unit 3 biochem

types of biosignals

autocrine, paracrine, endocrine

features of biosignaling systems

specificity, amplification, desensitization

major types of biosignaling systems

GPCR, RTK and kinase cascades, gated ion channels, nuclear receptor

life must be coordinated and homeostasis maintained

metabolism, growth, development, and reproduction

signal transduction/cell signaling/biosignaling

transmission of molecular signals from the environment (not membrane permeable) to the interior of a cell

signals cause changes in a cell’s composition and function

differentiation and antibody production

growth in size, shape, or strength

gene expression

ability to divide (sexual vs asexual division)

autocrine signaling

acting on same cell that produces them

paracrine signaling

acting on nearby cell

endocrine signaling

carried in bloodstream from producer cell to distant target cell

essential steps of cell signaling

signal reception, signal transduction, cellular response

signal reception

the signal must bind to a specific cellular protein called a receptor, which is often located on the surface of the cell

signal transduction

binding of the signal changes the receptor in some way, usually in conformation or shape.

the change in the receptor initiates a process converting the signal into a specific cellular response. the conversion may involve many steps

cellular response

the transduction system triggers a specific cellular response, such as the activation of an enzyme or altered gene expression

receptor

a membrane bound or soluble protein or protein complex which exerts a physiological effect after binding its natural ligand

types of receptors and an example

GPCR- epinephrine

enzyme linked- insulin

ligand gated ion channels- nicotinic acetylcholine

nuclear receptors- steroid receptor, not membrane bound

other membrane receptors- integrin receptor (crawling)

human genome has > GPCRs

-detect hormones, growth, as well as the bulk of smell and taste receptors

-20% of all cancers have a mutation in one of these genes

- >700 drugs approved by FDA that target GPCRs

- ~25% of new marketed drugs each year target a GPCR

eight features of signal-transducing systems

-specificity

-sensitivity

-amplification

-modularity

-desensitization/adaptation

-integration

-divergence

-localized response

specificity

signaling ligand fits binding site on its complementary receptor; other ligands do not fit

sensitivity

receptor has high affinity (low Ka) for signaling ligand (L)

amplification

when enzymes activate enzymes, the number of affected molecules increases geometrically in an enzyme cascade

use of enzymes (act on many targets) and multiple steps allows rapid amplification

modularity

proteins with multivalent affinities form diverse signaling complexes from interchangeable parts. phosphorylation provides reversible points of interaction

individual steps can act on multiple targets

desensitization/adaptation

receptor activation triggers a feedback circuit that shuts off the receptor or removes it from the cell surface

integration

when two signals have opposite effects on a metabolic characteristic such as the concentration of a second messenger X, or the membrane potential Vm, the regulatory outcome results from the integrated input from both receptors

divergence

when a receptor is activated by a signal, it activated two or more pathways with different end effects

localized response

when the enzyme that destroys an intracellular message is clustered with the message producer, the message is degraded before it can diffuse to distant points, so the response is only local and brief

typical ligands

small ions, organic molecules, polysaccharides, peptides, proteins

additional factors/concerns for ligand binding

weak interactions, is receptor cell-specific or found on all cells, high affinity of receptors for signal (Kd 10^-7 or less), coopertivity and clustering (lipid rafts + avidity)

avidity

play on affinity

did you hold on long enough to activate machinery?

• dont know, but once released, someone nearby can grab and also start to recruit

sum individual affinities for greater pictures

clustering makes higher chance of illiciting response

GPCRs

7-pass alpha helical integral membrane proteins

heterotrimic: alpha beta gamma

bind guanine nucleotides (GTP or GDP)

mediate signal transduction from GPCRs to other target proteins

primarily, signals are a stimulatory (Gs) or inhibitory (Gi) response

GPCR case study: epinephrine

hormone is made in adrenal glands

mediates stress response

mobilization of energy:

• binding to receptors in muscle or liver cells induces breakdown of glycogen

• binding to receptors in adipose cells induces lipid hydrolysis

  • binding to receptors in heart cells increases heart rate

steps in GPCRs sensing epinephrine

-epinephrine binds to its specific beta-adrenergic receptor

-allosteric change in hormone-receptor complex causes the GDP bound to Gsalpha to be replaced by GTP, activating Gsalpha

