MCB I Block 3
CELL MEMBRANE DISORDERS
- A possible cancer treatment is to attack tumor glycocalyx
- Liposomes can be used for Drug Delivery – 3 components needed within the liposome:
- Drug (either hydrophilic, or lipid-soluble)
- Antibody (homing peptide) to bring the entire liposome to the antigen
- PEG – protect from phagocytes.
- BAD, BAX in cytosol → poke hole in mitochondria (MPTP) → cyt c leaks out → apoptosome forms → caspase cascade → apoptosis. Bcl-2 prevents Apoptosis.
- mtDNA vs nuclear DNA
- Mito imports: RNA polymerase, amino acids, mitochondrial DNA replication enzymes, mitochondrial aminoacyl-tRNA synthases, and mitochondrial ribosomal proteins
- Mito makes its own: rRNA subunits, tRNA
- Hsp70 and signal translocation sequence target protein to TOM → TIM → signal sequence cleaved → hsp60 help fold the rest inside mito.
- SOD2 vs. GPX
- SOD2 before peroxide step, GPX after peroxide step.
- Similar to Oxidase (before) and Catalase (after) in Peroxisome
MITOCHONDRIAL GENETIC DISORDERS
- Mitochondrial Diseases Summary
- Diseases involved in deleted nuclear DNA that goes into mito (Alpers, PEO) – only one that’s not maternally transmitted)
- Diseases involved in deleted parts of mtDNA (KSS, Pearsons)
- Diseases involved in mutations in tRNA genes (i.e. MELAS, MERFF)
- Diseases involved in protein coding nuclear genes (i.e. LHON, NARP, Leigh)
- Phosphorylation vs. Dephosphorylation of lamin
- Phosphorylation disassembles lamin (by cdk1 during prophase)
- Dephosphorylation assembles lamin
- Lamin A binds to :
- Architectural partners
- Chromatin partners
- Gene-regulatory partners
- Signalling partners
- snRNP vs. snoRNP
- snRNP used in slicing
- snoRNP used in rRNA assembly
- PARTS OF THE NUCLEOLUS
- Fibrillar Center – inactive DNA (has “fibrils” of DNA)
- Dense fibrillar components (pars fibrosa) – active DNA (dense because you also got rRNAs here and snoRNPs)
- Granular Component (pars granulosa) – maturing ribosomal precursor particles here (ribosomal subunits look like “granules”)
- Direct vs. Indirect Pathway
- Direct Pathway = protein Golgi directly to the apical/basolateral side of plasma membrane
- Indirect Pathway = protein goes from golgi to some random side, then gets sorted to apical/basolateral later.
- Transcytosis is indirect pathway!
- Pinocytosis = only one that all cells do (constitutive); remodeling of cytoskeleton; vesicles travel via microtubules.
- Clathrin used in exocytosis and receptor-mediated endocytosis (remember not in phagocytosis or pinocytosis)
- M6P Tag is put on in Cis-Golgi by GlcNAc phosphotransferase. Mnemonic: M-“cis”-P
- V-Type ATPase maintains H+ content inside lysosome so Acid Hydrolases can stay active.
- Lysosomal membrane lipids & proteins are heavily glycosylated to protect them from digestion
- Polyubiquitination vs. Monoubiquitination
- Polyubiquitination is used for proteins that are made in the cell to go to proteasome.
- Monoubiquitination is used for proteins that are endocytosed from outside the cell to go to lysosome.
- Mucopolysaccharidoses vs. Sphingolipidoses vs. Mucolipidoses
- Mucopolysaccharidoses – accumulate GAG
- Sphingolipidoses – accumulate sphingolipid
- Mucolipidoses – accumulate glycoprotein and glycolipid
- Melanosome = specialized lysosome
- Plasmalogens (made in peroxisome) are the most abundant phospholipids in myelin. The products of VLCFA beta oxidation are used for biosynthesis of cholesterol, bile acids, and other compounds.
