Biochemistry: Notes & Objectives
Biochemistry: NOTES & OBJECTIVES (page 165 of 165)
Biomolecular Chemistry 704Human Biochemistry:
NOTES & OBJECTIVESFall 2005last updated 1/4/2007Christopher
B. Kolar
[email protected] study guide has been created in the course
of my studies at the University of Wisconsin School of Medicine and
Public Health. It is intended as an exam review of the required
learning objectives. It references a variety of course materials,
including lecture, Power Point, assigned readings, and sometimes
outside sources. While I have attempted to make it as thorough,
specific, and accurate as possible, I cannot guarantee this, so use
it at your own risk. If you have any questions or comments, or have
found an error within the text, please feel free to contact me.
COLOR KEY: red:
diseases
blue:
medications
orange:
enzymes and compounds
pink:
microorganisms
FORMAT KEY:
margins:1
tab stops:0.25
font:
Times New Roman
size:
10
1. Fundamentals of Protein StructureTABLE pK Values for Common
R-groupsgroupapproximate pK
-carboxyl (FREE)3 (C-terminal)
-carboxyl (Asp), -carboxyl (Glu)4
imidazole (His)6
sulfhydryl (Cys)8
1 -amino (FREE)8 (N-terminal)
2 -amino (Pro, FREE)9 (N-terminal)
-amino (Lys), phenolic hydroxyl (Tyr)10
guanido (Arg)12
- Henderson-Hasselbalch equation:
;
- modifications
- phosphorylation:attachment of a phosphoryl group to a hydroxyl
group, extruding water
- N-glycosylation:attachment of a sugar to an amine (commonly
with Asn)
- O-glycosylation:attachment of a sugar to an oxygen (commonly
on Ser, Thr, modified residues)
- hydroxylation:attachment of a hydroxyl group to the R group
(commonly on Pro, Lys)
- carboxylation:attachment of a carboxyl group to the R group
(commonly on Glu)
- nomenclature
- N-terminus to C-terminus
- substitute yl for ine, except for aspartyl, asparaginyl,
glutamyl, glutaminyl, cysteinyl, tryptophanyl- peptide bond
formation: carboxyl group + primary amine group ( peptide bond +
H2O- protein structure
- primary structure: sequence of amino acids
- secondary structure: common organizations of structure
- -helix
- 3.6 amino acids per helical turn, with each AA able to
participate in up to two H-bonds
- H-bonds: between =O, H-N- within helix, connecting i to
i+4
- little Pro due to incompatibility with helix angle; little Gly
due to being too free to form tight conformations
- -pleated sheet
- stretched polypeptide chains running either parallel or
antiparallel, depending on chain orientation
- H-bonds: between carbonyl and amide hydrogens of adjacent
chains
- turn
- allows protein backbone to make abrupt turns
- abundant Pro, Gly, due to stearic considerations
- tertiary structure: three-dimensional structure
- quaternary structure: interaction of tertiary domains
- classes of proteins
- enzymes:
accelerate the attainment of equilibrium
- structural:form biological structures
- transport:
carry biochemically important substances
- defense:
protect the body from foreign invaders
2. Enzyme Kinetics- general features of catalysts
- enzymes do not alter the final equilibrium ratio of substrates
and products
- enzymes act by lowering the activation energy of a
reaction
- enzymes do not determine the direction of a reaction
- catalysts are not used up during a reaction
- enzymes have an active site that positions AA R-groups in the
proper position for catalysis
- coenzymes and prosthetic groups
- coenzyme: loosely bound (KD of 10-5 to 10-7)
- prosthetic group: tightly bound (KD of 10-9, or covalently
bound)
- enzyme activity
- measurement of [product] vs. [time]
- slope changes with the factor of the enzyme (for arbitrary
units, m=2 with twice as much enzyme)
3. Factors Affecting Enzyme Activity- constants
- Vmax: maximum velocity of a reaction
- extensive property (more enzyme ( higher Vmax)
- Km: Michaelis-Menten constant
- intrinsic property (Km remains the same regardless of enzyme
concentration)
- substrate concentration at which enzyme is operating at half
of Vmax
- high Km: rate only approaches Vmax at high substrate
concentrations
- low Km: enzyme functions near Vmax even at lower substrate
concentrations
- equations
- Michaelis-Menten:
- Lineweaver-Burke (reciprocal plot):
- inhibition
- competitive inhibition: binds only to free enzyme (typically
at active site)
- Vmax:
no effect
- Km:
raises apparent Km
- saturation plot:shifts Km to the right, graph approaches Vmax
more slowly
- reciprocal plot:affects slope: rotates graph counterclockwise
on the y-intercept
- equation:
factor (1 + [I]/Ki) added to slope term
- non-competitive inhibition: binds to E or ES complex
(typically at distant binding site)
- Vmax:
decreases
- Km:
no effect
- saturation plot:flattens graph, but doesnt affect rate of
achieving Vmax
- reciprocal plot:affects slope and y-intercept: rotates graph
counterclockwise on the x-intercept
- equation:
factor (1 + [I]/Ki) added to slope AND intercept terms
- uncompetitive inhibition: binds only to ES complex (typically
at distant binding site); self titrating
- Vmax:
decreases
- Km:
decreases apparent Km
- saturation plot:flattens graph, makes it achieve Vmax much
more quickly
- reciprocal plot:affects y-intercept: shifts graph leftward on
a parallel line
- equation:
factor (1 + [I]/Ki) added to intercept terms
- irreversible inhibition: removes enzyme from the equation
- Vmax:
decreases stoichiometrically
- Km:
no effect
- kinetics:
- at [I] = KI, looks similar to non-competitive inhibition
- however, at [I] = 2KI, gives 100% inhibition/deactivation,
compared to 66.7%
4. Cellular Strategies of Enzyme Regulation- mechanisms of
enzyme regulation
- covalent modification
- proteolysis: destroys or activates enzyme
- R-group modification: phosphorylation, N-glycosylation,
O-glycosylation, hydroxylation, etc.
- feedback inhibition: final product inhibits first committed
step of the pathway
- allosteric regulation: changing enzyme shape through binding
on an allosteric site
- allosteric regulation
- features
- allosteric enzymes almost always have more than one
subunit
- saturation kinetics is generally sigmoidal, rather than
Michaelis-Menten (hyperbolic)
- inhibitors push curve right (more extreme sigmoid); activators
push curve left (more hyperbolic)
- K0.5: threshold concentration for regulation
- note that each individual enzyme is fully active or inactive-
aspartate transcarbamylase (ATCase): a model
- reaction: aspartate + carbamoyl phosphate (
N-carbamoylaspartate (ATCase, (Pi)
- function: introductory step in the production of CTP
- structure
- 12 subunits: 6 catalytic, 6 structural
- arranged in two states: T-state (compressed, inactive) and
R-state (open, active)
- inhibitor: CTP; stabilizes T-state (pushes sigmoid curve
right)
- activator: ATP; stabilizes R-state (pushes curve left)
- note that both ATP and CTP bind to the same site
5. Thermodynamics- general considerations
- thermodynamics indicates the favorability of a reaction given
the conditions, NOT the speed or pathway
- work can only be done if a system is not at equilibrium
- equilibrium constants
- Keq=1:reaction is at equilibrium
- Keq>1:products are favored
- Keq50, though most genes have several
- example: Factor VIII gene is 186 kbp in length, and 175 kbp is
contained in 25 introns
- first exon always contains at least some of the 5-UTR
- last exon always contains at least some of the 3-UTR,
including the poly(A) signal
- the genetic code
- overview
- 64 trinucleotide codons to specify each of the 20 amino
acids
- one start codon (AUG, or methionine), and three stop codons
(UAA, UAG, UGA)
- when an amino acid is specified by multiple codons, the 3rd
codon is often the only difference
- mutations
- silent mutation: does not change the sequence of the encoded
protein
- missense mutation: changes the codon to specify a different
amino acid
- nonsense mutation: changes a codon to a stop codon
- frameshift mutation: deletion of addition of nucleotides such
that the downstream reading frame shifts
point mutation in -thalassemias- thalassemias: an overview
- thalassemia: inherited deficiency in the production of or
globin (resulting in or thalassemia)
- the globin chain present in normal amounts tends to form
insoluble homotetramers that do not function well
- when either chain is nearly absent, severe anemia and death
usually occur before 10 without regular transfusions
- variance in severity
- 0: alleles that are completely inactive
- +: alleles that are partially active
- thalassemia minor: 0, asymptomatic
- thalassemia major: 00, requires regular blood transfusions
- intermediate: ++, shows intermediate symptoms
- mutations and consequences
- promoter regions
- cluster in two regions, about 90 and 30 base pairs upstream of
transcription start site
- CACCC at -88: regulatory protein
- ATA at -31: TATA box for binding TFIID
- point mutations generally result in + alleles, with
transcription reduced ~5-fold
- splice sites
- 5 splice site of intron 1
- mutation in initial GU sequence results in 0 allele
- mutation in more weakly-conserved site at position 5 results
in + allele
- 3 splice site of intron 2
- mutation in terminal AG to GG mutation results in 0 allele
- poly(A) signal: mutation of AAUAAA to AACAAA
- results in cleavage of pre-mRNA after another AAUAAA sequence
900 nt further downstream
- less -globin is made due to a loss in stability, resulting in
a + allele
- other mutations
- nonsense mutation in exon 2: 0 allele
- frameshift mutations: 0 allele
- missense mutations: rarely affect levels of -globin produced,
so do not result in thalassemia
- exception: Indianapolis -globin, which is highly unstable due
to a single AA substitution
- generally, may result in pathological effects, such as sickle
cell anemia
stable RNAs with biochemical functions- translated vs.
