205373753
SNAP-25 substrate peptide (residues 180-183) binds to but bypasses cleavage by catalytically active Clostridium botulinum neurotoxin E.
Clostridium botulinum neurotoxins are the most potent toxins to humans. The recognition and cleavage of SNAREs are prime evente in exhibiting their toxicity. We report here the crystal structure of the catalytically active full-length botulinum serotype E catalytic domain (BoNT E) in complex with SNAP-25 (a SNARE protein) substrate peptide Arg(180)-Ile(181)-Met(182)-Glu(183) (P1-P3'). It is remarkable that the peptide spanning the scissile bond binds to but bypasses cleavage by the enzyme and inhibits the catalysis fairly with K(i) approximately 69 microm. The inhibitory peptide occupies the active site of BoNT E and shows well defined electron density. The catalytic zinc and the conserved key residue Tyr(350) of the enzyme facilitate the docking of Arg(180) (P1) by interacting with its carbonyl oxygen that displaces the nucleophilic water. The general base Glu(212) side chain interacts with the main chain amino group of P1 and P1'. Conserved Arg(347) of BoNT E stabilizes the proper docking of the Ile(181) (P1') main chain, whereas the hydrophobic pockets stabilize the side chains of Ile(181) (P1') and Met(182) (P2'), and the 250 loop stabilizes Glu(183) (P3'). Structural and functional analysis revealed an important role for the P1' residue and S1' pocket in driving substrate recognition and docking at the active site. This study is the first of its kind and rationalizes the substrate cleavage strategy of BoNT E. Also, our complex structure opens up an excellent opportunity of structure-based drug design for this fast acting and extremely toxic high priority BoNT E.
12841382
Mode of VAMP substrate recognition and inhibition of Clostridium botulinum neurotoxin F
Clostridium botulinum neurotoxins (BoNTs) cleave neuronal proteins responsible for neurotransmitter release, causing the neuroparalytic disease botulism. BoNT serotypes B, D, F and G cleave and inactivate vesicle-associated membrane protein (VAMP), each at a unique peptide bond. The specificity of BoNTs depends on the mode of substrate recognition. We have investigated the mechanism of substrate recognition of BoNT F by determining the crystal structures of its complex with two substrate-based inhibitors, VAMP 22-58/Gln58D-cysteine and 27-58/Gln58D-cysteine. The inhibitors bind to BoNT F in the canonical direction (as seen for BoNTs A and E substrates) but are positioned specifically via three major exosites away from the active site. The cysteine sulfur of the inhibitors interacts with the zinc and exists as sulfinic acid in the inhibitor VAMP 27-58/Gln58D-cysteine. Arg133 and Arg171, which form part of two separate exosites, are crucial for substrate binding and catalysis.
19667728
Protection against malignant conversion in SENCAR mouse skin by all trans retinoic acid: inhibition of the ras p21-processing enzyme farnesyltransferase and Ha-ras p21 membrane localization.
Many studies have shown that all trans retinoic acid (RA) exhibits significant protective effects against mouse skin tumor promotion and spontaneous as well as enhanced malignant conversion. In a recently completed study, we showed that under treatments in which papillomas on SENCAR mouse skin are induced at low and high probabilities to convert to malignant carcinomas, RA affords significant protection against both tumor promotion and subsequent malignant conversion. More than 95% of these mouse skin papillomas and carcinomas have been shown to contain point mutation at the 61 codon of Ha-ras oncogene. The ras oncogene encodes a p21 protein that, in its mutated form, transforms mammalian cells only when p21 is at the inner surface of the plasma membrane, by a series of enzymatic reactions in which the initial step is catalyzed by farnesyltransferase (FTase). In this study, we assessed whether the protective effect of RA against malignant conversion involves the inhibition of ras p21 processing in those tumors that contain the activated ras oncogene. The FTase activity and the levels of cytosolic and membrane-bound Ha-ras p21 were determined in all papillomas and carcinomas obtained from acetone- or RA-treated animals. No matter how the data were analyzed and what comparisons were considered, in all the protocols used, compared with controls, papillomas and carcinomas obtained from RA-treated groups showed significantly decreased (P < 0.01-0.001) FTase activity. Furthermore, the tissue samples from RA-treated groups in different protocols also showed significantly diminished membrane localization of Ha-ras p21, with a concomitant increase in cytosolic Ha-ras p21 levels. The analysis of these data also showed that in all the protocols used, the increased FTase activity and membrane localization of Ha-ras p21 were associated with the induction of papillomas and their subsequent malignant conversion to squamous cell carcinomas. Taken together, these results indicate a strong correlation between the inhibition of ras p21 farnesylation because of a decrease in FTase activity by RA and its protective effect against malignant conversion of papillomas to carcinomas. Based on the results of this study, it is tempting to suggest that clinical trials evaluating the preventive or therapeutic potential of retinoids may be directed more toward those clinical malignancies that are known to contain the activated ras oncogene.
46117096
ras protein p21 processing enzyme farnesyltransferase in chemical carcinogen-induced murine skin tumors.
Farnesylation of ras protein p21 is crucial for the protein's membrane localization, which is essential for its cell-transforming activity, which in turn is thought to be critical for the ultimate induction of cancer. The cytosolic enzyme farnesyltransferase plays a major role in posttranslational modification of p21, but the level of farnesyltransferase activity in mammalian tumors and its relationship to the processing of cytosolic p21 that leads to tumorigenesis are unknown. We report here that farnesyltransferase activity was significantly higher in chemical carcinogen-induced benign skin papillomas in SENCAR mice than in the epidermises of control animals. The enzyme is primarily epidermal in origin, and kinetic studies with cytosol from epidermis and papillomas showed that the reaction was linear with respect to time, substrate concentration, and protein content. Skin papillomas showed significantly elevated levels of both cytosolic and membrane-bound Ha-ras p21, whereas far lesser cytosolic and almost negligible amounts of membrane-bound p21 were present in the epidermis of control mice. There was a positive correlation between increased enzyme activity in papilloma cytosol and the processing of overexpressed cytosolic Ha-ras p21 for its localization to membrane.
24288835
Toward a definition of masked hypertension and white-coat hypertension among hemodialysis patients.
BACKGROUND AND OBJECTIVES
Among people with essential hypertension, ambulatory BP measurement is superior to BP obtained in the clinic in predicting cardiovascular outcomes. In part, this is because it can detect white-coat hypertension and masked hypertension. Whether the same is true for hemodialysis patients is not known.


DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS
Using a threshold of 140/80 mmHg for median midweek dialysis-unit BP and 135/85 mmHg for 44-hour ambulatory BP, we defined four categories of BP: sustained normotension (SN), white-coat hypertension (WCH), masked hypertension (MHTN), and sustained hypertension (SHTN).


RESULTS
Among 355 long-term hemodialysis patients, the prevalence of SN was 35%, WCH 15%, MHTN 15%, and SHTN 35%. Over a mean follow-up of 29.6 (SD 21.7) months, 102 patients died (29%), yielding a crude mortality rate of 121/1000 patient-years. Unadjusted and multivariate-adjusted analyses showed increasing all-cause mortality with increasing severity of hypertension (unadjusted hazard ratios from SN, WCH, MHTN, SHTN: 1, 1.12, 1.70, 1.80, respectively [P for trend < 0.01]; adjusted hazard ratios: 1, 1.30, 1.36, 1.87, respectively [P for trend 0.02]). When a predialysis BP threshold of 140/90 mmHg was used to classify patients into BP categories, the prevalence of SN was 24%, WCH 26%, MHTN 4%, and SHTN 47%. Hazard ratios for mortality were similar when compared with median midweek dialysis-unit BP.


CONCLUSIONS
As in the essential hypertension population, MHTN and WCH have prognostic significance. The prognostic value of BP obtained in the dialysis unit can be refined with ambulatory BP monitoring.
1183339
Smoking, oxidative stress and inflammation: Impact on resting energy expenditure in diabetic nephropathy
BackgroundInflammation is associated with increased resting energy expenditure (REE) in patients with chronic kidney disease. Oxidative stress, on the other hand, appears not to increase REE. Smoking is a common mechanism for generating oxidative stress and inflammation. Whether smokers have increased REE and if so, whether it is accounted for by the pro-oxidant and inflammatory state is not known.MethodsA case control study of 11 smokers and 24 non-smokers with overt diabetic nephropathy was performed to evaluate the chronic effect of smoking on REE. REE (indirect calorimetry), glomerular filtration rate (iothalamate clearance), markers of oxidative stress (urinary and plasma malondialdehyde (MDA), and protein carbonyls) and inflammation (C-reactive protein, tumor necrosis factor-alpha, interleukin-6) were measured on two occasions four months apart.ResultsBiomarkers of inflammation (C-reactive protein) and oxidative stress (urinary and plasma MDA) were increased in smokers. REE was increased in smokers, 24.3 kcal/kg/day compared to 21 kcal/kg/day (p = 0.009) in non-smokers. After adjusting for age, GFR, MDA, C-reactive protein, and hemoglobin A1C the difference in REE between the two groups persisted (adjusted difference 3.51 kcal/kg/d, 95% confidence interval 0.59 – 6.45, p = 0.020).ConclusionPatients with overt diabetic nephropathy who smoke have a higher REE, oxidative and inflammatory state. Elevated REE is not attributable to heightened oxidative stress and inflammatory state. Smoking is an independent risk factor for elevated REE in patients with diabetic nephropathy and provides an additional mechanism by which it may lead to poor outcomes.
42453126
Allergic bronchopulmonary aspergillosis complicating chronic obstructive pulmonary disease.
Allergic bronchopulmonary aspergillosis (ABPA) is a well recognised entity which complicates the course of 1-2% of patients with asthma and 2-15% of cystic fibrosis. Also, current criteria generally require the presence of asthma or cystic fibrosis for recognition of ABPA. The occurrence of ABPA in conditions other than cystic fibrosis or bronchial asthma is rare. Patients with chronic obstructive pulmonary disease (COPD) have mucus hypersecretion and impaired mucociliary clearance but are not reported to develop ABPA. Herein, we report a 50-year-old male who presented with an exacerbation of COPD due to co-existent ABPA. To our knowledge, this is the first report of such an association. We also speculate on mechanisms of causality and impact of ABPA on disease progression in COPD.