![]() |
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
![]() |
![]()
TASK 1
Below you find the screenprints of databases that we are going to use in the search for information about biomolecules involved in the regulation of food intake. For the search of scientific publications: PubMed For the search of information about proteins (amino acid sequence, domains, function, essential articles and links to other sites : SwissProt To find out about chromosome location, genomic region (sequence, exons, introns) , transcripts (cDNA): Entrez Nucleotide For information about protein structure : PDB For information about pathologies linked to gene mutations: OMIM Work in pairs and search SwissProt for the following proteins Insulin homo sapiens
As you scroll down the Insulin page, answer the following questions:
NB Sequences are often presented in the FASTA format. This is a “clean script” presentation that can be used by FASTA programs to perform rapid sequence comparisons through alignments or structure predictions. Apart from the sequence, FASTA format also provides a single line description of the molecule in question (DNA or protein). FASTA stands for FAST ALL, reflecting that it can be used for a fast protein as well as nucleotide sequence comparison. It is a good idea to open FASTA sequence files in “bloc notes” , that way they stay “clean” (not annotated by Word or other word processing programs)
Leptin homo sapiens
As you scroll down the Leptin page, answer the following questions:
There are two types of single nucleotide polymorphism
Ghrelin homo sapiens
As you scroll down the Ghrelin page, answer the following questions:
End
TASK 2
In this TD we are going to explore the molecular composition of the insulin receptor. First we will explore the domain architecture of its ectodomain with the use of SwissProt and Pfam (protein families based on seed alignment and hidden Markov models ). Next we are going to employ the Protein Data Bank (PDB) to find the coordinates of the amino acids of the kinase domain of the insulin receptor both in its inactive and active state. Using the instructions provided in the exercise you will discover the highly conserved amino acid residues of protein kinases and you will learn how phosphorylation of the activation segment causes a shift in the conformation that renders the protein kinase competent for phosphorylating its substrates. Write a little report (either alone or in groups of two) of this activity including a description of:
Home-made images of your PyMol exercise are essential. Send the report in the form of a “ .doc “ file to i.kramer@iecb.u-bordeaux.fr. Deadline (date limite): 13 march 2009, 24h00 (weighting factor 0.2)
Start with Expasy SwissProt (http://expasy.org/) and search for the insulin receptor homo sapiens As you scroll down answer the following questions
Suggestion: Cut and paste the figure into this document and make a short list underneath to explain the abbreviated codes. Add the amino-acids numbers behind each domain. 1 2 3 4 4 5
We now are going to have a look at the insulin receptor ectodomain (extracellular component) Scroll up until you come across “Cross-references” and 3D structure databases; then select, at Entry, the protein segment which the largest amino acid sequence of the ectodomain (remember amino acids 28-957 are at the outside of the cell, the rest goes inside). Click on the appropriate PDB (protein data bank) “Entry” code You know have entered the PDB, an information portal to biological macromolecular structures You are first going to download the coordinates of the insulin receptor on your server, click on the telephone shown in the red-lined square above (figure 1). Call it “ InsR-ecto- 2dtg “ In order to find out what a PDB file is, click on the text icon
![]() Scroll down from the title and you find Molecular Description Asymmetric Unit with a list of “polymers” (meaning here polypeptide chains or segments of proteins)
Open PyMol (on the desktop of your computer) and import the 2dtg file In the PyMol tool bar (“The PyMol molecular graphics system”) you go to display and select “Sequence”, then click, and than select “sequence mode” and finally “chains” From the introductory page you have learned that chains A, B, C and D are part of an antibody so we like to remove them from the screen. Click, in PyMol Viewer, on A, B, C and D (the left hand corner of the screen). The selected chains become red. 1) change colour of the antibody Fab fragments by going to the right hand corner toolbar, at the level of “sele” (for select) and click on grey (at the bottom of the list) and then click on “grey80). 2) remove the Fab fragments from the screen by going to “sele” (for selected items) and click on H (for hide), then select “everything”. What is left on the screen is the ectodomain of the insulin receptor in green. You will see the chain indications A, B, C and D in grey20 wherease E is green (A B C D E) Select E and go to (sele) , select S (for show) and then click on “cartoon”, the ribbons should appear on your screen. To remove the lines you next click on H (for hide) and you select “lines”. You now see a long V-shaped protein, folded with lots of beta-sheets and some helices. Try all other forms of S (show) and do not forget to remove (Hide) the previous ones. This helps you to get a feel of how proteins can be represented. Also by playing around you familiarize with the action buttons of PyMol. Take your time for this exercise. Every time you have a new representation, you turn the protein around with the mouse in order to get a three dimensional feel of the ectodomain of the insulin receptor.
