scholarly journals Endoproteolytic Processing of RhoA by Rce1 Is Required for the Cleavage of RhoA by Yersinia enterocolitica Outer Protein T

2006 ◽  
Vol 74 (3) ◽  
pp. 1712-1717 ◽  
Author(s):  
Florian Fueller ◽  
Martin O. Bergo ◽  
Stephen G. Young ◽  
Klaus Aktories ◽  
Gudula Schmidt
Keyword(s):  
Amino Acids ◽  
Proteolytic Activity ◽  
Rho Gtpases ◽  
Mammalian Cells ◽  
Living Cells ◽  
Type I ◽  
Rho Proteins ◽  
Carboxyl Methylation ◽  
Carboxyl Terminal ◽  
Caax Motif

ABSTRACT The bacterial toxin Yersinia outer protein T (YopT) is a cysteine protease that cleaves Rho GTPases immediately upstream of a carboxyl-terminal isoprenylcysteine. By clipping off the lipid anchor, YopT releases Rho GTPases from membranes, resulting in rounding up of mammalian cells in culture. The proteolytic activity of YopT depends on the isoprenylation of the cysteine within the carboxyl-terminal CaaX motif, a reaction carried out by geranylgeranyltransferase type I. The CaaX motif (where “a” indicates aliphatic amino acids) of Rho proteins undergoes two additional processing steps: endoproteolytic removal of the last three amino acids (i.e., -aaX) by Rce1 (Ras-converting enzyme 1) and methylation of the geranylgeranylcysteine by Icmt (isoprenylcysteine carboxyl methyltransferase). In in vitro experiments, RhoA retaining -aaX cannot be cleaved by YopT. Nothing is known, however, about the influence of Rce1-mediated removal of -aaX on the activity of YopT in living cells. We hypothesized that Rce1-deficient mouse fibroblasts, in which the geranylgeranylated Rho proteins are not endoproteolytically processed, would be resistant to YopT. Indeed, this was the case. Microinjection of recombinant YopT into Rce1-deficient fibroblasts had no impact on the subcellular localization of RhoA and no impact on cell morphology. To determine if carboxyl methylation is also required for YopT action, we microinjected YopT into Icmt-deficient fibroblasts. In contrast to the results with Rce1-deficient cells, YopT cleaved RhoA and caused rounding up of the Icmt-deficient cells. Our data demonstrate that Rce1-mediated removal of -aaX from isoprenylated Rho GTPases is required for the proteolytic activity of YopT in living cells, whereas carboxyl methylation by Icmt is not.

scholarly journals Postprenylation CAAX Processing Is Required for Proper Localization of Ras but Not Rho GTPases

2005 ◽  
Vol 16 (4) ◽  
pp. 1606-1616 ◽  
Author(s):  
David Michaelson ◽  
Wasif Ali ◽  
Vi K. Chiu ◽  
Martin Bergo ◽  
Joseph Silletti ◽  
...  
Keyword(s):  
Posttranslational Modifications ◽  
Protein Trafficking ◽  
Rho Gtpases ◽  
Ras Proteins ◽  
Rho Proteins ◽  
Carboxyl Methylation ◽  
C Terminus ◽  
Individual Contributions ◽  
The Individual ◽  
And Function

The CAAX motif at the C terminus of most monomeric GTPases is required for membrane targeting because it signals for a series of three posttranslational modifications that include isoprenylation, endoproteolytic release of the C-terminal– AAX amino acids, and carboxyl methylation of the newly exposed isoprenylcysteine. The individual contributions of these modifications to protein trafficking and function are unknown. To address this issue, we performed a series of experiments with mouse embryonic fibroblasts (MEFs) lacking Rce1 (responsible for removal of the –AAX sequence) or Icmt (responsible for carboxyl methylation of the isoprenylcysteine). In MEFs lacking Rce1 or Icmt, farnesylated Ras proteins were mislocalized. In contrast, the intracellular localizations of geranylgeranylated Rho GTPases were not perturbed. Consistent with the latter finding, RhoGDI binding and actin remodeling were normal in Rce1- and Icmt-deficient cells. Swapping geranylgeranylation for farnesylation on Ras proteins or vice versa on Rho proteins reversed the differential sensitivities to Rce1 and Icmt deficiency. These results suggest that postprenylation CAAX processing is required for proper localization of farnesylated Ras but not geranygeranylated Rho proteins.

