Characterization of the promoter of the human farnesyltransferase beta subunit and the impact of the transcription factor OCT-1 on its expression.

Farnesyltransferase (FTase) enables about 100 proteins to interact with cellular membranes by catalyzing the posttranslational addition of a farnesyl group. Farnesylated proteins provide important functions and inhibitors against the ß-subunit of the heterodimer of FTase are intensively studied in clinical and preclinical trials. However, very little is known about the transcriptional regulation of the ß-subunit. The examined promoter region of the human FTase ß-subunit gene (FNTB) showed significant basal promoter activity in HEK-293 and in HeLa cells. We were able to locate the core promoter at -165 to -74. Ten potential binding sites of the transcription factor OCT-1 were detected. Three could be confirmed using EMSA super shift experiments. OCT-1 overexpression and knockdown confirmed it as an important regulator of FNTB expression. Our results provide a basis for further research on FNTB/OCT-1 regulation, its inhibitors and diseases influenced by both such as colon carcinoma or diabetes mellitus.

Impact of a conserved N-terminal proline-rich region of the α-subunit of CAAX-prenyltransferases on their enzyme properties.

BACKGROUND: The CAAX-prenyltransferases farnesyltransferase (FTase) and geranylgeranyltransferase I (GGTase I) are heterodimers with a common α- (FTα) and unique ß-subunits. Recently, α-subunits of species (e.g., human) that harbour an N-terminal proline-rich region (PRR) showed different dimerization behaviours than α-subunits without PRR (e.g., yeast). However, the specific function of the PRR has not been elucidated so far. METHODS: To determine whether the PRR is a conserved motif throughout eukaryotes, we performed phylogenetics. Elucidating the impact of the PRR on enzyme properties, we cloned human as well as rat PRR deficient FTα, expressed them heterologously and compared protein-protein interaction by pull-down as well as crosslinking experiments. Substrate binding, enzyme activity and sensitivity towards common FTase inhibitors of full length and PRR-deletion α-subunits and their physiological partners was determined by continuous fluorescence assays. RESULTS: The PRR is highly conserved in mammals, with an exception for marsupials harbouring a poly-alanine region instead. The PRR shows similarities to canonical SH3-binding domains and to profilin-binding domains. Independent of the PRR, the α-subunits were able to dimerize with the different physiological ß-subunits in in vitro as well as in yeast two-hybrid experiments. FTase and GGTase I with truncated FTα were active. The KM values for both substrates are in the single-digit µM range and show no significant differences between enzymes with full length and PRR deficient α-subunits within the species. CONCLUSIONS: Our data demonstrate that an N-terminal PRR of FTα is highly conserved in mammals. We could show that the activity and inhibitability is not influenced by the truncation of the N-terminal region. Nevertheless, this region shows common binding motifs for other proteins involved in cell-signalling, trafficking and phosphorylation, suggesting that this PRR might have other or additional functions in mammals. Our results provide new starting points due to the relevant but only partly understood role of FTα in eukaryotic FTase and GGTase I. Video Abstract.

Molecular and Pharmacological Characterization of the Interaction between Human Geranylgeranyltransferase Type I and Ras-Related Protein Rap1B.

Geranylgeranyltransferase type-I (GGTase-I) represents an important drug target since it contributes to the function of many proteins that are involved in tumor development and metastasis. This led to the development of GGTase-I inhibitors as anti-cancer drugs blocking the protein function and membrane association of e.g., Rap subfamilies that are involved in cell differentiation and cell growth. In the present study, we developed a new NanoBiT assay to monitor the interaction of human GGTase-I and its substrate Rap1B. Different Rap1B prenylation-deficient mutants (C181G, C181S, and ΔCQLL) were designed and investigated for their interaction with GGTase-I. While the Rap1B mutants C181G and C181S still exhibited interaction with human GGTase-I, mutant ΔCQLL, lacking the entire CAAX motif (defined by a cysteine residue, two aliphatic residues, and the C-terminal residue), showed reduced interaction. Moreover, a specific, peptidomimetic and competitive CAAX inhibitor was able to block the interaction of Rap1B with GGTase-I. Furthermore, activation of both Gαs-coupled human adenosine receptors, A2A (A2AAR) and A2B (A2BAR), increased the interaction between GGTase-I and Rap1B, probably representing a way to modulate prenylation and function of Rap1B. Thus, A2AAR and A2BAR antagonists might be promising candidates for therapeutic intervention for different types of cancer that overexpress Rap1B. Finally, the NanoBiT assay provides a tool to investigate the pharmacology of GGTase-I inhibitors.

