2004

Distinct stimulus-specific histone modifications at hsp70 chromatin targeted by the transcription factor heat shock factor-1.

A question of major current interest is whether histone modification at a given gene correlates simply with transcriptional status or if distinctive modifications appear depending on how that gene is activated. The stress-inducible gene Hsp70 is activated by heat shock or by sodium arsenite. Heat shock produces acetylation of histone H4 at Hsp70 chromatin, whereas arsenite produces both H4 acetylation and H3 phosphorylation at the gene. Histone H3 remains markedly hypoacetylated at Hsp70 under these conditions. Arsenite, but not heat shock, requires signaling via p38 MAP kinase for Hsp70 induction and histone H3 phosphorylation. However, independently of p38 MAP kinase, both stresses strongly activate the transcription factor Hsf1. Using Hsf1-/- cells, we show that this factor is responsible for targeting histone H4 acetylation to Hsp70 chromatin. We establish here that histone modifications at inducible genes are not simply a reflection of transcriptional activity, but are strictly dependent on the stimulus used for induction.

Animals↗
PubMed2004

Recruitment of histone modifications by USF proteins at a vertebrate barrier element.

The chicken beta-globin 5'HS4 insulator element acts as a barrier to the encroachment of chromosomal silencing. Endogenous 5'HS4 sequences are highly enriched with histone acetylation and H3K4 methylation regardless of neighboring gene expression. We report here that 5'HS4 elements recruit these histone modifications when protecting a reporter transgene from chromosomal silencing. Deletion studies identified a single protein binding site within 5'HS4, footprint IV, that is necessary for the recruitment of histone modifications and for barrier activity. We have determined that USF proteins bind to footprint IV. USF1 is present in complexes with histone modifying enzymes in cell extracts, and these enzymes specifically interact with the endogenous 5'HS4 element. Knockdown of USF1 expression leads to a loss of histone modification recruitment and subsequent encroachment of H3K9 methylation. We propose that barrier activity requires the constitutive recruitment of H3K4 methylation and histone acetylation at multiple residues to counteract the propagation of condensed chromatin structures.

Acetylation↗
PubMed2005

SUMO: a history of modification.

The small ubiquitin-like modifier (SUMO) is covalently linked to a variety of proteins and is deconjugated by SUMO-specific proteases. A characteristic of SUMO modification is that the biological consequences of conjugation do not appear proportionate to the small fraction of substrate that is modified. SUMO conjugation appears to alter the long-term fate of the modified protein even though the SUMO may be rapidly deconjugated. Thus an unmodified protein with a history of SUMO modification may have different properties from a protein that never has been modified. Here, the diverse effects of SUMO modification are discussed and models proposed to explain SUMO actions.

Animals↗
PubMed2005

Sulfhydryl modification of cysteine mutants of a neuronal glutamate transporter reveals an inverse relationship between sodium dependent conformational changes and the glutamate-gated anion conductance.

In the central nervous system, glutamate transporters remove the neurotransmitter from the synaptic cleft. The electrogenic transport of glutamate is coupled to the electrochemical sodium, proton and potassium gradients. Moreover, these transporters mediate a sodium- and glutamate-dependent uncoupled chloride conductance. In contrast to the wild type, the uptake of radiolabeled substrate of the G283C mutant is inhibited by [2-(trimethylammonium)ethyl]methanethiosulfonate, a membrane impermeant sulfhydryl reagent. In the wild type and the unmodified mutant, substrate-induced currents are inwardly rectifying and reflect the sum of the coupled electrogenic flux and the anion conductance. However, the sulfhydryl-modified G283C mutant exhibits currents that are non-rectifying and reverse at the equilibrium potential for chloride. These properties are similar to those of the I421C mutant after sulfhydryl modification. Importantly, in contrast to I421C, the modification of G283C does not cause an increase of the magnitude of the anion conductance and a decrease of the apparent substrate affinity. Moreover, in the G283C/I421C double mutant the phenotype of I421C is dominant. Sulfhydryl modification of I421C, but not of G283C, abolishes the sodium dependent transient currents. The results indicate the existence of multiple transitions between the coupled transport cycle and anion conducting states.

Amino Acid Transport System X-AG↗
PubMed2006

Post-Translational Modifications of the P2X(4) purinergic receptor subtype in the human placenta are altered in preeclampsia.

