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Histone deacetylases are key epigenetic regulators that control T cell-mediated immunity. A T cell-specific deletion ofHdac1(HDAC1cKO) protects mice against experimental autoimmune encephalomyelitis (EAE). However, it remains elusive whether inhibition of HDAC1 enzymatic activity, which could be achieved therapeutically by HDAC1 inhibitor treatment, is sufficient to block EAE induction. In order to address this question, we generated a novel mouse strain that expresses catalytically inactive HDAC1 (HDAC1Off) from theRosa26locus in HDAC1cKOCD4+T cells to mimic selective inhibition of HDAC1 enzymatic activityin vivo. Mice expressing wildtype HDAC1 in HDAC1cKOCD4+T cells (HDAC1On) were generated as corresponding controls. In contrast to HDAC1Onmice, HDAC1Offmice did not develop EAE, and this correlated with diminished leukocyte CNS infiltration. HDAC1OffCD4+T cells in the CNS displayed a severe reduction of IFNγ, IL-17A and TNFα proinflammatory cytokine expression, andin vivoactivated HDAC1OffCD4+T cells downregulated gene sets associated with T cell activation, cytokine expression and cell migration. This indicates impaired effector functions of HDAC1OffCD4+T cells. Taken together, our study demonstrates that the inhibition of the catalytic activity of HDAC1 in T cells is sufficient to achieve a clinical benefit in EAE disease development. This raises the translational perspective of pharmacological HDAC1 inhibition for treating human T cell-mediated autoimmune diseases.HighlightsSuccessful generation of a novel mouse model that expresses enzymatic-inactive HDAC1 to mimic HDAC1 inhibitor treatmentin vivo.Mice expressing enzymatically inactive HDAC1 instead of WT HDAC1 in T cells do not develop EAE and display diminished leukocyte CNS infiltration.In vivoactivated CD4+T cells expressing enzymatic inactive HDAC1 downregulate pathways important for T cell activation, cytokine expression and cell migration.Demonstrate the proof-of-principle that targeting the enzymatic activity of HDAC1 is a promising treatment strategy for autoimmune diseases.

α-synuclein (αS) is an intrinsically disordered protein whose functional ambivalence and protein structural plasticity are iconic. Coordinated protein recruitment ensures proper vesicle dynamics at the synaptic cleft, while deregulated oligomerization on cellular membranes contributes to cell damage and Parkinson’s disease (PD). Despite the protein’s pathophysiological relevance, structural knowledge is limited. Here, we employ NMR spectroscopy and chemical cross-link mass spectrometry on 14 N/ 15 N-labeled αS mixtures to provide for the first time high-resolution structural information of the membrane-bound oligomeric state of αS and demonstrate that in this state, αS samples a surprisingly small conformational space. Interestingly, the study locates familial Parkinson’s disease mutants at the interface between individual αS monomers and reveals different oligomerization processes depending on whether oligomerization occurs on the same membrane surface (cis) or between αS initially attached to different membrane particles (trans). The explanatory power of the obtained high-resolution structural model is used to help determine the mode-of-actionof UCB0599. Here, it is shown that the ligand changes the ensemble of membrane-bound structures, which helps to explain the success this compound, currently being tested in Parkinson’s disease patients in a phase 2 trial, has had in animal models of PD.

Post-translational modifications of histones are important regulators of the DNA damage response (DDR). By using affinity purification mass spectrometry (AP-MS) we discovered that genetic suppressor element 1 (GSE1) forms a complex with the HDAC1/CoREST deacetylase/demethylase co-repressor complex. In-depth phosphorylome analysis revealed that loss of GSE1 results in impaired DDR, ATR signalling and γH2AX formation upon DNA damage induction. Altered profiles of ATR target serine-glutamine motifs (SQ) on DDR-related hallmark proteins point to a defect in DNA damage sensing. In addition, GSE1 knock-out cells showed hampered DNA damage-induced phosphorylation on SQ motifs of regulators of histone post-translational modifications, suggesting altered histone modification. While loss of GSE1 does not affect the histone deacetylation activity of CoREST, GSE1 appears to be essential for binding of the deubiquitinase USP22 to CoREST and for the deubiquitination of H2B K120 in response to DNA damage. The combination of deacetylase, demethylase, and deubiquitinase activity makes the USP22-GSE1-CoREST subcomplex a multi enzymatic eraser that seems to play an important role during DDR. Since GSE1 has been previously associated with cancer progression and survival our findings are potentially of high medical relevance.

