Time-resolved in vivo ubiquitinome profiling by DIA-MS reveals USP7 targets on a proteome-wide scale.

Mass spectrometry (MS)-based ubiquitinomics provides system-level understanding of ubiquitin signaling. Here we present a scalable workflow for deep and precise in vivo ubiquitinome profiling, coupling an improved sample preparation protocol with data-independent acquisition (DIA)-MS and neural network-based data processing specifically optimized for ubiquitinomics. Compared to data-dependent acquisition (DDA), our method more than triples identification numbers to 70,000 ubiquitinated peptides in single MS runs, while significantly improving robustness and quantification precision. Upon inhibition of the oncology target USP7, we simultaneously record ubiquitination and consequent changes in abundance of more than 8,000 proteins at high temporal resolution. While ubiquitination of hundreds of proteins increases within minutes of USP7 inhibition, we find that only a small fraction of those are ever degraded, thereby dissecting the scope of USP7 action. Our method enables rapid mode-of-action profiling of candidate drugs targeting DUBs or ubiquitin ligases at high precision and throughput

A time travel through nematology in Germany – From the beginnings to the use of artificial intelligence

Das Nachrichtenblatt für den Deutschen Pflanzenschutzdienst, unser heutiges Journal für Kulturpflanzen, feiert seinen 100. Geburtstag. Seinem Ziel, „den im praktischen Pflanzenschutzdienst Tätigen Belehrung und Informationen (zu) übermitteln“, wie Otto Appel zur Einführung schrieb, ist es bis heute treu geblieben. Dies gilt auch für den Bereich der Nematologie. Die Themen haben sich dabei über die Zeit weiterentwickelt. Standen früher Biologie und Wirtspflanzenspektrum einzelner Arten pflanzenparasitärer Nematoden im Fokus des Interesses, so sind es heute Wirt-Parasit Interaktionen, Resistenz und Toleranz von Kulturpflanzen oder der Einfluss von Klimawandel und Globalisierung auf die Verbreitung und das Schadpotenzial der Nematoden. Der vorliegende Beitrag blickt zurück auf die Themen vor 100 Jahren, stellt am Beispiel laufender Arbeiten am Julius Kühn-Institut aktuelle Forschungsthemen vor und gibt einen Ausblick auf die Themen der Zukunft.The Nachrichtenblatt für den Deutschen Pflanzenschutzdienst, today's Journal of Cultivated Plants, celebrates its 100th birthday. It has remained true to its goal of “providing instruc­tion and information to those involved in the practical plant protection service” to this day as stated in its first issue by Otto Appel. This also applies to the field of nematology. The topics have expanded over time and developed further on an international level. While the main focus was initially on the biology and host plant spectrum of the various nematode species, today it is on host-parasite interactions, resistance and tolerance of cultivated plants and the influence of climate change and globalization on the distribution and harmful effects of nematodes. The present article looks back at the topics 100 years ago, presents current research topics using the example of ongoing work at the Julius Kühn Institute and provides an outlook on the topics of the future

Mps1 regulates kinetochore-microtubule attachment stability via the ska complex to ensure error-free chromosome segregation

The spindle assembly checkpoint kinase Mps1 not only inhibits anaphase but also corrects erroneous attachments that could lead to missegregation and aneuploidy. However, Mps1’s error correction-relevant substrates are unknown. Using a chemically tuned kinetochore-targeting assay, we show that Mps1 destabilizes microtubule attachments (K fibers) epistatically to Aurora B, the other major error-correcting kinase. Through quantitative proteomics, we identify multiple sites of Mps1-regulated phosphorylation at the outer kinetochore. Substrate modification was microtubule sensitive and opposed by PP2A-B56 phosphatases that stabilize chromosome-spindle attachment. Consistently, Mps1 inhibition rescued K-fiber stability after depleting PP2A-B56. We also identify the Ska complex as a key effector of Mps1 at the kinetochore-microtubule interface, as mutations that mimic constitutive phosphorylation destabilized K fibers in vivo and reduced the efficiency of the Ska complex’s conversion from lattice diffusion to end-coupled microtubule binding in vitro. Our results reveal how Mps1 dynamically modifies kinetochores to correct improper attachments and ensure faithful chromosome segregation

Identification of SRPK1 and SRPK2 as the Major Cellular Protein Kinases Phosphorylating Hepatitis B Virus Core Protein

Phosphorylation of hepatitis B virus (HBV) core protein has recently been shown to be a prerequisite for pregenomic RNA encapsidation into viral capsids, but the host cell kinases mediating this essential step of the HBV replication cycle have not been identified. We detected two kinases of 95 and 115 kDa in HuH-7 total cell lysates which interacted specifically with the HBV core protein and phosphorylated its arginine-rich C-terminal domain. The 95-kDa kinase was purified and characterized as SR protein-specific kinase 1 (SRPK1) by mass spectrometry. Based on this finding, the 115-kDa kinase could be identified as the related kinase SRPK2 by immunoblot analysis. In vitro, both SRPKs phosphorylated HBV core protein on the same serine residues which are found to be phosphorylated in vivo. Moreover, the major cellular HBV core kinase activity detected in the total cell lysate showed biochemical properties identical to those of SRPK1 and SRPK2, as examined by measuring binding to a panel of chromatography media. We also clearly demonstrate that neither the cyclin-dependent kinases Cdc2 and Cdk2 nor protein kinase C, previously implicated in HBV core protein phosphorylation, can account for the HBV core protein kinase activity. We conclude that both SRPK1 and SRPK2 are most likely the cellular protein kinases mediating HBV core protein phosphorylation during viral infection and therefore represent important host cell targets for therapeutic intervention in HBV infection

Proteomics analysis of protein kinases by target class-selective prefractionation and tandem mass spectrometry.

Protein kinases constitute a large superfamily of enzymes with key regulatory functions in nearly all signal transmission processes of eukaryotic cells. However, due to their relatively low abundance compared with the vast majority of cellular proteins, currently available proteomics techniques do not permit the comprehensive biochemical characterization of protein kinases. To address these limitations, we have developed a prefractionation strategy that uses a combination of immobilized low molecular weight inhibitors for the selective affinity capture of protein kinases. This approach resulted in the direct purification of cell type-specific sets of expressed protein kinases, and more than 140 different members of this enzyme family could be detected by LC-MS/MS. Furthermore the enrichment technique combined with phosphopeptide fractionation led to the identification of more than 200 different phosphorylation sites on protein kinases, which often remain occluded in global phosphoproteome analysis. As the phosphorylation states of protein kinases can provide a readout for the signaling activities within a cellular system, kinase-selective phosphoproteomics based on the procedures described here has the potential to become an important tool in signal transduction analysis

Exploiting features of adenovirus replication to support mammalian kinase production

Faced with the current wealth of genomic data, it is essential to have robust and reliable methods of converting DNA sequences into their functional gene products. We demonstrate here that when conditions are established that take advantage of the replication-associated virus amplification, the virus-induced shutdown of host protein synthesis as well as the activation of signalling pathways that normally occur during virus replication, adenovirus biology can be exploited to generate a potent kinase expression system. Residual virus in the protein production has always been a limitation for adenovirus systems and we describe a DNA intercalator/ultraviolet light treatment that eliminates residual adenovirus in protein preparations that has no deleterious effect on enzyme activity. The use of mammalian cells in combination with adenovirus generated a variety of active enzymes which could not be produced in Escherichia coli or baculovirus-infected insect cells. Thus, the utility of adenovirus-mediated enzyme expression as a versatile alternative to established protein production technologies is demonstrated