Enhanced Antiviral Function of Magnesium Chloride-Modified Heparin on a Broad Spectrum of Viruses.

Previous studies reported on the broad-spectrum antiviral function of heparin. Here we investigated the antiviral function of magnesium-modified heparin and found that modified heparin displayed a significantly enhanced antiviral function against human adenovirus (HAdV) in immortalized and primary cells. Nuclear magnetic resonance analyses revealed a conformational change of heparin when complexed with magnesium. To broadly explore this discovery, we tested the antiviral function of modified heparin against herpes simplex virus type 1 (HSV-1) and found that the replication of HSV-1 was even further decreased compared to aciclovir. Moreover, we investigated the antiviral effect against the new severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) and measured a 55-fold decreased viral load in the supernatant of infected cells associated with a 38-fold decrease in virus growth. The advantage of our modified heparin is an increased antiviral effect compared to regular heparin.

The small molecule Bcl-2/Mcl-1 inhibitor TW-37 shows single-agent cytotoxicity in neuroblastoma cell lines.

BACKGROUND: High-risk neuroblastoma with N-Myc amplification remains a therapeutic challenge in paediatric oncology. Antagonism of pro-death Bcl-2 homology (BH) proteins to pro-survival BH members such as Mcl-1 and Bcl-2 has become a treatment approach, but previous studies suggest that a combined inhibition of Bcl-2 and Mcl-1 is necessary. TW-37 inhibits Mcl-1 and Bcl-2 with almost the same affinity. However, single-agent cytotoxicity of TW-37 in neuroblastoma cell lines has not been investigated. METHODS: Cell viability, apoptosis, proliferation and changes in growth properties were determined in SKNAS, IMR-5, SY5Y and Kelly cells after treatment with TW-37. After transfection with Mcl-1 or Bcl-2 siRNA, apoptosis and proliferation were investigated in Kelly cells. Mice with Kelly cell line xenografts were treated with TW-37 and tumor growth, survival and apoptosis were determined. RESULTS: Cell lines with N-Myc amplification were more sensitive to TW-37 treatment, IC50 values for IMR-5 and Kelly cells being 0.28 µM and 0.22 µM, compared to SY5Y cells and SKNAS cells (IC50 0.96 µM and 0.83 µM). Treatment with TW-37 resulted in increased apoptosis and reduced proliferation rates, especially in IMR5 and Kelly cells. Bcl-2 as well as Mcl-1 knockdown induced apoptosis in Kelly cells. TW-37 led to a decrease in tumor growth and a favorable survival (p = 0.0379) in a Kelly neuroblastoma xenografts mouse model. CONCLUSION: TW-37 has strong single-agent cytotoxicity in vitro and in vivo. Therefore, combined inhibition of Bcl-2/Mcl-1 by TW-37 in N-Myc amplified neuroblastoma may represent an interesting therapeutic strategy.

Side Effects of Frequently Used Antihypertensive Drugs on Wound Healing in vitro.

