Degradation of GSPT1 causes TP53-independent cell death in leukemia whilst sparing normal hematopoietic stem cells

Targeted protein degradation is a rapidly advancing and expanding therapeutic approach. Drugs that degrade GSPT1 via the CRL4CRBN ubiquitin ligase are a new class of cancer therapy in active clinical development with evidence of activity against acute myeloid leukemia in early phase trials. However, other than activation of the integrated stress response, the downstream effects of GSPT1 degradation leading to cell death are largely undefined, and no murine models are available to study these agents. We identified the domains of GSPT1 essential for cell survival and show that GSPT1 degradation leads to impaired translation termination, activation of the integrated stress response pathway, and TP53-independent cell death. CRISPR-Cas9 screens implicated decreased translation initiation as protective to GSPT1 degradation, suggesting that cells with higher levels of translation are more susceptible to GSPT1 degradation. We defined two Crbn amino acids that prevent Gspt1 degradation in mice, generated a knock-in mouse with alteration of these residues, and demonstrated the efficacy of GSPT1-degrading drugs in vivo with relative sparing of numbers and function of long-term hematopoietic stem cells. Our results provide a mechanistic basis for the use of GSPT1 degraders for the treatment of cancer, including TP53-mutant AML

Opposing effects of cancer-type-specific SPOP mutants on BET protein degradation and sensitivity to BET inhibitors.

It is generally assumed that recurrent mutations within a given cancer driver gene elicit similar drug responses. Cancer genome studies have identified recurrent but divergent missense mutations affecting the substrate-recognition domain of the ubiquitin ligase adaptor SPOP in endometrial and prostate cancers. The therapeutic implications of these mutations remain incompletely understood. Here we analyzed changes in the ubiquitin landscape induced by endometrial cancer-associated SPOP mutations and identified BRD2, BRD3 and BRD4 proteins (BETs) as SPOP-CUL3 substrates that are preferentially degraded by endometrial cancer-associated SPOP mutants. The resulting reduction of BET protein levels sensitized cancer cells to BET inhibitors. Conversely, prostate cancer-specific SPOP mutations resulted in impaired degradation of BETs, promoting their resistance to pharmacologic inhibition. These results uncover an oncogenomics paradox, whereby mutations mapping to the same domain evoke opposing drug susceptibilities. Specifically, we provide a molecular rationale for the use of BET inhibitors to treat patients with endometrial but not prostate cancer who harbor SPOP mutations

Analysis of proteins and peptides on a chromatographic timescale by electron-transfer dissociation MS

Tutkimuksen tarkoituksena on selvittää, onko kaksikielisten lasten eri kielten sanastojen monipuolisuudessa eroavaisuuksia. Tätä lähden tutkimaan vertailemalla substantiivien ja verbien sanemääriä eri kielillä, selvittämällä sanastojen toisteisuutta sekä tutkimalla sanatoisteisuuden ja aineistossa kerran esiintyvien sanojen välistä riippuvuutta. Lisäksi haluan selvittää, onko sukupuolten välillä eroavaisuuksia sanaston monipuolisuutta tarkastellessa. Tutkimuskysymyksiä syntyi kaksi. Eroaako kaksikielisten lasten eri kielten sanastojen monipuolisuus toisistaan, ja löytyykö sukupuolten väliltä eroavaisuuksia sanastojen monipuolisuuden osalta? Ensimmäiseen tutkimuskysymykseen vastaan kolmella alakysymyksellä. Eroavatko lasten erikieliset tarinat sanemääriltään toisistaan, onko sanasto toisteisempaa toisella kielellä kuin toisella, ja onko sanojen toisteisuuden ja aineistossa kerran esiintyvien sanojen välillä yhteys? Tavoitteena on saada lisää tietoa kaksikielisten lasten sanaston monipuolisuudesta narratiivisella tutkimusmenetelmällä. On tärkeää saada tietoa kaksikielisten lasten sanaston monipuolisuudesta, sillä kaksi- ja monikielisten perheiden määrä on Suomessa koko ajan lisääntymässä. Tutkimus on monimenetelmällinen tutkimus, sillä aineisto kerättiin laadullisin menetelmin, mutta analysoitiin määrällisesti. Aineisto kerättiin sadutusmenetelmällä kevään ja syksyn aikana 2019. Tutkimushenkilöitä oli kahdeksan. Kahdeksan neljä-viisivuotiasta suomalaisranskalaista lasta, joiden toisen vanhemman äidinkieli on suomi ja toisen ranska. Tutkimushenkilöiden valinnan kriteerinä oli myös se, että lapsi asuu Suomessa ja käy ranskankielistä päiväkotia. Aineiston analyysissä käytettiin TTR-arvoa ja hapax legomenon -lekseemejä. Tämän lisäksi TTR-arvojen ja HL-lekseemien välille laskettiin korrelaatio, jotta voidaan nähdä, onko sanojen toisteisuuden ja aineistossa kerran esiintyvien sanojen välillä yhteys. Tutkimustuloksissa ilmeni, että suomen kielen sanasto on lapsilla monipuolisempaa kuin ranskan kielen sanasto. Sukupuolten välillä ei ilmennyt huomattavia eroavaisuuksia sanastojen monipuolisuuden osalta. Eri mittareilla saatujen tulosten välillä todettiin positiivinen korrelaatio molemmilla kielillä. Tämä tutkimus on kuvaileva tutkimus, joten tutkimustuloksia ei ole tarkoitus yleistää

Proximity Biotinylation as a Method for Mapping Proteins Associated with mtDNA in Living Cells

