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Results for “Post-translational modifications”

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PubMed1992

Lipid modification at the N terminus of photoreceptor G-protein alpha-subunit.

Myristate is a fatty acid (fourteen-carbon chain with no double bonds, C14:0) linked to the amino-terminal glycine of several proteins, including alpha-subunits of heterotrimeric (alpha/beta gamma) G proteins. We report here a novel modification at the N terminus of the alpha-subunit of the photoreceptor G protein transducin, T alpha, with heterogeneous fatty acids composed of laurate (C12:0), unsaturated C14:2 and C14:1 fatty acids, and a small amount (approximately 5%) of myristate. Both the GTPase activity of T alpha/T beta gamma and the T beta gamma-dependent ADP-ribosylation of T alpha catalysed by pertussis toxin were inhibited by the lauroylated and myristoylated N-terminal peptide of T alpha. The myristoylated peptide gave 50% inhibition at a 3.5 to approximately 4.5-fold lower concentration than the lauroylated peptide in each assay, indicating that the strength of the interaction between T alpha and T beta gamma is altered by heterogeneous fatty acids linked to T alpha. This suggests that a looser subunit interaction in transducin which is due to an abundance of N-linked fatty acids other than myristate would favour the rapid turnover and catalysis essential for the visual excitation in photoreceptor cells.

Adenosine Diphosphate Ribose↗

PubMed1992

Posttranslational modification and subcellular localization of the p12 capsid protein of herpes simplex virus type 1.

We have previously shown that the 12-kDa capsid protein (p12) of herpes simplex virus type 1 (HSV-1) is a gamma 2 (true late) gene product encoded by the UL35 open reading frame (D. S. McNabb and R. J. Courtney, J. Virol. 66:2653-2663, 1992). To extend the characterization of p12, we have investigated the posttranslational modifications and intracellular localization of the 12-kDa polypeptide. These studies have demonstrated that p12 is modified by phosphorylation at serine and threonine residues. In addition, analysis of p12 by acid-urea gel electrophoresis has indicated that the protein can be resolved into three components, designated p12a, p12b, and p12c. Using isotopic-labeling and alkaline phosphatase digestion experiments, we have determined that p12a and p12b are phosphorylated forms of the protein, and p12c is likely to represent the unphosphorylated polypeptide. The kinetics of phosphorylation was examined by pulse-chase radiolabeling, and these studies indicated that p12c can be completely converted into p12a and p12b following a 4-h chase. All three species of p12 were found to be associated with purified HSV-1 virions; however, p12b and p12c represented the most abundant forms of the protein within viral particles. We have also examined the intracellular localization of p12 by cell fractionation and indirect immunofluorescence techniques. These results indicated that p12 is predominantly localized in the nucleus of HSV-1-infected cells and appears to be restricted to specific regions within the nucleus.

Animals↗

PubMed2026

MARK1 suppresses infectious bursal disease virus replication via phosphorylating VP3.

Infectious bursal disease virus (IBDV) of the Birnaviridae family is a non-envelope, double-stranded RNA virus that encodes a VP3 protein with multiple functions, which controls viral genome replication, IFN-β production, and virus traffic in infected cells. Posttranslational modifications (PTMs), such as ubiquitination, of VP3 have been demonstrated for affecting its function and stability. To clarify the mechanism by which VP3 is regulated in IBDV infected cells, we focused on the phosphorylation of VP3. Mass spectrometry analysis identified that microtubule-affinity regulating kinases 1 (MARK1) was a kinase interacting protein of VP3. Inhibitory function of MARK1 in affecting viral replication was validated. We describe the phosphorylation event at the serine 130 (S130) and serine 163 (S163) residues of VP3 mediated by MARK1 via mass spectrometry analysis. Alanine replacement of the phosphorylation sites in VP3 significantly enhanced its RNA-binding activity. Additionally, the mutation of two serine residues led to remarkably improved in its polymerase-enhancing function. We then incorporated the two mutations to rescue recombinant IBDV. Viral growth curve analysis revealed that replication of mutant IBDV was significantly enhanced relative to wild type (WT) virus. In conclusion, we found that VP3 functions are specifically regulated by MARK1 mediated phosphorylation at S130 and S163 and that this regulation suppresses IBDV replication ultimately.

Infectious bursal disease virus↗

PubMed1992

Poly(ADP-ribosyl)ation: its role in inducible DNA amplification, and its correlation with the longevity of mammalian species.