BACKGROUND: The number of patients who has a daily intake of antihypertensive drugs is rising, due to an also rising prevalence of lifestyle diseases. Interestingly, knowledge about effects of these drugs in terms of wound healing is low. OBJECTIVE: Based on a few differing studies, the idea arose that antihypertensives may have side effects on wound healing. METHODS: Five antihypertensive drugs from different substance classes (metoprolol, amlodipine, ramipril, hydrochlorothiazide, candesartan) were investigated, in terms of possible impacts on cell metabolism and migration of human skin fibroblasts and keratinocytes. Additionally, histological and immunohistochemical analyses were performed in a 3-dimensional (3D) wound model addressing the influence on regeneration processes, such as cell migration, metabolic activity, apoptosis and epidermal thickness. RESULTS: Hydrochlorothiazide and ramipril exerted inhibiting effects in nearly all analyses, interestingly, in serum equivalent doses. In contrast, candesartan and amlodipine induced slight positive effects in 2D as well as in 3D models. The previously described positive effects of ß-blockers could only partially be confirmed by metoprolol. Antihypertensive drugs affected fibroblasts more than keratinocytes - whether positively or negatively. CONCLUSION: Antihypertensive drugs have an influence on keratinocytes and fibroblasts; they are not neutral. Candesartan has the most positive effects on skin cells. For angiotensin-converting enzyme inhibitors and thiazide diuretics, wound healing in a 3D model is delayed. ß-Receptor blockers seem to improve wound healing to a small extent just like calcium channel blockers. These results should be evaluated in a clinical trial to verify their clinical relevance.

Allosteric Activation of GDP-Bound Ras Isoforms by Bisphenol Derivative Plasticisers.

The protein family of small GTPases controls cellular processes by acting as a binary switch between an active and an inactive state. The most prominent family members are H-Ras, N-Ras, and K-Ras isoforms, which are highly related and frequently mutated in cancer. Bisphenols are widespread in modern life because of their industrial application as plasticisers. Bisphenol A (BPA) is the best-known member and has gained significant scientific as well as public attention as an endocrine disrupting chemical, a fact that eventually led to its replacement. However, compounds used to replace BPA still contain the molecular scaffold of bisphenols. BPA, BPAF, BPB, BPE, BPF, and an amine-substituted BPAF-derivate all interact with all GDP-bound Ras-Isoforms through binding to a common site on these proteins. NMR-, SOScat-, and GDI- assay-based data revealed a new bisphenol-induced, allosterically activated GDP-bound Ras conformation that define these plasticisers as Ras allosteric agonists.

Hypoxia Inducible Factor-2 Alpha and Prolinhydroxylase 2 Polymorphisms in Patients with Acute Respiratory Distress Syndrome (ARDS).

Hypoxia-inducible-factor-2α (HIF-2α) and HIF-2 degrading prolyl-hydroxylases (PHD) are key regulators of adaptive hypoxic responses i.e., in acute respiratory distress syndrome (ARDS). Specifically, functionally active genetic variants of HIF-2α (single nucleotide polymorphism (SNP) [ch2:46441523(hg18)]) and PHD2 (C/T; SNP rs516651 and T/C; SNP rs480902) are associated with improved adaptation to hypoxia i.e., in high-altitude residents. However, little is known about these SNPs' prevalence in Caucasians and impact on ARDS-outcome. Thus, we tested the hypotheses that in Caucasian ARDS patients SNPs in HIF-2α or PHD2 genes are (1) common, and (2) independent risk factors for 30-day mortality. After ethics-committee approval, 272 ARDS patients were prospectively included, genotyped for PHD2 (Taqman SNP Genotyping Assay) and HIF-2α-polymorphism (restriction digest + agarose-gel visualization), and genotype dependent 30-day mortality was analyzed using Kaplan-Meier-plots and multivariate Cox-regression analyses. Frequencies were 99.62% for homozygous HIF-2α CC-carriers (CG: 0.38%; GG: 0%), 2.3% for homozygous PHD2 SNP rs516651 TT-carriers (CT: 18.9%; CC: 78.8%), and 3.7% for homozygous PHD2 SNP rs480902 TT-carriers (CT: 43.9%; CC: 52.4%). PHD2 rs516651 TT-genotype in ARDS was independently associated with a 3.34 times greater mortality risk (OR 3.34, CI 1.09-10.22; p = 0.034) within 30-days, whereas the other SNPs had no significant impact (p = ns). The homozygous HIF-2α GG-genotype was not present in our Caucasian ARDS cohort; however PHD2 SNPs exist in Caucasians, and PHD2 rs516651 TT-genotype was associated with an increased 30-day mortality suggesting a relevance for adaptive responses in ARDS.

