EFL1 mutations impair eIF6 release to cause Shwachman-Diamond syndrome.

Shwachman-Diamond syndrome (SDS) is a recessive disorder typified by bone marrow failure and predisposition to hematological malignancies. SDS is predominantly caused by deficiency of the allosteric regulator Shwachman-Bodian-Diamond syndrome that cooperates with elongation factor-like GTPase 1 (EFL1) to catalyze release of the ribosome antiassociation factor eIF6 and activate translation. Here, we report biallelic mutations in EFL1 in 3 unrelated individuals with clinical features of SDS. Cellular defects in these individuals include impaired ribosomal subunit joining and attenuated global protein translation as a consequence of defective eIF6 eviction. In mice, Efl1 deficiency recapitulates key aspects of the SDS phenotype. By identifying biallelic EFL1 mutations in SDS, we define this leukemia predisposition disorder as a ribosomopathy that is caused by corruption of a fundamental, conserved mechanism, which licenses entry of the large ribosomal subunit into translation.

Detection of soluble co-factor dependent protein expression in vivo: application to the 4'-phosphopantetheinyl transferase PptT from Mycobacterium tuberculosis.

The need for early-on diagnostic tools to assess the folding and solubility of expressed protein constructs in vivo is of great interest when dealing with recalcitrant proteins. In this paper, we took advantage of the picomolar sensitivity of the bipartite GFP1-10/GFP11 system to investigate the solubility of the Mycobacterium tuberculosis 4'-phosphopantetheinyl transferase PptT, an enzyme essential for the viability of the tubercle bacillus. In vivo and in vitro complementation assays clearly showed the improved solubility of the full-length PptT compared to its N- and C-terminally truncated counterparts. However, initial attempts to purify the full-length enzyme overexpressed in Escherichia coli cells were hampered by aggregation issues overtime that caused the protein to precipitate within hours. The fact that the naturally occurring Coenzyme A and Mg(2+), essentials for PptT to carry out its function, could play a role in stabilizing the enzyme was confirmed using DSF experiments. In vitro activity assays were performed using the ACP substrate from the type I polyketide synthase PpsC from M. tuberculosis, a 2188 amino-acid enzyme that plays a major role in the virulence and pathogenicity of this microbial pathogen. We selected the most soluble and compact ACP fragment (2042-2188), identified by genetic selection of in-frame fragments from random library experiments, to monitor the transfer of the P-pant moiety from Coenzyme A onto a conserved serine residue of this ACP domain.

Experimental mapping of soluble protein domains using a hierarchical approach.

Exploring the function and 3D space of large multidomain protein targets often requires sophisticated experimentation to obtain the targets in a form suitable for structure determination. Screening methods capable of selecting well-expressed, soluble fragments from DNA libraries exist, but require the use of automation to maximize chances of picking a few good candidates. Here, we describe the use of an insertion dihydrofolate reductase (DHFR) vector to select in-frame fragments and a split-GFP assay technology to filter-out constructs that express insoluble protein fragments. With the incorporation of an IPCR step to create high density, focused sublibraries of fragments, this cost-effective method can be performed manually with no a priori knowledge of domain boundaries while permitting single amino acid resolution boundary mapping. We used it on the well-characterized p85α subunit of the phosphoinositide-3-kinase to demonstrate the robustness and efficiency of our methodology. We then successfully tested it onto the polyketide synthase PpsC from Mycobacterium tuberculosis, a potential drug target involved in the biosynthesis of complex lipids in the cell envelope. X-ray quality crystals from the acyl-transferase (AT), dehydratase (DH) and enoyl-reductase (ER) domains have been obtained.

The chemical landscape of the human ribosome at 1.67 Å resolution.

The ability of ribosomes to translate the genetic code into protein requires a finely tuned ion and solvent ecosystem. However, the lack of high-resolution structures has precluded accurate positioning of all the functional elements of the ribosome and limited our understanding of the specific role of ribosomal RNA chemical modifications in modulating ribosome function in health and disease. Here, using a new sample preparation methodology based on functionalised pristine graphene-coated grids, we solve the cryo-EM structure of the human large ribosomal subunit to a resolution of 1.67 Å. The accurate assignment of water molecules, magnesium and potassium ions in our model highlights the fundamental biological role of ribosomal RNA methylation in harnessing unconventional carbon-oxygen hydrogen bonds to establish chemical interactions with the environment and fine-tune the functional interplay with tRNA. In addition, the structures of three translational inhibitors bound to the human large ribosomal subunit at better than 2 Å resolution provide mechanistic insights into how three key druggable pockets of the ribosome are targeted and illustrate the potential of this methodology to accelerate high-throughput structure-based design of anti-cancer therapeutics.

Teaching old drugs new tricks.

Understanding the mechanism by which streptomycin binds to the small subunit of the mitoribosome may help researchers design less toxic derivatives of this antibiotic.

Direct targeted therapy for MLL-fusion-driven high-risk acute leukaemias.

