advanced exam 2 lecture 16

signal transduction; mito intro

signal transduction

Conversion of a signal from outside the cell into a functional change within the cell, typically through many steps from receipt of the signal to the response.

Contact-dependent, paracrine, synaptic, and endocrine.

List the four types of intercellular signaling.

contact-dependent signaling

Cells must be in direct membrane contact: a ligand on one membrane binds a receptor on the other.

paracrine signaling

Mediators are released into the extracellular space and act on neighboring cells.

synaptic signaling

Neurons transmit signals electrically along axons and release neurotransmitters at chemical synapses.

endocrine signaling

Endocrine cells release hormones into the bloodstream for distribution throughout the body, so target cells can be very far away.

Proteins, peptides, amino acids, nucleotides, steroids, retinoids, fatty acid derivatives, and dissolved gases.

What types of molecules can act as extracellular signals (ligands)?

Usually on the cell surface, but they can be intracellular.

Where are receptors for signaling molecules usually located?

phosphorylation of effector proteins and stimulation of cell secretions

rapid intracellular signaling responses

cell growth and division resulting from changes in gene expression

skow intracellular signaling responses

Different cells express different receptors; receptors are very specific for their targets; and pathways are multi-step with thresholds at each step.

How do cells achieve specificity amid environmental noise?

E.g., acetylcholine decreases firing rate in heart pacemaker cells, causes secretion in salivary gland cells, and causes contraction in skeletal muscle cells.

example of the same ligand causing different responses in different cells

Ion channel-coupled receptors, G-protein coupled receptors (GPCRs), and enzyme-coupled receptors.

What are the three major classes of cell-surface receptors?

The channel is typically closed and briefly opens when a signaling molecule (e.g., a neurotransmitter) binds, letting ions cross the membrane. Examples: glutamate receptors, including the NMDA receptor.

How do ion channel-coupled receptors work?

They indirectly regulate a separate membrane-bound enzyme or ion channel via a GTP-binding protein: receptor activation, GTP binds, G protein active, enzyme activation. The G protein is "on" with GTP and "off" with GDP.

How do G-protein coupled receptors work?

The concentration of small intracellular signaling molecules (second messengers) and the ion permeability of the plasma membrane.

What can GPCR signaling change in the cell?

A wide variety including light, odors, hormones, and neurotransmitters; it is a very large group of cell surface receptors.

What signals do GPCRs respond to?

They either function as enzymes or associate directly with enzymes they activate. They are usually single-pass transmembrane proteins with the ligand-binding site outside and the catalytic or enzyme-binding site inside; many are protein kinases.

How do enzyme-coupled receptors work?

Receptor tyrosine kinases bind growth factors and stimulate the RAS-MAPK pathway (cell growth, division, differentiation, wound healing, tissue repair, and integrin signaling/cell migration).

What is an example of an enzyme-coupled receptor pathway?

molecular switches

Intracellular proteins that relay signals by generating second messengers (cAMP, Ca2+) or by activating the next signaling/effector protein; most are activated or inactivated by phosphorylation.

protein kinases (serine/threonine or tyrosine)

add phosphate groups to specific amino acids

phosphatases

remove phosphate groups

WNT/SHH

signaling pathways: embryonic development - body axis patterning

TGF-beta

signaling pathways: growth, apoptosis, immune response.

NFkB

signaling pathways: responds to cellular stress (antigens, UV, heavy metals).

JAK-STAT

signaling pathways: responds to interleukins and interferons

Ras-MAPK and mTOR

signaling pathways: proliferation, apoptosis, differentiation

Ras

A monomeric GTPase attached to the lipid membrane that mediates signaling by most receptor tyrosine kinases. Active when bound to GTP.

HRAS, KRAS, and NRAS.

What are the three human forms of Ras?

Somatic defects are involved in cancer (~30% of human tumors have hyperactive Ras); germline defects cause RASopathies (NF1, Noonan, Costello, CFC).

What diseases involve the Ras pathway?

1. RTK activation by extracellular ligand

2. adapter proteins recruit proteins that activate Ras (GDP replaced by GTP)

3. active Ras recruits and activates Raf (MAPKKK)

4. Raf activates Mek (MAPKK)

5. Mek activates Erk (MAPK).

Describe the order of the RAS-MAPK cascade.

