Lecture 24: Learning objectives: Human genome<br />
1. The central Dogma says that DNA contains information that codes for proteins, yet only1.5% of human DNA serves direct c...
2. For a typical human gene, how much of the DNA composes exons and how much introns?<br /><ul><li>exons smaller than introns
gene 10-12 times larger than needed to encode information</li></li></ul><li>3. Describe how one gene can code for more tha...
 Tissue specific
Proteome is more complex than the genome
 Other species do not carry out alternative splicing to the same degree</li></li></ul><li>4. ~45% of our genome is repetit...
LINES (L1) – 20%
LTRs – 8%
There are also simple nucleotide repeats (sometimes referred to as Variable Number Tandem Repeats-VNTR or microsatellites)
These are highly variable among individuals and are used in DNA fingerprinting</li></ul>Transcripts – contain genes<br />p...
45% of genome are repetitive sequences
These include clustered structural elements (centromeres and telomeres), and repetitive sequences interspersed between and...
SNPs and disease-causing mutations: Not the same!<br />If you know what a point mutation is, then the description of a SNP...
7. What is the epigenome and how does it differ from the genome?<br />Genome<br />Blueprint for our phenotype<br />Epigeno...
8. Describe the differences between the human mitochondrial genome and the nuclear genome. Are there diseases associated w...
Lecture 25: Learning objectives: Overview of Molecular Genetics - DNA structure<br />
1. Describe the three component parts of the basic monomeric unit of a nucleic acid. In terms of these monomeric units, wh...
5C sugar
atoms designated with  (’)
2’deoxyribose-DNA
ribose in RNA </li></li></ul><li>Bases:<br /><ul><li>Purines Adenine & Guanine (A & G)
9 atom (atoms designated with regular numbers)
Pyrimidines
Cytosine (C), Thymine(T) in DNA, Uracil (U) replaces T in RNA
6 atoms</li></li></ul><li>2. Explain the key difference between a nucleotide and a nucleoside. How might that be important...
3. 5-flourouracil is a chemotherapeutic drug. Explain the basic reason why the patient in clinical correlation 1 had an ad...
4. Describe the covalent bond that link monomer units together in a DNA. Describe the noncovalentbonds that are the basis ...
4. Describe the covalent bond that link monomer units together in a DNA. Describe the noncovalent bonds that are the basis...
5. Explain what is meant by the two strands of DNA being ‘antiparallel’. What is the antiparallelfeature of the double hel...
6. Describe how complementary base pairs in RNA differ from those in DNA.<br />RNAs have secondary structure mediated by c...
7. Explain the notion of Tm in terms of DNA, and how it is related to the A+T or G+C content of the DNA.<br />DNA can be “...
Stability of double helix related to number of h-bonds
Tm is temperature at which 50% of DNA single stranded
Tm related to AT vs GC content (because of difference in # h-bonds in those base pairs)</li></li></ul><li>8. Describe the ...
Lectures 37 Purine metabolism<br />
R-5-P comes from pentose phosphate pathway<br />PRPP (5'-phosphoribosyl 1'-pyrophosphate) is made by adding two phosphates...
precursors<br />Amino acids<br />Glycine<br />Glutamine<br />Aspartate (aspartic acid)<br />Tetrahydrofolate (FH4) <br />A...
	3. Identify the rate limiting enzyme in de novo synthesis of purines. How is it regulated? What role does PRPP play in it...
Regulation of de novopurine synthesis<br />The single most important regulator of de novo purine synthesis is the AVAILABI...
Glutamine: PRPP amidotransferase has two allosteric sites allowing synergistic inhibition by GMP/IMP and AMP.<br />With su...
4. Describe the salvage pathway(s) for synthesis of purines. Explain the rationale behind Adenosine Kinase and why there i...
HGPRT
APRT
Adenosine kinase
Net effect
Recover purines prior to their complete degradation to uric acid</li></ul>3<br />1<br />
4. Describe the salvage pathway(s) for synthesis of purines. Explain the rationale behind Adenosine Kinase and why there i...
Why do we have an enzyme to salvage adenosine, but not the other base nucleosides?
Because there are higher levels of adenosine than other nucleosides
SAM /SAH
SAM=S-adenosylmethionine
These are molecules used in methylationrxns.. So there is a lot of it</li></ul>3<br />
5. Explain what happens if you don’t have the enzyme HGPRT. How does PRPP play a role in this?<br />HGPRT <br />hypoxanthi...
End product of purine degradation is uric acid<br />Pathway eliminates purines from<br />RNA/DNA degradation<br />Excess s...
Chronic Gout can be treated with allopurinol(trade name zyloprin), an inhibitor of xanthineoxidase.<br />Allopurinolhas a ...
7. Describe the factors that can contribute to gout.<br />The pKa of uric acid is 5.4. At physiological pH, nearly all uri...
Lecture 38 Pyrimidine metabolism<br />
1. Identify the enzyme that catalyses the first three steps in pyrimidine biosynthesis in mammals.<br />de novopyrimidine ...
Steps<br />111<br />C<br />A<br />D<br />Reaction 1:  Carbamoyl phosphate synthetase II (CPS II)<br />First, and regulated...
Steps<br />111<br />C<br />A<br />D<br />Reaction 2.  Aspartate transcarbamoylase (ATCase)<br /><ul><li>Adds aspartate to ...
The A in CAD
A separate enzyme in bacteria
Historical importance as first described allosteric enzyme
Bacterial enzyme inhibited by CTP, regulated enzyme in bacteria</li></li></ul><li>Steps<br />111<br />C<br />A<br />D<br /...
Atoms of base from <br />Glutamine (N3)<br />Aspartate (N1through C4)<br />CO2 (C2)<br />Sugar from PRPP<br />2. Identify ...
3. Identify the two enzymes that are critical to make thymidine from dUMP. Describe how the drugs methotrexate and 5-Flour...
4. Describe the synthesis of deoxynucleotides from ribonucleotides. What agents are used in the oxidation/reduction reacti...
Important enzymes in synthesis of pyrimidine nucleotides<br />RR <br />ribonucleotidereductase<br />NDP  dNDP<br />TS<br ...
As we saw in this case study, degradation of 5FU is an important consideration in its use. <br />The enzyme dihydropyrimid...
Folate and Vitamin B12summary (from notes)<br />Folate(the salt of folic acid)<br />water soluble vitamin found in green l...
1. Explain how the enzyme Dihydrofolatereductase (DHFR) is used in converting the vitamin folic enzyme to the active co-fa...
2. Explain the importance in the reaction that converts Methylene FH4 to Methyl FH4, and identify the enzyme that carries ...
