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PubMed1997

Isolation and molecular characterization of hepatitis B virus X-protein from a baculovirus expression system.

The X protein (HBx) of the human Hepatitis B Virus (HBV) is a regulatory protein that exercises a transcriptional activator function on a variety of regulatory elements and is therefore considered to be involved in the development of human hepatocellular carcinoma (HCC). So far, most attempts at elucidating HBx function have been undertaken at the genetic level, reflecting the difficulties in detecting the very low amounts of the protein in infected livers. Consequently, the questions of intracellular localization and posttranslational modification have not yet been completely answered. We therefore constructed recombinant baculoviruses that allowed expression of HBx and the hexa histidine HBx fusion protein HBxHis in insect cells. Cell fractionation experiments revealed that only a minor part of HBx is detectable in a soluble form in the cytosolic fraction, whereas most of the protein forms intracellular aggregates. These results could be confirmed by confocal laser immunofluorescence. The fusion of a hexa-histidine tag to the amino terminus of HBx allowed a rapid one-step purification by metal chelate affinity chromatography. The detailed analysis of purified HBxHis using electrospray ionization mass spectrometry uncovered two major components: the unmodified, monomeric, fully oxidized form with five intramolecular disulfide bridges, and its N-acetylated modification. Additionally, two minor peaks with mass differences of delta m = +80 da suggested that a small fraction of HBx becomes posttranslationally phosphorylated in insect cells. No further modifications could be observed, indicating that only phosphorylation might play a role in a possible posttranslational regulation of this viral activator.

Animals↗
PubMed2006

Proteomic technology for biomarker profiling in cancer: an update.

The progress in the understanding of cancer progression and early detection has been slow and frustrating due to the complex multifactorial nature and heterogeneity of the cancer syndrome. To date, no effective treatment is available for advanced cancers, which remain a major cause of morbidity and mortality. Clearly, there is urgent need to unravel novel biomarkers for early detection. Most of the functional information of the cancer-associated genes resides in the proteome. The later is an exceptionally complex biological system involving several proteins that function through posttranslational modifications and dynamic intermolecular collisions with partners. These protein complexes can be regulated by signals emanating from cancer cells, their surrounding tissue microenvironment, and/or from the host. Some proteins are secreted and/or cleaved into the extracellular milieu and may represent valuable serum biomarkers for diagnosis purpose. It is estimated that the cancer proteome may include over 1.5 million proteins as a result of posttranslational processing and modifications. Such complexity clearly highlights the need for ultra-high resolution proteomic technology for robust quantitative protein measurements and data acquisition. This review is to update the current research efforts in high-resolution proteomic technology for discovery and monitoring cancer biomarkers.

Biomarkers, Tumor↗
PubMed1993

N-terminal arginylation of proteins in explants of injured sciatic nerves and embryonic brains of rats.

Posttranslational modification of proteins by arginine and lysine has been demonstrated in crude extracts of vertebrate nerves and brain but not in intact cells. In the present experiments we have exploited the fact that Arg is added posttranslationally only at the N-terminus of target proteins, to demonstrate these reactions in intact cells of sciatic nerves and embryonic brains of rats. Sciatic nerves were crushed in anaesthesized rats and 2 hrs later segments of nerve, including the site of the crush, were removed and incubated in media containing [3H]Arg. Incorporation of [3H]Arg into total proteins was analyzed by acid precipitation and the presence of label at the N-terminus was determined by a modification of the Edman degradation procedure. Approximately 25% of protein bound [3H]Arg was released from the N-terminus by the Edman reaction indicating that it was added posttranslationally rather than through protein synthesis. N-terminal labeling was not detectable in nerves not crushed prior to explant and incubation. Slices of embryonic day 20 visual cortex, when incubated under similar conditions as injured sciatic nerves, also showed approximately 25% of the protein incorporated [3H]Arg at the N-terminus, while arginylation was not detectable in adult rat brain slices. Since Lys is not added posttranslationally to the N-terminus, we have attempted to observe lysylation of proteins in intact cells by using cycloheximide (Cx) to block protein synthesis without interfering with protein modification. The posttranslational incorporation of Arg/Lys into proteins was found to be insensitive to up to 2.0 mM Cx in tissue extracts (in vitro).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2005

Plasticity of histone modifications across the invertebrate to vertebrate transition: histone H3 lysine 4 trimethylation in heterochromatin.

