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"Stein, Frank"
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The Al-Rich Part of the Fe-Al Phase Diagram
The Al-rich part of the Fe-Al phase diagram between 50 and 80 at.% Al including the complex intermetallic phases Fe
5
Al
8
(ε), FeAl
2
, Fe
2
Al
5
, and Fe
4
Al
13
was re-investigated in detail. A series of 19 alloys was produced and heat-treated at temperatures in the range from 600 to 1100 °C for up to 5000 h. The obtained data were further complemented by results from a number of diffusion couples, which helped to determine the homogeneity ranges of the phases FeAl
2
, Fe
2
Al
5
, and Fe
4
Al
13
. All microstructures were inspected by scanning electron microscopy (SEM), and chemical compositions of the equilibrium phases as well as of the alloys were obtained by electron probe microanalysis (EPMA). Crystal structures and the variation of the lattice parameters were studied by x-ray diffraction (XRD) and differential thermal analysis (DTA) was applied to measure all types of transition temperatures. From these results, a revised version of the Al-rich part of the phase diagram was constructed.
Journal Article
Thermal proteome profiling for interrogating protein interactions
Thermal proteome profiling (TPP) is based on the principle that, when subjected to heat, proteins denature and become insoluble. Proteins can change their thermal stability upon interactions with small molecules (such as drugs or metabolites), nucleic acids or other proteins, or upon post‐translational modifications. TPP uses multiplexed quantitative mass spectrometry‐based proteomics to monitor the melting profile of thousands of expressed proteins. Importantly, this approach can be performed
in vitro
,
in situ
, or
in vivo
. It has been successfully applied to identify targets and off‐targets of drugs, or to study protein–metabolite and protein–protein interactions. Therefore, TPP provides a unique insight into protein state and interactions in their native context and at a proteome‐wide level, allowing to study basic biological processes and their underlying mechanisms.
Graphical Abstract
This tutorial explains the principles of thermal proteome profiling (TPP) and analyzes the different steps of a TPP experiment. It reviews the recent developments and current applications of this methodology, and provides an outlook of possible new applications.
Journal Article
Thermal proteome profiling in bacteria: probing protein state in vivo
Increasing antibiotic resistance urges for new technologies for studying microbes and antimicrobial mechanism of action. We adapted thermal proteome profiling (TPP) to probe the thermostability of
Escherichia coli
proteins
in vivo
.
E. coli
had a more thermostable proteome than human cells, with protein thermostability depending on subcellular location—forming a high‐to‐low gradient from the cell surface to the cytoplasm. While subunits of protein complexes residing in one compartment melted similarly, protein complexes spanning compartments often had their subunits melting in a location‐wise manner. Monitoring the
E. coli
meltome and proteome at different growth phases captured changes in metabolism. Cells lacking TolC, a component of multiple efflux pumps, exhibited major physiological changes, including differential thermostability and levels of its interaction partners, signaling cascades, and periplasmic quality control. Finally, we combined
in vitro
and
in vivo
TPP to identify targets of known antimicrobial drugs and to map their downstream effects. In conclusion, we demonstrate that TPP can be used in bacteria to probe protein complex architecture, metabolic pathways, and intracellular drug target engagement.
Synopsis
Thermal proteome profiling is adapted to
Escherichia coli
to probe the thermostability of proteins
in vivo
, yielding insights into protein complex architecture, protein activity, cellular metabolic state, intracellular drug target engagement and drug downstream effects.
The
E. coli
proteome is more thermostable than the human one, with protein thermostability depending on protein subcellular location.
Subunits of protein complexes residing in one compartment melt similarly, while protein complexes spanning compartments often have their subunits melting in a location‐wise manner.
Knockout of tolC led to the thermal destabilization of its interaction partners, the downregulation of a major porin (OmpF), and increased periplasmic stress.