3. What is the major source of 1 carbon units for tetrahydrofolateto use? Identify the tree major pathways (or reactions) ...
4. Describe the methyl trap and explain how it ties into deficiency of vitamin B12.<br />MTHFR<br />CH3<br />S-adenosylmet...
5. Describe how a vitamin B12 deficiency can occur, and explain why dietary deficiencies of B12 are rare.<br />B12 deficie...
6. Describe Pernicious Anemia, how it might occur, what specific populations seem to be at most risk, and how it is best t...
Lecture 43: Introduction to signal transduction<br />
1. 	Differentiate between chemical messengers that act via cell membrane receptors versusintracellular receptors in terms ...
Summary steps in steroid hormone/nuclear receptor activation:<br />1. ligand binds receptor.<br />2.Receptor activation- d...
1. N-terminal variable region containing transcriptional activation function 1 (transactivationdomain (TAF-1)- hormone-ind...
3. Describe the pathway activated by a chemical messenger binding to receptors that are tyrosine kinases or that bind tyro...
3. Describe the pathway activated by a chemical messenger binding to receptors that are tyrosine kinases or that bind tyro...
Fibroblast growth factor receptor (FGF-R) is a tyrosine kinase receptor. FGF-R mutations are involved in a variety of cran...
Insulin Receptor is a member of the tyrosine kinasefamily of receptors, but it exists as a pre-formed dimer in cell membra...
4. List the components of G-protein coupled receptors linked to activation of protein kinase A and phospholipase C and how...
4. List the components of G-protein coupled receptors linked to activation of protein kinase A and phospholipase C and how...
know that phosphorylation can either activate or inhibit the activity of an enzyme or other protein</li></ul>recall<br />C...
4. List the components of G-protein coupled receptors linked to activation of protein kinase A and phospholipase C and how...
5. List mechanisms by which chemical messenger signaling is terminated.<br />How are signals terminated?  <br />stimulus a...
Learning Objectives Block2 1
Learning Objectives Block2 1
Learning Objectives Block2 1
Learning Objectives Block2 1
Learning Objectives Block2 1
Learning Objectives Block2 1
Learning Objectives Block2 1
Learning Objectives Block2 1
Learning Objectives Block2 1
Learning Objectives Block2 1
Learning Objectives Block2 1
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Learning Objectives for Lectures 24,25, 30&31, 32, 37,38, 40, 43, 44, 45

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    1. 1. Lecture 24: Learning objectives: Human genome<br />
    2. 2. 1. The central Dogma says that DNA contains information that codes for proteins, yet only1.5% of human DNA serves direct coding functions. Explain why this is true and whatthe rest of our DNA is.<br />~ 1.5% of human DNA encodes information to make proteins (information found in mRNA)<br />(Introns do not contain protein coding information)<br />Remainder? Junk? <br />Structural (centromere, telomere) <br />Regulatory (promoters, regulatory elements)<br />Some genes don’t code for proteins (RNAs to help make proteins) <br />rRNAs, tRNAs, snRNAs<br />newly discovered non-coding RNAs (ncRNAs) <br />micro RNAs (miRNA)<br />small inhibitory RNAs (siRNA)<br />A lot of DNA of “unknown” function <br />transcribed into RNA (transcripts of unknown function)<br />
    3. 3. 2. For a typical human gene, how much of the DNA composes exons and how much introns?<br /><ul><li>exons smaller than introns
    4. 4. gene 10-12 times larger than needed to encode information</li></li></ul><li>3. Describe how one gene can code for more than one protein.<br />RNA transcript<br /><ul><li>Alternative splicing - multiple proteins from a single gene </li></ul>(old notion of one gene one protein doesn’t hold)<br /><ul><li> The rule rather than exception, estimate 75% of genes give rise to multiple proteins by alternative splicing
    5. 5. Tissue specific
    6. 6. Proteome is more complex than the genome
    7. 7. Other species do not carry out alternative splicing to the same degree</li></li></ul><li>4. ~45% of our genome is repetitive elements. Provide three examples of these.<br />Transposable elements- interspersed repetitive elements<br /><ul><li>SINES (Alu)- represent 13% of our genome
    8. 8. LINES (L1) – 20%
    9. 9. LTRs – 8%
    10. 10. There are also simple nucleotide repeats (sometimes referred to as Variable Number Tandem Repeats-VNTR or microsatellites)
    11. 11. These are highly variable among individuals and are used in DNA fingerprinting</li></ul>Transcripts – contain genes<br />portions of the DNA that code for proteins<br />Both strands (+)(-) code for proteins<br /><ul><li>Protein coding genes are ‘unique sequences’
    12. 12. 45% of genome are repetitive sequences
    13. 13. These include clustered structural elements (centromeres and telomeres), and repetitive sequences interspersed between and among genes.</li></li></ul><li>5. Explain what SNPs are and how they contribute to our individuality. Describe a cSNP and explain why they are less common than other SNPs. What is the relationship a SNP and a mutation? & 6. Explain what an allele is.<br />Every individual is genetically unique (even though we all are humans with ~23000 genes)<br />What makes us unique?<br />In the populations there can be many different alleles for a given gene, contributing to genetic diversity of the human species<br />An allele is a different form (sequence) of a particular gene<br />As diploids, we have two alleles of each gene (with the exception of genes on the X and Y chromosome)<br /> SNPs contribute to making alleles different<br />Polymorphisms are sequence differences between individuals, which account in part for these different alleles.<br />SNPs are the most common type of polymorphism<br />There are ~3-5 million sequence variations between two individuals <br />Note: 3.2E9 bp in total human genome<br />75% of these are SNPs<br />There are ~58,000 cSNPs in human genome (the rest are in the non-coding regions)<br />Most SNPs are outside of the protein coding region<br />Example: DYPD genes; 15 cSNPs (SNPs in coding region), 2506 in genes overall<br />cSNPs<br />Nonsynonymous- result in amino acid change in protein<br />More likely to be associated wwith disease phenotypes<br />synonymous- do not result in change in amino acid sequence of protein<br />Can be associated with disease phenotypes if they affect a process of gene expression (transcription, splicing, etc)- regulatory SNPs (rSNPS)<br />These affect the quantity of the gene that is expressed<br />
    14. 14. SNPs and disease-causing mutations: Not the same!<br />If you know what a point mutation is, then the description of a SNP might sound similar. True, both are single-nucleotide differences in a DNA sequence, but SNPs should not be confused with disease-causing mutations. The image to the right shows some tell-tale differences:<br />First, to be classified as a SNP, the change must be present in at least one percent of the general population. No known disease-causing mutation is this common.<br />Second, most disease-causing mutations occur within a gene's coding or regulatory regions and affect the function of the protein encoded by the gene. Unlike mutations, SNPs are not necessarily located within genes, and they do not always affect the way a protein functions.<br />
