1
Q
What parts makes up a nucleosome?
A
- Nucleosome = Histone + supercoiled DNA
2
Q
What are histones?
- What amino acids are they rich in?
- How many base pairs do they bind?
A
- Also known as 10-nm fibers
- A “bead” that contains 4 pairs of positively charged (rich in Arginine and Lysine) proteins that are supercoiled
- Binds ~146bp
3
Q
What is linker DNA?
A
free DNA between two nucleosomes
4
Q
What is linker histone and what is its function?
A
binds linker DNA to fold into 30-nm fibers, creating heterochromatin
5
Q
What are the two chromatin higher-order structures responsible for DNA compaction in interpahse nuclei and metaphase chromosomes?
A
30-nm Fibers and Loops
6
Q
What do nucleosomes fold into in terminally differentiated cells?
A
- 30-nm Fibers
- Nucleosomes fold into this in terminally differentiated cells because DNA is not needed
7
Q
What do nucleosomes fold into in cells undergoing mitosis or meiosis?
A
- Loops
- Nucleosomes fold into this in cells undergoing mitosis or meiosis to keep DNA in a more accessible form
8
Q
What are the two ATP-dependent chromatin remodelers?
A
SWI/SNF and ISWI
9
Q
How does SWI/SNF work?
A
- Uses ATP to open nucleosome so DNA can be more easily read
10
Q
How does ISWI work?
A
- Uses ATP to slide supercoiled DNA down, opening up more linker DNA
11
Q
What are the post-translational histone modifications and what effect do they have on the gene expression
A
- Methylation – silences gene
- Acetylation – activates gene
- Phosphorylation, ubiquitination, biotinylation
12
Q
What two enzymes are used in acetylation of histones and what do they do?
A
- HDAC (histone deacetylase) – promotes nucleosome folding → inactivating gene
- HAT (histone acetyltransferase) – destabilizes nucleosome folding → activating gene
13
Q
How is retinoic acid involved in the production of granulocytes and what defect in this process causes acute promyelocytic leukemia?
A
- Retinoic acid promotes the maturation of promyelocutes into granulocytes, one of the main types of WBCs
- In PML the promylocyte does not respond to retinoic acid induction, so promylocytes hyperproliferate.
14
Q
What is the pathway of normal proteomyelocute differentiation and what steps occur to acetylate a gene?
A
- Normal Pathway: retinoic acid binds retinoic acid receptor alpha (RARa) → conformational change → HDAC disassociates → HAT binds → acetylation → transcription of gene
15
Q
What is the defective pathway that ultimately causes the gene to be unexpresed in promyelocytic leukemia?
A
- Defective Pathway: retinoic acid receptor alpa (RARa) cannot bind retinoic acid → no conformational change → HDAC stays bound → gene stays silenced in nucleosome → no transcription
16
Q
What are the two main treatments for acute promyelocytic leukemia?
A
- Treatments
- Increase in [retinoic acid]
- HDAC inhibitors, such as trichostatin A (bad because not specific)
17
Q
What are the three distinct states of chromatin?
A
- Euchromatin
- Constituitive Heterochromatin
- Faculative Heterochromatin
18
Q
What state is euchromatin in and is it active or inactive DNA?
A
Euchromatin is active and open chromatin
19
Q
What is Constuitive Heterochromatin?
- Is it active or inactive DNA?
- Where is this usually located?
A
- Heterochromatin: repressed and condensed chromatin
- Constitutive: centromere region that occurs in all normal cell types during metaphase
20
Q
What is Faculative Heterochromatin?
- Is it active or inactive DNA?
- Where is this usually located?
A
- Heterochromatin: repressed and condensed chromatin
- Facultative: accumulated condensed chromatin that occurs in non-dividing cells (including X inactivation)
21
Q
What is the role of histone methylation?
A
- Histone methylation: does not affect histone charge, but promotes condensation and gene silencing
22
Q
What enzyme accomplishes methylation of histones and what is the pathway of how they work?
A
- Histone methyltransferases: one methylated histone recruits heterochromatin protein 1 (HP1) → HP1 recruits more histone methyltransferases in positive feedback loop
23
Q
Define epigenetics.
A
- Epigenetics: a change in the properties of a cell (phenotype) that is inherited but that does not represent a change in genetic information
24
Q
What are 4 processes that are considered to be epigenetic?