In this paper, we review our recent work on poly(ADP-ribosyl)ation and its relationships with DNA amplification and with the life span of different mammalian species. Poly(ADP-ribosyl)ation is a eukaryotic posttranslational protein modification catalyzed by poly(ADP-ribose) polymerase (PARP; EC 2.4.2.30). This enzyme is strongly activated by DNA strand breaks and apparently plays a role in DNA repair and other cellular responses to DNA damage. Our data from two different cell culture systems for inducible DNA amplification strongly suggest that poly(ADP-ribosyl)ation acts as a negative regulatory factor in the DNA amplification induced by carcinogens. Furthermore, we could show a strong positive correlation between directly stimulated PARP activities in mononuclear leukocytes of 13 mammalian species and the species' maximal life spans. The hypothesis is raised that a higher poly(ADP-ribosyl)ation capacity of long-lived species might contribute to the efficient maintenance of genome integrity and stability over their longer life span. Finally, we could show that the selectively overexpressed PARP DNA-binding domain efficiently inhibits poly(ADP-ribosyl)ation in a transdominant manner. This molecular genetic approach should permit further interventional studies on biological role(s) of poly(ADP-ribosyl)ation without application of low-molecular-weight PARP inhibitors, thus avoiding any of their possible side effects.

Animals↗

PubMed1992

Possible involvement of cathepsin L in processing of rat liver hexokinase to eliminate mitochondria-binding ability.

A previously found proteinase possibly involved in the modification of hexokinase to eliminate the mitochondria-binding ability without appreciable change in the catalytic activity (called hexokinase-processing enzyme hereafter), was purified by sequential chromatographies from rat liver and its properties were examined. The hexokinase-processing enzyme had carbohydrate moieties as evidenced by adsorption on immobilized concanavalin A, and had a molecular weight of about 23,000 as estimated by SDS-PAGE and gel filtration chromatography. Benzyloxycarbonyl-phenylalanyl-L-arginine-4-methylcoumaryl-7-amide (Z-Phe-Arg-MCA)-hydrolyzing activity was co-purified with this processing activity throughout the purification, while the hydrolyzing activity for benzyloxycarbonyl-L-arginyl-L-arginine-4-methylcoumaryl-7-amide (Z-Arg-Arg-MCA) was not. The processing activity, as well as Z-Phe-Arg-MCA hydrolyzing activity, was highly sensitive to cysteine proteinase inhibition, for example, by leupeptin and N-[N-3-(trans-carboxirane-2-carbonyl)-L-leucyl]agmatine (E-64). Furthermore, the enzyme preparation reacted with an antibody against cathepsin L purified from rat kidney. These results indicated that cathepsin L may be involved in the above-mentioned processing of hexokinase.

Animals↗

PubMed2026

Seed-type vacuolar processing enzymes recognize the 619th asparagine residue to posttranslationally cleave the HMW-GS 1Dy10-m619SN allele.

High molecular weight glutenin subunits (HMW-GSs) are critical grain storage proteins in wheat, which govern its unique processing quality. A HMW-GS 1Dy10 allele variant (1Dy10-m619SN), carrying a serine-to-asparagine substitution at the 619th residue, undergoes partial posttranslational cleavage. This modification leads to improved cookie-making quality. However, the enzymes mediating this cleavage remain unknown. In this study, we identified vacuolar processing enzymes (VPEs) as candidates for 1Dy10-m619SN processing using TurboID-based proximity labeling and RNA-seq analysis. In vitro cleavage assays confirmed that VPEs catalyzed 1Dy10-m619SN cleavage. Phylogenic analysis revealed that there are two seed-type VPEs in wheat, TaVPEI and TaVPEII, with TaVPEI being further subdivided into TaVPEI-1, TaVPEI-2, and TaVPEI-3. Despite sharing conserved catalytic domains, these isoforms display distinct temporal expression patterns, with TaVPEI-1 expression showing the strongest correlation with the posttranslational cleavage of 1Dy10-m619SN. TaVPEI-1 protein is localized to the vacuole, the well-known deposition site for HMW-GSs. Overexpression of TaVPEI-1 in wheat enhances the 1Dy10-m619SN cleavage. Collectively, these findings demonstrate that the seed-type VPEs in wheat are responsible for the posttranslational cleavage of 1Dy10-m619SN, which provides new insights into the molecular basis of wheat's unique processing quality.

Triticum↗

PubMed1992

Protein phosphorylation regulates the mouse sperm acrosome reaction induced by the zona pellucida.

