Block 5 Flashcards
RTK
Receptors with inherent kinase activity that bind peptide hormones such as EGF; monomeric in the plasma membrane but dimerize when bound which allows them to phosphorylate target molecules
What is the RTK signaling pathway?
EFG -> binds/dimerizes RTK -> phosphorylates SH2/PTB -> recruits Grb2 -> binds Sos on its SH3 domain -> activates Ras -> Raf -> MEK -> MAP kinase
Grb2
Recruited by dimerized form of SH2, has an SH3 domain that binds the GEF SOS
Sos
GEF for Ras G protein
Ras
G protein that is inactive and membrane bound when bound to GDP but is activated by GEFs that exchange GTP for GDP; activate the Mitogen Activated Protein (MAP) kinase pathway once activated
MAP kinase pathway
Common signaling pathway to activate transcription factors; Active Ras -> activates Raf -> phosphorylates MEK -> phosphorylates MAP kinase -> MAP kinase dimerizes and translocates to nucleus -> atcivates transcription factors for cell proliferatoin (such as c-fos which activates cyclin D)
Cyclin D
Gene involved in the first step of cell division
How is the RTK signal terminated
Usually by downregulation of the receptor via phosphatases
Why are mutated versions of RTK clinically significant?
Mutated RTK can result in a constitutively active receptor that activates the Ras-MAP kinase pathway which is associated with tumor growth
Why are mutated Ras proteins clinically significant?
Mutated Ras can bind but not hydrolyze GTP causing them to be trapped in the active state
Her2 receptor
Commonly mutated in breast CA- single amino acid mutation leads to dimerization of the receptor w/o a ligand which activates downstream target proteins
TGF-beta signaling pathway
Activates many cellular processes including modulating the immune response, inhibiting cell proliferation, and promoting cell differentiation by phosphorylating proteins that are able to translocate to the nucleus and regulate transcription
What are the TGF-beta isoforms and what do they do?
TGF-beta has 3 isoforms; R2 and R3 bind TGF-beta and recruit R1, R1 is activated by R2 and then phosphorylates Smad proteins
What are the steps in the TGF-beta signaling pathway?
TGF-beta binds to R2 and R3 isoforms -> recruits R1 -> R2 phosphorylates R1 -> active R1 phosphorylates Smad3 -> dimerizes and translocates to nucleus and binds DNA
What are the 2 ways that the TGF-beta signaling pathway terminated?
- Sno and Ski proteins bind Smad3 and recruit HDACs to downregulate trasncription
- Negative feedback- TGF-beta activates inhibitory Smads which block Smad from translocating to the nucleus
How do cytokines transmit signals?
Ctokines do not have inherent kinase activity so they have to bind to other kinases; they use the JAK-STAT pathway
What is the JAK-STAT pathway?
Cytokines binds to receptor -> JAKs dimerize and activate -> creates binding site for STAT -> STAT dimerizes via SH2 domains -> translocates to nucleus and bind DNA
How is the cytokine signal terminated?
SHP1 phosphatase or SOCS proteins
How does SHP1 phosphatase terminate cytokine signaling?
Removes phosphate from JAK; w/o phosphate, JAK has very weak kinase activity and can’t phosphorylate STAT proteins
How do SOCS proteins terminate cytokine signaling?
Competitive inhibition w/ JAK or STAT bc they have SH2 domains, can also recruit ubiquitin ligase to ubiquinate JAKs
Xeljanz
RA tx that inhibits JAK
What cellular processes use cytokine signaling?
Hematopoiesis and the immune response
Frizzled receptor
Receptor for Wnt that regulates the levels of beta-catenin protein; consists of 7 transmembrane domains that associates w/ co-receptor LRP
What happens when Wnt is not bound to Frizzled?
W/o ligand, beta-catenin is recruited to a destruction complex (includes APC and Axin), phosphorylated, and ubiquinated
Familial Adenomatous Polyposis
Familial form of colon CA caused by mutated APC gene
What are the steps in the Wnt signaling pathway?