    15. 15. 7. What is the epigenome and how does it differ from the genome?<br />Genome<br />Blueprint for our phenotype<br />Epigenome<br />Inheritable chemical marks not dependent on gene sequence that dictate how the blueprint should work to determine our phenotype<br />Contributes significantly to phenotype<br />The epigenome is more likely to be influence by environmental factors<br />Epigenomic marks play role in turning gene on and off<br />DNA methylation<br />Methyl groups attached to DNA backbone and regulate transcription<br />Histone modification<br />Chemical tags attach to histones – affect chromatin structure (how tightly DNA is wound)<br />
    16. 16. 8. Describe the differences between the human mitochondrial genome and the nuclear genome. Are there diseases associated with mutations in mitochondrial DNA? If so what are the characteristics of these diseases.<br />Mitochondrial DNA is tightly packed with information (unlike nuclear DNA where space does not seem to pose a problem)<br />These DNA are circular (like bacteria)<br />Human mitochondrial DNA is ~17,000 bp (recall nuclear DNA has 3.2e9 bp)<br />It codes for:<br />22 tRNAs<br />2 rRNAs<br />13 hydrophobic subunits of the ETC enzymes<br />All other mitochondrial proteins are encoded in the nuclear genome, translated in the cytoplasm, and the proteins imported into the mitochondria<br />Several genetic diseases of strict maternal inheritance are caused my mutations in mitochondrial DNA<br />The hallmark of these is:<br />1. Maternal inheritance<br />2. variability in severity of symptoms<br />Note: oxidative damage to somatic cell mitochondrial DNA has a cumulative effect with age and may be related to age-related pathologies<br />
    17. 17. Lecture 25: Learning objectives: Overview of Molecular Genetics - DNA structure<br />
    18. 18. 1. Describe the three component parts of the basic monomeric unit of a nucleic acid. In terms of these monomeric units, what are the chemical differences between RNA and DNA?<br /><ul><li>Base attached to 1’ position of sugar</li></ul>Nucleotide: component parts<br />(Monomeric unit of nucleic acids: DNA and RNA)<br /><ul><li>Nucleotide: 5C sugar, base, phosphate
    19. 19. 5C sugar
    20. 20. atoms designated with (’)
    21. 21. 2’deoxyribose-DNA
    22. 22. ribose in RNA </li></li></ul><li>Bases:<br /><ul><li>Purines Adenine & Guanine (A & G)
    23. 23. 9 atom (atoms designated with regular numbers)
    24. 24. Pyrimidines
    25. 25. Cytosine (C), Thymine(T) in DNA, Uracil (U) replaces T in RNA
    26. 26. 6 atoms</li></li></ul><li>2. Explain the key difference between a nucleotide and a nucleoside. How might that be important in using nucleotide analogs as chemotherapeutic drugs?<br />A purine or pyrimidine base linked to a sugar is a nucleoside.<br />Bases are linked to the sugar (ribose or deoxyribose) with a glycosidicbond between the 1' position of the sugar and the N1 (pyrimidine) or N9 (purine) of the base.<br />Addition of a phosphate to the sugar (usually at the 5' C) generates a nucleoside phosphate or nucleotide.<br />Nucleotides can contain one, two or three phosphates (designated α, β, γ). A single phosphate is a nucleoside monophosphate (e.g. AMP), two phosphates make a nucleoside diphosphate (e.g. ADP) and three phosphates make a nucleoside triphosphate (e.g. ATP).<br />The difference between a nucleoside and nucleotide is relevant to the clinical use of nucleoside analogs in chemotherapy, usually to inhibit DNA synthesis.<br />Nucleosides are uncharged and can get into and out of cells easily. <br />Nucleoside phosphates (nucleotides) cannot because the phosphate group is negatively charged at physiological pH, making it difficult for these molecules to cross the plasma membrane. Thus analog drugs that inhibit DNA synthesis as nucleotides are administered as the corresponding nucleoside, which can get across the cell membrane and be phosphorylated in the cell.<br />
    27. 27.
    28. 28. 3. 5-flourouracil is a chemotherapeutic drug. Explain the basic reason why the patient in clinical correlation 1 had an adverse drug reaction to 5FU.<br />5-Fluorouracil (5-FU) is a uracil analog widely used to treat solid tumors, such as colorectal and breast cancer. This agent has is still used as a major component of cancer chemotherapy. <br />5-FU is a pro-drug that requires activation to 5-fluoro-2-deoxyuridine monophosphate (5-FdUMP). <br />5-FdUMP inhibits tumor cell replication via inhibition of thymidylatesynthase (TS) activity, an enzyme required for de novo pyrimidine synthesis (see page 113).<br />Approximately 85% of an intravenous dose of 5-FU is inactivated in the liver by dihydropyrimidinedehydrogenase(DPYD [page 114,115]), an enzyme that exhibits up to 20-fold variation in activity among individuals due to polymorphisms in the gene. <br />Patients with low DPYD activity cannot effectively inactivate 5-FU, leading to excessive amounts of 5-FdUMP, causing gastrointestinal, and hematopoetictoxicities that are potentially fatal. (CC1)<br />This is a true pharmacogenetic problem, since for the most part patients with DPYD polymorphisms show no abnormal phenotype till challenged with the drug.<br />
    29. 29. 4. Describe the covalent bond that link monomer units together in a DNA. Describe the noncovalentbonds that are the basis for the double helix and indicate the basis of their specificity and their importance. How does this feature explain ‘Chargaff’s Rules’?<br />Phosphoanhydride bonds<br />“high energy”<br />ATP<br />N- Glycosidic<br />bond<br />5’ phosphate<br /> (ester bond)<br />Backbone Sugar phosphates <br />Linked by 3’-5’ phosphodiester bonds<br />Phosphodiester bond<br />Links nucleotide units together in a nucleic acid<br />The covalent bonds within <br />the monomeric unit in DNA<br />Negative charge<br />The phosphate linkage provides <br />a net negative charge to the <br />molecule at physiological pH<br />
    30. 30. 4. Describe the covalent bond that link monomer units together in a DNA. Describe the noncovalent bonds that are the basis for the double helix and indicate the basis of their specificity and their importance. How does this feature explain ‘Chargaff’s Rules’?<br />COMPLEMENTARY BASE PAIRING is the fundamental property of DNA allowing it to act as the repository of genetic information (be self-replicating, transcribed into RNA and have its information translated into the amino acid sequence of a protein)<br />Complementary base pairs are between specific bases on opposite strands of DNA in the double helix<br />A with T : G with C.<br />Pairing is based on the hydrogen bonding potential of specific groups (N, NH and C=O) of the bases.<br />Three factors contribute to stability of the base pairs.<br />1. The steric requirement for a purine:pyrimidinepair to fit inside the double helix.<br />2. The physical alignment of h-bond donor (+) and acceptor (-) groups.<br />3. “base stacking” interactions - negative free energy associated with maximizing hbondsand minimizing exposure of planar aromatic bases to water<br />The result of these forces are stable, specific base pairs, between an adenine base on one strand and a thymine base on the complementary strand, or a guanine base on one strand and a cytosine base on the complementary strand.<br />A - T base pairs contain two h-bonds, G-C base pairs three h-bonds; making G-C base pairs more thermally stable.<br />Prior to the description of the double helix by Watson and Crick, Erwin Chargaff determined that in DNA the molar amount of A always equaled the molar amount of T, and the molar amount of G always equaled the molar amount of C. <br />These became known as Chargaff’s rules. Watson and Crick’s model demonstrated the biochemical basis of Chargaff’s rules.<br />
