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PubMed2005

Human intervertebral disc aggrecan inhibits endothelial cell adhesion and cell migration in vitro.

STUDY DESIGN: The effect of human intervertebral disc aggrecan on endothelial cell growth was examined using cell culture assays. OBJECTIVE: To determine the response of endothelial cells to human intervertebral disc aggrecan, and whether the amount and type of aggrecan present in the intervertebral disc may be implicated in disc vascularization. SUMMARY OF BACKGROUND DATA: Intervertebral disc degeneration has been associated with a loss of proteoglycan, and the ingrowth of blood vessels and nerves. Neovascularization is a common feature also of disc herniation. Intervertebral disc aggrecan is inhibitory to sensory nerve growth, but the effects of disc aggrecan on endothelial cell growth are not known. METHODS: Aggrecan monomers were isolated separately from the anulus fibrosus and nucleus pulposus of human lumbar intervertebral discs, and characterized to determine the amount and type of sulfated glycosaminoglycan side chains present. The effects of these aggrecan isolates on the cellular adhesion and migration of the human endothelial cell lines, HMEC-1 and EAhy-926, were examined in vitro. RESULTS: Homogenous substrata of disc aggrecan inhibited endothelial cell adhesion and cell spreading in a concentration dependent manner. In substrata choice assays, endothelial cells seeded onto collagen type I migrated over the collagen until they encountered substrata of disc aggrecan, where they either stopped migrating, retreated onto the collagen, or, more commonly, changed direction to align along the collagen-aggrecan border. The inhibitory effect of aggrecan on endothelial cell migration was concentration dependent, and reduced by enzymatic treatment of the aggrecan monomers with a combination of chondroitinase ABC and keratinase/keratinase II. Anulus fibrosus aggrecan was more inhibitory to endothelial cell adhesion than nucleus pulposus aggrecan. However, this difference did not relate to the extent to which the different aggrecan isolates were charged, as determined by colorimetric assay with 1,9-dimethylmethylene blue, or to marked differences in the distribution of chondroitin sulfated and keratan sulfated side chains. CONCLUSIONS: Human intervertebral disc aggrecan is inhibitory to endothelial cell migration, and this inhibitory effect appears to depend, in part, on the presence of glycosaminoglycan side chains on the aggrecan monomer.

Aggrecans↗

PubMed2001

Purification and characterization of human laminin-8. Laminin-8 stimulates cell adhesion and migration through alpha3beta1 and alpha6beta1 integrins.

Recently identified laminin isoforms containing the alpha4 chain have been shown to be expressed in the basement membrane of restricted organs such as heart, skeletal muscle, and blood vessels, especially those in embryos. We screened 38 human cell lines for the expression of the laminin alpha4 chain by reverse transcriptase-polymerase chain reaction and found that T98G glioblastoma cells express only alpha4, but not other alpha chains. Laminin-8, an isoform containing the alpha4 and beta1 chains, was purified from conditioned medium of T98G cells by gel filtration and immunoaffinity chromatography using a monoclonal antibody against laminin beta1 chain. The purified laminin isoform was composed of disulfide-linked 230-, 220-, and 200-kDa subunits, which immunoblot analysis identified as the beta1, gamma1, and alpha4 chains. Purified laminin-8 had cell adhesive activity comparable to laminin-1 but significantly weaker than laminin-5 and laminin-10/11. T98G cells adhering to laminin-8 became more elongated than those adhering to other laminin isoforms and extended multiple pseudopods. Cell adhesion to laminin-8 was abolished by an antibody against the integrin beta1 subunit or a combination of antibodies against the integrin alpha3 and alpha6 subunits, but not by either anti-alpha3 or anti-alpha6 antibody alone, suggesting that both alpha3beta1 and alpha6beta1 integrins serve as adhesion receptors for laminin-8. Consistent with these observations, K562 erythroleukemic cells transfected with either integrin alpha3 or alpha6 cDNA were capable of adhering to laminin-8 when beta1 integrins were stimulated by the beta1-activating antibody 8A2. Despite its moderate cell adhesive activity, laminin-8 was significantly potent in promoting cell migration when compared with other laminin isoforms and fibronectin. Cell migration on laminin-8 was completely inhibited by a combination of antibodies against alpha3 and alpha6 integrins, and substantially inhibited by anti-alpha3 antibody alone, suggesting that laminin-8-mediated cell migration is predominantly mediated by alpha3beta1 integrin. Given its potency to stimulate cell migration and preferential localization to the basement membrane of capillaries and embryonic tissues, laminin-8 may play a role in processes requiring enhanced cell migration during development, wound healing, and angiogenesis.

