An image-based screen for secreted proteins involved in breast cancer G0 cell cycle arrest.

Secreted proteins regulate the balance between cellular proliferation and G0 arrest and therefore play important roles in tumour dormancy. Tumour dormancy presents a significant clinical challenge for breast cancer patients, where non-proliferating, G0-arrested cancer cells remain at metastatic sites, below the level of clinical detection, some of which can re-enter proliferation and drive tumour relapse. Knowing which secreted proteins can regulate entry into and exit from G0 allows us to manipulate their signalling to prevent tumour relapse. To identify novel secreted proteins that can promote breast cancer G0 arrest, we performed a secretome-wide, image-based screen for proteins that increase the fraction of cells in G0 arrest. From a secretome library of 1282 purified proteins, we identified 29 candidates that promote G0 arrest in non-transformed and transformed breast epithelial cells. The assay we have developed can be adapted for use in other perturbation screens in other cell types. All datasets have been made available for re-analysis and our candidate proteins are presented for alternative bioinformatic refinement or further experimental follow up.

A data science roadmap for open science organizations engaged in early-stage drug discovery.

The Structural Genomics Consortium is an international open science research organization with a focus on accelerating early-stage drug discovery, namely hit discovery and optimization. We, as many others, believe that artificial intelligence (AI) is poised to be a main accelerator in the field. The question is then how to best benefit from recent advances in AI and how to generate, format and disseminate data to enable future breakthroughs in AI-guided drug discovery. We present here the recommendations of a working group composed of experts from both the public and private sectors. Robust data management requires precise ontologies and standardized vocabulary while a centralized database architecture across laboratories facilitates data integration into high-value datasets. Lab automation and opening electronic lab notebooks to data mining push the boundaries of data sharing and data modeling. Important considerations for building robust machine-learning models include transparent and reproducible data processing, choosing the most relevant data representation, defining the right training and test sets, and estimating prediction uncertainty. Beyond data-sharing, cloud-based computing can be harnessed to build and disseminate machine-learning models. Important vectors of acceleration for hit and chemical probe discovery will be (1) the real-time integration of experimental data generation and modeling workflows within design-make-test-analyze (DMTA) cycles openly, and at scale and (2) the adoption of a mindset where data scientists and experimentalists work as a unified team, and where data science is incorporated into the experimental design.

Loss of feedback regulation between FAM3B and androgen receptor driving prostate cancer progression.

BACKGROUND: Although the fusion of the transmembrane serine protease 2 gene (TMPRSS2) with the erythroblast transformation-specific-related gene (ERG), or TMPRSS2-ERG, occurs frequently in prostate cancer, its impact on clinical outcomes remains controversial. Roughly half of TMPRSS2-ERG fusions occur through intrachromosomal deletion of interstitial genes and the remainder via insertional chromosomal rearrangements. Because prostate cancers with deletion-derived TMPRSS2-ERG fusions are more aggressive than those with insertional fusions, we investigated the impact of interstitial gene loss on prostate cancer progression. METHODS: We conducted an unbiased analysis of transcriptome data from large collections of prostate cancer samples and employed diverse in vitro and in vivo models combined with genetic approaches to characterize the interstitial gene loss that imposes the most important impact on clinical outcome. RESULTS: This analysis identified FAM3B as the top-ranked interstitial gene whose loss is associated with a poor prognosis. The association between FAM3B loss and poor clinical outcome extended to fusion-negative prostate cancers where FAM3B downregulation occurred through epigenetic imprinting. Importantly, FAM3B loss drives disease progression in prostate cancer. FAM3B acts as an intermediator of a self-governing androgen receptor feedback loop. Specifically, androgen receptor upregulates FAM3B expression by binding to an intronic enhancer to induce an enhancer RNA and facilitate enhancer-promoter looping. FAM3B, in turn, attenuates androgen receptor signaling. CONCLUSION: Loss of FAM3B in prostate cancer, whether through the TMPRSS2-ERG translocation or epigenetic imprinting, causes an exit from this autoregulatory loop to unleash androgen receptor activity and prostate cancer progression. These findings establish FAM3B loss as a new driver of prostate cancer progression and support the utility of FAM3B loss as a biomarker to better define aggressive prostate cancer.

