2. Introduction to Lipids and Lipoprotein Metabolism (Part II) Flashcards
1
Q
What is the transfer of cholesterol from cell membranes of peripheral cells to HDL driven by?
A
By the higher concentration of UC on peripheral cells
2
Q
What are the requirements for the synthesis of HDL?
A
- Apo A-1, made by hepatocytes, requires phosphatidylcholine
- Apo A-1 is associated with CM. As TG decreases, then the phospholipids and Apo-A1 pinch off and become HDL
3
Q
What are the effects of FH cells in response to lipoprotein addition?
A
- No effects on CE stores
- No effect on rate of UC synthesis
- No effect on HMG-CoA reductase activity
4
Q
What are your tracing with radioactive iodine125?
A
Allows us to follow the protein portion of a lipoprotein
5
Q
What are you tracing with radioactive hydrogen3?
A
- Fatty acyl chains
- Cholesterol moiety
6
Q
What are doubly labeled lipoproteins?
A
Labeled with radioactive tracers on the protein and on the lipid
7
Q
What is the dysfunction of FH cells?
A
FH cells are unable to bind, internalize, or metabolize LDL
8
Q
What are the effects of increasing LDL concentration in normal cells during binding, internalization, and hydrolysis?
A
- Binding: increase with concentration
- Internalization: increase with concentration
- Hydrolysis: increase with concentration
9
Q
What are the effects of increasing LDL concentration in FH cells during binding, internalization, and hydrolysis?
A
- Binding: no increase with concentration
- Internalization: slight increase with concentration
- Hydrolysis: slight increase with concentration
10
Q
What is hydrolyzed after internalization of LDL?
A
Apolipoprotein B
11
Q
What are the effects of increasing LDL concentration in normal cells during cholesterol synthesis, and cholesterol esterification?
A
- Cholesterol synthesis: decrease with concentration
- Cholesterol esterification: increase with concentration
12
Q
What are the effects of increasing LDL concentration in FH cells during cholesterol synthesis, and cholesterol esterification?
A
- Cholesterol synthesis: no decrease with concentration
- Cholesterol esterification: no increase with concentration
13
Q
What happens when you remove LDL in terms of HMG-CoA reductase activity in normal cells and FH cells?
A
- Normal cells: increase in HMG-CoA reductase activity
- FH cells: no response
14
Q
What happens when you add LDL in terms of HMG-CoA reductase activity in normal cells and FH cells?
A
- Normal cells: rapid decrease in HMG-CoA reductase activity
- FH cells: no response
15
Q
What is the cause of FH?
A
The absence of the LDL receptor
16
Q
What are the four classes of LDLR gene mutations?
A
- Synthesis
- Transport from ER to Golgi
- Binding of LDL
- Clustering in coated pits
17
Q
Which class of LDLR gene mutation can bind to LDL and be saturable as well?
A
Clustering in coated pits
18
Q
Why are there multiple factors that can be affected by gene variations in the LDL receptor?
A
Because it is a multi-domain protein, so there are many things that can be affected
19
Q
What apolipoprotein is the protein found in LDL? What kind of cholesterol?
A
- Apo B-100
- Cholesterol linoeoleate
20
Q
What is the pathway of cholesterol in a hepatocyte?
A
- Binds to a receptor
- Internalization
- Lysosomal hydrolysis
- Release of cholesterol to the ER
- Regulatory actions
21
Q
What are the regulatory actions caused by the internalization of cholesterol?
A
- Decrease in HMG-CoA
- Increase in ACAT for cholesterol esterification
- Decrease in LDL receptors
22
Q
What form is cholesterol under following esterification?
A
Cholesterol oleate
23
Q
Which apolipoprotein requires a APO B/E receptor in the liver?
A
LDL
24
Q
What receptor causes familial hypercholesterolemia?
A
APO B/E receptor
25
What can cause Metabolic Syndrome?
- Genetic disorders in Apo A-II in HDL
| - Genetic disorders in the CD36 receptor for fatty acids
26
What are the mechanisms for cellular homeostasis in cells other than hepatocytes if cholesterol concentration increases?
- ACAT converts cholesterol into CE
- Cholesterol travels to the membrane in hopes of being transferred to HDL
- Decrease in LDLR
- Decrease in HMG-CoA reductase
27
In which organelle is most cholesterol found in a cell?
The ER
28
Which transporter functions to help cells get rid of cholesterol from their membrane onto HDL?
ABCA1
29
What is the sterol response element?
A short and specific DNA sequence located in the promoter region of genes
30
What is the expression of SRE increased by? What is it decreased by?
- Increased by cholesterol depletion
| - Decreased by cholesterol excess
31
What is the sterol response element?
