Molecules and Cells in Human Disease

Cancer is driven by molecular changes in signaling proteins

Either by activating mutations in oncogenes or inactivating mutations in tumor suppressor genes

Src

Tyrosine kinase that was the first oncogene identified

Protein kinases

Catalyse a chemical reaction used in cells as a form of information transfer. This can control key enzymes in cell proliferation and can turn target proteins on/off

Kinase inhibition

Small molecules can be developed that inhibit enzymes like kinases

Philadelphia chromosome

Common translocation in chronic myologenous leukemia patients that generates a fusion BCR-ABL gene. ABL is a tyrosine kinase so imatinib is used to inhibit BCR-ABL.

Cell death regulation defects cause diseases

Neurodegenerative diseases involve loss of cells like neurons. Your immune system can kill your own cells. Cancer cells are resistant to apoptosis. Ischemia results in death of surrounding cells

Necrotic cell

Swelling of cell, loss of plasma membrane integrity, releases content into surrounding tissues.

Apoptotic cell

Shrinkage of cell, maintenance of plasma membrane integrity, and the cell is phagocytosed by macrophages.

Autophagy cells

Maintenance of plasma membrane integrity. Organelles are broken down and reused as nutrients

Cellular stress causes apoptosis

It protects from infected, damaged, or unwanted cells. Apoptosis will minimize collateral damage to the tissue. It achieves this as apoptotic cells are phagocytosed by other cells

Apoptosis morphological changes

Cell shrinkage, cytoskeleton collapses, loss of nuclear membrane, chromatin condenses and DNA is cleaved into fragments. The membrane blebs which breaks off into apoptotic bodies and the cell surface alters to attract phagocytes. This requires energy in form of ATP

Signalling pathway resulting in biological effect

A signal is detected by a receptor which activates enzyme 1 which activates enzyme 2 and causes a biological effect

Signalling pathway resulting in transcription factor

A signal is detected by a receptor and activates kinase 1 which activates kinase 2. This gets phosphorylated and so does the then activated transcription factor

Caspases

Endopeptidases which cleave within a protein. They cleave specific substrates and sites. They cleave proteins after an aspartate in the substrate

Caspase substrate cleavage

Substrate cleavage often results in activation of function of CAD. Makes caspase signalling enzymes acting on downstream effectors

CAD

Caspase activated DNAase

On/Off point for apoptosis

By activation of caspases

Executioner caspases

Have a small pro-domain

Initiator capsases

Have a large pro-domain

Caspases are expressed as inactive proenzymes

Become activated during apoptosis

Initiator caspases activate executioner caspases by proteolytic cleavage.

A signal is detected by a receptor and activates the initiator caspase which then activates the executioner caspase. This has an effect on the biological substrate.

Executioner dimers

Proenzyme executioner caspases are dimers. Cleavage by initiator caspases causes rearrangement of the active site

Initiator dimers

Exist as monomers but need to dimerize to activate via the pro-domain.

Caspase 9 is activated by the apoptosome

The apoptosome is a large multimeric complex.

APAF 1

A cytosolic monomer that assembles into a heptameter after binding to cytochrome c. The heptameric APAF1 can bind caspase 9 through CARD. Caspase 9 is now concentrated enough to dimerize

Cytochrome c release

It is released from the mitochondria right before caspase activation. It is the point of no return for a cell and is controlled by Bcl-2 protein family

MOMP

Bcl-2 family of proteins controls apoptosis by mediated mitochondrial outer membrane permeablisation

Bcl-2

Identified in follicular lymphoma - a cancer of B cells. It was the first identified in a family of related proteins. Some Bcl-2 proteins keep cells alive whilst some like Bax promote apoptosis

Bax

Bcl-2 associated X protein functions to promote cell death and antagonise Bcl-2

Anti-apoptotic Bcl-2

Have shared domain structure and block OMM permeabilisation. Includes Bcl-2, Bbl-X, Bbl-W, and Mcl-1

Pro-apoptotic Bcl-2s

Make holes in the OMM. Include Bax, Bak, and Bok

BH3-only proteins

Regulate pro and anti apoptotic proteins. They are activated by a range of damage signals a cell can receive.