Antibodies, Monoclonal↗

PubMed1992

Increase of migration of cultured endothelial cells by angiotensin-converting enzyme inhibitor derived from tuna muscle.

The influence of angiotensin-converting enzyme (ACE) inhibitory octapeptide derived from tuna muscle (tuna AI) on the bovine aorta endothelial cell (BAEC) migration was investigated, as compared with captopril. BAEC migration was quantitated 6 d after release from contact inhibition by a teflon fence assay. The culture grown in the presence of tuna AI (1 and 10 microM) clearly exhibited an increase in migration, compared with the control. The media collected from tuna AI (1 and 10 microM)-stimulated BAECs significantly exhibited the interleukin (IL) -1 activity that was detected by the thymocyte costimulation assay with phytohemagglutinin. Although tuna AI was a weaker ACE inhibitor than captopril, the increasing effect of tuna AI on the migration and the IL-1 generation in BAECs was slightly greater than that of captopril. In quiescent BAECs, tuna AI (1 microM) apparently induced c-myc and platelet derived growth factor (PDGF) A-chain messenger ribonucleic acid (mRNA) expressions within 30 min, which persisted for 6 h. In contrast, captopril induced a very low expression of c-myc mRNA, and had no relation to PDGF A-chain mRNA expression. These results suggest that the increase of BAEC migration by tuna AI, unlike captopril, is likely related to the induction or activation of IL-1, and c-myc and PDGF mRNAs, in addition to the inhibition of the conversion of endogenous angiotensin I to angiotensin II.

Amino Acid Sequence↗

PubMed1983

In vitro synthesis of laminin and entactin polypeptides.

Total RNA and poly(A+) RNA, isolated from 13.5-day-old mouse embryo parietal endoderm cells and from differentiated F9 teratocarcinoma cells that synthesize laminin and entactin, were translated in the reticulocyte lysate. Antiserum raised against purified and denatured laminin B chains specifically immunoprecipitated from the translation reaction polypeptides of Mr = 205,000, 200,000, and 185,000. Antiserum against the native complex of laminin and entactin also immunoprecipitated these polypeptides, although less efficiently. In addition, this antiserum immunoprecipitated polypeptides of Mr = 300,000, 270,000, and 140,000. Antiserum against purified and denatured entactin immunoprecipitated only the Mr = 140,000 polypeptide. In contrast, no polypeptides were immunoprecipitated from translation reactions programmed with RNA from undifferentiated F9 cells that produce only small amounts of laminin and entactin. The in vitro synthesized polypeptides migrate on NaDodSO4-polyacrylamide gel electrophoresis slower than the respective unglycosylated laminin and entactin chains isolated from cells treated with tunicamycin. Supplementing the reticulocyte lysate with dog pancreas microsomal membranes yields in vitro translation products which co-migrate with the respective glycosylated laminin and entactin chains of control cells. Taken together, these results suggest that the polypeptides described represent in vitro synthesized laminin and entactin chains.

Animals↗

PubMed1986

Cytoplasmic myosin from Drosophila melanogaster.

Myosin is identified and purified from three different established Drosophila melanogaster cell lines (Schneider's lines 2 and 3 and Kc). Purification entails lysis in a low salt, sucrose buffer that contains ATP, chromatography on DEAE-cellulose, precipitation with actin in the absence of ATP, gel filtration in a discontinuous KI-KCl buffer system, and hydroxylapatite chromatography. Yield of pure cytoplasmic myosin is 5-10%. This protein is identified as myosin by its cross-reactivity with two monoclonal antibodies against human platelet myosin, the molecular weight of its heavy chain, its two light chains, its behavior on gel filtration, its ATP-dependent affinity for actin, its characteristic ATPase activity, its molecular morphology as demonstrated by platinum shadowing, and its ability to form bipolar filaments. The molecular weight of the cytoplasmic myosin's light chains and peptide mapping and immunochemical analysis of its heavy chains demonstrate that this myosin, purified from Drosophila cell lines, is distinct from Drosophila muscle myosin. Two-dimensional thin layer maps of complete proteolytic digests of iodinated muscle and cytoplasmic myosin heavy chains demonstrate that, while the two myosins have some tryptic and alpha-chymotryptic peptides in common, most peptides migrate with unique mobility. One-dimensional peptide maps of SDS PAGE purified myosin heavy chain confirm these structural data. Polyclonal antiserum raised and reacted against Drosophila myosin isolated from cell lines cross-reacts only weakly with Drosophila muscle myosin isolated from the thoraces of adult Drosophila. Polyclonal antiserum raised against Drosophila muscle myosin behaves in a reciprocal fashion. Taken together our data suggest that the myosin purified from Drosophila cell lines is a bona fide cytoplasmic myosin and is very likely the product of a different myosin gene than the muscle myosin heavy chain gene that has been previously identified and characterized.

