Turnover of the carboxy-terminal tyrosine of alpha-tubulin and means of reaching elevated levels of detyrosination in living cells

1987 ◽  
Vol 88 (2) ◽  
pp. 185-203
Author(s):  
J. Wehland ◽  
K. Weber
Keyword(s):  
Primary Cultures ◽  
Neuroblastoma Cells ◽  
Cell Types ◽  
Morphological Transformation ◽  
Alpha Tubulin ◽  
Tubulin Antibodies ◽  
Cell Processes ◽  
Carboxy Terminal ◽  
Almost All ◽  
Different Cell Types

Monoclonal antibodies specific for either the tyrosinated (Tyr) or the detyrosinated (Glu) form of alpha-tubulin were elicited with synthetic peptides spanning the carboxy-terminal sequences of the two forms. While almost all microtubules (MTs) are usually of the Tyr-tubulin type (Tyr-rich MTs) some MTs containing noticeable amounts of Glu-tubulin (Glu-rich MTs) were found in many but not all cell lines studied. Glu-rich MTs seemed absent from proliferating CHO and N115 neuroblastoma cells. When differentiation of these cells was initiated by the addition of forskolin for CHO, or by serum deprivation for N115, elevated levels of microtubular Glu-tubulin were observed. In differentiated N115 cells Glu-tubulin was restricted to MT of elongated cell processes and was not found in growth cones and many MT of the cell body. Elevated levels of Glu-tubulin were also characteristic of other differentiated cell types, including neurones and myotubes but were not found in glial cells, astrocytes and fibroblasts in the same primary cultures. Additional experiments suggested that the restricted distribution of Glu-tubulin is the result of MT subsets with different stabilities. Results with mitotic drugs indicated that detyrosination occurs on MTs rather than on soluble tubulin and that stabilization of MTs usually favours the detyrosination process. Evidence for a functional alpha-tubulin cycle involving an inherent carboxypeptidase and a recharging ligase was apparent in 3T3 cells from the preponderance of Glu-rich MTs induced by taxol treatment or the micro-injection of certain antibodies either protecting the detyrosinated form (Glu-tubulin antibodies) or inhibiting retyrosination (ligase antibodies). As the same treatment of CHO cells resulted in comparable arrays of Glu-rich MTs only when forskolin was also present, different cell types may differ in the level of active carboxypeptidase. The results are discussed in terms of possible functions of the tyrosination/detyrosination cycle of alpha-tubulin. While most results can be explained on the basis of ‘older’ and, consequently, more detyrosinated MTs, others raise the possibility that cyclic-AMP-dependent events and certain environmental influences known to induce either a morphological transformation or a differentiation event may influence the carboxypeptidase inherent in the alpha-tubulin cycle.

scholarly journals Complex regulation and functional versatility of mammalian alpha- and beta-tubulin isotypes during the differentiation of testis and muscle cells.

1988 ◽  
Vol 106 (6) ◽  
pp. 2023-2033 ◽  
Author(s):  
S A Lewis ◽  
N J Cowan
Keyword(s):  
Cell Types ◽  
Beta Tubulin ◽  
Specific Expression ◽  
Alpha Tubulin ◽  
Tubulin Isotypes ◽  
Tubulin Isotype ◽  
A Cell ◽  
Complex Regulation ◽  
Carboxy Terminal ◽  
Different Cell Types

In the accompanying paper (Gu, W., S. A. Lewis, and N. J. Cowan. 1988. J. Cell Biol. 106: 2011-2022), we report the generation of three antisera, each of which uniquely recognizes a different mammalian alpha-tubulin isotype, plus a fourth antibody that distinguishes between microtubules containing the tyrosinated and nontyrosinated form of the only known mammalian alpha-tubulin gene product that lacks an encoded carboxy-terminal tyrosine residue. These sera, together with five sera we raised that distinguish among the known mammalian beta-tubulin isotypes, have been used to study patterns of tubulin isotype-specific expression in muscle and testis, two tissues in which characteristic developmental changes are accompanied by dramatic rearrangements in microtubule structures. As in the case of cells in culture, there is no evidence to suggest that there is subcellular sorting of different tubulin isotypes among different kinds of microtubule, even in a cell type (the developing spermatid) that simultaneously contains such functionally distinct structures as the manchette and the flagellum. On the other hand, the patterns of expression of the various tubulin isotypes show marked and distinctive differences in different cell types and, in at least one case, evidence is presented for regulation at the translational or posttranslational level. The significance of these observations is discussed in terms of the existence of the mammalian alpha- and beta-tubulin multigene families.

