Week 3 Flashcards
Cytoskeleton
- Dynamic intracellular structure establishes order in the cell, and organizes cells in their environment
- Formed by 3 families of proteins: microfilaments, microtubules, intermediate filaments
Microtubule Structure
- alpha/beta tubulin heterodimer
- forms long protofilaments that mind side by side and assemble into microtubules
- Protofilaments not stable → get broken down if they aren’t assembled into microtubules
B end =
A end =
B end = + end, grows and shrinks from this end, has GTP exposed here
A end = - end, anchor end, GTP not exposed and thus not hydrolyzed to GTP
Stabilizing end-binding proteins bind microtubules and…
keep protofilaments together even without GTP cap
Microtubule Function (4)
- Cellular cytoskeleton
- Intracellular transport - scaffold for cell organization and movement of organelles (including chromosomes)
- Cell division
- Cilia – movement of flagella and cilia
Microtubule motor proteins
Dynein
Kinesin
Microfilament structure
helical filament
composed of actin
Grows from + end, and shrinks from - end
- end has ATP binding pocket
- requires stepwise nucleation
Microfilament function
cell movement and contraction
Microfilament motor proteins
myosins
ATP favors _________, and GTP favors ___________
ATP –> Microfilament
GTP –> Microtubule
Intermediate Filament Structure
Complex rope
- a-helix monomers bound together in rope-like structure
- can be made from vimentin, keratin, neurofilament (NO mixing)
Function of intermediate filaments
mechanical stability
Microtubules polymerize/depolymerize from the _______ and where _______ is bound
+ end, where GTP is bound
GTP cap acts to…
stabilize microtubules, removal of cap or GTP hydrolysis –> depolymerization
DYNAMIC INSTABILITY
Microtubule severing enzymes structure
GTPases
6 subunits with active hole in the middle
Microtubule severing enzymes act to…
-Cause depolymerization by cutting Tubulin C-terminus tail
Hereditary Spastic Paraplesia
neuro-degenerative disease caused by mutation in severing enzyme (messes up neat organization of axonal microtubules)
Paclitaxel
- binds and stabilizes microtubules causing Tubulin aggregates → block mitosis
- Cancer treatment
Microtubule Molecular Motor
Organelles transported long distances within cells, using MTs as “tracks” and motor proteins
Kinesin transports cargo toward ______
Dynein transports cargo toward ________
Kinesin –> + end
Dynein –> - end
Kinesin uses _____ to walk along microtubules.
Kinesin domains (2)
ATP
Domains:
-Cargo-binding domain (uses adaptor molecules to carry lots of different cargos
-Head domain (motor domain) - hydrolyzes ATP and reversibly binds MTs with conformational change (power stroke)
Three types of MTs in mitosis
1) Astral MTs
2) Kinetochore MTs
3) Overlap MTs
Astral MTs
radiate out from centromeres
Kinetochore MTs
attached to kinetochore, formed at centromere of each duplicated chromosome
Overlap MTs
- interdigitate at equator of spindle
- Contain double headed kinesins that walk toward opposite + ends, pulling chromosomes apart
Microtubules in Mitosis act to…
Segregate replicated chromosomes during mitosis
Actin Filament Nucleation is the formation of _______ but can also use _____ and _____ to form pseudo nucleation centers
G-actin homotrimers (VERY slow)
Arp2/3 and FH2 (Foramin)
Arp2/3
mimics two actin monomers – allows polymerization to begin with only 1 actin binding to Arp2/3
-Generates branched actin filaments
FH2 (Foramin)
binds two actin monomers to form nucleation center and
-Generates straight bundle actin filaments
Arp2/3 and FH2 are regulated by ________. They are active when bound to _____ and inactive when bound to ______
small monomeric GTPases
GTP (active)
GDP (inactive)
Steps of Actin filament formation (2)
- Nucleation
2. Extension/Retraction (spontaneous)
Actin formation is regulated by (4)
1) G-actin concentration
2) ADP/ATP exchange
3) Capping
4) Depolymerization/Severing
Actin cytoskeleton determines epithelial cell _______.
