Covalent Modifications (covalent + modifications)

Distribution by Scientific Domains

Selected Abstracts

Structure, modifications and ligand-binding properties of rat profilin 2a

Teemu Haikarainen
Profilins are key regulators of the actin microfilament system and in neuronal tissues the profilin 2a isoform is the most abundant and important profilin. The high-resolution crystal structure of rat profilin 2a has been determined in the absence of ligands. By comparing the structure with those of peptide-liganded profilin 2a and unliganded profilin 2b, it can be concluded that the binding site for proline-rich peptides is pre-organized. The C-terminus of profilin 2a is also well ordered in the absence of ligand peptide, in contrast to the 2b isoform which is generated by alternative splicing. Covalent modifications of four cysteine residues were also detected in profilin 2a, as well as a number of other modifications in profilin 2 from rat brain; such modifications could significantly affect the function of profilin. It was also shown that profilin 2a binds to the neuronal protein palladin, including a synthetic palladin peptide; peptides from another profilin ligand, dynamin 1, failed to interact with both profilin 1 and profilin 2a. These results allow a better understanding of the structure,function relationships and ligand binding of mammalian profilin 2a. [source]

Epigenetic boundaries of tumour suppressor gene promoters: the CTCF connection and its role in carcinogenesis

Felix Recillas-Targa
Abstract Genetic and epigenetic regulations are essential mechanisms that ensure proper early and subsequent mammalian programming of diverse cellular processes. These mechanisms affect transcriptional regulation, stem cell determination and cell cycle control, including senescence and aging. It is not surprising that perturbation of the exquisite balance between genetic and epigenetic regulation can lead to diverse diseases, including cancer. Histone covalent modifications and DNA methylation do not explain all epigenetic phenomena. We describe a previously unsuspected epigenetic factor and propose the incorporation of the 11-zinc finger CCCTC-binding factor, known as CTCF as a novel and multifunctional epigenetic regulator. [source]

Site-specific proteolysis of cyclooxygenase-2: A putative step in inflammatory prostaglandin E2 biosynthesis

Arturo Mancini
Abstract Cyclooxygenase-2 (COX-2) catalyzes the rate-limiting step in inflammatory prostanoid biosynthesis. Transcriptional, post-transcriptional, and post-translational covalent modifications have been defined as important levels of regulation for COX-2 gene expression. Here, we describe a novel regulatory mechanism in primary human cells involving regulated, sequence-specific proteolysis of COX-2 that correlates with its catalytic activity and ultimately, the biosynthesis of prostaglandin E2 (PGE2). Proinflammatory cytokines induced COX-2 expression and its proteolysis into stable immunoreactive fragments of 66, 42,44, 34,36, and 28 kDa. Increased COX-2 activity (PGE2 release) was observed coincident with the timing and degree of COX-2 proteolysis with correlation analysis confirming a linear relationship (R2,=,0.941). Inhibition of induced COX-2 activity with non-steroidal anti-inflammatory drugs (NSAIDs) and COX-2 selective inhibitors also abrogated cleavage. To determine if NSAID inhibition of proteolysis was related to drug-binding-induced conformational changes in COX-2, we assayed COX-inactive NSAID derivatives that fail to bind COX-2. Interestingly, these compounds suppressed COX-2 activity and cleavage in a correlated manner, thus suggesting that the observed NSAID-induced inhibition of COX-2 cleavage occurred through COX-independent mechanisms, presumably through the inhibition of proteases involved in COX-2 processing. Corroborating this observation, COX-2 cleavage and activity were mutually suppressed by calpain/cathepsin protease inhibitors. Our data suggest that the nascent intracellular form of COX-2 may undergo limited proteolysis to attain full catalytic capacity. J. Cell. Biochem. 101: 425,441, 2007. © 2006 Wiley-Liss, Inc. [source]

Reactivity of Tyr,Leu and Leu,Tyr dipeptides: identification of oxidation products by liquid chromatography,tandem mass spectrometry

