This document discusses membrane proteins and the use of TDA 2.0TM technology to study them. TDA 2.0TM allows membrane proteins to be studied in a high-throughput manner while maintaining the membrane context. It works by attaching HIS-tagged membrane proteins to stable liposomes via Ni-NTA lipids. This allows the proteins to freely translate and interact as they would in a cell membrane. The technology bridges cellular and solution-based assays and reveals differences in enzyme behavior and compound screening results compared to solution alone. It enhances dimerization, substrate selection, activity, and lowers Km for several kinases. An example using insulin signaling proteins demonstrates replicating phosphorylation steps in a chemically defined system using TDA
Optimization of experimental protocols for cellular lysisExpedeon
In this project, we have compared existing sample preparation methods for proteomics studies against newly developed FASP method and our in-house developed SDS-TCA protocol. For our
preliminary studies, we have chosen a very well characterized soil microbe Pseudomonas putida.
Evaluation of LdhIsozymes Following the Treatment of Methyl Parathion in the ...inventionjournals
Lactate dehydrogenase converts lactate into pyruvate and has a very important role in carbohydrate metabolism. LDH activity depends on its isozymes and their activities change under pathological conditions. The increase in LDH activity suggests the increased anaerobic conditionsunder the influence of methylparathion to meet the energy demand when the aerobic oxidation is lowered. Following the treatment of methyl parathion there was a differential percentage in increase or decrease of different isozymes in the fish Labeo rohita. There were three distinct bands during the 96h of treatment almost parallel to LDH5 band of human serum suggesting liver damage. This was also confirmed by histopathological studies.
This course required us to present an article which prof gave us randomly. And my article is a review paper related to TLR signaling! I upload here just hope that it can be useful for someone who is interested in this approach for studyding TLR signaling dynamics based on Synthetic ligands!
Many thanks for your look at my presentation and leave some comments if I got mistakes inside!
Cell-free protein expression is performed without the use of living cells. Instead, all components needed to transcribe DNA to RNA and translate the RNA to protein (e.g. ribosomes, tRNAs, enzymes, cofactors, amino acids) are provided in solution for use in vitro. Generally, such solutions are obtained through making a cell lysate from a desired cell type. Cell-free mixtures have been made from both bacterial and eukaryotics cells.
ShRNA-specific regulation of FMNL2 expression in P19 cellsYousefLayyous
This video encompasses all the steps and data produced for my graduation project in BSc in Biopharmaceutical science. During the course of the project we modified mammalian cells using Short Hairpin RNA to inhibit the correct function of the cytoskelleton. In this way we studied the importance of FMNL2 for the activation and regulation of actin fibers. Among the methods used are Flourescent microscopy, mamallian cell culture, cloning and flow cytometry.
Optimization of experimental protocols for cellular lysisExpedeon
In this project, we have compared existing sample preparation methods for proteomics studies against newly developed FASP method and our in-house developed SDS-TCA protocol. For our
preliminary studies, we have chosen a very well characterized soil microbe Pseudomonas putida.
Evaluation of LdhIsozymes Following the Treatment of Methyl Parathion in the ...inventionjournals
Lactate dehydrogenase converts lactate into pyruvate and has a very important role in carbohydrate metabolism. LDH activity depends on its isozymes and their activities change under pathological conditions. The increase in LDH activity suggests the increased anaerobic conditionsunder the influence of methylparathion to meet the energy demand when the aerobic oxidation is lowered. Following the treatment of methyl parathion there was a differential percentage in increase or decrease of different isozymes in the fish Labeo rohita. There were three distinct bands during the 96h of treatment almost parallel to LDH5 band of human serum suggesting liver damage. This was also confirmed by histopathological studies.
This course required us to present an article which prof gave us randomly. And my article is a review paper related to TLR signaling! I upload here just hope that it can be useful for someone who is interested in this approach for studyding TLR signaling dynamics based on Synthetic ligands!
