Enhancing Human Spermine Synthase Activity by Engineered Mutations

Spermine synthase (SMS) is an enzyme which function is to convert spermidine into spermine. It was shown that gene defects resulting in amino acid changes of the wild type SMS cause Snyder-Robinson syndrome, which is a mild-to-moderate mental disability associated with osteoporosis, facial asymmetry, thin habitus, hypotonia, and a nonspecific movement disorder. These disease-causing missense mutations were demonstrated, both in silico and in vitro, to affect the wild type function of SMS by either destabilizing the SMS dimer/monomer or directly affecting the hydrogen bond network of the active site of SMS. In contrast to these studies, here we report an artificial engineering of a more efficient SMS variant by transferring sequence information from another organism. It is confirmed experimentally that the variant, bearing four amino acid substitutions, is catalytically more active than the wild type. The increased functionality is attributed to enhanced monomer stability, lowering the pKa of proton donor catalytic residue, optimized spatial distribution of the electrostatic potential around the SMS with respect to substrates, and increase of the frequency of mechanical vibration of the clefts presumed to be the gates toward the active sites. The study demonstrates that wild type SMS is not particularly evolutionarily optimized with respect to the reaction spermidine → spermine. Having in mind that currently there are no variations (non-synonymous single nucleotide polymorphism, nsSNP) detected in healthy individuals, it can be speculated that the human SMS function is precisely tuned toward its wild type and any deviation is unwanted and disease-causing

IN SILICO MODELING THE EFFECT OF SINGLE POINT MUTATIONS AND RESCUING THE EFFECT BY SMALL MOLECULES BINDING

Single-point mutation in genome, for example, single-nucleotide polymorphism (SNP) or rare genetic mutation, is the change of a single nucleotide for another in the genome sequence. Some of them will result in an amino acid substitution in the corresponding protein sequence (missense mutations); others will not. This investigation focuses on genetic mutations resulting in a change in the amino acid sequence of the corresponding protein. This choice is motivated by the fact that missense mutations are frequently found to affect the native function of proteins by altering their structure, interaction and other properties and cause diseases. A particular disease is the Snyder-Robinson syndrome (SRS), which is an X-linked mental retardation found to be caused by missense mutations in human spermine synthase (SMS). In this thesis, a rational approach to predict the effects of missense mutations on SMS wild-type characteristics was carried. Following this work, a structure-based virtual screening of small molecules was applied to rescue the disease-causing effect by searching the small molecules to stabilize the malfunctioning SMS mutant dimer

Characterization of molecular functions of polyamines in fruit development and ripening in tomato (Solanum lycopersicum)