scholarly journals Therapeutic Exosomes in Prognosis and Developments of Coronary Artery Disease

2021 ◽  
Vol 8 ◽  
Author(s):  
Ai-Qun Chen ◽  
Xiao-Fei Gao ◽  
Zhi-Mei Wang ◽  
Feng Wang ◽  
Shuai Luo ◽  
...  
Keyword(s):  
Coronary Artery Disease ◽  
Coronary Artery ◽  
Cell Types ◽  
Biological Functions ◽  
Stent Restenosis ◽  
Artery Disease ◽  
Almost All ◽  
Different Cell Types ◽  
Insight Into ◽  
Systematic Knowledge

Exosomes, with an diameter of 30~150 nm, could be released from almost all types of cells, which contain diverse effective constituent, such as RNAs, proteins, lipids, and so on. In recent years, exosomes have been verified to play an important role in mechanism, diagnosis, treatment, and prognosis of cardiovascular disease, especially coronary artery disease (CAD). Moreover, it has also been shown that exosomes derived from different cell types have various biological functions based on the cell stimulation and microenvironment. However, therapeutic exosomes are currently far away from clinical translation, despite it is full of hope. In this review, we summarize an update of the recent studies and systematic knowledge of therapeutic exosomes in atherosclerosis, myocardial infarction, and in-stent restenosis, which might provide a novel insight into the treatment of CAD and promote the potential clinical application of therapeutic exosomes.

scholarly journals Sodium Hydrogen Exchanger Regulatory Factor-1 (NHERF1) Regulates Fetal Membrane Inflammation

2020 ◽  
Vol 21 (20) ◽  
pp. 7747
Author(s):  
Ananth Kumar Kammala ◽  
Samantha Sheller-Miller ◽  
Enkhtuya Radnaa ◽  
Talar Kechichian ◽  
Hariharan Subramanian ◽  
...  
Keyword(s):  
Primary Cultures ◽  
Cell Types ◽  
Fetal Membrane ◽  
Regulatory Factor ◽  
Rna Targeting ◽  
Sodium Hydrogen Exchanger ◽  
Functional Relevance ◽  
Interfering Rna ◽  
Different Cell Types ◽  
Phosphate Buffered Saline

The fetal inflammatory response, a key contributor of infection-associated preterm birth (PTB), is mediated by nuclear factor kappa B (NF-kB) activation. Na+/H+ exchanger regulatory factor-1 (NHERF1) is an adapter protein that can regulate intracellular signal transduction and thus influence NF-kB activation. Accordingly, NHERF1 has been reported to enhance proinflammatory cytokine release and amplify inflammation in a NF-kB-dependent fashion in different cell types. The objective of this study was to examine the role of NHERF1 in regulating fetal membrane inflammation during PTB. We evaluated the levels of NHERF1 in human fetal membranes from term labor (TL), term not in labor (TNIL), and PTB and in a CD1 mouse model of PTB induced by lipopolysaccharide (LPS). Additionally, primary cultures of fetal membrane cells were treated with LPS, and NHERF1 expression and cytokine production were evaluated. Gene silencing methods using small interfering RNA targeting NHERF1 were used to determine the functional relevance of NHERF1 in primary cultures. NHERF1 expression was significantly (p < 0.001) higher in TL and PTB membranes compared to TNIL membranes, and this coincided with enhanced (p < 0.01) interleukin (IL)-6 and IL-8 expression levels. LPS-treated animals delivering PTB had increased levels of NHERF1, IL-6, and IL-8 compared to phosphate-buffered saline (PBS; control) animals. Silencing of NHERF1 expression resulted in a significant reduction in NF-kB activation and IL-6 and IL-8 production as well as increased IL-10 production. In conclusion, downregulation of NHERF1 increased anti-inflammatory IL-10, and reducing NHERF1 expression could be a potential therapeutic strategy to reduce the risk of infection/inflammation associated with PTB.

scholarly journals Predominance of the acylation route in the metabolic processing of exogenous sphingosine in neural and extraneural cells in culture

1999 ◽  
Vol 338 (1) ◽  
pp. 147-151 ◽  
Author(s):  
Laura RIBONI ◽  
Rosaria BASSI ◽  
Alessandro PRINETTI ◽  
Paola VIANI ◽  
Guido TETTAMANTI
Keyword(s):  
Cultured Cells ◽  
Primary Cultures ◽  
Calf Serum ◽  
Neuroblastoma Cells ◽  
Cell Types ◽  
Total Radioactivity ◽  
Metabolic Fate ◽  
Pulse Period ◽  
Human Neuroblastoma ◽  
Sphingosine 1 Phosphate