    31. 31. 5. Explain what is meant by the two strands of DNA being ‘antiparallel’. What is the antiparallelfeature of the double helix really based on?<br />Two strands of opposite polarity (anti-parallel)<br />[implications in replication and transcription]<br />Allows proper alignment of the complimentary base pairs– Essential for forming double helix<br />(recall analogy from lecture of putting pointer and middle finger together.. Don’t fit.. Flip one hand upside down, now they fit)<br />
    32. 32. 6. Describe how complementary base pairs in RNA differ from those in DNA.<br />RNAs have secondary structure mediated by complementary base pairing, but unlike DNA, the base pairing in RNA is intramolecular; the molecules still contain single 5’ and 3’ ends<br />
    33. 33. 7. Explain the notion of Tm in terms of DNA, and how it is related to the A+T or G+C content of the DNA.<br />DNA can be “melted”<br /><ul><li>Base pairs separated by breaking h-bonds with heat or chemicals
    34. 34. Stability of double helix related to number of h-bonds
    35. 35. Tm is temperature at which 50% of DNA single stranded
    36. 36. Tm related to AT vs GC content (because of difference in # h-bonds in those base pairs)</li></li></ul><li>8. Describe the fundamental basis for the hypochromic effect in double stranded DNA<br />Bases are aromatic and absorb UV light<br />Peak absorbance at 260 nm<br />Can be used to estimate concentration of nucleic acid in a solution<br />Hypochromic effect – amount of light absorbed by dsDNA ~50% less than by ssDNA<br />Related to the base-stacking interactions in double-stranded DNA, which decrease absorption of light<br />The change in absorbance when DNA goes from double-stranded to single stranded can be used in DNA melting experiment (figure) to monitor the % DNA that is denatured.<br />
    37. 37. Lectures 37 Purine metabolism<br />
    38. 38. R-5-P comes from pentose phosphate pathway<br />PRPP (5'-phosphoribosyl 1'-pyrophosphate) is made by adding two phosphates from ATP to the C1' position<br />energetically activates at C1' of the ribose where the purine ring will be assembled<br />PRPP is the immediate sugar precursor for both purineand pyrimidinebiosynthesis<br />The enzyme, PRPP synthetase, is highly regulatedby feedback inhibition by purine nucleotides (GDP and ADP)<br />Levels of PRPP are an important determinant of rate of purine biosynthesis<br />1. Describe where PRPP comes from and how it is used in nucleotide synthesis and its importance in determining the overall rate of de novo purine biosynthesis. What regulates PRPP synthetase?<br />
    39. 39. precursors<br />Amino acids<br />Glycine<br />Glutamine<br />Aspartate (aspartic acid)<br />Tetrahydrofolate (FH4) <br />Activated form of folate<br />CO2 (respiratory) <br />2. Identify the specific precursor molecules that contribute atoms to the purine ring.<br />
    40. 40. 3. Identify the rate limiting enzyme in de novo synthesis of purines. How is it regulated? What role does PRPP play in its activity and regulation?<br />First step unique to purine synthesis<br />The amide N from glutamine is attached to the C1' of ribose-5'-phosphate, displacing pyrophosphate to form phosphoribosylamine, glutamic acid and PPi (the N becomes N9 of the purine ring).<br />The enzyme, Glutamine: PRPP amidotransferase, is rate limiting and the regulated enzyme for de novo purine synthesis.<br />
    41. 41. Regulation of de novopurine synthesis<br />The single most important regulator of de novo purine synthesis is the AVAILABILITY of PRPP!!!!!<br />Pathway is regulated at 3 points:<br />1. PRPP synthetase is inhibited by GDP and ADP, end products of purine biosynthesis<br />a. availability of R-5-P from the pentose phosphate pathway and ATP are also important for nucleotide synthesis<br />b. PRPP used for both purine and pyrimidine biosynthesis<br />2. Glutamine: PRPP amidotransferase is feedback inhibited by GMP/IMP or AMP (other phosphorylatednucleotides), and activated by substrate PRPP<br />a. first unique step to purinesynthesis<br />3. at the branch point of IMP: AMP feedback inhibits its own synthesis; GMP feedback inhibits its own synthesis.<br />
    42. 42. Glutamine: PRPP amidotransferase has two allosteric sites allowing synergistic inhibition by GMP/IMP and AMP.<br />With substrate glutamine, a graph of reaction rate vs. substrate concentration is hyperbolic.<br />The Km for glutamine is close to normal physiological concentration of glutamine.<br />With substrate PRPP, the dependence of reaction rate to substrate concentration is sigmoidal.<br />When PRPP levels change, there is a more abrupt change in reaction rate.<br />Activation by substrate represents feed forward regulation by PRPP.<br />Normal physiological concentrations of PRPP are below its Km .<br />Binding of AMP, GMP or IMP to allostericsites shifts the curve to the right, and increases the Km for PRPP.<br />at high GMP/IMP or AMP and normal physiological concentrations of PRPP, very little enzymatic conversion to phosphoribosylamine occurs and the pathway is essentially shut down.<br />However, at high PRPP concentrations, inhibition by nucleotides is overcome<br />at the branch point of IMP: AMP feedback inhibits its own synthesis; GMP feedback inhibits its own synthesis.<br />Glutamine:PRPPamidotransferase2 substrates, 2 independent Kms<br />Gln + PRPP phosphoribosylamine + glu +PPi<br />
    43. 43. 4. Describe the salvage pathway(s) for synthesis of purines. Explain the rationale behind Adenosine Kinase and why there is this separate enzyme to phosporylate this nucleoside.<br />2<br />A salvage pathway for purine<br />biosynthesis is the primary source of purine nucleotides in cell types other than liver and brain. Salvage recaptures purine bases on their way to catabolic degradation, re-attaching them to PRPP generating IMP,<br />GMP and AMP.<br />Summary ofSalvage Pathway<br /><ul><li>3 reactions
    44. 44. HGPRT
    45. 45. APRT
    46. 46. Adenosine kinase
    47. 47. Net effect
    48. 48. Recover purines prior to their complete degradation to uric acid</li></ul>3<br />1<br />
    49. 49. 4. Describe the salvage pathway(s) for synthesis of purines. Explain the rationale behind Adenosine Kinase and why there is this separate enzyme to phosporylate this nucleoside.<br />Adenosine kinase<br /><ul><li>Phosphorylates adenosine nucleoside to AMP
    50. 50. Why do we have an enzyme to salvage adenosine, but not the other base nucleosides?