Putrescine (PUT), spermidine (SPD) and spermine (SPM) are three major polyamines (PAs) present in all living organisms. These biogenic amines have been implicated in diverse plant growth and development processes, including seed germination, tissue lignification, organogenesis, flowering, pollination, embryogenesis, fruit development, ripening, abscission, senescence, and stress responses. To elucidate molecular roles of PAs in fruit development and ripening, I characterized transgenic tomato plants ectopically expressing yeast spermidine synthase (ySpdSyn) or S-adenosylmethionine decarboxylase (ySAMdc) under constitutive CaMV 35S and/or fruit-specific SlE8 promoters. The ySpdSyn-expression enhanced PUT, SPD and SPM level in floral buds and fertilized developing ovaries by 2- to 3-fold compared to WT tissues with majority being sequestered as bound forms. Higher PA levels altered fruit shape of transgenic tomatoes to more obovoid than WT by regulating expression of fruit shape genes (SUN1 and OVATE), and cell division and expansion genes (CDKB2, CYCB2, KRP1 and CCS52B). Characterization of PA homeostasis during fruit growth and ripening revealed a strong correlation of conjugated PAs with transcripts abundance of PA biosynthesis (ODC, ADC, SAMdc3) and catabolizing genes (CuAO-like, PAO4-like) and the bound PAs to transcript levels of ySpdSyn and SAMdc2 suggesting a significant metabolic inter-conversion among the various forms of PAs. Co-expression of ySpdSyn and ySAMdc transgenes showed that SAMdc is the rate limiting step in biosynthesis of higher PAs with potential to alter PA homeostasis in fruit tissues. Characterization of ySpdSyn and ySAMdc transgenic and WT fruits showed that expression of transgenes was associated with higher firmness of ripened fruits both on-planta and after harvest up to 17 days after ripe stage. Free SPD/SPM levels were positively correlated with fruit firmness, accumulation of total solids and delay in fruit shriveling and inversely correlated with fresh fruit weight, juice pH and seed number in tomato fruits. Free PUT levels exhibited trends opposite to that seen with SPD/SPM confirming hypothesis that PUT and SPD/SPM ratios play significant roles in the outcome of biological functions of PAs. Evaluation of ySpdSyn lines under field conditions showed 50% increase in fruit yield per plant due to continued fruit set until late in the season and up to 60% increase in fruit fresh and dry weight much beyond the fruit breaker stage. The metabolomic changes in transgenic fruits were determined using the nuclear magnetic resonance spectroscopy (1H NMR) and compared to WT fruit metabolic profile during on-planta fruit ripening and post-ripening stages. Free SPD levels were positively correlated with Ile, Val, Glu, Gln, Trp, malate, citrate and trigonelline. The levels of Ala, Glu, Asp and UDP-NAcGLU were negatively correlated with free SPD levels but positively correlated with free PUT indicating differential function of these two PAs. Levels of fructose and AMP were also negatively correlated with free SPD. Conjugated and bound PAs exhibited a limited correlation with metabolome profiles. The node-edge network analyses among PAs, metabolites and their associated pathways showed that PAs upregulate many anabolic pathways, but negatively affect glycolysis, starch and sugar metabolism, and zeatin biosynthesis. Taken together these results indicate that SPD is associated with enhancing many metabolic pathways and delaying senescence-related processes leading to improved postharvest fruit quality. I have collated transcriptome of transgenic plants and mutants with altered PA levels. Its analyses revealed complex and differential relationships among PUT, SPD and SPM in regard to regulation of plant hormone biosynthesis and signaling. In summary, the use of transgenic plants with modified PA levels provide an insight into molecular functions of PAs in altering fruit architecture, improving fruit quality attributes, increasing fruit production and delaying ripening-related changes in tomatoes. Limited transcriptome profile suggest a complex crosstalk between PAs and plant growth hormones during fruit ripening. Metabolome profiles of transgenic fruits showed a significant impact of PAs on fruit quality improvement by restoring metabolic pathways during fruit ripening

Characterising ATP13A3 biological function and its role in pulmonary arterial hypertension

