LOH PP 3

What are the two broad pathways by which defective proteins bring about disease?

The two broad pathways are loss of function (LOF) and gain of function (GOF).

What are the two primary ways loss of function (LOF) can be caused by mutations?

Caused by mutations that cause proteins to lose thermodynamic stability (such that it cannot fold or folds to slowly, leading to excessive turnover), or lose its function without impairing its structure or stability.

What are examples of mutations that cause loss of function without impairing structure or stability?

Examples include “direct KO” mutations of a residue involved in catalytic activity, binding to another protein, or phosphorylation.

What does gain of function (GOF) mean in the context of protein folding and disease?

It can mean that the protein adopts an alternate, toxic fold that poisons the cell, nearby tissues, and organs, as seen in amyloid disease.

How else can gain of function (GOF) be brought on in cellular enzymes?

Through mutations of enzymes that cause them to be constitutively active meaning they are permanently active.

What are the three main types of alterations that occur in cancer?

• Inactivation of tumor suppressors (e.g., p53)

• Activation of oncogenic proteins (e.g., RAS)

• Dysregulation of DNA repair (e.g., BRCA1)

What does Knudson’s (1971) "Two-hit hypothesis" state regarding carcinogenesis?

Both alleles of a tumor suppressor must be inactivated—either through mutations or epigenetic silencing—to cause a malignant phenotype.

What typically accompanies the inactivation of tumor suppressors during carcinogenesis?

Additional activation of a proto-oncogene

How many alleles of p53 must be inactivated to see an effect, and why?

p53 only requires one allele to be inactivated due to a dominant negative effect, though the wild-type allele is often subsequently lost in cancer.

Why are certain animals like elephants and bats resistant to cancer?

They possess multiple copies of p53 and p53-like genes (e.g., elephants have 38 copies), whereas humans have only 2, meaning the loss of just 1 allele is enough to progress toward cancer.

What triggers p53 to jump into action,

Cell stress or DNA damage (from radiation, chemicals, or errors during division) activates p53.

What are p53’s primary protective functions?

It halts cell division to buy time for repair, triggers DNA repair genes, and induces apoptosis if damage is too severe to prevent tumor formation.

What are the stability and half-life properties of the p53 protein?

It is thermodynamically unstable Tm @ 42 degrees Celsius and has a short half-life in cells of approximately 15 minutes.

What nickname is given to p53, and what is its classification as a protein?

It is known as the "guardian of the genome" and functions as a transcription factor.

How does p53 behave in healthy cells versus when DNA damage occurs?

It is present in a latent conformation in normal cells and becomes activated by DNA damage, allowing it to bind specific target sites on chromosomes to activate expression of select proteins that drive cell-cycle arrest or apoptosis

What happens when mutations accumulate in DNA over time?

While individual mutations have a low probability of a catastrophic effect, accumulating enough mutations can lead to genomic instability and malignant tumors.

What are the three modular domains of the transcription factor p53?

The N-terminal transactivation domain, central DNA-binding domain, and C-terminal domain.

What is the function of the N-terminal transactivation domain of p53?

It recruits the transcriptional machinery to the site of the DNA binding and helps activate it

What is the function of the C-terminal domain of p53?

It contains nuclear localization, tetramerization, and other regulatory functions.

What is the function of the central DNA-binding domain of p53?

recognizing and binding specific DNA sequences

Is the single zinc ion in p53 part of a classical zinc-finger motif?

No its a metal ion that stabilizes the loops so that p53 can interact with DNA

What is the functional role of the beta-clam within the DNA binding domain?

Support for the helix and loop

How does the Lys120 mutation cause p53 to lose function?

They weaken p53's direct affinity for DNA because specific atomic interactions (like hydrogen bonds or electrostatic contacts) are lost

How does the Tyr220 hot spot mutation cause p53 to lose function?

They disrupt the internal hydrophobic packing by creating a surface cavity, which severely reduces p53's thermodynamic stability and causes the protein to unfold at physiological temperatures.

Which three classes does tumor p53 fall into

1. DNA contact 2. Destabilizing 3. Zinc binding

How does the Cys238 mutation cause p53 to lose function?

Removing the cysteine disrupts the coordination mechanisms holding the zinc ion in place, destabilizing the local structure and destroying p53's ability to properly contact DNA.

What abnormality regarding MDM2 is frequently observed in tumors that retain wild-type (WT) p53?

Tumors that have wild-type p53 often overexpress MDM2. Cancer that mutes tumor suppressor

What is the downstream cellular consequence of MDM2 overexpression in tumors with WT p53?

