Shared and specific functions of Arfs 1-5 at the Golgi revealed by systematic knockouts

ADP-ribosylation factors (Arfs) are small GTPases regulating membrane traffic in the secretory pathway. They are closely related and appear to have overlapping functions, regulators, and effectors. The functional specificity of individual Arfs and the extent of redundancy are still largely unknown. We addressed these questions by CRISPR/Cas9-mediated genomic deletion of the human class I (Arf1/3) and class II (Arf4/5) Arfs, either individually or in combination. Most knockout cell lines were viable with slight growth defects only when lacking Arf1 or Arf4. However, Arf1+4 and Arf4+5 could not be deleted simultaneously. Class I Arfs are nonessential, and Arf4 alone is sufficient for viability. Upon Arf1 deletion, the Golgi was enlarged, and recruitment of vesicle coats decreased, confirming a major role of Arf1 in vesicle formation at the Golgi. Knockout of Arf4 caused secretion of ER-resident proteins, indicating specific defects in coatomer-dependent ER protein retrieval by KDEL receptors. The knockout cell lines will be useful tools to study other Arf-dependent processes

Spatiotemporal variation of mammalian protein complex stoichiometries

Table reporting the summary of quantified complex members and their regulation. (XLSX 179 kb

CFIm-mediated alternative polyadenylation remodels cellular signaling and miRNA biogenesis

The mammalian cleavage factor I (CFIm) has been implicated in alternative polyadenylation (APA) in a broad range of contexts, from cancers to learning deficits and parasite infections. To determine how the CFIm expression levels are translated into these diverse phenotypes, we carried out a multi-omics analysis of cell lines in which the CFIm25 (NUDT21) or CFIm68 (CPSF6) subunits were either repressed by siRNA-mediated knockdown or over-expressed from stably integrated constructs. We established that >800 genes undergo coherent APA in response to changes in CFIm levels, and they cluster in distinct functional classes related to protein metabolism. The activity of the ERK pathway traces the CFIm concentration, and explains some of the fluctuations in cell growth and metabolism that are observed upon CFIm perturbations. Furthermore, multiple transcripts encoding proteins from the miRNA pathway are targets of CFIm-dependent APA. This leads to an increased biogenesis and repressive activity of miRNAs at the same time as some 3' UTRs become shorter and presumably less sensitive to miRNA-mediated repression. Our study provides a first systematic assessment of a core set of APA targets that respond coherently to changes in CFIm protein subunit levels (CFIm25/CFIm68). We describe the elicited signaling pathways downstream of CFIm, which improve our understanding of the key role of CFIm in integrating RNA processing with other cellular activities

Capturing protein communities by structural proteomics in a thermophilic eukaryote:Structural systems biology of lysates

The arrangement of proteins into complexes is a key organizational principle for many cellular functions. Although the topology of many complexes has been systematically analyzed in isolation, their molecular sociology in situ remains elusive. Here, we show that crude cellular extracts of a eukaryotic thermophile, Chaetomium thermophilum, retain basic principles of cellular organization. Using a structural proteomics approach, we simultaneously characterized the abundance, interactions, and structure of a third of the C. thermophilum proteome within these extracts. We identified 27 distinct protein communities that include 108 interconnected complexes, which dynamically associate with each other and functionally benefit from being in close proximity in the cell. Furthermore, we investigated the structure of fatty acid synthase within these extracts by cryoEM and this revealed multiple, flexible states of the enzyme in adaptation to its association with other complexes, thus exemplifying the need for in situ studies. As the components of the captured protein communities are known-at both the protein and complex levels-this study constitutes another step forward toward a molecular understanding of subcellular organization

Development of resistance to type II JAK2 inhibitors in MPN depends on AXL kinase and is targetable.

