Topic 2 extras Flashcards
1
Q
When MTP is defective, no Apo-__-containing particles are present in plasma, and only __ are present
A
When MTP is defective, no Apo-B-containing particles are present in plasma, and only HDLs are present
2
Q
Abetalipoproteinemia patients generally have mutations in ___
A
Abetalipoproteinemia patients generally have mutations in microsomal transfer protein (MTP)
3
Q
What are epigenetic modifications?
A
Epigenetic modifications, a mechanism for controlling the use of genetic information during the lifetime of the organism as well as the next generation
4
Q
What are inborn errors of metabolism?
A
o Inborn errors of metabolism are mistakes that occur in nature that have an impact on one specific gene
o Inborn errors of metabolism are rare genetic (inherited) disorders in which the body cannot properly turn food into energy.
5
Q
Which cells make cholesterol?
A
All cells in body capable of making cholesterol but the liver is the primary biosynthetic organ
6
Q
What is the primary source of fuel of the heart?
A
FA
7
Q
What happens to excess chylomicrons in endogenous lipid transport?
A
The excess chylomicrons make its way to the liver, where the triglycerides are repackaged into VLDLs and sent back out into the bloodstream to be transported to the adipose tissue, muscle and heart. These lipids under lipolysis in these different parts of the body and are used as a source of fuel/energy (ATP).
8
Q
What are the functions of Bile Acids?
A
- Emulsification of fats and fat-soluble nutrients
2. Activator of bile salt activated lipase
9
Q
How are bile acids made?
A
Are made from cholesterol by:
- Hydroxylation of steroid nucleus
- Epimerization of 3-beta-hydroxyl group into a 3-alpha hydroxyl group
- Saturation of the steroid nucleus
- Side chain cleavage
- Attachment to glycine or taurine
10
Q
Hydrolysis of Cholesterol
A
- The digestion of dietary cholesteryl esters involves release of the esterified FA through the action of a BS-dependent CE hydrolase secreted by the pancreas
- Free sterol then undergoes solubilization within mixed micelles in the upper small intestine.
11
Q
How is free cholesterol brought into enterocytes?
A
NPC1L1
12
Q
How are FA brought into enterocytes?
A
FATP4, CD36
13
Q
How are phospholipids brought into enterocytes?
A
are supposedly brought into the cell by MFSD2A transporter
14
Q
Where does assembly of chylomicrons take place?
A
The assembly of chylomicrons occurs within the lumen of the endoplasmic reticulum of the enterocytes
15
Q
Where is MTP found? Why?
A
Lipid-carrier protein like MTP (microsomal TG transfer proteins) exist in the ER and other organelles involved in assembly of chylomicrons
16
Q
What are lipoproteins made of?
A
All types of lipoproteins feature hydrophilic apolipoproteins, phospholipid polar head groups, and cholesterol hydroxyl groups facing outwards at the water interface, with phospholipid acyl tails and cholesterol steroid nuclei oriented towards the interior of the lipoprotein particle. Hydrophobic cholesteryl esters and triglyceride molecules form the core of the lipoprotein particle.
17
Q
Describe alpha-mobility
A
phospholipids, apo A, chylomicrons and HDL
18
Q
Describe beta-mobility
A
cholesterol, LDL, apo B
19
Q
Describe pre-beta-mobility
A
apo B, VLDL
20
Q
Where is Apo A found?
A
HDl, chylomicrons
21
Q
ApoB-48 vs ApoB-100
A
Apo B 48- Chylomicrons, doesn’t limit the max amount of lipids
Apo B-100- VLDL, LDL; limits the max amount of lipids;
22
Q
ApoB-100 is a ligand for which receptor?
A
LDLR
23
Q
ApoE is a ligand for which receptor?
A
ligand for LDL and VLDL receptors
24
Q
Function of ApoC
A
Lipoprotein lipase activator or inhibitor
25
What is the purpose of agarose gel electrophoresis in terms of lipoproteins?