A
- Developmentally regulated transcriptional factors
- Post-translational histone modifications
- Regulatory RNA transcription
- DNA methylation reproduced through cell divisions
25
How is DNA methylation maintained through several rounds of replication?
* After replication, CpG dinucleotide sequences maintain methylation by DNA-methyltransferases
26
* How does genomic imprinting work in terms of DNA methylation?
* What can a defect in this process lead to?
* Genomic Imprinting: parental-origin where certain alleles can be methylated (read: inactivated), leading to the expression of the other allele coding for a gene
* A defect in this process leads to Beckwith-Wiedeman Syndrome
27
How can DNA methylation lead to cancer?
* Hypomethylation: mitotic recombination, genomic instability
* Hypermethylation: loss of tumor suppressor gene activity
* 5-aza-2’deoxycytidine inhibits DNA methyltransferases
28
Define aneuploidy.
* organisms whose chromosome number differs from the WT by part of a single chromosome set
29
How does aneuploidy appear and name 5 disorders (the chromosome they effect and symptoms asssociated with them)?
* 2n-1 is monosomic
* 2n+1 is trisomic
* Disorders (13, 18, 21 → PED)
* Trisomy **13** – **P**atau Syndrome: 47,XX,+13
* Trisomy **18** – **E**dward Syndrome: 47,XX,+18
* Trisomy **21** – **D**own Syndrome: 47,XX,+21
* Monosomy X – Turner Syndrome: 45,X
* Female with infertility and adult stature
* Trisomy XXY – Klinefelter Syndrome: 47,XXY
* Male with tall stature, long extremities, hypogonadism, and breasts
30
Define polyploidy and what are some examples?
* Polyploidy – cells/organisms that have more than two pairs of all of the homologous chromosomes including sex chromosomes
* Examples: 69,XXX, 69,XXY, 69,XYY
31
Define balanced structural chromosomal abnormalities and the two types?
* Balanced Structural Chromosomal Abnormalities: do not change the number of alleles in affected areas
* Inversion
* Translocation
32
How does inversion occur on chromosomes?
33
How does translocation occur in chromosomes?
34
What are unbalanced structural abnormalities and what are the two types?
* Unbalanced Structural Chromosomal Abnormalities: change the number of alleles in the affected area
* Deletion
* Duplication
35
How does deletion work on chromosomes?
36
How does duplication work on chromosomes?
37
List two chromosomal deletion diseases.
* Cri-du-chat (5p deletion)
* Wolf Hirshhorn (4p deletion)
38
What mutation occurs in Cri-du-chat, where does it occur, and what are some characteristics of the disease?
* Cri-du-chat
* 5p monosomy – deletion of the short arm of chromosome 5
* Characteristics: cry similar to meowing kitten, widely spaced eyes, developmental delay
39
What mutation occurs in Wolf-Hirschhorn, where does it occur, and what are some characteristics of the disease?
* Wolf-Hirschhorn
* 4p- syndrome – deletion of the short arm of chromosome 4
* Characteristics: seizures, developmental delays, heart defects
40
How are chromosomal microdeletions detected?
* Chromosomal microdeletions cannot be detected by karyotyping but by FISH (uses WBCs to fluoresce chromosomes)
41
What is a type of chromosomal microdeletion disease?
* What chromosome and what gene is included in the deletion?
* What are some characteristics?
* Williams Syndrome
* Chromosomal 7 microdeletion, including the gene elastin
* Characteristics: “cocktail party” personality, circulatory system and heart defects
42
Compare and contrast the germline chromosomal mutation and somatic chromosomal mutations.
* Germline Chromosomal Mutations
* Mutations during meiosis that may affect all cells in the offspring
* Most large chromosomal deletions result in spontaneous abortion
* Somatic Chromosomal Mutations
* Mutations occur during mitosis of terminally differentiated cells that affects certain tissues
43
What can occur when tumor supressors are inactivated?
* When tumor suppressors are inactivated, somatic chromosomal abnormalities can lead to cancer
44
Define driver mutations and passenger mutations.
* Driver Mutations: small number of chromosomal abnormalities that cause cancer
* Passenger Mutations: many other chromosomal abnormalities that result from the proliferation of cancer cells
45
What occurs in chronic myeloid leukemia?