Hypoxia inducible factor-1 alpha and prolinhydroxlase 2 polymorphisms in patients with severe sepsis: a prospective observational trial.

BACKGROUND: Hypoxia-inducible-factor-1α (HIF-1α) and HIF-1 degrading prolyl-hydroxylases (PHD) are key regulators of the hypoxic-inflammatory response. Functionally active genetic variants in the HIF-1α (C/T; Single Nucleotide Polymorphism (SNP) rs11549465) and the PHD2 gene (EGLN1; C/T; SNP rs516651 and T/C; SNP rs480902) are associated with altered HIF-1α mRNA nuclear translocation and an altered adaptation to hypoxia. Furthermore, the HIF system is important in surviving inflammatory disorders and sepsis. Thus, we tested the hypotheses, that SNPs in the HIF-1α or PHD2 genes are (1) common in Caucasians, with 2) the HIF-1α genetic variant being associated with an altered HIF-1α mRNA expression; and 3) independent risk factors for 30-day mortality in severe sepsis. METHODS: After ethics approval, 128 septic patients (Caucasian descent) were included prospectively within 24 h after first diagnosing sepsis. Patients characteristics and severity of illness (simplified acute physiology score II), genotypes (Taqman assay), and their influence on leukocyte HIF-1α-mRNA-expression (Real-Time PCR) and 30-day mortality were determined. RESULTS: Frequencies were 0.8 % for homozygous HIF-1α TT-carriers (CT 17.6 %; CC 81.6 %), 2.5 % for homozygous PHD2 SNP rs516651 TT-allele carriers (CT 17.5 % and CC 80 %), and 9.4 % for homozygous PHD2 SNP rs480902 TT-allele carriers (CT 34.4 % and CC 56.3 %). While HIF-1α T-allele carriers had a borderline decrease in HIF-1α-mRNA-expression (p = 0.06) neither HIF-1α nor PHD2 SNPs were (independent) risk factors for 30-day mortality. CONCLUSIONS: Genetic variants in HIF-1α and PHD2 genes exist in Caucasians but do not appear to alter 30-day mortality in sepsis.

The FNTB promoter polymorphism rs11623866 as a potential predictive biomarker for lonafarnib treatment of ovarian cancer patients.

AIM: Despite promising preclinical findings regarding clinical utility of farnesyltransferase inhibitors (FTI), such as lonafarnib, success of clinical trials is limited. A multicentre AGO-OVAR-15 phase II trial reported an unfavourable effect of lonafarnib on the outcome of patients with advanced ovarian cancer. This study was performed as a genetic subgroup analysis of the AGO-OVAR-15 trial, and investigated the utility of the promoter polymorphism rs11623866 of the farnesyltransferase ß-subunit gene (FNTB) in predicting the clinical effectiveness of lonafarnib. METHODS: The influence of rs11623866 (c.-609G > C) on FNTB promoter activity was investigated by electrophoretic-mobility-shift assay, luciferase-reporter assay and RT-qPCR. A total of 57 out of 105 patients from the AGO-OVAR-15 trial, treated with carboplatin and paclitaxel ± lonafarnib, was genotyped for rs11623866 by restriction fragment length polymorphism analysis. Genotype-dependent survival analysis was performed by Kaplan-Meier analysis. RESULTS: The presence of the G allele was associated with increased FNTB promoter activity compared with the C allele. An unfavourable effect of lonafarnib was limited to patients carrying a GG genotype (HRPFS 6.2, 95%CI = 2.01, 19.41, P = 0.002; HROS 9.6, 95%CI = 1.89, 48.54, P = 0.006). Median progression free survival (PFS) for patients with the GG genotype in the lonafarnib treated arm was 10 months, whereas median PFS without FTI-treatment was 40 months. Median overall survival (OS) in the lonafarnib-treated group was 19 months, whereas median OS was not reached in the untreated group. CONCLUSIONS: Discrepancies between preclinical success and clinical failure may be due to the patients' genetic variability of FNTB. Therefore, our results may encourage retrospective evaluation of FNTB polymorphisms in previous FTI studies, especially those reporting positive FTI response.