Many thanks for your look at my presentation and leave some comments if I got mistakes inside!
Cell-free protein expression is performed without the use of living cells. Instead, all components needed to transcribe DNA to RNA and translate the RNA to protein (e.g. ribosomes, tRNAs, enzymes, cofactors, amino acids) are provided in solution for use in vitro. Generally, such solutions are obtained through making a cell lysate from a desired cell type. Cell-free mixtures have been made from both bacterial and eukaryotics cells.
ShRNA-specific regulation of FMNL2 expression in P19 cellsYousefLayyous
This video encompasses all the steps and data produced for my graduation project in BSc in Biopharmaceutical science. During the course of the project we modified mammalian cells using Short Hairpin RNA to inhibit the correct function of the cytoskelleton. In this way we studied the importance of FMNL2 for the activation and regulation of actin fibers. Among the methods used are Flourescent microscopy, mamallian cell culture, cloning and flow cytometry.
O termo “The Long Tail” (Cauda Longa em português) foi introduzido em Outubro de 2004 por Chris Anderson em um artigo na revista Wired para descrever certos modelos econômicos e de negócios como a Amazon.com ou a Netflix.
Master thesis of Sjierly Rodrigues Pereira, about the view of the youth in si...Denise van Keulen
Schooling for Life believes that all children in Sierra Leone and beyond have a right to develop themselves to their fullest potential. Schooling for Life fulfills its vision by supplying a programme with the unique combination of:
1. Scholarships for vocational education
2. Practical and Social skills trainings
3. Personal mentoring
The aim of the programme is to prepare the students for the job market and increase their chances for future job opportunities. To achieve this, Schooling for Life will collaborate with various businesses and NGOs. And did you know our programme is designed specifically upon local research carried out in 2008 and 2012, and is thus upon local demand? Read the research thesis of the founder of our organization Schooling for Life here:
Simplified receptor based pharmacophore approach to retrieve potent ptp lar i...rajmaha9
Simplified Receptor Based Pharmacophore Approach to Retrieve Potent PTP-LAR Inhibitors Using Apoenzyme
M. Elizabeth Sobhia*
Department of Pharmacoinformatics, National Institute of Pharmaceutical Education and Research (NIPER), S.A.S.
Nagar, Punjab 160062, India
Abstract: The design of biological active compounds from the apoenzyme is still a challenging task. Herein a simple yet efficient technique is reported to generate a receptor based pharmacophore solely using a ligand-free protein crystal structure. Human leukocyte antigen-related phosphatase (PTP-LAR) is an apoenzyme and a receptor like transmembrane phosphatase that has emerged as a drug target for diabetes, obesity and cancer. The prior knowledge of the active residues responsible for the mechanism of action of the protein was used to generate the LUDI interaction map. Then, the complement negative image of the binding site was used to generate the pharmacophore features. A unique strategy was
followed to design a pharmacophore query maintaining crucial interactions with all the active residues, essential for the enzyme inhibition. The same query was used to screen several databases consisting of the Specs, IBS, iniMaybridge, NCI and an in-house PTP inhibitor databases. In order to overcome the common bioavailability problem associated with phosphatases, the hits obtained were filtered by Lipinski’s Rule of Five, SADMET properties and validated by docking studies in Glide and GOLD. These docking studies not only suggest the essential ligand binding interactions but also the binding patterns necessary for the LAR inhibition. The ligand pharmacophore mapping studies further validated the
screened protocol and supported that the final screened molecules, presumably, showed potent inhibitory activity.
Subsequently, these molecules were subjected to Derek toxicity predictions and nine new molecules with different
scaffold were obtained as non-toxic PTP-LAR inhibitors. The present prospective strategy is a powerful technique to
identify potent inhibitors using the protein 3D structure alone and is a valid alternative to other structure-based and
random docking approaches.
