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Rhomboid peptidases are conserved intramembrane serine proteases with mitochondrial family members being involved in mitochondrial dynamics and apoptosis. The Saccharomyces cerevisiae mitochondrial rhomboid, Pcp1p, catalyzes the cleavage of two substrates: Ccp1p, which breaks down reactive oxygen species, and Mgm1p, a GTPase mediating mitochondrial fusion events. As an initial approach to determine the structural basis of Pcp1p activity, a screen to identify temperature sensitive alleles of PCP1 was performed using hydroxylamine mutagenesis. Eight mutants were identified from a pool of 30,000 colonies that exhibited either temperature sensitive growth or respiratory defects. These mutants also exhibited defects in Mgm1p and Ccp1p processing and some degree of abnormal mitochondrial morphology. The majority of amino acid changes occurred within the fourth and sixth transmembrane domains of Pcp1p, the location of the active site serine and histidine residues, supporting a role for these transmembrane helices in Pcp1p activity.

Rhomboids are intramembrane serine proteases that cleave transmembrane (TM) protein substrates within the phospholipid bilayer. Since the discovery of the first rhomboid protease, many homologous rhomboids have been identified in all kingdoms illustrating their biological significance. Rhomboids are key players in a variety of biological processes such as, cell signalling, protein degradation, mitochondria health, apoptosis, and pathogenicity. While the mechanism of substrate entry into the rhomboid active site is still not clear, it is thought to involve dynamics around the putative substrate gate, of which appears to be comprised of the fifth transmembrane a-helix. A powerful tool that can be used to investigate conformational dynamics around the substrate gate is solution-state nuclear magnetic resonance (NMR). However, due to the size restriction of solution-state NMR, only detergent micelles have been able to produce well- resolved 1H-15N HSQC spectra of rhomboids. However, the lipid membrane environment has a significant impact on rhomboid structure and function. The use of membrane-scaffolding proteins (MSPs) in the formation of nanodiscs has the potential to allow the study of rhomboid dynamics in lipid bilayers by solution-state NMR. Therefore, this thesis investigates the plausibility of incorporating rhomboid into nanodiscs that would be compatible with solution NMR with a focus on the E. coli rhomboid, ecGlpG. The formation of empty (no ecGlpG) and ecGlpG-encapsulated nanodiscs was attempted using two MSP variants. While some successful nanodisc formation was possible, MSP degradation and low yields were seen for all nanodisc samples. Further optimization or alternate nanodisc systems will be required to incorporate ecGlpG into more membrane-like environments in a state that is compatible with solution-state NMR.

While mitochondria are traditionally associated with energy production, recent studies identified its function in controlling the onset of apoptosis. Mitochondrial apoptotic control results from sequestering pro-apoptotic proteins, which are secreted following cellular stress. HtrA2/OMI is secreted from mitochondria to the cytosol following apoptotic induction, binds and degrades the inhibitor of apoptosis protein, XIAP in mammals, activating the caspase cascade. This study characterizes the expression of Drosophila HtrA2/OMI, a mitochondrial protein, its processing by Rhomboid-7 and demonstrates its pro-apoptotic function. Following exposure to apoptotic stress, dOMI is secreted from mitochondria and its expression profile displays an increase in a cleaved form consistent with Rhomboid-7 processing. dOMI expression resulted in sensitization of cells to apoptotic stress, observed through an increase in caspase activity. These data further validate the use of Drosophila in the study of mitochondrial driven apoptosis while implicating a potential role for Rhomboid-7 in apoptosis through proteolytic cleavage of dOMI.

While mitochondria are traditionally associated with energy production, recent studies identified its function in controlling the onset of apoptosis. Mitochondrial apoptotic control results from sequestering pro-apoptotic proteins, which are secreted following cellular stress. HtrA2/OMI is secreted from mitochondria to the cytosol following apoptotic induction, binds and degrades the inhibitor of apoptosis protein, XIAP in mammals, activating the caspase cascade. This study characterizes the expression of Drosophila HtrA2/OMI, a mitochondrial protein, its processing by Rhomboid-7 and demonstrates its pro-apoptotic function. Following exposure to apoptotic stress, dOMI is secreted from mitochondria and its expression profile displays an increase in a cleaved form consistent with Rhomboid-7 processing. dOMI expression resulted in sensitization of cells to apoptotic stress, observed through an increase in caspase activity. These data further validate the use of Drosophila in the study of mitochondrial driven apoptosis while implicating a potential role for Rhomboid-7 in apoptosis through proteolytic cleavage of dOMI.