Calcitonin gene-related peptide modulates the production of pro-inflammatory cytokines associated with periprosthetic osteolysis by THP-1 macrophage-like cells.

OBJECTIVE: An anti-resorptive impact of the neuropeptide calcitonin gene-related peptide (CGRP) on periprosthetic osteolysis, the leading cause of early prosthesis loosening, has been shown previously. In this study, the impact of CGRP on pro-inflammatory cytokine production associated with periprosthetic osteolysis was analysed using THP-1 macrophage-like cells. METHODS: Cells were stimulated with ultra-high-molecular-weight polyethylene (UHMWPE) particles (cell-to-particle ratios of 1:100 and 1:500) and lipopolysaccharides (LPS; 1 µg/ml) to establish osteolytic conditions, and simultaneously treated with CGRP (10(-8)M). Receptor activator of nuclear factor-κB (RANK), RANK ligand (RANKL) and tumour necrosis factor (TNF)-α mRNA expression were measured by quantitative RT-PCR. RANK protein was detected by Western blot. Secreted protein levels of TNF-α as well as interleukin (IL)-1ß and IL-6 were quantified in cell culture supernatants by ELISA and Bio-Plex cytokine assay, respectively. RESULTS: Activation of macrophage-like cells failed to enhance the production of RANK but led to a dose- and time-dependent increase of TNF-α mRNA and secreted protein levels of TNF-α, IL-1ß and IL-6. Application of CGRP time-dependently suppressed TNF-α mRNA expression induced by low-particle concentrations and LPS, while both particle- and LPS-induced secretion of TNF-α was inhibited. A pronounced inhibitory effect of CGRP on LPS-induced cytokine production at 24 h of incubation was also observed with IL-1ß and IL-6. CONCLUSIONS: CGRP shows a time-dependent inhibitory effect on the secretion of osteolysis-associated pro-inflammatory cytokines, indicating an indirect anti-resorptive influence of the neuropeptide on both aseptic prosthesis loosening and bacterially induced bone resorption which might enhance the life time of total joint replacements.

A 3'UTR polymorphism modulates mRNA stability of the oncogene and drug target Polo-like Kinase 1.

BACKGROUND: The Polo-like Kinase 1 (PLK1) protein regulates cell cycle progression and is overexpressed in many malignant tissues. Overexpression is associated with poor prognosis in several cancer entities, whereby expression of PLK1 shows high inter-individual variability. Although PLK1 is extensively studied, not much is known about the genetic variability of the PLK1 gene. The function of PLK1 and the expression of the corresponding gene could be influenced by genomic variations. Hence, we investigated the gene for functional polymorphisms. Such polymorphisms could be useful to investigate whether PLK1 alters the risk for and the course of cancer and they could have an impact on the response to PLK1 inhibitors. METHODS: The coding region, the 5' and 3'UTRs and the regulatory regions of PLK1 were systematically sequenced. We determined the allele frequencies and genotype distributions of putatively functional SNPs in 120 Caucasians and analyzed the linkage and haplotype structure using Haploview. The functional analysis included electrophoretic mobility shift assay (EMSA) for detected variants of the silencer and promoter regions and reporter assays for a 3'UTR polymorphism. RESULTS: Four putatively functional polymorphisms were detected and further analyzed, one in the silencer region (rs57973275), one in the core promoter region (rs16972787), one in intron 3 (rs40076) and one polymorphism in the 3'untranslated region (3'UTR) of PLK1 (rs27770). Alleles of rs27770 display different secondary mRNA structures and showed a distinct allele-dependent difference in mRNA stability with a significantly higher reporter activity of the A allele (p < 0.01). CONCLUSION: The present study provides evidence that at least one genomic variant of PLK1 has functional properties and influences expression of PLK1. This suggests polymorphisms of the PLK1 gene as an interesting target for further studies that might affect cancer risk, tumor progression as well as the response to PLK1 inhibitors.

Patients' and physicians' awareness of clinical symptoms and disease severity in tuberous sclerosis complex.

