Characterisation of a newly identified family of lipid transfer proteins at membrane contact sites

Non-vesicular intracellular lipid traffic is mediated by lipid transfer proteins (LTPs), which contain domains with an internal cavity that can solubilise and transfer lipids. One of the most widespread LTP folds is the Steroidogenic Acute Regulatory Transfer (StART) domain, which forms a hydrophobic pocket, and appears in proteins with different localisations and lipid specificities. The aim of this study was to characterise a new StART-like domain family, which we identified by a bioinformatics approach. I studied aspects of the localisations, functions and structural properties of six StART-like proteins in S. cerevisiae. The yeast StART-like proteins were endoplasmic reticulum (ER)-integral membrane proteins with transmembrane domains, and they localised at membrane contact sites: Lam1p/Lam3p, and Lam2p/Lam4p at junctions between ER and plasma membrane (PM); Lam5p/Lam6p at junctions between the ER and the vacuolar membrane, at nucleus-vacuole junction (NVJ) and at ER-mitochondria contacts. To study their functions, I purified the second StART-like domain of Lam4p, and I identified sterol as its lipid ligand from in vitro binding assays and in a spectroscopy approach with fluorescent ergosterol. We named the whole family LAM for Lipid transfer proteins Anchored at Membrane contact sites. The sterol binding property of the domains was related to a phenotype shared by LAM1, LAM2 and LAM3 delete strains, which showed an increased sensitivity to the sterol-sequestering polyene antifungal drug Amphotericin B (AmB). The two most sensitive strains (lam1∆ and lam3∆), displayed low sphingolipid levels, which is as yet unexplained. All AmB phenotypes were rescued by StART-like domains from the human LAMa, Lam2/4p and Lam5/6p, suggesting that these domains bind sterol. Simultaneous deletion of LAM1, LAM2, and LAM3 significantly reduced the extent of cortical ER-PM contacts, implying that they create the structure of the particularly punctate contact site they target. Finally, I started structural analysis of Lam4S2 to study the mechanism of sterol binding and to confirm our structural model

Functional analysis of a rare disease variant in human NRAS causing Noonan-Like syndrome

Background: Rare diseases are chronic and debilitating and while individually they affect less than 1 in 2,000 people, collectively they have a huge impact affect around 1.8 million Australians. Approximately 80% of these have a genetic origin, however only 30% of patients receive a formal molecular diagnosis. RASopathies are a group of rare disease that are caused by a mutations in the genes involved in the RAS-MAPK pathway, the most common of which is Noonan syndrome, which is characterised by heart defects, short stature, chest deformities, and specific craniofacial features. This study focuses on the effects of a novel c.173C>T (p.Thr58Ile) mutation, found in the NRAS gene of a patient diagnosed with Noonan-like syndrome, on the localisation and function of the NRAS protein. In doing so this study aimed to validate the role of mutations in the patient’s disease and provide information toward the creation of an experimental pipeline for the validation of other rare variants. Methods: U87-MG cells were transiently transfected with the NRAS constructs tagged with GFP2, and stained with specific antibody markers to determine localisation of the proteins using confocal microscopy. Functional studies included an Annexin V apoptosis assay, using flow cytometry to detect and quantify levels of apoptosis in transfected and untransfected populations, and the prediction of conserved domains using online bioinformatic tools. Results and Conclusions: Preliminary results from this study suggest that there is a difference between the localisation and function of the mutant and wild type proteins. The mutant protein was seen to co-localise with the Golgi apparatus as expected if the mutant protein was constitutively active. However the mutant protein was also observed to co-localise with the markers for the plasma membrane, nucleus and endoplasmic reticulum indicating that the mutation affects more than just the activation of the protein. An increase in apoptosis observed in NRASMUT transfected population, in comparison to both the wild type and untransfected populations, indicates that the mutation is engaging the pro-apoptotic function of NRAS associated with an increased activation of the RAS–RAF–MAPKK–MAPK pathway. Bioinformatic analyses identified that the mutation is location within a number of motifs involved in the binding of GTP and thus the activation and inactivation of NRAS, directing further studies toward the proliferation, activation and interaction of NRAS with effectors