untranslated RNA
- mRNA makes up a small fraction of a cells RNA (10%)
- most RNA in a cell is not translated, instead serving
important cellular functions
- ribosomal RNA (rRNA) (75% of total cellular RNA)
- the ribosome is 2/3 RNA and 1/3 protein
- catalysis of peptide bond formation by modern ribosomes is
carried out by rRNA
- transfer RNA (tRNA) (15% of total cellular RNA by weight)
- used by ribosome to read mRNA codon, provide the corresponding
amino acid
- one end: anticodon complementary to a given codon
- other end: amino acid corresponding to a codon
- undergo post-translational modification, giving them bases
other than A, C, G, U (e.g. T by methylation)
- due to redundancy, more than 20 different kinds of tRNAs
- small nuclear RNA (snRNA)
- small, 100-300 nucleotide RNAs that participate in processing
of pre-mRNA in the nucleus
- packaged with proteins into small nuclear ribonucleoprotein
particles (snRNPs)
- some autoimmune disorders (e.g. systemic lupus erythematosis)
produce antibodies that recognize snRNPs
- the disease significance of this is not known
- small nucleolar RNA (snoRNA)
- similar to snRNA, but found in nucleolus, which is dedicated
to ribosome synthesis
- base pair with newly-synthesized rRNA, direct processing and
modification of rRNA into mature form
- rRNA then assembles with ribosomal proteins to form the
ribosomal subunits, sent to cytoplasm
- micro RNA (miRNA)
- 20-22 nucleotides long, complementary to specific mRNA
- pairing of miRNA with mRNA targets mRNA for degradation by
ribonucleases
- have an important role in human gene expression
16. Genetic ScreeningStudy Guide
- detection of sickle cell anemia
- carrier detection: place blood droplets in low O2 environment,
microscopically look for sickle cells
- prenatal detection
- recognize that sickle cell mutation destroys an MstII
restriction enzyme site
- digest the allele DNA with MstII and place it in a size-based
gel electrophoresis
- normal individuals will have a long (1.15 kb) and a short (0.2
kb) fragment
- afflicted individuals will have a really long (1.35 kb)
fragment
- carriers will show all three fragments
- carrier detection of cystic fibrosis
- with the F508 mutation: allele-specific oligonucleotide (ASO)
detection
- radiolabeled ASO complementary to the normal sequence is used
to make a probe
- radiolabeled ASO complementary to the mutant sequence is used
to make another probe
- DNA (often PCR-amplified) is spotted on both probes
- carrier, normal, or afflicted is determined by where the
fluorescent spot occurs
- without the F508 mutation: RFLP linkage analysis
- an RFLP that is tightly linked to the mutation is found
- DNA from the family is digested, and the inheritance to an
afflicted child is analyzed
- from this, it can sometimes be deduced whether or not a given
unafflicted child is a carrier
- disease
- sickle cell anemia:
- cause: E6V (glutamate to valine) mutation in the -globin
allele
- effects: asymptomatic carriers; anemia and associated symptoms
in afflicted
- cystic fibrosis
- cause: any of numerous mutations in the cystic fibrosis
transmembrane regulator (CFTR)
- effects: defective Cl- transport; serious effects on
respiration and digestion, clogged pancreas, death ~25
you should understand:- RFLP markers
- RFLP analysis is based on the idea that tightly-linked traits
will segregate together during meiosis
- the RFLP trait and the disease trait are not causally
linked!
- presence of RFLP can only be used to deduce the segregation of
disease traits
- whether an RFLP is present or not in a diseased chromosome is
a matter of luck
- in parents where RFLP is present on one homolog, given an
afflicted child, carrier status can be determined
- in parents where either parent does or does not have the
marker in both homologues, deduction is difficultNotes: Lecture and
Reading
general considerations for genetic testing- testing vs.
screening
- genetic test: done in individuals considered likely to bear
the diseased allele
- genetic screening: application of a genetic test to a large
population
- most genetic screening is too expensive to justify wide
application
- instead, screening is typically limited to populations known
to be at risk
- goals of genetic testing
- identify adult carriers of debilitating or fatal diseases in
order to guide reproductive choices
- identify fetuses that will develop such diseases in order to
guide termination decisions
- identify inborn disorders that require prompt treatment or
prophylactic measures
- adults vs. newborns
- adults: relatively straightforward due to availability of
tissue
- fetuses: difficult due to the small amounts of tissue able to
be obtained from a fetus
direct detection of a mutation: sickle cell anemia- sickle cell
anemia: overview
- autosomal recessive disorder caused by a E6V mutation in
-globin
- HbS: formed of 2S2
- aggregates when deoxygenated, forms long fibers
- this alters cellular shape, causing cells to get stuck in
capillaries, and leading to tissue damage
- sickle cells are prone to lysis and last a few weeks in blood
rather than the usual 4 months
- identification of adult carriers: microscopic and
electrophoretic examination
- microscopic examination: some AS heterozygote RBCs in low O2
will result in sickle cell shape
- electrophoretic examination: HbS and HbA in a carrier can be
identified due to G, V charge differences
- identification of fetal carriers: restriction enzyme
analysis
- blood cannot be drawn, so a DNA test must be used
- mutation is A to T in the non-template strand that destroys a
MstII restriction enzyme recognition site
- flanking cleavage sites: 1150 bp and 200 bp away
- S allele: 1.35 kb fragment
- A allele: 1.15 kb, 0.2 kb fragments
- Southern blot analysis can be used to detect the fragments in
a digest of DNA extracted from fetal tissue
- note: MstII site destruction could be due to other mutations;
this test thus assesses presence or absence of A- identification by
sequence: allele-specific oligonucleotides (ASO)
- theory
- ASO, matching the normal DNA sequence and encompassing site of
mutation, is synthesized chemically
- under appropriate conditions of temperature and [salt], ASO
should hybridize only to the normal sequence
- can be used to detect any specific sequence change
- practice
- DNA from a tissue sample is spotted onto a membrane and
incubated with:
- strip 1: radiolabeled ASO complementary to normal sequence
- strip 2: radiolabeled ASO complementary to mutant sequence
- spots will glow based on which sequence is present
- note that PCR is often used to amplify the chromosomal segment
containing the sequence in question
inference of a mutation by linkage to an RFLP or SNP: cystic
fibrosis- restriction fragment length polymorphisms: an
overview
- precise identity of a mutation must be known in order for ASO
analysis to work
- sometimes the defective gene has not been identified
- sometimes many different mutant alleles exist within a given
population
- in such cases, inheritance can sometimes be traced using a
linked genetic marker
- RFLP: natural variations in DNA; no effect on phenotype, but
can be detected by restriction enzyme digestion
- cystic fibrosis: an overview
- autosomal recessive disorder caused by mutation in cystic
fibrosis transmembrane regulator (CFTR)
- CFTR: regulates transport of Cl- ions across cellmembranes
- symptoms usually include respiratory and digestive
problems
- lungs become clogged with mucous and are susceptible to
pneumonia
- pancreatic duct becomes clogged, and digestive enzymes fail to
reach the intestines
- 1/2000 U.S. newborns is afflicted
- median survival age for individuals with CF is about 25 YO
- diagnosis
- CF: causes excess salt in sweat
- CF carriers: no detectable phenotype, so a DNA test is
required
- mutations
- F508: 70% of carriers; deletion of phenylalanine at position
508
- blocks proteins transit from ER to cell membrane, thus
blocking its function in Cl- transport
- can be detected with ASO
- other 30% of carriers: more than 200 causative mutations,
making ASO much more difficult
- linkage analysis
- situation 1
- parents: disease alleles on chromosomes with site, normal
alleles on chromosomes lacking site
- children: homozygous uncut = normal, heterozygous = carrier,
homozygous cut = afflicted
- situation 2
- parents: disease alleles on chromosomes with site, one parents
normal allele also has site
- children: homozygous cut = afflicted OR carrier, heterozygous
= carrier OR normal
- situation 3
- parents: one parent has disease allele with site and normal
without, other parent has opposite
- children: homozygous cut/uncut = carrier, heterozygous =
afflicted OR normal
- situation 4
- parents: both parents have restriction sites on all four
chromosomes
- children: all will have restriction sites, and thus this site
is not useful
- there is no obligatory relationship between RFLP and a
disease17. Transcriptional Control of Gene ExpressionStudy Guidebe
able to:- eukaryotic protein-coding gene
- upstream elements
- upstream promoter elements: within a few hundred base pairs
upstream of initiation site
- TATA box: about 30 bp upstream of the initiation site
- transcription start site: found shortly after TATA box
- exons and introns: found downstream of the transcription start
site
- always an odd number of exons
- always an even number of introns
- enhancer locations: thousands of base pairs away in an
orientation-independent manner
- found within introns
- found far upstream or downstream of the gene
- steroid hormone mechanism of gene transcription regulation
- steroid hormones: cholesterol-derived molecular signal
- enter target tissue by diffusion through plasma membrane and
bind their nuclear receptor
- binding of hormone releases Hsp90, allowing hormone/receptor
complex to bind DNA
- this binding regulates (usually promotes) binding of RNAp,
along with other factors (TFIIB, TFIID)
- activation of PEPCK
- cortisol: adrenal steroid hormone; binds glucocorticoid
receptor, which binds GRE and promotes PEPCK
- glucagon: polypeptide hormone that signals low blood glucose,
promoting PEPCK
- adrenaline: adrenal hormone that signals need for glucose,
promoting PEPCK
- both cause an upregulation of cAMP, which binds CREB, and
complex binds CRE
you should know:- combinatorial control
- numerous genes may come together to repress or (more commonly)
activate gene transcription
- it is the combined effect of all elements that determines the
total regulation
- mechanism of Jun/Fos promoters
- -helices containing a leucine zipper and a basic region
- hydrophobic leucines at every 7th residue face the same side,
come together
- (+) charged basic regions oriented to fit into the grooves of
DNA, where they interact with (-) charged DNA
- bind at the AP-1 promoter element
- dimerization
- Jun/Jun: bind poorly
- Jun/Fos: bind extremely well
- Fos/Fos: do not bind at all
you should understand:- regulation
- general transcription factor: trans-acting elements required
for transcription of all protein-coding genes
- gene regulatory protein: modify basal level of transcription
by TFs, in a gene-specific manner
- domains of a transcriptional activator protein
- DNA-binding: specifically interacts with and binds DNA, about
8-10 bp long
- activation: interacts with general transcription factors
- effector: alters ability to activate transcription in response
to a cellular signal
Notes: Lecture and Readingoverview of regulation of gene
expression- cellular identity
- multicellular organisms must coordinate levels of gene
expression
- intercellular signals: hormones, growth factors, cell to cell
contact, amongst others
- controlling gene expression
- transcriptional control (most common)
- processing control
- translational control
- degradation control
- components of gene expression
- factors: proteins, RNA, or complexes thereof that act on
signals or elements present in DNA, RNA, or protein
- promoter elements: DNA sequences near the gene that aid in the
binding of RNAp II
- cis-acting elements: act on a local scale, with limited
expression
- example: sequence elements
- inherited defects in gene expression tend to be caused by
mutation of cis-acting elements
- trans-acting elements: act on a global scale, across numerous
molecules
- example: transcription factors
- mutations in transcription factors are typically lethal very
early on, and are often not recognized
control of gene expression: DNA sequence elements- overview
- DNA transcription level is generally controlled by the
interaction of trans-acting and cis-acting elements
- cis-acting sequence elements: collectively termed promoter
- trans-acting elements
- general transcription factors (TFs): required for
transcription of protein coding genes
- gene regulatory proteins: modify basal level of transcription
directed by TFs in a gene-specific manner
- activators: increase transcription
- repressors: decrease transcription
- RNAp II: initiation of transcription
- general transcription factors
- TFIID: binds sequence 5-TATAAA-3 (TATA box) at ~30 base pairs
upstream of transcription start site
- TFIIB: binds adjacent to TFIID
- transcription initiation: process
- TFIID and TFIIB bind to DNA, often joined by other factors
(such as TFIIA)
- RNAp II recognizes the DNA complex, binds, and begins
transcription
- TFIID and TFIIB stay bound to the promoter after initiation,
promoting additional recruitment of RNAp II
- process requires several activator proteins
- activators in human gene expression
- activator binding sites
- upstream elements: binding sites for activator proteins just
upstream of the promoter
- enhancers: binding sites located thousands of base pairs away;
orientation-independent
- activator proteins
- DNA-binding domain: recognizes a specific DNA sequence 8-10 bp
long
- activation domain: interacts with general transcription
factors
- effector domain: interacts with a cellular signal (e.g.