Short and specific DNA sequence located in the promoter region of genes whose expression is increased by cholesterol depletion, and decreased by cholesterol excess
32
What is the result of adding this sequence to the promoter region of other genes? Give an example.
Confers sensitivity to cellular cholesterol status in the same way as HMGR and LDLR genes
33
What is SREBP?
A protein that binds specifically to the SRE sequence and stimulates transcription (transcription factor)
34
What is the binding of SREBP to SRE dictated by?
By the concentration of unesterified cholesterol within the cell
35
When are SREBPs activated?
In response to decreased cellular cholesterol concentration
36
Where are SREBPs located?
On the ER membrane
37
What are the three isoforms of SREBP?
- SREBP-1a
- SREBP-1c
- SREBP-2
38
Which isoforms of SREBP are encoded by the same gene?
- SREBP-1a
| - SREBP-1c
39
What genes does SREBP-1a regulate?
Potent activator of all SREBP responsive genes
40
What genes does SREBP-1c regulate?
Regulates genes involved in fatty acid synthesis
41
How does SREBP-1c compare to SREBP-1a?
SREBP-1c is a weak activator compared to SREBP-1a
42
What genes do SREBP-2 regulate?
Regulates genes involved in cholesterol synthesis
43
What is the function of the transmembrane domain of SCAP?
Sterol-sensing transmembrane domain
44
What is the function of the C-terminal end of SCAP?
Interacts with SREBP
45
What is the function of Insig-1?
Retains the SREBP-SCAP complex in the ER when cholesterol is high
46
Explain what happens to SREBP when cholesterol is low.
1. Change in Insig-1 conformation, allows SREBP-SCAP complex to travel to the Golgi
2. Proteases on the Golgi cleave the DNA-binding domain from the complex
3. DNA-binding domain travels to the nucleus and binds to SRE
47
The DNA-binding domain of SREBP induces the expression of what responsive genes when it binds to SRE?
- HMG-CoA reductase
| - LDLR
48
What is the function of the S1 protease?
Cuts the luminal link of SREBP
49
What is the function of the S2 protease?
Cuts the linkage between the DNA-binding domain and the transmembrane domain
50
Which protein is an ER resident protein?
Insig
51
Which proteins in the SREBP molecular mechanism have cholesterol-sensing domains?
- Insig
| - SCAP
52
What can calcium modify in terms of cholesterol overall?
- The way cholesterol can regulate the activation of SREBP
| - Allows the cholesterol to move into the ER so that the cell can read the concentration of cholesterol
53
What characteristic allowed Brown and Goldstein to use skin fibroblasts?
The fact that cholesterol metabolism using the LDL receptor is present in all cells of the body
54
What is the role of cholesterol within the ER?
Plays a role in activating gene expression
55
The removal of cholesterol causes the expression of the ____ genes.
SRE
56
Do Insig, SCAP, or SREBP bind to cholesterol?
- Insig and SCAP do
| - Have cholesterol-sensing domains
57
What does mTORC1 regulate?
The function of protein and lipid metabolism (including SREBP)
58
Why is it logical that both lipids and proteins are regulate by the same component (mTORC1)?
Because when you increase the volume of the cell, you require both proteins and lipids
59
What is ABCA1?
- Codes for a transporter involved in the efflux of cholesterol from cells
- When high cholesterol concentration is present
- Onto HCL3
60
What is the role of ABCA1 when there is a high amount of cholesterol in the cell?
- The cholesterol moves from the ER to the cell membrane through a transporter encoded by ABCA1
- Starts reverse cholesterol transport
61
What is APOE?
- Codes for an apolipoprotein (Apo E) that can serve as a ligand for the LDL receptor
- Apo E is an exchangeable apolipoprotein
62
What is the role of Apo E when there is a high amount of cholesterol in the cell?
- An increase in Apo E promotes Apo E binding to the LDLR so that the cells can take it up
- As an exchangeable apolipoprotein, this results results in more chances for the apolipoproteins to be taken up by the liver LDL receptor
63
What is Cyp7a1?
- Codes for a microsomal enzyme that catalyzes the first and rate-limiting step in the classical bile acid biosynthetic pathway
- Promotes the conversion of cholesterol in the liver to bile acids for disposal
64
Is Cyp7a1 present in humans? How does it differ from mice Cyp7a1?
- Yes
| - BUT, it does not respond to excess cholesterol levels, as in mice
65
Differentiate the functions of ABCA1, Apo E, and Cyp7a1 when there is a high cholesterol concentration.
- ABCA1: starts reverse cholesterol transport
- Apo E: promotes internalization of the lipoproteins by the liver
- Cyp7a1: promotes the conversion of cholesterol in the liver to bile acids for disposal
66
Explain the reasoning behind the name "nuclear receptors."
