ABSTRACT
Aspirin as a possible treatment of cancer has been of increasing interest for over 50 years, but the balance of the risks and benefits remains a point of contention. We summarise the valid published evidence 'for' and 'against' the use of aspirin as a cancer treatment and we present what we believe are relevant ethical implications. Reasons for aspirin include the benefits of aspirin taken by patients with cancer upon relevant biological cancer mechanisms. These explain the observed reductions in metastatic cancer and vascular complications in cancer patients. Meta-analyses of 118 observational studies of mortality in cancer patients give evidence consistent with reductions of about 20% in mortality associated with aspirin use. Reasons against aspirin use include increased risk of a gastrointestinal bleed though there appears to be no valid evidence that aspirin is responsible for fatal gastrointestinal bleeding. Few trials have been reported and there are inconsistencies in the results. In conclusion, given the relative safety and the favourable effects of aspirin, its use in cancer seems justified, and ethical implications of this imply that cancer patients should be informed of the present evidence and encouraged to raise the topic with their healthcare team.
Subject(s)
Aspirin , Neoplasms , Humans , Aspirin/adverse effects , Gastrointestinal Hemorrhage/chemically induced , Gastrointestinal Hemorrhage/prevention & controlABSTRACT
BACKGROUND: We investigated the application of years of life lost (YLL) in routine cancer statistics using cancer mortality data from 1988 to 2017. METHODS: Cancer mortality data for 17 cancers and all cancers in the UK from 1988 to 2017 were provided by the UK Association of Cancer Registries by sex, 5-year age group, and year. YLL, age-standardised YLL rate (ASYR) and age-standardised mortality rate (ASMR) were estimated. RESULTS: The annual average YLL due to cancer, in the time periods 1988-1992 and 2013-2017, were about 2.2 and 2.3 million years, corresponding to 4510 and 3823 ASYR per 100,000 years, respectively. During 2013-2017, the largest number of YLL occurred in lung, bowel and breast cancer. YLL by age groups for all cancers showed a peak between 60-64 and 75-79. The relative contributions to incidence, mortality, and YLL differ between cancers. For instance, pancreas (in women and men) made up a smaller proportion of incidence (3%) but bigger proportion of mortality (6 and 5%) and YLL (5 and 6%), whereas prostate cancer (26% of incidence) contributed 13% mortality and 9% YLL. CONCLUSION: YLL is a useful measure of the impact different cancers have on society and puts a higher weight on cancer deaths in younger individuals.
Subject(s)
Breast Neoplasms , Prostatic Neoplasms , Male , Humans , Life Expectancy , United Kingdom/epidemiology , Breast Neoplasms/epidemiology , RegistriesABSTRACT
Background: Type 2 diabetes (T2D) is often regarded as a progressive, lifelong disease requiring an increasing number of drugs. Sustained remission of T2D is now well established, but is not yet routinely practised. Norwood surgery has used a low-carbohydrate programme aiming to achieve remission since 2013. Methods: Advice on a lower carbohydrate diet and weight loss was offered routinely to people with T2D between 2013 and 2021, in a suburban practice with 9800 patients. Conventional 'one-to-one' GP consultations were used, supplemented by group consultations and personal phone calls as necessary. Those interested in participating were computer coded for ongoing audit to compare 'baseline' with 'latest follow-up' for relevant parameters. Results: The cohort who chose the low-carbohydrate approach (n=186) equalled 39% of the practice T2D register. After an average of 33 months median (IQR) weight fell from 97 (84-109) to 86 (76-99) kg, giving a mean (SD) weight loss of -10 (8.9)kg. Median (IQR) HbA1c fell from 63 (54-80) to 46 (42-53) mmol/mol. Remission of diabetes was achieved in 77% with T2D duration less than 1 year, falling to 20% for duration greater than 15 years. Overall, remission was achieved in 51% of the cohort. Mean LDL cholesterol decreased by 0.5 mmol/L, mean triglyceride by 0.9 mmol/L and mean systolic blood pressure by 12 mm Hg. There were major prescribing savings; average Norwood surgery spend was £4.94 per patient per year on drugs for diabetes compared with £11.30 for local practices. In the year ending January 2022, Norwood surgery spent £68 353 per year less than the area average. Conclusions: A practical primary care-based method to achieve remission of T2D is described. A low-carbohydrate diet-based approach was able to achieve major weight loss with substantial health and financial benefit. It resulted in 20% of the entire practice T2D population achieving remission. It appears that T2D duration <1 year represents an important window of opportunity for achieving drug-free remission of diabetes. The approach can also give hope to those with poorly controlled T2D who may not achieve remission, this group had the greatest improvements in diabetic control as represented by HbA1c.