Does this in two ways:
polarity
Gate function: prevents movement of particles between cells
Sense Function: prevents proteins that need to be on apical side from diffusing across to basolateral side
Actin also plays a critical role in ________ formation
microcilli
extensions of apical surface, actin cytoskeleton runs into microvilli giving them support (Foramin dependent)
Examples of Asymmetric cell division (4)
o Red Blood Cells – no nucleus
o Generation of platelets
o Spermatogenesis
o Epithelial cells
rho
monomeric GTPase, activates formin, that forms long bundles of actin that makes contractile actinomysin ring
-regulated by astral microtubules
Actinomysin ring
o Key during last stages of cell division (Cytokinesis)
o Contraction of ring drives formation of cleavage furrow and separation of daughter cells
o Timing of contraction and ring formation are highly regulated and important for symmetry of cell division
Cell movement
- Lemellipodium (leading edge) senses extracellular signal that binds cell surface → activate monomeric GTPases → activate Arp2/3 → polymerize branched actin → pushes plasma membrane forward
- Extending one end, contracting/depolymerizing other end
Muscle sliding filament theory
- Myosin (thick filament) assembles with actin (thin filament)
- Myosin in middle
- Myosin heads bind to actin and pull forward, causing distance between Z discs to contract and causes muscle to contract
-ATP-driven walk of myosin heads along actin filaments
Myosin
actin-binding motor proteins
-Structurally similar to kinesins
- Neck region - ATPase activity and actin binding site
- Coiled coil domain – where dimerization occurs
Classes of signaling molecules (2)
Lipophilic
Hydrophilic
Lipophilic
- EX) Steroid Hormones
- Can penetrate the membrane → Can have intra-cellular receptors
- No cellular storage because can cross membrane
- Release controlled by synthesis only
- Slow
Hydrophilic
- EX) peptides, protines, amino acids
- Cannot penetrate the membrane → receptor must be on cell surface
- Can be stored intracellularly in vesicles
- Release is controlled via vesicle release
- Fast
Tools of signaling pathways (5)
1) Receptors
2) Second Messengers
3) Protein modification
4) protein-protein binding
5) GTP/GDP exchange
Phosphodiesterases (PDEs)
- Constitutive signal terminator
- Hydrolyze cAMP or cGMP → AMP/GMP
PKG
cGMP-dependent protein kinase, phosphorylates PDES and enhances cGMP binding
Cooperative binding of PDEs
Cooperative binding - catalytic and non-catalytic binding domains (binding at one increases affinity for cGMP at the other)
Mechanisms for signal termination (5)
1) Extracellular signaling molecule taken up, reused, broken down, or diffuse to reach a too low concentration
2) Termination initiated by another signal
3) Enzymes dedicated to turn off signals
4) Signaling proteins with built in terminators
• EX) G-proteins (ras-like proteins) – slow GTPases that reverse their active GTP-bound state
5) Receptor desensitization, receptor internalization
Nodes (EX)
-Points in a network that receive multiple inputs and/or multiple outputs
- Ca2+ : most extensive node in signaling
- Involved in linking “specific” upstream signals with “specific” downstream outputs in many different pathways
Acquired resistance to TKIs occurs by…
- Second site mutations: in EGFR arising or selected in patients who initially benefit from therapy, but then acquire resistance and disease progression
- Up regulation of different RTK pathways: can allow cancer to bypass block by using a different RTK pathway → drug no longer effective
EGFR
Promotes cell growth and proliferation
- Target for cancer therapeutics