The metabolic fate of exogenous [3H]sphingosine was investigated in five types of cultured cells: primary cultures of neurons and astrocytes, murine and human neuroblastoma cells and human skin fibroblasts. After administration of 40 nM [3-3H]sphingosine into a cell-conditioned medium containing fetal calf serum, all cell types rapidly and efficiently incorporated the long-chain base in a time-dependent fashion. In all cases, after a 120 min pulse, the amount of radioactivity taken up was in the range of the endogenous sphingosine content. However, unchanged [3H]sphingosine represented only a very minor portion of the label incorporated into cells throughout the pulse period (10–120 min), indicating rapid and efficient sphingosine metabolism in these cells. Most of the [3H]sphingosine taken up was metabolically processed, either by degradation (assessed as 3H2O release into the culture medium) or by N-acylation (mainly to radioactive ceramide, sphingomyelin, neutral glycolipids and gangliosides). [3H]Sphingosine 1-phosphate accounted for less than 2% of the total radioactivity incorporated in all cases. Throughout the pulse period and in all cell types, 3H-labelled organic metabolites largely prevailed over 3H2O, indicating that N-acylation is the major metabolic fate of sphingosine in these cells under apparently physiological conditions. These results are consistent with the notion that sphingosine has a rapid turnover in the cells studied, and indicate that regulation of the basal level of this bioactive molecule occurs mainly through N-acylation.

scholarly journals Targeting GRK5 for Treating Chronic Degenerative Diseases

2021 ◽  
Vol 22 (4) ◽  
pp. 1920
Author(s):  
Federica Marzano ◽  
Antonio Rapacciuolo ◽  
Nicola Ferrara ◽  
Giuseppe Rengo ◽  
Walter J. Koch ◽  
...  
Keyword(s):  
Neurological Diseases ◽  
Cell Types ◽  
Cardiac Cells ◽  
Multifunctional Protein ◽  
Surface Receptors ◽  
Extracellular Signals ◽  
Degenerative Disorders ◽  
Almost All ◽  
Related Proteins ◽  
Different Cell Types

G protein-coupled receptors (GPCRs) are the largest family of cell-surface receptors and they are responsible for the transduction of extracellular signals, regulating almost all aspects of mammalian physiology. These receptors are specifically regulated by a family of serine/threonine kinases, called GPCR kinases (GRKs). Given the biological role of GPCRs, it is not surprising that GRKs are also involved in several pathophysiological processes. Particular importance is emerging for GRK5, which is a multifunctional protein, expressed in different cell types, and it has been found located in single or multiple subcellular compartments. For instance, when anchored to the plasma membrane, GRK5 exerts its canonical function, regulating GPCRs. However, under certain conditions (e.g., pro-hypertrophic stimuli), GRK5 translocates to the nucleus of cells where it can interact with non-GPCR-related proteins as well as DNA itself to promote “non-canonical” signaling, including gene transcription. Importantly, due to these actions, several studies have demonstrated that GRK5 has a pivotal role in the pathogenesis of chronic-degenerative disorders. This is true in the cardiac cells, tumor cells, and neurons. For this reason, in this review article, we will inform the readers of the most recent evidence that supports the importance of targeting GRK5 to prevent the development or progression of cancer, cardiovascular, and neurological diseases.

Platelet Toll-Like Receptors: Bridging Inflammation and Immunity

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. SCI-45-SCI-45
Author(s):  
Paul Kubes
Keyword(s):  
Immune Cells ◽  
Conflicts Of Interest ◽  
Detection System ◽  
Vascular Dysfunction ◽  
Cell Types ◽  
Neutrophil Extracellular Traps ◽  
Extracellular Traps ◽  
Almost All ◽  
Different Cell Types

Abstract Since the discovery that there is a series of pattern recognition receptors that allow the immune system to detect pathogens, there has been a lot of work to elucidate the signaling molecules that contribute to this detection system. Although the cell types involved would seem to be just as important, our understanding of which cells are critical remains less well explored. Using intravital imaging to visualize the different cell types, we were surprised to find that in almost all conditions that we examined, platelets were rapidly recruited to afflicted tissues. Under some conditions, platelets bound vascular macrophage including Kupffer cells and helped contain bacteria. These were instantaneous responses. At later times, platelets bound neutrophils and induced the production of neutrophil extracellular traps (NETs) that helped to catch bacteria as well as viruses but did induce some local vascular injury. In some scenarios, platelets bound endothelial cells and whether this was to wall off and contain infections or a hijacking of platelets by bacteria to induce vascular dysfunction and poor perfusion remains unclear. Visualizing platelets in sterile injury also revealed important contributions to helping recruit other immune cells that help to heal. The role of the platelet as an effector in infections and inflammation will be discussed. Disclosures No relevant conflicts of interest to declare.

