Alkaline ceramidases (ACERs) are a class of poorly understood transmembrane enzymes controlling the homeostasis of ceramides. They are implicated in human pathophysiology, including progressive ...leukodystrophy, colon cancer as well as acute myeloid leukemia. We report here the crystal structure of the human ACER type 3 (ACER3). Together with computational studies, the structure reveals that ACER3 is an intramembrane enzyme with a seven transmembrane domain architecture and a catalytic Zn
binding site in its core, similar to adiponectin receptors. Interestingly, we uncover a Ca
binding site physically and functionally connected to the Zn
providing a structural explanation for the known regulatory role of Ca
on ACER3 enzymatic activity and for the loss of function in E33G-ACER3 mutant found in leukodystrophic patients.
To assess the safety and local recurrence-free survival in patients after cryoablation for treatment of pulmonary metastases.
This multicenter, prospective, single-arm, phase 2 study included 128 ...patients with 224 lung metastases treated with percutaneous cryoablation, with 12 and 24 months of follow-up. The patients were enrolled on the basis of the outlined key inclusion criteria, which include one to six metastases from extrapulmonary cancers with a maximal diameter of 3.5 cm. Time to progression of the index tumor(s), metastatic disease, and overall survival rates were estimated using the Kaplan–Meier method. Complications were captured for 30 days after the procedure, and changes in performance status and quality of life were also evaluated.
Median size of metastases was 1.0 plus or minus 0.6 cm (0.2–4.5) with a median number of tumors of 1.0 plus or minus 1.2 cm (one to six). Local recurrence-free response (local tumor efficacy) of the treated tumor was 172 of 202 (85.1%) at 12 months and 139 of 180 (77.2%) at 24 months after the initial treatment. After a second cryoablation treatment for recurrent tumor, secondary local recurrence-free response (local tumor efficacy) was 184 of 202 (91.1%) at 12 months and 152 of 180 (84.4%) at 24 months. Kaplan–Meier estimates of 12- and 24-month overall survival rates were 97.6% (95% confidence interval: 92.6–99.2) and 86.6% (95% confidence interval: 78.7–91.7), respectively. Rate of pneumothorax that required pleural catheter placement was 26% (44/169). There were eight grade 3 complication events in 169 procedures (4.7%) and one (0.6%) grade 4 event.
Percutaneous cryoablation is a safe and effective treatment for pulmonary metastases.
7TM proteins are not necessarily GPCRs Vasiliauskaité-Brooks, Ieva; Healey, Robert D.; Granier, Sébastien
Molecular and cellular endocrinology,
07/2019, Letnik:
491
Journal Article
Recenzirano
Odprti dostop
In this review article, we summarize the current knowledge on a large and diverse superfamily of seven-pass transmembrane proteins functionally independent from the GPCR superfamily. We include the ...newest research findings about their physiological roles and their mechanism of action. In particular, we concentrate on the structural basis for the newly discovered amide hydrolase activity, with a focus on adiponectin receptors for which structures are available. Finally, we discuss the remaining challenges in understanding the activation and signaling of these intramembrane proteins and suggest how regulation of the amide hydrolase activity may help in development of new therapeutic agents.
•Structure of adiponectin receptors revealed a seven-transmembrane domain endowed with intramembrane lipid amidase activity.•These data highlighted the existence of a seven-pass transmembrane protein fold which are functionally distinct from the GPCR superfamily.
Sphingolipid metabolism is tightly controlled by enzymes to regulate essential processes in human physiology. The central metabolite is ceramide, a pro‐apoptotic lipid catabolized by ceramidase ...enzymes to produce pro‐proliferative sphingosine‐1‐phosphate. Alkaline ceramidases are transmembrane enzymes that recently attracted attention for drug development in fatty liver diseases. However, due to their hydrophobic nature, no specific small molecule inhibitors have been reported. We present the discovery and mechanism of action of the first drug‐like inhibitors of alkaline ceramidase 3 (ACER3). In particular, we chemically engineered novel fluorescent ceramide substrates enabling screening of large compound libraries and characterized enzyme:inhibitor interactions using mass spectrometry and MD simulations. In addition to revealing a new paradigm for inhibition of lipid metabolising enzymes with non‐lipidic small molecules, our data lay the ground for targeting ACER3 in drug discovery efforts.