ABSTRACT
Norbin (Neurochondrin, NCDN) is a highly conserved 79â kDa adaptor protein that was first identified more than a quarter of a century ago as a gene up-regulated in rat hippocampus upon induction of long-term potentiation. Most research has focussed on the role of Norbin in the nervous system, where the protein is highly expressed. Norbin regulates neuronal morphology and synaptic plasticity, and is essential for normal brain development and homeostasis. Dysregulation of Norbin is linked to a variety of neurological conditions. Recently, Norbin was shown to be expressed in myeloid cells as well as neurons. Myeloid-cell specific deletion revealed an important role of Norbin as a suppressor of neutrophil-derived innate immunity. Norbin limits the ability of neutrophils to clear bacterial infections by curbing the responsiveness of these cells to inflammatory and infectious stimuli. Mechanistically, Norbin regulates cell responses through binding to its interactors, in particular to a wide range of G protein-coupled receptors (GPCRs). Norbin association with GPCRs controls GPCR trafficking and signalling. Other important Norbin interactors are the Rac guanine-nucleotide exchange factor P-Rex1 and protein kinase A. Downstream signalling pathways regulated by Norbin include ERK, Ca2+ and the small GTPase Rac. Here, we review the current understanding of Norbin structure, expression and its roles in health and disease. We also explore Norbin signalling through its interactors, with a particular focus on GPCR trafficking and signalling. Finally, we discuss avenues that could be pursued in the future to increase our understanding of Norbin biology.
Subject(s)
Neuropeptides , Rats , Animals , Neuropeptides/metabolism , Signal Transduction , Receptors, G-Protein-Coupled/metabolism , Guanine Nucleotide Exchange Factors/metabolism , Adaptor Proteins, Signal Transducing/metabolismABSTRACT
Food addiction, specifically ultra-processed food addiction, has been discussed in thousands of peer-reviewed publications. Although 20% of adults meet criteria for this condition, food addiction is not a recognized clinical diagnosis, leading to a dearth of tested treatment protocols and published outcome data. Growing numbers of clinicians are offering services to individuals on the basis that the food addiction construct has clinical utility. This audit reports on clinical teams across three locations offering a common approach to programs delivered online. Each team focused on a whole food low-carbohydrate approach along with delivering educational materials and psychosocial support relating to food addiction recovery. The programs involved weekly sessions for 10-14 weeks, followed by monthly support. The data comprised pre- and post- program outcomes relating to food addiction symptoms measured by the modified Yale Food Addiction Scale 2.0, ICD-10 symptoms of food related substance use disorder (CRAVED), mental wellbeing as measured by the short version of the Warwick Edinburgh Mental Wellbeing Scale, and body weight. Sample size across programs was 103 participants. Food addiction symptoms were significantly reduced across settings; mYFAS2 score -1.52 (95% CI: -2.22, -0.81), CRAVED score -1.53 (95% CI: -1.93, -1.13) and body weight was reduced -2.34 kg (95% CI: -4.02, -0.66). Mental wellbeing showed significant improvements across all settings; short version Warwick Edinburgh Mental Wellbeing Scale 2.37 (95% CI: 1.55, 3.19). Follow-up data will be published in due course. Further research is needed to evaluate and compare long-term interventions for this complex and increasingly burdensome biopsychosocial condition.
ABSTRACT
Evidence on aspirin and cancer comes from two main sources: (1) the effect of aspirin upon biological mechanisms in cancer, and (2) clinical studies of patients with cancer, some of whom take aspirin. A series of systematic literature searches identified published reports relevant to these two sources. The effects of aspirin upon biological mechanisms involved in cancer initiation and growth appear to generate reasonable expectations of effects upon the progress and mortality of cancer. Clinical evidence on aspirin appears overall to be favourable to the use of aspirin, but evidence from randomized trials is limited, and inconsistent. The main body of evidence comes from meta-analyses of observational studies of patients with a wide range of cancers, about 25% of whom were taking aspirin. Heterogeneity is large but, overall, aspirin is associated with increases in survival and reductions in metastatic spread and vascular complications of different cancers. It is important that evaluations of aspirin used as an adjunct cancer treatment are based upon all the available relevant evidence, and there appears to be a marked harmony between the effects of aspirin upon biological mechanisms and upon the clinical progress of cancer.
