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PubMed1989

Lipid modification of proteins and their membrane transport.

An effective method for artificial attachment of lipid anchors to water-soluble proteins has been developed. To this end, a protein molecule is modified in a system of reversed micelles by a water-insoluble reagent, e.g. fatty acid chloride. Fatty acylated proteins acquire an ability to translocate across lipid membranes and penetrate intact cells. This principle of imparting transmembrane properties to water-soluble proteins makes it possible to realize in vivo a direct transport of antibodies across the hemato-encephalic barrier into the brain and to develop a method for virus suppression by fatty acylated anti-viral antibodies capable of penetrating infected cells. The effect of a drastic increase in the biological activity of exogenous protein factors, e.g. Staphylococcus aureus enterotoxin A, as a result of their artificial fatty acylation has been discovered. The above-mentioned phenomena are discussed in relation to the in vivo data, indicating that post-translational modification of proteins by fatty acids and phospholipids is very widespread in nature and evidently plays an important role in protein transport and sorting. In this connection, lipid modification of proteins is regarded as a possible general step of protein transport in vivo.

Animals↗
PubMed2002

N-Terminal peptide labeling strategy for incorporation of isotopic tags: a method for the determination of site-specific absolute phosphorylation stoichiometry.

Determining the phosphorylation stoichiometry at specific sites in a phosphoprotein is a very challenging task. We describe here a novel mass spectrometry based method that is capable of measuring the absolute phosphorylation stoichiometry at specific sites without the need for specific internal standards, phospho-site antibodies or radioactivity. The method is based on a gentle chemical labeling strategy which specifically and differentially labels the N-terminus of all peptides in a sample with either a D(5)- or D(0)-propionyl group and measures the ratio of the abundance of the D(5)/D(0) peptide pairs simultaneously using mass spectrometry. Using matrix-assisted laser desorption/ionization (MALDI), the method can measure absolute stoichiometry to within at least 10% and can be applied to both in vitro and in vivo phosphorylated peptides and proteins. Furthermore, this method can potentially be applied to the quantitative study of other types of protein post-translational modifications, and the profiling of protein expression on the proteome level.

Amino Acid Sequence↗
PubMed2003

KaiB functions as an attenuator of KaiC phosphorylation in the cyanobacterial circadian clock system.

In the cyanobacterium Synechococcus elongatus PCC 7942, the KaiA, KaiB and KaiC proteins are essential for generation of circadian rhythms. We quantitatively analyzed the intracellular dynamics of these proteins and found a circadian rhythm in the membrane/cytosolic localization of KaiB, such that KaiB interacts with a KaiA-KaiC complex during the late subjective night. KaiB-KaiC binding is accompanied by a dramatic reduction in KaiC phosphorylation and followed by dissociation of the clock protein complex(es). KaiB attenuated KaiA-enhanced phosphorylation both in vitro and in vivo. Based on these results, we propose a novel role for KaiB in a regulatory link among subcellular localization, protein-protein interactions and post-translational modification of Kai proteins in the cyanobacterial clock system.

Bacterial Proteins↗
PubMed2003

The new frontier in muscular dystrophy research: booster genes.

More than 30 different forms of muscular dystrophy (MD) have been molecularly characterized and can be diagnosed, but progress toward treatment has been slow. Gene replacement therapy has met with great difficulty because of the large size of the defective genes and because of difficulties in delivering a gene to all muscle groups. Cell replacement therapy has also been difficult to realize. Will it even be possible to design specific therapy protocols for all MDs? Or is a more realistic goal to treat some of the secondary manifestations that are common to several forms of MD, such as membrane instability, necrosis, and inflammation, and to promote regeneration? As reviewed here, enhanced expression of a range of proteins provides a boost for degenerating dystrophic muscle in mouse models. Expression of a mini-agrin promotes basement membrane formation instead of laminin alpha2; integrin alpha7, GalNac transferase, and ADAM12 promote cell adhesion and muscle stability in the absence of dystrophin; calpastatin prevents muscle necrosis; and nitric oxide synthase prevents inflammation. ADAM12, IGF-I, and myostatin blockade promote regeneration and reduce fibrosis. One can envision numerous other candidate booster genes which encode proteins that promote survival and/or regeneration of the compromised muscle or proteins that affect post-translational modifications of critical proteins. Finally, fibrosis, which is the curse of many human diseases, may also be attacked. Once the mechanisms of the boosters are better understood, drugs may be developed to provide the boost to muscle. Some of the experiences in models of muscular dystrophy may inspire new approaches in other genetic degenerative diseases as well.