Evaluation of LdhIsozymes Following the Treatment of Methyl Parathion in the ...inventionjournals
Lactate dehydrogenase converts lactate into pyruvate and has a very important role in carbohydrate metabolism. LDH activity depends on its isozymes and their activities change under pathological conditions. The increase in LDH activity suggests the increased anaerobic conditionsunder the influence of methylparathion to meet the energy demand when the aerobic oxidation is lowered. Following the treatment of methyl parathion there was a differential percentage in increase or decrease of different isozymes in the fish Labeo rohita. There were three distinct bands during the 96h of treatment almost parallel to LDH5 band of human serum suggesting liver damage. This was also confirmed by histopathological studies.
Evaluation of LdhIsozymes Following the Treatment of Methyl Parathion in the ...inventionjournals
Lactate dehydrogenase converts lactate into pyruvate and has a very important role in carbohydrate metabolism. LDH activity depends on its isozymes and their activities change under pathological conditions. The increase in LDH activity suggests the increased anaerobic conditionsunder the influence of methylparathion to meet the energy demand when the aerobic oxidation is lowered. Following the treatment of methyl parathion there was a differential percentage in increase or decrease of different isozymes in the fish Labeo rohita. There were three distinct bands during the 96h of treatment almost parallel to LDH5 band of human serum suggesting liver damage. This was also confirmed by histopathological studies.
Seminario basado en el artículo "Structural determination of group A Streptococcal surface
dehydrogenase and characterization of its interaction with
urokinase-type plasminogen activator receptor"
Most bacteria are free-living organisms that grow by increasing
in mass and then divide by binary fission.
Growth and division are controlled by genes, the expression
of which must be regulated appropriately. Genes
whose activity is controlled in response to the needs of a
cell or organism are called regulated genes. All organisms
also have a large number of genes whose products
are essential to the normal functioning of a growing and
dividing cell, no matter what the conditions are. These
genes are always active in growing cells and are known as
constitutive genes or housekeeping genes; examples include
genes that code for the enzymes needed for protein
synthesis and glucose metabolism. Note that all genes are
regulated on some level. If normal cell function is impaired
for some reason, the expression of all genes, including
constitutive genes, is reduced by regulatory
mechanisms. Thus, the distinction between regulated
and constitutive genes is somewhat arbitrary.
2. There are several classes of membrane proteins: single- and multi- pass
transmembrane proteins, proteins which are associated with the membrane via lipid
anchors (such as myrisoylation, palmitoylation or GPI anchors) or electrostatic
interactions, and proteins which are normally cytosolic but form complexes with
membrane proteins. TDA 2.0™ is not suitable for use with multi-pass transmembrane
proteins in general, however, all other membrane proteins which have distinct
domains on one side of the membrane would work with TDA 2.0™, regardless of
which subcellular membrane, or face of the membrane, the protein is associated
with.
2
3. [LEFT PANEL] Typically membrane proteins are assayed by expressing recombinant
fragments, often containing only the active site of the enzyme, and are interrogated
in a high-throughput solution-based assay. While cost-effective and expeditious, this
format ignores any organization, structure and topology imparted by membrane.
[RIGHT PANEL] An alternative assay is to examine endogenous or over-expressed
enzymes in a living cellular system. While this system faithfully replicates the
membrane environment and contains the full compliment of every other relevant
animal protein, it is slow, very expensive, and not readily adaptable to high-
throughout testing of a chemical library.
Importantly, efficacy of compounds identified in a solution assay tends to correlate
poorly with the efficacy in cellular assays.
*MIDDLE PANEL+ TDA 2.0™ is an enabling technology which bridges the gap between
these two formats, providing the context afforded by a biological membrane in a
platform fully compatible with HTS and all detection formats.