Subject(s)
Cardiovascular Diseases , Neoplasms , Aspirin/pharmacology , Aspirin/therapeutic use , Cardiovascular Diseases/drug therapy , Humans , Neoplasms/drug therapyABSTRACT
BACKGROUND: More deprived populations typically experience higher cancer incidence rates and smoking prevalence compared to less deprived populations. We calculated the proportion of cancer cases attributable to smoking by socio-economic deprivation in England and estimated the impact smoking has on the deprivation gap for cancer incidence. METHODS: Data for cancer incidence (2013-2017), smoking prevalence (2003-2007) and population estimates (2013-2017) were split by sex, age-group and deprivation quintile. Relative risk estimates from meta-analyses were used to estimate the population attributable fraction (PAF) for 15 cancer types associated with smoking. The deprivation gap was calculated using age-specific incidence rates by deprivation quintile. RESULTS: Smoking-related cancer PAFs in England are 2.2 times larger in the most deprived quintile compared to the least deprived quintile (from 9.7% to 21.1%). If everyone had the same smoking prevalence as the least deprived quintile, 20% of the deprivation gap in cancer incidence could have been prevented. If nobody smoked, 61% of the deprivation gap could have been prevented. CONCLUSIONS: The majority of the deprivation gap in cancer incidence could have been prevented in England between 2013-2017 if nobody had smoked. Policy makers should ensure that tobacco control policies reduce overall smoking prevalence by tackling smoking inequalities.
Subject(s)
Neoplasms , Poverty , England/epidemiology , Humans , Incidence , Neoplasms/epidemiology , Neoplasms/etiology , Smoking/adverse effects , Smoking/epidemiology , Socioeconomic FactorsABSTRACT
BACKGROUND: Cancer incidence variation between population groups can inform public health and cancer services. Previous studies have shown cancer incidence rates vary by ethnic group in England. Since their publication, the completeness of ethnicity recording in cancer data has improved, and relevant inequalities (e.g. risk factor prevalence and healthcare access) may have changed. METHODS: Age-standardised incidence rates were calculated for Asian, Black, Mixed/Multiple and White ethnic groups in England in 2013-2017, using almost 3 million diagnoses across 31 cancer sites. Rate ratios were calculated with the White ethnic group as reference. Sensitivity analyses used imputed ethnicity for cases with missing data and perturbed population estimates. RESULTS: Incidence rates for most cancer sites and ethnic group and sex combinations were lower in non-White minority ethnic groups compared with the corresponding White group, with particularly low rate ratios (below 0.5) for melanoma skin cancer and some smoking-related cancers (lung, bladder and oesophageal cancers). Exceptions included prostate cancer (2.1 times higher in males of Black ethnicity), myeloma (2.7-3.0 times higher in people of Black ethnicity), several gastrointestinal cancers (1.1-1.9 times higher in people of Black ethnicity, 1.4-2.2 times higher in people of Asian ethnicity), Hodgkin lymphoma (1.1 times higher in males of Asian ethnicity, 1.3 times higher in males of Black ethnicity) and thyroid cancers (1.4 times higher in people of Asian ethnicity, 1.2 times higher in people of Black ethnicity). Sensitivity analyses did not materially alter these results (rate ratios changed by a maximum of 12 percentage points, the direction and significance of results were unchanged in all but two cancer site/sex/ethnic group combinations). CONCLUSIONS: People of non-White minority ethnicity in England generally have lower cancer risk than the White population, though there are a number of notable exceptions. These results should galvanise efforts to better understand the reasons for this variation, and the possible impact on cancer services, patient experiences and outcomes.