Animals↗

PubMed1996

Inflammation-induced changes in the phenotype and cytokine profile of cells migrating through skin and afferent lymph.

In the present study, we have localized cytokine-secreting cells within an ectoparasite-induced inflammatory lesion and monitored the phenotype and cytokine profile of cells migrating from the inflammatory lesion to the local draining lymph node via the afferent lymphatics. Interleukin (IL)-8-producing cells were first detected in skin within 6 hr of infection, with increased numbers observed at 24 and 48 hr post infection. While these cells were concentrated within the neutrophil influx, adjacent to disrupted epidermis; they were also found scattered throughout the surrounding dermis in areas where significant cellular infiltration was not apparent. IL-1 alpha- and IL-1 beta-producing cells could not be detected until 24 hr after infection and were restricted to areas of intense neutrophil accumulation. Concurrent with the onset of inflammation was a threefold increase in the total number of cells migrating through the draining afferent lymph. This increase in cellularity was due primarily to increased migration of CD4 and gamma delta T cells. Cytokine mRNA synthesis by migrating afferent lymph cells was examined by reverse transcription-polymerase chain reaction (RT-PCR) analysis of RNA extracted prior to, and at regular intervals during the course of the inflammatory response. IL-1 beta and IL-8, but not IL-1 alpha or IL-6 mRNA, was detected in migrating afferent lymph cells. Tumour necrosis factor (TNF)-alpha-specific mRNA was present in migrating afferent lymph cells at all time points both prior to, and following infection. Soluble IL-8 protein, but not IL-1 alpha, IL-1 beta or TNF-alpha protein, could be detected in lymph, with the amount of IL-8 detected increasing as the infection progressed. mRNA coding for cytokines associated with T-cell activation, such as IL-2, IL-4 or interferon (IFN)-gamma, was also detected in migrating cells, although the cytokine profiles of different experimental animals were extremely variable.

Animals↗

PubMedDate not supplied

[Lateral diffusion of the protein components of the respiratory assembly of Microsoccus lysodeikticus].

Preparations with a selectively decreased (by 85-90%) content of NADH dehydrogenase were isolated by means of heating treatment of M. lysodeikticus isolated membranes. The degree of the reduction of the NADH dehydrogenase nearest neighbour in the respiration chain of cytochrome b556 in heated membranes is similar to that in intact membranes. It is concluded that cytochrome b556 and (or) NADH dehydrogenase are capable to lateral migration in the membrane of M. lysodeikticus, resulting in the inter-chain electrone transport. A coefficient of their lateral diffusion is calculated (D equals 8-10(-10)-2-10(-9) CM2SEC-1 At 30 degrees C) on the basis of kinetics of cytochrome reduction by NADH dehydrogenase. The electron transport, due to a diffusion of respiration carriers from one assambly to another, proceeds 100 times as slow as the electrone transport in the respiratory chain. The data obtained allow to consider the aggregation of respiration enzymes as a dynamic formation.

Biological Transport↗

PubMed1994

A genetically engineered, protein-based optical biosensor of myosin II regulatory light chain phosphorylation.

Myosin II is an important motor in the contraction of smooth and striated muscle as well as in a variety of non-muscle cell motile events including cytokinesis, cortical contractions during migration of fibroblasts, and capping of receptors. Phosphorylation of the 20-kDa light chain by myosin light chain kinase is part of the regulation of smooth muscle and mammalian nonmuscle myosin II. We designed, characterized, and tested the use of a protein-based optical biosensor to monitor this phosphorylation "switch." A regulatory light chain was genetically engineered to contain a single cysteine at amino acid position 18. The mutant light chain (Cys18.LC20), reacted with the fluorophore acrylodan, responded to phosphorylation of serine 19 with a fluorescence emission quenching of 60% and a 28-nm red-shift. When the acrylodan-labeled mutant light chain (AC-Cys18.LC20) was exchanged into turkey gizzard myosin II, it exhibited a 25% fluorescence emission quenching and a 10-nm red-shift upon phosphorylation of serine 19. The myosin II optical biosensor exhibited nearly control levels of the rate of phosphorylation, K+ATPase activity, and in vitro motility. The acrylodan-labeled light chain was exchanged into the A-bands of chicken pectoralis myofibrils in situ to demonstrate the localization and activity of the biosensor in a highly ordered contractile system. Fluorometry and quantitative fluorescence microscopic imaging experiments demonstrated that AC-Cys18.LC20 exchanged myofibrils expressed a phosphorylation-dependent fluorescence change. Labeled light chains were also incorporated into stress fibers of living fibroblasts and smooth muscle cells. This general approach of combining molecular biology and fluorescence spectroscopy to create novel protein-based optical biosensors should provide valuable tools for investigations with model systems and solution studies and ultimately yield important information about temporal-spatial chemical and molecular changes in live cells.