A 33 kDa protein with sequence homology to the ‘laminin binding protein’ is associated with the cytoskeleton in hydra and in mammalian cells

1991 ◽  
Vol 100 (4) ◽  
pp. 789-797 ◽  
Author(s):  
E. Keppel ◽  
H.C. Schaller
Keyword(s):  
Amino Acids ◽  
Monoclonal Antibody ◽  
Mammalian Cells ◽  
Binding Protein ◽  
Cell Fractionation ◽  
Calculated Molecular Mass ◽  
Intracellular Location ◽  
Carboxyl Terminal ◽  
Laminin Binding Protein ◽  
Laminin Binding

In hydra and in mammalian cells the monoclonal antibody V recognises an epitope which colocalises with cytoskeletal structures. Using this antibody for expression screening, a cDNA clone (955 bp) was isolated from hydra, which covers an open reading frame for a protein of 294 amino acids with a calculated molecular mass of 32.8 kDa. Northern blot analysis of hydra RNA resulted in a single mRNA species of 1.2 kb, and primer extension experiments proved this to be the full length message. 218 residues at the amino terminus of the hydra protein show extensive homology (73.5%) to a human protein designated ‘laminin binding protein’. The carboxyl-terminal 76 amino acids possess no significant similarity (20%). The monoclonal antibody V, which recognises an epitope in this carboxyl-terminal part, reacts in Western blots, both in hydra and in mammalian cells, with a protein of 33 kDa and not with the 45 kDa ‘laminin binding protein’. The 33 kDa protein is not extracellular or transmembrane, but has a strictly intracellular location as indicated by its amino acid sequence and by immunocytochemical and cell fractionation studies. In non-dividing mammalian cells the 33 kDa protein colocalises with filamentous structures; in dividing cells it dissociates from it and concentrates centrally. Presence of the SPLR-sequence, which is the consensus phosphorylation motif for the p34cdc2 kinase, links this 33 kDa protein to events occurring during the cell cycle.

scholarly journals Rce1 deficiency accelerates the development of K-RAS–induced myeloproliferative disease

Blood ◽  
2006 ◽  
Vol 109 (2) ◽  
pp. 763-768 ◽  
Author(s):  
Annika M. Wahlstrom ◽  
Briony A. Cutts ◽  
Christin Karlsson ◽  
Karin M. E. Andersson ◽  
Meng Liu ◽  
...  
Keyword(s):  
Amino Acids ◽  
Bone Marrow ◽  
Bone Marrow Cells ◽  
Hematopoietic Cells ◽  
Converting Enzyme ◽  
Myeloproliferative Disease ◽  
Ras Proteins ◽  
Carboxyl Terminal ◽  
Caax Motif

Abstract The RAS proteins undergo farnesylation of a carboxyl-terminal cysteine (the “C” of the carboxyl-terminal CaaX motif). After farnesylation, the 3 amino acids downstream from the farnesyl cysteine (the -aaX of the CaaX motif) are released by RAS-converting enzyme 1 (RCE1). We previously showed that inactivation of Rce1 in mouse fibroblasts mislocalizes RAS proteins away from the plasma membrane and inhibits RAS transformation. Therefore, we hypothesized that the inactivation of Rce1 might inhibit RAS transformation in vivo. To test this hypothesis, we used Cre/loxP recombination techniques to simultaneously inactivate Rce1 and activate a latent oncogenic K-RAS allele in hematopoietic cells in mice. Normally, activation of the oncogenic K-RAS allele in hematopoietic cells leads to rapidly progressing and lethal myeloproliferative disease. Contrary to our hypothesis, the inactivation of Rce1 actually increased peripheral leukocytosis, increased the release of immature hematopoietic cells into the circulation and the infiltration of cells into liver and spleen, and caused mice to die more rapidly. Moreover, in the absence of Rce1, splenocytes and bone marrow cells expressing oncogenic K-RAS yielded more and larger colonies when grown in methylcellulose. We conclude that the inactivation of Rce1 worsens the myeloproliferative disease caused by oncogenic K-RAS.

scholarly journals A Short Peptide Derived from the ZorO Toxin Functions as an Effective Antimicrobial

