Proteomics/Signal Transduction

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Proteomics/Signal Transduction
2012-08-28 00:50:18
Med school

Proteomics and Ahktar lectures
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  1. The study of the genome of an organism is based on DNA, RNA, and/or protein? What methods are used in genomics?
    nucleic acids (DNA and RNA); S. blot, N. blot, and microarrays
  2. What is the study of all the proteins produced in an animal? Does this include modified proteins? What techniques are used to study this proteins in lab setting?
    Proteomics; Yes; MS, MS/MS, SDS-PAGE, W. Blot, HPLC, LC-MS
  3. When to use genomics vs. proteomics?
    Analyzing normal vs. diseased tissue, drug treated cells vs. control
  4. Genomics is not useful in measuring samples that do not contain nucleic acids. 4 examples include?
    Saliva, urine, CSF, and plasma
  5. What is the purpose of using a microarray analysis? How does it work?
    to measure changes in mRNA; purify RNA then convert to cDNA and hybridize them to a probe on microarray to observe changes in gene expression
  6. Relationship between average proteome of humans, plants, and yeasts?
    Plants > Humans > Yeast
  7. 2-D Gel Electrophoesis takes advantage of a protein's ____ and molecular weight to observe proteins?
    Isoelectric point
  8. Trypsin cuts on the ______ side of these two amino acids?
    Caroxyl side of Lysine and Arg.
  9. How does MALDI-ToF or MS work?
    Protein samples is fragmented via trypsin, sample is mixed in a matrix for assistance, laser turns peptides into more fragments, desorption ionizes them to propel them in a dector where the time of flight is measured (time proportional to size)....larger the peptide the longer it takes
  10. How does MS/MS work?
    Same as MS but includes a collision cell and electronic gate that only allows certain peptides in (for example peptides over 1700 KD). Then that peptide is broken down into more fragments. 
  11. What does MS/MS allow you to determine that MS doesnt?
    It allows you to determine AA sequence and molecular weight. 
  12. What are 4 advantages of using MS?
    Fast, Sensitive (can detect small quantities), Simultaneous testing (for testing for numerous diseases), Discriminating (can detect molecules in mixtures)
  13. What is PKU? Symptoms?
    Increase in Phe levels b/c cant convert it to Tyr.
  14. How can noroviruses be detected (using what method) and how can MS help detect norovirus?
    Using PCR; MS can detect capside of virus in human stool
  15. What is the best method for measuring whole-cell effects a drug has?
    A.) microarray
    B.) W. Blot
    C.) Isoelectric Focusing
    D.) Proteomics
    D, you want to use methods that look at proteins
  16. Early diagnosis of cancer can be aided using _____ as a potential identifier at its earliest stages
  17. Three types of extracellular messengers and their subcategories?
    Hormones (peptides, catecholamines, thyriods, steriods); neurotransmitters, ecisanoids
  18. Compare and contrast hyrophilic hormones vs. lipophilic hormone in:

    transport in blood
    receptor site
    mechanism of action
    • transport in blood - H: free, L: transport protein involved 
    • half-life- H: short, L: long
    • receptor site- H: plasma membrane, L: nucleus
    • mechanism of action- H: 2nd messenger, L: transcription factor
  19. Protein Kinase A (cAMP- dependent Kinase)
    Ser/Thr kinase
  20. Protein Kinase G (cGMP dependent kinase)
    Ser/the kinase
  21. Protein Kinase C
    ser/thr kinase
  22. Ca/CaM dependent kinase
  23. Mitogen activated (MAP kinase)
  24. EGF receptor
  25. PDGF
  26. FGF
  27. Insulin
  28. Src Protein
    non-receptor tyrosine kinase
  29. Types of Protein Phosphatases that are Ser/Thr and Tyrosine?
    • Ser/Thr: Type I, IIA, IIB, IIC
    • Tyro: CD45
  30. Autocrine Signaling
    messengers act on cells that released them (cancer)
  31. Paracrine
    the released messengers act on nearby cells (adjacent cells)
  32. Endocrine
    messengers act on cells distant from their site of production
  33. Characteristics of G-protein coupled receptor i?n plasma membrane?
    Extra- and intra-cellular domains with 7 transmembrane domains
  34. G-protein characteristics (subunits that make it up, which has GTPase activity)
    alpha, beta, gamma; alpha can hydrolyze and bind to GTP
  35. Four types of G-proteins, and what differentiates them from each other.
    Gs (stimulatory), Gi(inhibitory), Gq (stimulated phospholipase C-beta), and Gt (aka transducin which stimulates cGMP-dependent phosphodiesterase).

