Exam 2 Flashcards
The type of intercellular signaling in which one cell can communicate with another over long distances is called ___________.
endocrine
Describe autocrine signaling
one cell type is both the sender and the target
Which type of signaling is contact dependent?
juxtacrine
Synaptic signaling is a type of paracrine signaling used by __________.
nerve cells
In _________ signaling, the cells are in immediate vicinity of eachother.
paracrine
Describe intracellular receptors (4)
- usually bind hydrophobic ligands
- may be located either in the cytosol or nucleus in unbound state
- when bound to their ligand, regulate gene transcription
- when bound to their ligand, function as dimeric complexes binding to specific DNA sequences
Describe 3 functions of cell surface receptors
- when bounds to its ligand, could result in activation of an enzymatic cascade
- always opens an ion channel when bounds to its ligand
- must produce a second messenger when it binds to its ligand
Describe 3 methods in which cells can terminate signal transduction by cell surface receptors
- reducing agonist availability in the vicinity of the target cell
- internalizing and degrading the receptor-agonist complex
- modifying the receptor so that it is inactive or desensitized
What is calmodulin?
a protein that binds Ca2+
What are 4 downstream effects of the activation of phospholipase C?
- increase in intracellular Ca2+ concentration
- release of diacylglycerol (DAG) from a phospholipid
- activation of protein kinase C
- phosphorylation of certain cytoplasmic proteins
The a-subunit of G proteins may be ___________ because it has two forms.
- stimulatory or inhibitory
- Which form is released depends on the specific hormone and receptor that have interacted.
How can you increase cyclic AMP?
activate adenylate cyclase by a-subunit of Gs protein
Why would low GTPase activity result in constitutive activation of Gs and adenylate cyclase?
The GTP-bound a-subunit does not reform the aBy trimer.
Describe tyrosine kinases
- The N-terminal end is extracellular and site of binding of the ligand
- The catalytic site must be on an intracellular domain
- Dimerization causes causes its activation
- Growth factor binding to the receptor triggers dimerization which activates the kinase activity
- The first protein phosphorylated is the receptor itself, which then attracts other proteins to be phosphorylated
Ras protein is a critical regulator in cell proliferation, and its activity is enhanced by activated tyrosine kinase. Describe its action.
- adaptor proteins binding to phosphorylated tyrosines on receptor tyrosine kinase
- recruitment and stimulation of Ras-activating protein
- exchange of GDP for GTP on the Ras protein
- initiation of a cascade in which several kinases are activated sequentially by phosphorylation
How does the elevation of cyclic AMP in eukaryotic cells lead to altered transcription of certain genes?
- cAMP binding to protein kinase A causes dissociation of catalytic from regulatory subunits, exposing nuclear localization sequences on catalytic subunits.
- these can translocate onto the nucleus where they can phosphorylate and activate cAMP-regulated gene regulatory proteins (CREBs), which control genes containing cAMP-sensitive regulatory elements (CREs).
How do excitatory and inhibitor neurotransmitters differ in their effects on ligand-gated ion channels?
- Excitatory neurotransmitters (acetylcholine, glutamate) bind to cation-selective receptors and allow ions like Na+ to enter, depolarizing the membrane
- Inhibitory neurotransmitters (GABA, glycine) bind to anion-selective receptors and allow anions like Cl to enter, hyperpolarizing the membrane.
What mechanisms contribute to termination of GPCR-mediated signal transduction?
- Acceleration of GTPase activity by RGS proteins
- Phosphorylation of the GPCR by GRK
- Breakdown of cAMP by PDE
- β-Arrestin binding to the GPCR
cAMP is a second messenger that _________
leads to phosphorylation of cytoplasmic and nuclear proteins by PKA
List 4 characteristics of cytokine receptors:
- can have long disordered regions ranging over 600 residues
- can bind more than one kind of cytokine, even as many as ten different cytokines
- diverse number of extracellular domains
- transmembrane proteins with extracellular, intracellular and transmembrane regions
Cytokine are proteins that….
have diverse function, including one protein that can be either anti-inflammatory or pro-inflammatory.
If the common gamma chain acquired a mutation that inhibited PI3K/Akt signaling, the cell would be more likely to ____ .
undergo apoptosis
In Gq signaling, cytoplasmic calcium levels increase as a result of ……
passive diffusion of calcium from the ER through gated channels
Gleevec (imatinib) treatment produces a prolonged remission (but not a cure) of most cases of CML. In all the responsive cases, the Abl tyrosine protein kinase activity in the cancer cells has been activated by…….
aberrant recombination that caused loss of the inhibitory domain from the catalytic domain
Which enzymes phosphorylate licensing factors to initiate replication?
