Cell Exam 1

Transformative medicine

created from cell biology breakthroughs, useful in diagnosis and treatment of human disease

How long did eukaryotes take?

~1850 million years

Why are eukaryotes so complicated compared to prokaryotes?

• extensive compartmentalization

• multicellular requires better cell-cell communication

• limited metabolic flexibility and environmental adaptability

• primarily adapted for specialized functions

Consequences of compartmentalization?

• typical eukaryotic cells can synthesize ~10,000 protein types with 30 destinations

• organelles have distinct functions due to unique protein components

• organelles need to communicate

• specific amino acids target proteins to their destiination

• correct localization requires proper folding

• misfolded and mislocalized proteins cause numerous genetic diseaeses

Molecular chaperones

aid in folding of proteins

Conservative substitutions

less deleterious as the original amino acid is replaced with a functional equivalent

Nonconservative substitutions

more deleterious as the amino acid is replaced with a non-functional equivalent

What extrinsic factors can cause protein misfolding?

Heat, reactive oxygen

Most common cause of pathogenic protein misfolding

missense mutations

What are the other two less common causes of pathogenic protein misfolding?

Nonsense mutations and deletions

Some missense mutations may result in

gain of function (oncogenes) or loss of function

Why do organelles have distinct calcium environments?

Calcium acts as a signaling molecule, when increased it can change the activity of the cell

How does calcium enter the cell?

Open channel in membrane, driven by thermodynamics as calcium is going down a gradient (-G), but getting it out is much harder

How does calcium go against its gradient to exit the cell?

An ATP pump

—% of ATP in cells is consumed by the sodium-potassium ATPase, which pumps - sodium - and - potassium -

30%, 3 out, 2 in

Why do organelles have distinct pH environments?

Some carrier proteins bind in certain pH and release in others, pumps to move protons

How many steps could it have taken to go from unicellular to multicellular?

5 steps (Chlamydomonas to Volvox)

What mutations in yeast make multicellularity possible?

ACE2 for septum destruction after cytokinesis, CLB2 & CLN3 prevents cells from getting too large before they divide, GIN4 required for bud growth and assembly of septin ring

Multicellularity is how many years in the making?

~1200 million years

Why is multicellularity an interesting feat for cells?

Give up independence for specialized functions and curtail reproduction for the greater good

How does ancestral gene regulatory networks drive cancer?

Regression of cells to be selfish and reproduction focused

What is the atavistic theory of cancer?

Origin of cancer could be found in the early transitional phase form unicellular to multicellular, cells have reactivated cell proliferation genes from unicellularity that are normally controlled

Why are different model organisms used?

• different but complementary approaches

• choose context and experimental approach

• application to human biology through conserved proportions of protein-encoding genes

Why are model organisms applicable to humans?

• proportion of protein-encoding genes required for cellular processes is evolutionarily conserved

• biochemical functions of shared proteins are conserved and largely but not always interchangeable

Why do multicellular organisms have so many more genes?

• roughly half of protein-coding genes have a known function

• multiple genes that carry out the same function for specialized functions and needs in human cells

• insert genes from certain models to test if it functions in other models

How do human orthologs work in yeast genomes?

insertion revealed half could be humanized and still function

Replaceable genes are mostly

throw-back or ancestral unicellular genes whose expression is normally upregulated in cancer cells, while more sophisticated options are not replaceable

Some of the most replaceable genes effect

metabolism

Least replaceable genes affect

replication and repair, cell growth and death

programmed cell death

regulated killing of cells that eliminates damaged or unneeded cells from the body

Plasma membrane is?

• multifunctional boundary

• contains proteins, lipids, carbs and RNA

What does the plasma membrane do (7)?

• selective barrier and transport conduit

• dynamic sensor for environment

• enables communication

• provides a scaffold for intracellular signaling

• interacts with cytoskeleton

• dynamic during cell migration and cell division

• final destination for most proteins that move through the secretory pathway

Phospholipid bilayer determines

fluidity, thickness, curvature, permeability

Fluidity

measure of solid/liquid, determined by phospholipids and cholesterol

Membrane thickness

determined by FA chain lengths and size of polar heads, typical bilayer ~4nm (2 from tails and 1 from each head)

Unsaturated FA

have a double bond which creates a bend, reducing compactness and increasing fluidity

Saturated FA

have increased van de Waals and decreased fluidity/increased thickness from compactness

Cholesterol

must abundant sterol in membrane, amphipathic, influences thickness and fluidity

At low levels of cholesterol,

cholesterol disperses and the planar sterol ring intercalates between the hydrocarbon chains which reduces van Der Waals interactions and increases fluidity

