Biochem: Trisomy 21 - Develop Signaling Flashcards
What kinds of phosphorylation take place regarding regulation of the cell cycle?
Both activating and inhibiting phosphorylation are involved in cell cycle regulation. Ex. During S phase, the helicase (that was put in position on the DNA during G1) goes through activating phosphorylation to begin replication. In order for this to happen, the ORC (origin recognition complex) must be go through inhibitory phosphorylation, thus be inactivated.
What is the ubiquitin-proteasome system? How is it used in regulating the cell cycle?
a pathway that marks target proteins with polyubiquitin protein chains and then delivers that polyubiquinated target protein to the proteasome for degradation. Think of proteasomes as a barrel with a cap that only recognize certain keys in order to open. Polyubiquitination = that key. Thus, making it regulated degradation.
What is the anaphase promoting complex (APC) and what (2) substrates does it target?
APC is an E3 ubiquitin ligase that targets M-phase-cyclins for destruction by ubiquitinating the M-cyclin. M-Cdk (activated complex of M-cycle + its cyclin dependent kinase) controls (in part) the transition from metaphase to anaphase.
APC also marks securin for destruction, which results in the release of separase. This allows sister chromatids to be separated during anaphase. Failure of this separation can lead to nondisjunction and trisomy.
- Securin: keeps separase inactive
- Separase: separates sister chromatids for anaphase
What does the metaphase/anaphase transition check for? Name a ubiquitin ligase that plays an important role in this checkpoint.
this checkpoint asks “are all chromosomes attached to the spindle?” i.e. Ready to start pulling sister chromatids apart?
The anaphase promoting complex (APC) is a ubiquitin ligase that marks securin for destruction. Securin keeps separase inactive. Once separase is no longer inactivated by securin, it separates sister chromatids for anaphase = disjuncion.
proto-oncogene v. oncogene. What does “gain of function” mean?
proto-oncogenes: regulate proliferation only when proliferation is needed.
once the proto-oncogene is associated with a mutational gain of function (meaning always on), it becomes an oncogene
What is Myc? Rb? How are gain of functions and loss of functions in these associated with cancer?
Myc is a major accelerator (proto-oncogene) of cell proliferation. It is a transcription factor that induces the synthesis of G1 cyclin, which forms a C1-cdk complex. Active C1-cdk phosphorylates Rb. Rb is a tumor suppressor that puts the brakes on the cell cycle by binding and repressing the function of E2F proteins. When Rb is phosphorylated, it releases E2F allowing it to function as transcription factors.
Gain of function mutations in Myc (making it always on) overrides normal controls on cell growth. Loss of function, or inactivation, of Rb removes a negative regulator of cell growth, favoring unregulated proliferation of cancer.
Interpret this pathway. What kinds of mutations in this pathway would be associated with cancer?
You have growth factor signaling (mitogen) activating Myc. Myc activates synthesis of G1 cyclin, which interacts with its cdk. That activate kinase phosphorylates Rb. As a consequence of this phosphorylation, the E2F transcription factor that Rb is wrapped around is released and is now active.
An activating mutation of the proto-oncogene Myc and an inactivating mutation of the tumor suppressor Rb would both favor unregulated proliferation.
What is meant by the term regulators of regulators with regards to the cell cycle?
ex. M-phase cyclin starts to rise before M in G2 and then levels fall after M. This rise and fall is one layer of regulation. But during the rise, need to keep the M-Cdk complex under wraps (inactive) until ready to enter mitosis. Wee1 is a Cdk-inhibitory kinase that works by negative phosphorylation. By keeping M-Cdk inactive, the levels can build up and then all be activated at once when the regulatory negative phosphate is removed by CDC25 phosphatase.
Thus, M-cyclin regulated by Cdk. M-Cdk regulated by Wee1 and Wee1 regulated by Cdc25.
What is Wee1? How did it get its name?
Wee1 is an inhibitory kinase that negatively phosphorylates the M-phase-Cdk complex, keeping it inactive until the cell is ready for M phase. Mutational inactivation of Wee1 allows cells to progress through the cell cycle too quickly without letting growth catch up with division. Thus, the cells are smaller, or wee.
Which regulatory is the key to the G2/M transition?
Activation of the CDC25 phosphatase. When CDC25 is activated, it removes the inhibitory Wee1 from the M-Cdk complex, allowing M-Cdk to be active.
The regulatory of CDC25 is the cell asking itself: “Am I large enough to divide?”
What effects can DNA damage have on cell cycle progression (what 3 kinases specifically)?
Signals of DNA damage will inhibit the pro-proliferative action of the G1/S-Cdk and S-Cdk kinases going into S-phase. This is b/c you don’t want to start any synthesis until you’ve repaired the DNA damage. DNA damage will also inhibit the activating function of CDC25 on M-Cdk. Don’t want to go through mitosis with damaged DNA. ALL this regulation is done through kinases and phosphatases.