Lipoproteins can be separated by agarose gel electrophoresis
3 predominant bands and thereby were used to separate the lipoproteins into three categories called the alpha mobility, pre-B and beta mobility lipoproteins
26
density of the lipoproteins in ascending order
chylomicrons < VLDL < LDL < HDL
27
lipoproteins in ascending order of size
HDL < LDL < VLDL < chylomicrons
28
Lipoproteins
| Enterocytes make __ and liver cells (hepatocytes) make __
Enterocytes make chylomicrons and liver cells (hepatocytes) make VLDL
29
Do enterocytes or hepatocytes mae LDL?
No
LDL is a metabolic byproduct of VLDL, LDLs are made as VLDLs get smaller in size because the lipids are being hydrolyzed by Lipoprotein Lipase as they travel through the bloodstream.
30
Chylomicron
| major lipid?
Triglycerides
31
VLDL
| major lipid?
Triglycerides
32
IDL
| major lipid?
Cholesteryl esters
33
LDL
| major lipid?
Cholesteryl esters
34
HDL
| major lipid?
Phospholipids
35
Where and how is HDL produced?
HDL is produced by lipolysis (removal of lipids) of lipoproteins (mostly VLDL and chylomicrons) as well as being secreted directly by liver in the form of apoA1 (protein) with phospholipids
36
Steps of exogenous transport system
Step 1: Dietary lipids enter the small intestine, which triggers bile acids to be secreted from the gallbladder
Step 2: dietary lipids are hydrolyzed in the lumen of the small intestine and then taken up by the transporters found on the apical surface of the enterocytes lining the small intestine
Step 3: in the enterocytes, the triglycerides and cholesterol are re-esterified and packaged into chylomicrons
Step 4: chylomicrons released from enterocytes circulate first through the intestinal lymphatic system and then enter systemic circulation. Once chylomicrons are released into the blood circulation, then triglyceride hydrolysis occurs at the capillary surface of tissues by lipoprotein lipase (LPL)
Step 5: chylomicron remnants are taken in by liver cells and the lipids (i.e. CE)
37
How can bile acids be lost?
Some bile acids are lost as fecal steroids
38
How is chylomicron assembly regulated?
Chylomicron assembly within the intestinal enterocyte is tightly regulated by the production of apolipoprotein B (apoB) and the activity of microsomal TG transfer protein (MTP), which transfers lipids onto emerging Apo-B particles
39
Apolipoproteins associated with chylomicrons?
Apo A and Apo B
40
What are the 2 Endogenous Transport Systems
1) reverse cholesterol transport
| 2) Movement of lipid from liver to peripheral tissues
41
Describe movement of lipids from liver to tissues
1) In the hepatocytes triacylglycerols and cholesteryl esters are assembled to form nascent VLDL particles. Nascent VLDL particles are released into the bloodstream
2) Again, like chylomicrons, VLDL particles circulate and encounter lipoprotein lipase (LPL) expressed on endothelial cells. LPL hydrolyzes TGs and releases free FAs and glycerol. These products can be absorbed from the blood by peripheral tissues, principally adipose and muscle. They can be used for energy or converted back to Tgs for storage.
3) Through TG depletion, the VLDL particle is converted to a denser, smaller LDL and
TRL (Triglyceride-rich lipoprotein).
4) LDLs can be picked up by the liver via LDL receptor and catabolized.
Modified or oxidized LDL can also be taken up by a scavenger receptor on macrophages in various tissues
LDL can also donate cholesterol to HDL which would carry it to the liver via HDL receptor. LCAT would transfer cholesterol to HDL from LDL
42
Reverse Cholesterol Transport Pathway: direct
• Step 1: Unesterified cholesterol is located on the membrane of peripheral cells
• Step 2: Unesterified cholesterol will move from membrane of peripheral cells onto the surface of HDL
• Step 3: LCAT is an enzyme located on the surface of HDL that will convert unesterified cholesterol into cholesteryl esters
o When the cholesterol becomes esterified (cholesteryl ester), it becomes more hydrophobic and forcing it in the middle of HDL, this means that the surface of the HDL is now empty and able to accept more unesterified cholesterol from the surface of the peripheral cells because there is a gradient produced. As the HDL accepts more unesterified cholesterol, it gets bigger in size.