    Only their alpha subunits are different
  36. G-protein coupled receptor mechanism?
    in resting state, alpha/beta/gamma subunits of G-protein are in tact. binding of stimulatory ligand causes alpha subunit to bind to GTP, which causes conformational change. This Gs protein stimulated ADENYL CYCLASE (AC) who then stimulates production of cAMP form ATP. cAMP phosphorylates Protein Kinase A's (PKA) R-R subunits which release two C-C subunits. Once alpha-GTP is hydrolyzed to alpha-GDP, it binds to beta/gamma and the signal stops.
  37. Mechanism of ATP/cAMP production and breakdown using enzymes?
    • ATP (AC) --> cAMP 
    • cAMP (PDE) --> AMP
  38. Methylxanthine examples and fxn?

    Forskolin fxn?
    Methylxanthine = caffeine, theophylline (they inhibit PDE, i.e. they increase cAMP levels)

    Forskolin activates AC (i.e. increase cAMP levels)
  39. Example of stimulatory and inhibitory hormones that affect cAMP levels?
    • Stim: Epi (Beta-1 adrenergic)
    • Inhib: Acetylcholine (muscarinic), and Epi (Alpha-2 adrenergic)
  40. Examples of proteins phosphorylated by PKA?
    Activated Proteins: Phosphoryl kinase, hormone-sensitive lipase, phosphatase inhibitor I, tyrosine hydroxylase (nerve)

    Inhibited proteins: glycogen synthase, pyruvate kinase (liver)
  41. 4 ways of turning of cAMP signaling cascade?
    • 1.) Receptor densensitization (i.e. the longer the R is exposed to ligand, the more densensitized it gets)
    • 2.) Receptor is internalized and degraded
    • 3.) Receptor synthesis is down-regulated
    • 4.) The receptor is densensitized due to phosphorylation via PKA and/or Beta-adrenergic receptor kinase (BARK) and subsequent binding of arrestin to the R
  42. How does Cholera affect cAMP production, Gs- subunit, symptoms?
    Cholera toxin causes ADP-ribosylation of Gs subunit (meaning it can bind GTP but lost its GTPase activity). Therefore, Gs is LOCKED in active mode and stimulates AC who stimulated cAMP. Increase in cAMP causes large efflux of Na+/water ---> severe diarrhea
  43. How does morphine treat symptoms of cholera?
    Morphine inhibits AC via activation of endorphin R's 

    ATP (AC) --> cAMP
  44. Whooping Cough (Pertussis Toxin) mechanism of action?
    Toxin ADP-ribosylates Gi subunit (causing it to say in INACTIVE form b/c cant exchange GDP for GTP). Thus, large increase in cAMP and muscous secretion. 
  45. Diptheria?
    Toxin ADP-ribosylates EF-2, a G-protein that participates in mRNA dependent protein synthesis. Toxin inhibits protein synthesis causing cell death.
  46. Is Ca2+ more inside or outside cell?
  47. Large influx of Ca2+ during stimulation from ligand can be restored by:
    • 1. Ca2+ pump in plasma membrane
    • 2. Ca2+ pumps in mito. and ER that pumps it into these organelles
    • 3. Na/Ca exchanger on plasma membrane
  48. Mechanism for IP3 synthesis?
    PIP2 (Phospholipase C) --> IP3 + DAG