Recently, the ligand-receptor signal transduction mechanism has been implicated in mediating the zona pellucida (ZP)-induced acrosome reaction. Little is known about the role of protein phosphorylation in this specific event. We examine whether modification of protein phosphorylation and dephosphorylation affects the kinetics of the acid-solubilized ZP-induced acrosome reaction of mouse sperm. Mouse epididymal sperm were incubated in modified Krebs-Ringer bicarbonate medium for a period of 90 to 120 min and then treated with 2 acid-solubilized ZP/microliters for an additional 60 min. The chlortetracycline fluorescence assay was used to monitor the acrosome reaction. Capacitated sperm were inhibited from undergoing acid-solubilized ZP-induced acrosome reaction in the presence of an inhibitor of cyclic nucleotide-dependent protein kinase, H8; activators of the Ca(2+)- and phospholipid-dependent protein kinase (protein kinase C); an inhibitor of phosphatases 1 and 2A, okadaic acid; or an inhibitor of protein tyrosine kinases, genistein. The addition of inhibitors of protein kinase C, such as staurosporine, H7, and protein kinase C [19-36] pseudosubstrate, inhibited the phorbol ester-dependent inhibition of the acid-solubilized ZP-induced acrosome reaction. The present study suggests that protein phosphorylation and dephosphorylation play a regulatory role in the process of the ZP-induced acrosome reaction.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗

PubMed2026

A posttranslational modification of fimbriae drives pathogenicity in Klebsiella pneumoniae.

Antimicrobial resistance is a severe public health burden. Especially concerning are multidrug resistant (MDR) infections, which restrict treatment options and significantly increase mortality risk. A major cause of MDR infections worldwide is carbapenem-resistant Klebsiella pneumoniae (CRKp). The predominant CRKp sequence type worldwide is ST258. However, the factors underlying ST258's epidemic success are not well defined. Genomic analyses of clinical isolates of CRKp have found that the two-component regulatory system CrrAB is a genomic feature of ST258, suggesting that it may contribute to its global dominance. Despite this, the molecular details underpinning CrrAB's contribution to ST258 Kp biology and pathogenicity are poorly understood. We used RNA-sequencing to identify the regulon of CrrA and found that CrrAB induces the expression of a gene, encoding Crr-regulated fimbriae modifying protein (CfmP), that is essential for pathogenesis driven by this two-component system. We performed mass spectrometry analyses of fimbriae purified from Kp expressing or lacking cfmP and found that CfmP induces a novel oxidation to a histidine residue in the major pilin subunit of fimbriae, FimA. We demonstrate that this oxidation significantly increases host cell adhesion and high bacterial loads within the host. CrrAB also drives high antibiotic resistance in CRKp. Thus, our results place CrrAB at the intersection of pathogenicity and antibiotic resistance supporting its function as an important regulatory system driving the global dominance of ST258.

Klebsiella pneumoniae↗

PubMed2025

Characterization and chemoproteomic profiling of protein O-GlcNAcylation in SOD1-G93A mouse model.

BACKGROUND: Amyotrophic lateral sclerosis (ALS) is a devastating motor neuron disease. Protein O-linked β-N-acetylglucosamine (O-GlcNAc) modification has been found to affect the processing of several important proteins implicated in ALS. However, the overall level and cellular localization of O-GlcNAc during ALS progression are incompletely understood, and large-scale profiling of O-GlcNAcylation sites in this context remains unexplored. METHODS: By using immunostaining analysis and chemoenzymatic labeling-based quantitative chemoproteomics, we assayed O-GlcNAcylation dynamics of lumbar spinal cords from SOD-G93A mice and their non-transgenic (NTG) littermates, the most widely used animal model for studying ALS pathogenesis. RESULTS: We discovered that the global O-GlcNAcylation was significantly reduced at the disease end stage. Correlatively, a great increase of OGA was observed. Immunohistochemistry and immunofluorescence analysis showed a higher proportion of O-GlcNAc-positive neurons in the NTG group, while O-GlcNAc colocalization with astrocytes/microglia was elevated in SOD1-G93A mice. Moreover, we reported the identification of 568 high-confidence O-GlcNAc sites from end-stage SOD1-G93A and NTG mice. Of the 568 sites, 226-many of which occurred on neuronal function and structure-related proteins-were found to be dynamically regulated. CONCLUSION: These data provide a valuable resource for dissecting the functional role of O-GlcNAcylation in ALS and shed light on promising therapeutic avenues for ALS. The chemoenzymatic labeling-based chemoproteomic approach is applicable for probing O-GlcNAc dynamics in various pathological processes.