Step 5: HDL will carry the cholesterol to the liver
43
Reverse Cholesterol Transport Pathway: indirect
CETP is an enzyme that will exchange:
o Cholesteryl esters from HDL -> lower density lipoproteins, such as chylomicron reminants, VLDL and VLDL reminants
o Triglycerides from lower density lipoproteins -> HDL
o Cholesteryl esters will make its way to lower and lower density lipoproteins, until it reaches LDLs where it then can interact with LDL receptors on the surface of liver cells and be taken into the hepatocytes and hydrolyzed back into unesterified (free) cholesterol.
44
What is GPI protein?
* Endothelial cells make up the wall (face the lumen) of capillaries. The endothelial cells express the protein GPI on their surface.
* GPI protein is attached to the plasma membrane of the endothelial cells lining blood vessels and has an acidic domain sticking outwards.
* Chylomicrons interact with the acidic domain of one GPI protein at the same time as LPL interacts with another GPI protein right next to it.
* Both GP1 proteins bring the chylomicron and the LPL together so that the triglycerides in the chylomicrons can be hydrolyzed and the FA can be released into the bloodstream.
45
What is HDL3
HDL precursor
46
What is the effect of saturated fats consumption?
Consumption of saturated fats increase circulating total lipoprotein and LDL CH levels (especially myristic and palmitic acid)
47
Consumption of trans FAs raises __ and lowers __ levels
Consumption of trans FAs raises LDL and lowers HDL levels
48
Effects of n-6 and n-3 PUFAs consumption
* Consumption of n-6 PUFAs lowers circulatory CH levels
| * Consumption of n-3 PUFAs lowers circulatory TG levels (only minor impact on circulatory CH levels)
49
Fates of CH in the liver
1. Esterifying free/unesterified cholesterol: into cholesteryl esters
o The cell prefers cholesteryl esters because they are not bioactive
2. VLDL assembly: using cholesterol to assemble VLDL decreases the unesterified cholesterol poor. VLDL can then be transported out of the liver cell
3. Secretion into bile: the unesterified cholesterol from liver cells can be secreted into bile as a way to decrease unesterified cholesterol pool.
4. Used for bile acid synthesis: bile acids are produced with excess cholesterol and the rate limiting enzyme in the pathway is Cyp7a1. Cholesterol used to make bile is gone forever as bile synthesis is irreversible
50
Ways for peripheral cells to decrease CH levels
the only ways that peripheral cells are able to decrease the levels of unesterified cholesterol is to store as cholesteryl esters or efflux them out of the cell and allow them to circulate throughout the entire body.
51
WHat is the acceptor of excess cholesterol in the cells?
HDL
52
What is CHD?
• CHD is caused by atherosclerosis, a process in which the coronary arteries as well as other arteries become occluded.
53
What is CVD?
• CHD and stroke together are known as cardiovascular diseases (CVD)
54
What is MI?
o A heart attack or myocardial infarction (MI) occurs when one or more of the three major coronary arteries become blocked.
55
What is a stroke?
A stroke occurs when the arteries supplying the brain become occluded.
56
Familial Hypercholesterolemia
| Characteristics and Treatment
Characteristics:
• Results from delayed clearance of LDL associated with defects in the LDL receptor or Apo-B genes
• LDL-C levels in excess
Treatment:
Combination of statin and ezetimibe
57
Acquired vs genetic causes of dislipidemias
o Acquired: malnutrition – deficiency or abnormally high intake of lipids
o Genetic: variations/mutations in genes, which cause low or high lipid concentration in the blood or low/high of one type of lipid in the blood
58
Describe Abetalipoproteinia
An example of hypocholesterolemia
Low to undetectable circulating chylomicrons
Caused by mutations in MTP
Malabsorption of fats and fat-soluble vitamins (e.g. vitamin A, E)
59
What is the function of MTP?
o MTP allows for the combining of Apo-B48 with triglyceride for the secretion of chylomicron particles in the intestine and Apo-B-100 with triglyceride for secretion of VLDL in the liver
Necessary for production of chylomicrons- > MTP is required for the transport of triglycerides, cholesteryl esters, and phosphatidylcholine between intracellular membranes
60
What are the clinical feature of abetalipoproteinemia
* Chronic diarrhea (steatorrhea)
* Retinitis pigmentosa (poor absorption of vitamin A)
* Ataxia
* Star-shaped RBCs (acanthocytosis)
61
What are the dietary management prescriptions in abetalipoproteinemia
1. Restriction of long-chain dietary TG (<15 g/day), use medium chain TG as an alternate source of fat
2. Supplementation of fat-soluble vitamins (A and E)
62
What is ezetimibe?