The rhomboid family of intramembrane serine proteases can catalyze proteolysis of substrates that are normally embedded in the cell membrane, making them key players in a diverse range of biological processes. While X-ray crystal structures provide detailed insights into the mechanism of intramembrane hydrolysis, questions remain concerning how transmembrane (TM) substrates are able to gain access to the rhomboid active site, and whether interactions with the membrane environment can influence its structure and function. In this thesis, these questions were investigated using the E. coli rhomboid ecGlpG. In Chapter 3, the effect of hydrophobic mismatch between lipid and protein was investigated using families of amphiphiles with saturated alkyl chains. While ecGlpG displayed maximal activity against a water-soluble model substrate when solubilized in detergents containing 10-12 carbon atoms, shorter and longer chain detergents led to loss of activity. An even larger effect was observed when ecGlpG was reconstituted into phospholipid bicelles, with no proteolytic activity being detected in 14-carbon lipids. These results suggest that mismatch between the hydrophobic regions of the catalytic TM domain (TMD) and the local membrane environment is detrimental to proteolysis. To obtain further insight into the structure and dynamics of ecGlpG, sample conditions were identified in Chapter 4 that enabled, for the first time, the acquisition of NMR spectra showing signals from the ecGlpG TMD. While significant peak broadening prevented chemical shift assignment, the sensitivity and resolution of peaks corresponding to the tryptophan indole NH group allowed their use as structural probes. These were employed in Chapter 5 to characterize the open conformation of ecGlpG that is postulated to facilitate substrate entry. These spectra showed evidence of an open conformation in which the intact α5 is laterally displaced. Interactions with a substrate-derived peptide also appeared to stimulate gate opening; however, activity assays suggested that formation of the open state could compromise catalytic activity against water-soluble substrates, and that interactions with TM substrates could counter this effect. Taken together, these results provide new insight into the role of both the local membrane environment and α5-conformational dynamics on intramembrane proteolysis, and suggest a mechanism to prevent cleavage of off-target rhomboid substrates in vivo.

The rhomboid protease, Pcp1p, localizes to the mitochondrial inner membrane in Saccharomyces cerevisiae. The 346 amino acid protein contains an N-terminal mitochondria targeting signal followed by 6 transmembrane helices. Pcp1p substrates include, Ccp1p and Mgm1p; both soluble intermembrane space proteins after Pcp1p cleavage. Haploid pcp1 mutants grow slow, lose mitochondrial DNA, and have abnormal mitochondrial morphology; phenotypes similar to mgm1 mutants. Processing of Mgm1p depends on Pcp1p activity, matrix ATP levels, and a functional Tim23-Pam complex. This suggests a potential link between Pcp1p and the Tim23 complex. To test this hypothesis, epitope tagged versions of Pcp1p, Tim23p, Tim21p, and Pam18p were generated for use in immunoprecipitation and sucrose gradient ultracentrifugation experiments. The data suggest that Pcp1p exists in a higher order protein complex that may contain multiple Pcp1p subunits and components of the Tim23 translocon. The importance of this interaction in the processing of precursor proteins remains to be determined.

Rhomboid intramembrane serine proteases cleave polypeptide chains within lipid bilayer. Rhomboid proteases were originally discovered in Drosophila melanogaster where they regulate ontogenesis of the fly, but they are present in all domains of life. Nowadays, various diseases, such as malaria, amoebiasis, Parkinson's disease, various tumour malignancies, and diabetes, have been linked with rhomboid proteases. However, natural substrates and function of most rhomboids remain elusive. Cell biology tools are needed for unravelling functions of rhomboids, as well as for potential pharmacological applications, and this together fuels the effort to develop specific rhomboid inhibitors. The inhibitors known to date always bear an electrophilic warhead attacking the nucleophilic serine of the atypical serine-histidine catalytic dyad of rhomboid. From the various developed inhibitors, peptidyl -ketoamides substituted at the ketoamide nitrogen by hydrophobic groups, discovered in our laboratory, hold the biggest potential. They are potent, reversible, selective, tunable, and are built around a pharmacophore already approved for medical use. Here, I set out to improve peptidyl -ketoamides by exploring the chemical space in the active site of rhomboid and testing substituents of the ketoamide nitrogen of increasing...

