Деформационное упрочнение начально-изотропных металлов при деформировании по траекториям малой кривизны

На примере стали мартенситного класса исследованы закономерности деформационного упрочнения при нагружении по траекториям, имеющим вид двухзвенных ломаных, которым соответствуют траектории деформирования малой кривизны. Показано, что поверхность нагружения, разделяющая области упругого и упругопластического деформирования, смещается в направлении вектора, который соединяет центр поверхности нагружения и изображающую точку на траектории нагружения, при этом не изменяется форма ее фронтальной части. Зависимость величины смещения центра поверхности нагружения от интенсивности накопленных пластических деформаций описывается кривой, инвариантной к виду траектории нагружения.На прикладі сталі мартенситного класу досліджено закономірності деформаційного зміцнення при навантаженні по траєкторіях, що мають вигляд дволанкових ламаних, яким відповідають траєкторії деформування малої кривини. Показано, що поверхня навантаження, яка розділяє області пружного та пружнопластичного деформування, зміщується у напрямку вектора, який з ’єднує центр поверхні навантаження та відображуючу точку на траєкторії навантаження, при цьому форма фронтальної частини не змінюється. Залежність величини зміщення центра поверхні навантаження від інтенсивності накопичених пластичних деформацій описується кривою, яка є інваріантною відносно траєкторії навантаження.By the example of martensitic steel we study regularities of strain hardening under loading along two-section broken lines corresponding to slightly curved strain paths. It is shown that the loading surface separating domains of elastic and elastoplastic strains (yield surface) is displaced in the direction of a vector connecting the surface center with the loading trajectory image point, while the shape of its frontal part remains unchanged. The yield surface center displacement versus the intensity of accumulated plastic strains is described by a curve invariant to the loading trajectory

Confident and sensitive phosphoproteomics using combinations of collision induced dissociation and electron transfer dissociation

We present a workflow using an ETD-optimised version of Mascot Percolator and a modified version of SLoMo (turbo-SLoMo) for analysis of phosphoproteomic data. We have benchmarked this against several database searching algorithms and phosphorylation site localisation tools and show that it offers highly sensitive and confident phosphopeptide identification and site assignment with PSM-level statistics, enabling rigorous comparison of data acquisition methods. We analysed the Plasmodium falciparum schizont phosphoproteome using for the first time, a data-dependent neutral loss-triggered-ETD (DDNL) strategy and a conventional decision-tree method. At a posterior error probability threshold of 0.01, similar numbers of PSMs were identified using both methods with a 73% overlap in phosphopeptide identifications. The false discovery rate associated with spectral pairs where DDNL CID/ETD identified the same phosphopeptide was < 1%. 72% of phosphorylation site assignments using turbo-SLoMo without any score filtering, were identical and 99.8% of these cases are associated with a false localisation rate of < 5%. We show that DDNL acquisition is a useful approach for phosphoproteomics and results in an increased confidence in phosphopeptide identification without compromising sensitivity or duty cycle. Furthermore, the combination of Mascot Percolator and turbo-SLoMo represents a robust workflow for phosphoproteomic data analysis using CID and ETD fragmentation. Biological significance Protein phosphorylation is a ubiquitous post-translational modification that regulates protein function. Mass spectrometry-based approaches have revolutionised its analysis on a large-scale but phosphorylation sites are often identified by single phosphopeptides and therefore require more rigorous data analysis to unsure that sites are identified with high confidence for follow-up experiments to investigate their biological significance. The coverage and confidence of phosphoproteomic experiments can be enhanced by the use of multiple complementary fragmentation methods. Here we have benchmarked a data analysis pipeline for analysis of phosphoproteomic data generated using CID and ETD fragmentation and used it to demonstrate the utility of a data-dependent neutral loss triggered ETD fragmentation strategy for high confidence phosphopeptide identification and phosphorylation site localisation

Current challenges in software solutions for mass spectrometry-based quantitative proteomics

This work was in part supported by the PRIME-XS project, grant agreement number 262067, funded by the European Union seventh Framework Programme; The Netherlands Proteomics Centre, embedded in The Netherlands Genomics Initiative; The Netherlands Bioinformatics Centre; and the Centre for Biomedical Genetics (to S.C., B.B. and A.J.R.H); by NIH grants NCRR RR001614 and RR019934 (to the UCSF Mass Spectrometry Facility, director: A.L. Burlingame, P.B.); and by grants from the MRC, CR-UK, BBSRC and Barts and the London Charity (to P.C.