Human protein function prediction: application of machine learning for integration of heterogeneous data sources

Experimental characterisation of protein cellular function can be prohibitively expensive and take years to complete. To address this problem, this thesis focuses on the development of computational approaches to predict function from sequence. For sequences with well characterised close relatives, annotation is trivial, orphans or distant homologues present a greater challenge. The use of a feature based method employing ensemble support vector machines to predict individual Gene Ontology classes is investigated. It is found that different combinations of feature inputs are required to recognise different functions. Although the approach is applicable to any human protein sequence, it is restricted to broadly descriptive functions. The method is well suited to prioritisation of candidate functions for novel proteins rather than to make highly accurate class assignments. Signatures of common function can be derived from different biological characteristics; interactions and binding events as well as expression behaviour. To investigate the hypothesis that common function can be derived from expression information, public domain human microarray datasets are assembled. The questions of how best to integrate these datasets and derive features that are useful in function prediction are addressed. Both co-expression and abundance information is represented between and within experiments and investigated for correlation with function. It is found that features derived from expression data serve as a weak but significant signal for recognising functions. This signal is stronger for biological processes than molecular function categories and independent of homology information. The protein domain has historically been coined as a modular evolutionary unit of protein function. The occurrence of domains that can be linked by ancestral fusion events serves as a signal for domain-domain interactions. To exploit this information for function prediction, novel domain architecture and fused architecture scores are developed. Architecture scores rather than single domain scores correlate more strongly with function, and both architecture and fusion scores correlate more strongly with molecular functions than biological processes. The final study details the development of a novel heterogeneous function prediction approach designed to target the annotation of both homologous and non-homologous proteins. Support vector regression is used to combine pair-wise sequence features with expression scores and domain architecture scores to rank protein pairs in terms of their functional similarities. The target of the regression models represents the continuum of protein function space empirically derived from the Gene Ontology molecular function and biological process graphs. The merit and performance of the approach is demonstrated using homologous and non-homologous test datasets and significantly improves upon classical nearest neighbour annotation transfer by sequence methods. The final model represents a method that achieves a compromise between high specificity and sensitivity for all human proteins regardless of their homology status. It is expected that this strategy will allow for more comprehensive and accurate annotations of the human proteome

KiPIK Screening: A Novel Method to Identify Kinases Responsible for Phosphorylation events of Interest.

Ph. D. ThesisProtein phosphorylation is one of the most abundant forms of post-translational modification and regulates nearly every aspect of cell biology. Understanding the function of a particular phosphorylation event depends to a great extent on identifying the kinase responsible for catalysing it. However, while advances in mass spectrometry based phosphoproteomics have seen an explosion in the ability to detect phosphorylation events occurring in cells, methodological limitations make identifying the kinase responsible for specific phosphorylation events challenging. This thesis explores this problem, beginning with a discussion of the determinants of kinase-substrate specificity in a cell. This is followed by a review of methodologies currently available for identifying kinases responsible for specific phosphorylation events, and a chapter exploring the utility of one of these techniques (siRNA kinome screening) for identifying kinases required for specific histone phosphorylation events in mitosis. We then report the development of KiPIK screening (Kinase Inhibitor Profiling to Identify Kinases), a novel general method for identifying the kinase responsible for a phosphorylation event of interest. The method exploits the fact that in recent years large numbers of kinase inhibitors have been profiled for inhibitory activity on near-kinome-wide panels of recombinant kinases. The method treats the inhibitory information for each kinase as a ‘fingerprint’ for the identification of kinases acting on target phosphorylation sites in cell extracts. In this thesis we detail the development of the technique and validate it on diverse known kinase-phosphosite pairs, including two mitotic histone phosphorylations carried out by Haspin and Aurora B, EGFR autophosphorylation, and the phosphorylation of integrin 1 by Src-family kinases. Finally, we use it to identify the kinase responsible for an as yet unassigned mitotic phosphosite on the Chromosomal Passenger Complex component INCENP. KiPIK screening is broadly applicable and technically straightforward. In addressing the methodological insufficiency in this fundamental area, it has the potential to benefit research widely