Ras-GEFs (guanine nucleotide exchange factors) swap GDP for GTP to activate Ras.

Ras-GAPs (GTPase-activating proteins) increase GTP hydrolysis (GTP to GDP) to inactivate Ras.

How is Ras activated and inactivated?

They are resistant to GAP-mediated GTPase stimulation, so there is little GTP hydrolysis and Ras stays bound to GTP (constitutively active). Often found in tumors of the pancreas, lung, colon, and thyroid.

Why are hyperactive Ras mutations oncogenic?

A-RAF, B-RAF, and C-RAF.

Somatic B-RAF variants (usually V600E missense) are found in up to 7% of tumors and constitutively stimulate Mek and Erk (oncogene).

A-RAF and C-RAF require extra activation steps and play less of a role in cancer.

What are the forms and cancer role of Raf?

Mek (MAPKK): MEK1 and MEK2, phosphorylated by Raf, which phosphorylates Erk.

Erk (MAPK): Erk1 and Erk2, which regulate protein activity (fast response) and gene expression via transcription factors (slow response).

What are the forms of Mek and Erk?

RASopathies

A group of syndromes resulting from errors in Ras-MAPK pathway

Craniofacial features, cardiac malformations, cutaneous/musculoskeletal (short stature)/ocular abnormalities, neurocognitive impairment, hypotonia, and increased cancer risk. Combined incidence is about 1/1000.

What features are shared among RASopathies?

Clinical trials of MEK inhibitors, based on knowledge of the pathway from cancer drug trials.

What treatments are being tried for RASopathies?

Multiple cafe au lait spots, axillary and inguinal freckling, multiple cutaneous neurofibromas, and iris Lisch nodules.

What are the skin findings in neurofibromatosis 1?

Learning difficulties (at least 50%), plexiform neurofibromas, optic nerve and other CNS gliomas, malignant peripheral nerve sheath tumors, tibial dysplasia, and vasculopathy.

What are other (non-skin) findings in NF1?

Autosomal dominant, about 50% de novo, with highly variable expressivity even within a family. Caused by NF1 (neurofibromin), a tumor suppressor that normally acts as a Ras-GAP; the mutant form cannot inactivate Ras.

What is the inheritance and cause of NF1?

Short stature, congenital heart defects (pulmonary valve stenosis, hypertrophic cardiomyopathy, ASD), and developmental delay.

What are the major findings in Noonan syndrome?

Broad or webbed neck, pectus carinatum/excavatum, cryptorchidism, wide-set downslanted eyes/ptosis, low-set posteriorly rotated ears, coagulation defects, lymphatic dysplasias, ocular abnormalities, and slightly increased childhood cancer risk.

What are other findings in Noonan syndrome?

Autosomal dominant, 25-70% de novo. Genes: PTPN11 (50%), SOS1 (13%), RAF1 (3-17%), KRAS (<5%), and NRAS, BRAF, MAP2K1 (each <1%).

What are the inheritance and genes of Noonan syndrome?

Short stature, ID, curly or sparse fine hair, diffuse hypotonia and joint laxity with ulnar deviation of wrists and fingers, macrocephaly, and cardiac involvement (hypertrophy, valvar pulmonic stenosis, arrhythmia).

What are the major findings in Costello syndrome?

15% lifetime risk of malignant tumors (rhabdomyosarcoma and neuroblastoma in young children; transitional cell carcinoma of the bladder in adolescents/young adults).

Autosomal dominant, almost always de novo, caused by HRAS; rare recurrence via gonadal mosaicism.

What cancer risks and genetics are associated with Costello syndrome?

FGFR3 encodes a fibroblast growth factor receptor acting upstream of RAS-MAPK. Activating variants permanently turn the receptor on, constantly inhibiting chondrocyte development and reducing bone growth.

What does FGFR3 do and how does it cause achondroplasia?

Achondroplasia (ACH), hypochondroplasia (HCH), thanatophoric dysplasia (TDI/TDII), and several craniosynostosis syndromes.

What conditions are caused by FGFR3 variants?

A few FGFR3 variants that change a single amino acid; over 95% are G380R.