BACKGROUND: Improving the poor prognosis of infant leukaemias remains an unmet clinical need. This disease is a prototypical fusion oncoprotein-driven paediatric cancer, with MLL (KMT2A)-fusions present in most cases. Direct targeting of these driving oncoproteins represents a unique therapeutic opportunity. This rationale led us to initiate a drug screening with the aim of discovering drugs that can block MLL-fusion oncoproteins. METHODS: A screen for inhibition of MLL-fusion proteins was developed that overcomes the traditional limitations of targeting transcription factors. This luciferase reporter-based screen, together with a secondary western blot screen, was used to prioritize compounds. We characterized the lead compound, disulfiram (DSF), based on its efficient ablation of MLL-fusion proteins. The consequences of drug-induced MLL-fusion inhibition were confirmed by cell proliferation, colony formation, apoptosis assays, RT-qPCR, in vivo assays, RNA-seq and ChIP-qPCR and ChIP-seq analysis. All statistical tests were two-sided. RESULTS: Drug-induced inhibition of MLL-fusion proteins by DSF resulted in a specific block of colony formation in MLL-rearranged cells in vitro, induced differentiation and impeded leukaemia progression in vivo. Mechanistically, DSF abrogates MLL-fusion protein binding to DNA, resulting in epigenetic changes and down-regulation of leukaemic programmes setup by the MLL-fusion protein. CONCLUSION: DSF can directly inhibit MLL-fusion proteins and demonstrate antitumour activity both in vitro and in vivo, providing, to our knowledge, the first evidence for a therapy that directly targets the initiating oncogenic MLL-fusion protein.

eIF6 rebinding dynamically couples ribosome maturation and translation.

Protein synthesis is a cyclical process consisting of translation initiation, elongation, termination and ribosome recycling. The release factors SBDS and EFL1-both mutated in the leukemia predisposition disorder Shwachman-Diamond syndrome - license entry of nascent 60S ribosomal subunits into active translation by evicting the anti-association factor eIF6 from the 60S intersubunit face. We find that in mammalian cells, eIF6 holds all free cytoplasmic 60S subunits in a translationally inactive state and that SBDS and EFL1 are the minimal components required to recycle these 60S subunits back into additional rounds of translation by evicting eIF6. Increasing the dose of eIF6 in mice in vivo impairs terminal erythropoiesis by sequestering post-termination 60S subunits in the cytoplasm, disrupting subunit joining and attenuating global protein synthesis. These data reveal that ribosome maturation and recycling are dynamically coupled by a mechanism that is disrupted in an inherited leukemia predisposition disorder.

Hemocyte siRNA uptake is increased by 5' cholesterol-TEG addition in Biomphalaria glabrata, snail vector of schistosome.

Biomphalaria glabrata is one of the snail intermediate hosts of Schistosoma mansoni, the causative agent of intestinal schistosomiasis disease. Numerous molecular studies using comparative approaches between susceptible and resistant snails to S. mansoni infection have helped identify numerous snail key candidates supporting such susceptible/resistant status. The functional approach using RNA interference (RNAi) remains crucial to validate the function of such candidates. CRISPR-Cas systems are still under development in many laboratories, and RNA interference remains the best tool to study B. glabrata snail genetics. Herein, we describe the use of modified small interfering RNA (siRNA) molecules to enhance cell delivery, especially into hemocytes, the snail immune cells. Modification of siRNA with 5' Cholesteryl TriEthylene Glycol (Chol-TEG) promotes cellular uptake by hemocytes, nearly eightfold over that of unmodified siRNA. FACS analysis reveals that more than 50% of hemocytes have internalized Chol-TEG siRNA conjugated to Cy3 fluorophores, 2 hours only after in vivo injection into snails. Chol-TEG siRNA targeting BgTEP1 (ThioEster-containing Protein), a parasite binding protein, reduced BgTEP1 transcript expression by 70-80% compared to control. The level of BgTEP1 protein secreted in the hemolymph was also decreased. However, despite the BgTEP1 knock-down at both RNA and protein levels, snail compatibility with its sympatric parasite is not affected suggesting functional redundancy among the BgTEP genes family in snail-schistosoma interaction.

Somatic genetic rescue of a germline ribosome assembly defect.

Indirect somatic genetic rescue (SGR) of a germline mutation is thought to be rare in inherited Mendelian disorders. Here, we establish that acquired mutations in the EIF6 gene are a frequent mechanism of SGR in Shwachman-Diamond syndrome (SDS), a leukemia predisposition disorder caused by a germline defect in ribosome assembly. Biallelic mutations in the SBDS or EFL1 genes in SDS impair release of the anti-association factor eIF6 from the 60S ribosomal subunit, a key step in the translational activation of ribosomes. Here, we identify diverse mosaic somatic genetic events (point mutations, interstitial deletion, reciprocal chromosomal translocation) in SDS hematopoietic cells that reduce eIF6 expression or disrupt its interaction with the 60S subunit, thereby conferring a selective advantage over non-modified cells. SDS-related somatic EIF6 missense mutations that reduce eIF6 dosage or eIF6 binding to the 60S subunit suppress the defects in ribosome assembly and protein synthesis across multiple SBDS-deficient species including yeast, Dictyostelium and Drosophila. Our data suggest that SGR is a universal phenomenon that may influence the clinical evolution of diverse Mendelian disorders and support eIF6 suppressor mimics as a therapeutic strategy in SDS.