Secretome screening reveals immunomodulating functions of IFNα-7, PAP and GDF-7 on regulatory T-cells.

Regulatory T cells (Tregs) are the key cells regulating peripheral autoreactive T lymphocytes. Tregs exert their function by suppressing effector T cells. Tregs have been shown to play essential roles in the control of a variety of physiological and pathological immune responses. However, Tregs are unstable and can lose the expression of FOXP3 and suppressive functions as a consequence of outer stimuli. Available literature suggests that secreted proteins regulate Treg functional states, such as differentiation, proliferation and suppressive function. Identification of secreted proteins that affect Treg cell function are highly interesting for both therapeutic and diagnostic purposes in either hyperactive or immunosuppressed populations. Here, we report a phenotypic screening of a human secretome library in human Treg cells utilising a high throughput flow cytometry technology. Screening a library of 575 secreted proteins allowed us to identify proteins stabilising or destabilising the Treg phenotype as suggested by changes in expression of Treg marker proteins FOXP3 and/or CTLA4. Four proteins including GDF-7, IL-10, PAP and IFNα-7 were identified as positive regulators that increased FOXP3 and/or CTLA4 expression. PAP is a phosphatase. A catalytic-dead version of the protein did not induce an increase in FOXP3 expression. Ten interferon proteins were identified as negative regulators that reduced the expression of both CTLA4 and FOXP3, without affecting cell viability. A transcriptomics analysis supported the differential effect on Tregs of IFNα-7 versus other IFNα proteins, indicating differences in JAK/STAT signaling. A conformational model experiment confirmed a tenfold reduction in IFNAR-mediated ISG transcription for IFNα-7 compared to IFNα-10. This further strengthened the theory of a shift in downstream messaging upon external stimulation. As a summary, we have identified four positive regulators of FOXP3 and/or CTLA4 expression. Further exploration of these Treg modulators and their method of action has the potential to aid the discovery of novel therapies for both autoimmune and infectious diseases as well as for cancer.

High throughput generation of a resource of the human secretome in mammalian cells.

The proteins secreted by human tissues and blood cells, the secretome, are important both for the basic understanding of human biology and for identification of potential targets for future diagnosis and therapy. Here, a high-throughput mammalian cell factory is presented that was established to create a resource of recombinant full-length proteins covering the majority of those annotated as 'secreted' in humans. The full-length DNA sequences of each of the predicted secreted proteins were generated by gene synthesis, the constructs were transfected into Chinese hamster ovary (CHO) cells and the recombinant proteins were produced, purified and analyzed. Almost 1,300 proteins were successfully generated and proteins predicted to be secreted into the blood were produced with a success rate of 65%, while the success rates for the other categories of secreted proteins were somewhat lower giving an overall one-pass success rate of ca. 58%. The proteins were used to generate targeted proteomics assays and several of the proteins were shown to be active in a phenotypic assay involving pancreatic ß-cell dedifferentiation. Many of the proteins that failed during production in CHO cells could be rescued in human embryonic kidney (HEK 293) cells suggesting that a cell factory of human origin can be an attractive alternative for production in mammalian cells. In conclusion, a high-throughput protein production and purification system has been successfully established to create a unique resource of the human secretome.

The interleukin-like epithelial-mesenchymal transition inducer ILEI exhibits a non-interleukin-like fold and is active as a domain-swapped dimer.

