Investigations of the MceIJ-Catalyzed Posttranslational Modification of the Microcin E492 C-Terminus: Linkage of Ribosomal and Nonribosomal Peptides To Form “Trojan Horse” Antibiotics†

Biochemistry ◽  
2008 ◽  
Vol 47 (35) ◽  
pp. 9289-9299 ◽  
Author(s):  
Elizabeth M. Nolan ◽  
Christopher T. Walsh
Keyword(s):  
Posttranslational Modification ◽  
Trojan Horse ◽  
Nonribosomal Peptides ◽  
C Terminus

scholarly journals Single‐residue posttranslational modification sites at the N‐terminus, C‐terminus or in‐between: To be or not to be exposed for enzyme access

PROTEOMICS ◽  
2015 ◽  
Vol 15 (14) ◽  
pp. 2525-2546 ◽  
Author(s):  
Fernanda L. Sirota ◽  
Sebastian Maurer‐Stroh ◽  
Birgit Eisenhaber ◽  
Frank Eisenhaber
Keyword(s):  
Posttranslational Modification ◽  
C Terminus ◽  
N Terminus

scholarly journals Development of a Qualitative Sequential Immunoassay for Characterizing the Intrinsic Properties of Circulating Cardiac Troponin I

2010 ◽  
Vol 56 (8) ◽  
pp. 1307-1319 ◽  
Author(s):  
Yixin Lin ◽  
Qin Fu ◽  
Jie Zhu ◽  
Julie M Miller ◽  
Jennifer E Van Eyk
Keyword(s):  
Real Time ◽  
Posttranslational Modification ◽  
Cardiac Troponin ◽  
Troponin I ◽  
Direct Detection ◽  
Cardiac Troponin I ◽  
Diffractive Optics ◽  
Intrinsic Properties ◽  
C Terminus ◽  
Modified Forms

BACKGROUND With myocardial infarction (MI), cardiac troponin is released from the heart into circulation, where it can be detected with immunoassays independently quantifying cardiac troponin I (cTnI) or cTnT. There is, however, no single immunoassay that sequentially probes the posttranslational modification status of cTnI or directly characterizes whether circulating cTnI is bound to cTnC and/or cTnT. Here we describe the development of a qualitative immunoassay to directly probe the primary and ternary structure of circulating cTnI through diffractive optics technology (dotLab® System, Axela). METHODS Anti-cTnI antibody 8I-7 was immobilized on a patterned sensor to capture cTnI. One or more detector antibodies were sequentially introduced to probe for amino acid sequence integrity or phosphorylation status of cTnI, or its association with cTnC and/or cTnT. Respective immunocaptures were recorded as real-time diffractive intensities (DIs), and the DI differences were analyzed. Each immunodetection was independent of the others but was done in a single sequential assay. RESULTS This diffraction-based immunoassay successfully characterized cTnI. The unamplified assay determined whether cTnI was degraded at N-terminus and/or C-terminus or phosphorylated. Sequential application of multiple detector antibodies without an antibody-stripping step enables real-time interrogation of 5 different epitopes of cTnI, or direct detection of the cTn complex (cTnI–cTnC–cTnT) in a single sequential assay. Finally, this assay was optimized with amplification to directly detect circulating cTnI bound to cTnC and cTnT in serum from an MI patient. CONCLUSIONS The dot® Immunoassay is the first qualitative sequential immunoassay to address the direct interactions of the troponin subunits and various modified forms of cTnI.

scholarly journals Mechanistic basis for ubiquitin modulation of a protein energy landscape

2020 ◽  
Author(s):  
Emma C. Carroll ◽  
Naomi R. Latorraca ◽  
Johanna M. Lindner ◽  
Brendan C. Maguire ◽  
Jeff G. Pelton ◽  
...  
Keyword(s):  
Quality Control ◽  
Posttranslational Modification ◽  
Energy Landscape ◽  
26S Proteasome ◽  
Dynamics Simulation ◽  
Structural Motif ◽  
Protein Energy ◽  
C Terminus ◽  
Model Protein ◽  
Mechanistic Basis