Histone posttranslational modifications mediate establishment of structurally and functionally distinct chromatin compartments of eukaryotic nuclei. The association of different histone modifications with euchromatic and heterochromatic compartments is relatively conserved in highly divergent model organisms such as Drosophila and mammals. However, some differences between these model systems have been uncovered while limited data are available from organisms nearer the invertebrate-vertebrate transition. We identified a chromatin compartment in both diploid and endocycling cells of the urochordate, Oikopleura dioica, enriched in heterochromatic histone modifications and DNA methylation. Surprisingly, this compartment also contained high levels of histone H3 trimethylated at lysine 4 (H3 Me(3)K4), a modification thus far associated with actively transcribed sequences. Although in Drosophila and mouse cells, H3 Me(3)K4 was prevalently associated with euchromatin, we also detected it in their pericentromeric heterochromatin. We further showed that H3 Me(3)K4 abundance was not necessarily proportional to local levels of transcriptional activity in either euchromatin or heterochromatin. Our data indicate greater plasticity across evolution in the association of histone lysine methylation with functionally distinct chromatin domains than previously thought and suggest that H3 Me(3)K4 participates in additional processes beyond marking transcriptionally active chromatin.

Animals↗
PubMed2005

Evaluating triggers and enhancers of tau fibrillization.

Alzheimer's disease is characterized in part by the aggregation of tau protein into filamentous inclusions. Because tau filaments form in brain regions associated with memory retention, and because their appearance correlates well with the degree of dementia, they have emerged as robust markers of disease progression. Yet the discovery that mutations in tau protein can lead directly to filament and tangle formation in humans, and that filament formation is linked to neurodegeneration in model biological systems, suggests that tau aggregation may also contribute directly to degeneration in affected neurons. In this context, the mechanism of tau filament formation and its modulation by mutation and posttranslational modification is of fundamental importance. Here, recent progress on the molecular mechanisms underlying tau aggregation deduced from in vivo and in vitro experimentation is reviewed and a model rationalizing the effect of posttranslational and other structural modifications on assembly kinetics and thermodynamics is presented. We hypothesize that tau aggregation can be described as a heterogeneous nucleation reaction, where exogenous effectors, tau gene mutations, or other modifications that stabilize assembly-competent conformations of tau act to trigger the fibrillization reaction. In contrast, those that modulate postnuclear equilibria can enhance fibrillization by increasing the free energy difference between polymers and unincorporated monomers, resulting in stabilization of filaments at low bulk protein concentrations.

Alzheimer Disease↗
PubMed1980

Characterization of the subunit composition of HGPRTase from human erythrocytes and cultured fibroblasts.

Hypoxanthine-guanine phosphoribosyltransferase is a ubiquitous human enzyme, the inherited deficiency of which leads to a specific metabolic-neurological syndrome. Native acrylamide isoelectric focusing revealed that the human enzyme consists of different numbers of isoenzymes depending on the tissue of origin. The erythrocytic enzyme has the most isoenzymes while the enzyme from cultured fibroblasts has only a single isoenzyme. The isoenzyme pattern of the erythrocytic enzyme changes on storage of the crude hemolysate at 4 C. Treatment of the stored crude hemolysate with 4.5 M urea and 0.35 mM beta-mercaptoethanol results in an isoenzyme pattern similar to that of the fresh crude extract. Thus the additional isoenzymes are generated on storage not by covalent modification of the enzyme but probably by binding of small molecules to the enzyme or to association of the enzyme molecules. Hypoxanthine-guanine phosphoribosyltransferase has been purified to 80% homogeneity in three steps, DEAE Sephadex chromatography, heat treatment at 85 C for 5 min, and hydroxylapatite chromatography. Denaturing two-dimensional gel electrophoresis of the erythrocytic enzyme revealed that the erythrocytic enzyme is composed of three major types of subunits (1-3) with the same molecular weight but different isoelectric points. In contrast, the fibroblast enzyme is composed of only a single type of subunit, which comigrates with subunit 1 of the erythrocytic enzyme. Since there is a single genetic locus in humans for HGPRTase (the enzyme is X linked) (Nyhan et al., 1967), the observed subunit modification of the erythrocyte enzyme appears to be the result of posttranslational modification. These findings provide a simple explanation for the observed electrophoretic properties of human HGPRTase. A patient with 0.5% of HGPRTase activity in his erythrocytes was found to have small amounts (greater than 0.5% but less than 5% of normal) of the erythrocytic HGPRTase subunits.

Autoradiography↗
PubMed1995

Complete amino acid sequence of Proteus mirabilis PR catalase. Occurrence of a methionine sulfone in the close proximity of the active site.