* Chronic Myeloid Leukemia: translocation and subsequent fusion of genes on chromosome 9 and chromosome 22 →increased expression of tyrosine kinase → cancer
* Imatinib binds to tyrosine kinase → inactivation
46
What occurs in Burkitt's Lymphoma?
* Burkitt’s Lymphoma: translocation and subsequent fusion of genes on chromosome 8 and 14 → overexpression of c-myc → transcription of genes that stimulate cell proliferation
47
Define the process of transcription.
* Transcription – synthesis of RNA from a DNA template
48
What are the different types of RNA polymerases in eukaryotes?
* Where are they located?
* What do they synthesize?
RNA Polymerases R MT (are empty).
## Footnote
I -R
II - M
III - T
49
What two drugs act on RNA polymerases and what polymerases do they act on?
* Drugs
* Alpha-amanitin (from a mushroom) – affects RNA polymerase II and III
* Rifampicin – inhibits prokaryotic RNA polymerase
50
What are the three steps in transcription and what occurs in each step?
* Initiation – binds to promoter site
* Rate of gene expression is controlled by the ability of RNA polymerase II and transcription factors and mediator proteins to bind to DNA
* Elongation – nucleotides added to 3’ end
* Termination – termination site is reached
51
Define promoters in transcription and where is it located on a gene.
* Promoters: recognize specific transcription factors that allow for expression of a gene or set of co-genes
* Always upstream from gene
52
Define ehancers in transcription and where is it located on a gene.
* Enhancers: bind transcription factors that brings enhancer and promoter into close proximity by looping DNA activating transcription
* Can be upstream or downstream from gene
53
Define insulators in transcription.
* Insulators: CTCF protein can stabilize loop domains of DNA and can act to either enhance a group of genes or silence them
54
What occurs to produce mRNA from pre-mRNA?
1. 5’ Cap
* eIF4 binds to initiate protein synthesis
2. Splicing
* Introns: non-coding region
* Exons: codes for AAs
3. 3’ End Formation with PolyA Tail
4. Export to Cytosol
55
What signals for splicing of introns to occur?
* Mutation here causes what?
* Introns begin and end with highly conserved nucleotides
* 5’ GU…AG 3’ for RNA
* Mutations in these four nucleotides will result in disease pathologies
56
How do splicesomes work to remove introns?
* Spliceosome removes introns of pre-mRNA using ATP hydrolysis
1. Initiated at an A residue with hydroxylation within the intron
2. —OH attacks the first 5’ G phosphate of intron to create a lariat structure
3. Free exon attacks the first 5’ phosphate at beginning of next exon
4. Exons combine, releasing excised intron
57
What is the composition and function of splicing regulator (SR) proteins?
* Splicing Regulator Proteins (**SR** Proteins)
* Composition
* Serine and arginine rich proteins (**SR**)
* Function
* mRNA splicing regulation
* Recognizing the correct splice sites
58
What is the mechansim of splicing regulator proteins?
* mRNA strand contains regions of pre-mRNA that act as enhancers and silencers (ESSs, ESEs, ISSs, ISEs) → detected by SR proteins → recruitment of spliceosome depends on balance between the silencers and enhancers of SR proteins determines whether a particular exon will be included in transcript
59
How does splicing produce different mRNA variants from a single gene?
* What's an example?
* A single gene contains multiple exons that can be spliced in different conformations creating different functional proteins
* Tropomyosin
* 4 different genes code for many different forms of tropomyosin
60
What are the two types of splicing mutations that can occur and provide examples of diseases that may result from these mutations?
* Invariant GT and AG dinucleotides in the 5’ and 3’ splice site boundaries causing the intron to be maintained in mature mRNA
* CFTR
* Beta thalassemia
* Mutations in an ESE can alter dsmRNA splicing by affecting binding of splicing proteins
* Spinal Muscular Atrophy
61
What kind of therapeutics can be used to treat splicing mutations?
* What is an example that treats the premature stop codon in Duchenne's Muscular Dystrophy?
* Therapeutics
* Oligonucleotides repress splicing mutations: cover up cryptic splice sites and direct splicing patterns back to wild type
* Premature Stop Codon: DMD
* Eteplirsen: an oligonucleotide therapeutic can cover up stop codon and help make limited functional proteins (BMD)
62
Where do mutations occur for mRNA turnover to be affected?
* Identify the effects of these mutations.