A selective and orally bioavailable VHL-recruiting PROTAC achieves SMARCA2 degradation in vivo

Targeted protein degradation offers an alternative modality to classical inhibition and holds the promise of addressing previously undruggable targets to provide novel therapeutic options for patients. Heterobifunctional molecules co-recruit a target protein and an E3 ligase, resulting in ubiquitylation and proteosome-dependent degradation of the target. In the clinic, the oral route of administration is the option of choice but has only been achieved so far by CRBN- recruiting bifunctional degrader molecules. We aimed to achieve orally bioavailable molecules that selectively degrade the BAF Chromatin Remodelling complex ATPase SMARCA2 over its closely related paralogue SMARCA4, to allow in vivo evaluation of the synthetic lethality concept of SMARCA2 dependency in SMARCA4-deficient cancers. Here we outline structure- and property-guided approaches that led to orally bioavailable VHL-recruiting degraders. Our tool compound, ACBI2, shows selective degradation of SMARCA2 over SMARCA4 in ex vivo human whole blood assays and in vivo efficacy in SMARCA4-deficient cancer models. This study demonstrates the feasibility for broadening the E3 ligase and physicochemical space that can be utilised for achieving oral efficacy with bifunctional molecules

Interconversion between active and inactive TATA-binding protein transcription complexes in the mouse genome

The TATA binding protein (TBP) plays a pivotal role in RNA polymerase II (Pol II) transcription through incorporation into the TFIID and B-TFIID complexes. The role of mammalian B-TFIID composed of TBP and B-TAF1 is poorly understood. Using a complementation system in genetically modified mouse cells where endogenous TBP can be conditionally inactivated and replaced by exogenous mutant TBP coupled to tandem affinity purification and mass spectrometry, we identify two TBP mutations, R188E and K243E, that disrupt the TBP–BTAF1 interaction and B-TFIID complex formation. Transcriptome and ChIP-seq analyses show that loss of B-TFIID does not generally alter gene expression or genomic distribution of TBP, but positively or negatively affects TBP and/or Pol II recruitment to a subset of promoters. We identify promoters where wild-type TBP assembles a partial inactive preinitiation complex comprising B-TFIID, TFIIB and Mediator complex, but lacking TFIID, TFIIE and Pol II. Exchange of B-TFIID in wild-type cells for TFIID in R188E and K243E mutant cells at these primed promoters completes preinitiation complex formation and recruits Pol II to activate their expression. We propose a novel regulatory mechanism involving formation of a partial preinitiation complex comprising B-TFIID that primes the promoter for productive preinitiation complex formation in mammalian cells

O-GlcNAcylation and oxidation of proteins: is signalling in the cardiovascular system becoming sweeter?

O-GlcNAcylation is an unusual form of protein glycosylation, where a single-sugar [GlcNAc (N-acetylglucosamine)] is added (via β-attachment) to the hydroxyl moiety of serine and threonine residues of nuclear and cytoplasmic proteins. A complex and extensive interplay exists between O-GlcNAcylation and phosphorylation. Many phosphorylation sites are also known glycosylation sites, and this reciprocal occupancy may produce different activities or alter the stability in a target protein. The interplay between these two post-translational modifications is not always reciprocal, as some proteins can be concomitantly phosphorylated and O-GlcNAcylated, and the adjacent phosphorylation or O-GlcNAcylation can regulate the addition of either moiety. Increased cardiovascular production of ROS (reactive oxygen species), termed oxidative stress, has been consistently reported in various chronic diseases and in conditions where O-GlcNAcylation has been implicated as a contributing mechanism for the associated organ injury/protection (for example, diabetes, Alzheimer's disease, arterial hypertension, aging and ischaemia). In the present review, we will briefly comment on general aspects of O-GlcNAcylation and provide an overview of what has been reported for this post-translational modification in the cardiovascular system. We will then specifically address whether signalling molecules involved in redox signalling can be modified by O-GlcNAc (O-linked GlcNAc) and will discuss the critical interplay between O-GlcNAcylation and ROS generation. Experimental evidence indicates that the interactions between O-GlcNAcylation and oxidation of proteins are important not only for cell regulation in physiological conditions, but also under pathological states where the interplay may become dysfunctional and thereby exacerbate cellular injury

Mdm20 Stimulates PolyQ Aggregation via Inhibiting Autophagy Through Akt-Ser473 Phosphorylation

