Malaria is an infectious disease caused by parasites of the genus Plasmodium, which leads to approximately one million deaths per annum worldwide. Chemical validation of new antimalarial targets is ...urgently required in view of rising resistance to current drugs. One such putative target is the enzyme N-myristoyltransferase, which catalyses the attachment of the fatty acid myristate to protein substrates (N-myristoylation). Here, we report an integrated chemical biology approach to explore protein myristoylation in the major human parasite P. falciparum, combining chemical proteomic tools for identification of the myristoylated and glycosylphosphatidylinositol-anchored proteome with selective small-molecule N-myristoyltransferase inhibitors. We demonstrate that N-myristoyltransferase is an essential and chemically tractable target in malaria parasites both in vitro and in vivo, and show that selective inhibition of N-myristoylation leads to catastrophic and irreversible failure to assemble the inner membrane complex, a critical subcellular organelle in the parasite life cycle. Our studies provide the basis for the development of new antimalarials targeting N-myristoyltransferase.
The organization of a cell emerges from the interactions in protein networks. The interactome is critically dependent on the strengths of interactions and the cellular abundances of the connected ...proteins, both of which span orders of magnitude. However, these aspects have not yet been analyzed globally. Here, we have generated a library of HeLa cell lines expressing 1,125 GFP-tagged proteins under near-endogenous control, which we used as input for a next-generation interaction survey. Using quantitative proteomics, we detect specific interactions, estimate interaction stoichiometries, and measure cellular abundances of interacting proteins. These three quantitative dimensions reveal that the protein network is dominated by weak, substoichiometric interactions that play a pivotal role in defining network topology. The minority of stable complexes can be identified by their unique stoichiometry signature. This study provides a rich interaction dataset connecting thousands of proteins and introduces a framework for quantitative network analysis.
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•Human interactome dataset connecting 5,400 proteins with 28,500 interactions•Three quantitative dimensions measure specificities, stoichiometries, and abundances•Stable complexes are rare but stand out by a signature of balanced stoichiometries•Weak interactions dominate the network and have critical topological properties
Weak interactions shape the cellular protein interaction network as determined from proteomic measures of cellular interaction specificities, the strength of those interactions, and the cellular copy numbers of the proteins involved.
To examine global changes in breast heterogeneity across different states, we determined the single‐cell transcriptomes of > 340,000 cells encompassing normal breast, preneoplastic BRCA1+/– tissue, ...the major breast cancer subtypes, and pairs of tumors and involved lymph nodes. Elucidation of the normal breast microenvironment revealed striking changes in the stroma of post‐menopausal women. Single‐cell profiling of 34 treatment‐naive primary tumors, including estrogen receptor (ER)+, HER2+, and triple‐negative breast cancers, revealed comparable diversity among cancer cells and a discrete subset of cycling cells. The transcriptomes of preneoplastic BRCA1+/– tissue versus tumors highlighted global changes in the immune microenvironment. Within the tumor immune landscape, proliferative CD8+ T cells characterized triple‐negative and HER2+ cancers but not ER+ tumors, while all subtypes comprised cycling tumor‐associated macrophages, thus invoking potentially different immunotherapy targets. Copy number analysis of paired ER+ tumors and lymph nodes indicated seeding by genetically distinct clones or mass migration of primary tumor cells into axillary lymph nodes. This large‐scale integration of patient samples provides a high‐resolution map of cell diversity in normal and cancerous human breast.
Synopsis
To examine global changes in breast heterogeneity across different states, this gene expression resource integrates large‐scale patient samples from diverse tissue states and breast cancer subtypes, offering a refined high‐resolution map of cell diversity in the normal and cancerous human mammary gland.
Single‐cell transcriptome analyses profile > 340,000 cells encompassing normal breast, preneoplastic BRCA1+/– tissue, the major breast cancer subtypes, and metastatic lymph nodes.
Pre‐ to post‐menopause transition is associated with marked stromal changes, with decreased PDGFRb and matrix‐associated genes in fibroblasts.
Progression from preneoplasia to tumors correlates with increased immune infiltration in BRCA1 mutation carriers.
Tumor epithelial compartments show comparable diversity in different breast cancer subtypes.
Cycling CD8+ T‐cells are reduced in estrogen receptor (ER)+ tumors, suggesting different immunoregulatory patterns.
Both clonal selection and mass migration contribute to lymph node metastases in patients with ER+ cancer.
A large‐scale gene expression resource integrates diverse tissue samples and reveals unexpected heterogeneity of breast cancer subtypes.
Genetic interaction (GI) maps, comprising pairwise measures of how strongly the function of one gene depends on the presence of a second, have enabled the systematic exploration of gene function in ...microorganisms. Here, we present a two-stage strategy to construct high-density GI maps in mammalian cells. First, we use ultracomplex pooled shRNA libraries (25 shRNAs/gene) to identify high-confidence hit genes for a given phenotype and effective shRNAs. We then construct double-shRNA libraries from these to systematically measure GIs between hits. A GI map focused on ricin susceptibility broadly recapitulates known pathways and provides many unexpected insights. These include a noncanonical role for COPI, a previously uncharacterized protein complex affecting toxin clearance, a specialized role for the ribosomal protein RPS25, and functionally distinct mammalian TRAPP complexes. The ability to rapidly generate mammalian GI maps provides a potentially transformative tool for defining gene function and designing combination therapies based on synergistic pairs.
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► Ultracomplex shRNA library minimizes false positives/negatives in genome-wide screens ► Pooled double-shRNA strategy systematically maps genetic interactions between hits ► Application of two-step strategy identifies pathways controlling ricin susceptibility ► The resulting map uncovers functionally distinct mammalian TRAPP complexes
A high-throughput method that relies on the use of ultracomplex shRNA libraries makes it possible to create genetic interaction maps in mammalian cells. This approach will be applicable to many cellular processes and conditions, as illustrated by the discovery of distinct TRAPP complexes involved in endocytosis.