* Mutations that affect mRNA turnover
* 5’ UTR
* Affects initiation of translation
* 3’ UTR
* PolyA Tail
* Polyadenylation signal sequence mutations → reduced stability
* Stop Codon
* Chain termination mutations → elongated mRNA
63
What are the 4 cytoplasmic decay pathways?
* Deadenylation/decapping-dependent pathways
* Nonsense mediated decay (quality control)
* 3’ UTR AU-Rich Elements (AREs) Mediated mRNA Turnover
* Micro RNA / RNAi-dependent pathway
64
When is mRNA turnover by Deadenylation/decapping-dependent pathways?
This is how most RNAs are removed.
65
When does nonsense mediated decay occur?
Think of this as quality control - Elimination of mRNAs with premature stop codons (PTCs) – nonfunctional mRNAs
66
When/how does 3’ UTR AU-Rich Elements (AREs) Mediated mRNA Turnover occur?
* What is an example?
* Rapid, regulated turnover of specific mRNAs
* ARE regions can be bound by destabilizing or stabilizing factors which can either degrade mRNA or extend mRNA’s half-life for more translation
* Example: TTP destabilizes mRNA allowing for deadenylation/decapping and HuR stabilizes mRNA, prolonging its half-life
67
Provide an example of how mRNA binding protens affect mRNA stability.
Ferritin and Transferrin Receptor Regulation
68
Define the function of ferretin and transferrin receptors.
* Ferritin: large intracellular iron storage protein
* Transferrin Receptor (TfR): recognizes iron-bound transferrin and transport it into cell
69
How/when does Ferritin and Transferrin Receptor Regulation occur?
* Low intracellular Fe+2: Iron Response Protein (IRP) has no Fe+2 bound
* Binds 5’ UTR at Iron Response Element (IRE) region → stops initiation in translation → no synthesis of ferritin
* Binds 3’ UTR at IRE region → stabilizes mRNA → synthesis of TfR
* High intracellular Fe+2: IRP has Fe+2 bound → change in conformation of protein → cannot bind IRE regions
70
What are miRNAs and what are they used as?
* small RNAs with only 22 nucleotides → very stable structure
* Used as:
* Act as biomarkers because they are produced in response to upregulation of specific mRNA sequences
* Act as therapeutics to silence specific mRNAs
71
How are miRNA's processed?
* **Drosha** recognizes Pri-miRNA and removes 3’ and 5’ ends → **exportin** exports into cytoplasm → **Dicer** splits ds-miRNA into ss-miRNA sequences → ss-miRNA sequences complex with RNA-induced silencing complex (RISC) → **RISC** uses ss-miRNA as a template to bind cytosolic mRNA → signals for mRNA cleavage or mRNA translational repressor
72
What is the significance of mRNA localization?
* How does it occur?
* Provide an example of a protein that takes part in this process
* mRNA can be translated at different sites across a cell by transportation via microtubule
* Translation of mRNA in dendrites can be activated by depolarization of membrane
* FMRP: acts as a chaperone to repress mRNA translation until it reaches target location
73
What bond connects tow AAs?
Peptide bond formation: hydrolysis reaction between two AAs
74
Describe mRNA in eukaryotes and prokaryotes?
* mRNA – single strand sequence that codes for AAs
* Prokaryotic mRNAs are often encode for multiple proteins (multicistronic) and eukaryotic mRNAs often encode for one (monocistronic)
75
Describe rRNA.
rRNA (ribozyme RNA) – found on the catalytic site of peptide formation in peptidyl transferase center
76
Describe tRNA structure.
tRNA – inverse L shaped loop structure that contains anticodon and binds AA to 3’ end
77
Describe tRNA formation.
What enzyme is used?
What are the two functions of this enzyme?
* Formation
* tRNA charged with an AA using 2 high energy bonds
* AA + ATP → aminoacyl-AMP + PPi
* Aminoacyl-tRNA Synthetase matches AA with anticodon on tRNA
* Aminoacyl-tRNA Sythetase proofreads match
78
Explain the significance of the wobble base.
* First and second bases of a codon have high fidelity/specificity to anticodon
* The wobble base – the third base – has low fidelity, and accounts for silent mutations due to 61 codon combinations and limited tRNAs
* A or G with U
* C or U with G
79
How is iniation of translation different in prokaryotes?