2-Naphthylamine↗

PubMed1998

A computer-assisted system for reconstructing and interpreting the dynamic three-dimensional relationships of the outer surface, nucleus and pseudopods of crawling cells.

Newly developed software additions to the three-dimensional dynamic image analysis system, 3D-DIAS, are described for simultaneously reconstructing and motion analyzing in three dimensions the outer surface, nucleus and pseudopods of living, crawling cells. This new system is then used to describe for the first time a nuclear behavior cycle in translocating Dictyostelium discoideum amoebae and to investigate the role of pseudopod extension in this process. The nuclear behavior cycle is tuned to the two phases of the general cell behavior cycle [Wessels et al., 1994], and includes nuclear migration both in the z- and in the x,y-axes from the proximal border of the prior anterior pseudopod to the proximal border of a newly expanding anterior pseudopod. Nuclear migration is cued by pseudopod-substratum contact, achieves velocities in excess of 50 microm/min, and is accompanied by characteristic changes in nuclear shape. The rules and characteristics of nuclear behavior are demonstrated to be intact in two mutants affecting pseudopod formation, a myosin IB null mutant (myoB-) and a myosin II heavy chain phosphorylation mutant (3XALA). The rules and characteristics of nuclear migration, however, are disrupted upon dissolution of microtubules by colcemid. Together the above results demonstrate that the newly developed 3D-DIAS system can be used to gain new insights into the dynamic changes in the intracellular 3D architecture associated with cellular translocation.

Animals↗

PubMed2003

Canine ileal motor activity after a model of jejunoileal autotransplantation.

OBJECTIVE: To determine mechanisms by which extrinsic innervation to the jejunoileum controls ileal motility. SUMMARY BACKGROUND DATA: Small bowel transplantation is complicated by diarrhea and delayed gastric emptying, possibly secondary to altered motility. Ileal motility after small bowel transplantation is poorly characterized. METHODS: Motor activity was recorded from four dogs during fasting and after feeding small (64 Kcal) or large (256 Kcal) meals. Short-chain fatty acids known to induce unique ileal motor patterns were administered into the distal ileum during fasting. Dogs were studied before and after jejunoileal denervation simulating autotransplantation. RESULTS: After jejunoileal denervation, the ileal migrating motor complex (MMC) persisted but was no longer temporally coordinated with duodenal MMCs. Spontaneous giant migrating contractions occurred more frequently after denervation and more commonly originated proximally in the jejunum, but the velocity of migration did not differ. In contrast, the incidence and characteristics of spontaneous discrete clustered contractions (DCCs) did not differ. Short-chain fatty acids reproducibly initiated giant migrating contractions and discrete clustered contractions in the distal ileum without differences before and after denervation. Large but not small meals inhibited the ileal MMC after denervation. CONCLUSIONS: Extrinsic innervation and/or intrinsic neural continuity with the duodenum and/or colon control temporal coordination of ileal motility with the duodenum and modulate postprandial inhibition of fasting motility and presence of giant migrating contractions. These changes in motility patterns may prove important in mediating enteric dysfunction after small bowel transplantation.

Animals↗

PubMed1984

[On the description of migration using counting processes].

"In this paper modern counting process theory is used to describe the migrations of inhabitants of an area between the regions of this area, e.g. between the 11 federal states of the Federal Republic of Germany.... It is assumed that the migration behaviour of an individual inhabitant is described by a Markov process." The advantages of using counting process theory are discussed. (summary in ENG)

Demography↗

PubMed2005

Ca2+ influx through L-type Ca2+ channels controls the trailing tail contraction in growth factor-induced fibroblast cell migration.

