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Enzymes by Sukhmeet Sachal, Robyn-Lynn Fur, Yemina Chung

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Sukhmeet Sachal

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Transcript of Enzymes by Sukhmeet Sachal, Robyn-Lynn Fur, Yemina Chung

This study identifies BACE2 as a potent amyloid ß-degrading protease (AßDP).
Based on its high catalytic efficiency and its ability to degrade amyloid ß-protein (Aß) intracellularly BACE2 represents a particularly strong therapeutic candidate for the treatment or prevention of Alzheimer’s disease (AD). By Rohan Sachal Enzymes http://www-news.uchicago.edu/releases/06/images/061011.enzyme.jpg Catalysts
Specific protein molecules that influence rate of chemical reaction
Not created or destroyed in a reaction
Composed of one or more polypeptides
numerous peptide bonds form a chain-like structure What are enzymes? http://www.edinformatics.com/biotechnology/500px-Two_substrates.png Substrate
substance upon which an enzyme acts as a catalyst

Lock-key model

Active site
where a shape specific substrate attaches to the enzyme
gives the properties of specificity and catalysis to an enzyme Enzyme Specificity http://karimedalla.wordpress.com/2012/10/17/7-6b-enzymes/ Enzyme Inhibition https://wikispaces.psu.edu/pages/viewpage.action?pageId=112526688&navigatingVersions=true Factors that influence the rate of reaction
Substrate and Enzyme concentration
Presence of inhibitors Enzyme Kinetics To observe enzymes in action.
Understand the concept of enzyme specificity
Understand the basic concepts of spectrophotometry.
Understand the concepts of enzyme kinetics and inhibition. Lab Objectives Catalase (enzyme) catalyzes the breakdown of hydrogen peroxide to water and oxygen
Bubbles will form if catalase is present

Hydrogen Peroxide is very harmful
Must be broken down by enzymes such as catalase 1) Observing Enzyme Activity: Catalase 2 cubes of fresh pear (1 cm)
1 cube of cooked pear (1 cm)
3 test tubes
1 knife
Beaker with water and transfer pipette
Flask with 3% hydrogen peroxide and transfer pipette Materials H2O H2O2 H2O2 C B A Catalase Results Enzyme is denatured, no reaction takes place Oxygen gas released CONTROL H2O H2O2 H2O2 C B A Controls Lactase hydrolyzes its disaccharide substrate, into glucose and galactose monomers 2. Enzyme Specificity: Lactase
4 test tubes (Wash and re-use the tubes from experiment 1)
Flask with lactase and transfer pipette
Flask with lactose and transfer pipette
Flask with sucrose and transfer pipette
Flask with maltose and transfer pipette
4 glucose-testing strips Materials CONTROL - - - + Add lactase to each tube
Test for the presence of glucose using the strips None D Maltose Sucrose Lactose C B A Controls + _ _ _ Results technique for measuring light absorption by a solution at any chosen wavelength of light.
Measures concentrations of any dissolved substance that absorbs energy in the visible or UV range
Does not change the substance measured
Ideal for following the changes in solute concentration during a reaction Spectrophotometry Spectrophotometry Basic Spectrophotometry Different molecules absorb different wavelengths of light
eg) a molecule appears red if it absorbs all light except red OR if it absorbs the wavelength corresponding to the light of the colour that is complementary to red (ie. Blue-green) 3. Spectrophotometer Practice Spectrovis spectrophotometer
Spectrophotometer cuvettes filled with food dye (4 colours to be shared)
Water-filled spectrophotometer cuvette (1 per group)
Beaker with water and transfer pipette Materials Determining the appropriate wavelength for each color (blue, green, red, yellow) Spectrophotometer Results Succinate dehydrogenase catalyses the reaction of succinate to fumarate to transport electrons and hydrogen atoms to the electron carrier FAD. FAD is then reduced to FADH2.
4. Enzyme kinetics and competitive inhibition: Succinate dehydrogenase

Succinate dehydrogenase also catalyzes this interaction, but instead of reducing FAD in this case DCIP is reduced. Succinate dehydrogenase