Subject(s)
Ethnicity , Gastrointestinal Neoplasms , Humans , Incidence , Male , Minority Groups , Risk FactorsABSTRACT
PURPOSE OF REVIEW: People with T2 Diabetes (T2D) who follow a low carbohydrate diet (LCD) may increase their dietary protein intake. Dietary protein can modulate renal function so there is debate about its role in renal disease. There is concern that higher protein intakes may promote renal damage, and that LCDs themselves may impact on cardiovascular risk. We review the evidence around LCDs, renal and cardiovascular risk factors and compare to results obtained in a real-world, primary care setting. RECENT FINDINGS: Chronic kidney disease (CKD) is a well-recognised microvascular complication of T2D caused in part by; chronically increased glomerular pressure, hyperfiltration, increased blood pressure and advanced glycation end products. Hyperglycemia can be seen as central to all of these factors. A LCD is an effective first step in its correction as we demonstrate in our real-world cohort. SUMMARY: We found evidence that LCDs for people with T2D may improve many renal and cardiovascular risk factors. In our own LCD cohort of 143 patients with normal renal function or only mild CKD, over an average of 30âmonths the serum creatinine improved by a significant mean of 4.7 (14.9) µmol/L. What remains to be shown is the effect of the approach on people with T2D and moderate/severe CKD.
Subject(s)
Diabetes Mellitus, Type 2 , Renal Insufficiency, Chronic , Diabetes Mellitus, Type 2/epidemiology , Diet, Carbohydrate-Restricted , Dietary Proteins , Humans , Kidney/physiology , Primary Health CareABSTRACT
BACKGROUND: Despite the accumulation of research papers on aspirin and cancer, there is doubt as to whether or not aspirin is an acceptable and effective adjunct treatment of cancer. The results of several randomised trials are awaited, and these should give clear evidence on three common cancers: colon, breast and prostate. The biological effects of aspirin appear likely however to be of relevance to cancer generally, and to metastatic spread, rather than just to one or a few cancers, and there is already a lot of evidence, mainly from observational studies, on the association between aspirin and survival in a wide range of cancers. AIMS: In order to test the hypothesis that aspirin taking is associated with an increase in the survival of patients with cancer, we conducted a series of systematic literature searches to identify clinical studies of patients with cancer, some of whom took aspirin after having received a diagnosis of cancer. RESULTS: Three literature searches identified 118 published observational studies in patients with 18 different cancers. Eighty-one studies report on aspirin and cancer mortality and 63 studies report on all-cause mortality. Within a total of about a quarter of a million patients with cancer who reported taking aspirin, representing 20%-25% of the total cohort, we found aspirin to be associated with a reduction of about 20% in cancer deaths (pooled hazard ratio (HR): 0.79; 95% confidence intervals: 0.73, 0.84 in 70 reports and a pooled odds ratio (OR): 0.67; 0.45, 1.00 in 11 reports) with similar reductions in all-cause mortality (HR: 0.80; 0.74, 0.86 in 56 studies and OR: 0.57; 0.36, 0.89 in seven studies). The relative safety of aspirin taking was examined in the studies and the corresponding author of every paper was written to asking for additional information on bleeding. As expected, the frequency of bleeding increased in the patients taking aspirin, but fatal bleeding was rare and no author reported a significant excess in fatal bleeds associated with aspirin. No author mentioned cerebral bleeding in the patients they had followed. CONCLUSIONS: There is a considerable body of evidence suggestive of about a 20% reduction in mortality in patients with cancer who take aspirin, and the benefit appears not to be restricted to one or a few cancers. Aspirin, therefore, appears to deserve serious consideration as an adjuvant treatment of cancer, and patients with cancer, and their carers, have a right to be informed of the available evidence.
ABSTRACT
BACKGROUND: Obesity is a risk factor for complications from SARS-CoV-2 infection, increasing the need for effective weight management measures in primary care. However, in the UK, COVID-19 restrictions have hampered primary care weight management referral and delivery, and COVID-19 related weight gain has been reported. The present study evaluated outcomes from a multicomponent weight loss and health promotion programme in UK primary care, delivered remotely due to COVID-19 restrictions. METHOD: Patients with obesity, type 2 diabetes or pre-diabetes attended six 90 min sessions over 10 weeks on Zoom. The dietary component comprised a low-carbohydrate 'real food' approach, augmented with education on physical activity, intermittent fasting, gut health, stress management, sleep and behaviour change. Anthropometric and cardiometabolic data were self-reported. Mental well-being was assessed with the Warwick Edinburgh Mental Wellbeing Scale. Subjective outcomes and participant feedback about the programme were collected with an anonymous online survey. RESULTS: Twenty participants completed the programme. Weight loss and improvements in body mass index, waist circumference, systolic and diastolic blood pressure and mental well-being achieved statistical and clinical significance. Mean weight loss (5.8 kg) represented a 6.5% weight loss. Participants' subjective outcomes included weight loss without hunger (67%) and increased confidence in their ability to improve health (83%). All participants reported the usage of Zoom to access the programme as acceptable with 83% reporting it worked well. CONCLUSION: A multicomponent weight loss and health promotion programme with a low-carbohydrate dietary component, clinically and statistically significantly improved health outcomes including weight status, blood pressure and mental well-being in a group of primary care patients when delivered remotely. Further research is warranted.