The catalase of Proteus mirabilis PR, a peroxide-resistant (PR) mutant of Proteus mirabilis, binds strongly NADPH, which is a unique property among known bacterial catalases. The enzyme subunit consists of 484 amino acid residues for a mass of 55,647 daltons. The complete amino acid sequence was resolved through the combination of protein sequencing, mass spectrometry, and nucleotide sequencing of a PCR fragment. The sequence obtained was compared with that of other known catalases. Amino acids of the active site are all conserved as well as essential residues involved in NADPH binding. Among the amino acids interacting with the heme, a methionine sulfone was found at position 53, in place of a valine in most other catalases. The origin of oxidation of this methionine is unknown, but the presence of this modification could change iron accessibility by large substrates or inhibitors. This posttranslational modification was also demonstrated in the wild-type P. mirabilis catalase.

Amino Acid Sequence↗
PubMed1988

Protein modification in aging.

The age-related accumulation of abnormal forms of enzymes is attributable to posttranslational modification of protein structure and to a progressive loss with age of proteases that preferentially degrade the modified forms. The protein modifications include, but are not limited to: the oxidation of amino acid side chains (especially, side chains of prolyl, arginyl, lysyl and histidinyl residues) by mixed-function oxidation systems; the deamidation of asparaginyl and glutaminyl residues; the racemization and isomerization of aspartyl and asparaginyl residues; the isomerization of prolyl residues; the oxidation of cysteine sulfhydryl groups; and spontaneous changes in protein conformation that are apparently unlinked to changes in amino acid composition. Evidence supporting the roles of these protein modifications and of the proteases that degrade abnormal enzymes during aging is discussed, as well as a consideration of some technical limitations of the methods used in their study.

Aging↗
PubMedDate not supplied

Interaction of eukaryotic initiation factor 5A with the human immunodeficiency virus type 1 Rev response element RNA and U6 snRNA requires deoxyhypusine or hypusine modification.

Hypusine formation on the eukaryotic initiation factor 5A (eIF-5A) precursor represents a unique posttranslational modification that is ubiquitously present in eukaryotic cells and archaebacteria. Specific inhibition of deoxyhypusine synthase leads to growth arrest and cell death. The precise cellular function of eIF-5A and the physiological significance of hypusine modification are not clear. Although the methionyl-puromycin synthesis has been suggested to be the functional assay for eIF-5A activity in vitro, the role of eIF-5A in protein synthesis has not been established. Recent studies have suggested that eIF-5A may be the cellular target of the human immunodeficiency virus type 1 Rev and human T cell leukemia virus type 1 Rex proteins. Motif analysis suggested that eIF-5A resembles a bimodular RNA-binding protein in that it contains a stretch of basic amino acids clustered at the N-terminal region and a leucine-rich stretch at the C-terminal region. Using Rev target RNA, RRE, as a model, we tested the hypothesis that eIF-5A may be an RNA-binding protein. We found that both deoxyhypusine and hypusine-containing eIF-5A can bind to the 252-nt RRE RNA, as determined by a gel mobility shift assay. In contrast, the unmodified eIF-5A precursor cannot. Deoxyhypusine-containing eIF-5A, but not its precursor, could also cause supershift of the Rev stem-loop IIB RRE complex. Preliminary studies also indicated that eIF-5A can bind to RNA such as U6 snRNA and that deoxyhypusine modification appears to be required for the binding. The ability of eIF-5A to directly interact with RNA suggests that deoxyhypusine formation of eIF-5A may be related to its role in RNA processing and protein synthesis. Our study also suggests the possibility of using a gel mobility shift assay for eIF-5A-RNA binding as a functional assay for deoxyhypusine and hypusine formation.

Gene Products, rev↗
PubMed1992

Isoprenoid modification of proteins distinct from membrane acyl proteins in the prokaryote Acholeplasma laidlawii.

Isoprenylation is an important posttranslational modification that affects the activity, subunit interactions and membrane anchoring of different eukaryotic proteins. The small, cell-wall-less prokaryote Acholeplasma laidlawii has more than 20 membrane acyl-proteins enriched in myristoyl and palmitoyl chains. Radioactive mevalonate, a precursor to isoprenoids, was incorporated into several specific membrane proteins of 20 to 45 kDa and two soluble proteins of 23-25 kDa, respectively. No acyl proteins and none of the polar acyl lipids became labelled but these are all labelled by radioactive fatty acids. Mevalonate was incorporated mainly into a minor neutral, non-saponifiable lipid which migrated just above a C30-isoprenoid (squalene) on TLC-plates. The isoprenoid chains could not be released by mild alkaline hydrolysis from most of the isoprenylated proteins, although this procedure releases acyl chains from lipids and all acylated proteins. Isoprenylated proteins were enriched in the detergent phase upon partition with the non-ionic detergent Triton X-114. This behaviour is similar to the acyl proteins of this organism and indicates that the isoprenoid chains give the proteins a hydrophobic character.

