Cell Bio Exam 2

Both

1957 Ribonuclease Experiment

• Denature by heat (breaks noncovalent bonds)

• Denature by reducing agents (breaks disulfide bonds)

• Protein spontaneously refolds when these elements are removed

• Significance: folds in vitro, solely by primary amino acid seq interactions

Can you predict the structure of proteins?

With AI models like Alphafold2

Chaperones use ATP hydrolysis to drive protein folding of hydrophobic domains

does not drive, more passive

Chaperones

• Bind hydrophobic regions

• Protein misfolding, aggregation

• Prevent premature interactions/binding

• Co-translational binding, before interacting domains are synthesized

• Bound before subcellular targeting is initiated

Hsp70

• Bind hydrophobic parts of unfolded regions

• Release and rebind with ATP hydrolysis

• Cycles on and off until hydrophobic regions find each other in the correct fold

• When hydrophobic domains are no longer available, Hsp70 no longer binds

• Found in cytosol and in subcellular organelles

• The unfolded polypeptide will then be transferred from Hsp70 to chaperonin to fold

Chaperones definition

• Proteins that facilitate the folding of other proteins

• Act as catalysts that facilitate assembly w/o being part of the assembled complex

How does chaperones function?

By binding to and stabilizing unfolded or partially folded polypeptide chains that are intermediates along the pathway leading to the final correctly folded state

This binding stabilizes the amino-terminal portion in an unfolded conformation until the rest of the polypeptide chain is synthesized

Chaperonins

• Hsp60 Family

• Chamber protein within which protein folding takes place

• Consists of multiple protein subunits arranged in 2 stacked rings to form a double-chambered structure

• Shields unfolded polypeptide chains from the cytosol within its chamber

• ATP binding is required for

  • Association of cap

  • Removal of cap and release of protein

How does the chaperonin work?

E. Coli GroEL subunits rotate hydrophobic to hydrophilic surfaces, promoting protein folding

cytosol

the aq component of the cytoplasm of a cell, within which various organelles and particles are suspended

Ubiquitin

• Small protein

• Links to lysine in proteins

• Attachment can mark proteins for degradation

Ubiquitin-Proteasome Pathway

• Major mediator of regulated protein degradation

  1. Ubiquitin is activated by E1 with ATP

  2. The ubiquitin is then transferred to E2

  3. The ubiquitin is then transferred to the target protein by E2 complexed with a third protein, called ubiquitin ligase (E3).

  4. Proteins targeted for degradation are marked by the addition of multiple ubiquitins to form a polyubiquitin chain, which is catalyzed by some E3s. These are recognized and degraded by the proteasome (a large, multisubunit protease complex)

How many E3s in mammalian cells?

About 600 to confer target specificity

Proteasome lid

• Binds polyubiquitin chain

• Removes ubiquitin subunits (ATP hydrolysis)

• Unfolds and translocates protein into center chamber

Central barrel complex

hollow core lined with proteases that cleave peptide bonds to chop into peptide fragments

Recycling

peptides later digested by cytosolic peptidases into amino acids for cellular recycling

Angelman’s Syndrome:

• Genetic disorder of nervous system

• Developmental and cognitive disabilities, lack of speech, overly happy syndrome

• Cause:

  • Ch 15 deletion, includes UBE3A

  • E3 ligase needed for targeted protein degradation in neuron development

Protein activity regulation

• Binding small molecule regulators

• Post-translational modifications

Protein-protein interactions

• Complexes

• Subcellular organization: condensates membrane-less organelles (ex: nucleoulus)

Protein Kinase A

• Inactive: when regulatory subunits (R) are bound to the catalytic subunit (C)

• Active: when 2nd messenger molecule, cAMP binds the regulator and it changes conformation/lets go of the now active catalytic subunits

• cAMP dependent protein kinase

• A tetramer consisting of 2 regulatory and 2 catalytic subunit

• Cyclic AMP binds to the regulatory subunits, leading to their dissociation from the catalytic subunits. The free catalytic subunits are then enzymatically active and able to phosphorylate serine residues on their target proteins.