Pulmonary arterial hypertension (PAH) is a rare but devastating disease characterised by the progressive remodelling of the small pulmonary vessels. Although the causes may vary, similar pathobiological features are shared among different forms of PAH, with endothelial dysfunction, the hyperproliferation of smooth muscle cells and mesenchymal cells in the vascular wall, as well as inflammation contributing to this process. Despite the availability of licensed therapies, 5-year survival for patients remains less than 60%. To uncover additional fundamental mechanisms of PAH pathobiology, our group undertook whole genome sequencing in 1038 idiopathic or heritable PAH patients and identified disease-associated heterozygous mutations in ATP13A3, a P5B-type ATPase. P5 ATPases, namely ATP13A1-5, are putative transmembrane proteins that generate and maintain significant chemical gradients across biological membranes by active transport of cations. However, to date, studies of their functions are sparse. Therefore, the focus of my project was to characterise ATP13A3 in the pulmonary vasculature and to uncover the underlying mechanism of how mutations in this gene contribute to PAH pathogenesis. Although the biological functions of ATP13A3 are elusive, its homologue (CATP-5) in C. elegans was previously reported as a polyamine transporter. Polyamines are a group of naturally existing polycations essential for many cellular processes. Therefore, my working hypothesis was that ATP13A3 loss of function might disrupt the polyamine homeostasis and hence contribute to the abnormal vascular remodelling in PAH. To address this, I first confirmed ATP13A3 as residing in the recycling endosome of endothelial cells. This localisation of ATP13A3 suggested it might be related to polyamine transport since the endocytic pathway was recognised as a crucial route for polyamine trafficking. To expand this finding, I developed molecular tools to knockdown or to overexpress ATP13A3 in vascular cells and analysed how these affected polyamine transport. This led to the discovery of putrescine as the preferred substrate for ATP13A3 and also revealed a basal reduction of other polyamines in endothelial cells. Further investigation highlighted an essential role of ATP13A3 in pulmonary vasculature with its deficiency restrained cell growth and predisposed endothelial cells to apoptosis. To validate these findings, blood outgrowth endothelial cells (BOECs) were isolated from a PAH patient bearing an LK726X ATP13A3 frameshift mutation (LK726X). Intriguingly, ATP13A3 causing ATP13A3 reduction, impaired polyamine uptake and induced apoptosis in BOECs. This provides the first evidence for the pathogenic effects of a PAH associated ATP13A3 mutation. In addition, using a lentiviral overexpression system, I also demonstrated impairments of other PAH- associated mutations on ATP13A3 mediated polyamine uptake, reinforcing the involvement of ATP13A3 in PAH pathobiology. To explore whether ATP13A3 mutations can cause PAH, our laboratory has generated a mouse model harbouring a human ATP13A3 mutation (P452Lfs) via the MRC Harwell Gene editing service. My colleague, Dr Ekaterina Legchenko and I have discovered that the Atp13a3P452Lfs/P452Lfs mice spontaneously develop PAH-like hemodynamic changes at 6-months of age. Preliminary data also revealed lower Atp13a3 level and reduced polyamine content in the lungs of the mutant mice, suggesting that Atp13a3 frameshift mutation (P452L) may contribute to PAH pathogenesis via the disruption of polyamine homeostasis in vivo. In conclusion, my work characterises for the first time that ATP13A3 is a polyamine transporter in vascular cells with putrescine as its preferable transport substrate. ATP13A3 mutations identified from PAH patients are highly likely to impair polyamine homeostasis and contribute to PAH progression. These findings open up a new field for investigating PAH pathobiology and also highlight the potential role of rebalancing polyamine homeostasis in PAH treatment

Modulation of Coxsackievirus Protease Activity by Polyamines

Coxsackievirus type B (CVB3) is one of the six serotypes of the Coxsackievirus family of non-enveloped, linear, and positive-sense single-stranded RNA viruses. It is a pathogenic enterovirus that belongs to the same genus as the notable pathogen poliovirus. CVB3 can cause a range of illnesses from a fever to gastrointestinal distress but is most noteworthy for the ability to cause viral myocarditis, a swelling of the heart muscle. Coxsackievirus, like all RNA viruses, tends to develop mutations rapidly due to its error prone polymerase and lack of proofreading activity. These mutations can be advantageous for the virus, allowing it to develop traits that enhance replication and pathogenesis. Due to its major clinical importance and a lack of available vaccine and antivirals, there is an impetus to identify effective antivirals against CVB3. In that regard, the virus\u27 ability to mutate poses a major obstacle to successful antiviral treatment and must be explored further to better understand the mechanisms of antiviral resistance and improve drug development.Polyamines are small positively charged molecules that are present in all cells and have crucial roles in processes such as transcription, translation, DNA replication and signaling. They are also fundamental to a virus\u27 ability to infect a host. When drugs that diminish polyamines are added to cells, the virus\u27 ability to replicate tapers. One drug that acts in this way is difluoromethylornithine (DFMO), which diminishes the production of polyamines by inhibiting the first step in polyamine synthesis: the conversion of ornithine to putrescine. In order to better understand how CVB3 could evolve resistance to antiviral treatments, CVB3 was seriallypassaged in the presence of DFMO. The resultant mutations that arose in CVB3 may correlate with its ability to infect a host who is undergoing antiviral therapy.Passaging CVB3 in the presence of DFMO illuminated 3 mutations: VP3- 234R, which is a site in the viral capsid; 2A- Q29K and 3C- Q52R both of which are sites in viral proteases. These proteases are crucial for multiple stages of infection including protein processing and packaging. Due to the vital importance of the viral proteases, these mutations and their seeming tie with polyamines, the focus of this study will revolve around these mutations. From these initial findings, we hypothesize that polyamines modulate viral protease activity and CVB3 gains resistance to polyamine depletion via mutation of its protease(s)