Remember, the insulin receptor precursor is cut into two chains. Go back to your SwissProt entry (see above) and find out where exactly the protein is cut. Then go the display and select “sequence mode” and then “residue codes” . What you see are soft grey (grey80) letters, which are the amino-acids of the Fab fragment of the antibodies (which we ignore) and green letters, which are amino acids of the insulin ectodomain. You now search for the cleavage site. The numbers of the amino acids on top of PyMol viewer should correspond to the numbers in the SwissProt database but always verify the identity of the amino acids as a “backup”. Select the alpha-chain amino acids and colour them “orange”. Then select the amino acids of the beta-chain and colour them “yellow-orange”. Now find the representation that according to you shows best that the protein is made up of two chains. You may change colours if you like. You may also change the black background. You can this this by going to the Display in the PyMol Molecular Graphics System (see figure 2) and click on background, then choose another colour).
Go to the casier in Ulysse and bring up the article of “Insulin Receptor Structure McKern” and learn how the receptor is made of two subunits with only one operational insulin binding site. End
TASK 3
In this practical you are going to explore the molecular composition of the intracellular kinase domain of the insulin receptor and in particular how phosphorylation changes its structure and as a consequence its activity. You will finish the TD by showing how the tyrosine phosphatase PTP1B exerts a negative control on the insulin signalling pathway. You are going to employ the Protein Data Bank (PDB) to study the positioning (coordinates) of the amino acids of the kinase domain both in its inactive and active state. Using the instructions provided in the exercise you will discover where highly conserved amino acids in kinase domains are situated and you will get a rough impression how they are involved in the the regulation of kinase activity. Write a little report (either alone or in groups of two) of this activity including a description of:
Home-made images of your PyMol exercise are essential. Send the report in the form of a “ .doc “ file to i.kramer@iecb.u-bordeaux.fr. Deadline (date limite): 13 march 2009, 24h00 (weighting factor 0.2)
Start with Expasy SwissProt (http://expasy.org/) and search for the insulin receptor homo sapiens As you scroll down answer the following questions
Choose 1IRK ( 1irk) and click on it to be redirected to the PDB-pages concerned. Download the PDF file. Section I inactive insulin receptor kinase
Open the file in PyMol In “PyMol Molecular Graphics System” (menu) In “PyMol Viewer” Then start to select the following amino-acids and present them as “sticks” with “colours by element” K1030 (lysine), E1047 (glutamate), D1150 (aspartate) and D1132 (aspartate catalytic residue). D1132 is located in the VHRD, valine-histidine-arganine-asparate sequence which is part of the protein kinase sequence signature Next you select the following amino-acids and present them as sticks with red colour Y1158, Y1162, Y1163 (all tyrosines) Next you select the following amino acids and present them as “cartoon” with “yellow “ colour G1003, Q1004, G1005, S1006, F1007, G1008, M1009
Next you select the following amino acids and present them as “cartoon” with “yellow “ colour D1150 -> P1172
In this segment you find a highly conserved sequence DFG, Aspartate-Phenylalanine-Glycine, which is part of the protein kinase sequence signature. After this segment you find an APE (arginine-proline-glutamate sequence which you often find in protein kinases (sometimes ALE) Finally, you select the proline in the sequence LPVRWMAPE, just after the activation segment, present it as stick and colour it blue. This proline is another highly conserved residue but only for tyrosine protein kinases, the serine/threonine have a threonine instead and this plays, in part, a role in substrate selectivity (tyrosine or serine/threonine) Get an orientation that shows nicely all the amino-acids you have selected. Saving your work In “PyMol Molecular Graphics System” (menu) Pay special attention to tyrosine Y1162, proline P1172 and aspartate D1132 Section II active insulin receptor kinase
Go to “File” -> reinitialize Go to “Plugin” -> PDB loader service and enter 1ir3 (this gives the same result as 1IR3) Repeat exactly what you have done before but notice that the tyrosines in the activation segment are now indicated as PTR, meaning phosphotyrosine This structure has to components that were not present in the previous one
Represent ANP as “Sticks” and colour by “Element” ( CHNOS ) with carbon en rose (fourth from top of list)