CDK2
DDK
Cell surface receptors with seven transmembrane domains signal by….
acting as a guanine nucleotide exchange factor
Inositol tri-phosphate, IP3, is produced by which enzyme activity?
phospholipase C
List characteristics of replication
- owes its accuracy to 3’ and 5’ exonucleolytic activity of DNA polymerases or associated proteins. This can remove a mismatched terminal nucleotide (proofreading)
- involves Okazaki fragments because synthesis occurs only in the 5’ to 3’ direction
- begins with the formation of an RNA primer
- requires proteins like primase, ligase, helicase, and others
In eukaryotic DNA replication….
Topoisomerases catalyze changes in the linking number, facilitating untwisting of the parental DNA strands. Since changing the linking number is a transesterification, it protects the integrity of the DNA and occurs without the need of additional energy.
How many initiation sites are there in eukaryotic DNA replication?
multiple
In eukaryotic replication, helicases open the replication fork. How is this powered?
requires the hydrolysis of ATP
In eukaryotic DNA replication, which strand requires the formation of Okazaki fragments?
The lagging strand requires the formation of Okazaki fragments because it has the 5’-end oriented toward the fork
What is the role of ligase in DNA replication?
Ligase simply forms the phosphodiester bond between adjacent nucleotides after the gap has been filled.
What is the function of the RNA component of telomerase?
The RNA component acts as a template for the synthesis of a segment of DNA.
Where are telomeres located?
Telomeres are at the 3’-end of each strand so that the 5’-ends can be replicated
Telomerase is a reverse transcriptase that recognizes…
a G-rich single strand of DNA
Define transition mutation
- mutation that results from substitution of one purine for another or of one pyrimidine for another
- point mutations
Define transversion
occurs when a purine is substituted for a pyrimidine or vice versa
Define frame shift
results from the insertion of one or two bases into the DNA chain
What mutation is frequently caused by chemicals (like acridine) that intercalate into DNA?
frame shift mutation
A missense mutation…
codes for a different amino acid
A nonsense mutation…
is when the code was changed to a stop signal
Homologous recombination involves….
a four-stranded intermediate (Holliday junction) which can be cut in either of two ways
What are the functions of chaperones?
- assist the covalent folding or unfolding of proteins
- prevent formation of aggregates
- responsive to cell stress
- assist in protein degradation by leading proteins to protease systems
What are most chaperones?
heat shock proteins
Disorders associated with mutations in
chaperones can affect….
muscle, bone, and/or the central nervous system
List 4 examples of PTM functions:
recognition signal, protein degradation, histone/DNA modifications, activity regulation
What is the function of proteolytic processing and conformational change?
activation
What is the function of PTM-dependent proteolysis?
Degredation
What are the functions of PTM-dependent recognition?
activation, interaction, localization and secretion
What are the functions of reversible multi-site PTMs?
dynamic regulation or modulation of protein activity and protein-protein and protein-DNA interactions
What diseases do large aggregates cause?
Alzheimer’s, Parkinson’s, Huntington’s, amyotrophic lateral sclerosis
How is protein degradation a part of protein homeostasis?
- controls certain cellular processes
- getting rid of, and recycling garbage (misfolds, damaged proteins, large aggregates)
ubiquitin-proteasome proteolysis
for proteins that are targeted for degradation as part of cellular regulation
Lysosomal digestion
membrane-bound organelles containing proteases that can degrade exogenous proteins or aged/damaged organelles
autophagy
maintains normal cellular functioning by protein degradation and turnover of damaged organelles – turned up during stress
autophagy – process
1) a double membraned structure forms by vesicle nucleation around cytoplasmic contents and becomes an autophagosome
2) Fusion of the autophagosome with the lysosome results in the autolysosome
3) Cytoplasmic substrates are degraded
How does defective autophagy lead to alzheimer’s?
The accumulation of AB production in collaboration with decreased AB clearance leads to an increased amount of intracellular AB oligomers. This leads to neurodegeneration.
Function of E1 (activating)
- ATP hydrolysis to add ubiquitin to a cysteine
- ATP dependent
Function of E2 (conjugating)
- receives ubiquitin on a cysteine
Function of E3 (ligase)
- specific recognition of protein to be degraded
- catalyzes transfer from E2 to substrate
- needs at least 4 Ub
- have specificity
- recognize degrons on their substrates
What activity does 20S proteasome have?
trypsin, caspase, chymotrypsin
What is a degron?
recognition sequence or structure for an E3 ligase
Destabilizing residues
Arg, Leu, Phe
PEST
proline (P), glutamic acid (E), serine (S), threonine (T)
What was the first approved proteasome inhibitor?
bortezomib
PROTAC
Proteolysis-targeting chimera
What us the purpose of signal transduction?
