Extensive molecular tinkering in the evolution of the membrane attachment mode of the Rheb GTPase

Rheb is a conserved and widespread Ras-like GTPase involved in cell growth regulation mediated by the (m)TORC1 kinase complex and implicated in tumourigenesis in humans. Rheb function depends on its association with membranes via prenylated C-terminus, a mechanism shared with many other eukaryotic GTPases. Strikingly, our analysis of a phylogenetically rich sample of Rheb sequences revealed that in multiple lineages this canonical and ancestral membrane attachment mode has been variously altered. The modifications include: (1) accretion to the N-terminus of two different phosphatidylinositol 3-phosphate-binding domains, PX in Cryptista (the fusion being the first proposed synapomorphy of this clade), and FYVE in Euglenozoa and the related undescribed flagellate SRT308; (2) acquisition of lipidic modifications of the N-terminal region, namely myristoylation and/or S-palmitoylation in seven different protist lineages; (3) acquisition of S-palmitoylation in the hypervariable C-terminal region of Rheb in apusomonads, convergently to some other Ras family proteins; (4) replacement of the C-terminal prenylation motif with four transmembrane segments in a novel Rheb paralog in the SAR clade; (5) loss of an evident C-terminal membrane attachment mechanism in Tremellomycetes and some Rheb paralogs of Euglenozoa. Rheb evolution is thus surprisingly dynamic and presents a spectacular example of molecular tinkering

In Silico Identification of Protein S‑Palmitoylation Sites and Their Involvement in Human Inherited Disease

S-Palmitoylation is a key regulatory mechanism controlling protein targeting, localization, stability, and activity. Since increasing evidence shows that its disruption is implicated in many human diseases, the identification of palmitoylation sites is attracting more attention. However, the computational methods that are published so far for this purpose have suffered from a poor balance of sensitivity and specificity; hence, it is difficult to get a good generalized prediction ability on an external validation set, which holds back the further analysis of associations between disruption of palmitoylation and human inherited diseases. In this work, we present a reliable identification method for protein S-palmitoylation sites, called SeqPalm, based on a series of newly composed features from protein sequences and the synthetic minority oversampling technique. With only 16 extracted key features, this approach achieves the most favorable prediction performance up to now with sensitivity, specificity, and Matthew’s correlation coefficient values of 95.4%, 96.3%, and 0.917, respectively. Then, all known disease-associated variations are studied by SeqPalm. It is found that 243 potential loss or gain of palmitoylation sites are highly associated with human inherited disease. The analysis presents several potential therapeutic targets for inherited diseases associated with loss or gain of palmitoylation function. There are even biological evidence that are coordinate with our prediction results. Therefore, this work presents a novel approach to discover the molecular basis of pathogenesis associated with abnormal palmitoylation. SeqPalm is now available online at http://lishuyan.lzu.edu.cn/seqpalm, which can not only annotate the palmitoylation sites of proteins but also distinguish loss or gain of palmitoylation sites by protein variations

A synthetic biology toolbox for examining and engineering strigolactone biosynthesis

Strigolactones (SLs) are plant hormones and rhizosphere-signalling molecules that control plant architecture and environmental adaption, promote symbioses with soil organisms, and mediate root parasitism. These diverse activities give SLs great promise as agrochemicals, with potential applications in optimising plant architecture, controlling parasitic weeds, enhancing nutrient uptake, and improving tolerance to drought and salinity. However, agricultural use is currently unfeasible as there are no economically viable sources of SLs or SL analogues. Microbes engineered for SL biosynthesis could provide a cheap, renewable, and scalable production method that could overcome current supply challenges, and enable SLs to be deployed as agrochemicals.Synthetic biology – characterised by modularity, standardisation, interoperability of biological parts, and engineering principals such as the design-build-test-learn cycle – offers an approach to transform microbial engineering into an engineering discipline. This thesis describes the development of a synthetic biology toolbox for studying and engineering SL biosynthesis, comprising a SL production module, a detection module, and characterisation of one of the least understood enzymes in the pathway.\ua0Saccharomyces cerevisiae was selected as the initial host organism due to its favourable industrial properties, and previous work demonstrating that it is a suitable host for multiple elements in the SL biosynthetic pathway. However, conversion of β-carotene to CL was achieved at very low titres, with CL produced at approximately 1,000,000-fold lower concentration than b-carotene. This was at least in part due to poor conversion of all-trans-β-carotene to 9-cis-β-carotene through DWARF27 (D27), prompting further investigation into this enzyme.Characterization of enzymes increases the reliability of their deployment as a biological part in an engineered system, and provides background information for enzyme engineering. As little is known about the D27 structure-function relationship, the localization and activity of rice (Oryza sativa) D27 (OsD27) were investigated, and efforts were made to elucidate its three-dimensional structure. In a transient Nicotiana benthamiana system, OsD27 was found in stromal and thylakoid membrane-bound forms, demonstrating the sub-organellar localisation of this enzyme for the first time. A maltose binding protein (MBP) fusion of OsD27 had activity in E. coli, and the purified fusion of OsD27 catalysed the reversible isomerisation of β-carotene around the C9-C10 double bond in vitro, with Km = 3.3­ ± 1.2 mM for all-trans-β-carotene and Km = 5.4 ± 1.4 mM for 9-cis-β-carotene. Extensive efforts to crystallise OsD27 for structural characterization did not yield results. Purified MBP fusions of OsD27 aggregated into soluble oligomers ranging from 10-150 nm in hydrodynamic radius. The aggregates were not compatible with crystal formation, and could not be completely dissociated using detergents. A non-aggregating D27 homologue from Ziziphus jujuba was identified, and future crystallization trials using this protein may provide insight into the structure-function relationship of D27.As D27 showed activity in Escherichia coli, E. coli was investigated as an alternative production host for SL. Introduction of rice (Oryza sativa) OsD27ΔTP, pea (Pisum sativum) PsCCD7 and PsCCD8 resulted in production of the SL precursor, CL. A combinatorial screen of enzyme fusion partners identified thioredoxin (Trx)-OsD27ΔTP, Trx-PsCCD7 and MBP-PsCCD8 produced 147 ± 17 µg/L (mean ± standard deviation) CL in shake flask cultures after 72 hours. Enhancing precursor supply by introduction of a lower mevalonate pathway supplemented with mevalonate as a substrate increased production to 221 ± 22 µg/L (mean ± standard deviation). The CL-production strain provides a valuable source of CL for research, will act as a chassis for investigating downstream SL diversification pathways, and serves as a starting strain for engineering production of SLs.High-throughput strain construction is rapidly becoming available through automation, and dedicated software and machine-learning approaches are accelerating design and learn phases of metabolic engineering. These developments leave the test phase as a rate-limiting step in many design-build-test-learn cycles. To debottleneck the test phase for SL strain engineering, genetically encoded fluorescent SL biosensors were developed as a detection method compatible with high throughput analysis. The biosensors used domain insertion of circularly permuted GFP into the SL receptors DAD2 from Petunia hybrida, and HTL7 from Striga hermonthica, such that binding of SLs resulted in loss of fluorescence in vitro or in an in vivo protoplast system. In addition to applications in high-throughput screening, these biosensors may have utility for studying SL biology.This toolkit is expected to enable investigations into SL diversification, and expedite future strain engineering for production of SLs, ultimately supporting the development of SLs as agrochemicals to address current and future challenges in agriculture