Animals↗
PubMed1991

Effect of lactation on the decline of brush border lactase activity in neonatal pigs.

It has been shown that during the early phase of lactation porcine milk contains high concentrations of hormones and growth factors. The aim of the present investigation was to examine the hypothesis that the temporal coordination of intestinal maturation in piglets can be extrinsically regulated through changes in the composition of milk during the suckling period. Gut morphology and the ontogeny of brush border lactase activity were investigated in piglets reared on two suckling regimens designed to expose the animals to compositionally distinct milk. The first group of animals were cross-fostered onto postcolostrum sows and thereafter suckled normally for up to eight weeks. These normally suckled (N) animals consequently received both early and late lactation products. The second group of piglets were cross-fostered each week, for up to eight weeks, onto newly farrowed sows which were postcolostrum. As a result of this repeated cross-fostering (CF) these animals received only early lactation products. Animals were sacrificed at one, three, five, seven, and eight weeks postpartum. Biochemically active lactase decreased significantly (p less than 0.001) in both groups over eight weeks, but the rate of loss of activity was greater in the CF animals than in the N pigs by approximately 50% at week 3 and 25% at week 8. Quantitative histochemical analysis of lactase activity corroborated the biochemical data. At three weeks maximal enzyme activity was observed approximately 400 microns from the villus/crypt junction. Histochemically detected lactase decreased throughout the suckling period, but the intensity of reaction product was consistently weaker over the entire villus surface in the CF animals. Immunocytochemically detectable lactase was identified at the same sites as the histochemical reaction products. In addition, immunofluorescence microscopy showed the presence of histochemically undetectable enzyme on the basolateral and brush border membranes of both villus and crypt cells. Villus/crypt ratios were significantly lower (p<0.001) in the CF animals than in the N pigs between weeks 3 and 5. The results of this study suggest that lactation products can accelerate the loss of brush border lactase activity. The observed decline in biochemically and histochemically detected lactase was considered to be a consequence of reduced enterocyte lifespan, decreased synthesis of enzyme protein, or altered post-translational modification of enzyme protein, or a combination of there.

Animals↗
PubMed1999

Regulation of chondrocyte gene expression.

Extracellular influences known to affect the regulation of chondrocyte biosynthetic and catabolic activity have been shown to include soluble factors, extracellular matrix and mechanical stress. A balance of these numerous extracellular influences is required for normal function of articular cartilage. It is likely that OA is the result of an imbalance of regulatory influences, ultimately resulting in deleterious changes in gene expression, altered extracellular matrix (ECM) and tissue degeneration. Molecular signaling via soluble mediators has been shown to be crucial to cartilage homeostasis. A number of vitamins, hormones, growth/differentiation factors and cytokines have been implicated in chondrocyte differentiation and cartilage metabolism. During normal maintenance, as well as in aging and pathology, these soluble factors can significantly influence the physical properties and the function of cartilage. Chondrocytes, like cells in other tissues, exist within an information-rich extracellular environment consisting of ECM molecules, a milieu which interacts with and modulates the activity of growth factors, hormones and ECM remodeling enzymes. Cell surface matrix receptors, including a family of proteins known as integrins, connect structural information in the ECM to a complex cellular response mechanism in the cell's interior. Integrins on cell surfaces detect and transduce signals in a cooperative manner with other adhesion receptor classes and/or growth factor receptors. The effects of mechanical stress upon a number of chondrocyte biological parameters has been examined in several laboratories. Other investigations have addressed the mechanism by which mechanical force affects biochemical and biosynthetic processes in chondrocytes, in particular synthesis of aggrecan, a major component of the cartilage ECM. Each of these extracellular influences upon chondrocyte metabolism may affect regulation of chondrocyte ECM biosynthesis at many levels, including mRNA transcription, RNA splicing, nuclear transport, protein translation, post-translational modification, intracellular vesicular transport, and protein secretion. Transcriptional regulation of some of the major protein and proteoglycan components of the cartilage ECM has been examined in a number of species, and promoters have been characterized for aggrecan, link protein and collagen type II genes. There is evidence that gene expression may be altered in OA cartilage, providing clues as to which subsets of genes expressed in chondrocytes may be considered relevant to OA pathophysiology.

Aggrecans↗
PubMed2006

Regulation of Wnt signaling by protein-protein interaction and post-translational modifications.