The structure of succinate compared to its competitive inhibitor, malonate. Spectrovis spectrophotometer
Spectrophotometer cuvettes
Assay medium
Mitochondrial suspension on ice
Pipettes (2 x 2 mL, 1 x 5 mL) Materials Procedure Tube 1: Blank
Tube 2: Low dose of mitochondrial suspension
Tube 3: Blank
Tube 4: Medium dose of mitochondrial suspension
Tube 5: Medium dose + malonate
Tube 6: Medium dose - succinate

Tube 7: Blank
Tube 8: High Dose of mitochondrial suspension Controls Enzyme Continuous-Flow Enzyme Assay on a Microfluidic Chip for Monitoring Glycerol Secretion from Cultured Adipocytes http://en.wikipedia.org/wiki/File:Glycerol-3D-balls.png http://www.cnrs.fr/cnrs-images/sciencesdelavieaulycee/cellule/intro.htm Adipocytes: fat/ lipid cells

Glycerol: syrupy liquid (C3H8O3) produced by adipocytes

Enzyme Assay: Methods for measuring enzymatic activity Vocabulary * Developed a dual-chip microfluidic system for culturing adipocytes, perfusing them, and then monitoring glycerol secretion using a continuous-flow enzyme assay
- One chip is a cell perfusion chamber
- Second chip is a continous-flow
enzyme assay modified for
fluorescence-based detection

*Used to demonstrate transient increases in glycerol secretion during exposure of the cells to isoproterenol, Beta-adrenergic antagonist What? http://trialx.com/curetalk/2012/12/obesity-is-not-your-fault-who-should-be-blamed-then/ The ability to monitor changes in cellular secretion over time may provide insight into adipocyte metabolism and the dis-regulation that occurs with obesity- related disorders. Why? http://pubs.acs.org/doi/pdfplus/10.1021/ac8026965 Schematics of the two microfluidic devices used Experiment
*Glycerol standards were pumped through cell perfusion chip and into the enzyme assay chip where they were mixed with reagents for the glycerol assay, while monitoring fluorescence
-glycerol standards were pumped
through the perfusion chip at 80 μL
- Limit of detection (LOD) of the on-
line fluorescence-based enzyme
assay, was 4 μM for glycerol

*The assay gave a linear response from the detection limit to 50 μM glycerol
Signal generated by fluorescence-based glycerol assay yielded similar RSDs over 3 hour period indicating good stability and relatively long-term operation On-Line Enyme Assay Characterization Experiment
* test for monitoring dynamic changes in glycerol release from adipocytes- monitored glycerol secretion during treatment of adipocytes with the beta-adrenergic agonist isoproterenol
Cells were loaded into the cell chamber and perfused with glucose

*Cells remained adherent during transfer and perfusion
Basal measurements were collected for at least 60 min and yielded an average concentration of 28 μM
*When switching to a perfusion fluid containing 20 μM isoproterenol, glycerol secretion increased by approx. 3-fold over basal secretion followed by a sustained release that was ∼40% over basal levels
*Results indicate the reliability of the on-line measurements Glycerol Secretion from Adipocytes Performed several control experiments to ensure that the glycerol recordings were not due to artifacts

-Secretion from preadipocytes was used as a negative control

-Assay was performed in the absence and presence of 20 μM
isoproterenol and 25 or 50 μM glycerol standards