- Zellweger vs. XALD
- Zellweger = peroxisome can’t import peroxisomal enzymes
- XALD = peroxisome can’t import VLCFA
SIGNAL TRANSDUCTION DISORDERS
- G-Proteins (and what they’re involved in):
- Ran = nuclear translocation
- Arf = vesicle budding
- Rab = vesicle fusion
- Ras = signal transduction (as in GRB2-Sos-Ras)
- Rho = actin polymerisation – stress fibers
- Rac = actin polymerisation – lamellipodia
- Cdc42 = actin polymerisation – filipodia
- RTK Signalling Defects:
- VEGF – Colorectal cancer, NSCLC
- EGFR (HER1) → Breast Cancer, Colorectal Cancer
- EGFR2 (HER2) → Breast Cancer
- G-PROTEIN RECEPTORS
- Gs, Gi: effector = adenyl cyclase, 2nd messenger= cAMP , activates = PKA
- Gq: effector = PLC, 2nd messenger= DAG/IP3, activates = PKC
- RTK RECEPTORS
- RTK – PLC: eff = PLC, 2nd = DAG/IP3, activates = PKC
- RTK – PI3K: eff = PI3K, 2nd= PIP2/3, activates PKB (i.e. AKT/PTEN)
- (PI3K: PIP2→PIP3 whereas PTEN: PIP3→PIP2 )
- (AKT is like PKB)
- Insulin signalling uses the PI3K-PKB pathway → effect vesicles with Glut4 receptors to merge with PM
- RTK – GRB2-SOS-Ras-MapK-TF(Fos/Jun)-GF Pathway
- NON-TK RECEPTORS
- Cytokine Receptors: 1st messenger= cytokine, Eff=JAK Phosph’d → STAT Phosph’d →STAT dimerized → transcription
- Integrin receptors: 1st messenger=collagen → Eff=ILK (integrin-linked kinase) or FAK (focal adhesion kinase) →prolif, growth, differentiation, apoptosis
- SERINE-THREONINE KINASE RECEPTOR
- Serine-Threonine Kinase Receptor: 1st messenger=TGFbeta → Eff=S/T domain → 2nd messenger → Smad2 or Smad3 dimerase with Smad4 → transcription
- INTRACELLULAR RECEPTORS
- Steroid Hormone Receptors: Steroid and Zinc → Receptor → HRE → transcription
- NO Receptors: Acetylecholine →NO synthase → make NO from arginine → NO then goes to smooth muscle cell to convert GTP to cGMP, which relaxes it.
- MTOC = place where microtubules grow from… so find in migrating cells, mitotic cells, nerve cells, base of cilia and flagella, etc.
- gamma-tubulin rings in MTOC “seed” sphere
- Basal bodies = MTOCs under cilia and flagella
- Dynactin complex attach dynein or kinesin to vesicle.
- Viruses can travel on dynein (+→-) or kinesin(-→+) to and fro cell body.
- MAP vs. Kinesin13
- MAP (microtubule associated protein) stabilizes microtubules. i.e. tau
- Kinesin 13 (catastrophe factor) destabilizes microtubules
- Notice you have 2 inner and 3 outer dynein arms in cilia.
- Colchicine: inhibit polymerization of MT → Cell Death. Treat Gout
- Vincristine/Vinblastine: inhibit polymerization of MT → Cell Death. Treat cancers w/ high mitotic index
- Paclitaxel (Taxol): inhibit depolymerization → blocks mitosis → Cell Death. Treat breast, prostate, lung cancer. ODDBALL (only one that inhibits depolymerization).
INTERMEDIATE FILAMENT DISORDERS
- MT vs. IF vs. Actin in assembly energy
- MT: need GTP to assemble
- IF: don’t need GTP or ATP to assemble
- Actin: need ATP to assemble
- Neurofilament defects found in Alzheimer’s, Parkinson’s, and ALS (amyotrophic lateral sclerosis)
- Different Types of Tangles:
- Glial Tangles = tangles of GFAP
- Lewy Bodies = tangles of neurofilament
- Neurofibrilary Tangles = hyperphosphorylated tau in AD (mitochondria!)
- MT vs Actin
- MT: + end = grow; – end = breakdown
- Actin: + end = fast grow; – end = slow grow
- Notice that ATP is in g-actin. ADP is in f-actin
- MT seed: tubulin gamma
- Seed for branched actin: ARP (actin-related protein) (- end)
- Seed for unbranched actin: Formin (+ end)
- Profilin exchanges ADP for ATP on the G-actin, making it ready for polymerization.
