BICD 110 Midterm 2

What is phagocytosis

an ancient, actin-dependent pathway that enables cells to engulf particles/cells/pathogens > 500nm (induced mechanism)

What are the steps of detection of opsonized target in phagocytosis?

pathogen that is tagged with Fab/Fc antibody is recognized by Fc region (constant) by Fc receptor on effector cell

What are the steps of the formation of phagocytic cup in phagocytosis?

1. probing: membrane ruffles and extends to interact with tagged pathogen

2. early signaling and cup formation: once enough Fc receptors engage, cup forms and induces downstream signaling

3. pseudopod extension: extends even more to facilitate engulfment

4. phagosome closure: pathogen is fully enclosed

What are the steps of the phagosome maturation in phagocytosis?

1. engulfed bacteria acquires factors: Rab5 and GTPases

2. fuse with early endosome, where proton pump V-ATPase lowers pH to 5.5-6

3. conversion of Rab5-Rab7 + late endosome promotes conversion to late phagosome. pH dec to 4.5

4. fusion with lysosome brings degradative enzyme (now phagolysosome)

5. NADPH oxidase complex recruited to produce reactive oxygen species (ROS) to bleach interior of phagolysosome

6. after degradation, phagolysosome is exocytosed

What is macropinocytosis?

an ancient, actin dependent pathway that enables cell to engulf fluids. macropinosomes range from 200nm-10um. membrane “ruffles” can be induced by growth factors

Is phagocytosis and macropinocytosis induced or constitutive?

phagocytosis: induced
macropinocytosis: constitutive

Steps for clatherin-dependent, receptor-mediated endocytosis

1. specific ligands bind to transmembrane receptors on PM

2. AP2 (locked) recognizes NPXY or LL motifs and binds to cargo receptor tails on PM and PIP2

3. AP2 recruits clatherin triskelion, which curves membrane into CCP (pit)

4. pit buds off into vesicle, AP and clathierin triskelion dissociate

What happens to lysosomal enzymes when the pH is 5 and why?

it becomes active, safeguarding mechanism

How do lysosomes decrease their pH

V-type ATPase are lysosomal membrane pumps, they pump protons (H+) into the lumen of the lysosome

LDL particle structure

the shell is a single apolipoprotein wrapped around and phospholipid monolayer. the core is hydrophobic with neutral lipids. the particle, ApoB, is taken from the bloodstream and transported

Mechanism for receptor-mediated endocytosis of LDL

LDLr: B-propeller domain in PM, with NPXY sorting signal in cytosol. Cys-rich “hook” binds to ApoB (LDL) at pH 7. after endocytosis, pH 5 in late endosome weakens interaction (+ charged), and LDL is released. LDLr is recycled to PM

How does SRE-binding proteins monitor ER cholesterol levels?

when cholesterol levels are low, the ER sends SREBP in COPII vesicles to Golgi, which sends SREBP to nucleus. SREBP activates SRE on DNA and activates cholesterol synthesis pathways

What are mutivesicular bodies (MVB)

they degrade cytosolic portions of membrane proteins

Mechanism for degradation of PM receptor

1. lysosomal enzymes TGN → late endosome

2. endosome carrying PM receptor fuse with late endosome

3. vesicles containing PM receptor bud inward (MVB)

4. MVB fuse with lysosome. lysosomal enzyme activate and degrade PM receptor

Mechanism for MVB formation

1. transmembrane proteins for degradation are ubiquitinated

2. Hrs protein sorts ubiquitinated cargo into inward buds

3. ubiquitin attracts ESCRT machinery and forms inward vesicles

4. ATP is invested into Vps4 to disassemble ESCRT from membrane

Mechanism for autophagy

1. ATG proteins induce formation of cup shaped structure around target in two bilayers

2. Atg8 specifies membrane growth

3. fusion of autophagosome and lysosome

4. degradation of target

What are caveolae?

stable membrane domains that depend on cholesterol (SDS PAGE cannot break apart) and contribute to exocytic and endocytic events

What is transcytosis?

the transfer of macromolecules from the apical to basolateral membrane and vise versa via endo and exocytosis.

What type of junction are apical and basolateral membrane separated by?

tight junction

What are the 5 major endocytic pathways?

phagocytosis, macropinocytosis, clatherin-dependent endocytosis, caveolae, clatherin/caveolin-indepdent

What does chemical and electrical gradients depend on?

chemical: concentration of molecules on either side of mem

electrical: ratio of ions (charge) on either side of mem

What is facilitated transport?

transport of a single type of molecule along its concentration gradient

What is co-transport, secondary active transport?

energy available from ion down an electrochemical gradient drives movement of a molecule against its concentration gradient

What is a ATP-powered pump?

energy released by ATP hydrolysis drives movement of specific ions or small molecules against their electrochemical gradient

What three transporters cooperate at the PM?