It results in the rapid degradation of p53 before it can successfully activate transcription and get rid of cancer cells

Which class of inhibitors is designed to target the p53-MDM2 axis in these types of cancers?

The Nutlin-class of inhibitors, which target the p53-MDM2 axis

How do Nutlin-class inhibitors work against cancers with wild-type p53 and overexpressed MDM2?

They target the p53-MDM2 axis by blocking the interaction between the two proteins, preventing MDM2 from degrading p53 and allowing p53 to accumulate and destroy the tumor cells.

What are RAS proteins?

small signaling proteins that function as molecular switches Because they switch between an active ON state and an inactive OFF state depending on whether they are bound to GTP or GDP.

What happens when RAS is in its GTP-bound state?

RAS becomes active and interacts with other proteins in pro-growth signaling networks.

What happens to RAS when GTP is hydrolyzed?

GTP is converted to GDP, causing RAS to switch from the ON state to the OFF state.

How do guanine nucleotide exchange factors (GEFs) reactivate RAS?

GEFs turn RAS back on by binding to it and removing GDP, allowing cellular GTP to spontaneously bind to RAS.

What is the functional consequence of mutations that impair RAS's ability to hydrolyze GTP or impair a GAP's ability to enhance hydrolysis?

They lead to a permanent "always ON" state that promotes cancerous growth.

What is the G12C mutation in RAS, and what is its context in cancer?

It is a mutation that locks RAS in an active state, is caused by smoking, and is particularly prevalent in lung cancers.

How do G-protein coupled receptors (GPCRs) act as GEFs in signaling pathways?

When a ligand binds to the GPCR, the receptor changes shape. This allows it to help remove GDP from RAS and replace it with GTP, turning RAS ON.

What are the three main components of a Proteolysis Targeting Chimera (PROTAC) drug?

A warhead ,

a variable length linker,

E3 ligase binding drug (such as Von Hippel-Lindau)

What is the primary mechanism and advantage of a PROTAC compared to a traditional inhibitor?

The target protein is not just inhibited; it is completely gotten rid of (degraded) via the proteasome

What functional requirement does a PROTAC warhead have regarding the target protein?

The warhead only has to bind the target protein, not inhibit it or change its biological function,

What are Transmissible Spongiform Encephalopathies (TSEs), and what constitutes their infectious agent?

Fatal, progressive, degenerative diseases of the central nervous system, and their infectious agent is a prion—an alternate conformation of a normal brain protein.

What are the different transmission modes and disease origins associated with TSEs?

via direct implantation of diseased brain tissue,

consuming infected tissue,

transplanted organs,

growth hormones from infected donors (iatrogenic means),

occur sporadically (with no mutation in PrP) or be linked to genetic mutations in PrP

What are some notable animal examples of transmissible spongiform encephalopathies?

: Scrapie (sheep and goats), bovine spongiform encephalopathy (BSE / 'mad cow disease'), transmissible mink encephalopathy, and elk/deer wasting disease.

What are some notable human examples of transmissible spongiform encephalopathies?

Kuru, Creutzfeldt-Jakob disease (CJD), Gerstmann-Sträussler-Scheinker (GSS) disease, and fatal familial insomnia (FFI).

What happens to TSE infectivity after irradiation or heat treatment?

Infectivity is not completely eliminated by normal irradiation or heat.

What substances can reduce TSE infectivity?

Protein-denaturing chemicals such as NaOH, SDS

What are examples of inherited prion diseases?

Fatal familial insomnia (FFI) and familial Creutzfeldt-Jakob disease (CJD).

What do mutations tell us about prion diseases?

They show that changes in the prion protein gene can cause inherited prion diseases.

What did Prusiner and his colleagues isolate in 1982?

A protein fragment that could cause spongiform encephalopathy. No nucleic acid was detected with it.

What did Prusiner's discovery support?

The idea that an infectious protein, called a prion, can cause disease without DNA or RNA.

What is PrPᶜ?

The normal cellular form of the prion protein. attached to the outside of the cell membrane.

Charactertiscs of PrPᶜ

non-pathogenic (normal).

Soluble.

protease-sensitive.

Mostly α-helix.

Charactertiscs of PrPsᶜ

pathogenic.

Insoluble.

protease-insensitive (resistant).

β-sheet and less α-helix than PrPᶜ.

How does the formation of a "nucleus" or "seed" drive the progression of prion pathology?

When multiple PrP^Sc} molecules come into contact, they bind via a beta-sheet interaction to form a stable complex that does not readily dissociate, serving as a template that rapidly recruits and makes more

Why are TSEs normally very rare in healthy people?

Because PrPᶜ is much more stable than PrPˢᶜ, so there usually isn't enough PrPˢᶜ to form a seed that starts the disease process.