Toxins ◽  
2019 ◽  
Vol 11 (7) ◽  
pp. 392 ◽  
Author(s):  
Otsuka ◽  
Ishikawa ◽  
Takahashi ◽  
Masuda
Keyword(s):  
Amino Acids ◽  
Antimicrobial Peptides ◽  
Mammalian Cells ◽  
Membrane Damage ◽  
Type I ◽  
Gram Positive ◽  
Short Peptide ◽  
E Coli ◽  
Gram Positive Bacteria ◽  
Endogenous Expression

Antimicrobial peptides are potential molecules for the development of novel antibiotic agents. The ZorO toxin of a type I toxin–antitoxin system in Escherichia coli O157:H7 is composed of 29 amino acids and its endogenous expression inhibits E. coli growth. However, little is known about its inhibitory mechanism. In this study, we demonstrate that the ZorO localized in the inner membrane affects the plasma membrane integrity and potential when expressed in E. coli cells, which triggers the production of cytotoxic hydroxyl radicals. We further show that five internal amino acids (Ala–Leu–Leu–Arg–Leu; ALLRL) of ZorO are necessary for its toxicity. This result prompted us to address the potential of the synthetic ALLRL peptide as an antimicrobial. Exogenously-added ALLRL peptide to Gram-positive bacteria, Staphylococcus aureus and Bacillus subtilis, and a fungus, Candida albicans, trigger cell membrane damage and exhibit growth defect, while having no effect on Gram-negative bacterium, E. coli. The ALLRL peptide retains its activity under the physiological salt concentrations, which is in contrast to natural antimicrobial peptides. Importantly, this peptide has no toxicity against mammalian cells. Taken together, an effective and short peptide, ALLRL, would be an attractive antimicrobial to Gram-positive bacteria and C. albicans.

scholarly journals Endolyn is a mucin-like type I membrane protein targeted to lysosomes by its cytoplasmic tail

2000 ◽  
Vol 345 (2) ◽  
pp. 287-296 ◽  
Author(s):  
Gudrun IHRKE ◽  
Sally R. GRAY ◽  
J. Paul LUZIO
Keyword(s):  
Amino Acids ◽  
Membrane Proteins ◽  
Membrane Protein ◽  
Mammalian Cells ◽  
Core Protein ◽  
Rat Kidney ◽  
Cytoplasmic Tail ◽  
Lysosomal Membrane ◽  
Type I ◽  
Lysosomal Membrane Proteins

Endolyn (endolyn-78) is a membrane protein found in lysosomal and endosomal compartments of mammalian cells. Unlike ‘classical’ lysosomal membrane proteins, such as lysosome-associated membrane protein (lamp)-1, it is also present in a subapical compartment in polarized WIF-B hepatocytes. The structural features that determine sorting of endolyn are unknown. We have identified a rat endolyn cDNA by expression screening. The cDNA encodes a ubiquitously expressed type I membrane protein with a short cytoplasmic tail of 13 amino acids and many putative sites for N- and O-linked glycosylation in the predicted luminal domain. Endolyn is closely related to two human mucin-like proteins, multi-glycosylated core protein (MGC)-24 and CD164 (MGC-24v), expressed in gastric carcinoma cells and bone marrow stromal and haematopoietic precursor cells respectively. The predicted transmembrane and cytoplasmic tail domains of endolyn, as well as parts of its luminal domain, also show some similarities with lamp-1 and lamp-2. Like these and other known lysosomal membrane proteins, endolyn contains a YXXØ motif at the C-terminus of its cytoplasmic tail (where Ø is a bulky hydrophobic amino acid), but with no preceding glycine. Nonetheless, the last ten amino acids of this tail, when transplanted on to human CD8, caused efficient targeting of the chimaeric protein to endosomes and lysosomes in transfected normal rat kidney cells.

Methionine-Specific Staining of Collagen

1982 ◽  
Vol 40 ◽  
pp. 294-295
Author(s):  
E.M. Kuhn ◽  
K.D. Marenus ◽  
M. Beer
Keyword(s):  
Amino Acids ◽  
Collagen Fibers ◽  
Type Iii Collagen ◽  
Type I ◽  
Electron Microscopic ◽  
Good Correspondence ◽  
Specific Staining ◽  
Type Iii ◽  
Different Types ◽  
Sulfur Containing

Fibers composed of different types of collagen cannot be differentiated by conventional electron microscopic stains. We are developing staining procedures aimed at identifying collagen fibers of different types.Pt(Gly-L-Met)Cl binds specifically to sulfur-containing amino acids. Different collagens have methionine (met) residues at somewhat different positions. A good correspondence has been reported between known met positions and Pt(GLM) bands in rat Type I SLS (collagen aggregates in which molecules lie adjacent to each other in exact register). We have confirmed this relationship in Type III collagen SLS (Fig. 1).

