Proteins Post Midterm Flashcards
Cyclin
(Cyclin -> Cell Cycle)
It’s concentration works as the cell timer
CDK (Cyclin Dependent Kinase Binds to it, and gets activated)
Made continuously and destroyed periodically
CDK (Cyclin Dependent Kinase)
Binds to Cyclin and defines the “active” molecule
CDK activity forms the tresholds that activate the DNA synthesis (S phase) and Mitosis (M phase)
Wee1
Kinase that inhibits the Cyclin-CDK combo until the cell has grown enough (opp of Cdc25)
excess Wee1 = Too big of cells
Lack Wee1= Too small of cells
Cdc25
Activates Cyclin-CDK initiating cell division (opp of Wee1)
Excess Cdc25 = Too small of cells
Lack Cdc25 = Too big of cells
Cyclin-CDK activity
- Phosphorylate DNA replication machinery
- Breaks down the nuclear envelope (phosphorylation of lamin filaments )
basically it phosphorylates key mechanisms
Kinesin-5
tetrameric kinesins that slide anti-parallel microtubules
+ end directed
When both + end are reached the microtubules are spread apart, driving the 2 spindle poles away from each other
pushes the centrosome apart
When inhibited = monopolar spindle + chromosome bouquet
Dynein (anchored)
- end-directed motor protein
carries cargo towards - en on microtubule
positions the spindle
if dynein is anchored to the cortex, the microtubule is forcibly smashed into the cortex by the anchored dynein
Disruption of Dynein anchoring = aberrant spindle positioning
Kinesin-13
create “poleward flux” of microtubules,
located at spindle poles
depolarizing microtubules from - end (gamma TURCs have been lost), pulling them inwards towards the pole,
bringing the chromosomes toward the poles
also present at the kinetochore where it triggers catastrophies and depolymerization which drives the chromosome movement
APC (Anaphase Promoting Complex)
Poly-ubiquitinates M-Phase Cyclins,
(It ligates ubiquitin to the polypeptide chain) to then feed them to the proteasome (trash can)
gets its target-specificity and activation from binding partners
if no APC = no cyclin degradation, no progression to anaphase
Cdc20
binding partner to APC
binds to APC, will then ubiquitinate various proteins (Securin) triggering anaphase transition
Inhibiting Cdc20 = WAIT for transition
Release of Cdc20 = GO!
Cohesin
protein ring that physically ties the two sister chromatids together
needs to be cleaved by (Separase) for chromatids to separate
Separase
cleaves Cohesin and allows for the separation of sister chromatids
kept secured by Securin (which is ubiquitinated by Cdc20-APC)
Mad2
(Mitotic Arrest Defect)
exists in 2 conformations (Open and Closed)
its conversion is catalyzed by unaligned chromosomes
Open = Cannot inhibit Cdc20
Closed = Inhibits Cdc20 (Blocking the Anaphase transition)
Mad1
Binds to unaligned chromosomes and serves as catalyst for Mad2 confrmation change
p31
Protein in the cytoplasm
interacts with Mad1 and closed Mad2 tetramer when released from kinetochore
destroys Closed Mad2 & Mad1 complex
Destroys Closed Mad2 and Cdc20 complex
(Silences the wait signal)
Aurora B
Kinase in the kinetochore
when phosphorylated, destabilizes the kinetochore attachments
When bi-directional, kinetochore gets stretched and aurora B get separated, releasing Mad1/Mad2
Netrin
A signaling molecule that guides the neuron
will attract and repel some growth cones
(discovered by McGill graduate)
Semaphorin
A type of Netrin (guidance cue)
Is a repelling signal for the growth cones
CAM (Cell-adhesion molecules)
membrane proteins that mediate cell-cell adhesion and cell-matrix adhesions
Cell-cell = Cadherins
Cell-matrix = Integrins
Tight adhesion from many weak links (like Velcro)
Combination of Cis (lateral) and Trans (vertical) interactions
Adapter proteins
what adhesion receptors recruit
Acts as a linker between receptor and cytoskeleton
can recruit intracellular molecules for signaling pathways
Multi-adhesive matrix proteins + 1 example
Long and flexible components of the ECM that have many repeats
Regulates cell-matrix adhesion and thus cell shape and behavior!