- coordinate cell metabolism, growth, and development
- ensure homeostasis among tissues and organs
- respond to external stimuli
What do the 5 modes of cell-cell communication differ in?
response time, receptor affinity, local ligand concentration
Three steps of signal transduction
receive signal, transduction, response
How many surface proteins bind for juxtacrine signaling?
two surface proteins
What is the response time, ligand concentration, and receptor affinity for endocrine signaling?
- minutes to hours
- low concentration
- high affinity
What is the response time, ligand concentration, and receptor affinity for paracrine signaling?
- seconds to minutes
- high concentration
- low affinity
What is the response time, ligand concentration, and receptor affinity for synaptic signaling?
- milliseconds
- very high concentration
- very low affinity
What are first messengers and ligands for receptors?
secreted molecules
cell surface receptors proteins bind…
hydrophilic or large ligands
intracellular receptor proteins bind….
hydrophobic ligands such as steroid hormones
Tyrosine, when phosphorylated, forms interaction with _________
lysine and arginine
What controls enzyme activity and protein-protein interactions?
protein phosphorylation
How do cell-surface receptors initiate signaling?
second messengers, change membrane potential, receptor enzyme activation
What are the four major cell-surface receptors?
1) ligand-gated ion channel receptors
2) enzyme-linked or catalytic receptors
3) cytokine family receptors
4) G protein-coupled receptors (GPCR)
Besides receptor internalization, another way to terminate the signaling from receptor tyrosine kinases could be:
protein phosphatase as part of a protein phosphorylation cycle
Which subunit of the trimeric (aBy) G protein should be similar in structure to the monomeric G protein?
Ga
Cytokines are involved in ________________ and are therefore a possible approach for therapeutic treatment of pathological pain from inflammation such as peripheral nerve injury
anti-inflammatory
In neutrophil migration, which interaction is most important to ensure neutrophils can cross the endothelium?
LFA-1 and ICAM-1
What are two downstream effectors of RAS?
RAF complex, PI3K
What is the distinguishing feature of a chemokine?
Chemokines are chemoattractants that are responsible for directing the migration of immune cells (e.g. neutrophils) along the concentration gradient of chemokine
What does it mean if a cytokine is pleiotropic?
same cytokine acting on two different cell types
Explain the difference between the rolling adhesion stage and the tight binding stage of neutrophil migration:
Rolling adhesion is low-affinity interactions of s-Lex and selectin on endothelium
Tight binding is high affinity interactions of LFA-1 integrin with adhesion protein ICAM-1 on endothelium guided by CXCL8 receptor on neutrophils binding to CXCL8 on endothelium
Describe how IL-6 promotes the formation of the heterohexameric complex of the receptor?
IL-6 binding to IL-6R alpha promotes gp130 association to form a trimer, which then dimerizes giving two copies of (gp130, IL-6 R alpha, IL-6)
What different strategies have been used to target IL-6 and the IL-6 receptor complex by drugs?
Antibody biologics exist against IL-6 (Siltuximab) and against the IL-6 receptor (Tocilizumab and Sarilumab) in soluble and membrane bound forms. In all cases, the antibody prevents protein- protein association and IL-6 signaling.
Regulator of G protein Signaling (RGS) proteins deactivate G proteins through its _______________ activity.
GTPase accelerating protein (GAP)
Explain Ga deactivation.
The gamma phosphate is hydrolyzed off GTP, converting it back to GDP
The promoter region of most genes does not contain a bona fide TATA box. Explain how transcription can
still be initiated for these genes:
TBP is loaded onto “compatible” (presumably AT-rich) DNA by the 16 transcription accessory factor (TAFs)
The combined energy from weak DNA binding by TBP and strong DNA binding by the TAFs and TAF-associated TFs make the interaction of TBP with the DNA sufficiently strong to cause the kink in the DNA.
The bend in the TBP-bound DNA is self-stabilizing as it forms, and the kinked-DNA-TBP complex is further stabilized by binding of TFIIA and TFIIB
What is the effect on transcription following histone acetylation?
Activation
What is the effect on transcription following DNA methylation?
Repression
Describe the mechanism of action for Cisplatin, a commonly used cancer drug.