PURPOSE Myeloproliferative neoplasms (MPN) dysregulate JAK2 signaling. Since clinical JAK2 inhibitors have limited disease-modifying effects, type II JAK2 inhibitors such as CHZ868 stabilizing inactive JAK2 and reducing MPN clones, gain interest. We studied whether MPN cells escape from type ll inhibition. METHODS MPN cells were continuously exposed to CHZ868. We used phosphoproteomic analyses and ATAC-/RNA-sequencing to characterize acquired resistance to type II JAK2 inhibition, and targeted candidate mediators in MPN cells and mice. RESULTS MPN cells showed increased IC50 and reduced apoptosis upon CHZ868 reflecting acquired resistance to JAK2 inhibition. Among >2500 differential phospho-sites, MAPK pathway activation was most prominent, while JAK2-STAT3/5 remained suppressed. Altered histone occupancy promoting AP-1/GATA binding motif exposure associated with upregulated AXL kinase and enriched RAS target gene profiles. AXL knockdown resensitized MPN cells and combined JAK2/AXL inhibition using bemcentinib or gilteritinib reduced IC50 to levels of sensitive cells. While resistant cells induced tumor growth in NSG mice despite JAK2 inhibition, JAK2/AXL inhibition largely prevented tumor progression. Since inhibitors of MAPK pathway kinases such as MEK are clinically used in other malignancies, we evaluated JAK2/MAPK inhibition with trametinib to interfere with AXL-MAPK-induced resistance. Tumor growth was halted similarly to JAK2/AXL inhibition and in a systemic cell line-derived mouse model, marrow infiltration was decreased supporting dependency on AXL-MAPK. CONCLUSIONS We report on a novel mechanism of AXL-MAPK-driven escape from type II JAK2 inhibition, which is targetable at different nodes. This highlights AXL as mediator of acquired resistance warranting inhibition to enhance sustainability of JAK2 inhibition in MPN

Zmiany w kompozycji kompleksu NuRD i ich funkcjonalne konsekwencje

Zmiany w kompozycji kompleksu NuRD i ich konsekwencje funkcjonalne.Kompleks NuRD (ang. Nucleosome remodeling and deacetylase) odpowiada za regulację chromatyny. Unikalną cechą tego kompleksu jest jego podwójna aktywność enzymatyczna. Spośród sześciu głównych podjednostek, NuRD zawiera białka z aktywnością zarówno ATP-zależnej helikazy jak i deacetylazy histonowej. Jednym z dodatkowych komponentów jest przedstawiciel rodziny białek wiążących metylowane DNA: MBD2 lub MBD3. Pomimo ich wysokiego podobieństwa w sekwencjach, różnią się one znacznie w powinowactwie względem metylowanego DNA. W pojedynczej cząsteczce kompleksu tylko jedna wersja białka MBD jest obecna. Zostało dowiedzione, że ilość białek MBD jest specyficzna dla poszczególnych typów komórek i zazwyczaj jedna z podjednostek jest obecna na znacznie wyższym poziome niż druga. Sugeruje się, że komórkowo specyficzna kompozycja kompleksu NuRD wpływa istotnie na jego funkcje.Linia komórkowa HEK293 jest przykładem w którym wersja kompleksu MBD3-NuRD występuje na znacznie wyższym poziomie niż MBD2-NuRD. W niniejszej pracy analizowano funkcjonalne konsekwencje zmian w ilości białek MBD w linii komórkowej HEK293. W celu zmniejszenia stosunku między wersjami kompleksu, stabilna linia komórkowa z indukowalnym wyciszeniem genu MBD3 została wyprowadzona. Używając metod opartych na spektrometrii masowej, analizowano zmiany w proteomie jądrowym uzyskanych komórek. Analiza porównawcza wykazała że spadkowi poziomu MBD towarzyszył wzrost poziomu MBD2, skutkując tym samym ponad 50% obniżeniem stosunku ich ilości. Zmiany w poziomie białek MBD połączone były ze znaczącymi zmianami w ilości pozostały komponentów kompleksu NuRD. Dodatkowo, zaobserwowano, że zaindukowane zmiany w kompleksie NuRD skutkowały istotnym spadkiem ilości białek zaangażowanych w edytowanie RNA.The nucleosome remodeling and deacetylase (NuRD) complex regulates the organization of chromatin. An unique feature of the NuRD is the dual enzymatic activity. Among its six core subunit, the NuRD contains proteins with activity of both ATP-dependent helicase and histone deacetylase. One of the additional components is a member of MBD (methyl-CpG binding domain) family : MBD2 or MBD3. Despite high sequence similarity, they differ in respect to affinity for methylated DNA. In a single NuRD complex only one version of MBD protein is presented. It has been shown that abundance of MBD proteins is cell type specific and usually one of these paralagous subunits is more abundant than the other one. Cell-type specific composition of NuRD has been proposed to have implications in the complex functionality.HEK293 cell line is an example where the MBD3-NuRD version of the complex is much more abundant than the MBD2-NuRD. In this study, functional consequences of changes in MBD proteins abundance in HEK293 cell line have been investigated. To decrease the ratio of two version of the complex a stable cell line with inducible silencing of MBD3 was generated. Using mass spectrometry methods, changes in the nuclear proteome of engineered cell line were analyzed. Comparative analysis of proteomes revealed that decrease of MDB3 was accompanied by an increase of MBD2, therefore their ratio was decreased over 50%. Alterations in the level of MBD proteins were connected with significant changes in abundance of other NuRD components as well. Additionally, it was observed that induced changes in NuRD complex resulted in decrease of RNA editing protein abundance