Wnt -> binds frizzled -> recruits Axin away from destruction complex -> beta-catenin is not degraded and translocates to nucleus -> associates with TCF -> activates genes for cell growth
How is Wnt clinically significant?
Wnt is critical for embryonic development and for regulating growth of colon/mammary epithelial cells
APC mutation
Common in colon CA; APC is mutated an unable to form a destruction complex which leads to overexpression of beta-catenin
Receptor cross talk
The input a cell receives from different receptors are integrated to trigger a particular pattern of changes in enzyme activity or gene expression
Beta-arrestin complex
While it normally shuts down GPCRs, it can recruit Src to activate MAP kinase which explains why cardiac hypertrophy is a feature of prolonged epi exposure
Cross talk between GPCRs an MAP kinase pathway
Prolonged epi exposure can lead to cardiac hypertrophy because beta arrestin complex recruits Src, which then activates the MAP kinase pathway
Mitogen
Any molecules that stimulates cell proliferation
What is the cell cycle?
The process that controls cell division
What are the 5 phases of the cell cycle?
G0- quiescent G1- initial commitment point for cell division S- DNA replication G2- preparation for mitosis M- mitosis
Which cells go through the cell cycle?
All cells go through the cell cycle but at varying rates, eg cells of the GI tract are continually dividing while liver cells only divide if exposed to a specific stimulus and neurons only divide under unusual circumstances
Mitosis promoting factor (MPF)
Cyclin B-CDK1 complex that stimulates mitosis/mieosis; stimulated by progesterone
Cyclins
Proteins that are synthesized and degraded w/ the cell cycle to activate specific cyclin-dependent kinases (CDKs)
CDKs
Proteins that regulate the cell cycle by allowing for ordered recruitment and expression of genes during cell cycle progression
Cyclin D-CDK4,6
Activated by GFs during G1 to initiate the process of cell division to inhibit Rb ie induce EF2
Cyclin E-CDK2
Expressed later in G1 after the cell passes the restriction point; increases phosphorylation of Rb so that EF2 is fully activated and assemble to pre-replication complex proteins ORC, mcm helicase, CDC6, and CDT1
How do cyclins activate CDKs?
Cyclin binding causes a conformational change that facilitates ATP binding and exposes a phosphorylation site on the T-loop of the CDK; phosphorylation of the T-loop is what activates the CDK
At what point is the cell committed to divide?
Once it passes the restriction point in the G1 phase
What is the restriction point?
Once a cell has accumulated enough cyclin E
Rb
Tumor suppressor that inhibits the EF2 transcription factor; inactivated by cyclin D-CDK4,6
EF2
Transcription factor that induces DNA replication enzymes, cyclin D, cyclin E, and cyclin A which are required for cell to proceed to the next stage of the cell cycle
ORC
Origin replication complex
What happens during the S phase?
Cyclin E is degraded so CDK2 can associate with cycin A which triggers recruitment of DNA polymerase and activates mcm helicase
Why can DNA only be replicated once?
Origins that have fired once cannot be relicensed until they pass through the following mitosis; CDC6 is degraded or exported
What happens during the G2 phase?
Cyclin A and B accumulate and form complexes with CDK1
What determines whether the cell progresses from G2 to mitosis?
Cell size (ie whether it has sufficient cell components) and some external features or DNA repair pathways
What do cdc25 and Wee1 do?
Control progression from G2 to mitosis
cdc25
Phosphatase that activates the Cyclin B-CDK1 (MPF) complex so that the cell can continue to mitosis
Wee1
Kinase that adds an inhibitory phosphate to tyrosine 15 on the Cyclin b-CDK1 complex so that the cell is unable to progress to mitosis
How do mitotic cyclins (MPF) trigger mitosis?
Phosphorylate several proteins to induce mitosis including lamins, condensins, microtubule associated proteins, and Anaphase Promoting Complex/Cyclosom (APC/C)
What happens when MPF phosphorylates lamins?
Lamin network is disrupted and the nuclear envelope disassembles
What happens when MPF phosphorylates condensins?