Production and secretion of pro-metastatic proteins is a feature of many tumor cells. The FAM3C interleukin-like epithelial-to-mesenchymal-transition (EMT) inducer (ILEI) has been shown to be strongly up-regulated in several cancers and to be essential for tumor formation and metastasis in epithelial cells, correlating with a significant decrease in overall survival in colon and breast cancer patients. ILEI has been seen to interact with the γ-secretase presenilin 1 subunit (PS1). However, not much is known about the mechanism-of-action or the detailed ILEI structure. We present here the crystal structures of FAM3C ILEI and show that it exists as monomers but also as covalent dimers. The observed ILEI ß-ß-α fold confirmed previous indications that the FAM3C proteins do not form classical four-helix-bundle structures as was initially predicted. This provides the first experimental evidence that the interleukin-like EMT inducers are not evolutionarily related to the interleukins. However, more surprisingly, the ILEI dimer structure was found to feature a trans-linked domain swap, converting an intramolecular disulfide to intermolecular. Interestingly, dimeric but not monomeric ILEI was subsequently found to cause a dose-dependent increase in EpRas cell invasiveness comparable with TGF-ß, indicating that the dimer might be the active ILEI species. This is in line with a parallel study showing that covalent oligomerization of ILEI is essential for EMT and tumor progression in vivo The structures and the activity data give some first insight into the relationship between dimerization and ILEI function as well as indicate an intriguing link between ILEI, the PS1-protease, TGF-ß, and the TGF-ß receptor 1.

FAM3B PANDER and FAM3C ILEI represent a distinct class of signaling molecules with a non-cytokine-like fold.

The FAM3 superfamily is predicted to contain classical four-helix bundle cytokines, featuring a typical up-up-down-down fold. Two members of FAM3 have been extensively studied. FAM3B PANDER has been shown to regulate glucose homeostasis and ß cell function, whereas the homologous FAM3C ILEI has been shown to be involved in epithelial-mesenchymal transition and cancer. Here, we present a three-dimensional structure of a FAM3 protein, murine PANDER. Contrary to previous suggestions, PANDER exhibits a globular ß-ß-α fold. The structure is composed of two antiparallel ß sheets lined by three short helices packing to form a highly conserved water-filled cavity. The fold shares no relation to the predicted four-helix cytokines but is conserved throughout the FAM3 superfamily. The available biological data and the unexpected new fold indicate that FAM3 PANDER and ILEI could represent a new structural class of signaling molecules, with a different mode of action compared to the traditional four-helix bundle cytokines.

Biochemical discrimination between selenium and sulfur 1: a single residue provides selenium specificity to human selenocysteine lyase.

Selenium and sulfur are two closely related basic elements utilized in nature for a vast array of biochemical reactions. While toxic at higher concentrations, selenium is an essential trace element incorporated into selenoproteins as selenocysteine (Sec), the selenium analogue of cysteine (Cys). Sec lyases (SCLs) and Cys desulfurases (CDs) catalyze the removal of selenium or sulfur from Sec or Cys and generally act on both substrates. In contrast, human SCL (hSCL) is specific for Sec although the only difference between Sec and Cys is the identity of a single atom. The chemical basis of this selenium-over-sulfur discrimination is not understood. Here we describe the X-ray crystal structure of hSCL and identify Asp146 as the key residue that provides the Sec specificity. A D146K variant resulted in loss of Sec specificity and appearance of CD activity. A dynamic active site segment also provides the structural prerequisites for direct product delivery of selenide produced by Sec cleavage, thus avoiding release of reactive selenide species into the cell. We thus here define a molecular determinant for enzymatic specificity discrimination between a single selenium versus sulfur atom, elements with very similar chemical properties. Our findings thus provide molecular insights into a key level of control in human selenium and selenoprotein turnover and metabolism.

Crystal structure of conserved domains 1 and 2 of the human DEAD-box helicase DDX3X in complex with the mononucleotide AMP.