3
4. How does template directed assembly work? Engineered recombinant HIS-tagged
proteins are produced such that the HIS-tag is on the correct terminus of the protein
to reflect the polarity of the enzyme with respect to the membrane (for example,
ecto-domains of a receptor would be C-terminally tagged while intracellular domains
are N-terminally tagged). TDA 2.0™ is a soluble stable liposome which is made from
derivitized lipids which have Ni-NTA covalently attached to the lipid head group. This
allows the HIS-tagged proteins to bind to the liposome creating an environment much
like a cellular membrane. Unlike a sepharose or agarose bead where HIS-tagged
proteins must detach and reattach to translate across the surface, the lipids are fully
fluid within the 2-dimensional surface of the liposome, so associated proteins can
translate and rotate freely. The spatial and relational organization provided by the
membrane surface, combined with this fluidity, promotes the formation of higher-
order structures, such as homo- or hetero- dimers or multimers, and allows
recruitment of accessory factors.
4
6. Insulin receptor is activated by dimerization which leads to trans-phosphorylation on
several tyrosines. The phosphorylated dimer is the active RTK. Typically, in order to
see activation in solution, manganese is included in the reaction, which creates a
non-physiological environment. Above, the left panel shows a radiometric filter
binding assay for autophosphorylation, while the right panel shows
autophosphorylation and phosphorylation of an IRS1-derived peptide substrate. As
you can see, addition of 10mM MnCl2 increases autophosphorylation as well as
substrate phosphorylation. However, addition of TDA 2.0™ in a physiological relevant
buffer without MnCl2 shows robust activation. We interpret this data to show
enhanced functional dimerization of InR on TDA 2.0™ in a physiologic buffer.
6
7. Zap-70 is a T-Cell receptor effector that is normally activated by recruitment to the
phosphorylated ITAM domains of CD3-zeta. In vitro, recombinant Zap-70 is difficult
to screen as it has been reported to have a very long and variable lag phase. We see
this same effect as shown in the top three panels where after two hours we see
dramatically different activity of Zap-70 in three different experiments. However,
addition of TDA 2.0™ reduces the lag time significantly and increases the
predictability of activation, creating a very robust assay for Zap-70. This data was
generated using a Caliper EZ reader.
7
8. Many enzymes show enhanced activity when assayed in the presence of TDA 2.0™.
While secondary in importance to the effects TDA 2.0™ has on improving the biology
of an enzyme, this is nonetheless another very beneficial feature of TDA 2.0™.
8
9. When looking at the ability of an enzyme to phosphorylate a peptide substrate,
researchers often use synthetically derived peptides, such as poly-Glu(4)-Tyr. We
noticed that when examining activity of ErbB4, a receptor tyrosine kinase (RTK),
towards PolyGlu, there was no improvement in activity in the presence of TDA 2.0™.
However, when examining activity towards peptides derived from natural substrates
of ErbB4, such as Abl and Src, a considerable enhancement in activity is noted in the
presence of TDA 2.0™. We interpret this as an indication that the substrate
preference of the enzyme is altered when in the membrane context, perhaps
selecting more biologically relevant substrates.
9
10. To investigate that systematically, we used a different RTK, TrkB, and contracted
Molecular Devices to screen a library of peptides in the absence (red) and presence
(blue) of TDA 2.0™. The first observation is that the best substrates of TrkB are
completely different in the presence and absence of TDA 2.0™. This indicates to us
that a key biological property of the enzyme, namely substrate selection, is
significantly altered by TDA 2.0™. The second observation from this data set comes
from analysis of the sequences of the peptides substrates above. In the absence of
TDA 2.0™, comparison of substrate sequence to the non-redundant protein database
reveals these substrates either fail to match anything in the database (they are
synthetic peptides) or they match viral proteins, which are not likely to be natural
substrates of TrkB. In the presence of TDA 2.0™ many synthetic or non-relevant
substrates are also identified, however, peptides derived from IRS1 and EGFR, known
substrates of TrkB, are identified as substrates. This indicates that presentation of
TrkB in the context of TDA 2.0™ biases the substrate selectivity towards relevant
substrates of the enzyme.