ex: Laminin
Cadherins
Confer species-specificity
calcium dependant
connects to the cytoskeleton through adapter proteins (connecting the cell to the extracellular matrix)
the layered recruitement of the adapter proteins eventually connect to actin filaments and ARP
ECM (Extra cellular matrix)
diverse crosslinked of polymers
components:
- Collagen
- Laminin (multi-adhesive ECM protein)
- entactin
- perlecan
Collagen IV
found in the ECM
triple helix structure similar to ropes
collagen fibrils (fibers) are built from propeptides that are cleaved and cross-linked
(the main problem with it is how to prevent polymerization of the fibers inside their ER (endoplasmic reticulum))
Integrins
cell-matrix adhesion molecule
straighten when activated
Bent-over = intracellular domains alpha helices stuck to each other
Straight= physically separated -> opens 2 binding sites (Talin and Kindlin)
cluster in focal adhesion complexes
they have 5 distinct layers and are connected to signaling pathways
Talin and Kindlin
proteins that bind to the b-integrin tail when straight (activated) and signal the cell that it is now stuck
Cell-surface receptors
receives chemical signals
ex: hormones released from the endocrine glands
ligands will bind to receptors according to chemical equilibrium
chemical equilibrium and physiological response
Ligands will bind to receptors according to chemical equilibrium
but the response of cells is often more sensitive than anticipated
reason: signal amplification
short term signals
modification of cellular metabolism, function, movement
ramp up and down quickly
not all or nothing
long term signals
modification of gene expression, development
there is a signal amplification!
all in or nothing
(cell division, diferenciation…)
endocrine signaling
endocrine glands release hormones that affect long distance targets
here is the functioning pathway:
hormone secretion into blood by endocrine gland -> goes through blood vessel -> gets out and onto distant target cells (where cell surface receptors are)
Adrenaline (epinephrine)
hormone that triggers short-term response
discovered twice and used as vasoconstrictive medicine
b-adrenergic receptors (family A GPCR)
GPCR (G Protein Coupled Receptors)
made of:
- 7 transmembrane alpha helices
- extracellular segments
- cytosolic segments
polypeptides that weave through the helices, forming a basket
acts like a GEF (Guaninne exchange factor)
when a ligand binds = change in conformation (signal to the cell that ligand is bound) in receptor -> allows the cytosolic segment to bind and activate heterotrimeric G protein
G proteins
trimeric (a, b, y) GTPase that transduces hormone signals
Ga and Gb have (antipathic helices)
Similar to Rho family GTPases (on and off states)
dissociates within seconds of ligand binding (GTP hydrolysis rate)
GDP bound = Off state
GTP bound = On state
antipathic helices
found on a and b subunits of G proteins
hydrophobic helices that, inserted in leaflet of plasma membrane
allows close G protein and GPCR proximity
GPCR signal transduction pathway (4 parts needed)
1) membrane imbedded receptor (7 alpha helices)
2) heterotrimeric G protein
3) membrane-bound effector protein
4) proteins for amplification and adaptation (ex; cAMP, Ca2+)
Steps to heterotrimeric G protein activation and deactivation (6)
hint: bouncing of Ga
- hormone binding induces conf change of the receptor
- conf change allows for Ga subunit to bind and activate
- G protein becomes activated (GDP -> GTP)
- GTP binding to Ga = dissociation from Gby and GPCR
- hormone dissociates,
Ga binds to the effector (activates it) - Hydrolysis of GTP ->GDP
Ga dissociates from the effector,
reassociates with Gby
FRET (Förster resonance energy transfer)
allows to show dissociation through the use of fluorescent molecules impacting each other
allows us to measure the physical distance between 2 proteins
Adenylyl cyclase
a common effector of activated G proteins (GDP)
makes cAMP (secondary messenger)
cAMP
made by Adenylyl cyclase effector
secondary messenger
activates PKA (protein Kinase A)
Inactive PKA is made of catalytic subunits and regulatory subunits, when cAMP is present, it fills crevasses in the regulatory subunit which releases the catalytic subunits
PKA (Protein Kinase A)
activates by cAMP
directly controls the molecules of glycogen metabolism
signal amplification
multi-step activation
use of secondary messengers
reason for superior physiological reaction compared to chemical balance
RTK (Receptor Tyrosine Kinases)
receive signals from growth factors (NGF, EGF) and trigger cells to proliferate
key elements:
- extracellular domain (ligand binding site) receptor
- cytosolic segment (with tyrosine kinase activity) Kinase + activation arm
- C-terminal (with tyrosine residue to become phosphorylated by receptor’s kinase)
Aberrant RTK signaling is found in all human cancers!