Growth factor-induced cell migration underlies various physiological and pathological processes. The mechanisms by which growth factors regulate cell migration are not completely understood. Although intracellular elevation of Ca2+ is known to be critical in cell migration, the source of this Ca2+ elevation and the mechanism by which Ca2+ modulates this process in fibroblast cells are not well defined. Here we show that increase of cellular Ca2+ through Ca2+ influx, rather than Ca2+ release from intracellular stores, is essential for growth factor-induced fibroblast cell migration. Voltage-gated L-type Ca2+ channels, previously known to exist in excitable cells such as neurons and muscle cells, are shown here to be present in fibroblasts as well. Furthermore, these channels are responsible for the Ca2+ influx. L-type Ca2+ channel inhibitors block growth factor-induced Ca2+ influx and fibroblast cell migration. One mechanism by which Ca2+ signals control cell migration is to regulate the contraction of the trailing edge of migrating fibroblasts; this process is controlled by the small GTPase Rho in fast migrating cells such as leukocytes. Downstream of Ca2+, both calmodulin and myosin light chain kinase, but not calcineurin, are involved leading to phosphorylation of the myosin light chain at the trailing end. Thus, trailing edge contraction is critically regulated by Ca2+ influx through L-type Ca2+ channels in growth factor-induced fibroblast cell migration.

Animals↗

PubMed2005

Oxidative stress produced with cell migration increases synthetic phenotype of vascular smooth muscle cells.

Phenotypic modulation of vascular smooth muscle cells (VSMC) and reactive oxygen species (ROS) is important in vascular pathogenesis. Understanding how these factors relate to cell migration can improve design of therapeutic interventions to control vascular disease. We compared the proliferation, protein content and migration of cultured aortic VSMC from wild type (WT) versus transgenic mice (Tgp22phox), in which overexpression of p22phox was targeted to VSMC. Also, we compared H2O2 generation and expression of specific phenotypic markers of non-migrating with migrating WT versus Tgp22phox VSMC in an in vitro wound scratch model. Enhanced H2O2 production in Tgp22phox versus WT VSMC (p < 0.005) significantly correlated with increased protein content, proliferation, and migration. VSMC migrating across the wound edge produced more H2O2 than non-migrating VSMC (p < 0.05). The expression of synthetic phenotypic markers, tropomyosin 4 and myosin heavy chain embryonic (SMemb), was enhanced significantly, while the expression of contractile marker, smooth muscle alpha-actin, was reduced significantly in migrating versus non-migrating cells, and also in Tgp22phox versus WT (p < 0.005) VSMC. These results are consistent with increased production of ROS accelerating the switch from the contractile to the synthetic phenotype, characterized by increases in proliferation, migration, and expression of TM4 and SMemb and decreased alpha-actin.

Animals↗

PubMed2006

Laminin alpha2 chain-positive vessels and epidermal growth factor in lung neuroendocrine carcinoma: a model of a novel cooperative role of laminin-2 and epidermal growth factor in vessel neoplastic invasion and metastasis.

Capillaries expressing the laminin alpha2 chain in basement membranes may be considered early developing vessels in normal and neoplastic human tissues. Therefore, we investigated whether up-regulation of this extracellular matrix protein favors transendothelial migration of neoplastic cells and then metastasis. In lung small and large cell neuroendocrine carcinomas, which exhibit a stronger metastatic tendency among carcinomas, laminin alpha2 chain-positive vessels were more numerous than in carcinoid tumors and supraglottis, breast, and lung non-small cell carcinomas, suggesting a direct relationship between these vessels and metastasis. In vitro studies showed that epidermal growth factor (EGF) induced a more efficient migration of the AE-2 lung neuroendocrine carcinoma cell line through the purified laminin alpha2 chain rather than through the laminin beta1 chain and fibronectin. AE-2 cells constitutively expressed all EGF receptors and the alpha6beta1 integrin, which is one of the laminin alpha2 chain receptors. EGF up-regulated alpha6beta1 expression in several tumors. In this regard, we show that EGF increased the chemo-kinetic migration of AE-2 cells through EAHY endothelial monolayers, which was inhibited by the anti-alpha6 integrin chain monoclonal antibody. These data indicate that laminin alpha2 chain and alpha6beta1 may be mutually involved in EGF-dependent migration of AE-2 cells and that laminin alpha2 chain-positive vessels may favor metastasis of EGF-dependent tumors.

Antibodies, Monoclonal↗

PubMed1998

Co-migration of RAPD-PCR amplicons from Aeromonas hydrophila.