Azides are versatile bioorthogonal reporter moieties that are commonly used for site-specific labeling and functionalization of RNA to probe its biology. The preparation of azido modified nucleic acids by solid-phase synthesis is problematic due to the inherent reactivity of P(III) species with azides according to the Staudinger reaction. Various strategies have been developed to bypass this limitation and are often time-consuming, low-yielding and labor-intensive. In particular, the synthesis of RNA with internal 2'-azido modifications is restricted to a single approach that employs P(V) chemistry instead of the widely used P(III) phosphoramidite chemistry. To fill this methodological gap, we present a novel convenient path toward 2'-azido RNA from readily accessible 2'-amino RNA through treatment with the diazotizing reagent fluorosulfuryl azide (FSO

Lysosomes are highly dynamic organelles that regulate metabolic signaling pathways by recruiting cytosolic molecules to protein platforms on the lysosomal membrane. We performed a proximity-dependent labeling screen to identify novel proteins recruited to the LAMTOR complex, which regulates lysosome positioning and key signaling pathways such as mTORC1, AMPK, and MEK/ERK. We identified a network of proteins involved in actin remodelling, including Pleckstrin homology domain-containing family G member 3 (PLEKHG3), an actin-binding Rho guanine nucleotide exchange factor enriched in protrusions. We show that GFP-PLEKHG3 accumulates in focal adhesion sites, where it colocalizes with peripheral lysosomes. Peripheral accumulation of lysosomes concentrates PLEKHG3 below the plasma membrane, inhibits protrusion formation and limits cell motility. Thus, subcellular positioning of lysosomes impacts PLEKHG3 subcellular localization and the cell’s protrusion activity, shape, and motility. The results shed new light on the interplay between lysosomes and actin dynamics and provides insights into the mechanisms controlling cellular processes such as shape regulation and motility of the plasma membrane.

Acetylation is one of the most abundant post-translational protein modifications that regulates all cellular compartments ranging from chromatin to cytoskeleton and Golgi. The dynamic acetylation of the Golgi stacking protein GRASP55 was shown to regulate Golgi reassembly after mitosis. Here we provide a detailed protocol for the analysis of Golgi acetylation including in vitro assays to detect protein acetylation and mass spectrometry analysis to identify specific acetylation sites and their relative abundance.

Background The Hydra head organizer acts as a signaling center that initiates and maintains the primary body axis in steady state polyps and during budding or regeneration. Wnt/beta-Catenin signaling functions as a primary cue controlling this process, but how Wnt ligand activity is locally restricted at the protein level is poorly understood. Here we report a proteomic analysis of Hydra head tissue leading to the identification of an astacin family proteinase as a Wnt processing factor. Results Hydra astacin-7 (HAS-7) is expressed from gland cells as an apical-distal gradient in the body column, peaking close beneath the tentacle zone. HAS-7 siRNA knockdown abrogates HyWnt3 proteolysis in the head tissue and induces a robust double axis phenotype, which is rescued by simultaneous HyWnt3 knockdown. Accordingly, double axes are also observed in conditions of increased Wnt activity as in transgenic actin::HyWnt3 and HyDkk1/2/4 siRNA treated animals. HyWnt3-induced double axes in Xenopus embryos could be rescued by coinjection of HAS-7 mRNA. Mathematical modelling combined with experimental promotor analysis indicate an indirect regulation of HAS-7 by beta-Catenin, expanding the classical Turing-type activator-inhibitor model. Conclusions We show the astacin family protease HAS-7 maintains a single head organizer through proteolysis of HyWnt3. Our data suggest a negative regulatory function of Wnt processing astacin proteinases in the global patterning of the oral-aboral axis in Hydra.

