There are now numerous examples of post-translational modification with geranylgeranyl or farnesyl substituents. Once thought of as solely a mechanism for association of proteins with membranes, other functional aspects of protein prenylation have come to be appreciated. Although, in almost all instances, such proteins are membrane associated, they are often found to also engage in protein-protein interactions. In some instances, such interactions are critical aspects of prenylated protein trafficking. In this review, the role of prenylation in mediating protein-protein interactions will be considered. The hypothesis will be developed that such interactions occur through recognition of the prenyl group and a second domain, on the prenylated protein, by a heterodimeric protein partner.

The work presented herein details post-translational modifications (PTMs) in Staphylococcus aureus that are involved in mediating the stress response and normal cellular processes. The first PTM that was investigated is regulated intramembrane proteolysis (RIP) for the activation of the ECF sigma factor σS. We achieved this by analyzing the role of the site-1 protease, which we termed “putative regulator of sigmaS” (PrsS), as it is predicted to be the first enzyme in the RIP cascade, leading to the activation of σS. It was determined that the putative site-1 protease, prsS, mimics transcriptional profiles of sigS; with expression low in all strains examined other than in the highly mutagenic strain RN4220. Moreover, up-regulation of the protease was observed in response to cell wall-targeting antibiotics, DNA-damaging agents, and during infection in human serum and RAW 264.7 cells, similar to that previously demonstrated for sigS. It was further determined that prsS mutants, like sigS mutants, are more sensitive to cell wall-targeting antibiotics and DNA-damaging agents, which is explained, in part, by alterations in altered abundance of proteins in the prsS mutant that mediate antibiotic resistance (Pbp2a, FemB, and HmrA) and the response to DNA damage (BmrA, Hpt, and Tag). Importantly, transcriptional analyses of proteins affected in the protease mutant, revealed that their expression is decreased in both prsS and sigS mutants, suggesting that this is a result of sigS-mediated regulation. Lastly, it was determined that PrsS, similar to σS, is required for infection in whole human blood and murine models of virulence. Next, since the abundance of a stress response protease, HtrA1, was altered in prsS mutants, we aimed to assess the roles of this enzyme, and its homolog HtrA2 in S. aureus. Interestingly, we first determined that unlike that previously described for the HtrA enzymes, these proteases do not have a role in Agr-mediated virulence regulation. We attribute this finding to unintended mutations likely introduced during strain construction, which is common for S. aureus strains. We next used transcription profiling of the htrA genes in order to understand their role in the cell, and found that they are moderately expressed under standard conditions, and are up-regulated in response to both in vitro and ex vivo stressors that lead to cell protein, DNA, and cell envelope damage. Further to this, the protease mutants are more sensitive to numerous conditions that affect macromolecular stability, including elevated temperature, alterations in pH, reactive oxygen species, DNA damage, and antimicrobial stress. In order to further explore these sensitivities and gain insight into putative substrates, we employed a yeast-2 hybrid screen, and identified numerous proteins that interact with HtrA1 and HtrA2, including those that mediate the response to stress and normal cellular homeostasis. Taken together, we provide evidence to suggest the HtrA proteases in S. aureus are required both during standard conditions and in stress-inducing environments to mediate protein folding and proteolysis of a broad range of substrates. Finally, we performed the first examination of prenylation in a bacterial organism. Prenylation is a well-studied post-translational modification (PTM) in eukaryotes, wherein a prenyl group is added to a metabolite or the C-terminal “CAAX” motif of a protein. Interestingly, the machinery exists for this PTM in a wide variety of prokaryotic species, thus we set out to investigate its impact in S. aureus. To achieve this, we disrupted prenyl group synthesis by inactivating ispA, the gene encoding a prenyl synthetase. The abrogation of prenylation ensued in striking alterations in the cell, including lack of pigmentation and smaller colony size, similar to small-colony variants (SCVs) of S. aureus. In addition to this, the ispA mutant displayed a growth defect, as a result of lower ATP levels. Moreover, the prenylation mutant displayed alterations in resistance to antibiotics, including increased resistance to aminoglycosides and antimicrobial peptides (AMPs), yet elevated sensitivity to cell wall-targeting antibiotics. These differences in susceptibility to cell envelope targeting antibiotics are a result of alterations in cell envelope architecture, including variations in fatty acid composition and increased membrane fluidity. Collectively, the pleotropic consequences of the disruption of prenylation indicate that this process is key to maintaining cellular homeostasis in S. aureus, and perhaps other bacterial species.

