Complex genetic approaches to neurodegenerative diseases.

Neurodegenerative diseases are fatal disorders in which disease pathogenesis results in the progressive degeneration of the central and/or the peripheral nervous systems. These diseases currently affect -2% of the population but are expected to increase in prevalence as average life expectancy increases. The majority of these diseases have a complex genetic basis. The work presented in this thesis aimed to investigate the genetic basis of two neurodegenerative diseases, amyotrophic lateral sclerosis (ALS) and the human prion diseases kuru and sporadic Creutzfeldt-Jakob disease (sCJD), using novel complex genetic approaches. ALS is a fatal neurodegenerative disease in which motor neurons are seen to degenerate. It is a complex disease with 10% of individuals having a family history and the remaining 90% of non-familial cases having some genetic component. The gene DYNC1H1 is involved in retrograde axonal transport and is a good candidate for ALS. In this thesis the genetic architecture of DYNC1H1 was elucidated and a mutation screen of exons 8, 13 and 14 was undertaken in familial forms of ALS and other motor neuron diseases. No mutations were found. A linkage disequilibrium (LD) based association study was conducted using two tagging single nucleotide polymorphisms (tSNPs) which were identified as sufficient to represent genetic variation across DYNC1HI. These tSNPs were tested for an association with sporadic ALS (SALS) in 261 cases and 225 matched controls but no association was identified. Kuru is a devastating epidemic prion disease which affected a highly geographically restricted area of the Papua New Guinea highlands, predominantly affected adult women and children. Its incidence has steadily declined since the cessation of its route of transmission, endocannibalism, in the late 1950's. Kuru imposed strong balancing selection on codon 129 of the prion gene (PRNP). Analysis of kuru-exposed and unexposed populations showed significant deviations from Hardy-Weinberg equilibrium (HWE) consistent with the known protective effect of codon 129 heterozygosity. Signatures of selection were investigated in the surviving populations, such as deviations from HWE and an increasing cline in codon 129 valine allele frequency, which covaried with disease exposure. A novel PRNP G127V polymorphism was detected which, while common in the area of highest kuru incidence, was absent from kuru patients and unexposed population groups. Genealogical analysis revealed that the heterozygous PRNP G127V genotype confers strong prion disease resistance, which has been selected by the kuru epidemic. Finally, PRNP copy number was investigated as a possible genetic mechanism for susceptibility to kuru and sCJD. No conclusive copy number changes were identified

Expression of Secondary Metabolite Gene Clusters and Production of Secondary Metabolites in Three Nostoc strains Subjected to Deprivation on Nutrients and Competition

Cyanobacteria are a unique source of natural products, where most of them are synthesized by non-ribosomal peptide synthase, polyketide synthase, or a hybrid of these pathways. The identification of such biosynthetic genes responsible for the production of secondary metabolites is still a relatively unexplored area, and it remains many natural products for which a biosynthetic origin is unknown. Secondary metabolites from marine cyanobacteria have gained much attention the last decades, however few comprehensive studies on secondary metabolites and their biosynthetic gene clusters from terrestrial cyanobacteria have been conducted. Three terrestrial Nostoc spp. KVJ20, KVJ2, and KVJ10 were recently sequenced which allowed us to conduct genome-wide predictions by AntiSMASH of their potential to produce secondary metabolites. These strains were subjected to various cultivation conditions including nutrient limitations and competition. Gene expression analysis by RT-qPCR of predicted gene clusters and the production of secondary metabolites by UPLC-HR-MS were conducted. Analysis of gene expression patterns revealed higher expression of several NRPS, PKS and RiPP genes in nutrient-deprived media, as well as confirming the present known function of the housekeeping genes; NifH, GvpC, and PilT. Most of the secondary metabolites found by UPLC-HR-MS were not identified, however a variety of Nostocyclopeptides, Suomilide/Banyaside-like peptides, Anabaenopeptins, as well as Aeruginosin, Hapalosin, and Nosperin were recorded from extracts of the respective strains. The results from this thesis give valuable knowledge for further cultivation of terrestrial cyanobacteria, with the purpose of awaking cryptic gene clusters and identifying novel secondary metabolites. We have also suggested conditions most suitable for enrichment for identified compounds

Molecular characterization of the tetratricopeptide repeat-mediated interactions of murine stress-inducible protein 1 with major heat shock proteins