What is p53? How has it earned its name of guardian of the genome? How fdoes MDM2 relate to this?
p53 is the “granddaddy” tumor suppressor gene, which under normal circumstances remains is kept at low levels by MDM2. MDM2 is ubiquitin ligase that ubiquinates p53 resulting in its degradation. But, when p53 is phosphorylated in response to DNA damage, it is no longer recognized by MDM2, letting p53 levels build up in an activated state.
Active p53 is a transcription factor that induces the expression of p21 and other proteins that suppress cell cycle progression. This action of p53 gives cells time to fix DNA damage before replication or mitosis = guardian of the genome.
What happens to p53 in the absence of DNA damage?
p53 is still being made, but its being degraded by the ubiquitin ligase MDM2. Only in response to DNA damage is p53 phosphorylated such that MDM2 no longer recognizes it for degradation. Then it can go on to inhibit the proliferative activity of various kinases, to give the cell time to repair DNA damage.
What’s the cell’s first response to DNA damage? What happens if the damage is too great to repair?
First thing that happens in response to DNA damage is to arrest S-phase to give cell time to repair damage (p53 action). If that doesn’t work, it’s cell death.
Describe the abnormal differentiation of myeloid progenitor cells in chronic myelogenous leukemia (CML).
in CML, the common myeloid progenitors (CMPs) take a mutational hit that activates the Brc-Abl oncogene. Brc-Abl transforms CMPs into blast cells that have 1) disregulated proliferation and 2) inability to differentiate into erythrocytes and megakaryocytes, but still able to differentiate into mature WBCs.
blast cells: transformed immature precursors.
In chronic myelogenous leukemia (CML), what lineages of the myeloid progenitor cells are disrupted and which remain intact? What sx are related to CML?
In CML, the Brc-Abl oncogene transforms the common myeloid progenitor (CMP) into blast cells, which are unable to differentiate into erythroid or megakaryocytic lineages, but can still differentiate into mature WBCs.
This leads to sx of fever, fatigue, and bleeding.
What is the Philadelphia (Ph) chromosome? How does the Ph chromosome cause chronic myelogenous leukemia (CML)?
The Ph chromosome is formed from a balanced, reciprocal translocation between chromosomes 9 and 22. This mutation takes a proto-oncogene, the Abl-kinase, and converts it into an oncogene, Brc-Abl. Fusion of Brc and Abl results in a dis-regulated Abl kinase that drives the proliferation of the CML clone at the early stages of the disease (chronic myelogenous leukemia).
Sketch a karyotype of an individual with the Philadelphia chromosome.
Balanced, reciprocal translocation between chromosomes 9 and 22.
What is Imatinib and what is its target? How does it work?
Imatinib is a highly specific inhibitor of the Bcr-Abl kinase, the oncogene that is the major driving force behind the abnormal properties of the CML clone at the early stages of the disease.
A protein kinase, such as Bcr-Abl, has two substrates it works on: ATP and the target protein. Though you might expect Imatinib to target the protein substrate binding, it instead blocks ATP binding. And you get enough selectivity with this for clinical benefit.
What laboratory test would be used to detect complete molecular remission of chronic myelogenous leukemia?
PCR (polymerase chain reaction) = doing DNA synthesis in a test tube. Involves multiple rounds of DNA denaturation (melting), annealing, and then extension. The boundaries of the Bcr and Abl segments have been defined by bound primers, so by the time you’re in the 3rd cycle of PCR, you’re only replicating the single, small fragment.
Idea is to produce enough of this fragment to be able to visualize it on electrophoresis gel. A major molecular response = 10,000-fold reduction in the philadelphia chrom (detectable BcrAbl). Means you have knocked that clonal cell responsible for the mutation way, way down.
What is a nucleosome?
Nucleosomes are the primary unit of DNA packaging in the human nucleus. Each nucleosome consists of 8 histone proteins arounjd which the DNA wraps ~1.5 times. The tails of each histone project out of the nucleosome and are subject to various modifications, including methylation and acetylation. Nucleosomes are linked by 60-80 base-pairs of DNA bound by histone H1. H1 plays an important role in bringing nucleosomes together in more compact, higher order structures.
Using +/-, conceptualize the interactions of histones with DNA. What effect does acetylation have on histones? Deacetylation?
Histones are highly basic, with 20-50% of them composed of Lys and Arg. Lys and Arg both have high pKs and will be in the conjugated acid state at physiological pH, which gives them a + charge. Ex. Lys end will be -NH3+
Acetylation of histones adds an acetyl grp (C=OCH3) to the N of Lys. This neutralizes the + charge, reducing the # of ionic bonds stabilizing the interactions of the histones with DNA. Thus, histone acetylation = loosening DNA = transcription activation. Conversely, histone deactylases stabilize nucleosomes and reduce gene expression.
