Etude structurale et fonctionnelle de la polykétide synthase PpsC et de son activateur PptT chez Mycobacterium tuberculosis

PpsC est une polykétide synthase mono-modulaire de type I essentielle à la synthèse de facteurs de virulence chez Mycobacterium tuberculosis appelés dimycocérosates de phthiocérol. Afin d'aider à la découverte de nouveaux anti-tuberculeux et de mieux comprendre le mécanisme employé par les polykétide synthases, nous avons entrepris l'étude structurale et fonctionnelle de PpsC par cristallographie des rayons X. Les polykétide synthases de type I sont composées de plusieurs domaines, chacun possédant une activité catalytique différente. Nous avons utilisé la méthode du domain trapping pour identifier des fragments solubles représentant chaque domaine de PpsC. L'étude de ces fragments a permis de déterminer la structure des domaines acyl-transférase, déshydratase, et enoyl-reductase de PpsC. Nous avons aussi caractérisé le mécanisme catalytique du domaine déshydratase en se basant sur la structure d'un complexe enzyme-substrat et de tests enzymatiques in vitro. Les polykétide synthases doivent être activées par le transfert d'un groupe prosthétique sur leur domaine porteur d'acyl. Chez Mycobacterium tuberculosis, PptT est responsable de l'activation et est donc essentielle à la virulence. En utilisant la technologie split-GFP, nous avons mis au point un protocole de production et purification de PptT où des cofacteurs jouent un rôle essentiel à sa stabilité in vivo et in vitro. L'activité de l'enzyme a été caractérisée par le transfert in vitro du groupe prosthétique sur le domaine porteur d'acyl de PpsC. Enfin, nous avons résolu la structure tridimensionnelle de PptT à 1,4 A par cristallographie aux rayons X, permettant ainsi la conception rationnelle d'inhibiteurs.PpsC is a type-I mono-modular polyketide synthase essential for the synthesis of phthiocerol dimycocerosates virulence factors in Mycobacterium tuberculosis. In an attempt to both help the search for new antituberculosis drugs, and decipher the complex mechanism used by polyketide synthases, we have undertaken the functional and structural study of PpsC using X-ray crystallography. Type-I polyketide synthases are made of several domains, each possessing a different catalytic activity. We have used the recently developed domain trapping method to identify soluble fragments representing each domain of PpsC. The study of these fragments has led to the structural determination of the acyltransferase, dehydratase, and enoylreductase domains of PpsC. In addition, we have deciphered the catalytic mechanism of the dehydratase domain, based on the structure of a substrate-enzyme complex and in vitro enzymatic tests. Polyketide synthases need to be activated by the transfer of a prosthetic group onto their acyl carrier protein domain. In Mycobacterium tuberculosis, PptT is responsible for the activation of polyketide synthases and is thus essential to its virulence. Using the split-GFP technology, we have designed a protocol for the production and purification of PptT, where its co-factors play an essential role for its stability both in vivo and in vitro. Activity of the enzyme was assessed by in vitro transfer of the prosthetic group onto the acyl carrier protein domain of PpsC. Finally, we have solved the structure of PptT at 1.4 A resolution using X-ray crystallography, which will allow the structure-based design of inhibitors

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

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.Medical Research Council, Bloodwise, Wellcome Trust, Ted’s Gang, The Connor Wright Shwachman Diamond Projec

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

A conservation roadmap for the subterranean biome

The 15th UN Convention on Biological Diversity (CBD) (COP15) will be held in Kunming, China in October 2021. Historically, CBDs and other multilateral treaties have either alluded to or entirely overlooked the subterranean biome. A multilateral effort to robustly examine, monitor, and incorporate the subterranean biome into future conservation targets will enable the CBD to further improve the ecological effectiveness of protected areas by including groundwater resources, subterranean ecosystem services, and the profoundly endemic subsurface biodiversity. To this end, we proffer a conservation roadmap that embodies five conceptual areas: (1) science gaps and data management needs; (2) anthropogenic stressors; (3) socioeconomic analysis and conflict resolution; (4) environmental education; and (5) national policies and multilateral agreements.Peer reviewe

Teaching old drugs new tricks.

Peer reviewed: TrueUnderstanding the mechanism by which streptomycin binds to the small subunit of the mitoribosome may help researchers design less toxic derivatives of this antibiotic

Serious Issues with Cryo-EM Structures of Human Prothrombinase

Thrombin is generated from prothrombin through sequential cleavage at two sites by the enzyme complex prothrombinase, composed of a serine protease, factor (f) Xa, and a cofactor, fVa, on phospholipid membranes. The strict order of cleavage is first at Arg320 to produce the active intermediate meizothrombin, followed by cleavage at Arg271 to release thrombin from its membrane-anchoring pro-domains. Since thrombin is the effector enzyme in blood coagulation, a structural understanding of the assembly and function of prothrombinase is of critical importance. The affinity of fXa for fVa is low, with assembly dependent on a phospholipid membrane surface, which makes structural studies challenging. In a recent paper published in the journal Blood, Ruben and colleagues from the group of Enrico Di Cera reported a major breakthrough in the field (DOI: 10.1182/blood.2022015807): the cryo-EM structures of human prothrombinase on nanodiscs at 5.5Å resolution (7TPQ) and of a catalytically inert human prothrombinase with its substrate prothrombin in the absence of any membrane at 4.1Å resolution (7TPP). The breakthrough warranted a commentary in Blood and featured as a State of the Art lecture at the ISTH conference in 2022, including an accompanying article in the Journal of Thrombosis and Haemostasis (DOI:https://doi.org/10.1002/rth2.12830). As is the norm in structural biology, the original paper was reviewed without access to the deposited coordinates and maps, and it was therefore not possible for referees to assess the validity of the structures or their interpretations. In this article we provide a post hoc analysis of the quality of the reported coordinates and maps, and look closely at the claimed intermolecular contacts on which the supposed breakthrough depends. We demonstrate that the work is deeply flawed, with not a single claimed intermolecular contact supported by the map. We initially hypothesized that the low information content of the map EMD26060 and its unusual features were evidence of extreme reference bias, sometimes called ‘Einstein from noise’. However, the authors acceded to our request to deposit their data on the EMPIAR server (11615) and it is now clear that this is not the case (https://pubpeer.com/publications/1A47110B82A77E2902BFC5BE8E97B0). Rather, the pathology of the map is due to extreme orientation bias, resulting in poor quality, striation and discontinuity in the map, coupled with an over-estimation of resolution. We conclude that the map EMD26060 and coordinates 7TPP are deeply flawed and do not contain any useful information regarding the assembly or function of the prothrombinase complex

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

International audienceThe 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 0-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

Insights into Substrate Modification by Dehydratases from Type I Polyketide Synthases

International audienceDehydration 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