Murine stress-inducible protein 1 (mSTI1) is a co-chaperone that is homologous with the human heat shock protein 70 (Hsp70)/heat shock protein 90 (Hsp90)-organizing protein (Hop). The two proteins are homologues of the highly conserved stress-inducible protein 1 (STI1) family of co-chaperones. The STI1 proteins interact directly and simultaneously at some stage, with Hsp70 and Hsp90 in the formation of the hetero-multi-chaperone complexes that facilitate the folding of signal transducing kinases and functional maturation of steroid hormone receptors. The interactions of mSTI1 with both Hsp70 and Hsp90 is mediated by a versatile structural protein-protein interaction motif, the tetratricopeptide repeat (TPR). The TPR motif is a degenerate 34-amino acid sequence a-helical structural motif found in a significant number of functionally unrelated proteins. This study was aimed at characterizing the structural and functional determinants in the TPR domains of mSTI1 responsible for binding to and discriminating between Hsp70 and Hsp90. Guided by data from Hop's crystal structures and amino acid sequence alignment analyses, various biochemical techniques were used to both qualitatively and quantitatively characterize the contacts necessary for the N-terminal TPR domain (TPR1) of mSTI1 to bind to the C-terminal EEVD motif of heat shock cognate protein 70 (Hsc70) and to discriminate between Hsc70 and Hsp90. Substitutions in the first TPR motif of Lys⁸ or Asn¹² did not affect binding of mSTI1 to Hsc70, while double substitution of these residues abrogated binding. A substitution in the second TPR motif of Asn⁴³ lowered but did not abrogate binding. Similarly, a deletion in the second TPR motif coupled with a substitution of Lys⁸ or Asn¹² reduced but did not abrogate binding. Steady state fluorescence and circular dichroism spectroscopies revealed that the double substitution of Lys⁸ and Asn¹² resulted in perturbations of inter-domain interactions in mSTl1. Together these results suggest that mSTI1-Hsc70 interaction requires a network of electrostatic interactions not only between charged residues in the TPR1 domain of mSTI1 and the EEVD motif of Hsc70, but also outside the TPR1 domain. It is proposed that the electrostatic interactions in the first TPR motif collectively made by Lys⁸ and Asn¹² define part of the minimum interactions required for successful mSTI1-Hsc70 interaction. In the first central TPR domain (TPR1A), single substitution of Lys³°¹ was sufficient to abrogate the mSTI1-Hsp90 interaction. Using a truncated derivative of mSTI1 incapable of binding to Hsp90, residues predicted by crystallographic data to determine Hsp70 binding specificity were substituted in the TPR1 domain. The modified protein had reduced binding to Hsc70, but showed significant binding capacity for Hsp90. In contrast, topologically equivalent substitutions on a truncated derivative of mSTI1 incapable of binding to Hsc70 did not confer Hsc70 specificity on the TPR2A domain. These data suggest that binding of Hsc70 to the TPR1 domain is more specific than binding of Hsp90 to the TPR2A domain. In addition, residues C-terminal of helix A in the second TPR motif of mSTI1 were shown to be important in determining specific binding to Hsc70. Binding assays using surface plasmon resonance spectroscopy showed that the affinities of binding of mSTI1 to Hsc70 and Hsp90 were 2 μM and 1.5 μM respectively. Preliminary in vivo studies revealed differences in the dynamics of binding of endogenous and exogenous recombinant mSTI1 with Hsc70 and Hsp90. The outcome of this study poses serious implications for the mechanisms of mSTI1 interactions with Hsc70 and Hsp90 in the cell

Genomics approaches to exploit the biotechnological potential of marine sponge-derived Streptomyces spp. isolates