Mass spectrometry analysis of circulating breast cancer cells from a Xenograft mouse model

Circulating tumor cells (CTCs) are precursors of metastasis in various cancer types. Many aspects regarding CTC biology remain poorly understood. Here, we describe mass spectrometric analysis of CTCs from a breast cancer xenograft mouse model, including procedures comprising CTC enrichment, separation of different CTC subpopulations, and their quantitative proteomic assessment. This protocol aims to facilitate the identification of protein content dynamics in human CTCs that are physiologically shed from tumor-bearing xenografts, providing a framework for investigating metastasis biology. For complete details on the use and execution of this protocol, please refer to Donato et al. (2020)

Changes in the Physicochemical and Bioactive Properties of Yerba Mate Depending on the Brewing Conditions

Yerba Mate drink made from dried and crushed leaves and twigs of Paraguayan holly (Ilex paraguariensis A. St.-Hil.), which is a valuable source of bioactive substances, in particular antioxidants. The available literature lacks data on changes in the content and profile of bioactive compounds such as tannins, caffeine, the phenolic acid profile of flavonoids and carotenoids, as well as total polyphenol content and antioxidant activity in Yerba Mate infusions depending on different brewing conditions, and how different brewing conditions affect the physicochemical properties of these infusions. Therefore, this study evaluated the physicochemical properties of dried and Yerba Mate infusions prepared via single and double brewing processes at 70 °C and 100 °C. The organoleptic evaluation, as well as the instrumental color measurement, showed significant changes in the total color difference (ΔE) and the L*a*b* chromatic coordinates of dried Yerba Mate samples and their infusions. Moreover, the research showed higher contents of tannins (mean 1.36 ± 0.14 g/100 g d.m.), caffeine (mean 17.79 ± 3.49 mg/g d.m.), carotenoids (mean 12.90 ± 0.44 μg/g d.m.), phenolic acids (mean 69.97 ± 7.10 mg/g d.m.), flavonoids (mean 5.47 ± 1.78 mg/g d.m.), total polyphenols (mean 55.26 ± 8.51 mg GAE/g d.m.), and antioxidant activity (mean 2031.98 ± 146.47 μM TEAC/g d.m.) in single-brewed Yerba Mate infusions compared to double-brewed (0.77 ± 0.12 g/100 g d.m., 14.28 ± 5.80 mg/g d.m., 12.67 ± 0.62 μg/g d.m., 57.75 ± 8.73 mg/g d.m., 3.64 ± 0.76 mg/g d.m., 33.44 ± 6.48 mg GAE/g d.m. and 1683.09 ± 155.34 μM TEAC/g d.m., respectively). In addition, infusions prepared at a lower temperature (70 °C) were characterized by a higher content of total polyphenols and higher antioxidant activity, in contrast to the tannin and carotenoid contents, the levels of which were higher at 100 °C than at 70 °C. Considering the high amount of bioactive ingredients, in particular antioxidants, and a wide range of health benefits, it is worth including Yerba Mate in the daily diet