scholarly journals Lipid Profiling of Mouse Intestinal Organoids for studying APC Mutations

2021 ◽  
Author(s):  
Zoë Jukes ◽  
Anne Freier ◽  
Maria Glymenaki ◽  
Richard Brown ◽  
Lee Parry ◽  
...  
Keyword(s):  
Lipid Metabolism ◽  
High Performance ◽  
Three Dimensional ◽  
Reversed Phase ◽  
Apc Gene ◽  
Dimensional Structure ◽  
Lipid Profiling ◽  
Intestinal Organoids ◽  
Apc Mutations

Inactivating mutations including both germline and somatic mutations in the adenomatous polyposis coli (APC) gene drives most familial and sporadic colorectal cancers. Understanding the metabolic implications of this mutation will aid to establish its wider impact on cellular behaviour and potentially inform clinical decisions. However, to date, alterations in lipid metabolism induced by APC mutations remain unclear. Intestinal organoids have gained widespread popularity in studying colorectal cancer and chemotherapies, because their three-dimensional structure more accurately mimics an in vivo environment. Here, we aimed to investigate intra-cellular lipid disturbances induced by APC gene mutations in intestinal organoids using a reversed-phase ultra-high-performance liquid chromatography mass spectrometry (RP-UHPLC-MS)-based lipid profiling method. Lipids of the organoids grown from either wildtype (WT) or mice with Apc mutations (Lgr5–EGFP-IRES-CreERT2Apcfl/fl) were extracted and analysed using RP-UHPLC-MS. Concentrations of phospholipids (e.g. PC(16:0/16:0), PC(18:1/20:0), PC(38:0), PC(18:1/22:1)), ceramides (e.g. Cer(d18:0/22:0), Cer(d42:0), Cer(d18:1/24:1)) and hexosylceramide (e.g. HexCer(d18:1/16:0), HexCer(d18:1/22:0))   were higher in Apcfl/fl organoids, whereas levels of sphingomyelins (e.g. SM(d18:1/14:0), SM(d18:1/16:0) ) were lower compared to WT. These observations indicate that cellular metabolism of sphingomyelin was upregulated, resulting in the cellular accumulation of ceramides and production of HexCer due to the absence of Apcfl/fl in the organoids. Our observations demonstrated lipid profiling of organoids and provided an enhanced insight into the effects of the APC mutations on lipid metabolism, making for a valuable addition to screening options of the organoid lipidome.

Tomographic and Topographic Investigation of Poly-D,L-Lactide-Co-Glycolide Microspheres Loaded with Prostaglandine E2 for Extended Drug Release Applications

2010 ◽  
Vol 89-91 ◽  
pp. 687-691 ◽  
Author(s):  
Rolf Zehbe ◽  
Bernhard Watzer ◽  
Rainer Grupp ◽  
Sven Halstenberg ◽  
Heinrich Riesemeier ◽  
...  
Keyword(s):  
High Performance ◽  
Three Dimensional ◽  
Active Agent ◽  
Dimensional Structure ◽  
Binary Solvent ◽  
Bioactive Substances ◽  
Water Emulsion ◽  
Wide Range

Polymeric, biodegradable microspheres represent a good reliable system to investigate the release of bioactive substances in both in vitro and in vivo applications. Common biomaterials for the synthesis of these microspheres are aliphatic polyesters of the poly(α-hydroxy)acids, especially poly-L-lactides (PLA) and polyglycolides (PGA) or their copolymers poly-D,L-lactide-co-glycolides (PLGA). In our own previous studies we have developed PLGA microspheres with integrated PGE2 as model substance for a wide range of biomedical applications, especially in angiogenesis, fracture healing and cartilage repair. The synthesis is based on a binary solvent in water emulsion approach, where two different solvents are used to dissolve the active agent and the polymer, while being miscible in each other (CHCl3, ethyl acetate). Both, the degradation of the material and the release profiles were investigated using SEM and mass spectrometry coupled with gas- or high performance liquid chromatography. SEM and AFM measurements indicated a porous structure of the microspheres but could not resolve the true three dimensional structure of the microspheres. Therefore, synchrotron radiation-based µCT (SR-µCT) investigations were performed to link the release profile to the structural design of the microspheres. As a result, we were able to cross validate the experimental data from SEM and AFM with SR-µCT, demonstrating both micro-porosity and nano-porosity. The polymer itself appears to consist of 200 nm – 300 nm sized particles.