* Prokaryotic Cells
* Initiation: binds to Shine Dalgarno Sequence (eight nucleotides telling where ribosomes are to bind)
80
What are the steps of iniation of translation in eukaryotes?
1. eIF3 is bound to small subunit of ribosome (prevents large subunit from attaching)
2. eIF2 → eIF2B dissociation triggers replacement of GDP by GTP on eIF2 by eIF5 → allows for binding to met-tRNA → binds to eIF3
3. eIF4E domain binds the m7GTP cap
4. eIF4 complex with mRNA binds to eIF3 complex
5. eIF4A domain unwinds secondary structures in 5’ region until AUG is found
6. GTP is hydrolyzed to GDP, releasing initiation factors
7. Large subunit binds to small subunit
8. Translation
81
What are Kozak sequences?
Kozak Consensus Sequence: ribosome will ignore AUG sequences until A or G is at -3 position upstream and G at +4 position downstream
82
What are internal ribosome entry sites?
Internal Ribosome Entry Sites: sites where ribosome attaches to mRNA because 5’ UTR has too many secondary structures and is too far from start codon
83
Steps of elongation of translation in eukaryotes?
* E-site is exit, P-site is peptidyl, A-site is acceptor
* Steps
1. eEF1 binds GTP and aminoacyl-tRNA → transfers to A-site
2. Anticodon-codon binding causes GTP hydrolysis
3. Release of eEF1
4. Growing peptide in P-site is transferred to AA on tRNA in A-site
1. The ribosome acts as ribozyme to catalyze reaction
5. eEF2 binds GTP → hydrolysis to GDP → movement of ribosome by three nucleotides
84
Steps of termination of translation in eukaryotes?
* Steps
1. RF1 mimics tRNA structure and binds to stop codon in A-site
2. RF3 terminates translation by disassociating ribosome subunits from mRNA via GTP hydrolysis
85
How many ATP are used in a single peptide bond formation?
4
86
What is the regulation pathway of translation at eIF2?
What causes this?
* Phosphorylation of eIF2 → eIF2B stays bound to eIF2 → decreased GDP/GTP exchange → inactivates eIF2 → blocks binding of met-tRNA → stops initiation of translation
* Stress Response: cause phosphorylation of eIF2, blocking initiation
* Nutrient deprivation, oxidative stress, ER stress, dsRNA
87
How does mTOR regulate translation?
How is it related to cancer?
What drug targets mTOR?
* mTOR phosphorylates “blocking” proteins on eIF4E (4E-BP) and eIF4A (PDCD4) → eIF4E and eIF4A are free to bind to eIF4G → form eIF4F (entire eIF4 complex) → initiation of translation
* Since mTOR promotes cell growth and proliferation it can be targeted for cancer drugs therefore reducing cell growth
* Rapamycin
88
What is personalized/precision medicine?
Using genotype to predict phenotype
89
How genomics can impact the day-to-day practice of medicine.
Genomics can identify and help treat patients with germ line pathogenic variants
Used in diagnosing and treating patients with somatic mutations (i.e. cancer)
90
The nature and extent of single gene (Mendelian) disease susceptibility.
Name the disease patterns? (4)
* Inheritance patterns
* Autosomal dominant – one copy of defective gene manifests in phenotype
* Autosomal recessive – both copies of gene are defective
* X-linked
* De novo
* 8,000 known single-gene Mendelian diseases
* Diseases
* Marfan’s
* Tay Sachs
* CF
91
Using family history to identify patterns of inheritance of Mendelian diseases.
* De novo – spontaneous mutations
* X-linked – mainly males
92
What are polygenic genes?
Explain how GWAS relates?
* Multiple genes have the ability to affect phenotypic pathologies
* GWAS
* Maps single nucleotide polymorphisms across the human genome to look for statistically significant differences in nucleotides
* Statistically significant SNPs can correlate to mutation of genes nearby
93
What is the application of genome sequencing in the neonatal intensive care unit?
* Standard genetic diagnosis can be weeks, resulting in severe defects
* Identify risk → perform genomic sequencing → diagnosis
94
What is the role of pharmacogenomics in drug therapy?
Example of CF?
* Genetic testing can distinguish specific mutations in a disease, allowing for unique drug treatments
* Example
* G551D mutation of cystic fibrosis can be specifically treated with Ivacaftor
95
What is the role of somatic mutations in cancer onset and progression?