P2X(4) receptors are activated by extracellular ATP to raise intracellular calcium, thus altering cell signalling. ATP release occurs under pathophysiological, stress and adverse cell conditions; these are all increased in preeclampsia. Although P2X(4) is abundantly expressed in normal placenta neither the differences in the amount of protein nor its post-translational modifications have been studied in placentae from pregnancies complicated by preeclampsia. Thus we examined P2X(4) protein expression, localization and post-translational modifications in normotensive controls, term and preterm preeclamptic placentae. Densitometric analysis of Western blots showed a significant increase in P2X(4) protein expression in both term (p=0.002) and preterm preeclamptic (p=0.0008) placental samples compared to normotensive controls however the tissue localization of this receptor subtype was unaltered across the groups. Our data showed that P2X(4) is a nitrated protein in the placenta and this nitration is upregulated in preterm preeclamptic placenta compared to normotensive controls (p=0.03). We also demonstrated that P2X(4) is heavily glycosylated in the placenta by deglycosylation with PNGase F which reduced the protein product size by 23 kDa. We propose that P2X(4) acts within the syncytiotrophoblast to alter intracellular calcium and subsequent signalling pathways thereby restoring placental cell homeostasis following ATP-induced changes during pathophysiological conditions such as preeclampsia. We also propose that the post-translational modifications of nitration and glycosylation are required for the normal functioning of P2X(4).

Adult↗
PubMed2005

Structure-activity studies on different modifications of nociceptin/orphanin FQ: identification of highly potent agonists and antagonists of its receptor.

Nociceptin/orphanin FQ (N/OFQ) and its receptor system modulate a variety of biological functions and further understandings of physiological and pathological roles of this system require new potent agonists and antagonists of its receptor. Two series of N/OFQ related analogues were synthesized to investigate the relationship of different modifications. We combined modifications including: (a) Phe(4)-->(pF)Phe(4); (b) Ala(7), Ala(11)-->Aib(7), Aib(11); (c) Leu(14), Ala(15)-->Arg(14), Lys(15). Compared with the first series, N-terminus of the second series was changed from Phe(1) to Nphe(1). All the analogues were amidated at C-terminus. These compounds were tested in binding studies on rat brain membranes and mouse vas deferens assay. Results indicated that the compounds of the first series showed higher affinity and potency than N/OFQ (pK(i)=9.33; pEC(50)=7.50). In particular, [(pF)Phe(4), Aib(7), Aib(11), Arg(14), Lys(15)] N/OFQ-NH(2) was found to be a highly potent agonist with pK(i)=10.78 in binding studies and pEC(50)=9.37 in mouse vas deferens assay. The second series all competitively antagonized the effects of N/OFQ in mouse vas deferens assay. [Nphe(1), (pF)Phe(4), Aib(7), Aib(11), Arg(14), Lys(15)] N/OFQ-NH(2) was the best antagonist with pA(2)=8.39 and showed high binding affinity with pK(i)=9.99. Thus modifications which increase the potency of agonist have synergistic effect on biological activity and a replacement of N-terminus leads to shift of analogues from agonist to antagonist.

Animals↗
PubMed2005

Structure and activity of enzymes that remove histone modifications.

The post-translational modification of histones plays an important role in chromatin regulation, a process that insures the fidelity of gene expression and other DNA transactions. Equally important as the enzymes that generate these modifications are the enzymes that remove them. Recent studies have identified some of the enzymes that remove histone modifications and have characterized their activities. In addition, structural and biochemical studies of these enzymes have focused on the histone lysine deacetylases HDAC8 and sirtuins, and on the arginine and lysine demethylases PAD and BHC110/LSD1, respectively. These new findings may be used as a context to present new information that contributes to our understanding of chromatin regulation, and to pose remaining questions pertaining to the activities of these enzymes and the roles they play in chromatin regulation.

Animals↗
PubMed2006

Posttranslational modifications of NEMO and its partners in NF-kappaB signaling.

NEMO, the regulatory subunit of the IkappaB kinase (IKK) complex that controls the activation of the transcription factor NF-kappaB, is required for IKK function in most situations, but its exact mode of action has remained elusive until recently. A series of publications now provides information about how posttranscriptional modifications of NEMO, such as ubiquitination, sumoylation or phosphorylation, regulate its function in the IKK complex. These modifications might also regulate a cytosolic pool of free NEMO that controls the activation of NF-kappaB induced by genotoxic stress. Together with a better identification of the modifications controlling partners of NEMO, a clearer picture of how IKK becomes activated upon cell stimulation is starting to emerge, providing new clues for how the NF-kappaB pathway could be modulated for therapeutic purposes.