-activated Gsalpha separates from Gsbetagamma, moves to adenylyl cyclase, and activates it. many Gsalpha subunits may be activated by one occupied receptor

-adenylyl cyclase catalyzes the formation of cAMP

-cAMP activates PKA

-phosphorylation of cellular proteins by PKA causes the cellular response to epinephrine

-cAMP is degraded, reversing the activation of PKA

trimeric G protein dissociation leads to interactions with effectors

effector = adenylyl cyclase

cyclic nucleotide phosphodiesterase

breaks bond to make cAMP → AMP

synthesis of cAMP contributes to rapid amplification

cAMP is common secondary messenger

a large number of GPCRs mediate their effects via cAMP

allosterically activates cAMP-dependent protein kinase A (PKA)

PKA activation leads to activation of enzymes that produce glucose from glycogen

generally, protein kinases are enzymes that catalyze the phosphorylation of

ser, thr, or tyr hydroxyl groups in target proteins

what does target selection depend on

the presence of an amino acid sequence within the target protein that can be recognized by the kinase

importance of anchoring/scaffold proteins

cAMP is able to mediate multiple signals due to localization of PKA

PKA is localized to particular structures by an anchoring protein

different anchors are expressed in different cell types to determine the downstream affect of cAMP

AKAP5

anchoring/scaffolding protein

can change depending on cell type

not an enzyme

yay for rapid amplification!

signal amplification in GPCRs

activation of a few GPCRs leads to the activation of few adenylyl cyclase enzymes

each active adenylyl cyclase enzyme makes many cAMP molecules quickly, thus activating several PKA enzymes

these activate thousands of glycogen-degrading enzymes in the liver tissue

at the end, tens of thousands of glucose molecules are released to the bloodstream

self-inactivation of GPCRs

down regulation of cAMP levels occurs by the hydrolysis of GTP in the subunit of the G protein

• inherent GTPase activity to alpha

cyclic nucleotide phosphodiesterase

• gets rid of cAMP

GEFs and GAPs

guanine exchange factor (GEF)- help exchange GDP for a new GTP

GTPase activating proteins (GAPs)- increase the rate of GTP hydrolysis

the beta adrenergic receptor is an example of a GEF

desensitization to turn of GPCR signaling

binding of epi to beta-adrenergic receptor triggers dissociation of Gsbetagamma from Gsalpha

Gsbetagamma recruits betaARK to the membrane where it phosphorylates ser residues at the carboxyl terminus of the receptor

beta-arrestin (beta arr) binds to the phosphorylated carboxyl-terminal domain of the receptor

receptor-arrestin complex enters the cell by endocytosis

in endocytic vesicle, arrestin dissociates; receptor is dephosphorylated and returned to cell surface

beta ARK

adds phosphate to GPCR

changes shape and charge

creates new binding site

beta arrestin can bind

beta arrestin

helps initiates endocytosis and once binds receptor, blocks more G protein activation

agonist

structural analogs that bind to a receptor and mimic the effect of the natural ligand

want relatively low Kd, not super important

antagonist

structural analogs that bind to a receptor without triggering the natural effect

need low Kd

activated G proteins can transduce a variety of cellular effects via “effectors”

adenylyl cyclase- cAMP

phospholipases- DAG and IP3 (lipid second messengers)

activation of Ca2+ and K+ channels leading to an increase in these ions in the cytosol

enzyme linked membrane receptors

most consist of: extracellular ligand-binding domain, intracellular catalytic domain

when ligands bind to two receptor polypeptides, the polypeptides aggregate, forming a dimer

this activated the tyrosine-kinase activity of both monomers

these add phosphates to the tyrosine tails of the other polypeptide

note: single ligand, or two, can be needed. RTKs, but in some receptors it isn’t alw

the most common catalytic domains have tyrosine kinase activity (RTKs)

intrinsic kinase activity is low

ligand binding and receptor conformation changes increases activity and possible substrates

cross/autophosphorylate (P comes from ATP) leads more conformational changes allowing binding and catalytic phosphorylation of specific target proteins

recruit scaffold, or target proteins (adds a phosphate group to specific tyrosine residues)

fully activated receptor proteins allow

multi docking proteins that bind to specific phosphorylated tyrosine molecules on the receptor

one tyrosine kinase receptor dimer may activate ten or more different intracellular proteins simultaneously