Mutations in RHBDF2, the gene encoding inactive rhomboid protein iRHOM2, result in the dominantly inherited condition Tylosis with oesophageal cancer (TOC). TOC causes plamoplantar keratoderma, oral precursor lesions and up to a 95 % life-time risk of oesophageal squamous cell carcinoma (SCC). The role of iRHOM2 in the epidermis is not well characterised, although we previously showed dysregulated epidermal growth factor receptor (EGFR) signalling and accelerated migration in TOC keratinocytes, and a role for iRHOM2 was shown in trafficking the metalloproteinase ADAM17. Substrates of ADAM17 include EGFR ligands and adhesion molecules. iRHOM2 localisation and function were investigated in frozen sections and keratinocyte cell lines from control and TOC epidermis. Although iRHOM2 was predicted to be an ER-membrane protein, it showed cell-surface expression in control epidermis, with variable localisation in TOC. Increased processing and activation of ADAM17 was seen in TOC keratinocytes compared with control cells, suggesting that increased ADAM17-mediated processing of EGFR ligands may cause the changes in EGFR signalling. Downstream of iRHOM2-ADAM17, Eph/Ephrin and NOTCH signalling also appeared affected. Additionally, desmosomes in TOC epidermis lacked the electron-dense midline of the mature desmosomes seen in normal skin; this was accompanied by increased processing of desmoglein 2, a substrate of ADAM17. Expression and localisation of iRHOM2 was also investigated in TOC and sporadic SCC. iRHOM2 expression varied between SCC cell lines, and appeared to correlate with ADAM17 and NOTCH1 expression in oesophageal SCC and head and neck SCC cells. In summary, iRHOM2 mutations in TOC appear to be gain-of-function in nature, resulting in increased ADAM17 processing and enhanced EGFR signalling. Questions remaining include the reason why iRHOM2 is found at the plasma membrane. Future study of the iRHOM2-ADAM17 pathway may provide additional insight into the mechanism of epidermal wound healing and the pathogenesis of oesophageal SCC.

Of the organisms with sequenced genomes, plants appear to possess the most rhomboid protease-encoding genes. However, our knowledge of processes in plants that involve Regulated Intramembrane Proteolysis (RIP) and rhomboid proteases remains low. As expressed recently by other researchers, finding a natural substrate for a rhomboid protease represents the biggest experimental challenge. Using yeast mitochondria-based assays, a potential link between the plastid translocon component Tic40 and organellar rhomboid proteases was recently uncovered. In this particular link, rhomboid proteases appear capable of influencing the pattern of imported Tic40 in yeast mitochondria. Tic40 may thus represent a natural plant target of organellar rhomboid proteases. Here, we obtained further motif-oriented evidence supporting Tic40 as a natural plant rhomboid substrate. A comparative analysis of sequences revealed that Tic40 may also possess similar TMD motifs found in the model substrate, Spitz. Rhomboid proteases often require these motifs to cleave substrates within intramembrane environments. Using site-directed mutagenesis and yeast mitochondria assays, the impact of mutations occurring in the motifs ASISS, GV, QP, and GVGVG of Tic40 was assessed. In terms of cleavage and changing the pattern of imported Tic40, some of the mutations showed decreased activities and a few showed enhancements. More importantly, the overall observed pattern associated with select Tic40 mutations resembled the characteristics reported for the model substrates. In particular, mutations in the Tic40 GV motif produced similar results as that observed with Spitz, by drastically decreasing or increasing cleavage as a function of amino acid sequence. / Thesis (Master, Biology) -- Queen's University, 2010-05-30 10:22:07.72