As a front-line treatment for cardiovascular disease, statins are among some of the most widely prescribed drugs worldwide. Statins are effective at lowering cholesterol, but approximately 7-29% of patients report some form of adverse muscle effect during the course of treatment. The severity of these side effects ranges from low-level to life-threatening myopathy. The mechanism of statin myopathy remains ill-defined, but muscle-specific E3 ubiquitin ligases have been implicated. In addition, statins have been shown to activate caspase-1 (and increase IL-1β) in immune cells, which is a key effector of the NLRP3 inflammasome. The relevance of this inflammatory response in statin myopathy remains unknown. Using C2C12 myotubes, an in vitro model of statin-induced myopathy was developed to test the impact of NLRP3 inflammasome activation on markers of statin myopathy. Gene expression of the muscle-specific E3 ubiquitin ligases atrogin-1 and MuRF-1 (atrogenes) were used as markers of statin-induced myopathy. Lipopolysaccharide priming of the NLRP3 inflammasome was found to lower the effective dose of fluvastatin required to augment atrogene expression. This effect correlated with reduced phosphorylation of Akt and FOXO3a, a transcription factor regulating atrogene expression. Statin-induced atrogene expression was also found to be dependent on an isoprenoid that is required for protein prenylation rather than cholesterol biosynthesis pathways. Fluvastatin increased caspase-1 activity in a prenylation-dependent manner and selective inhibitors of NLRP3 and caspase-1 were able to prevent increased atrogene expression with fluvastatin treatment. Therefore, the NLRP3 inflammasome contributes to markers of statin-induced myopathy through a prenylation-dependant pathway in muscle cells. This work presents a novel mechanism involved in statin myopathy, and has shown that the inflammasome may represent a new drug target to mitigate muscle symptoms in patients taking statins. / Thesis / Master of Science (MSc) / Statins are a class of widely prescribed cholesterol-lowering drugs that reduce the risk of heart attack and stroke. However, many patients often complain of statin-induced muscle side effects (myopathy) that impact their quality of life. Symptoms of this statin-induced myopathy can manifest as muscle pain and weakness. The underlying biology causing this condition is still not well understood. Independent of its cholesterol-lowering effect, statins can activate an immune receptor called the NLRP3 inflammasome, indicating that inflammation may contribute to myopathy. Therefore, the primary goal of this study was to determine if this immune response contributes to statin-induced myopathy. It was found that inhibition of the NLRP3 inflammasome lowers markers of statin myopathy. Results from this study will provide further insight into mechanisms regulating this myopathy, and may lead to new treatments that can help alleviate statin side effects in muscle.