Intrinsically disordered regions

polar and charged domains, available for interactions

bind other proteins, RNA Concentrate and separate into discrete domains

Participate in membrane-less compartments

Phospholipids

• Amphipathic molecules consisting of two hydrophobic fatty acid chains linked to a phosphate-containing hydrophilic head

• Spontaneously form bilayers in aq solutions bc their fatty acid tails are poorly soluble in water so the hydrophobic tails will be buried in the interior of the membrane and the polar head grps will be exposed on both sides in contact w water

Amyloids

• Fibrous aggregates of misfolded proteins

• Misfolded proteins aggregate to form insoluble amyloid fibrils characterized by beta-sheet structure

Alzheimer’s disease

• Characterized by amyloid plaques in the brains of patients. These form due to aggregation of amyloid beta protein

• Neurodegenerative, common cause of dementia

• Alzheimer’s may be caused from the toxicity of soluble forms of ABeta oligomers rather than accumulation of amyloid plaque

• The amyloid beta protein plaques were a correlation w alz

• The mutations in amyloid beta protein genes cause disease (causation)

Prions

• Misfolded proteins that are capable of self-replication

• Misfolded proteins that stimulate further misfolding

Inherited Alzheimer’s Disease may be caused by…

• Mutations in amyloid precursor protein gene

• Mutations in proteinase (catalyzes proteins into smaller polypeptides, so w/o this amyloid proteins are less regulated)

Aduhelm Trial

• Cleared plaques

• Did not effect symptoms (as in cognitive decline)

• Very expensive

Donanemab

• Clears plaque and slows cognitive decline

• Verrrry expensive, serious side effects

So what can we learn from all the Alzheimer’s Disease research mishaps?

• We should critically evaluate

• We should be more open to other hypotheses outside of the amyloids

• Can look at Tau protein

  • Stabilizes microtubules in neurons

  • Mutations can cause dementia

• Mouses may not be a relevant model for human disease

Transmissible spongiform encephalopathy

AKA prion diseases

Kuru

• To shake

• 1950s New Guinea unusual human disease, rare and fatal

• Cannibalism practice of eating tissue of deceased elders

• Long incubation period

• Prion disease

Scrapie

• Sheep

• Progressive

• Fatal

• Brain deterioration

• Persistent scratching

• Related to chronic wasting disease

Creutzeldt-Jakob Disease

• Human

• Rare inherited and sporadic disease

Mad Cow Disease

• Contaminated feed (probably containing scrapie sheep tissues)

• 4 million cows killed to prevent spread to other cattle and humans

• 1980s-1990s spread to humans

Stanley Prusiner: Basis for Prion Diseases

• So at first they thought it was a virus, BUT it was resistant to nucleases

• Sensitive to proteases → proteins only hypothesis

• Once sequence identified → single proteins → prions (encoded in genome)

PrPC

• Prion protein normal fold

• Susceptible to proteases

• Remains monomeric

• Not disease related

• α-helical form

PrPSc

• Forms a misfolded amyloid structure

• Can propagate by inducing the misfolding of PrPcs to the amyloid PrPSc

• Can infect a cell and replicate by inducing autocatalytic amyloid formation of endogenous PrPc

• More resistant to proteases (an enzyme which breaks down proteins and peptides)

• Polymeric

• Disease-related

Phospholipid Bilayer

• Forms a stable barrier bw 2 aq compartments and represent the basic structure of all biological membranes

Phospholipid Structure

• Hydrophilic head

• Hydrophobic tail - fatty acid tail

• Fatty acid chain length and degree of saturation varies

• Typically, one chain is saturated, one is unsaturated

• Net negative charge inner surface of bilayer due to PS in inner leaflet

How are phospholipid membranes built and distributed?