They exist and are normally found in the nucleus bound to their target sequences
67
How are nuclear receptors bound to their ligand and DNA?
They can exist bound to DNA with OR without a ligand
68
What are the multiple domains of nuclear receptors?
- Ligand binding domain
| - DNA binding domain
69
```
The HRE half-site is:
A) 5'-ACCTGA-3'
B) 5'-ATGACC-3'
C) 5'-CCAGTA-3'
D) 5'-AGGTCA-3'
E) 5'-ACTGAC-3'
```
D) 5'-AGGTCA-3'
70
What is the DNA that nuclear receptors bind to called?
Hormone response element (HRE)
71
The DNA sequence that NRs bind to is characterized by how many sites?
Two half sites
72
What is a direct repeat?
One direction of the half-sites (from left to right or from right to left)
73
What is an everted repeat?
Pointing in opposite directions to the outside
74
What is an inverted repeat?
Pointing towards the middle
75
How many nucleotides could DNA half-sites for NRs contain?
0 to 12 nucleotides
76
Which receptor is the only NR receptor that can exist outside of the nucleus?
Estrogen; arrives to the nucleus when estrogen enters the cell
77
What are orphan receptors?
Refers to the fact that we are unsure what they bind
78
What are monomeric receptors?
- Only requires ONE receptor to be bound to the HRE
| - Still bounds as a homodimer, but only ONE needs to be contacting the DNA
79
How do RXR heterodimers function?
- One binds to the compound
| - The other allows it to bind to DNA
80
Steroid hormones function as a ________.
homodimer
81
T3R, RAR, VDR, CAR, LXR are examples of ligands for which receptor?
RXR heterodimers
82
RXR, COUP, HNF-4 are examples of ligands for which receptor?
Dimeric orphan receptors
83
GR, MR, PR, AR, and ER are examples of ligands for which receptors?
Steroid receptors
84
ROR, ERR, NGFI-B, SF-1 are examples of ligands for which receptor?
Monomeric orphan receptors
85
What are the four types of hormone receptors?
- Steroid receptors
- RXR heterodimers
- Dimeric orphan receptors
- Monomeric orphan receptors
86
Where are NRs usually found in DNA?
- In the promoter region
| - Upstream of the start site
87
What are the two possibilities when a ligand binds to a nuclear receptor?
- Ligand binding
| - Ligand release
88
What can cause ligand release to an NR receptor?
When there is not enough ligand, it releases
89
Ligand binding (or release) causes what two consequences?
- Recruitment/release of co-activators
| - Recruitment/release of co-repressors
90
The recruitment/release of co-activators/repressors causes what two consequences?
- Stimulation of gene transcription
| - Repression of gene transcription
91
What are the two isoforms of LXR?
- Alpha
| - Beta
92
What are LXRs activated by? When is that generated?
- Oxysterols
| - Generated during the synthesis and metabolism of cholesterol
93
Where is LXR-alpha predominantly expressed?
- In tissues and cells that are important in maintaining lipid homeostasis
- Liver, intestine, adipose tissue, macrophages
94
Where is LXR-beta expressed?
Ubiquitous distribution
95
Are LXR-alpha and LXR-beta encoded by the same gene?
No, they are encoded by separate genes
96
Explain how ABCA1 expression is increased when cholesterol is increased in the cell.
- Oxysterols are produced
- Activates LXR-alpha
- Stimulates the transcription of the ABCA1 gene
- Facilitation of the efflux of cholesterol from cells
97
What molecular mechanism explains why mice are capable of increasing their synthesis of bile acids when there is an increase in cholesterol?
- Mice have LXRs on their promoter elements of Cyp7a1, which are activated by oxysterols (increased during high cholesterol)
- Humans do not; but they still possess Cyp7a1
98
SREBP-1c regulates genes involved in what?
- Fat metabolism (involved in TG biosynthesis)
| - Ex: steroyl CoA desaturase
99
What nuclear receptor regulates SREBP-1c?
LXR
100
What are synonymous single nucleotide polymorphisms?
- Distinct SNPs at the same position lead to the SAME polypeptide sequence
- Due to differences in the Wobble position
101
What are non-synonymous single nucleotide polymorphisms?
Distinct SNPs at the same position lead to DIFFERENT polypeptide sequences
102
Which allele leads to hypertriglyceridemia and diabetes (Japanese population)?
Apo E7 (E244K, E245K)
103
Which allele leads to Alzheimer's disease?
Apo E4 (C112R)
104
Which allele leads to optic neuropathy? How?
- SNP-219G
| - It is NOT in the protein coding region of the gene, but in the promoter region
105
What are expressed sequence tags (ESTs)?
