Reversible Lysine Acetylation Regulates Activity of Human Glycine N-Acyltransferase-like 2 (hGLYATL2): Implications for Production of Glycine-conjugated Signaling Molecules

Background: Lysine acetylation is a major post-translational modification of proteins. Results: Human glycine N-acyltransferase-like 2 is regulated by acetylation/deacetylation of lysine 19. Conclusion: Reversible lysine acetylation is important in regulating the activity and function of human glycine N-acyltransferases. Significance: Our study links post-translational modification of proteins with the production of lipid signaling molecules, the N-acyl glycines

Differential binding of tropomyosin isoforms to actin modified with m-maleimidobenzoyl-N-hydroxysuccinimide ester and fluorescein-5-isothiocyanate

AbstractDifferential interactions of tropomyosin (TM) isoforms with actin can be important for determination of the thin filament functions. A mechanism of tropomyosin binding to actin was studied by comparing interactions of five αTM isoforms with actin modified with m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) and with fluorescein-5-isothiocyanate (FITC). MBS attachment sites were revealed with mass spectrometry methods. We found that the predominant actin fraction was cross-linked by MBS within subdomain 3. A smaller fraction of the modified actin was cross-linked within subdomain 2 and between subdomains 2 and 1. Moreover, investigated actins carried single labels in subdomains 1, 2, and 3. Such extensive modification caused a large decrease in actin affinity for skeletal and smooth muscle tropomyosins, nonmuscle TM2, and chimeric TM1b9a. In contrast, binding of nonmuscle isoform TM5a was less affected. Isoform’s affinity for actin modified in subdomain 2 by binding of FITC to Lys61 was intermediate between the affinity for native actin and MBS-modified actin except for TM5a, which bound to FITC–actin with similar affinity as to actin modified with MBS. The analysis of binding curves according to the McGhee–von Hippel model revealed that binding to an isolated site, as well as cooperativity of binding to a contiguous site, was affected by both actin modifications in a TM isoform-specific manner

Search for new synthetic immunosuppressants II. Tetrazole analogues of hymenistatin I

Linear and cyclic hymenistatin I (HS I) analogues with dipeptide segments Ile2-Pro3, Pro3-Pro4 and Val6-Pro7 replaced by their tetrazole analogues Ile-Ψ[CN4]-Ala3, Pro3-Ψ[CN4]-Ala4 and Val6-Ψ[CN4]-Alawere synthesized by the solid phase peptide synthesis method and cyclized with the TBTU and/or HATU reagent. The peptides were examined for their immunosuppressive activity in the lymphocyte proliferation test (LPT)

Desorption/ionization on silicon for small molecules : a promising alternative to MALDI TOF

A method has been developed for laser desorption/ionization of catecholamines from porous silicon. This methodology is particularly attractive for analysis of small molecules. MALDI TOF mass spectrometry, although a very sensitive technique, utilizes matrices that need to be mixed with the sample prior to their analysis. Each matrix produces its own background, particularly in the low-molecular mass region. Therefore, detection and identification of molecules below 400 Da can be difficult. Desorption/ionization of samples deposited on porous silicon does not require addition of a matrix, thus, spectra in the low-molecular mass region can be clearly readable. Here, we describe a method for the analysis of catecholamines. While MALDI TOF is superior for proteomics/peptidomics, desorption/ionization from porous silicon can extend the operating range of a mass spectrometer for studies on metabolomics (small organic molecules and their metabolites, such as chemical neurotransmitters, prostaglandins, steroids, etc.)

Big Data in der Lehre in den Sozialwissenschaften. Schlussbericht im Auftrag der Schweizerischen Akademie der Geistes- und Sozialwissenschaften

Spätestens seit der Lancierung des Nationalen Forschungsprogramms 75 im Jahr 2015 hat das Thema zu Big Data breitenwirksam Einzug in die akademische Forschung gehalten: In der Informatik, der Physik, der Mathematik. Auch die Wirtschaftswissenschaften befassen sich häufig mit Big Data – die anderen sozialwissenschaftlichen Disziplinen hingegen kaum. Wie sieht es in der Lehre aus? Der Bericht «Big Data in der Lehre in den Sozialwissenschaften» gibt klare Hinweise. Er basiert auf einer Online-Befragung von rund 400 Mitarbeiterinnen und Mitarbeitern an Schweizer Universitäten und Hochschulen, die in der sozialwissenschaftlichen Forschung und Lehre tätig sind: Er zeigt, dass in den Sozialwissenschaften und verwandten Disziplinen Big Data häufiger in der Forschung als in der Lehre Verwendung findet. In der Lehre ist Big Data zwar ebenfalls angekommen, jedoch nicht hinreichend institutionalisiert und wenig auf die akademischen Curricula abgestimmt. Es sei an der Zeit, den Mehrwert von Big Data «aufzuzeigen sowie die Kombination von unterschiedlichen Datentypen und weiteren möglichen Forschungsdesigns zu thematisieren», schreiben Markus Zürcher und Peter Farago in der Einführung zum Bericht

Metabolism of Cryptic Peptides Derived from Neuropeptide FF Precursors: The Involvement of Insulin-Degrading Enzyme