hormone, phosphorylation)
- found only in certain activator proteins
- other gene regulatory proteins are always on, thus activity is
determined primarily by their concentration
- combinatorial control: phosphoenolpyruvate carboxykinase
(PEPCK)
- definitions
- combinatorial control: level of synthesis determined by net
effect of all bound regulators
- PEPCK: key role in gluconeogenesis; produced primarily in the
liver
- PEPCK structure
- TATA box:
-30
- CRE:
-100
- AP-1 promoter:
-125, -250, -275
- GRE:
-360
- HNF4 binding site:
-400
- receptors
- cyclic AMP response element (CRE)
- glucagon and adrenaline (which signal need for glycolysis)
stimulate production of cAMP
- cAMP stimulates a protein kinase that activates the protein
CREB
- cAMP binds CREB, complex binds CRE, promoting
transcription
- AP-1 promoter: bind Jun/Fos general activators, regulated in
some part by their synthesis
- glucocorticoid response element (GRE)
- DNA sequence element
- binds hormone/glucocorticoid receptor (GR) complex, which
increases transcription
- hepatocyte nuclear factor 4 (HNF4)
- tissue-specific activator, present primarily in the kidney and
liver
- absence of this factor restricts synthesis in other tissues,
even if cortisol, glucagon, or adrenaline is high
nuclear receptors- nuclear receptors: overview
- nuclear receptors: gene regulatory proteins that bind small,
hydrophobic molecules in their effector domains
- steroid hormone receptors: have a steroid-derived hormone
receptor, such as cortisol, estrogens, or androgens
- steroids are lipid soluble, and can thus diffuse through cell
membrane to bind a nuclear receptor
- this allows direct action, as opposed to the indirect use of
second messengers such as cAMP
- other examples of molecules using nuclear receptors
- thyroxine (thyroid hormone), vitamin D, retinoic acid (derived
from vitamin A)
- ligand for these is currently unknown
- nuclear receptor structure
- structure
- variable N-terminal receptor (transcription activator)
- DNA-binding domain
- C-terminal ligand-binding domain
- ligand binding
- Hsp90: inhibitory protein that complexes nuclear receptors
without bound ligand
- upon binding of ligand, Hsp90 is released, and complex can
bind to DNA to regulate transcription
- combinatorial control: HNF4
- DNA binding: the effect of mutation
- amino acids of DNA-binding domain of protein make highly
specific contacts with DNA bases
- this allows the domains to precisely read the sequence
- mutation of even a single base pair can significantly disrupt
this contact
- Factor IX
- overlapping receptors
- androgen receptor: binds testosterone to activate
transcription
- HNF4: orphan nuclear receptor and tissue-specific
activator
- mutations in Factor IX
- Leyden mutation
- occurs at -20, impacting the HNF4 binding site
- this causes hemophilia in young children, but males improve
after puberty due to androgen receptor
- Brandenburg mutation
- occurs at -26, disrupting both the HNF4 and androgen receptor
binding sites
- this causes lifelong hemophelia, as both binding sites are
disrupted
- HNF4 in the kidney and pancreas: effects of mutation
- maturity-onset diabetes of the young, type 1 (MODY1): rare
autosomal dominant; caused by mutation
- type 2 diabetes: increased risk based on single nucleotide
polymorphisms in HNF4
- underscores the importance of HNF4 in sugar metabolism
Jun and Fos: leucine zipper- structure
- long -helices with two domains
- leucine zipper: Leu side chains at every seventh position,
forming a hydrophobic stripe on one side
- basic region: positively charged, can interact with
negatively-charged DNA
- two of these helices come together, forming a dimer stabilized
by hydrophobic Leu contacts
- after dimerization, basic region contacts DNA
- activation
- heterodimers vs. homodimers
- Jun/Jun: bind AP-1 site to some degree
- Fos/Fos: do not bind DNA
- Jun/Fos: bind DNA better than Jun homodimers
- Fos
- increases transcriptional activation by Jun
- stimulated by growth factors; may help initiate cell
division
- overexpression: can cause cancer
18. Protein SynthesisStudy Guidedo the following:-
structures
- aminoacyl AMP: AMP with amino acid attached (via
carboxyphosphate linkage) to 5 C
- aa-tRNA: amino acid attached (via esterification) to 3 C or 2C
of tRNA N-terminal adenylate residue
- structure of a translating ribosome
- large (60S) and small (40S) subunit
- 40S subunit: decoding of mRNA, directly on mRNA
- 60S subunit: carries out peptidyl transferase reaction
- ribosomal sites
- A site: aminoacyl tRNA site, containing the incoming peptide;
3-most structure
- P site: peptidyl tRNA site, containing the growing polypeptide
chain; middle site
- E site: exit site, where empty tRNA molecules leave the
ribosome; 5-most structure
- components
- aminoacyl tRNA: located in A site, contains amino acid to be
added
- peptidyl tRNA: located in T site, contains growing polypeptide
chain (N-terminus distal to ribosome)
- codon: three letter code located on DNA
- anticodon: complementary three letter code on RNA
- peptidyl-transferase site: located between ends of the
aminoacyl and peptidyl tRNAs
you should know:- components of translation initiation
- start codon: AUG (methionine)
- tRNA: methionyl-tRNAMet(i) (Meti is specific to
initiation)
- recognition of the start codon: ribosome looks for first AUG
sequence downstream of the 5-mGppp cap
- EF-1 and protein elongation
- function: binds GTP, binds an aminoacyl-tRNA, and brings it to
the aminoacyl site in the ribosome
- molecular clock
- peptidyl transferase can only work after EF-1 has left the
site, which requires hydrolysis of GTP
- binding of aa-tRNA anticodon signals hydrolysis of GTP
- proofreading: if anticodon does not match, tRNA will
dissociate before GTP hydrolysis is complete
you should understand:- diphtheria toxin
- EF-2 is a GTP-binding protein that is required for
translocation of peptidyl tRNA from A site to P site
- diphtheria toxin ADP-ribosylates (from NAD+) a specific amino
acid residue in EF-2, inactivating it
- one molecule of toxin is potent enough to kill an entire
cell
- tRNAs and the genetic code
- there are 20 amino acids, 20 aminoacyl-tRNA synthetases and 64
possible amino acid codons
- redundancy: multiple codons must be recognized by a single
aminoacyl-tRNA synthetase
- as such, many synthetases must use structural features other
than tRNA codon in order to bind
Notes: Lecture and ReadingtRNA activation: aminoacylation-
components
- definitions
- aminoacyl-tRNA synthetase: enzyme that activates tRNA by
attaching amino acid
- tRNAamino acid: recognizes RNA codon, involved in transferring
it to a growing polypeptide
- anticodon: sequence by which an activated tRNA recognizes and
binds DNA
- there are 20 AA-tRNA synthetases, one for each amino acid
- more than 20 tRNA molecules required for all codons (some tRNA
can recognize multiple anticodons)
- some synthetases must therefore recognize multiple tRNA
molecules
- isoacceptors: tRNAs that have different anticodon sequences
but become charged with the same amino acid
- some synthetases recognize tRNA molecules by their anticodon
sequence
- synthetases that charge multiple tRNAs must recognize other
structural features of the tRNA
- enzymatic process: two steps, both catalyzed by aminoacyl-tRNA
synthetase
- adenylation
- amino acid + ATP ( aminoacyl-AMP + PPi
- this reaction activates the amino acid for use in the next
step
- fidelity
- this reaction gives the synthetase another opportunity to
proofread the amino acid, increasing fidelity 100X
- if aminoacyl-AMP does not fit properly, adenylate is
hydrolyzed and amino acid is discarded
- aminoacylation
- aminoacyl-AMP + tRNA ( aminoacyl-tRNA + AMP
- 2 or 3 OH of terminal adenine in tRNA attacks the
carboxyphosphate bond formed in adenylation reaction
- this attaches the amino acid to the tRNA, activating it for
use in polypeptide elongation
- note that every tRNA has an adenylate residue on the 3 end
- net energy used: 2 phosphates
- PPi generated in aminoacylation step is hydrolyzed to 2Pi by
pyrophosphatase
- this makes the net reaction more exothermic, driving it
forward by mass actionribosome structure and function-
definitions
- peptidyl transferase: enzyme that transfers amino acids from
aa-tRNA to the growing polypeptide chain
- decoding: interaction of tRNA with mRNA wherein tRNA
anticodons read RNA codons and add amino acids- structure
- large subunit (60S) and small subunit (40S)
- 40S subunit: decoding of mRNA
- 60S subunit: carries out peptidyl transferase reaction
- three sites
- A site: aminoacyl tRNA site, containing the incoming
peptide
- P site: peptidyl tRNA site, containing the growing polypeptide
chain
- E site: exit site, where empty tRNA molecules leave the
ribosome
- the process of translation
- initiation
- 40S and 60S subunits are brought together at the first codon
to be translated, forming the A, P, and E sites
- initiation codon: AUG (methionine)
- in rare cases, codon is GUG or UUG, but methionine is still
incorporated
- in bacteria, the AUG used is somewhat variable, depending on
the sequence context
- in eukaryotes, the first AUG downstream of the 5 cap is almost
always used for initiation
- initiator tRNA: methionyl-tRNAMet (tRNAMet(i))
- this is a special methylated form of methionine that is used
specifically to initiate transcription
- binds directly in the P site, rather than the A site
- often cleaved off later by an N-terminal protease
- elongation
- aa-tRNAaa binds in the A site of the ribosome, immediately
downstream of the previous codon
- peptidyl transferase activity of 60S subunit
- catalyzes attack of A-site free amino group to the P-site