Molecular Basis for Extender Unit Specificity of Mycobacterial Polyketide Synthases.

Mycobacterium tuberculosis is the causative agent of the tuberculosis disease, which claims more human lives each year than any other bacterial pathogen. M. tuberculosis and other mycobacterial pathogens have developed a range of unique features that enhance their virulence and promote their survival in the human host. Among these features lies the particular cell envelope with high lipid content, which plays a substantial role in mycobacterial pathogenicity. Several envelope components of M. tuberculosis and other mycobacteria, e.g., mycolic acids, phthiocerol dimycocerosates, and phenolic glycolipids, belong to the "family" of polyketides, secondary metabolites synthesized by fascinating versatile enzymes-polyketide synthases. These megasynthases consist of multiple catalytic domains, among which the acyltransferase domain plays a key role in selecting and transferring the substrates required for polyketide extension. Here, we present three new crystal structures of acyltransferase domains of mycobacterial polyketide synthases and, for one of them, provide evidence for the identification of residues determining extender unit specificity. Unravelling the molecular basis for such specificity is of high importance considering the role played by extender units for the final structure of key mycobacterial components. This work provides major advances for the use of mycobacterial polyketide synthases as potential therapeutic targets and, more generally, contributes to the prediction and bioengineering of polyketide synthases with desired specificity.

Mechanism of completion of peptidyltransferase centre assembly in eukaryotes.

During their final maturation in the cytoplasm, pre-60S ribosomal particles are converted to translation-competent large ribosomal subunits. Here, we present the mechanism of peptidyltransferase centre (PTC) completion that explains how integration of the last ribosomal proteins is coupled to release of the nuclear export adaptor Nmd3. Single-particle cryo-EM reveals that eL40 recruitment stabilises helix 89 to form the uL16 binding site. The loading of uL16 unhooks helix 38 from Nmd3 to adopt its mature conformation. In turn, partial retraction of the L1 stalk is coupled to a conformational switch in Nmd3 that allows the uL16 P-site loop to fully accommodate into the PTC where it competes with Nmd3 for an overlapping binding site (base A2971). Our data reveal how the central functional site of the ribosome is sculpted and suggest how the formation of translation-competent 60S subunits is disrupted in leukaemia-associated ribosomopathies.

Binding of ISRIB reveals a regulatory site in the nucleotide exchange factor eIF2B.

The integrated stress response (ISR) is a conserved translational and transcriptional program affecting metabolism, memory, and immunity. The ISR is mediated by stress-induced phosphorylation of eukaryotic translation initiation factor 2α (eIF2α) that attenuates the guanine nucleotide exchange factor eIF2B. A chemical inhibitor of the ISR, ISRIB, reverses the attenuation of eIF2B by phosphorylated eIF2α, protecting mice from neurodegeneration and traumatic brain injury. We describe a 4.1-angstrom-resolution cryo-electron microscopy structure of human eIF2B with an ISRIB molecule bound at the interface between the ß and δ regulatory subunits. Mutagenesis of residues lining this pocket altered the hierarchical cellular response to ISRIB analogs in vivo and ISRIB binding in vitro. Our findings point to a site in eIF2B that can be exploited by ISRIB to regulate translation.

Insights into Substrate Modification by Dehydratases from Type I Polyketide Synthases.

Dehydration reactions play a crucial role in the de novo biosynthesis of fatty acids and a wide range of pharmacologically active polyketide natural products with strong emphasis on human medicine. The type I polyketide synthase PpsC from Mycobacterium tuberculosis catalyzes key biosynthetic steps of lipid virulence factors phthiocerol dimycocerosates and phenolic glycolipids. Given the insolubility of the natural C28-C30 fatty acyl substrate of the PpsC dehydratase (DH) domain, we investigated its structure-function relationships in the presence of shorter surrogate substrates. Since most enzymes belonging to the (R)-specific enoyl hydratase/hydroxyacyl dehydratase family conduct the reverse hydration reaction in vitro, we have determined the X-ray structures of the PpsC DH domain, both unliganded (apo) and in complex with trans-but-2-enoyl-CoA or trans-dodec-2-enoyl-CoA derivatives. This study provides for the first time a snapshot of dehydratase-ligand interactions following a hydration reaction. Our structural analysis allowed us to identify residues essential for substrate binding and activity. The structural comparison of the two complexes also sheds light on the need for long acyl chains for this dehydratase to carry out its function, consistent with both its in vitro catalytic behavior and the physiological role of the PpsC enzyme.