Animals↗

PubMed1992

Identification of a novel type of processing sites in the precursor for the sea anemone neuropeptide Antho-RFamide (<Glu-Gly-Arg-Phe-NH2) from Anthopleura elegantissima.

Neuropeptides are synthesized as large precursor proteins that undergo posttranslational cleavages and modifications to produce bioactive peptides. Here, we have cloned two closely related precursor proteins for the sea anemone neuropeptide Antho-RFamide (<Glu-Gly-Arg-Phe-NH2) from Anthopleura elegantissima. The first precursor (435 amino acids long) contains 13 copies of immature Antho-RFamide (Gln-Gly-Arg-Phe-Gly) and nine other, Antho-RFamide-related neuropeptide sequences that are in the C-terminal part of the protein. The second precursor (429 amino acid residues) harbors 14 copies of immature Antho-RFamide and eight other related peptide sequences. Each copy of Antho-RFamide or Antho-RFamide-related peptide is followed, at its C-terminal side, by a single Arg residue, which is an established signal for posttranslational cleavage. At the N terminus of each Antho-RFamide sequence, however, basic residues are lacking, and instead one or more acidic residues occur. These acidic residues are the cleavage sites for a new type of processing enzyme occurring in neurons. This enzyme could either be an amino- or endopeptidase hydrolyzing at the C-terminal side of Asp or Glu residues. The N-terminal regions of the two precursor proteins harbor eight copies of the putative neuropeptide sequence Pro-Gln-Phe-Trp-Lys-Gly-Arg-Phe-Ser and three additional, closely related sequences. The total number of all established and putative neuropeptides that may be cleaved from the precursors is 33. Thus, the Antho-RFamide precursors beong to the most complex peptide precursor proteins known so far.

Amino Acid Sequence↗

PubMed1992

Cell cycle modification during the transitions between meiotic M-phases in mouse oocytes.

When metaphase II-arrested mouse oocytes (M II) are activated very soon after ovulation, they respond abortively by second polar body extrusion followed by another metaphase arrest (metaphase III, M III; Kubiak, 1989). The M II/M III transition resembles the natural transition between the first and second meiotic metaphases (M I/M II). We observed that a similar sequence of events takes place during these two transitions: after anaphase, a polar body is extruded, the microtubules of the midbody disappear rapidly and a new metaphase spindle forms. The MPM-2 monoclonal antibody (which reacts with phosphorylated proteins associated with the centrosome during M-phase) stains discrete foci of peri-centriolar material only in metaphase arrested oocytes; during both transitional periods, a diffuse staining is observed, suggesting that these centrosomal proteins are dephosphorylated, as in a normal interphase. However, the chromosomes always remain condensed and an interphase network of microtubules is never observed during the transitional periods. Incorporation of 32P into proteins increases specifically during the transitional periods. Pulse-chase experiments, after labeling of the oocytes in M phase with 32P, showed that a 62 kDa phosphoprotein band disappears at the time of polar body extrusion. Histone H1 kinase activity (which reflects the activity of the maturation promoting factor) drops during both transitional periods to the level characteristic of interphase and then increases when the new spindle forms. Both the M I/M II and M II/M III transitions require protein synthesis as demonstrated by the effect of puromycin. These results suggest that the two M-phase/M-phase transitions are probably driven by the same molecular mechanism.

Animals↗

PubMed2024

Integrative Multi-PTM Proteomics Reveals Dynamic Global, Redox, Phosphorylation, and Acetylation Regulation in Cytokine-Treated Pancreatic Beta Cells.

Studying regulation of protein function at a systems level necessitates an understanding of the interplay among diverse posttranslational modifications (PTMs). A variety of proteomics sample processing workflows are currently used to study specific PTMs but rarely characterize multiple types of PTMs from the same sample inputs. Method incompatibilities and laborious sample preparation steps complicate large-scale physiological investigations and can lead to variations in results. The single-pot, solid-phase-enhanced sample preparation (SP3) method for sample cleanup is compatible with different lysis buffers and amenable to automation, making it attractive for high-throughput multi-PTM profiling. Herein, we describe an integrative SP3 workflow for multiplexed quantification of protein abundance, cysteine thiol oxidation, phosphorylation, and acetylation. The broad applicability of this approach is demonstrated using cell and tissue samples, and its utility for studying interacting regulatory networks is highlighted in a time-course experiment of cytokine-treated β-cells. We observed a swift response in the global regulation of protein abundances consistent with rapid activation of JAK-STAT and NF-κB signaling pathways. Regulators of these pathways as well as proteins involved in their target processes displayed multi-PTM dynamics indicative of complex cellular response stages: acute, adaptation, and chronic (prolonged stress). PARP14, a negative regulator of JAK-STAT, had multiple colocalized PTMs that may be involved in intraprotein regulatory crosstalk. Our workflow provides a high-throughput platform that can profile multi-PTMomes from the same sample set, which is valuable in unraveling the functional roles of PTMs and their co-regulation.

