Regulation of mitochondria by proteolysis

Proteolysis is increasingly documented as a method of regulation of mitochondrial function. Our studies of rhomboid-family proteins' roles in organelles show that this is also the case in the social amoeba Dictyostelium discoideum, in which four of these membrane-bound, evolutionarily ubiquitous, serine proteases are found. Rhomboid proteases act on disparate substrates in different organisms so far studied, but their mode of action is conserved: their location in the membrane means that their membrane-tethered substrates can act in signalling upon release, or be activated, by rhomboid-mediated cleavage. Among eukaryotic rhomboids is the mitochondrial protease 'PARL', which ensures the maintenance of the structural and functional integrity of mitochondria and plastids, but we have found that other Dictyostelium rhomboids also affect the organelle. Studying the development and behaviour of Dictyostelium, a microbial model organism with a complex life cycle that includes uni- and multicellular stages, allowed investigation of the role of rhomboids in unicellular vegetative growth, multicellular development and sporulation, phagocytosis, and response to the environment. We found that two rhomboid-null mutants gave rise to changes in development, rhmA altering the response to chemoattractants and demonstrating decreased motility in general, whereas rhmB null cells had slower growth rates with decreased response to folic acid. RhmA, although located in the contractile vacuole, affects the ultrastructure of mitochondria, and RhmB-GFP fusions protein was localised to the mitochondrion. qPCR analysis revealed RhmA and RhmB transcript levels peaking during the multicellular growth phase and transcriptional networks suggest the Dictyostelium rhmA is regulated along with the orthologues of Saccharomyces cerevisiae mitochondrial rhomboid substrates

Dictyostelium discoideum: a model for testing novel inhibitors of urokinase-type plasminogen activator

The social amoeba Dictyostelium discoideum is a useful non-animal eukaryote for testing novel compounds and dissecting cell regulatory molecular networks. We used this model organism to investigate the effect of a series of arylboronic acids and pinacol esters on development, chemotaxis and viability. These compounds were studied in parallel by collaborators for serine protease and urokinase-type plasminogen activator (uPA) inhibition, both in vitro and in vivo. In those biochemical assays, three compounds, BC11, SR3 and BC57, displayed micromolar (50 µM) inhibition of uPA, with an excellent selectivity profile over related proteases (Smith et al., 2012). Notably, the same compounds disturbed cell adhesion and migration in Dictyostelium, without any effect on viability. Compound BC11 was chemotoxic rather than just chemostatic, proving to be the most potent and selective inhibitor of uPA in these independent Dictyostelium and biochemical assays. Smith E, Spencer J, Ali M, et al. (2012) Elucidating novel urokinase-type plasminogen activator inhibitors. In Journal of Thrombosis and Haemostasis, ppe10–e24

Book review: "Reinventing the wheel: Milk, Microbes and the Fight for Real Cheese" by Bronwen and Francis Percival

N/

Regulated intramembrane proteolysis in the plastid envelope

Rhomboid proteins were the most recently identified of the four families of intramembrane proteases. Found in almost all organisms, these serine proteases operate in a diverse range of pathways. They regulate Drosophila growth factor signalling (Urban et al., 2002), permit infection by apicomplexans (Dowse et al., 2005), play a key role in mitochondrial dynamics (Herlan et al., 2004), and allow bacterial quorum sensing via channel activation (Stevenson et al., 2007). In plants, as in other eukaryotes, rhomboids are encoded by a multigene family. These are little researched to date but we documented previously that A. thaliana rhomboid RBL10 was situated in the plastid envelope and has roles in fertility and photosynthesis (Thompson et al., 2012)

Identifying the transporters of different flavonoids in plants

We recently identified a new component of flavonoid transport pathways in Arabidopsis. The MATE protein FFT (Flower Flavonoid Transporter) is primarily found in guard cells and seedling roots, and mutation of the transporter results in floral and growth phenotypes. The nature of FFT’s substrate requires further exploration but our data suggest that it is a kaempferol diglucoside. Here we discuss potential partner H+-ATPases and possible redundancy among the close homologues within the large Arabidopsis MATE family

Investigating ‘rhomboid’ membrane proteases’ roles in development and signalling in Dictyostelium

The rhomboid family of intramembrane serine proteases is almost ubiquitous across all kingdoms of life. The enzymes are poorly conserved at sequence level but are similar in their structures and active-site motifs. They cleave disparate substrates, and it is the role of rhomboids across evolution that seems to be better conserved, since a membrane location means they are ideally placed for signalling and proteolytic activation events. Thus, we are investigating rhomboid function in the development and chemotaxis of Dictyostelium discoideum, the microbial, biomedical model organism that is capable of unicellular vegetative growth and multicellular development. A small group, including four apparently enzymatically active, rhomboids was identified in Dictyostelium. We found that development was unaltered following deletion of rhmC, whereas attempts to knock out the putative mitochondrial rhmD proved lethal. rhmA and rhmB null mutants give rise to changes in development, the lack of RhmA altering the response to chemoattractants and decreasing motility of the multicellular ‘slug’. rhmB null cells have lower viability, a smaller spore-sorus and a decreased response to folic acid stimulation. These results correspond with qRTPCR analysis, in which rhmA and rhmB transcript levels are highest during the multicellular growth phase. TEM intriguingly suggests a role in mitochondrion ultrastructure for RhmA, supported by bioinformatic interaction networks. These reveal Dictyostelium RhmA cotranscription with homologues of the Saccharomyces cerevisiae mitochondrial rhomboid’s substrates, which also regulate mitochondrial morphology