- Removed enzyme reagent during a cellular secretion experiment Control Experiments for Secretion Data Characterized the perfusion chip with simulations and experiments to determine the shear stress on cells and uniformity of drug application to the cells
- Perfusion chip has a recessed cell chamber area relative to the inlet and
outlet flow stream to reduce shear stress and permit laminar flow of fluid
over the cells
Estimated shear stress upon the cells is 0.006 N m-2
- This low value, plus the observation that the flow did not cause cells to
detach or deform, suggests that the chip design prevents excessive shear on
the cells Characterization of Cell Perfusion Chip *The microfluidic platform was able to detect changes in glycerol levels from ∼50 000 adipocytes under basal conditions and upon pharmaco- logical treatment with isoprotereno
*Results proved that the use of microfluidic device for on-line mixing and detection reduced the consumption of costly reagents to less than 1% of off-line volumes and decreased labor by automating the mixing and detection on-chip
*Culturing adipocytes on coverslips increased the throughput of cells available for study as well as decreased biological variability as multiple experiments could be tested from cells cultured in the original Petri dish
*This experiment proves that this device may be applied to studying the oscillatory nature of glycerol release and long-term changes in metabolism. Conclusion Continuous-Flow Enzyme Assay on a Microfluidic Chip for Monitoring Glycerol Secretion from Cultured Adipocytes
Anna M. Clark, Kyle M. Sousa, Colin Jennings, Ormond A. MacDougald, and Robert T. Kennedy
Analytical Chemistry 2009 81 (6), 2350-2356 Bibliography Results Main Reaction Succinate will turn into fumarate and DCIP will turn into DCIP-H2 (colourless) in the presence of succinate dehydrogenase Tube 2 4 8 Amount of mitochondrial suspension increase from more enzyme being present. Expected: Faster Fastest With the presence of the competitive inhibitor, Malonate Tube 5 expect: Remain Blue Absence of succinate Tube 6 Expect: no reaction A farnesyltransferase inhibitor prevents both theonset and late progression of cardiovasculardisease in a progeria mouse model
Identification of BACE2 as an avid ß-amyloid-degrading protease Terminology Yemina Chung Alzheimer disease (AD)
amyloid β-protein (Aβß)
amyloid precursor protein (APP)
amyloid β-protein degrading protease (AßDP)
ß-site APP-cleaving enzyme-1 (BACE1)
ß-site APP-cleaving enzyme-2 (BACE2)  http://learn.genetics.utah.edu/content/disorders/whataregd/alzheimers/images/AD_brain.jpg Alzheimer disease (AD): a progressive and presently incurable neurodegenerative disorder
Affects the brain regions important for mnemonic and cognitive functions WHAT Hutchinson-Gilford progeria syndrome (HGPS) is the most dramatic form of human premature aging. Death occurs at a mean age of 13 years, usually from heart attack or stroke. One per 4 - 8 million http://upload.wikimedia.org/wikipedia/commons/c/c6/APP_processing.png abnormal accumulation of the amyloid ß -protein (Aßβ) in the brain region
Amyloid β-protein (Aß): mixture of peptides ranging from 37 to 43 amino acids produced via sequential cleavage of the amyloid precursor protein (APP)
Amyloid Precursor Protein (APP): a membrane protein expressed in many tissues and concentrated in the synapses of neurons. HOW Mutations in 3 genes—APP and presenilin-1 and −2—are known to cause Alzheimer’s disease
increase production of all forms of Amyloid ßβ-protein
The precise mechanisms underlying Alzheimer disease remains to be determined. PROBLEM By Rohan Sachal SOLUTION Proteases: enzymes that catalyze the hydrolytic degradation of proteins to smaller amino acid polymers
Important enzyme: Amyloid βß- protein degrading protease (AßDP)

degrade the amyloid ß-protein (Aß)
potential treatment of Alzheimer’s disease

To discover new amyloid ß-degrading proteases (AßDPs):
A functional cDNA screen was designed to identify proteases capable of lowering net amyloid ß-protein levels using an array of downstream assays. ß-site amyloid precursor protein-cleaving enzyme 2 (BACE2)
Found to be the best enzyme from the cDNA Screening
capable of lowering amyloid ß-protein
Unexpected finding: WHY?
homolog BACE1, very well studied enzyme, does not degrade amyloid ß-protein (Aß) efficiently
150 times less efficient than BACE2. DISCOVERED… 1) Overview and outcome of functional screening for Amyloid ß-protein lowering proteases. 
A, Diagram illustrating the overall design of the screen.
B, Results of the screen, expressed as [Aß]/AP ratios. The largest decrease in Aß levels was achieved by BACE2.
C, BACE2 and its homolog, BACE1, were transfected together into CHO cells. BACE2, but not BACE1, expression resulted in significant decreases in the levels of both Aß40 and Aß42. RESULTS 2) BACE2 and BACE1 degrade Aß in vitro
A, Percent Aß remaining following incubation with different concentrations of recombinant BACE2 for various lengths of time.
B, Comparison the relative Aß-degrading ability of recombinant BACE2 vs. BACE1. Note that 24 h incubation with BACE1 was required to achieve approximately the same extent of degradation as effected by BACE2 in 10 min. RESULTS 3) BACE2-mediated Aß degradation is pH-dependent
Catalytic efficiency of BACE2 depends on pH value.
The rate of hydrolysis of Aß40 across a range a pH values was compared.
BACE2 was found to be maximally effective at pH 3.5 and decreases effectiveness at higher pH values.
These findings strongly suggest that BACE2 would not be operative at the cell surface or within the extracellular space. RESULTS 4) BACE2 degrades Aß more efficiently than well-established AßDPs
 A, Degradation of Aß in vitro by equivalent concentrations (5 nM) of recombinant BACE2, IDE, NEP and plasmin. BACE2 degrades Aß at a faster rate than NEP and plasmin, but not IDE. 