Proteomics↗

PubMed1992

Analysis of the glycosylation and phosphorylation of the alpha-subunit of the lysosomal enzyme, beta-hexosaminidase A, by site-directed mutagenesis.

The glycosylation and subsequent phosphorylation of mannose residues is a pivotal modification during the biosynthesis of lysosomal enzymes. We have identified the sites of N-linked glycosylation and oligosaccharide phosphorylation on the alpha-subunit of beta-hexosaminidase and have determined the influence of the oligosaccharides on the folding and transport of the enzyme. The potential glycosylation sequences, either singly or in combination, were eliminated through site-directed mutagenesis of the cDNA. By expression of the mutant cDNAs in COS-1 cells, each of the three glycosylation sites on the alpha-subunit was found to be modified by an oligosaccharide. One of the three oligosaccharides was the preferred site of phosphorylation. The absence of any individual oligosaccharide did not diminish the expression of the catalytic activity associated with the alpha-chain, implying proper folding and assembly of subunits. A profound effect was observed, however, when all three oligosaccharides were absent. The unglycosylated alpha-subunit, resulting from genetic alteration of all three glycosylation sites or synthesis of the wild-type protein in the presence of tunicamycin, was catalytically inactive. It was found to be improperly folded into an insoluble aggregate, linked through inappropriate disulfide bonds. The unglycosylated protein was trapped in the lumen of the endoplasmic reticulum and was found in a complex with the Ig heavy chain-binding protein, BiP. The properties of the nonglycosylated, misfolded alpha-subunit were similar to some mutant alpha-subunits in Tay-Sachs disease patients. The results indicate that the oligosaccharides are essential, although not in a site-specific manner, for proper folding and cellular transport of the alpha-subunit.

Amino Acid Sequence↗

PubMed1992

Gag proteins of the highly replicative MN strain of human immunodeficiency virus type 1: posttranslational modifications, proteolytic processings, and complete amino acid sequences.

The MN strain of human immunodeficiency virus type 1 was grown in H9 cells, concentrated by centrifugation, and disrupted, and proteins were purified by reversed-phase high-pressure liquid chromatography. Complete amino acid sequences were determined for the mature Gag proteins, showing natural proteolytic cleavage sites and the order of proteins (p17-p24-p2-p7-p1-p6) in the Gag precursors. At least two sequence variants of p24 and eight sequence variants of p17 were detected. The two most abundant variants of p24 and p17 represented at least 50% +/- 5% and 20% +/- 5% of their totals, respectively. These data suggest heterogeneity in the virus population, with 50% of the total virus containing the most abundant forms of p17 and p24 and 20% of the virus containing the second most abundant forms. The Gag precursors of these suggested viruses differ from each other by only 3 amino acid residues but differ from the precursors predicted by the published MN proviral DNA sequence by 10 residues. Electrospray ionization mass spectrometry analysis of the purified p24 forms showed that the measured molecular weight of the protein was 200 +/- 50 atomic mass units greater than the calculated molecular weight. The source of additional mass for the p24 forms was not determined, but the observation is consistent with previous suggestions that the protein is phosphorylated. Greater than 98% of the total recovered p17 was myristylated at the N-terminal glycine residue, and the measured molecular weights (as determined by electrospray ionization mass spectrometry) of the most abundant forms were within 3 atomic mass units of the calculated molecular weights (15,266).

Amino Acid Sequence↗

PubMed1992

Myristyl acylation of the tumor necrosis factor alpha precursor on specific lysine residues.