- Over expressed in tumors
- Increased EGFR correlates with poorer clinical outcome in breast, lung, head, and neck
Two main mechanisms of RTK-targeted anti-cancer agents
antibodies
TKIs
Antibodies in RTK-targeted anti-cancer drugs
Primary role of antibodies is to block ligand binding to EGFR and prevent receptor dimerization
Tyrosine Kinase Inhibitors (TKIs) in RTK-targeted anti-cancer drugs
(TKIs) inhibit catalytic activity (usually) by binding (and blocking) in ATP binding site
Mechanism of Receptor Tyrosine Kinase (RTK) activation
1) Ligand binds extracellular domain
2) dimerization of RTKs
3) Activates catalytic activity of kinase resulting in autophosphorylation of receptor
4) Causes binding by SH2 domain containing protein
5) Grb2 binds phosphorylated RTK
6) binds Sos
7) proximity of Sos with membrane-bound ras –> activation of Ras (GDP –> GTP)
Grb 2
- adaptor protein
- consists of SH2 and SH3 domains, recognizes RTK only when it’s phosphorylated
- binds phosphorylated RTK and recruits Sos
Sos
- Exchange factor
- bound to Grb2 and in close proximity with membrane-bound Ras and activates Ras via nucleotide exchange GDP –> GTP
Ras
Activated by Sos
- GTPases, membrane bound switches
- GTP/GDP binding determines activation/inactivation
- Switch themselves off
- Regulated by GAPs and GEFs
-Ras+GTP = active → Multiple downstream pathways that promote cell growth, survival, motility
GAP –>
GEF –>
GEF: takes GDP off, allows GTP to bind
GAP: increases speed at which Ras is switched off
SH2 domain binds…
binds to phospho-tyrosine containing peptides
SH3 domain binds…
binds to Proline containing peptides
Inactive hetero-trimeric G protein
a-GDPBy heterotrimer
Agonist binds receptor ->
active conformation of receptor turns receptor into enzyme (Guanine nucleotide exchange factor)
Rate limiting step in G protein activation
Receptor coupled G-protein alpha subunit nucleotide exchange (rate limiting step) GDP -> GTP -> Active
Effectors
Enzymes that generate second messengers and ion channels that control membrane permeability
Ga-GTP undergoes spontaneous ______
nucleotide hydrolysis (GTPase built in to G-protein signals) -> Inactive
M3 Muscarinic Cholinergic Receptor antagonist in lung
ipratropium
B1-Adrenergic Receptor agonists
Norepniphrine, epinephrine, or isoproterenol
B1-Adrenergic Receptor activates
Adenyl Cyclase
Adenyl cyclase-> ____ -> ____ -> ____
AC -> cAMP -> PKA -> Ca2+ channels
Ca2+ influx in heart ->
increased heart rate and contraction
cAMP production in lungs ->
Bronchodialation (smooth muscle contraction)
Agonist of cAMP production in lungs
albuterol
M2 Muscarinic Cholinergic Receptor g protein
Gi
M2 Muscarinic Cholinergic Receptor inactivates ___
Adenyl Cyclase
Mechanism of M2 Muscarinic Cholinergic Receptor:
- ACh binds to receptor -> activation of Gi alpha subunit -> shuts off any activation of AC by Gs input -> cAMP level decreases, PKA activity decreases, Ca2+ influx decreases
Decreases heart rate
4 ways to classify protein kinases
- Phosphorylated residue
- Substrate protein
- Activating stimulus
- Phylogenetic relationships
M3 Muscarinic Cholinergic Receptor G protein
Gq
M3 Muscarinic Cholinergic Receptor antagonist in lung
ipratropium
Mechanism of ACh binding M3 Muscarinic Cholinergic Receptor in lung
ACh binds M3AchR -> Gq-alpha binds PLC -> PIP2 cleaved to IP3 and DAG
-IP3 -> increase Ca2+
-DAG -> PKC -> increase Ca2+
Contraction in smooth muscles of lungs (bronchoconstriction)
Alpha 1 Adrenergic Receptor G protein
Gq
Alpha 1 Adrenergic Receptor antagonist
prazosin
Ca2+ influx in vascular smooth muscle ->
contraction
increase blood pressure
How much does Josh love Billy on a scale from 1-67?