• Synthesized on the Endoplasmic Reticulum - cytosolic leaflet where most phospholipids are made

• Scramblases distribute lipids symmetrically bw leaflets

Cholesterol

• Abundant in animal cell membranes

• Rigid ring structure

• Hydrophilic hydroxyl head grp

• Hydrophobic tail

Fluid Mosaic Model

• Proteins are inserted into the lipid bilayer

• Membrane proteins carry out the specific functions

• Integral membrane proteins: embedded directly within the lipid bilayer

  • Single pass

  • Multi-pass

• Peripheral membrane proteins: membrane association is indirect, through protein-protein interactions

How do we dissociate integral membrane proteins?

• Via detergents

• Hydrophobic tails bind hydrophobic regions of integral membrane proteins, forming detergent-protein complexes that are soluble in aq solution

How do we dissociate peripheral membrane proteins?

Dissociate from membrane by extreme pH or high salt

Soluble in aq solutions

Alpha helixes and beta barrel folds

• Integral membrane protein transmembrane domains

• Rich in hydrophobic amino acids

• Typically folded as alpha helix or beta barrels

• Folds that shied polar peptide bond thru H bonding

Hydropathy Analysis

Can predict potential alpha-helical transmembrane domains from primary aa seq, rich in hydrophobic amino acids

Temperature and Membrane Fluidity

• 👆 thermal energy = 👆 fluidity

  • Fatty acid chains associate less tightly, more fluid

Saturation and membrane fluidity

• Lipids containing unsaturated fatty acids increases kinks in the fatty acid chains due to the dbl bonds ▶ more fluid membrane

Fatty Acid Chain Length and Fluidity

• Interactions bw shorter fatty acids chains are weaker than those w longer chains ▶ membranes w shorter fatty acid chains are less rigid and more fluid

Cholesterol and Membrane Fluidity

• At low temp, increases fluidity (separates phospholipid fatty acid chains, loosens packing)

• At high temp, decreases fluidity (rigid structure among fatty acids, restricts fluidity)

How do we know proteins are mobile in membranes?

• 1970s researchers

• Used anti-human and anti-mouse antibodies labeled w diff fluorescent dyes

• Formed human/mouse hybrids

• Labelled human and mouse proteins intermingled over cell surface within 40 min of incubation

• “This lateral movement of membrane proteins was first shown directly by Larry Frye and Michael Edidin in 1970. Frye and Edidin fused human and mouse cells in culture to produce human–mouse cell hybrids (Figure 15.5). They then analyzed the distribution of proteins in the membranes of these hybrid cells using antibodies that specifically recognize proteins of human and mouse origin. These antibodies were labeled with different fluorescent dyes, so the human and mouse proteins could be distinguished by fluorescence microscopy. Within 40 minutes after fusion, the mouse and human proteins became intermixed over the surface of hybrid cells, indicating that they moved freely through the plasma membrane”

FRAP

• Fluorescence Recovery After Photobleaching

• “. In this technique, a region of interest in a cell expressing a GFP-labeled protein is bleached by exposure to high-intensity light. Fluorescence recovers over time due to the movement of unbleached GFP-labeled molecules into the bleached region, allowing the rate at which the protein moves within the cell to be determined”

The Fate of a newly synthesized protein

Heat Shock Proteins

• Chaperones & chaperonins are actually heat-shock proteins

• They accumulate in response to exposing cells to high-temperature

• “Many HSPs function as molecular chaperones to protect thermally damaged proteins from aggregation, unfold aggregated proteins, and refold damaged proteins or target them for efficient degradation.”

Explain the 2 different possible consequences if protein folding fails in cells, and how each could contribute to disease

• Misfolded protein aggregation → neurodegenerative diseases like Alzheimer’s

• Loss of protein function → cystic fibrosis

  • “Cystic fibrosis is caused by mutations in CFTR (responsible for the transport of chloride ions across the plasma membranes of several types of cells, such as those lining the respiratory tract). “Defective chloride transport as a result of these mutations leads to obstruction of the respiratory tract by thick plugs of mucus, leading to recurrent infections and the death of most patients from lung disease.”