Humans↗
PubMed2006

Screening for post-translational modifications in conotoxins using liquid chromatography/mass spectrometry: an important component of conotoxin discovery.

Mass spectrometry has emerged as an important technique for conotoxin analysis due to its capacity for selective, sensitive, information-rich analyses. Using liquid chromatography/mass spectrometry, Conus venom can be fractionated and the peptides surveyed for specific post-translational modifications, indicating those toxin components likely to have an important biological function. With Conus striatus and Conus victoriae venom as models, bromination, carboxylation and glycosylation modifications are identified through characteristics such as isotopic distribution and labile losses observed during mass spectrometric analysis. This modification screening approach enables the identification of a C. victoriae bromo-carboxy-conotoxin, designated vc5c, as a candidate for detailed mass spectrometric analysis. Using a cDNA sequence coupled with liquid chromatography/mass spectrometry and nanoelectrospray ionization-ion trap-mass spectrometry, the sequence of vc5c is determined to be ICCYPNXWCCD, where W is 6-bromotryptophan, X is gamma-carboxy glutamate and C is disulfide-linked cysteine. This represents the ninth T-superfamily (-CC-CC- scaffold) toxin that has been isolated from venom and characterized.

Amino Acid Sequence↗
PubMed2005

Technical modifications of laparoscopic donor nephrectomy: improved results with refinements in technique that mimic open nephrectomy.

INTRODUCTION: We have performed laparoscopic donor nephrectomy (lap Nx) since 2000. In June 2002, we modified the technique to minimize the known disadvantages of ATN and delayed graft function. We review our series of lap Nx before and after introduction of these modifications, respectively. METHODS: Four technical modifications of lap Nx were introduced following the initial previously described 28 cases (Group I) and applied to the consecutive 42 cases described herein (Group II): (1) decreasing the intra-abdominal pressure from 15 mm Hg to 8 mm Hg; (2) early dissection of the ureter and gonadal vein followed by vascular dissection (sharp and blunt using hydrodissection); (3) leaving the left gonadal vein in continuity with the left renal vein; and (4) early introduction of the Endocatch bag. RESULTS: Operative time was 276.6 +/- 67.1 min vs 210.0 +/- 38.0 min for groups I and II, respectively (P = .04). Warm ischemia was 4.9 +/- 1.9 min vs 1.5 +/- 0.9, min for groups I and II, respectively (P < .01). ATN occurred in 3/28 (10.7%) in group I vs 2/42 (4.8%) in-group II. There was one-vascular insult in the initial 5 Rt lap Nx, namely, transection of a segmental artery, that was reconstructed by bench repair without an impact on the outcome. Conversion was needed in 3/28 (10.7%) group I, but none of group II donors. CONCLUSION: The outcome of lap Nx can be significantly improved by applying technical modifications that simulate open Nx.

Abdomen↗
PubMed2006

Decreased inhibitory activity of prothrombin to calcium oxalate crystallization by specific chemical modification of its gamma-carboxyglutamic acid residues.

OBJECTIVES: To investigate the inhibitory role of gamma (gamma)-carboxyglutamic acid (Gla) in the Gla domain of urinary prothrombin in the formation of calcium oxalate crystals. METHODS: Morpholine and formaldehyde at different concentrations were added to the solution of prothrombin to cause the conversion of Gla to gamma-methyleneglutamic acid (MGlu). The extent of the modification was controlled by the relative amount of modification reagents to prothrombin. The 100-fold molar excess of modification reagents to prothrombin produced the prothrombin molecule containing 2gamma-MGlu residues, and the 10,000-fold molar excess produced the molecule containing 8gamma-MGlu residues. The inhibitory activities of prothrombin and modified prothrombin to calcium oxalate crystallization were evaluated by the seeded crystallization technique. RESULTS: The inhibitory index of Gla prothrombin to calcium oxalate crystallization was 14.2%, that of 2gamma-MGlu prothrombin was 12.8%, decreased by about 10%, and that of 8gamma-MGlu prothrombin was 5.0%, decreased by about 65%. CONCLUSIONS: The Gla in the Gla domain of urinary prothrombin may play an important role in inhibiting the formation of renal calcium oxalate calculus.

1-Carboxyglutamic Acid↗

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