scholarly journals Evaluation of Nonenzymatic Posttranslational Modification–Derived Products as Biomarkers of Molecular Aging of Proteins

2010 ◽  
Vol 56 (9) ◽  
pp. 1401-1412 ◽  
Author(s):  
Stéphane Jaisson ◽  
Philippe Gillery
Keyword(s):  
Posttranslational Modifications ◽  
Posttranslational Modification ◽  
New Technologies ◽  
Clinical Chemistry ◽  
Routine Practice ◽  
Potential Contribution ◽  
Age Related ◽  
Specificity And Sensitivity ◽  
Molecular Aging

BACKGROUND During their biological life, proteins are exposed in a cumulative fashion to irreversible nonenzymatic, late posttranslational modifications that are responsible for their molecular aging. It is now well established that these damaged proteins constitute a molecular substratum for many dysfunctions described in metabolic and age-related diseases, such as diabetes mellitus, renal insufficiency, atherosclerosis, or neurodegenerative diseases. Accordingly, the specific end products derived from these reactions are considered potentially useful biomarkers for these diseases. CONTENT The aim of this review is to give an overview of nonenzymatic posttranslational modifications of proteins and their influence in vivo, take inventory of the analytical methods available for the measurement of posttranslational modification–derived products, and assess the potential contribution of new technologies for their clinical use as biological markers of protein molecular aging. SUMMARY Despite their clinical relevance, biomarkers of posttranslational modifications of proteins have been studied only in the context of experimental clinical research, owing to the analytical complexity of their measurement. The recent implementation in clinical chemistry laboratories of mass spectrometry–based methods that provide higher specificity and sensitivity has facilitated the measurement of these compounds. These markers are not used currently by clinicians in routine practice, however, and many challenges, such as standardization, have to be confronted before these markers can be used as efficient tools in the detection and monitoring of long-term complications of metabolic and age-related diseases.

scholarly journals The CTCF Insulator Protein Is Posttranslationally Modified by SUMO

2008 ◽  
Vol 29 (3) ◽  
pp. 714-725 ◽  
Author(s):  
Melissa J. MacPherson ◽  
Linda G. Beatty ◽  
Wenjing Zhou ◽  
Minjie Du ◽  
Paul D. Sadowski
Keyword(s):  
Posttranslational Modifications ◽  
Posttranslational Modification ◽  
Zinc Finger Protein ◽  
Finger Protein ◽  
Ability To Act ◽  
Polycomb Protein ◽  
P2 Promoter ◽  
Polycomb Bodies

ABSTRACT The CTCF protein is a highly conserved zinc finger protein that is implicated in many aspects of gene regulation and nuclear organization. Its functions include the ability to act as a repressor of genes, including the c-myc oncogene. In this paper, we show that the CTCF protein can be posttranslationally modified by the small ubiquitin-like protein SUMO. CTCF is SUMOylated both in vivo and in vitro, and we identify two major sites of SUMOylation in the protein. The posttranslational modification of CTCF by the SUMO proteins does not affect its ability to bind to DNA in vitro. SUMOylation of CTCF contributes to the repressive function of CTCF on the c-myc P2 promoter. We also found that CTCF and the repressive Polycomb protein, Pc2, are colocalized to nuclear Polycomb bodies. The Pc2 protein may act as a SUMO E3 ligase for CTCF, strongly enhancing its modification by SUMO 2 and 3. These studies expand the repertoire of posttranslational modifications of CTCF and suggest roles for such modifications in its regulation of epigenetic states.

scholarly journals Posttranslational modification of tubulin by palmitoylation: I. In vivo and cell-free studies.

1997 ◽  
Vol 8 (4) ◽  
pp. 621-636 ◽  
Author(s):  
J M Caron
Keyword(s):  
Posttranslational Modifications ◽  
Posttranslational Modification ◽  
Hydrophobic Interactions ◽  
Covalent Attachment ◽  
Protein Structure Analysis ◽  
Free System ◽  
Intracellular Membranes ◽  
Cell Free System ◽  
Cytoplasmic Proteins