    51. 51. Because there are higher levels of adenosine than other nucleosides
    52. 52. SAM /SAH
    53. 53. SAM=S-adenosylmethionine
    54. 54. These are molecules used in methylationrxns.. So there is a lot of it</li></ul>3<br />
    55. 55. 5. Explain what happens if you don’t have the enzyme HGPRT. How does PRPP play a role in this?<br />HGPRT <br />hypoxanthine-guanine phosphoribosyltransferase<br />Attach Hx or G to PRPP<br />The importance of HGPRT is evidenced by the pathology associated with Lesch-Nyan syndrome (genetic defect in HGPRT), severe hyperuricemia.<br />results in breakdown of excess Hx and G to uric acid<br />failure to use PPRP stimulates synthesis of more purines, which are ultimately degraded to uric acid.<br />
    56. 56. End product of purine degradation is uric acid<br />Pathway eliminates purines from<br />RNA/DNA degradation<br />Excess synthesis<br />Dietary sources<br />Overall<br />Remove phosphates<br />Remove sugars<br />Oxidize bases <br />removes exocyclic amino groups<br />The key enzyme in purine catabolism is xanthineoxidase. It is a non-hemeiron containing enzyme that uses FAD and molybdenum (VI) as co-factors and converts hypoxanthine to xanthine, and xanthineto uric acid.<br />6. Which enzyme of purine degradation is inhibited by the drug Allopurinol? Why is this drug given for treatment of chronic gout?<br />
    57. 57. Chronic Gout can be treated with allopurinol(trade name zyloprin), an inhibitor of xanthineoxidase.<br />Allopurinolhas a very high affinity for xanthineoxidase.<br />It is also a weak substrate, but the product, alloxanthine, is also an inhibitor<br />Allopurinolis effective in two ways.<br />First by inhibiting xanthineoxidase, it favors excretion of the more soluble products hypoxanthine and xanthine.<br />Second, allopurinol is converted by HGPRT to the corresponding nucleotide that feedback inhibits PRPP synthesis and purine synthesis.<br />Allopurinolmay decrease frequency of gouty attacks, but does nothing to lessen the pain of an acute attack.<br />Also addresses overproduction, not under-excretion<br />6. Which enzyme of purine degradation is inhibited by the drug Allopurinol? Why is this drug given for treatment of chronic gout?<br />
    58. 58. 7. Describe the factors that can contribute to gout.<br />The pKa of uric acid is 5.4. At physiological pH, nearly all uric acid is in the form of sodium urate.<br />urateis insoluble at concentrations near the normal physiological range. Excess uric acid (hyperuricemia), occurs in Gout. Urate crystals precipitate, particularly in the joints, causing severe inflammation and pain.<br />Etiology of gout is not always clear. Familial Xlinked gout may be caused by<br />1. mutations resulting in mis-regulation of PRPP synthetaseresulting in failure to decrease activity in presence of excess purines<br />2. partial inactivation of HGPRT<br />3. factors governing uric acid re-uptake and excretion (80 % of uric acid is excreted in the urine, 20 % in the feces).<br />Secondary gout can be caused by tissue destruction (which increases purine degradation and uric acid production) or diuretics for used for treatment of hypertension<br />excess consumption of alcohol exacerbates gout by acting as a diuretic, and causing lactic acidosis, which interferes with the ability to excrete uric acid.<br />diet rich in organ meats<br />
    59. 59. Lecture 38 Pyrimidine metabolism<br />
    60. 60. 1. Identify the enzyme that catalyses the first three steps in pyrimidine biosynthesis in mammals.<br />de novopyrimidine biosynthesis-Make the pyrimidine ring first, then attach to PRPP<br />C A D catalyzes first three steps in pyrimidine ring synthesis<br />CPS II<br />ATCase<br />Dihydrooratase<br />See next three slide taken from lecture<br />
    61. 61. Steps<br />111<br />C<br />A<br />D<br />Reaction 1: Carbamoyl phosphate synthetase II (CPS II)<br />First, and regulated step in eukaryotes<br />Condensation of CO2, an NH2 from glutamine, and a phosphate from ATP to form carbamoyl phosphate<br />Feedback inhibited by UTP<br />Similar reaction catalyzed by CPS I in mitochondria – entry of NH3+ into urea cycle: CPS II in the cytoplasm<br />In mammals, CPS II part of a single multifunctional protein called CAD catalyzes the first three reactions in pyrimidine biosynthesis. <br />Named for the first three activities, C = CPSII<br />
    62. 62. Steps<br />111<br />C<br />A<br />D<br />Reaction 2. Aspartate transcarbamoylase (ATCase)<br /><ul><li>Adds aspartate to carbamoyl phosphate
    63. 63. The A in CAD
    64. 64. A separate enzyme in bacteria
    65. 65. Historical importance as first described allosteric enzyme
    66. 66. Bacterial enzyme inhibited by CTP, regulated enzyme in bacteria</li></li></ul><li>Steps<br />111<br />C<br />A<br />D<br />Reaction 3. Dihydrooratase<br />the D in CAD<br />Ring closing by dehydration<br />
    67. 67. Atoms of base from <br />Glutamine (N3)<br />Aspartate (N1through C4)<br />CO2 (C2)<br />Sugar from PRPP<br />2. Identify the specific precursor molecules that contribute atoms to the pyrimidine ring.<br />
    68. 68. 3. Identify the two enzymes that are critical to make thymidine from dUMP. Describe how the drugs methotrexate and 5-FlouroUracil used in cancer chemotherapy, inhibit thymidinesynthesis. Why is thymidine synthesis a good chemotherapeutic target? How does the compound folinic acid factor into the inhibition of thymidine synthesis by FOLFOX 4?<br />Thymidine synthesis from dUMP by adding a methyl group to the base at the 5 position.<br />1. The methyl group comes from N5,N10 methyleneFH4 (tetrahydrofolate) – Another important use of folic acid<br />2. The reaction is catalyzed by thymidylatesynthase.<br />key enzyme because thymidine is uniquely needed for DNA synthesis making it an excellent target for drugs used to inhibit DNA synthesis and cell growth. (5-fluorouracil (5FU) clinical correlation 1)<br />3. N5,N10 methylene FH4 is converted to dihydrofolate (FH2) in the reaction.<br />FH4 must be regenerated for continued thymidine synthesis.<br />catalyzed by dihydrofolatereductase (DHFR)<br />also a key enzyme for thymidine synthesis and a target for chemotherapy.<br />Methotrexateis a folate analog and a competitive inhibitor of DHFR commonly used to metabolically inhibit DNA synthesis.<br />