Merkel cell carcinoma (MCC) is an uncommon, but highly malignant, cutaneous tumor. Merkel cell polyoma virus (MCV) has been implicated in a majority of MCC tumors; however, viral-negative tumors have ...been reported to be more prevalent in some geographic regions subject to high sun exposure. While the impact of MCV and viral T-antigens on MCC development has been extensively investigated, little is known about the etiology of viral-negative tumors. We performed targeted capture and massively parallel DNA sequencing of 619 cancer genes to compare the gene mutations and copy number alterations in MCV-positive (n = 13) and -negative (n = 21) MCC tumors and cell lines. We found that MCV-positive tumors displayed very low mutation rates, but MCV-negative tumors exhibited a high mutation burden associated with a UV-induced DNA damage signature. All viral-negative tumors harbored mutations in RB1, TP53, and a high frequency of mutations in NOTCH1 and FAT1. Additional mutated or amplified cancer genes of potential clinical importance included PI3K (PIK3CA, AKT1, PIK3CG) and MAPK (HRAS, NF1) pathway members and the receptor tyrosine kinase FGFR2. Furthermore, looking ahead to potential therapeutic strategies encompassing immune checkpoint inhibitors such as anti-PD-L1, we also assessed the status of T-cell-infiltrating lymphocytes (TIL) and PD-L1 in MCC tumors. A subset of viral-negative tumors exhibited high TILs and PD-L1 expression, corresponding with the higher mutation load within these cancers. Taken together, this study provides new insights into the underlying biology of viral-negative MCC and paves the road for further investigation into new treatment opportunities.
Protein N-myristoylation is a ubiquitous co- and post-translational modification that has been implicated in the development and progression of a range of human diseases. Here, we report the global ...N-myristoylated proteome in human cells determined using quantitative chemical proteomics combined with potent and specific human N-myristoyltransferase (NMT) inhibition. Global quantification of N-myristoylation during normal growth or apoptosis allowed the identification of >100 N-myristoylated proteins, >95% of which are identified for the first time at endogenous levels. Furthermore, quantitative dose response for inhibition of N-myristoylation is determined for >70 substrates simultaneously across the proteome. Small-molecule inhibition through a conserved substrate-binding pocket is also demonstrated by solving the crystal structures of inhibitor-bound NMT1 and NMT2. The presented data substantially expand the known repertoire of co- and post-translational N-myristoylation in addition to validating tools for the pharmacological inhibition of NMT in living cells.
The nucleolus is a key organelle that coordinates the synthesis and assembly of ribosomal subunits and forms in the nucleus around the repeated ribosomal gene clusters. Because the production of ...ribosomes is a major metabolic activity, the function of the nucleolus is tightly linked to cell growth and proliferation, and recent data suggest that the nucleolus also plays an important role in cell-cycle regulation, senescence and stress responses. Here, using mass-spectrometry-based organellar proteomics and stable isotope labelling, we perform a quantitative analysis of the proteome of human nucleoli. In vivo fluorescent imaging techniques are directly compared to endogenous protein changes measured by proteomics. We characterize the flux of 489 endogenous nucleolar proteins in response to three different metabolic inhibitors that each affect nucleolar morphology. Proteins that are stably associated, such as RNA polymerase I subunits and small nuclear ribonucleoprotein particle complexes, exit from or accumulate in the nucleolus with similar kinetics, whereas protein components of the large and small ribosomal subunits leave the nucleolus with markedly different kinetics. The data establish a quantitative proteomic approach for the temporal characterization of protein flux through cellular organelles and demonstrate that the nucleolar proteome changes significantly over time in response to changes in cellular growth conditions.
The Human Viral Challenge (HVC) model has, for many decades, helped in the understanding of respiratory viruses and their role in disease pathogenesis. In a controlled setting using small numbers of ...volunteers removed from community exposure to other infections, this experimental model enables proof of concept work to be undertaken on novel therapeutics, including vaccines, immunomodulators and antivirals, as well as new diagnostics.Crucially, unlike conventional phase 1 studies, challenge studies include evaluable efficacy endpoints that then guide decisions on how to optimise subsequent field studies, as recommended by the FDA and thus licensing studies that follow. Such a strategy optimises the benefit of the studies and identifies possible threats early on, minimising the risk to subsequent volunteers but also maximising the benefit of scarce resources available to the research group investing in the research. Inspired by the principles of the 3Rs (Replacement, Reduction and Refinement) now commonly applied in the preclinical phase, HVC studies allow refinement and reduction of the subsequent development phase, accelerating progress towards further statistically powered phase 2b studies. The breadth of data generated from challenge studies allows for exploration of a wide range of variables and endpoints that can then be taken through to pivotal phase 3 studies.We describe the disease burden for acute respiratory viral infections for which current conventional development strategies have failed to produce therapeutics that meet clinical need. The Authors describe the HVC model's utility in increasing scientific understanding and in progressing promising therapeutics through development.The contribution of the model to the elucidation of the virus-host interaction, both regarding viral pathogenicity and the body's immunological response is discussed, along with its utility to assist in the development of novel diagnostics.Future applications of the model are also explored.
Mn(CO)(4){S(2)CNMe(CH(2)CO(2)H)}, 1, is shown to be a CO releasing molecule providing at least three moles CO per mole of compound. The mechanism of CO loss is dissociative and reversible and was ...investigated using Gaussian 09 calculations. The reversible binding of CO results in a relatively stable solution of the compound, while in the presence of a CO receptor or a ligand to prevent the rebinding of CO, the CO is lost rapidly. The X-ray structure was determined.
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