It is well established that microtubules interact with intracellular membranes of eukaryotic cells. There is also evidence that tubulin, the major subunit of microtubules, associates directly with membranes. In many cases, this association between tubulin and membranes involves hydrophobic interactions. However, neither primary sequence nor known posttranslational modifications of tubulin can account for such an interaction. The goal of this study was to determine the molecular nature of hydrophobic interactions between tubulin and membranes. Specifically, I sought to identify a posttranslational modification of tubulin that is found in membrane proteins but not in cytoplasmic proteins. One such modification is the covalent attachment of the long chain fatty acid palmitate. The possibility that tubulin is a substrate for palmitoylation was investigated. First, I found that tubulin was palmitoylated in resting platelets and that the level of palmitoylation of tubulin decreased upon activation of platelets with thrombin. Second, to obtain quantities of palmitoylated tubulin required for protein structure analysis, a cell-free system for palmitoylation of tubulin was developed and characterized. The substrates for palmitoylation were nonpolymerized tubulin and tubulin in microtubules assembled with the slowly hydrolyzable GTP analogue guanylyl-(alpha, beta)-methylene-diphosphonate. However, tubulin in Taxol-assembled microtubules was not a substrate for palmitoylation. Likewise, palmitoylation of tubulin in the cell-free system was specifically inhibited by the antimicrotubule drugs Colcemid, podophyllotoxin, nocodazole, and vinblastine. These experiments identify a previously unknown posttranslational modification of tubulin that can account for at least one type of hydrophobic interaction with intracellular membranes.

scholarly journals Age-related Increases in Plasma Phosphatidylcholine Hydroperoxide Concentrations in Control Subjects and Patients with Hyperlipidemia

2000 ◽  
Vol 46 (6) ◽  
pp. 822-828 ◽  
Author(s):  
Mikio Kinoshita ◽  
Shinichi Oikawa ◽  
Kyoko Hayasaka ◽  
Akihiro Sekikawa ◽  
Tazuko Nagashima ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Total Cholesterol ◽  
Plasma Lipids ◽  
Lipid Peroxide ◽  
Lipid Peroxides ◽  
In Vivo Evaluation ◽  
Age Related ◽  
Specificity And Sensitivity ◽  
The Mean

Abstract Background: The basal lipid peroxide concentration in the plasma of patients with hyperlipidemia may be related to atherosclerosis. Quantitative determination of lipid peroxides in the plasma is an important step in the overall evaluation of the biochemical processes leading to oxidative injury. Unfortunately, the currently available methods for lipid peroxidation lack specificity and sensitivity. Methods: Hyperlipidemic patients (44 males and 50 females), ages 12–82 years (mean ± SE, 53 ± 2.3 years for males, 58 ± 2.0 years for females, and 56 ± 14 years for total cases), and normolipidemic volunteers (controls, 32 males and 15 females), ages 13–90 years (49 ± 4 years for males, 65 ± 4 years for females, and 55 ± 24 years for total cases), were recruited in the present study. Plasma phosphatidylcholine hydroperoxide (PCOOH) was determined by chemiluminescence-HPLC (CL-HPLC). Results: Plasma PCOOH concentrations increased with age in both controls and hyperlipidemic patients. However, the mean plasma PCOOH concentration in patients with hyperlipidemia (331 ± 19 nmol/L; n = 94) was significantly (P <0.001) higher than in the controls (160 ± 65 nmol/L; n = 47). Plasma PCOOH concentrations were similar in three hyperlipidemic phenotypes: hypercholesterolemia (IIa), hypertriglyceridemia (IV), and combined hyperlipidemia (IIb). The mean plasma PCOOH in patients with treatment-induced normalized plasma lipids was 202 ± 17 nmol/L. There was no significant correlation between plasma PCOOH concentration and total cholesterol, triglycerides, or phospholipids in hyperlipidemic patients. For all subjects, there was a significantly positive correlation between plasma PCOOH and each lipid (total cholesterol, P = 0.0002; triglycerides, P = 0.0137; and phospholipids, P <0.0001). Analysis of fatty acids composition of plasma phosphatidylcholine showed significantly low concentrations of n-6 fatty acids moieties (linoleic acid and arachidonic acid) in patients compared with controls. Conclusions: Our results suggest that an increase in plasma PCOOH in patients with hyperlipidemia may be related to the development and progression of atherosclerosis, particularly in the elderly. Measurement of plasma PCOOH is useful for in vivo evaluation of oxidative stress.

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.