B,C, Effects on Aß40 (A) and Aß42 (C) levels following BACE2, NEP, ECE1b and IDE. BACE2 lowers the levels of both Aß species to an extent exceeding NEP and ECE1b, but comparable to IDE. RESULTS http://possibilistic.org/presentation/alzheimers-cell-phys/img/comparison1.jpg CONCLUSION http://www.alz.org/downloads/facts_figures_2012.pdf SOCIETAL RELEVANCE Cannon, Catherine. “The protein GM-CSF cure arthritis and can fight Alzheimer's.” Julienews.it. 24 Aug. 2010. University of Florida. 20 Feb. 2013. <http://www.sciencebuddies.org/science-fair-projects/project_mla_format_examples.shtml>

Genetic Science Learning Center. "Alzheimer’s Disease." Learn Genetics. 21 February 2013 <http://learn.genetics.utah.edu/content/disorders/whataregd/alzheimers/>

Grad, Jon. “PBB Protein APP.” Photo. Wikipedia.org. 08 March 2007. 20 Feb. 2013. <http://en.wikipedia.org/wiki/File:PBB_Protein_APP_image.jpg>

Hamel, Gregory . “How Do People Get Alzheimer's Disease?” eHow Health. 21 Feb. 2013. Demand Media, Inc. <http://www.ehow.com/how-does_5147737_do-people-alzheimers-disease.html?ref=Track2&utm_source=ask>

Leissring, Malcolm A. “Identification of BACE2 as an avid ß-amyloid-degrading protease.” Molecular Neurodegeneration. 17 Sep. 2012. BioMed Central Ltd . 15 Feb. 2013. <http://www.molecularneurodegeneration.com/content/7/1/46#>

“Mayo Clinic Researchers Identify New Enzyme To Fight Alzheimer's Disease.” Mayo Clinic. 17 Sep. 2012. Mayo Foundation for Medical Education and Research. 15 Feb. 2013. <http://www.mayoclinic.org/news2012-jax/7087.html>

Peltan, I. “APP processing.” Photo. Wikipedia.org. 04 Oct. 2006. 20 Feb. 2013. <http://en.wikipedia.org/wiki/File:APP_processing.png>.

“2012 Alzheimer’s Disease Facts and Figures.” Alzheimer’s & Dementia. 8.2 (2012): 16,17. Web. 20 Feb. 2013. <http://www.alz.org/downloads/facts_figures_2012.pdf>

