New developments in probing and targeting protein acylation in malaria, leishmaniasis and African sleeping sickness

Infections by protozoan parasites, such as Plasmodium falciparum or Leishmania donovani, have a significant health, social and economic impact and threaten billions of people living in tropical and sub-tropical regions of developing countries worldwide. The increasing range of parasite strains resistant to frontline therapeutics makes the identification of novel drug targets and the development of corresponding inhibitors vital. Post-translational modifications (PTMs) are important modulators of biology and inhibition of protein lipidation has emerged as a promising therapeutic strategy for treatment of parasitic diseases. In this review we summarize the latest insights into protein lipidation in protozoan parasites. We discuss how recent chemical proteomic approaches have delivered the first global overviews of protein lipidation in these organisms, contributing to our understanding of the role of this PTM in critical metabolic and cellular functions. Additionally, we highlight the development of new small molecule inhibitors to target parasite acyl transferases

Surface and Subsurface Attenuation of Trenbolone Acetate Metabolites and Manure-Derived Constituents in Irrigation Runoff on Agro-Ecosystems

Although studies have evaluated the ecotoxicity and fate of trenbolone acetate (TBA) metabolites, namely 17α-trenbolone (17α-TBOH), 17β-trenbolone (17β-TBOH), and trendione (TBO), their environmental transport processes remain poorly characterized with little information available to guide agricultural runoff management. Therefore, we evaluated TBA metabolite transport in representative agricultural systems with concurrent assessment of other manure-derived constituents. Leachate generated using manure from TBA-implanted cattle was applied to a subsurface infiltration plot (4 m) and surface vegetative filter strips (VFSs; 3, 4, and 5 m). In the subsurface experiment, 17α-TBOH leachate concentrations were 36 ng L−1 but decreased to 12 ng L−1 in initial subsurface discharge. Over 75 minutes, concentrations linearly increased to 23 ng L−1 (C/Co = 0.32–0.64). In surface experiments (n = 4), 17α-TBOH leachate concentrations ranged from 11–150 ng L−1, remained nearly constant with time, but were attenuated by ∼70–90% after VFS treatment with no statistical dependence on the VFS length. While attenuation clearly occurred, the observations of a highly mobile fraction of all constituents in both surface runoff and subsurface discharge suggest that these treatment strategies may not always be capable of achieving threshold discharge concentrations. To attain no observed adverse effect levels (NOAELs) in receiving waters, concurrent assessment of leachate concentrations and available dilution capacities can be used to guide target treatment performance levels for runoff management. Dilution is usually necessary to achieve NOAELs, and receiving waters with less than 70–100 fold dilution capacity are at the highest risk for steroidal endocrine disruption

Coping with strong translational non-crystallographic symmetry and extreme anisotropy in molecular replacement with Phaser: human Rab27a

Data pathologies caused by effects such as diffraction anisotropy and translational non-crystallographic symmetry (tNCS) can dramatically complicate the solution of crystal structures of macromolecules. Such problems were encountered in determining the structure of a mutant form of Rab27a, a member of the Rab GTPases. Mutant Rab27a constructs that crystallise in the free form were designed for use in the discovery of drugs to reduce primary tumour invasiveness and metastasis. One construct, hRab27aMut, crystallised within 24 hours and diffracted to 2.82 Å with a unit cell possessing room for a large number of protein copies. Initial efforts to solve the structure using molecular replacement by Phaser were not successful. Analysis of the dataset revealed that the crystals suffered from both extreme anisotropy and strong tNCS. As a result, large numbers of reflections had estimated standard deviations much larger than their measured intensities and their expected intensities, revealing problems with the use of such data at the time in Phaser. By eliminating extremely weak reflections with the largest combined effects of anisotropy and tNCS, these problems could be avoided, allowing a molecular replacement solution to be found. Lessons learned in solving this structure have guided improvements in numerical analysis used in Phaser, particularly in identifying diffraction measurements conveying very little information content. The calculation of information content could also be applied as an alternative to ellipsoidal truncation. The post mortem analysis also revealed an oversight in accounting for measurement errors in the fast rotation function. While the crystal of mutant Rab27a is not amenable to drug screening, the structure can guide new modifications to obtain more suitable crystal forms

Global Profiling and Inhibition of Protein Lipidation in Vector and Host Stages of the Sleeping Sickness Parasite Trypanosoma brucei