    69. 69. 4. Describe the synthesis of deoxynucleotides from ribonucleotides. What agents are used in the oxidation/reduction reaction? What inhibits the reaction?<br />Deoxynucleotides:<br />Made from ribonucleosidediphosphates (NDPs)<br />Catalyzed by Ribonucleotidereductase(nucleoside diphosphatereductase)<br />Removal of O at 2’ position<br />Reduction reaction; something must be oxidized<br />Immediate reducing agent for the reaction <br />Pair of sulfhydril group on small redox proteins (thioredoxinor glutaredoxin)<br />Oxidized to disulfides<br />Regenerate -SH via thioredoxinreductase<br />Use NADPH – ultimate reducing agent<br />Regulation<br />Ribonucleotidereductase inhibited by dATP<br />Hydroxyurea (drug), also inhibits RR<br />There is little need for dNTPs when cells are not actively synthesizing DNA. The key enzymes for DNA synthesis, (RR, TS, DHFR, TK (ribonucleotidereductase, thymidylatesynthetase, dihydrofolatereductase and thymidinekinase)are cell cycle regulated such that their expression is increased as cells pass from G1 to S phase, when DNA synthesis occurs.<br />
    70. 70. Important enzymes in synthesis of pyrimidine nucleotides<br />RR <br />ribonucleotidereductase<br />NDP  dNDP<br />TS<br />thymidylatesynthetase<br />dUMP dTMP<br />(FH4FH2)<br />DHFR<br />dihydrofolatereductase<br />FH2FH4<br />TK<br />thymidinekinase<br />TMPTTP<br />TS<br />DHFR<br />TK<br />
    71. 71. As we saw in this case study, degradation of 5FU is an important consideration in its use. <br />The enzyme dihydropyrimidinedehydrogenase (DPYD) is an enzyme in the pyrimidine degradation pathway. <br />The gene is polymorphic in the population with a significant number of people containing mutations that lead to loss of function. <br />However, since failure to degrade pyrimidines does not ordinarily lead to pathology, the polymorphism goes unnoticed until a person is challenged with high doses of the pyrimidine 5FU.<br />5. Describe the role of DPYD (dihydropyrimidinedehydrogenase) in pyrimidine degradation. Why are the levels of this activity only problematic when you treat a patient with 5FU?<br />pyrimidine degradation<br />DPYD<br />
    72. 72. Folate and Vitamin B12summary (from notes)<br />Folate(the salt of folic acid)<br />water soluble vitamin found in green leafy vegetables. <br />participates in a select set of 1 carbon transfers as the active co-factor tetrahydrofolate (FH4)<br />One of its major uses is in the synthesis of nucleotides and deoxynucleotides to make RNA and DNA. <br />Folatedeficiency in pregnant women is the major cause of neural tube defects. <br />This may be related to decreased ability to make DNA and RNA, but <br />could also be caused by a decreased ability to methylate DNA. <br />Although folate does not directly donate methyl groups to DNA, it does donate a methyl group to Vitamin B12 which is used to generate S-adenosylmethionine, the direct DNA methylating agent. <br />Vitamin B12, (cobalamin), is another water soluble vitamin. <br />One of its two uses is in re-methylation of homocysteine to methionine, a reaction requiring methylated vitamin B12. <br />The ultimate source of the methyl group is N5-methyltetrahydrofolate. <br />Formation of N5-methyltetrahydrofolate is irreversible and the only way to regenerate FH4 is for methylated-FH4 to donate its methyl group to vitamin B12. <br />Pernicious anemia is the major symptom of vitamin B12 deficiency and results from a secondary deficiency in folate related to its being trapped in the otherwise unusable form, N5-methyltetrahydrofolate. <br />This is referred to as the methyl trap. <br />Since we store large amounts of B12 in the liver and B12 is abundant in the food supply, dietary deficiencies per se are rare. <br />However, there are defects in uptake of B12 secondary to stomach or intestinal surgery that result in B12 deficiency. <br />More commonly there is an age-related decrease in B12 uptake that results in B12 deficiency in the elderly.<br />
    73. 73. 1. Explain how the enzyme Dihydrofolatereductase (DHFR) is used in converting the vitamin folic enzyme to the active co-factor Tetrahydrofolate (FH4). What other pathway is DHFR important for?<br />Folate= folic acid (a vitamin!)<br />DHFR required to convert folic acid to its active co-factor form FH4<br />2 rxns, both utilized NADHP + H+ as a source of reducing equivalents<br />DHFR is also important in pyrimidine synthesis <br />Thymidine synthesis from dUMP by adding a methyl group to the base at the 5 position.<br />N5,N10 methylene FH4 is converted to dihydrofolate (FH2) in the reaction.<br />See Lecture 38 #3<br />DHFR reduces <br />these 2 double <br />bonds<br />
    74. 74. 2. Explain the importance in the reaction that converts Methylene FH4 to Methyl FH4, and identify the enzyme that carries out the reaction.<br />FH4 carries carbons in several oxidation states. <br />In humans, the oxidation states are reversible up to methylene, but not from methyleneto methyl. <br />This is an important part of the methyl trap hypothesis.<br />The conversion of methylene FH4 to methyl FH4 is catalyzed by methylenetetrahydrofolatereductase (MTHFR)<br />
    75. 75. 3. What is the major source of 1 carbon units for tetrahydrofolateto use? Identify the tree major pathways (or reactions) that use FH4.<br />Thymidinesythesis<br />(thymidylatesynthase)<br />TS<br />DHFR<br />Major source of 1C units for thransfer thru FH4 is serine<br />3 major recipients that us FH4:<br />1. purines(rxn5)<br />2. pyrimidine (just thymidine) <br />deoxyuridineto form deoxythymidine (using N5,N10 methylene FH4) (rxn6)<br />3. vitamin B12 (cobalamin) to form CH3 - Vit B12 (methylcobalamin) (using N5-methyl FH4). (rxn 8)<br />This is the ONLY reaction which uses methyl FH4<br />A deficiency in B12 traps folate as N5-methyl FH4 this is sometimes referred to as the methyl trap<br />
    76. 76. 4. Describe the methyl trap and explain how it ties into deficiency of vitamin B12.<br />MTHFR<br />CH3<br />S-adenosylmethionine (SAM) is the major methyl donor used in methylating DNA and RNA<br />When SAM donates a methyl it is converted to SAH which loses its adensoine (see Lec 37 #4- why purine salvage pathway has a separate enzyme [adenylatekinase] to salvage this nucleoside and we have no mechanism to salvage any other nucleosides).<br />Methylation of Vitamin B12only reaction that uses N5methyl FH4<br />Formation of N5methyl FH4 from N5N10methylene FH4 is irreversible<br />B12 deficiency – (acceptor for methyl group), traps folate in an unusable form, leading to secondary deficiency of folate (diminished nucleotide synthesis)<br />MTHFR (methylenetetrahydrofolatereductase) is regulated (inhibited) by SAM<br />B12 deficiency- lots of homocysteine, cant make nucleotides, cant regenete SAM, MTHFR is not inhibited--- methylFH4 accumulates and can’t be used for anyhting (secondary deficiency to folate)<br />