AbstractUbiquitin is a common posttranslational modification canonically associated with targeting proteins to the 26S proteasome for degradation and also plays a role in numerous other non-degradative cellular processes. Ubiquitination at certain sites destabilizes the substrate protein, with consequences for proteasomal processing, while ubiquitination at other sites has little energetic effect. How this site specificity—and, by extension, the myriad effects of ubiquitination on substrate proteins—arises remains unknown. Here, we systematically characterize the atomic-level effects of ubiquitination at various sites on a model protein, barstar, using a combination of NMR, hydrogen-deuterium exchange mass spectrometry, and molecular dynamics simulation. We find that, regardless of the site of modification, ubiquitination does not induce large structural rearrangements in the substrate. Destabilizing modifications, however, increase fluctuations from the native state resulting in exposure of the substrate’s C terminus. Both of the sites occur in regions of barstar with relatively high conformational flexibility. Destabilization, however, appears to occur through different thermodynamic mechanisms, involving a reduction in entropy in one case and a loss in enthalpy in another. By contrast, ubiquitination at a non-destabilizing site protects the substrate C terminus through intermittent formation of a structural motif with the last three residues of ubiquitin. Thus, the biophysical effects of ubiquitination at a given site depend greatly on local context. Taken together, our results reveal how a single post-translational modification can generate a broad array of distinct effects, providing a framework to guide the design of proteins and therapeutics with desired degradation and quality-control properties. (248 words)Significance StatementFluctuations on a protein energy landscapes encode the mechanistic basis for vital biological processes not always evident from static structures alone. Ubiquitination, a key posttranslational modification, can affect a protein’s energy landscape with consequences for proteasomal degradation, but the molecular mechanisms driving ubiquitin-induced energetic changes remain elusive. Here, we systematically characterize the energetic effects of ubiquitination at three sites on a model protein. We find that distinct thermodynamic mechanisms can produce the same outcome of ubiquitin-induced destabilization at sensitive sites. At a non-sensitive site, we observe formation of a substrate–ubiquitin interaction that may serve to protect against destabilization. This work will enable development of predictive models of the effect of ubiquitin at any given site on a protein with implications for understanding and engineering regulated ubiquitin signaling and protein quality control in vivo.

scholarly journals Mechanistic basis for ubiquitin modulation of a protein energy landscape

2021 ◽  
Vol 118 (12) ◽  
pp. e2025126118
Author(s):  
Emma C. Carroll ◽  
Naomi R. Latorraca ◽  
Johanna M. Lindner ◽  
Brendan C. Maguire ◽  
Jeffrey G. Pelton ◽  
...  
Keyword(s):  
Posttranslational Modification ◽  
26S Proteasome ◽  
Dynamics Simulation ◽  
Structural Motif ◽  
Local Context ◽  
Exchange Mass Spectrometry ◽  
Substrate Protein ◽  
Protein Energy ◽  
C Terminus ◽  
Mechanistic Basis

Ubiquitin is a common posttranslational modification canonically associated with targeting proteins to the 26S proteasome for degradation and also plays a role in numerous other nondegradative cellular processes. Ubiquitination at certain sites destabilizes the substrate protein, with consequences for proteasomal processing, while ubiquitination at other sites has little energetic effect. How this site specificity—and, by extension, the myriad effects of ubiquitination on substrate proteins—arises remains unknown. Here, we systematically characterize the atomic-level effects of ubiquitination at various sites on a model protein, barstar, using a combination of NMR, hydrogen–deuterium exchange mass spectrometry, and molecular dynamics simulation. We find that, regardless of the site of modification, ubiquitination does not induce large structural rearrangements in the substrate. Destabilizing modifications, however, increase fluctuations from the native state resulting in exposure of the substrate’s C terminus. Both of the sites occur in regions of barstar with relatively high conformational flexibility. Nevertheless, destabilization appears to occur through different thermodynamic mechanisms, involving a reduction in entropy in one case and a loss in enthalpy in another. By contrast, ubiquitination at a nondestabilizing site protects the substrate C terminus through intermittent formation of a structural motif with the last three residues of ubiquitin. Thus, the biophysical effects of ubiquitination at a given site depend greatly on local context. Taken together, our results reveal how a single posttranslational modification can generate a broad array of distinct effects, providing a framework to guide the design of proteins and therapeutics with desired degradation and quality control properties.

scholarly journals Prenylation of mammalian Ras protein in Xenopus oocytes.

1990 ◽  
Vol 10 (11) ◽  
pp. 5945-5949 ◽  
Author(s):  
R Kim ◽  
J Rine ◽  
S H Kim
Keyword(s):  
Biological Activity ◽  
Posttranslational Modification ◽  
Xenopus Oocytes ◽  
Cholesterol Biosynthesis ◽  
Mevalonic Acid ◽  
Xenopus Laevis Oocytes ◽  
Ras Protein ◽  
C Terminus