Tuberous sclerosis complex (TSC) is a rare inherited disease with the potential to affect virtually every organ system. Clinical presentation is age- and partly sex-dependent and varies broadly with respect to disease manifestations including treatment-refractory epilepsy, intellectual disability and TSC-associated neuropsychiatric disorders, chronic kidney disease or progressive lung function decline. Given the complexity of this disease, multidisciplinary care in specialized TSC centres is recommended. We aimed to elucidate the state of knowledge of patients/caregivers and physicians on individual disease manifestations. We further examined whether the association to a TSC centre has an impact on the comprehensive consideration of potential disease manifestations. Therefore, a survey was performed in a cohort of German TSC patients and their physicians. Complete information was available for 94 patients with a median age of 18 years [range 1-55] and a sex distribution of 53.2% (male): 48.8% (female). Using almost identical questionnaires for patients/caregivers and their respective physician, there was a good correlation for disease assessments associated with relevant morbidity and mortality like epilepsy, renal angiomyolipoma, cardiac rhabdomyomas or intellectual disability. Correlation was moderate for several neuropsychiatric disorders and only poor for hypomelanotic macules, dental pits or retinal achromic patches. Estimation of overall disease severity using a numeric rating scale correlated highly significantly (Pearson correlation coefficient = 0.767; p < 0.001) between patients/caregivers and physicians. In general, physicians more likely quoted items as 'unknown' than patients (822 answers vs. 435 answers in the respective groups). Questionnaires completed by physicians who were associated with a specialized TSC centre declared a significantly lower proportion of items as unknown (mean 8.7% vs. 20.5%; p < 0.001). These findings indicate that patients treated by specialized TSC centres seem to obtain a more comprehensive surveillance. Furthermore, it shows that there were reasonable surveillance strategies in general and sufficient patient/caregiver interaction and education in the examined cohort. However, for the most prominent disease characteristics there was a good awareness within both the patients/caregivers and the physicians group.

The BCL2-938 C > A promoter polymorphism is associated with risk group classification in children with acute lymphoblastic leukemia.

BACKGROUND: Acute lymphoblastic leukemia (ALL) is the most common pediatric cancer. While current treatment regimens achieve almost 80% overall survival, long-term side effects of chemotherapeutic agents can be severe. The functional BCL2-938C > A promoter polymorphism is known to influence the balance between survival and apoptosis of malignant hematolymphoid cells. We investigated its usefulness as a marker for treatment stratification for children with ALL. METHODS: We analyzed DNA from 182 children suffering from ALL in this study to determine genotypes of the -938 C > A polymorphism by "slow-down" PCR. RESULTS: ALL patients with the BCL2-938CC genotype had an approximately 3-fold higher risk of belonging to a high-risk group. Within the high-risk group, 50% of BCL2-938CC patients were classified as high-risk due to poor prednisone response whereas only 33% of patients with AC and AA genotypes were classified as high-risk for the same reason. CONCLUSIONS: Our results suggest that BCL2-938C > A genotyping may be beneficial for therapy response prediction in ALL patients, and warrant examination in a larger cohort to validate its usefulness for treatment stratification of pediatric ALL patients.

Expression of aquaporin 5 and the AQP5 polymorphism A(-1364)C in association with peritumoral brain edema in meningioma patients.