Mouse Model for Alzheimer’s Disease

• They did a mouse model w mutant human APP (amyloid precursor protein) gene

• Observe plaques

• Neurodegenerative symptoms

• Test and develop alz therapeutics

• Vaccination against ABeta peptide

• But does this really represent humans w alz?

Describe why transmembrane domains frequently have alpha-helical or beta barrel structures.

1. Alpha-Helical Transmembrane Domains

Most common in eukaryotic and prokaryotic membrane proteins

🔹 Why Alpha-Helices?

• Hydrophobic Amino Acid Side Chains: The outer surface of the helix contains nonpolar residues that interact favorably with the lipid bilayer.

• Internal Hydrogen Bonding: Backbone hydrogen bonds (between C=O and N-H groups) are satisfied within the helix, avoiding unfavorable interactions with the hydrophobic membrane.

• Versatility: Multiple helices can assemble into channels, transporters, or receptors, allowing for diverse functions.

🔹 Examples:

• G protein-coupled receptors (GPCRs) – Seven transmembrane helices for signaling.

• Ion channels (e.g., K⁺ channels, aquaporins) – Helices form selective pores.

Nucleus

A key feature that distinguishes bw eukaryotic and prokaryotic cells

Functions of the Nucleus

• Hosts/Protects the DNA genome

• Specialization of nuclear vs cytoplasmic functions provides opportunity for transcription & translation regulation (ex: alternative splicing)

• Mechanical stability

Nucleus Structure

• Double membrane (nuclear envelope)

• Nuclear pores (composed of proteins)

• Nuclear lamina (nuclear skeleton) composed of lamins (intermediate filaments)

• Subnuclear organization and membrane-less organelles (nucleolus)

What’s the evolutionary advantage of having a nucleus? Examples?

• Protection of the genome

• Separate synthesis/processing of RNA from cytoplasmic events; provides opportunities for gene expression regulations

• Examples:

  • Protein nuclear import & export enables transcriptional regulation

  • Splicing & alternative splicing enables greater complexity from a single gene

  • Regulation of RNA transport enables translational regulation

Evolutionary origin of the nucleus

• Membrane invagination → double membrane w pores that formed at membrane junctions

• Interior of nucleus and cytosol originated from the same compartment

Topological Relationship of the Cell 🤪

• Nuclear Compartment → topologically related to the cytosol

  • The cytoplasm and the nucleus are said to be topologically equivalent because the outer and inner nuclear membranes are continuous with one another, so that the flow of material between the nucleus and cytosol occurs without crossing a lipid bilayer.

• The ER, Golgi, lysosome, & vesicular compartments → topologically related to cell exterior

Nuclear Envelope

• Outer Membrane

  • Continuous w rough ER membrane

• Inner Membrane

  • Integral membrane proteins bind nuclear lamine proteins

Cell exterior

Nuclear Pore Complexes

• Only channels for import and export

• Selective and regulated

• Huge molecular machine

• Abundant (~3-4k of em)

• Formed by nucleoporin proteins (NUPs)

• Asymmetric: distinct cytoplasmic & nuclear features, important for directional trasnport

• Central Channel

  • Lined by FG-rich proteins

  • NUPs are highly conserved in eukaryotes

  • IDR

  • Interact w transport machinery, restrict diffusion, promote selective transport

Molecular Traffic through Nuclear Pore Complexes

• Two Transport Mechanisms:

  • Passive diffusion: small molecules pass freely thru the nuclear pore and equilibrate across envelope

  • Selective transport: most proteins and RNAs, recognized by specific signals, selectively transported across nuclear pore and accumulate in one compartment

Nuclear Localization Signals

• Selective nuclear transport relies on restriction of passive diffusion by Nuclear Pore Complex Interior

• Specific import and export seqs on cargo protein:

  • Import: Nuclear Localization Seqs (NLS)

  • Export: Nuclear Export Seqs (NES)

• Specific receptor proteins function as importers/exporters:

  • Bind and release NLS (or NES) in appropriate locations

  • Direct transport thru the Nuclear Pore Complex

Discovery of NLS

• 1980s

• Butel and Lanford noted that SV40 T- Antigen viral protein that normally is transported to the nucleus stays in the cytoplasm if a particular sequence is mutated

• Alan Smith’s grp identified a small essential region by deletion studies in SV40 T Antigen

• They fused their presumptive NLS to a normally cytosolic protein

  • NLS was linked to gold particles and injected into the cytosol

  • Cells were fixed @ various times

  • Gold particles visualized in EM

  • NLS is sufficient for nuclear import!!!

