Vocab/Major Concepts Exam 1

functional outputs of gene that expresses trait:

protein

steps to get functional output of gene

1) transcription (DNA → mRNA copy)

2) mRNA processing (5’ cap, poly-A tail, splice out introns)

3) mRNA from nucleus → cytoplasm

4) translation (mRNA → aa chain)

5) polypeptide folding and transport to cellular destination

3 parts making up amino acids

amino group, carboxyl group, R group/side chain (determines which aa)

non-covalent factors contributing to tertiary structure of protein

hydrogen bonding, ionic bonds, van der waals (dipole-dipole and london dispersion forces), hydrophobic interactions

Anfinson, Sela, White 1957 experiment showed… general steps

showed that amino acids are all that is required to yield functional, folded proteins

1) pure RNase enzyme was treated with heat (breaks non-covalent bonds) and beta-mercaptoethanol (breaks disulfide bonds) to denature

2) conditions reversed (regular heat, beta-mercaptoethanol removed via dialysis bag)

3) enzyme activity tested to see if protein had refolded → activity was present therefore protein had refolded in the presence of nothing but its aa sequence

primary structure of protein

its amino acid sequence

secondary structure of protein

folded structure of backbone but not r-groups/residues, typically alpha helices and beta sheets

tertiary structure of protein

overall 3D structure of protein, including r groups and secondary structures

quaternary structure of protein

interaction of multiple polypeptide chain subunits

why are alpha helices and beta sheets so common (found in almost all proteins)

they depend on amino/carbonyl interactions, which are present in every amino acid

both are ways to ‘hide’ hydrophobic groups inside protein → hydrophobic interactions

chaperone proteins, why they’re needed

proteins that bind directly to polypeptides as they come off the ribosome to prevent them from forming incorrect noncovalent bonds with surrounding ‘mosh pit’ of distractions in the cell → brings nascent polypeptides to isolated environment for folding

they act as catalysts → chaperone information helps polypeptide folding, but its information does not at all contribute to how the protein will form (since that’s based on aas)

chaperonins

isolated environments for protein folding - chamber made of 2 stacked protein rings (subunits)

chaperones drop nascent polypeptides off here so they can fold without distraction/incorrect interactions

common chaperone family

HSP70, binds to hydrophobic stretches of nascent polypeptides and transfers to chaperonins

basis of many neurodegenerative diseases

misfolded proteins that aggregate

Alzheimer’s associated with…

amyloid plaques, tau tangles

amyloid plaques

contribute to alzheimer’s

alzheimer’s precursor protein (APP) proteolytic product is Abeta42 cleavage fragment. These fragments aggregate and create amyloid plaques.

tau tangles

tau is a microtubule protein that stabilizes motor proteins that ‘walk’ along microtubules; when they misfold and aggregate, we get tau tangles

what are prion diseases, some examples

proteins, not viruses, act as an infectious agent: misfolded proteins can bind to and induce misfolding in native (correct) versions of that prion protein - allows transmission between members of species too

examples - mad cow, Cretzfeldt-Jakob

what causes cystic fibrosis

autosomal recessive mutation in CFTR gene, which codes for Cl- channels in lungs. mutations typically occur in phe58, resulting in loss of a chaperone binding site. this mutation reduces Cl- channels in lungs so mucus builds up in lungs and lung function is lost.

proteolytic cleavage

translation may yield inactive form of a protein (for instance, if its function is unwanted in location of translation → proteases in ER, for example) and proteases cleave polypeptide into its final functional form

glycosylation, main kinds

addition of carbohydrates to proteins to form glycoproteins

N-linked: N to Asn

O-linked: O to Ser or Thr

glycolipids

lipids linked to oligosaccharides

glycosylation with glycolipids

mainly done to localize proteins in membrane. glycolipids can be added to proteins’ c-terminus. then attached to membrance by GPI anchors, leading to membrane localization