Members of the Streptomyces genus are widely known for their capability in producing compounds of pharmacological, clinical, and biotechnological interest, being the source of approximately a third of all the antibiotics that have been identified to date. However, the discovery of natural products with antimicrobial activities has declined following the so-called “Golden Age of Antibiotics” (1940s-1950s), particularly due to the common re-discovery of previously known compounds. Thus, natural products discovery research has shifted towards investigating diverse environmental niches, such as marine ecosystems, mangroves, and symbiotic communities of insects and sponges, resulting in the discovery of a variety of previously unidentified compounds of pharmacological interest; including those isolated from marine-derived Streptomyces species. However, in despite of their relevance as producers of potentially novel bio-active molecules with pharmacological, clinical and biotechnological interest, marine-derived Streptomyces isolates are still rather underexplored and under-characterized, particularly those found in association with marine sponges. In the studies presented in this thesis, various state-of-the-art methodologies related to genome mining and bioinformatics-based pipelines, together with molecular and synthetic biology, were employed and proved to be extremely useful in helping to uncover the biotechnological potential of marine sponge-derived Streptomyces isolates. These studies essentially aimed at a) genetically characterizing marine sponge-derived Streptomyces spp. isolates and their potential to produce novel secondary metabolites, as shown in Chapter 2; b) to in silico identify, isolate, and quantify a secondary metabolite produced by a marine sponge-derived Streptomyces isolate, together with genetically characterizing its genome-encoded biosynthetic gene cluster (BGC), as reported in Chapter 3; and c) to perform an in silico screening of a novel polyesterase from a marine sponge-derived Streptomyces isolate, followed by heterologous protein expression in an E. coli host, as demonstrated in Chapter 4. In Chapter 2, two of the first complete genomes from marine sponge-derived Streptomyces spp. isolates were determined, namely from Streptomyces sp. SM17 and Streptomyces sp. SM18. The high-quality data provided in this study allowed for a reliable prediction of secondary metabolites biosynthetic gene clusters (BGCs) in their genomes, which determined that these isolates possess a variety of BGCs potentially encoding for the production of known compounds, and also potentially new molecules. Differential growth assessment determined that the marine isolates SM17 and SM18 grew and differentiated better in the presence of salts in the culture medium, when compared to their phylogenetically determined closely-related terrestrial relatives, namely S. albidoflavus J1074 (referred to as S. albus J1074 in Chapter 2) and S. pratensis ATCC 33331, respectively. Comparative genomics allowed for the identification of a proposed environmental niche adaptations (ENA) gene pool, which included genes related to osmotic stress defence, transcriptional regulation; symbiotic interactions; antimicrobial compound production and resistance; ABC transporters; together with horizontal gene transfer and defence-related features. These results shed new light on some of the genetic traits possessed by these marine sponge-derived isolates, and on how these might be linked to secondary metabolites production, and further highlighted their importance for the discovery of potentially novel natural products. In Chapter 3, the previously unreported capability of the Streptomyces sp. SM17 to produce surugamides has been described. Surugamides are a family of compounds that have been previously reported to possess antitumor and antifungal activities. This was performed employing genome mining, which allowed for the identification of the surugamides BGC (sur BGC) in the SM17 genome, and analytical chemistry techniques for compound isolation and quantification. Phylogenomics analyses provided novel insights with respect to the distribution and conservation of the sur BGC at a genetic level, and provided evidence that the sur BGC might have had a marine origin. Additionally, when comparing the surugamide A production capabilities of a marine isolate (strain SM17) with a terrestrial relative (strain J1074) employing a “One Strain Many Compounds” (OSMAC)-based cultivation approach, the Streptomyces sp. SM17 isolate was shown to produce higher levels of surugamide A in all the conditions tested for. These findings may provide important insights towards a better characterisation, improved production and industrial development of this family of compounds. In Chapter 4, the capability of marine sponge-derived Streptomyces spp. isolates to degrade synthetic polyesters was investigated. This was based on the fact that these microorganisms might have developed mechanisms to assimilate components of micro-plastics, which are now believed to be ubiquitous in marine ecosystems and pose as one of the top environmental problems that society faces today. Using 15 known PET hydrolases (PETases) as references, including the Ideonella sakaiensis 201-F6 PETase, in silico screening was performed to determine the presence of homologs to these reference PETase enzymes in 52 Streptomyces genome sequences (of which 29 were derived from marine ecosystems). The best candidate identified, namely the SM14est protein from the marine sponge-derived Streptomyces sp. SM14, was in silico characterised with respect to its amino acid sequence and predicted three dimensional structure, and was subsequently heterologously expressed in an E. coli host. This allowed for the confirmation of the polyesterase activity possessed by the SM14est enzyme, via a polycaprolactone (PCL) plate-clearing assay. Better characterising, identifying sources, and determining methods for improved protein expression are essential steps towards the development of biotechnological applications and industrial processes employing this family of enzymes, such as new plastic waste processing technologies