Robust, efficient, and reproducible protein extraction and sample processing is a key step for bottom-up proteomics analyses. While many sample preparation protocols for mass spectrometry have been described, selecting an appropriate method remains challenging since some protein classes may require specialized solubilization, precipitation, and digestion procedures. Here, we present a comprehensive comparison of the 16 most widely used sample preparation methods, covering in-solution digests, device-based methods, and commercially available kits. We find a remarkably good performance of the majority of the protocols with high reproducibility, little method dependency, and low levels of artifact formation. However, we revealed method-dependent differences in the recovery of specific protein features, which we summarized in a descriptive guide matrix. Our work thereby provides a solid basis for the selection of MS sample preparation strategies for a given proteomics project.

Candida albicans is a normal component of the human microflora that colonizes mucosal/epithelial surfaces and the gastrointestinal tract as a commensal organism. However, in an immunocompromised host, it can cause life-threatening infections of high mortality and morbidity. Virulence as well as antifungal drug resistance of C. albicans is often regulated by posttranslational modifications (PTM) of proteins via lysine acetylation by lysine acetyltransferases. Here, we report an experimental approach using tandem mass tag (TMT) labeling for the detection and quantification of lysine acetylation of histone and nonhistone proteins in C. albicans.

Multiplex proteomics using isobaric labeling tags has emerged as a powerful tool for the simultaneous relative quantification of peptides and proteins across multiple experimental conditions. However, the quantitative accuracy of the approach is largely compromised by a phenomenon termed ion interference, causing fold changes to appear compressed. The degree of compression is generally unclear, and the contributing factors are poorly understood. In this study, we thoroughly characterize ion interference at the MS2 level by using a defined two-proteome experimental system. We address the poor agreement between the apparent precursor purity in the isolation window and the actual level of interference in MS2-scans, a discrepancy that is only effectively resolved by considering potential co-fragmenting peptide ions hidden within the “noise”. We further propose a comprehensive modeling strategy for obtaining accurate, feature-wise assessments of interference. Finally, we present a simple algorithm to calculate interference-corrected reporter ion intensities of peptides and proteins, thereby successfully removing ratio compression.

The ClpC1:ClpP1P2 protease is a core component of the proteostasis system in mycobacteria. To improve the efficacy of antitubercular agents targeting the Clp protease, we characterized the mechanism of the antibiotics cyclomarin A and ecumicin. Quantitative proteomics revealed that the antibiotics cause massive proteome imbalances, including upregulation of two unannotated yet conserved stress response factors, ClpC2 and ClpC3. These proteins likely protect the Clp protease from excessive amounts of misfolded proteins or from cyclomarin A, which we show to mimic damaged proteins. To overcome the Clp security system, we developed a BacPROTAC that induces degradation of ClpC1 together with its ClpC2 caretaker. The dual Clp degrader, built from linked cyclomarin A heads, was highly efficient in killing pathogenic Mycobacterium tuberculosis, with >100-fold increased potency over the parent antibiotic. Together, our data reveal Clp scavenger proteins as important proteostasis safeguards and highlight the potential of BacPROTACs as future antibiotics.