GPI anchors

glycosylphosphatidylinosital → contains similar phosphatidylinosital group as oligosaccharide of glycolipid attached to a protein. These two will associate and GPI answer (glycosylphos…) brings to membrane.

intrinsically disordered region

area of protein with both stable and unstable secondary/tertiary structures; variable regions. usually polar region (more interactions with itself/other proteins) and lackes hydrophobic core (these provide stability)

intrinsically disordered proteins

protein whose entire secondary/tertiary structure is dynamic → can act as a ‘key’ to several ‘locks’

liquid-liquid phase separation, LLPS

IDRs of several proteins interact and separate from surrounding liquid environment into a more concentrated but still liquid phase, often called a biomolecular condensate → compartment with specific proteins, RNAs, etc

how do fluorescent molecules function/fluoresce

fluorophore is excited by photon. an electron goes from low → high energy, emitting light. the electron will spontaneously return to (near) ground state, emitting a light with a longer wavelength (lower energy), which can be visualized/measured

how do fluorescent microscopes work

light goes through a ‘filter’ to select absorption wavelength of sample (to excite it). a dichroic mirror reflects the light onto the specimen. emitted light from the specimen goes back through the dichroic mirror at a different wavelength, which can be visualized with an eyepiece or computer. will appear as a black background, and light appears where fluorescent molecule is present.

enzymes

molecules, often proteins, that function as catalysts to reduce activation energy and speed up reactions without being used up by the reaction

how enzymes work, generally

may bind 2 substrates together at active site in correct position/orientation to react so molecules don’t have to ‘float around’ until they happen to find the correct reaction position

AND/OR

induced fit → substrate binding distorts substrate and enzyme conformations so they’re closer to the transition state conformation

AND/OR

stabilize high energy intermediate of reaction (often with antibodies, bind to antigens)

ligands, receptors

ligands = substrates acted upon, converted to product

(one type of) receptors = enzymes, things that encourage ligand → product

Kd, dissociation constant

concentration of ligand at which 50% of receptors are occupied at equilibrium, allows us to predict which interactions are taking place in a cell given concentrations of ligand/receptor. a lower Kd indicates stronger affinity for ligand for receptor.

Kd = [R][L] / [RL]

fraction occupancy (theta)

gives amount of receptors with bound ligand at a given ligand concentration

theta = 1 / (1+ Kd/[L])

Tau paper → general question being addressed based on background information

in Alzheimer’s brains, tau tangles have been observed in the entorhinal cortex (EC) and extra-hippocampal areas. the question is whether the tau originates in the EC then spreads transynaptically to other regions, or if tau is appearing via independent mechanisms in these other regions.

Tau paper → what was the main experiment used?

transgenic mice were created that expressed (misfolded/Alz) human tau in the EC only, and fluorescent imaging/dyes were used to visualize the location of tau in mice after disease progression in young (10 m/o) and old (22 m/o) mice. dyes used were MC1, which shows abnormal conformation of human tau and cp27, which shows human specific tau and antibody (normal)

the transgenic mice were a cross between neuropsin-tTA mice and TauTg mice. The neuropsin-tTa mice express the transcription factor tTa in the entorhinal cortex. Tau Tg mice express human tau if induced, but this induction requires tTa. A cross of the mice produces about ¼ progeny expression both tTa transcription factor and tau in the EC, thus producing mice with tau in EC only.

Tau paper → where was tau spread seen in young mice? Regions of brain and part of neurons?

MC1 in EC, DG acons, CA3 axons, CA1 axons

Cp27 in EC, DG axons

Tau paper → where was tau spread seen in old mice? Regions of brain and part of neurons?