Short sequences that are generated by sequencing either one or both ends of an expressed gene (i.e. sequences from cDNAs of mRNAs)
106
Are all ESTs identified?
- No, some are (mapped to correspond fully characterized genes)
- Many correspond to unidentified genes
107
Why would you want to increase Apo E if there is high cellular cholesterol concentration?
For the liver to increase uptake of chylomicron remnants, IDL, VLDL, and HDL
108
Why does C. elegans have much more nuclear receptors than humans?
Because they need to respond to their environment extremely quickly
109
Do small nucleotide polymorphisms exist in twins?
Yes
110
Why do we turn RNA into cDNA for sequencing?
Because RNA is extremely difficult to work with as it is very labile
111
How was the Human Genome Project completed?
ESTs (from RNA to cDNA) were mapped back to the genome
112
What does Q-PCR mean?
Quantitative polymerase chain reaction
113
What are you trying to observe from the cells or tissues in Q-PCR?
- The abundance of each mRNA
| - If you have a lot of mRNA, it is likely that the protein being encoded by the RNA is also increased
114
Describe the Q-PCR process.
1) Acquire tissues or cells
2) DNase treatment
3) Reverse transcription + primers + specific probe(s)
4) Fluorescent cDNA product and relative quantitation
115
What is the downside of Q-PCR?
- Only good for one target at a time
| - If there are 20 000 distinct kinds of mRNA, you will need 20 000 experiments
116
What process is faster than Q-PCR?
Microarray
117
What can Q-PCR and microarray be used for?
Study WHICH and how SPECIFIC mRNA species are altered by changes in state of nutrition or metabolic status, or in the state of disease
118
Describe the microarray process.
1) Acquire cells or tissues to acquire the total RNA
2) Reverse transcription to cDNA
3) Transcription to cRNA and addition of fluorescence
4) Fragmentation and hybridization to target gene sequences in microarray
5) Scan and quantification
119
What is present in each slide of a microarray?
Printed, immobilized pieces of complimentary mRNA (complimentary to a specific gene) on a glass surface
120
What do the colours indicate in a microarray analysis?
- Green: increase in experimental mRNA
- Red: decrease in experimental mRNA
- Yellow: experimental mRNA is the same as the control
- Black: no target gene present (gene is not expressed)
121
What is hybridization?
RNA pairing with complimentary DNA, which forms a hybrid duplex
122
What is the problem with microarray analysis? What is used instead?
- It deals with fragmented pieces of RNA
| - Instead, we use RNA-Seq
123
What does RNA-Sequencing provide?
Provides sequence information, including RNA sequences translated into genomic sequences, mapping genes that have not yet been determined
124
What does a RNA expression level vs. nucleotide position graph for RNA sequencing tell us?
Tells us which regions of the genes express a lot of mRNA
125
Which technique allows us a much better method to identify SNPs?
RNA-Sequencing
126
Assuming that the rate of degradation of all RNA is equal, what does that tell you?
- An increase in transcription rate EQUALS an increase in RNA abundance
- So, you could make more protein from RNA
127
In real life, which rates change based on nutrient regulation?
- Rates of initiation of transcription
- Rates of decay
- Rate of degradation
128
How can you separate proteins?
- SDS-Page
| - HPLC
129
How can you measure the molecular mass of a protein? What does this allow?
- Mass spectrometer
- Because we know the sequence of every protein from the genes we own, we can predict what kinds of genes cause the expression of these proteins
130
What are transgenic mice?
Mice in which there is an addition of new genes (e.g. human genes)
131
What are gene "knock-out" mice?
Mice in which there is the creation of specific variations
132
Give examples of inbred strains of mice.
- C57BL/6J
| - Swiss albino
133
What do variations in inbred strains of mice allow?
Allows you to compare how these mice respond to a similar challenge, and to track down the differences using bioinformatics
134
What are disadvantages of mice as an experimental model for human physiology?
- Metabolic differences
- Nutrient requirement differences
- Genetic differences
135
How can bioluminescence be used in transgenesis?
As a tracer to figure out where the genes are expressed
136
Describe the transgenesis protocol in mice.
1) Transgene construction
2) Cloning
3) Evaluation in vitro
4) Microinjection into the zygote
5) Embryo transfer
6) Genomic analysis
137
Describe the process of pronuclear microinjection.
- The zygote is held by a glass needle with a gentle suction (immobilized)
- A fine glass needle is used to inject a solution of DNA inside the male pro-nucleus
- DNA inserted integrates with the genome of the mouse
138
What is transgenesis?
Natural or synthetic gene(s) introduced into another genome for permanent propagation
139
Differentiate natural and synthetic genes.
- Natural: genes obtained from another species
| - Synthetic: contains components from other genes or species