Conceição Fonseca
Abstract The exposure of peptides and proteins to reactive hydroxyl radicals results in covalent modifications of amino acid side-chains and protein backbone. In this study we have investigated the oxidation the isomeric peptides tyrosine,leucine (YL) and leucine,tyrosine (LY), by the hydroxyl radical formed under Fenton reaction (Fe2+/H2O2). Through mass spectrometry (MS), high-performance liquid chromatography (HPLC-MS) and electrospray tandem mass spectrometry (HPLC-MSn) measurements, we have identified and characterized the oxidation products of these two dipeptides. This approach allowed observing and identifying a wide variety of oxidation products, including isomeric forms of the oxidized dipeptides. We detected oxidation products with 1, 2, 3 and 4 oxygen atoms for both peptides; however, oxidation products with 5 oxygen atoms were only present in LY. LY dipeptide oxidation leads to more isomers with 1 and 2 oxygen atoms than YL (3 vs 5 and 4 vs 5, respectively). Formation of the peroxy group occurred preferentially in the C -terminal residue. We have also detected oxidation products with double bonds or keto groups, dimers (YL,YL and LY,LY) and other products as a result of cross-linking. Both amino acids in the dipeptides were oxidized although the peptides showed different oxidation products. Also, amino acid residues have shown different oxidation products depending on the relative position on the dipeptide. Results suggest that amino acids in the C -terminal position are more prone to oxidation. Copyright © 2009 John Wiley & Sons, Ltd. [source]

A tandem MS precursor-ion scan approach to identify variable covalent modification of albumin Cys34: a new tool for studying vascular carbonylation

Giancarlo Aldini
Abstract We developed a liquid chromatography electrospray ionisation multi-stage mass spectrometry (LC-ESI-MS/MS) approach based on precursor-ion scanning and evaluated it to characterize the covalent modifications of Cys34 human serum albumin (HSA) caused by oxidative stress and reactive carbonyl species (RCS) adduction. HSA was isolated and digested enzymatically to generate a suitable-length peptide (LQQCPF) containing the modified tag residue. The resulting LQQCPF peptides were identified by LC-ESI-MS/MS in precursor-ion scan mode and further characterized in product-ion scan mode. The product ions for precursor-ion scanning were selected by studying the MS/MS fragmentation of a series of LQQCPF derivatives containing Cys34 modified with different ,,,-unsaturated aldehydes and di and ketoaldehydes. We used a Boolean logic to enhance the specificity of the method: this reconstitutes a virtual current trace (vCT) showing the peaks in the three precursor-ion scans, marked by the same parent ion. The method was first evaluated to identify and characterize the Cys34 covalent adducts of HSA incubated with 4-hydroxy-hexenal, 4-hydroxy- trans -2-nonenal (HNE) and acrolein (ACR). Then we studied the Cys34 modification of human plasma incubated with mildly oxidized low-density lipoproteins (LDL), and the method easily identified the LQQCPF adducts with HNE and ACR. In other experiments, plasma was oxidized by 2,2'-azobis(2-amidinopropane) HCl (AAPH) or by Fe2+/H2O2. In both conditions, the sulfinic derivative of LQQCPF was identified and characterized, indicating that the method is suitable not only for studying RCS-modified albumin, but also to check the oxidative state of Cys34 as a marker of oxidative damage. Copyright © 2008 John Wiley & Sons, Ltd. [source]

Functionalizing the interior of dendrimers: Synthetic challenges and applications

Stefan Hecht
Abstract Chemists' fascination with dendrimers mainly originates from their unique architecture and its exploitation for the design of well-defined functional macromolecules. Depending on the nature of the synthesis, functionalization is traditionally introduced at the core, the periphery, or both. However, the specific incorporation of functional groups at the interior layers, i.e., generations, represents a considerable synthetic hurdle that must be overcome for the full potential of dendrimers to be realized. This review covers recent advances in this emerging frontier of dendrimer science with a particular focus on covalent modifications. Monomer design, syntheses, and properties of various dendritic backbone types are discussed. Internal functionalization dramatically increases the degree of complexity that can be implemented into a dendrimer macromolecule and, therefore, promises to lead to smart materials for future applications in bio- and nanotechnologies. © 2003 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 41: 1047,1058, 2003 [source]