Na/K ATPase: 321 NOKIA
K+ channel: K out
Na/lysine symporter: Na in (down gradient), lysine in (up gradient)

Fill in the table

Structure of aquaporin

homotetramer (4 identical subunits), highly conserved hydrophilic AA channel, arrangement of H-bonds and 0.28nm diameter pore, no conformational change during transport

What is Vmax and what is it dependent on?

maximum velocity at which you can transport a substrate across bilayer is dependent on the NUMBER of transporters that is working at max rate and the gradient is large

What is Km and what is it dependent on?

concentration at which rate of uptake is half-maximal (mM) is dependent on affinity of transporter

How does GLUT1 work in simple terms?

1. outward open conf binds glucose

2. ligand bound occluded conf

3. inward open conf releases glucose

4. ligand free occluded conf

• cycle will work in reverse if conc changes

• 14 highly homologous GLUT proteins

What are P-class pumps?

• type of ATP-powered pump

• generate ion gradients across membranes

• catalytic a subunit is phosphorylated

• ex: Na/K ATPase

Mechanism for Na/K ATPase

1. high affinity for Na+, low aff for K, ATP binds

2. phosphorylation of Asp, D, ADP released

3. conformational change, Na+ low aff, released. high aff for K+

4. dephosphorylation of Asp, conformational change

5. low aff for K+, high aff for Na+

What are V-class pumps?

• type of ATP-powered pump

• couples ATP hydrolysis to transport protons against conc. gradient

• general low pH of plant vacuole & lysosome

• NOT phosphorylated

Mechanism of V-class pump

• V1 subunit binds ATP

• ATP hydrolysis triggers 120° rotation of V1

• rotation of V0 drives proton binding from cytosol to lumen via de/protonation of glutamate and aspartate

How do V-class pump + Cl- channels acidify cell

Cl- facilitated transport thru channel balances/neutralized pumped H+ so there is no electric potential. this allows for even more H+ to be pumped into lysosome to reach desired pH=5

What are F-class pumps?

• most transport only H+

• NO phosphoprotein intermediate

• work in reverse to use energy in proton conc. or voltage gradient to drive ATP synthesis in mito or bacteria

What are ABC superfamily pumps?

• have two transmembrane (T) domains + two cytosolic ATP-binding (A) domains

• couple ATP hydrolysis to solute movement

• each ABC protein is specific for single substrate

• export wide variety of toxins and drugs

Mechanism for transcellular transport of glucose from intestinal lumen into blood

1. Na/K pump establishes concentration gradient so cell has negative membrane potential

2. glucose is taken from intestine lumen by 2xNa/glc symporter

3. glucose transported out of cell by low aff uniporter GLUT2

Which type of membrane transport proteins has the highest rate of transport: channels, uniporters, symporter/antiporter, ATP-powered pumps

channels

What is the 4 types of extracellular signaling?

1. endocrine

2. paracrine

3. autocrine

4. cell-cell (juxtacrine)

What is endocrine signaling?

signaling molecules are synthesized and secreted by signaling cells and transported thru circulatory system. affects distant target cells expressing specific receptor. ex. epinephrine, insulin

What is paracrine signaing?

signaling molecules are synthesized and secreted by cell and affect only nearby target cells expressing specific receptor. ex: NT, growth factor

What is autocrine signaling?

cells respond to signals they secrete (tumor cells may overproduce). ex: growth factors

What is cell-cell contacts (juxtacrine)?

direct contact with surface receptors of neighboring cells

Properties of hydrophobic signaling molecules

• diffuse across PM

• bind to cytosolic receptors

• receptor-signal complex moves into nucleus

• binds promoters in DNA to activate/repress gene exp

• ex: steroids (testosterone, estrogen, cortisol)

Properties of hydrophilic signaling molecules

• cannot cross PM

• binds to cell surface receptor

• activates downstream signal protein/secondary messenger

• activate effector proteins

General mechanism of signal transduction pathway

1. receptor (R) binds ligand (H)

2. triggers conf change (R*), activated receptor binds signal transduction protein (S1)

3. amplification: S1 binds to other proteins S2, activates/inhibits

4. S2 activates more proteins S3-S5, different targets/locations in cell

5. signaling proteins bind and activate effector protein (E)

   1. can be enzyme, trxn factor, ion channel etc

6. feedback loop: signal transduction proteins/effector can inhibit upstream mechanism

Structure of GPCR

• N-term out, C term in cytosol

• 7 transmembrane domains

• 4 extracellular segments (E)

• 4 intracellular segments (C)

How does GPCR detect ligand and send signals inside?

use extracellular loops to detect ligand and send signals inside using intracellular loops

What does Family A of GPCR detect?