Pro and anti apoptotic Bcl-2 proteins interact

On the OMM to regulate MOMP. BH3-only proteins will either inhibit anti-apoptotic proteins or activate pro-apoptotic proteins

Mutations affect Bcl-2 threshold

Mutations in oncogenes and tumour suppressor genes can shift the threshold for Bcl-2 regulation of MOMP.

BH3-only protein regulation

Some are regulated by growth factor signalling. Some are regulated by DNA damage

PUMA regulation by p53

DNA damage can regulate PUMA by p53, a tumour suppressor which gets mutated in 50% of cancers. Chemotherapy often tries to activate BH3-only proteins to kill cancer cells but if p53 is mutated then the tumour cells don't respond

Mimic BH3 domain

NMR spectroscopy used to identify small molecules that bound in the same region as the BH3 domain. Compound joined to generate high affinity binding. Lead to a compound being produced which was the first BH3-mimetic

Further modification

To generate an orally active variant, and further to increase target specificity and reduce side effects.

Caspase 8 activation

By extrinsic signals like receptors on the cell surface. Needs death domain and death effector domain

CD95

Also called Fas it binds to Fas ligand. It is a trimeric receptor with a single transmembrane spanning domain

FADD

Fas associated death domain containing protein

DISC

Death inducing signalling complex

Caspase 8 cleavage

It undergoes autocatalytic cleavage to stabilise the active dimer

ALPS

Autoimmune condition where mutations mean there is an accumulation of T-lymphocytes which are normally controlled by apoptosis. Causes autoimmune symptoms and lymphoid tumours. Caused by inactivating mutations in CD95, FasL, or caspase 9

Non-apoptotic caspase activation

Same as apoptotic caspases but the signal and substrate are different

PAMPs

Pathogen associated molecular patterns recognise bacterial cell walls or microbial DNA

DAMPs

Damage associated molecular patterns

Necrosis characteristics

Cells and organelles swell, and the plasma membrane ruptures. This releases DAMPs so can induce inflammation

Repurfusion damage

Restoration of blood flow can affect movement of many ions and deplete cellular energy levels by activating enzymes like PARP. This can be even more damaging

Autophagy

Cells eating themselves. They form large vacuoles in the cytoplasm but the plasma membrane stays intact. There is no membrane blebbing, nuclear condensation, DNA fragmentation, or caspase activation. The autophagosome fuses with lysosomes and the contents get degraded

Nutrient deprivation

Can initiate autophagy as cells try to survive. Turns off mTOR so the ATG13 complex is active and causes autophagy

Cancer cells can enter autophagy

If resources are limited cells enter autophagy. Tumour cells resistant to apoptosis stay alive using autophagy by making tumour cells dormant and hard to kill. Removal of stress allows dormant cells to activate again

Mitophagy

Specialised form of autophagy that protects cells from damaged mitochondria

PINK and Parkin

Label defective mitochondria and target them to autophagosomes for destruction

Kidney connective tissue

Majority of the kidney is made of connective tissue, not cells

Cells and ECM are linked

Helps to resist mechanical forces and give mechanical integrity.

Collagen

Most abundant protein in the body. Needed to hold tissues together

Collagen isoforms

There are 28 different types but collagen I is most common and makes up over 80%. It makes up tendons and ligaments but different types are used to form different ECM structures

Collagen structure

All have a repeating sequence with proline, glycine, and another amino acid. These form chains and three chains form a tight helical structure

Proline

Causes tight wrapping of the three chains around each other

Glycine

Gives flexibility

Proline and glycine hydroxylation

Forms hydroxyproline and hydroxyglycine which form antrachain hydrogen bonds to stabilise the triple helix.

Hydroxylase

Need vitamin C as a cofactor. Converts proline or lysine to their hydroxy version

Basement membrane

Specialised to organise cell layers in metazoans. First recognisable ECM to form during metazoan development. Mutations in genes for early BM cause no development beyond this stage.

Basement membrane strength

It's very mechanically strong and diseases in it cause loss in mechanical strength.

Collagen IV

Specific to basement membranes. It assembles into 2D mats rather than fibres. This is because the N and C terminus don't get cleaved.

Collagen IV N terminus

Called 7S domain and is rich in cysteine and leucine. Domains interact to form a teamer of collagen trimers which contain extensive crosslinks holding the tetramer together.