Meiotic CENP-C is a shepherd: bridging the space between the centromere and the kinetochore in time and space

2020 ◽  
Vol 64 (2) ◽  
pp. 251-261
Author(s):  
Jessica E. Fellmeth ◽  
Kim S. McKim
Keyword(s):  
Chromosome Segregation ◽  
New Technologies ◽  
Early Prophase ◽  
Unique Role ◽  
Kinetochore Assembly ◽  
M Phase ◽  
In Vivo Analysis ◽  
Meiosis I

Abstract While many of the proteins involved in the mitotic centromere and kinetochore are conserved in meiosis, they often gain a novel function due to the unique needs of homolog segregation during meiosis I (MI). CENP-C is a critical component of the centromere for kinetochore assembly in mitosis. Recent work, however, has highlighted the unique features of meiotic CENP-C. Centromere establishment and stability require CENP-C loading at the centromere for CENP-A function. Pre-meiotic loading of proteins necessary for homolog recombination as well as cohesion also rely on CENP-C, as do the main scaffolding components of the kinetochore. Much of this work relies on new technologies that enable in vivo analysis of meiosis like never before. Here, we strive to highlight the unique role of this highly conserved centromere protein that loads on to centromeres prior to M-phase onset, but continues to perform critical functions through chromosome segregation. CENP-C is not merely a structural link between the centromere and the kinetochore, but also a functional one joining the processes of early prophase homolog synapsis to late metaphase kinetochore assembly and signaling.

Optical imaging probes and their potential contribution to radiotracer development

2016 ◽  
Vol 55 (02) ◽  
pp. 51-62 ◽  
Author(s):  
S. Hermann ◽  
M. Schäfers ◽  
C. Höltke ◽  
A. Faust
Keyword(s):  
Optical Imaging ◽  
Fluorescent Dyes ◽  
Great Influence ◽  
Potential Contribution ◽  
Preclinical Evaluation ◽  
Guided Surgery ◽  
Fluorescence Guided Surgery ◽  
Imaging Probes ◽  
Unspecific Binding

SummaryOptical imaging has long been considered a method for histological or microscopic investigations. Over the last 15 years, however, this method was applied for preclinical molecular imaging and, just recently, was also able to show its principal potential for clinical applications (e.g. fluorescence-guided surgery). Reviewing the development and preclinical evaluation of new fluorescent dyes and target-specific dye conjugates, these often show characteristic patterns of their routes of excretion and biodistribution, which could also be interesting for the development and optimization of radiopharmaceuticals. Especially ionic charges show a great influence on biodistribution and netcharge and charge-distribution on a conjugate often determines unspecific binding or background signals in liver, kidney or intestine, and other organs.Learning from fluorescent probe behaviour in vivo and translating this knowledge to radio-pharmaceuticals might be useful to further optimize emerging and existing radiopharmaceuticals with respect to their biodistribution and thereby availability for binding to their targets.

The Synergy between CRISPR and Chemical Engineering

2019 ◽  
Vol 19 (3) ◽  
pp. 147-171
Author(s):  
Cia-Hin Lau ◽  
Chung Tin
Keyword(s):  
Viral Vectors ◽  
Chemical Engineering ◽  
Target Genes ◽  
New Technologies ◽  
Rapid Development ◽  
Genetic Circuits ◽  
Viral Packaging ◽  
Conditional Control ◽  
Logic Operations

Gene therapy and transgenic research have advanced quickly in recent years due to the development of CRISPR technology. The rapid development of CRISPR technology has been largely benefited by chemical engineering. Firstly, chemical or synthetic substance enables spatiotemporal and conditional control of Cas9 or dCas9 activities. It prevents the leaky expression of CRISPR components, as well as minimizes toxicity and off-target effects. Multi-input logic operations and complex genetic circuits can also be implemented via multiplexed and orthogonal regulation of target genes. Secondly, rational chemical modifications to the sgRNA enhance gene editing efficiency and specificity by improving sgRNA stability and binding affinity to on-target genomic loci, and hence reducing off-target mismatches and systemic immunogenicity. Chemically-modified Cas9 mRNA is also more active and less immunogenic than the native mRNA. Thirdly, nonviral vehicles can circumvent the challenges associated with viral packaging and production through the delivery of Cas9-sgRNA ribonucleoprotein complex or large Cas9 expression plasmids. Multi-functional nanovectors enhance genome editing in vivo by overcoming multiple physiological barriers, enabling ligand-targeted cellular uptake, and blood-brain barrier crossing. Chemical engineering can also facilitate viral-based delivery by improving vector internalization, allowing tissue-specific transgene expression, and preventing inactivation of the viral vectors in vivo. This review aims to discuss how chemical engineering has helped improve existing CRISPR applications and enable new technologies for biomedical research. The usefulness, advantages, and molecular action for each chemical engineering approach are also highlighted.