• biological amines

• light/odorants

• peptides

• purines

• lipids

What does Family B of GPCR detect?

peptides (like glucagon)

What does Family C of GPCR detect?

• biological amines

• glutamate

• Ca2+

Mechanism for general GPCR signal transduction pathway

1. ligand binding induces receptor activation and conf change

2. activated receptor binds to trimeric G protein (alpha subunit)

3. GPCR GEF activity stimulate Ga to release GDP

4. GTP binding changes Ga conf and activates

5. Gby dissociates from Ga

6. Ga-GTP activates effector enzyme

7. Ga GTPase activity: GTP → GDP

8. Ga dissociates from effector enzyme (deactivates)

Adenylyl cyclase + ATP → ? → activates ?

cAMP → activates protein kinase A (PKA)

Guanylyl cyclase + GTP → ? → activates ?

cGMP → activates protein kinase G (PKG) and cation channels

phospholipase C + PIP2 → ?? → activates ??

DAG → +Ca2+, activates protein kinase C (PKC)
IP3 → opens Ca2+ channels in ER

How does cAMP active PKA?

cAMP releases inhibitory subunits

-cAMP: bind and inhibit catalytic subunit

+cAMP: release catalytic subunit

What does PKA do?

has two catalytic kinase subunits that catalyze phosphorylation of target specific Ser/Thr

Mechanism for how PKA controls gene expression through CREB

1. GPCR activates Adenylyl cyclase, which produces cAMP

2. cAMP activates PKA

3. PKA catalytic subunits move to nucleus

4. PKA phosphorylates CREB transcription factor

5. CREB forms complex with co-activator CBP/P300, stimulates transcription of genes controlled by CRE (CREB responsive element)

Mechanism for IP3/DAG pathway

1. GPCR activation activates PLC

2. PLC cleaves PIP2 → IP3 + DAG

3. IP3 diffuses through cytosol and opens Ca2+ ER channel

4. Ca2+ ions released, binds PKC, activation by DAG on PM

5. activated PKC leaves membrane to phosphorylate subcellular targets

How does arrestin inactivate GPCR via endocytosis?

longer the signal, the more phosphorylation of GPCR. phosphorylated receptor binds arrestin, and AP2 is recruited for clatherin coated endocytosis. either dephosphorylated or degraded

Mechanism for receptor tyrosine kinases (RTK)

1. + ligand → RTK dimerizes

2. cytosolic domain kinases phosphorylate each other on Tyr

3. phosphorylated Tyr residues provide docking site for SH2 and other proteins

What does RTK do?

activate small GTPase Ras via adapter proteins by binding SH2 domain of GRB2, which binds to SH3, which binds to SOS, which has GEF activity towards Ras, forming active Ras-GTP

Mechanism for MAP Kinase cascade

1. after Ras is activated, Raf binds to Ras-GTP

2. Raf releases its 14-3-3 protein

3. Ras GTP hydrolysis to Ras-GDP releases active Raf

4. Raf phosphorylates and activates MEK

5. MEK phosphorylates MAP kinase on Tyr

6. MAP kinase phosphorylates transcription factors and others

Mechanism for induction of gene transcription by MAP kinase

1-3. dimeric MAP kinase phosphorylates p90RSK, which moves into nucleus, phosphorylating SRF
4-5. MAP kinase moves to nucleus, phosphorylates TCF
6. phosphorylated TCF, SRF stimulate transcription of c-fos and other genes

Hydrophobic signaling molecules require:
receptor at PM, receptor in cytosol, signal transduction mol, amplification via second messengers

receptor in cytosol

Cleavage of PIP2 gives rise to which second messenger?
cAMP, IP3, cGMP, IP4

IP3

Which subunit of trimeric G proteins bind GDP/GTP? alpha, beta, or gamma?

alpha

Activation of PKC requires: cAMP, cGMP, K+, Ca2+?

Ca2+

What are membraneless compartments?