    77. 77. 5. Describe how a vitamin B12 deficiency can occur, and explain why dietary deficiencies of B12 are rare.<br />B12 deficiency not dietary, due to failure to absorb<br />VitB12 is only needed in small amounts<br />It is very abundant in food supply<br />Also, mammals store large amounts (mg quantities ) in the liver<br />True dietary deficiencies rare <br />Severe, chronic malnutrition (alcoholics) <br />Strict vegans??<br />B12 deficiency not dietary, due to failure to absorb<br />Process of absorption of B12 in ileum leads to its storage in the liver:<br />B12binds to R-binders<br />Protein made by salivary gland and/or gastric cells <br />(transcobalamin I)<br />B12 transferred from R-binder to Intrinsic Factor<br />glycoprotein made by gastric parietal cells<br />R-binder degraded by pancreatic proteases, releases B12<br />B12 binds to IF (protease resistant)<br />B12 absorbed in the ileum, in complex with IF, [by IF receptor]<br />B12 transferred to transcobalamin II for transport in the blood<br />Stored in large amounts in liver<br />
    78. 78. 6. Describe Pernicious Anemia, how it might occur, what specific populations seem to be at most risk, and how it is best treated.<br />Pernicious anemia<br />from defects in B12 absorption<br />Surgical resection of stomach or ileum<br />Malabsorption disorder Crohn’s disease; Celiac Sprue<br />High intestinal pH<br />Decreased degradation of R-binder and B12 release<br />Low acid secretion in stomach (long term use of proton pump inhibitors for acid reflux)<br />Age related decrease in synthesis of Intrinsic Factor<br />pernicious anemia common in the elderly: 10% of those over 75 clinical deficiency<br />Pernicious anemia<br />Symptoms - megaloblastic anemia<br />Maturation of RBC requires adequate folate for RNA and DNA synthesis<br />B12 deficiency results in secondary folate deficiency due to methyl/folatetrap<br />Pernicious anemia is treated with injections of B12<br />Folate supplementation treats anemia but can mask neurological deficits.<br />B12 deficiency can also cause neurological symptoms<br />
    79. 79. Lecture 43: Introduction to signal transduction<br />
    80. 80. 1. Differentiate between chemical messengers that act via cell membrane receptors versusintracellular receptors in terms of ligand transport, general receptor structure, mechanism ofaction, and intracellular effects.<br />
    81. 81. Summary steps in steroid hormone/nuclear receptor activation:<br />1. ligand binds receptor.<br />2.Receptor activation- dissociation of chaperone proteins, Heat Shock Proteins, e.g., hsp90, hsp70, and dimerization<br />3. receptor binds to specific DNA sequences called hormone response elements (HREs)- usually located in 5’ enhancer region of a target gene<br />5. hormone-receptor-DNA complex is recognized by coactivatorsthat have histoneacetyltransferase (HAT) activity that help “open” chromatin and interact with components of the RNA polymerase II transcription initiation complex (see Dr. Geoghegan’s lectures 27-9)<br />6. increase in transcription of target gene – mRNA synthesis(5’-3’)<br />7. translation of mRNA into protein<br />8. protein activates cellular response(s) – inside cell or communication with adjacent cells<br />Location of intracellular receptors:<br />Cytoplasmic and/or nuclear – then act in the NUCLEUS <br />
    82. 82. 1. N-terminal variable region containing transcriptional activation function 1 (transactivationdomain (TAF-1)- hormone-independent;<br />2. central DNA binding domain (DBD)– receptor has 2 zinc fingers that physically interacts directly with DNA in the major groove and along the phosphate backbone;<br />3. C-terminal ligand binding domain (LBD) – where hormone binds non-covalently in the “ligandbinding cavity” or “pocket” – LBD has AF-2 that is hormonedependent<br />2. Name the 3 major domains of steroid/nuclear receptors and describe the function.<br />Steroid receptors are hormone-dependent transcriptional activators<br />
    83. 83. 3. Describe the pathway activated by a chemical messenger binding to receptors that are tyrosine kinases or that bind tyrosine kinases.<br />Receptors that are kinases or that bind kinases:<br />Growth factor and cytokine receptors share the common feature that the intracellular domain of the receptor or an associated protein is a protein kinase that when the growth factor/cytokine (ligand) binds the extracellular domain, becomes active. <br />The activated receptor kinasephosphorylates an amino acid residue on the receptor itself (autophosphorylation) or an associated protein. <br />The message is propagated through signal transducer proteins that bind the activated receptor complex, e.g., STAT or Smad.<br />Tyrosine Kinase Receptors are monomers in the cell membrane with a single transmembrane spanning region). <br />When the ligand, i.e., a growth factor, binds the receptor, the receptor dimerizes which allows activation of the intracellular tyrosine kinase domain in each receptor monomer which then phosphorylate each other on certain tyrosine residues (autophosphorylation). <br />The tyrosine-phosphorylated intracellular domain forms specific binding sites for signal transducer proteins.<br />
    84. 84. 3. Describe the pathway activated by a chemical messenger binding to receptors that are tyrosine kinases or that bind tyrosine kinases.<br />Ras and the MAP kinase pathway. <br />The activated tyrosine kinase domain forms a binding site for proteins with a SH2 domain. <br />The adaptor protein Grb2, which is bound to a membrane phophoinositide, has an SH2 domain.<br />When the SH2 domain of Grb2 binds the activated (tyrosine-phosphorylated) domain of an activated growth factor receptor, a conformational change occurs in another domain of the Grb2 protein called the SH3 domain which is a binding site for the protein SOS – do not worry about this name. <br />SOS is a guanine nucleotide exchange factor (GEF) for Ras, a monomeric G protein located in the plasma membrane. <br />SOS activates exchange of GTP for GDP on Rascausing a conformational change in Ras that promotes Raf binding to Ras. <br />Rafis a serine protein kinasethat is a MAPKKK (mitogen activated protein kinasekinasekinase). <br />Rafinitiates a sequence of successive phosphorylationsof intracellular protein kinases called a phosphorylation cascade. <br />The MAP kinase cascade terminates at the phosphorylation of a number of transcription factors, which one or ones depends on the cell type. <br />The phosphorylation of the transcription factor regulates its activity, for example stimulating its activity and increasing transcription of genes involved in cell growth and survival.<br />You do not need to memorize every protein in this pathway, but know the concept.<br />KNOW that activation of Tyr-kinase<br />Receptors leads to activation of<br />Ras (a guanine nucleotide exchg. factor)<br />And consequently of the MAPK pathway<br />