ABSTRACT
BACKGROUND: In a single general practice (GP) surgery in England, there was an eightfold increase in the prevalence of type 2 diabetes (T2D) in three decades with 57 cases and 472 cases recorded in 1987 and 2018, respectively. This mirrors the growing burden of T2D on the health of populations round the world along with healthcare funding and provision more broadly. Emerging evidence suggests beneficial effects of carbohydrate-restricted diets on glycaemic control in T2D, but its impact in a 'real-world' primary care setting has not been fully evaluated. METHODS: Advice on a lower carbohydrate diet was offered routinely to patients with newly diagnosed and pre-existing T2D or prediabetes between 2013 and 2019, in the Norwood GP practice with 9800 patients. Conventional 'one-to-one' GP consultations were used, supplemented by group consultations, to help patients better understand the glycaemic consequences of their dietary choices with a particular focus on sugar, carbohydrates and foods with a higher Glycaemic Index. Those interested were computer coded for ongoing audit to compare 'baseline' with 'latest follow-up' for relevant parameters. RESULTS: By 2019, 128 (27%) of the practice population with T2D and 71 people with prediabetes had opted to follow a lower carbohydrate diet for a mean duration of 23 months. For patients with T2D, the median (IQR) weight dropped from of 99.7 (86.2, 109.3) kg to 91.4 (79, 101.1) kg, p<0.001, while the median (IQR) HbA1c dropped from 65.5 (55, 82) mmol/mol to 48 (43, 55) mmol/mol, p<0.001. For patients with prediabetes, the median (IQR) HbA1c dropped from 44 (43, 45) mmol/mol to 39 (38, 41) mmol/mol, p<0.001. Drug-free T2D remission occurred in 46% of participants. In patients with prediabetes, 93% attained a normal HbA1c. Since 2015, there has been a relative reduction in practice prescribing of drugs for diabetes leading to a T2D prescribing budget £50 885 per year less than average for the area. CONCLUSIONS: This approach to lower carbohydrate dietary advice for patients with T2D and prediabetes was incorporated successfully into routine primary care over 6 years. There were statistically significant improvements in both groups for weight, HbA1c, lipid profiles and blood pressure as well as significant drug budget savings. These results suggest a need for more empirical research on the effects of lower carbohydrate diet and long-term glycaemic control while recording collateral impacts to other metabolic health outcomes.
ABSTRACT
Hypertension is the second biggest known global risk factor for disease after poor diet; perhaps lifestyle interventions are underutilized? In a previous small pilot study, it was found that a low carbohydrate diet was associated with significant improvements in blood pressure, weight, 'deprescribing' of medications and lipid profiles. We were interested to investigate if these results would be replicated in a larger study based in 'real world' GP practice. 154 patients with type 2 diabetes or impaired glucose tolerance were recruited into an observational cohort study in primary care. The effects of a low carbohydrate diet sustained for an average of two years (interquartile range 10-32 months) on cardiovascular risk factors were examined. Results demonstrate significant and substantial reductions in blood pressure (mean reduction of systolic BP 10.9 mmHg (interquartile range 0-22 mmHg) (p < 0.0001), mean reduction in diastolic BP 6.3 mmHg (interquartile range 0-12.8 mmHg) (p < 0.0001) and mean weight reduction of 9.5 Kg (interquartile range 5-13 Kg) (p < 0.0001) together with marked improvement in lipid profiles. This occurred despite a 20% reduction in anti-hypertensive medications. This novel and potentially highly effective dietary modification, done very cheaply alongside routine care, offers hope that should be tested in a large prospective trial.