Use of synthetic fluorescent ceramide molecules allows the discovery of the first selective drug‐like small molecule inhibitors for alkaline ceramidase 3, an intra‐membrane enzyme involved in sphingolipid metabolism in health and disease. These inhibitors represent a new paradigm for controlling lipid metabolism with drug‐like small molecules targeting conformationally dynamic membrane proteins.
•Co-expression of thaumatin and a molecular chaperone increases secretion 2-fold.•An optimised purification methodology of recombinant thaumatin is reported.•Production of highly purified recombinant ...thaumatin variants is detailed.•Thaumatin variants for thaumatin:receptor interaction studies have been produced.
The sweetest tasting molecule known is the protein thaumatin, first isolated from the katemfe fruit, Thaumatococcus daniellii. Thaumatin is used in the food and beverage industry as a low-calorie sugar substitute. Thaumatin interacts with taste receptors in the oral cavity eliciting a persistent sweet taste and a bitter, liquorice flavor. Recombinant thaumatin was expressed in Pichia pastoris and through a co-expression strategy with a molecular chaperone, yields of one engineered thaumatin variant increased by greater than two-fold. A detailed purification strategy for thaumatin is reported resulting in a homogenous sample recovered at a yield of 42%. The recombinant thaumatins were extensively characterised using size exclusion chromatography for homogeneity, reversed-phase HPLC for purity (99%), peptide digest LC–MS/MS for sequence determination, and circular dichroism and tryptophan fluorescence spectroscopies for conformational characterisation. These new thaumatin variants are amenable for bioconjugation, providing chemical biology tools for thaumatin:taste receptor interaction studies.
The polyhistidine (6XHis) motif is one of the most ubiquitous protein purification tags. The 6XHis motif enables the binding of tagged proteins to various metals, which can be advantageously used for ...purification with immobilized metal affinity chromatography. Despite its popularity, protein structures encompassing metal-bound 6XHis are rare. Here, we obtained a 2.5 Å resolution crystal structure of a single chain Fv antibody (scFv) bearing a C-terminal sortase motif, 6XHis and TwinStrep tags (LPETGHHHHHHWSHPQFEKG
S
WSHPQFEK). The structure, obtained in the presence of cobalt, reveals a unique tetramerization motif (TetrHis) stabilized by 8 Co
ions. The TetrHis motif contains four 6 residues-long β-strands, and each metal center coordinates 3 to 5 residues, including all 6XHis histidines. By combining dynamic light scattering, small angle x-ray scattering and molecular dynamics simulations, We investigated the influence of Co
on the conformational dynamics of scFv 2A2, observing an open/close equilibrium of the monomer and the formation of cobalt-stabilized tetramers. By using a similar scFv design, we demonstrate the transferability of the tetramerization property. This novel metal-dependent tetramerization motif might be used as a fiducial marker for cryoelectron microscopy of scFv complexes, or even provide a starting point for designing metal-loaded biomaterials.
Membrane proteins are central to many pathophysiological processes, yet remain very difficult to analyze structurally. Moreover, high-throughput structure-based drug discovery has not yet been ...exploited for membrane proteins because of lack of automation. Here, we present a facile and versatile platform for in meso membrane protein crystallization, enabling rapid atomic structure determination at both cryogenic and room temperatures. We apply this approach to human integral membrane proteins, which allowed us to identify different conformational states of intramembrane enzyme-product complexes and analyze by molecular dynamics simulations the structural dynamics of the ADIPOR2 integral membrane protein. Finally, we demonstrate an automated pipeline combining high-throughput microcrystal soaking, automated laser-based harvesting, and serial crystallography, enabling screening of small-molecule libraries with membrane protein crystals grown in meso. This approach brings needed automation to this important class of drug targets and enables high-throughput structure-based ligand discovery with membrane proteins.