• Ezetimibe is a drug that blocks the absorption of cholesterol without affecting the absorption of other lipids
o Ezetimibe is a competitive inhibitor of NPC1L1, therefore it binds to the transporter and does not allow cholesterol (the natural substrate) to bind, thereby inhibiting cholesterol absorption
63
Effect of plant sterols on cholesterol absorption
Plant sterols are normally poorly absorbed but they are able to interact with NPC1L1 cholesterol transporter. Therefore, if the concentration of plant sterols increases, the absorption of cholesterol will decrease because plant sterols are competing for the same transporter
64
What did FH study allowed to discover?
The study of FH led to the discovery of low density lipoprotein (LDL) receptor, which undergoes a mechanism by which this receptor mediates feedback control of cholesterol synthesis
65
What is FH caused by?
FH was shown to be caused by inherited defected in the gene encoding LDL receptor; these defects disrupt the normal control of cholesterol metabolism
o The different mutations of the LDL receptor, cause:
1. No receptor synthesized
2. Receptor synthesized, but transported slowly from ER to Golgi
3. Receptors are processed and reach cell surface, but fail to bind LDL normally
4. Receptors reach cell surface and bind LDL, but fail to cluster in coated pits (therefore fail to undergo receptor-mediated endocytosis, in order to bring LDL into the cell)
66
Most of the genome is comprised of __
Most of the genome is comprised of regulatory elements
67
WHat are the observable signs of FH?
High concentration of cholesterol in the blood
| Presence of xanthelasmas and tendon xanthomas
68
How can FH be diagnosed?
Fibroblasts (skin cells) of FH patients can be readily obtained, and can be easily grown and studied in the laboratory.
Lipoproteins can be reliably isolated from the blood of FH patients.
The metabolism of cholesterol can observed.
69
FH cells vs normal cells in terms of cellular cholesterol concentration (as CE), rate of cholesterol (UC) synthesis, and HMG-CoA reductase activity
Normal cells have low cellular cholesterol concentration (as CE), low rate of UC synthesis and low HMG-CoA activity
FH cells have high cellular cholesterol concentration (as CE), high rate of UC synthesis and high HMG-CoA activity
70
Normal vs FH fibroblasts - response to Lp depletion
Normal: Mobilization of CE stores, Increase rate of UC synthesis, Increase in HMG-CoA reductase activity
FH cells
No effect on CE stores (high), No effect on rate of UC synthesis (high), No effect on HMG-CoA reductase activity (high)
71
Normal vs FH fibroblasts - response to Lp addition
Normal cells
Slight increase in cellular CE concentration
Decrease in rate cholesterol synthesis
Decrease in HMG-CoA reductase activity
FH cells
No effect on CE stores (high)
No effect on rate of UC synthesis (high)
No effect on HMG-CoA reductase activity (high)
72
How can lipoproteins be traced?
• Apolipoprotein can be labeled with radioactive tracer (such as I125)
o Use a procedure where a radioactive element such as iodine125 (I125) is added to the apolipoproteins.
o This allows us to track the movement of apolipoproteins along with tracking the lipoproteins themselves as they move throughout the body to cells
73
How can lipids be labeled?
• Lipids can also be labeled with radioactive tracers (such as H3; may be placed on the fatty acyl chains, or cholesterol moiety, etc.)
74
FH fibroblast cells are unable to __, __ or __ LDL
FH fibroblast cells are unable to bind, internalize or metabolize LDL
75
What is a multidomain protein?
one protein with many different parts therefore there are many things that can be affected—different variations
76
what is Apolip of LDL?
ApoB 100
77
What is the process of LDL internalization?