Proximity-dependent protein labeling provides a powerful in vivo strategy to characterize the interactomes of specific proteins. We previously optimized a proximity labeling protocol for C. elegans using the highly active biotin ligase TurboID. A significant constraint on the sensitivity of TurboID is the presence of abundant, endogenously biotinylated proteins that take up bandwidth in the mass spectrometer, notably carboxylases that use biotin as a co-factor. In C. elegans, these comprise POD-2/acetyl-CoA carboxylase alpha, PCCA-1/propionyl-CoA carboxylase alpha, PYC-1/pyruvate carboxylase and MCCC-1/methylcrotonyl-CoA carboxylase alpha. We developed ways to remove these carboxylases prior to streptavidin purification and mass spectrometry, by engineering their corresponding genes to add a C-terminal His10 tag. This allows us to deplete them from C. elegans lysates using immobilized metal affinity chromatography (IMAC). To demonstrate the method’s efficacy, we use it to expand the interactome map of the presynaptic active zone protein ELKS-1. We identify many known active zone proteins, as well as previously uncharacterized potentially synaptic proteins. Our approach provides a quick and inexpensive solution to a common contaminant problem in biotin- dependent proximity labeling. The approach may be applicable to other model organisms and will enable deeper and more complete analysis of interactors for proteins of interest.

Robust, efficient and reproducible protein extraction and sample processing is a key step for bottom-up proteomics analyses. While many sample preparation protocols for mass spectrometry have been described, selecting an appropriate method remains challenging, since some protein classes may require specialized solubilization, precipitation, and digestion procedures. Here we present a comprehensive comparison of 16 most widely used sample preparation methods, covering in-solution digests, device-based methods, as well as commercially available kits. We find a remarkably good performance of the majority of the protocols with high reproducibility, little method dependencies and low levels of artifact formation. However, we revealed method-dependent differences in the recovery of specific protein features, which we summarized in a descriptive guide-matrix. Our work thereby provides a solid basis for the selection of MS sample preparation strategies for a given proteomics project.

Ammonia oxidation as the first step of nitrification constitutes a critical process in the global nitrogen cycle. However, fundamental knowledge of its key enzyme, the copper-dependent ammonia monooxygenase is lacking, in particular for the environmentally abundant ammonia oxidizing archaea (AOA). Here, the structure of the enzyme is investigated by blue-native gel electrophoresis and proteomics from native membrane complexes of two AOA. Beside the known AmoABC subunits and the earlier predicted AmoX, two new protein subunits, AmoY and AmoZ, were identified. They are unique to AOA, highly conserved and co-regulated, and their genes are linked to other AMO subunit genes in streamlined AOA genomes. Modelling and in gel cross-link approaches support an overall protomer structure similar to the distantly related bacterial particulate methane monooxygenase indicating that AmoY and AmoZ serve an important structural and functional role. These data open avenues for further structure-function studies of this ecologically important key nitrification complex.

Patient-derived organoid (PDO) cancer models are generated from epithelial tumor cells. Although they reflect the molecular tumor characteristics, they lack the complexity of the tumor microenvironment, which is a key driver of tumorigenesis and therapy response. Here, we present a colorectal cancer (CRC) organoid model that incorporates epithelial cells and stromal fibroblasts from the same patient. Molecular characterization of primary cancer associated fibroblasts (CAFs) and matched normal fibroblasts (NF) revealed proteomic, secretome and gene expression differences in pathways associated with tumor related fibroblast function. Further, CAFs retained higher motility compared to NFs in vitro. Importantly, both CAFs and NFs supported cancer cell proliferation in 3D co-cultures, without the addition of classical niche factors. PDOs grown together with fibroblasts displayed a larger cellular heterogeneity of tumor cells compared to mono-cultures, and closely resembled the in vivo tumor morphology. This was also confirmed by the calculation of cellular proportions of epithelial cell subtypes in organoid mono-versus co-cultures, which were inferred through bioinformatics deconvolution of bulk RNA sequencing data using published single cell RNA sequencing datasets from CRC tissues. Additionally, we observed a mutual crosstalk between tumor cells and fibroblasts in the co-cultures. This was manifested by majorly deregulated pathways such as cell-cell communication and extracellular matrix remodeling in the organoids. For the fibroblasts, we observed enhanced expression of tumor induced marker genes and cytokines characteristic for myo- and immunogenic fibroblasts. This model will be vital as a physiological personalized tumor model to study disease mechanisms and therapy response in CRC.One Sentence SummaryPatient matched fibroblasts support tumor organoid growth in 3D co-culture and maintain intratumoral cellular heterogeneity and histo-morphology.