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•A platform for rapid in meso structures by serial crystallography (SSX)•Insights into ADIPOR2 receptor-ligand dynamic interactions•A web-based application for remote user-guided experimental design and execution•An automated SSX-based ligand discovery pipeline for membrane proteins is introduced
Membrane proteins are key regulators of most physiological processes and represent attractive targets for drug discovery programs. One of the most successful methods to obtain high-resolution structures of membrane proteins relies on in meso crystallization in combination with serial synchrotron crystallography. However, this remains a difficult process, with challenges at every step, including complex manual sample recovery protocols leading to limited throughput and sample loss and the difficulty in carrying out high-throughput ligand screening experiments. We have developed a new approach enabling rapid, automated structural analysis of membrane proteins in meso based on the CrystalDirect technology that addresses these issues, enabling high-throughput drug discovery with membrane proteins.
Membrane proteins control many biological processes and represent attractive targets for drug discovery, but are difficult to study structurally. Healey et al. present an automated approach, combining the CrystalDirect technology and serial crystallography, for rapid structural analysis of membrane proteins and opening new opportunities for high-throughput drug discovery.
Objective
To identify and evaluate the measurement properties of self‐report physical activity instruments suitable for patients with osteoarthritis (OA).
Methods
We conducted a comprehensive 2‐stage ...systematic review using multiple electronic databases, from inception until July 2018. In the stage 1 review, we sought to identify all self‐report physical activity instruments used in individuals with joint pain attributable to OA in the foot, knee, hip, or hand. In the stage 2 review, we searched for and appraised studies investigating the measurement properties of the instruments identified. In both stages of the review, we screened all articles for study eligibility criteria, completed data extraction using the Qualitative Attributes and Measurement Properties of Physical Activity questionnaire checklist, and conducted methodology quality assessments using a modified COSMIN (COnsensus‐based Standards for the selection of health Measurement INstruments) checklist. Measurement properties for each physical activity instrument were evaluated and combined, using narrative synthesis.
Results
In the stage 1 review, we identified 23 unique self‐report physical activity instruments. In the stage 2 review, we identified 54 studies that evaluated the measurement properties of 13 of the 23 instruments identified. Instrument reliability varied from inadequate to adequate (intraclass correlation coefficient ≥0.7). Instrument construct and criterion validity assessment showed small to moderate correlations with direct measures of physical activity. Instrument responsiveness was assessed in only 1 instrument and was unable to detect changes in comparison to accelerometers.
Conclusion
Although many instruments were identified as being potentially suitable for use in patients with OA, none demonstrated adequate measurement properties across all domains of reliability, validity, and responsiveness. Further high‐quality assessment of self‐report physical activity instruments is required before such measures can be recommended for use in OA research.
Climate change is warming the temperatures and lengthening the Arctic growing season with potentially important effects on plant phenology. The ability of plant species to acclimate to changing ...climatic conditions will dictate the level to which their spatial coverage and habitat-type dominance is different in the future. While the effect of changes in temperature on phenology and species composition have been observed at the plot and at the regional scale, a systematic assessment at medium spatial scales using new noninvasive sensor techniques has not been performed yet. At four sites across the North Slope of Alaska, changes in the Normalized Difference Vegetation Index (NDVI) signal were observed by Mobile Instrumented Sensor Platforms (MISP) that are suspended over 50 m transects spanning local moisture gradients. The rates of greening (measured in June) and senescence (measured in August) in response to the air temperature was estimated by changes in NDVI measured as the difference between the NDVI on a specific date and three days later. In June, graminoid- and shrub-dominated habitats showed the greatest rates of NDVI increase in response to the high air temperatures, while forb- and lichen-dominated habitats were less responsive. In August, the NDVI was more responsive to variations in the daily average temperature than spring greening at all sites. For graminoid- and shrub-dominated habitats, we observed a delayed decrease of the NDVI, reflecting a prolonged growing season, in response to high August temperatures. Consequently, the annual C assimilation capacity of these habitats is increased, which in turn may be partially responsible for shrub expansion and further increases in net summer CO2 fixation. Strong interannual differences highlight that long-term and noninvasive measurements of such complex feedback mechanisms in arctic ecosystems are critical to fully articulate the net effects of climate variability and climate change on plant community and ecosystem processes.
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