The Wnt signaling pathway is conserved in various species from worms to mammals, and plays important roles in cellular proliferation, differentiation, and migration. Wnt stabilizes cytoplasmic beta-catenin and then the accumulated beta-catenin is translocated into the nucleus, where it activates the transcriptional factor T-cell factor (Tcf)/lymphoid enhancer factor (Lef), and thereby stimulates the expression of genes including c-myc, c-jun, fra-1, and cyclin D1. Tight regulation of this response involves post-translational modifications of the components of the Wnt signaling pathway. Phosphorylation, ubiquitination, and sumoylation have been shown to affect the half-life of beta-catenin and the transcriptional activity of Tcf/Lef. The precise spatio-temporal patterns of these multiple modifications determine the driving force of various cellular responses.

Animals↗
PubMed1996

Isolation of the murine S100 protein MRP14 (14 kDa migration-inhibitory-factor-related protein) from activated spleen cells: characterization of post-translational modifications and zinc binding.

MRP14 (macrophage migration-inhibitory factor-related protein of molecular mass 14 kDa) is an S100 calcium binding protein constitutively expressed in human neutrophils which may be associated with cellular activation/inflammation. Murine MRP14 expression was up-regulated following concanavalin A activation of spleen cells, and the protein was isolated from conditioned medium in high yield (approx. 500 ng/ml). MRP14 had a mass of 12972 +/- 2 Da by electrospray ionization MS, whereas the theoretical mass derived from the cDNA sequence, after removal of the initiator Met, was 12918 Da, suggesting that the protein was post-translationally modified. We identified four post-translational modifications of MRP14: removal of the N-terminal Met, N-terminal acetylation, disulphide bond formation between Cys79 and Cys90, and 1-methylation of His106; the calculated mass was then 12971.8 Da. Methylation of His106 was further characterized after incubation of spleen cells with L-[methyl-3H]Met during concanavalin A stimulation. Sequential analysis of a peptide (obtained by digestion with Lys C) containing methylated His indicated that > 80% of the label in the cycle corresponded to His106, suggesting that the methyl residue was transferred from S-adenosyl-L-methionine. Comparison of the C18 reverse-phase HPLC retention times of phenylthiocarbamoyl derivatives of a hydrolysed digest peptide of MRP14 with those of standards confirmed methyl substitution on the 1-position of the imidazole ring. MRP14 bound more 85Zn2+ than the same amounts of the 10 kDa chemotactic protein (CP10) or S100 beta. Ca2+ decreased Zn2+ binding in S100 beta but it did not influence binding to MRP14, suggesting that the Zn2+ binding site was distinct from and independent of the two Ca2+ binding domains.

Acetylation↗
PubMed1994

PIG-tailed membrane proteins.

Some membrane proteins are associated with the plasma membrane solely through a glycolipid moiety (GPI anchor). The GPI anchor is composed of a core structure of phosphatidylinositol attached to a glycan chain which, in turn, is attached to the C-terminus of the protein. The GPI-anchored protein can be released from the cell surface by the action of GPI-specific phospholipases C and D. In protozoa, GPI anchors represent the predominant mechanism for integrating cell-surface proteins into the lipid bilayer. Addition of a glycolipid anchor to a nascent protein requires a C-terminal hydrophobic signal sequence on the protein which is rapidly exchanged for a pre-assembled anchor. GPI anchors may have roles in protein targeting, cell signalling and in the uptake of small molecules (potocytosis). The human disease 'paroxysmal nocturnal haemoglobinuria' represents a defect in biosynthesis of the GPI anchor. Other lipid post-translational modifications of proteins are also recognized as important in regulating protein function (myristoylation, palmitoylation, prenylation).

Amino Acid Sequence↗
PubMed2003

Glycosylation defects: a new mechanism for muscular dystrophy?

Recently, post-translational modification of proteins has been defined as a new area of focus for muscular dystrophy research by the identification of a group of disease genes that encode known or putative glycosylation enzymes. Walker-Warburg Syndrome (WWS) and muscle-eye-brain disease (MEB) are caused by mutations in two genes involved in O-mannosylation, POMT1 and POMGnT1, respectively. Fukuyama muscular dystrophy (FCMD) is due to mutations in fukutin, a putative phospholigand transferase. Congenital muscular dystrophy type 1C and limb girdle muscular dystrophy type 2I are allelic, both being due to mutations in the gene-encoding fukutin-related protein (FKRP). Finally, the causative gene in the myodystrophy (myd) mouse is a putative bifunctional glycosyltransferase (Large). WWS, MEB, FCMD and the myd mouse are also associated with neuronal migration abnormalities (often type II lissencephaly) and ocular or retinal defects. A deficiency in post-translational modification of alpha-dystroglycan is a common feature of all these muscular dystrophies and is thought to involve O-glycosylation pathways. This abnormally modified alpha-dystroglycan is deficient in binding to extracellular matrix ligands, including laminin and agrin. Selective deletion of dystroglycan in the central nervous system (CNS) produces brain abnormalities with striking similarities to WWS, MEB, FCMD and the myd mouse. Thus, impaired dystroglycan function is strongly implicated in these diseases. However, it is unlikely that these five glycosylation enzymes only have a role in glycosylation of alpha-dystroglycan and it is important that other protein targets are identified.