scholarly journals Structure of the Lectin Mannose 6-Phosphate Receptor Homology (MRH) Domain of Glucosidase II, an Enzyme That Regulates Glycoprotein Folding Quality Control in the Endoplasmic Reticulum

2013 ◽  
Vol 288 (23) ◽  
pp. 16460-16475 ◽  
Author(s):  
Linda J. Olson ◽  
Ramiro Orsi ◽  
Solana G. Alculumbre ◽  
Francis C. Peterson ◽  
Ivan D. Stigliano ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Three Dimensional ◽  
Binding Pocket ◽  
Dimensional Structure ◽  
Mannose Residue ◽  
Glucosidase Ii ◽  
Mannose 6 Phosphate ◽  
First Time ◽  
Conserved Residue

Here we report for the first time the three-dimensional structure of a mannose 6-phosphate receptor homology (MRH) domain present in a protein with enzymatic activity, glucosidase II (GII). GII is involved in glycoprotein folding in the endoplasmic reticulum. GII removes the two innermost glucose residues from the Glc3Man9GlcNAc2 transferred to nascent proteins and the glucose added by UDP-Glc:glycoprotein glucosyltransferase. GII is composed of a catalytic GIIα subunit and a regulatory GIIβ subunit. GIIβ participates in the endoplasmic reticulum localization of GIIα and mediates in vivo enhancement of N-glycan trimming by GII through its C-terminal MRH domain. We determined the structure of a functional GIIβ MRH domain by NMR spectroscopy. It adopts a β-barrel fold similar to that of other MRH domains, but its binding pocket is the most shallow known to date as it accommodates a single mannose residue. In addition, we identified a conserved residue outside the binding pocket (Trp-409) present in GIIβ but not in other MRHs that influences GII glucose trimming activity.

scholarly journals Protein Folding: Search for Basic Physical Models

2003 ◽  
Vol 3 ◽  
pp. 623-635 ◽  
Author(s):  
Ivan Y. Torshin ◽  
Robert W. Harrison
Keyword(s):  
Protein Folding ◽  
Tertiary Structure ◽  
Three Dimensional ◽  
Physical Models ◽  
Dimensional Structure ◽  
Computational Techniques ◽  
Linear Sequence ◽  
Physical Forces

How a unique three-dimensional structure is rapidly formed from the linear sequence of a polypeptide is one of the important questions in contemporary science. Apart from biological context ofin vivoprotein folding (which has been studied only for a few proteins), the roles of the fundamental physical forces in thein vitrofolding remain largely unstudied. Despite a degree of success in using descriptions based on statistical and/or thermodynamic approaches, few of the current models explicitly include more basic physical forces (such as electrostatics and Van Der Waals forces). Moreover, the present-day models rarely take into account that the protein folding is, essentially, a rapid process that produces a highly specific architecture. This review considers several physical models that may provide more direct links between sequence and tertiary structure in terms of the physical forces. In particular, elaboration of such simple models is likely to produce extremely effective computational techniques with value for modern genomics.

scholarly journals Computational Protein Stabilization Can Affect Folding Energy Landscapes and Lead to Domain-Swapped Dimers

2021 ◽  
Author(s):  
Klara Markova ◽  
Antonin Kunka ◽  
Klaudia Chmelova ◽  
Martin Havlasek ◽  
Petra Babkova ◽  
...  
Keyword(s):  
Protein Folding ◽  
Protein Design ◽  
Energy Landscape ◽  
Single Point ◽  
Three Dimensional ◽  
Protein Stabilization ◽  
Dimensional Structure ◽  
Evolutionary Analysis ◽  
Folding Energy