IRS1

insulin receptor substrate 1

binds one of the phosphorylated sites on receptor on its own

it itself can then by phosphorylated and recruit additional proteins

conserved protein fold domains

important for scaffolding, and docking, during RTK signaling

domain that specifically looks for phosphorylated tyrosines

PTB domain (phospho tyrosine binding)

the phosphotyrosines of activated RTKs bind PTB domains as well as

SH2 domains

SH2 domains in proteins that couple activated RTKs to downstream components of signal transduction pathways

RTK example: insulin

insulin is a peptide hormone produced in pancreas

insulin is produced and released from the pancreas in response to nutrients such as glucose

insulin reaches target cells, such as liver, muscle, or fat tissue cells via bloodstream

binding of insulin to the insulin receptor initiates a cascade of events that leads to increased glucose uptake and metabolism

binding pocket of RTK

inactive: negatively charged AA in binding pocket, activation loop blocks substrate-binding site

active: triply phosphorylated activation loop moves dramatically to make room for target protein, exposed phosphorylated tyrosines

once phosphorylated, IRS-1 becomes

the point of nucleation for a complex of proteins that carry the message from the insulin receptor to end targets in the cytosol and nucleus through a long series of intermediate proteins

the phosphotyrosines of IRS-1 serve as

docking sites for SH2-containing signaling proteins

SH2 domains have affinity for

phosphorylated tyrosine residues

Grb2

adaptor protein that contains an SH2 domain, and therefore, binds to IRS-1

Grb2 brings together IRS-1 and Sos

Sos binds

Ras and promotes GTP exchange

Sos is a GEF

Ras is a monomeric G protein

the insulin signaling cascade

insulin receptor binds insulin and undergoes autophosphorylation on its carboxyl-terminal Tyr residues

insulin receptor phosphorylates IRS1 on its Tyr residues

SH2 domain of Grb2 binds to P-Tyr of IRS1. Sos binds Grb2, then to Ras causing GDP release and GTP binding to Ras

activated Ras binds and activates Raf1

Raf1 phosphorylates MEK on two Ser residues, activating it. MEK phosphorylates ERK on a Thr and a Tyr residue, activating it

ERK moves into the nucleus and phosphorylates nuclear transcription factors such as Elk1, activating them

phosphorylated Elk1 joins SRF to stimulate the transcription and translation of a set of genes needed for cell division

simple insulin signaling cascade

insulin binds, phosphorylation, IRS1 phosphorylation, Grb2, Sos, Ras, Raf, MEK, ERK, phosphorylates TF for GLUT4

more immediate glucose import

glucose transporters “stored” within cell in membrane vesicles

when insulin interacts with its receptor, vesicles move to surface and fuse with the PM, increasing number of glucose transporters in the PM

when insulin level drops, glucose transporters are removed from the PM by endocytosis, forming small vesicles

the smaller vesicles fuse with larger endosome

patches of the endosome enriched with glucose transporters bud off to become small vesicles, ready to return to the surface when insulin levels rise again

insulin _____ the metabolic effects of epinephrine in most tissues

opposes

dampens signaling through betaAR system

specifically, the insulin receptor kinase phosphorylates two Tyr residues in the cytoplasmic tail of a betaAR, resulting in the internalization of the receptor, lowering cells sensitivity to epinephrine

nicotinic acetylcholine receptor

receptor = channel

5 proteins, 2 are alpha

subunits fold into four transmembrane alpha helices, M2 amphipathic helices surround channel

cooperative binding of acetylcholine

bulky hydrophobic Leu side chains of M2 helices close the channel

binding of two acetylcholine molecules causes twisting of M2 helices

M2 helices now have smaller, polar residues lining the channel

steroid receptors

hormone diffuses across plasma membrane, binds specific receptor protein in the nucleus