tRNA-amino acid ester linkage
- this displaces the growing polypeptids chain from the P-site
to the A-site, leaving the P-site empty
- reaction is favorable because the aminoacyl linkage has higher
energy than the nascent peptide bond
- note: the catalysis is performed by the RNA of the large
subunit, NOT the protein component
- translocation
- ribosome moves three nucleotides downstream on the mRNA,
ejecting the uncharged tRNA from the E-site
- this moves the peptidyl-tRNA to the P-site, opening up the A
site for the next aminoacyl-tRNA
- repetition: moves ribosome in 5(3 direction, with concurrent
synthesis of polypeptide in N to C direction
- termination
- UAA, UAG, UGA are not recognized by tRNAs, but instead by
protein release factors
- directs ribosome to stop synthesis, and peptidyl transferase
activity hydrolyzes the last bond to the tRNA
- ribosome and the novel protein are released
- polysome: mRNA with several attached,
simultaneously-translating ribosomes
- ribosomes near 5 end: polypeptide is short, incomplete
- ribosomes near 3 end: polypeptide is longer, closer to
complete
accessory factors in translation: EF-1 and EF-2- G proteins
- G proteins: GTP-binding required for activity
- both EF-1 and EF-2 are G proteins
- G proteins used in numerous cellular processes, such as
vesicle transport, protein and RNA transport, cell signals
- EF-1
- EF-1: GTP-regulated protein that binds aminoacyl-tRNA and
delivers it to the A site
- upon binding of tRNA anticodon to mRNA codon, GTP is
hydrolyzed to GDP + Pi
- after hydrolysis, EF-1-GDP is released from the ribosome, and
peptide bond formation can occur
- because this process takes time, GTP is acting as a molecular
clock
- peptide bond formation cannot occur until GTP is hydrolyzed
and the complex has left the site
- incorrect AA-tRNA molecules, which bind the codon more weakly,
usually dissociate before hydrolysis
- correct AA-tRNA molecules will remain until hydrolysis
occurs
- this is thus a proofreading step that, along with aa-tRNA
synthetase, reduces translational error to 1/10,000
- EF-2
- EF-2: GTP-regulated protein required for translocation of
peptidyl-tRNA from A site to P site
- diphtheria
- diphtheria: toxin in certain strains of Corynebacterium
diphtheria
- acts by catalyzing transfer of ADP-ribose from NAD+ to a
specific amino acid in EF-2, inactivating the protein
- this blocks protein synthesis by halting the translocation
step
- a single molecule of toxin can kill an entire cell (which
contains half a million EF-2 molecules)
- erythromycin
- erythromycin: antibiotic that binds large subunit RNA in
bacterial ribosome, inhibits translocation
- because this does not affect eukaryotic translocation, it is
an effective antibiotic
- some bacterial strains are emerging that have a resistance to
this
energy of protein synthesis- addition of each amino acid residue
to a growing polypeptide chain requires 4 high energy phosphate
bonds
- aminoacyl-tRNA synthetaseamino acid activationATP ( AMP + PPi
( 2Pi
- EF-1
molecular clock
GTP ( GDP + Pi
- EF-2
translocation
GTP ( GDP + Pi19. Protein TargetingStudy Guide- protein
pathways
- cytosol
- ribosome: synthesizes protein and releases into cytosol
(default path)
- certain sequences can cause import into the nucleus or
mitochondria
- endoplasmic reticulum
- ribosome synthesizes protein, and a signal sequence is
recognized by SRP
- SRP arrests translation, binds an SRP membrane receptor, and
docks ribosome with translocon
- protein is extruded into the ER, becoming membrane-bound or
soluble based on stop/start sequences
- KDEL receptors maintain the protein in the ER, returning them
from vesicles via retrograde transport
- lysozome
- ribosome synthesized protein, and a signal sequence is
recognized by SRP
- SRP arrests translation, binds an SRP receptor, and docks
ribosome with translocon
- protein is extruded into the ER, becoming membrane-bound or
soluble based on stop/start sequences
- phosphomannose in N-linked oligosaccharides targeted by
receptors protein for lysosome
- cell surface
- ribosome synthesized protein, and a signal sequence is
recognized by SRP
- SRP arrests translation, binds an SRP receptor, and docks
ribosome with translocon
- protein is extruded into the ER, becoming membrane-bound or
soluble based on stop/start sequences
- protein exits trans-Golgi and is brought to the cell surface
(default ER path)
- polypeptide fates: signal sequences
- signal peptidase, no stop transfer:
soluble protein secreted outside the cell
- internal uncleaved signal peptidase, no stop transfer:
IMP on cellular membrane, with N-terminus inside, C-terminus
outside cell
- internal uncleaved signal peptidase, one stop transfer:
IMP on cellular membrane, with one extracellular loop
- folding assistants
- Hsp90: molecular chaperone that permits proteins to fold
without aggregating
- protein disulfide isomerase: catalyzes disulfide bond
cleavage, allowing proteins to attain lowest energy state
know the following:- signal recognition particle (SRP)
- binds ribosomes from which a signal sequence has emerged
- arrests translation (by blocking the A site) and brings
ribosome to ER membrane
- after binding a receptor, SRP docks the ribosome over a
translocon
- SRP leaves, allowing translation to continue into the
translocon
- N-linked glycosylation
- ER-mediated attachment of sugars to asparagine residues of
nascent polypeptides
- because this only happens within the ER, IMPs that are
N-glycosylated will only have extracellular sugars
- recall: extracellular, ER lumen, lysosomal lumen are all
topologically equivalent
- I-cell disease
- mannose phosphokinase, which N-glycosylates certain proteins,
is defective
- phosphomannose receptors target proteins for the lysosome
- proteins that should be targeted for the lysosome are instead
secreted by the default pathway
- lysosomes are unable to do their job; this leads to severe
psychomotor retardation, skeletal defects
Notes: Lecture and Reading
localization signals in protein transport: overview- signal
sequence
- signal sequence: localization signal that specifies synthesis
into lumen of ER
- usually found at N-terminus of a protein
- consists of basic amino acid (Lys or Arg) followed by a
stretch of hydrophobic residues
- proteins with signal sequence
- synthesized into ER, where default path is through Golgi
apparatus and constitutive secretion on the cell surface
- some specific signals can cause proteins to be secreted in a
regulated fashion from secretory vesicles
- other specific signals can cause proteins to be sent to the
lysosome
- retention signals for organelle-specific proteins of ER, Golgi
allow reuse and recycling of those proteins
- ER import of proteins is co-transcriptional
- proteins lacking a signal sequence
- synthesized and secreted into the cytosol, where default path
is to remain there
- nuclear import signals
- direct proteins to nucleus
- usually contain several basic residues
- mitochondrial import signals
- direct proteins to mitochondria
- usually amphipathic helices with basic residues on one face,
hydrophobic residues on other
- cytosolic import of proteins is post-transcriptional
the endoplasmic reticulum: entrance to vesicular transport
pathway- protein translation
- signal recognition particle (SRP): binds ER signal in
polypeptides emerging from ribosome
- consists of RNA and several proteins
- causes translation to stop, and docks with an SRP receptor
located on the membrane
- translocon: pore through ER membrane
- when inactive, contains a plug in the ER lumen that prevents
free pass of molecules
- SRP/SRP receptor/ribosome complex binds to translocon
- translation begins again, and elongating protein is extruded
into ER
- post-translation
- SRP is released from elongating ribosome, floats away to find
another signal peptide
- ribosome dissociates from translocon, and mRNA becomes soluble
again
- mRNA usually stays attached to the membrane via other
ribosomes in the polysome
- protein positioning
- components
- stop sequences
- hydrophobic sequence in polypeptide that signals a stop in
translocation
- this gets stuck in the translocon, and is extruded to become
part of the membrane of the ER
- start sequences
- hydrophobic sequence in polypeptide that signals the beginning
of translocation
- these are brought to a translocon similar to signal
sequences
- signal peptidase
- cleaves the signal peptide, creating a new N-terminal end that
faces the inner ER lumen
- signal sequence remains in the ER lumen until it is degraded
by other enzymes
- function
- use of signal peptidases, stop sequences, and start sequences
alters the orientation and position of the protein
- each odd-numbered hydrophobic segment acts as a signal peptide
or start transfer sequence
- each even-numbered hydrophobic segment acts as a stop transfer
sequence
- polarity of integral membrane proteins
- signal sequence, when cleaved by peptidase, places the
N-terminal region within the ER lumen
- if signal sequence is further into the polypeptide and is not
cleaved by peptidase, N-terminus in cytosol
- knowing this, protein orientation can be predicted
- because of how vesicles work, the ER lumen is topographically
equivalent to extracellular membrane
- proper folding: molecular chaperones
- binding protein (BiP): molecular chaperone protein present in
high concentration in ER lumen
- molecular chaperone: protein specialized to guide the folding
and assembly of other proteins
- proteins are extruded into the ER lumen in an extended state,
and are not properly folded
- this promotes non-specific aggregation of exposed hydrophobic
regions
- molecular chaperones shield forming polypeptides, giving them