<p>The functionality of a protein depends on its unique three-dimensional structure, which is a result of the folding process when the nascent polypeptide follows a funnel-like energy landscape to reach a global energy minimum. Computer-encoded algorithms are increasingly employed to stabilize native proteins for use in research and biotechnology applications. Here, we reveal a unique example where the computational stabilization of a monomeric α/β-hydrolase enzyme (<i>T</i><sub>m</sub> = 73.5°C; Δ<i>T</i><sub>m</sub> > 23°C) affected the protein folding energy landscape. Introduction of eleven single-point stabilizing mutations based on force field calculations and evolutionary analysis yielded catalytically active domain-swapped intermediates trapped in local energy minima. Crystallographic structures revealed that these stabilizing mutations target cryptic hinge regions and newly introduced secondary interfaces, where they make extensive non-covalent interactions between the intertwined misfolded protomers. The existence of domain-swapped dimers in a solution is further confirmed experimentally by data obtained from SAXS and crosslinking mass spectrometry. Unfolding experiments showed that the domain-swapped dimers can be irreversibly converted into native-like monomers, suggesting that the domain-swapping occurs exclusively <i>in vivo</i>. Our findings uncovered hidden protein-folding consequences of computational protein design, which need to be taken into account when applying a rational stabilization to proteins of biological and pharmaceutical interest.</p>

scholarly journals Computational Protein Stabilization Can Affect Folding Energy Landscapes and Lead to Domain-Swapped Dimers

2021 ◽  
Author(s):  
Klara Markova ◽  
Antonin Kunka ◽  
Klaudia Chmelova ◽  
Martin Havlasek ◽  
Petra Babkova ◽  
...  
Keyword(s):  
Protein Folding ◽  
Protein Design ◽  
Energy Landscape ◽  
Single Point ◽  
Three Dimensional ◽  
Protein Stabilization ◽  
Dimensional Structure ◽  
Evolutionary Analysis ◽  
Folding Energy

<p>The functionality of a protein depends on its unique three-dimensional structure, which is a result of the folding process when the nascent polypeptide follows a funnel-like energy landscape to reach a global energy minimum. Computer-encoded algorithms are increasingly employed to stabilize native proteins for use in research and biotechnology applications. Here, we reveal a unique example where the computational stabilization of a monomeric α/β-hydrolase enzyme (<i>T</i><sub>m</sub> = 73.5°C; Δ<i>T</i><sub>m</sub> > 23°C) affected the protein folding energy landscape. Introduction of eleven single-point stabilizing mutations based on force field calculations and evolutionary analysis yielded catalytically active domain-swapped intermediates trapped in local energy minima. Crystallographic structures revealed that these stabilizing mutations target cryptic hinge regions and newly introduced secondary interfaces, where they make extensive non-covalent interactions between the intertwined misfolded protomers. The existence of domain-swapped dimers in a solution is further confirmed experimentally by data obtained from SAXS and crosslinking mass spectrometry. Unfolding experiments showed that the domain-swapped dimers can be irreversibly converted into native-like monomers, suggesting that the domain-swapping occurs exclusively <i>in vivo</i>. Our findings uncovered hidden protein-folding consequences of computational protein design, which need to be taken into account when applying a rational stabilization to proteins of biological and pharmaceutical interest.</p>

Hepatocyte Aggregate Culture Technique for Bioreactors in Hybrid Liver Support Systems

1993 ◽  
Vol 16 (12) ◽  
pp. 843-846 ◽  
Author(s):  
J.C. Gerlach ◽  
K. Klöppel ◽  
C. MÜller ◽  
N. Schnoy ◽  
M.D. Smith ◽  
...  
Keyword(s):  
High Performance ◽  
Large Scale ◽  
Support Systems ◽  
Three Dimensional ◽  
Culture Technique ◽  
Dimensional Structure ◽  
Cell Aggregates ◽  
Liver Support ◽  
Aggregate Culture