- High vs. Low concentration of ATP-G-Actin
- High conc ATP-G-Actin: monomers added at both ends, but faster at +
- Low conc ATP-G-Actin: monomers added to + and come off – → treadmilling
- TYPES OF ARFs!!
- ARFs that bind to G-actin
- Thymosin: binds G-actin→ prevents F-actin assembly (“thanatopsis”)
- Profilin: Binds G-actin → promotes F-actin assembly (“promotes filament”)
- ARFs that bind to G-actin
- ARFs that bind to F-actin
- Cofilin: disassemble at – end (disassemble) (“cuts filament”)
- Gelsolin: caps + end (prevent further growth) (“gel sol cap”)
- Besides being in muscles, actin are in:
- Microvilli – they just are.
- Stress Fibers – contractile bundles of actin
- Lamellipodia/Filipodia – contract to move cell
- Cytokinesis – contract to pinch cells apart
- Two types of cross-linking:
- To form parallel cables (“bundles”) –
- alpha actinin (contractile, because loose so myosin can insert in it, i.e. stress fibers),
- fimbrin (non-contractile, because dense, i.e. microvilli), villin
- To form filament webs (“gel”) –
- spectrin (remember in RBC),
- filamin (“filament”)
- To form parallel cables (“bundles”) –
- Stress fibers (remember they are contractile, so they are crosslinked by alpha-actinin) connect to ECM via focal adhesions (integrin). Remember they are used in cell signaling (collagen effect integrin →effect ILK or FAK → proliferation)
- Stress fibers vs. Microvilli
- Stress fibers: alpha-actinin and focal adhesions cross-linking/attaching actins to integrins (in ECM)
- Microvilli: Fimbrin and Villin cross-linking actins
- G-Proteins: Rho vs Rac vs Cdc42
- Rho = actin polymerisation (via formin “unbranched cables”) – stress fibers
- Rac = actin polymerisation (via WASP then arp2/3 because branched) – lamellipodia
- Cdc42 = actin polymerisation (via WASP then arp 2/3 because branched) – filipodia
- Mnemonic: “Rhostress,” “Raclam,” Cd. Fili”
- Drugs that Affect Actin Polymerisation:
- Cytochalasin D: bind to + end, ends polymerization, depolymerisation at – end
- Latrunculin: like sponge – soaks up free monomers, prevent polymerization
- Phalloidin: binds to filament, stops polymerization. Remember Red Texas Phalloidin?
- I has I head + calmodulin (“I” looks like a microvilli)
- II has II heads + essential + regulatory – II looks like squeezing (contractile)
- V has 2 heads + calmodulin (V for Vesicle!)
- Proteins that attach to Actin/Myosin
- Cap Z = cap at the Z disc
- Titin = pulls myosin tight
- Tropomodulin = “tropo minus end”
- CapZ vs. Tropomodulin
- CapZ @ + end
- Tropomodulin @ – end
- Z disc = CapZ + alpha-actinin
- Mechanism for Muscle Contraction
- ATP-binding detaches myosin head (the reasoning behind rigor mortis)
- ATP→ADP moves it into the myosin-binding site
- ADP-detachment does powerstroke
- Actin – dystrophin – (beta dystroglycan-GRB2-alpha dystroglycan) – laminin2
- Satellite Cell (in basal lamina of mature muscle) → myoblast → +myotube +myofibers → mature muscle
- Stem Cell “Side Population Cells” (in bone marrow) → differentiate into satellite cells
- Muscular Dystrophy → repair cannot keep up with damage → connective tissue + fat accumulation. Detect w/ blood test – find increased serum creatine kinase. Or do PCR deletion screening.
- Absence of dystrophin → loss of DAP (dystrophin associated proteins) at sarcolemma→Absence of physical link → fragile sarcolemma
- Dystrophin = largest gene therefore prone to many mutations. Binds actin cytoskeleton to sarcoglycan-dystroglycan complex.
| X-LINKED MUSCULAR DYSTROPHIES:
AUTOSOMAL MUSCULAR DYSTROPHIES:
- Muscular Dystrophy Treatments: physical therapy, pacemaker
- Treatment of DMD:
- Inject donor myoblast? Difficult because muscle cells don’t divide, so need to inject a lot.