Graphical Abstract
Thermal proteome profiling is adapted to
Escherichia coli
to probe the thermostability of proteins
in vivo
, yielding insights into protein complex architecture, protein activity, cellular metabolic state, intracellular drug target engagement and drug downstream effects.
Journal Article
The functional landscape of the human phosphoproteome
Protein phosphorylation is a key post-translational modification regulating protein function in almost all cellular processes. Although tens of thousands of phosphorylation sites have been identified in human cells, approaches to determine the functional importance of each phosphosite are lacking. Here, we manually curated 112 datasets of phospho-enriched proteins, generated from 104 different human cell types or tissues. We re-analyzed the 6,801 proteomics experiments that passed our quality control criteria, creating a reference phosphoproteome containing 119,809 human phosphosites. To prioritize functional sites, we used machine learning to identify 59 features indicative of proteomic, structural, regulatory or evolutionary relevance and integrate them into a single functional score. Our approach identifies regulatory phosphosites across different molecular mechanisms, processes and diseases, and reveals genetic susceptibilities at a genomic scale. Several regulatory phosphosites were experimentally validated, including identifying a role in neuronal differentiation for phosphosites in SMARCC2, a member of the SWI/SNF chromatin-remodeling complex.
Phosphorylation sites are ranked for functional relevance using a comprehensive, high-quality human phosphoproteome.
Journal Article
Laves phases: a review of their functional and structural applications and an improved fundamental understanding of stability and properties
Laves phases with their comparably simple crystal structure are very common intermetallic phases and can be formed from element combinations all over the periodic table resulting in a huge number of known examples. Even though this type of phases is known for almost 100 years, and although a lot of information on stability, structure, and properties has accumulated especially during the last about 20 years, systematic evaluation and rationalization of this information in particular as a function of the involved elements is often lacking. It is one of the two main goals of this review to summarize the knowledge for some selected respective topics with a certain focus on non-stoichiometric, i.e., non-ideal Laves phases. The second, central goal of the review is to give a systematic overview about the role of Laves phases in all kinds of materials for functional and structural applications. There is a surprisingly broad range of successful utilization of Laves phases in functional applications comprising Laves phases as hydrogen storage material (Hydraloy), as magneto-mechanical sensors and actuators (Terfenol), or for wear- and corrosion-resistant coatings in corrosive atmospheres and at high temperatures (Tribaloy), to name but a few. Regarding structural applications, there is a renewed interest in using Laves phases for creep-strengthening of high-temperature steels and new respective alloy design concepts were developed and successfully tested. Apart from steels, Laves phases also occur in various other kinds of structural materials sometimes effectively improving properties, but often also acting in a detrimental way.
Journal Article
Computationally-driven engineering of sublattice ordering in a hexagonal AlHfScTiZr high entropy alloy
Multi-principle element alloys have enormous potential, but their exploration suffers from the tremendously large range of configurations. In the last decade such alloys have been designed with a focus on random solid solutions. Here we apply an experimentally verified, combined thermodynamic and first-principles design strategy to reverse the traditional approach and to generate a new type of hcp Al-Hf-Sc-Ti-Zr high entropy alloy with a hitherto unique structure. A phase diagram analysis narrows down the large compositional space to a well-defined set of candidates. First-principles calculations demonstrate the energetic preference of an ordered superstructure over the competing disordered solid solutions. The chief ingredient is the Al concentration, which can be tuned to achieve a D0
19
ordering on the hexagonal lattice. The computationally designed D0
19
superstructure is experimentally confirmed by transmission electron microscopy and X-ray studies. Our scheme enables the exploration of a new class of high entropy alloys.