* Somatic mutations: genetic mutation during mitosis
* Multiple homeostatic processes must be disrupted in order for a normal cell to become malignant
* 6 independent events in order for cancer to occur
96
Explain how driver mutations can help target caner therapy?
* In order for cancer to occur, one disruption must occur in each respective pathway
* You almost never see two genes in one pathway affected
97
Describe the RAS pathway?
* Ras Activation Pathway
* EGF binds to one subunit of EGFR → cross-phosphorylation of EGFR dimer → recruitment of Grb and SOS complex → complex acts on Ras-GDP to replace with Ras-GTP → Ras-GTP recruit Raf → phosphorylation cascade on MEK, ERK, and eventually cyclin D
98
How can genomic analysis can facilitate cancer diagnosis?
Explain using the ALL/AML example.
* ALL and AML look very similar under the microscope, but treatments require different approaches → genomic analysis used to distinguish between the two different leukemia
* Tumor DNA can also be detected in blood serum
99
How does genomic analysis contributes to cancer prognosis?
* Cancer classification by microarray analysis yields distinct subcategories of tumor types
* Various tumor types have different prognoses
100
What is therole of genomic analysis in selecting the right therapy?
Pathway analysis yields mutations causing cancer, which dictates drug selection
101
What are the potential and limits of immunotherapy?
Limits: individuals treated with antibodies to recognize tumor cells sometimes do not know how to recognize the tumor → no tumor-specific T cells
Potential: antibodies can allow T cells to respond and label tumor cells for degradation
102
Define
* Signal-regulated transcription factor:
* Enhancer element:
* Silencer element:
* Response element (RE):
* Co-regulator:
* Transrepression:
* **Signal-regulated transcription factor**: a protein that binds to DNA in a sequence-specific manner and mediates transcriptional activation OR repression
* **Enhancer element**: a DNA sequence that binds transcriptional activators
* **Silencer element**: a DNA sequence that binds transcriptional repressors
* **Response element (RE)**: a DNA sequence bound by signal-regulated TFs, which can function as an enhancer or a silencer
* **Co-regulator**: a TF interacting protein that functions with DNA-bound TFs to enhance or to repress transcription
* **Transrepression**: the process by which nuclear hormone receptors antagonize several signal-transduction pathways through various DNA-dependent and independent mechanisms
103
Explain how GATA regulates genes involved with hematopoiesis?
* GATA TFs are developmental regulators of genes involved in hematopoiesis
* GATA2 is elevated in early erythroid progenitors
* High levels of GATA2 → enhancement of GATA1 expression → high levels of GATA1 → GATA1 competitively binds to FOG1 → GATA2 suppression
* GATA1 has higher affinity for GATA sequences, so it can no longer go backwards in development → destined to be an erythrocyte
* GATA upregulates various forms of beta-globin synthesis needed at different points in development by binding LCR (locus control region = enchancer) and respective promoter
104
Structure of of NR (nuclear receptors)?
* Nuclear Receptor (NR) Transcription Factors
* Steroid hormone receptors (homodimer)
* Zinc Finger TF
* Steroid-binding pocket binds ligand → changes conformation → two zinc fingers bind major groove of DNA
* Heterodimeric nuclear receptors
105
Explain how transcription factors can both activate and repress transcription.
Ligand binds to NR protein → binds co-regulator to form co-regulator complex → either activates or represses transcription
106
Compare the mechanisms and effects of drugs (3) that target transcription of Estrogen Receptors.
* Estrogen Receptors (ER)
* In ER+ breast cancer, the ER is implicated in its pathogenesis
* Selective Estrogen Receptor Modulators (SERMs) competitively inhibit ERs, not allowing for necessary conformational change to bind co-regulators
* Tamoxifen
* Raloxifene
* Selective Estrogen Receptor Downregulators (SERDs) covalently bind ERs, causing degradation
* Fulvestrant
* Aromatase Inhibitors
* Aromatase converts testosterone to estrogen
107
What are Androgen Receptors?
What drug blocks an ARs action? How?
* Androgen Receptors (ARs)
* Androgen is critical in male sexual development and in pathogenesis of prostate cancer
* Ligand is dihydrotestosterone (DHT)
* Enzalutamide
* Three targets: competitively binds to AR, inhibits translocation of activated AR, and inhibits AR binding to DNA
108
What are glucocorticoid receptors?
Ligands? Functions?