time to fold individually
- proper folding: disulfide bonds
- protein disulfide isomerase (PDI): catalyzes cleavage of
disulfide bonds
- disulfide bonds
- cytosol: reducing environment (favors removal of disulfide
bonds)
- ER lumen: oxidizing environment (favors addition of disulfide
bonds)
- upon entry into ER, cysteine disulfide bonds form
spontaneously, often incorrectly
- PDIcleaves bonds, allowing protein to continue towards its
lowest energy (most stable) state
- protein glycosylation
- N-linked glycosylation: attachment of sugars to amino group of
certain asparagine residues
- most proteins entering ER are covalently modified in this
way
- glycosyl transferase: catalyzes this process on luminal side
of ER membrane
- only happens within the ER lumen
- secretory vesicles work such that ER side of integral membrane
proteins (IMPs) will face outside cell
- because of this, only extracellular portions of IMPs are
glycosylated
the Golgi apparatus and beyond- Golgi apparatus structure
- Golgi apparatus consists of 4-8 cisternae organized in a
stacked fashion
- cisternae: disk-shaped membrane bound vesicles
- movement through the Golgi
- cis face: cisterna closest to ER (also called transitional
ER)
- trans face: cisterna closest to plasmalemma
- proteins move stepwise through cisterna in vesicles that bud
off each face and merge with the next
- from the trans face, vesicles move to the lysosome, secretory
granules, or directly to the plasmalemma
- retrograde transport: returns proteins to ER
- occurs through the use of retrograde vesicles that bud off and
return to ER
- proteins to be returned to ER (e.g. BiP or PDI) are recognized
by KDEL receptor
- KDEL receptor: Lys-Asp-Glu-Leu conserved sequence- Golgi
function
- localization: proteins are sent to proper places within the
cell
- this often occurs by enzymes binding to specific receptors on
Golgi membranes
- example: lysosome
- N-linked oligosaccharides of lysosome enzymes are recognized
by phosphomannose receptors
- phosphomannose receptors bind, package enzymes into vesicles
bound for lysosome
- I-cell disease: autosomal recessive disorder characterized by
psychomotor and skeletal difficulties
- defect in kinase that phosphorylates mannose in N-linked
oligosaccharides of lysosomal enzymes
- phosphomannose receptor fails to recognize this, and proteins
are secreted instead
- lysosome is unable to complete its digestion, leading to large
cellular inclusions
- glycosylation
- N-linked sugars can be trimmed, and different sugars can be
added in order to modify oligosaccharides
- other proteins are glycosylated directly in the Golgi
- O-linked glycosylation: oligosaccharides added to OH of Ser,
Thr residues
- less prevalent than N-linked glycosylation
- glycosylation functions: proper folding, stability, and
cell-cell interactions
- cleavage
- in trans-Golgi and beyond, some proteins are cleaved into
mature form
- example: insulin
- ribosomal synthesis:
preproinsulin
- ER signal peptide removal:
proinsulin
- secretory vesicle internal peptide removal:insulin
- numerous other peptide hormones and neuropeptides are made in
a similar fashion
20. Posttranscriptional Control of Gene ExpressionStudy Guidedo
the following:- absorption and transport of iron
- divalent metal ion transporter 1 (DMT1) channel protein brings
iron into the enterocyte
- ferroportin channel protein secretes iron into the
bloodstream
- apotransferrin (Tf) binds plasma iron, becoming transferrin,
and brings it to a target cell
- transferrin receptor (TfR) on the target cell binds and
internalizes Tf within endosomes (cellular vesicles)
- acidic endosomes cause release of iron, which enters cytosol
through DMT1
- apotransferrin and TfR are returned to plasma membrane and
reused
- ferritin stores any excess Fe
- regulation of ferritin, transferrin
- iron response element (IRE): promoter loop found in 5 end of
ferrritin, 3 end of TfR
- iron regulatory protein 1 (IRP1): cytoplasmic aconitase
without bound iron; binds and masks IRE
- high [Fe]
- IRP1 ( cytoplasmic aconitase, leaving IREs in mRNA
unoccupied
- ferritin: exposed IRE at 5 end promotes translation
initiation, leading to higher [ferritin]
- transferrin receptor: exposed IREs at 3 end promote
degradation, leading to lower [TfR]
- enterocytes: promotes uptake of iron and loss to feces as
enterocytes are sloughed
- other cells: promotes iron storage, limits further uptake of
iron, decreasing intracellular [Fe]
- low [Fe]
- cytoplasmic aconitase ( IRP1, which binds IREs in mRNA
- ferritin: blocks initiation of translation, leading to lower
[ferritin]
- transferrin receptor: blocks 3 end and exonuclear degradation,
leading to higher [TfR]
- enterocytes: iron capture is limited, shunting iron into the
bloodstream
- other cells: low ferritin and high TfR increase uptake and
intracellular [Fe]
you should know:- splice site recognition: U1 of spliceosome,
finding a 5-GUAAGU-3 mRNA sequence (GU most important)
- forming different sizes of apolipoprotein B
- B100 (liver): full protein is translated
- B-48 (intestine): post-transcriptional deamination of cytosine
(forming uracil) forms a premature stop codon
- stability of Fos
- destabilization elements found in coding region, 3-UTR
region
- both lead to more rapid 3 exonuclease activity
- this is critical, as overexpression of Fos is linked to
cancer
- iron: primarily used for oxygen transport
you should understand:- alternative splicing
- mutually exclusive exons
- differential use of splicing sites, as in smooth/striated
muscle
- allows spliceosome to skip or include certain exons
- alternative 3 terminal exons
- use of optional intron containing a poly(A) site, as in Ig
heavy chains
- if the optional intron is used, the first poly(A) site is
where translation ends
- if the optional intron is not used, the terminal poly(A) site
is used
Notes: Lecture, Readingadvantages of posttranscriptional control
of gene expression- multiple proteins from one gene
- happens through alternative splicing, RNA editing
- increases repertoire of proteins that can be made from a fixed
number of genes
- 1/3 to 1/2 of all human genes produce pre-mRNAs that are
subject to alternative splicing
- faster regulation
- transcriptional regulation is on the order of hours because of
the amount of time required
- rapid modulation can occur at the level of the mature mRNA
through changes in stability, translation efficiency
- takeover during quiescent transcription
- at some stages in life (e.g. embryogenesis), translation does
not occur
- it is still important for proteins present to be accountable
to regulation
alternative splicing: mechanism and examples- mechanism of
splicing
- splice site: intron/exon junctions that are the site of
splicing
- 5 splice site: GU followed by a number of preferred
nucleotides
- 3 splice site: AG preceded by a polypyrimidine (U, C) rich
sequence
- deviations from these sequences increase likelihood of
overlooking splice site
- U1 snRNA: one of 5 snRNAs required for splicing
- U1 snRNP recognizes and binds 5 splice site
- protein splicing factors recognize, bind 3 splice site
- reaction
- factors excise the intron, attach 5 splice site to an internal
branch point, making a lariat shaped structure
- lariat is degraded by nucleases
- alternative splicing
- alternative splicing: use of different splicing sites to
modify identity of introns and exons
- mutually exclusive exons: -tropomyosin (-TM)
- -tropomyosin: regulatory protein in muscle contraction,
blocking binding of actin to myosin
- alternative splicing creates a slightly different molecule,
based on tissue location
- smooth muscle: exon 3 is skipped, and only 1, 2, and 4 are
included
- striated muscle: exon 2 is skipped, and only 1, 3, and 4 are
included
- mechanism: tissue-specific splicing regulatory factors bind
and mask alternative splice sites
- alternative 3-terminal exons: immunoglobin (Ig)
- immune response: causes antibodies to change from
membrane-bound to soluble forms
- alternative terminal exons in the Ig change the C-terminus
from hydrophobic to hydrophilic
- membrane-bound
- optional intron is spliced out
- terminal exon is hydrophobic, with poly(A) site 2
- soluble
- optional intron remains, and an internal poly(A) site (1)
causes premature termination
- terminal exon is hydrophilic
- like with mutually-exclusive exons, tissue- or stage-specific
factors modify the splicing
RNA editing: mechanism and example- mechanism
- RNA editing: post-transcriptional insertion, deletion, or
adjustment of individual nucleotides
- much less common form of RNA processing
- examples
- apolipoprotein B: protein involved in fat transport
- intestinal mucosa: produces a 240 kD protein
- liver: produces a 500 kD protein
- in the intestine, a cytosine base is deaminated, converting it
to uracil
- this results in a stop codon and truncation of the intestinal
protein
- APOBEC-1: enzyme that edits apo B mRNA
- related enzyme, APOBEC-3G, defends cells against viruses by
heavily modifying their DNA
- unfortunately, HIV produces a protein (Vif) that protects the
virus from this
- AMPA receptors: glutamate receptors required for CNS
function
- two sites are edited, causing deamination of adenosine to
inosine (guanine analog)
- both result in amino acid substitution
- one is critical, as mice unable to edit the site develop
seizures and die by 3 weeks
- editing at the other site increases with a developing brain,
and may influence neuron function
RNA stability: changing the rate of protein synthesis- overview
of mRNA stability
- rate of mRNA degradation is a means of controlling protein
levels
- mRNA stabilities vary greatly, with half lives from less than