Mdm20 is an auxiliary subunit of the NatB complex, which includes Nat5, the catalytic subunit for protein N-terminal acetylation. The NatB complex catalyzes N-acetylation during de novo protein synthesis initiation; however, recent evidence from yeast suggests that NatB also affects post-translational modification of tropomyosin, which is involved in intracellular sorting of aggregated proteins. We hypothesized that an acetylation complex such as NatB may contribute to protein clearance and/or proteostasis in mammalian cells. Using a poly glutamine (polyQ) aggregation system, we examined whether the NatB complex or its components affect protein aggregation in rat primary cultured hippocampal neurons and HEK293 cells. The number of polyQ aggregates increased in Mdm20 over-expressing (OE) cells, but not in Nat5-OE cells. Conversely, in Mdm20 knockdown (KD) cells, but not in Nat5-KD cells, polyQ aggregation was significantly reduced. Although Mdm20 directly associates with Nat5, the overall cellular localization of the two proteins was slightly distinct, and Mdm20 apparently co-localized with the polyQ aggregates. Furthermore, in Mdm20-KD cells, a punctate appearance of LC3 was evident, suggesting the induction of autophagy. Consistent with this notion, phosphorylation of Akt, most notably at Ser473, was greatly reduced in Mdm20-KD cells. These results demonstrate that Mdm20, the so-called auxiliary subunit of the translation-coupled protein N-acetylation complex, contributes to protein clearance and/or aggregate formation by affecting the phosphorylation level of Akt indepenently from the function of Nat5

Proteomics of transcription factors

Peptide mass spectrometry (MS) is an invaluable analytical method in biological and medical research. It is the only technique that, when integrated with liquid chromatography (LC) and database search tools, allows a highly sensitive qualitative characterization and highly accurate quantitative comparison of proteomes. Although many proteomes are much more complex than their corresponding genomes, due to, for example, extreme differences in protein abundance and post-translational modifications, continuous technical advances in MS instrumentation and peptide pre-fractionation techniques lead to increasing fractions of proteomes that can be covered. Nevertheless, the targeted analysis of subsets of proteomes defined by post-translational modifications (PTMs), for example phosphorylation, acetylation, or glycosylation, using specialized enrichment techniques, is required to gain insight into cellular processes that would be inadequately covered by analysis of the full proteome alone. The technological progression in proteomics also benefits the analysis of protein complexes and other relatively small ensembles of proteins. With modern MS instrumentation, a targeted analysis is mostly not required to create a comprehensive picture of protein complexes, including PTMs and protein isoforms. Selected core technologies of proteomics are introduced in Chapter 1. It is mainly focused on MS instrumentation and database searching, but also covers aspects like peptide fragmentation and methods in quantitative proteomics. In this chapter we also give a brief introduction to the general transcription factors (GTFs) TFIID and SAGA and put them into their broader biological context

Planare Primaerstrahler mit integriertem, rauscharmen Empfangskonverter

Within the reported investigation an aperture-coupled patch-element was used for arrayelements because of its high gain, symmetry of the radiation pattern and good ability of integrating electronics. Components like low losses phase shifters and switches for the Ku-band and a digital control unit were designed. An LNC with a simplified planar patch antenna was designed using modern highly integrated chips for automatic assembly. An electronically steerable antennasystem consisting of a solid reflector and a phased array feed, has a smaller gain compared to a conventionally antenna system. This gainreduction is caused by the oversized reflectordiameter due to the prevention of spill over loss. (orig./RHM)Im Rahmen der dokumentierten Forschungsarbeiten wurden aperturgekoppelte Patchelemente als Grundelemente fuer das Array verwendet, da sie hohen Gewinn, Symmetrie des Strahlungsdiagramms und gute Eigenschaften hinsichtlich Integration von Elektronik aufwiesen. Auch sind Komponenten, wie Phasenschieber und Schalter fuer das Ku-Band, sowie eine digitale Steuerung entworfen worden. Ein LNC wurde entwickelt, der im wesentlichen vollautomatisch bestueckbar ist und eine Integration planarer Erreger ermoeglicht. Es fanden moderne, hoch integrierte Bauteile Verwendung. Ein elektronisch schwenkbares Antennensystem, mit einem elektronisch schwenkbarem Erreger und einem starren Reflektor zeigt gegenueber einem klassischen Antennensystem eine deutliche Gewinnreduzierung, da der Reflektor zur Vermeidung von Ueberstrahlung von Schwenken ueberdimensioniert werden muss. (orig./RHM)Available from TIB Hannover: F94B0488+a / FIZ - Fachinformationszzentrum Karlsruhe / TIB - Technische InformationsbibliothekSIGLEBundesministerium fuer Forschung und Technologie (BMFT), Bonn (Germany)DEGerman