Significance The pharmaceutical targeting of mutated BRAF has shown promising clinical outcomes in patients with melanoma. However, more than 300 reported BRAF patient mutations and the occurrence of a kinase-drug resistance mechanism hamper patient-oriented therapies. We have developed an extendable and cell-based kinase conformation reporter platform (KinCon) to predict and compare the effect of kinase drugs. We systematically evaluated drug efficacies by assessing KinCon reporter dynamics in response to different drugs and patient mutations. Our findings suggest that FDA-approved melanoma inhibitors may have the potential to block non-V600E–mutated BRAF activities also in non–small-cell lung cancers (NSCLC). We assume that widespread analyses of KinCon:drug interactions may assist in the future in identifying patient mutation–specific and thus more effective kinase inhibitors., Kinase-targeted therapies have the potential to improve the survival of patients with cancer. However, the cancer-specific spectrum of kinase alterations exhibits distinct functional properties and requires mutation-oriented drug treatments. Besides post-translational modifications and diverse intermolecular interactions of kinases, it is the distinct disease mutation which reshapes full-length kinase conformations, affecting their activity. Oncokinase mutation profiles differ between cancer types, as it was shown for BRAF in melanoma and non–small-cell lung cancers. Here, we present the target-oriented application of a kinase conformation (KinCon) reporter platform for live-cell measurements of autoinhibitory kinase activity states. The bioluminescence-based KinCon biosensor allows the tracking of conformation dynamics of full-length kinases in intact cells and real time. We show that the most frequent BRAF cancer mutations affect kinase conformations and thus the engagement and efficacy of V600E-specific BRAF inhibitors (BRAFi). We illustrate that the patient mutation harboring KinCon reporters display differences in the effectiveness of the three clinically approved BRAFi vemurafenib, encorafenib, and dabrafenib and the preclinical paradox breaker PLX8394. We confirmed KinCon-based drug efficacy predictions for BRAF mutations other than V600E in proliferation assays using patient-derived lung cancer cell lines and by analyzing downstream kinase signaling. The systematic implementation of such conformation reporters will allow to accelerate the decision process for the mutation-oriented RAF-kinase cancer therapy. Moreover, we illustrate that the presented kinase reporter concept can be extended to other kinases which harbor patient mutations. Overall, KinCon profiling provides additional mechanistic insights into full-length kinase functions by reporting protein–protein interaction (PPI)-dependent, mutation-specific, and drug-driven changes of kinase activity conformations.

During particular stress conditions, transfer RNAs (tRNAs) become substrates of stress-induced endonucleases, resulting in the production of distinct tRNA-derived small RNAs (tsRNAs). These small RNAs have been implicated in a wide range of biological processes, but how isoacceptor and even isodecoder-specific tsRNAs act at the molecular level is still poorly understood. Importantly, stress-induced tRNA cleavage affects only a few tRNAs of a given isoacceptor or isodecoder, raising the question as to how such limited molecule numbers could exert measurable biological impact. While the molecular function of individual tsRNAs is likely mediated through association with other molecules, addressing the interactome of specific tsRNAs has only been attempted by using synthetic RNA sequences. Since tRNAs carry post-transcriptional modifications, tsRNAs are likely modified but the extent of their modifications remains largely unknown. Here, we developed a biochemical framework for the production and purification of specific tsRNAs using human cells. Preparative scale purification of tsRNAs from biological sources should facilitate experimentally addressing as to how exactly these small RNAs mediate the multitude of reported molecular functions.