Subject(s)
Diabetes Mellitus, Type 2/diet therapy , Diet, Carbohydrate-Restricted , Hypertension/diet therapy , Primary Health Care , Biomarkers/blood , Diabetes Mellitus, Type 2/blood , Diabetes Mellitus, Type 2/complications , Female , General Practice , Humans , Hypertension/blood , Hypertension/complications , Insulin Resistance , Lipids/blood , Male , Middle Aged , Retrospective Studies , Treatment Outcome , Weight LossABSTRACT
Protein prenylation is a widespread post-translational modification in eukaryotes that plays a crucial role in membrane targeting and signal transduction. RabGTPases is the largest group of post-translationally C-terminally geranylgeranylated. All Rabs are processed by Rab geranylgeranyl-transferase and Rab escort protein (REP). Human genetic defects resulting in the loss one of two REP isoforms REP-1, lead to underprenylation of RabGTPases that manifests in retinal degradation and blindness known as choroideremia. In this study we used a combination of microinjections and chemo-enzymatic tagging to establish whether Rab GTPases are prenylated and delivered to their target cellular membranes with the same rate. We demonstrate that although all tested Rab GTPases display the same rate of membrane delivery, the extent of Rab prenylation in 5 hour time window vary by more than an order of magnitude. We found that Rab27a, Rab27b, Rab38 and Rab42 display the slowest prenylation in vivo and in the cell. Our work points to possible contribution of Rab38 to the emergence of choroideremia in addition to Rab27a and Rab27b.
Subject(s)
Choroideremia/metabolism , rab GTP-Binding Proteins/metabolism , Choroideremia/genetics , Escherichia coli , Humans , Prenylation , Time FactorsABSTRACT
Rab geranylgeranyl transferase (RabGGTase) catalyzes the attachment of geranylgeranyl isoprenoids to Rab guanine triphosphatases, which are key regulators in vesicular transport. Because geranylgeranylation is required for proper function and overexpression of Rabs has been observed in various cancers, RabGGTase may be a target for novel therapeutics. The development of selective inhibitors is, however, difficult because two related enzymes involved in other cellular processes exist in eukaryotes and because RabGGTase recognizes protein substrates indirectly, resulting in relaxed specificity. We report the synthesis of a peptidic library based on the farnesyl transferase inhibitor pepticinnamin E. Of 469 compounds investigated, several were identified as selective for RabGGTase with low micromolar IC(50) values. The compounds were not generally cytotoxic and inhibited Rab isoprenylation in COS-7 cells. Crystal structure analysis revealed that selective inhibitors interact with a tunnel unique to RabGGTase, implying that this structural motif is an attractive target for improved RabGGTase inhibitors.
Subject(s)
Alkyl and Aryl Transferases/antagonists & inhibitors , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Oligopeptides/chemistry , Oligopeptides/pharmacology , Alkyl and Aryl Transferases/chemistry , Animals , COS Cells , Catalytic Domain , Chlorocebus aethiops , Enzyme Inhibitors/chemical synthesis , Kinetics , Models, Molecular , Oligopeptides/chemical synthesis , Peptide Library , Structure-Activity Relationship , Substrate SpecificityABSTRACT
Protein prenylation is a widespread phenomenon in eukaryotic cells that affects many important signaling molecules. We describe the structure-guided design of engineered protein prenyltransferases and their universal synthetic substrate, biotin-geranylpyrophosphate. These new tools allowed us to detect femtomolar amounts of prenylatable proteins in cells and organs and to identify their cognate protein prenyltransferases. Using this approach, we analyzed the in vivo effects of protein prenyltransferase inhibitors. Whereas some of the inhibitors displayed the expected activities, others lacked in vivo activity or targeted a broader spectrum of prenyltransferases than previously believed. To quantitate the in vivo effect of the prenylation inhibitors, we profiled biotin-geranyl-tagged RabGTPases across the proteome by mass spectrometry. We also demonstrate that sites of active vesicular transport carry most of the RabGTPases. This approach enables a quantitative proteome-wide analysis of the regulation of protein prenylation and its modulation by therapeutic agents.