Protein farnesyltransferase (FTase, E. C. 2.5.1.21) post-translationally modifies regulatory proteins involved in controlling cell growth, division, and differentiation. Recently, a cDNA clone (PsFTb) encoding a pea (Pisum sativum) FTase b subunit was isolated. Initial studies led to the hypothesis that FTase plays a role in the regulation of plant cell division. To gain insight into FTase function in plants, a detailed study of the expression pattern of FTase genes was carried out. A cDNA (NgFTb) encoding the b subunit of tobacco FTase was cloned from a Nicotiana glutinosa cDNA library to initiate studies in tobacco. In tobacco BY-2 suspension culture, levels of NgFTb mRNA and FTase activity transiently increased at the early log phase of cell growth and rapidly declined before cells entered stationary phase. These data, along with inhibitor studies in the BY-2 system, support our hypothesis. To understand the expression and regulation of pea FTase subunit genes, 5'-upstream sequences of both pea FTase subunit genes (PsFTb and PsFTa) were cloned from a pea genomic library. The 5'-upstream sequence (~2 kb) of PsFTa was fused to GUS (b-glucuronidase) and GFP (green fluorescent protein) reporter genes and introduced into tobacco plants. This 2 kb upstream region appears insufficient to provide PsFTa promoter function. On the other hand, 3.2 kb of PsFTb 5'-upstream sequence expressed as a PsFTb:GUS construct is fully functional in transgenic tobacco plants. GUS expression was most prominent in actively growing cells supporting FTase involvement in plant cell cycle control. GUS activity was also found in mature and imbibed embryos but not premature embryos, consistent with the role of FTase in abscisic acid (ABA) signaling. An unexpected pattern of GUS activity, not correlated with dividing cells or ABA signaling, was also observed in the transgenic plants. GUS activity was detected in vascular bundles adjacent to actively-growing tissues and in regions that connect two organs, e.g., junctions between stems and leaf petioles, cotyledons and hypocotyls, roots and hypocotyls. Auxin promotes PsFTb expression while light and sucrose inhibit expression. These spatial and temporal expression patterns strongly suggest that FTase has a broader role associated with regulation of nutrient transportation or allocation in plants. / Ph. D.

Huntington’s disease (HD) is a heritable, neurodegenerative disorder characterized by motor, cognitive, and psychiatric disturbances. The progressive disease is caused by an unstable CAG expansion within the gene that normally encodes for the huntingtin protein (Htt). The expanded mutant form of Htt (mHtt) is expressed ubiquitously throughout patients’ bodies; however, neuronal degeneration is prominent only in the corpus striatum and, to a lesser extent, the cortex. The Ras homolog Rhes is also preferentially localized to the striatum. The putative co-factor Rhes has been shown to act with mHtt to cause neuronal death. Simvastatin, a lipid lowering drug, and zoledronate, a nitrogen bisphosphonate, act on the mevalonate pathway, which gives both Rhes and its target cells, binding sites. The current study aimed to interrupt the mevalonate pathway and inactivate, via de-prenylation, Rhes in CD-1 mice exposed to 3-nitroproprionic acid, a neurotoxin that mimics HD mitochondrial dysfunction and striatal degeneration. Results suggest that drug treatment does not rescue motor impairments and may potentiate 3-NP damage. The persistent motor deficits are discussed in relation to possible Rhes de-prenylation.

Diss. (sammanfattning) Göteborg : Göteborgs universitet, 2009. / Härtill 3 uppsatser.

Post-translational modification of proteins with isoprenoids was first recognized as a general phenomenon in 1984. In recent years, our understanding, including mechanistic studies, of the enzymatic reactions associated with these modifications and their physiological functions has increased dramatically. Of particular functional interest is the role of prenylation in facilitating protein-protein interactions and membrane-associated protein trafficking. The loss of proper localization of Ras proteins when their farnesylation is inhibited has also permitted a new target for anti-malignancy pharmaceuticals. Recent advances in the enzymology and function of protein prenylation are reviewed in this article.