Chromosomes condense
What happens when MPF phosphorylates microtubule associated proteins?
Mitotic spindle can form
What happens when MPF phosphorylates the APC/C?
Chromosomes are able to separate
APC/C
Ubiquitin ligase that degrades the anaphase inhibitor securin and later degrades MPF which causes degradation of cyclin B so that the cell can re-enter G1
cdc20
Binds to APC/C and triggers it to ubiquinate securin
cdh1
Binds to APC/C and triggers it to ubiquinate cyclin B
Which 2 proteins regulate the APC/C?
cdc20 and cdh1
What is the separase enzyme?
Cleaves the links holding sister chromatids together; inhibited by securin
Securin
Enzyme that inhibits anaphase by inhibiting the separase enzyme
Interphase
Encompasses G0, G1, S, and G2 phases
Prophase
Chromosomes condense and spindle begins to assemble
Prometaphase
Nuclear envelope disassembles, spindle assembly is completed, chromosomes begin to align b/w the two spindle poles
Metaphase
Chromosomes completely align to trigger anaphase
Anaphase
Chromosomes physically separate, sister chromatids move to opposite spindle poles and poles then move apart
Telophase
Nuclear envelope reforms and cytokinesis occurs
What % of the human genome accounts for individual differences?
0.1%
Endogenous (spontaneous) mutagenesis
DNA replication errors caused by normal cellular processes
Genetic defects
Defects in the endogenous DNA repair and detox machineries that increase risk of developing certain types of CAs
Exogenous mutagenesis
Mutations resulting from exposure to carcinogenic agents
What are the 5 types of DNA damage caused by endogenous mutagenesis?
- oxidation of bases
- alkylation of bases
- hydrolysis of bases ie deamination, depurination, etc
- bulky adduct formation
- mismatch of bases
Oxidation of bases
ROS oxidize bases and interrupt the DNA
Alkylation of bases
Addition of alkyl groups
Deamination
Loss of amino group from C, methyl C, G, or A (deamination of A or G is more rare)
What happens when C is deaminated?
Deaminated C produces U and causes a C to T point mutation
What happens when A is deaminated?
Deaminated A produces hypoxanthine (HX) and causes an A to G point mutation
What happens when methyl C is deaminated?
Deaminated methyl C is the same as T and the cell cannot differentiate between the mutated T and normal T so this causes an irreversible G-C to A-T mutation
Nitrous acid, HNO2
Potent mutagen formed from nitrates (NO3) or nitrites (NO2) that stimulates deamination
Vitamin C
Inhibits nitrosamine formation in the stomach
How do BRCA1 & 2 contribute to risk of breast CA?
These are DNA repair machinery that increase risk of breast CA when they are mutated
HPV
Causes cervical CA
HBV
Causes liver CA
Epstein-Barr virus
Causes lymphoma
H. pylori
Causes gastric CA
UVB light
Directly damages DNA by creating pyridine dimers by cross-linking adjacent C and T bases
UVA light
Indirectly damages DNA by producing free radicals
Ionizing radiation
Causes breaks in DNA strand
Thermal disruption at elevated temperature
Increases the rate of depurination and single strand breaks in DNA
How does ionizing radiation produce single or double stranded breaks in DNA?
ROS formation which interact with C4 and create unstable intermediates
UV irradiation
Causes formation of pyridine-pyridine dimers (ie T-T, C-C, or T-C) which are difficult to repair
Intercalating agents
Chemicals that slide b/w stacked bases of the DNA duplex and disrupt replication and transcription
What are naturally occurring intercalating agents excreted by organisms
Actinomysin D, aflatoxin, and echinomysin
Aflotoxin B1
Toxic metabolite produced by mold that can lead to liver CA if not detoxified
Alkylating agents
DNA cross-linking agents containing reactive alkyl groups that prevent DNA synthesis and separation, inhibit transcription, and induct mutagenesis
MNNG and nitrogen mustard
Alkylating agents
What are the 3 main types of chemo?