How do inherited TSEs promote disease differently from sporadic TSEs?

Mutations make it easier for PrPᶜ to change into PrPˢᶜ, increasing the chance that a harmful seed will form.

What do transgenic mouse studies show about the protein-only model of prion diseases?

They show that more copies of the PrP gene lead to faster prion replication and earlier disease, supporting the protein-only model.

How many PrP gene copies does a wild-type mouse have?

2 copies.

How long does a wild-type mouse take to die?

130 days.

How fast is prion replication in wild-type mice?

Moderate (++).

How many PrP gene copies does a knockout mouse have?

0 copies. Disease is extremely slow or absent, with death taking more than 475 days.

What is prion replication like in knockout mice?

Virtually no replication (0*).

How many PrP gene copies does a heterozygous mouse have?

1 copy.

How long does a heterozygous mouse take to die?

About 220 days.

How fast is prion replication in heterozygous mice?

Slow (+).

How many PrP gene copies does the transgenic mouse have?

More than 50 copies.

What happens when a mouse has more than 50 PrP gene copies?

Prion replication becomes much faster, and disease develops much sooner.

How long does the transgenic mouse take to die?

About 55 days.

How fast is prion replication in transgenic mice?

Very rapid (+++).

What do Scott et al.'s experiments show about the species barrier in prion transmission?

Greater similarity = easier transmission.

How does a wild-type hamster respond to hamster amyloid vs mouse?

It develops disease quickly, in about 90 days vs Disease is much slower, taking about 450 days, showing a strong species barrier.

How does a wild-type mouse respond to mouse amyloid vs hamster

It develops disease in about 130 days vs It remains alive/unaffected, showing a strong species barrier.

What does the transgenic mouse express?

Two hamster PrP genes.

How does the transgenic mouse respond to hamster amyloid?

It develops disease quickly, in about 80 days.

How does the transgenic mouse respond to mouse amyloid?

It remains alive/unaffected.

: What does the amyloid cascade hypothesis say about Alzheimer's disease?

Abeta42 forms aggregates that contribute to neuron death and Alzheimer's disease.

How is Abeta42 produced?

APP is cut by secretase enzymes, producing Abeta42.

Why does Abeta42 aggregate more easily than Abeta40?

Abeta42 has an extra Ile-Ala sequence at its end.

What is the strongest genetic risk factor for Alzheimer's disease?

The ApoE allele.

Which ApoE allele increases Alzheimer's risk?

ApoE4.

Which ApoE allele is associated with protection from Alzheimer's?

ApoE2.

How does ApoE4 increase Alzheimer's risk?

: ApoE4 binds to Abeta42 oligomers and promotes disease.

What is the secretase complex?

A protein complex in the cell membrane that cuts APP and other proteins.

What does the alpha-secretase pathway do?

It cuts inside the Aβ region, so Aβ42 cannot be produced.

What does the beta-secretase (BACE1) pathway do?

It cuts APP outside the Aβ region, leaving a fragment that can be cut by gamma-secretase.

What does gamma-secretase do?

It cuts the remaining APP fragment to produce Aβ40 or Aβ42.

What are the main components of gamma-secretase?

Presenilin, nicastrin, APH-1, and PS2.

: Why did beta- and gamma-secretase inhibitors cause problems in clinical trials?

They also cut other important proteins, causing serious side effects and toxicity.

How do newer drugs target gamma-secretase?

They modulate gamma-secretase instead of completely blocking it. It shifts gamma-secretase toward producing more Aβ40 and less Aβ42.

What type of structure does the Aβ42 fibril have?

Parallel, in-register β-sheets. The peptide strands all point in the same direction.

What interactions stabilize the Aβ42 fibril?

Side-chain interactions and hydrogen bonds stabilize the fibril.

What type of side-chain interactions are common in Aβ42 fibrils?

Many are hydrophobic interactions.

How many intertwined filaments make up a full-length Aβ42 fibril?

Two intertwined filaments.

What does Aducanumab target?

Highly aggregated Aβ42 fibrils.

What does Lecanemab target?

: Pre-fibrillar Aβ oligomers.

What are AD plaques mostly made of?

Aβ42.

What happens to the Aβ42:Aβ40 ratio in Alzheimer's disease?

It is generally higher.

How can APP gene duplication lead to Alzheimer's disease?

Extra copies of APP can increase Aβ production and contribute to early-onset AD.

Where are known AD-related genetic mutations found?

In APP, secretase-related genes, or APOE.

How do secretase mutations affect the Aβ42:Aβ40 ratio?

Mutations linked to early-onset AD increase the ratio, while protective mutations decrease the ratio.