  • Import occurs thru nuclear pores!!

Characteristics of NLS

• Can be

  • A continguous stretch of amino acids

  • Bipartite patch

Importins

Nuclear import receptor

In the cytosol, importins bind: NLs of cargo then outer NPC cytoplasmic filaments

Transported thru pore/channel proteins

At the nuclear side of the envelope, importin releases cargo protein into the nucleus

Problem: So importins have to bind cargo outside the nucleus but it also has to release cargo inside the nucleus. How’s it gonna get back out?

Solution: Ran-GTP in nucleus binds importin, makes importin release cargo (after conformational change), and transports cargo less importin back to the cytosol

Ran has 2 forms…

• RanGTP in the nucleus

  • ABUNDANT IN NUCLEUS

• RanGDP in the cytosol

  • ABUNDANT IN CYTOPLASM

  • Importin binds cargo

RanGEF

• Guanine Nucleotide Exchange Factors swaps GDP out allowing GTP in

• Bound to chromatin

RanGAP

Activates hydrolysis of GTP to GDP

Bound to the outer pore

Nuclear Export of Proteins

• Exportins bind to NES (nuclear export signals) of cargo

• In presence of Ran-GTP

• Exportins release:

  • NES-cargo

  • In presence of Ran-GDP

• Ran-GDP and exportins are then returned to nucleus

Ran-GTP would accumulate in the nucleus

Regulation of Nuclear Import of Transcription Factors: Masking the NLS (not yeast)

• Mask NLS by protein binding:

  • NF-kB transcription factor is bound by a protein (IkB) which masks NLS in the cytoplasm

• Extracellular signals cause IkB phosphorylation recognized by an E3 ligase, causing ubiquitin-mediated proteolysis, allowing NF-kB nuclear import

Regulation of Nuclear Import of Transcription Factors: Masking the NLS (Yeast)

• Yeast transcription factor Pho4 is kept in cytoplasm by phosphorylation near/at nuclear localization signal

• Signal: phosphate depletion

• Need for cellular phosphate, results in dephosphorylation, exposes the NLS, and Pho4 transported to nucleus

mRNA export

• Independent of Ran

• Once spliced and polyadenylated, mRNAs are bound by exporter complex proteins (quality control for fully proceeds mRNAs)

• A helicase, on cytoplasmic face of nuclear pore complex, releases the mRNA from exporter complex

• Helicase bound cytosolic face of NPC provides directionality

RNAs that function in nucleus either retained or moved out and back in:

• snoRNAs for rRNA processing in nucleus - not exported

• lncRNAs stay in the nucleus

• snRNAs exported, associate w proteins, and reimported as RNPs for splicing

RNAs that function in cytoplasm:

• tRNAs & miRNAs exported directly via specific exportins; participate in translation and RNA regulation cytoplasm

• rRNAs are first bound by ribosomal proteins, in nucleolus, then exported as ribosome subunits for translation functions in cytoplasm

Nuclear Lamina

• Lines the inner nuclear membrane

• Structural support (links to cytoplasmic cytoskeleton)

• Attachment points for chromatin (affects chromatin organization)

• Participates in nuclear envelope breakdown and reassembly

• Intermediate filament

• Interact w both

  • Chromatin

  • Inner membrane proteins

• Bind to protein complexes that bind to the cytoskeleton in the cytosol. The inner lamina provides a link bw chromatin and forces in cytoplasm.