The LDL particle will bind to the receptor, then internalize and in the process of internalization, the endosome is broken down and the CE are broken down and ApoB broken down into amino acids.
78
Response of cells to increased cellular cholesterol concentration
1. Decreased HMG CoA reductase activity
2. Increased ACAT activity to make CE
3. Decreased number of LDL receptors
79
What happens when there is a need for cholesterols in the cell
If there is a need for cholesterol in the cells- CE are broken down by cholesterol esterase so the cholesterol is brought from storage into the ER
80
Danger of high Apo(a) levels?
Apo(a) is associated with high Lipoprotein(a) levels
Genetic studies and numerous epidemiologic studies have identified Lp(a) as a risk factor for atherosclerotic diseases such as coronary heart disease and stroke
Lp(a) plasma concentrations are highly heritable and mainly controlled by the apo(a) gene LPA
81
CETP is associated with elevated __
CETP is associated with elevated HDL
82
Cellular cholesterol homeostasis steps at high cholesterol levels
1) Decreased LDLR
2) Decreased HMGR
3) Increased ACAT (UC->CE)
4) Increased ABCA1 to increase efflux of CE by transporting it to HDL
83
What is SRE?
A short and specific DNA sequence located in the promoter region of genes whose expression is increased by cholesterol depletion and decreased by cholesterol excess
84
What is SREBP?
• SREBP is a protein that binds specifically to the SRE sequence and stimulates transcription of the corresponding genes
85
What is the binding of SREBP to SRE dictated by?
The binding of SREBP to SRE is dictated by the concentration of unesterified cholesterol within the cell
86
SREBPs are activated in response to __ cellular cholesterol concentration
SREBPs are activated in response to DECREASED cellular cholesterol concentration
87
Where is SREBP located?
On the ER membrane
88
What are the domains of SREBP?
Transmembrane domain
| Cytoplasmic domain
89
WHat are the isoforms of SREBP?
SREBP-1a, SREBP-1c, encoded by one gene;
| SREBP-2, encoded by another gene
90
What are the roles of each of the isoforms of SREBP?
SREBP-1a is a potent activator of ALL SREBP responsive genes
SREBP-1c regulates genes involved in fatty acid synthesis rather than cholesterol synthesis; weaker activator compared to SREBP-1a
SREBP-2 preferentially regulates genes involved in cholesterol synthesis
91
What is SCAP? How does it function?
SREBP cleavage-activating protein which is required for proteolytic cleavage of SREBP
o SCAP has a sterol-sensing transmembrane domain.
If it senses a decrease in cholesterol levels, it will activate SREBP.
o SCAP forms a complex with SREBPs in the membrane of the ER
The c-terminal end of SCAP interacts with SREBPs
92
What is INSIG?
| How does it work?
o INSIGs are ER resident proteins with two isoforms. Bund to SCAP
o High cholesterol levels in the cell causes INSIG to retain SREBP-SCAP complex in the ER
93
Give an example of genes whose expression is increased by cholesterol depletion and decreased by cholesterol excess.
HMG-CoA reductase (HMGR) and low density lipoprotein receptor (LDLR) genes
94
Describe SREBP action at low cholesterol
1. When the level of cholesterol decreases in the cell, it is sensed by SCAP.
2. INSIG undergoes a conformational change that allows it to release its hold on SCAP-SREBP complex due to weaker interaction of INSIG with SCAP therefore allowing this complex to be more mobile in the ER membrane
3. The SCAP-SREBP complex will continue to move until it reaches the golgi
4. In the golgi, two proteases will act on the SREBP-SCAP complex
o S1P: cuts the luminal portion of SREBP
o S2P: cuts between the DNA binding domain and transmembrane domain of SREBP
5. The DNA binding domain of SREBP will be released from the golgi and travel to the nucleus in order to bind SRE (steroid response element) located in the promoter region of the responsive genes
6. This will induce/stimulate transcription of the responsive genes in order to increase the levels of cholesterol in the cell
95
Which domain of SREBP is considered to be the nuclear SREBP?