    85. 85. Fibroblast growth factor receptor (FGF-R) is a tyrosine kinase receptor. FGF-R mutations are involved in a variety of craniosyntoses and chondrodysplasias<br />Diseases associated with mutations in FGF-R receptor<br />Apert’s syndrome<br />Craniofacial abnormalities<br />Mental retardation<br />Achondroplasia<br />Most common form of dwarfism<br />
    86. 86. Insulin Receptor is a member of the tyrosine kinasefamily of receptors, but it exists as a pre-formed dimer in cell membranes. <br />Each monomer consists of 2 domains: α and β with the α forming the binding surface for insulin and the β subunits having a single transmembraneregion plus tyrosine kinase activity. <br />When insulin binds to the α subunits, the β subunits become active and autophosphorylate each other. <br />The activated tyrosine kinasephosphorylatesproteins with IRS (insulin receptor substate) at multiple sites thus creating binding sites for other proteins with SH2 domains, e.g., Grb2, PLCγ, and PI3 kinase.<br />Thus, insulin binding to the insulin receptor can activate MAPKsignalingvia Grb2 activation, DAG and IP3 through activation of PLCγ, and downstream targets of PI3K including protein kinase B (also called AKT) which in turn, phosphorylatesother proteins<br />Activation of the insulin receptor leads to phosphorylation of target proteins that either activate or inhibit the activity of these proteins, resulting in physiological responses.<br /> insulin receptor: present in virtually all tissues, but the major sites of insulin action are liver, muscle, and adipose tissue<br />insulin has distinct effects in other tissues including pancreas, kidneys, brain, lungs, immune system, platelets, nervous system, and bone.<br />You do not need to memorize every intermediary protein, but know the concept, i.e., that insulin activates an intracellular tyrosine kinase pathway.<br />ras<br />MAPK<br />
    87. 87. 4. List the components of G-protein coupled receptors linked to activation of protein kinase A and phospholipase C and how cAMP, DAG, and IP3 activate intracellular signaling.<br />Clinical Relevance: 27% of all FDA-approved drugs target GPCRs<br />G-protein coupled receptors (GPCRs) <br />7 transmembrane regions<br />C-terminus in cytoplasm<br />E = effector: e.g.,adenylatecyclase, phospholipase C, <br />Trimeric<br />G protein:<br />a, b-g subunits<br />GDP inactive GTP active<br />Heptahelical Receptors (7 transmembrane spanning α- helices): These are also called G-protein-coupled receptors and they are the most common type of membrane receptor. Binding of the hormone activates the receptor via a G-protein-coupled mechanism to generate second messengers, which are small non-protein compounds such as cAMP inside the cell’s cytoplasm. Second messengers are present in low concentrations so that the message can be rapidly initiated and terminated. Different heptahelical receptors bind different G proteins that exert different effects on their target proteins.<br />G-proteins are heterotrimers: α,β,γ subunits When ligandbinds to a heptahelicalreceptor, G- proteins, which are anchored in the cell membrane, are activated causing the release of GDP and binding of GTP to the Gα-protein that activates adenylatecyclase which produces Camp, the second messenger molecule.<br />
    88. 88. 4. List the components of G-protein coupled receptors linked to activation of protein kinase A and phospholipase C and how cAMP, DAG, and IP3 activate intracellular signaling.<br />cAMP activates PKA (protein kinase A)<br />PKA phosphorylates intracellular targets<br />cAMP<br />phosphodiesterase<br />Activation of Protein Kinase A (PKA) by cAMPresults in PKA activation and PKA phosphorylatesintracellular target proteins. cAMPis hydrolyzed to AMP (inactive) by phosphodiesterase.<br />know 3 aa that can be phosphorylated- Ser, Thr, Tyr<br /><ul><li>know that phosphorylation is reversible
    89. 89. know that phosphorylation can either activate or inhibit the activity of an enzyme or other protein</li></ul>recall<br />CFTR<br />
    90. 90. 4. List the components of G-protein coupled receptors linked to activation of protein kinase A and phospholipase C and how cAMP, DAG, and IP3 activate intracellular signaling.<br />Phosphatidylinositol pathway: <br />Phosphatidylinositol 4’,5’ bisphosphate (PI-4,5-bisP, PIP2) is cleaved by phospholipase Cto generate 2 second messengers in the cytoplasm: diacylglycerol (DAG) and inositoltrisphosphate (IP3) <br />IP3 activates release of intracellular Ca++ that activates Ca++- calmodulinkinase that phosphorylatesintracellular proteins<br />DAG activates protein kinase C that phosphorylatesintracellular proteins.<br />This diagram shows a G-protein coupled with phospholipase C<br />know this pathway<br />
    91. 91. 5. List mechanisms by which chemical messenger signaling is terminated.<br />How are signals terminated? <br />stimulus abates: messenger no longer released<br /> messenger may be rapidly degraded, e.g., peptide hormones<br />Receptor may be desensitized by hydrolysis of phosphate residues by phosphatases<br />Degradation of 2nd messenger, e.g., cAMP, IP3 – GTPase – GDP by phosphodiesterases<br />Internalization of receptor- degradation- recycling, e.g. insulin receptor<br />Signal Termination<br />Some signals needed to regulate metabolic responses or transmit nerve impulses must be rapidly turned off when the hormone is no longer being produced or released. On the other hand, signals that stimulate cell proliferation may turn off more slowly. Many chronic diseases are caused by failure to terminate a response at the appropriate time.<br />

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