Acholeplasma laidlawii↗
PubMedDate not supplied

[Changes in immunochemical properties of serum ferritin after its post-translation modification with glucose in patients with diabetes mellitus].

The paper is concerned with experimental and clinical problems. Posttranslation modification of ferritin with glucose has been shown to cause change of its immunochemical properties. Changes in iron metabolism and in the level of glycosylated hemoglobin A1c were noted in patients with different forms of diabetes mellitus.

Ceruloplasmin↗
PubMed1998

The microtubular system and posttranslationally modified tubulin during spermatogenesis in a parasitic nematode with amoeboid and aflagellate spermatozoa.

Using transmission electron microscopy and immunologic approaches with various antibodies against general tubulin and posttranslationally modified tubulin, we investigated microtubule organization during spermatogenesis in Heligmosomoides polygyrus, a species in which a conspicuous but transient microtubular system exists in several forms: a cytoplasmic network in the spermatocyte, the meiotic spindle, a perinuclear network and a longitudinal bundle of microtubules in the spermatid. This pattern differs from most nematodes including Caenorhabditis elegans, in which spermatids have not microtubules. In the spermatozoon of H. polygyrus, immunocytochemistry does not detect tubulin, but electron microscopy reveals two centrioles with a unique structure of 10 singlets. In male germ cells, microtubules are probably involved in cell shaping and positioning of organelles but not in cell motility. In all transient tubulin structures described in spermatocytes and spermatids of H. polygyrus, detyrosination, tyrosination, and polyglutamylation were detected, but acetylation and polyglycylation were not. The presence/absence of these posttranslational modifications is apparently not stage dependent. This is the first study of posttranslationally modified tubulin in nematode spermatogenesis.

Animals↗
PubMed2006

Chromatin structure and epigenetics.

In eukaryotic cells, the DNA molecule is found in the form of a nucleoprotein complex named chromatin. The basic unit of the chromatin is the nucleosome, which comprises 147 base pairs of DNA wrapped around an octamer of core histones (made of two molecules of each H2A, H2B, H3, and H4 histones). Each nucleosome is linked to the next by small segments of linker DNA. Most chromatin is further condensated by winding in a polynucleosome fibre, which may be stabilized through the binding of histone H1 to each nucleosome and to the linker DNA. The modulation of the structure of the chromatin fibre is critical for the regulation of gene expression since it determines the accessibility and the sequential recruitment of regulatory factors to the underlying DNA. Depending on the different transcriptional states, the structure of the chromatin may be altered in its constituents (e.g. the presence of repressors, activators, chromatin remodelling complexes, and/or incorporation of histone variants), and in covalent modifications of its constituents (such as DNA methylation at cytosine residues, and posttranslational modifications of histone tails). Here, we give an overview of the molecular mechanisms involved in chromatin regulation and the epigenetic transmission of its state, both in normal and pathological scenarios.

Animals↗
PubMed2007

Max is acetylated by p300 at several nuclear localization residues.

Max is a ubiquitous transcription factor with a bHLHZip [basic HLH (helix-loop-helix) leucine zipper] DNA-binding/dimerization domain and the central component of the Myc/Max/Mad transcription factor network that controls cell growth, proliferation, differentiation and apoptotic cell death in metazoans. Max is the obligatory DNA-binding and dimerization partner for all the bHLHZip regulators of the Myc/Max/Mad network, including the Myc family of oncoproteins and the Mad family of Myc antagonists, which recognize E-box DNA elements in the regulatory regions of target genes. Max lacks a transcription regulatory domain and is the only member of the network that efficiently homodimerizes. Binding of Max homodimers to E-box elements suppresses the transcription regulatory functions of its network partners and of other non-network E-box-binding regulators. In contrast with its highly regulated partners, Max is a constitutively expressed and phosphorylated protein. Phosphorylation is, however, the only Max post-translational modification identified so far. In the present study, we have analysed Max posttranslational modifications by MS. We have found that Max is acetylated at several lysine residues (Lys-57, Lys-144 and Lys-145) in mammalian cells. Max acetylation is stimulated by inhibitors of histone deacetylases and by overexpression of the p300 co-activator/HAT (histone acetyltransferase). The p300 HAT also directly acetylates Max in vitro at these three residues. Interestingly, the three Max residues acetylated in vivo and in vitro by p300 are important for Max nuclear localization and Max-mediated suppression of Myc transactivation. These results uncover novel post-translational modifications of Max and suggest the potential regulation of specific Max complexes by p300 and reversible acetylation.