Alkylating agents, anti-metabolites, and organic drugs/natural products
Cyclophosphamide, cisplatin, and carboplatin
Alkylating chemo drug that cross-links DNA and forms adducts
Methotrexate
Anti-metabolite chemo drug that mimics tetrahydrofolate and inhibits DNA synthesis
5-flourouracil (5-FU)
Anti-metabolite chemo drug that inhibits DNA synthesis by competing w/ uracil
Doxorubicin & andriamycin
Organic chemo drugs that inhibit topoisomerase II
Taxol & Paclitaxel
Organic chemo drugs that bind beta-tubulin and stabilize microtubule assembly
Vincristine & Vinblastine
Organic chemo drugs that bind to tubulin and inhibit MT assembly
Radiation therapy
CA tx that produces double-stranded DNA breaks and generates massive amounts of ROS which causes the cells to commit apoptosis
Missing base
Caused by acid or heat depurination
Altered base
Caused by IR and alkylating agents
Incorrect base
Caused by spontaneous deaminations
Deletion-insertion
Caused by intercalating agents
Dimer formation
Caused by UV radiation
Double stranded DNA breaks
Caused by ionizing radiation and chemicals eg bleomycin
Interstrand cross-links
Psoralen derivatives; Mitomycin C
Transition mutation
BP substitutions from a purine to purine or pyrimidine to pyrimidine
Transversion mutation
BP substitutions from a purine to a pyrimidine or vice versa
What are the 4 types of BP mutations?
BP substitutions (transitions or transversions), insertions, and deletions
What type of mutations causes SCA?
BP substitution
What type of mutation causes CF?
BP deletion
What type of mutation causes Fragile X mental retardation?
BP insertion
What are the 3 types of DNA repair?
Excision repair, mismatch repair, repair DNA strand breaks
Base excision repair (BER)
Pathway for repairing non-helix-distorting base lesions; glycolysases recognize and remove aberrant base, endonucleases remove the sugar phosphate, DNA polymerase beta fills in the gap, and ligase joins the strands
Nucleotide excision repair (NER)
Pathway for repairing bulky-helix-distorting lesions (ie from pyridine dimers; damaged DNA is recognized, unwound, incised, and repaired
global genomic NER and transcription-coupled NER
2 different methods of NER that differ in how they recognize damaged DNA
Xeroderma pigmentosa (XP)
Genetic defect in genes responsible for NER pathway causing severe sensitivity to UV light and can lead to premature aging, skin CAs, and eye or neurological problems
Mismatch Repair (MMR) System
Corrects mismatches of normal base-pairing and inhibits homologous recombination b/w non-identical sequences
MSH2
Codes for protein that recognizes mismatched bps
MLH1
Codes for enzymes that cut out the mismatched bp
What mutations are involved in hereditary colon CA?
Mutations in MSH2 or MLH1 that code for proteins needed for the MMR System
How do MMR proteins know which is the correct nucleotide?
In bacteria, certain A residues become methylated after a new strand is synthesized. The MMR system acts quickly and it assumes that the methylated (parent) strand is the correct bp and then it corrects the daughter strand. We aren’t sure how this process works in eukaryotes
Hereditary nonpolyposis colon CA
Most involve genetic defects in the MMR system
How are double stranded breaks (DSBs) repaired?