How codon choice determines evolvability and evolutionary robustness in short linear motifs

Short linear motifs, made up of 2-10 amino acids in linear sequence space, are a central component of cellular decision making through proteins. They form a modular system in cells where combinations of domains and motifs are used as basic functional building blocks through interactions. Functions mediated through these motifs include cellular localisation, post-translational modifications, degradation and general protein-protein interactions. Since motifs are made up of a small number of amino acids they have unusual evolutionary properties, for instance they can evolve de novo, or be lost, through a small number of substitutions. This is of particular importance in pathogens such as viruses. Many viruses evolve new host-like motifs to interact with the host and change the regulation and signalling landscape within host cells to mediate infection. In this body of work, I have used influenza as a model to elucidate aspects of the evolutionary properties of motifs. I have been able to leverage recent progress made in determining nucleotide mutation rates and have developed a model for motif evolution that is defined from the nucleotide and codon levels. Simulations using this methodology suggested that different codons have varying propensities to evolve into amino acids within a linear motif. In other words, some sequences have higher motif evolvability. The simulations also indicated a fitness benefit to use some codons over others to encode linear motifs, due to the varying propensity to evolve. These findings suggest that motifs that are encoded by specific codons have higher motif evolutionary robustness, i.e. they can tolerate more mutations without affecting function. I went on to investigate if these predicted properties have played a role in motif evolution in influenza. I found that conserved motifs in influenza use the codons inferred to have higher evolutionary robustness. This would lead to increased fitness, as motifs are less often lost through mutations. I also found that this mutational robustness acts on stop codon usage in influenza, suggesting an explanation for an old observation of predominant use of TAA in many organisms. Interestingly, it also appears that evolutionary robustness of a motif can be varied to tune the rate of motif change, which influenza utilises in glycosylation motifs that interface with the host immune system. Finally, I investigated whether the codon choice and evolvability at early stages of viral host shifts could be used to predict the emergence of functional motifs. I have found that motif evolvability can aid the prediction of motif emergence. For influenza strains H1N1 and H3N2, which were introduced in the human population from birds during the 1900s, the sequence of the early strains could be used to predict the majority of the glycosylation sites that would emerge the following decades. The predictability of motif emergence could have important implications for vaccination efforts. The methodologies developed here, and the observations made about how motif evolution is shaped by codon choices in a predictable way will be important for a better understanding of the evolution of complexity and regulation involving motifs. This may have implications for complex diseases such as cancers, and for our understanding of the evolution of pathogen innovations and functionality

SiteSeek: Post-translational modification analysis using adaptive locality-effective kernel methods and new profiles

<p>Abstract</p> <p>Background</p> <p>Post-translational modifications have a substantial influence on the structure and functions of protein. Post-translational phosphorylation is one of the most common modification that occur in intracellular proteins. Accurate prediction of protein phosphorylation sites is of great importance for the understanding of diverse cellular signalling processes in both the human body and in animals. In this study, we propose a new machine learning based protein phosphorylation site predictor, SiteSeek. SiteSeek is trained using a novel compact evolutionary and hydrophobicity profile to detect possible protein phosphorylation sites for a target sequence. The newly proposed method proves to be more accurate and exhibits a much stable predictive performance than currently existing phosphorylation site predictors.</p> <p>Results</p> <p>The performance of the proposed model was compared to nine existing different machine learning models and four widely known phosphorylation site predictors with the newly proposed PS-Benchmark_1 dataset to contrast their accuracy, sensitivity, specificity and correlation coefficient. SiteSeek showed better predictive performance with 86.6% accuracy, 83.8% sensitivity, 92.5% specificity and 0.77 correlation-coefficient on the four main kinase families (CDK, CK2, PKA, and PKC).</p> <p>Conclusion</p> <p>Our newly proposed methods used in SiteSeek were shown to be useful for the identification of protein phosphorylation sites as it performed much better than widely known predictors on the newly built PS-Benchmark_1 dataset.</p