Utilizing a modified culture technique for hepatocytes, a high performance suspension culture is possible in which hepatocytes spontaneously form cell aggregates. The aggregates of 20-100 cells have been histologically confirmed to hold a three-dimensional structure, they show a long-term external metabolism and a survival time comparable with standard adhesion cultures. This technique has several advantages in the construction of large scale bioreactors for hybrid liver support systems.

scholarly journals Antibodies against Thrombospondin-Related Anonymous Protein Do Not Inhibit Plasmodium Sporozoite Infectivity In Vivo

2000 ◽  
Vol 68 (6) ◽  
pp. 3667-3673 ◽  
Author(s):  
Soren Gantt ◽  
Cathrine Persson ◽  
Keith Rose ◽  
Ashley J. Birkett ◽  
Ruben Abagyan ◽  
...  
Keyword(s):  
Metal Ion ◽  
Competitive Inhibition ◽  
Polyclonal Antibodies ◽  
Three Dimensional ◽  
Calcium Ionophore ◽  
Plasmodium Yoelii ◽  
Gliding Motility ◽  
Vaccine Antigen ◽  
Dimensional Structure

ABSTRACT Thrombospondin-related anonymous protein (TRAP), a candidate malaria vaccine antigen, is required for Plasmodiumsporozoite gliding motility and cell invasion. For the first time, the ability of antibodies against TRAP to inhibit sporozoite infectivity in vivo is evaluated in detail. TRAP contains an A-domain, a well-characterized adhesive motif found in integrins. We modeled here a three-dimensional structure of the TRAP A-domain of Plasmodium yoelii and located regions surrounding the MIDAS (metal ion-dependent adhesion site), the presumed business end of the domain. Mice were immunized with constructs containing these A-domain regions but were not protected from sporozoite challenge. Furthermore, monoclonal and rabbit polyclonal antibodies against the A-domain, the conserved N terminus, and the repeat region of TRAP had no effect on the gliding motility or sporozoite infectivity to mice. TRAP is located in micronemes, secretory organelles of apicomplexan parasites. Accordingly, the antibodies tested here stained cytoplasmic TRAP brightly by immunofluorescence. However, very little TRAP could be detected on the surface of sporozoites. In contrast, a dramatic relocalization of TRAP onto the parasite surface occurred when sporozoites were treated with calcium ionophore. This likely mimics the release of TRAP from micronemes when a sporozoite contacts its target cell in vivo. Contact with hepatoma cells in culture also appeared to induce the release of TRAP onto the surface of sporozoites. If large amounts of TRAP are released in close proximity to its cellular receptor(s), effective competitive inhibition by antibodies may be difficult to achieve.

scholarly journals DUF3380 Domain from a Salmonella Phage Endolysin Shows PotentN-Acetylmuramidase Activity

2016 ◽  
Vol 82 (16) ◽  
pp. 4975-4981 ◽  
Author(s):  
Lorena Rodríguez-Rubio ◽  
Hans Gerstmans ◽  
Simon Thorpe ◽  
Stéphane Mesnage ◽  
Rob Lavigne ◽  
...  
Keyword(s):  
Enzymatic Activity ◽  
Thermal Resistance ◽  
High Performance ◽  
Antimicrobial Agents ◽  
Biochemical Characterization ◽  
Specific Activity ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Gram Negative ◽  
Content Type