Homologous recombination (HR) or non-homologous end joining (NHEJ)
BRCA1
Recruited to sites of DSBs and directs NHEJ
BRCA2
Regulates HR
DnaA
Prokaryotic origin binding protein
ORC + cdt1
Eukaryotic origin binding protein
SSB
Prokaryotic ssDNA binding protein
RPA
Eukaryotic ssDNA binding protein
DnaB
Prokaryotic helicase
MCM 2-7, cdc 45, GINS (CMG)
Eukaryotic helicase
DnaC
Prokaryotic helicase loader
DnaG
Prokaryotic primase
Polymerase primase
Eukaryotic primase
Delta complex of DNA Pol III
Prokaryotic polymerase clamp loader
RFC
Eukaryotic polymerase clamp loader
Beta subunit of DNA Pol III
Prokaryotic polymerase sliding clamp
PCNA
Eukaryotic polymerase sliding clamp
DNA Pol III
Prokaryotic polymerase
Pol Delta
Eukaryotic polymerase for lagging strand
Pol Epsilon
Eukaryotic polymerase for leading strand
Pol I and RNase H
Prokaryotic RNA repair and replacement
RNase H1, FEN 1, and Pol delta
Eukaryotic RNA repair and replacement
DNA ligase
Prokaryotic and eukaryotic gap joining protein
Tus protein
Prokaryotic termination protein
Telomerase
Eukaryotic termination protein
Topisomerase and DNA gyrase
Prokaryotic supercoil relaxer proteins
Topoisomerase
Eukaryotic supercoil relaxer proteins
Fork protein complex
Link helicase and polymerase in eukaryotes
Autophagy
Mildest form of cell death for organelle turnover in which the cell is engulfed from within; can be pro-life or pro-death mechanism )either via autophagy or induction of apoptosis)
Autophagosome
Double membrane vacuole formed from engulfed portions of cytoplasm that will fuse with lysosomes to make an autolysosome for autophagy
Which diseases feature autophagy?
Huntington’s dz, prion dx, breast CA cells tx’d w/ Tamoxifen
Extrinsic apoptosis pathway
Triggered by immune response ie killer T cells or TNF-alpha –> FASR/TNFR –> oligomerizes receptor and recruits death domain containing FADD/TRADD –> recruits and activates caspase 8 –> activates effector caspases ie caspase 3 –> apoptosis
Intrinsic apoptosis pathway
Activated by stress/DNA damage
Bcl-2, bcl-xl
Anti-apoptotic proteins located in the mitochondrial membrane that inhibit apoptotic proteins such as Bax and Bak
BH3-Only proteins
Bid and Bim; stimulate pore formation by pro-apoptotic Bcl proteins (Bax/Bak) and inhibit function of anti-apoptotic proteins (Bcl-2/bcl-xl)
Bax, Bak
Pro-apoptotic Bcl proteins that dimerize and release cytochrome C through pores
How do Bcl proteins control apoptosis
Bcl proteins are located in the mitochondrial membrane; they come in 3 varieties- pro and anti apoptotic and BH3-only. The ration of pro to anti apoptotic proteins determines whether the cell will commit apoptosis
Anti-apoptotic Bcl proteins
Bcl-2, bcl-xl
Pro-apoptotic Bcl proteins
Bax, Bak
BH3-only Bcl proteins
Bid, Bim, Bad
Apoptosome
Forms by binding of cytochrome c and Apaf-1 when they are released from the mitochondrial membrane in the intrinsic apoptosis pathway; binds and activates caspase 9
Caspase 9
Initiator caspase in the intrinsic apoptosis pathway
Effector caspases
Activated by initiator caspases and result in apoptosis
How is the external apoptotic pathway linked to the internal pathway?
Caspase 8 can cleave and activate the BH3-only protein Bid, which promotes pore formation by pro-apoptotic proteins and thus apoptosis
Anoikis
A specific type of apoptosis triggered by detachment from the ECM; tumor cells must learn to overcome this in order to metastasize
Akt kinase
Kinase stimulated integrin-ECM binding that adds inhibitory phosphate to Bad which inhibits apoptosis
How is anoikis activated?
W/o integrin-ECM binding, Akt is not activated so it can’t inhibit Bad and thus can’t inhibit apoptosis
UPR
Unfolded protein response; cell death pathway activated by ER stress and unfolded proteins that leads to apoptosis via activation of caspase 12, which activates caspase 3
BiP
Chaperone protein that senses ER stress
Necrosis
“Extreme apoptosis” triggered by toxin exposure, ischemia, or hypoxia that causes the cell to swell and the membrane to break down, releasing intracellular contents and triggering an inflammatory response
Repurfusion injury
Further injury/induction of necrosis following influx of immune cells into injury site during the inflammatory response which lead to accumulation of ROS/NGO
What controls whether a cell will undergo necrosis vs apoptosis?