An investigation of RNR regulation in fission yeast by confocal laser scanning FRET and near-TIRF microscopy

For genome integrity, adequate levels of deoxyribonucleotide (dNTPs) are essential to maintain faithful DNA replication and repair via the regulation of ribonucleotide reductase (RNR). In the fission yeast, RNR is composed of two subunits: Cdc22 and Suc22. The importance of Spd1 (RNR inhibitor) in Cdc22-Suc22 complex formation has been demonstrated by imaging of S. pombe containing fluorescent protein (FP) modified RNR subunit proteins in the presence of Spd1 and absence of Spd1 cells using confocal laser scanning microscopy. To investigate further the significant role of Spd1 in the regulation of RNR, 41 mutants created by Nestoras group. We used fluorescence resonance energy transfer (FRET) by acceptor photobleaching to investigate the RNR subunit interaction and provide evidence for a new model for the role of Spd1 in RNR regulation. Different treatments such as HU, 4NQO and heat shock have been used to investigate the effect of radical scavenging on the inhibition of RNR activity and induced DNA damage on S. pombe cell viability to elucidate further the role of Spd1 in the regulation of RNR. Finally a novel imaging technique, near-total internal reflection microscopy has been developed and applied with dual-view detection. The technique has been applied to image, simultaneously, the donor CFP and acceptor YFP channels of the FP-tagged RNR complex in the wild-type S. pombe cells and perform FRET measurements that are consistent with the confocal fluorescence results. In conclusion, a new hypothesis for the role of Spd1 has been drawn from the results, which is that the inhibitory role of Spd1 mediates the Suc22-Cdc22 (R1-R2) interaction to form a FRET competent but immature and inactive RNR complex, while with Spd1 deleted RNR is clearly active in a conformation that lacks FRET

Association Rate Constants of Ras-Effector Interactions Are Evolutionarily Conserved

Evolutionary conservation of protein interaction properties has been shown to be a valuable indication for functional importance. Here we use homology interface modeling of 10 Ras-effector complexes by selecting ortholog proteins from 12 organisms representing the major eukaryotic branches, except plants. We find that with increasing divergence time the sequence similarity decreases with respect to the human protein, but the affinities and association rate constants are conserved as predicted by the protein design algorithm, FoldX. In parallel we have done computer simulations on a minimal network based on Ras-effector interactions, and our results indicate that in the absence of negative feedback, changes in kinetics that result in similar binding constants have strong consequences on network behavior. This, together with the previous results, suggests an important biological role, not only for equilibrium binding constants but also for kinetics in signaling processes involving Ras-effector interactions. Our findings are important to take into consideration in system biology approaches and simulations of biological networks

Regulatory post-translational modifications and protein-protein interactions involved in function and proteostasis of aromatic amino acid hydroxylases