Ras protein requires an intermediate of the cholesterol biosynthetic pathway for posttranslational modification and membrane anchorage. This step is necessary for biological activity. Maturation of Xenopus laevis oocytes induced by an oncogenic human Ras protein can be inhibited by lovastatin or compactin, inhibitors of the synthesis of mevalonate, an intermediate of cholesterol biosynthesis. This inhibition can be overcome by mevalonic acid or farnesyl diphosphate, a cholesterol biosynthetic intermediate downstream of mevalonate, but not by squalene, an intermediate after farnesyl pyrophosphate in the pathway. This study supports the idea that in Xenopus oocytes, the Ras protein is modified by a farnesyl moiety or its derivative. Furthermore, an octapeptide with the sequence similar to the C-terminus of the c-H-ras protein inhibits the biological activity of Ras proteins in vivo, suggesting that it competes for the enzyme or enzymes responsible for transferring the isoprenoid moiety (prenylation) in the oocytes. This inhibition of Ras prenylation by the peptide was also observed in vitro, using both Saccharomyces cerevisiae and Xenopus oocyte extracts. These observations show that Xenopus oocytes provide a convenient in vivo system for studies of inhibitors of the posttranslational modification of the Ras protein, especially for inhibitors such as peptides that do not penetrate cell membranes.

scholarly journals Does the Importance of the C-Terminal Residues in the Maturation of RgpB from Porphyromonas gingivalis Reveal a Novel Mechanism for Protein Export in a Subgroup of Gram-Negative Bacteria?

2006 ◽  
Vol 189 (3) ◽  
pp. 833-843 ◽  
Author(s):  
Ky-Anh Nguyen ◽  
James Travis ◽  
Jan Potempa
Keyword(s):  
Outer Membrane ◽  
Porphyromonas Gingivalis ◽  
Posttranslational Modification ◽  
Secretory Pathway ◽  
Outer Membrane Proteins ◽  
Gram Negative Bacteria ◽  
Wild Type ◽  
C Terminus ◽  
In The Wild ◽  
Representative Member

ABSTRACT The mature 507-residue RgpB protein belongs to an important class of extracellular outer membrane-associated proteases, the gingipains, from the oral pathogen Porphyromonas gingivalis that has been shown to play a central role in the virulence of the organism. The C termini of these gingipains along with other outer membrane proteins from the organism share homologous sequences and have been suggested to function in attachment of these proteins to the outer membrane. In this report, we have created a series of truncated and site-directed mutants of the C terminus from a representative member of this class, the RgpB protease, to investigate its role in the maturation of these proteins. Truncation of the last two residues (valyl-lysine) from the C terminus is sufficient to create an inactive version of the protein that lacks the posttranslational glycosylation seen in the wild type, and the protein remains trapped behind the outer membrane. Alanine scanning of the last five residues revealed the importance of the C-terminal motif in mediating correct posttranslational modification of the protein. This result may have a wider implication in a novel secretory pathway in distinct members of the Cytophaga-Flavobacterium-Bacteroidetes phylum.

scholarly journals Functional Analysis of the GPI Transamidase Complex by Screening for Amino Acid Mutations in Each Subunit

Molecules ◽  
2021 ◽  
Vol 26 (18) ◽  
pp. 5462
Author(s):  
Si-Si Liu ◽  
Fei Jin ◽  
Yi-Shi Liu ◽  
Yoshiko Murakami ◽  
Yukihiko Sugita ◽  
...  
Keyword(s):  
Functional Analysis ◽  
Posttranslational Modification ◽  
Homologous Proteins ◽  
Gpi Anchor ◽  
Purification Method ◽  
C Terminus ◽  
Precursor Proteins ◽  
Amino Acid Mutations ◽  
Gpi Transamidase ◽  
Particle Images

Glycosylphosphatidylinositol (GPI) anchor modification is a posttranslational modification of proteins that has been conserved in eukaryotes. The biosynthesis and transfer of GPI to proteins are carried out in the endoplasmic reticulum. Attachment of GPI to proteins is mediated by the GPI-transamidase (GPI-TA) complex, which recognizes and cleaves the C-terminal GPI attachment signal of precursor proteins. Then, GPI is transferred to the newly exposed C-terminus of the proteins. GPI-TA consists of five subunits: PIGK, GPAA1, PIGT, PIGS, and PIGU, and the absence of any subunit leads to the loss of activity. Here, we analyzed functionally important residues of the five subunits of GPI-TA by comparing conserved sequences among homologous proteins. In addition, we optimized the purification method for analyzing the structure of GPI-TA. Using purified GPI-TA, preliminary single particle images were obtained. Our results provide guidance for the structural and functional analysis of GPI-TA.