15 minutes to more than 10 hours
- default state: long half life
- unstable mRNA: contains cis-acting elements that direct their
rapid degradation
- removal of poly(A) tail
- stabilizing factors: increase average length of poly(A)
tails
- by default, 3 exonucleases progressively shorten the poly(A)
tail of mRNAs
- degradation from ~200 to 10 g iron in the body, sometimes as
much as 50 g
- serum transferrin is >50% saturated (compared to 30% for
normal)
- hereditary hemochromatosis (HH): most common form of inherited
disease
- causative mutation in HFE gene, but the function of that
protein is not well understood
- homozygotes are relatively common (1/200)
- symptoms: at middle age; including cirrhosis, bronze
pigmentation, diabetes mellitus, cardiomyopathy
- treatment: weekly bleedings
- iron overload as a complication of sickle cell anemia,
thalassemia major
- intestinal absorption of iron increased as the body attempts
to make more RBCs
- blood transfusions bring even more iron in
- treatment: chelating agents (e.g. deferoxamine) that bind
iron, promote its disposal in the urine
21. Posttranscriptional Control of Gene ExpressionStudy Guidebe
able to:- mutation-inducing radiation
- ionizing radiation (e.g. X-rays)
- initial effect: cause double-stranded break in DNA
- mutations: DNA rearrangement such as inversions,
translocations
- ultraviolet (UV) radiation
- initial effect: dimerization of adjacent pyrimidine bases,
especially T=T
- mutations: require DNA repair processes that can sometimes be
error-prone
- nucleotide excision repair pathway
- target: chemically-modified DNA, such as T=T dimers
- mechanism
- proteins recognize, bind DNA
- endonucleases make 5, 3 incisions in the damaged strand
- helicase removes damaged strand (to be digested by
nucleases)
- common repair pathway seals DNA, using undamaged strand as a
template
- defect consequences: xeroderma pigmentosum, which includes
lesioning and cancers in the skin
- mismatch repair pathway
- target: mismatched bases or slippage loops
- mechanism
- proteins recognize newly-synthesized strand, bind
- endonucleases make 5, 3 cuts
- exonucleases directly digest DNA
- common repair pathway seals DNA, using single strand
template
you should understand:- inherited cancer defects
- proto-oncogenes: accelerate replication and cell growth,
oncogenes inherited dominantly
- tumor suppressor genes: inhibit replication and cell growth;
defects inherited recessively
- xeroderma pigmentosum
- inherited defect in the nucleotide excision pathway
- A, C: defect in recognition (pyrimidine dimer binding)
- F, G: defect in incision (exonucleases)
- B, D: defect in excision (helicases)
- V: defect in error-free bypass
- defects in any of these increase likelihood of use of
error-prone bypass
- in error-prone bypass, defects are repaired essentially at
random, causing numerous mutations
- MSH2 gene
- codes for a gene in mismatch repair
- inheritance of repair defect: autosomal recessive
- inheritance of predisposition: autosomal dominant
- receiving one bad copy increases likelihood that other will be
knocked out, yielding cancerNotes: Lecture and ReadingDNA damage
and mutations- DNA damage is usually repaired prior to
replication
- consequences of replicating damaged DNA
- cell death: DNAp stops, synthesis is arrested, and cell
undergoes apoptosis
- error-free bypass: DNAp falls off, specialized high fidelity
DNAp repairs mutation, and replication resumes
- error-prone bypass: specialized DNAp is error-prone, inserts
nucleotides at random, leading to mutations
- error-prone bypass is the most carcinogenic
mechanisms of DNA damage- deamination: removal of an amine group
(frequently on C)
- deaminated bases: altered pairing properties
- deamination of C gives T, which pairs with A instead of G
- depurination: removal of a purine base by spontaneous
hydrolysis of the glycosidic linkage (frequently G)
- alkylating agents: induce depurination (examples: some
chemotherapeutic drugs)
- Rev1: inserts dCMP opposite a missing base in the template
strand
- dimerization: covalent linkage of adjacent pyrimidine bases
(especially T=T)
- caused by ultraviolet (UV) radiation
- stop normal replicative polymerase, can be bypassed by a
specialized DNAp
- double-stranded breaks: separation of DNA across both
strands
- caused by X-ray radiation
- leads to highly reactive free ends of DNA
- inversions: flipping orientation of DNA without changing
position
- translocations: fusion of DNA from two different
chromosomes
common features of DNA repair pathways- 99.9 % of DNA damage is
repaired prior to mutation
- common pathway of repair
- removal of a portion of the damaged strand, leaving
single-stranded DNA
- polymerization over the gap by DNAp, using the undamaged
strand as a template
- sealing of the DNA by DNA ligase, using ATP as the source of
energy
- specialized enzymes
- apurinic (AP) endonuclease: recognizes, excises depurinated
nucleotide
- uracil-DNA glycosylase: recognizes, hydrolyzes uracil base,
allowing AP endonuclease to excise abasic site
DNA repair defects and cancer- inherited defects in repair
pathways increase susceptibility to cancer
- exponential growth: a single defect increases likelihood of
next defect, which increases likelihood of next defect
- typically, defects in 5-6 DNA repair pathway genes are
required before cancers begin to appear
- oncogenes: genes that promote unregulated cell growth
(accelerate growth)
- proto-oncogenes: promote regulated growth of normal cells
- dominant mutations: only one defective allele is required for
manifestation of disease
- analogy: cellular gas pedal
- tumor suppressor genes: genes that allow unregulated cell
growth only when deactivated (repress growth)
- normal function: repress cell proliferation
- recessive mutations: both alleles must be deactivated for
manifestation of disease
- analogy: cellular brake pedal
nucleotide excision repair: xeroderma pigmentosum- xeroderma
pigmentosum
- cause: defect in nucleotide excision repair pathway
- symptoms: hypersensitivity to sunlight, 2000-fold increase in
skin cancer frequency
- nucleotide excision repair (NER) pathway
- process
- specialized proteins identify, bind the particular lesion
- DNA endonucleases, directed by bound proteins, create single
strand breaks 5 and 3 of lesion
- helicase displaces lesioned oligodeoxynucleotide
- gap is paired as in the common repair pathway
- this process acts on a number of different DNA damages
- specific defects in XP
- XP-A through XP-G
- A, C:pyrimidine dimer recognition
- F, G:DNA endonucleases (5, 3 respectively)
- B, D:DNA helicases
- XP-V gene: specialized DNAp error-free bypass that bypasses
thymine dimers with AMP residues
- with homozygous defect in this gene, only error-prone bypass
or cell death can occur
- as skin absorbs most UV radiation, that is most susceptible to
disease (cell death, cancer)
mismatch repair: colorectal cancer- colorectal cancer: cancer of
the epithelium lining the colon and rectum
- accounts for 10% of all cancer defects in the US
- contain mutations in several of the known proto-oncogenes and
tumor supressor genes
- most are spontaneous, though 1 in 8 are hereditary
non-polyposis colorectal cancer (HNPCC)
- inheritance of HNPCC: defect in MSH2
- mutation: autosomal recessive inheritance
- predisposition: autosomal dominant inheritance
- having one defective allele (inherited dominantly) increases
predisposition to disease (manifests recessively)
- MSH2: mismatched base pair repair sequence
- substrate: single base mismatches and/or small mismatched
insertions that form single-stranded loops
- directs assembly of protein complex that contains a single
stranded endonuclease
- this endonuclease recognizes the newly-synthesized strand,
which is the more likely defective strand
- exonuclease excises defective DNA, and repair proceeds as in
the common pathway
- note that this is an exonuclease rather than a helicase, as
found in NER
- this may happen because the DNA to be removed is not
chemically altered
22. Nucleotide Metabolism and its Control with DrugsStudy
Guidedo the following:- phosphate attachments
- ribose 5-P: attached to 5C
- PRPP: P attached to 5C, PP attached to 1C on bottom- AMP
catabolism to uric acid
- schematic pathway (blood cells)
- AMP ( adenosine (H2O ( Pi; nucleotide phosphatase)
- adenosine ( inosine (I) (H2O ( NH3; adenosine deaminase)
- inosine ( hypoxanthine (Pi ( ribose 1-phosphate; nucleoside
phosphorylase)
- hypoxanthine ( xanthine (xanthine oxidase)
- xanthine ( uric acid (xanthine oxidase
- base salvage pathway: utilizes hypoxanthine for purine
synthesis
- summary: adenine vs. guanine
- adenine: AMP ( A ( I ( hypoxanthine ( xanthine ( uric acid
- guanine: GMP ( G ( ( guanine ( xanthine ( uric acid
you should know:- inosine monophosphate (IMP): precursor to both
AMP, GMP
- ribonucleotide reductase: catalyzes ribonucleotides to
deoxyribonucleotides, with NDPs as substrates
- thymidilate synthase: catalyzes dUMP ( dTMP
you should understand:- gout
- caused by an excess of uric acid which crystallizes in the
joints rather painfully
- allopurinol
- competitive inhibitor of xanthine oxidase, resulting in more
soluble xanthine or hypoxanthine
- xanthine and hypoxanthine can simply be excreted
- 5-fluoro-dUMP
- incorporation pathway
- 5-fluorouracil ( 5-f-UMP (PRPP ( PPi, pyrimidine PRT)
- 5-f-UMP ( 5-f-UDP (ATP ( ADP, nucleotide kinase)
- 5-f-UDP ( 5-f-dUDP (XH2 ( X + H2O, ribonucleotide
reductase)
- 5-f-dUDP ( 5-f-dUMP (H2O ( Pi, nucleotide phosphatase)
- mechanism
- covalently binds thymidilate synthase, halting conversion of
dUMP ( dTMP
- this halts cell growth and slows growth of the tumorNotes:
Lecture and Readingoverview of nucleotide metabolism- functions of
nucleotides
- genetic information:
DNA, RNA
- energy metabolism:
ATP
- intracellular signals:
cAMP, cGMP, GTP
- coenzymes:
adenosine derivatives
- activating metabolic intermediates:aminoacyl-AMP
- nucleotide metabolism
- amino acids ( ribonucleotides
- ribonucleotides ( RNA (NTP, RNAp) ( ribonucleotides (NMPs,
nuclease, kinase)
- ribonucleotides ( deoxyribonucleotides
- deoxyribonucleotides ( DNA ( deoxyribonucleotides
- deoxyribonucleotides ( purine, pyrimidine bases
- purine, pyrimidine bases ( ribonucleotides (salvage
pathway)
- purine, pyrimidine bases ( waste products
- ribonucleotides ( purine, pyrimidine bases ( waste
products
- nucleotide synthesis
- de novo: from scratch, using amino acids and other small
molecules (highly expensive)
- base salvage: from body or diet; more efficient (relatively
inexpensive)
recycling of nucleosides- mechanism
- NTPs ( mRNA ((PPi; RNA polymerase)
- mRNA ( NMPs (H2O(, nuclease)
- NMPs ( NDPs (ATP ( ADP, kinase)
- NDPs ( NTPs (ATP ( ADP, kinase)
- enzymes
- nucleotide kinase: add phosphate groups to NMPs and NDPs,
often using ATP as a Pi donor
- nucleoside kinase: converts ribonucleosides to ribonucleotides
as NMPs
catabolism (degradation) of nucleotides- excess nucleotides:
will be degraded into ribose and nucleotide bases, and bases will
be excreted
- nucleotide degradation: NMP to hypoxanthine
- pathway: AMP to hypoxanthine (blood)
- AMP ( adenosine, or A (H2O(, (Pi; phosphatase)
- adenosine ( inosine, or I (H2O(, (NH3; adenosine
deaminase)
- inosine ( hypoxanthine (Pi(, (ribose 1-P; nucleoside
phosphorylase)
- pathway: AMP to hypoxanthine (other tissues, such as
muscle)
- AMP ( adenosine, or A (H2O(, (Pi; phosphatase)
- inosine ( hypoxanthine (Pi(, (ribose 1-P; nucleoside
phosphorylase)
- adenosine ( inosine, or I (H2O(, (NH3; adenosine
deaminase)
- CMP, GMP, UMP degradation
- similar pathways, but base is not modified
- cytosine, guanine, and uracil are released
- adenosine deaminase defect: severe combined immunodeficiency
disease (SCID)
- ADA defect decreases conversion of adenosine to inosine
- this is particularly damaging to lymphocytes, though the
reasons are not well understood
- SCID: first disease with which gene therapy was attempted
- nucleotide degradation: hypoxanthine ( uric acid
- pathway
- hypoxanthine ( xanthine (xanthine oxidase)
- xanthine ( uric acid (xanthine oxidase)
- uric acid
- beneficial anti-oxidant maintained near its solubility limit
in blood by active re-absorption from kidney
- gout: caused by excess uric acid, which crystallizes painfully
in the joints and kidneys
- allopurinol: competitively inhibits xanthine oxidase; soluble
xanthine and hypoxanthine are secreted
base salvage pathways- base salvage pathway: overview
- bases can freely cross cell membrane
- in tissues where they are in excess, they can be degraded
- they can then diffuse to a tissue where they are needed, and
then be reincorporated into nucleotides
- this is also how ingested bases, and pharmaceutical analogs,
can be used as nucleotides
- base salvage pathway
- ribose 5-phosphate: the backbone of base salvage and de novo
nucleotide synthesis
- enzymes in the base salvage pathway
- activation: requires ribose 5-phosphate conversion to
5-phosphoribosyl-1-pyrophosphate (PRPP)
- conversion to nucleotide: requires a phosphoribosyl
transferase (PRT)
- general pathway
- ribose 5-phosphate ( 5-phosphoribosyl-1-pyrophosphate (ATP (
AMP, Mg2+; PRPP synthetase)
- PRPP + nucleotide base ( NMP ((PPi; nucleotide PRT)
- NMP ( NDP ( NTP (ATP ( ADP, nucleotide kinase)
- specific pathways: hypoxanthine-guanine phosphoribosyl
transferase (HGPRT)
- guanine: guanine ( GMP (PRPP ( PPi; HGPRT)
- inosine: hypoxanthine ( IMP (PRPP ( PPi; HGPRT)
- hypoxanthine is intermediate in structure between adenine,
guanine
- it can therefore be used as a precursor to both AMP and
GMP
- clinical problems: HGPRT deficiency
- partial decrease: increased uric acid, susceptibility to
gout
- complete deficiency: Lesch-Nyhan syndrome
- characterized by mental retardation, spastic cerebral palsy,
and propensity for self mutilation
- also leads to gout, usually death by kidney failure
- X-linked recessive trait: essentially restricted to males
deoxyribonucleotide synthesis from ribonucleotides- general
pathway
- NDP ( dNDP (ribonucleotide reductase)
- note: ribonucleotide reductases only work on NDPs
- dNDP ( dNTP (nucleotide kinase)
- specific pathway: formation of dTTP from dUDP
- dUDP ( dUMP (nucleotide phosphatase)
- dUMP ( dTMP (thymidilate synthase)
- dTMP (( dTTP (nucleotide kinase)
- keeping dUTP levels low
- because DNA polymerase does not distinguish between dUTP,
dTTP, high [dUTP] is toxic to the cell
- dUTPase: expressed by cells to hydrolyze dUTP to dUMP, thereby
promoting thymidilate conversion to dTMP
- binding affinities for dUTP
- DNAp: Km = 10 m
- dUTPase: Km = 1 m
- the higher dUTPase affinity for dUTP helps assure dUTP will be
held too low for common use by DNAp
- 5-fluoro-dUMP: cancer drug
- function: irreversibly inhibit thymidilate synthase,
indirectly halting cell division and slowing tumor growth
- pathway: base salvage
- 5-flurouracil ( 5-F-UMP (PRPP ( PPi; pyrimidine PRT)
- 5-F-UMP ( 5-F-UDP (ATP ( ADP; nucleotide kinase)
- 5-F-UDP ( 5-F-dUDP (XH2 ( X + H2O; ribonucleotide
reductase)
- 5-F-dUDP ( 5-F-dUMP (H2O ( Pi; nucleotide phosphatase)
- 5-F-dUMP covalently binds thymidilate synthase during reaction
with the enzyme, halting it
23. Retroviruses and Gene TherapyStudy Guidedo the following:-
infective cycle of a simple retrovirus
- virus recognized, taken into cell, and reverse transcribed
from single RNA to double DNA
- double-stranded DNA provirus is integrated into host
chromosome, where it is transcribed
- protein-coding genes used to make encapsidation proteins,
which encapsidate the genomic RNA
- encapsidated virus particles exit cell, look for more
targets
- SCID and ADA: first trial
- surviving T cells removed from patient, infected with
retroviruses containing functional ADA genes
- recombinant T cells infused into patient, giving a marked
improvement in clinical condition and T cell count
- improvements: infect progenitor cells, rather than
differentiated T cells, to reduce the need for repeated
infusions
- HSV thymidine kinase and ganclovir
- ganciclovir cannot be recognized by cellular nucleoside
kinases in order to be activated
- HSV tk does recognize it, however, converting it to a dGMP
analog which cellular kinases will recognize
- by using gene therapy, the HSV tk gene can be incorporated
directly into tumor cells
- with infusion of ganciclovir, tumor cells take substance in,
where it works to halt DNA replication
you should know:- creation of a vector
- recombinant DNA technology is used to make a viral plasmid
containing encapsidation signal, foreign gene
- helper cells are created with helper plasmids containing
encapsidation proteins, defective encapsidation signal
- helper cells are infected with vector plasmids
- helper plasmid proteins encapsidate vector genomic DNA,
creating mature virus particles that can be collected
- advantages and disadvantages of retroviruses
- small genome: easily manipulated, but cant accept large
genes
- infects wide range of tissues: greater target possibilities,
but more difficult to be specific
- incorporation into host DNA: more stable expression of foreign
gene, but can also modify normal expression
- requires cell division: easier to target cancers, but harder
to work in differentiated tissues
Notes: Lecture and Readinggene therapy: general considerations-
use
- gene therapy: introduction of genes into a patient as a means
of treating or preventing a disease
- targets: somatic cells
- hereditary mutations would still be passed on to progeny
- genetic modification of germ cells in humans is currently
non-viable
- technique: insert the gene into a viral genome and use it to
infect target cells
- vector: virus that has been engineered for the purpose of
infecting cells with specific genes
- retroviruses
- contain an RNA genome that is copied into DNA (reverse
transcriptase) after entering a cell
- commonly used vector in gene therapy
- DNA cloning: used in gene therapy to isolate large amounts of
a gene, insert it into an appropriate vector
infective cycle of the retrovirus and its inhibition by AZT-
characterization based on regulatory mechanisms
- simple: Moloney Murine Leukemia Virus (MMLV); widely
understood, commonly used
- complex: Human Immunodeficiency Virus (HIV); regulation poorly
understood, but could be useful in the future
- infective cycle of the retrovirus
- entrance: virus uses cell surface receptors to gain entrance
into the cell
- reverse transcription: reverse transcriptase; single-stranded
RNA genome ( double-stranded DNA (provirus)
- integration: provirus enters cell nucleus, recombines into
host chromosome to become part of host DNA
- transcription: cellular RNAp II generates viral genomic RNA
and viral pre-mRNA
- splicing: viral pre-mRNA is used to generate mature mRNA
- translation: viral proteins are made from mRNA
- encapsidation: viral proteins encapsulate viral genome,
forming virus particles (RNA core, protein capsule)
- budding: virus particles bud from cell surface, becoming
surrounded in membrane envelope
- HIV retrovirus: inhibition by AZT
- ddNTP: dideoxy nucleotide triphosphates that, when
incorporated into DNA, halt synthesis
- HIV reverse transcriptase, a sloppy enzyme, has an unusually
high affinity (low Km) for certain ddNTPs
- AZT: 3-azido-2,3-dideoxythymidine
- DDI: 2,3-dideoxyinosine
- cellular DNAp has a much higher Km for AZT, DDI, and is thus