The non-heme iron and (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin (BH4) dependent aromatic amino acid hydroxylases (AAAHs) family of enzymes include phenylalanine hydroxylase (PAH), tyrosine hydroxylase (TH), and tryptophan hydroxylase 1 and 2 (TPH1 and TPH2). PAH catalyses the rate-limiting step in the catabolism of phenylalanine (L-Phe) that mainly takes place in the liver. TH catalyses the first and rate-limiting step in the biosynthesis of catecholamine neurotransmitters and hormones dopamine, norepinephrine and epinephrine in the brain and periphery. TPHs catalyse the first and rate-limiting step in the biosynthesis of serotonin in the peripheral (TPH1) and the central (TPH2) nervous systems. The AAAHs are of physiological and clinical importance. Dysfunctional PAH results in phenylketonuria (PKU), characterised by elevated levels of L-Phe in the blood, which can lead to brain damage. Catecholamine deficiency, due to dysfunctional TH, leads to motor dysfunction and neuropsychiatric disorders, such as TH deficiency (THD) and Parkinson’s disease. Reduced level of serotonin has been linked to anxiety disorder, depression, posttraumatic stress disorder and attention deficit hyperactivity disorder. Hence, the reactions catalysed by the AAAHs are important and tightly regulated. The aim of this thesis was to study the regulation of the AAAHs PAH and TH both in physiological and pathological states. We focused on regulatory mechanisms by selected post-translational modifications and protein-protein interactions and phosphorylation, investigating their role in the function, localisation and proteostasis of these enzymes using cellular and animal models. We investigated the role of DNAJC12, a type III member of the HSP40/DNAJ family, in the folding and degradation of wild-type (Wt) and mutant PAH. We observed a positive correlation between DNAJC12 and Wt and mutant PAH protein levels in the soluble cellular fractions. Detailed characterisations in liver lysates of the hyperphenylalaninemic Enu1 mouse (p.V106A-PAH mutation) revealed increased ubiquitination, instability, and aggregation of mutant PAH compared with Wt PAH. Furthermore, we showed that in the liver lysates, DNAJC12 interacts with both Wt and mono-ubiquitinated PAH; also, PAH mutation did not alter mRNA expression of DNAJC12. Our results support the role of DNAJC12 not only in proper folding but also in the processing of misfolded ubiquitinated PAH. We characterised a new custom-made Pah-R261Q knock-in mouse carrying mutation c.782G>A in the Pah gene. The homozygous Pah-R261Q mice exhibited reduced PAH activity and BH4 responsive hyperphenylalaninemia. Moreover, the mutant mice presented a reduced BH4 content in the liver, altered lipid metabolism, and increased oxidative stress, including increased mRNA expression of DNAJC12. Furthermore, the Pah-R261Q mice displayed large amyloid-like ubiquitinated PAH aggregates. The colocalisation of mutant PAH with selective autophagy markers indicated the involvement of the autophagic pathway in the clearance of mutant aggregates. These findings indicate a paradigm shift from a loss-of-function disorder to a toxic gain-of-function in PKU pathology. We next investigated the functional role of Ser31 phosphorylation in the regulation of TH in the cellular models. We observed that the perinuclear distribution of THpSer31 was concomitant with Golgi complex and synaptic vesicle marker in rat and human dopaminergic cells. The co-distribution of THpSer31 with vesicular monoamine transporter 2 (VMAT2) and α-synuclein (α-syn) in cells and their detection as co-immunoprecipitant in mouse brain lysate indicated an association of TH with vesicles. Furthermore, disruption of the microtubules caused accumulation of TH in the cell soma. Our study revealed that Ser31 phosphorylation regulates the subcellular localisation of TH by facilitating protein-protein interaction with VMAT2 and α-syn and enabling its transport toward axon terminals along microtubules. Finally, using SH-SY5Y cells, we sought to investigate the relationship between phosphorylation at different phosphosites and the nuclear distribution of TH, which was earlier proposed to be associated with Ser19 phosphorylation. We indeed observed that THpSer19 was predominantly nuclear, yet the phospho-null mutant of Ser19 (V5-TH-S19A) surprisingly accumulated significantly higher in the nuclear fraction when compared to Wt. Moreover, other phosphosites (Ser31 and Ser40) did not seem to influence the nuclear distribution of TH. When the phospho-null mutant of Thr8 (V5-TH-T8A) was expressed in SH-SY5Y cells, recombinant TH in the nuclear fraction was significantly reduced compared to Wt and the phospho-mimicking mutant V5-THT8E, indicating the potential role of Thr8 phosphorylation in the nuclear distribution of TH. In addition, inhibition of importin-β also reduced the amount of recombinant TH in the nucleus suggesting the involvement of the importin-β/RanGTP system in the nuclear localisation of TH in SH-SY5Y cells. To conclude, this study has brought new insights on the short-term regulation of AAAHs (PAH and TH) in physiological and pathological conditions by interacting with partners and by post-translational modifications, such as ubiquitination and phosphorylation (for TH), which ultimately affect their abundance, function and availability in different compartments of cells. Thus, this study has shed light on some of the molecular mechanisms involved in the proteostasis of AAAHs. Together, these findings open new research avenues to better understand disorders associated with the AAAHs.Doktorgradsavhandlin