- Step Cell Therapy – either upregulate utrophin (homologous to dystrophin but in fetus), or inject stem cells that would differentiate into myoblasts, or exon-skipping
EXTRACELLULAR MATRIX DISORDERS
- Matrix Metalloproteinases
- MMP I = Collagenase I (so breaks down collagen I)
- MMP II = Collagenase IV (so breaks down collagen IV, used by cell to invade basal lamina, as well as chemotaxis)
- Glycoprotein vs. Proteoglycan
- Glycoproteins = adhesive (Gl for Glue)
- Proteoglycan/GAG = space filler, compression (Pr for Press)
- Hyaluronan is a type of “space filler – produced during wound healing, which need a lot of cell-free space for repair cells to migrate. Its length is humongous – to create space filler! It is not covalently linked to core protein so it is not compact.
- Gel of Vitreous Humor = collagen suspended in hyaluronan + lots of water → ultimate shock absorber!
- Hyaluronan (a type of proteoglycan) vs. Other Proteoglycan
- Remember proteoglycan is usually a GAG + core protein
- Hyaluronan is a special exception! Is a GAG without core protein!
- Proteoglycan complexes can be Aggrecan (Cartilage) or Versican.
- Aggrecan proteoglycans attach to hyaluronan proteoglycan! (like paintbrushes on a string)
- After growth factor binds to Perlecan or Syndecan (heparan sulfate), it has a better and more convenient binding access to receptor on cell surface
- Collagen 6 cross-links Collagen 1
- Collagen 9 cross-links Collagen 2
- Fibrillin cross-links elastin
- Basal Lamina is a Specialized type of ECM:
- Integrin – Laminin – Perlecan/Entactin – Collagen IV
- ECM Composed of:
- Structural: collagen, elastin, fibrillin
- Glycoprotein Adhesives: laminin, fibronectin, entactin
- Proteoglycan Filler: proteoglycan (perlecan/syndecan), Hyaluron
- Two Organizations:
- Interstitial Matrix
- Basal Lamina
- Basal Lamina vs. Other types of ECM!
- Basal Lamina: Integrin II – (Glycoprotein) Laminin – Perlecan (Proteoglycan) – CollagenIV (Structure)
- Unspecialized ECM or stress fibers: Actin – Vinculin/Talin – Integrin I – (Glycoprotein) Fibronectin – Syndecan (Proteoglycan) – CollagenI/III/V (Structure)
- Fibronectin has these binding sites:
- For collagen
- For cell surface integrins (binding sequence is RGD)
- For heparin (Proteoglycan)
- For Fibrin
- Plasma membrane is weak, so need strong matrix on each side, hence ECM on the outside of the cell membrane and actin matrix on the inside.
- Interaction between fibronectin and actin
- Integrin creates interaction between actin (inside cell) with fibronectin (outside cell). So actin and fibronectin can align in same direction!
- So fibronectin is involved in cell spreading and migration (in the right direction), as it creates a “paste” of cells that are properly aligned. Embryogenesis and wound healing both need this direction and “paste” created by fibronectin.
- Fibronectin: 2 forms:
- Soluble = for blood coagulation
- Insoluble = for the “paste” that guide cell migration and growth (w/ alignment)
CELL-MATRIX INTERACTIONS DISORDERS
- Remember Vinculin is a focal adhesion (“foot hold”) protein, on the cytosolic side of the cell – connect actin filament of stress fibers to ECM
- Integrins attach glycoproteins via its RGD sequence
- Integrin is the telephone between ECM and Cell, but they’re also the connection. So if signals tell them to cluster, they will adhese the cell more and cause anchorage-dependent growth. The reverse will cause cell motility.
- Remember glycoprotein IIb and IIIa in Glanzmann’s Thrombasthenia? They are platelet integrins!!(β3)
- Focal Adhesion vs. Hemidesmosome:
- Focal Adhesion: actin stress fibers – vinculin/talin – integrin – fibronectin – collagen (I, III, V)
- Hemidesmosome: keratin – plectin (“plaque”) – integrin – laminin – collagen VII – Collagen IV (of basal lamina)
- Filapodia vs. Lamellipodia
- Notice filopodia are “filament feet”
- Lamellipodia are “lamellar feet”
- Old actin filaments are disassembled in back, and new ones are created in front → crawling of cell. Contraction at back propels cell body forward.
- It’s interesting how the MMP and TIMPs are stored in ECM (Basal Lamina), as if ECM has the keys to its own doors to allow cell to enter.