At normal physiological amounts, cholesterol

aggregates into lipid rafts, the short tail forms van Der Waals interactions with fatty acids, reducing fluidity and increasing thickness

Integral membrane proteins

one or more membrane-spanning alpha-helices

Lipid anchored proteins

covalently attaches to the lipid that inserts into one leaflet, mainly face cytosol, involved in cell signaling

Peripheral proteins

interact noncovalently with membrane spanning or lipid anchored proteins

Alpha helix

carbonyl oxygen of each peptide bond is H bonded to the amide acid 4 residues towards the C terminus

Why can proline never be in an alpha helix?

it has a built in bend that makes it a helix breaker

Most membrane spanning alpha helices are comprised of

amino acids with hydrophobic R groups

Amphipathic alpha helices

Alternating pairs of hydrophobic and hydrophilic R groups, essential to create channels and transporters

Alpha helices minimally have - amino acids but typically have

16, 20

Channels made of alpha helices

arranged in cylinder for potassium or glucose with hydrophobic R groups

Glycophorin A

• single pass membrane spanning protein

• certain S, T, N on extracellular domain are glycosylated

• positively charged K and R stabilize association of GYPA with PPM by electrostatic interactions with phosphate heads

GYPs are

• most abundant integral proteins in the erythrocyte PM

• attach the PM to the cytoskeleton through adapter proteins including Spectrin

• receptors for the Plasmodium parasites that cause malaria

• homodimers within PM due to adjacent alpha helices

A mutant GYPB confers

malaria resistance

How does mutant GYPB work?

a chromosomal rearrangement replaces GYPB with a GYPA/B fusion that has a lower binding affinity to the parasite, which uses a lock and key mechanism

• one copy - 40%

• two copies - 70%

• Dantu region

Simple or passive diffusion is

limited across a phospholipid bilayer

Rate-limiting step is

movement of polar molecules into the hydrophobic interior of the bilayer

Receptor-mediated endocytosis

Import of proteins, lipoproteins, reactive ions all require cell-surface receptors

The two major functions of integral membrane proteins

• enable transport of polar molecules, H2O and ions

• enable transport of molecules against concentration gradients

Membrane is permeable to - and semipermeable to -

gases and ethanol, water

Channels

only transport H2O or ions down gradients, often regulated or gated (facilitated transport)

Transporters

uniporters, antiporters and symporters

ATP powered pump

transport against electrochemical gradients directly coupled to ATP hydrolysis (active transport)

Speed of transport mechanisms, fastest to slowest

channels, transporters, ATP-powered pumps

Uniporter

one molecule moves down a gradient, ex. GLUT1

Symporter

two different molecules move in the same direction

Antiporters

2 different molecules move in opposite directions

Symporters and antiporters are what type of transport?

Secondary active transport

SLC transporters are associated with

human disease

GLUT1 Transporter mutations

result in compromised transport activities for glucose, are associated with disease as a result of lack of energy supply to the brain and cancers

Km is the measure of

binding affinity for half-maximal transport, lower = better

Vmax

maximal rate of transport, higher = better

GLUT transporters have

12 amphipathic membrane spanning alpha helices arranged cylindrically to form a hydrophilic conduit that undergo conformational change each transport cycle

Rapid conversion of glucose to glucose-6-phosphate

keeps glucose levels low

Mutations in the glucose binding site of GLUT1

increase Km and are associated with the GLUT1 deficiency syndrome

GLUT1 binding

strictly 6-7 hydrogen bond, not all conservative mutations would still work, depending on conformation

Can GLUT1 move other hexoses?

Yes, but with increased Km as other sugars don’t fit as well as glucose so there are fewer hydrogen bonds

GLUT5 transporters

on apical membrane of intestinal epithelial cell has a higher affinity for fructose, certain leukemic cells use GLUT5 to preferentially import fructose

Electrochemical gradients are present when

electrical gradient is the same direction as chemical/different

Gc

free energy due to chemical concentration gradient

Gm

free energy due to membrane potential

Gt

Gc +Gm

Artificial cells with a solid phospholipid bilayer cannot

move sodium and potassium

If the artificial cell has sodium channels

only sodium is moving, so the cell will develop charge separation → membrane potential developed

When enough sodium enters the artificial cell

the concentrations tarts repelling sodium from coming in and the negative charge outside starts slowly pulling them out, so the cell is in equilibrium

Resting membrane potential is typically

-70 mV primarily by K+ export of non-neurons

What helps maintain resting membrane channels?

resting potassium channels used to maintain as sodium comes into the cell down the gradient

Cells use energy of sodium import to

couple it with another molecule moving against its gradient