It is the DNA binding domain of SREBP that is considered the “nuclear SREBP
96
Which organs secretes PCSK9 protein?
mainly the liver
97
CSK9 gene expression predominantly regulated by __
CSK9 gene expression predominantly regulated by SREBP2
98
What is PCSK9?
- PCSK9 is an enzyme produced mainly by the liver.
- It secretes a protein that promotes LDL receptor degradation in internalized endosome. This lead to lower LDL receptor concentration on the surface of the cell membrane-> Higher LDL particle blood circulation and lower cholesterol uptake
99
How is PCSK9 regulated in our bodies?
- PCSK9 expression is mainly regulated by SREBP-2
100
Connection of hypo/hypercholesterolemia and PCSK9?
- Hypercholesterolemia is associated with gain of function variants of PCSK9
- Hypocholesterolemia associated with loss of function variants of PCSK9
101
How do PCSK9 inhibitors work?
PCSK9 inhibitor drugs are based on immunity- Monoclonal antibodies (MAB). They target PCSK9 and bind to it, making it less able to breakdown LDL receptors
102
How is SREBPS degraded?
If SREBP is bound to the promoter as it gets older it gets modified by ubiqiunation which becomes a signal that this protein has been around for a while (the time is judged from the length of UB tail) which will signal for it to be escorted for degradation in the lysosome
103
What is the effect on Cyp7a1 that is only observed in mice?
Increased CH concentration stimulate the transcription of CyP7a1-> allows for increased removal of cholesterol
This only occurs in mice
104
What are the genes that are stimulated by high cholesterol concentration?
APOE
ABCA1
Cyp7a1
105
What is the role of ABCA1?
codes for a transporter involved in the efflux of cholesterol from cells
106
What is the effect of increased cholesterol concentration on ABCA1?
It's expression is increased
107
What is deficiency of ABCA1 associated with?
Tangier disease- People cannot offload CH from peripheral tissue to HDL due to mutation in ABCA1 -> they accumulate cholesterol in the tissue
108
What is the effect of high cholesterol on APOE? Consequences?
High cholesterol concentration induces increased expression of APOE which codes for Apo-E
Apo-E is found CM, VLDL, LDL, IDL
Apo-E serves as a ligand for VLDL and LDL receptors
Increase in APOE-> more Apo-E in VLDL and LDL-. Faster uptake of them from the circulation-> increase in cholesterol uptake by the liver-> cholesterol movement to the liver is increased
109
What are the domains of nuclear receptors?
ligand binding domain and DNA binding domain
110
What is the name of the DNA section bound by nuclear receptors?
HRE half site
111
What are the possible arrangements of half-sites?
direct, everted or inverted repeat
112
Nuclear receptors bind to DNA as a _ or __ or __
Nuclear receptors bind to DNA as a monomer or homodimer or heterodimer
113
Variations of How NRs regulate gene expression
1) Ligand binding or release
2) Recruitment/release of co-activators or co-repressors
3) Stimulation or repression of gene transcription
114
Where are NR target elements normally found?
• Normally found in the promoter region upstream from the start site, close to the start site
115
What is LXR?
A member of the NR superfamily of transcription factors
116
What are the isoforms of LXR?
alpha and beta
LXR-alpha is predominantly expressed in tissues and cells that are important in maintaining lipid homeostasis (e.g., liver, intestine, adipose tissue, macrophages); Has higher rate of expression
LXR-beta is ubiquitous (found everywhere) distribution
- They have different sensitivity to oxysterols
117
What activates LXR?
activated by oxysterols (generated during the synthesis
| of cholesterol, and during the metabolism of cholesterol)
118
Are LXR-a and LXR-b identical?
LXRalpha and LXRbeta are not identical but similar (and therefore different biological importance) but likely have overlapping target genes and ligand preference.
119
What are the genes regulated by LXR?
* ABCA1, facilitates the efflux of cholesterol from cells
* CYP7A1
* SREBP-1c
120
Synonymous vs Nonsynonymous SNPs
o Synonymous – distinct SNPs at the same position lead to the same polypeptide sequence
The wobble position can change, different DNA sequence but the same protein produced
o Nonsynonymous – distinct SNPs at the same position lead to different polypeptide sequences
Different sequence leads to amino acid substitution or stop codon
121
What was a unique finding about Apo-E SNPs?