    IP3 and DAG are secondary messenger molecules
  49. Mechanism of hormone-stimulated IP3/DAG system?
    Gq-GTP protein stimulates PIP2 + PLC-beta to create DAG and IP3. DAG stimulates Protein Kinase C and IP3 binds to Ca2+ channels on ER to stimulate release of Ca2+ intracellularly.
  50. IP3 synthesis degradation cycle?
    Treatment for manic-depressive illness?
    • IP4
    •                   ^
    •                    |
    • ...PIP2 --> IP3 --> IP2 --> IP1 --> Free Inositol --> PIP2.... 
    •                                                 INHIBIT IP1 to Free Inositol b/c using Lithium decreases Free Inositol which means less PIP2 to create IP3
  51. What are the second messenger functions of Ca2+ in the IP3/DAG system?
    1. Activation of Ca2+-Calmodulin-dependent protein kinase II (CaM Kinase II), a multifunctional enzyme that phosphorylates proteins at Ser/Thr

    2. Ca2+-ATPase stimulation, which is maintains low intracellular Ca2+ via pump

    3. Myosin light-chain kinase (MLC-Kinase): which phosphorylates MLC for smooth muscle contraction; dephosphorylation of MLC causes smooth muscle relaxation.
  52. Second messenger functions of DAG in IP3 system?
    DAG does not leave the plasma membrane, unlike IP3, and in presence of Ca2+ and Phos. serine (PS), stimulates protein kinase C (PKC). Phorbol esters and tumor supressors also stimulate PKC.
  53. DAG can be converted into what 3 compounds?
    • 1. Phosphatidic Acid: which acts as a Ca2+ ionophore (allows Ca2+ to pass through membrane through a channel)
    • 2. Lyso-PA: stimulat cell growth
    • 3. Arachidonic Acid: which generates prostaglandins, thromboxanes, and leukotrienes
  54. Cyclic GMP Second Messenger Mechanism?
    • GTP (GC) --> cGMP
    • cGMP (cGMP PDE) --> GMP

    **GC exists in both soluble and membrane bound form
  55. Rhodopsin consists of what? Where is it located?
    11-cis retinal (derived from Vitamin A) and Opsin (7 alpha-helices transmembrane protein); around the membraneous disc
  56. Describe phototransduction seq. of events for dark?
    -cGMP levels are high (so they bind to Na+/Ca2+ channels allowing Na+/Ca2+ to flow in cell causing depolarization)

    • -Glutamate is released at synaptic terminal into retinal neuron
  57. Describe phototransduction seq. of events for light?
    • -Light strikes rhodopsin causing 11-cis retinal to isomerize to all-trans retinal
    • -MetaRhodopsin activates Gt in which Gt-alpha binded to GTP activated cGMP PDE which decreases cGMP levels
    • -MetaRhodopsin splits into all-trans retinal + opsin
    • -As a result, Na+/Ca2+ channels close (keeps Ca inside, Na outside)
    • -Decrease in Na+ hyperpolarizes the cell
    • -Glutamate release is suppressed from cell

  58. Intermediate products of rhodopsin during its activation?
    Rhodopsin (+light) --> Bathorhodopsin --> Lumirhodopsin --> Metarhodopsin --> opsin + all-trans retinal

    ** Metarhodopsin activates transducin
  59. Mechanism of rod cells from light back to dark state? 4 ways.
    Rhodopsin Kinase: it phosphorylates rhodopsin at Ser/Thr  and makes it inactive. Arrestin binds and prevents binding and activation of rhodopsin so PDE is not active

    GTPase Activity: Gt-alpha hydrolyzes GTP for GDP

    Resynthesis of cGMP: When Ca2+ levels fall in cell, this stimulates G.C. to create cGMP which opens Na+/Ca2+ channel

    Return of 11-cis retinal: all-trans retinal is converted back into 11-cis retinal which combines with opsin to create rhodopsin
  60. Receptors with enzymatic activities? Answer in this format: 