Palmitoylation and regulation of the funny current HCN4 channel

The sinoatrial node (SAN) acts as the primary pacemaker of the heart as it spontaneously generates electrical activity that propagate through the cardiac conduction system, underpinning automaticity of the heart. A network of surface membrane ion currents (“membrane clock”) and the rhythmic oscillation of local Ca²⁺ release from the sarcoplasmic reticulum (“calcium clock”) work interdependently to form a coupled-clock system that drives pacemaker automaticity and its regulation on a beat-to-beat basis. The “funny current” (If) is a key component of the membrane clock contributing to the diastolic depolarisation of the SAN. Hyperpolarisation-activated cyclic nucleotide-gated channel HCN4 is the predominant isoform responsible for almost 70% of the sinoatrial If. HCN4 channels localise to lipid rafts in the SAN and disorganisation of these raft membrane microdomains result in channel redistribution, thus altering its kinetic properties. Ion channels are an integral component of the complex sinoatrial pacemaking network, and their regulation is therefore central to controlling the heart rate. S-palmitoylation is a form of lipidation that involves the covalent addition of a 16-carbon palmitate to a thiol group of a cysteine residue in a protein. Unlike most lipid modifications, palmitoylation is unique due to its reversible nature, allowing the dynamic regulation of both soluble and integral proteins. In recent years, palmitoylation has emerged as an important regulator of cardiac electrophysiology as it influences the function and membrane microdomain localisation of key cardiac Na⁺ and Ca²⁺ handling proteins. The present in-vitro study was adopted to characterise palmitoylation of HCN4 channels and to establish its functional consequences. Site-specific resin assisted capture (acyl-RAC) was used to assess palmitoylation of HCN4 in human embryonic kidney (HEK) cells as well as endogenous HCN4 in isolated neonatal rat whole heart and atrial myocytes. HCN4 was sub-stoichiometrically palmitoylated in all experimental systems examined. Truncated HCN4 intracellular amino and carboxyl termini fused to YFP and cysteine-to-alanine mutations of the palmitoylation sites in HEK-293 cells mapped HCN4 palmitoylation sites to a pair of cysteines (C93 and C179) in the HCN4 N-terminus domain. A double cysteine-to-alanine mutation C93/179AA of both palmitoylation sites reduced palmitoylation of full-length HCN4 by ~67% in comparison to wild type HCN4. Membrane impermeable biotinylation of cell surface HCN4 revealed that palmitoylation did not influence its trafficking to the cell surface or cell surface turnover rate. Standard discontinuous sucrose gradient indicated that HCN4 channels did not require palmitoylation to localise to lipid rafts in HEK-293 cells. Whole-cell patch clamp was used to investigate IHCN4 in HEK-293 cells engineered to stably express wild type and mutant HCN4. Loss of palmitoylation at the N-terminus significantly reduced HCN4 current magnitude by ~5 to 8-fold across a range of voltages. However, it did not alter its half-maximal activation voltage (V₀.₅: -90.4 ± 2.5 mV for WT vs -90.4 ± 1.6 mV for C93/179AA), nor its activation slope factor (k: 7.1 ± 0.5 mV for WT vs 6.0 ± 0.2 mV for C93/179AA). Phylogenetic analysis was used to evaluate the evolutionary acquisition of HCN4 palmitoylation within the pre-metazoan and metazoan lineage. While cysteine 93 was broadly conserved within all classes of HCN4 vertebrate orthologs, conservation of cysteine 179 was confined to placental mammals. Together, this study demonstrated the importance of palmitoylation as a regulator of HCN4 channel function by enhancing HCN4-mediated currents. Palmitoylation of the HCN4 amino terminus is likely to significantly enhance If in the SAN, accelerating diastolic depolarisation, and increasing heart rate