scholarly journals Phosphoprotein enriched in astrocytes-15 is expressed in mouse testis and protects spermatocytes from apoptosis

Reproduction ◽  
2007 ◽  
Vol 133 (4) ◽  
pp. 743-751 ◽  
Author(s):  
S C Mizrak ◽  
F Renault-Mihara ◽  
M Párraga ◽  
J Bogerd ◽  
H J G van de Kant ◽  
...  
Keyword(s):  
Sertoli Cells ◽  
Meiotic Prophase ◽  
Primary Cultures ◽  
Cell Types ◽  
Mouse Testis ◽  
C Terminus ◽  
Specific Step ◽  
Different Cell Types

Phosphoprotein enriched in astrocytes (PEA-15) is a 15 kDa acidic serine-phosphorylated protein expressed in different cell types, especially in the CN. We initially detected the expression of PEA-15 in primary cultures of Sertoli cells. To assess the presence and localization of PEA-15 in the mouse testis, we studied the expression pattern of the PEA-15 protein by immunohistochemistry and mRNA byin situhybridization. Both the protein and the mRNA of PEA-15 were localized in the cytoplasm of Sertoli cells, all types of spermatogonia, and spermatocytes up till zygotene phase of the meiotic prophase. Subsequently, with ongoing development of the spermatocytes, the expression decreased and was very low in the cytoplasm of diplotene spermatocytes. To analyze the possible role of PEA-15 in the developing testis, null mutants for PEA-15 were examined. As the PEA-15 C terminus contains residues for ERK binding, we studied possible differences between the localization of the ERK2 protein in wild type (WT) andPEA-15−/−mice. In the WT testis, ERK2 was localized in the cytoplasm of Sertoli cells, B spermatogonia, preleptotene, leptotene, and zygotene spermatocytes, whereas in the KO testis, ERK2 was primarily localized in the nuclei of these cells and only little staining remained in the cytoplasm. Moreover, in PEA-15-deficient mice, significantly increased numbers of apoptotic spermatocytes were found, indicating an anti-apoptotic role of PEA-15 during the meiotic prophase. The increased numbers of apoptotic spermatocytes were not found at a specific step in the meiotic prophase.

scholarly journals Balance between S-nitrosylation and denitrosylation modulates myoblast proliferation independently of soluble guanylyl cyclase activation

2017 ◽  
Vol 313 (1) ◽  
pp. C11-C26 ◽  
Author(s):  
Aline M. S. Yamashita ◽  
Maryana T. C. Ancillotti ◽  
Luciana P. Rangel ◽  
Marcio Fontenele ◽  
Cicero Figueiredo-Freitas ◽  
...  
Keyword(s):  
Guanylyl Cyclase ◽  
Primary Cultures ◽  
Soluble Guanylyl Cyclase ◽  
Cell Types ◽  
Myoblast Fusion ◽  
No Synthase ◽  
Myoblast Proliferation ◽  
Cgmp Signaling ◽  
Different Cell Types

Nitric oxide (NO) contributes to myogenesis by regulating the transition between myoblast proliferation and fusion through cGMP signaling. NO can form S-nitrosothiols (RSNO), which control signaling pathways in many different cell types. However, neither the role of RSNO content nor its regulation by the denitrosylase activity of S-nitrosoglutathione reductase (GSNOR) during myogenesis is understood. Here, we used primary cultures of chick embryonic skeletal muscle cells to investigate whether changes in intracellular RSNO alter proliferation and fusion of myoblasts in the presence and absence of cGMP. Cultures were grown to fuse most of the myoblasts into myotubes, with and without S-nitrosocysteine (CysNO), 8-Br-cGMP, DETA-NO, or inhibitors for NO synthase (NOS), GSNOR, soluble guanylyl cyclase (sGC), or a combination of these, followed by analysis of GSNOR activity, protein expression, RSNO, cGMP, and cell morphology. Although the activity of GSNOR increased progressively over 72 h, inhibiting GSNOR (by GSNOR inhibitor – GSNORi – or by knocking down GSNOR with siRNA) produced an increase in RSNO and in the number of myoblasts and fibroblasts, accompanied by a decrease in myoblast fusion index. This was also detected with CysNO supplementation. Enhanced myoblast number was proportional to GSNOR inhibition. Effects of the GSNORi and GSNOR knockdown were blunted by NOS inhibition, suggesting their dependence on NO synthesis. Interestingly, GSNORi and GSNOR knockdown reversed the attenuated proliferation obtained with sGC inhibition in myoblasts, but not in fibroblasts. Hence myoblast proliferation is enhanced by increasing RSNO, and regulated by GSNOR activity, independently of cGMP production and signaling.

scholarly journals In vivo microtubules are copolymers of available beta-tubulin isotypes: localization of each of six vertebrate beta-tubulin isotypes using polyclonal antibodies elicited by synthetic peptide antigens.