Random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) uses arbitrary primers and low stringency annealing conditions to amplify anonymous DNA fragments which are then depicted in agarose gels. RAPD-PCR fingerprints have been used for typing and differentiation of bacteria and, increasingly, for the study of genetic relationships between strains and species of microorganisms, plants and animals. The analysis of such fingerprints is based upon the assumption that co-migration of amplicons does not occur and that any given band contains a single amplicon. This report shows that co-migration of fragments of nearly identical size, but different nucleotide sequences, occurs between different isolates and within single RAPD-PCR bands from Aeromonas hydrophila. The possibility of the same phenomenon occurring for other prokaryotic or eukaryotic genomes argues for caution in the interpretation of RAPD-PCR fingerprints.

Aeromonas hydrophila↗

PubMedDate not supplied

[Analysis of the dynamics of transitions in Leptomonas collosoma spliced leader RNA and a possible role of gaps upon splicing].

In [1], the existence of two competing secondary structures at the 5'-end of Leptomonas collosoma spliced leader RNA was shown. On going from one steady-state secondary structure to the other, and vice versa, RNA passes through several states in which the chain gets tied up into nodes. If the transitions are reversible, the nodes are figurative both in the forth and back directions although the secondary structures that arise in this process are quite different, and the forth and back routs do not coincide. It is assumed that under certain conditions, the chain can get tied into the next node of series 5(2), which most likely will not get untied. This node is tightened around GG nucleotides. In this case, the migration of the gap is possible, as a result of which the chain can break. Independent experiments indicate that the RNA chain breaks just at this site upon splicing. Based on these results, a possible role of nodes in the enzymatic activity is discussed.

Animals↗

PubMed2006

Detection of kappa and lambda light chain monoclonal proteins in human serum: automated immunoassay versus immunofixation electrophoresis.

Recently, turbidimetric immunoassays for detecting and quantifying kappa and lambda free light chains (FLC) have become available and are promoted as being more sensitive than immunofixation electrophoresis (IFE) in detecting FLC monoclonal proteins. In this study, we assessed the ability of these turbidimetric assays to detect serum monoclonal proteins involving both free and heavy-chain-bound kappa and lambda light chains compared to standard immunofixation electrophoresis. Sera demonstrating a restricted band of protein migration (other than a definite M spike) by serum protein electrophoresis (SPE), which may represent early monoclonal proteins, were also examined. When compared to IFE, percent agreement, sensitivity, and specificity for the kappa-FLC and lambda-FLC were 94.6, 72.9, and 99.5% and 98.5, 91.4, and 99.7%, respectively, in detecting monoclonal proteins involving free and heavy-chain-bound light chains. The majority of sera (73.7%) demonstrating a restricted band of protein migration on SPE demonstrated abnormal IFE patterns suggestive of multiple myeloma or monoclonal gammopathy of unknown significance, but gave normal kappa/lambda FLC ratios using the turbidimetric immunoassays. In conclusion, the kappa and lambda FLC assays are significantly less sensitive (72.9 to 91.4%) than IFE, but specific in detecting serum monoclonal proteins. Moreover, the kappa/lambda ratio has little value in routine screening since the majority of sera with abnormal IFE patterns had normal kappa/lambda FLC ratios.

Antibodies, Monoclonal↗

PubMed1998

Evidence that oxidized proteins are substrates for N-terminal arginylation.

While the posttranslational N-terminal arginylation of proteins has been demonstrated in a variety of eukaryotic cells including neurons and their axons, the targets of the reaction are poorly understood. Several lines of evidence suggest that arginylation may be a cytoprotective mechanism used by cells to target oxidatively damaged (and thus potentially toxic) proteins for degradation. In the present experiments, we have begun to test this hypothesis by incubating oxidized test proteins in a rat brain extract capable of arginylating endogenous proteins. Bovine serum albumin, pancreatic ribonuclease-A and the A-chain of insulin were chosen as test proteins and either oxidized by metal catalyzed oxidation or purchased in their oxidized forms and incubated with the extract and [3H]Arg. SDS PAGE of the incubation product showed [3H]Arg migrating with the oxidized forms of BSA and RNase but not with the un-oxidized form of BSA. Following incubation with the oxidized A-chain of insulin, analysis of the [3H]product by SDS PAGE and HPLC showed co-migration of [3H]Arg with A-chain standards and amino acid sequencing showed [3H]Arg at the N-terminus of the A-chain of insulin. The data suggest that oxidative damage to a protein may be a signal for its N-terminal arginylation.

Animals↗