The nuclear lamins form a karyoskeleton providing structural rigidity to the nucleus. One member of the lamin family, lamin A, is first synthesized as a 74 kDa precursor, prelamin A. After the endopeptidase and methylation reactions which occur after farnesylation of the CAAX-box cysteine, there is a second endoproteolysis that occurs 15 amino acids upstream from the C-terminal farnesylated cysteine residue. Studies with knockout mice have implicated the enzyme Zmpste24 (Face-1) as a suitable candidate to perform one or both of these proteolytic reactions. Evidence has been presented elsewhere establishing that Zmpste24 possesses a zinc-dependent CAAX endopeptidase activity. In the present study, we confirm this CAAX endopeptidase activity with recombinant, membrane-reconstituted Zmpste24 and show that it can accept a prelamin A farnesylated tetrapeptide as substrate. To monitor the second upstream endoproteolytic cleavage of prelamin A, we expressed a 33 kDa prelamin A C-terminal tail in insect cells. We demonstrate that this purified substrate possesses a C-terminal farnesylated and carboxyl-methylated cysteine and, therefore, constitutes a valid substrate for assaying the second endoproteolytic step in lamin A maturation. With this substrate, we demonstrate that insect cell membranes bearing recombinant Zmpste24 can also catalyse the second upstream endoproteolytic cleavage.

In an attempt to synthesize a photoreactive geranylcysteine derivative, A, with an appropriate photoprobe to be studied as a mimic for farnesylated protein, B, the following syntheses were carried out. The hydroxyl group of geraniol was protected with DHP/PPTs to generate 1 (98.1%). Allylic oxidation of 1 by using TBHP/SeO2 yielded 2 (31.5%). A modified oxidation increased the yield of 2 (34.4%). Treatment of 2 with BTC/pyridine afforded 3 (86.0%). Reaction of 3 with NaN3/DMF gave 8 and 9 (49.4%). Deprotection of this mixture under PPTs/EtOH afforded 10 and 11 (64.1%). Because of the unexpected reaction of 3 with N3-, we focused on alternative target molecules 12 and 13 (Figure 7). Our attempts to synthesize the first intermediates 14 and 15 in the syntheses of 12 and 13 (Scheme 14 and Scheme 15) have resulted in the isolation of the unreacted starting material.

The nuclear lamins form a karyoskeleton providing structural rigidity to the nucleus. One member of the lamin family, lamin A, is first synthesized as a 74 kDa precursor, prelamin A. Following the endopeptidase and methylation reactions which occur after farnesylation of the CAAX-box cysteine, there is a second endoproteolysis that occurs 15 amino acids upstream from the C-terminal farnesylated cysteine residue. Studies with knockout mice have implicated the enzyme Zmpste24 as a candidate to carry out one or both of these proteolytic reactions. In this body of work, the CAAX endopeptidase activity of recombinant, membrane reconstituted, Zmpste24 is demonstrated using a prelamin A farnesylated tetrapeptide as substrate. To monitor the second upstream endoproteolytic cleavage a 33 kDa prelamin A carboxyl terminal tail of prelamin A was expressed in insect cells. This purified substrate possesses a fully processed CAAX box, and, therefore, constitutes a valid substrate for assaying the second endoproteolytic step in lamin A maturation. In vitro reactions with this substrate and insect cell membranes bearing recombinant Zmpste24 demonstrate that Zmpste24 may possess the ability to directly catalyze the second endoproteolytic cleavage. Previous studies on nuclear envelope fractions have ascribed this second activity to a chymotrypsin like protease. However, Zmpste24 contains the canonical HEXXH domain, a common characteristic of zinc metalloproteinases. Experiments on Zmpste24 in this work demonstrate that inactivating the HEXXH domain by site directed mutagenesis results in a loss of the first endoproteolysis reaction, while the second endoproteolytic activity is retained. Supporting these data is the observation that a truncated mutant of Zmpste24 (residues: Met1 - Pro230) that does not contain the HEXXH motif, loses the first endoproteolytic activity while retaining the second. Furthermore, this second activity is not sensitive to the metalloproteinase inhibitors EDTA and 1,10-orthophenanthroline, but is sensitive to the chymotrypsin inhibitor TPCK and its fluorescent analogue, FFCK. The fact that Zmpste24 can be affinity labeled with FFCK suggests that this second activity is directly caused by a second, yet unidentified, active site with a chymotrypsin-like catalytic mechanism.