Journal Article
Discovery of RNA-binding proteins and characterization of their dynamic responses by enhanced RNA interactome capture
Following the realization that eukaryotic RNA-binding proteomes are substantially larger than anticipated, we must now understand their detailed composition and dynamics. Methods such as RNA interactome capture (RIC) have begun to address this need. However, limitations of RIC have been reported. Here we describe enhanced RNA interactome capture (eRIC), a method based on the use of an LNA-modified capture probe, which yields numerous advantages including greater specificity and increased signal-to-noise ratios compared to existing methods. In Jurkat cells, eRIC reduces the rRNA and DNA contamination by >10-fold compared to RIC and increases the detection of RNA-binding proteins. Due to its low background, eRIC also empowers comparative analyses of changes of RNA-bound proteomes missed by RIC. For example, in cells treated with dimethyloxalylglycine, which inhibits RNA demethylases, eRIC identifies m6A-responsive RNA-binding proteins that escape RIC. eRIC will facilitate the unbiased characterization of RBP dynamics in response to biological and pharmacological cues.
RNA interactome capture allows the detailed investigation of RNA-bound proteomes. Here the authors describe enhanced RNA-interactome capture using LNA-modified probes for increased sensitivity and specificity.
Journal Article
Dendritic autophagy degrades postsynaptic proteins and is required for long-term synaptic depression in mice
The pruning of dendritic spines during development requires autophagy. This process is facilitated by long-term depression (LTD)-like mechanisms, which has led to speculation that LTD, a fundamental form of synaptic plasticity, also requires autophagy. Here, we show that the induction of LTD via activation of NMDA receptors or metabotropic glutamate receptors initiates autophagy in the postsynaptic dendrites in mice. Dendritic autophagic vesicles (AVs) act in parallel with the endocytic machinery to remove AMPA receptor subunits from the membrane for degradation. During NMDAR-LTD, key postsynaptic proteins are sequestered for autophagic degradation, as revealed by quantitative proteomic profiling of purified AVs. Pharmacological inhibition of AV biogenesis, or conditional ablation of atg5 in pyramidal neurons abolishes LTD and triggers sustained potentiation in the hippocampus. These deficits in synaptic plasticity are recapitulated by knockdown of atg5 specifically in postsynaptic pyramidal neurons in the CA1 area. Conducive to the role of synaptic plasticity in behavioral flexibility, mice with autophagy deficiency in excitatory neurons exhibit altered response in reversal learning. Therefore, local assembly of the autophagic machinery in dendrites ensures the degradation of postsynaptic components and facilitates LTD expression.
Pruning dendritic spines requires autophagy. Here, the authors show that autophagy is required for long-term depression (LTD), a major form of synaptic plasticity. LTD induces the biogenesis of autophagic vesicles locally in dendrites to facilitate the degradation of postsynaptic proteins.
Journal Article
Structural analysis of human ARS2 as a platform for co-transcriptional RNA sorting
ARS2 is a highly conserved metazoan protein involved in numerous aspects of nuclear RNA metabolism. As a direct partner of the nuclear cap-binding complex (CBC), it mediates interactions with diverse RNA processing and transport machineries in a transcript-dependent manner. Here, we present the human ARS2 crystal structure, which exhibits similarities and metazoan-specific differences to the plant homologue SERRATE, most notably an additional RRM domain. We present biochemical, biophysical and cellular interactome data comparing wild type and mutant ARS2 that identify regions critical for interactions with FLASH (involved in histone mRNA biogenesis), NCBP3 (a putative cap-binding protein involved in mRNA export) and single-stranded RNA. We show that FLASH and NCBP3 have overlapping binding sites on ARS2 and that CBC–ARS2–NCBP3 form a ternary complex that is mutually exclusive with CBC–ARS–PHAX (involved in snRNA export). Our results support that mutually exclusive higher-order CBC–ARS2 complexes are critical in determining Pol II transcript fate.
Arsenic resistance protein 2 (ARS2) plays an important role in nuclear RNA metabolism and interacts with the nuclear cap-binding complex (CBC). Here the authors present the human ARS2 structure and identify regions important for its interactions with binding partners supporting that mutually exclusive higher order CBC-ARS2 complexes are formed.
Journal Article