• coherent assembly of biological macromolecules

• lack membrane

• round/spherical shape (surface tension)

What phase is membraneless compartments most like?

liquid

Properties of liquids

short range positional order, high mobility

Properties of solids

long range positional order, molecules trapped, doesn’t rearrange

What % of all proteins have intrinsically disordered regions?

50%

Multivalent scaffolding proteins can undergo what process?

liquid-liquid phase separation

What is the implications of membraneless compartments?

1. reaction specificity: concentration of reactants inside condensates can inc rxn kinetics

2. reaction kinetics: increased viscosity can slow rxn kinetics

3. reaction inhibition by sequestration: prevent rxns

4. concentration buffer: buffer for molecules in bulk phase

Without energy input, condensates will?

harden out, turn into solid. to pry apart, use ATP dependent machinery (chaperone, disaggregases)

What are stress granules and their function?

ribonucleoprotein that is phase separated, membraneless that is formed due to stress on cell. they promote cell survival by condensing translocationally stalled mRNA, RBP, ribosomal components etc

Maturation of stress granule

1. SSRNA is released

2. dephosphorylated Ras GTPase-activating PBP1/2 (G3BP1/2) associates w/ released RNA and assembles into liquid condesates

3. caprin 1 and other RBP promote maturation of stress granule

Which factors impact the critical/saturation concentration of LLPS? PTM, chemical environment, time, all of the above

all of the above

What residues does SH2 on Grb2 recognize?

phospho-tyrosine residues

What residues does SH3 on Grb2 recognize?

poly-proline residues

What are the 3 distinct zones within nucleoli?

FC (fibrillar center): transcription of rDNA

DFC (dense fibrillar center): rRNA processing

GC (granular components): ribosome assembly, storage for unfolded proteins

What’s the mechanism to get through the nuclear pore complex (NPC) for proteins > 40kDa and <40kDa?

<40kDa: diffusion

>40kDa: nuclear localization/nuclear export signal or nuclear transport receptor

What fills the nucleoporin?

FG-repeat gel-like condensate that allows diffusion of small molecules

Mechanism for nuclear import

1. importin binds to NLS of cargo protein

2. importin-cargo diffuses thru NPC (work w/ FG-nucleoproins)

3. Ran-GDP releases GDP, binds GTP

4. Ran-GTP binds to importin, releases cargo

5. importin-Ran-GTP complex transported to cytoplasm

6. GTP hydrolysis, Ran-GDP conf change releases importin

GEF in nucleus, GAP in cytoplasm

Mechanism for Ran-dependent nuclear export

1. exportin 1 binds to NES-cargo protein and Ran-GTP

2. complex diffuses through NPC

3. Ran-GTP → Ran-GDP conformational change releases NES cargo into cytosol

4. exportin 1 and Ran-GDP transported back into nucleus

What kind of amino acids are NES (nuclear export signals) rich in?

hydrophobic

Mechanism for Ran-indepdendent nuclear export

1. NXF1/NXT1 nuclear export receptor complex binds to mRNA protein complex

2. complex diffuses through NPC by interactions with FG

3. RNA helicase (Dbp5) located on cytoplasmic side uses ATP to remove NXF1/NXT1

4. NXF1/NXT diffuse back into nucleus

What % of human DNA encodes proteins? What does the rest do?

1.5%, the rest are regulatory sequences that control gene expression

What does the SMC complex do?

it clamps chromatin strands together

What structure stabilizes the nucleoporins

Y-complex

Is the concentration of Ran-GDP is higher in the cytosol or nucleoplasm?

cytosol, because more GAP in cytoplasmic filaments

Is nuclear localization signal (NLS) acidic, basic, hydrophobic?

basic

Mechanism for >500kDa proteins to mitochondrial matrix

1. amphipathic N-terminal targeting seq target proteins to mito matrix

2. Tom20/22 outer membrane import receptor binds to seq

3. target signal inserted into translocon Tom40

4. protein moves through Tom40 and inserts into inner membrane translocon Tim

5. protein binds to matrix Hsp70 to “pull” peptide through

6. Hsp70 ATP hydrolysis releases protein

7. protein folds

Mechanism of targeting proteins to peroxisomes

1. peroxisomal targeting seq PTS1 on C-term: S-K-L binds Pex5

2. complex binds to Pex14r in membrane

3. matrix protein dissociates into peroxsomal matrix

4. ubiquitinylation of Pex5 by Pex2/10/12 complex

5. ATP-dependent removal of Pex5 by Pex1/Pex6