Aquaporins (AQP) are a growing family of water-channel proteins, numbering 13 to date. Recent studies have reported AQP1 and AQP4 to be involved in the development and resorption of brain edemas of different origin. Other AQPs have also been detected in brain tissue, but their impact on brain edema remains to be shown. To evaluate a possible role of AQP5 in brain edema, we investigated the association of AQP5 expression and the functional AQP5 promoter polymorphism A(-1364)C with occurrence and intensity of peritumoral edema in meningioma patients. Peritumoral edema was classified in three degrees based on preoperative imaging in 89 meningioma patients treated at the University Hospital Essen between 2003 and 2006. AQP5 expression was assessed immunohistochemically in tumor tissue obtained during neurosurgical tumor resection. Genotypes of the A(-1364)C polymorphism were determined using the "slowdown" polymerase chain reaction. Higher levels of AQP5 expression were significantly correlated with the AQP5-1364 AA genotype (P = 0.02). AQP5 expression was positively correlated with edema (P = 0.04). AQP5 genotypes were not significantly associated with the occurrence, but with the intensity of peritumoral brain edema (P = 0.04). In our cohort, 40 % of patients with grade I, 66.7 % with grade II, and 76.5 % with grade III edema possessed at least one A allele. Development and intensity of peritumoral edema in meningiomas are associated with AQP5 expression. The intensity of edema correlates with the AQP5 A(-1364)C genotype. This suggests AQP5 as an interesting new candidate involved in peritumoral brain edema in meningioma patients.

Regulation of protein prenylation.

Prenyltransferases (PTases) are known to play a role in embryonic development, normal tissue homeostasis and cancer by posttranslationally modifying proteins involved in these processes. They are being discussed as potential drug targets in an increasing number of diseases, ranging from Alzheimer's disease to malaria. Protein prenylation and the development of specific PTase inhibitors (PTIs) have been subject to intense research in recent decades. Recently, the FDA approved lonafarnib, a specific farnesyltransferase inhibitor that acts directly on protein prenylation; and bempedoic acid, an ATP citrate lyase inhibitor that might alter intracellular isoprenoid composition, the relative concentrations of which can exert a decisive influence on protein prenylation. Both drugs represent the first approved agent in their respective substance class. Furthermore, an overwhelming number of processes and proteins that regulate protein prenylation have been identified over the years, many of which have been proposed as molecular targets for pharmacotherapy in their own right. However, certain aspects of protein prenylation, such as the regulation of PTase gene expression or the modulation of PTase activity by phosphorylation, have attracted less attention, despite their reported influence on tumor cell proliferation. Here, we want to summarize the advances regarding our understanding of the regulation of protein prenylation and the potential implications for drug development. Additionally, we want to suggest new lines of investigation that encompass the search for regulatory elements for PTases, especially at the genetic and epigenetic levels.

Prenyltransferase gene expression reveals an essential role of prenylation for the inflammatory response in human gingival fibroblasts.

BACKGROUND: Prenyltrasferases (PTases) are a class of enzymes known to be responsible for promoting posttranslational modification at the carboxyl terminus of proteins containing a so-called CaaX-motif. The process is responsible for proper membrane localization and the appropriate function of several intracellular signaling proteins. Current research demonstrating the pathomechanistic importance of prenylation in inflammatory illnesses emphasizes the requirement to ascertain the differential expression of PT genes under inflammatory settings, particularly in periodontal disease. METHODS: Telomerase-immortalized human gingival fibroblasts (HGF-hTert) were cultured and treated with either inhibitors of prenylation (PTI) lonafarnib, tipifarnib, zoledronic acid, or atorvastatin at concentrations of 10 µM in combination with or without 10 µg Porphyromonas gingivalis lipopolysaccharide (LPS) for 24 h. Prenyltransferase genes FNTB, FNTA, PGGT1B, RABGGTA, RABGGTB, and PTAR1 as well as inflammatory marker genes MMP1 and IL1B were detected using quantitative real-time polymerase chain reaction (RT-qPCR). Immunoblot and protein immunoassay were used to confirm the results on the protein level. RESULTS: RT-qPCR experiments revealed significant upregulation of IL1B, MMP1, FNTA, and PGGT1B upon LPS treatment. PTase inhibitors caused significant downregulation of the inflammatory cytokine expression. Interestingly, FNTB expression was significantly upregulated in response to any PTase inhibitor in combination with LPS, but not upon LPS treatment only, indicating a vital role of protein farnesyltransferase in the proinflammatory signaling cascade. CONCLUSIONS: In this study, distinct PTase gene expression patterns in pro-inflammatory signaling were discovered. Moreover, PTase inhibiting drugs ameliorated inflammatory mediator expression by a significant margin, indicating that prenylation is a major pre-requisite for innate immunity in periodontal cells.