NH2-terminal glycine myristyl acylation is a cotranslational modification that affects both protein localization and function. However, several proteins that lack NH2-terminal glycine residues, including the interleukin 1 (IL-1) precursors, also contain covalently linked myristate. To date, the site(s) of acylation of these proteins has not been determined. During an evaluation of IL-1 acylation, it was observed that [3H]myristate-labeled human monocyte lysates contained a prominent 26-kD myristylated protein, which was identified as the tumor necrosis factor alpha (TNF) precursor protein on the basis of specific immune precipitation. Radioimmunoprecipitates from the supernates of labeled monocytes indicated that the processed or mature 17-kD form of TNF does not contain myristate, suggesting that the site of acylation occurs within the 76-amino acid propiece of the precursor molecule. As the TNF precursor does not contain an NH2-terminal glycine, we hypothesized that myristyl acylation occurs on the N-epsilon-NH2 groups of lysine, of which two are present in the propiece (K19K20). Synthetic peptides were designed to include all seven lysine residues present within the entire 26-kD TNF precursor, and used in an in vitro myristyl acylation assay containing peptide, myristyl-CoA, and monocyte lysate as a source of enzyme. Analysis of reaction products by reverse phase high performance liquid chromatography and gas phase sequencing demonstrated the exclusive myristyl acylation of K19 and K20, consistent with the presence in monocytes of a specific lysyl N-epsilon-NH2-myristyl transferase activity. The acylated lysine residues are located immediately downstream from a hydrophobic, probable membrane-spanning segment of the propiece. Specific myristyl acylation of the TNF propiece may facilitate membrane insertion or anchoring of this critical inflammatory mediator.

Acylation↗

PubMed1992

Expression and modification of Hox 2.1 protein in mouse embryos.

A polyclonal antibody, alpha Hox 2.1a, has been generated and used to immunolocalize Hox 2.1 protein in mouse embryos. Protein is present in nuclei of all tissues previously shown to express Hox 2.1 RNA. In addition, protein is seen in somites and proximal regions of the limb buds, tissues in which Hox 2.1 RNA expression was not clearly detected previously by in situ hybridization. At the 7 somite stage, protein is detectable in the neural tube up to the level of somite 1, but later retracts to a more posterior position. Immunoblot, in vitro translation, and immunoprecipitation experiments were carried out to characterize the Hox 2.1 protein. The results show that the Hox 2.1 gene produces at least two related phosphorylated proteins present in different proportions in different tissues.

Animals↗

PubMed1992

Control of biosynthesis and post-transcriptional modification of the progesterone receptor.

The rabbit progesterone receptor undergoes dual regulation at the level of transcription: positive by estrogens and negative by progestins. The two aspects of this regulation are mediated by a single intragenic estrogen-responsive element. Estrogen receptor binding to this element has been demonstrated but progestin down-regulation does not proceed through DNA binding of the progesterone receptor. This result suggests some kind of protein-protein interaction--direct or indirect--between estrogen and progesterone receptors. At the post-transcriptional level, the progesterone receptor undergoes a hormone-dependent hyperphosphorylation of serine residues localized in the N-terminal region. Studies of progesterone receptor mutants have determined the influence of the different receptor domains in the phosphorylation mechanism. A casein kinase copurifies with the receptor. The role of this phosphorylation remains to be determined.

Animals↗

PubMed1992

Cultured aortic smooth muscle cells from newborn and adult rats show distinct cytoskeletal features.

It is well known that arterial smooth muscle cells (SMC) of adult rats, cultured in a medium containing fetal calf serum (FCS), replicate actively and lose the expression of differentiation markers, such as desmin, smooth muscle (SM) myosin and alpha-SM actin. We report here that compared to freshly isolated cells, primary cultures of SMC from newborn animals show no change in the number of alpha-SM actin containing cells and a less important decrease in the number of desmin and SM myosin containing cells than that seen in primary cultures of SMC from adult animals; moreover, contrary to what is seen in SMC cultured from adult animals, they show an increase of alpha-SM actin mRNA level, alpha-SM actin synthesis and expression per cell. These features are partially maintained at the 5th passage, when the cytoskeletal equipment of adult SMC has further evolved toward dedifferentiation. Cloned newborn rat SMC continue to express alpha-SM actin, desmin and SM myosin at the 5th passage. Thus, newborn SMC maintain, at least in part, the potential to express differentiated features in culture. Heparin has been proposed to control proliferation and differentiation of arterial SMC. When cultured in the presence of heparin, newborn SMC show an increase of alpha-SM actin synthesis and content but no modification of the proportion of alpha-SM actin total (measured by Northern blots) and functional (measured by in vitro translation in a reticulocyte lysate) mRNAs compared to control cells cultured for the same time in FCS containing medium. This suggests that heparin action is exerted at a translational or post-translational level. Cultured newborn rat aortic SMC furnish an in vitro model for the study of several aspects of SMC differentiation and possibly of mechanisms leading to the establishment and prevention of atheromatous plaques.

Actins↗