MC1 in EC, DG soma

Cp27 in EC, DG soma, CA1 soma

where are proteins made

in cytosol where ribosomes are → same kinds of ribosomes synthesize all proteins

possible transport mechanisms for proteins to get to final destination

gated transport: cytosol → inside nucleus via nuclear pores

transmembrane transport: cytosol → inside ER → other locations OR cytosol → mitochondria, chloroplasts, peroxisomes

vesicular transport: cytosol → ER → golgi, lysosomes, plasma membrane → CAN come back to ER

basic transmembrane transport progression/elements (general, for cytosol → golgi)

polypeptides are synthesized by ribosomes actively transporting proteins into ER

signal sequence on N-terminal is recognized by SRP, which associates with SRP receptor on golgi membrane and brings polypeptide into translocon. Polypeptide in translocon forms hairpin as it continues translating, removing the plug. ss placed into membrane through lateral gate. fully synthesized polypeptide’s ss is cleaved with signal peptidase, and protein released into ER lumen.

signal sequence

sequence of amino acid residues on N-terminal of nascent polypeptide that indicates where that protein should go → for instance, into ER, to be retained in ER lumen, into/out of nucleus

signal recognition particle

recognizes and binds to nascent polypeptides’ signal sequences; also associates with SRP receptors on membrane of ER (or other location), thus guiding polypeptide to its destination and essentially dropping it off

SRP receptor

signal recognition particle receptor in ER membrane where SRP attached to nascent polypeptides go to bind, thus associating the polypeptide with adjacent translocon

translocon

channel between intracellular space and ER lumen where nascent polypeptides will go to finish translation before being inserted into the ER lumen. Contains a plug that is displaced by hairpin structures of growing polypeptides, and has two gates: transmembrane into ER luman, and lateral gate where signal sequence goes into lipid bilayer and is ultimately cleaved

signal peptidase

enzyme that cleaves the signal sequence from a polypeptide that has finished translation in the translocon, allowing it to enter the ER lumenal space

how are transmembrane proteins inserted into the ER membrane (or other membrane)

same transmembrane pathway as above, but polypeptide contains a second signal sequence: stop sequence → this triggers the transfer of the transmembrane protein into the phosphoplipid bilayer rather than cleavage of ss and transport into ER lumen

how are proteins with transmembrane domains inserted into the ER (or other) membrane

Proteins have internal signal sequences rather than at their N-terminal. SRP/SRP receptor/translocon will recognize it as normal, but the signal sequence will remain as a transmembrane domain rather than being cleaved. The N-terminal (beginning) of the polypeptide will be left in the cytoplasm. Opposite configuration can happen too → N terminal can be directed into ER lumen and C-terminal left in cytoplasm.

This pattern also allows multipass proteins (with multiple transmembrane domains) to be inserted appropriately into a membrane. Multiple start and stop-transfers will determine sequences inserted into the membrane, while intermediate parts will ‘loop’ inside ER or in cytoplasm.

SDS-PAGE

Dpi/beta-mercaptoethanol break noncovalent bonds in proteins, while sodium dodecyl sulfate creates uniformly charged particles. Proteins then move through gel based on their size → lager molecules don’t move as far as fast. Rf is distance travelled/total length, and can be plotted to a standard curve to determine the size of the proteins.

GET pathway (& steps)

pathway for tail-anchored proteins to become inserted in ER membrane; transmembrane sequence located at C-terminal.

C-terminal ss is bound by chaperone Sgt2, which interacts with GET3-5. Protein transfers to GET3 in its ATP bound state. ATP hydrolysis of GET3 released protein to bind to GET1-2 insertase complex. GET 1-2 insertase activity places protein’s transmembrane domain into the ER membrane.

trafficking

direction and targeting of vesicular traffic by coat proteins and elaborate molecular mechanisms to match vesicles with target membrane

general trafficking pathway

proteins & lipids go from the ER to golgi in transport vesicles

transport vesicles bud from ER exit sites (ERES)

transport vesicles fuse with one another and form vesicles and tubules of ER-Golgi intermediate comples (ERGIC)

cargo moves from ERGIC to cis golgi complex

peptide and carbohydrate signals determine if transmembrane proteins should go to golgi/elsewhere