SNP in the coding region of a sequence normally coding for a protein needed in lipid metabolism can lead to a neurological disorder
SNP in the promoter region of the gene (not in the protein coding region)-> optic neuropathy
Therefore, ApoE plays a diverse role
122
__ LXR activity--> __ SREBP activity
Increased LXR activity--> increased SREBP activity
123
What can ER stress be caused by and what are the consequences?
ER stress due to imbalance of nutrients and energy homeostasis prevents ER from doing it's job properly
124
What are EST?
Expressed Sequence tags
Short sequences that are generated by sequencing either one or both ends of an expressed gene (i.e. sequences form cDNAs of mRNAs)
125
Are all of EST known to us?
• Some ESTs are identified (correspond to fully characterized genes); many correspond to unidentified genes
o We have all the sequences but we do not know if they code mRNA
o By sequencing mRNA, we can map them and determine the sequence of DNA and see that we have characterized/sequenced this gene before and now we know the mRNA that is associated with it.
126
What have we determined after determining the sequence of mRNA?
By determining the sequence of mRNA we came to realize that one gene can give 2 different mRNA and 2 different protein products
E.g. SRBP1a and 1c- they are encoded by the same gene-> different protein
127
WHat are the 2 ways of studying changes in gene expression using RNA?
- Q-PCR
| - Microarray
128
WHat are the steps of Q-PCR?
1. Reverse transcribe the mRNA. In order to detect the transcripts by hybridization, they need to be labeled, and because starting material maybe limited, an amplification step is also used. Perform a reverse transcription (RT) reaction to produce a complementary DNA strand (cDNA)
2. Transcribe the cDNA and add a fluorescent label to get labeled cRNA
3. Hybridize the labeled target to the microarray. The squares contain specific sequence that corresponds to a specific gene.
• We have a piece of DNA that has been immobilized onto glass surface.
4. Scan the microarray and quantitate the signal.
129
When is qPCR used?
Choose qPCR for your experiment if you have to analyze the expression of a few genes with a known sequence
130
When is microarray analysis used?
If we wanted to measure a lot of genes at once, or we are not sure which ones we are analyzing we do microarray analysis-> possible because we know identities of different genes so we create a way of putting them on a slide and finding what kinds of cDNAs we make from a mRNA
131
What is the purpose of RNA-Seq?
Combines EST and SNP
Provides sequence information; including genomic sequences that have not yet been determined
Provides information regarding the relative abundance of transcripts
Reveals the presence and quantity of RNA in a biological sample
Specifically, RNA-Seq facilitates the ability to look at alternative gene spliced transcripts, post-transcriptional modifications, gene fusion, mutations/SNPs and changes in gene expression over time, or differences in gene expression in different groups or treatments.
132
What are the downsides of microarray and EST?
* In microarray we rely on the sequences we know
* EST we now the sequence, but not the gene. If we wouldn't know the sequence-> no probe can be determined-> doesn't work
133
What is qPCR used for?
to measure the amount of a specific RNA
134
What is the use of EST? Downside
ESTs may be used to identify gene transcripts, and are instrumental in gene discovery and in gene-sequence determination
Downside: we may know the sequence but not it's purpose
135
What are the steps of RNA-seq
1) Isolate RNAs
| 2) Generate cDNA using RT, fragment, size select, add linkers
136
What is proteomic analysis used for?
Can be used to study which and how proteins/enzymes are altered by changes in state of nutrition or metabolic status.
137
Why is male pronucleus used for injections?
Larger-> easier to be precise
138
Describe steps of manipulation male/female mice genome
Solution containing DNA is used
it is injected into male pronucleus as it Is larger and easier to hit
This DNA will be integrated into the genome due to the repair process
139
Natural vs synthetic genes
o Natural genes: obtained from another species
| o Synthetic genes: contains components from other genes or species
140
Steps of transgenesis
1) Transgene construction
2) Cloning
3) evaluation in vitro
4) microinjection
f) genomic analysis
141
What are knock outs
Creation of specific mutations
142
Overexpression of LDL receptors
| in transgenic mice, how was it achieved
Synthetic transgene to direct overexpression of LDL
receptors in the liver
LDLR transgenic mice fed a low cholesterol diet display
low blood LDL concentration (lower than wild-type mice)
LDLR transgenic mice exhibit resistance to diet-induced
hypercholesterolemia
143
What is size exclusion chromatography
Large particles cannot enter gel and are excluded, they have less volume to traverse and elute sooner.