Genetic engineering of rice for improved agronomic characteristics

This thesis describes the production of three populations of transgenic rice plants using particle bombardment method altered in two main traits: (1) polyamine content and (2) insect pest resistance. The expression of antisense heterologous oat arginine decarboxylase (ADC) cDNA in transgenic rice plants suppressed endogenous ADC enzyme activity, and decreased putrescine and spermidine levels in a tissue/organ dependent manner, with no concomitant changes in the expression of other polyamine biosynthetic genes. The second population of transgenic rice plants engineered with a homologous spermidine synthase (SpdSyn) transgene, and observed through two generations, showed increased expression of both endogenous and transgene mRNAs. However, no significant accumulation of spermidine level in transgenic rice plants when compared to wild type control plants was observed. Putrescine levels were significantly increased in these transgenic plants. The study suggested the possible presence of an inter-conversion process from spermidine to putrescine in transgenic plants, triggered by over-expression of SpdSyn mRNAs. Novel insect resistance gene constructs encoding fusion proteins, including (1) rice thioredoxin h fused with snowdrop lectin-GNA (TRX-GNA), (2) the first domain of Bt toxin gene-Cry 1 Ac fused with GNA (Ac-GNA) and (3) Cry 1 Ac fused with ricin B chain-RTB (Ac-RTB) were assembled. When expressed in transgenic plant, these fusion proteins displayed an additive effect as insect toxins by maintaining the functional properties of the individual proteins. Artificial diet bioassays against insect pests showed that using these fusion proteins could enhance toxicity, insecticidal spectrum and possibly durability of resistance to insect pests. Our results clearly showed that transgenic rice plants expressing these fusion protein genes are resistant to brown planthopper, an important insect pest in tropical rice growing areas. These rice plants behave as horizontally resistant cultivars that are suitable for integrated pest management (IPM) networks

Cellular and process engineering to improve mammalian membrane protein expression

Improving the expression level of recombinant mammalian proteins has been pursued for production of commercial biotherapeutics in industry, as well as for biomedical studies in academia, as an adequate supply of correctly folded proteins is a prerequisite for all structure and function studies. Presented in this dissertation are different strategies to improve protein functional expression level, especially for membrane proteins. The model protein is neurotensin receptor 1 (NTSR1), a hard-to-express G protein-coupled receptor (GPCR). GPCRs are integral membrane proteins playing a central role in cell signaling and are targets for most of the medicines sold worldwide. Obtaining adequate functional GPCRs has been a bottleneck in their structure studies because the expression of these proteins from mammalian cells is very low. The first strategy is the adoption of mammalian inducible expression system. A stable and inducible T-REx-293 cell line overexpressing an engineered rat NTSR1 was constructed. 2.5 million Functional copies of NTSR1 per cell were detected on plasma membrane, which is 167 fold improvement comparing to NTSR1 constitutive expression. The second strategy is production process development including suspension culture adaptation and induction parameter optimization. A further 3.5 fold improvement was achieved and approximately 1 milligram of purified functional NTSR1 per liter suspension culture was obtained. This was comparable yield to the transient baculovirus-insect cell system. The third strategy is high throughput miRNA screening. MiRNAs are a novel class of small, non-coding RNAs that can simultaneously silence multiple genes. The NTSR1-expressing cell line was subjected to human miRNA mimic library screening and nine miRNA mimics were identified to improve functional expression of NTSR1 by as much as 48%. Interestingly, five out of nine identified miRNA mimics were effective in improving the functional expression of other proteins, including luciferase (cytosolic protein), serotonin transporter (membrane protein) and glypican-3 hFc protein (secreted protein). These indicated that the identified miRNAs could have a wide role in enhancing production of proteins with biomedical interest. As genome-wide siRNA screens has emerged to be a powerful methodology for deducing gene functions in various diseases, we applied this technology on HEK293 cells constitutively expressing luciferase reporter to generate a genome-wide profile for recombinant protein expression process. Top 10 genes leading to greatest improvement of luciferase production were validated and tested with secreted and membrane proteins. Investigation of these genes/pathways may provide profound information to understanding protein biosynthesis process in mammalian cells

Dual Delivery Systems Based On Polyamine Analog Benspm As Prodrug And Gene Delivery Vectors