1987 ◽  
Vol 105 (4) ◽  
pp. 1707-1720 ◽  
Author(s):  
M A Lopata ◽  
D W Cleveland
Keyword(s):  
Polyclonal Antibodies ◽  
Cell Types ◽  
Amino Acid Residues ◽  
Beta Tubulin ◽  
Tubulin Isotypes ◽  
Peptide Antigens ◽  
Tubulin Isotype ◽  
Carboxy Terminal ◽  
Different Cell Types

beta-Tubulin is encoded in the genomes of higher animals by a small multigene family comprising approximately seven functional genes. These genes produce a family of closely related, but distinct polypeptide isotypes that are distinguished principally by sequences within the approximately 15 carboxy-terminal amino acid residues. By immunizing rabbits with chemically synthesized peptides corresponding to these variable domain sequences, we have now prepared polyclonal antibodies specific for each of six distinct isotypes. Specificity of each antiserum has been demonstrated unambiguously by antibody binding to bacterially produced, cloned proteins representing each isotype and by the inhibition of such binding by preincubation of each antiserum only with the immunizing peptide and not with heterologous peptides. Protein blotting of known amounts of cloned, isotypically pure polypeptides has permitted accurate quantitative measurement of the amount of each beta-tubulin isotype present in the soluble and polymer forms in various cells, but has not revealed a bias for preferential assembly of any isotype. Localization of each isotype in three different cell types using indirect immunofluorescence has demonstrated that in vivo each class of microtubules distinguishable by light microscopy is assembled as copolymers of all isotypes expressed in a single cell.

scholarly journals p107 is active in the nucleolus in non-dividing human granulosa lutein cells

2000 ◽  
Vol 25 (3) ◽  
pp. 275-286 ◽  
Author(s):  
C Green ◽  
R Chatterjee ◽  
HH McGarrigle ◽  
F Ahmed ◽  
NS Thomas
Keyword(s):  
Cell Cycle ◽  
Cell Types ◽  
Quiescent State ◽  
Cdk Inhibitor ◽  
Proliferating Cells ◽  
Dividing Cells ◽  
E2f Transcription Factors ◽  
Almost All ◽  
Different Cell Types ◽  
P27 Kip1

Cells are maintained in a quiescent state by members of the retinoblastoma protein family, pRb and p130. Both are phosphoproteins and hypophosphorylated forms of pRb and p130 bind and repress the activity of E2F transcription factors, thereby preventing entry into the cell cycle. Mitogenic stimulation causes activation of cyclin dependent kinases (cdk) that phosphorylate both pRb and p130, thereby releasing E2F factors which stimulate the transcription of a number of genes that are required for DNA synthesis and for regulating the cell cycle. In non-dividing cells, cdks are maintained in an inactive state by cdk inhibitor proteins such as p27(Kip1). The aim of our study was to determine how E2F complexes are regulated during the differentiation of human primary granulosa lutein cells (GLC) of the corpus luteum (CL). The CL is formed in the ovary after ovulation at the terminal stage of folliculogenesis after completion of maturation and differentiation of Graafian follicles. As shown by flow cytometry GLC are not dividing, being predominantly in the G(0)/G(1) phase of the cell cycle and, consistent with this, they contain the cdk inhibitor protein, p27(Kip1), but not E2F-1 which is normally expressed only in proliferating cells. The GLC do express E2F-4, hypophosphorylated pRb, p130 forms 1 and 2 and, surprisingly, hypophosphorylated p107. p107 is normally present only in dividing cells where it regulates E2F activity during the cell cycle. These forms of pRb, p130 as well as p107, together with E2F-4 are all active in that they can bind an E2F DNA-binding site in a pull-down assay. Immunocytochemistry shows that these proteins are expressed in almost all GLC but have different sub-cellular distribution: p107 is concentrated in nucleoli, while p130 and E2F-4 show relatively even nuclear and cytoplasmic distributions. Both pRb and p130 have been implicated previously in repressing E2F activity in many different cell types during cell cycle arrest in G(0)/G(1). We conclude that p107 is active in human primary GLC but its nucleolar localisation would suggest that it represses ribosomal RNA synthesis rather than E2F activity.

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