Subject(s)
Biotin/analogs & derivatives , Isotope Labeling , Polyisoprenyl Phosphates/chemistry , Protein Prenylation/physiology , Terpenes/chemistry , Animals , Biotin/chemistry , Biotin/metabolism , COS Cells , Catalytic Domain , Chlorocebus aethiops , Models, Molecular , Molecular Structure , Polyisoprenyl Phosphates/metabolism , Protein Binding , Protein Conformation , Protein Engineering/methods , Signal Transduction , Terpenes/metabolismABSTRACT
Post-translational isoprenylation of proteins is carried out by three related enzymes: farnesyltransferase, geranylgeranyl transferase-I, and Rab geranylgeranyl transferase (RabGGTase). Despite the fact that the last one is responsible for the largest number of individual protein prenylation events in the cell, no structural information is available on its interaction with substrates and products. Here, we present structural and biophysical analyses of RabGGTase in complex with phosphoisoprenoids as well as with the prenylated peptides that mimic the C terminus of Rab7 GTPase. The data demonstrate that, unlike other protein prenyl transferases, both RabGGTase and its substrate RabGTPases completely 'outsource' their specificity for each other to an accessory subunit, the Rab escort protein (REP). REP mediates the placement of the C terminus of RabGTPase into the active site of RabGGTase through a series protein-protein interactions of decreasing strength and selectivity. This arrangement enables RabGGTase to prenylate any cysteine-containing sequence. On the basis of our structural and thermodynamic data, we propose that RabGGTase has evolved from a GGTase-I-like molecule that 'learned' to interact with a recycling factor (GDI) that, in turn, eventually gave rise to REP.
Subject(s)
Alkyl and Aryl Transferases/chemistry , Alkyl and Aryl Transferases/physiology , Alkyl and Aryl Transferases/metabolism , Amino Acid Sequence , Animals , Cysteine/chemistry , Models, Biological , Models, Molecular , Molecular Sequence Data , Prenylation , Protein Binding , Protein Conformation , Protein Interaction Mapping , Protein Processing, Post-Translational , Protein Structure, Tertiary , Rats , ThermodynamicsSubject(s)
Alkyl and Aryl Transferases/antagonists & inhibitors , Alkyl and Aryl Transferases/chemistry , Drug Design , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Alkyl and Aryl Transferases/genetics , Animals , COS Cells , Chlorocebus aethiops , Crystallography, X-Ray , Humans , Protein ConformationSubject(s)
Phosphatidic Acids/metabolism , Phosphotransferases (Alcohol Group Acceptor)/metabolism , Amino Acid Sequence , Animals , Binding Sites , COS Cells , Chlorocebus aethiops , Feedback, Physiological , Models, Molecular , Molecular Sequence Data , Phosphotransferases (Alcohol Group Acceptor)/genetics , Point MutationABSTRACT
Posttranslational modification of proteins with farnesyl and geranylgeranyl isoprenoids is a widespread phenomenon in eukaryotic organisms. Isoprenylation is conferred by three protein prenyltransferases: farnesyl transferase (FTase), geranylgeranyl transferase type-I (GGTase-I), and Rab geranylgeranyltransferase (RabGGTase). Inhibitors of these enzymes have emerged as promising therapeutic compounds for treatment of cancer, viral and parasite originated diseases, as well as osteoporosis. However, no generic nonradioactive protein prenyltransferase assay has been reported to date, complicating identification of enzyme-specific inhibitors. We have addressed this issue by developing two fluorescent analogues of farnesyl and geranylgeranyl pyrophosphates {3,7-dimethyl-8-(7-nitro-benzo[1,2,5]oxadiazol-4-ylamino)-octa-2,6-diene-1}pyrophosphate (NBD-GPP) and {3,7,11-trimethyl-12-(7-nitro-benzo[1,2,5]oxadiazo-4-ylamino)-dodeca-2,6,10-trien-1} pyrophosphate (NBD-FPP), respectively. We demonstrate that these compounds can serve as efficient lipid donors for prenyltransferases. Using these fluorescent lipids, we have developed two simple (SDS-PAGE and bead-based) in vitro prenylation assays applicable to all prenyltransferases. Using the SDS-PAGE assay, we found that, in contrast to previous reports, the tyrosine phosphatase PRL-3 may possibly be a dual substrate for both FTase and GGTase-I. The on-bead prenylation assay was used to identify prenyltransferase inhibitors that displayed nanomolar affinity for RabGGTase and FTase. Detailed analysis of the two inhibitors revealed a complex inhibition mechanism in which their association with the peptide binding site of the enzyme reduces the enzyme's affinity for lipid and peptide substrates without competing directly with their binding. Finally, we demonstrate that the developed fluorescent isoprenoids can directly and efficiently penetrate into mammalian cells and be incorporated in vivo into small GTPases.