In mass-spectrometry-based interaction proteomics on-bead digestion protocols are commonly applied after affinity-enrichment due to their simplicity and high efficiency. However, on-bead digestion often leads to strong background signals due to co-digestion of the bead-bound ligands such as streptavidin or antibodies. We present an effective, rapid and low-cost method to specifically reduce the peptide signals from co-digested matrix ligands. A short pre-incubation of matrix beads with Sulfo-NHS-Acetate (S-NHS-Ac) leads to acetylation of free amines on lysine side-chains of the bead-bound ligands making them resistant to Lys-C-mediated proteolysis. After binding of bait proteins to the acetylated beads we employ a two-step digestion protocol with the sequential use of Lys-C protease for on-bead digestion followed by in-solution digestion with trypsin. The strong reduction of interfering ligand peptides improves signal strength and data quality for the peptides of interest in liquid chromatography mass spectrometry (LC-MS).

In mass-spectrometry-based interaction proteomics on-bead digestion protocols are commonly applied after affinity-enrichment due to their simplicity and high efficiency. However, on-bead digestion often leads to strong background signals due to co-digestion of the bead-bound ligands such as streptavidin or antibodies. We present an effective, rapid and low-cost method to specifically reduce the peptide signals from co-digested matrix ligands. A short pre-incubation of matrix beads with Sulfo-NHS-Acetate (S-NHS-Ac) leads to acetylation of free amines on lysine side-chains of the bead-bound ligands making them resistant to Lys-C-mediated proteolysis. After binding of bait proteins to the acetylated beads we employ a two-step digestion protocol with the sequential use of Lys-C protease for on-bead digestion followed by in-solution digestion with trypsin. The strong reduction of interfering ligand peptides improves signal strength and data quality for the peptides of interest in liquid chromatography mass spectrometry (LC-MS).

Transition from the stem/progenitor cell fate to meiosis is mediated by several redundant posttranscriptional regulatory pathways in Caenorhabditis elegans . Interfering with all three branches causes tumorous germ lines. SCF PROM-1 comprises one branch and mediates a scheduled degradation step at entry into meiosis. prom-1 mutants show defects in the timely initiation of meiotic prophase I events, resulting in high rates of embryonic lethality. Here, we identify the phosphatase PPM-1.D/Wip1 as crucial substrate for PROM-1. We report that PPM-1.D antagonizes CHK-2 kinase, a key regulator for meiotic prophase initiation, including DNA double-strand breaks, chromosome pairing, and synaptonemal complex formation. We propose that PPM-1.D controls the amount of active CHK-2 via both catalytic and noncatalytic activities; notably, noncatalytic regulation seems to be crucial at meiotic entry. PPM-1.D sequesters CHK-2 at the nuclear periphery, and programmed SCF PROM-1 –mediated degradation of PPM-1.D liberates the kinase and promotes meiotic entry.

The green alga Chlamydomonas reinhardtii is one of the most studied microorganisms in photosynthesis research and for biofuel production. A detailed understanding of the dynamic regulation of its carbon metabolism is therefore crucial for metabolic engineering. Post-translational modifications can act as molecular switches for the control of protein function. Acetylation of the ɛ-amino group of lysine residues is a dynamic modification on proteins across organisms from all kingdoms. Here, we performed a mass spectrometry-based profiling of proteome and lysine acetylome dynamics in Chlamydomonas under varying growth conditions. Chlamydomonas liquid cultures were transferred from mixotrophic (light and acetate as carbon source) to heterotrophic (dark and acetate), or photoautotrophic (light only) growth conditions for 30 h before harvest. In total, 5863 protein groups and 1376 lysine acetylation sites were identified with a FDR < 1 %. As a major result of this study, our data shows that dynamic changes in abundance of lysine acetylation on various enzymes involved in photosynthesis, fatty acid metabolism, and the glyoxylate cycle are depending on acetate and light. Exemplary determination of acetylation site stoichiometries revealed particularly high occupancy levels on K175 of the large subunit of RuBisCO and K99 and K340 of peroxisomal citrate synthase, respectively, under heterotrophic conditions. The lysine acetylation stoichiometries correlated with increased activities of cellular citrate synthase, and the known inactivation of the Calvin-Benson cycle under heterotrophic conditions. In conclusion, the newly identified dynamic lysine acetylation sites may have a great potential for genetic engineering of metabolic pathways in Chlamydomonas.