* Glucocorticoid Receptors (GRs)
* Ligand-activated TF
* Natural ligand: cortisol
* Synthetic ligand: dexamethasone
* Functions
* Activates transcription of anti-inflammatory genes
* Block transcription of inflammation genes
109
What is the pro-inflammatory pathway?
What are its target genes?
* Cytokine (pro-inflammatory) binds TNFR (cell surface receptor) → TNFR activates IKK → phosphorylating IKB → release of NK-kB (TF) → pro-inflammatory transcription
* Target genes include pro-inflammatory cytokines, interleukins (IL variants), nitric oxide synthase, and COX-2
* Positive feedback loop due to further transcription of pro-inflammatory cytokines
110
In the anti-inflammatory pathway, what are these key components called:
* Transcription Factor?
* Inhibitor?
* Regulator?
* Transcription Factor: **NF-kB**
* Inhibitor: **IkB**
* Regulator: **IKK**
111
How is activation of NF-kB is blocked by glucocorticoids?
* Sequesters NF-kB in nucleus
* Promotes transcription of IKB, which sequesters NF-kB in cytosol
112
How is TNFR targeted for anti-inflammatory therapies?
* TNFR antibodies block inflammation by blocking TNFR pathway, blocking phosphorylation of IKB, therefore sequestering NK-kB in cytosol
113
How does the kidney respond to normal and low oxygen levels?
* Normoxia: normal oxygen supply → HIF-alpha’s proline residues are hydroxylated → VHL binds → ubiquitinates HIF-alpha → degradation
* Hypoxia: inadequate oxygen supply → nuclear translocation of HIF-alpha → dimerizes with ARNT → binds to hypoxic response element (HRE) → transcription of various genes (i.e HPO for erythropoiesis, transferrin, angiogenesis)
114
What is Von Hippel-Lindau Disease?
pathway?
* Von Hippel-Lindau (VHL) Disease
* Autosomal dominant
* Mutations inhibit HIF-alpha degradation → overexpression of HIF-target genes → cell proliferation → cancer
115
What are possible treatments for anemia using the HIF-alpha pathway?
Prolyl hydroxylase inhibitors → block VHL binding → no ubiquination → no degradation → elevated HIF-alpha → more erythropoiesis → more RBCs
116
Compare and contrast three categories of stem cells.
1. **pluripotent** stem cell (can give rise to all tissue types)
1. i.e. embryonic stem cells
2. **multipotent** stem cell (can give rise to more limited tissue types)
1. i.e. Hematopoietic stem cells
3. **adult** stem cell (can give rise to only one tissue type)
1. i.e. Muscle stem cells
117
Describe how stem cells can be used as therapeutics. (3) ways?
* Direct Treatment
* Transplantation of tissue specific stem cells (i.e. lymphomas)
* Regenerative Medicine – restores partial tissue function (i.e. after ischemic event)
* Genetic Defects – repair small mutations in genome (i.e. CFTR)
118
What is the general technique for gene therapy?
* Introduce “new” genetic material within stem cell (viral vectors)
* Directly corrects a genetic defect (uses DNA editing/CRISPR)
119
What are barrier to gene therapy?
* Delivery to appropriate location
* Avoidance of deleterious response
* Achievement of sustained expression
120
What makes a good target gene?
* Structural defect in gene that is translated into the protein product (CFTR)
* Promoter mutation affecting gene expression levels (Beta-Thalassemia)
* Splice site mutations or termination codons affecting RNA turnover/stability (Duchenne’s Muscular Dystrophy)
121
Explain iPSC.
* Harvest patient’s somatic diseased cells → generate iPSCs via pluripotency transcription factors (OSKM) → correct disease-causing mutation via nucleases → differentiate specific cell types → inject into patient
122
Explain virsus-mediated gene introduction.
* Virus-mediated Gene Introduction
* Extract patient’s HSCs (hematopoietic stem cells) → treated with lentivirus with non-mutated genes → non-mutated gene is inserted into patients stem cell → inserted back into the patient
* Patient’s previous mutated somatic cell is eradicated by chemotherapy
123
Explain CRISPR.
* CRISPR
* CRISPR/Cas are RNA guided site-specific DNA nucleuses
* CRISPR located mutated gene via RNA sequence
* Nuclease activity removes mutated gene and replaces with donor non-mutated DNA
SPM
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