Extend of mitochondrial membrane permeability ie leve of energy depletion
Beclin/Atg proteins
Important for forming the autophagosome
Necroptosis
“Programmed necrosis”; an alternative caspase indepedent pathway from the activation of TNF-alpha receptor that involves RIP1 & 3 proteins and results in release of metabolic enzymes, increased ROS, release of cytotoxic mitochondrial proteins, and membrane damage
Entosis
Cellular catabolism observed in tumor cells
Parthanatos
Cell death involving Poly-ADP-ribose polymerase-1
Proto-oncogene
Genes that code for protein that promotes cell proliferation or inhibits apoptosis
Oncogene
Mutated proto-oncogene or one w/ altered expression that confer gain of function to the cell
Tumor supressor gene
Gene coding for proteins that inhibit cell proliferation or promote apoptosis; mutation causes loss of function
Tumorigenesis in colon CA
Loss of APC -> hyperplastic epithelium
DNA hypomethylation -> early adenomas
Activation of K-ras -> intermediate adenomas
Loss of 18q TSG -> late adenomas
Loss of p53 -> carcinoma -> invasion and mets
Types of proto-oncogenes
Mutation (point or deletion), chromosomal translocation, amplification ie overexpression, GFs and GFRs, signaling proteins, TFs, cell cycle proteins, anti-apoptotic protiens
How do GFs function as proto-oncogenes?
Trigger entry into G1
IL-2 and IL2R
T cell GF that is overexpressed in T-cell leukemia
Her2 receptor mutations
Feature of breast CA that causes GF receptor to autodimerize
Trastuzumab
Her2 Ab used to tx breast CA pts w/ Her2 to Neu mutation
Gefitinib
EGFR tyrosine kinase receptor used to tx non-small cell lung CA w/ EGFR mutations
Erb2 oncoprotein
Oncogenic form of EGFR
B-raf mutation
MAP kinase kinase kinase that is common in metastatic melanoma;
Vemurafenib
Mutant Raf kinase inhibitor used to tx melanoma pts however pts can develop resistance
bcr-abl
“philadelphia chromosome”; common chromosomal translocation seen in CLL where c-abl kinase is fused to the bcr gene which alters the specificity of the abl kinase and promotes tumorgenicity
Gleevac
Abl kinase inhibitor used to tx CML and other CAs
myc
TF that activates cyclin D, E2F; overexpression is seen in late-stage neuroblastomas and B-cell lymphomas
CLL
Chromosomal translocation results in increased expression of anti-apoptotic protein bcl-2
Types of tumor supressors
Receptors/signal transducers involved in inhibiting cel cycle, cell cycle inhibitors, check-point control proteins, pro-apoptotic proteins, DNA repair enzymes
What is the primary anti-growth signaling pathway?
TGF-beta; stimulates p15 and PAI-1
p15
TGF-beta responsive protein that inhibits cyclin D-CDK4
PAI-1
TGF-beta responsive protein that regulates ECM proteins
Hereditary retinoblastoma
Pts are heterozygous for Rb but tumor cells have lost both Rb alleles (loss of heterozygosity)
p21-CIP family
Bind cyclin-CDK complexes and block activity
INK4 family
CDK inhibitors specific for cyclin D-CDK4,6; includes p15 and p16
p15 & p16
INK4 family CDK inhibitor that specifically inhibit cyclin D-CDK4,6
p16 mutations
Seen in families genetically disposed to melanoma
Check point control proteins
Regulate progression through cell cycle; include p21-CIP family and INK4 family proteins
p53
Key check point control protein that is the most commonly mutated protein in CA; activates CDK inhibitors and pro-apoptotic proteins once it is stabilized by DNA damage
BRCA1
Functions in DNA-damaged induced cell cycle checkpoins, and as a scaffold protein, enhances p53 phosphorylation and stabilization after DNA damage, regulates Wee1 kinase and cdc25 phosphatase
Telomerase
Enzyme that extends telomeres; lacking in normal adult cells