Represent Mg Mg as “Dots”and colour -> Cyans -> Pale Cyan Turn around and try to find the differences between 1ir3 and 1irk (use the image in the casier to get some help) Save your image both as a .pse file and, after having applied “Ray”, as a .png file You can again play around with representations to get a feel of what the active protein kinase and its substrate peptide looks like Further reading: find a short article describing the insulin receptor kinase and its mode of activation in the casier (structure of the insulin receptor tyrosine kinase - SR Hubbard) Section III tyrosine protein phosphatase bound to the phosphorylated
insulin receptor kinase domain Start with PDB and enter 1g1f (1G1F) Find out which proteins are presented. In order to find which phosphotyrosine residue is the first target of the tyrosine protein phosphatase, take into account that cysteine 215 normally is the catalytic residue (which, importantly, is replaced by an alanine in this structure in order to render the phosphatase inactive, otherwise you would never be able to fix the tyrosine-phosphate substrate!) Save your file as a .pse and, after having RAYed, save it as a .png for your report End
TASK 4 Antropometrics practical
In this TD you are going to be confronted with the type of body measurements that are performed around the world in order to get an impression of the general (as well as abdominal) adiposity state of humanity. The tests are simple because they have to be employed in large populations (thousands of individuals). The measurements therefore only provide a rough and indirect estimate of fat content, but numerous comparisons have shown that the outcome of these tests are comparable to more sophisticated methods (density measurements, and others). Such measurement serve to a better understanding the relationship between longevity and adiposity, essential for insurance companies (to calculate premium rates) as well as for public health authorities (to estimate budgets). One distinguishes two types of adiposity; gluteofemoral (buttocks and thighs, also referred to as lower-body fat), which is poorly associated with chronic diseases that may reduce lifespan, and abdominal (waist, also referred to as upper-body fat), which is associated with chronic diseases (Pischon T, Boeing H, Hoffmann K, Bergmann M , Schultze MB et al. General and abdominal adiposity and risk of death in Europe. N Engl J Med 2008;359:2105-2119, Haslam DW, James WP. Obesity. Lancet 2005;366:1197-1209; Wang Y, Rimm EB, Stampfer MJ, Willet WC, Hu FB. Comparison of abdominal adiposity and overall obesity in predicting risk of type 2 diabetes among men. Am J Clin Nutr 2005;81:555-563). This afternoon you are going to estimate your body mass index (BMI) to get an impression of your relative adiposity (do you belong to the skinny or the not-so-skinny group), your waist-to-hip ratio, to get an estimate of what type of adiposity it concerns (affecting your life expectation or not) and finally you are going to measure two representative skin-folds in order to estimate your fat- and your lean-body mass. It should be stressed that the association between adiposity and public health only applies to large populations and not to individuals, meaning that people with a high BMI may outlive those with an “ideal BMI” (estimated around 25) and that the skinny ones may “cost” a great deal more in hospital charges than the not-so-skinny ones. It should also be stressed that you do not necessarily live healthy and happily (and then instantly die with a smile on your face at the age of 85) when you follow the instructions of health-care authorities to the letter. Firstly there is more to life than BMI or waist-to-hip ratios and secondly, with respect to physiology and pathology, it is insane to consider men all equal in view of the enormous heterogeneity of its genetic composition (certain statements of health-politics have the sole purpose to enforce hegemony of the ruling majority). However, it is certainly not unwise to follow health-care instructions; it may improve the quality of life in the five years that precede your last heart beat. In particular, whilst acknowledging a certain bias, it has been proven that coming to the SVI632 Signal Transduction course on bicycle extends a healthy and happy life span with a considerable number of years. BMI
Where do you stand? Classification de l'état nutritionnel chez l'adulte en fonction de
l'indice de masse corporelle (IMC)
selon l'OMS et l'International Obesity Task Force (1998)
Waist-to-hip ratio
Considered at “risk”:
ratio > 1.0 in men Ratio > 0.86 in women (see below) From Pischon et al. N Engl J Med 2008;359:2105 Body fat estimation
Fat content is: % of fat x body weight Lean body mass equals: body weight minus fat content (this measure is sometimes used to estimate doses of medicaments or doses of general anesthesia) Catagories of body fat content According to Health Check Systems, The American Council on Exercise
To find out more about anthropometric calculations & values, consult the following sites:
End of practical
|
![]()
|
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
back to top | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Last Updated July 7, 2009 10:17 PM | admin news | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||