- Remember MMP is collagenase. MMPI digests Collagen I, MMPII digests Collagen IV (basal lamina)
CELL-CELL INTERACTIONS DISORDERS
- Cell Junctions:
- Tight Jxns Occlude
- Adherens and Desmosomes anchor
- Gap junctions communicate
- TADG = from apex to base = tight jxn → adherens jxn → desmosomes → gap jxn
- Adherens vs. Desmosomes
- Adherens – connect via actin. A for “Actin”
- Desmosomes – connect via intermediate filaments
- Cadherins = “Calcium-Dependent Adherens”
- T vs A vs D vs G
- Tight Junction: Claudin/Occludin – ZO – actin
- Adherins: Cadherin – catenin/alpha actinin – actin
- Desmosomes: Cadherins (desmoglein/desmocollin) – Plakoglobin/Desmoplakin – intermediate filaments
- Gap Junctions: 1000s channels = 2 connexons = 6 connexins
- So both desmosome and hemidesmosome have “plaques” (Have parts of it named plec-something or plak-something)
- You can tell which side of the cell is which by looking at where the zonula occludens is.
- Paracellular pathway = passage of free molecules between the cells. Zonula occludens is the door to this pathway.
- Bullous Pemphigoid vs. Pemphigus
- Bullous Pemphigoid – autoimmune attack on hemidesmosomes
- Pemphigus – autoimmune attack on desmosomal cadherins
- Gap Junction:
- Electrical: Peristalsis, Coordinated depolarization of heart muscles, uterine contraction
- Chemical: cAMP passes between cells so can respond the same way to a hormone
- High Calcium level (from damaged cell) → closes gap junction, to prevent damage.
- Connexin 26 Mutation (in cochlea): deafness
- Connexin 50 Mutation (in lens): cataracts → blindness
- Connexin 32 Mutation (in peripheral myelin): Chargot-Marie-Tooth → degeneration of peripheral nerves → atrophy of distant muscles, decreased deep tendon reflex, “foot drop,” “high-stepping gait,” “high-arched foot,” “hammer toes” (really mean nicknames for CMT!)
- Connexin 32 is unique in that it is actually an INTRAcellular gap junction, because it is the communication between the different onion layers of schwann cells.
- L-Selectin for leukocyte – for lymphocyte homing (addressins on lymphocytes attach to the L-selectins on lymphoid organs, homing)
- P-Selectin for platelet
- E-Selectin for endothelial cell – leukocyte can “roll” on the E-selectin in the bloodstream.
- Lymphocyte Rolling, Trapping, and Extravasation
- Rolls on E-selectin – WEAK ADHESION SO IT CAN ROLL
- Then, eventually rolls onto ICAM , which binds to the lymphocyte’s integrin – STRONG ADHESION, so it can stop and migrate through
- Collagen Structure:
- 3 residues/turn, L hand helix
- 3 stranded helix, R hand helix
- PXG repeats:
- Proline (P) – remember that it is bulky from Block 1?
- Hydroxyproline (X) – remember hydroxylated proline from Block 2?
- Glycine – connects all 3 strands with each other via H-bonding.
- MAKING A COLLAGEN:
- Make 2 a1 & 1 a2 chains + signal peptide (preprocollagen)
- Cleave signal peptide (procollagen)
- Hydroxylate proline and lysine (via Vitamin C)
- Glycosylate hydroxylysine
- Disulfide bond aligns chain and initiates triple helix formation from C→N
- H bonds form between hydroxyproline of procollagens.
- Procollagen Peptidase removes Propeptides on both C and N end.
- Collagen I = skin/bone/tendon
- Collagen II = Cartilage
- Collagen III = blood vessels, fetal skin
- Collagen IV (sheet-forming) = Basal Lamina
- Collagen VII = Anchoring
- Other Collagenopathies:
- Achondrogenesis type II
- Kniest dysplasia – dwarf, enlarged joints
- Spondyloepimetaphyseal dysplasia
- Stickler syndrome – underdeveloped facial bones
- Campomelic Dysplasia – Mutation in SOX9 → decrease Collagen II
- Thanatophoric Dysplasia – born dead. FGFR3 mutation, like achondroplasia
- Hypochondroplasia – also FGFR3 mutation, like achondroplasia