The term “cryptome” refers to the subset of cryptic peptides with bioactivities that are often unpredictable and very different from the parent protein. These cryptic peptides are generated by proteolytic cleavage of proteases, whose identification in vivo can be very challenging. In this work, we show that insulin-degrading enzyme (IDE) is able to degrade specific amino acid sequences present in the neuropeptide pro-NPFFA (NPFF precursor), generating some cryptic peptides that are also observed after incubation with rat brain cortex homogenate. The reported experimental findings support the increasingly accredited hypothesis, according to which, due to its wide substrate selectivity, IDE is involved in a wide variety of physiopathological processes

Synthesis and characterisation of PEG-peptide surfaces for proteolytic enzyme detection

Peptide surfaces were obtained by the covalent immobilisation of fluorescently labelled pentapeptides carboxyfluorescein–glycine–arginine–methionine–leucine–glycine, either directly or through a poly(ethylene glycol) (PEG) linker on modified silicon wafers. Each step during the preparation of the peptide surfaces was confirmed by several surface characterisation techniques. Time-of-flight secondary ion mass spectrometry (ToF-SIMS) and X-ray photoelectron spectroscopy were used to determine the surface composition, the wafers philicity was measured by contact angle and atomic force microscopy was used to investigate the surface morphology. Exposure of the peptide surfaces to trypsin resulted in the release of a fluorescently labelled peptide product, which allowed the kinetics of the enzymatic reaction to be followed with the aid of fluorescence spectroscopy. The electrospray ionisation mass spectrometry analysis of the post-digestion solution confirmed that the pentapeptides attached to the solid support undergo specific trypsin hydrolysis at the C-terminus of the arginine residues. Detailed surface analyses before and after the enzyme action was performed using ToF-SIMS. Because of the limited accessibility of the short peptide directly attached to the surface, a quantitative yield of enzymatic hydrolysis was observed only in case when the peptide was bound through the PEG linker. The insertion of the PEG linker increased the number of immobilised peptides and the rate of enzymatic digestion which consequently improved the quality of the enzyme assays. The described approach may be used for different peptide sequences designed for other proteases. Figure Monitoring of trypsin hydrolysis on PEG-peptide surfac

Constant activity of glutamine synthetase after morphine administration versus proteomic results

Glutamine synthetase is a key enzyme which has a regulatory role in the brain glutamate pool. According to previously published proteomic analysis, it was shown that the expression level of this enzyme is affected by morphine administration. In our study, we examined the activity of glutamine synthetase in various structures of rat brain (cortex, striatum, hippocampus and spinal cord) that are biochemically and functionally involved in drug addiction and antinociception caused by morphine. We were not able to observe any significant changes in the enzyme activity between morphine-treated and control samples despite previously reported changes in the expression levels of this enzyme. These findings stressed the fact that changes observed in the expression of particular proteins during proteomic studies may not be correlated with its activity

Stripping voltammetric detection of insulin at liquid–liquid microinterfaces in the presence of bovine albumin

Electrochemistry at the interface between two immiscible electrolyte solutions (ITIES) provides a platform for label-free detection of biomolecules. In this study, adsorptive stripping voltammetry (AdSV) was implemented at an array of microscale ITIES for the detection of the antidiabetic hormone insulin. By exploiting the potential-controlled adsorption of insulin at the ITIES, insulin was detected at 10 nM via subsequent voltammetric desorption. This is the lowest detected concentration reported to-date for a protein by electrochemistry at the ITIES. Surface coverage calculations indicate that between 0.1 and 1 monolayer of insulin forms at the interface over the 10 – 1000 nM concentration range of the hormone. In a step toward assessment of selectivity, the optimum adsorption potentials for insulin and albumin were determined to be 0.900 V and 0.975 V, respectively. When present in an aqueous mixture with albumin, insulin was detected by tuning the adsorption potential to 0.9 V, albeit with reduced sensitivity. This provides the first example of selective detection of one protein in the presence of another by exploiting optimal adsorption potentials. The results presented here provide a route to the improvement of detection limits and achievement of selectivity for protein detection by electrochemistry at the ITIES

Aptamer-based multiplexed proteomic technology for biomarker discovery

Interrogation of the human proteome in a highly multiplexed and efficient manner remains a coveted and challenging goal in biology. We present a new aptamer-based proteomic technology for biomarker discovery capable of simultaneously measuring thousands of proteins from small sample volumes (15 [mu]L of serum or plasma). Our current assay allows us to measure ~800 proteins with very low limits of detection (1 pM average), 7 logs of overall dynamic range, and 5% average coefficient of variation. This technology is enabled by a new generation of aptamers that contain chemically modified nucleotides, which greatly expand the physicochemical diversity of the large randomized nucleic acid libraries from which the aptamers are selected. Proteins in complex matrices such as plasma are measured with a process that transforms a signature of protein concentrations into a corresponding DNA aptamer concentration signature, which is then quantified with a DNA microarray. In essence, our assay takes advantage of the dual nature of aptamers as both folded binding entities with defined shapes and unique sequences recognizable by specific hybridization probes. To demonstrate the utility of our proteomics biomarker discovery technology, we applied it to a clinical study of chronic kidney disease (CKD). We identified two well known CKD biomarkers as well as an additional 58 potential CKD biomarkers. These results demonstrate the potential utility of our technology to discover unique protein signatures characteristic of various disease states. More generally, we describe a versatile and powerful tool that allows large-scale comparison of proteome profiles among discrete populations. This unbiased and highly multiplexed search engine will enable the discovery of novel biomarkers in a manner that is unencumbered by our incomplete knowledge of biology, thereby helping to advance the next generation of evidence-based medicine