The class I histone deacetylases are essential regulators of cell fate decisions in health and disease. While pan- and class-specific HDAC inhibitors are available, these drugs do not allow a comprehensive understanding of individual HDAC function, or the therapeutic potential of isoform-specific targeting. To systematically compare the impact of individual catalytic functions of HDAC1, HDAC2 and HDAC3, we generated human HAP1 cell lines expressing catalytically inactive HDAC enzymes. Using this genetic toolbox we compare the effect of individual HDAC inhibition with the effects of class I specific inhibitors on cell viability, protein acetylation and gene expression. Individual inactivation of HDAC1 or HDAC2 has only mild effects on cell viability, while HDAC3 inactivation or loss results in DNA damage and apoptosis. Inactivation of HDAC1/HDAC2 led to increased acetylation of components of the COREST co-repressor complex, reduced deacetylase activity associated with this complex and derepression of neuronal genes. HDAC3 controls the acetylation of nuclear hormone receptor associated proteins and the expression of nuclear hormone receptor regulated genes. Acetylation of specific histone acetyltransferases and HDACs is sensitive to inactivation of HDAC1/HDAC2. Over a wide range of assays, we determined that in particular HDAC1 or HDAC2 catalytic inactivation mimics class I specific HDAC inhibitors. Importantly, we further demonstrate that catalytic inactivation of HDAC1 or HDAC2 sensitizes cells to specific cancer drugs. In summary, our systematic study revealed isoform-specific roles of HDAC1/2/3 catalytic functions. We suggest that targeted genetic inactivation of particular isoforms effectively mimics pharmacological HDAC inhibition allowing the identification of relevant HDACs as targets for therapeutic intervention.

Changing environmental cues lead to the adjustment of cellular physiology by phosphorylation signaling networks that typically center around kinases as active effectors and phosphatases as antagonistic elements. Here, we report a signaling mechanism that reverses this principle. Using the hyperosmotic stress response in Saccharomyces cerevisiae as a model system, we find that a phosphatase‐driven mechanism causes induction of phosphorylation. The key activating step that triggers this phospho‐proteomic response is the Endosulfine‐mediated inhibition of protein phosphatase 2A‐Cdc55 (PP2ACdc55), while we do not observe concurrent kinase activation. In fact, many of the stress‐induced phosphorylation sites appear to be direct substrates of the phosphatase, rendering PP2ACdc55 the main downstream effector of a signaling response that operates in parallel and independent of the well‐established kinase‐centric stress signaling pathways. This response affects multiple cellular processes and is required for stress survival. Our results demonstrate how a phosphatase can assume the role of active downstream effectors during signaling and allow re‐evaluating the impact of phosphatases on shaping the phosphorylome., The phosphatase PP2ACdc55 assumes the role of an active downstream effector during hyperosmotic stress signaling in Saccharomyces cerevisiae. Inhibition of the phosphatase is the key event resulting in increased phosphorylation, while induction of kinases affecting the same substrates appears not to be required.