Epigenetic control of plant immunity

SUMMARY In eukaryotic genomes, gene expression and DNA recombination are affected by structural chromatin traits. Chromatin structure is shaped by the activity of enzymes that either introduce covalent modifications in DNA and histone proteins or use energy from ATP to disrupt histone,DNA interactions. The genomic ,marks' that are generated by covalent modifications of histones and DNA, or by the deposition of histone variants, are susceptible to being altered in response to stress. Recent evidence has suggested that proteins generating these epigenetic marks play crucial roles in the defence against pathogens. Histone deacetylases are involved in the activation of jasmonic acid- and ethylene-sensitive defence mechanisms. ATP-dependent chromatin remodellers mediate the constitutive repression of the salicylic acid-dependent pathway, whereas histone methylation at the WRKY70 gene promoter affects the activation of this pathway. Interestingly, bacterial-infected tissues show a net reduction in DNA methylation, which may affect the disease resistance genes responsible for the surveillance against pathogens. As some epigenetic marks can be erased or maintained and transmitted to offspring, epigenetic mechanisms may provide plasticity for the dynamic control of emerging pathogens without the generation of genomic lesions. [source]

Photo-activity induced by amyloidogenesis

Olga Tcherkasskaya
Abstract Accumulation of chemically altered proteins is a noted characteristic of biological aging, and increasing evidence suggests a variety of deleterious cellular developments associated with senescence. Concomitantly, the "aging" of protein deposits associated with numerous neurological disorders may involve covalent modifications of their constituents. However, the link between disease-related protein aggregation and chemical alterations of its molecular constituents has yet to be established. The present study of amyloidogenic ,-synuclein protein points to a decisive change in the biophysical behavior of growing protein aggregates with progressive photo-activity in the visible range of the electromagnetic spectrum. I hypothesize that the photo-activity induced by filament formation is governed by the same mechanism as seen for the intrinsic chromophore of 4-(p-hydroxybenzylidene)-5-imidazolinone-type in the family of green fluorescent proteins. This type of the covalent alterations is initiated concurrently with amyloid elongation and involves a complex multi-step process of chain cyclization, amino acid dehydration, and aerial oxidation. Given that different stages in filament formation yield distinct optical characteristics, the photo-activity induced by amyloidogenesis may have application in molecular biology by enabling in vivo visualization of protein aggregation and its impact on cellular function. [source]

Epigenetic changes in cancer,

APMIS, Issue 10 2007
A cancer develops when a cell acquires specific growth advantages through the stepwise accumulation of heritable changes in gene function. Basically, this process is directed by changes in two different classes of genes: Tumor suppressor genes that inhibit cell growth and survival and oncogenes that promote cell growth and survival. Since several alterations are usually required for a cancer to fully develop, the malignant phenotype is determined by the compound status of tumor suppressor genes and oncogenes. Cancer genes may be changed by several mechanisms, which potentially alter the protein encoding nucleotide template, change the copy number of genes, or lead to increased gene transcription. Epigenetic alterations, which, by definition, comprise mitotically and meiotically heritable changes in gene expression that are not caused by changes in the primary DNA sequence, are increasingly being recognized for their roles in carcinogenesis. These epigenetic alterations may involve covalent modifications of amino acid residues in the histones around which the DNA is wrapped, and changes in the methylation status of cytosine bases (C) in the context of CpG dinucleotides within the DNA itself. Methylation of clusters of CpGs called "CpG-islands" in the promoters of genes has been associated with heritable gene silencing. The present review will focus on how disruption of the epigenome can contribute to cancer. In contrast to genetic alterations, gene silencing by epigenetic modifications is potentially reversible. Treatment by agents that inhibit cytosine methylation and histone deacetylation can initiate chromatin decondensation, demethylation and reestablishment of gene transcription. Accordingly, in the clinical setting, DNA methylation and histone modifications are very attractive targets for the development and implementation of new therapeutic approaches. Many clinical trials are ongoing, and epigenetic therapy has recently been approved by the United States Food and Drug Administration (US FDA) for use in the treatment of myelodysplastic syndrome (MDS) and primary cutaneous T-cell lymphoma (CTCL). [source]