hyperactive = tumor grows
no RTK = no cell growth
when a ligand binds to the receptor it undergoes a dimerization
SH2
domains that bind to phosphorylated tyrosine residues
HER2
Has a kinase activity that facilitates all EGF family signaling
more HER2 = cell is more sensitive to signaling by many EGF family member
HER2 is over-expressed in 25% of breast cancer
GRB2
links active RTK to Sos
has:
(1) SH2 domain (binds to phosphorylated tyrosine on RTK)
(2) SH3 domains (binds to Sos, to proline-rich sequences)
Sos
Links Ras-GDP to GRB2 (which is linked to RTK)
acts as a GEF for Ras (catalyzes Ras-GDP to Ras-GTP)
Ras
small GTPase (on and off state)
most common mutation in human cancer
discovered in Rat Sarcoma virus
similar to Ga protein but does not have a GAP domain (need to recruit GAP protein to accelerate its hydrolysis rate, which is otherwise slow)
if always “on” ->tumor
active Ras triggers a kinase cascade
SH3
domain that binds to proline-rich peptides
creates a kink in the polypeptide chain
proline for 2 reasons:
- assumes an extended conformation
- binding specificity, fits into SH3 binding pocket
Raf
Phosphorylates MEK
activated by ras-GDP
which induces a change in Raf conformation, partially activating it
it gets fully activated when Ras GDP to Ras GTP hydrolysis releases Raf from Ras
released Raf forms a dimer with another which increases its kinase activity
MEK
kinase that phosphorylates target protein
Dual specificity
mainly phosphorylates MAP kinases in the activation loop
MAPK (MAP kinase)
can translocate into nucleus to phosphorylate different proteins (Transcription factors)
growth factors
stimulate cell proliferation and survival
NGF (Nerve Growth factor)
EFG (Epidermal Growth Factor)
Rita Levi-montalcini and Stanley Cohen (chicken egg and snake venom exp)
How does ligand binding to RTKs or EGF receptors convey signaling
Ligand binding induces dimerization of RTKs
causes a conformation change that brings the 2 intracellular kinase domains into close proximity
the kinase start to phosphorylate each other’s activation loops (phosphorylation in trans)
this is the signal
activation of the RTKs is the start of cascade events!
3 ways cells know what is happening in their environment
- Integrins, C-terminal tail separate aka physical separation (signal)
- GPCR, 7 helices conformation change (signal) when the ligand binds
- RTK, coming together phosphorylation in trans (signal)
additional signaling proteins recruited by phosphorylated RTKs
GRB2 (1 SH2 domain and 2 SH3 domains), Sos, Ras
what is the Kinase Cascade (just the order of role)
RTK -> GRB2 ->Sos -> Ras -> Raf ->MEK ->MAPK
what are the steps to the Ras/MAP kinase signal Transduction Pathway
(6 steps)
- Ras activated by exchange of GDP for GTP (thanks to Sos acting as Ras GEF)
- active Ras recruits bind and activate Raf
- GTP hydrolysis leads to dissociation of Ras from Raf
- Raf activates MEK
- MEK activates MAPK
- Active MAPK translocates into the nucleus and activates many transcription factors (related to proliferation)
Metastasis
requires cells to break connections with their neighbors so that it can round up and split chromosomes
focal adhesions are constantly remodeled by metastatic migration
contact inhibition
when you are bound to other cells it inhibits proliferation
Endoplasmic Reticulum
- site for protein and lipid synthesis
- single membrane bilayer organelle
- interconnected tubules and sheets , forming a network
- has a single internal space known as ER lumen
it is highly dynamic (constantly reorganized)
is functionally organized as:
Rough ER (sheets)
Smooth ER (tubules)
Smooth ER
Lipid biosynthesis
Contain ER exit sites (involved in ER-Golgi traffic)
mostly exists as tubules
Rough ER
protein biosynthesis
protein target and translocation into ER
mostly sheets
microsomes
when cells are homogenized, the rough ER breaks into small microsomes
- still functional (still has ribosomes)
- Easily purified
are necessary for ER import of proteins (with signal sequence)
reticulons
class of proteins
have a W-shaped structure that generates curvature
necessary for the formation of ER tubules and ER sheets
Atlastins
class of dynamin-like GTPases
undergo GTP-dependent oligomerization
necessary for the fusion of different ER tubules
CLIMP63
ER luminal spacer for sheet formation
ribosomes provide pressure to flatten the ER sheet and CLIMP63 prevents it from touching and keeps space for the lumen
What are the 3 proteins needed for ER shaping?