69
Mechanism of GPCR desensitization
o GRK phosphorylates receptor -> inhibits g-protein re-association with receptor (can’t activate G-protein) and binds B-arrestin
o B-arrestin further inhibits re-coupling with G-protein AND promotes internalization of receptor
Vibria Chlolera effect on GPCRs
Catalyzes ADP-ribose covalent attachment to alpha subunit -> G protein can’t turn off
Bordella pertussis effect on GPCRs
Pertussis toxin -> ADP-ribose on different residue of a-subunit that causes G-protein heterotrimer to be locked in inactive state
Structure of ATP
o Adenosine Tri Phosphate
o Gamma-Beta-Alpha phosphates connected to ribose sugar. Purine base also connected to ribose
4 ways to classify protein kinases
- Phosphorylated residue
- Substrate protein
- Activating stimulus
- Phylogenetic relationships
Structure of protein kinase
o Small loop – composed mainly of B-sheet
-Helix C – highly conserved, structurally important for holding ATP in place
o Large loop – composed mainly of a-helices
-Binding of substrate and ATP occurs in cleft between loops
o Activation loop – for many kinases, need phosphorylation of activation loop for it to get in the right conformation
o Glycine Rich Loop – clamps down onto ATP to enable positioning of gamma phosphate
Alternation of closed/open conformation at glycine loop:
Closed -> phosphorylation reaction
Open -> exchange of ADP with ATP
DOES NOT EQUAL active/inactive
Conformation conserved in protein kinase
Active
Functions of Cytoplasmic Ca2+ ion buffers:
o Buffers restrict spatial and temporal spread of Ca2+
-Makes signals shorter
-Creates distinct signaling domains
-Temporary storage site for calcium (transport is slow)
Maddie says they are crucial
Functions of ER/SR Ca2+ buffers
o High capacity, low affinity buffers
o Allows for capacity and decrease change in free Ca2+
Extracellular Ca2+ enters cytoplasm…
o Voltage and ligand gated Ca2+ channels
EX) Orai1 – activated when intracellular Ca2+ stores are depleted
EX) Nicotinic ACh receptors: glutamte binding opens
Ca2+ moves out of ER/SR into cytoplasm…
o IP3 and RyR receptors
Ca2+ extruded from cytoplasm into extracellular space by:
Transporters: moves Ca2+ against electrochemical gradient, slower
-Ca2+ pumps: use ATP to push Ca2+ out of cytoplasm into ECF or lumen of ER
Active transport
EX) SERCA
-3 Na+ in / 1 Ca2+ out exchanger
Stored Na+ gradient used to extrude Ca2+ out
Ca2+ extruded from cytoplasm into the lumen of ER/SR…
Ca2+ pumps
Proteins that contain EF domain hands
o Calmodulin - Contains four EF-hand Ca2+ binding sites
-Ca2+ can bind EF hands with high affinity
o Parvalbumin (cellular Ca2+ buffer)
o Calpain (Ca2+ activated protease)
o Troponin
Proteins that contain C2 domain
o Protein Kinase C - When PKC wants to associate with plasma membrane -> PKC phosphorylates effector
o Synaptotagmin - Responsible for Ca2+ dependent fusion of synaptic vesicles (release of NT)
Embryonic stem cells
have potential to generate all tissue types
Adult Stem Cells:
have limited potential – only renewing the tissue they were derived from
*unless reprogrammed
Niche in hemolytic stem cells
Osteoblast
Niche in intestinal stem cells
Paneth cells
Stem cell commitment (differentiation)
- Cells become differentiated by suppressing all genetic information by modifying chromatin so it is in a locked-down non-expressible form
-Reprogramming opens up chromatin so they can be expressed
Adult stem cell plasticity
Bone marrow transplants with adult stem cells are used to treat diseases
Reprogramming Adult Somatic Cells into Induced Pluripotent Stem Cells or Embryonic-Like Stem Cells
o Can use RNA vectors or modified mRNA to reprogram skin cells into IPS
o Use patient’s own cells so you don’t need immunosuppressant
Epidermal stem cells are target of carcinogenesis since they are ______
the only cells to permanently reside in epithelial tissues
Therapies directed against rapidly dividing cells
Tumor with stem cells, most cells die after treatment, but cancer stem cells survive -> tumors recur
Therapies directed against cancer stem cells
Tumor can regress!