The enzyme N-myristoyltransferase (NMT) catalyzes the essential fatty acylation of substrate proteins with myristic acid in eukaryotes and is a validated drug target in the parasite Trypanosoma brucei, the causative agent of African trypanosomiasis (sleeping sickness). N-Myristoylation typically mediates membrane localization of proteins and is essential to the function of many. However, only a handful of proteins are experimentally validated as N-myristoylated in T. brucei. Here, we perform metabolic labeling with an alkyne-tagged myristic acid analogue, enabling the capture of lipidated proteins in insect and host life stages of T. brucei. We further compare this with a longer chain palmitate analogue to explore the chain length-specific incorporation of fatty acids into proteins. Finally, we combine the alkynyl-myristate analogue with NMT inhibitors and quantitative chemical proteomics to globally define N-myristoylated proteins in the clinically relevant bloodstream form parasites. This analysis reveals five ARF family small GTPases, calpain-like proteins, phosphatases, and many uncharacterized proteins as substrates of NMT in the parasite, providing a global view of the scope of this important protein modification and further evidence for the crucial and pleiotropic role of NMT in the cell

Mouse Stbd1 is N-myristoylated and affects ER–mitochondria association and mitochondrial morphology

Starch binding domain-containing protein 1 (Stbd1) is a carbohydrate-binding protein that has been proposed to be a selective autophagy receptor for glycogen. Here, we show that mouse Stbd1 is a transmembrane endoplasmic reticulum (ER)-resident protein with the capacity to induce the formation of organized ER structures in HeLa cells. In addition to bulk ER, Stbd1 was found to localize to mitochondria-associated membranes (MAMs), which represent regions of close apposition between the ER and mitochondria. We demonstrate that N-myristoylation and binding of Stbd1 to glycogen act as major determinants of its subcellular targeting. Moreover, overexpression of non-myristoylated Stbd1 enhanced the association between ER and mitochondria, and further induced prominent mitochondrial fragmentation and clustering. Conversely, shRNA-mediated Stbd1 silencing resulted in an increase in the spacing between ER and mitochondria, and an altered morphology of the mitochondrial network, suggesting elevated fusion and interconnectivity of mitochondria. Our data unravel the molecular mechanism underlying Stbd1 subcellular targeting, support and expand its proposed function as a selective autophagy receptor for glycogen and uncover a new role for the protein in the physical association between ER and mitochondria

Molecules incorporating a benzothiazole core scaffold inhibit the N-myristoyltransferase of Plasmodium falciparum.

Recombinant N-myristoyltransferase of Plasmodium falciparum (termed PfNMT) has been used in the development of a SPA (scintillation proximity assay) suitable for automation and high-throughput screening of inhibitors against this enzyme. The ability to use the SPA has been facilitated by development of an expression and purification system which yields considerably improved quantities of soluble active recombinant PfNMT compared with previous studies. Specifically, yields of pure protein have been increased from 12 microg x l(-1) to >400 microg x l(-1) by use of a synthetic gene with codon usage optimized for expression in an Escherichia coli host. Preliminary small-scale 'piggyback' inhibitor studies using the SPA have identified a family of related molecules containing a core benzothiazole scaffold with IC50 values 80% at a concentration of 10 microM

Tipifarnib prevents development of hypoxia-induced pulmonary hypertension

Aims. RhoB plays a key role in the pathogenesis of hypoxia - induced pulmonary hypertension. Farne sylated RhoB promotes growth responses in cancer cells and we investigated whether inhibition of protein farnesylation will have a protective effect. Methods and Results. The analysis of l ung tissues from rodent models and pulmonary hypertensive patients showed increased levels of protein farnesylation. Oral farnesyltransferase inhibitor tipifarnib prevented development of hypoxia - induced pulmonary hypertension in mice. Tipifarnib reduced hypoxia - induced vascular cell proliferation, increased endothelium - dependent vasodilatation and reduced vasoconstriction of intrapulmonary arteries without affecting cell viability. Protective effects of tipifarnib were associated with inhibition of Ras and RhoB, actin depolymerisation and increased eNOS expression in vi tro and in vivo . Farnesylated - only RhoB (F - RhoB) increased proliferative responses in cultured pulmonary vascular cells, mimicking the effects of hypoxia, while both geranylgeranylated - only RhoB (GG - RhoB) and tipifarnib had an inhibitory effect. Label - fre e proteomics linked F - RhoB with cell survival, activation of cell cycle and mitochondrial biogenesis. Hypoxia increased and tipifarnib reduced the levels of F - RhoB - regulated proteins in the lung, reinforcing the importance of RhoB as a signalling mediator. Unlike simvastatin, tipifarnib did not increase the expression levels of Rho proteins. Conclusions. Our study demonstrates the importance of protein farnesylation in pulmonary vascular remodeling and provides a rationale for selective targeting of this pa thway in pulmonary hypertension