Reticulons
Atlastins
CLIMP63
ER signal sequence
guides ribosome-mRNA-nascent peptide complex to the ER
when properly attached to the ER, peptides get translated and translocated into the lumen of the ER (co-translational)
around 24 aa long
has 10 hydrophobic aa in its middle (important)
necessary for ER import
- ER signal sequence is cleaved off when imported into the ER
Signal Recognition Particle (SRP)
recognizes the ER signal sequence thanks to its P54 hydrophobic binding group that binds to it
it docs the nascent polupeptide-ribosome-mRNA onto the ER by its interaction with SRP receptor
SRP receptor (signal Recognition Particle)
when it interacts with SRP (which binds to the signal ssequence) it binds the polypeptide-ribosome-mRNA to the ER
it physically interacts with the translocon by having the 2 subunits GTP-bound which brings the complex closer to the ER
has an a and b subunit
a-subunit interacts with P54 of SRP
P54
key structure of the SRP (Signal Recognition Particle)
hydrophobic binding group that binds to the signal sequence
Signal Peptidase
once translation in the ER lumen is completed, it cleaves off the signal sequence, leaving it to be degraded
What are the steps (8) to the targeting of secretory proteins into the ER lumen ?
- ER signal sequence emerges from the ribosome
- bound by a SRP
- SRP and nascent polypeptide chain ribosome complex bind to SRP receptor (strengthened by GTP binding of SRP and its receptor)
- complex properly docked to ER translocator
- Signal sequence opens translocator and enters
- Both P54 and a subunit of SRP receptor hydrolyze GTP to GDP so they dissociate (SRP recycles back) - The polypeptide chain enters and continues to elongate into the ER lumen as mRNA is translated
- ER signal sequence is cleaved by Signal peptidase
- entire polypeptide into the ER lumen
- translocator closes and ribosome is released, folding of peptide starts
What are the 2 models for ER motility on microtubules?
- Slide along microtubules
- Growth on the + end
Na+ / K+ ATP pump and action potentials
establishes concentration gradients of ions
action potentials are controlled by the opening of Na+ channels
Na+ ions diffuse outward, opening neighboring Na+ channels
Action potentials are unidirectional
neurotransmitters
small chemical compounds
diffuse across the synaptic cleft
packaged into synaptic vesicles by H+ gradients
binding causes depolarization of the target cell
Ca2+ influx triggers exocytosis of neurotransmitters
Ca 2+ channels at axon terminals
Ca2+ influx triggers exocytosis of neurotransmitters
neuroreceptors
- ligand-gated (like acetylcholine receptor)
- GPCR
V-class H+ pumps
packages neurotransmitters into the synaptic vesicle
looks like ATP synthases (basically works in its reverse form)
uses ATP hydrolysis energy
let’s in a H+ into the synaptic vesicle
SNARE complexes
docks synaptic vesicles at the plasma membrane
bundles of a-helix that hold 2 plasma membranes in close proximity to one another
the target of botox!