hormone binding changes the conformation of the receptor. forms homo or heterodimers with other hormone receptor complexes and binds to specific regulatory regions called hormone response elements (HREs) in the DNA adjacent to specific genes

receptor attracts coactivator or corepressor proteins, and, with them, regulates transcription of the adjacent genes, increasing or decreasing the rate of mRNA formation

altered levels of the hormone-regulated gene product produce the cellular response to the hormone

tumor suppressor gene

encode proteins that normally restrain cell division

oncogenes

cancer causing genes. mutant genes that cause cells to exhibit rapid. uncontrollable proliferation

proto oncogenes

a normal gene that can become an oncogene due to mutations or increased expression. code for proteins that help to regulate cell growth and differentiation

examples of apoptosis

development of embryo (fingers)

anti-self antibodies present

stressed cells

some cancers result from mutations in these paths

caspases involved in apoptosis

caspase 8, caspase 9, caspase 3, 6, 7

carbohydrates in food are important sources of energy

plant starch consists of chains of linked glucose molecules. these chains are broken down into individual glucose molecules for eventual generation of ATP and precursors for other molecules

carbohydrates in cell walls and as protective coating in shells

proteoglycan, chitin

carbs as marker molecules

oligosaccharides for cell-cell recognition

what biological molecules are carbs precursors for

amino acids, lipids, purines, pyrimidines

where is more than half of all organic carbon found

carbs

general formula for carbs

C_n(H₂O)_n

what is sweetness

subjective interaction between a chemical and taste receptor in your mouth

encoded by two genes called TAS1R2 and TAS1R3 which encode for GPCRs

what ligands need to bind both TAS1R2 AND TAS1R3

glucose, sucrose, fructose

what ligands only need to bind TAS1R2 OR TAS1R3

alitame, neotame, aspartame, sucralose

what gets recruited once ligand binds TAS1R2 and 3

brazzein

aldose

carbonyl carbon is on end (aldehyde)

if there are at least three carbons, you have a chiral carbon

ketose

carbonyl carbon is not on end (ketone)

need at least four carbons to have a chiral carbon

monosaccharides

aldose and ketose

have one carbonyl group

have one or more hydroxyl groups

colorless, crystalline

nomenclature based on number of carbons; triose, tetrose, pentose, hexose

stereoisomers

same composition (MW) but different arrangements

enantiomers and diastereoisomers

enantiomers

non superimposable mirror images

e.g. D and L conformations

diastereoisomers

isomers that are not mirror images e.g. D-altrose and D-glucose

epimers and anomers

epimers

diastereoisomers that differ at one of several asymmetric carbon atoms

e.g. D-glucose and D-mannose

anomers

diastereoisomers that differ at a new asymmetric carbon atom formed on ring closure

e.g. alpha-D-glucose and beta-D-glucose

how to assign D or L in enantiomers

refers to the configuration of the highest numbered chiral carbon

aldehyde is C1, proceed from there

if carbonyl carbon is not at end, C1 is C closest to it

OH on highest number chiral C faces right? D form. faces left? L form.

what configuration do natural sugars have

D

diastereoisomers have different

physical properties

glucose

six carbon aldehyde containing sugar (aldohexose)

primary fuel for living cells

exclusive energy source for brain cells, erythrocytes, and cells of the eye

dietary sources include plant starch and the disaccharides lactose, maltose, and sucrose

fructose

fruit sugar

ketose sugar

twice as sweet as sucrose and is often used in food products in smaller amounts. produces the same sweetening effect with fewer calories (high fructose corn syrup)

why is lots of high fructose corn syrup bad

our bodies can’t process lots and lots of fructose

can lead to fatty acid deposits on liver

galactose

necessary to synthesize a variety of biomolecules

• lactose

• glycolipids/phospholipids

• proteoglycans/glycoproteins

galactosemia

genetic disorder in which an enzyme required to metabolize galactose is missing. accumulation causes liver damage, cataracts, and severe brain damage during development

fisher projection

straight line

Haworth projection

cyclic