Prenylation of mammalian Ras protein in Xenopus oocytes

1990 ◽  
Vol 10 (11) ◽  
pp. 5945-5949
Author(s):  
R Kim ◽  
J Rine ◽  
S H Kim
Keyword(s):  
Biological Activity ◽  
Posttranslational Modification ◽  
Xenopus Oocytes ◽  
Cholesterol Biosynthesis ◽  
Mevalonic Acid ◽  
Xenopus Laevis Oocytes ◽  
Ras Protein ◽  
C Terminus

Ras protein requires an intermediate of the cholesterol biosynthetic pathway for posttranslational modification and membrane anchorage. This step is necessary for biological activity. Maturation of Xenopus laevis oocytes induced by an oncogenic human Ras protein can be inhibited by lovastatin or compactin, inhibitors of the synthesis of mevalonate, an intermediate of cholesterol biosynthesis. This inhibition can be overcome by mevalonic acid or farnesyl diphosphate, a cholesterol biosynthetic intermediate downstream of mevalonate, but not by squalene, an intermediate after farnesyl pyrophosphate in the pathway. This study supports the idea that in Xenopus oocytes, the Ras protein is modified by a farnesyl moiety or its derivative. Furthermore, an octapeptide with the sequence similar to the C-terminus of the c-H-ras protein inhibits the biological activity of Ras proteins in vivo, suggesting that it competes for the enzyme or enzymes responsible for transferring the isoprenoid moiety (prenylation) in the oocytes. This inhibition of Ras prenylation by the peptide was also observed in vitro, using both Saccharomyces cerevisiae and Xenopus oocyte extracts. These observations show that Xenopus oocytes provide a convenient in vivo system for studies of inhibitors of the posttranslational modification of the Ras protein, especially for inhibitors such as peptides that do not penetrate cell membranes.

Neurofilaments and Paired Helical Filaments

1985 ◽  
Vol 43 ◽  
pp. 740-743
Author(s):  
J. Metuzals
Keyword(s):  
Animal Model ◽  
Posttranslational Modifications ◽  
Posttranslational Modification ◽  
Giant Axon ◽  
Paired Helical Filaments ◽  
Squid Giant Axon ◽  
In Vitro Experiments ◽  
Thin Sections ◽  
Structural Relationships

It has been demonstrated that the neurofibrillary tangles in biopsies of Alzheimer patients, composed of typical paired helical filaments (PHF), consist also of typical neurofilaments (NF) and 15nm wide filaments. Close structural relationships, and even continuity between NF and PHF, have been observed. In this paper, such relationships are investigated from the standpoint that the PHF are formed through posttranslational modifications of NF. To investigate the validity of the posttranslational modification hypothesis of PHF formation, we have identified in thin sections from frontal lobe biopsies of Alzheimer patients all existing conformations of NF and PHF and ordered these conformations in a hypothetical sequence. However, only experiments with animal model preparations will prove or disprove the validity of the interpretations of static structural observations made on patients. For this purpose, the results of in vitro experiments with the squid giant axon preparations are compared with those obtained from human patients. This approach is essential in discovering etiological factors of Alzheimer's disease and its early diagnosis.

Immunocytochemical localization of β1-4 galactosyltransferase in endometrial carcinoma cells

1990 ◽  
Vol 48 (3) ◽  
pp. 944-945
Author(s):  
Ichiro Yamamoto ◽  
Toshiaki Tachibana ◽  
Hiroko Maruyama ◽  
Noriyuki Komatsu ◽  
Hiroyuki Kuramoto ◽  
...  
Keyword(s):  
Endometrial Carcinoma ◽  
Cell Lines ◽  
Endometrial Adenocarcinoma ◽  
Protein A ◽  
Rabbit Antiserum ◽  
Carcinoma Cells ◽  
Affinity Column ◽  
Antibody Technique ◽  
Galactosyl Transferase ◽  
C Terminus

We have paid attention to the alteration of glycosyltransferase in carcinoma cells, because it might be related to the malignancy of the cells. In this connection, localization of β1-4 galactosyl transferase (β1-4 Gal T) in human endometrial carcinoma cells was examined immunocytochemically using two kinds of cell lines, each of which showed different degree of differentiation.An antibody was purified from the rabbit antiserum against the synthetic peptide, IFNRLVFRGMSC (W89) of human β1-4 Gal T coupled with KLH (keyhole limpet hemocyanine) by protein A column and peptide-affinity column chromatography. The anti-W89 serum reacts to the C-terminus of human β 1-4 Gal T and to both membrane-bound and soluble forms of the enzyme. Cell line of well differentiated endometrial adenocarcinoma (I) and that of poorly differentiated endometrial adenocarcinoma (50B) were cultivated respectively in MEM medium containing 15% FCS and 2 mM glutamine for 4 d at 37°C under 5% CO2. The cells were fixed in a mixture of 4% paraformaldehyde and 0.1% glutaraldehyde in 0.1 M Soerensen’s phosphate buffer (pH 7.4) at 4°C for 30 min, washed with PBS, then freezed and thawed. The indirect method of the peroxidase- labeled antibody technique was used for immunocytochemistry of both LM and TEM on the cell lines. The cells were dehydrated in ethanol and embedded in TAAB 812. Ultrathin sections were observed under a TEM, JEM-100S.

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