Botox (Botulinum toxin)
degrades SNARE complexes
undocks synaptic vesicles and prevents acetylcholine (muscle contraction) release
but! ur body still produces SNARE proteins so over time the botox fades
Synaptotagmin
Ca2+ sensor
Ca2+ influx causes membrane fusion and neurotransmitter release
Ca2+ binds to synaptotagmin which displaces complexin
complexin
Inhibits SNARE (which makes membranes fuse)
Synaptotagmin binds to Ca2+ -> displaces complexin ->SNARE conformation change (smashes membranes together)
Na+ symporters
reuptakes neurotransmitters
uses the energy of Na+ moving down its concentration gradient into the cell
antidepressants inhibit that reuptake, allowing serotonin to persist longer in the synaptic cleft
GTPase Dynamin
needed for synaptic vesicle recycling
collar that pinches the vesicle off
only 10% of vesicles are docked and used per firing (allows for multiple firing rounds)
Steps to the cycling of neurotransmitters and of synaptic vesicles (6 steps)
- Import of neurotransmitter
- Movement of vesicle to the active zone
- vesicle docking at the plasma membrane (SNARE complex)
- exocytosis of neurotransmitter triggered by influx of Ca2+
- reuptake of neurotransmitter (Na+ symporters)
- recovery of synaptic vesicles via endocytosis (GTPase Dynamin)
Growth cone
leads to axon formation
contain complex cytoskeletal machinery
immature neurite
small protrusions in early neuron formation will become dendrites or axons (growth cone)
problems for brain wiring (2)
- Making the right connections
- Getting neurons to the right place
ventricular zone
origin of neurons, which then migrate outward toward the cerebral cortex
cerebral cortex and neurons
neurons migrate radially long distances to pattern the cortex
needs guidance cues
microtubule role in cell migration
MT = front ->activate Rac (leading edge)
MT = back ->Inhibits Rho (stress fibers)
no MT = Active Rho (stress fibers)
Doublecortin DCX
mutation in DCX causes defects in neuronal migration
single amino acid mutation in microtubule-associated protein
female phenotype = excess gray matter near the cortex
male phenotype = smooth brain, early death
scratch assay
has shown that depolymerization of microtubules stops migration
cells wander around randomly
without MT, lamellopodia extends but has nowhere to go
Guidance cues
connect to Rho family GTPases
Attracts and repels growth cones
(netrin (attract and repel) Semaphorins (repel)
how do microtubules inhibit Rho
MT sequester and inactivate Rho-GEF
MT depolymerizes = release of Inactive Rho-GEF = activates Rho = Stress fiber formation
sarcomere
the basic unit of the “sliding filament theory”
tightly packed arrays of actin filaments and myosin filaments
separated by Z-disks
undergo rapid contraction upon muscle stimulation
structure is set by nebulin, titin and other capping proteins
Myosin (as a general protein type)
a protein that produces force (converts ATP into mechanical force)
Myosin II
type of myosin
forms bundles that pull actin filaments inward
the bundles contract the actin arrays (these contractile bundles are what muscles are made of)
the neck domain of myosin acts as the lever arm
Z disk
caps at the end of sarcomeres
The Crossbridge Cycle (5 steps)
it couples 1 ATP hydrolysis to 1 power stroke
- Binds ATP, head released from actin
ATP hydrolysis puts myosin into a strained conformation
2. Hydrolysis of ATP to ADP + Pi myosin head rotates into a cocked state
myosin binds the actin filament in the ADP+Pi state
3. Myosin head binds actin filament
phosphate release relaxes the strain in the myosin head
4. “power stroke”: release of phosphate and elastic energy straightens myosin, moves actin filament left
New ATP binding releases myosin from actin filament
5. ADP released, ATP bound, head released from actin
what causes rigor mortis
failure of myosin to detach in the absence of ATP
what are the 3 design challenges of muscles
- prevent continuous contractions
- activate contraction
- freeze the structure of the sarcomere
tropomyosin
blocks the binding site for myosin on the actin filament
troponin
binds calcium and pulls tropomodulin out of the way
sarcoplasmic reticulum
calcium storing organelle
this calcium is needed for muscle contraction
motor neurons
transmit signals from the brain that activate muscle contractions
stimulation by motor neurons causes a rapid spike in Ca2+ muscle fibers
muscle contraction is stimulated by the presence of calcium
what are the 2 central requirements for cell division
- increase your size
- duplicate your DNA
what is the one thing cells can measure?
concentrations :)
Cdr2 and Pom1
(protein gradient measuring cell length im S.pombe)
small cells:
Cdr 2 wants to inhibit Wee1
is itself inhibited by Pom1
large cells:
Pom1 stays at the poles and is “far enough”
Cdr2 can inhibit Wee1
what are the 2 things that a cell has to do during mitosis?
- segregate your chromosomes (perfectly)
- degrade your cyclins
Sec61
BiP
N-linked glycosylation
Type I membrane proteins
Type II membrane proteins
Type III membrane proteins
Type IV membrane proteins
How do cells prevent polymerization of collagen fibers inside their ER