10
11. Initial rates were measured and Km for ATP determined for various enzymes in the
presence and absence of TDA 2.0™ using the Caliper EZ reader platform. All enzymes
examined to date show significantly lower Km ATP in the presence of TDA 2.0™.
11
12. Data presented by Dr. Kathleen Seyb (Marcie Glicksman’s group at Harvard
Neuroscience/Brigham and Women’s) at the Society for Biomolecular Sciences annual
meeting. They had a grant-driven project to screen Lyn kinase through their library of
75,000 compounds. In order to get high enough signal in their solution-based assay,
high concentrations of enzyme (>200 nM) had to be used which made the screen
financially impossible. Addition of TDA 2.0™ reduced the amount of enzyme required
for a good signal over 25-fold and reduced the cost per well by 50%, leading to
successful completion of the screen.
12
13. This graph shows a subset of the compounds which were screened both in the
presence and absence of TDA 2.0™. It’s not a direct comparison as the enzyme
concentration required to get data in the absence of TDA 2.0™ is not the same, and
the signal was much lower, but is instructive nevertheless. The graph plots %
inhibition in the absence of TDA 2.0™ along the X-axis and in the presence along the
Y. Noticeably, TDA 2.0™ alters the pharmacology of Lyn as there are hits unique to
each condition. We’re in the process now of examining these compounds in follow-
up cellular assays to try to determine if using TDA 2.0™ leads to better quality lead
compounds and is more predictive of cell-based assays. Regardless, this data shows
that TDA 2.0™ reveals differences in compound SAR (structure-activity relationship),
13
14. Insulin signal in the cell initiates upon receptor dimerization and activation by
transphosphorylation on specific tyrosines. The phosphotyrosines serve as binding
sites for PI3k which is recruited and propagates the signal by converting PIP2 to PIP3,
in turn recruiting PDK1 and AKT to the membrane. Activation of Akt by PDK1 and
mTOR leads to phosphorylation of many AKT-substrates when ultimately lead to
biological effects such as lipolysis, glucose update, growth or proliferation. We are
developing an assay which replicates many of these steps in a chemically defined
system.
14
15. The first step we’re replicating is the phosphorylation of AKT by PDK1. We’ve made a
HIS tagged construct of AKT, and well as a HIS-tagged form of PDK1 to deliver these
enzymes to the membrane without PIP3. To extend the utility of this assay format, we
included GST-tagged mTOR which phosphorylates AKT on S473. When AKT is
phosphorylated on both S473 and T308, AKT kinase activity is increased several
orders of magnitude.
15
16. This figure shows Western blots of reactions containing different combinations of
PDK, mTOR and AKT with and without TDA 2.0™.
The top panels are anti-GST Westerns showing consistent loading of GST-tagged
mTOR.
The second set of panels show consistent loading of AKT (lower band) and PDK1
(upper band) using an anti-HIS tag antibody.
The third set of panels employs a phospho-specific anti-AKT-pS473 antibody to show
phosphorylation of AKT by GST-tagged mTOR.
The lower set of panels employs a phospho-specific anti-AKT-pT308 antibody to show
phosphorylation of AKT by HIS-tagged PDK1.
In the presence of TDA 2.0™, when AKT and PDK are combined, phosphorylation of
AKT increases 2-3 fold (compare lanes 5 to 6). This is not solely due to co-localization
of AKT and PDK as the same result is obtained using a FLAG-tagged version of PDK1
(data not shown). Further, when AKT and mTOR are combined in the presence of
TDA 2.0™, phosphorylation on S473 increases 4-5 fold (compare lanes 7 to 8). Since
mTOR is GST-tagged and not co-localized, this confirms our result with FLAG-tagged
PDK1 and indicates that AKT is a better substrate for it’s upstream activators when
associated with a membrane such as TDA 2.0™.
16
17. Finally, when examining the ability of AKT to phosphorylate CROSSTide™, only when
AKT, PDK1 and mTOR are combined in the presence of TDA 2.0™ do we see robust
kinase activity.
17