Small particles can enter gel and have more volume to traverse. They elute later
144
Types of "knock-outs"
Mutation can be global (whole animal) or local (specific
tissue, or even specific cells)
Mutations can also be introduced at certain stages of
development
145
ES Targeted gene disruption:steps ( how to make editing of the gen
harvesting stem cells from agouti mice at blastocyst stage
Multiplication of these cells in vitro
Adding targeting vector (DNA)
Screen, isolate and inject into blastocyte (albino)
Now Black coat is modified DNA and white coat is not.
Injected into the mice and given hormones as if it was pregnant
From this egg a chimera mice is born: some parts are black, some parts are white
Then breeded with albino mice. If a white mice is born-> failure only wild type genome
Black->modified genome
146
Crispr/Cas9 gene editing
The CRISPR-Cas9 system works similarly in the lab. Researchers create a small piece of RNA with a short"guide" sequence that attaches (binds) to a specific target sequence of DNA in a genome. The RNA also binds to the Cas9 enzyme. As in bacteria, the modified RNA is used to recognize the DNA sequence, and the Cas9 enzyme cuts the DNA at the targeted location.
Once the DNA is cut, researchers use the cell's own DNA repair machinery to add or delete pieces of genetic material, or to make changes to the DNA by replacing an existing segment with a customized DNA sequence.
147
Result of targeted disruption of the LDLR gene in mice
Blood cholesterol elevated ->Human FHelevated-> LDLR -
Response to dietary - hypercholesterolemia ->LDLR-hypercholesterolemia->Human FH
Affected lipoprotein
species
LDL in both LDLR- and human FH
Atherosclerosis susceptible-> LDLR-
(cholesterol-fed)
premature->Human FH
148
What happens to lipid profile is LXRalpha is disrupted
Increased liver mass, increased hepatic cholesterol(with 0.2% and 2% cholesterol in the diet), decreased TGs
No Cyp7a activation-> liver does not get rid of Cholesterol
149
Does ezetimibe stop cholesterol absorption completely
No, there is still 12%
150
How do chylomicrons activate lipoprotein lipase (LPL)? what is the function of LPL?
Via apolipoprotein C-II, mature chylomicrons activate lipoprotein lipase (LPL),
It is an enzyme on endothelial cells lining the blood vessels. which catalyzes the hydrolysis of triacylglycerol which ultimately releases glycerol and fatty acids from the chylomicron
151
What happens to LDL in terms of Atheroma, adipose and muscle tissueÉ
LDL modifications can occur
LDL yields in free cholesterol
- Can be converted to CE via ACAT
Or can be donated to HDL3 which will be turned into HDL2 when UC is converted to CE
152
How many phospholipid leaflets are there in chylomicrons?
1
153
Lipoproteins have __ mobility
Lipoproteins have electrostatic/charge mobility
154
Why can chylomicron get so big
It has ApoB-48 that doesn't really have a size limit
155
What is atheroma?
degeneration of the walls of the arteries caused by accumulated fatty deposits and scar tissue, and leading to restriction of the circulation and a risk of thrombosis.
156
How does specific binding of chylomicron and LDL occur
Via interaction of acidic domain of the receptor and the basic protein domain of the apolipoprotein on the chylomicron
LDL also binds the same way
Binding via electrostatic charge
157
What are fibrates?
Fibrates is a class of drugs that bind bile acids-> more excretion and also CyP7a1 is released from negative feedback by bile acids
158
Which gene gets affected in the metabolic syndrome? What are the consequences?
CD36
Metabolic syndrome is a cluster of biochemical and physiological abnormalities associated with the development of cardiovascular disease and type 2 diabetes.
159
Can nuclear receptors bind DNA without a ligand?
yes