DExD-box helicases are involved in all aspects of cellular RNA metabolism. Conserved domains 1 and 2 contain nine signature motifs that are responsible for nucleotide binding, RNA binding and ATP hydrolysis. The human DEAD-box helicase DDX3X has been associated with several different cellular processes, such as cell-growth control, mRNA transport and translation, and is suggested to be essential for the export of unspliced/partially spliced HIV mRNAs from the nucleus to the cytoplasm. Here, the crystal structure of conserved domains 1 and 2 of DDX3X, including a DDX3-specific insertion that is not generally found in human DExD-box helicases, is presented. The N-terminal domain 1 and the C-terminal domain 2 both display RecA-like folds comprising a central beta-sheet flanked by alpha-helices. Interestingly, the DDX3X-specific insertion forms a helical element that extends a highly positively charged sequence in a loop, thus increasing the RNA-binding surface of the protein. Surprisingly, although DDX3X was crystallized in the presence of a large excess of ADP or the slowly hydrolyzable ATP analogue ATPgammaS the contaminant AMP was seen in the structure. A fluorescent-based stability assay showed that the thermal stability of DDX3X was increased by the mononucleotide AMP but not by ADP or ATPgammaS, suggesting that DDX3X is stabilized by AMP and elucidating why AMP was found in the nucleotide-binding pocket.

The flexible N-terminal domain of ribosomal protein L11 from Escherichia coli is necessary for the activation of stringent factor.

The stringent response is activated by the binding of stringent factor to stalled ribosomes that have an unacylated tRNA in the ribosomal aminoacyl-site. Ribosomes lacking ribosomal protein L11 are deficient in stimulating stringent factor. L11 consists of a dynamic N-terminal domain (amino acid residues 1-72) connected to an RNA-binding C-terminal domain (amino acid residues 76-142) by a flexible linker (amino acid residues 73-75). In vivo data show that mutation of proline 22 in the N-terminal domain is important for initiation of the stringent response. Here, six different L11 point and deletion-mutants have been constructed to determine which regions of L11 are necessary for the activation of stringent factor. The different mutants were reconstituted with programmed 70 S(DeltaL11) ribosomes and tested for their ability to stimulate stringent factor in a sensitive in vitro pppGpp synthesis assay. It was found that a single-site mutation at proline 74 in the linker region between the two domains did not affect the stimulatory activity of the reconstituted ribosomes, whereas the single-site mutation at proline 22 reduced the activity of SF to 33% compared to ribosomes reconstituted with wild-type L11. Removal of the entire linker between the N and C-terminal domains or removal of the entire proline-rich helix beginning at proline 22 in L11 resulted in an L11 protein, which was unable to stimulate stringent factor in the ribosome-dependent assay. Surprisingly, the N-terminal domain of L11 on its own activated stringent factor in a ribosome-dependent manner without restoring the L11 footprint in 23 S rRNA in the 50 S subunit. This suggests that the N-terminal domain can activate stringent factor in trans. It is also shown that this activation is dependent on unacylated tRNA.

Mapping the interaction of SmpB with ribosomes by footprinting of ribosomal RNA.

In trans-translation transfer messenger RNA (tmRNA) and small protein B (SmpB) rescue ribosomes stalled on truncated or in other ways problematic mRNAs. SmpB promotes the binding of tmRNA to the ribosome but there is uncertainty about the number of participating SmpB molecules as well as their ribosomal location. Here, the interaction of SmpB with ribosomal subunits and ribosomes was studied by isolation of SmpB containing complexes followed by chemical modification of ribosomal RNA with dimethyl sulfate, kethoxal and hydroxyl radicals. The results show that SmpB binds 30S and 50S subunits with 1:1 molar ratios and the 70S ribosome with 2:1 molar ratio. SmpB-footprints are similar on subunits and the ribosome. In the 30S subunit, SmpB footprints nucleotides that are in the vicinity of the P-site facing the E-site, and in the 50S subunit SmpB footprints nucleotides that are located below the L7/L12 stalk in the 3D structure of the ribosome. Based on these results, we suggest a mechanism where two molecules of SmpB interact with tmRNA and the ribosome during trans-translation. The first SmpB molecule binds near the factor-binding site on the 50S subunit helping tmRNA accommodation on the ribosome, whereas the second SmpB molecule may functionally substitute for a missing anticodon stem-loop in tmRNA during later steps of trans-translation.