Provide a rationale for how different cell types arise from a common DNA blueprint.
The concept of genomic equivalence is that every cell in the human body (excluding gametes) has the same set of genes as every other cell.
Genomic equivalence was proven in 1997 when Dolly the sheep was cloned by inserting the nucleus of a somatic adult sheep cell into an enucleated egg. Proof that DNA within the differentiated cell contained all necessary info to create every cell in a new animal!
What is the initiation codon of mRNA? What amino acid does it code for?
AUG = methionine
What affects which trans-acting factors will be expressed in a given cell? What is the significance of this? Give an example.
Trans-acting factors are expressed in a tissue-specific manner, so the variety of trans-acting factors that are expressed differ per the specific tissue and cell.
It is the different transcription factors binding to regulatory elements in DNA that dictate whether a gene is turned on or off in a particular cell.
ex. gene with both brain and limb specific enhancers. In limb cell, its no longer expressing the brain-specific protein and only expressing the limb-specific protein.
What does RNA polymerase II do? What are promoter sequences? What does the mediator complex do?
Responsible for synthesis (transcription) of mRNA in a eukaryotic cell. RNA Pol II recognizes promoter sequences–such as the TATA box and the INR (initiation region).
The mediator complex is a series of proteins that helps RNA Pol II to bind. The mediator complex links enhancers with promoter sequences.
What is histone acetyltransferase? How is it related to cis-acting sequences and trans-acting factors?
histone acetyltransferase is a chromatin remodeling enzyme that makes regulatory regions of mRNA more accessible
What are the (5) overall steps of transcription?
- Trans-acting factors bind to cis-acting sequences of the enhancer
- These factors recruit histone acetyltransferase enzymes to open up the chromatin, making the promoter accessible
- The mediator binds to the enhancer, linking the enhancer and promoter
- RNA pol II–assisted by the mediator–binds to the promoter
- stable transcription pre-initiation complex is formed
What are carbohydrates? Identify glucose in its ring conformation. How do you identify whether glucose is in its alpha- or beta-anomeric state?
Carbohydrates are carbons that are hydrated (CHOH or CH2O) and have either an aldehyde or ketone.
Glucose has 6 C’s and an aldehyde group (C’1) that can be attacked by C’5 to form the ring structure. Once in the ring structure, C’1 is called the anomeric carbon. It is asymmetric. If the hydroxyl grp is:
- down = alpha
- up = beta
What is distinguishable about the anomeric carbon in a glucose ring? How can you use that to identify it as glucose?
The anomeric carbon (C’1) is the only carbon in the ring bonded to two Oxygens.
If you see the anomeric carbon on the right, the orientation of the hydroxyl groups on C2-C5 will be down, up, down, up.
What are epimers of carbohydrates? Identify the C2 and C4 epimers of glucose.
epimers of carbohydrates just have a single position inverted.
Glucose C’s = 2-down, 3-up, 4-down, 5-up.
galactose = C4 epimer of glucose. -OH is up
mannose = C2 epimer of glucose. -OH is up
Distinguish D-glucuronic acid from L-iduronic acid.
These 2 are epimers with a single inversion at the C’5 position. Product of when glucose is oxidized aT the C’6 grp to COO-.
D-Glucuronic acid (GlcUA) (left) has COO- grp up. Glucuronic acid is found in the liver; used to modify bilirubin.
L-iduronic acid (IdUA) (right) has C’5 inversion; COO- = down.
Describe the role of dolichol moiety in the rxn catalyzed by oligosaccharide transferase.
Dolichol is a lipid that is extremely hydrophobic (note the pyrophosphate on the end). This dolichol anchors the core oligosacchraride in the ER membrane.
Oligosaccharide transferase waits on lumenal side of the ER membrane for an Asp-X-Ser and an Asn-X-Thr (beginning of polypeptide chain) to come through. It’s substrate is the 14-residue core oligosaccharide + the dolichol. This enzyme transfers the core oligosacchraide from dolichol to the growing polypeptide chain.
What are the terminal antigens in the ABO blood system?
- Type A = N-Acetyl galactosamine (GlcNAc).
- Type B = galactose. Remember! Galactose is a C4 epimer of glucose
- Type O = no antigen on O
General structure of glycosaminoglycans
glycosaminoglycans are a disaccharide repeat (two sugars repeated over and over again). In position A is usually a sugar acid (carbohydrate acid) (ex. glucuronic acid). In B position, it’s typically an amino sugar (ex. N-acetyl galactosamine).
The acid component will have a caboxylic acid grp and the amino sugar often has a sulfate group. At phys pH, this makes glycosaminoglycans highly negatively charged.