Acetylation↗
PubMed2002

Intrinsic and extrinsic pathway signaling during neuronal apoptosis: lessons from the analysis of mutant mice.

Trophic factor deprivation (TFD)-induced apoptosis in sympathetic neurons requires macromolecular synthesis-dependent BAX translocation, cytochrome c (cyt c) release, and caspase activation. Here, we report the contributions of other intrinsic and extrinsic pathway signals to these processes. Sympathetic neurons expressed all antiapoptotic BCL-2 proteins examined, yet expressed only certain BH3-only and multidomain proapoptotic BCL-2 family members. All coexpressed proapoptotic proteins did not, however, exhibit functional redundancy or compensatory expression, at least in the Bax-/-, Bak-/-, Bim-/-, Bid-/-, and Bad-/- neurons examined. Although the subcellular distribution or posttranslational modification of certain BCL-2 proteins changed with TFD, neither transcriptional nor posttranslational mechanisms regulated the expression or subcellular localization of BID, BAD, or BAK in this paradigm. Despite modest induction of Fas and FasL expression, Fas-mediated signaling did not contribute to TFD-induced apoptosis in sympathetic neurons. Similar findings were obtained with K+ withdrawal-induced apoptosis in cerebellar granule neurons, a model for activity-dependent neuronal survival in the CNS. Thus, expression alone does not guarantee functional redundancy (or compensation) among BCL-2 family members, and, at least in some cells, extrinsic pathway signaling and certain BH3-only proteins (i.e., BID and BAD) do not contribute to BAX-dependent cyt c release or apoptosis caused by TFD.

Animals↗
PubMed1999

Differential stimulation of PKC phosphorylation of potassium channels by ZIP1 and ZIP2.

Targeting of protein modification enzymes is a key biochemical step to achieve specific and effective posttranslational modifications. Two alternatively spliced ZIP1 and ZIP2 proteins are described, which bind to both Kvbeta2 subunits of potassium channel and protein kinase C (PKC) zeta, thereby acting as a physical link in the assembly of PKCzeta-ZIP-potassium channel complexes. ZIP1 and ZIP2 differentially stimulate phosphorylation of Kvbeta2 by PKCzeta. They also interact to form heteromultimers, which allows for a hybrid stimulatory activity to PKCzeta. Finally, ZIP1 and ZIP2 coexist in the same cell type and are elevated differentially by neurotrophic factors. These results provide a mechanism for specificity and regulation of PKCzeta-targeted phosphorylation.

Alternative Splicing↗
PubMed1999

Covalent modification of the homeodomain-interacting protein kinase 2 (HIPK2) by the ubiquitin-like protein SUMO-1.

Posttranslational modifications such as ubiquitination and phosphorylation play an important role in the regulation of cellular protein function. Homeodomain-interacting protein kinase 2 (HIPK2) is a member of the recently identified family of nuclear protein kinases that act as corepressors for homeodomain transcription factors. Here, we show that HIPK2 is regulated by a ubiquitin-like protein, SUMO-1. We demonstrate that HIPK2 localizes to nuclear speckles (dots) by means of a speckle-retention signal. This speckle-retention signal contains a domain that interacts with a mouse ubiquitin-like protein conjugating (E2) enzyme, mUBC9. In cultured cells, HIPK2 is covalently modified by SUMO-1, and the SUMO-1 modification of HIPK2 correlates with its localization to nuclear speckles (dots). Thus, our results provide firm evidence that the nuclear protein kinase HIPK2 can be covalently modified by SUMO-1, which directs its localization to nuclear speckles (dots).

Amino Acid Sequence↗
PubMed2000

Lantibiotic biosynthesis: interactions between prelacticin 481 and its putative modification enzyme, LctM.

Class AII and AIII lantibiotics and mersacidin are antibacterial peptides containing unusual residues obtained by posttranslational modifications of prepeptides, presumably catalyzed by LanM. LctM, the LanM for lacticin 481, is essential for the production of this class AII lantibiotic. Using the yeast two-hybrid system, we showed direct contact between the prelacticin 481 and LctM, supporting the proposed LctM function. Sixteen domains are conserved between the 10 known LanM proteins, whereas three additional domains were found only in class AII LanM proteins and in MrsM, the LanM for mersacidin. All the truncated LctM proteins that we tested presented impaired LctA-binding activity.

Amino Acid Sequence↗