3 main steps of vesicular transport

vesicle budding

transport of vesicle through cytoplasm

fusion of vesicle to target membrane

coat proteins

include COPI, COPII, clathrin

physically shape and pinch off vesicles, and bind to appropriate adaptor proteins that will bind to cargo receptors

COPII coat protein

coats vesicles carrying cargo from ER → Golgi

COPI coat protein

coats vesicles retrieving ER-resident proteins that have ‘escaped’ to the ERGIC or cis golgi

clathrin coat protein

coats vesicles going outwards from the trans-golgi network OR coming back from the plasma membrane to endosomes or other organelles

has a triskelion shape made of heavy and light chains

adaptin

proteins that mediate interactions between clathrin coats and membrane proteins

dynamin

a G protein that physically pinches off coated vesicles from ER membrane

lumenal ER proteins → Golgi

bound by transmembrane receptor proteins that selectively package cargo into its vesicle

KDEL retrieval sequence

amino acid sequence at the C-terminus of resident ER proteins that are destined to stay in the ER lumen. If these proteins escape the ER to the ERGIC or Golgi, recycling receptors recognize the KDEL sequence and return that protein to the ER

Rabs

a family of GTPases/G proteins that are present on vesicle membranes and bind to tethering factors on target membranes to help facilitate vesicle/membrane fusion

tethering factors

transmembrane proteins present on target membranes that Rabs will recognize and bind to, bringing a vesicle of cargo with it

SNAREs

vSNAREs present on vesicles, tSNAREs present on target membranes, both possess coiled-coil domains that associate when rab/tethering factor bring vesicle/membrane together. Coiled-coil domains zip and bring vesicle/membrane close and membrane fusion occurs

Rab proteins act as a molecular switch - active/inactive forms?

active when GTP bound

inactive when GDP bound

GTP-for-GDP binding in rabs

switches rab to active form

GTPase activity

GTP hydrolysis to GTP in Rabs switches rab to inactive form

GAPS

GTPase activating proteins that often facilitate GTPase activity of rabs

GEFs

guanine nucleotide exchange factors that often facilitate activating step of rabs

elements of neurotransmitter release

vesicle SNARE = synaptobrevin

target SNARE = syntaxin and SNAP-25

calcium sensor = synaptotagmin

how does transport from the golgi generally work

fused cargo travels from cis to trans golgi (modification in between). in the trans-golgi network, proteins are sorted and then sent to their final destination in vesicles

golgi → plasma membrane

vesicles sent either directly from golgi to PM or via recycling endosomes

golgi → regulated secretion

vesicles sorted into distinct secretory granules

how are proteins targeted to lysosomes

sent from golgi to late endosomes that will develop into lysosomes. lumenol proteins are marked by mannose-6-phosphates after entering gogli, and transmembrane receptors recognize this - concentrate and target lysosome proteins for transport to late endosome

glycosylation

glycoprotein created when 14-sugar oligosaccharide is added to acceptor Asn residue and 3 glucose residues are removed, occurs in ER

N-linked oligpsaccharides of glycoproteins are transported from ER → Golgi and further modified in golgi by sequence of reactions catalyzed by various enzymes

detailed clathrin coats/how they work for transport

clathrin coated vesicles mediate traffic: golgi → out, PM → in

1) proteins delivered to trans-golgi membrance. Arf1/GDP activated to Arf1/GTP by guanine nucleotide exchange factor ArfGEF

2) Arf/GTP recruits an adaptor protein (which also serves as a binding site for clathrin coat assembly)

3) adaptor binds to transmembran receptor’s cytosilic tail with its lumenol cargo attached

4) the G-protein dynamin constricts the vesicle neck, causing membrance fission

how clathrin coats also mediate endocytosis

1) molecules taken up from outside the cell with endocytic vesicles

2) endocytic vesicles fuse with early endosome

3) membrane receptors are recycled to plasma membrane through recycling endosomes