BIBLIOGRAPHY http://www-news.uchicago.edu/releases/06/images/061011.enzyme.jpg Catalysts
Specific protein molecules that influence rate of chemical reaction
They are not created or destroyed in a reaction
Composed of one or more polypeptides
numerous peptide bonds form a chain-like structure What are enzymes? This study identifies BACE2 as an amyloid ß-degrading protease (AßDP).
High catalytic efficiency and ability to degrade amyloid ß-protein (Aß) intracellularly
BACE2 represents a strong therapeutic candidate for the treatment or prevention of Alzheimer’s disease. Progeria http://newspaper.li/progeria/ Almost all cases of HGPS are caused by a de novo point mutation in the lamin A (LMNA) gene that results in production of a mutant lamin A protein termed progerin.
Without lamin A, the nuclear lamina is unable to provide the nuclear envelope with adequate structural support, causing it to take on an abnormal shape.
Since the support that the nuclear lamina normally provides is necessary for the organizing of chromatin during mitosis, weakening of the nuclear lamina limits the ability of the cell to divide. inhibit the activity of an enzyme needed in order to make a link between progerin proteins and farnesyl groups.
so progerin cannot remain attached to the nucleus rim and it has now a more normal state
Use has been mostly limited to animal models
Phase II clinical trial using the FTI lonafarnib began in May 2007 Farnesyltransferase inhibitors (FTIs) Untreated cells from children with the genetic disease progeria (left) compared to similar cells treated with farnesyltransferase inhibitors (FTIs). In the test tube, FTIs reverse the nuclear damage caused by the disease. Does not directly "cure" the underlying cause of progeria.
Prevents prelamin A from going to the nucleus in the first place so that no prelamin A can build up on the nuclear membrane, but equally, there is no production of normal lamin A in the nucleus. Lamin A does not appear to be necessary for life; mice in which the LMNA gene is knocked out show no embryological symptoms
This implies that it is the buildup of prelamin A in the wrong place, rather than the loss of the normal function of lamin A, that causes the disease. Transgenic mice carrying a copy of the human HGPS mutation (LMNA G608G) display a phenotype characterized by a dramatic and progressive loss of vascular smooth muscle cells (VSMCs) in the media of large arteries
similar to the cardiovascular disease observed in autopsy studies of patients with HGPS.
Another group of Wild type control mice Mice First Part
Start treatment when mice are one month old
150 mg/kg/day, 450 mg/kg/day (comparable to doses delivered to human patients), and untreated mice as control for both wild type and transgenic mice All study mice are
represented on a plot demonstrating the increasing levels of non-farnesylated
HDJ-2 with increasing dose of FTI. FTI prevents the cardiovascular phenotype when treatment is begun immediately at weaning Summary Children with HGPS often display relatively stable cardiovascular disease for the majority of their lives before a dramatic acceleration in symptoms later in life
They will display varying severities of cardiovascular disease.

Does FTIs have an effect on pre-existing disease ? However… Part II:
allowed mice to reach the age of 9 months before beginning a 6-month course of oral FTI treatment.
Either 450 mg/ kg/day or vehicle only. FTI tipifarnib can successfully prevent both the appearance and the late progression of cardiovascular disease.
highly significant improvements in VSMC loss and proteoglycan accumulation in the treated mice.
highest dose (450 mg/kg/day)
Most significant improvements
Exhibits the highest levels of FTI activity Results Treated mice displayed a highly significant arrest in the severe late progression of the loss of VSMCs seen in the untreated transgenic mice (Fig 4B)
This halting of disease progression was dramatic and affected all vessels: descending aorta, ascending aorta, carotid artery, and abdominal aorta (Fig. 4C)
suggests that FTIs may also induce disease regression in mice that already shows phenotypic change May have implications well beyond just this rare condition since all individuals produce small amounts of the mutant progerin protein FTL’s potential Capell, Brian C., Michelle Olive, and Michael R. Erdos. "A Farnesyltransferase Inhibitor Prevents Both The Onset And Late Progression Of Cardiovascular Disease In A Progeria Mouse Model." Proceedings Of The National Academy Of Sciences Of The United States Of America 105.41 (2008): 15902-15907. General Science Full Text (H.W. Wilson). Web. 21 Feb. 2013.
K. Cao, J. J. Graziotto, C. D. Blair, J. R. Mazzulli, M. R. Erdos, D. Krainc, F. S. Collins, Rapamycin Reverses Cellular Phenotypes and Enhances Mutant Protein Clearance in Hutchinson-Gilford Progeria Syndrome Cells. Sci. Transl. Med. 3, 89ra58 (2011).
Meta M, Yang SH, Bergo MO, Fong LG, Young SG (2006). "Protein farnesyltransferase inhibitors and progeria".
http://stm.sciencemag.org/content/3/89/89ra58.abstract Bibliography Factors that lead to Alzheimer's Disease.
Genetics (Family history with Alzheimer's)
Heart disease increases risk
Person's level of education
more education --> less affected by Alzheimer's (have more healthy brain synapses)
People with cognitive problems To reduce risk:
avoid head injuries
keep your heart healthy This protein is permanently modified by a lipid farnesyl group, and acts as a dominant negative, disrupting nuclear structure.
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