less sensitive to these drugs
retroviral vectors and their use in gene transfer- simple
retrovirus genome
- three protein
- gag: capsid protein
- pol: reverse transcriptase
- env: membrane envelope protein
- cis-acting elements
- used in reverse transcription, integration, proviral
transcription, RNA processing, translation, encapsidation
- typically found in long terminal repeats (LTRs) near the end
of the genome
- construction of a vector provirus
- elements
- DNA cloning: process by which large quantities of a specific
DNA fragment can be made in bacterial cells
- plasmids: small, circular, extragenomic DNA naturally found in
bacteria
- restriction enzymes: used to cut DNA at specific sites
- DNA ligase: used to ligate digested DNA
- process
- plasmid is digested at specific restriction enzyme sites
- proviral DNA is inserted into the plasmid, and DNA ligase is
used to ligate the fragments
- therapeutic gene is inserted in place of the viral genes in
further steps of digestion, ligation
- recombinant plasmid is introduced into bacterial cells, and
cells are cultured
- construction of an encapsidation-defective helper provirus
- vector provirus lacks the proteins for encapsulation, though
it contains the receptors to be encapsulated
- encapsulation signal: cis-acting genomic element by which
viral genome is recognized
- in helper cells, the original provirus has its encapsulation
signal knocked out
- thus the helper provirus can create the packaging proteins,
but cannot itself be packaged
- construction of a vector
- proviral DNA form of genome is inserted into a bacterial
plasmid
- the encapsidation proteins are removed, a foreign gene is
inserted, and the DNA is ligated
- helper cells are made by inserting the E- helper provirus
plasmid into bacteria
- vector plasmid is inserted into helper cells
- E- helper provirus directs expression of encapsidation
proteins, which encapsidate vector genome to make virus
- virus particles are collected from cell culture, used for gene
transfer
- thus, for the sake of avoiding out of control infection, the
vector virus is initially split
- vector genome: contains foreign gene, encapsidation signal,
and can only be used for genetic transfer
- helper provirus: contains encapsidation genes, no
encapsidation signal, can only be used for initial
encapsidation
- advantages and disadvantages of retroviral vectors
- small genome: easily manipulated, but cannot accept large
genes
- infects broad range of tissues: can be used for many diseases,
but is harder to target with specificity
- host integration of proviral DNA: stable maintenance of
therapeutic gene, but can alter cellular gene expression
- integration requires cell division: helps to target cancer
cells, but limits use in differentiated tissues
gene transfer trials- first trial: severe combined
immunodeficiency disease (SCID) due to inherited adenosine
deaminase (ADA) defect
- process
- surviving T lymphocytes were isolated
- retroviral vector was used to transfer a functional ADA gene
into T cells
- T cells were reintroduced to bloodstream
- results
- gave a marked improvement in clinical condition, T cell
count
- however, required repeated infusions, as T cells typically
survive 3-6 months
- desire: incorporate ADA into progenitor cells, so that
repeated infusions would not be necessary
- subsequent trials: SCID due to X-linked absence of functional
gamma chain of CD3
- results: 9 of 11 boys successfully treated
- complications: 2 of these developed T cell leukemia 30 34
months after therapy
- in one cell, gene had incorporated near LMO2, a proto-oncogene
involved in hematopoiesis
- this acted as a transcription activator, leading the cell to
grow more frequently and be selected for
- through normal mechanisms of cancer, it picked up other
mutations and became cancerous
- cancer treatments: ganciclovir
- function: halts DNA replication when incorporated into
replicating DNA
- problem
- active form of ganciclovir (dGTP analog) cannot be taken in by
cells
- however, mammalian nucleoside kinases do not recognize
inactive form, cannot activate it
- solution: herpes simplex virus (HSV) thymidine kinase (tk)
- HSVtk has a broad substrate specificity, and can convert
ganciclovir to a dGMP analog
- cellular nucleotide kinases can then recognize, convert to
dGTP analog, where it can be used
- trial use in gene therapy
- brain tumors were directly injected with a retrovirus
containing an HSV tk gene
- after integration into tumor DNA, ganciclovir was
administered
- only tumor cells expressing HSV tk used ganciclovir
- results: now in phase III trials with 200 patients
enrolled
25. Membrane Structure and TransportStudy Guide
do the following:- phosphoglyceride structure
- glycerol 3-phosphate
- head group: polar alcohol, phosphodiester linkage
- fatty chains: fatty acids, ester linkage
- major classes of lipids and membrane proteins
- phosphoglycerides: glycerol 3-phosphate linked to two fatty
acids and a polar alcohol group
- sphingolipids: sphingosine ester-linked to polar head group,
amide-linked to fatty acid
- sphingomyelin: phospocholine head group
- glycosphingolipids: sugar residues in head groups; often
involved in cell-cell signaling
- cerebrosides: glucose, galactose as a head group
- gangliosides: complex oligosaccharide head groups
- cholesterols: small polar hydroxyl head group, nonpolar
steroid rings, nonpolar hydrocarbon tail
understand the following:- membranes
- membrane structure: aliphatic lipids forming a bilayer, with
hydrophobic tails congregating at the center
- membrane fluidity: determined by lipid composition, with
increased saturation leading to decreased fluidity
- membrane asymmetry: lipids and proteins are unable to
spontaneously flip within the bilayer
- transport proteins
- channels:
gated pores with access regulated by specific signals
diffusion-limited
- transporters:physical binding and facilitated transport
protein kinetics
- transport
- passive: solute down an electrochemical gradient
- active: transport against an electrochemical gradient,
requiring coupling to energy source
Notes: Lecture and Readingthe composition of membranes-
composition
- primary component: lipids, proteins
- secondary component: carbohydrate (usually as glycolipid or
glycoprotein)
- fluid mosaic model: lipid bilayer
- function:barrier to most macromolecules, as well as common
nutrients (glucose, amino acids)
- lipids:bilayer structure of membrane, as well as fluid
nature
- proteins:diffuse in two dimensions, incapable of passive
directionality changes, responsible for transport
lipid components: phosphoglycerides, sphingolipids, and
cholesterol- lipid components: overview
- amphipathic: molecules containing both polar (hydrophilic) and
nonpolar (hydrophobic) regions
- polar lipids: amphipathic lipids such as those in biological
membranes
- in aqueous solutions, hydrophobic tails oriented away from
water, with polar ends interacting with water
- three classes: phosphoglycerides, sphingolipids,
cholesterol
- phosphoglycerides
- general structure: glycerol 3-phosphate (G3P) linked to two
fatty acids (R1, R2) and a polar alcohol group (X)
- fatty acids:
ester linkage
OC(=O)R
- polar alcohol group:phosphodiester linkageOP(=O, OH)OX
- fatty acyl groups
- fatty acid composition
- R1: usually saturated
- R2: usually unsaturated
- examples
- palmitic:C16
- stearic:
C18
- oleic:
C189
- chain length
- non-polarity: longer chain length increases
- melting point: increases with increasing non-polarity (chain
length)
- membrane fluidity: influenced by melting point of lipids
within the lipid bilayer
- degree of unsaturation
- n: double bond at position n to (n+1)
- melting point: decreases with increasing degree of
unsaturation
- animal fats: high melting point (numerous saturated acids)
- plant fats: lower melting point (unsaturated fatty acids)
- common fatty acids in human phosphoglycerides
common namesaturationcarbonsabbreviationmelting point (C)
palmiticsaturated16C1663
stearicsaturated18C1870
oleicmonounsaturated18C18913
linoleicpolyunsaturated18C189,125
- memorization: PSOL (16, 18, 18 9, 189,12)
- polar alcohol groups
- choline
- ethanolamine
- serine
- glycerol
- inositol
- sphingolipids
- localization: found in all tissues, especially abundant in
neural tissue (defects in metabolism give retardation)
- general structure: sphingosine ester-linked to polar head
group, amide-linked to fatty acid
- sphingosine: long structure with a non-polar tail
- overall appearance: similar to phospholipid, but without
glycerol, and only one varying tail
- sphingomyelin: phosphocholine head group
- phosphocholine ester-linked to terminal alcohol moiety of
sphingosine
- structure, properties similar to phosphatidylcholine
- glycosphingolipids: one or more sugar residues in head
group
- cerebrosides: glucose or galactose as a head group
- gangliosides: large, polar head groups composed of complex
oligosaccharides
- involved in recognition events at cell surface
- ABO blood type specified bye glycosphingolipids, other
glycoproteins present on RBC surface
- cholesterol
- general structure: small polar hydroxyl head group, nonpolar
steroid rings, nonpolar hydrocarbon tail
- steroid rings: rigid, increase membrane rigidity
effects of lipid composition on membrane structure and fluidity-
asymmetry of lipids in bilayer
- lateral movement: free within plane of bilayer
- vertical movement: lipids do not flip flop from one side to
another
- this gives membranes asymmetrical surface based on
synthesis
- membrane fluidity
- two states: liquid, gelatin
- biological membranes, ph
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