4) early endosomes mature to late endosomes

5) transport vesicles carrying acid hydrolases (come from trans-golgi network) fuse with late endosomes that then mature into lysosomes

nuclear pore complexes

large structures forming transport channels through the nuclear envelope, which are the only channels where small polar molecules, ions, or macromolecules (proteins/RNA) can go betwen nucleus and cytoplasm

nucleoporins/NUPs

a group of about 30 proteins that assemble to form the nuclear pore complex

FG-NUPs

proteins that make up nuclear pore channels → nucleoporins with domains composed of short, repeated motifs rich in phenylalanine and glycine residues (FG repeats) that form selective barrier within the pore

nuclear localization signals

an amino acid sequence that target proteins for transportation from the cytoplasm to the nucleus

nuclear transport receptors

a protein that recognizes nuclear localization signals and mediates transport across the nuclear envelope; karypharins, include importins and exportins

importin

a karyopherin (transport protein) that recognizes nuclear localization signals and directs nuclear import. directs protein cargo from cytoplasm to nucleus through nuclear pore. binds with FG motifs in pore, which enables cargo to penetrate and pass through

Ran

a small GTP-binding protein (G-protein) involved in nuclear import and export. Ran/GTP binds importin once inside the nucleus and disrupts the importin/cargo complex and releasing cargo into the nucleus.

exportins

karyopherins that recognize nuclear export signals and direct transport from the nucleus to the cytosol through the nuclear pore complex

autophagosome

A vesicle containing internal organelles enclosed by fragments of cytoplasmic membranes that fuses with lysosomes. Has a double membrane, used by cells to ‘clean up’ own waste products

apical domain

The exposed free surface of a polarized epithelial cell.

basolateral domain

The surface region of a polarized epithelial cell that is in contact with adjacent cells or the extracellular matrix.

Atg proteins

autophagy-related proteins. A family composed approximately 20 proteins that function together to carry out autophagy.

cis, medial, trans golgi

cis golgi is where vesicles from ER fuse/enter. medial golgi in the middle, facilitates cargo modification. trans golgi is nearest the nucleus and is where some vesicles are sent out to other locations

how does ran/GTP cycle work

Ran/GTP binds importin once inside the nucleus and disrupts the importin/cargo complex and releasing cargo into the nucleus. Importin-ran/GTP complex exits nucleus. Ran-Gap (GTPase-activating) protein stimulates hydrolysis of GTP to GDP. importin is released and can be reused, and ran/GDP goes back to nucleus with its import receptor NTF2. In the nucleus, Ran GEF (bound to chromatin) stimulates the exchange of GDP bound to Ran for GTP, leading to the conversion of Ran/GDP to Ran/GTP and it can be reused

human mitochondrial genome contains

small circular genome of about 16 kilobases, 13 protein coding sequences

human mitochondrial-coded proteins mainly function in

respiratory complexes I, III, IV, V, or electron transport chain

most proteins found in the mitochondria…

are produced by nucleus genome, but had to be transported into mitochondria (about 1500 total proteins)

translation/folding of mitochondrial proteins & location

synthesized by free ribosomes in cytosol and bound by cytosolic chaperones, stabilizing polypeptide in unfolded configuration. the partly unfolded polypeptide is transported through cytosol into mitochondria through transmembrane transport, mediated by mitochondrial chaperones → also facilitate polypeptide folding to final form once in mitochondria

presequence

signal sequence directing proteins to mitochondria, usually 15-55 amino acids with (+) residues, often alpha helical, cleaved after transport

Tom complex

transmembrane protein in outer mitochondrial membrane that is targeted by the presequence, brings mito proteins to mitochondria

Tim23 complex

transmembrane protein complex in inner mitochondrial membrane where mito proteins bind after entering intermembrane space through Tom complex

how do proteins enter mitochondrial matrix from IM space

bound to Tim23 complex; import motor complex containing Hsp70 chaperones uses ATP hydrolysis to drive protein translocation across innner mito membrane into matrix