Significance Akt is an essential protein kinase that controls cell growth, survival, and metabolism. Akt is activated by the lipid second messengers PIP3 and PI(3,4)P2 and by phosphorylation. However, the relative contributions of lipid binding and phosphorylation to Akt activity in the cell are controversial. Here, we have determined the structure of autoinhibited Akt1, which reveals how the lipid-binding PH domain maintains the kinase domain in an inactive conformation in the absence of PIP3. Despite stoichiometric phosphorylation, Akt adopts an autoinhibited conformation with low basal activity in the absence of PIP3. Our work reveals the mechanistic basis of Akt hyperactivation in cancer and overgrowth diseases and unambiguously establishes that Akt depends on lipids for activity in the cell., The protein kinase Akt is one of the primary effectors of growth factor signaling in the cell. Akt responds specifically to the lipid second messengers phosphatidylinositol-3,4,5-trisphosphate [PI(3,4,5)P3] and phosphatidylinositol-3,4-bisphosphate [PI(3,4)P2] via its PH domain, leading to phosphorylation of its activation loop and the hydrophobic motif of its kinase domain, which are critical for activity. We have now determined the crystal structure of Akt1, revealing an autoinhibitory interface between the PH and kinase domains that is often mutated in cancer and overgrowth disorders. This interface persists even after stoichiometric phosphorylation, thereby restricting maximum Akt activity to PI(3,4,5)P3- or PI(3,4)P2-containing membranes. Our work helps to resolve the roles of lipids and phosphorylation in the activation of Akt and has wide implications for the spatiotemporal control of Akt and potentially lipid-activated kinase signaling in general.

The protein kinase Akt is one of the primary effectors of growth factor signaling in the cell. Akt responds specifically to the lipid second messengers phosphatidylinositol-3,4,5-trisphosphate [PI(3,4,5)P3] and phosphatidylinositol-3,4-bisphosphate [PI(3,4)P2] via its PH domain, leading to phosphorylation of its activation loop and the hydrophobic motif of its kinase domain, which are critical for activity. We have now determined the crystal structure of Akt1, revealing an autoinhibitory interface between the PH and kinase domains that is often mutated in cancer and overgrowth disorders. This interface persists even after stoichiometric phosphorylation, thereby restricting maximum Akt activity to PI(3,4,5)P3- or PI(3,4)P2-containing membranes. Our work helps to resolve the roles of lipids and phosphorylation in the activation of Akt and has wide implications for the spatiotemporal control of Akt and potentially lipid-activated kinase signaling in general.

SummaryThe transition from the stem cell/progenitor fate to meiosis is mediated by several redundant post-transcriptional regulatory pathways in C. elegans. Interfering with all three branches causes tumorous germlines. SCFPROM-1 comprises one branch and mediates a scheduled degradation step at entry into meiosis. prom-1 mutants show defects in timely initiation of events of meiotic prophase I, resulting in high rates of embryonic lethality. Here, we identify the phosphatase PPM-1.D/Wip1 as crucial substrate for PROM-1. We report that PPM-1.D antagonizes CHK-2 kinase, a key regulator for meiotic prophase initiation e.g., DNA double strand breaks, chromosome pairing and synaptonemal complex formation. We propose that PPM-1.D controls the amount of active CHK-2 by both catalytic and non-catalytic activities, where strikingly the non-catalytic regulation seems to be crucial at meiotic entry. PPM-1.D sequesters CHK-2 at the nuclear periphery and programmed SCFPROM-1 mediated degradation of PPM-1.D liberates the kinase and promotes meiotic entry.

SummaryHijacking the cellular protein degradation system offers unique opportunities for drug discovery, as exemplified by proteolysis targeting chimeras (PROTACs). Despite their superior properties over classical inhibitors, it has so far not been possible to reprogram the bacterial degradation machinery to interfere with microbial infections. Here, we develop small-molecule degraders, so-called BacPROTACs, that bind to the substrate receptor of the ClpC:ClpP protease, priming neo-substrates for degradation. In addition to their targeting function, BacPROTACs activate ClpC, transforming the resting unfoldase into its functional state. The induced higher-order oligomer was visualized by cryo-EM analysis, providing a structural snapshot of activated ClpC unfolding a protein substrate. Finally, degradation assays performed in mycobacteria demonstrate in vivo activity of BacPROTACs, highlighting the potential of the technology to provide next generation antibiotics.