Cisplatin is an alkylating agent, that causes breaks in DNA. This will inhibit replication and lead to cell cycle arrest – inhibit proliferation
Name the 3 transport mechanisms for Ca2+ out of the cytosol:
NCX: Sodium-Calcium exchanger
Ca2+ ATPase
SERCA: Sarco/endoplasmic reticulum Ca2+ ATPase
What is the function of RNA polymerase II?
RNA polymerase II transcribes the protein-encoding genes to produce mRNA
Which transcription factors form the DAB complex?
TFIID
TFIIA
TFIIB
Explain how the pre-initiation complex is formed:
- TFIID (including TBP) binds at the TATA box and bends DNA
- DAB complex is formed upon TFIID associating with TFIIA and TFIIB
- TFIIF and RNA polymerase II join the complex
- Additional transcription factors join.
- TFIIE recruits TFIIH to the complex.
Which transcription factor recruits TFIIH?
TFIIE
What are the functions of TFIIH?
Helicase, ATPase, kinase
How many polypeptides does the final pre-initiation complex have?
27 polypeptides
How is TF specificity achieved by?
modularity
What are complex promoters?
Complex promoters provide addition regulation for either enhancing or repressing transcription. They accelerate and provide specialized regulation.
What are the DNA response elements?
- SRE (sterol response element)
- CRE (cAMP) response element
How does chromatin remodeling play a central role in transcriptional control?
It regulates the accessibility of condensed DNA to proteins.
What is a HAT and what effect does it have?
- Histone acetyl transferase
- acetylation of K (Lysine)
- weakens histone-DNA contacts
- Increases nucleosome mobility
What is an HDAC and what affect does it have?
- histone deacetylase
- histone modification that restores contacts
- decreases mobility
How does DNA methylation play a role in repressing transcription?
DNA methylation forms a binding site for methyl-CpG domains that block transcription factors from binding DNA.
What is an ADNR and what effect does it have?
- ATP-dependent nucleosome remodeling factors
- alters nucleosome sliding
- weaken histone-DNA contacts and generate greater access to DNA for interacting proteins
Where does the first messenger bind on ligand-gated ion channels?
extracellular ligand binding domain
What forms the channel of a ligand-gated ion channel?
transmembrane helices
Ligands of ligand-gated ion channels are ___________. Gating leads to depolarization of ____________.
neurotransmitters, neuronal membranes
How are ligand-gated ion channels clinically relevant?
site for anesthetics, BP regulation, CVD
RTKs (receptor tyrosine kinases) ______________ intracellular domains for docking in initial steps of signal pathways.
autophosphorylate
What does dysfunction of RTKs lead to?
cancer
One agonist-bound GPCR can activate _________ G proteins
10-100
cascade effect
cytokines can stimulate the production of other cytokines
Cytokine types
interleukins, chemokines, tumor necrosis factor, interferons
Interleukins (structure and function)
- some have 4-helix bundle structure
- secreted in inflammatory response
- regulate growth/differentiation of hematopoietic cells
Chemokines (structure and function)
- less than 100 residues, monomeric or homodimeric
- cell migration during development, immune response,
tumor necrosis factor (structure and function)
- beta-pleated sandwich, bell shaped trimer
- released upon infection by macrophages
When TNF-a is in the membrane bound form, what kind of signaling is it involved in?
juxtacrine
Interferons (structure and function)
- five helices
- regulate signaling among cells infected with viruses or bacterial pathogens
- activate macrophages and NK cells
Cytokines are involved in _____________ pathways and are therefore a possible approach for therapeutic treatment of pathological pain from inflammation such as peripheral nerve injury.
anti-inflammatory
Ig (cytokine receptor structure)
single transmembrane helix with a cytoplasmic domain
Hemopoietic Growth factor (cytokine receptor structure)
motif on extracellular domain
TNF (cytokine receptor structure)
cysteine-rich extracellular binding domain
Intracellular domains (cytokine receptor structure)
- remarkably atypical
- long disordered region
Communication between immune cells is important to orchestrate a _____________
strong immune response
What key actions do cytokines control to elicit a strong immune response?
proliferation, differentiation, migration, apoptosis
Macrophage release of chemokines induces _______.
neutrophil migration
What inflammatory cytokines do resident macrophages release at the site of infection?
IL-1B, TNF-a, IL-6, CXCL8, IL-12
CXCL8 function
directs neutrophils where to cross the endothelium and subsequent migration to the infected tissues by moving along a concentration gradient of CXCL8
Canakinumab
- drug against IL-1
- antibody that sequesters the IL-1B cytokines
Secukinumab
- antibody against IL-17A that has FDA approval against psoriasis