A recurring challenge in cell biology is to define the molecular components of macromolecular complexes of interest. The predominant method, immunoprecipitation, recovers only strong interaction partners that survive cell lysis and repeated detergent washes. We explored peroxidase-catalyzed proximity biotinylation, APEX, as an alternative, focusing on the mitochondrial nucleoid, the dynamic macromolecular complex that houses the mitochondrial genome. Via 1-min live-cell biotinylation followed by quantitative, ratiometric mass spectrometry, we enriched 37 nucleoid proteins, seven of which had never previously been associated with the nucleoid. The specificity of our dataset was very high, and we validated three hits by follow-up studies. For one novel nucleoid-associated protein, FASTKD1, we discovered a role in downregulation of mitochondrial complex I via specific repression of ND3 mRNA. Our study demonstrates that APEX is a powerful tool for mapping macromolecular complexes in living cells, and can identify proteins and pathways that have been missed by traditional approaches

Prostate cancer: Ubiquitylome analysis identifies dysregulation of effector substrates in SPOP-mutant prostate cancer

Cancer genome characterization has revealed driver mutations in genes that govern ubiquitylation; however, the mechanisms by which these alterations promote tumorigenesis remain incompletely characterized. Here, we analyzed changes in the ubiquitin landscape induced by prostate cancer-associated mutations of SPOP, an E3 ubiquitin ligase substrate-binding protein. SPOP mutants impaired ubiquitylation of a subset of proteins in a dominant-negative fashion. Of these, DEK and TRIM24 emerged as effector substrates consistently up-regulated by SPOP mutants. We highlight DEK as a SPOP substrate that exhibited decreases in ubiquitylation and proteasomal degradation resulting from heteromeric complexes of wild-type and mutant SPOP protein. DEK stabilization promoted prostate epithelial cell invasion, which implicated DEK as an oncogenic effector. More generally, these results provide a framework to decipher tumorigenic mechanisms linked to dysregulated ubiquitylation

Lso2 is a conserved ribosome-bound protein required for translational recovery in yeast.

Ribosome-binding proteins function broadly in protein synthesis, gene regulation, and cellular homeostasis, but the complete complement of functional ribosome-bound proteins remains unknown. Using quantitative mass spectrometry, we identified late-annotated short open reading frame 2 (Lso2) as a ribosome-associated protein that is broadly conserved in eukaryotes. Genome-wide crosslinking and immunoprecipitation of Lso2 and its human ortholog coiled-coil domain containing 124 (CCDC124) recovered 25S ribosomal RNA in a region near the A site that overlaps the GTPase activation center. Consistent with this location, Lso2 also crosslinked to most tRNAs. Ribosome profiling of yeast lacking LSO2 (lso2Δ) revealed global translation defects during recovery from stationary phase with translation of most genes reduced more than 4-fold. Ribosomes accumulated at start codons, were depleted from stop codons, and showed codon-specific changes in occupancy in lso2Δ. These defects, and the conservation of the specific ribosome-binding activity of Lso2/CCDC124, indicate broadly important functions in translation and physiology

Proteomic mapping in live Drosophila tissues using an engineered ascorbate peroxidase

Characterization of the proteome of organelles and subcellular domains is essential for understanding cellular organization and identifying protein complexes as well as networks of protein interactions. We established a proteomic mapping platform in live Drosophila tissues using an engineered ascorbate peroxidase (APEX). Upon activation, the APEX enzyme catalyzes the biotinylation of neighboring endogenous proteins that can then be isolated and identified by mass spectrometry. We demonstrate that APEX labeling functions effectively in multiple fly tissues for different subcellular compartments and maps the mitochondrial matrix proteome of Drosophila muscle to demonstrate the power of APEX for characterizing subcellular proteomes in live cells. Further, we generate “MitoMax,” a database that provides an inventory of Drosophila mitochondrial proteins with subcompartmental annotation. Altogether, APEX labeling in live Drosophila tissues provides an opportunity to characterize the organelle proteome of specific cell types in different physiological conditions

H2A.Z.1 Monoubiquitylation Antagonizes BRD2 to Maintain Poised Chromatin in ESCs

Summary: Histone variant H2A.Z occupies the promoters of active and poised, bivalent genes in embryonic stem cells (ESCs) to regulate developmental programs, yet how it contributes to these contrasting states is poorly understood. Here, we investigate the function of H2A.Z.1 monoubiquitylation (H2A.Z.1ub) by mutation of the PRC1 target residues (H2A.Z.1K3R3). We show that H2A.Z.1K3R3 is properly incorporated at target promoters in murine ESCs (mESCs), but loss of monoubiquitylation leads to de-repression of bivalent genes, loss of Polycomb binding, and faulty lineage commitment. Using quantitative proteomics, we find that tandem bromodomain proteins, including the BET family member BRD2, are enriched in H2A.Z.1 chromatin. We further show that BRD2 is gained at de-repressed promoters in H2A.Z.1K3R3 mESCs, whereas BRD2 inhibition restores gene silencing at these sites. Together, our study reveals an antagonistic relationship between H2A.Z.1ub and BRD2 to regulate the transcriptional balance at bivalent genes to enable proper execution of developmental programs. : Shedding light on the contrasting functions of the histone variant H2A.Z.1 in gene regulation, Surface et al. show that H2A.Z.1 monoubiquitylation is required for the transcriptional repression of developmental promoters in mESCs by antagonizing downstream transcriptional activators including the BET bromodomain family member BRD2