ABSTRACTBacteriophage-encoded endolysins are highly diverse enzymes that cleave the bacterial peptidoglycan layer. Current research focuses on their potential applications in medicine, in food conservation, and as biotechnological tools. Despite the wealth of applications relying on the use of endolysin, little is known about the enzymatic properties of these enzymes, especially in the case of endolysins of bacteriophages infecting Gram-negative species. Automated genome annotations therefore remain to be confirmed. Here, we report the biochemical analysis and cleavage site determination of a novelSalmonellabacteriophage endolysin, Gp110, which comprises an uncharacterizeddomain ofunknownfunction (DUF3380; pfam11860) in its C terminus and shows a higher specific activity (34,240 U/μM) than that of 14 previously characterized endolysins active against peptidoglycan from Gram-negative bacteria (corresponding to 1.7- to 364-fold higher activity). Gp110 is a modular endolysin with an optimal pH of enzymatic activity of pH 8 and elevated thermal resistance. Reverse-phase high-performance liquid chromatography (RP-HPLC) analysis coupled to mass spectrometry showed that DUF3380 hasN-acetylmuramidase (lysozyme) activity cleaving the β-(1,4) glycosidic bond betweenN-acetylmuramic acid andN-acetylglucosamine residues. Gp110 is active against directly cross-linked peptidoglycans with various peptide stem compositions, making it an attractive enzyme for developing novel antimicrobial agents.IMPORTANCEWe report the functional and biochemical characterization of theSalmonellaphage endolysin Gp110. This endolysin has a modular structure with an enzymatically active domain and a cell wall binding domain. The enzymatic activity of this endolysin exceeds that of all other endolysins previously characterized using the same methods. A domain of unknown function (DUF3380) is responsible for this high enzymatic activity. We report that DUF3380 hasN-acetylmuramidase activity against directly cross-linked peptidoglycans with various peptide stem compositions. This experimentally verified activity allows better classification and understanding of the enzymatic activities of endolysins, which mostly are inferred by sequence similarities. Three-dimensional structure predictions for Gp110 suggest a fold that is completely different from that of known structures of enzymes with the same peptidoglycan cleavage specificity, making this endolysin quite unique. All of these features, combined with increased thermal resistance, make Gp110 an attractive candidate for engineering novel endolysin-based antibacterials.

scholarly journals Purification, Characterization, and Gene Sequence of Michiganin A, an Actagardine-Like Lantibiotic Produced by the Tomato Pathogen Clavibacter michiganensis subsp. michiganensis

2006 ◽  
Vol 72 (9) ◽  
pp. 5814-5821 ◽  
Author(s):  
I. Holtsmark ◽  
D. Mantzilas ◽  
V. G. H. Eijsink ◽  
M. B. Brurberg
Keyword(s):  
Amino Acids ◽  
Three Dimensional ◽  
Reversed Phase ◽  
Dimensional Structure ◽  
Sequence Information ◽  
Type B ◽  
Clavibacter Michiganensis ◽  
Ring Rot ◽  
A Genome ◽  
Tomato Pathogen

ABSTRACT Members of the actinomycete genus Clavibacter are known to produce antimicrobial compounds, but so far none of these compounds has been purified and characterized. We have isolated an antimicrobial peptide, michiganin A, from the tomato pathogen Clavibacter michiganensis subsp. michiganensis, using ammonium sulfate precipitation followed by cation-exchange and reversed-phase chromatography steps. Upon chemical derivatization of putative dehydrated amino acids and lanthionine bridges by alkaline ethanethiol, Edman degradation yielded sequence information that proved to be sufficient for cloning of the gene by a genome-walking strategy. The mature unmodified peptide consists of 21 amino acids, SSSGWLCTLTIECGTIICACR. All of the threonine residues undergo dehydration, and three of them interact with cysteines via thioether bonds to form methyllanthionine bridges. Michiganin A resembles actagardine, a type B lantibiotic with a known three-dimensional structure, produced by Actinoplanes liguriae, which is a filamentous actinomycete. The DNA sequence of the gene showed that the michiganin A precursor contains an unusual putative signal peptide with no similarity to well-known secretion signals and only very limited similarity to the (only two) available leader peptides of other type B lantibiotics. Michiganin A inhibits the growth of Clavibacter michiganensis subsp. sepedonicus, the causal agent of ring rot of potatoes, with MICs in the low nanomolar range. Thus, michiganin A may have some potential in biological control of potato ring rot.

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