Collagen IV C terminus

Called NC1 and is 230aa long. Drives triple helix formation and hexamer formation through end-end interactions

Crosslinking

4 triple helices crosslink through disulfide and lysine/hydroxylysine crosslinks

Interruptions

Interruptions in Gly-X-Y repeats cause increased flexibility and sites for cross linking

NC1 groove

All NC1 monomers have a groove which fits a finger-like projection from a collagen monomer

Collagen IV genes

There are 6 types of collagen IV genes which means there are 56 possible trimers. However only 3 are made

Glomerular BM

Specialised BM in the kidney that acts as a filter. It forms a thicker BM and is between the endothelial cells of blood vessels, and podocytes which are a specialised epithelial cell

Different parts of the kidney express different isoforms

The Bowmans capsule and globular basement membrane are made of different collagen isoforms. Bowmans has α1α1α2 whilst GBM has α3α4α5

Alport syndrome

Discovered by Albert Alport in 1927 as he witnessed physical symptoms of a hereditary form of nephritis. Found a loss of a3a4a5 in the 90s

Col α3α4α5 vs Col α1α1α2

α3α4α5 has more inter and intra chain crosslinks which protect it from increased pressure, and proteolysis from proteases in serum/the filtrate. If the wrong collagen is in the GBM it will undergo proteolytic degradation

Collagen mutations

X-linked Alport's are most common and caused by a mutation in the Col Iv α5 gene. It is severe in hemizygous males but varies depending on nature of mutations. It can be mild to non-existent in heterozygous females

Lysil Oxidase

Deaminates hydroxylysine and lysine to generate reactive aldehyde groups. These form covalent bonds with other lysines and hydroxylysines to form intermolecular crosslinks

Hydroxylysine and Hydroxyproline

Form intrachain hydrogen bonds that stabilise the triple helix

Collagen IV heterotrimers

α1α1α2

α3α4α5

α5α5α6

Most basement membranes

Made of collagen IV made of α1α1α2

BM protein isoforms

Different isoforms are found in different tissues and give these BM specific properties. In development α1α1α2 is expressed in the kidney but as the kidney matures it switches to expression of α3α4α5

Collagen IV gene pairs

Genes are in pairs because they have arisen through gene duplication. They share a common bi-directional promoter. 1 and 2, 3 and 4, and 5 and 6 are paired

α3 α4 mutations

Associated with autosomal recessive form of Alpert's

Collagen IV alport mutations

Can occur throughout the molecule but are mostly in the coiled domain.

Early onset disease mutations

Include large gene rearrangements. Non-sense mutations and splice sites lead to truncations

Late onset adult type mutations

Include missense mutations disrupting Gly-X-Y repeats. Conserved cysteine residues affect cross linking.

α3 knockout in mice

Develop progressive glomerulopathy. Phenocopies human autosomal dominant Alports

Alports mouse model

Been used to evaluate ACE inhibitors to reduce onset of symptoms. They reduce pressure in the glomerulus by vasodilation of efferent vessels to increase the life span of Alports mice.

Cure Alport's

Genetically engineered mice where α3 has been turned back on the glomerulus has been developed. Results show repair and return of function to defective GBM when added to Col IV

Laminin

A major component of BMs that self assembles into a network to present binding sites for cells. It is a high MW glycoprotein with a cruciform structure.

Laminin structure

Made of three protein chains from three separate genes. They assemble into the cruciform structure. The α chain is the longest, and then there are β and γ chains. The β and γ wrap around the α chain, with their N terminus forming the short arms. The globular part is the C terminus of the α chain

Globular domains

On the α chain and are spread out by spacers which are comprised of EGF repeats

N terminal globular domains

Promote polymerisation into a network. Laminin 1 spontaneously forms a network in vitro

α chain globular domain

At the C terminus there are 5 LG domains that interact with cell surface receptors

Integrin binding site

LG1 LG2 LG3

Dystroglycan and Heparin binding site

LG4 LG5

11 laminin genes

There are 5α 3β and 3γ chains but only 15 different heterotrimeric combinations are formed. Different laminin isoforms show tissue specific expression

α1β1γ1

Present in embryonic BM so if there is a mutation and these isoforms aren't expressed, the embryo will not continue developing

Pierson syndrome

Caused by loss of laminin β2 isoform. Causes congenital nephrotic syndrome progressing to end stage renal disease, eye abnormalities, and severe muscular hypotonia. Laminin 11 containing β2 is expressed in the GBM, eye, and synaptic BM so explains symptoms.