What is the common cause of achondroplasia?

Short stature, rhizomelic (disproportionately short) arms and legs, bowed legs, large head with prominent forehead, flattened nasal bridge, crowded teeth, trident hands, joint stiffness/pain, spinal stenosis, sleep apnea, and ear infections.

What are the features of achondroplasia?

Voxzogo

An FDA-approved drug for achondroplasia that blocks the inhibition of bone growth and increases growth rate, started as early as 6 months.

Part of the Ras GTPase superfamily that regulates the cytoskeleton (shape, polarity, motility, adhesion), cell-cycle progression, and transcription (also signaling via GTP binding and hydrolysis)

what is the Rho family?

1. A signaling molecule activates a receptor tyrosine kinase

2. which activates PI 3-kinase

3. which activates two protein kinases (PDK1 and AKT/PKB)

4. which activate target proteins including mTOR (acting a few steps downstream of PI3K) to promote cell growth.

Describe the PI3K-mTOR pathway.

It phosphorylates phosphatidylinositol to PI(3,4,5)P3, which binds several intracellular signaling proteins and brings them together to assemble a signaling complex.

What does activated PI 3-kinase do to lipids?

Energy factories: most of the cell's ATP is made there (about 30 ATP/glucose vs 2 ATP/glucose from glycolysis). A high percentage of mitochondrial mass is internal membranes where ATP production occurs.

What is the main function of mitochondria?

The outer membrane, the intermembrane space, the inner membrane, and the matrix.

What are the four regions of a mitochondrion?

Fatty acid oxidation and the citric acid cycle, which breaks down small molecules from fats and carbohydrates to produce high-energy electrons.

What happens in the mitochondrial matrix?

Oxidative phosphorylation: high-energy electrons are transferred through a series of compounds, using their energy to generate a proton gradient across the membrane, ultimately producing ATP.

What happens at the inner mitochondrial membrane?

• Breaks down carbohydrates and fatty acids to acetyl CoA and then CO2

• electron transfer to convert NAD+ to NADH.

• NADH electrons go to the ETC and ultimately to O2, forming water and ATP.

citric acid (Krebs) cycle

Citrate (fatty acids and sterols)

Alpha-ketoglutarate (amino acids and purines)

Succinyl CoA (porphyrins and heme)

Oxaloacetate (amino acids, purines, pyrimidines, and glucose)

What biosynthetic precursors does the citric acid cycle provide?

1. Each NADH carries a pair of high-energy electrons that reduce O2 to H2O.

2. Electrons flow through protein complexes, pumping protons across the membrane;

3. the proton gradient powers ATP synthesis (Complex V).

How does OXPHOS work overall?

Complexes I, III, and IV. Complex II brings in FADH2 from the citric acid cycle without pumping protons.

Which ETC complexes pump protons?

NADH, cytochrome C, and coenzyme Q (ubiquinone/ubiquinol); CoQ10 is the most common form in mammals.

What are the electron carriers in the ETC?

NADH dehydrogenase: a massive complex (>900 kD, 46 subunits) encoded by both nuclear and mitochondrial DNA. It transfers electrons from NADH to coenzyme Q, pumping four protons from the matrix to the intermembrane space.

What is Complex I?

Succinate dehydrogenase: functions in both the citric acid cycle and OXPHOS. Electrons from FADH2 go to CoQ, with no protons pumped.

What is Complex II?

Cytochrome C reductase: ubiquinol (QH2) transfers electrons to it (and on to cytochrome C), with two protons transferred.

What is Complex III?

Cytochrome c oxidase: the last step, where electrons are transferred to oxygen and 2 protons are pumped out.

What is Complex IV?

ATP synthase: makes ATP using the proton gradient (more than 23 subunits). A "stick" embedded in the membrane contains the proton channel, and a "ball" protruding into the matrix has catalytic activity.

What is Complex V?

chemiosmotic hypothesis

The proton gradient developed by electron transfer stores potential energy (the proton motive force) that is used to make ATP. It has a chemical component (pH difference) and an electrical component (intermembrane space side is positive).

Protons moving from high to low concentration are coupled to conformational changes in the enzyme subunits, resulting in ATP synthesis.

How does ATP synthase make ATP?