Glycosylation↗
PubMed2006

Enhanced peptide mass fingerprinting through high mass accuracy: Exclusion of non-peptide signals based on residual mass.

Peptide mass fingerprinting (PMF) is among the principle methods of contemporary proteomic analysis. While PMF is routinely practiced in many laboratories, the complexity of protein tryptic digests is such that PMF based on unrefined mass spectrometric peak lists is often inconclusive. A number of data processing strategies have thus been designed to improve the quality of PMF peak lists, and the development of increasingly elaborate tools for PMF data reduction remains an active area of research. In this report, a novel and direct means of PMF peak list enhancement is suggested. Since the monoisotopic mass of a peptide must fall within a predictable range of residual values, PMF peak lists can in principle be relieved of many non-peptide signals solely on the basis of accurately determined monoisotopic mass. The calculations involved are relatively simple, making implementation of this scheme computationally facile. When this procedure for peak list processing was used, the large number of unassigned masses typical of PMF peak lists was considerably attenuated. As a result, protein identifications could be made with greater confidence and improved discrimination as compared to PMF queries submitted with raw peak lists. Importantly, this scheme for removal of non-peptide masses was found to conserve peptides bearing various post-translational and artificial modifications. All PMF experiments discussed here were performed using Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS), which provided the high mass resolution and high mass accuracy essential for this application. Previously reported equations relating the nominal peptide mass to the permissible range of fractional peptide masses were slightly modified for this application, and these adjustments have been illustrated in detail. The role of mass accuracy in application of this scheme has also been explored.

Algorithms↗
PubMedDate not supplied

Plasma desorption mass spectrometry of natural and recombinant peptides and proteins.

Plasma desorption mass spectrometry (PDMS), which utilizes the fission fragments from the spontaneous decay of californium-252 to ionize large biomolecules, has emerged as a powerful analytical tool in the field of protein chemistry. Because of its high mass range and sensitivity, PDMS is an excellent method for direct molecular weight determination of peptides and small proteins, with much better accuracy than that of the most commonly used classical methods of gel permeation chromatography or SDS-gel electrophoresis. Furthermore, PDMS analysis of the peptide mixture resulting from a specific enzymatic or chemical cleavage of the protein, allows rapid and facile checking of DNA-derived protein sequences and protein structures produced by recombinant DNA technology. The relatively nondestructive nature of the PD mass spectrometric analysis allows further confirmation of the sequence assignments of individual peptide fragments through additional chemical or enzymatic reactions on the PDMS matrix-bound peptides. This PD mapping approach, combined with classical gas phase sequencing, can be used to identify and locate post-translational modifications in proteins, such as glycosylation, phosphorylation, disulfide linkages, and also detect the presence of peptide and protein variants in synthetic, native and recombinant peptides and proteins.

Amino Acid Sequence↗
PubMed1991

Production of recombinant protein C in serum-containing and serum-free perfusion culture.

For the development of a perfusion culture producing recombinant human protein C, the effects of fetal calf serum and growth factors on cell growth and recombinant protein production were investigated. Although the growth of recombinant cells was stimulated by serum in a dose-dependent manner, a lower concentration of serum (2%) could support both synthesis and post-translational modification of protein C as efficiently as 10% serum. Among the growth factors tested, transferrin enhanced protein C production to the level comparable with 10% serum, while insulin was effective in maintaining cellular metabolism. Based on these results, a perfusion culture for a scale-up production of recombinant protein C was done using an Opticell culture system. A good productivity of the recombinant protein was obtained in low serum or serum-free medium for more than one month.

Cell Division↗
PubMed1999

Tissue transglutaminase: apoptosis versus autoimmunity.