Rhomboids and proteolysis in the Dicty mitochondrion

Proteolytic regulation in Dictoystelium mitochondria Proteolysis is increasingly recognised as a key regulatory mechanism in cell biology, proteases comprising 2-5% of organism genomes across evolution. They govern protein activation, localisation, exposure of cryptic binding sites and release of neoproteins: indeed, altered protease expression and substrate-proteolysis are important in pathogenesis, including Parkinson’s and other neurodegenerative diseases. Proteolysis takes place within cell membranes via families of evolutionarily well-conserved intermembrane proteases. Amongst these are the 'rhomboid' serine proteases, enzymes known to influence development, signalling and infection in a range of eukaryotes and prokaryotes. Of four predicted enzymatically-active rhomboids in D. discoideum, we have found three to be important in the mitochondrion. The conserved eukaryotic, mitochondrial 'PARL'-type protease (RhmD) cannot be inactivated but the rhmA-rhomboid knockout responds poorly to chemoattractant, shows decreased motility and displays aberrant mitochondrial ultrastructure and altered respiration. A further rhomboid knockout, rhmB-, has reduced growth rates and folate chemotaxis and, interestingly, a double A/B-mutant is unable to phagocytose prey bacteria. Whereas RhmA-GFP is visualised at the contractile vacuole, RhmB-GFP colocalises with MitoTracker to the mitochondrion, but transcription of A and B both peak during multicellular growth and, in qPCR, rhmB transcription is differentially regulated in rhmA- cells. Since transcription levels are also altered of our predicted Dictyostelium orthologues of Saccharomyces cerevisiae mitochondrial rhomboid-substrates, we are examining (co-)localisation of orthologue-RFP in GFP-expressing and rhmA and B- cells, while conducting reporter studies with the remaining rhomboids C and D

Mapping connections in the neonatal brain with magnetic resonance imaging

The neonatal brain undergoes rapid development after birth, including the growth and maturation of the white matter fibre bundles that connect brain regions. Diffusion MRI (dMRI) is a unique tool for mapping these bundles in vivo, providing insight into factors that impact the development of white matter and how its maturation influences other developmental processes. However, most studies of neonatal white matter do not use specialised analysis tools, instead using tools that have been developed for the adult brain. However, the neonatal brain is not simply a small adult brain, as differences in geometry and tissue decomposition cause considerable differences in dMRI contrast. In this thesis, methods are developed to map white matter connections during this early stage of neurodevelopment. First, two contrasting approaches are explored: ROI-constrained protocols for mapping individual tracts, and the generation of whole-brain connectomes that capture the developing brain's full connectivity profile. The impact of the gyral bias, a methodological confound of tractography, is quantified and compared with the equivalent measurements for adult data. These connectomes form the basis for a novel, data-driven framework, in which they are decomposed into white matter bundles and their corresponding grey matter terminations. Independent component analysis and non-negative matrix factorisation are compared for the decomposition, and are evaluated against in-silico simulations. Data-driven components of dMRI tractography data are compared with manual tractography, and networks obtained from resting-state functional MRI. The framework is further developed to provide corresponding components between groups and individuals. The data-driven components are used to generate cortical parcellations, which are stable across subjects. Finally, some future applications are outlined that extend the use of these methods beyond the context of neonatal imaging, in order to bridge the gap between functional and structural analysis paradigms, and to chart the development of white matter throughout the lifespan and across species

Role of a rhomboid protease in a CO2-concentrating mechanism

Photosynthetic efficiency of some of the most important global crop plants is limited by the low affinity for CO2 of ribulose bisphosphate carboxylase–oxygenase (Rubisco) and the wastage of energy from photosynthesis via the release of fixed CO2 in a side reaction with oxygen. Experimental increases in the CO2 concentration around C3 plants did enhance the level of photosynthesis and yield, so it is of interest to investigate the crop-improvement potential of the CO2-concentrating mechanism (CCM) utilised by cyanobacteria to overcome the limitations of Rubisco. Utilising this mechanism of improving Rubisco efficiency and thus crop yield is attractive as it does not necessarily require major changes to leaf anatomy. The cyanobacterial CCM allows these photosynthetic prokaryotes to increase CO2 concentration up to 1000-fold near carboxysomes, protein-walled compartments of localised Rubisco, and increased CO2 levels also activate Rubisco and repress photorespiration. Within a complex network of CCM regulation is control at the level of transcription for genes encoding CO2-uptake and bicarbonate transport. Amongst the regulatory components are LysR-type transcription factors acting as activators or repressors, and the protease FtsH2. We report here how a rhomboid protease also plays a pivotal role in transcriptional control of CCM genes in Synechocystis sp. PCC6803