    Receptor     |     Enzymatic activity
    ANP | Guanylate Cyclase

    Insulin | Tyr Kinase

    EGF | Tyr Kinase

    PDGF | Tyr Kinase

    FGF | Tyr Kinase

    TGF-Beta | Ser/Thr Kinase

    Leukocyte CD45 Protein | Tyrosine Phosphatase
  61. Atrial Natriuretic Peptide (ANP) Mechanism?
    ANP binds to ANP-R which triggers intracellular domain of receptor G.C. activity (i.e. GTP --> cGMP) which activates Protein Kinase G (PKG)
  62. What does ANP do?
    ANP regulates blood pressure, salt, and water balance in body fluids.
  63. Biosynthesis of Nitric Oxide (NO)?
    L-arginine + O2 (Nitric Oxide Synthase) --> Citrulline + NO
  64. NO's function?
    To activate soluble G.C. to synthesize cGMP to relax smooth muscle
  65. Sildenafil (Viagra) mechanism?
    Inhibits cGMP-dependent PDE (i.e. prevents degradation of cGMP to 5'-GMP). Thus, it keeps cGMP levels high for muscle relaxation and maintainence of blood flow. 

  66. Difference between GF (i.e. EGF, FGF) and Insulin Tyrosine kinase receptor activity?
    Growth factor: ligan binds to R; R dimerizes and intracellular domains phosphorylate each other allowing other proteins (i.e. PLC-gamma, PI 3-kinase, etc.) to bind. All these proteins contain Src homology (SH2) meaning they allow other proteins to bind at docking site.

    Insulin: A tetramer whose beta-subunits are phosphorylated via ligand binding. Then, Insulin Receptor Substrate 1 (IRS-1) binds to serve as a docking site for SH2-containing proteins like PLC-gamma, PI 3-kinase, GRB/SOS, others.
  67. Acetylcholine NMJ mechanism?
    Ace bind to R and allows Na+/K+ channel to open and depolarize cell. Depolarization of cell causes Ca2+ influx which causes muscle contraction.
  68. Acetylcholine fxn (in muscarinic and nicotinic Rs)? 
    GABA Fxn?
    Glutamate Fxn?
    Ace Muscarinic: 7-transmembrane Rs coupled to IP3/DAG pathway

    Ace Nicotinic: opens Na+/K+ ion channel

    GABA: Functions as chloride channel

    Glutamate: Opens Ca2+ channel
  69. IRS-1 and EGF can activate what three enzymes/proteins?
    • PLC-Gamma1 
    • PI 3-Kinase
    • Ras
  70. PI 3-Kinase mechanism?
    PIP2 ( PI 3-Kinase) --> PIP3 ---> PDK1 ---> Akt (aka Protein Kinase B) 
  71. Ras signaling mechanism?
    • IRS-1 and/or EGF activate Ras (which has weak ATPas activity). With help of SOS it can bind GTP and activate Raf, MEK, ERK, Transcription Factor, Gene activation. It can shut off using GAP. 
  72. 4 Examples of Intracellular R's?
    • Steriod Hormone R's (glucocorticoids, mineralcorticoids, progestins, androgens, sex hormones)
    • Thyroid hormones R's (thyroxine T4, tri-iodothyronin T3)
    • Retinoic Acid R's
    • Vitamind D R's
  73. How much does Shabnam love you? 
    a) not at all 
    b) a little 
    c) a lot 
    d) more than anything
    d) more than anything! :) 
  74. Steriods (i.e. glucocorticoids) intracellular mechansim of activating transcription?
    Steriods bind to receptor in cytosol at the C-terminal of receptor. In the inactive form the DNA binding domain was covered with Heat Shock Protein (HSP), but when steriod binds to R, HSP dissociates and the Ligand-R complex enters nucleus. In the nucleus the ligand-R complex binds to DNA at the DNA binding domain known as the Hormone Response Element (HRE).
  75. Which compounds bind intracellularly to their receptor in the nucleus and then bind to hormone response element (a specific DNA region)?
    Thyriod, Vitamin D, and Retinoic Acid 

    **there is no Heath Shock Protein with these individuals