Autoimmune diseases are characterized by multiple autoantibodies and/or autoreactive T cells that recognize a large number of antigens. Many of these antigens undergo extensive post-translational modifications during apoptosis and act as substrates for the proapoptotic cystein proteases. Here, Mauro Piacentini and Vittorio Colizzi discuss the effects on autoimmunity produced by post-translational modifications of proteins catalysed by the proapoptotic enzyme tissue transglutaminase.

Animals↗
PubMed1993

Modulation of insulin secretion from normal rat islets by inhibitors of the post-translational modifications of GTP-binding proteins.

Many GTP-binding proteins (GBPs) are modified by mevalonic acid (MVA)-dependent isoprenylation, carboxyl methylation or palmitoylation. The effects of inhibitors of these processes on insulin release were studied. Intact pancreatic islets were shown to synthesize and metabolize MVA and to prenylate several candidate proteins. Culture with lovastatin (to inhibit synthesis of endogenous MVA) caused the accumulation in the cytosol of low-M(r) GBPs (labelled by the [alpha-32P]GTP overlay technique), suggesting a disturbance of membrane association. Concomitantly, lovastatin pretreatment reduced glucose-induced insulin release by about 50%; co-provision of 100-200 microM MVA totally prevented this effect. Perillic acid, a purported inhibitor of the prenylation of small GBPs, also markedly reduced glucose-induced insulin secretion. Furthermore, both N-acetyl-S-trans,trans-farnesyl-L-cysteine (AFC), which inhibited the base-labile carboxyl methylation of GBPs in islets or in transformed beta-cells, and cerulenic acid, an inhibitor of protein palmitoylation, also reduced nutrient-induced secretion; an inactive analogue of AFC (which did not inhibit carboxyl methylation in islets) had no effect on secretion. In contrast with nutrients, the effects of agonists that induce secretion by directly activating distal components in signal transduction (such as a phorbol ester or mastoparan) were either unaffected or enhanced by lovastatin or AFC. These data are compatible with the hypothesis that post-translational modifications are required for one or more stimulatory GBPs to promote proximal step(s) in fuel-induced insulin secretion, whereas one or more inhibitory GBPs might reduce secretion at a more distal locus.

Acetates↗
PubMed2005

Recent advances in the molecular basis of Lafora's progressive myoclonus epilepsy.

Lafora's disease (LD) is an autosomal recessive and fatal form of progressive myoclonus epilepsy with onset in late childhood or adolescence. LD is characterised by the presence of intracellular polyglucosan inclusions, called Lafora bodies, in tissues including the brain, liver and skin. Patients have progressive neurologic deterioration, leading to death within 10 years of onset. No preventive or curative treatment is available for LD. At least three genes underlie LD, of which two have been isolated and mutations characterised: EPM2A and NHLRC1. The EPM2A gene product laforin is a protein phosphatase while the NHLRC1 gene product malin is an E3 ubiquitin ligase that ubiquitinates and promotes the degradation of laforin. Analyses of the structure and function of these gene products suggest defects in post-translational modification of proteins as the common mechanism that leads to the formation of Lafora inclusion bodies, neurodegeneration and the epileptic phenotype of LD. In this review, we summarise the available information on the genetic basis of LD, and correlate these advances with the rapidly expanding information about the mechanisms of LD gained from studies on both cell biological and animal models. Finally, we also discuss a possible mechanism to explain the locus heterogeneity observed in LD.

Humans↗
PubMed2005

The mRNA encoding tauCstF-64 is expressed ubiquitously in mouse tissues.

Polyadenylation is a process of endonucleolytic cleavage of the mRNA, followed by addition of up to 250 adenosine residues to the 3' end of the mRNA. Polyadenylation is essential for eukaryotic mRNA expression, and CstF-64 is a subunit of the CstF polyadenylation factor that is required for accurate polyadenylation. We discovered that there are two forms of the CstF-64 protein in mammalian male germ cells, one of which (CstF-64) is expressed in all tissues, the other of which (tauCstF-64) is expressed only in male germ cells and in brain (albeit at significantly lower levels in the brain). Therefore, we were surprised to find that, using reverse transcription-PCR, cDNA cloning, and RNA blot analyses, tauCstF-64 mRNA was expressed at higher levels in brain than in testis. Also, tauCstF-64 mRNA was expressed at lower but detectable levels in all tissues tested, including epididymis, heart, kidney, liver, lung, muscle, ovary, spleen, thymus, and uterus. These results suggest the hypothesis that tauCstF-64 mRNA is regulated at the translational or post-translational level.

3' Untranslated Regions↗