scholarly journals Effect of Posttranslational Modifications on the Structure and Activity of FTO Demethylase

2021 ◽  
Vol 22 (9) ◽  
pp. 4512
Author(s):  
Michał Marcinkowski ◽  
Tomaš Pilžys ◽  
Damian Garbicz ◽  
Jan Piwowarski ◽  
Damian Mielecki ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Size Exclusion Chromatography ◽  
Posttranslational Modifications ◽  
Size Exclusion ◽  
Saxs Data ◽  
Wide Range ◽  
Exclusion Chromatography ◽  
Demethylase Activity ◽  
Structure And Activity

The FTO protein is involved in a wide range of physiological processes, including adipogenesis and osteogenesis. This two-domain protein belongs to the AlkB family of 2-oxoglutarate (2-OG)- and Fe(II)-dependent dioxygenases, displaying N6-methyladenosine (N6-meA) demethylase activity. The aim of the study was to characterize the relationships between the structure and activity of FTO. The effect of cofactors (Fe2+/Mn2+ and 2-OG), Ca2+ that do not bind at the catalytic site, and protein concentration on FTO properties expressed in either E. coli (ECFTO) or baculovirus (BESFTO) system were determined using biophysical methods (DSF, MST, SAXS) and biochemical techniques (size-exclusion chromatography, enzymatic assay). We found that BESFTO carries three phosphoserines (S184, S256, S260), while there were no such modifications in ECFTO. The S256D mutation mimicking the S256 phosphorylation moderately decreased FTO catalytic activity. In the presence of Ca2+, a slight stabilization of the FTO structure was observed, accompanied by a decrease in catalytic activity. Size exclusion chromatography and MST data confirmed the ability of FTO from both expression systems to form homodimers. The MST-determined dissociation constant of the FTO homodimer was consistent with their in vivo formation in human cells. Finally, a low-resolution structure of the FTO homodimer was built based on SAXS data.

scholarly journals Regorafenib-Attenuated, Bleomycin-Induced Pulmonary Fibrosis by Inhibiting the TGF-β1 Signaling Pathway

2021 ◽  
Vol 22 (4) ◽  
pp. 1985
Author(s):  
Xiaohe Li ◽  
Ling Ma ◽  
Kai Huang ◽  
Yuli Wei ◽  
Shida Long ◽  
...  
Keyword(s):  
Pulmonary Fibrosis ◽  
Signaling Pathway ◽  
Transforming Growth Factor ◽  
Kinase Inhibitor ◽  
In Vivo Experiments ◽  
Age Related ◽  
And Migration ◽  
Tgf Β1

Idiopathic pulmonary fibrosis (IPF) is a fatal and age-related pulmonary disease. Nintedanib is a receptor tyrosine kinase inhibitor, and one of the only two listed drugs against IPF. Regorafenib is a novel, orally active, multi-kinase inhibitor that has similar targets to nintedanib and is applied to treat colorectal cancer and gastrointestinal stromal tumors in patients. In this study, we first identified that regorafenib could alleviate bleomycin-induced pulmonary fibrosis in mice. The in vivo experiments indicated that regorafenib suppresses collagen accumulation and myofibroblast activation. Further in vitro mechanism studies showed that regorafenib inhibits the activation and migration of myofibroblasts and extracellular matrix production, mainly through suppressing the transforming growth factor (TGF)-β1/Smad and non-Smad signaling pathways. In vitro studies have also indicated that regorafenib could augment autophagy in myofibroblasts by suppressing TGF-β1/mTOR (mechanistic target of rapamycin) signaling, and could promote apoptosis in myofibroblasts. In conclusion, regorafenib attenuates bleomycin-induced pulmonary fibrosis by suppressing the TGF-β1 signaling pathway.

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