Combination drug and gene therapy shows promise in cancer treatment. However, the success of such strategy requires careful selection of the therapeutic agents, as well as development of efficient delivery vectors. BENSpm (N1, N11-bisethylnorspermine), a polyamine analogue targeting the intracellular polyamine pathway, draws our special attention because of the following reasons: (1) polyamine pathway is frequently dysregulated in cancer; (2) BENSpm exhibits multiple functions to interfere with the polyamine pathway, such as to up-regulate polyamine metabolism enzymes and down-regulate polyamine biosynthesis enzymes. Therefore BENSpm depletes all natural polyamines and leads to apoptosis and cell growth inhibition in a wide range of cancers; (3) preclinical studies proved that BENSpm can act synergistically with various chemotherapy agents, making it a promising candidate in combination therapy; (4) multiple positive charges in BENSpm enable it as a suitable building block for cationic polymers, which can be further applied to gene delivery. In this dissertation, our goal was to design dual-function delivery vector based on BENSpm that can function as a gene delivery vector and, after intracellular degradation, as an active anticancer agent targeting dysregulated polyamine metabolism. We first demonstrated strong synergism between BENSpm and a potential therapeutic gene product TRAIL. Strong synergism was obtained in both estrogen-dependent MCF-7 breast cancer cells and triple-negative MDA-MB-231 breast cancer cells. Significant dose reduction of TRAIL in combination with BENSpm in MDA-MB-231 cells, together with the fact that BENSpm rendered MCF-7 cells more sensitive to TRAIL treatment verified our rationale of designing BENSpm-based delivery platform. This was expected to be beneficial for overcoming drug resistance in chemotherapy, as well as boosting the therapeutic effect of therapeutic genes. We first designed a lipid-based BENSpm dual vector (Lipo-SS-BEN) capable of intracellular release of BENSpm using thiolytically sensitive dithiobenzyl carbamate linker. Similar activity on SSAT enzyme induction by Lipo-SS-BEN compared with BENSpm free drug verified the success of this prodrug design. Biodegradability of Lipo-SS-BEN contributed to decreased toxicity compared with nondegradable control LipoBEN. However, decreased enhancement of TRAIL activity was observed for Lipo-SS-BEN when compared with BENSpm, indicating that the lipid-related toxicity diminished the synergism. In addition, compared with LipoBEN and DOTAP, decreased transfection efficiency of Lipo-SS-BEN demonstrated instability of Lipo-SS-BEN in extracellular environment. In order to design a dual delivery vector with reduced vector toxicity and improved linker stability, we employed dendritic polyglycerol (PG) as a safe carrier backbone, onto which BENSpm was conjugated through carbamate linkage (PG-BEN). Polymers with norspermine (PG-Nor) shell and amine-terminated PG (PG-NH2) were synthesized as controls. The BENSpm dual vector PG-BEN demonstrated superior gene delivery function, and showed decreased toxicity compared with the control polymers. However, compared with BENSpm, which depleted all natural polyamines, PG-BEN only down-regulated intracellular putrescine levels. In addition, no free BENSpm was detected in PG-BEN treated cells. These results suggested that in order to take full advantage of BENSpm anticancer activity, alternative linker chemistry needs to be further explored. We then incorporated bis(2-hydroxyethyl) disulfide as a self-immolative linker to synthesize polymer prodrugs of BENSpm (DSS-BEN). The proposed mechanism of BENSpm release from DSS-BEN contains two steps: disulfide bond is first cleaved in the reducing intracellular space, then the intermediate further undergoes slow intramolecular cyclization to release free BENSpm. Cell line-dependent BENSpm release after DSS-BEN treatment was observed using HPLC analysis, demonstrating the success of our linker strategy. DSS-BEN showed comparable transfection efficiency as polyethylenimine and showed decreased toxicity in several cell lines compared with the nondegradable control DCC-BEN. We further demonstrated that DSS-BEN could act synergistically with several therapeutic agents, making it a promising delivery platform for combination therapy in cancer. In all, we have successfully developed a dual delivery vector based on BENSpm, which fulfills its function as a gene delivery vector as well as a prodrug of BENSpm, and possesses synergistic potential to augment the effect of the co-delivered agents