3 source of androgen
1) Testis (90-95% of testosterone)
2) Adrenal glands (5-10% of testosterone)
3) Intracrine androgen production
- Cancer cells themselves produce testosterone
Structure of Androgen Receptor
- N-terminus transactivation domain
- DNA binding domain
- Hinge Region
- C-terminus ligand binding domain
Mechanism of Androgen receptor effecting transcription
Androgen binds AR in cytoplasm → moves to nucleus → homodimerization in nucleus → co-activators bind → AR binds DNA, activates transcription
4 Possible Mechanisms for Resistance to Endocrine Therapy for Prostate Cancer
1) AR activation via non-gonadal testosterone (Testosterone comes from adrenal or intracrine sources)
2) Over expression of AR
3) AR mutation leading to promiscuous (slutty) AR activation
4) Truncated form of AR, with constitutive activation of the ligand binding domain
Abiraterone
inhibits CYP 17 (key in androgen production)
-Block testosterone production from all 3 sources
Enzalutamide
inhibits AR activity
-Inhibits nuclear translocation, coactivator recruitment, and DNA binding of AR
Four Major classes of ECM components
1) Glycosamnioglycans (GAGs)
2) Fibrous proteins
3) Multidomain Adaptor Proteins
4) Water and Solutes
Fibrous proteins include:
Collagen and Elastin
Multidomain Adaptor Proteins include
Fibronectin and Lamin
Glycosaminoglycans (GAGs) function
- form “gel” as space-filling molecules in the cell
- Determine stiffness and hydration of ECM
GAGs vs. Proteoglycans
GAG= large unbranced disaccharide polymers
Proteoglycans = GAGs + “core Protein” (4 sugar linker)
Collagen
most abundant, tough polymers that provide tensile strength in connective tissues
Elastin
elastic protein found in a variety of tissues
Fibronectin
- found all over
- Can bind a variety of proteins (collagen, heparan/GAGs, each other)
- Can also bind integrins – principal adhesion molecule on cell surfaces
Lamin
- Found on Basal Lamina
- Provides “ground” for epithelial cells
- Heterotrimeric form in the shape of a cross
- Cross-links with itself
Role of of Matrix Metaloproteases (MMPs) in ECM Remodeling
-ECM needs to be broken down and remade frequently, this job falls to extracellular proteases (MMP)
Role of Adhesion in cell function and survival
- Cells need a substance to which they can attach, both for motile purposes and also because non-carcinogenic cells can’t grow without something to attach to.
- Adhesion NOT glue-like
- Cells ‘stick’ to very specific types of materials, and different cells stick to different things
- Combination of mechanical adhesion and cellular communication
- Cell knows what it is attaching to and can respond appropriately
Three types of Cell Adhesion Molecules (CAMs)
1) Cadherin
2) Immunoglobulin
3) Integrin
Cadherin
- attaches to other cadherins
- homodimer
- Calcium-activated “zipping” between cadherins on one surface and another
Immunoglobulin
- attaches to other Igs
- Does not require calcium
- monomer
Integrin
- Attaches to ECM proteins and has intracellular signaling end
- Heterodimer (alpha/beta subunits) - lots of combinations
- ECM binding domain that binds lamin, fibronectin, collagen, etc.
- Intracellular “tails” of integrins attach to varius things inside the cell (catenins, cytoskeleton, etc.)
Cell Adhesion Molecules (CAMs) in signaling
CAM extracellular portions can interact with signal proteins to tell the cell what it’s attached to.
Proteins associated intracellularly with CAM
-Cytoskeletal proteins to anchor the CAMs in the cell membrane (mechanical linkage): often actin-binding proteins.
-Signaling proteins: often protein kinases or GTPases.
EX) Cadherins with catenins
Cell adhesion and leukocytes
Selectin CAMs “catch” leukocytes coming out of the bloodstream
Epithelial Cancer dispersal and cell adhesion
mutation or down-regulation in Cadherin (CAM) can cause problems with catenin and thus promote dispersal of epithelium cancer cells
(cell free to float around, no longer attached to other cells)