Subnuclear Organization

• Heterochromatin (inactive): near nuclear membrane (via lamins interactions) & periphery of nucleolus

• Euchromatin (active): preferentially localizes to the rest of the nucleus

Evidence for the location of active/inactive genes

• EM of hetero vs euchromatin

• FISH of chromosomal DNA/RNA

• Chromatin IP/ Lamin Association

Heterochromatin in interphase nuclei

• Euchromatin (active) is distributed throughout the nucleus

• Heterochromatin (inactive) is associated w the nuclear envelope (nuclear lamina) and the nucleolus

Nuclear bodies

• Membrane-less organelles, condensates

• Domains within nucleus, maintained by interactions bw proteins or protein-RNA

Nucleolus

rRNA synthesis & processing, ribosome assembly (needed in large qty)

larger in metabolically active cells

Functions in rRNA synthesis, processing and ribosome assembly

Maintained by interactions between protein and RNA components

Contains 100s of copies of rRNA encoding genes

Subdomains form a multilayered biomolecular condensate within nucleolus

Multilayered biocondensate: Ribosome Assembly

Domains for:

• rRNA gene transcription

• rRNA processing

• Ribosome subunit assembly

Ribosomal Proteins

• Synthesized in cytosol

• Imported

• Assembled into 40S and 60S subunits

• Exported as ribosomal subunits (ribosome subunits are not to scale, are 30x smaller than nuclear pores)

Nuclear envelope breaks down at mitosis to enable formation of 2 nuclei

• Lamin phosphorylation: envelope fragmentation

• Nuclear envelope assembly: dephosphorylated lamins bind to chromatin and bound to inner membrane, reassemble nuclear envelope

What happens to the nuclear proteins after cell division?

During cell division, the nuclear envelope breaks down, and the proteins within are scattered.

Cytosol contains

• Many metabolic pathways

• Protein synthesis

  • Cytoskeleton

Nucleus contains

• Main genome

• DNA synthesis

• RNA synthesis and splicing

• Ribosome assembly

Rough ER Functions

• Protein synthesis, modification, and transport to many organelles and plasma membrane

Smooth ER Functions

• Lipid and steroid synthesis, detoxification

ER Structure

• A continuous network of membrane-enclosed tubules and sacs (cisternae)

• Nuclear envelope is part of the ER!

• Typically largest organelle

• Rough ER covered by ribosomes on its cytosolic surface

Key Players in ER Function:

• Signal recognition particle and translocon

  • “As they emerge from the ribosome, signal sequences are recognized and bound by the signal recognition particle (SRP) consisting of six polypeptides and a small cytoplasmic RNA (SRP RNA). The SRP binds the ribosome as well as the signal sequence, inhibiting further translation and targeting the entire complex (the SRP, ribosome, mRNA, and growing polypeptide chain) to the rough ER by binding to the SRP receptor on the ER membrane (Figure 12.6). Binding to the receptor triggers the hydrolysis of GTP bound to the SRP, releasing the SRP from both the ribosome and the signal sequence of the growing polypeptide chain. The ribosome then binds to a protein translocation complex or translocon in the ER membrane, and the signal sequence is inserted into a membrane channel”

• Chaperones (BiP), protein modification (S-S, lipid anchors)

  • “[…] chaperones that facilitate the folding of

    polypeptide chains (see Chapter 10). The Hsp70 chaperone, BiP, is thought to

    bind to the unfolded polypeptide chain as it crosses the membrane and then

    mediate protein folding within the ER (Figure 12.14). Correctly assembled

    proteins are released from BiP (and other chaperones) and are available for

    transport to the Golgi apparatus”

• Quality Control (ERAD, UPR)

  • ERAD - ER Associated Degradation ~ misfolded proteins are identified, returned from the ER to the cytosol, and degraded by the ubiquitin-proteasome system.

  • UPR - Unfolded protein response ~ activated if an excess of unfolded proteins accumulates in the ER

    • Activation of the UPR pathway leads to expansion of the ER and production of additional chaperones to meet the need for increased protein folding as well as a reduction in the amount of newly synthesized proteins entering the ER

Secretory Pathway

Rough ER → Golgi → secretory vesicles → cell