Plasma and skeletal muscle free amino acids in type I, insulin-treated diabetic subjects

Diabetes ◽  
1985 ◽  
Vol 34 (8) ◽  
pp. 812-815 ◽  
Author(s):  
L. Borghi ◽  
R. Lugari ◽  
A. Montanari ◽  
P. Dall'Argine ◽  
G. F. Elia ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Amino Acids ◽  
Free Amino Acids ◽  
Free Amino ◽  
Type I

scholarly journals Autophagy Deficiency by Atg4B Loss Leads to Metabolomic Alterations in Mice

Metabolites ◽  
2021 ◽  
Vol 11 (8) ◽  
pp. 481
Author(s):  
Gemma G. Martínez-García ◽  
Raúl F. Pérez ◽  
Álvaro F. Fernández ◽  
Sylvere Durand ◽  
Guido Kroemer ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Amino Acids ◽  
Mammalian Cells ◽  
Tissue Homeostasis ◽  
In Vivo Studies ◽  
Protective Mechanism ◽  
Cardiac Damage ◽  
Wide Range ◽  
First Time

Autophagy is an essential protective mechanism that allows mammalian cells to cope with a variety of stressors and contributes to maintaining cellular and tissue homeostasis. Due to these crucial roles and also to the fact that autophagy malfunction has been described in a wide range of pathologies, an increasing number of in vivo studies involving animal models targeting autophagy genes have been developed. In mammals, total autophagy inactivation is lethal, and constitutive knockout models lacking effectors of this route are not viable, which has hindered so far the analysis of the consequences of a systemic autophagy decline. Here, we take advantage of atg4b−/− mice, an autophagy-deficient model with only partial disruption of the process, to assess the effects of systemic reduction of autophagy on the metabolome. We describe for the first time the metabolic footprint of systemic autophagy decline, showing that impaired autophagy results in highly tissue-dependent alterations that are more accentuated in the skeletal muscle and plasma. These changes, which include changes in the levels of amino-acids, lipids, or nucleosides, sometimes resemble those that are frequently described in conditions like aging, obesity, or cardiac damage. We also discuss different hypotheses on how impaired autophagy may affect the metabolism of several tissues in mammals.

scholarly journals A Novel Nanobody Precisely Visualizes Phosphorylated Histone H2AX in Living Cancer Cells under Drug-Induced Replication Stress

Cancers ◽  
2021 ◽  
Vol 13 (13) ◽  
pp. 3317
Author(s):  
Eric Moeglin ◽  
Dominique Desplancq ◽  
Audrey Stoessel ◽  
Christian Massute ◽  
Jeremy Ranniger ◽  
...  
Keyword(s):  
Cancer Cells ◽  
Mammalian Cells ◽  
Chromatin Modification ◽  
Replication Stress ◽  
Living Cells ◽  
Single Step ◽  
Dna Double Strand Breaks ◽  
Drug Induced ◽  
Histone H2ax ◽  
C Terminus

Histone H2AX phosphorylated at serine 139 (γ-H2AX) is a hallmark of DNA damage, signaling the presence of DNA double-strand breaks and global replication stress in mammalian cells. While γ-H2AX can be visualized with antibodies in fixed cells, its detection in living cells was so far not possible. Here, we used immune libraries and phage display to isolate nanobodies that specifically bind to γ-H2AX. We solved the crystal structure of the most soluble nanobody in complex with the phosphopeptide corresponding to the C-terminus of γ-H2AX and show the atomic constituents behind its specificity. We engineered a bivalent version of this nanobody and show that bivalency is essential to quantitatively visualize γ-H2AX in fixed drug-treated cells. After labelling with a chemical fluorophore, we were able to detect γ-H2AX in a single-step assay with the same sensitivity as with validated antibodies. Moreover, we produced fluorescent nanobody-dTomato fusion proteins and applied a transduction strategy to visualize with precision γ-H2AX foci present in intact living cells following drug treatment. Together, this novel tool allows performing fast screenings of genotoxic drugs and enables to study the dynamics of this particular chromatin modification in individual cancer cells under a variety of conditions.

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